Antibodies binding to C5aR2 membrane protein and preparation methods thereof

Abstract

The present invention provides an antibody that binds to the C5aR2 membrane protein, as well as methods for preparing and using the antibody. This invention utilizes VLPs to display the C5aR2 membrane protein or its epitope for the first time. Through mammalian cell expression, the full-length membrane protein and its epitope undergo functionally significant post-translational modifications. Immunizations using this antigenic protein yielded monoclonal antibodies with high binding affinity for C5aR2. The antibodies of the present invention can be used in various assays, including ELISA and FACS, and have potential for antibody drug development.

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 within the G protein-coupled receptor (GPCR) superfamily. C5aR1 and C5aR2 are two subtypes of the C5aR complement receptor, co-expressed on a variety of cell types, including neutrophils, macrophages, and astrocytes. Both are receptors for the inflammatory mediator complement C5a. C5aR1 and C5aR2 bind to C5a with comparable affinity, with C5aR2 binding to C5a-des-Arg with higher affinity than C5aR1. C5aR1 is a typical GPCR, mediating downstream signaling pathways through G protein-coupled activation or G protein-independent β-arrestin signaling. C5aR2, on the other hand, is an atypical GPCR that lacks G protein-coupled activity and exclusively signals through β-arrestins. The third and seventh transmembrane regions of C5aR2 lack the conserved DRY and NPXXY motifs, but instead contain D131LC and N287PLMF. Its intracellular third domain is truncated, and these structural features are likely to have a significant impact on its function. C5aR2 is a hot topic in drug development for inflammatory diseases. Other studies have shown that blocking antibodies targeting C5aR2 can reduce the number of cancer stem cells and enhance tumor chemotherapy sensitivity in the field of cancer treatment [1,2].

[0003] Due to the low expression level of GPCR membrane proteins, it is difficult to prepare C5aR2 antigen proteins for immunization and antibody screening. VLPs displaying the full-length membrane protein or specific membrane protein epitopes are excellent antigens for immunization. Envelope VLPs display the full-length membrane protein in its native conformation and act as immune adjuvants, stimulating a host immune response against the membrane protein and facilitating the screening of membrane protein-specific antibodies. Non-enveloped VLPs display key epitopes of the membrane protein, facilitating the activation of a host immune response against specific epitopes, thereby screening for functional antibodies. The development of antibodies that bind to the C5aR2 membrane protein can serve as a tool for GPCR research and also has potential for drug development. Summary of the Invention

[0004] The object of the present invention is to provide an antibody that binds 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 shown in SEQ ID NO: 7; H-CDR2 shown in SEQ ID NO: 8; H-CDR3 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 shown in SEQ ID NO: 17; H-CDR2 shown in SEQ ID NO: 18; H-CDR3 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 with a sequence shown in LVS; and L-CDR3 shown in SEQ ID NO: 24.

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

[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 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.

[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 to promote 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 a 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 embodiment, 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 according to 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 group consisting of DNA, RNA, viral vector, plasmid, transposon, other gene transfer systems, 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, comprising:

[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 group consisting of 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, detection plate or detection kit for detecting C5aR2 molecules;

[0065] (2) Drugs for preventing and / or treating diseases or conditions 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 immunoblotting (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, 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 is provided, 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 a 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 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;

[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 VLPs to multivalently display C5aR2 protein or C5aR2 protein epitopes and expressing them in mammalian cells to obtain C5aR2 antigen-VLPs 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, the spleen of the immunized animal is collected to prepare hybridoma clones;

[0083] (S4) performing preliminary screening of hybridoma clones using the C5aR2 antigen-VLP to obtain antigen-binding-positive hybridoma clones;

[0084] (S5) negatively screening the clones that are positive for antigen binding using empty VLPs without C5aR2 antigen to select positive hybridoma clones that only bind to C5aR2 antigen;

[0085] (S6) limiting dilution of the positive hybridoma clone that binds only to the C5aR2 antigen to a monoclonal state, repeatedly verifying the binding of the monoclonal hybridoma cell supernatant to the C5aR2 antigen, thereby obtaining a monoclonal hybridoma cell line capable of producing an anti-C5aR2 antibody that specifically binds to the C5aR2 antigen.

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

[0087] In another preferred example, the N-terminal sequence of human C5aR2 has the amino acid sequence 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) both adopt 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 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, or the 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 described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1 Figure 1 shows ELISA assays of antisera obtained from immunization with the C5aR2 antigen and antigenic proteins. A shows the binding of antisera obtained from immunization with full-length human C5aR2 / VLP to both full-length human C5aR2 / VLP and human C5aR2 NTD-VLP; B shows the binding of antisera obtained from immunization with human C5aR2 NTD-VLP to both antigens.

[0097] Figure 2 Figure 1 shows the binding of 17A2D9 and 30F10E9 recombinant antibodies to human and cynomolgus monkey C5aR2. 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, as well as C5a ligand, to human C5aR2NTD-VLPs is shown.

[0099] Figure 4 The results show that the 17A2D9 and 30F10E9 recombinant antibodies compete with the C5a ligand for binding to human C5aR2NTD-VLP.

[0100] Figure 5 Figure 1 shows the results of FACS flow cytometric analysis of recombinant monoclonal antibodies binding to Hela cells and competing with the C5a ligand. A shows the results of 17A2D9 recombinant antibody binding to Hela cells and competing with the C5a ligand; B shows the results of 30F10E9 recombinant antibody binding to Hela cells and competing with the C5a ligand. DETAILED DESCRIPTION

[0101] After extensive and in-depth research, extensive screening, and validation, the inventors ultimately obtained two antibodies that bind to the membrane protein C5aR2. They also proposed, for the first time, a method for preparing antibodies that specifically bind to the C5aR2 membrane protein by immunizing animals using VLPs displaying the C5aR2 membrane protein or its epitopes as antigens. The present invention employed enveloped VLPs displaying the full-length human C5aR2 membrane protein and non-enveloped VLPs displaying the N-terminal sequence of the membrane protein C5aR2, respectively, to generate two high-affinity murine monoclonal antibodies, 17A2D9 and 30F10E3, that bind to different epitopes of the C5aR2 membrane protein. Both antibodies exhibit cross-species binding activity to human and monkey C5aR2 membrane proteins and can be used for membrane protein detection using Elisa and FACS. The murine monoclonal antibody 30F10E3 competitively blocks the binding of the ligand C5a to the receptor human C5aR2 membrane protein.

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

[0103] the term

[0104] In order to make the present invention easier to understand, certain 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 generally 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 change. 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 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 "optionally" 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 regions of a specific sequence may have but need not have, and may have 1, 2, or 3.

[0109] As used herein, "sequence identity" 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 a sequence described herein and a sequence to which it is 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%, and 100%.

[0110] As used herein, the term "non-enveloped VLP multivalent display" or "non-enveloped VLP display" refers to the fusion of antigen epitopes to VLP (Virus Like Particle) capsid monomer subunits through structural design, expression in mammalian cells, self-assembly of VLP monomers to form VLP particles and display of antigen epitopes on the VLP surface.

[0111] As used herein, the term "enveloped VLP multivalent display" or "enveloped VLP display" refers to the expression of enveloped VLPs (Enveloped VLPs) in mammalian HEK293 cells. After the protein subunits assemble into particles, the particles bud from the cells, bringing out the recombinantly expressed full-length membrane protein on the cell surface and displaying it on the particle surface, forming membrane protein / VLP (lipoparticles) (see CN117003876B). C5aR2 / VLPs prepared using this method display multi-molecule, conformationally intact C5aR2 membrane protein directly on the VLP surface without the need for additional surfactants. This is a method for obtaining membrane proteins, and VLPs act as immune adjuvants. C5aR2 / VLPs are suitable for immunization as membrane protein antigens.

[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 antibody variable region 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 four sections of FR sequences and three sections of CDR sequences arranged at intervals (see Kabat et al., NIH Publ. No. 91-3242, Volume 1, 647-669 pages (1991)). The sequence and spatial structure conformation of the heavy chain and light chain variable regions determine the specific binding of the antibody to the antigen epitope. The antibody constant region 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 chains" of vertebrate antibodies (immunoglobulins) can be classified into either κ or λ based on the amino acid sequence of their constant regions. Immunoglobulins can be divided into five main classes (IgA, IgD, IgE, IgG, and IgM) based on the amino acid sequence of their heavy chain constant regions, as well as antibody subtypes (isotypes). For example, mouse IgG includes IgG1, IgG2a, and IgG2b subtypes. The subunit structures and three-dimensional configurations of different immunoglobulin classes are well known 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 character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring any particular method for production of the antibody.

[0117] The present invention also includes monoclonal antibodies having the corresponding amino acid sequences of the anti-C5aR2 monoclonal antibodies, monoclonal antibodies having the variable region chains of the anti-C5aR2 monoclonal antibodies, 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 light and heavy chains containing variable regions (complementarity determining regions, CDRs), as long as the variable regions are identical to or have at least 90% homology, preferably at least 95% homology, with the variable regions of the light and heavy chains of the present invention.

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

[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 at the hinge region; and (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 with 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 the 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 murine antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies can be prepared using recombinant DNA techniques well known in the art. The term "murine antibody" as used herein refers to a monoclonal antibody against the C5aR2 membrane protein prepared using the knowledge and skill of the art.

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

[0127] As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably. The terms "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 contributes 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, the present invention provides an anti-C5aR2 antibody or antigen-binding fragment thereof. Two anti-C5aR2 antibodies were obtained through mouse immune screening. One is a monoclonal antibody, clone 17A2D9, obtained by immunizing mice with envelope VLPs expressing the full-length human C5aR2 membrane protein; the other is a monoclonal antibody, clone 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 portion at the N-terminus is the signal peptide sequence (SEQ ID NO: 5); the middle portion is the heavy chain variable region (SEQ ID NO: 6), and the underlined portions 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 italicized bold portion 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 LQGTHQPWT FGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0135] Among them, the bold underlined portion at the N-terminus is the signal peptide sequence (SEQ ID NO: 11); the middle portion is the light chain variable region (SEQ ID NO: 12), and the underlined portions 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 rule; the italicized bold portion is the light chain constant region (mouseIg kappa, SEQ ID NO: 15).

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

[0137] MEWSWIFLFLLSGTAGVHS QVQLKESGPGLVAPSQSLSITCTVS GFSLMDYG VSWIRQPPGKGLEWLGV IWGDGKT NYNSALKSRLNIREDKSKSQVVLKMSSLQTDDTAMYYC ARWGFLY WGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFV DDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0138] Among them, the bold underlined portion at the N-terminus is the signal peptide sequence (SEQ ID NO: 5); the middle portion is the heavy chain variable region (SEQ ID NO: 16), and the underlined portions 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 italicized bold portion is the heavy chain constant region (mouse IgG1, SEQ ID NO: 20).

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

[0140] MRVLAELLGLLLFCFLGVRC DVVMTQTPLTLSVTFGQPASISCKSS QSLLHSDGRTY LNWLLQRPGQSPKRLIY LVS KLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC WQGTHFPHT FGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0141] Among them, the bold underlined portion at the N-terminus is the signal peptide sequence (SEQ ID NO: 21); the middle portion is the light chain variable region (SEQ ID NO: 22), and the underlined portions 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 rule; the italicized bold portion 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 and heavy chain variable region sequences and the antibody structural conformation, enabling specific binding to the C5aR2 membrane protein. Using the antibody variable region genes or complementarity-determining region (CDR) genes, various genetically engineered antibodies can be engineered and produced in any expression system utilizing 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 chemiluminescent compound, e.g., 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 antibodies of the present invention refer to polypeptides that have C5aR2 membrane protein binding activity and include the aforementioned CDR regions. The term also encompasses variants of polypeptides that have the same function as the antibodies of the present invention and include the aforementioned CDR regions. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more (generally 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, as well as additions of one or more (generally within 20, preferably within 10, and more preferably within 5) amino acids to the C-terminus and / or N-terminus. The term also encompasses 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 present 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 polynucleotide molecules encoding the above-mentioned antibodies or fragments thereof or fusion proteins thereof. The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or artificially synthesized DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical to the coding region sequence shown in SEQ ID NOs: 25-28 or a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence encoding an amino acid sequence identical to the polypeptide of the present invention, but differing from the coding region sequence shown in SEQ ID NOs: 25-28.

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

[0151]

[0152] Nucleotide sequence of the light chain of the recombinant 17A2D9 antibody (SEQ ID NO: 26)

[0153] ATGAATCTTCCCGTGCACCTGCTTGTATTGCTGCTTTTCTGGATACCCGCATCAAGGGGAGACGTGGTGGTCACCCAAACACCGTTGTCCCTTCCAGTTTCCTTTGGGAACCAAGTGAGCATCAGCTGTCGCTCATCCCAAAGTCTGGCAAACTCATACGGCAACACTTACCTTTCATGGTATCTTCATAAGCCCGGACAATCTCCACAGCTTTTGATCTATGGAATCTCCAACAGGTTTAGCGGCGTGCCTGATCGATTTTCAGGATCTGGCTCAGGGACCGACTTCACACTGAAGATCAGCGCCATAAAGCCCGAAGACCTCGGAATTTATTACTGTCTCCAGGGAACTCATCAGCCGTGGACTTTTGGAGGGGGAACAAAACTGGAGATTAAACGCGCCGATGCCGCTCCTACAGTGAGCATCTTTCCTCCTTCCTCCGAGCAGCTGACAAGCGGCGGCGCCAGCGTGGTGTGTTTCCTGAACAACTTCTATCCTAAGGACATCAATGTGAAGTGGAAGATCGACGGCAGCGAGAGACAGAACGGCGTGCTGAACTCCTGGACCGACCAGGATTCCAAGGACTCCACCTACTCCATGTCCTCCACACTGACCCTGACCAAGGATGAGTACGAGAGGCACAACAGCTACACATGCGAGGCCACACACAAGACCTCCACCAGCCCTATCGTGAAGAGCTTCAATAGAAACGAGTGC

[0154] Nucleotide sequence of the heavy chain of the 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 the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0159] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide or a polynucleotide further including additional coding and / or non-coding sequences. The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can generally be obtained by PCR amplification, recombinant methods, or synthetic methods. In addition, the heavy chain or light chain can be fused with a protein or tag sequence (e.g., fluorescent protein, Flag tag) to form a fusion protein.

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

[0161] Currently, DNA sequences encoding proteins of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely by chemical synthesis. This 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 sequences 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; 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 such as Drosophila S2 or Sf9; and animal cells such as CHO, COS7, and 293 cells.

[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 isolated 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 comprising the above-mentioned antibody or active fragment thereof, or fusion protein thereof, or immunoconjugate thereof, and a pharmaceutically acceptable carrier. Typically, 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 used to directly 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 (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned antibody of the present invention (or its conjugate) 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 mg / 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 present invention, when using a pharmaceutical composition, a safe and effective amount of an antibody or immunoconjugate of the present invention is administered to a mammal, wherein the safe and effective amount is generally at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight. Preferably, the dose 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 of a skilled physician.

[0170] application

[0171] The antibodies of the present invention have broad biological and clinical applications, encompassing a variety of fields, including the diagnosis and treatment of C5aR2-related diseases, basic medical research, and biological research. A preferred application is in the clinical diagnosis and targeted therapy of C5aR2-related diseases, such as inflammatory diseases, or cancer or tumor diagnosis and treatment.

[0172] Antibody preparation method of the present invention

[0173] In one aspect of the present invention, a method for screening and preparing anti-C5aR2 antibodies is provided, which comprises the steps of preparing VLPs multivalently displaying C5aR2 antigens (C5aR2 antigen-VLPs, such as the enveloped VLPs displaying full-length human C5aR2 (human C5aR2 / VLPs) of the present invention, or non-enveloped VLPs displaying the N-terminal sequence of human C5aR2 (human C5aR2 NTD-VLPs)), immunizing animals with the C5aR2 antigen-VLPs, and subsequently screening monoclonal hybridoma cells to obtain 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 VLP surface and expresses it using mammalian cells, so that the antigen or antigen epitope has post-translational modification. At the same time, the VLP has the function of an immune adjuvant, so that the prepared C5aR2 antigen-VLP can more effectively activate the immune animal to produce antibodies.

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

[0176] 1. The present invention uses VLPs to display membrane proteins or membrane protein epitopes as antigens for immunization, stimulating a host immune response. Human C5aR2 membrane proteins or epitopes thereof are multivalently displayed on VLPs and expressed in mammalian cells, resulting in post-translational modifications of the antigens or epitopes. Furthermore, the VLPs act as immune adjuvants, and the displayed epitopes are capable of generating a robust immune response.

[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 murine monoclonal antibody 17A2D9 of the present invention was obtained by immunization and screening of VLPs displaying the full-length C5aR2 membrane protein; the murine monoclonal antibody 30F10E3 was obtained by immunization and screening of VLPs displaying the N-terminal sequence of C5aR2. Both can specifically bind to the C5aR2 membrane protein; and 30F10E3 has the function of competitive blocking with the C5a ligand.

[0180] 5. The murine monoclonal antibodies 17A2D9 and 30F10E3 of the present invention can be used for Elisa and FACS detection of C5aR2 membrane protein and cell sorting. Furthermore, the murine 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 will be 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 stated, percentages and parts are weight percentages and parts by weight. 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: Immunization was performed using enveloped VLPs displaying the full-length human C5aR2 membrane protein and non-enveloped VLPs displaying the N-terminal sequence of the membrane protein C5aR2 (uniport: Q9P296) (1-38aa, MGNDSVSYEYGDYSDLSDRPVDCLDGACLAIDPLRVAP, SEQ ID NO: 29). Both C5aR2 VLP antigens were expressed in HEK293 mammalian cells. For the primary immunization, 50 μl of C5aR2 VLP antigen (estimated total protein concentration 0.5 mg / ml) was mixed with an equal volume of aluminum hydroxide adjuvant and injected intramuscularly into six-week-old female Balb / C mice at multiple sites. Booster immunizations were performed with the same dose as the primary immunization, with two weeks between immunizations 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 (2.5 μg / ml), respectively. The antiserum was diluted 300-fold and then added to the Elisa plate in a 3-fold serial dilution to bind to the antigen. Figure 1 Center A shows the binding of antisera obtained by immunization with full-length human C5aR2 / VLP to two antigens. Figure 1 Center B shows the binding of antisera obtained from immunization with human C5aR2NTD-VLP to two antigens. Both antigens yielded high titers of antisera, allowing for further screening of specific antibodies binding to membrane proteins.

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

[0186] 2.1 Antibody Screening after Immunization with Full-Length Human C5aR2 / VLP

[0187] Prior to mouse cell fusion, 50 μl of full-length human C5aR2 / VLP (envelope VLP, estimated total protein concentration 0.5 mg / ml) was diluted with 1× PBS to 300 μl and injected intraperitoneally for booster immunization. Mouse spleens were mixed with SP2 / 0 cells and fused using PEG1500 as a fusion agent. After 10 days of culture, 100 μl / well (2.5 μg / ml) of full-length human C5aR2 / VLP was coated onto polyvinyl chloride (PVC) ELISA plates. Hybridomas positive for antigen binding were screened by indirect ELISA. Hybridomas were then negatively screened using VLPs that do not display membrane proteins, coated onto PVC plates at 20 μl / ml, and negatively screened by indirect ELISA. Hybridomas that bind to the membrane protein were removed, resulting in polyclonal hybridomas that bind to the membrane protein. After limiting dilution to monoclonal status, the hybridomas were expanded and cryopreserved. The supernatant of the 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 the human C5aR2 membrane protein.

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

[0189] Prior to mouse cell fusion, 50 μl of human C5aR2 NTD-VLP (non-enveloped VLP, estimated total protein concentration 0.5 mg / ml) was diluted with 1X PBS to 300 μl and injected intraperitoneally for booster immunization. Mouse spleens were mixed with SP2 / 0 cells and fused using PEG1500 as a fusion agent. After 10 days of culturing, 100 μl / well (2.5 μg / ml) of human C5aR2 NTD-VLP was coated onto polyvinyl chloride (PVC) ELISA plates. Hybridoma clones positive for antigen binding were screened by indirect ELISA. Non-enveloped VLPs that do not display the NTD epitope were then used for counterscreening. Hybridoma clones were negatively screened by indirect ELISA using 20 μl / ml of non-enveloped VLP coated onto the plates. Hybridoma clones that bind to the membrane protein NTD were removed and isolated. Furthermore, 100 μl / well (2.5 μg / ml) of human C5aR2 NTD-VLP was coated onto an ELISA plate. 50 μl of the supernatant from the selected positive polyclonal clones was mixed with 50 μl of 20 nM biotinylated ligand C5a and added to the ELISA plate to compete for binding to human C5aR2 NTD-VLP. Binding of biotinylated ligand C5a alone to human C5aR2 NTD-VLP was used as a control. This screened polyclonal clone, 30F10, was identified as competitively blocking ligand binding to human C5aR2 NTD-VLP. The 30F10 polyclonal clone was then subjected to limiting dilution to a monoclonal state, expanded for culture, and cryopreserved. The monoclonal hybridoma supernatant was then validated using an indirect ELISA, resulting in the identification of the 30F10E3 monoclonal hybridoma cell line capable of competitively blocking the binding of the ligand C5a to the human C5aR2 receptor.

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

[0191] Monoclonal hybridoma cell lines were cultured to a cell density of 1E+07 cells / ml, and hybridoma monoclonal antibody sequencing was performed (Bio-Ying Bio). The 17A2D9 antibody had an IgG2a heavy chain (SEQ ID NO: 1) and a mouse Ig kappa light chain (SEQ ID NO: 2). The 30F10E3 antibody had an IgG1 heavy chain (SEQ ID NO: 3) and a mouse Ig kappa light chain (SEQ ID NO: 4). Gene sequences encoding the signal peptide, variable region, and constant region of the heavy and light chains (SEQ ID NOs: 25 and 26; SEQ ID NOs: 27 and 28) were synthesized and inserted into mammalian cell expression vectors. Commercial mammalian cell expression vectors such as pTT5 and pCDNA3.1 can be used. The recombinant plasmids were transfected into Expi293 mammalian cells, where the antibodies were secreted. The cell expression supernatant was affinity purified with protein A to obtain recombinant anti-human C5aR2 antibodies.

[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 bound to C5aR2 / VLP for Elisa.

[0194] The results are as follows Figure 2 Figure A shows that 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 and 30F10E9 monoclonal antibodies (120nM) and biotinylated ligand C5a (100nM) were diluted 3-fold and bound to C5aR2 NTD-VLP 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 1 ug / ml. Recombinant 17A2D9 and 30F10E9 monoclonal antibodies (120 nM) were diluted two-fold and mixed with 50 nM biotin-labeled ligand C5a. The mixture was then added to the coated ELISA plate and bound 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 C5a ligand 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 ligand

[0199] HeLa cells, which overexpress the C5aR2 membrane protein, were prepared into a single-cell suspension (3% FBS in PBS) at a cell density of 2E+06 cells / ml. Antibodies 17A2D9 and 30F10E3, as well as biotinylated C5a ligand, were diluted to 0.1 μM and allowed to bind to the HeLa cells. The suspension was incubated at 4°C for 30 minutes. FITC-conjugated goat anti-mouse (binding to mouse monoclonal antibodies 17A2D9 and 30F10E3) and SA-FITC (binding to biotinylated C5a ligand) were then added and incubated at 4°C for 30 minutes. HeLa cells were washed three times with 1× PBS and then gently pipetted to homogenize into single cells. FACS analysis was performed for antibody-cell binding and ligand-cell binding assays. The other group was an antibody-ligand competition assay. Recombinant antibodies 17A2D9 and 30F10E3 were mixed with biotin-labeled C5a ligand to a final concentration of 0.1 μM. The cells were then incubated at 4°C for 30 minutes. SA-FITC was then added and allowed to bind for 30 minutes at 4°C. HeLa cells were washed three times with PBS and then gently pipetted into single cells for analysis by FACS.

[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 middle 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 biotin-labeled C5a ligand binds to Hela cells (positive control); Figure 5 Curve 4 in middle 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 Figure 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 this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

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 shown in SEQ ID NO: 7; H-CDR2 shown in SEQ ID NO: 8; H-CDR3 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 shown in SEQ ID NO: 17; H-CDR2 shown in SEQ ID NO: 18; H-CDR3 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 shown in the sequence 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% 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% 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% 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% sequence identity thereto.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody binds to human C5aR2 and cynomolgus monkey C5aR2, and has human / monkey cross-species binding activity.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody competes with the C5a ligand for binding to C5aR2, thereby blocking the binding of the C5a ligand to C5aR2.

6. 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 any one of claims 1 to 5; and (ii) signal peptides that promote secretory expression of the antibody and / or tag sequences for purification and detection.

7. The recombinant antibody according to claim 6, wherein 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.

8. The recombinant antibody according to claim 6, wherein 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.

9. A polynucleotide molecule encoding a polypeptide selected from the group consisting of: (1) the anti-C5aR2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5; or (2) The recombinant antibody according to claim 6.

10. An expression vector comprising the polynucleotide molecule according to claim 9.

11. A host cell comprising the vector according to claim 10, or a host cell having the polynucleotide molecule according to claim 9 integrated into its genome.

12. An antibody conjugate, characterized in that: The antibody conjugate comprises: (a) the anti-C5aR2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or the recombinant antibody according to claim 6; and (b) a conjugated moiety selected from the group consisting of a detectable label, an enzyme, a cytokine, a radionuclide, and a nanoparticle / nanorod.

13. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: (a) the anti-C5aR2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the recombinant antibody according to claim 6, or the antibody conjugate according to claim 12; and (b) a pharmaceutically acceptable carrier.

14. Use of the anti-C5aR2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the recombinant antibody according to claim 6, or the antibody conjugate according to claim 12 for preparing: Detection reagent for detecting C5aR2 molecules.

15. Use of the anti-C5aR2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the recombinant antibody according to claim 6, or the antibody conjugate according to claim 12 for preparing a detection plate for detecting C5aR2 molecules.

16. Use of the anti-C5aR2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the recombinant antibody according to claim 6, or the antibody conjugate according to claim 12 for preparing: Detection kit for detecting C5aR2 molecules.

17. The use according to any one of claims 14 to 16, characterized in that The detection includes flow cytometry (FACS), cell immunofluorescence detection, ELISA detection, and protein immunoblotting (WB) detection.

18. An in vitro non-diagnostic and non-therapeutic method for detecting C5aR2 protein in a sample, the method comprising the steps of: (1) contacting a sample with the anti-C5aR2 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the recombinant antibody according to claim 6, or the antibody conjugate according to claim 12; (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of C5aR2 protein in the sample.

19. A method for screening anti-C5aR2 antibodies, comprising the steps of: (S1) using VLPs to multivalently display a full-length human C5aR2 protein or a C5aR2 protein epitope, and expressing it in mammalian cells to obtain C5aR2 antigen-VLPs for immunization, wherein the amino acid sequence of the full-length human C5aR2 protein is shown as uniport: Q9P296, and the C5aR2 protein epitope is the N-terminal sequence of human C5aR2 shown as the amino acid sequence of SEQ ID NO: 29; (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, obtaining the spleen of the immunized animal and preparing hybridoma clones; (S4) performing preliminary screening of hybridoma clones using the C5aR2 antigen-VLP to obtain antigen-binding positive hybridoma clones; (S5) negatively screening the clones that are positive for antigen binding using empty VLPs without C5aR2 antigen to select positive hybridoma clones that only bind to C5aR2 antigen; (S6) limiting dilution of positive hybridoma clones that bind only to the C5aR2 antigen to a monoclonal state, repeatedly verifying the binding of the monoclonal hybridoma cell supernatant to the C5aR2 antigen, thereby obtaining a monoclonal hybridoma cell line capable of producing anti-C5aR2 antibodies that specifically bind to the C5aR2 antigen.

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