CN112521499A

CN112521499A - anti-CXCL 13 antibodies and uses thereof

Info

Publication number: CN112521499A
Application number: CN202011552066.3A
Authority: CN
Inventors: 勾蓝图; 杨金亮; 吴梦丹
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2020-12-24
Filing date: 2020-12-24
Publication date: 2021-03-19
Anticipated expiration: 2040-12-24
Also published as: CN112521499B

Abstract

The present invention relates to the technical field of antibodies, in particular to anti-CXCL13 antibodies or fragments thereof and their uses. The technical problem to be solved by the present invention is to provide an anti-CXCL13 antibody or antibody fragment with good affinity, stability and specificity. The technical solution of the present invention is to provide an antibody or a fragment thereof that can bind to CXCL13, which is obtained by humanization transformation of the CDR region of the preferred murine monoclonal antibody. The anti-CXCL13 antibody of the invention has good affinity, specificity and stability, can neutralize CXCL13, thereby inhibiting the biological function of CXCL13, and shows good application prospects in detecting CXCL13 and treating CXCL13-related diseases.

Description

anti-CXCL 13 antibodies and uses thereof

Technical Field

The invention relates to the technical field of antibodies, in particular to an anti-CXCL 13 antibody and a fragment thereof and application thereof.

Background

Antibodies are biological macromolecules composed of heavy chains and light chains, and play an important role in humoral immunity. The heavy chain or light chain of the antibody molecule is composed of a variable region and a constant region respectively, wherein the variable region mainly plays a role in combining a target antigen, and the constant region mainly plays a role in immune regulation. Antibodies can be classified into IgG, IgM, IgE, IgA, IgD, etc., and can be further subdivided into subtypes, depending on structural and sequence characteristics. For example, human IgG heavy chains can be classified into IgG1, IgG2, IgG3, and IgG4, and light chains can be classified into κ and λ. The antibody can be efficiently and specifically combined with a target molecule to regulate and control a signal path at the downstream of the target molecule, so that the antibody can be developed into a medicament to achieve the purpose of treating diseases. Currently, antibodies have been developed as an important biotechnological drug, and monoclonal antibodies are mostly IgG-type antibodies, so that the monoclonal antibodies are often referred to as IgG-type antibodies.

An antibody molecule of the IgG type is a tetramer of 2 heavy and 2 light chains, with a molecular weight of about 150kD, formed by interchain disulfide bonds. Antibody molecules can be divided into variable and constant regions according to structural and functional characteristics, with the variable region primarily serving for antigen binding and the constant region primarily serving for immunological effects. The variable regions of an antibody can be further divided into Complementarity Determining Regions (CDRs) and Framework Regions (FRs), wherein each of the heavy or light chains contains 3 CDR regions (heavy chain VH-CDR1, VH-CDR2, VH-CDR3, light chain VL-CDR1, VL-CDR2, VL-CDR 3.) and 4 FR regions flanking each CDR region (FR1, FR2, FR3, FR 4). The loop formed by the CDR region is the main part for combining the antibody molecule with the antigen, and the FR region forms the support structure of the CDR region through space folding. The specific recognition of different antigen molecules by antibodies is mainly realized by the amino acid polymorphism of 6 CDR regions (VH-CDR1, 2, 3 and VL-CDR1, 2, 3) and the conformation polymorphism of loop. Because the structural similarity of the FR regions of different antibodies is higher, after the CDR region of one antibody is replaced with the CDR region of other antibody molecules, if the FR regions of different antibody molecules are matched properly, the conformational change of the CDR region before and after replacement is smaller, so that the new variable region formed after replacement can still retain the antigen binding capacity, and the characteristic is the basis of CDR grafting (CDR grafting) technology. CDR regions of a mouse antibody can be replaced with CDRs of a human antibody by CDR grafting techniques, and thus recombined with human FR regions to form a humanized antibody (humanized antibody) which retains the antigen-binding ability if the FR regions of the human-mouse antibody are properly matched.

Hybridoma technology is currently an important technology in the antibody discovery process. After the antigen immunization of a mouse, B cells of the mouse develop in lymphoid tissues to form germinal centers, and the affinity of the antibody is gradually improved through somatic high-frequency mutation (SHM). Separating mouse spleen cells, fusing with mouse myeloma cells in vitro to form hybridoma cells, and measuring the antibody activity in hybridoma culture supernatant to screen out the hybridoma cells producing the target antibody. The hybridoma cells are gradually monocloned after subcloning, mRNA of the subcloned cells is extracted for antibody variable region gene sequencing, and the amino acid sequence of the antibody variable region can be deduced. Currently, most of the antibodies on the market are screened by hybridoma technology. Since the antibody molecules derived from hybridoma technology complete the affinity maturation process in mice, those B cells that are cross-reactive with mouse self-proteins are eliminated in the mouse bone marrow by "negative selection", and thus the antibody molecules can effectively reduce the cross-reactivity with self-proteins or similar proteins.

Due to differences in amino acid sequences encoded by antibody genes between different species, Human Anti-mouse antibodies, i.e., HAMA (Human Anti-mouse antibody) reactions, are generated when murine antibodies are used for Human therapy, resulting in the development of ADA (Anti-drug antibody). The neutralizing anti-drug antibody not only can influence the target accessibility of the antibody drug and reduce the treatment effect of the antibody drug, but also can increase the risk of side effects due to immune complexes formed by the anti-drug antibody. The adoption of CDR grafting technology to transform murine antibody into humanized antibody can reduce HAMA reaction and reduce ADA incidence rate, which has been proved by clinical research. At present, dozens of humanized antibodies are on the market, including trastuzumab, pembrolizumab and bevacizumab, which indicates that the humanized technology is a reliable recombinant antibody technology for developing therapeutic antibodies.

Monoclonal antibodies can exert pharmacological effects through a variety of mechanisms. The antibody variable region can bind to soluble ligand outside cells, block the binding of the ligand and a receptor, and cut off downstream signal transmission induced by the ligand, so that antibody drugs developed by taking immune cytokines as targets can improve inflammatory diseases, such as adalimumab, belimumab, siltuximab and other antibodies are approved to be on the market. Antibody constant regions can exert immune regulatory effects including antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and the like, and thus antibody drugs developed targeting tumor cell surface molecules can kill tumor cells, such as commercially available antibodies like rituximab, trastuzumab, cetuximab, and the like.

Autoimmune diseases are a large group of diseases that cause damage by an immune reaction with the body's own tissues. The pathogenesis of many autoimmune diseases is quite complex and some are unclear. In the course of these diseases, the immune regulation process is disturbed, and various cytokines or immune cells can participate in the immune response of self tissues. Antibodies directed against a particular target may modulate a cytokine or immune cell of interest, inhibiting the progression of the immune response, and thereby ameliorate the symptoms of autoimmune disease. For example, adalimumab, which is a TNF- α -targeted antibody, can treat rheumatoid arthritis, belimumab, which is a BAFF-targeted antibody, can treat systemic lupus erythematosus, and ixekizumab, which is an IL-17A-targeted antibody, can treat psoriasis.

B cells are important immune cells and are involved in various autoimmune diseases, such as systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease, immune thrombocytopenic purpura, primary sjogren's syndrome, autoimmune thyroid disease, dermatomyositis, myasthenia gravis, and the like. Chemokine 13(C-X-C motif 13, CXCL13) plays an important role in B cell development. CXCL13 is also known as B Lymphocyte Chemokine (BLC) or B cell-attracting chemokine 1 (BCA-1) and belongs to the CXC chemokine family. CXCL13 is mainly secreted from follicular dendritic cells and is highly expressed in lymphoid tissues such as spleen and lymph nodes. The receptor of CXCL13 is chemokine receptor 5(C-X-C chemokine type 5, CXCR5), belongs to 7 transmembrane protein receptor, is mainly expressed on the surface of B cell, and recently is also reported to be expressed in partial T lymphocyte. B cells are attracted by CXCL13 and migrate to lymphoid follicles, which have important regulatory effects on the formation of the hair center. After CXCL13/CXCR5 signal axis is blocked, germinal center in lymph tissue is reduced, B cell dysgenesis is reduced, inflammation is reduced, and symptoms of autoimmune diseases are improved. In addition to autoimmune diseases, CXCL13 has also been shown to be associated with the proliferation and metastasis of some tumors, and can induce the proliferation and metastasis of CXCR5 positive tumors, so blocking the CXCL13/CXCR5 signaling axis can also inhibit the development of some tumor diseases. In addition, it has also been reported that CXCL 13-induced tumor microenvironment breg (regulation B cell) can be involved in the development of tumor diseases through immunosuppression.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a novel anti-CXCL 13 antibody or antibody fragment, which has good affinity, stability and specificity.

The technical scheme for solving the technical problem is to provide an anti-CXCL 13 antibody or a fragment thereof. The amino acid sequences of the heavy chain complementarity determining regions VH-CDR1, VH-CDR2 and VH-CDR3 of the antibody or the fragment thereof are respectively selected from SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, and the amino acid sequences of the light chain complementarity determining regions VL-CDR1, VL-CDR2 and VL-CDR3 are respectively selected from SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.

Further, the anti-CXCL 13 antibody or a fragment thereof contains a heavy chain variable region VH comprising the VH-CDR1, VH-CDR2 and VH-CDR3 linked to the framework region FR of a human antibody.

Preferably, the amino acid sequence of the VH of the heavy chain variable region of the antibody or the fragment thereof is shown as SEQ ID NO. 7.

Further, the anti-CXCL 13 antibody or a fragment thereof contains a light chain variable region VL formed by linking the VL-CDR1, VL-CDR2 and VL-CDR3 to the framework region FR of a human antibody.

Preferably, the amino acid sequence of the light chain variable region VL of the anti-CXCL 13 antibody or fragment thereof is represented by SEQ ID No. 8.

Further, the heavy chain constant region of the anti-CXCL 13 antibody or fragment thereof may be selected from the heavy chain constant regions of human immunoglobulin IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA, or IgD.

Further, the light chain constant region of the anti-CXCL 13 antibody or fragment thereof may be selected from the light chain constant regions of human immunoglobulin kappa or lambda.

The invention also provides nucleic acid molecules encoding the anti-CXCL 13 antibodies or fragments thereof described above.

The invention also provides a recombinant vector comprising a nucleic acid molecule encoding the anti-CXCL 13 antibody described above.

The invention also provides a cell containing the recombinant vector.

Further, the invention also provides application of the anti-CXCL 13 antibody in preparing a CXCL13 detection reagent.

Further, the invention also provides application of the anti-CXCL 13 antibody in preparing a medicament for blocking the binding of CXCL13 and CXCL13 receptors to inhibit the biological function of CXCL13

The antibody of the invention can be prepared into various forms of pharmaceutical preparations according to the conventional pharmaceutical technology, and liquid injections and freeze-dried injections are preferred.

The antibodies of the invention may be combined with other drugs to form pharmaceutical compositions that may be used to treat diseases in conjunction with other therapeutic methods, including chemotherapy, radiation therapy, biological therapy, and the like.

The invention has the beneficial effects that:

the invention provides an anti-CXCL 13 antibody, which is a humanized antibody formed by splicing the CDR region of a preferred murine monoclonal antibody and the FR region of a human antibody by adopting a CDR grafting technology, wherein the portability of the CDR region is better, and the CDR region shows enough affinity after being spliced and recombined with the FR regions of a plurality of human antibody germline members. The anti-CXCL 13 has good affinity, specificity and stability, can neutralize CXCL13 and block the combination of CXCL13 and a receptor such as CXCR5, thereby inhibiting the cell migration and growth induced by CXCL13, and has good application prospect in the aspects of detecting CXCL13 and treating CXCL13 related diseases and the like.

Drawings

FIG. 1: transwell experiments showed that neutralizing hybridoma antibodies inhibited cell migration.

FIG. 2: reduced SDS-PAGE analysis of recombinant monoclonal antibodies.

FIG. 3: the binding activity of the recombinant monoclonal antibody to CXCL13 was verified by ELISA.

FIG. 4: the Transwell experiment verifies the inhibition effect of the recombinant monoclonal antibody on cell migration.

FIG. 5: immunohistochemistry detected the expression of CXCL13 in tonsil tissues.

FIG. 6: and comparing the relative expression amount of the humanized antibody.

FIG. 7: plasma concentration-time profiles of humanized antibodies hNo5-1 and hNo5-4 in mice.

FIG. 8: cIEF analyzes the isoelectric point and charge isomer distribution of humanized antibody hNo 5-4.

FIG. 9: SEC-HPLC detection of humanized antibody hNo5-4 at various time points after treatment under stress conditions.

FIG. 10: detection of cIEF at various time points after treatment with stress conditions for humanized antibody hNo 5-4.

FIG. 11: humanized antibody hNo5-4 inhibited the migration of CXCR5 positive cells.

FIG. 12: humanized antibody hNo5-4 inhibited the growth of CXCR5 positive lymphoma cells.

Detailed Description

The invention provides a novel anti-CXCL 13 antibody or antibody fragment, which has good affinity, stability and specificity.

The anti-CXCL 13 antibody of the invention is obtained by screening the original murine antibody by hybridoma technology. Mice were immunized by mixing human CXCL13 protein and hemocyanin conjugated with human CXCL13 (CXCL13-KLH) with adjuvant. And after the serum titer is qualified, separating the spleen cells of the mice, fusing the spleen cells with myeloma cells of the mice in vitro and culturing the spleen cells, thereby obtaining cell culture supernatant containing the antibody. First, affinity antibodies with CXCL13 binding activity were obtained by ELISA screening, then neutralizing antibodies were further screened by cell-based competitive ELISA, antibodies with good binding kinetics were further screened by SPR, and finally cell functional antibodies were screened by Transwell assay. Preferred antibodies can competitively block binding of CXCL13 to cells positive for its receptor (e.g., human prostate cancer cell PC-3 positive for CXCR5), and thus can inhibit CXCL 13-induced cell migration. To ensure the specificity of the antibodies, receptor-independent antibodies with positive signals in cell lysate ELISA and flow cytometry were discarded by negative sieves. Preferred hybridoma antibodies are sub-cloned for conservation.

In order to obtain the amino acid sequence of the hybridoma antibody, the invention sequences the antibody mRNA of the hybridoma cell, obtains the variable region sequence of the hybridoma antibody, and prepares a recombinant monoclonal antibody for functional verification. The mRNA gene of the antibody variable region is amplified by PCR through an upstream signal peptide primer and a downstream constant region primer, and the antibody variable region gene is obtained by further sequencing. An expression vector was constructed by fusing the murine antibody variable region with the antibody constant region. The expression plasmid containing the antibody gene is transfected into HEK293 cells for transient expression, purified by a protein A affinity column, and the purity and the content of the antibody are confirmed by SDS-PAGE and spectrophotometry to meet the required requirements.

The obtained high-purity recombinant monoclonal antibody is used for verifying the binding activity of the recombinant antibody through ELISA, and further verifying the cell migration blocking effect of the antibody on CXCL13 induction through a Transwell experiment. To examine whether the antibodies have cross-reactivity, on one hand, whether the antibodies have cross-reactivity with IL8, CXCL6, CXCL10 and CXCL12, which have high homology to human CXCL13, was examined by ELISA, and on the other hand, whether the antibodies can simultaneously recognize human and monkey CXCL 13. Through the screening, an antibody 3-D2-2-B10 which has good functional activity and is combined with both human and monkey CXCL13 is obtained. In order to ensure the specificity of the antibody 3-D2-2-B10, the antibody did not show a significant binding signal as confirmed by Western blot of cell lysates, ELISA of cell lysates, and flow cytometry of various cells. Subsequently, the binding kinetics of the antibody 3-D2-2-B10 was precisely determined by SPR, and immunohistochemistry was used to confirm that the antibody can recognize native CXCL13 in tonsil tissues.

To achieve humanization of antibody 3-D2-2-B10, the variable regions of antibody 3-D2-2-B10 were modeled homologously according to antibody structure in the PDB database, and CDR regions were determined based on amino acid primary sequence features and spatial conformation of the variable regions. The variable region of 3-D2-2-B10 is compared with the amino acid sequences encoded by human antibody germline genes V and J, and based on the factors of identity, similarity, conservation, etc. of the framework region FR, 5V genes and 1J gene in total in the IGHV library are preferably grafted and spliced with the heavy chain CDR of 3-D2-2-B10 to form 5 different humanized heavy chain variable regions, wherein the V genes are IGHV1-69 x 08, IGHV1-46 x 01, IGHV1-24 x 01, IGHV1-18 x 01, IGHV1-69-2 x 01, and the J genes are preferably grafted and spliced with the heavy chain CDR of 3-D2-2-B10.

Similarly, it is preferable that 4V genes and 1J gene in total of IGKV1-39 × 02, IGKV1/OR-3 × 01, IGKV1-12 × 01, and IGKV1-9 × 01 in the IGKV library are graft-spliced with the light chain CDRs of 3-D2-2-B10 to constitute 4 different humanized light chain variable regions.

The humanized heavy and light chain variable regions were fused to the heavy and light chain constant regions of human IgG1 κ, respectively, to form complete heavy and light chains, and 5 heavy chains were combined with 4 light chains to construct 20 different humanized antibodies.

Then, recombinant plasmids containing the 20 humanized antibody genes are constructed, and HEK293 cells are transfected for transient expression. Cell culture supernatants were collected for SPR binding kinetics screening. The constructed 20 humanized antibodies all show good affinity, and the CDR regions of the anti-CXCL 13 antibody have good portability. The transient expression supernatant of 6 strains of antibody is collected, purified by protein A affinity column and concentrated by ultrafiltration, and the uniformity, purity and content of the antibody are respectively determined by SEC-HPLC, reduced SDS-PAGE and NanoDrop 2000.

Furthermore, the 6-strain humanized antibody is subjected to stability analysis, and a humanized antibody having high stability is preferable in terms of melting temperature (Tm) and aggregation initiation temperature (Tagg). hNo5-4 was initially determined to be a preferred humanized antibody by comparing the half-life of the humanized antibody in mice.

Multiple concentrations of antigen were set by SPR to determine in detail the binding kinetics of hNo5-4 to CXCL 13. Antibody hNo5-4 was detected to have an isoelectric point of 8.9 and small acidic and basic peaks using isoelectric focusing capillary electrophoresis (cIEF). Under the conditions of repeated freeze thawing and long-time temperature and pressure, the charge distribution and the monomer component of the antibody hNo5-4 can be well kept stable, which indicates that the antibody has good stability.

The anti-CXCL 13 antibody of the present invention can be modified into antibody fragments such as Fab (anti-binding fragment), scFv (single-chain fragment variable) and the like by conventional gene recombination techniques. Antibody fragments such as Fab, scFv and the like have small volume and strong tissue permeability, and have unique advantages in some application fields. Fab is a heterodimer consisting of heavy chain variable region-constant region 1(VH-CH1) and light chain variable region-constant region (VL-CL) and has a molecular size of 1/3 that of an IgG molecule. Due to the absence of the Fc segment, Fab-induced immune effects were significantly reduced compared to IgG and cytokine release was weaker. Currently, antibody drugs with Fab as the structure, such as abciximab, ranibizumab and the like, have been approved to be on the market. The scFv is formed by fusing VH and VL and a connecting peptide linker between the VH and VL, has the molecular size of 1/6 of IgG, has the characteristics of strong tissue permeability, short half-life and the like, has unique advantages in the fields of imaging diagnosis and some treatment, and a bispecific antibody blinatumomab based on the scFv is also approved to be marketed. The antibody fragment can be further fused with other proteins or coupled with other small molecules, and can be used for diagnosis and treatment of diseases through targeted delivery.

The anti-CXCL 13 antibody of the present invention can further improve affinity by mutating amino acids in CDR regions by genetic engineering techniques. The CDR regions of antibodies play a key role in the binding of antibodies to antigens, where amino acids can interact with the amino acids of the antigen through hydrogen bonds, ionic bonds, van der waals forces, and the like. By mutating amino acids in the CDR regions of an antibody, the interaction of the CDRs with the antigen can be further enhanced, thereby increasing the affinity of the antibody. The application of the antibody library technology in the aspect of antibody affinity evolution is mature, an antibody mutation library can be established through strategies such as alanine hot spot mutation, error-prone PCR and the like, high-throughput screening of a mutant antibody is carried out, and the antibody affinity evolution is realized in vitro.

The antibody of the invention can be expressed by stable cell lines, so that the antibody can be used for large-scale production of a large amount of protein. The gene coding the amino acid of the antibody can be obtained by the conventional gene recombination technology, and can be inserted into an expression vector after DNA sequence optimization, synthesis and PCR amplification. The vector used may be a plasmid, virus or gene fragment commonly used in molecular biology. The front end of the DNA sequence for coding the antibody is added with a protein secretion signal peptide gene to ensure that the antibody can be secreted to the outside of the cell. The vector sequence contains elements such as a promoter for gene expression, protein translation initiation and termination signals, and poly A (PolyA). The vector contains an antibiotic resistance gene to facilitate replication of the vector in a host cell, such as a bacterium, for use in vector preparation. In addition, the vector can also contain a selective gene to facilitate the selection of a stable transfection host cell for constructing a stably expressed cell strain.

After the vector containing the antibody-encoding DNA sequence is constructed, the vector can be used to transfect or transform a host cell to express the corresponding protein. There are many expression systems that can be used to express antibodies, including eukaryotic cells and prokaryotic cells, including mammalian cells, insect cells, yeast, bacteria, and the like. Mammalian cells are the preferred system for expressing the protein, since prokaryotic cells readily form inclusion bodies when expressing intact antibodies. There are various mammalian cells that can be used for large-scale expression of antibodies, such as CHO cells, HEK293 cells, NS0 cells, COS cells, etc., and they are included in the list of cells that can be used in the present invention. The recombinant vector containing the gene encoding the above antibody can be transfected into host cells by various methods including electroporation, lipofection, calcium phosphate transfection and the like.

A preferred method of protein expression is by expression using stably transfected host cells containing the selectable gene. For example, after stably transfecting host cells lacking Neomycin resistance with a recombinant vector containing a Neomycin (Neomycin) resistance gene, the concentration of Neomycin may be increased in a cell culture solution to select a stable cell line with high expression; alternatively, for example, after stable transfection of host cells lacking DHFR with a recombinant vector containing the dihydrofolate reductase (DHFR) gene, a cell culture medium can be enriched for Methotrexate (MTX) to select for stable cell lines with high expression.

Expression systems other than mammalian cells, such as insect cells, yeast, bacteria, and the like, may also be used to express the antibodies or fragments thereof of the present invention, which are also encompassed by host cells that can be used with the present invention. These expression systems express proteins in higher amounts than mammalian cells in some cases, but are likely to form inclusion bodies, and therefore further protein renaturation is required.

The antibodies of the present invention may also be carried and expressed using viral vectors, including, but not limited to, adenoviral vectors (adenovirus vectors), adeno-associated viral vectors (adeno-associated viral vectors), retroviral vectors (retroviral vectors), herpes simplex viral vectors (herpes simplex viral-based vectors), lentiviral vectors (lentivirus vectors), and the like.

The anti-CXCL 13 antibody can be used for detection of CXCL13, including Western blot, ELISA and immunohistochemistry, and has a wide application range. The sample of the cell lysate containing CXCL13 is separated by SDS-PAGE under the reducing condition and detected by Western blot, and only shows a positive signal at the theoretical molecular weight part, which shows that the anti-CXCL 13 antibody of the invention is not combined with other proteins in the cell lysate, has good specificity, and simultaneously prompts that the anti-CXCL 13 antibody of the invention recognizes a linear epitope, so the CXCL13 can be still recognized under the denaturing condition. Immunohistochemistry of paraffin tissue sections shows that the CXCL13 can be detected to be specifically distributed in lymph follicles by the antibody, and the antibody is consistent with literature reports. These results show that the antibody of the present invention has wide application foreground as detection probe, and has the features of wide use, specific signal, etc.

Transwell experiments prove that the humanized antibody hNo5-4 preferably used in the invention can inhibit CXCL 13-induced migration of Raji, SU-DHL-6 and PC-3 of CXCR5 positive cells. In addition, cytological experiments also demonstrated that humanized antibody hNo5-4 was able to inhibit CXCL 13-induced growth of Raji and SU-DHL-6 cells and showed concentration dependence. This demonstrates that the anti-CXCL 13 antibodies of the invention can block CXCL 13-induced cell proliferation and migration, and have good cell functional activity. The antibody can regulate and control the biological function of CXCL13, and shows the application prospect of treating CXCL13 related diseases.

The following examples illustrate the discovery, preparation, testing and use of antibodies in accordance with the present invention. The content and use of the invention is not limited to the scope of the embodiments.

Example 1 immunization of mice

40 μ g of human CXCL13 protein, CXCL13-KLH (human CXCL 13-coupled hemocyanin), and an immunoadjuvant were mixed together in equal volume under sterile conditions to prepare 100 μ L of suspension, which was injected into the hind leg and calf muscle of BALB/c mice. On days 21 and 35, immunization was performed in the same manner for 1 needle, and the tail vein was sampled 2 weeks later. CXCL13 was coated onto ELISA plates (50 ng/well), and the antibody titers of mouse serum were measured by ELISA at a ratio of 1:300000, followed by antigen challenge and spleen cell fusion 3 days later.

Example 2 spleen cell fusion

After euthanization of mice, spleens were separated under sterile conditions, spleen cell suspensions were prepared using a 70 μm screen and washed 2 times with basal medium for cell counting. SP2/0 was mixed with splenocytes at a ratio of 1:3, centrifuged and the supernatant discarded, 1mL of 37 ℃ preheated PEG was added dropwise over 1min, allowed to stand at 37 ℃ for 90s, followed by 20mL of 37 ℃ preheated basal medium over 6 min. The cells were harvested by centrifugation, and 20mL of HAT medium pre-warmed at 37 ℃ was added to resuspend the cells in a 1X 10 format⁵And (3) adding the fused cells into a 96-hole cell culture plate according to the density of each splenocyte/hole, placing the 96-hole cell culture plate in a carbon dioxide cell culture box for culture, and taking culture supernatant for ELISA detection when the cell confluency reaches more than 70%.

Example 3 affinity screening

CXCL13 solution was prepared at 1. mu.g/mL using PBS, and the plate (50. mu.L/well) was added and coated overnight at 4 ℃. The plates were washed 3 times with PBST, 5% BSA blocking solution (300. mu.L/well) was added and incubated at 37 ℃ for 2 h. The plates were washed 3 times with PBST, hybridoma supernatant (50. mu.L/well) was added and incubated at 37 ℃ for 1.5 h. The plates were washed 3 times with PBST, added with a 1:5000 dilution of HRP-goat anti-mouse solution (50. mu.L/well), and incubated at 37 ℃ for 1 h. The plates were washed 3 times with PBST, ready-to-use TMB color developing solution (100. mu.L/well) was added, and incubated at 37 ℃ for 10min in the absence of light. 2M H was added₂SO₄The color development was stopped (100. mu.L/well) and the OD value was measured at 450 nm. OD value of mouse IgG (1. mu.g/mL) control well<0.3, taking the positive well (OD value)>2.0) corresponding hybridoma culture supernatants (total 150 wells) were screened for neutrality.

Example 4 neutralization screening

PC-3 cells stably expressing human CXCR5 were plated and cultured to a confluency of 90% or more. PBST was washed twice, and cells were fixed for 15min at room temperature by adding 4% paraformaldehyde (200. mu.L/well). PBST was washed twice, 5% BSA blocking solution (200. mu.L/well) was added, and blocking was performed at 4 ℃ for 2 h. PBST plates were washed twice (200. mu.L/well), supernatants were discarded, equal volumes of mixed sample (100. mu.L/well) of CXCL13-hFc fusion protein (1. mu.g/mL) and hybridoma culture supernatant previously incubated at 4 ℃ for 1h, and incubated at 4 ℃ for 1 h. Discard the supernatant and wash the plate 2 times with PBST. HRP-goat anti-human IgG (100. mu.L/well) was added at a 1:5000 dilution and incubated at 4 ℃ for 1 h. Discard the supernatant and wash the plate 2 times with PBST. Ready-to-use TMB developing solution (100. mu.L/well) was added thereto, and the mixture was incubated at room temperature for 15 min. 2M H was added₂SO₄The color development was stopped (100. mu.L/well) and the OD value was measured at 450 nm. Compared with mouse IgG control without neutralization (5 mu g/mL), the neutralizing antibody (total 100 wells) can obviously inhibit the binding of CXCL13 to PC-3 cells (inhibition rate)>50％)。

Example 5 subcloning of hybridoma cells

The hybridoma cells were subcloned by limiting dilution. Collecting hybridoma cells secreting neutralizing antibody, counting, diluting the hybridoma cells with complete culture medium, adding the hybridoma cells into a 96-well cell culture plate at the cell density of 0.5 per well, continuously culturing, and preserving the seeds of the remaining cells after expanding culture. After 10 days of subclone culture, the culture supernatant of the monoclonal hole is taken for affinity ELISA and neutralization ELISA verification respectively, the positive clone is taken for secondary subclone, and the remaining cells are preserved after enlarged culture. And continuously verifying the secondary subclones according to the screening mode of the primary subclones. Finally, 15 strains of neutralizing subclones are further preferably selected from 50 strains of subclones with good affinity, and are subjected to the next SPR binding kinetics screening.

Example 6 SPR screening

The appropriate amount of coupling was calculated according to the formula RL ═ Rmax × MWligand d)/(Sm × MWanalyte), hCXCL13-HSA-His was captured to the NTA chip, and the response of different hybridoma supernatants in the mobile phase was examined using Biacore T200. Data fitting was performed by Evaluation Software to obtain binding curves and kinetic parameters. The results show that 14 hybridoma antibodies showed good binding kinetics (see table 1).

Table 1: hybridoma antibody binding kinetic detection

Example 7 cell function screening

Resuspend PC-3 cells stably expressing human CXCR5 in complete medium and add 100. mu.L of cell suspension (1X 10) to the upper layer of the Transwell chamber⁴One/well), 600. mu.L of a mixed sample of CXCL13 (1. mu.g/mL) and neutralizing antibody hybridoma culture supernatant of equal volume previously incubated at room temperature for 30min was added to the lower layer, and the culture was continued for 8 h. The Transwell chamber was removed, transferred to a well containing methanol, and fixed at room temperature for 10 min. The Transwell cell was removed and transferred to a well containing crystal violet stain at room temperatureAnd dyeing for 15 min. The Transwell chamber was removed, washed 2 times with deionized water, photographed under an upright microscope and the number of cells migrating to the lower chamber was counted. Cell migration inhibition by the 13-strain hybridoma antibody compared to control group (medium) without antibody addition>50% (see fig. 1).

Example 8 specificity screening

Culturing and collecting various CXCL13 negative cell lines, adding RIPA lysate (weak) to prepare a cell protein sample, and coating the cell protein sample on an enzyme label plate (500 ng/hole). Different hybridoma supernatants were used as primary antibodies and HRP-goat anti-mouse antibodies as secondary antibodies, and ELISA was used to detect whether the hybridoma supernatants bound the cell lysis fraction. In addition, different hybridoma supernatants were used as primary antibody, FITC-goat anti-mouse as secondary antibody, and Flow Cytometry (FCM) was used to determine whether the hybridoma supernatants bound to the cell surface. The results showed that of the 13 strains of functional antibody, 9 strains showed no binding signal and good specificity (see table 2).

Table 2: hybridoma antibody specificity detection

Antibodies	ELISA	FCM
			3-G4-C7-B11	●	●
8-G11-2-D9	●	●
			6-F1-F1	●	●
37-H5-C7	—	—
			1-F8-F8	●	●
7-D7-B10	—	—
			3-D2-2-B10	—	—
25-E11-C5	—	—
			13-F11-B6	—	—
5-E7-C4-E12	—	—
			18-C1-B3-A2	—	—
10-H7-B5-G10	—	—
			26-A8-D12	—	—

Note: "●" represents binding, "-" represents not binding

Example 9 obtaining antibody variable region sequences

Collecting the hybridoma subclones, and extracting RNA by using a Trizol method. And carrying out reverse transcription by taking the extracted RNA as a template to obtain cDNA. The heavy and light chain variable regions of the antibody were PCR amplified using degenerate primers (Novagen Ig-Primer Sets), respectively, and the PCR amplification products were detected by agarose gel electrophoresis. And obtaining a target DNA fragment by adopting a gel recovery kit, and then carrying out TA cloning to construct a recombinant plasmid. The recombinant plasmid is transformed into competent cell DH5 alpha by heat shock method, and plated for blue-white screening. Picking white single colony to 0.5mL LB liquid culture medium, shaking culturing at 37 deg.C and 220rpm for 3h, sampling bacterial liquid, and sequencing to obtain 6 antibody sequences.

Example 10 construction and preparation of recombinant monoclonal antibodies

And splicing the heavy chain variable region gene segments and the light chain variable region gene segments with the signal peptide and the mouse heavy chain constant region gene segments and the mouse light chain constant region gene segments respectively by adopting overlapping PCR (polymerase chain reaction), and sequencing and identifying. The genes of the heavy chain and the light chain of the antibody with correct splicing are respectively inserted into pTT5 plasmid, the recombinant plasmid is transfected into HEK293 cells by adopting a PEI method, serum-free suspension culture (30mL) is carried out, and the antibody is transiently expressed. Cell supernatants from 6 days of culture were collected, filtered through a 0.22 μm filter and the antibody purified by protein G affinity chromatography. The antibody was ultrafiltered and replaced into PBS solution, the purity of the antibody was verified by reducing SDS-PAGE, the antibody concentration was determined by NanoDrop 2000, and the antibody was stored at-80 ℃ after dispensing (see FIG. 2). Except that the expression level of the 1-strain antibody was too low, 5-strain recombinant monoclonal antibodies (SEQ ID NO: No.2, No.3, No.5, No.6, No.11) were obtained in total.

Example 11 affinity and cell function verification of recombinant monoclonal antibodies

According to the detection method of hybridoma screening, the recombinant monoclonal antibody is respectively verified for ELISA affinity (see figure 3) and Transwell cell function (see figure 4). The results show that the affinity and the cell function of the 5-strain recombinant monoclonal antibody are in accordance with expectations.

Example 12 Cross-reactivity of recombinant monoclonal antibodies

Human IL-8, CXCL6, CXCL10 and CXCL12 which have higher homology with CXCL13 are respectively coated on an enzyme label plate (50 ng/hole), and ELISA detection shows that no positive signal is detected by 5 recombinant monoclonal antibodies. In addition, human and monkey CXCL13 were coated on the ELISA plates (50 ng/well), and ELISA detection showed that only antibody 3-D2-2-B10 (sequencing No.5) exhibited binding ability to human and monkey CXCL 13.

Example 13 specificity verification of antibody 3-D2-2-B10

Culturing and collecting various CXCL13 negative cell lines, and adding RIPA lysate (weak) to prepare a cell protein sample. One part of the cell protein sample is separated by reducing SDS-PAGE (40 mu g/hole) electrophoresis, the other part of the sample is coated on an enzyme label plate (500 ng/hole), an antibody 3-D2-2-B10 is used as a primary antibody, an HRP-goat anti-mouse antibody is used as a secondary antibody, and whether the antibody is combined with a cell lysis component or not is detected by Western blot and ELISA respectively. In addition, the antibody 3-D2-2-B10 was used as a primary antibody, FITC-goat anti-mouse was used as a secondary antibody, and whether the antibody bound to the cell surface was detected by Flow Cytometry (FCM). The results showed that no positive signal was detected by antibody 3-D2-2-B10, showing good specificity.

Example 14 determination of the binding kinetics of antibody 3-D2-2-B10

The appropriate amount of coupling was calculated according to the formula RL ═ Rmax × MWligand)/(Sm × MWanalyte), antibody 3-D2-2-B10 was captured to CM5 chip by anti-mouse IgG, and human, monkey CXCL13 responses under concentration gradient conditions in the mobile phase were examined using Biacore 8K. Data fitting is carried out through Software Evaluation Software to obtain a binding curve and kinetic parameters. The results show that 3-D2-2-B10 shows good binding kinetics with human and monkey CXCL13 (see Table 3).

Table 3: antibody 3-D2-2-B10 binding kinetics assay

	ka(10⁴M^-1s^-1)	kd(10^-7s^-1)	KD(pM)
				Human CXCL13	18.6	63.4	3.41
Cynomolgus CXCL13	1.73	8.68	5.03

Example 15 immunohistochemical analysis recognition of native CXCL13 in tissues by antibody 3-D2-2-B10

Formalin-fixed paraffin-embedded tonsil tissue sections were obtained. Immunohistochemistry was used to detect the expression of CXCL13 in tonsil tissues with 3-D2-2-B10 as primary antibody and Mab801(AF801, R & D) antibody as positive control antibody. Analysis showed that CXCL13 was mainly distributed in lymphoid follicles, and 3-D2-2-B10 had good specificity (see fig. 5).

Example 16 antibody humanization engineering

Antibody 3-D2-2-B10 variable regions were homologously modeled based on antibody structure in the PDB database, and CDR regions were determined based on amino acid primary sequence features and variable region spatial conformation. The variable region of 3-D2-2-B10 is compared with the amino acid sequences encoded by human antibody germline genes V and J, and based on the factors of identity, similarity, conservation, etc. of the framework region FR, 5V genes and 1J gene in total in the IGHV library are preferably grafted and spliced with the heavy chain CDR of 3-D2-2-B10 to form 5 different humanized heavy chain variable regions, wherein the V genes are IGHV1-69 x 08, IGHV1-46 x 01, IGHV1-24 x 01, IGHV1-18 x 01, IGHV1-69-2 x 01, and the J genes are preferably grafted and spliced with the heavy chain CDR of 3-D2-2-B10. Similarly, it is preferable that 4V genes and 1J gene in total of IGKV1-39 × 02, IGKV1/OR-3 × 01, IGKV1-12 × 01, and IGKV1-9 × 01 in the IGKV library are graft-spliced with the light chain CDRs of 3-D2-2-B10 to constitute 4 different humanized light chain variable regions. The humanized heavy and light chain variable regions were fused to the heavy and light chain constant regions of human IgG1 κ, respectively, to form complete heavy and light chains, and 5 heavy chains were combined with 4 light chains to construct 20 different humanized antibodies. The amino acid sequences of VH-CDR1, VH-CDR2 and VH-CDR3 of the heavy chain complementarity determining regions of the 20 humanized antibodies are GYTFTGYWIE (SEQ ID NO.1), EILPGRGSTNYNEKFKG (SEQ ID NO.2) and ADDDYGYFDV (SEQ ID NO.3) respectively, and the amino acid sequences of VL-CDR1, VL-CDR2 and VL-CDR3 of the light chain complementarity determining regions are RASKSISKYLA (SEQ ID NO.4), SGSILHS (SEQ ID NO.5) and QQHNEYPYT (SEQ ID NO.6) respectively. The direction of each amino acid sequence involved in the present invention is from N-terminus to C-terminus.

Example 17 humanized antibody SPR screening

pTT5 recombinant plasmid containing humanized antibody gene is constructed, the recombinant plasmid is transfected into HEK293 cells by PEI method, serum-free suspension culture is carried out, and the humanized antibody is expressed in small amount (2mL) in a transient mode. The supernatant was captured on a Protein A chip and the affinity was determined using Biacore 8K. The results showed that all 20 constructed humanized antibodies showed good affinity (see table 4). The results show that the CDR of the murine antibody 3-D2-2-B10 can still maintain good affinity after being spliced with the FR of various human germline antibodies, and the CDR region of the anti-CXCL 13 antibody has good portability.

Table 4: humanized antibody binding kinetics assay

Example 18 comparison of expression level of humanized antibody

The recombinant plasmid is transfected into HEK293 cells by a PEI method, serum-free suspension culture (30mL) is carried out, and the antibody is transiently expressed. Cell culture supernatants were collected for 6 days, filtered through 0.22 μm filters, and the antibodies were purified by protein G affinity chromatography. The antibody was ultrafiltered and replaced with PBS solution, and the homogeneity, purity and content of the antibody were measured by SEC-HPLC, reduced SDS-PAGE and NanoDrop 2000, respectively. The results showed that hNo5-4 was expressed in a significantly higher amount than the other humanized antibodies (see FIG. 6).

Example 19 analysis of thermal stability of humanized antibody

The 6-strain antibody having the superior affinity was selected, the antibody concentration was adjusted to 1mg/mL, and the melting temperature (Tm) and aggregation initiation temperature (Tagg) of the antibody were measured using UNcle. Tm is determined by reflecting the conformational changes of the protein by the fluorescence changes of the endogenous aromatic amino acids of the protein. And detecting aggregates with different sizes by adopting two wavelengths of 266nm and 473nm, monitoring the aggregation condition of the protein in the process of heating, and determining Tagg. Comprehensive analysis of UNcle anlysis showed that hNo5-1 and hNo5-4 have superior thermal stability (see Table 5).

Table 5: detection of thermal stability of humanized antibody

hNo5-1

hNo5-2

hNo5-3

hNo5-4

hNo5-5

hNo5-6

Tm(℃)

72.57±0.06

67.02±0.01

72.33±0.03

72.13±0.06

63.24±0.01

70.45±0.03

Tagg(℃)

82.75±0.02

66.3±0.03

81.68±0.01

83.5±0.03

64.71±0.01

77.56±0.03

Example 20 half-Life of humanized antibodies in mice

The NOD/scid mice were given 10mg/kg each of humanized antibodies hN05-1 and hNo5-4 by a single tail vein injection, and control mice were not given antibody. Blood was collected at 0.5h, 1h, 6h, 1d, 7d, 14d, 21d, 28d time points and serum was prepared by orbital bleeding. Human CXCL13 was coated on an ELISA plate, and serum at different time points was used as a primary antibody, and HRP-goat anti-human was used as a secondary antibody, and the humanized antibody concentration in mouse serum at different time points was detected by ELISA, and a blood concentration-time curve was drawn (see FIG. 7). The results showed that hNo5-1 had a half-life in mice of 10.5 days and hNo5-4 of 11.5 days.

Preferably hNo5-4 is used for detailed analysis of affinity and isoelectric point, and further analysis of biological effect, by combining factors such as binding kinetics, expression amount, stability, half-life period, etc.

The amino acid sequence of the heavy chain of antibody hNo5-4 is (SEQ ID NO. 7):

QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYWIEWVRQAPGQGLEWMGEILPGRGSTNYNEKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARADDDYGYFDVWGQGTTVTVSS。

the amino acid sequence of the light chain of antibody hNo5-4 is (SEQ ID NO. 8):

DIQMTQSPSSVSASVGDRVTITCRASKSISKYLAWYQQKPGKAPKLLIYSGSILHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHNEYPYTFGGGTKVEIK。

example 21 determination of affinity and isoelectric Point of antibody hNo5-4

Preferably, hNo5-4 is used for detailed affinity and isoelectric point analysis, combining the factors of binding kinetics, expression level, stability, half-life and the like. hNo5-4 was captured to a Protein A chip and affinity assays were performed at multiple concentrations of human CXCL13 using Biacore 8 k. The results show that hNo5-4 and human CXCL13 show good binding kinetics, wherein KD is 3.24 × 10^-13M，kd＝2.49×10^-5(s^-1)，ka＝7.67×10⁷(M^-1s^-1). The isoelectric point and charge isomer status of hNo5-4 was measured by cIEF (Agilent 7100 CE). The results showed hNo5-4 had an isoelectric point of 8.9, and the acidic and basic peaks were small (see FIG. 8).

Example 22 evaluation of stability under pressure conditions

To further examine the stability of humanized antibody hNo5-4 under stress, the antibody was freeze-thawed 5 times repeatedly, and then placed at 40 ℃ for 0d, 1d, 3d, and 5 d. Antibody samples were taken at each time point for SEC-HPLC (see FIG. 9) and cIEF (see FIG. 10) detection. The results show that the monomeric components and charge distribution of antibody hNo5-4 remain well stabilized, indicating that the antibodies of the invention have good stability.

Example 23 antibody hNo5-4 inhibits migration of CXCR5 positive cells

Resuspending CXCR5 positive PC-3, Raji, Ramos cells in complete medium and adding 100. mu.L of cell suspension (1X 10) to the upper layer of the Transwell chamber⁴One/well), 600. mu.L of pre-mix was added to the lower layerA sample of equal volumes of CXCL13 (1. mu.g/mL) and hNo5-4 antibody (5. mu.g/mL) was first incubated at room temperature for 30min and incubation continued for 8 h. For PC-3 cells, the Transwell chamber was removed, transferred to a well containing methanol, fixed at room temperature for 10min, transferred to a well containing crystal violet stain, stained at room temperature for 15min, and finally removed, washed 2 times with deionized water, photographed under an upright microscope and counted for the number of cells that migrated to the lower chamber. For Raji and Ramos cells, cell counts were performed directly on the sub-chamber media. The results show that hNo5-4 can inhibit migration of different types of CXCR5 positive cells (see fig. 11).

Example 24 antibody hNo5-4 inhibits the growth of CXCR5 positive lymphoma cells

Raji and SU-DHL-6 cell suspensions were prepared separately from complete medium and 50. mu.L of cell suspension (3X 10) was added to 96-well cell culture plates³pieces/mL) and cultured for 24 h. Configuring a concentration gradient hNo5-4 antibody by using a complete culture medium, mixing hNo5-4 antibody with different concentrations with 200ng/mL human CXCL13 solution in equal volumes, adding the mixture into the cultured cell holes, and continuing culturing for 72 hours. And detecting ATP of the cells by using a CellTiter-Glo luminescence method cell viability detection kit, and analyzing the cell viability. The results showed that hNo5-4 inhibited the growth of Raji and SU-DHL-6 cells and showed concentration dependence (see FIG. 12).

Sequence listing

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Claims

1. An antibody or fragment thereof against CXCL13, characterized in that: the amino acid sequences of the heavy chain complementarity determining regions VH-CDR1, VH-CDR2 and VH-CDR3 of the antibody or the fragment thereof are SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively; the amino acid sequences of the light chain complementarity determining regions VL-CDR1, VL-CDR2 and VL-CDR3 of the antibody or the fragment thereof are SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, respectively.

2. The anti-CXCL 13 antibody or fragment thereof according to claim 1, wherein: contains a heavy chain variable region VH which is spliced by the VH-CDR1, the VH-CDR2 and the VH-CDR3 and a human antibody framework region FR.

3. The anti-CXCL 13 antibody or fragment thereof according to claim 2, wherein: the amino acid sequence of the antibody heavy chain variable region VH is shown in SEQ ID NO. 7.

4. The anti-CXCL 13 antibody or fragment thereof according to claim 1, wherein: contains light chain variable region VL which is spliced by the VL-CDR1, VL-CDR2 and VL-CDR3 and human source antibody framework region FR.

5. The anti-CXCL 13 antibody or fragment thereof according to claim 4, wherein: the amino acid sequence of the antibody light chain variable region VL is shown in SEQ ID NO. 8.

6. The anti-CXCL 13 antibody or fragment thereof according to any one of claims 1 to 5, wherein the heavy chain constant region of the antibody is derived from the heavy chain constant region of human immunoglobulin IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA, IgD.

7. The anti-CXCL 13 antibody or fragment thereof according to any one of claims 1 to 5, wherein the light chain constant region of the antibody is derived from the light chain constant region of human immunoglobulin kappa or lambda.

8. A nucleic acid molecule encoding the anti-CXCL 13 antibody or fragment thereof according to claims 1 to 7.

9. A recombinant vector comprising the nucleic acid molecule of claim 8.

10. A cell comprising the recombinant vector of claim 9.

11. Use of an anti-CXCL 13 antibody or fragment thereof according to any one of claims 1 to 7 in the preparation of a CXCL13 detection reagent.

12. Use of an anti-CXCL 13 antibody or fragment thereof according to any one of claims 1 to 7 in the preparation of a medicament for blocking CXCL13 binding to a CXCL13 receptor to inhibit CXCL13 biological function.