IL-2 mutant as well as construction method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to an IL-2 mutant and a construction method and application thereof, and the IL-2 mutant comprises a mutation site for enhancing IL-2R alpha binding and a mutation site for weakening IL-2R beta / gamma binding. The mutation site for enhancing IL-2R alpha binding comprises 1, 2, 3, 4, 5, 6, 7, 8 or 9 mutation sites selected from the group consisting of T37R, V69Y, E68Q, N71W, Y107D, M104F, L66G, E60H and L63W; the mutation site for weakening IL-2R beta / gamma binding comprises 1, 2, 3, 4 or 5 mutation sites selected from the group consisting of H16D, N88L, Q126V, L18E and Q126L. According to the application, the IL-2 antagonist is designed, the binding affinity of the IL-2 antagonist to IL-2R alpha is enhanced, and meanwhile, the binding affinity to IL-2R beta and IL-2R gamma is reduced, so that the proliferation of Tregs is inhibited, the anti-tumor immune response is enhanced, and the systemic toxicity and immunogenicity are reduced. According to the application, a molecular dynamics simulation and calculation method is also utilized to accurately identify and regulate the binding affinity of IL-2 and different receptor subunits, an IL-2 antagonist with multi-target specific binding is designed, and accurate regulation of an IL-2 signal channel is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to an IL-2 mutant and a construction method and application thereof. BACKGROUND

[0002] Interleukin 2 (IL-2) is a four-helical bundle pleiotropic cytokine secreted by activated T cells, and its immune activity is regulated by the IL-2 receptor family CD25, CD122 and CD132. At a low level, IL-2 inhibits the immune system by binding to high-affinity receptors (activating regulatory T cells Tregs). At a high level, IL-2 activates effector T cells by binding to medium-affinity receptors, promotes their proliferation and differentiation, and enhances the immune response. IL-2 has high selectivity for the trimeric high-affinity receptor, mainly based on the unique contribution of CD25. The dimer after IL-2 and CD25 binding forms a stable tetramer structure with CD122-CD132, which transmits signals to the intracellular regulation of T cell proliferation and function.

[0003] High-dose recombinant IL-2 can be used to treat patients with metastatic renal cell carcinoma and metastatic melanoma. Although great progress has been made in immunotherapy, the immunosuppressive effect of CD4+CD25 regulatory T cells interferes with the anti-tumor immune response in cancer patients, and high-dose IL-2 can induce severe toxicity, including vascular leakage syndrome, pulmonary edema, hypotension and cardiotoxicity, which limits its clinical application. In order to solve the toxicity, IL-2 is mainly modified in the early stage by fusing with other proteins or with specific antibodies to target IL-2 to tumor sites. The development of selective IL-2 mutants mainly changes the interaction of IL-2 with interleukin 2 receptor (IL-2R). Such IL-2 mutants mainly include: PEGylation modification, fusion FC, IL-2 mutant protein design, bispecific antibody design, combined immune checkpoint antibody drug strategy, etc. Although there are many methods to reduce toxicity or enhance anti-tumor immune response by modifying IL-2, no engineered IL-2 has been approved for marketing through clinical trials. SUMMARY

[0004] The present application provides an IL-2 mutant and a construction method and application thereof. By designing an IL-2 antagonist, the binding affinity to IL-2Rα is enhanced, while the binding affinity to IL-2Rβ and IL-2Rγ is reduced, thereby inhibiting the proliferation of Tregs, enhancing the anti-tumor immune response, and reducing systemic toxicity and immunogenicity.

[0005] To solve the above technical problems, in a first aspect, the embodiments of the present application provide an IL-2 mutant, comprising: a mutation site for enhancing IL-2Rα binding and a mutation site for weakening IL-2Rβ / γ binding; wherein the mutation site for enhancing IL-2Rα binding comprises 1, 2, 3, 4, 5, 6, 7, 8 or 9 selected from the following: T37R, V69Y, E68Q, N71W, Y107D, M104F, L66G, E60H, L63W; and the mutation site for weakening IL-2Rβ / γ binding comprises 1, 2, 3, 4 or 5 selected from the following: H16D, N88L, Q126V, L18E, Q126L.

[0006] In a second aspect, the embodiments of the present application further provide a construction method of the IL-2 mutant as described in the above embodiments, which is used to achieve the construction of the IL-2 mutant as described in the above embodiments, and comprises the following steps: obtaining an initial crystal structure of an IL-2 and IL-2R complex, and performing molecular dynamics simulation on the initial crystal structure; based on the simulated crystal structure, calculating the binding free energy to screen the target mutant.

[0007] In some exemplary embodiments, obtaining the initial crystal structure of the IL-2 and IL-2R complex comprises: obtaining the initial crystal structure of the IL-2 and IL-2R complex from the Protein Data Bank, respectively obtaining an IL-2 complex system and an IL-2R complex system.

[0008] In some exemplary embodiments, after obtaining the initial crystal structure of the IL-2 and IL-2R complex, and before performing the molecular dynamics simulation on the initial crystal structure, the method further comprises: removing all water molecules and ions in the initial crystal structure.

[0009] In some exemplary embodiments, performing the molecular dynamics simulation on the initial crystal structure comprises the following steps: using ff14SB force field parameters in the AMBER force field, and generating the required topological parameters through the Tleap module of the AMBER18 software package; placing each complex system in a truncated octahedral periodic box, and using a TIP3P water model to ensure that the minimum buffer distance between any atom of the complex and the box surface is at least To avoid boundary effects, Na + and Cl - ions are added to the complex system to neutralize the total charge of the system; a thermostat is used to control the temperature, the collision frequency is set to 1.0 ps -1 , and a constant pressure device is used to control the system pressure, and the pressure is maintained at 1.0 atm; the long-range electrostatic interaction is calculated, and the cutoff distance of the non-bonding interaction is set to

[0010] In some example embodiments, based on the simulated crystal structure, the binding free energy is calculated, and the target mutant is screened, including: using an alanine scanning method to identify the binding interface The contributions of all the residue pairs to the binding free energy are added to obtain the total binding free energy.

[0011] In some example embodiments, after the target mutant is screened, the calculation of the protein-protein binding affinity is further included.

[0012] In some example embodiments, the calculation of the protein-protein binding affinity is achieved by thermodynamic integration calculation.

[0013] In some example embodiments, the formula of the thermodynamic integration calculation is:

[0014]

[0015] wherein, Wt represents a wild type state; Mut represents a mutant type state; ΔG Wt represents the free energy of the wild type state; ΔG Mut represents the free energy of the mutant type state; λ is a coupling parameter; V(λ) is a coupling potential function; when λ = 0, the complex system is completely in the wild type state, and when λ = 1, the complex system is completely in the mutant type state.

[0016] In a third aspect, the embodiments of the present application also provide an application of the IL-2 mutant in a drug for inhibiting the proliferation of regulatory T cells, a drug for enhancing an anti-tumor immune response, and a drug for treating cancer.

[0017] The technical solutions provided by the embodiments of the present application have at least the following advantages:

[0018] The embodiments of the present application provide an IL-2 mutant and a construction method and application thereof. The IL-2 mutant includes a mutant site for enhancing IL-2Rα binding and a mutant site for weakening IL-2Rβ / γ binding. The mutant site for enhancing IL-2Rα binding includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 selected from the following: T37R, V69Y, E68Q, N71W, Y107D, M104F, L66G, E60H, and L63W. The mutant site for weakening IL-2Rβ / γ binding includes 1, 2, 3, 4, or 5 selected from the following: H16D, N88L, Q126V, L18E, and Q126L.

[0019] In one aspect, the present application enhances the binding affinity of IL-2 antagonists to IL-2Ra while reducing the binding affinity to IL-2Rβ and IL-2Rγ, thereby inhibiting the proliferation of Tregs, enhancing anti-tumor immune responses, and reducing systemic toxicity and immunogenicity. In another aspect, the present application uses molecular dynamics simulation and computational methods to accurately identify and regulate the binding affinity of IL-2 to different receptor subunits, and designs IL-2 antagonists with multi-target specific binding to achieve precise regulation of the IL-2 signaling pathway. Moreover, the present application also accelerates the design and optimization process of IL-2 antagonists by combining computational methods and experimental verification, reduces research and development costs, shortens development cycles, and improves the efficiency of protein engineering. BRIEF DESCRIPTION OF DRAWINGS

[0020] One or more embodiments are illustrated by way of example in the figures that are part of this disclosure and which are illustrative, but not restrictive, of the present embodiments, unless otherwise specified, the figures in the drawings do not constitute a proportional limitation.

[0021] Figure 1 A flowchart of the construction method of the IL-2 mutant provided by an embodiment of the present application. DETAILED DESCRIPTION

[0022] As known from the background art, there is currently no engineered IL-2 that has been clinically approved for marketing.

[0023] Although there are currently some IL-2 antagonists, such as H9-RETR and H9T, there is still a need for mutation analysis of IL-2 to develop an IL-2 antagonist that is most suitable for the patient's physical condition, which can not only inhibit Treg cells to a certain extent, but also reduce IL-2 toxicity. The reason why it is said to inhibit Treg cells to a certain extent rather than eliminate Treg cells is that Treg cells play an important role in the human immune system, such as immune tolerance, anti-inflammatory effects, and maintaining immune balance. IL-2 antagonists are IL-2 mutant proteins, which are also IL-2 analogs, inhibit Treg cell proliferation while minimizing immunogenicity and systemic toxicity.

[0024] Existing IL-2 mutants, IL-2 exerts its immunomodulatory effects by binding to three subunits of the IL-2 receptor (IL-2R). At high physiological concentrations, IL-2 mainly activates effector T cells by binding to the IL-2Rβγ complex; while at low physiological concentrations, IL-2 activates Tregs by binding to the CD25-CD122-CD132 complex. This complex receptor binding mechanism makes it difficult to precisely regulate the therapeutic effect of IL-2. Existing IL-2 engineering methods are difficult to achieve precise regulation of the binding affinity of IL-2 to different receptor subunits, resulting in ineffective inhibition of Treg activation while enhancing effector T cell activation.

[0025] Moreover, the high cost and low efficiency of protein engineering also limit IL-2 engineering. Although there have been studies that have designed high-affinity IL-2 mutants through methods such as yeast display technology, these methods have high costs, long cycles, and low efficiency. For example, although K. Dane Wittrup et al. successfully developed IL-2 antagonists, these methods face many challenges in practical applications. Existing protein engineering methods lack efficient and low-cost computational methods when designing and optimizing IL-2 mutants, resulting in high research and development costs, long development cycles, and difficulty in quickly screening ideal mutants.

[0026] To solve the above technical problems, the embodiments of the present application provide an IL-2 mutant and its construction method and application. The IL-2 mutant comprises: a mutation site that enhances IL-2Rα binding and a mutation site that weakens IL-2Rβ / γ binding; wherein the mutation site that enhances IL-2Rα binding comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 selected from the following: T37R, V69Y, E68Q, N71W, Y107D, M104F, L66G, E60H, L63W; the mutation site that weakens IL-2Rβ / γ binding comprises 1, 2, 3, 4, or 5 selected from the following: H16D, N88L, Q126V, L18E, Q126L. The embodiments of the present application provide an IL-2 mutant and its construction method and application. By designing an IL-2 antagonist, the binding affinity to IL-2Rα is enhanced, while the binding affinity to IL-2Rβ and IL-2Rγ is reduced, thereby inhibiting the proliferation of Tregs, enhancing the anti-tumor immune response, and reducing systemic toxicity and immunogenicity.

[0027] The embodiments of the present application will be described in detail below with reference to the accompanying drawings. However, those of ordinary skill in the art can understand that in the embodiments of the present application, many technical details are presented in order to enable the reader to better understand the present application. However, the technical solutions claimed by the present application can be implemented even without these technical details and various changes and modifications based on the following embodiments.

[0028] The IL-2 mutant provided by the embodiments of the present application comprises a mutation site for enhancing IL-2Rα binding and a mutation site for weakening IL-2Rβ / γ binding; wherein the mutation site for enhancing IL-2Rα binding comprises 1, 2, 3, 4, 5, 6, 7, 8 or 9 selected from the following: T37R, V69Y, E68Q, N71W, Y107D, M104F, L66G, E60H, L63W; and the mutation site for weakening IL-2Rβ / γ binding comprises 1, 2, 3, 4 or 5 selected from the following: H16D, N88L, Q126V, L18E, Q126L.

[0029] The present application provides an IL-2 mutant (IL-2 competitive antagonist) having multi-target specific binding ability, which can be used to inhibit the proliferation of regulatory T cells (Treg), enhance anti-tumor immune response, and reduce systemic toxicity. The IL-2 mutant designed in the present application can enhance the binding ability with CD25 and reduce the binding ability with CD122 or CD132; the IL-2 mutant can competitively block the binding of wild-type IL-2 with the trimeric receptor, but does not activate the downstream signaling pathway; the IL-2 mutant can also inhibit the proliferation of Treg, enhance the activity of effector T cells, and be used for cancer immunotherapy.

[0030] The full sequence of the IL-2 mutant is the wild-type amino acid sequence, which is the amino acid sequence shown in SEQ ID NO. 1 (APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT).

[0031] Referring to Figure 1 The embodiments of the present application also provide a construction method of the IL-2 mutant as described in the above embodiments, which is used to realize the construction of the IL-2 mutant as described in the above embodiments, and comprises the following steps:

[0032] Step S1, obtaining an initial crystal structure of an IL-2 and IL-2R complex, and performing molecular dynamics simulation on the same.

[0033] Step S2, calculating the binding free energy based on the simulated crystal structure, and screening to obtain a target mutant.

[0034] In some embodiments, obtaining the initial crystal structure of the IL-2 and IL-2R complex in step S1 includes: obtaining the initial crystal structure of the IL-2 and IL-2R complex from a protein data bank, thereby obtaining the IL-2 complex system and the IL-2R complex system, respectively.

[0035] In some embodiments, after obtaining the initial crystal structure of the IL-2 and IL-2R complex and before performing molecular dynamics simulations on the initial crystal structure, the method further includes removing all water molecules and ions from the initial crystal structure.

[0036] In some embodiments, molecular dynamics simulations of the initial crystal structure include the following steps: using the ff14SB force field parameters in the AMBER force field and generating the required topological parameters via the Tleap module of the AMBER18 software package; placing each complex system in a truncated octahedral periodic box and using the TIP3P water model to ensure that the minimum buffer distance between any atom of the complex and the box surface is at least [value missing]. To avoid boundary effects, Na is added to the complex system. + and Cl - Ions are used to neutralize the total charge of the system; a thermostat is used to control the temperature, and the collision frequency is set to 1.0 ps. -1 A constant pressure regulator was used to control the system pressure, maintaining it at 1.0 atm; long-range electrostatic interactions were calculated, and the cutoff distance for non-bonded interactions was set to...

[0037] Specifically, this application first retrieved the initial crystal structure of the IL-2 and IL-2R complexes (PDB ID: 2ERJ) from the Protein Data Bank (PDB). Prior to simulation preparation, all water molecules and ions were removed from the crystal structures. The required topological parameters were generated using the ff14SB force field parameters in the AMBER force field and via the Tleap module of the AMBER18 software package. Each complex system was placed in a truncated octahedral periodic box, and the TIP3P water model was used to ensure that the minimum buffer distance between any atom of the complex and the box surface was at least [value missing]. To avoid boundary effects, Na+ and Cl- ions were added to neutralize the total charge of the system. A thermostat was used to control the temperature, and the collision frequency was set to 1.0 ps. -1 This application uses a constant pressure regulator to control the system pressure, maintaining it at 1.0 atm. For calculating long-range electrostatic interactions, this application sets the cutoff distance for non-bonded interactions to be... In the simulation process, the constraints are related to the bonds of hydrogen atoms. The system is first minimized using the steepest descent method, and then further optimized using the conjugate gradient method to solve potential atomic collision problems. During the minimization process, the coordinates of the protein atoms are constrained, with a constraint force constant of to allow the solvent molecules to adapt to the geometry of the complex. Then the constraints are removed to optimize the entire system, especially to solve the collision that the mutation may introduce. In the NVT ensemble, the system is subjected to a slow heating process for 300 ps to raise the temperature to 300 K, and all protein atoms are constrained. Subsequently, the simulation is balanced for 20 ns under the NPT ensemble, and no constraint is added to limit the molecular dynamics simulation, with a step time of 1 ps. 100 snapshots are regularly extracted from the final 10 ns trajectory for calculating the binding free energy. To ensure statistical reliability, five independent trajectories are generated for the MD simulation of each system.

[0038] In some embodiments, the binding free energy is calculated based on the simulated crystal structure in step S2, and the target mutant is screened, comprising: using the alanine scanning method to identify the contribution of the residue pairs in the binding interface to the binding free energy, and adding the contributions of all residue pairs to the binding free energy to obtain the total binding free energy.

[0039] Specifically, the process of calculating the binding free energy is as follows:

[0040] The binding free energy (ΔG bind ) is composed of the gas phase contribution (ΔG gas ) and the solvation (ΔG sol ) contribution, and the calculation formula is as follows:

[0041] ΔGbind=ΔG gas +ΔG sol

[0042] Where the gas phase contribution includes electrostatic interaction, van der Waals interaction and entropy change:

[0043] ΔG gas =ΔE vdw +ΔE ele -TΔS

[0044] Where ΔE vdw +ΔE ele represents the sum of van der Waals interaction energy and electrostatic interaction energy between the two proteins, and -TΔS represents the entropy change contribution in the binding process. The solvation free energy (ΔG sol ) is usually calculated by implicit solvation method, such as generalized Born (GB) method.

[0045] ΔGsol = AG GB + AG np

[0046] where AG GB represents the electrostatic contribution in the solvation free energy, while AG np represents the non-polar contribution, usually assessed by the Solvent Accessible Surface Area (SASA).

[0047] AG np = k - SASA + b

[0048] Here, the proportionality constant k is set to while the SASA (Solvent Accessible Surface Area) is calculated by the Linear Combination Overlap (LCPO) algorithm.

[0049] By mutating a specific residue (x) to alanine (a), the difference in free energy before and after the mutation represents the contribution of that single residue to the binding free energy, which can be written as:

[0050]

[0051] where, represents the binding free energy after mutating the residue to alanine, represents the binding free energy of the wild type. which can be written as:

[0052]

[0053] The gas-phase energy contribution can be written as:

[0054]

[0055] where, represents the gas-phase free energy component of the interaction between residue x and the receptor B, and similarly, represents the gas-phase free energy component between residue a and the receptor B. It can be obtained that:

[0056]

[0057] where, and -TAS x→a represent the contributions of van der Waals interactions, electrostatic interactions, and entropy to the binding free energy in the system. which can be written as:

[0058]

[0059] where, G sol (A(a)^B), G sol(A(x)^B), G sol (A(a)) and G sol (A(x)) represent the solvation energies of the mutant complex, wild-type complex, mutant monomer, and wild-type monomer, respectively. The above equation can be decomposed into different components:

[0060]

[0061] In the calculations, the dielectric constants of nonpolar, polar, and charged amino acids were set to 1, 3, and 1040, respectively. In this study, an alanine scanning method was used to identify the binding interface. The contribution of residues within the range to the binding free energy, assuming it exceeds... The interaction between the residues and the receptor is negligible. The contributions of all residues to the binding free energy are summed to obtain the total binding free energy:

[0062]

[0063] Entropy change (-TΔS) is typically calculated using conventional model analysis. However, due to high computational costs, most GB or PB calculations neglect entropy calculation. Here, this application uses the IE method to calculate the entropy contribution in the alanine scan method. The formula for the IE method is as follows:

[0064]

[0065] Where K is the ideal gas constant (set to 1.9872 cal·mol⁻¹). -1 K -1 T is the temperature (usually set to room temperature, 298.15 K for calculation), and β represents 1-KT. and The values ​​represent the interaction energy fluctuations between the alanine mutant and the wild type, respectively. To minimize computational statistical errors, five independent molecular dynamics (MD) runs were performed on each system, each simulation lasting 20 nanoseconds. The ASGB and IE values ​​were analyzed using the trajectory data from the last 10 nanoseconds of each simulation, excluding the highest and lowest values, which may represent outliers or statistical fluctuations. The average of the remaining three simulation runs was used as the final analytical data.

[0066] In some embodiments, after the target mutant is obtained through screening, the method further includes: calculating protein-protein binding affinity.

[0067] In some embodiments, protein-protein binding affinity is calculated through thermodynamic integral calculation.

[0068] In some embodiments, the formula for calculating the thermodynamic integral is:

[0069]

[0070] where Wt represents the wild-type state; Mut represents the mutant state; AG Wt represents the free energy of the wild-type state; AG Mut represents the free energy of the mutant state; λ is the coupling parameter; V(λ) is the coupling potential function; when λ = 0, the complex system is completely in the wild-type state, while λ = 1, the complex system is completely in the mutant state.

[0071] Thermodynamic integration (TI) is used to describe the free energy landscape of the system as a function of λ by introducing a coupling parameter λ and a coupling potential function V(λ), which allows a smooth transition from the WT state to the Mut state. When λ = 0, the system is completely in the WT state, while λ = 1 corresponds to the Mut state. The TI method calculates the free energy difference between the two states through a carefully designed thermodynamic cycle, connecting a series of TI calculations and physical observables. For each transition, a single-step simulation is performed, in which the WT state disappears while the Mut residue appears. Process 1 represents the transition from the unbound wild-type protein to the wild-type protein bound to the receptor protein. Process 2 represents the transition from the unbound mutant protein to the mutant protein bound to the receptor protein. Process 3 describes the transition from the unbound wild-type protein to the unbound mutant protein. Process 4 represents the transition from the wild-type protein bound to the receptor protein to the mutant protein bound to the receptor protein.

[0072] The initial structure of the IL-2 / CD25 complex comes from PDB. A series of mutant structures are constructed by introducing point mutations on selected residues using the Rosetta software suite. The Amber 18 software package and ff14SB force field are used to describe the protein. The tiMerge function in the parmed.py tool is used to generate new topology and coordinate files suitable for simulation, ensuring that the mutant residues are correctly connected to the protein backbone. Each complex system is placed in a TIP3P water model and uses a truncated octahedral periodic box to ensure that the minimum buffer distance between any atom in the complex and the surface of the box is to avoid boundary effects. To neutralize the total charge of the system, an appropriate amount of counterions is added to the simulation system. The temperature is controlled using a Langevin heat bath with a collision frequency of 2 ps -1 , the time step is 2 fs, and the pressure is controlled using a Berendsen pressure controller to maintain the pressure in the system at 1.0 atm. The PME algorithm is used to handle long-range electrostatic interactions in the system, and the settings in this application are This represents the cutoff distance for non-bonded interactions. The SHAKE algorithm was used to constrain hydrogen bonds during the MD simulations. All simulations used a 2fs time step. For simulations of mutant residues, a soft nucleus potential was employed, with parameters α and β set to 0.5 and 0.5, respectively. This application sets 22 λ windows for the IL-2 / IL-2-CD25 complex, with λ values ​​of 0.0, 0.025, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, and 1.0. Each λ window is simulated for 5 ns to calculate the free energy change.

[0073] In addition, this application also performs performance tests on the target mutants through protein expression and purification, and surface plasmon resonance (SPR) combined with affinity detection.

[0074] The IL-2 mutant was expressed in HEK293 cells using a transient transfection system. First, primers were designed to amplify the IL-2 mutant gene (Ala21-Thr153), which was then cloned into the pCDNA3.1(+) vector, ensuring the addition of a C-terminal His tag for purification. After construction, the plasmid was transfected into HEK293 cells using polyethyleneimine (PEI) at a DNA:PEI ratio of 1:3 (w / w). When cells reached 70%-80% confluence, the transfection complex was added to the HEK293 cells. Cells were cultured at 37°C and 5% CO2 for 6 hours, then replaced with fresh growth medium and cultured for another 48-72 hours to promote protein secretion. After expression, the culture supernatant was collected, and the His-tagged fusion protein was purified by Ni-NTA affinity chromatography. Subsequently, the protein was further purified using ion exchange chromatography (IEX) and gel filtration chromatography (SEC). Purity was analyzed and validated by SDS-PAGE and Western blot, and endotoxin levels were determined using the Limulus Amebocyte Lysate (LAL) kit to ensure they were below 1.0 EU / μg.

[0075] In the surface plasmon resonance (SPR) binding affinity assay, the binding interactions were characterized using the sensor chip on a Biacore 8K instrument. Lyophilized protein powder was reconstituted and aliquoted according to the Certificate of Analysis (COA) to concentrations greater than 10 μg per vial to avoid repeated freeze-thaw cycles. Buffer exchange was performed using a desalted column. The run buffer contained 10 mM N-(2-hydroxyethyl)piperazine-N′-2-sulfonic acid (HEPES), 150 mM sodium chloride (NaCl), 3 mM EDTA, and 0.005% Tween-20, with the pH adjusted to 7.4. Protein concentrations after buffer exchange were determined using a SPECTROstar Nano. Human IL-2RA protein was diluted to 10 μg / mL and injected into the Protein A capture chip at a flow rate of 10 μL / min until the experimental flow cell (Fc2) reached a capture level of approximately 200 response units (RU), while the reference flow cell (Fc1) remained unchanged. Analytes were prepared by serial two-fold dilutions or single-concentration dilutions. Solutions obtained in the run buffer were sequentially injected into the experimental and reference flow cells at a flow rate of 30 μL / min for binding and dissociation phases, both performed within the run buffer. After each concentration analysis, the sensor chip was regenerated using a glycine-HCl solution at pH 1.5 at a flow rate of 20 μL / min for 30 seconds to remove captured ligands and undissociated analytes. The same amount of ligand was recaptured into the experimental flow cell before the next concentration analysis. Data acquisition was performed using Biacore 8KControl Software 4.0.8.20368. All experimental procedures were performed within the run buffer, and SPR analytical reagents were filtered and degassed before use to ensure data accuracy.

[0076] In summary, this application provides an IL-2 mutant, comprising: mutation sites that enhance IL-2Rα binding (T37R, V69Y, E68Q, N71W, Y107D, M104F, L66G, E60H, L63W) and mutation sites that weaken IL-2Rβ / γ binding (H16D, N88L, Q126V, L18E, Q126L).

[0077] The IL-2 mutant designed in this application contains nine mutation sites: T37R, V69Y, E68Q, N71W, Y107D, M104F, L66G, E60H, and L63W; optionally, it further includes H16D, N88L, Q126V, L18E, and Q126L.

[0078] This application also provides the use of the IL-2 mutant as described in the above embodiments in drugs for inhibiting the proliferation of regulatory T cells, drugs for enhancing anti-tumor immune responses, and drugs for treating cancer.

[0079] The IL-2 mutant provided in this application has an IL-2 mutant protein that can be used to prepare drugs for inhibiting the proliferation of regulatory T cells (Tregs). The IL-2 mutant protein can also be used to prepare drugs for enhancing anti-tumor immune responses; it can also be used to prepare drugs for treating cancers (such as melanoma and renal cell carcinoma).

[0080] The IL-2 mutants designed in this application have been validated through computational simulation and in vitro experiments. The technical solution is feasible, the results are clear, and it has the potential for clinical translation. SPR (surface plasmon resonance) assays showed that the mutants with the best affinity for CD25 (N71W, E88Q, Q146V) had an 8.9-fold increase in affinity, while their affinity for CD132 was reduced by approximately 70.9-fold.

[0081] Table 1. Affinity test results of CD25 with multiple concentrations of IL-2, single concentrations of IL-2, and seven mutant proteins.

[0082] Receptor Analyte Analyte concentration Association rate constant (1 / Ms) Dissociation rate constant (1 / s) Equilibrium dissociation constant (M) CD25 IL-2 0.391 - 50 nM 4.85E+06 9.72E-02 2.00E-08 CD25 IL-2 50 nM 1.05E+07 1.84E-01 1.75E-08 CD25 IL-2 (T37R, V69Y) 50 nM 9.25E+07 1.35E+00 1.46E-08 CD25 IL-2 (N71W, E68Q) 50 nM 1.70E+07 3.40E-02 2.00E-09 CD25 IL-2 (T37R, V69Y, E88Q) 50 nM 1.77E+07 5.85E-02 3.31E-09 CD25 IL-2 (N71W, E68Q, V69Y) 50 nM 1.04E+07 9.32E-02 8.98E-09 CD25 IL-2 (V69A, Q74P, 1128T) 50 nM 1.99E+07 2.34E-02 1.18E-09 CD25 IL-2 (T37R, N71W, E68Q, V69Y) 50 nM 6.98E+06 4.54E-02 6.51E-09 CD25 IL-2 (E61Q) 50 nM 8.14E+06 6.06E-01 7.45E-08

[0083] In Table 1, the three mutants consisting of IL-2 mutants N71W, E68Q, and V69Y were used as positive controls, while E61Q was used as a negative control. All of these results are consistent with the literature reports, indicating that the experimental results of this application are accurate.

[0084] Table 2. Affinity test results of CD122-CD132 with multiple concentrations of IL-2, single concentrations of IL-2, and four mutant proteins.

[0085] Receptor Analyte Analyte concentration Association rate constant (1 / Ms) Dissociation rate constant (1 / s) Equilibrium dissociation constant (M) CD122-CD132 IL-2 1.953 - 125 nM 4.40E+05 3.97E-04 9.03E-10 CD122-CD132 IL-2 125 nM 5.38E+05 3.50E-04 6.50E-10 CD122-CD132 IL-2 (N71W, E68Q, Q126V) 125 nM 1.26E+06 5.80E-02 4.61E-08 CD122-CD132 IL-2 (N71W, E68Q, L18E) 125 nM 5.01E+05 2.70E-03 5.39E-09 CD122-CD132 IL-2 (Q126T) 125 nM 1.66E+06 9.49E-02 5.70E-08 CD122-CD132 IL-2 (N71W, E68Q, Q126L) 125 nM 1.06E+06 9.33E-02 8.79E-08

[0086] In Table 2, IL-2 (Q126T) serves as the control group, which is consistent with literature reports, demonstrating the accuracy of the experimental detection results in this application.

[0087] Based on the above technical solutions, this application provides an IL-2 mutant, its construction method, and its application. The IL-2 mutant includes: a mutation site that enhances IL-2Rα binding and a mutation site that weakens IL-2Rβ / γ binding; wherein, the mutation sites that enhance IL-2Rα binding include 1, 2, 3, 4, 5, 6, 7, 8, or 9 selected from the following: T37R, V69Y, E68Q, N71W, Y107D, M104F, L66G, E60H, L63W; and the mutation sites that weaken IL-2Rβ / γ binding include 1, 2, 3, 4, or 5 selected from the following: H16D, N88L, Q126V, L18E, Q126L.

[0088] On the one hand, this application designs IL-2 antagonists to enhance their binding affinity for IL-2Rα while reducing their binding affinity for IL-2Rβ and IL-2Rγ, thereby inhibiting Treg proliferation, enhancing anti-tumor immune responses, and reducing systemic toxicity and immunogenicity. On the other hand, this application utilizes molecular dynamics simulations and computational methods to precisely identify and regulate the binding affinity of IL-2 with different receptor subunits, designing IL-2 antagonists with multi-target specific binding, achieving precise regulation of the IL-2 signaling pathway. Furthermore, this application combines computational methods and experimental verification to accelerate the design and optimization process of IL-2 antagonists, reduce R&D costs, shorten the development cycle, and improve the efficiency of protein engineering.

[0089] Those skilled in the art will understand that the above-described embodiments are specific examples of implementing this application, and in practical applications, various changes in form and detail may be made without departing from the spirit and scope of this application. Any person skilled in the art can make their own modifications and alterations without departing from the spirit and scope of this application; therefore, the scope of protection of this application should be determined by the scope defined in the claims.

Claims

1. An IL-2 mutant, characterized in that, These include mutation sites that enhance IL-2Rα binding and mutation sites that weaken IL-2Rβ / γ binding; among which, Mutations that enhance IL-2Rα binding include 1, 2, 3, 4, 5, 6, 7, 8 or 9 of the following: T37R, V69Y, E68Q, N71W, Y107D, M104F, L66G, E60H, L63W; Mutations that weaken IL-2Rβ / γ binding include 1, 2, 3, 4, or 5 of the following: H16D, N88L, Q126V, L18E, and Q126L.

2. A method for constructing an IL-2 mutant, the method being used to construct the IL-2 mutant as described in claim 1, characterized in that, Includes the following steps: The initial crystal structure of the IL-2 and IL-2R complex was obtained, and molecular dynamics simulations were performed on it. Based on the simulated crystal structure, the binding free energy was calculated, and the target mutants were screened out.

3. The method for constructing the IL-2 mutant according to claim 2, characterized in that, Obtain the initial crystal structure of the IL-2 and IL-2R complex, including: The initial crystal structures of the IL-2 and IL-2R complexes were obtained from the protein data bank, resulting in the IL-2 complex system and the IL-2R complex system, respectively.

4. The method for constructing the IL-2 mutant according to claim 2, characterized in that, After obtaining the initial crystal structure of the IL-2 and IL-2R complex, and before performing molecular dynamics simulations on the initial crystal structure, the method further includes: Remove all water molecules and ions from the initial crystal structure.

5. The method for constructing the IL-2 mutant according to claim 3, characterized in that, Molecular dynamics simulations of the initial crystal structure include the following steps: Use the ff14SB force field parameters from the AMBER force field and generate the required topology parameters using the Tleap module of the AMBER18 software package; Each complex system was placed in a truncated octahedral periodic box, and the TIP3P water model was used to ensure that the minimum buffer distance between any atom of the complex and the box surface was at least [value missing]. To avoid boundary effects; Add Na to the complex system + and Cl - Ions, to neutralize the total charge of the system; A thermostat is used to control the temperature, and the collision frequency is set to 1.0 ps. -1 A constant pressure device is used to control the system pressure and maintain it at 1.0 atm. Calculate long-range electrostatic interactions, setting the cutoff distance for non-bonded interactions as...

6. The method for constructing the IL-2 mutant according to claim 2, characterized in that, Based on the simulated crystal structure, the binding free energy was calculated, and target mutants were screened, including: Alanine scanning method was used to identify the binding interface. The contribution of residues within the range to the binding free energy is summed to obtain the total binding free energy.

7. The method for constructing the IL-2 mutant according to claim 2, characterized in that, After the target mutant is obtained through screening, the process also includes calculating protein-protein binding affinity.

8. The method for constructing the IL-2 mutant according to claim 7, characterized in that, Thermodynamic integral calculations are used to calculate protein-protein binding affinity.

9. The method for constructing the IL-2 mutant according to claim 7, characterized in that, The formula for calculating the thermodynamic integral is as follows: Where Wt represents the wild-type state; Mut represents the mutant state; ΔG Wt Represents the free energy in the wild-type state; ΔG Mut V(λ) represents the free energy of the mutant state; λ is the coupling parameter; V(λ) is the coupling potential function; when λ = 0, the complex system is completely in the wild state, while when λ = 1, the complex system is completely in the mutant state.

10. The use of the IL-2 mutant as described in claim 1 in a drug for inhibiting the proliferation of regulatory T cells, a drug for enhancing anti-tumor immune responses, and a drug for treating cancer.

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