A monoclonal antibody specifically binding to ccl5 and uses thereof

Abstract

This invention discloses a monoclonal antibody that specifically binds to CCL5 and its applications, belonging to the field of biomedical technology. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 1-3 or 6-8, and the light chain variable region comprises CDR1, CDR3, and CDR2 (SAS or RAS) as shown in SEQ ID NO: 4, 5, 9, or 10. The heavy chain and light chain sequences are shown in SEQ ID NO: 11-12 or 13-14, respectively. The monoclonal antibody of this invention can be used to specifically block the differentiation process of CCL5-mediated decoy TRAIL receptor 1 (dcTR1)-expressing immunosuppressive tumor-associated neutrophils (TANs) and to restore the antigen-presenting function of dendritic cells in the tumor microenvironment, showing good application potential in anti-tumor therapy.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a monoclonal antibody that specifically binds to CCL5 and its applications. Background Technology

[0002] Existing CCL5-related antibodies and tumor immunotherapy technologies mainly include: antibodies targeting CCL5 or chemokines, oncolytic virus systems expressing anti-CCL5 antibodies, combination therapy using CCL5 as a tumor immunomodulatory factor, and anti-tumor immunotherapy enhanced based on the CCL5 / chemokine axis. However, most of these technologies focus on immune cell chemotaxis regulation, changes in the inflammatory microenvironment, viral delivery systems, and tumor immune enhancement. They do not yet address CCL5-induced immunosuppressive neutrophil differentiation, the mechanism of CCL5-mediated immunosuppressive tumor-associated neutrophil (TAN) formation mediated by decoy TRAIL receptor 1 (DcTRAILR1, dcTR1), the inhibition of cDC1 antigen presentation by CCL5, and the development of anti-CCL5 antibodies based on functional immune differentiation screening. Existing CCL5-related technologies have the following main shortcomings:

[0003] (1) The main focus is on chemotaxis. Most existing technologies consider CCL5 as a chemokine, focusing only on its role in immune cell migration. However, current technologies have not yet found that CCL5 can drive the formation of dcTR1, which has immunosuppressive functions. + TANs.

[0004] (2) Lack of functional screening for immunosuppressive neutrophil differentiation. Existing anti-CCL5 antibodies are mainly screened based on ELISA binding ability, chemotactic blocking ability, and receptor competition ability. The above screening methods cannot identify functional antibodies that can inhibit immunosuppressive TAN differentiation.

[0005] (3) cDC1 function cannot be restored. Current technology has not proven that CCL5 blockade can restore cDC1 maturation, CCR7 expression and cross-antigen presentation ability, and its synergistic effect on dendritic cell therapy (DCtherapy) is limited.

[0006] In summary, current interventions targeting CCL5 mainly focus on blocking its chemotactic function or inhibiting signal transduction of the CCL5 / CCR5 axis, but have not yet addressed the mechanism by which CCL5 regulates neutrophil fate determination and its role in cDC1 functional remodeling. Existing anti-CCL5 antibodies have significant functional limitations. Summary of the Invention

[0007] Therefore, the main objective of this invention is to provide a monoclonal antibody that specifically binds to CCL5, which neutralizes CCL5 and inhibits the differentiation of immunosuppressive neutrophils. It is the first discovery that CCL5 can induce the formation of dcTR1, which has immunosuppressive function. + TANs further inhibit cDC1 maturation and cross-antigen presentation, and screen and obtain anti-CCL5 monoclonal antibodies that can specifically regulate CCL5-mediated immunosuppressive neutrophil differentiation and enhance cDC1 function.

[0008] Another object of the present invention is to provide the use of the monoclonal antibody that specifically binds to CCL5 in the preparation of a medicament for treating tumors.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A first aspect of the present invention provides a monoclonal antibody or its antigen-binding moiety that specifically binds to CCL5, comprising a heavy chain variable region and a light chain variable region, wherein:

[0011] The heavy chain variable region includes CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 1-3, and the light chain variable region includes CDR1, CDR3 as shown in SEQ ID NO: 4 and 5 and CDR2 with the sequence SAS;

[0012] Alternatively, the heavy chain variable region may contain CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 6-8, and the light chain variable region may contain CDR1, CDR3 as shown in SEQ ID NO: 9 and 10, and CDR2 with the sequence RAS.

[0013] Specifically, the CDR sequences of the heavy chain variable region and the light chain variable region are as follows:

[0014] 2E5 heavy chain CDR:

[0015] CDR1: GFSLSSYT (SEQ ID NO: 1), CDR2: IYPSGSS (SEQ ID NO: 2), CDR3: ARGGYDSYNVGTLDL (SEQ ID NO: 3);

[0016] 2E5 Light Chain CDR:

[0017] CDR1: QSISRY (SEQ ID NO: 4), CDR2: SAS, CDR3: QNNYGPASYGGA (SEQ ID NO: 5).

[0018] 7H5 heavy chain CDR:

[0019] CDR1: GFSLSTYT (SEQ ID NO: 6), CDR2: LDNYGNT (SEQ ID NO: 7), CDR3: ARGGYGNYNIGTLDL (SEQ ID NO: 8).

[0020] 7H5 Light Chain CDR:

[0021] CDR1: QSISSY (SEQ ID NO: 9), CDR2: RAS, CDR3: QNNYGPINYGAA (SEQ ID NO: 10).

[0022] Preferably, the heavy chain and light chain sequences of the monoclonal antibody that specifically binds to CCL5 or its antigen-binding portion are as shown in SEQ ID NO: 11 and 12, or as shown in SEQ ID NO: 13 and 14, respectively.

[0023] Preferably, the monoclonal antibody that specifically binds to CCL5 or its antigen-binding portion specifically binds to the CCL5 protein, with a binding affinity KD of 1-10 nM.

[0024] Preferably, the monoclonal antibody that specifically binds to CCL5 or its antigen-binding portion specifically blocks the differentiation process of CCL5-mediated immunosuppressive tumor-associated neutrophils (TANs) expressing decoy TRAIL receptor 1 (DcTRAILR1, abbreviated as dcTR1) and is used to restore the antigen-presenting function of dendritic cells in the tumor microenvironment.

[0025] In a second aspect, the present invention provides an isolated nucleic acid molecule that encodes the heavy chain variable region and light chain variable region of the monoclonal antibody or its antigen-binding moiety that specifically binds to CCL5, or that encodes the monoclonal antibody or its antigen-binding moiety that specifically binds to CCL5.

[0026] A third aspect of the present invention provides an expression vector comprising the nucleic acid molecule.

[0027] A fourth aspect of the invention provides a host cell comprising the expression vector.

[0028] A fifth aspect of the invention provides a pharmaceutical composition comprising the monoclonal antibody that specifically binds to CCL5 or its antigen-binding portion, and a pharmaceutically acceptable carrier.

[0029] A sixth aspect of the invention provides the use of the monoclonal antibody that specifically binds to CCL5 or its antigen-binding portion in the preparation of a medicament for treating tumors.

[0030] Preferably, the drug is used in combination with dendritic cell immunotherapy, or in combination with immune checkpoint inhibitors, chemotherapy drugs, or radiotherapy.

[0031] Preferably, the drug exerts its effects by inhibiting CCL5-induced differentiation of dcTR1⁺ immunosuppressive tumor-associated neutrophils, restoring the maturation and cross-antigen presentation function of cDC1 dendritic cells, and / or enhancing the anti-tumor immune response of CD8⁺ T cells.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] 1. This invention provides a neutralizing antibody targeting CCL5, which can be used to specifically block CCL5-mediated immunosuppressive neutrophil (dcTR1) activity. + TAN) differentiation process, and used to restore the antigen presentation function of dendritic cells (especially cDC1) in the tumor microenvironment, including upregulating the expression of CCR7, CD80, CD86 and MHC-I, and enhancing cDC1-mediated CD8+. + T cell activation capacity, thereby enhancing anti-tumor immune response.

[0034] 2. This invention reveals for the first time the key role of CCL5 in regulating the differentiation of immunosuppressive neutrophils and the functional remodeling of cDC1, providing new targets and strategies for tumor immunotherapy.

[0035] 3. Compared with existing commercial anti-CCL5 antibodies, the monoclonal antibody that specifically binds to CCL5 in this invention has the following advantages: significantly inhibiting dcTR1. + TAN differentiation restores cDC1 maturation and CCR7 expression, enhancing cross-antigen presentation ability and the anti-tumor effect of DCtherapy.

[0036] 4. The monoclonal antibodies that specifically bind to CCL5 in this invention, especially monoclonal antibody 2E5, are functionally different from traditional CCL5 blocking antibodies. They can not only inhibit CCL5-mediated neutrophil migration, but also specifically inhibit immunosuppressive dcTR1. + The differentiation process of TANs reshapes the tumor immune microenvironment.

[0037] 5. In various tumor models, the monoclonal antibody specifically binding to CCL5 in this invention, when used in combination with DC therapy, significantly inhibited tumor growth, prolonged survival, and reduced distant metastasis, demonstrating superior overall efficacy compared to commercial anti-CCL5 antibodies. Furthermore, this antibody did not exhibit significant organ toxicity or serum biochemical abnormalities in vivo, demonstrating good safety and promising application potential in anti-tumor therapy. Attached Figure Description

[0038] Figure 1 The above figures show the results of secondary screening of antibody clones targeting mouse CCL5 in this example. The horizontal axis represents different antibody clones, and the vertical axis represents the absorbance (OD) measured at a wavelength of 450 nm. 450 )value.

[0039] Figure 2 In this example, enzyme-linked immunosorbent assay (ELISA) was used to detect the binding ability of candidate antibodies 2E5 and 7H5 to human and mouse CCL5 proteins. The horizontal axis represents different detection groups, including human CCL5, mouse CCL5, and the negative control group (NC), and the vertical axis represents the absorbance (OD) measured at 450 nm. 450 )value.

[0040] Figure 3 In this example, cell migration assays were used to evaluate the effects of different antibodies on CCL5-mediated neutrophil chemotaxis. The assays included a CCL5-free group, a CCL5 group combined with an isotype control antibody (IgG), a CCL5 group combined with a commercially available anti-CCL5 antibody (ComAb), a CCL5 group combined with a 2E5 antibody, and a CCL5 group combined with a 7H5 antibody. The horizontal axis represents different treatment groups, and the vertical axis represents the number of migrating Ly6G-positive neutrophils.

[0041] Figure 4 Ly6G under different processing conditions in the examples + dcTR1 + The proportion of neutrophils was used to evaluate the effect of candidate antibodies on neutrophil differentiation. The horizontal axis represents different treatment groups, and the vertical axis represents Ly6G. + dcTR1 + Cells in total Ly6G + The percentage (%) in cells was used to assess the regulatory effect of antibodies on the differentiation of immunosuppressive neutrophils.

[0042] Figure 5 The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified 2E5 antibody under reducing and non-reducing conditions are shown in the examples to evaluate antibody purity and integrity. The chromatogram of the purified 2E5 antibody by size exclusion chromatography (SEC) was used to detect the antibody aggregation state and purity, and the results showed that 2E5 mainly exists in monomeric form. The sensor spectrum of the binding kinetics between 2E5 and CCL5 was detected by biofilm layer interferometry (BLI), and the binding kinetic parameters between the antibody and antigen were obtained by fitting, and the dissociation constant (KD) was 3.88 nM.

[0043] Figure 6The figures show tumor growth curves of mice with tumor models 4T1 and MC38 in the examples after different treatments. The horizontal axis represents the time after drug administration, and the vertical axis represents the tumor volume. Different curves represent different treatment groups and are used to evaluate the in vivo antitumor activity of antibody 2E5.

[0044] Figure 7 Ly6G levels in tumor-associated neutrophils (TAN) from different treatment groups in the 4T1, MC38, and E0771 tumor models described in this example are... + dcTR1 + The proportion of cells is shown on the x-axis, representing different treatment groups, including the IgG control group, the commercial anti-CCL5 antibody group (ComAb), and the 2E5 treatment group; the y-axis represents Ly6G. + dcTR1 + The percentage of cells in total TAN cells (%) was used to evaluate the inhibitory effect of antibody 2E5 on the formation or accumulation of immunosuppressive dcTR1+ TAN subsets.

[0045] Figure 8 XCR1 in the tumor models 4T1, MC38, and E0771 in the examples + CD103 + The proportion of conventional dendritic cell type 1 (cDC1), with the x-axis representing different treatment groups and the y-axis representing XCR1. + CD103 + The percentage of cDC1 cells in total dendritic cells (%) was used to evaluate the ability of antibody 2E5 to promote the recovery and infiltration of cDC1 cells in tumor tissue.

[0046] Figure 9 In this example, serum biochemical parameters, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), and urea levels, were detected in BALB / c mice and C57BL / 6 mice after treatment with IgG control or 2E5. The horizontal axis represents different treatment groups, and the vertical axis represents the detection values ​​of the corresponding biochemical parameters, which are used to evaluate the in vivo safety and potential hepatotoxicity of antibody 2E5. Detailed Implementation

[0047] To more fully understand and demonstrate the technical solutions, objectives, and advantages of the present invention, the technical effects produced by the present invention will be further described in detail and completely below with reference to the accompanying drawings and specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. It should be noted that other embodiments obtained by those skilled in the art without departing from the concept of the present invention are all within the protection scope of the present invention.

[0048] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0049] The following examples provide a monoclonal antibody or its antigen-binding portion that specifically binds to CCL5, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 1-3, and the light chain variable region comprises CDR1, CDR3 as shown in SEQ ID NO: 4 and 5 and CDR2 with the sequence SAS;

[0050] Alternatively, the heavy chain variable region may contain CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 6-8, and the light chain variable region may contain CDR1, CDR3 as shown in SEQ ID NO: 9 and 10, and CDR2 with the sequence RAS.

[0051] In some implementations, the monoclonal antibodies that specifically bind to CCL5 are monoclonal antibodies 2E5 and 7H5, and the CDR sequences of the heavy chain variable region and the light chain variable region are as follows:

[0052] Heavy chain CDR of 2E5:

[0053] CDR1: GFSLSSYT (SEQ ID NO: 1), CDR2: IYPSGSS (SEQ ID NO: 2), CDR3: ARGGYDSYNVGTLDL (SEQ ID NO: 3);

[0054] 2E5 light chain CDR:

[0055] CDR1: QSISRY (SEQ ID NO: 4), CDR2: SAS, CDR3: QNNYGPASYGGA (SEQ ID NO:).

[0056] Heavy chain CDR of 7H5:

[0057] CDR1: GFSLSTYT (SEQ ID NO: 6), CDR2: LDNYGNT (SEQ ID NO: 7), CDR3: ARGGYGNYNIGTLDL (SEQ ID NO: 8).

[0058] 7H5 light chain CDR:

[0059] CDR1: QSISSY (SEQ ID NO: 9), CDR2: RAS, CDR3: QNNYGPINYGAA (SEQ ID NO: 10).

[0060] In some embodiments, the monoclonal antibodies that specifically bind to CCL5 are monoclonal antibodies 2E5 and 7H5, the heavy chain and light chain sequences of monoclonal antibody 2E5 are shown in SEQ ID NO: 11 and 12, respectively, and the heavy chain and light chain sequences of monoclonal antibody 7H5 are shown in SEQ ID NO: 13 and 14, respectively.

[0061] Specifically, the antibody 2E5 heavy chain sequence (SEQ ID NO: 11):

[0062] METDTLLLWVLLLWVPGSTGQSVKESEGGLFKPTNTLTLTCTVSGFSLSSYTISWVRQAPGNGLEWIGIIYPSGSSYYATWAKSRSTITRNTSLNTVTLKMASLTAADTATYFCARG GYDSYNVGTLDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0063] Antibody 2E5 light chain sequence (SEQ ID NO: 12):

[0064] METDTLLLWVLLLWVPGSTGADVVMTQTPASVSEPVGGTVTIKCQASQSISRYLAWYQQKPGQPPKLLIYSASTLASGVPSRFKGSGSGTEFTLTISDLECADAATYYCQNNYGPASYG GAFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0065] Antibody 7H5 heavy chain sequence (SEQ ID NO: 13):

[0066] METDTLLLWVLLLWVPGSTGQSVKESEGGLFKPTDTLTLTCTVSGFSLSTYTISWVRQAPGNGLEWIGILDNYGNTYCASWAKSRSTITRNTNLNAVTLKMTSLTAADTATYFCARG GYGNYNIGTLDLWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0067] Antibody 7H5 light chain sequence (SEQ ID NO: 14):

[0068] METDTLLLWVLLLWVPGSTGADVVMTQTPASVSEPVGGTVTIKCQASQSISSYLSWYQQKPGQPPKLLIYRASTLASGVPSRFKGSGSGTEFTLTISDLECADAATYYCQNNYGPINYG AAFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0069] In some embodiments, a monoclonal antibody that specifically binds to CCL5 or its antigen-binding moiety specifically binds to the CCL5 protein, with a binding affinity KD of 1-10 nM.

[0070] In some implementations, a monoclonal antibody that specifically binds to CCL5 or its antigen-binding moiety specifically blocks the differentiation process of CCL5-mediated immunosuppressive tumor-associated neutrophils (TANs) expressing decoy TRAIL receptor 1 (dcTR1) and is used to restore the antigen-presenting function of dendritic cells in the tumor microenvironment.

[0071] The use of the aforementioned monoclonal antibodies or antigen-binding moieties that specifically bind to CCL5 in the preparation of drugs for treating tumors, by inhibiting CCL5-induced dcTR1 + Immunosuppressive tumor-associated neutrophil differentiation, restoration of cDC1 dendritic cell maturation and cross-antigen presentation function, and / or enhancement of CD8 + T cells play a role in the anti-tumor immune response.

[0072] In some implementations, the above-mentioned drugs are used in combination with dendritic cell immunotherapy, or in combination with immune checkpoint inhibitors, chemotherapy drugs, or radiotherapy.

[0073] The technical solutions used in the following embodiments mainly include:

[0074] (1) Antigen design and immunization preparation

[0075] Recombinant human CCL5 protein (amino acid sequence 24-91, with a His tag) was used as an immunogen to immunize Japanese white rabbits and obtain polyclonal antibody serum against CCL5.

[0076] Immunization may include: ① initial immunization with Freund's complete adjuvant; ② booster immunization with Freund's incomplete adjuvant; ③ an immunization cycle of 4 weeks; ④ subcutaneous multi-site injection. Serum was obtained after immunization, and its binding capacity to human CCL5 and cross-reactivity with mouse CCL5 were detected by ELISA.

[0077] (2) Single B cell sorting and antibody screening methods

[0078] B cells were isolated from the spleen of immunized rabbits and sorted for antigen-specific B cell selection using the following two methods:

[0079] (a) Flow cytometry sorting based on biotin-labeled CCL5 antigen;

[0080] (b) Sorting based on antigen plating;

[0081] The sorted single B cells were cultured and the supernatant was collected. The cells were then screened by ELISA, including: ① human CCL5 binding detection; ② mouse CCL5 cross-reactivity detection; ③ capture ELISA detection. Candidate clones with cross-reactivity were screened, including 2E5 and 7H5.

[0082] (3) Antibody molecule construction

[0083] The selected candidate antibodies were genetically engineered to construct chimeric antibodies. The heavy chain contained a rabbit variable region (VH) and a human IgG1 constant region (CH1-CH3), and the light chain contained a rabbit variable region and a rabbit constant region (CL). The antibody coding sequence was cloned into the expression vector pcDNA3.4.

[0084] (4) Antibody expression and purification

[0085] The above expression vector was transfected into Expi293F cells for transient expression to obtain recombinant antibodies. The antibodies were purified by Protein A affinity chromatography, and the following quality control measures were performed: ① Purity was determined by SDS-PAGE; ② Monomer content was determined by SEC-HPLC (>95%); ③ Endotoxin content was determined (<1 EU / μg).

[0086] (5) Functional screening and core feature limitation

[0087] Among the antibodies screened, monoclonal antibody 2E5 has the following characteristics: ① It can bind to both human and mouse CCL5 simultaneously; ② Its binding affinity to CCL5 is at the nanomolar level; ③ It can dose-dependently inhibit CCL5-induced dcTR1. + ④ Significantly reduced dcTR1 in the tumor microenvironment; neutrophil differentiation; + Neutrophil percentage.

[0088] (6) Application

[0089] Monoclonal antibodies are used to inhibit immunosuppressive neutrophil differentiation, enhance dendritic cell antigen presentation, and boost CD8. + T-cell immune responses are used to prepare anti-tumor drugs, especially in combination with dendritic cell therapy.

[0090] Example 1: Construction, expression, and activity verification of CCL5 recombinant antigen

[0091] (1) CCL5 gene design and synthesis

[0092] To obtain recombinant CCL5 protein for antibody immunization and screening, mature peptide coding sequences of human and mouse CCL5 were designed and synthesized, including: Human CCL5 (24-91aa) and Mouse CCL5 (24-91aa), with a corresponding coding sequence length of 204 bp. The genes were synthesized commercially and constructed into prokaryotic expression vectors, respectively.

[0093] (2) Construction of recombinant expression vector

[0094] The following recombinant expression plasmids were constructed: pET-28a-SUMO-Human CCL5 (24-91aa), pET-32a-Human CCL5 (24-91aa), pET-28a-SUMO-Mouse CCL5 (24-91aa), and pET-32a-MouseCCL5 (24-91aa). The pET-28a vector was used to construct the SUMO fusion protein, and the pET-32a vector was used to construct the TrxA fusion protein. These different tagging systems were used to improve protein solubility, reduce non-specific tag recognition during screening, and establish a cross-validation system.

[0095] (3) Expression and purification of recombinant CCL5 protein

[0096] The above expression vector was transformed into an *E. coli* expression system for induced expression. After small-scale expression screening, the following results were obtained:

[0097] Immunogen: HumanCCL5 (24-91aa)-SUMO-His;

[0098] Proteins detected: HumanCCL5(24-91aa)-TrxA-His, MouseCCL5(24-91aa)-TrxA-His.

[0099] The recombinant protein was then purified, and its purity and integrity were analyzed by SDS-PAGE.

[0100] (4) Label removal verification

[0101] To verify the native conformation and activity of the recombinant protein, HumanCCL5(24-91aa)-SUMO-His was digested with SUMO protease. After digestion, the changes in the target protein band were detected by SDS-PAGE, confirming that the fusion tag could be effectively removed.

[0102] Example 2: Establishment of an antigen screening system

[0103] (1) Establishment of a dual-label antigen screening system

[0104] To avoid obtaining non-specific antibodies against the fusion tag, this embodiment establishes a dual-tag cross-screening system, including a SUMO-tagged immunogen and a TrxA-tagged detectiongen. By alternating between different tag systems for ELISA screening, the accuracy of screening antibodies against the CCL5 natural epitope is improved.

[0105] (2) Validation of the Biotin-based detection system

[0106] Further ELISA validation was performed on Human CCL5-biotin and Mouse CCL5-biotin.

[0107] The results showed that the biotin-labeled protein maintained good binding activity and could be used for subsequent single B cell screening and Capture ELISA detection.

[0108] Example 3: Preparation and Screening of Rabbit Anti-CCL5 Polyclonal Antibodies

[0109] (1) Animal immunization

[0110] Recombinant HumanCCL5 (24-91aa) protein was used as an immunogen to immunize Japanese white rabbits.

[0111] The immunogen is: HumanCCL5(24-91aa)-TrxA-His;

[0112] The immunization procedure included: multiple subcutaneous injections, initial immunization with Freund's complete adjuvant, booster immunization with Freund's incomplete adjuvant, a total of 5 immunizations, with an immunization cycle of approximately 63 days, and serum collection for subsequent testing after the immunization period.

[0113] (2) Serum ELISA detection

[0114] The binding capacity of immune serum to CCL5 protein was detected by ELISA.

[0115] Coating antigen: Human CCL5 (24-91aa)-His;

[0116] Detection method: The immune serum was initially diluted 1:1000 and then serially diluted 3-fold. HRP-labeled goat anti-rabbit IgG was used as the secondary antibody, and the TMB colorimetric system was used for detection.

[0117] The results showed that the immunized rabbit serum could effectively recognize human CCL5 protein and had a high antibody titer.

[0118] (3) Detection of cross-reactivity of mouse CCL5

[0119] Cross-reactivity was detected using Mouse CCL5 (24-91aa) protein.

[0120] The results showed that some immune sera could simultaneously recognize human and mouse CCL5 proteins, suggesting that the induced antibodies have cross-species binding capabilities.

[0121] (4) Purification of polyclonal antibodies

[0122] Protein A affinity chromatography was used to purify the immune serum, and pre-immune serum was purified in parallel and used as a negative control.

[0123] Purified antibody: Endotoxin level less than 1 EU / μg, used for subsequent functional validation and single B cell screening.

[0124] Example 4: Sorting of anti-CCL5 single B cells and screening of candidate clones

[0125] (1) Antigen-specific single B cell sorting

[0126] Rabbits with high serum titers after immunization and exhibiting cross-reactivity with mouse CCL5 were selected as subjects for monoclonal antibody development. After isolating rabbit spleen cells, antigen-specific single B cell sorting was performed using flow cytometry.

[0127] The sorting antigen used was: biotin-Human CCL5 (24-91aa)-TrxA-His;

[0128] Two rounds of sorting were conducted, resulting in the sorting of a total of 8 96-well plates.

[0129] (2) Single B cell culture and initial screening

[0130] The sorted single B cells were cultured in 96-well plates.

[0131] B cell supernatant was collected after culture and preliminarily screened using ELISA.

[0132] Initial screening coating antigen: Human CCL5 (24-91aa)-TrxA-His;

[0133] A positive clone is defined as: ELISA OD 450 Value ≥ 5 times the background value.

[0134] After screening, a total of 67 positive clones were obtained, such as Figure 1 As shown.

[0135] Example 5: Cross-selection of candidate clones

[0136] (1) Human CCL5 combined screening

[0137] Human CCL5 (24-91aa)-His was used as the coating antigen for ELISA screening of single B cell culture supernatants. Positive clones were defined as those with OD450-630 > 0.7.

[0138] The results showed that a total of 67 positive clones that could specifically bind to Human CCL5 were obtained.

[0139] (2) Capture ELISA rescreening

[0140] Further validation was performed using Biotinylated Human CCL5 (24-91aa)-His using Capture ELISA.

[0141] The results showed that among the 67 positive clones, 59 were able to effectively capture and bind to Biotinylated HumanCCL5.

[0142] (3) Screening for cross-reactivity of mouse CCL5

[0143] Cross-reactivity was further detected using MouseCCL5(24-91aa)-TrxA-His.

[0144] The results showed that only two clones (2E5 and 7H5) were able to bind to both human and mouse CCL5 proteins simultaneously.

[0145] The above results indicate that the hierarchical screening system of this embodiment can effectively screen from a large number of candidate clones to obtain candidate functional antibodies that possess human CCL5 binding ability, mouse CCL5 cross-reactivity, and stable antigen capture ability.

[0146] Example 6: Construction, expression, and physicochemical property analysis of anti-CCL5 chimeric antibody

[0147] (1) Candidate clone selection

[0148] Based on the aforementioned cross-reactivity and Capture ELISA screening results, 2E5 and 7H5 were selected as candidate clones for subsequent recombination expression.

[0149] (2) Construction of antibody expression vector

[0150] The variable region sequence of the antibody was amplified from positive B cells, and a recombinant chimeric antibody expression vector was constructed.

[0151] The heavy chain contains a rabbit-derived VH region and a human IgG1 constant region, and the light chain contains a rabbit-derived VL region and a human κ light chain constant region. The sequence was cloned into the pcDNA3.4 expression vector.

[0152] (3) Expression and purification of recombinant antibodies

[0153] The above expression vector was transfected into HEK293F or Expi293F cells for transient expression. After culture, the supernatant was collected and purified using Protein A affinity chromatography. At least 3 mg of purified antibody was obtained from each antibody strain.

[0154] (4) ELISA combined with validation

[0155] The binding ability of purified antibodies to Human CCL5 and Mouse CCL5 proteins was detected by ELISA.

[0156] The binding affinity of candidate antibodies 2E5 and 7H5 to human and mouse CCL5 proteins was detected using ELISA. Figure 2 As shown, the left figure shows the binding of antibody 2E5 to human and mouse CCL5 proteins, respectively, and the right figure shows the binding of antibody 7H5 to human and mouse CCL5 proteins, respectively. It shows that both 2E5 and 7H5 can bind to both Human CCL5 (24-91aa) and Mouse CCL5 (24-91aa) at the same time, indicating that the obtained antibodies have cross-species binding ability.

[0157] (5) Capture ELISA verification

[0158] Further validation using Biotinylated Human CCL5 was performed using a Capture ELISA.

[0159] The results showed that both 2E5 and 7H5 were able to stably capture and bind the Human CCL5 protein.

[0160] (6) SDS-PAGE purity analysis

[0161] The purified antibody 2E5 was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under both reducing and non-reducing conditions to determine its integrity and purity.

[0162] The results are as follows Figure 5 As shown in the upper left, under non-reducing conditions, 2E5 mainly appears as an intact antibody band of about 150 kDa; under reducing conditions, a heavy chain band of about 50 kDa and a light chain band of about 25 kDa can be observed, with no obvious impurities, indicating that the prepared antibody has high purity and good integrity.

[0163] (7) Antibody affinity analysis

[0164] The binding affinity between the antibody and Human CCL5 was detected using biolayer interference (BLI) technology.

[0165] Experimental results are as follows Figure 5 As shown in the upper right, the binding affinity constant (KD) of 2E5 with Human CCL5 is 3.88 nM, and the binding affinity constant (KD) of 7H5 with Human CCL5 is 2.44 nM, indicating that both antibodies have nanomolar binding affinity.

[0166] (8) SEC-HPLC analysis: The purified antibody was analyzed by SEC-HPLC.

[0167] The results are as follows Figure 5 As shown below, the antibody mainly exists in monomeric form, with a monomeric proportion of more than 95%, indicating that it has good stability and purity.

[0168] (9) Endotoxin detection

[0169] The purified antibodies were analyzed using an endotoxin detection method.

[0170] The results showed that the endotoxin levels of 2E5 and 7H5 were both below 0.25 EU / mg, meeting the requirements for subsequent in vivo experiments.

[0171] Example 7: Functional screening of 2E5 on CCL5-induced immunosuppressive neutrophil differentiation

[0172] (1) Neutrophil chemotaxis experiment

[0173] The Transwell assay was used to detect the inhibitory effects of different anti-CCL5 antibodies on CCL5-mediated neutrophil migration. Neutrophils derived from the bone marrow of Ccl5⁻ / ⁻ mice were seeded into the upper chamber of a Transwell assay, while recombinant mouse CCL5 was added to the lower chamber. The cells were then pre-incubated with IgG control antibody, commercial anti-CCL5 antibody (ComAb), 2E5, and 7H5, respectively. After 2 hours of incubation, the number of migrating cells was counted.

[0174] The results are as follows Figure 3 As shown, ComAb exhibits the strongest inhibitory effect on CCL5-mediated neutrophil chemotaxis, followed by 7H5, while 2E5 has a relatively weak inhibitory effect on chemotaxis.

[0175] (2) dcTR1 + Neutrophil differentiation inhibition experiment

[0176] Neutrophils derived from mice were cultured in tumor conditioned medium (TCM) and recombinant CCL5 protein was added to induce dcTR1. + Neutrophil differentiation was induced. ComAb, 2E5, and 7H5 were then added, respectively. After 48 hours of culture, Ly6G was detected by flow cytometry. + dcTR1 + Neutrophil percentage.

[0177] The results are as follows Figure 4 As shown, only 2E5 can significantly inhibit CCL5-induced Ly6G. + dcTR1 +Neutrophil production was inhibited, but neither 7H5 nor ComAb showed a significant inhibitory effect.

[0178] The above results indicate that although 2E5 is not the most potent chemokine blocking antibody, it can specifically inhibit CCL5-induced immunosuppressive dcTR1. + The neutrophil differentiation process demonstrates that 2E5 possesses functional characteristics distinct from existing anti-CCL5 antibodies. Based on this unique functional advantage, 2E5 was identified as the lead clone for subsequent research.

[0179] Example 8: In vivo safety evaluation of 2E5

[0180] (1) Drug administration and sample collection

[0181] Balb / c and C57BL / 6 mice were treated with 2E5 antibody, respectively. Serum and tissue samples from major organs were collected after treatment for safety analysis.

[0182] (2) Serum biochemical analysis

[0183] The following indicators were detected using a fully automated biochemical analyzer: ALT, AST, CREA, and UREA.

[0184] The results are as follows Figure 9 As shown, none of the above indicators showed significant abnormal changes after the 2E5 treatment.

[0185] (3) Histopathological analysis

[0186] Histological analysis was performed on major organs such as the heart, liver, spleen, lungs, and kidneys.

[0187] The results showed that no obvious tissue damage or inflammatory pathological changes were observed after 2E5 treatment.

[0188] The above results indicate that 2E5 has good in vivo tolerability and safety.

[0189] Example 9: 2E5 enhances the in vivo antitumor activity of DC therapy

[0190] (1) Establishment of tumor treatment model

[0191] 4T1, MC38 and E0771 tumor-bearing mouse models were established respectively.

[0192] Starting from day 7 after tumor inoculation, mice received DC treatment in combination with: IgG control antibody, commercial anti-CCL5 antibody (ComAb) and 2E5. The antibody was administered 3 times a week for a total of 9 times.

[0193] (2) Tumor growth inhibition analysis

[0194] The results are as follows Figure 6 As shown, 2E5 significantly inhibited tumor growth in the 4T1, MC38, and E0771 models. Compared with the IgG and ComAb groups, 2E5 exhibited a stronger and more durable anti-tumor effect. The endpoint tumor weight analysis results were consistent with the tumor growth curves.

[0195] (3) Survival and migration analysis

[0196] In the 4T1 model: 2E5 significantly prolonged the overall survival of tumor-bearing mice and reduced the burden of lung metastases.

[0197] The above results indicate that 2E5 can significantly enhance the anti-tumor effect of DC therapy in various tumor models, and is superior to existing commercial anti-CCL5 antibodies.

[0198] Example 10: Regulatory effect of 2E5 on the function of immunosuppressive tumor-associated neutrophils (TAN) and cDC1

[0199] (1) Tumor-associated neutrophil analysis

[0200] dcTR1 in the tumor microenvironment was analyzed by flow cytometry. + TANs.

[0201] The results are as follows Figure 7 As shown, 2E5 significantly reduces dcTR1 in the 4T1, MC38, and E0771 models. + The proportion of TANs.

[0202] (2) In vitro dose-dependent verification

[0203] Neutrophils derived from Ccl5⁻ / ⁻ mice were cultured in TCM supplemented with recombinant CCL5 and different concentrations of 2E5 were added.

[0204] The results showed that 2E5 could reduce dcTR1 in a dose-dependent manner. + Neutrophil percentage.

[0205] (3) Analysis of cDC1 ratio and maturity status

[0206] Further analysis was conducted on dendritic cells in the tumor and its draining lymph nodes.

[0207] The results are as follows Figure 8 As shown, this illustrates MHC-II after 2E5 treatment. + CD11c + The proportions of DC and cDC1 were increased, and CCR7 expression was upregulated. In addition, the expression of CD80, CD86, and MHC-I on the cDC1 surface was significantly enhanced.

[0208] (4) Analysis of cDC1 cross-antigen presentation function

[0209] Detection of MHC-I-SIINFEKL complex expression and OT-ICD8 + T-cell activation capacity.

[0210] The results showed that 2E5 significantly enhanced the cDC1 cross-antigen presentation ability, and OT-ICD8 + T cell proliferation and T cell effector function.

[0211] The above results indicate that 2E5 inhibits the immunosuppressive dcTR1 + The formation and maintenance of TANs can relieve their inhibition of cDC1 development and cross-antigen presentation, thereby enhancing the anti-tumor immune response.

[0212] The above are merely preferred embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A monoclonal antibody that specifically binds to CCL5, or its antigen-binding moiety, characterized in that, It contains a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region includes CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 1-3, and the light chain variable region includes CDR1, CDR3 as shown in SEQ ID NO: 4 and 5 and CDR2 with the sequence SAS; Alternatively, the heavy chain variable region may contain CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 6-8, and the light chain variable region may contain CDR1, CDR3 as shown in SEQ ID NO: 9 and 10, and CDR2 with the sequence RAS.

2. The monoclonal antibody or its antigen-binding moiety that specifically binds to CCL5 according to claim 1, characterized in that, The heavy and light chain sequences of the monoclonal antibody that specifically binds to CCL5 or its antigen-binding moiety are shown in SEQ ID NO: 11 and 12, or in SEQ ID NO: 13 and 14, respectively.

3. The monoclonal antibody or its antigen-binding moiety that specifically binds to CCL5 according to claim 1, characterized in that, The monoclonal antibody or its antigen-binding moiety that specifically binds to CCL5 specifically binds to the CCL5 protein, with a binding affinity KD of 1-10 nM.

4. The monoclonal antibody or its antigen-binding moiety that specifically binds to CCL5 according to claim 1, characterized in that, The monoclonal antibody that specifically binds to CCL5 or its antigen-binding portion is used to specifically block the differentiation process of CCL5-mediated immunosuppressive tumor-associated neutrophils (TANs) expressing decoy TRAIL receptor 1 (dcTR1) and to restore the antigen-presenting function of dendritic cells in the tumor microenvironment.

5. A nucleic acid molecule, characterized in that, It encodes the heavy chain variable region and light chain variable region of the monoclonal antibody or its antigen-binding moiety that specifically binds to CCL5 as described in any one of claims 1 to 4, or encodes the monoclonal antibody or its antigen-binding moiety that specifically binds to CCL5 as described in any one of claims 1 to 4.

6. An expression carrier, characterized in that, It comprises the nucleic acid molecule as described in claim 5.

7. A host cell, characterized in that, It comprises the expression vector as described in claim 6.

8. Use of the monoclonal antibody or antigen-binding portion thereof that specifically binds to CCL5 as claimed in any one of claims 1 to 4 in the preparation of a medicament for treating tumors.

9. The application according to claim 8, characterized in that, The drug may be used in combination with dendritic cell immunotherapy, or with immune checkpoint inhibitors, chemotherapy drugs, or radiotherapy.

10. The application according to claim 8 or 9, characterized in that, The drug works by inhibiting CCL5-induced dcTR1. + Immunosuppressive tumor-associated neutrophil differentiation, restoration of cDC1 dendritic cell maturation and cross-antigen presentation function, and / or enhancement of CD8 + T cells play a role in the anti-tumor immune response.

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