A mutant of the cytokine interleukin-10, its preparation method and application

Abstract

This invention discloses a mutant of the cytokine interleukin-10, its preparation method, and its applications. The mutant of interleukin-10 is based on the amino acid sequence SEQ ID NO.1 and involves the following mutations: any one or a combination of at least two of T13W, H14F, N18W, N21M, N92M, N92L, K99F, K99M, K99Y, or L103W. This invention creatively discovers that by designing mutations at multiple different sites on the amino acid sequence, the affinity of IL-10 can be increased by 1-5 orders of magnitude compared to the native sequence, thereby enhancing T-cell anti-tumor and antiviral immune responses.

Description

[0001] This application claims priority to patent application number 2022116277856 (the earlier application was filed on December 16, 2022, and its invention is entitled "A mutant of interleukin-10 and its preparation method and application"). Technical Field

[0002] This invention belongs to the field of biotechnology and relates to a mutant of the cytokine interleukin-10, its preparation method, and its application. Background Technology

[0003] Cytokines are a class of small protein molecules with broad biological activities, mainly secreted by immune cells, and have wide applications in clinical practice. Interleukin-10 (IL-10) is one such cytokine, primarily secreted by macrophages and T helper cells. In T cell signal transduction, IL-10 must first bind to the IL-10 receptor α on the T cell surface. The resulting complex then binds to the IL-10 receptor β, ultimately activating intracellular JAK1 phosphorylation and downstream STAT3 protein phosphorylation. The phosphorylated STAT3 dimer enters the nucleus and acts as a transcription factor to regulate the expression of related genes, thus modulating cellular function. Natural IL-10 has a relatively low affinity for the IL-10 receptor β; its dissociation constant (Kd) is only 234 μM in the presence of the IL-10 receptor α, and its affinity is negligible in the absence of IL-10 receptor α. Therefore, optimizing the design of IL-10 to enhance its affinity and specificity for its corresponding receptor is essential.

[0004] CN114163514A discloses an IL-10 mutant and its applications. The amino acid sequence of the mutant is SPGQGTQSENSCTHFPGNLPNMLRDRRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYL GCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCH. The mutation site of the mutant protein is L26R. This IL-10 mutant reduces its affinity for antibodies. When purifying specific IL-10 antibodies, affinity chromatography columns prepared from this IL-10 mutant are easier to elute and separate under the premise that the binding rate of the two does not change much, and the purified antibody has high purity. However, this IL-10 mutant only undergoes single point mutation, and the mutation site is singular.

[0005] CN108025040A discloses a method for treating diseases and conditions using interleukin-10. This method combines a PEG-IL-10 agent with an IL-15 agent to treat subjects suffering from or prevent the occurrence of a cancer-related disease, condition, or symptom. The method includes a specific dosing regimen and provides the opportunity for additive or synergistic effects in the treatment and / or prevention of the cancer-related disease, condition, or symptom described in the patent. Furthermore, such combination therapies typically allow for reduced dosage and / or frequency of PEG-IL-10 and / or the IL-15 agent combined with it, which can minimize or eliminate any adverse effects. However, the PEG-IL-10 formulation failed to prolong patient survival in a phase III clinical trial for pancreatic cancer, resulting in a failure.

[0006] In summary, IL-10 currently suffers from several drawbacks, including low affinity for its receptor, the need for high-dose administration, potential toxicity in tumor immunotherapy, and a limited number of mutation sites. Developing an IL-10 mutant with diverse mutation sites and high receptor affinity to reduce dosage and potential toxicity has become one of the most pressing issues to be addressed in the field of biotechnology. Summary of the Invention

[0007] To address the shortcomings of existing technologies and practical needs, this invention provides a mutant of the cytokine interleukin-10, its preparation method, and its application. This invention solves the problems of low affinity of IL-10 to its receptor, the need for high-dose use, potential toxic side effects in tumor immunotherapy, and the single mutation site, thereby improving the affinity of IL-10 and enhancing T-cell anti-tumor and antiviral immune responses.

[0008] To achieve this objective, the present invention employs the following technical solution:

[0009] In a first aspect, the present invention provides a mutant of interleukin-10, wherein the mutant of interleukin-10 is based on the amino acid sequence SEQ ID NO.1 and undergoes the following mutations: any one or a combination of at least two of T13W, H14F, N18W, N21M, N92M, N92L, K99F, K99M, K99Y or L103W.

[0010] This invention creatively discovers that by designing mutations at multiple different sites of amino acids, the affinity of IL-10 can be improved by 1-5 orders of magnitude compared with the natural sequence, thereby enhancing the ability of T cells to proliferate and activate in vivo and in vitro.

[0011] SEQ ID NO.1:

[0012] SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0013] In a second aspect, the present invention provides a nucleic acid molecule containing the coding sequence of the mutant interleukin-10 described in the first aspect.

[0014] Thirdly, the present invention provides a recombinant vector containing the nucleic acid molecules described in the second aspect.

[0015] Fourthly, the present invention provides a recombinant cell containing the nucleic acid molecules described in the second aspect and / or the recombinant vector described in the third aspect.

[0016] Fifthly, the present invention provides a method for preparing a mutant of interleukin-10 as described in the first aspect, the method comprising the following steps:

[0017] (1) The interleukin-10 protein sequence with a specific mutation combination was reverse-translated into a nucleic acid sequence, and the codons were optimized for the expression system used. The obtained nucleic acid sequence was then synthesized into a whole gene and inserted into a plasmid vector to obtain a recombinant plasmid.

[0018] (2) The recombinant plasmid obtained in step (1) is transformed into an expression system for cell culture. Then, the cell culture supernatant is collected for protein purification to obtain the mutant of the cytokine interleukin-10.

[0019] Preferably, the plasmid vector comprises pcDNA3.4;

[0020] Preferably, the expression system comprises mammalian cells HEK293.

[0021] In a sixth aspect, the present invention provides a method for obtaining a mutant of interleukin-10 according to the fifth aspect, the method being based on an AI model and the computational chemistry method FlexdGG, and obtaining the sequence of the mutant of interleukin-10 according to the first aspect through an iterative algorithm.

[0022] The AI ​​model uses geometric and chemical features as input and outputs a descriptor vector.

[0023] The computational chemistry method FlexdGG performs main-chain perturbation sampling on the interleukin-10 mutant based on Rosetta's energy function and calculates the binding free energy between proteins before and after the mutation design.

[0024] In a seventh aspect, the present invention provides a pharmaceutical composition comprising a mutant of interleukin-10 as described in the first aspect.

[0025] Preferably, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0026] Preferably, the excipients include any one or a combination of at least two of the following: carrier, wetting agent, solubilizer, osmotic pressure regulator, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0027] Eighthly, the present invention provides the use of the mutant of interleukin-10 described in the first aspect in the preparation of immunotherapy products.

[0028] Preferably, the immunotherapy product includes products for the prevention and / or targeted treatment of tumors.

[0029] Preferably, the tumor includes any one of melanoma, colon cancer, lung cancer, esophageal cancer, ovarian cancer, cervical cancer, or breast cancer.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] This invention creatively discovers that by designing mutations at multiple different sites of amino acids, the affinity of IL-10 can be improved by 1-5 orders of magnitude compared with the natural sequence, thereby enhancing T cell immune responses such as anti-tumor and anti-viral activity. Attached Figure Description

[0032] Figure 1 The graph shows the BLI affinity test curves for the full concentration gradient of the IL-10 mutant N18W / K99F / L103W.

[0033] Figure 2 The graph shows the BLI affinity test curves for the full concentration gradient of the IL-10 mutants N18W / K99F / N92M.

[0034] Figure 3 The graph shows the BLI affinity test curves for the full concentration gradient of the IL-10 mutants N18W / T13W / N92L.

[0035] Figure 4 The graph shows the BLI affinity test curves for Super IL-10 at full concentration gradients.

[0036] Figure 5The image shows the results of the in vitro activation capacity assay for IL-10 mutant T cells. Detailed Implementation

[0037] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0038] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0039] Example 1

[0040] (1) The mutant protein sequences shown in SEQ ID NO.2-9 were reverse-translated into nucleic acid sequences, and codon optimization was performed for the expression system used. The resulting gene sequences are shown in SEQ ID NO.10-17. The obtained nucleic acid sequences were synthesized into a whole gene and inserted into a pcDNA3.4 plasmid vector containing the Fc Tag to obtain a recombinant plasmid.

[0041] (2) The recombinant plasmid obtained in step (1) is transformed into the HEK293 expression system of mammalian cells for cell culture. Then, the cell culture supernatant is collected for protein purification to obtain the mutant of the cytokine interleukin-10.

[0042] SEQ ID NO.2: IL-10N18W / K99F mutant sequence

[0043] SPGQGTQSENSCTHFPGWLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDF KGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLFTLRLRLRRCHRFLPCEN KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN.

[0044] SEQ ID NO.3: IL-10N18W / K99F / L103W mutant sequence

[0045] SPGQGTQSENSCTHFPGWLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDF KGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLFTLRWRLRRCHRFLPCEN KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN。

[0046] SEQ ID NO.4: IL-10 N18W / K99F / N21M mutant sequence

[0047] SPGQGTQSENSCTHFPGWLPMMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDF KGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLFTLRLRLRRCHRFLPCEN KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN。

[0048] SEQ ID NO.5: IL-10 N18W / K99F / N92M mutant sequence<00001​​​​​​​​​​​​​SPGQGTQSENSCTHFPGWLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDF KGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLYTLRLRLRRCHRFLPCEN KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN。

[0054] SEQ ID NO.8: IL-10 N-terminus 18W / K99Y / H14F mutant sequence

[0055] SPGQGTQSENSCTFFPGWLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFK GYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLYTLRLRLRRCHRFLPCENK SKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN。

[0056] SEQ ID NO.9: IL-10 N-terminus 18W / T13W / N92L mutant sequence

[0057] SPGQGTQSENSCWHFPGWLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDF KGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVLSLGENLKTLRLRLRRCHRFLPCEN KSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN。

[0058] SEQ ID NO.1 : IL-10 N-terminus 18W / K99F mutant nucleic acid sequence

[0059] It should be noted that there seems to be a small error in your original text where "SEQ ID NO.10" in is likely a miswriting as it is later referred to as "SEQ ID NO.1" in the translation. I've corrected it in the translation for better consistency. If this is not what you intended, please let me know.AGCCCTGGACAGGGAACCCAGAGCGAGAATAGCTGCACCCACTTCCCCGGATGGCTGCCTAATATGCTGAGAGACTTGAGAGACGCCTTCAGCAGAGTGAAAACTTTTTTTCAGATGAAGGACCAGCTGGACAACCTGCTGCTGAAGGAGAGCCTGCTGGAGGACTTCAAGGGCTACCTGGGCTGCCAGGCCCTGAGCGAGATGATCCAGTTCTACCTGGAGGAGGTGATGCCCCAGGCCGAGAACCAGGACCCCGACATCAAGGCCCACGTGAACAGCCTGGGCGAGAACCTGTTCACCCTGAGACTGAGACTGAGAAGATGCCACAGATTCCTGCCCTGCGAGAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAACGCCTTCAACAAGCTGCAGGAGAAGGGCATCTACAAGGCCATGAGCGAGTTCGACATCTTCATCAACTACATCGAGGCCTACATGACCATGAAGATCAGAAAC。

[0060] SEQ ID NO.11: Nucleic acid sequence of IL-10 N18W / K99F / L103W mutant

[0061] AGCCCTGGACAGGGAACCCAGAGCGAGAATAGCTGCACCCACTTCCCCGGATGGCTGCCTAATATGCTGAGAGACTTGAGAGACGCCTTCAGCAGAGTGAAAACTTTTTTTCAGATGAAGGACCAGCTGGACAACCTGCTGCTGAAGGAGAGCCTGCTGGAGGACTTCAAGGGCTACCTGGGCTGCCAGGCCCTGAGCGAGATGATCCAGTTCTACCTGGAGGAGGTGATGCCCCAGGCCGAGAACCAGGACCCCGACATCAAGGCCCACGTGAACAGCCTGGGCGAGAACCTGTTCACCCTGAGATGGAGACTGAGAAGATGCCACAGATTCCTGCCCTGCGAGAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAACGCCTTCAACAAGCTGCAGGAGAAGGGCATCTACAAGGCCATGAGCGAGTTCGACATCTTCATCAACTACATCGAGGCCTACATGACCATGAAGATCAGAAAC。

[0062] SEQ ID NO.12: Nucleic acid sequence of IL-10 N18W / K99F / N21M mutant

[0063] AGCCCTGGACAGGGAACCCAGAGCGAGAATAGCTGCACCCACTTCCCCGGATGGCTGCCTATGATGCTGAGAGACTTGAGAGACGCCTTCAGCAGAGTGAAAACTTTTTTTCAGATGAAGGACCAGCTGGACAACCTGCTGCTGAAGGAGAGCCTGCTGGAGGACTTCAAGGGCTACCTGGGCTGCCAGGCCCTGAGCGAGATGATCCAGTTCTACCTGGAGGAGGTGATGCCCCAGGCCGAGAACCAGGACCCCGACATCAAGGCCCACGTGAACAGCCTGGGCGAGAACCTGTTCACCCTGAGACTGAGACTGAGAAGATGCCACAGATTCCTGCCCTGCGAGAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAACGCCTTCAACAAGCTGCAGGAGAAGGGCATCTACAAGGCCATGAGCGAGTTCGACATCTTCATCAACTACATCGAGGCCTACATGACCATGAAGATCAGAAAC。

[0064] SEQ ID NO.13: Nucleic acid sequence of IL-10 N18W / K99F / N92M mutant

[0065] AGCCCTGGACAGGGAACCCAGAGCGAGAATAGCTGCACCCACTTCCCCGGATGGCTGCCTAATATGCTGAGAGACTTGAGAGACGCCTTCAGCAGAGTGAAAACTTTTTTTCAGATGAAGGACCAGCTGGACAACCTGCTGCTGAAGGAGAGCCTGCTGGAGGACTTCAAGGGCTACCTGGGCTGCCAGGCCCTGAGCGAGATGATCCAGTTCTACCTGGAGGAGGTGATGCCCCAGGCCGAGAACCAGGACCCCGACATCAAGGCCCACGTGATGAGCCTGGGCGAGAACCTGTTCACCCTGAGACTGAGACTGAGAAGATGCCACAGATTCCTGCCCTGCGAGAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAACGCCTTCAACAAGCTGCAGGAGAAGGGCATCTACAAGGCCATGAGCGAGTTCGACATCTTCATCAACTACATCGAGGCCTACATGACCATGAAGATCAGAAAC。

[0066] SEQ ID NO.14: Nucleic acid sequence of IL-10 N18W / K99M mutant

[0067] AGCCCTGGACAGGGAACCCAGAGCGAGAATAGCTGCACCCACTTCCCCGGATGGCTGCCTAATATGCTGAGAGACTTGAGAGACGCCTTCAGCAGAGTGAAAACTTTTTTTCAGATGAAGGACCAGCTGGACAACCTGCTGCTGAAGGAGAGCCTGCTGGAGGACTTCAAGGGCTACCTGGGCTGCCAGGCCCTGAGCGAGATGATCCAGTTCTACCTGGAGGAGGTGATGCCCCAGGCCGAGAACCAGGACCCCGACATCAAGGCCCACGTGAACAGCCTGGGCGAGAACCTGATGACCCTGAGACTGAGACTGAGAAGATGCCACAGATTCCTGCCCTGCGAGAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAACGCCTTCAACAAGCTGCAGGAGAAGGGCATCTACAAGGCCATGAGCGAGTTCGACATCTTCATCAACTACATCGAGGCCTACATGACCATGAAGATCAGAAAC。

[0068] SEQ ID NO.15: Nucleic acid sequence of IL-10 N18W / K99Y mutant

[0069] AGCCCTGGACAGGGAACCCAGAGCGAGAATAGCTGCACCCACTTCCCCGGATGGCTGCCTAATATGCTGAGAGACTTGAGAGACGCCTTCAGCAGAGTGAAAACTTTTTTTCAGATGAAGGACCAGCTGGACAACCTGCTGCTGAAGGAGAGCCTGCTGGAGGACTTCAAGGGCTACCTGGGCTGCCAGGCCCTGAGCGAGATGATCCAGTTCTACCTGGAGGAGGTGATGCCCCAGGCCGAGAACCAGGACCCCGACATCAAGGCCCACGTGAACAGCCTGGGCGAGAACCTGTACACCCTGAGACTGAGACTGAGAAGATGCCACAGATTCCTGCCCTGCGAGAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAACGCCTTCAACAAGCTGCAGGAGAAGGGCATCTACAAGGCCATGAGCGAGTTCGACATCTTCATCAACTACATCGAGGCCTACATGACCATGAAGATCAGAAAC。

[0070] SEQ ID NO.16: Nucleic acid sequence of IL-10 N18W / K99Y / H14F mutant

[0071] AGCCCTGGACAGGGAACCCAGAGCGAGAATAGCTGCACCTTCTTCCCCGGCTGGCTGCCTAATATGCTGAGAGACTTGAGAGACGCATTCAGCAGAGTGAAAACCTTCTTCCAGATGAAAGACCAGCTCGACAACCTCCTGCTGAAAGAGAGCCTGCTGGAGGACTTCAAGGGCTACCTGGGATGCCAGGCTCTGTCAGAAATGATTCAGTTCTATCTCGAAGAGGTGATGCCCCAGGCCGAAAACCAGGACCCTGATATCAAAGCCCACGTGAACAGCCTGGGAGAGAATCTGTACACACTGAGACTGCGGCTGAGACGGTGCCATAGATTCCTGCCTTGTGAGAACAAAAGCAAAGCCGTGGAGCAGGTGAAGAACGCCTTCAACAAGCTGCAGGAGAAGGGGATCTATAAGGCCATGAGCGAATTTGACATCTTCATCAACTACATCGAGGCCTACATGACCATGAAAATCAGAAAT。

[0072] SEQ ID NO.17: Nucleic acid sequence of IL-10 N18W / T13W / N92L mutant

[0073] AGCCCTGGACAGGGAACCCAGAGCGAGAATAGCTGCTGGCACTTCCCCGGATGGCTGCCTAATATGCTGAGAGACTTGAGAGACGCATTCAGCAGAGTGAAAACCTTCTTCCAGATGAAGGACCAGCTGGACAACCTCCTGCTGAAGGAGAGCCTGCTGGAGGATTTTAAGGGCTACCTGGGCTGCCAGGCCCTGAGCGAGATGATCCAGTTCTATCTCGAAGAGGTGATGCCCCAGGCC GAGAATCAGGACCTGATATCAAGGCACACGTGCTGAGCCTGGGCGAAAATCTGAAAACACTGAGACTGAGACTGAGGAGGTGCCACAGGTTCCTCCCTTGTGAGAACAAATCCAAGGCCGTCGAGCAGGTCAAGAACGCATTCAACAAGCTGCAGGAAGGGCATCTACAAAGCCATGAGCGAGTTCGATATCTTCATCAACTACATCGAGGCCTACATGACATGAAGATCCGAAAT.

[0074] Example 2

[0075] Affinity assay of IL-10 mutants binding to their receptors.

[0076] (1) The purified IL-10 mutant obtained in Example 1, expressed as an Fc fusion protein, was used to measure the dissociation constant K between IL-10 and IL-10Rβ under the condition of co-incubation with IL-10Rα. D First, 10 μg / mL biotin-labeled IL-10Rβ was immobilized on an SA chip. Then, 500 nM IL-10 mutant and 500 nM IL-10Rα were co-incubated at 25 °C for 1 hour. The resulting complex solution was analyzed using Gator biofilm layer interferometry (BLI) to detect binding and dissociation curves, and the dissociation constant K was calculated. D The experimental results are shown in Table 1. The dissociation constant K of the natural type IL-10 is... D =234μM, the results showed that the affinity of all expressed mutants in the examples was superior to that of non-mutant IL-10;

[0077] (2) After preliminary experiments, the mutants N18W / K99F / L103W, N18W / K99F / N92M and N18W / T13W / N92L showed relatively higher affinity, which was close to the affinity of Super IL-10, which performed best in the literature (Table 1). Therefore, a more accurate full-concentration gradient affinity determination was carried out. Super IL-10 is a mutant obtained by Christopher Garcia et al. in 2021 (Saxton et al., Science 371, 1222 (2021)). First, biotin-labeled IL-10Rβ at a concentration of 10 μg / mL was immobilized on an SA chip. Then, serially diluted IL-10 mutants at concentrations of 500 nM, 166.67 nM, 55.56 nM, 18.52 nM, 6.17 nM, and 2.06 nM were co-incubated with IL-10Rα at a concentration of 500 nM at 25 °C for 1 hour. The resulting complex solution was analyzed using Gator biofilm layer interferometry (BLI) to detect binding and dissociation curves, and the dissociation constant K was calculated. D More accurate fitting of full-concentration gradient BLI measurement data and dissociation constant K D The statistical results are shown in Table 2. The full-concentration gradient BLI affinity test curves for the IL-10 mutant N18W / K99F / L103W are shown in the figure. Figure 1 As shown in the figure, the BLI affinity test curves for the IL-10 mutants N18W / K99F / N92M at full concentration gradients are as follows. Figure 2 As shown in the figure, the BLI affinity test curves for the IL-10 mutants N18W / T13W / N92L at full concentration gradients are as follows. Figure 3 As shown in the figure, the BLI affinity test curves for Super IL-10 across the full concentration gradient are as follows: Figure 4 As shown, Figures 1-4 The dissociation constants obtained from the fitting are shown in Table 2.

[0078] Table 1

[0079]

[0080]

[0081] Table 2

[0082] IL-10 mutant <![CDATA[K on (1 / Ms)]]> <![CDATA[K off (1 / s)]]> <![CDATA[K D (nM)]]> <![CDATA[FullR 2 ]]> N18W / K99F / L103W 4.07E+005 3.85E-003 9.46 0.9680 N18W / K99F / N92M 3.14E+005 2.54E-003 8.11 0.9754 N18W / T13W / N92L 1.41E+005 3.11E-003 22.1 0.9514 SuperIL-10 3.95E+004 6.88E-004 17.4 0.9978

[0083] The results showed that mutants N18W / K99F / L103W and N18W / K99F / N92M exhibited lower dissociation constants than Super IL-10, indicating higher affinity, while mutant N18W / T13W / N92L achieved an affinity similar to Super IL-10.

[0084] Example 3

[0085] Assay of the in vitro activation capacity of IL-10 mutant T cells.

[0086] Jurkat T cells were placed in 96-well plates, and native and mutant IL-10 were added and cultured at 37°C for 20 min to activate T cells. Then, paraformaldehyde was added for fixation at room temperature for 10 min. Cells were permeabilized with ice-cold methanol at -20°C for 30 min to facilitate subsequent intracellular staining. Cells were then resuspended in autoMACS buffer and co-incubated with phosphorylated STAT3 antibody at room temperature for 1 h for staining. Finally, flow cytometry was used to detect and compare fluorescence intensity.

[0087] The results showed that T cells treated with mutant IL-10 had stronger fluorescence intensity, indicating that mutant IL-10 could activate T cells better than the natural type.

[0088] Example 4

[0089] Assay of the in vitro activation capacity of IL-10 mutant T cells.

[0090] The biological activity of recombinant interleukin-10 (IL-10) was monitored by using commercially available HEK-Blue™ IL-10 cells from Invivogen. The specific experimental procedure is as follows: 1. When HEK Blue IL-10 cells reached 80% confluence, they were washed with PBS, centrifuged at 1000 rpm for 5 min, and counted. Cells were then transferred to 96-well plates at 5 × 10⁻⁶ cells / well. 4 1. Spread cells / wells at a density and volume of 150 μL / well. 2. Add IL-10-Fc at a serial dilution of 80 ng / mL to 8 wells (50 μL / well) and incubate at 37°C with 5% CO2 for 20 h. 3. Prepare QB solution in advance. Add 160 μL of QB solution to each well of a new 96-well plate. Remove the incubated plate from the incubator and aspirate 40 μL of supernatant from each well into the corresponding QB solution well. Incubate at 37°C in the dark for 2 h. 4. Read the absorbance at 630 nm using a microplate reader, evaluate the curve, and calculate EC50.

[0091] Experimental results are as follows Figure 5As shown, WT represents the wild-type IL-10 sequence, sample SM1 corresponds to the N18W / K99F / L103W mutant, sample SM2 corresponds to the N18W / K99F / N92M mutant, sample SM3 corresponds to the N18W / K99Y / H14F mutant, and sample SM4 corresponds to the N18W / T13W / N92L mutant. The results show that cells treated with mutant IL-10 have lower EC50 values, indicating that mutant IL-10, by binding to IL-10R, can better activate the downstream JAK1-STAT3 signaling pathway, thereby enhancing the proliferation and tumor-killing activity of CD8+ T cells.

[0092] In summary, this invention creatively discovers that by designing mutations at multiple different sites of amino acids, the affinity of IL-10 can be improved by 1-5 orders of magnitude compared to the natural sequence, thereby enhancing the ability of T cells to proliferate and activate in vivo and in vitro.

[0093] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A mutant of interleukin-10, characterized in that, The mutant of interleukin-10 undergoes any one of the following combinations of mutations (1)-(5) based on the amino acid sequence SEQ ID NO.1: (1) A combination of N18W with either K99F or K99Y; (2) A combination of N18W and N92M with any one of K99F or K99Y; (3) Combinations of N18W and T13W with any one of N92M and N92L; (4) Combinations of N18W and L103W with any one of K99F or K99Y; (5) A combination of N18W and H14F with any one of K99F or K99Y.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule contains the coding sequence of the mutant interleukin-10 as described in claim 1.

3. A recombinant vector, characterized in that, The recombinant vector contains the nucleic acid molecule as described in claim 2.

4. A recombinant cell, characterized in that, The recombinant cells contain the nucleic acid molecules of claim 2 and / or the recombinant vector of claim 3.

5. A method for preparing a mutant of interleukin-10 according to claim 1, characterized in that, The method includes the following steps: (1) The interleukin-10 protein sequence with a specific mutation combination was reverse-translated into a nucleic acid sequence, and the codons were optimized for the expression system used. The obtained nucleic acid sequence was then synthesized into a whole gene and inserted into a plasmid vector to obtain a recombinant plasmid. (2) The recombinant plasmid obtained in step (1) is transformed into an expression system for cell culture. Then, the cell culture supernatant is collected for protein purification to obtain the mutant of interleukin-10.

6. The method for producing a mutant of interleukin-10 according to claim 5, characterized in that, The method is based on an AI model and the computational chemistry method FlexdGG, and obtains the sequence of the interleukin-10 mutant as described in claim 1 through an iterative algorithm; The AI ​​model uses geometric and chemical features as input and outputs a descriptor vector. The computational chemistry method FlexdGG performs main-chain perturbation sampling on the interleukin-10 mutant based on Rosetta's energy function and calculates the binding free energy between proteins before and after the mutation design.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a mutant of interleukin-10 as described in claim 1.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

9. The pharmaceutical composition according to claim 8, characterized in that, The excipients are selected from any one or a combination of at least two of the following: carrier, wetting agent, solubilizer, osmotic pressure regulator, coating material, colorant, pH regulator, antioxidant, antibacterial agent or buffer.

10. The use of the mutant of interleukin-10 of claim 1 in the preparation of a medicament for the prevention and / or treatment of cancer, wherein the cancer is selected from any one of melanoma, colon cancer, lung cancer, ovarian cancer, or breast cancer.

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