Antigenic polypeptides of cxcr5 protein, anti-cxcr5 protein antibodies, and uses thereof

Abstract

The application discloses a polypeptide, the amino acid sequence of which is shown as SEQ ID NO: 1 or 2. The inventors find that the polypeptide has the following advantages: the recognized antigen epitope is clear, the cross-reaction is low, the selection of the antigen domain is more flexible and easy to synthesize, the cost is low, and the synthesis period is short by predicting the CXCR5 protein sequence. In addition, the polypeptide can be used for screening an anti-CXCR5 protein antibody, the obtained anti-CXCR5 protein antibody has strong binding capacity with the CXCR5 protein, the cost and period of developing the CXCR5 antibody drug can be effectively reduced, and a new strategy is provided for tumor and autoimmune disease treatment and disease diagnosis.

Description

Technical Field

[0001] This application belongs to the field of biomedicine, specifically relating to an antigenic polypeptide of CXCR5 protein, an anti-CXCR5 protein antibody and its uses, and more specifically to a polypeptide, recombinant protein, reagent or kit, anti-CXCR5 protein antibody or its antigen-binding fragment, nucleic acid molecule, expression vector, recombinant cell, product, and its uses. Background Technology

[0002] CXCR5, also known as Burkitt's lymphoma receptor 1 (BLR1) or CD185 (differentiation cluster 185), is a member of the CXC family of chemokine receptors, belonging to the G protein-coupled receptor superfamily. Its structural features include seven transmembrane α-helices, which form the main part of the receptor, enabling it to embed into the cell membrane. It contains an extracellular N-terminus and an intracellular C-terminus, and three loops both extracellularly and intracellularly. The unprocessed CXCR5 precursor contains 372 amino acids and has a molecular weight of 42 kDa. The N-terminal region is crucial for ligand recognition; this region is often missing a complete N-terminus in reported chemokine receptor structures. Studies have shown that soluble CXCR5 molecules are essentially composed of the extracellular region of CXCR5, generally including the first 60 amino acids of the molecule; the extracellular transmembrane domains are involved in the specific recognition and binding of ligands, and these domains stabilize the receptor-ligand complex through interactions such as hydrogen bonds, ionic bonds, and hydrophobic interactions.

[0003] CXCR5 is primarily expressed on mature B cells, follicular helper T cells (Tfh cells), and some dendritic cells. Through binding to its ligand CXCL13, it participates in various biological processes, including B cell migration and localization in specific regions, helper T cell differentiation, and lymphoid organ development. Studies have shown that CXCR5 and its ligand CXCL13 are essential for B / T cell migration from the B / T boundary region to secondary lymphoid organs (GCs, or germinal centers), and that B / T cell interactions in the B / T region are necessary for BCR affinity maturation and B cell proliferation. In the absence of CXCR5, B cells cannot migrate from T-cell-rich areas to spleen B cell follicles, resulting in the failure to form functional germinal centers. CXCR5 is also a crucial factor enabling B lymphocytes to return to lymph node follicles, promoting lymphoid follicle aggregation, and facilitating lymph node development. Furthermore, CXCR5 is expressed on all mature peripheral B cells and a small number of T cell subtypes, playing a crucial role not only in tumor proliferation but also closely associated with autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis, making it a potential therapeutic target. Therefore, developing an antigenic peptide of the CXCR5 protein and an anti-CXCR5 antibody is of great importance. Summary of the Invention

[0004] This application aims to at least partially address one of the technical problems existing in the prior art. To this end, this application provides an antigenic polypeptide of the CXCR5 protein, an anti-CXCR5 protein antibody, and its uses.

[0005] This application is based on the following discoveries of the inventors:

[0006] Currently, CXCR5 target development primarily utilizes recombinant proteins and peptides as immunogens. Commercially available CXCR5 proteins are mainly recombinant proteins obtained from *E. coli* or 293 cell expression systems. However, CXCR5 is a seven-transmembrane protein, and the expression and purification of recombinant CXCR5 proteins may face challenges, including proper protein folding, solubility, stability, and avoiding aggregate formation. These challenges typically require optimization of expression conditions, use of appropriate fusion tags, adjustment of buffer conditions, and purification strategies. In some cases, CXCR5 may need to be co-expressed or co-purified with its ligand CXCL13 to ensure proper ligand-receptor interactions and signal transduction functions. However, the preparation of recombinant CXCR5 proteins is time-consuming, expensive, and difficult to purify. Furthermore, the recombinant CXCR5 antigens currently used in China are mainly imported, resulting in high prices, long procurement cycles, and an unstable supply, which is detrimental to supporting domestic scientific and clinical research. Additionally, the imported antigen reagents have complex components and cannot meet the needs of applications requiring further conjugation and modification.

[0007] The inventors of this application used computer-aided simulation to predict antigen-binding sites, obtained key amino acid residues for CXCR5 binding, and designed a polypeptide sequence that mimics CXCR5 binding. This polypeptide sequence is relatively short and bypasses the complex process of multiple transmembrane protein expression and purification, making its expression and purification relatively simple and easy. The obtained polypeptide contains only the antigen-binding site, enabling targeted development of specific antibodies and saving antibody development time. Therefore, the CXCR5 polypeptide of this application clearly recognizes antigen epitopes, has low cross-reactivity, allows for more flexible selection of antigen domains, is easier to synthesize, has low cost, and a short synthesis cycle. Furthermore, it can effectively reduce the cost and cycle of CXCR5 antibody drug development, providing a new strategy for the treatment and diagnosis of tumors and autoimmune diseases.

[0008] In a first aspect, this application provides a polypeptide. According to embodiments of this application, the amino acid sequence of the polypeptide is as shown in SEQ ID NO:1 or 2. The above-described anti-CXCR5 protein antibody or its antigen-binding fragment of this application has a strong binding affinity to the CXCR5 protein and can be used for the detection of CXCR5 protein, or for the treatment and diagnosis of tumors and autoimmune diseases.

[0009] In a second aspect of this application, a recombinant protein is provided. According to embodiments of this application, the recombinant protein comprises the polypeptide described in the first aspect and a carrier protein; the carrier protein and the polypeptide are linked. As previously known, the polypeptide in the first aspect can serve as a CXCR5 antigen. Therefore, the recombinant protein containing the aforementioned polypeptide has the advantage of high antigenicity, can stimulate an immune response in antigen-immunized animals, and generate specific antibodies, and can be used to screen for anti-CXCR5 protein antibodies.

[0010] In a third aspect of this application, a reagent or kit is provided. According to embodiments of this application, the reagent or kit comprises: a polypeptide as described in the first aspect, or a recombinant protein as described in the second aspect. As is known prior, the polypeptide of the first aspect and the recombinant protein of the second aspect can serve as CXCR5 antigens. Therefore, the reagent or kit containing the aforementioned polypeptide has the advantage of high antigenicity, can stimulate an immune response in antigen-immunized animals, and generate specific antibodies, and can be used to screen for anti-CXCR5 protein antibodies.

[0011] In a fourth aspect of this application, the use of the polypeptide described in the first aspect, the recombinant protein described in the second aspect, or the reagent or kit described in the third aspect in the preparation of anti-CXCR5 protein antibodies is disclosed. As is known prior, the polypeptide of the first aspect, the recombinant protein described in the second aspect, or the reagent or kit described in the third aspect can serve as a CXCR5 antigen. Therefore, recombinant proteins containing the aforementioned polypeptides have the advantage of high antigenicity, can stimulate antigen-immunized animals to produce an immune response and generate specific antibodies, and can be used to screen for anti-CXCR5 protein antibodies.

[0012] In a fifth aspect of this application, a method for preparing anti-CXCR5 protein antibodies is provided. According to an embodiment of this application, the method includes: immunizing a target animal with the polypeptide described in the first aspect, the recombinant protein described in the second aspect, or the reagent or kit described in the third aspect to obtain serum from the immunized target animal; and screening the antibodies in the serum to obtain anti-CXCR5 protein antibodies. As is known, the polypeptide of the first aspect, the recombinant protein of the reagent or kit described in the second or third aspect can serve as a CXCR5 antigen. Therefore, the aforementioned polypeptide has the advantage of high antigenicity, and using the aforementioned polypeptide or a recombinant protein, reagent, or kit containing the aforementioned polypeptide can stimulate an antigen-immunized animal to produce an immune response and generate specific antibodies, which can be used to screen for anti-CXCR5 protein antibodies.

[0013] In a sixth aspect, this application discloses an anti-CXCR5 protein antibody or its antigen-binding fragment. According to embodiments of this application, the anti-CXCR5 protein antibody or its antigen-binding fragment comprises HCDR1 to 3; wherein, 1) the amino acid sequence of HCDR1 is as shown in SEQ ID NO:5; the amino acid sequence of HCDR2 is as shown in SEQ ID NO:6; and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:7; or 2) the amino acid sequence of HCDR1 is as shown in SEQ ID NO:8; the amino acid sequence of HCDR2 is as shown in SEQ ID NO:9; and the amino acid sequence of HCDR3 is as shown in SEQ ID NO:10. The above-mentioned anti-CXCR5 protein antibody or its antigen-binding fragment of this application has a strong binding ability to CXCR5 protein and can be used for the detection of CXCR5 protein, or for the treatment and diagnosis of tumors and autoimmune diseases.

[0014] In a seventh aspect, this application provides a nucleic acid molecule. According to embodiments of this application, the nucleic acid molecule encodes the polypeptide described in the first aspect, the recombinant antigen described in the second aspect, or the anti-CXCR5 protein antibody or antigen-binding fragment described in the sixth aspect. In an eighth aspect, this application provides an expression vector. According to embodiments of this application, the expression vector carries the nucleic acid molecule described in the seventh aspect. When linking the above-mentioned nucleic acid molecule to the expression vector, the nucleic acid molecule can be directly or indirectly linked to control elements on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the expression vector itself, or they can be exogenous, i.e., not derived from the expression vector itself. Naturally, the nucleic acid molecule and the control elements need to be operably linked.

[0015] In a ninth aspect of this application, a recombinant cell is provided. According to embodiments of this application, the recombinant cell carries the nucleic acid molecule described in the seventh aspect or the expression vector described in the eighth aspect, or expresses the polypeptide described in the first aspect, the recombinant antigen described in the second aspect, the anti-CXCR5 protein antibody described in the sixth aspect, or an antigen-binding fragment. Using this recombinant cell, under suitable conditions, the aforementioned polypeptide, recombinant antigen, anti-CXCR5 protein antibody, or antigen-binding fragment can be effectively expressed intracellularly.

[0016] In a tenth aspect of this application, a product is provided. According to embodiments of this application, the composition comprises the anti-CXCR5 protein antibody or antigen-binding fragment described in the sixth aspect. The product of this application has CXCR5 binding affinity and can be used for the detection of CXCR5, the detection of CXCR5-expressing cells, or for the prevention and / or treatment of CXCR5-mediated diseases.

[0017] In the eleventh aspect of this application, the use of the anti-CXCR5 protein antibody or antigen-binding fragment described in the sixth aspect in the preparation of a product or medicament, wherein the product is used to detect CXCR5 protein, and the medicament is used to prevent and / or treat diseases associated with pathogenic cells expressing CXCR5.

[0018] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0019] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0020] Figure 1 The results of serum titer detection before and after CXCR5 immunization of alpacas in Example 2 of this application;

[0021] Figure 2 This describes the phage washing and enrichment process in Example 3 of this application.

[0022] Figure 3 The results of ELISA detection of the binding ability of four peptides and recombinant CXCR5 protein in pairs 3-24 and 1-48 in Example 4 of this application are shown.

[0023] Figure 4 The results of ELISA detection of the binding activity of recombinant CXCR5 at 3-24 and 1-48 in Example 4 of this application are shown.

[0024] Figure 5 This demonstrates the specificity of the cross-ELISA validation of 3-24 and 1-48 in Example 4 of this application;

[0025] Figure 6 The results of ELISA detection of the binding ability of 3-24-mFc and 1-48-mFc to four peptides and recombinant CXCR5 protein in Example 5 of this application are shown.

[0026] Figure 7 ECGs of antibodies 3-24-mFc and 1-48-mFc in Example 5 of this application 50 value;

[0027] Figure 8 The results of cell binding activity assay for the CXCR5 bivalent antibody Raij in Example 6 of this application are shown. Detailed Implementation

[0028] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0029] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0030] This application details

[0031] Definitions and General Terms

[0032] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this application but do not exclude other contents.

[0033] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0034] In this paper, the terms “identity,” “homology,” or “similarity” are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including: Needleman et al. (1970) J. Mol. Biol. 48: 443, a homology alignment algorithm; Smith et al. (1981) Adv. Appl. Math. 2: 482, a local homology algorithm; Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444, a similarity search method; and the Smith-Waterman algorithm (Meth. Mol. Biol). .70:173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J.Mol.Biol. 215:403-410). Computer programs utilizing these algorithms are also available, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth.Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0035] Without substantially affecting antibody activity (retaining at least 90% of the activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the sequence of this application to obtain variants of the antibody or its functional fragment sequence. These are all considered to be included within the scope of protection of this application. For example, amino acids with similar properties can be substituted in the variable region. The variant sequences described in this application can have at least 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence. Sequence identity described in this application can be measured using sequence analysis software, such as the computer program BLAST using default parameters, especially BLASTP or TBLASTN. The amino acid sequences mentioned in this application are shown in N-terminus to C-terminus format.

[0036] In this paper, the term "at least 80% homology" refers to at least 80% homology with each reference sequence, which can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%. The term "at least 90% homology" refers to at least 90% homology with each reference sequence, which can be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0037] In this paper, the term “variant” or “mutant” can refer to any naturally occurring or engineered molecule that contains one or more nucleotide or amino acid mutations.

[0038] In this document, the terms "recombinant protein" and "fusion protein" generally refer to a protein obtained by the fusion of two or more proteins or polypeptides. Genes or nucleic acid molecules encoding the two or more proteins or polypeptides may be linked together to form a fusion gene or fused nucleic acid molecule that encodes the fusion protein. Translation of the fusion gene produces a single polypeptide having the properties of at least one, or even each, of the two or more proteins or polypeptides prior to fusion. Recombinant fusion proteins are artificially created using recombinant DNA technology for biological research or therapy. Recombinant fusion proteins are proteins created through genetic engineering of a fusion gene. This application relates to recombinant fusion proteins, and the terms fusion protein and recombinant fusion protein are used herein with the same meaning. The fusion proteins described herein generally contain at least two domains (A and C) and optionally include a third component, a linker between the two domains. The generation of recombinant fusion proteins is known in the art and generally involves removing the stop codon from a cDNA sequence encoding a first protein or polypeptide, and then attaching a cDNA sequence of a second protein in a frame-compliant manner via ligation or overlap extension PCR. This DNA sequence is then expressed by a cell as a single protein. The protein can be engineered to include the complete sequence of either of the original proteins or polypeptides, or just a portion of them.

[0039] The fusion protein described in this application is typically prepared using biosynthetic methods. Based on the nucleotide sequence described in this application, those skilled in the art can readily obtain the encoding nucleic acid of this application using various known methods. These methods include, but are not limited to, PCR, artificial DNA synthesis, etc., and specific methods can be found in J. Sambrook, *Molecular Cloning: A Laboratory Manual*. As one embodiment of this application, the encoding nucleic acid sequence of this application can be constructed by segmenting and synthesizing the nucleotide sequence followed by overlap extension PCR.

[0040] In this document, the term "expression vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule to host cells and / or between host cells. The expression vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The expression vector also includes vectors having multiple of the aforementioned functions. The expression vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the expression vector, the expression vector can produce the desired expression product.

[0041] In this document, the term "recombinant cell" generally refers to a cell in which the genetic material of a host cell is modified or recombined using genetic engineering or cell fusion techniques to obtain a unique trait with stable inheritance. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant expression vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acids (e.g., vectors) into cells using various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of this application and can be used for the expression and / or secretion of target proteins. Examples of suitable host cells that can be used in this application include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

[0042] In this document, the term "pharmaceutical composition" generally refers to a unit dose form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with a carrier constituting one or more adjunct components. Typically, compositions are prepared by uniformly and adequately combining an active antibody or antigen-binding fragment with a liquid carrier, a finely fragmented solid carrier, or both.

[0043] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a specific target dosage form. The use of any conventional excipients is also within the scope of consideration for this application, except for any range of incompatibility between the excipient and the antibody or antigen-binding fragment of this application, such as any adverse biological effects or harmful interactions with any other component of the pharmaceutically acceptable composition.

[0044] In this document, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The antibody or antigen-binding fragment, or pharmaceutical composition of this application, may be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are foreseeable, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but this application is not limited to these exemplified routes of administration. Preferably, the composition of this application is administered via intravenous or subcutaneous injection.

[0045] In this document, the term "treatment" refers to the use of a drug or antibody or antigen-binding fragment to achieve a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of disease or its symptoms, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects caused by disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of disease in individuals susceptible to disease but not yet diagnosed with the disease; (b) suppression of disease, such as inhibiting disease progression; or (c) alleviating disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any medication that administers a drug or antibody or antigen-binding fragment to an individual to treat, cure, alleviate, improve, reduce, or suppress the individual's disease, including but not limited to administering a drug containing the antibody or antigen-binding fragment described herein to an individual in need.

[0046] This application provides a detailed description of the antigenic peptide of CXCR5 protein, the anti-CXCR5 protein antibody, and their uses.

[0047] This application discloses a polypeptide, a recombinant protein, an anti-CXCR5 protein antibody or an antigen-binding fragment thereof, a method for preparing an anti-CXCR5 protein antibody or an antigen-binding fragment thereof, a fusion protein, a nucleic acid molecule, an expression vector, a recombinant cell, a conjugate, a composition, a reagent, a kit, a method for detecting CXCR5 protein, and their uses, which will be described in detail below.

[0048] Peptides, recombinant proteins and their uses

[0049] In a first aspect, this application proposes a polypeptide. According to embodiments of this application, the amino acid sequence of the polypeptide is as shown in SEQ ID NO:1 or 2. The inventors, through prediction of the CXCR5 protein sequence, discovered that the above-mentioned polypeptide has a clearly defined antigenic epitope, low cross-reactivity, more flexible selection of antigenic domains, is easier to synthesize, has low cost, and a short synthesis cycle. Furthermore, the above-mentioned polypeptide can be used to screen for anti-CXCR5 protein antibodies. The resulting anti-CXCR5 protein antibodies have a strong binding affinity to the CXCR5 protein, effectively reducing the cost and cycle time of CXCR5 antibody drug development, and providing a new strategy for the treatment and diagnosis of tumors and autoimmune diseases.

[0050] NYPLTLEMDLENLEDLFC(SEQ ID NO:1);

[0051] FWELDRLDNYNDTSLVENHLC (SEQ ID NO: 2).

[0052] It should be noted that in this application, "the amino acid sequence as shown in SEQ ID NO:A" includes the amino acid sequence of SEQ ID NO:A or the amino acid sequence of SEQ ID NO:A with conservative modifications, all of which are within the scope of protection of this application. For example, "the amino acid sequence of the polypeptide as shown in SEQ ID NO:1 or 2" means that the polypeptide has the amino acid sequence shown in SEQ ID NO:1 or 2, or has the amino acid sequence of SEQ ID NO:1 or 2 with conservative modifications, all of which are within the scope of protection of this application.

[0053] In this document, "conserved modified amino acid sequences" refers to amino acid modifications that do not significantly affect or alter the properties of the amino acid sequence (e.g., do not change the binding properties of the aforementioned peptide to the CXCR5 antibody). These modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the protein sequences (e.g., the CXCR5 protein) of this application using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conserved amino acid substitutions involve replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acidic side chains (such as aspartic acid and glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (such as threonine, valine, and isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, and histidine). Therefore, one or more amino acid residues in the protein sequence of this application (e.g., the CXCR5 protein) can be substituted with other amino acid residues from the same side chain family, and the retained function of the altered protein sequence (e.g., the CXCR5 protein) can be tested using the functional assay methods described herein. Exemplarily, conservative modifications are limited to no more than 80% of the total number, preferably no more than 90%. In this article, "conserved modified amino acid sequences" also include naturally occurring amino acid modifications. "Naturally occurring mutations" refer to mutations caused by changes in alleles due to individual differences or other reasons during the natural mutation process.

[0054] This application discloses a polypeptide. According to embodiments of this application, the amino acid sequence of the polypeptide, as shown in SEQ ID NO: 1 or 2, has at least 90% sequence similarity.

[0055] In a second aspect of this application, a recombinant protein is provided. According to embodiments of this application, the recombinant protein comprises the polypeptide described in the first aspect and a carrier protein; the carrier protein and the polypeptide are linked. As previously known, the polypeptide in the first aspect can serve as a CXCR5 antigen. Therefore, the recombinant protein containing the aforementioned polypeptide has the advantage of high antigenicity, can stimulate an immune response in antigen-immunized animals, and generate specific antibodies, and can be used to screen for anti-CXCR5 protein antibodies.

[0056] According to embodiments of this application, the recombinant protein may further include at least one of the following technical features:

[0057] According to embodiments of this application, the carrier protein includes at least one of protein tags, keyhole limpethemacyanin (KLH), bovine serum albumin (BSA), and ovalbumin (OVA).

[0058] According to embodiments of this application, the carrier protein is selected from at least one of hemocyanin, bovine serum albumin, and ovalbumin. This improves the immunogenicity and immune response of the CXCR5 protein antigen.

[0059] According to embodiments of this application, the C-terminus of the carrier protein is linked to the N-terminus of the polypeptide, or the N-terminus of the carrier protein is linked to the C-terminus of the polypeptide.

[0060] In an optional embodiment of this application, the carrier protein is a His tag. This facilitates protein purification and promotes protein solubility.

[0061] According to embodiments of this application, the recombinant antigen further includes a linker peptide.

[0062] According to embodiments of this application, the C-terminus of the carrier protein is connected to the N-terminus of the linker peptide, the C-terminus of the linker peptide is connected to the N-terminus of the polypeptide, or the C-terminus of the polypeptide is connected to the N-terminus of the linker peptide, and the C-terminus of the linker peptide is connected to the N-terminus of the carrier protein.

[0063] In a third aspect of this application, a reagent or kit is provided. According to embodiments of this application, the reagent or kit comprises: a polypeptide as described in the first aspect, or a recombinant protein as described in the second aspect. As is known prior, the polypeptide of the first aspect and the recombinant protein of the second aspect can serve as CXCR5 antigens. Therefore, the reagent or kit containing the aforementioned polypeptide has the advantage of high antigenicity, can stimulate an immune response in antigen-immunized animals, and generate specific antibodies, and can be used to screen for anti-CXCR5 protein antibodies.

[0064] In a fourth aspect of this application, the use of the polypeptide described in the first aspect, the recombinant protein described in the second aspect, or the reagent or kit described in the third aspect in the preparation of anti-CXCR5 protein antibodies is disclosed. As is known prior, the polypeptide of the first aspect, the recombinant protein described in the second aspect, and the reagent or kit described in the third aspect can serve as CXCR5 antigens. Therefore, recombinant proteins containing the aforementioned polypeptides have the advantage of high antigenicity, can stimulate antigen-immunized animals to produce an immune response and generate specific antibodies, and can be used to screen for anti-CXCR5 protein antibodies.

[0065] In a fifth aspect of this application, a method for preparing anti-CXCR5 protein antibodies is provided. According to an embodiment of this application, the method includes: immunizing a target animal with the polypeptide described in the first aspect, the recombinant protein described in the second aspect, or the reagent or kit described in the third aspect to obtain serum from the immunized target animal; and screening the antibodies in the serum to obtain anti-CXCR5 protein antibodies. As is known, the polypeptide of the first aspect, the recombinant protein described in the second aspect, or the reagent or kit described in the third aspect can be used as a CXCR5 antigen. Therefore, the aforementioned polypeptide has the advantage of high antigenicity. Using the aforementioned polypeptide or a recombinant protein containing the aforementioned polypeptide, reagent, or kit can stimulate an antigen-immunized animal to produce an immune response and generate specific antibodies, which can be used to screen for anti-CXCR5 protein antibodies.

[0066] According to an embodiment of this application, the method includes:

[0067] (1) Synthesize the polypeptide described in the first aspect or the recombinant protein described in the second aspect;

[0068] (2) Immunize the target animal with the polypeptide;

[0069] (3) The antibody titer in the serum of immunized animals was detected by ELISA. After the titer reached the threshold, the serum of immunized animals was collected, PBMCs were extracted from the serum, a phage library was constructed, and the antibodies in the PBMCs were screened to obtain the target antibody.

[0070] (4) Purify the target antibody and perform ELISA and flow cytometry on the purified target antibody.

[0071] According to an embodiment of this application, the method includes:

[0072] (1) Synthesize the polypeptide described in the first aspect or the recombinant protein described in the second aspect;

[0073] (2) The polypeptide and recombinant protein are used to cross-immunize animals (for example, the polypeptide is used for the first three immunizations, and then the polypeptide is mixed with the recombinant antigen for each subsequent immunization).

[0074] (3) The antibody titer in the serum of immunized animals was detected by ELISA. After the titer reached the threshold, the serum of immunized animals was collected, PBMCs were extracted from the serum, a phage library was constructed, and the antibodies in the PBMCs were screened to obtain the target antibody.

[0075] (4) Purify the target antibody and perform ELISA and flow cytometry on the purified target antibody.

[0076] Anti-CXCR5 protein antibody or its antigen-binding fragment and its preparation method

[0077] In a sixth aspect of this application, an anti-CXCR5 protein antibody or an antigen-binding fragment thereof is provided. According to an embodiment of this application, the anti-CXCR5 protein antibody or its antigen-binding fragment comprises HCDR1 to 3; wherein, 1) the amino acid sequence of HCDR1 is as shown in SEQ ID NO:5;

[0078] The amino acid sequence of HCDR2 is shown in SEQ ID NO:6;

[0079] The amino acid sequence of HCDR3 is shown in SEQ ID NO:7; or

[0080] 2) The amino acid sequence of HCDR1 is shown in SEQ ID NO:8;

[0081] The amino acid sequence of HCDR2 is shown in SEQ ID NO:9;

[0082] The amino acid sequence of HCDR3 is shown in SEQ ID NO:10.

[0083] The anti-CXCR5 protein antibody or its antigen-binding fragment described in this application has a strong binding ability to the CXCR5 protein and can be used to detect the CXCR5 protein, or for the treatment and diagnosis of tumors and autoimmune diseases.

[0084] GRTFSTWR(SEQ ID NO:5);

[0085] ISNSGAYT (SEQ ID NO:6);

[0086] AAAVNFLMYRNVIASERRGYDY(SEQ ID NO:7);

[0087] GLSFSSYA (SEQ ID NO:8);

[0088] IGWSGTTT(SEQ ID NO:9);

[0089] ATSRIRPELTATAYRY (SEQ ID NO:10).

[0090] In this article, the term "antibody" is used in the broadest sense, and can include monoclonal antibodies, multispecific antibodies, and chimeric antibodies. Their specific structures are not limited, as long as they exhibit the desired biological activity. They typically consist of a heavy chain (H chain). The amino acid sequence at the amino terminus (N-terminus) of the peptide chain varies considerably and is called the variable region (V-terminus); the carboxyl terminus (C-terminus) is relatively stable and changes very little and is called the constant region (C-terminus).

[0091] In this paper, the heavy chain complementarity determination region (heavy chain variable region CDR) is referred to as "HCDRs" or "HCDR", which includes HCDR1 (also known as CDR-H1), HCDR2 (also known as CDR-H2), and HCDR3 (also known as CDR-H3). Commonly used CDR definition schemes in this field include: Kabat definition, Chothia definition, IMGT definition, Contact definition, and AbM definition.

[0092] As stated in this article, the “Kabat definition” refers to the definition system described by Kabat et al., USDept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983). For the “Chothia definition”, see Chothia et al., J Mol Biol 196: 901-917 (1987).

[0093] Kabat et al. also defined a numbering system applicable to the variable region sequence of any antibody. Those skilled in the art can readily map this Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As described herein, "Kabat number" refers to the numbering system described in Kabat et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983), and the HCDRs and LCDRs of the antibody or antigen-binding fragments of this application are numbered using the aforementioned numbering system. It should be noted that the polypeptide sequences described above in this application are numbered according to the Kabat numbering system. However, those skilled in the art are fully capable of converting the sequence Kabat numbers in the sequence listing to "HCDRs" under other numbering systems (including but not limited to AbM, IMGT, and Chothia). CDRs determined based on the heavy chain variable region disclosed in this application using other rules disclosed in the art also fall within the scope of protection of this disclosure.

[0094] In this document, the term "antigen-binding fragment" refers to a fragment containing part or all of an antibody that lacks at least some of the amino acids present in the full-length chain but still possesses the performance activity of specifically binding to an antigen. For example, the fragment may contain part or all of the antibody's CDR. Such fragments are biologically active because they bind to the antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Such fragments are selected from single-domain antibodies or VHH-Fc fusion proteins. Such fragments can be generated using recombinant nucleic acid technology or through enzymatic or chemical cleavage of antigen-binding molecules (including intact antibodies).

[0095] This application discloses a polypeptide. According to an embodiment of this application, the anti-CXCR5 protein antibody or its antigen-binding fragment comprises HCDR1-3; wherein, 1) the amino acid sequence of HCDR1 is as shown in SEQ ID NO:5 or an amino acid sequence having at least 90% sequence similarity thereto;

[0096] The amino acid sequence of HCDR2 is shown in SEQ ID NO:6 or an amino acid sequence having at least 90% sequence similarity to it.

[0097] The amino acid sequence of HCDR3 is as shown in SEQ ID NO:7 or an amino acid sequence having at least 90% sequence similarity to it; or

[0098] 2) The amino acid sequence of HCDR1 is as shown in SEQ ID NO:8 or an amino acid sequence having at least 90% sequence similarity to it;

[0099] The amino acid sequence of HCDR2 is shown in SEQ ID NO:9 or an amino acid sequence having at least 90% sequence similarity to it.

[0100] The amino acid sequence of HCDR3 is shown in SEQ ID NO:10 or an amino acid sequence having at least 90% sequence similarity to it.

[0101] It should be noted that, in this application, "the amino acid sequence of HCDR1 as shown in SEQ ID NO:5" includes the amino acid sequence of SEQ ID NO:5 or the conservatively modified amino acid sequence of SEQ ID NO:5, all of which are within the scope of protection of this application. Similarly, "the amino acid sequence of HCDR2 as shown in SEQ ID NO:6" includes the amino acid sequence of SEQ ID NO:6 or the conservatively modified amino acid sequence of SEQ ID NO:6, all of which are within the scope of protection of this application. Likewise, "the amino acid sequence of HCDR3 as shown in SEQ ID NO:7" includes the amino acid sequence of SEQ ID NO:7 or the conservatively modified amino acid sequence of SEQ ID NO:7, all of which are within the scope of protection of this application. The amino acid sequence of HCDR1 as shown in SEQ ID NO:8 includes the amino acid sequence of SEQ ID NO:8 or the conservatively modified amino acid sequence of SEQ ID NO:8, all of which are within the scope of protection of this application. Finally, "the amino acid sequence of HCDR2 as shown in SEQ ID NO:9" includes the amino acid sequence of SEQ ID NO:9 or the conservatively modified amino acid sequence of SEQ ID NO:9, all of which are within the scope of protection of this application. In this application, "the amino acid sequence of HCDR3 is as shown in SEQ ID NO:10" includes the amino acid sequence of SEQ ID NO:10 or the amino acid sequence of SEQ ID NO:10 in a conservative modified form, and both are within the scope of protection of this application.

[0102] According to embodiments of this application, the above-mentioned anti-CXCR5 protein antibody or its antigen-binding fragment may further include at least one of the following technical features:

[0103] According to embodiments of this application, the anti-CXCR5 protein antibody or antigen-binding fragment includes a heavy chain framework region.

[0104] According to embodiments of this application, at least a portion of the heavy chain framework region is derived from at least one of mouse antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof.

[0105] According to embodiments of this application, at least a portion of the heavy chain framework region is derived from at least one of a rabbit antibody or a mutant thereof, a mouse antibody or a mutant thereof, and a human antibody or a mutant thereof.

[0106] In an optional embodiment of this application, the heavy chain frame region includes HFR1 to 4; wherein:

[0107] 1) The amino acid sequence of HFR1 is shown in SEQ ID NO:20 or an amino acid sequence having at least 90% sequence similarity to it;

[0108] The amino acid sequence of HFR2 is shown in SEQ ID NO:21 or an amino acid sequence having at least 90% sequence similarity to it.

[0109] The amino acid sequence of HFR3 is shown in SEQ ID NO:22 or an amino acid sequence having at least 90% sequence similarity to it.

[0110] The amino acid sequence of HFR4 is as shown in SEQ ID NO:23 or an amino acid sequence having at least 90% sequence similarity to it; or

[0111] 2) The amino acid sequence of HFR1 is shown in SEQ ID NO:24 or an amino acid sequence having at least 90% sequence similarity to it;

[0112] The amino acid sequence of HFR2 is shown in SEQ ID NO:25 or an amino acid sequence having at least 90% sequence similarity to it.

[0113] The amino acid sequence of HFR3 is shown in SEQ ID NO:26 or an amino acid sequence having at least 90% sequence similarity to it.

[0114] The amino acid sequence of HFR4 is shown in SEQ ID NO:27 or an amino acid sequence having at least 90% sequence similarity to it.

[0115] QVQLQESGGGLVQAGSSLLRLSCAAS(SEQ ID NO:20);

[0116] MGWFRQAPGKEREFVAA(SEQ ID NO:21);

[0117] SHADFVKGRFTISRDNAKNRVYLQMNSLKPEDTAVYYC(SEQ ID NO:22);

[0118] WGQGTQVTVSS(SEQ ID NO:23);

[0119] QVQLQESGGGVVQAGGSLLRLSCAAS(SEQ ID NO:24);

[0120] MAWFRQAPGKEREFVAT(SEQ ID NO:25);

[0121] AYADSAKGRFTITRDNAKSTGYLQMTSLKPEDTAVYYC(SEQ ID NO:26);

[0122] WGQGTQVTVSS (SEQ ID NO:27).

[0123] In an optional embodiment of this application, the heavy chain frame region includes HFR1 to 4; wherein:

[0124] 1) The amino acid sequence of HFR1 is shown in SEQ ID NO:20;

[0125] The amino acid sequence of HFR2 is shown in SEQ ID NO:21;

[0126] The amino acid sequence of HFR3 is shown in SEQ ID NO:22;

[0127] The amino acid sequence of HFR4 is shown in SEQ ID NO:23; or

[0128] 2) The amino acid sequence of HFR1 is shown in SEQ ID NO:24;

[0129] The amino acid sequence of HFR2 is shown in SEQ ID NO:25;

[0130] The amino acid sequence of HFR3 is shown in SEQ ID NO:26;

[0131] The amino acid sequence of HFR4 is shown in SEQ ID NO:27.

[0132] According to embodiments of this application, the antibody or antigen-binding fragment includes: a heavy chain variable region with an amino acid sequence such as SEQ ID NO:11 or having at least 90% sequence similarity thereto; or a heavy chain variable region with an amino acid sequence such as SEQ ID NO:12 or having at least 90% sequence similarity thereto.

[0133] QVQLQESGGGLVQAGSSLRLSCAASGRTFSTWRMGWFRQAPGKEREFVAAISNSGAYTSHADFV KGRFTISRDNAKNRVYLQMNSLKPEDTAVYYCAAAVNFLMYRNVIASERRGYDYWGQGTQVTVSS (SEQ ID NO: 11);

[0134] QVQLQESGGGVVQAGGSLRLSCAASGLSFSSYAMAWFRQAPGKEREFVATIGWSGTTTAYADSAK GRFTITRDNAKSTGYLQMTSLKPEDTAVYYCATSRIRPELTATAYRYWGQGTQVTVSS (SEQ ID NO: 12).

[0135] According to embodiments of this application, the antibody or antigen-binding fragment includes: a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:11; or a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:12.

[0136] It should be noted that, in this application, "the amino acid sequence as shown in the heavy chain variable region of SEQ ID NO:11" includes the amino acid sequence of SEQ ID NO:11 or the conservatively modified amino acid sequence of SEQ ID NO:11, all of which are within the scope of protection of this application. Similarly, in this application, "the amino acid sequence as shown in the heavy chain variable region of SEQ ID NO:12" includes the amino acid sequence of SEQ ID NO:12 or the conservatively modified amino acid sequence of SEQ ID NO:12, all of which are within the scope of protection of this application.

[0137] According to embodiments of this application, the anti-CXCR5 protein antibody or its antigen-binding fragment further includes a heavy chain constant region.

[0138] According to embodiments of this application, at least a portion of the heavy chain constant region is derived from at least one of mouse antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof.

[0139] According to embodiments of this application, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

[0140] According to embodiments of this application, the heavy chain constant regions are all derived from at least one of rabbit antibodies or mutants thereof, mouse antibodies or mutants thereof, and human antibodies or mutants thereof.

[0141] According to an embodiment of this application, the N end of the heavy chain constant region is connected to the C end of the heavy chain variable region.

[0142] According to embodiments of this application, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO:13 or an amino acid sequence having at least 90% sequence similarity thereto.

[0143] PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIE RTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO:13).

[0144] According to an embodiment of this application, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:13.

[0145] It should be noted that the phrase "the amino acid sequence of the heavy chain constant region as shown in SEQ ID NO:13" in this application includes the amino acid sequence of SEQ ID NO:13 or the amino acid sequence of SEQ ID NO:13 in a conservative modified form, and both are within the scope of protection of this application.

[0146] According to embodiments of this application, the anti-CXCR5 protein antibody includes at least one of polyclonal antibodies and single-domain antibodies; or the antigen-binding fragment includes, but is not limited to, the VHH-Fc fusion protein.

[0147] In this document, the terms "polyclonal antibody" and "multispecific antibody" are synonymous, both referring to antibodies that can recognize multiple antigenic epitopes. For example, antibodies that recognize two antigenic epitopes (bispecific antibodies, abbreviated as biantibodies), three antigenic epitopes, or four antigenic epitopes are used in a broad sense, and their specific structures are not limited, as long as they can recognize multiple antigenic epitopes. In this application, at least one of the multiple antigenic epitopes is derived from CXCR5.

[0148] In this paper, the terms “single-domain antibody,” “nanobody,” and “VHH antibody” are used interchangeably. The antibody was originally described as an antigen-binding immunoglobulin (variable) domain of a “heavy chain antibody” (i.e., “antibody lacking light chains”) containing a heavy chain variable region (VH) and conventional CH2 and CH3 regions, which specifically binds to antigen proteins (e.g., CXCR5) through the heavy chain variable region.

[0149] According to embodiments of this application, the anti-CXCR5 protein antibody or its antigen-binding fragment is obtained using the polypeptide described in the first aspect, the recombinant protein described in the second aspect, or the reagent or kit described in the third aspect.

[0150] According to embodiments of this application, the anti-CXCR5 protein antibody or antigen-binding fragment comprises:

[0151] The heavy chain with an amino acid sequence as shown in SEQ ID NO:14; or the heavy chain with an amino acid sequence as shown in SEQ ID NO:15.

[0152] QVQLQESGGGLVQAGSSLRLSCAASGRTFSTWRMGWFRQAPGKEREFVAAISNSGAYTSHADFVKGRFTISRDNAKNRVYLQMNSLKPEDTAVYYCAAAVNFLMYRNVIASERRGYDYWGQGTQVTVSSPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO:14);

[0153] QVQLQESGGGVVQAGGSLRLSCAASGLSFSSYAMAWFRQAPGKEREFVATIGWSGTTTAYADSAKGRFTITRDNAKSTGYLQMTSLKPEDTAVYYCATSRIRPELTATAYRYWGQGTQVTVSSPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO:15).

[0154] Methods for preparing antibodies, fusion proteins, conjugates

[0155] In another aspect of this application, a method for preparing the anti-CXCR5 protein antibody or its antigen-binding fragment as described in the sixth aspect is proposed. According to embodiments of this application, the method includes: immunizing animals with the polypeptide described in the first aspect, the recombinant protein described in the second aspect, or the reagents or kits described in the third aspect, to obtain the anti-CXCR5 protein antibody or its antigen-binding fragment. The method described above for preparing the anti-CXCR5 protein antibody or its antigen-binding fragment results in an anti-CXCR5 protein antibody or its antigen-binding fragment exhibiting a strong binding affinity to the CXCR5 protein. This effectively reduces the cost and time required for CXCR5 antibody drug development, providing a new strategy for the treatment and diagnosis of tumors and autoimmune diseases.

[0156] In another aspect of this application, a fusion protein is proposed. According to embodiments of this application, the fusion protein comprises: the anti-CXCR5 protein antibody or antigen-binding fragment described in the sixth aspect, or the anti-CXCR5 protein antibody or antigen-binding fragment prepared according to the above method; and a bioactive protein or fragment thereof. The fusion protein of this application has a strong binding affinity to the CXCR5 protein and can be used for the detection of the CXCR5 protein, or for the treatment and diagnosis of tumors and autoimmune diseases.

[0157] According to embodiments of this application, the fusion protein may further include at least one of the following technical features:

[0158] According to embodiments of this application, the bioactive protein or fragment thereof includes at least one selected from protein tags, albumin or fragment thereof, and Fc fragments.

[0159] In this article, "protein tag" generally refers to a polypeptide or protein fused together with a target protein (antibody or antigen-binding fragment) for expression, detection, retrieval, or purification of the target protein. Examples include, but are not limited to, His tags (also known as His-Tag, sequence HHHHHH), Flag tags (also known as Flag-Tag, sequence DYKDDDDK), GST tags (also known as GST-Tag, glutathione thiotransferase tag), MBP tags (also known as MBP-Tag, maltose-binding protein tag), SUMO tags, and C-Myc tags.

[0160] In this document, "Fc fragment" generally refers to the Fc region of IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM, including CH2, CH3 regions and optionally hinge regions. Preferably, the IgG, IgA1, IgA2, IgD, IgE, or IgM is derived from mouse, human, primate, or alpaca sources.

[0161] According to embodiments of this application, the albumin or a fragment thereof is serum albumin or a fragment thereof.

[0162] According to embodiments of this application, the albumin or a fragment thereof is human serum albumin.

[0163] In another aspect of this application, a conjugate is proposed. According to an embodiment of this application, the conjugate comprises: the anti-CXCR5 protein antibody or antigen-binding fragment described in the sixth aspect, the anti-CXCR5 protein antibody or antigen-binding fragment prepared according to the above method, or the above-described fusion protein; and a coupling portion linked to the anti-CXCR5 protein antibody or antigen-binding fragment, or the fusion protein. The conjugate of this application has CXCR5 binding affinity and can be used to detect CXCR5, detect cells expressing CXCR5, or for the prevention and / or treatment of CXCR5-mediated diseases.

[0164] According to embodiments of this application, the above-mentioned coupling may further include at least one of the following technical features:

[0165] According to embodiments of this application, the coupling portion includes at least one selected from carriers, drugs, toxins, cytokines, protein tags, modifiers, and chemotherapeutic agents.

[0166] In this document, the carrier can be a substance capable of suspension or dispersion in a liquid phase (e.g., solid-phase carriers such as particles and magnetic beads), or a solid phase capable of containing or carrying a liquid phase (e.g., supports such as plates, membranes, and test tubes, as well as containers such as well plates, microfluidic paths, glass capillaries, nanopillars, and monolithic columns); it can also be a labeling carrier for labeling antibodies or antigen-binding fragments or fusion proteins against CXCR5 protein, such as enzymes (e.g., peroxidase, alkaline phosphatase, luciferin, β-galactosidase), affinity substances (e.g., one of streptavidin and biotin, one of complementary sense and antisense nucleic acids), fluorescent dyes (e.g., fluorescein, fluorescein isothiocyanate, rhodamine, green fluorescent protein, red fluorescent protein), luminescent substances (e.g., luciferin, aequorin, acridinium ester, tris(2,2'-bipyridine)ruthenium, luminol), and radioactive isotopes (e.g., 3 H, 14 C 32 P, 35 S, 125 I) and gold colloids, etc.

[0167] According to embodiments of this application, the carrier comprises a fluorescent dye.

[0168] According to an embodiment of this application, the conjugate is a staining reagent, and the conjugate portion is a fluorescent dye.

[0169] According to embodiments of this application, the fluorescent dye includes any one or more of the following: FITC, PE, Cy5, PI, 7-AAD, APC, AlexaFluor, eFluor, PE-Cy7, APC-Cy7, PerCP, PerCPcy5.5, PB, BV series, QDot series, and BUV series dyes.

[0170] According to embodiments of this application, the drug is a small molecule drug that can bind to antibodies or antigen-binding fragments, fusion proteins, or multispecific antibodies.

[0171] According to embodiments of this application, the protein tag includes, but is not limited to, His tag, Flag tag, GST tag, MBP tag, SUMO tag, and C-Myc tag.

[0172] According to embodiments of this application, the term "modifier" should be interpreted broadly and may refer to substances used to modify proteins. Exemplarily, it may be polyethylene glycol or a derivative thereof.

[0173] It should be noted that the binding method between the conjugated portion and the antibody or antigen-binding fragment, or the fusion protein, can use methods known in the art. For example, methods such as physical adsorption, covalent binding, methods using affinity substances (e.g., biotin, streptavidin), and ion binding can be used.

[0174] Nucleic acid molecules, expression vectors, recombinant proteins

[0175] In a seventh aspect of this application, a nucleic acid molecule is provided. According to embodiments of this application, the nucleic acid molecule encodes the polypeptide described in the first aspect, the recombinant antigen described in the second aspect, the anti-CXCR5 protein antibody or antigen-binding fragment described in the sixth aspect, the anti-CXCR5 protein antibody or antigen-binding fragment prepared according to the above-described method, or the above-described fusion protein. The nucleic acid molecule of this application may encode the above-described polypeptide, the above-described recombinant antigen, the above-described anti-CXCR5 protein antibody or antigen-binding fragment, or the above-described fusion protein.

[0176] According to an embodiment of this application, the nucleic acid molecule is DNA.

[0177] It should be noted that those skilled in the art will understand that the nucleic acid molecules mentioned herein actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases in this specification and claims, the other complementary strand is also disclosed. Furthermore, the nucleic acid sequences in this application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0178] In an eighth aspect, this application provides an expression vector. According to an embodiment of this application, the expression vector carries the nucleic acid molecule described in the seventh aspect. When linking the nucleic acid molecule to the expression vector, the nucleic acid molecule can be directly or indirectly connected to control elements on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the expression vector itself, or they can be exogenous, i.e., not derived from the expression vector itself. Naturally, the nucleic acid molecule and the control elements only need to be operably linked.

[0179] In this article, "operable ligation" refers to ligating a foreign gene into an expression vector so that the control elements within the expression vector, such as transcriptional and translational control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used expression vectors include plasmids and bacteriophages.

[0180] According to some specific embodiments of this application, after the expression vector is introduced into suitable recipient cells, it can effectively realize the expression of the aforementioned peptides, recombinant antigens, anti-CXCR5 protein antibodies or antigen-binding fragments, and fusion proteins under the mediation of the regulatory system, thereby achieving the in vitro large-scale acquisition of peptides, recombinant antigens, anti-CXCR5 protein antibodies or antigen-binding fragments, and fusion proteins.

[0181] In some specific embodiments of this application, the above-mentioned vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus, or a bacteriophage.

[0182] In some specific embodiments of this application, the above-mentioned vector is a lentiviral vector.

[0183] In one optional embodiment of this application, the expression vector is a plasmid expression vector.

[0184] In a ninth aspect of this application, a recombinant cell is provided. According to embodiments of this application, the recombinant cell carries the nucleic acid molecule described in the seventh aspect or the expression vector described in the eighth aspect, or expresses the polypeptide described in the first aspect, the recombinant antigen described in the second aspect, the anti-CXCR5 protein antibody or antigen-binding fragment described in the sixth aspect, the anti-CXCR5 protein antibody or antigen-binding fragment prepared according to the above methods, or the above-described fusion protein. Using this recombinant cell under suitable conditions, the aforementioned polypeptide, recombinant antigen, anti-CXCR5 protein antibody or antigen-binding fragment, and fusion protein can be effectively expressed intracellularly.

[0185] According to some specific embodiments of this application, the recombinant cells can efficiently and extensively express polypeptides, recombinant antigens, anti-CXCR5 protein antibodies or antigen-binding fragments, and fusion proteins under suitable conditions, wherein the aforementioned anti-CXCR5 protein antibodies or their antigen-binding fragments have stronger specificity.

[0186] It should be noted that "suitable conditions" refers to conditions suitable for the expression of the polypeptides, recombinant antigens, anti-CXCR5 protein antibodies or antigen-binding fragments, and fusion proteins described in this application. Those skilled in the art will readily understand that suitable conditions for the expression of the polypeptides, recombinant antigens, anti-CXCR5 protein antibodies or antigen-binding fragments, and fusion proteins include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the aforementioned polypeptides, recombinant antigens, anti-CXCR5 protein antibodies or antigen-binding fragments, and fusion proteins according to the specific environment of their laboratory.

[0187] According to an embodiment of this application, the recombinant cells are obtained by introducing the expression vector described in the eighth aspect into a host cell.

[0188] It should be noted that the recombinant cells described in this application are not particularly limited and can be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, etc. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosoma, and Trichoderma; insect cells such as armyworms; plant cells such as tobacco; and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells. In some embodiments, the recombinant cells described in this application are preferably mammalian cells, including BHK cells, CHO cells, NSO cells, or COS cells, and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0189] According to embodiments of this application, the recombinant cells are eukaryotic cells, preferably mammalian cells.

[0190] product

[0191] In a tenth aspect of this application, a product is provided. According to embodiments of this application, the composition comprises the anti-CXCR5 protein antibody or antigen-binding fragment described in the sixth aspect, the anti-CXCR5 protein antibody or antigen-binding fragment prepared according to the above method, the above-described fusion protein, or the above-described conjugate. The product of this application has CXCR5 binding affinity and can be used to detect CXCR5, detect cells expressing CXCR5, or for the prevention and / or treatment of CXCR5-mediated diseases.

[0192] According to embodiments of this application, the product is selected from compositions, reagents, or kits.

[0193] According to embodiments of this application, the composition is a pharmaceutical composition.

[0194] According to embodiments of this application, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0195] The pharmaceutical compositions of this application can be administered via any acceptable method of administration. The pharmaceutical compositions of this application can be formulated into solid, semi-solid, liquid, or gaseous forms, such as injections or lyophilized powders, as currently known or readily apparent to those skilled in the art. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection, or infusion techniques. The pharmaceutical compositions of this application are formulated to ensure that the bioactive components contained therein are bioavailable after administration to a patient.

[0196] According to embodiments of this application, the product is selected from reagents or kits. The reagents or kits have CXCR5 binding affinity and can be used to detect CXCR5 and to detect cells expressing CXCR5.

[0197] As previously mentioned, the anti-CXCR5 protein antibodies or antigen-binding fragments of some specific embodiments of this application can effectively bind to human CXCR5 protein. Therefore, reagents or kits containing the aforementioned anti-CXCR5 protein antibodies or antigen-binding fragments can effectively perform qualitative or quantitative detection of human CXCR5 protein. The reagents or kits provided in this application can be used, for example, in immunoblotting, immunoprecipitation, and other reagents or kits involving the detection of human CXCR5 using the specific binding of antibodies. These kits may contain any one or more of the following: antagonists, anti-CXCR5 antibodies, or drug reference materials; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents; instructions or literature, etc. Anti-CXCR5 antibodies can be used for different types of diagnostic tests, such as detecting the presence of various diseases, drugs, toxins, or other proteins in vitro or in vivo. For example, they can be used to test for related diseases by detecting the serum or blood of a subject, or for scientific research, using the kits to detect human CXCR5 protein in the sample to be tested. Such related diseases may include CXCR5-related diseases, such as cancer. The binding molecules are also applicable to the above applications and will not be elaborated further here.

[0198] According to some specific embodiments of this application, the kit may also include commonly used ingredients for detecting CXCR5, such as coating solutions.

[0199] use

[0200] In the eleventh aspect of this application, this application proposes the use of the anti-CXCR5 protein antibody or antigen-binding fragment described in the sixth aspect, the anti-CXCR5 protein antibody or antigen-binding fragment prepared according to the above method, the above fusion protein, or the above conjugate in the preparation of products or drugs, wherein the products are used to detect CXCR5 protein, and the drugs are used to prevent and / or treat diseases related to pathogenic cells expressing CXCR5.

[0201] According to embodiments of this application, the pathogenic cells expressing CXCR5 are selected from pathogenic mature B cells, memory B cells, pathogenic T cells, and follicular helper T cells.

[0202] According to embodiments of this application, the diseases associated with pathogenic cells expressing CXCR5 are selected from B-cell-derived lymphoproliferative disorders and T-cell-derived lymphoproliferative disorders.

[0203] According to embodiments of this application, the B-cell-derived lymphoproliferative disorders are selected from acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), and diffuse large B-cell lymphoma (DLBCL).

[0204] According to embodiments of this application, the T-cell-derived lymphoproliferative disease is selected from angioimmunoblastic T-cell lymphoma, cutaneous T-cell lymphoma, and T-cell lymphoma with leukemia dissemination.

[0205] method

[0206] In a twelfth aspect of this application, a method for detecting CXCR5 protein is provided. According to an embodiment of this application, the method includes: contacting a sample to be tested with an anti-CXCR5 protein antibody or antigen-binding fragment as described in the sixth aspect, an anti-CXCR5 protein antibody or antigen-binding fragment prepared according to the above method, the above-described fusion protein, the above-described conjugate, or the above-described reagent or kit to form an immune complex.

[0207] According to embodiments of this application, based on the signal of the immune complex, it is determined whether the sample to be tested contains CXCR5 protein or whether it contains cells expressing CXCR5 protein, or the content of CXCR5 protein or cells expressing CXCR5 protein in the sample to be tested.

[0208] According to embodiments of this application, the signal of the immune complex is detected by at least one of ELISA, Western Blot, and IHC.

[0209] According to embodiments of this application, the immune complex further includes a second antibody, which binds to the anti-CXCR5 protein antibody or antigen-binding fragment.

[0210] According to embodiments of this application, the signal includes a fluorescence signal.

[0211] In a thirteenth aspect of this application, a method for preventing and / or treating diseases associated with pathogenic cells expressing CXCR5 is provided. According to embodiments of this application, the method comprises administering to a subject a pharmaceutically acceptable dose of the anti-CXCR5 protein antibody or antigen-binding fragment described in the sixth aspect, the anti-CXCR5 protein antibody or antigen-binding fragment prepared according to the above method, the above-described fusion protein, or the above-described conjugate.

[0212] The effective amount of the anti-CXCR5 protein antibody or antigen-binding fragment, pharmaceutical composition, or conjugate described in this application may vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0213] The anti-CXCR5 protein antibody or antigen-binding fragment, pharmaceutical composition, or conjugate of this application may be incorporated into a medicament suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These medicaments may be prepared in various forms, such as liquid, semi-solid, and solid dosage forms, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions) or lyophilized powders. Typical medicaments are in the form of injection solutions or infusion solutions. The aforementioned anti-CXCR5 protein antibody or antigen-binding fragment, pharmaceutical composition, or conjugate may be administered via intravenous infusion or injection, or intramuscular or subcutaneous injection.

[0214] In this document, the term "subject" refers to a vertebrate, preferably a mammal, and most preferably a human. Mammals include, but are not limited to, rodents, apes, humans, livestock, racing animals, and pets. Tissues, cells, and their progeny from biological entities obtained in vivo or cultured in vitro are also included.

[0215] According to embodiments of this application, the pathogenic cells expressing CXCR5 are selected from pathogenic mature B cells, memory B cells, pathogenic T cells, and follicular helper T cells.

[0216] According to embodiments of this application, the diseases associated with pathogenic cells expressing CXCR5 are selected from B-cell-derived lymphoproliferative disorders and T-cell-derived lymphoproliferative disorders.

[0217] According to embodiments of this application, the B-cell-derived lymphoproliferative disorders are selected from acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), and diffuse large B-cell lymphoma (DLBCL).

[0218] According to embodiments of this application, the T-cell-derived lymphoproliferative disease is selected from angioimmunoblastic T-cell lymphoma, cutaneous T-cell lymphoma, and T-cell lymphoma with leukemia dissemination.

[0219] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0220] Example 1: Synthesis of CXCR5 peptide

[0221] To obtain antibodies that specifically recognize the extracellular domain of CXCR5, antigen-binding sites were predicted for this target before immunization. The CXCR5 sequence (P32302) downloaded from the uniprot database was entered into the website https: / / www.detaibio.com / tools / epitope-prediction.html for prediction. The Parker algorithm was selected in the output, resulting in 13 types of sequences (score > 5). Among them, 7 types of sequences belonged to the extracellular region. Furthermore, the amino acid sequence of CXCR5 was predicted using the GenScript antigen peptide design tool, yielding 7 types of peptides. Combining these two methods for antigen-binding site prediction, 4 types of peptides were designed, synthesized, and expressed by GenScript.

[0222] The amino acid sequence information of the CXCR5 polypeptide is as follows:

[0223] name sequence Peptide 1 NYPLTLEMDLENLEDLFC(SEQ ID NO:1) Peptide 2 ENHLSPATEGPLMASFKAVFC(SEQ ID NO:3) Polypeptide 3 FWELDRLDNYNDTSLVENHLC(SEQ ID NO:2) Peptide 4 LARLKAVDNTSKLNGSLPC(SEQ ID NO:4)

[0224] Example 2: Animal Immunization and Plasma Titer Detection

[0225] (1) Animal Immunization

[0226] To enrich specific antibodies from alpaca plasma, an immunization strategy was employed to cross-immunize the CXCR5 peptides (peptide 1 with amino acid sequence as shown in SEQ ID NO:1, peptide 2 with amino acid sequence as shown in SEQ ID NO:3, peptide 3 with amino acid sequence as shown in SEQ ID NO:2, and peptide 4 with amino acid sequence as shown in SEQ ID NO:4) designed in Example 1 with recombinant protein (Synthetium oxyphylla, 18301-H82H). The immunization strategy was as follows: the first three immunizations used four types of CXCR5 peptides; thereafter, each immunization involved cross-immunization with a mixture of peptides and recombinant antigen, every two weeks. Before each immunization, 2-5 mL of peripheral blood was collected for serum titer testing to assess the enrichment of CXCR5-specific antibodies in the alpaca.

[0227] (2) Plasma titer testing

[0228] 100 ng of recombinant CXCR5 protein, peptide 1, peptide 2, peptide 3, and peptide 4 were coated onto an ELISA plate and incubated overnight at 4°C. The next day, 2% BSA (diluted with PBST) was added, and the plate was blocked at room temperature for 2 hours. Plasma samples before and after immunization were analyzed 10-fold. ～ 10 5 Dilute 1:10000 and incubate at room temperature for 1 hour; wash 5 times with PBST, add 1:10000 diluted Goat Anti-Alpaca IgG H&L(HRP) (AlpVHHs, 053-404-005) antibody, and incubate at room temperature for 1 hour; wash 5 times with PBST, add chromogenic solution (abcam, ab171522), react for 5 minutes, and then add stop solution (abcam, ab171529). Read the absorbance on a microplate reader. The results are as follows. Figure 1 As shown.

[0229] The serum titer after immunization reached as high as 10. 5 This indicates that the alpaca produced CXCR5-specific antibodies. Compared with pre-immunization plasma, the plasma titer significantly increased to over 1000 after peptide immunization. On day 14 after immunization, 30 mL of peripheral blood was collected from the alpaca for PBMC enrichment.

[0230] Example 3: Screening positive clones using phage display technology

[0231] (1) Construction of phage-display nanobody library

[0232] 30 mL of alpaca blood sample was collected after immunization in Example 2. PBMC enrichment was performed according to the instructions for lymphocyte separation medium (GE, 17-1440-02). TRIzol (INVITROGEN, 15596-018) was added to induce complete cell lysis. RNA was extracted using the phenol-chloroform method. The concentration of the obtained RNA was determined by Nanodrop 2000, and its purity was identified by agarose gel electrophoresis. The procedure was performed according to SuperScript. TM III. First-Strand Synthesis SuperMix (Invitrogen, 18080-044) kit was used for cDNA synthesis. The reverse transcription product cDNA was used as a template to amplify the V region fragment (VHH) of the heavy chain antibody using Nest-PCR.

[0233] The first round of PCR amplification reaction system consisted of 10 μL cDNA, 1 μL Primer 1 (10 μm, Sangon Biotech), 1 μL Primer 2 (10 μm, Sangon Biotech), and 12.5 μL 2×tans Taq Mix (NEB, Y1527), brought to a total of 25 μL with NF-H2O (Invitrogen, AM9937). The sequence of Primer 1 was: GTCCTGGCTGCTCTTCTACAAGG (SEQ ID NO:16); the sequence of Primer 2 was: GGTACGTGCTGTTGAACTGTTCC (SEQ ID NO:17). The first round amplification program was: 95℃, 5 min, (95℃, 45 s, 56℃, 45 s, 72℃, 45 s) for 18 cycles, followed by 72℃, 5 min.

[0234] The second-round PCR amplification reaction system consisted of 3 μL of the first-round amplification product, 1 μL of Primer 3 (10 μm, bioengineered), 1 μL of Primer 4 (10 μm, bioengineered), and 25 μL of 2×tans Taq mix (NEB), brought to a total of 50 μL with NF-H2O. The Primer 3 sequence was: GATGTGCAGCTGCAGGAGTCTGGRGGAGG (SEQ ID NO:18); the Primer 4 sequence was: CTAGTGCGGCCGCTGAGGAGACGGTGACCTGGGT (SEQ ID NO:19). The second-round amplification program was: 95℃ for 5 min, (95℃, 45 s, 56℃, 45 s, 72℃, 45 s) for 15 cycles, followed by 72℃ for 5 min.

[0235] PCR products were analyzed by agarose gel electrophoresis at 120V for 40 min. The target fragment band was ~750bp in the first round of PCR and ~500bp in the second round. The target fragment was recovered using the QIAgen gel purification kit (Qiagen, 28706). After determining the concentration and purity of the recovered products using Nanodrop 2000, the obtained VHH fragment and pMECS phage vector were digested with restriction endonucleases NotI (NEB, R0189L) and PstI (NEB, R0140L), respectively. The next day, the digested products of the VHH fragment and phage vector were recovered by agarose gel electrophoresis and agarose gel recovery kit (QIAGEN, 20051), respectively, and ligated using T4 DNA ligase (NEB, M0202S). The ligation product was purified using a PCR Purification Kit (QIAGEN, 28106). The purified product was then transformed into *E. coli* TG1 (LUCIGEN, 60502-2) by electroporation. The resulting bacterial culture was the original phage-displayed nanobody library, with a volume of 2 × 10⁻⁶. 7 / mL.

[0236] (2) Screening of CXCR5 nanobodies

[0237] The electroporated bacterial culture was inoculated into 100 mL of culture medium and cultured at 37 °C and 220 rpm until the logarithmic growth phase. Infection was then performed by adding M13K07 helper phage with an MOI of 20. The culture was incubated at room temperature for 30 min, centrifuged at 3,000 × g for 10 min, and the precipitate was resuspended. The precipitate was then inoculated into 200 mL of 2TY medium and cultured overnight at 30 °C and 220 rpm. The next day, the phage supernatant was collected by centrifugation at 5,000 × g for 30 min. PEG6000 / NaCl was added to precipitate the phage, and the culture was incubated on ice for 30 min. The precipitate was then centrifuged at 5,000 × g and 4 °C for 30 min. The precipitate was resuspended in 1 mL of PBS, and its titer was calculated to be 4 × 10⁻⁶. 14 pfu / mL.

[0238] 1 μg of each of peptides 1-4 was used to coat ELISA plates overnight. The next day, 200 μL of 2% skim milk was added, and the plates were blocked at room temperature for 2 hours. The plates were then washed 5 times with 200 μL of PBST. 2 × 10⁻⁶ ppm of each peptide were added to the plates. 11PFU phage suspension was incubated at room temperature for 1 hour, followed by 5 washes with 100 μL PBST. 100 μL of 1.5% triethylamine was added, and the mixture was incubated at room temperature for 15 minutes for phage elution. The eluted phage was neutralized with 100 μL of Tris HCl (pH 7.4). 10 μL of the eluted phage was used to infect TG1 cells in logarithmic growth phase, and the cells were plated to assess the eluted phage titer. The remaining eluted phage was then amplified and rescued. The newly rescued phage was further washed, gradually reducing the amount of antigen added. The enrichment of phage after two rounds of washing is shown below. Figure 2 As shown.

[0239] The results showed that after two rounds of phage screening, the specific phages for peptide 1 were enriched by 1000 times, and peptide 3 was enriched by 100 times.

[0240] Furthermore, antibodies 3-24 and 1-48 were obtained from the second round of phage screening. The heavy chain variable region of antibody 3-24 is shown in SEQ ID NO:11, and the heavy chain variable region of antibody 1-48 is shown in SEQ ID NO:12.

[0241] Example 4: Assay of CXCR5 Nanobody Activity

[0242] (1) Combining activity detection

[0243] To verify whether antibodies 3-24 and 1-48 screened in Example 3 could bind to the CXCR5 peptide and recombinant protein, 100 ng of peptides 1, 2, 3, and 4 from Example 1, along with recombinant CXCR5 protein (Yiqiao, 18301-H82H), were coated onto ELISA plates and incubated at 37°C for 2 h. The plates were washed twice with PBST, and 2% BSA (diluted with PBST) was added, followed by blocking at room temperature for 2 h. Then, 1.5 μg / mL of antibodies 3-24 and 1-48 screened in Example 3 were added, and the plates were incubated at room temperature for 1 h. The plates were washed five times with PBST, and a 1:3000 dilution of Anti-HA (HRP) (abcam, ab128131) antibody was added, followed by incubation at room temperature for 1 h. The plates were washed five times with PBST, and the chromogenic substrate was added. After reacting for 5–10 min, the stop solution was added. The absorbance was read on an ELISA reader, and the results are as follows: Figure 3 As shown.

[0244] The results showed that antibody 3-24 exhibited good binding ability to peptide 3, while antibody 1-48 was able to bind to peptide 1. Furthermore, both 3-24 and 1-48 were able to bind to recombinant CXCR5 protein.

[0245] (2) Affinity test

[0246] 200 ng of recombinant CXCR5 protein (Yiqiao, 18301-H82H) was coated onto an ELISA plate and incubated overnight at 4°C. The next day, 2% BSA (diluted with PBST) was added, and the plate was blocked at room temperature for 2 h. Four-fold serial dilutions of antibodies 3-24 and 1-48 selected in Example 3 were added, with an initial concentration of 100 nM, and incubated at room temperature for 1 h. After washing 5 times with PBST, 1:3000 dilution of Anti-HA (HRP) antibody was added, and the plate was incubated at room temperature for 1 h. After washing 5 times with PBST, the chromogenic substrate was added, and after reacting for 5 min, stop solution was added. The absorbance was read on an ELISA reader, and the results are as follows: Figure 4 As shown in the figure. The results showed that both antibodies 3-24 and 1-48 had good binding ability to recombinant CXCR5 protein. The EC50 was calculated based on the binding curve. 50 The values ​​all reached the nanomolar level.

[0247] To further detect the affinity of antibodies 3-24 and 1-48 for recombinant CXCR5 protein, the recombinant CXCR5 protein was coupled to a CM chip, and an affinity dissociation process was run on a Biacore T100. Antibodies 3-24 and 1-48 were serially diluted 2-fold, respectively, at an inflow rate of 30 μl / min, with binding time of 120 s and dissociation time of 180 s. The results are shown in Table 1.

[0248] The results showed that antibody 3-24 had an affinity of 3.03E-09(M) for recombinant CXCR5 protein, and antibody 1-48 had an affinity of 1.06E-08(M) for recombinant CXCR5 protein, indicating that both candidate antibodies 3-24 and 1-48 had good affinity.

[0249] Table 1: SPR test for CXCR5 nanobody affinity

[0250] Nanobody numbering ka(1 / Ms) kd(1 / s) KD(M) Rmax(RU) 1-48 4.36E+05 0.00462 1.06E-08 31 3-24 6.55E+05 0.00198 3.03E-09 163

[0251] (3) Specific detection

[0252] To verify whether antibodies 3-24 and 1-48 screened in Example 3 can specifically recognize recombinant human CXCR5 protein (sigma, 18301-H82H), cross-binding tests were performed on antibodies 3-24 and 1-48 with different recombinant proteins, using 200 ng of each antibody. Recombinant CXCR5 protein (Yiqiao, 18301-H82H) and other recombinant proteins, including human CD27 protein (ACRO, CD7-H5254), human CD40 protein (Yiqiao, 10774-H08H), human CD117 protein (ACRO, CD7-H52H4), and human CD3 protein (Yiqiao, 10977-H08S), were coated onto ELISA plates and incubated overnight at 4°C. The next day, 2% BSA was added for blocking at room temperature for 2 hours. Antibody 3-24 and 1-48 (1.5 μg / mL) were added and incubated at room temperature for 1 hour. After washing five times with PBST, diluted Anti-HA (HRP) antibody was added and incubated at room temperature for 1 hour. After washing five times with PBST, chromogenic buffer was added, and stop buffer was added within 10 minutes of reaction. The absorbance was read on a microplate reader, and the results are as follows: Figure 5 As shown.

[0253] The results showed that both antibodies 3-24 and 1-48 could specifically recognize human CXCR5 protein, exhibiting high target specificity.

[0254] Example 5: Modification of CXCR5 chimeric bivalent antibody

[0255] To obtain antibody products that match indirect immunoassay, antibodies 3-24 and 1-48 were bivalently modified. The sequences of antibodies 3-24 and 1-48 were directly constructed into a pcDNA3.1 vector containing the Fc of mouse IgG1 (amino acid sequence as shown in SEQ ID NO:22) via gene synthesis. The vector was then transfected into HEK 293T cells and cultured at 37°C, 8% CO2, relative humidity ≥80%, and shaking at 150 rpm for 5-6 days. The supernatant was collected and purified with protein A to obtain recombinant chimeric bivalent antibodies, namely antibody 3-24-mFc (amino acid sequence as shown in SEQ ID NO:14) and antibody 1-48-mFc (amino acid sequence as shown in SEQ ID NO:15).

[0256] (1) Detection of binding activity of bivalent antibodies

[0257] To verify whether the bivalent antibody retained its binding activity to CXCR5, binding capacity and affinity were determined. 100 ng of peptides 1, 2, 3, and 4 from Example 1, along with recombinant CXCR5 protein (Yiqiao, 18301-H82H), were coated onto ELISA plates and incubated at 37°C for 2 h. The plates were washed twice with PBST, and 2% BSA (diluted with PBST) was added, followed by blocking at room temperature for 2 h. 1.5 μg / mL of antibody 3-24-mFc and antibody 1-48-mFc were added, and the plates were incubated at room temperature for 1 h. The plates were washed five times with PBST, and a 1:3000 dilution of Anti-mouse Fc(HRP) (Invitrogen, 31437) antibody was added, followed by incubation at room temperature for 1 h. The plates were washed five times with PBST, and the chromogenic substrate was added. After reacting for 5–10 min, stop solution was added. Absorbance values ​​were read on an ELISA reader. The OD values ​​of the experimental groups were... 450 The value of nm minus the OD of the negative control group 450 A bar chart was plotted using the values ​​of nm, and the results are as follows: Figure 6 As shown.

[0258] The results showed that both antibodies 3-24-mFc and 1-48-mFc could bind to recombinant CXCR5 protein and exhibited good binding activity. Antibody 3-24-mFc could also bind to four peptides, with the highest binding activity observed with peptide 3.

[0259] (2) Affinity detection of bivalent antibodies

[0260] The microplate was coated with 200 ng of CXCR5 recombinant protein and incubated overnight at 4°C. ELISA was performed as above. The next day, diluted antibodies 3-24-mFc and 1-48-mFc were added and incubated at room temperature for 1 h. Anti-mouse Fc (HRP) secondary antibody diluted 1:3000 was added and incubated at room temperature for 1 h. The colorimetric reaction was terminated within 10 min of developing the chromogenic solution, and the absorbance was read on the microplate reader. Results are shown below. Figure 7 .

[0261] The results showed that the affinity of antibody 3-24-mFc was 0.04 nM and that of antibody 1-48-mFc was 0.04 nM.

[0262] Furthermore, the affinity of antibodies 3-24-mFc and 1-48-mFc for the recombinant CXCR5 protein was detected on a Biacore T200 instrument, and the results are shown in Table 2.

[0263] The results showed that the affinity of the 3-24-mFc antibody reached 3.24E-10(M), while the affinity of the 1-48-mFc antibody reached 6.79E-10(M), which is more than 10 times higher than that of the nanobody. Therefore, this further demonstrates that the antibody has good development potential and application value.

[0264] Table 2: SPR test for affinity of CXCR5 bivalent antibody

[0265] Nanobody numbering ka(1 / Ms) kd(1 / s) KD(M) Rmax(RU) 3-24-mFc 2.31E+06 7.48E-04 3.24E-10 5.124 1-48-mF 1.11E+06 7.52E-04 6.79E-10 8.101

[0266] Example 6: Flow cytometry assay of 3-24-mFc and 1-48-mFc binding activity

[0267] To confirm whether antibodies 3-24-mFc and 1-48-mFc could recognize the cell surface CXCR5 antigen and to apply them to flow cytometry detection, Raji cells were washed with PBS-F (PBS containing 5% FBS), centrifuged at 400×g for 5 min, and the cells were collected and added with 2% BSA, then blocked at 4℃ for 1 h. 2.5E+05 cells were then bound to antibodies 3-24-mFc and 1-48-mFc, respectively. A positive control was used with Anti-CXCR5 antibody (abcam, ab272936), and the cells were incubated at 4℃ for 1 h. Cells were washed twice with PBS-F, and then incubated with a 1:200 diluted fluorescent secondary antibody at 4℃ in the dark for 1 h. After washing twice with PBS-F and centrifuging at 400×g for 5 min, the cells were resuspended in 200 μL of working buffer and analyzed by flow cytometry. Results are shown below. Figure 8 .

[0268] Flow cytometry results showed that the cell binding rate of antibody 3-24-mFc was 89.05%, and that of antibody 1-48-mFc was 98.71%, comparable to commercially available antibodies. Therefore, this further demonstrates that these two antibodies (3-24-mFc and 1-48-mFc) can bind well to the CXCR5 target on the cell surface and can be used as candidate antibody molecules for flow cytometry-ready antibody development.

[0269] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0270] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:1 or 2.

2. A recombinant protein, characterized in that, The invention comprises the polypeptide and carrier protein of claim 1, wherein the carrier protein comprises at least one of protein tag, hemocyanin, bovine serum albumin and ovalbumin.

3. A reagent or kit, characterized in that, Includes the polypeptide of claim 1 or the recombinant protein of claim 2.

4. Use of the polypeptide of claim 1, the recombinant protein of claim 2, or the reagent or kit of claim 3 in the preparation of anti-CXCR5 protein antibodies.

5. A method for preparing anti-CXCR5 protein antibody, characterized in that, include: The target animal is immunized with the polypeptide of claim 1, the recombinant protein of claim 2, or the reagent or kit of claim 3 to obtain serum from the immunized target animal; The antibodies in the serum were screened to obtain anti-CXCR5 protein antibodies.

6. An anti-CXCR5 protein antibody or its antigen-binding fragment, characterized in that, The anti-CXCR5 protein antibody or its antigen-binding fragment includes HCDR1-3; wherein... 1) The amino acid sequence of HCDR1 is shown in SEQ ID NO:5; The amino acid sequence of HCDR2 is shown in SEQ ID NO:6; The amino acid sequence of HCDR3 is shown in SEQ ID NO:7; or 2) The amino acid sequence of HCDR1 is shown in SEQ ID NO:8; The amino acid sequence of HCDR2 is shown in SEQ ID NO:9; The amino acid sequence of HCDR3 is shown in SEQ ID NO:

10.

7. The anti-CXCR5 protein antibody or its antigen-binding fragment according to claim 6, characterized in that, The anti-CXCR5 protein antibody or antigen-binding fragment includes a heavy chain framework region; Optionally, at least a portion of the heavy chain framework region is derived from at least one of mouse antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof; Optionally, at least a portion of the heavy chain framework region is derived from at least one of a rabbit antibody or a mutant thereof, a mouse antibody or a mutant thereof, and a human antibody or a mutant thereof; Optionally, the antibody or antigen-binding fragment comprises: The amino acid sequence is as shown in the heavy chain variable region of SEQ ID NO:11; or The amino acid sequence is shown in the heavy chain variable region of SEQ ID NO:12; Optionally, the anti-CXCR5 protein antibody or its antigen-binding fragment further includes a heavy chain constant region; Optionally, at least a portion of the heavy chain constant region is derived from at least one of mouse antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof; Optionally, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; Optionally, the heavy chain constant region is derived from at least one of a rabbit antibody or a mutant thereof, a mouse antibody or a mutant thereof, and a human antibody or a mutant thereof; Optionally, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:13; Optionally, the anti-CXCR5 protein antibody comprises at least one of polyclonal antibodies and single-domain antibodies; or The antigen-binding fragment includes the VHH-Fc fusion protein.

8. The anti-CXCR5 protein antibody or its antigen-binding fragment according to claim 6, characterized in that, The anti-CXCR5 protein antibody or its antigen-binding fragment is obtained using the polypeptide of claim 1, the recombinant protein of claim 2, or the reagent or kit of claim 3; And / or, the anti-CXCR5 protein antibody or antigen-binding fragment comprises: The amino acid sequence is as shown in the heavy chain of SEQ ID NO:14; or The amino acid sequence is shown in SEQ ID NO:15 for the heavy chain.

9. A nucleic acid molecule, expression vector, or recombinant cell, characterized in that, The nucleic acid molecule encodes the polypeptide of claim 1, the recombinant antigen of claim 2, or the anti-CXCR5 protein antibody or antigen-binding fragment of any one of claims 6 to 8; The expression vector carries the aforementioned nucleic acid molecules; The recombinant cells carry the aforementioned nucleic acid molecules or the aforementioned expression vector, or The recombinant cells express the polypeptide of claim 1, the recombinant antigen of claim 2, or the anti-CXCR5 protein antibody or antigen-binding fragment of any one of claims 6 to 8.

10. A product characterized in that, include: The anti-CXCR5 protein antibody or antigen-binding fragment according to any one of claims 6 to 8; Optionally, the product is selected from compositions, reagents, or kits, preferably pharmaceutical compositions.

11. Use of the anti-CXCR5 protein antibody or antigen-binding fragment according to any one of claims 6 to 8 in the preparation of a product or drug, wherein the product is used to detect CXCR5 protein, and the drug is used to prevent and / or treat diseases associated with pathogenic cells expressing CXCR5; Optionally, the pathogenic cells expressing CXCR5 are selected from pathogenic mature B cells, memory B cells, pathogenic T cells, and follicular helper T cells; Optionally, the diseases associated with pathogenic cells expressing CXCR5 are selected from B-cell-derived lymphoproliferative disorders and T-cell-derived lymphoproliferative disorders. Optionally, the B-cell-derived lymphoproliferative disorders are selected from acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), mantle cell lymphoma (MCL), and diffuse large B-cell lymphoma (DLBCL). Optionally, the T-cell-derived lymphoproliferative disease is selected from angioimmunoblastic T-cell lymphoma, cutaneous T-cell lymphoma, and T-cell lymphoma with leukemia dissemination.

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