Interleukin 2 mutants and uses thereof

Abstract

The application discloses interleukin 2 mutants and application thereof, and the application introduces at least one amino acid mutation to change the binding of IL-2 to different receptors by structural analysis of IL-2 and its three receptors, computer-aided design technology, modeling and molecular docking. In the construction of the designed different IL-2 mutants, expression, purification and function analysis are carried out to confirm that the mutants do not bind to IL-2R alpha receptor, but basically do not affect the binding to IL-2R beta / gamma receptor complex. The mutants provided by the application can better activate Teff cells and NK cells, instead of preferentially activating Treg cells with inhibitory function like wild-type IL-2, and the mutants of IL-2 can better play an anti-tumor role in vivo.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to interleukin-2 mutants and their applications. Background Technology

[0002] Interleukin-2 (IL-2) is a cytokine belonging to the chemokine family. It was the first interleukin factor to be cloned and the first to be approved for cancer treatment. It is derived from multiple cells (primarily activated CD4+). + T cells produce cytokines, which have multi-directional effects (mainly promoting lymphocyte growth, proliferation, and differentiation); they play an important role in the body's immune response and antiviral infection, stimulating the proliferation of T cells initiated by specific antigens or mitogenic factors; activating T cells and promoting cytokine production; stimulating NK cell proliferation, enhancing NK killing activity and producing cytokines, inducing LAK cell production; promoting B cell proliferation and antibody secretion; and activating macrophages.

[0003] The IL-2 receptor (IL-2R) is a heteromer composed of three chains: α, β, and γ. Specifically, IL-2R consists of IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132). β / γ are essential for activation of downstream signaling pathways, while α primarily promotes their binding. When the IL-2 receptor α subunit (IL-2RA) exists alone, its affinity for IL-2 is only one percent of that of the complete IL-2 receptor. Therefore, IL-2R is classified into high, medium, and low affinity receptors (see...). Figure 1 , Rosanne Spolski et al. Nat RevImmunol 2018 Oct; 18(10):648-659.).

[0004] Regulatory T cells (Tregs) express a trimeric IL-2 receptor composed of α, β, and γ chains. In contrast, naive CD8+ T cells (Teffs) and natural killer (NK) cells carry a dimer receptor composed only of β and γ chains. Because the trimeric receptor has approximately 100 times higher affinity for IL-2 than the dimer receptor, IL-2 preferentially binds to Treg cells in vivo. Tregs are a subset of T cells with significant immunosuppressive activity, characterized by the expression of Foxp3, CD25, and CD4. They suppress the immune responses of other cells and are the main controllers of self-tolerance. Normally, their absence or dysfunction leads to autoimmune diseases. Effector CD8+ T cells (Teffs) and natural killer cells (NK cells) have strong target cell killing capabilities. In cancer patients, the balance between Tregs and Teffs is disrupted, with an increased proportion of Tregs. If wild-type IL-2 is used, Treg cells will be stimulated first, which is detrimental to the anti-tumor effect of IL-2. With the progress of basic research, the activation mechanism of IL-2 on Teff and Treg cells has become clearer. More and more companies are modifying IL-2 to reduce the low affinity of IL-2 mutants or derivatives for IL-2Rα, while maintaining or increasing the affinity for IL-2Rβ. This allows IL-2 mutants or derivatives to selectively activate Teff and NK cells, rather than preferentially activating Treg cells, thus enabling them to exert better anti-tumor effects in vivo. Summary of the Invention

[0005] The technical problem to be solved by this invention is: how to reduce the binding of IL-2 to the IL-2Rα subunit without affecting the binding of IL-2 to the IL-2Rβ / γ subunit, thereby improving the anti-tumor effect of IL-2 and providing IL-2 with better anti-tumor effect.

[0006] To solve the above-mentioned technical problems, in a first aspect, the present invention provides an IL-2 mutant, which is a protein obtained by mutating IL-2 with the amino acid sequence SEQ ID No.1. The affinity of the IL-2 mutant for IL-2Rα is lower than that of IL-2 for IL-2Rα.

[0007] Furthermore, in the aforementioned IL-2 mutant, the mutation is the addition of one amino acid between positions 40 and 41 of SEQ ID No. 1, wherein the one amino acid is selected from A or P.

[0008] Furthermore, in the above-mentioned IL-2 mutants, the IL-2 mutant is a protein whose amino acid is any of the following: SEQ ID No. 5, SEQ ID No. 12, SEQ ID No. 16, SEQ ID No. 18, SEQ ID No. 10 and SEQ ID No. 8.

[0009] To address the aforementioned technical problems, in a second aspect, the present invention provides biomaterials related to the aforementioned IL-2 mutant, wherein the biomaterials may be any of the following:

[0010] B1) The nucleic acid molecule encoding the IL-2 mutant;

[0011] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0012] B3) A recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2);

[0013] B4) Recombinant microorganisms containing the nucleic acid molecule described in B1), or the expression cassette described in B2), or the recombinant vector described in B3);

[0014] B5) An animal cell line containing the nucleic acid molecule described in B1), or the expression cassette described in B2), or the recombinant vector described in B3);

[0015] B6) Host cells that produce the IL-2 mutant.

[0016] Furthermore, in the aforementioned biological materials, the nucleotide sequence of the nucleic acid molecule described in B1) is selected from any one of the following: positions 7-408 of SEQ ID No. 23, positions 7-411 of SEQ ID No. 30, positions 7-411 of SEQ ID No. 34, positions 7-408 of SEQ ID No. 36, positions 7-411 of SEQ ID No. 28, or positions 7-408 of SEQ ID No. 26.

[0017] To address the aforementioned technical problems, in a third aspect, the present invention provides a method for preparing the IL-2 mutant, the method comprising culturing the recombinant microorganism described in B4), the animal cell line described in B5), or the host cell described in B6) to obtain the IL-2 mutant.

[0018] To address the aforementioned technical problems, in a fourth aspect, the present invention provides the use of the aforementioned IL-2 mutant or the aforementioned biological material in the preparation of drugs or formulations for treating diseases.

[0019] Furthermore, in the above applications, the diseases are selected from cancer and viral infections.

[0020] Furthermore, the cancer can be selected from non-small cell lung cancer, lung cancer, breast cancer, colorectal cancer, prostate cancer, stomach cancer, liver cancer, various hematological malignancies, pancreatic cancer, skin cancer, ovarian cancer, head and neck tumors, myeloma, melanoma, and other cancers.

[0021] Furthermore, the viral infections mentioned include viral infections such as HIV and hepatitis viruses.

[0022] To address the aforementioned technical problems, in a fifth aspect, the present invention provides the use of the IL-2 mutant or the biological material in the preparation of compositions for stimulating the immune system of an individual.

[0023] To address the aforementioned technical problems, in a fifth aspect, the present invention provides a drug or drug composition containing the IL-2 mutant.

[0024] The drug or drug composition may be a composition for treating cancer and / or viral infections.

[0025] In this invention, the drug or drug composition further includes a pharmaceutically acceptable carrier.

[0026] The carrier materials include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric-coated carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). These materials can be used to formulate various dosage forms, including but not limited to tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, suppositories, lyophilized powder injections, etc. These can be conventional formulations, sustained-release formulations, controlled-release formulations, and various microparticle delivery systems. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into tablets. Examples of carriers include diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and aluminum silicate; humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and polyvinylpyrrolidone; and disintegrants. Examples of carriers include dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid esters, sodium dodecyl sulfate, methylcellulose, and ethylcellulose; disintegration inhibitors include sucrose, tristearate, cocoa butter, and hydrogenated oil; absorption enhancers include quaternary ammonium salts and sodium dodecyl sulfate; and lubricants include talc, silica, corn starch, stearates, boric acid, liquid paraffin, and polyethylene glycol. Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets. Various carriers known in the art can be widely used to formulate unit-dose dosage forms into pills. Examples of carriers include diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, kaolin, and talc; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and disintegrants such as agar powder, dried starch, alginate, sodium dodecyl sulfonate, methylcellulose, and ethylcellulose. For preparing unit-dose dosage forms into suppositories, a wide variety of carriers known in the art can be used. Examples of carriers include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides. For preparing unit-dose dosage forms into injectable formulations such as solutions, emulsions, lyophilized powders for injection, and suspensions, all diluents commonly used in the art can be used, such as water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxyethylene isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. In addition, to prepare isotonic injection solutions, appropriate amounts of sodium chloride, glucose, or glycerol can be added to the injectable formulation. Furthermore, conventional solubilizers, buffers, pH adjusters, etc., can also be added.In addition, colorants, preservatives, flavorings, tasters, sweeteners or other materials may be added to pharmaceutical preparations if necessary.

[0027] The above dosage forms can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection, and intracavitary injection.

[0028] Because wild-type IL-2 has an affinity for IL-2Rα / β / γ that is about two orders of magnitude higher than that for IL-2Rβ / γ, wild-type IL-2 preferentially binds to repressive Treg cells, which is not conducive to the activation of Teff cells and NK cells. Therefore, the anti-tumor effect of wild-type IL-2 is limited.

[0029] This invention provides a mutant of IL-2 that reduces binding to the IL-2Rα subunit without affecting binding to the IL-2Rβ / γ subunit; this can better activate Teff cells and NK cells, rather than preferentially activating Treg cells with inhibitory functions as in wild-type IL-2. This mutant of IL-2 can exert a better anti-tumor effect in vivo. Attached Figure Description

[0030] Figure 1 The affinity of the three subunits of the IL-2 receptor for IL-2.

[0031] Figure 2 Example of a plasmid map for an IL-2 expression vector.

[0032] Figure 3 Example 1: Expression of IL-2 protein before and after bacterial induction.

[0033] Figure 4 Example 2 of IL-2 protein expression before and after bacterial induction.

[0034] Figure 5 Example 1: SDS-Electrophoresis analysis of purified IL-2 protein.

[0035] Figure 6 Example 2: SDS electrophoresis analysis of purified IL-2 protein.

[0036] Figure 7 Comparison of the proliferative stimuli of CTLL-2 by some IL-2 mutants.

[0037] Figure 8 This study compares the proliferative stimuli of Mo7e by some IL-2 mutants.

[0038] Figure 9 Comparison of the efficacy of some IL-2 mutants in tumor-bearing mouse models Detailed Implementation

[0039] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. CTLL-2 cells in the following examples were purchased from Suzhou Dingdian Biomedical Co., Ltd., catalog number TCM-C724. Mo7e cells in the following examples were purchased from Zhejiang Meisen Cell Technology Co., Ltd., catalog number CTCC-001-0368. pET-21a(+) was purchased from Baosai Biotechnology Co., Ltd., catalog number D38.

[0041] This application, through the study of the crystal structure of IL-2 binding to its three receptors, and using computer-aided design technology, designed a series of IL-2 mutants that may disrupt the binding of IL-2Rα without affecting the binding of IL-2Rβ / γ. Through screening at the molecular and cellular levels, a series of mutants that meet the requirements were obtained, as follows.

[0042] Example 1: Construction and expression of prokaryotic expression plasmids for different IL-2 mutants

[0043] Based on the known structural information of IL-2 and its receptors (PNAS, 2006: 103(8)2788-2793), different IL-2 mutants were designed using computer-aided design, and the corresponding DNA sequences were obtained through codon optimization (specific sequence codes and descriptions are shown in Table 1, and specific amino acid and nucleotide sequences are shown in Table 2). Taking the Aldesleukin coding gene as an example, the corresponding nucleotide sequence was commercially synthesized (an NdeI restriction site was added to the 5' end of the coding gene DNA molecule, and an EcoR1 restriction site was added to the 3' end). The DNA molecule with the nucleotide sequence SEQ ID No. 19 and the pET 21a(+) plasmid were double-digested with NdeI and EcoRI restriction endonucleases. The digested products were ligated with T4 ligase and transfected into JM109 competent cell line. After restriction digestion and sequencing verification, the pET 21a(+)-HL002-00 expression plasmid was obtained. The plasmid map is shown below. Figure 2 As shown.

[0044] Sequencing results showed that the pET 21a(+)-HL002-00 expression plasmid was obtained by replacing the fragment between the NdeI and EcoRI restriction endonuclease recognition sites of pET-21a(+) with the DNA molecule whose nucleotide sequence is shown in positions 7-408 of SEQ ID No. 19, while keeping the other nucleotide sequences of pET-21a(+) unchanged. The amino acid sequence of pET 21a(+)-HL002-00 expressed is the protein (HL002-00 or Aldesleukin) of SEQ No. 1.

[0045] Other plasmid construction methods refer to HL002-00, the only difference being the substitution of the target coding gene.

[0046] The pET 21a(+)-HL002-01 expression plasmid is a recombinant expression vector obtained by replacing the fragment between the NdeI and EcoRI restriction endonuclease recognition sites of pET-21a(+) with the DNA molecule whose nucleotide sequence is shown in positions 7-408 of SEQ ID No. 20, while keeping the other nucleotide sequences of pET-21a(+) unchanged. The amino acid sequence of pET 21a(+)-HL002-01 expressed is the protein of SEQ No. 2 (i.e., HL002-01).

[0047] The pET 21a(+)-HL002-02 expression plasmid is a recombinant expression vector obtained by replacing the fragment between the NdeI and EcoRI restriction endonuclease recognition sites of pET-21a(+) with the DNA molecule whose nucleotide sequence is shown in positions 7-408 of SEQ ID No. 21, while keeping the other nucleotide sequences of pET-21a(+) unchanged. The pET 21a(+)-HL002-02 expresses the protein (i.e., HL002-02) with the amino acid sequence of SEQ No. 3.

[0048] The pET 21a(+)-HL002-03 expression plasmid was obtained by replacing the fragment between the NdeI and EcoRI restriction endonuclease recognition sites of pET-21a(+) with the DNA molecule whose nucleotide sequence is shown in positions 7-408 of SEQ ID No. 22, while keeping the other nucleotide sequences of pET-21a(+) unchanged. The pET 21a(+)-HL002-03 expresses the protein (i.e., HL002-03) with the amino acid sequence of SEQ No. 4.

[0049] The pET 21a(+)-HL002-04 expression plasmid is a recombinant expression vector obtained by replacing the fragment between the NdeI and EcoRI restriction endonuclease recognition sites of pET-21a(+) with the DNA molecule whose nucleotide sequence is shown in positions 7-408 of SEQ ID No. 23, while keeping the other nucleotide sequences of pET-21a(+) unchanged. The pET 21a(+)-HL002-04 expresses the protein (i.e., HL002-04) with the amino acid sequence of SEQ No. 5.

[0050] The pET 21a(+)-HL002-05 expression plasmid is a recombinant expression vector obtained by replacing the fragment between the NdeI and EcoRI restriction endonuclease recognition sites of pET-21a(+) with the DNA molecule whose nucleotide sequence is shown in positions 7-408 of SEQ ID No. 24, while keeping the other nucleotide sequences of pET-21a(+) unchanged. The pET 21a(+)-HL002-05 expresses the protein (i.e., HL002-05) with the amino acid sequence of SEQ No. 6.

[0051] The pET 21a(+)-HL002-06 expression plasmid is a recombinant expression vector obtained by replacing the fragment between the NdeI and EcoRI restriction endonuclease recognition sites of pET-21a(+) with the DNA molecule whose nucleotide sequence is shown in positions 7-408 of SEQ ID No. 25, while keeping the other nucleotide sequences of pET-21a(+) unchanged. The pET 21a(+)-HL002-06 expresses the protein (i.e., HL002-06) with the amino acid sequence of SEQ No. 7.

[0052] The pET 21a(+)-HL002-07 expression plasmid is a recombinant expression vector obtained by replacing the fragment between the NdeI and EcoRI restriction endonuclease recognition sites of pET-21a(+) with the DNA molecule whose nucleotide sequence is shown in positions 7-408 of SEQ ID No. 26, while keeping the other nucleotide sequences of pET-21a(+) unchanged. The pET 21a(+)-HL002-07 expresses the protein (i.e., HL002-07) with the amino acid sequence of SEQ No. 8.

[0053] The pET 21a(+)-HL002-08 expression plasmid is a recombinant expression vector obtained by replacing the fragment between the NdeI and EcoRI restriction endonuclease recognition sites of pET-21a(+) with the DNA molecule whose nucleotide sequence is shown in positions 7-408 of SEQ ID No. 27, while keeping the other nucleotide sequences of pET-21a(+) unchanged. The pET 21a(+)-HL002-08 expresses the protein (i.e., HL002-08) with the amino acid sequence of SEQ No. 9.

[0054] The pET 21a(+)-HL002-09 expression plasmid is a recombinant expression vector obtained by replacing the fragment between the NdeI and EcoRI restriction endonuclease recognition sites of pET-21a(+) with the DNA molecule whose nucleotide sequence is shown in positions 7-411 of SEQ ID No. 28, while keeping the other nucleotide sequences of pET-21a(+) unchanged. The amino acid sequence of the pET 21a(+)-HL002-09 expressed protein is the protein in SEQ No. 10 (i.e., HL002-09).

[0055] The pET 21a(+)-HL002-10 expression plasmid is a recombinant expression vector obtained by replacing the fragment between the NdeI and EcoRI restriction endonuclease recognition sites of pET-21a(+) with the DNA molecule whose nucleotide sequence is shown in positions 7-411 of SEQ ID No. 29, while keeping the other nucleotide sequences of pET-21a(+) unchanged. The pET 21a(+)-HL002-10 expresses the protein (i.e., HL002-10) with the amino acid sequence of SEQ No. 11.

[0056] The pET 21a(+)-HL002-11 expression plasmid is a recombinant expression vector obtained by replacing the fragment between the NdeI and EcoRI restriction endonuclease recognition sites of pET-21a(+) with the DNA molecule whose nucleotide sequence is shown in positions 7-411 of SEQ ID No. 30, while keeping the other nucleotide sequences of pET-21a(+) unchanged. The pET 21a(+)-HL002-11 expresses the protein (i.e., HL002-11) with the amino acid sequence of SEQ No. 12.

[0057] The pET 21a(+)-HL002-12 expression plasmid is a recombinant expression vector obtained by replacing the fragment between the NdeI and EcoRI restriction endonuclease recognition sites of pET-21a(+) with the DNA molecule whose nucleotide sequence is shown in positions 7-411 of SEQ ID No. 31, while keeping the other nucleotide sequences of pET-21a(+) unchanged. The pET 21a(+)-HL002-12 expresses the protein (i.e., HL002-12) with the amino acid sequence of SEQ No. 13.

[0058] The pET 21a(+)-HL002-13 expression plasmid is a recombinant expression vector obtained by replacing the fragment between the NdeI and EcoRI restriction endonuclease recognition sites of pET-21a(+) with the DNA molecule whose nucleotide sequence is shown in positions 7-411 of SEQ ID No. 32, while keeping the other nucleotide sequences of pET-21a(+) unchanged. The pET 21a(+)-HL002-13 expresses the protein (i.e., HL002-13) with the amino acid sequence of SEQ No. 14.

[0059] The pET 21a(+)-HL002-14 expression plasmid is a recombinant expression vector obtained by replacing the fragment between the NdeI and EcoRI restriction endonuclease recognition sites of pET-21a(+) with the DNA molecule whose nucleotide sequence is shown in positions 7-411 of SEQ ID No. 33, while keeping the other nucleotide sequences of pET-21a(+) unchanged. The pET 21a(+)-HL002-14 expresses the protein (i.e., HL002-14) with the amino acid sequence of SEQ No. 15.

[0060] The pET 21a(+)-HL002-15 expression plasmid is a recombinant expression vector obtained by replacing the fragment between the NdeI and EcoRI restriction endonuclease recognition sites of pET-21a(+) with the DNA molecule whose nucleotide sequence is shown in positions 7-411 of SEQ ID No. 34, while keeping the other nucleotide sequences of pET-21a(+) unchanged. The pET 21a(+)-HL002-15 expresses the protein (i.e., HL002-15) with the amino acid sequence of SEQ No. 16.

[0061] The pET 21a(+)-HL002-16 expression plasmid is a recombinant expression vector obtained by replacing the fragment between the NdeI and EcoRI restriction endonuclease recognition sites of pET-21a(+) with the DNA molecule whose nucleotide sequence is shown in positions 7-411 of SEQ ID No. 35, while keeping the other nucleotide sequences of pET-21a(+) unchanged. The pET 21a(+)-HL002-16 expresses the protein (i.e., HL002-16) with the amino acid sequence of SEQ No. 17.

[0062] The pET 21a(+)-HL002-17 expression plasmid is a recombinant expression vector obtained by replacing the fragment between the NdeI and EcoRI restriction endonuclease recognition sites of pET-21a(+) with the DNA molecule whose nucleotide sequence is shown in positions 7-408 of SEQ ID No. 36, while keeping the other nucleotide sequences of pET-21a(+) unchanged. The pET 21a(+)-HL002-17 expresses the protein (i.e., HL002-17) with the amino acid sequence of SEQ No. 18.

[0063] Different mutant plasmids were transfected into BL21(DE3) competent bacteria. Single colonies were picked, cultured overnight, and on the third day, inoculated at a 1:100 ratio. The culture was continued until the OD value reached approximately 0.6-1.0, then IPTG was added to a final concentration of 1 mM for induction for 4 hours. SDS-PAGE was used to compare protein levels before and after induction to preliminarily determine the expression of the target protein. Figure 3 , Figure 4 The image shows the expression of some proteins. Figure 3 and Figure 4 The first band in the image is a protein molecular weight marker; "-" indicates no induction, while "+" indicates IPTG induction for 4 hours. Figure 3 and Figure 4 As can be seen in the induced bacterial protein, there is an additional protein (indicated by the arrow), with a molecular weight between 10KD and 15KD, which is consistent with the protein molecular weight of IL-2; this indicates that the target protein is expressed.

[0064] Table 1: Codes and corresponding sequence numbers of IL-2 and its mutants

[0065] code name mutation site amino acid sequence number Nucleotide sequence number HL002-00 none SEQ No.1 SEQ No. 19 HL002-01 K43V SEQ No.2 SEQ No.20 HL002-02 Y45A SEQ No.3 SEQ No.21 HL002-03 R38A / F42A SEQ No.4 SEQ No. 22 HL002-04 R38A / F42A / Y45A SEQ No. 5 SEQ No.23 HL002-05 F42L SEQ No.6 SEQ No.24 HL002-06 E61V SEQ No.7 SEQ No. 25 HL002-07 P65L SEQ No. 8 SEQ No. 26 HL002-08 P65V SEQ No. 9 SEQ No. 27 HL002-09 Add an A before the 41st digit SEQ No. 10 SEQ No. 28 HL002-10 Add G before 41 SEQ No.11 SEQ No. 29 HL002-11 Add a P before the 41st digit SEQ No. 12 SEQ No.30 HL002-12 Add N before the 41st bit SEQ No. 13 SEQ No.31 HL002-13 Add an A before the 65th digit SEQ No. 14 SEQ No.32 HL002-14 Add G before 65-bit SEQ No. 15 SEQ No.33 HL002-15 Add a P before the 65th digit SEQ No. 16 SEQ No.34 HL002-16 Add N before 65 bits SEQ No. 17 SEQ No.35 HL002-17 R38A / F42A / Y45A / P65V SEQ No. 18 SEQ No.36

[0066] Table 2: Specific sequences (N-terminus to C-terminus or 5' to 3')

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073] Example 2: Purification of IL-2 mutant protein

[0074] The purification steps for the IL-2 mutant protein are as follows:

[0075] S1) Collect bacterial cells by centrifugation, wash twice with PBS, and resuspend in buffer solution. The ratio of bacterial cells to buffer solution is 1:50, that is, 1g of bacterial cells are added to 50ml of buffer solution, and the precipitate is resuspended by pipetting. The bacteria are sonicated to disrupt the bacteria, centrifuged at 10000g for 20 minutes, and the precipitate is collected to obtain inclusion bodies.

[0076] S2) Resuspend the inclusion bodies in buffer solution, sonicate the inclusion bodies, centrifuge at 10000g for 20 minutes to collect the inclusion body precipitate, and repeat this step once.

[0077] The buffer solutions in S1) and S2) consist of 20 mM Tris-HCl, 1 mM EDTA, 0.1 M NaCl, 0.5% (by mass) Tritium-X-100, 2 M Urea, and the remainder is water, with a pH of 8.0.

[0078] S3) Dissolve the precipitate obtained in S2) in a solution (100mM Tris-HCl containing 6M GuHCl, 2mM EDTA, 10mM DTT, and the remainder being water, pH 8.0). Add 50ml of solution to 1g of precipitate, stir gently at room temperature for 1h, incubate in a 50℃ water bath for 30min, dilute the 6M GuHCl concentration to 4.8M with water for injection, centrifuge to collect the supernatant, dilute the GuHCl concentration to 3.5M again with water for injection, adjust the pH to 5.0 with acetic acid, incubate at 4℃ for 60min, and then centrifuge to collect the precipitate.

[0079] S4) Resuspend the precipitate collected in S3) in a solution containing 20 mM NaAc, 3.5 M GuHCl, and 5 mM DTT, pH 5.0. Wash and centrifuge to retain the precipitate. Dissolve the protein in a solution (0.1 M Tris-Cl containing 6 M GuHCl, pH 8.0) at a mass-to-volume ratio of 1:50. Transfer the solution to a dialysis bag (Viskase, Cat#MD34-3.5, molecular weight cutoff 3.5 KD). Dialyze with 0.1 M Tris-Cl containing 4.8 M GuHCl and 0.1 mM CuCl2 at pH 8.0. After changing the solution twice, transfer the dialysis bag to a solution containing 2 M GuHCl and 0.1 M Tris-Cl at pH 8.0. After 4 hours, place it in a 10 mM NaAc solution (pH 5.0). 4.5) Dialyze the protein in the solution and change the medium more than twice to obtain proteins HL002-00, HL002-01, HL002-02, HL002-03, HL002-04, HL002-05, HL002-06, HL002-07, HL002-08, HL002-09, HL002-10, HL002-11, HL002-12, HL002-13, HL002-14, HL002-15, HL002-16, and HL002-17. The purity and concentration of the proteins were then determined. Figure 5 and Figure 6 This is a partial result of the protein purity analysis using SDS-PAGE. The electrophoresis results show that the relevant proteins have reached electrophoretic purity.

[0080] Example 3: Affinity detection of IL-2 mutants with IL-2Rα or IL-2Rβ / γ

[0081] The affinity of IL-2 mutants for receptors IL-2Rα and IL-2Rβ / γ was measured using surface plasmon resonance (Biacore T-100) and compared with clinically used IL-2 (Aldesleukin).

[0082] Human IL2-Rβ&Rγ-Fc Protein (Cat.#ILG-H5254, AcroBiosystems) was coupled to a CM5 chip using an amino-coupling method. The affinity between the samples (IL-2 mutant and IL2Rβ&Rγ) was analyzed using a pH 7.4 buffer (solvent: water, solutes: 10 mM HBS-EP, 150 mM NaCl, 0.05% Tween 20 and 3 mM EDTA) as the working buffer. The samples (HL002-00, HL002-01, HL002-02, HL002-03, HL002-04, HL002-05, HL002-06, HL002-07, HL002-08, HL002-09, HL002-10, HL002-11, HL002-12, HL002-13, HL002-14, HL002-15, HL002-16, and HL002-17 obtained from Example 2) were diluted with buffer to prepare protein solutions of 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5625 nM, 0.78125 nM, and 0.390625 nM, respectively. Human IL2-Rβ&Rγ-Fc (Cat.#ILG-H5254) was diluted to a 10 μg / mL solution, with a coupling level of approximately 200 RU. The affinity constant (K0) was analyzed using kinetic models. D ).

[0083] Human IL2-Rα Protein, His Tag (Cat.#ILA-H52H9, AcroBiosystems) was coupled to a CM5 chip (Cat.#BR100530, Cytiva) using an amino-coupling method. The working buffer consisted of 10 mM HBS-EP + 150 mM NaCl + 0.05% Tween 20 + 3 mM EDTA, pH 7.4. The affinity between the samples (IL-2 mutant and Tag) was analyzed. The samples (IL-2 and mutant) were diluted with buffer to prepare protein solutions of 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5625 nM, 0.78125 nM, and 0.390625 nM, respectively. Human IL2-Ra Protein, His Tag (Cat.#ILA-H52H9) was diluted to 10 μg / mL, with a coupling level of approximately 200 RU. The affinity constant (KA) was analyzed using kinetic models. D ).

[0084] The results are shown in Table 3, where K is... DThe higher the value, the lower the affinity (binding ability). NB (No Binding) is a result of Biacore indicating that there is no binding.

[0085] Regulatory T cells (Tregs) express a trimeric IL-2 receptor composed of α, β, and γ chains. Naive CD8+ T cells (Teffs) and natural killer (NK) cells carry a dimeric receptor composed only of β and γ chains. Therefore, reducing binding to IL2-Rα can decrease Treg cell proliferation (Tregs have an inhibitory effect on tumors), while maintaining the ability to bind to IL2-Rβ / γ allows IL2 mutants to maintain their ability to inhibit the proliferation of Teff and NK cells. Mechanistically, a low affinity for IL2-Rα combined with maintaining the ability to bind to IL2-Rβ / γ is more conducive to the anti-tumor effect of IL2.

[0086] As shown in Table 3, although the binding affinity of each mutant to IL2-Rβ / γ differs from that of Aldesleukin, for example, the affinity of HL002-11, HL002-15, HL002-16, and HL002-17 is lower, all of them are within the same order of magnitude as the control. The affinity of each mutant for IL2-Rα differed from that of Aldesleukin. Among them, HL002-02 and HL002-05 showed virtually no change in affinity for IL2-Rα compared to Aldesleukin, failing to achieve the expected effect. The affinity of HL002-03, HL002-04, HL002-09, HL002-010, HL002-11, HL002-12, and HL002-17 differed from that of Aldesleukin by nearly three orders of magnitude or more. In particular, HL002-4, HL002-11, and HL002-17, based on Biacore data, showed virtually no binding to IL2-Rα.

[0087] Table 3: Affinity Constant (K) D Measurement results

[0088] code name mutation site KD(M)E-8(IL2Rα) KD(M)E-10(IL2Rβ / γ) HL002-00 Aldesleukin 2.50 2.497 HL002-01 K43V 34.4 3.189 HL002-02 Y45A 6.32 3.637 HL002-03 R38A / F42A 3200 3.144 HL002-04 R38A / F42A / Y45A NB 3.620 HL002-05 F42L 5.46 2.151 HL002-06 E61V 26.2 4.709 HL002-07 P65L 106 2.374 HL002-08 P65V 50.2 7.160 HL002-09 Add an A before the 41st digit 5700 4.182 HL002-10 Add G before 41 4200 3.247 HL002-11 Add a P before the 41st digit NB 15.329 HL002-12 Add N before the 41st bit 1500 4.073 HL002-13 Add an A before the 65th digit 210 8.027 HL002-14 Add G before 65-bit 128 6.140 HL002-15 Add a P before the 65th digit 350 20.47 HL002-16 Add N before 65 bits 30.8 11.58 HL002-17 R38A / F42A / Y45A / P65V NB 13.14

[0089] Example 4: Assay of the effect of IL-2 mutant on the proliferation of CTLL-2 and Mo7e cells

[0090] According to the literature Mol Cancer Ther (2012) 11(6):1279–1288, CTLL-2 cells have an IL2-Rα / β / γ complex, similar to Treg cells; while Mo7e cells only have IL2-β / γ receptors on their surface and lack IL2-Rα receptors, similar to Treg cells. CD8+ T cells and NK cells. If the IL-2 mutant has a weaker affinity for the IL2-Rα receptor, then its ability to stimulate the proliferation of CTLL2 (Treg cells) is weaker; while if the IL-2 mutant has a similar affinity for the IL2-β / γ receptor to Aldesleukin, then its ability to stimulate Mo7e (CD8+ T and NK cells) is similar. Based on the results in Table 3 of Example 3, we selected several mutants (HL002-04, HL002-07, HL002-09, HL002-11, HL002-15, HL002-17) that met the criteria of significantly reduced affinity for the IL2-Rα receptor and relatively small reduction in affinity for the IL-β / γ receptor for analysis of the proliferation activity of CTLL-2 and Mo7e cells.

[0091] 4.1 Assay of the effect of IL-2 mutant on CTLL-2 cell proliferation

[0092] Cytokine-dependent growth-dependent CTLL-2 cells (mouse T cells) were cultured in RPMI 1640 medium supplemented with 200 IU / ml IL-2 (Beijing Sihuan Pharmaceutical Co., Ltd., National Drug Approval Number S20040020) and 10% fetal bovine serum at 37°C and 5.0% CO2 until the logarithmic growth phase was reached. The cells were harvested by centrifugation at 1000 rpm for 5 minutes and washed three times with phosphate-buffered saline (Gibco, Cat#10010023). Cells were then resuspended in RPMI 1640 medium containing 10% fetal bovine serum and added to 96-well cell culture plates at a density of 5000 cells per well. After 4 hours of culture (cytokine starvation), serially diluted Aldesleukin or mutant samples (100 ng / ml, 33.33 ng / ml, 11.11 ng / ml, 3.70 ng / ml, 1.23 ng / ml, 0.46 ng / ml, 0.14 ng / ml, 0.046 ng / ml, 0.015 ng / ml, 0.0051 ng / ml, 0.0017 ng / ml, 0 ng / ml) were added to the wells of the 96-well tissue culture plates. After another 2 days of culture, 20 μl of CCK8 (Dojindo, Cat#CK04-500tests) was added to each well, and the plates were incubated at 37°C and 5.0% CO2 for 2 hours. Then, absorbance was read at 450nm and 630nm to check cell growth.

[0093] See results Figure 7 , Figure 7The ordinate represents the difference in absorbance at 450 nm and 630 nm, and the abscissa represents the commonly used logarithmic value of IL-2 mutant concentration (base of 10). The results showed that compared with HL002-00 (Aldesleukin), all selected IL-2 mutants reduced the ability to stimulate CTLL-2 cell proliferation. Among them, HL002-11 and HL002-17 showed the weakest ability to stimulate CTLL-2 cell proliferation, followed by HL002-04, HL002-09, HL002-07, and HL002-15.

[0094] 4.2. Determination of the effect of IL-2 mutant on Mo7e cell proliferation

[0095] The assay method for stimulating the proliferation of Mo7e cells (giant cell leukemia lineage) with IL-2 mutants is similar to that for CTLL-2, the only difference being that Mo7e cells are prepared using RPMI supplemented with 8 ng / ml GM-CSF (PeproTech, Cat#300-03-50UG) and 10% fetal bovine serum. Cells were cultured in RPMI 1640 medium to the logarithmic growth phase. Cells were collected and washed, then resuspended in GM-CSF-free RPMI 1640 medium and added to 96-well plates at a density of 20,000 cells per well. After 4 hours of culture, 20,000 cells were added to each well of a 96-well tissue culture plate with serially diluted Aldesleukin or mutant samples (concentrations of 500 ng / ml, 166.67 ng / ml, 55.56 ng / ml, 18.52 ng / ml, 6.17 ng / ml, 2.06 ng / ml, 0.69 ng / ml, 0.23 ng / ml, 0.076 ng / ml, and 0 ng / ml, respectively). The plates were then incubated at 37°C and 5.0% CO2 for 4 days. After 4 days, 20 μl of CCK8 (Dojindo, Cat#CK04-500tests) was added to each well, and the plates were incubated at 37°C and 5.0% CO2 for 2 hours. Cell growth was then assessed by taking readings at 450 nm and 630 nm.

[0096] See results Figure 8 , Figure 8 The ordinate represents the difference in absorbance at 450 nm and 630 nm, and the abscissa represents the common logarithm of the IL-2 mutant concentration (base 10). The results showed that HL002-09 was slightly more effective than HL002-00 in stimulating Mo7e cell proliferation, while the others were slightly weaker. HL002-04 and HL002-15 exhibited relatively weaker proliferative activity in stimulating Mo7e cells, but the activity of all molecules stimulating Mo7e was not significantly different.

[0097] from Figure 7 and Figure 8 As can be seen from the figures, the mutants mainly affect CTLL-2 proliferation, while having little effect on Mo7e cell proliferation. Combined with Table 3 and literature reports, this indicates that the mutants primarily affect the binding of IL-2 to the IL-2Rα / β / γ complex, while having little effect on the binding of IL-2 to IL-2Rβ / γ. The mutants with the weakest CTLL-2 proliferative activity but the strongest ability to stimulate Mo7e proliferation have better anti-tumor effects mechanistically. For example, HL002-11, HL002-17, and HL002-04 show better effects. While HL002-09, HL002-15, and HL002-07 are better than Aldesleukin, they are inferior to HL002-11, HL002-17, and HL002-04.

[0098] Example 5: Antitumor effect of IL-2 mutant in tumor-bearing mouse model

[0099] Lymphoblastic B lymphocyte TC-1 (#ATCC:CRL-2785) cell line (preserved by Crown Bioscience, Inc.) was cultured in DMEM medium containing 10% fetal bovine serum (FBS, Sigma) at 37°C and 5% CO2. C57BL / 6J mice (7-9 weeks old, female, weighing 17-23g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were subcutaneously inoculated with 1×10⁻⁶ cells / mL. 6 TC-1 cells were used to monitor tumor growth daily. Mice were randomly assigned to six groups (eight tumor-bearing mice per group) when the tumor volume reached 60-100 mm³. Day 1 of drug administration was designated as Day 1. The experimental groups received injections of recombinant purified IL-2 protein diluted in sterile 0.9% saline at a final dose of 0.5 mg / kg, administered twice weekly for four weeks. The control group received 0.9% saline injections. The administration volume was 5 μl / g body weight, and tumor volume was measured twice weekly. The tumor volume was calculated using the formula: Tumor volume (mm³) = 1 / 2 × (a × b²) (where a represents the major axis and b represents the minor axis).

[0100] The first group was the 0.9% saline control group; the second group was HL-002-11; the third group was HL-002-00 (wild-type IL-2); the fourth group was HL-002-02; the fifth group was HL-002-04; and the sixth group was HL-002-15.

[0101] See results Figure 9 .Depend on Figure 9It can be seen that under the same conditions, HL-002-02 and HL-002-00 (wild-type IL-2) have similar tumor suppression effects, and the mutant of HL-002-02 did not improve the tumor suppression effect of IL-2; HL-002-04, HL-002-11 and HL-002-15 have better tumor suppression effects than the HL-002-00 group, and HL-002-04 has the best tumor growth suppression effect.

[0102] Example 6: Stimulating effect of IL-2 mutant on the proliferation of CD8+ T cells and CD4+ T cells

[0103] CD8+ T cells and CD4+ T cells were isolated from human peripheral blood cells using CD8+ T cell (CD3+, CD8+) and CD4+ T cell (CD3+, CD8+) isolation kits, respectively. The isolated cells were cultured overnight in RPMI 1640 medium containing 10% fetal bovine serum at 37°C and 5.0% CO2. Cells were collected and stained with carboxyfluorescein diacetate succinimide ester (CFSE) and incubated at 37°C for 15 minutes. After centrifugation and washing, the cells were resuspended in RPMI 1640 medium containing 10% fetal bovine serum. 10,000 cells per well were added to 96-well cell culture plates containing pre-diluted IL-2 mutant solutions (HL-002-00, HL-002-02, HL-002-4, HL-002-11, HL-002-15, and HL-002-17 solutions). After 3 days of culture, the number of CFSC-positive cells was analyzed by flow cytometry to determine the effect of different IL-2 mutants on the growth of different cells.

[0104] In the CD4+ cell proliferation assay, the experiment was divided into 6 groups: IL-2 mutants HL-002-00, HL-002-02, HL-002-4, HL-002-11, HL-002-15, and HL-002-17, serially diluted with PBS at concentration gradients of 100 ng / ml, 33.33 ng / ml, 11.11 ng / ml, 3.70 ng / ml, 1.23 ng / ml, 0.46 ng / ml, 0.14 ng / ml, 0.046 ng / ml, 0.015 ng / ml, 0.0051 ng / ml, and 0 ng / ml, respectively. The experiment was performed in triplicate. A negative control (0 ng / ml) with 10 μl of PBS was added to each well, and replicates were performed for each concentration.

[0105] In the CD8+ cell proliferation assay, the experiment was divided into three groups based on the type of dimeric protein: IL-2 mutants HL-002-00, HL-002-02, HL-002-4, HL-002-11, HL-002-15, and HL-002-17, serially diluted with PBS at concentrations of 100 ng / ml, 33.33 ng / ml, 11.11 ng / ml, 3.70 ng / ml, 1.23 ng / ml, 0.46 ng / ml, 0.14 ng / ml, 0.046 ng / ml, 0.015 ng / ml, 0.0051 ng / ml, and 0 ng / ml. The experiment was performed in triplicate. A negative control (0 ng / ml) was also included, with 10 μl of PBS added to each well, and replicates were provided for the negative control.

[0106] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. An IL-2 mutant, characterized by: The IL-2 mutant is a protein obtained by mutating IL-2 with the amino acid sequence SEQ ID No.

1. The mutation is the addition of one amino acid between positions 40 and 41 of SEQ ID No.

1. The one amino acid is selected from A or P. The affinity of the IL-2 mutant for IL-2Rα is lower than that of IL-2 for IL-2Rα.

2. A nucleic acid molecule, characterized by: The nucleic acid molecule is the nucleic acid molecule encoding the IL-2 mutant of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that: The nucleotide sequence of the nucleic acid molecule is shown as positions 7-411 of SEQ ID No. 30 or positions 7-411 of SEQ ID No.

28.

4. An expression box, characterized in that: The expression cassette is an expression cassette containing the nucleic acid molecule described in claim 2 or 3.

5. A recombinant vector, characterized in that: The recombinant vector is a recombinant vector containing the nucleic acid molecule of claim 2 or 3 or the expression cassette of claim 4.

6. Recombinant microorganisms, characterized by: The recombinant microorganism is a recombinant microorganism containing the nucleic acid molecule of claim 2 or 3, the expression cassette of claim 4, or the recombinant vector of claim 5.

7. A recombinant animal cell line, characterized by: The recombinant animal cell line is a recombinant animal cell line containing the nucleic acid molecule of claim 2 or 3, the expression cassette of claim 4, or the recombinant vector of claim 5.

8. A host cell, characterized by: The host cell is the host cell that produces the IL-2 mutant of claim 1.

9. A method for preparing the IL-2 mutant of claim 1, the method comprising culturing the recombinant microorganism of claim 6, the recombinant animal cell line of claim 7, or the host cell of claim 8 to obtain the IL-2 mutant.

10. A drug, characterized in that: The drug contains the IL-2 mutant as described in claim 1.

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