An antibody targeting the human interleukin-6 receptor, its applications and products
By developing a specific amino acid sequence-targeting antibody against the human interleukin-6 receptor, the problems of poor blocking efficacy and insufficient safety of existing IL6R antibodies have been solved, achieving highly efficient blocking of the IL6 receptor, which is suitable for the treatment of a variety of inflammatory and immune diseases.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- REYOUNG SUZHOU BIOLOGY SCI & TECH CO LTD
- Filing Date
- 2024-12-30
- Publication Date
- 2026-06-30
AI Technical Summary
Existing IL6R antibodies have limited blocking efficacy and insufficient safety in treating autoimmune diseases such as rheumatoid arthritis.
An antibody targeting the human interleukin-6 receptor was developed, containing specific HCDR and LCDR amino acid sequences. The heavy and light chain variable regions have high sequence similarity. The antibody was expressed in host cells via a biological expression vector to form an antibody-drug conjugate to improve the blocking effect and safety.
This antibody exhibits high affinity, effectively blocking the binding of IL6 to its receptor, reducing inflammatory responses, and has good safety profile, making it suitable for the treatment of various inflammatory and immune-related diseases.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of protein engineering technology, specifically relating to an antibody targeting the human interleukin-6 receptor and its applications and products. Background Technology
[0002] Interleukin-6 (IL6) has a wide range of effects on both immune and non-immune cells, and often exhibits hormone-like properties that influence homeostatic processes. IL6 possesses environment-dependent pro-inflammatory and anti-inflammatory properties and is considered a major target for clinical intervention. It affects the proliferation, differentiation, and activation of various cell types by binding to the IL6 receptor (IL6R) and activating the JAK / STAT signaling pathway. In autoimmune diseases such as rheumatoid arthritis (RA), overexpression of IL6 leads to chronic inflammation and tissue damage.
[0003] IL6 can be transduced through the IL6 receptor (IL6R) (in both transmembrane and soluble forms), allowing it to exert anti-inflammatory or pro-inflammatory effects through both classical and transsignal pathways, thereby driving its various roles in immune regulation, inflammatory diseases, and tumor diseases.
[0004] To date, the most widely studied approach to inhibiting IL6 activity is the use of monoclonal antibodies against IL6R, such as tocilizumab. Tocilizumab neutralizes IL6 activity by binding to IL6R at its IL6-binding site and is used to treat rheumatoid arthritis, systemic arthritis, juvenile idiopathic arthritis, adult paroxysmal quiescent disease, polyarteritis, giant cell arteritis, and cytokine release syndrome.
[0005] In summary, the development of IL6R antibodies is of great significance for treating a variety of diseases, modulating immune responses, and developing new therapeutic approaches. These antibodies reduce inflammatory responses and immune-mediated tissue damage by blocking the interaction between IL6 and its receptor. With further clinical research and drug development, IL6R antibodies hold the promise of providing more effective treatment options for a wide range of inflammatory and immune-related diseases. Summary of the Invention
[0006] To address the aforementioned problems, this invention provides an antibody targeting the human interleukin-6 receptor, along with its applications and products.
[0007] On the one hand, the present invention provides an antibody that targets the human interleukin-6 receptor.
[0008] Specifically, the antibody comprises: (1) Containing any one or more of HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively; or any one or more amino acid sequences that have at least 50% sequence similarity to SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively; and / or (2) Contains any one or more of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively, or any one or more amino acid sequences that have at least 50% sequence similarity to SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6 respectively.
[0009] SEQ ID NO.1: DTYMY.
[0010] SEQ ID NO.2: WIDPANVNTKYDPKFQG.
[0011] SEQ ID NO.3: DYGA.
[0012] SEQ ID NO.4: SSQSVLYSSNQKNYLA.
[0013] SEQ ID NO.5: WASTRES.
[0014] SEQ ID NO.6: HQYLSSRT.
[0015] More specifically, the antibody (1) Contains a heavy chain variable region as shown in SEQ ID NO.7, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO.7; and / or (2) Contains a light chain variable region as shown in SEQ ID NO.8, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO.8.
[0016] SEQ ID NO.7: QVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMYWVKQRPEQGLEWIGWIDPANVNTKYDPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCASDYGAWGQGTTLTVSS.
[0017] SEQ ID NO. 8: DIVMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQHKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCHQYLSSRTFGGGTKLEIKR.
[0018] More specifically, the antibody (1) Contains a heavy chain with a sequence as shown in SEQ ID NO.9, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO.9; and / or Contains a light chain as shown in SEQ ID NO.10, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO.10.
[0019] SEQ ID NO.9: QVQLQQSGAELVKPGASVKLSCTASGFNIKDTYMYWVKQRPEQGLEWIGWIDPANVNTKYDPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCASDYGAWGQG TTLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.
[0020] SEQ ID NO.10: DIVMTQSPSSLAVSAGEKVTMSCKSSQSVLYSSNQKNYLAWYQHKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCHQYLSSRTFGGGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.
[0021] In some specific embodiments of the present invention, the sequence of the heavy chain of the antibody is shown in SEQ ID NO.9, and the sequence of the light chain is shown in SEQ ID NO.10.
[0022] On the other hand, the present invention provides a nucleic acid molecule that encodes the heavy chain and / or light chain of the aforementioned antibody.
[0023] Specifically, the nucleic acid molecule contains a nucleic acid sequence as shown in SEQ ID NO.11, or any one or more nucleic acid sequences that have at least 85% sequence similarity to SEQ ID NO.11; and / or It contains a nucleic acid sequence as shown in SEQ ID NO.12, or any one or more nucleic acid sequences that have at least 85% sequence similarity to SEQ ID NO.12.
[0024]
[0025] SEQ ID NO.12:
[0026] In certain specific embodiments of the present invention, the sequence of the heavy chain encoding gene of the antibody is shown in SEQ ID NO.11; and the sequence of the light chain encoding gene is shown in SEQ ID NO.12.
[0027] In another aspect, the present invention provides a biological expression vector comprising the above-mentioned nucleic acid molecules.
[0028] Specifically, the expression vectors include, but are not limited to: pET, pCMV, pEGFP, pSV2, or CMV4.
[0029] In another aspect, the present invention provides a host cell comprising the aforementioned antibodies, nucleic acid molecules, and / or biological expression vectors.
[0030] Specifically, the host cells include, but are not limited to: Escherichia coli, Saccharomyces cerevisiae, Kluyveromyces martensii, CHO, SP2 / 0, HEK293, or BHK.
[0031] In another aspect, the present invention provides a conjugate comprising the aforementioned antibody.
[0032] Specifically, the conjugates include, but are not limited to, immune conjugates, fusion proteins, or multi-target ADCs.
[0033] In another aspect, the present invention provides a composition comprising the above-described antibody.
[0034] More specifically, the composition further includes pharmaceutically acceptable excipients.
[0035] In another aspect, the present invention provides the use of the above-mentioned antibodies, nucleic acid molecules, biological expression vectors, host cells, conjugates and / or compositions in the preparation of medicaments for treating and / or preventing viral infections, cardiovascular diseases, nervous system diseases, cytokine release syndromes, autoimmune diseases and / or tumor diseases.
[0036] Specifically, the autoimmune diseases include, but are not limited to, rheumatoid arthritis and / or systemic lupus erythematosus.
[0037] Specifically, the tumor-related diseases include, but are not limited to: breast cancer, colorectal cancer, lung cancer, ovarian cancer, cervical cancer, laryngeal cancer, nasopharyngeal cancer, and / or liver cancer.
[0038] Compared with the prior art, the present invention has the following advantages: The antibody targeting the human interleukin-6 receptor provided by this invention has high affinity, good blocking effect, and good safety. Detailed Implementation
[0039] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.
[0040] Example 1 Antigen Preparation Method 1. Antigen preparation 1.1 Preparation of IL6 receptor protein The human IL6 receptor α subunit (human IL6RA) was purchased from acrobiosystems, Cat# ILR-H4223.
[0041] 1.2 Construction of a stable cell pool for the human IL6 receptor The full-length sequence encoding human IL6RA was cloned into the pcDNA3.1(-) vector. The constructed expression vector was transformed into HEK293F cells using Lipofectamine™ 3000 Transfection Reagent (Thermo, Cat# L3000150). After screening with 400 μg / mL G418 (Gibco, Cat#10131-027) and FACS verification, HEK293F_human IL6RA cells that stably expressed human IL6RA were obtained.
[0042] 2. Animal immunization To obtain high-affinity and high-specificity antibodies against the human IL6 receptor, protein immunization was performed using the extracellular region fragment of IL6RA. Several Balb / c and C57BL / 6 mice were immunized according to a classic immunization schedule to induce an immune response. Mouse serum was collected, and serum titers were determined using ELISA or FACS with HEK293F_human IL6RA cells, using the IL6RA extracellular protein as an antigen. Mice with high titers were selected for fusion.
[0043] 3. Hybridoma fusion and screening Prior to fusion, the status of mouse myeloma cells SP2 / 0 was adjusted to ensure that their growth density did not exceed 1×10⁻⁶. 6 Cells / mL. Animals were immunized 3 days in advance, and feeder cells were prepared one day in advance, with a plate number of 2.0 × 10⁶ cells / mL. 4 Cells / wells are fused using an electrofusion apparatus to ensure that the ratio of spleen cells or lymph node cells to SP2 / 0 cells is between 10:1 and 5:1, and the number of spleen cells seeded in each well does not exceed 1×102. 5 Seven days after fusion, the supernatant was harvested and the culture medium was replaced. The collected supernatant was used to obtain one hybridoma cell line that showed a positive result in the screening experiment by ELISA and flow cytometry to determine cell binding activity.
[0044] A. ELISA binding method Dilute IL6RA to 0.5 μg / mL with PBS (pH 7.4) and coat 96-well plates. Add 100 μL to each well. After blocking, add hybridoma culture supernatant and incubate. After washing, add Peroxidase-Affini Pure Goat Anti-Mouse IgG, Fcγ Fragment Specific (minX Hu, Bov, HrsSrProt) and incubate. After washing, add chromogenic substrate and incubate. After stopping the reaction, read the OD. 450 The absorbance value.
[0045] B. Flow cytometry determination of hybridoma supernatant cell binding activity Hybridoma supernatant was subjected to flow cytometry binding assays with HEK293F_human IL6RA and HEK293F cells. The clones that specifically bound HEK293F_human IL6RA cells were the target clones.
[0046] Each reaction used 50 μL, with a total volume of 2.5 × 10⁻⁶. 5 Cells were prepared using hybridoma supernatant as the primary antibody, along with positive antibody, isotype control, and PBS. After incubation with the primary antibody, the cells were washed and incubated with Goat anti-Mouse IgG (H+L) iFlour 647 (GenScript, 3 μg / mL) as the secondary antibody. After washing, the fluorescence signal was read by flow cytometry.
[0047] Example 2: Preparation and activity identification of human-mouse chimeric antibodies 1. Acquisition of mouse resistance genes Total RNA was extracted from hybridoma cell lines using the Trizol (Ambion 15596-026) kit, and then cDNA was prepared by reverse transcription of total RNA using the Takara PrimeScript 1st Strand cDNA Synthesis Kit. The heavy and light chain variable regions of the antibody were amplified using degenerate primers.
[0048] 2. Construction of human-mouse chimeric antibodies The mouse anti-light chain variable region sequence was spliced with the human IgG kappa CL region sequence to obtain the full-length light chain sequence. The light chain expression vector was then constructed into a pcDNA3.1 vector containing a nitrogen-terminal signal peptide through whole-genome synthesis.
[0049] The variable region sequence of the mouse anti-heavy chain was spliced with the CH1, CH2 and CH3 region sequences of human IgG4 to obtain the full-length heavy chain sequence. The heavy chain expression vector was obtained by synthesizing the entire gene and constructing it into a pcDNA3.1 vector containing a nitrogen-terminal signal peptide.
[0050] 3. Expression and purification of human-mouse chimeric antibodies The light and heavy chain expression vectors were used to transiently transfect HEK293F cells. After transfection, the 293F cells were cultured for 7 days, and the culture supernatant was collected. The chimeric antibody in the supernatant was then purified using a protein A affinity chromatography column and dialyzed into PBS at pH 7.4. The concentration was determined using the A280 method, and SDS-PAGE analysis was performed. The antibody sequence is shown in SEQ ID NO. 9 and SEQ ID NO. 10, and it was named 116D2D3.
[0051] 4. Flow cytometry was used to determine the binding ability of human and mouse chimeric antibodies to cells. The binding ability of human-mouse chimeric antibodies to HEK293F_human IL6RA cells and HEK293F blank control cells was determined. Each reaction used 100 μL of 2.0 × 10⁻⁶ cells. 7 Cells were collected at 100 μL / well. The primary antibody consisted of 3 µg / mL purified human-mouse chimeric antibody 116D2D3 (SEQ ID NO.9 and SEQ ID NO.10), 3 µg / mL positive antibody (Anti-IL-6R alphaAntibody, R&D systems, Catalog #: MAB227), 3 µg / mL isotype control (Invitrogen, Catalog #026100), and PBS containing 1% BSA. After incubation with the primary antibody for 2 h, the cells were washed, and 100 μL of 1:200 diluted Goat anti-human IgG Fc (FITC) (abcam, Cat# ab97224) was added to each well as a secondary antibody and incubated for 1 h. After washing, the fluorescence signal was read by flow cytometry. The experimental results are shown in Table 1.
[0052] Table 1
[0053] 5. Determination of the neutralizing activity of the antibody against human IL6 binding to the human IL6 receptor. A. ELISA method Human IL6 protein (Acrobiosystems, Cat.No.IL6-H4218) was diluted to a final concentration of 5 μg / mL using Na2CO3-NaHCO3, pH 9.6 coating buffer. 100 μL was added to each well, and the mixture was incubated overnight at 4 °C. The serially diluted chimeric antibody was mixed with 200 ng / mL human IL6RA, with 200 ng / mL human IL6RA without chimeric antibody serving as a control. The mixture was incubated at room temperature for 2 h. The incubated mixture or human IL6RA alone was added to the wells coated with human IL6. After binding at room temperature for 1 h, each well was washed, and 100 μL of a 1:5000 diluted His-tag antibody coupled with HRP (Abcam, Cat#ab1269) was added. After washing, 100 μL of TMB chromogenic solution (Aladdin, Cat#T117926) was added for color development. After stopping the reaction, the OD was read. 450 The absorbance value is used to calculate the IC50 of the antibody. 50 Value, IC 50 The value represents the antibody concentration added when the binding signal between human IL6RA and coated human IL6 decreases by 50%. (Calculated using GraphPad Prism software under Dose response-inhibition: Log(inhibitor) vs Responses-Variable slope (Four parameters)). The hIL6 / hIL6R binding inhibition IC50 value represents the antibody concentration added when the binding signal between human IL6RA and coated human IL6 decreases by 50%. 50 The experimental results are shown in Table 2.
[0054] B. SPR method His antibody chip surface was prepared using a CM5 chip (GE, Cat# 29149603) and a His capture kit (GE, Cat# 29234602). Human IL6RA protein was captured onto the His antibody chip surface through the binding of the His tag to the His-tagged antibody. The binding signal of 50 nM human IL6 flowing through the chip surface served as a positive control. The test sample was first flowed through the chip surface containing captured Human IL6RA, followed by 50 nM human IL6, and the binding signal was measured. The percentage decrease in IL6 binding signal caused by antibody binding was used as an indicator of whether the antibody had a blocking effect. The results of the hIL6 / hIL6 receptor binding inhibition experiment are shown in Table 2.
[0055] C. In vitro activity blocking experiment The inhibition assay was performed using human IL-6-dependent XG-1 cells. XG-1 cells were cultured in complete medium containing human IL-6 (90% (v / v) 1640 basal medium, 10% (v / v) fetal bovine serum). After washing the cells twice, they were cultured in medium without human IL-6 for 24 h, and then resuspended at 4 × 10⁶ cells / mL. 5 A suspension of 20,000 cells / mL was added to each well of a 96-well plate. Serially diluted purified antibody was added to the wells, followed by 8 pM of human IL-6, and mixed thoroughly. Cells were cultured at 37 °C for 72 h at 5% CO2. The IC50 of the antibody was then determined using a CCK-8 assay kit. 50 Value, IC50 value of XG-1 cell proliferation inhibition 50 The experimental results are shown in Table 2.
[0056] Table 2
[0057] 6. Determination of antibody species cross-binding activity The chimeric antibody to be tested was captured using a Protein A chip. Human IL6RA and mouse IL6RA (Acrobiosystems, Cat# ILR-M82E9) were serially diluted and used as analytical streams. The antibody was immobilized on the chip surface, and the affinity between the antibody and IL6RA was measured. The experimental results are shown in Table 3.
[0058] Table 3
[0059] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.
Claims
1. An antibody, characterized in that, (1) Containing any one or more of HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively; or any one or more amino acid sequences that have at least 50% sequence similarity to SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively; and / or (2) Contains any one or more of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively, or any one or more amino acid sequences that have at least 50% sequence similarity to SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6 respectively.
2. The antibody according to claim 1, characterized in that, (1) Contains a heavy chain variable region as shown in SEQ ID NO.7, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO.7; and / or (2) Contains a light chain variable region as shown in SEQ ID NO.8, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO.
8.
3. The antibody according to any one of claims 1-2, characterized in that, (1) Contains a heavy chain with a sequence as shown in SEQ ID NO.9, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO.9; and / or It contains a light chain with a sequence as shown in SEQ ID NO.10, or any one or more amino acid sequences that have at least 85% sequence similarity to SEQ ID NO.
10.
4. The antibody according to claim 3, characterized in that, The sequence of the heavy chain of the antibody is shown in SEQ ID NO.9, and the sequence of the light chain is shown in SEQ ID NO.
10.
5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the heavy chain and / or light chain of the antibody according to any one of claims 1-4.
6. The nucleic acid molecule according to claim 5, characterized in that, Contains a nucleic acid sequence as shown in SEQ ID NO.11, or any one or more nucleic acid sequences that have at least 85% sequence similarity to SEQ ID NO.11; and / or It contains a nucleic acid sequence as shown in SEQ ID NO.12, or any one or more nucleic acid sequences that have at least 85% sequence similarity to SEQ ID NO.
12.
7. A biological expression vector, characterized in that, The biological expression vector comprises the nucleic acid molecule as described in any one of claims 5-6.
8. A host cell, characterized in that, The host cell comprises the antibody as described in any one of claims 1-4, the nucleic acid molecule as described in any one of claims 5-6, and / or the biological expression vector as described in claim 7.
9. A coupling, characterized in that, It includes the antibody according to any one of claims 1-4.
10. The coupling according to claim 9, characterized in that, The conjugates include: immunoconjugates, fusion proteins, or multi-target ADCs.
11. A composition, characterized in that, It includes the antibody according to any one of claims 1-4.
12. The composition according to claim 11, characterized in that, The composition also includes pharmaceutically acceptable excipients.
13. The use of the antibody according to any one of claims 1-4, the nucleic acid molecule according to any one of claims 5-6, the biological expression vector according to claim 7, the host cell according to claim 8, the conjugate according to claims 9-10, and / or the composition according to any one of claims 11-12 in the preparation of medicaments for treating and / or preventing viral infections, cardiovascular diseases, nervous system diseases, cytokine release syndromes, autoimmune diseases, and / or tumor diseases.
14. The application according to claim 13, characterized in that, The autoimmune diseases mentioned include rheumatoid arthritis and / or systemic lupus erythematosus.
15. The application according to claim 13, characterized in that, The tumor-related diseases include: breast cancer, colorectal cancer, lung cancer, ovarian cancer, cervical cancer, laryngeal cancer, nasopharyngeal cancer, and / or liver cancer.