L103g site-modified anti-ifn-gamma autoantibody escape mutants and uses thereof

Abstract

The application relates to an L103G site modified anti-IFN-gamma autoantibody escape mutant and application thereof, and relates to the technical field of biological medicine and immunotherapy. The L103G site modified anti-IFN-gamma autoantibody escape mutant is a mutant of IFN-gamma, can effectively escape the attack of anti-IFN-gamma autoantibody compared with a wild type, and retains biological activity. For a part of patient groups which are not sensitive to other mutation sites, the mutant can effectively fill the treatment blind area, and can also be used as a probe to identify patients sensitive to the mutant in an AIGA patient group, so as to lay a foundation for guiding precise medication in the clinic.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and immunotherapy, and to an escape mutant of anti-IFN-γ autoantibody modified at the L103G site and its application. Background Technology

[0002] Anti-interferon-γ autoantibody (AIGA) syndrome, also known as adult-onset immunodeficiency syndrome (AOID), is an acquired immunodeficiency disease. Its treatment faces many challenges and seriously affects the prognosis of patients.

[0003] The core issue of this disease is the immunodeficiency caused by autoantibodies neutralizing IFN-γ. Therefore, even with targeted therapy, some patients' bone marrow plasma cells continue to secrete antibodies, leading to recurrent infections, making complete eradication difficult with conventional drugs. Furthermore, patients with this disease are prone to severe concurrent infections such as disseminated nontuberculous mycobacteria and fungi, requiring long-term combination anti-infective therapy. However, because the immunodeficiency is not effectively corrected, the disease is prone to recurrence, prolonged treatment courses, numerous adverse reactions, and poor patient compliance.

[0004] Therefore, there is a need to develop a novel IFN-γ molecule that can evade the neutralizing effect of anti-IFN-γ autoantibodies while maintaining its ability to bind to the IFN-γ receptor (IFNGR1), thereby maintaining normal immune activation. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides an L103G site-modified anti-IFN-γ autoantibody escape mutant. This mutant exhibits superior neutralization escape capability, preventing attack by anti-IFN-γ autoantibodies while retaining biological activity. For some patient groups insensitive to other mutation sites, this mutant can effectively fill treatment gaps and solve the technical problem in existing technologies where exogenous IFN-γ is easily neutralized and rendered ineffective by anti-IFN-γ autoantibodies in AIGA patients.

[0006] This invention provides an L103G site modified anti-IFN-γ autoantibody escape mutant, wherein the 103rd amino acid of the L103G site modified anti-IFN-γ autoantibody escape mutant is glycine.

[0007] The aforementioned anti-IFN-γ autoantibody escape mutants exhibit reduced binding affinity or neutralization to autoantibodies derived from the plasma of AIGA patients compared to wild-type IFN-γ, meaning they can escape the neutralizing effect of anti-IFN-γ autoantibodies while retaining their activity binding to IFN-γ receptor 1 (IFNGR1). This invention first used computer-aided structural simulation to predict the B-cell antigen epitopes and receptor-binding interface on the IFN-γ surface, screening for mutations in a series of amino acid residues located in highly immunogenic regions but not at key receptor-binding sites. Experimental validation of several potential sites confirmed that the mutant with glycine at amino acid position 103 exhibited significant neutralization escape ability in the plasma of AIGA patients, particularly in patient populations with specific antibody profiles, where its escape effect was exceptionally strong. Furthermore, this mutant retained its biological activity binding to IFN-γ, suggesting its important application value in personalized precision medicine. It should be clearly pointed out that, limited by the complexity of antigen-antibody interactions, simple computer prediction cannot confirm the dual function of the mutant (preservation of activity and escape neutralization). Empirical studies have found that even high-scoring predicted sites have a large number of negative results: for example, the D63 site (D63A), which is adjacent to the region of interest in this invention, failed to effectively escape neutralization; while the predicted site F15A, located at the N-terminal helix, tends to form insoluble inclusion bodies in prokaryotic expression systems, and despite multiple attempts at renaturation, a uniform soluble protein could not be obtained, thus preventing subsequent functional verification. The significant differences between the above predictions and actual results fully demonstrate that the L103G mutant finally screened in this invention has achieved unexpected technical effects. It successfully achieves soluble expression while also possessing high antibody escape activity, which can be easily predicted by those not skilled in the art, and therefore has outstanding substantive characteristics and significant progress.

[0008] In one embodiment, the L103G site-modified anti-IFN-γ autoantibody escape mutant has at least 95% sequence identity with the amino acid sequence of wild-type IFN-γ, except for amino acid at position 103, as shown in SEQ ID NO:1.

[0009] In one embodiment, the L103G site modified anti-IFN-γ autoantibody escape mutant has the same amino acid sequence as wild-type IFN-γ except for amino acid at position 103. The amino acid sequence of the L103G site modified anti-IFN-γ autoantibody escape mutant is shown in SEQ ID NO:2.

[0010] The present invention also provides an expression gene encoding an anti-IFN-γ autoantibody escape mutant modified at the L103G site.

[0011] The present invention also provides an expression vector comprising the aforementioned expression gene.

[0012] The present invention also provides a recombinant cell comprising the aforementioned expression vector.

[0013] The present invention also provides a method for preparing the L103G site modified anti-IFN-γ autoantibody escape mutant, comprising the following steps: inserting the expression gene into an initial vector to construct an expression vector, transforming the expression vector into a host cell, inducing expression, and obtaining the L103G site modified anti-IFN-γ autoantibody escape mutant.

[0014] The present invention also provides a drug comprising a pharmaceutically active ingredient, wherein the pharmaceutically active ingredient comprises the L103G site-modified anti-IFN-γ autoantibody escape mutant.

[0015] The working principle of the above-mentioned drugs is as follows: When the L103G site-modified anti-IFN-γ autoantibody escape mutant is used as the active ingredient of the immune reconstitution drug, it utilizes its dual characteristics of neutralization escape and preservation of biological activity. On the one hand, it can escape the recognition of high-titer anti-IFN-γ autoantibodies in the patient's body and avoid being neutralized and inactivated; on the other hand, it can still effectively bind to the IFN-γ receptor (IFNGR1), thereby maintaining the normal immune activation state in the patient's body, thus achieving immune reconstitution in AIGA patients.

[0016] In one embodiment, the drug further includes pharmaceutically acceptable excipients.

[0017] The present invention also provides a kit comprising a solid-phase carrier, a detection reagent, and the L103G site-modified anti-IFN-γ autoantibody escape mutant, wherein the L103G site-modified anti-IFN-γ autoantibody escape mutant is coated on the solid-phase carrier, the L103G site-modified anti-IFN-γ autoantibody escape mutant is used to capture anti-IFN-γ autoantibodies in a sample, and the detection reagent is used to detect the captured anti-IFN-γ autoantibodies.

[0018] The aforementioned kit detects the binding signal between anti-IFN-γ autoantibodies and L103G site-modified anti-IFN-γ autoantibody escape mutants in a sample, and compares this signal with the binding signal of wild-type IFN-γ. This allows the kit to determine the suitability (or clinical sensitivity) of the patient providing the sample for the L103G site-modified anti-IFN-γ autoantibody escape mutant. Therefore, this kit can be used to detect the presence of a missing binding epitope between anti-IFN-γ autoantibodies and L103G site-modified anti-IFN-γ autoantibody escape mutants, or to screen target patient populations where in vivo anti-IFN-γ autoantibodies cannot effectively neutralize the mutants.

[0019] The present invention also provides the application of the L103G site-modified anti-IFN-γ autoantibody escape mutant in pharmaceuticals or kits.

[0020] The above kit can be used to detect the specificity of binding of anti-IFN-γ autoantibodies to the IFN-γ autoantibody escape mutant and / or to screen sensitive patients. In this invention, the aforementioned sensitive patients refer to those whose anti-IFN-γ autoantibodies, upon testing, cannot effectively recognize or bind to the L103G site-modified anti-IFN-γ autoantibody escape mutant, or whose L103G site-modified anti-IFN-γ autoantibody escape mutant can effectively evade the neutralizing effect of their anti-IFN-γ autoantibodies. That is, sensitive patients whose anti-IFN-γ autoantibodies do not bind or bind at low levels to the L103G site-modified anti-IFN-γ autoantibody escape mutant.

[0021] Compared with the prior art, the present invention has the following beneficial effects: This invention discloses an L103G site-modified anti-IFN-γ autoantibody escape mutant and its application. Compared with wild-type IFN-γ, this mutant can effectively evade attack by anti-IFN-γ autoantibodies while retaining biological activity. Therefore, for some patient groups insensitive to other mutation sites, the L103G site-modified anti-IFN-γ autoantibody escape mutant has unique and superior escape characteristics, effectively filling treatment blind spots. Simultaneously, this mutant can be expressed in a prokaryotic system and renature into a soluble dimer, maintaining the basic structural characteristics of cytokines and overcoming the technical defects of similar designs (such as F15A) that easily form inclusion bodies and are difficult to renature. Furthermore, probes prepared based on this mutant can detect the recognition of this specific site by antibodies in AIGA patients, thereby screening out patient groups sensitive to this mutant and guiding precision clinical medication. Attached Figure Description

[0022] Figure 1 This is a comparative analysis diagram of IFN-γ antigen epitope prediction and receptor binding interface in this invention; Figure 2 This is a schematic diagram of the design of mutation sites in this invention, where the yellow arrows indicate the mutation sites of this invention. L103G is the key site with neutralization escape function verified by this invention; F15A and D63A are the predicted sites designed at the same time (as negative controls, showing that they failed to achieve the expected function). Figure 3 This is a flowchart of the expression and purification process of the L103G mutant protein in this invention, which covers the entire process from plasmid construction, E. coli expression, inclusion body washing, denaturation and refolding to finally obtaining soluble protein; Figure 4 The graph shows the effect of the mutant L103G on the induction of HLA-DR expression in THP-1 cells by flow cytometry. The first histogram from left to right shows the shift in HLA-DR fluorescence intensity on the cell surface after treatment with the mutant L103G; the second table shows the mean fluorescence intensity (MFI) of each group. Figure 5 The graph shows the neutralization and inhibition curves of CSG plasma from AIGA patients against wild-type IFN-γ, the mutant L103G of this invention, and the control mutant D63A. Detailed Implementation

[0023] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0025] Unless otherwise specified, all reagents, materials, and equipment used in this embodiment are commercially available; unless otherwise specified, all test methods are conventional test methods in this field.

[0026] Example 1 Prediction of IFN-γ neutralizing epitopes and design of L103G mutants For ease of description, the proteins and illustrations involved in this embodiment and subsequent embodiments are numbered and described as follows: Mutant of the present invention: Mutant 2 (L103G); Control mutant: Mutant 3 (F15A); Mutant 5 (D63A); Wild-type human IFN-γ: WT.

[0027] I. In order to obtain mutants that can both evade antibodies and retain activity, this embodiment employs a computer-aided design strategy: Structure acquisition: The crystal structure of human IFN-γ (PDB ID: 1FG9) was downloaded from the PDB database, and the A and B chains were extracted as a dimer model.

[0028] Epitope prediction: Linear and conformational epitopes on the protein surface were analyzed using the SEPPA 3.0 server (with parameters set to Secreted, Homo) and the IEDB B-cell epitope prediction tool. The prediction results showed that the molecular surface region containing leucine at position 103 (L103) had an extremely high antigenicity score.

[0029] Screening strategy: Compare the predicted antigenic epitopes with known IFN-γ / IFNGR1 binding interfaces (e.g., ... Figure 1 (As shown). Key residues that directly participate in receptor binding are removed, and residues located at the ends of surface loops or helices are screened out.

[0030] Site Identification: Based on the above predictions, this invention focuses on designing the L103 site located on the molecular surface for mutation verification. Simultaneously, to verify the accuracy of the prediction strategy and as a control, two other predicted sites, F15 and D63, were selected. Subsequent experiments confirmed that only the mutant L103G successfully achieved the design goal (high activity retention and escape neutralization). The specific mutation design is as follows (site distribution is shown in the figure). Figure 2 (as shown) (1) L103G (preferred in this invention): located on the molecular surface, it mutates hydrophobic leucine to glycine, which is predicted to significantly change the local hydrophobic environment and thus disrupt antibody binding.

[0031] (2) F15A (control 1): located at the N-terminal helix, is a potential epitope (later confirmed to have poor drug-like properties).

[0032] (3) D63A (control 2): ​​Located in the acidic residue region on the surface, the change in charge can theoretically affect the antibody affinity (later confirmed to be poor escape effect).

[0033] Sequence of mature wild-type human IFN-γ protein: QDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRG (SEQ IDNO: 1).

[0034] The sequence of L103G (preferred in this invention): QDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDGNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRG (SEQ IDNO: 2) The sequence of F15A (control 1): QDPYVKEAENLKKYANAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRG (SEQ IDNO: 3) The sequence of D63A (control 2): QDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDAQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRG (SEQ IDNO: 4) Example 2 Expression and purification of IFN-γ mutant L103G This embodiment describes the preparation process of the recombinant mutant protein. The flowchart is shown below. Figure 3 .

[0035] 1. Plasmid Construction: Using a plasmid containing the full-length coding sequence of human IFN-γ as a template, specific mutations were introduced using site-directed mutagenesis. Specific primer pairs containing the target mutated bases were designed and synthesized for the key mutation sites screened in this invention. The site-directed mutagenesis primer sequences used to construct the mutants of this invention are as follows (all directions are 5' -> 3'): (1) Constructing the L103G mutant (in this invention, leucine at position 103 is mutated to glycine): L103G-F (forward): TACAGCGTGACCGATGGCAACGTGCAGCGC (SEQ ID NO: 5) L103G-R (reverse): GCGCTGCACGTTGCCATCGGTCACGCTGTA (SEQ ID NO: 6).

[0036] (2) In order to make parallel comparisons, control mutants D63A and F15A were also constructed in this embodiment, and the construction method was the same as above.

[0037] Mutant D63A is formed by mutating aspartic acid at position 63 to alanine. D63A-F (forward): AAGAACTTCAAAGACGCGCAGTCTATCCAGAAA (SEQ ID NO:7) D63A-R (reverse): TTTCTGGATAGACTGCGCGTCTTTGAAGTTCTT (SEQ ID NO: 8); Mutant F15A is formed by mutating phenylalanine at position 15 to alanine. F15A-F (Forward): ACCTGAAGAAATACGCGAACGCAGGTCACTC (SEQ ID NO: 9) F15A-R (reverse): GAGTGACCTGCGTTTCGCGTATTTCTTCAGGT (SEQ ID NO: 10).

[0038] PCR amplification was performed using the primers described above. After DpnI digestion to remove the methylated parental template plasmid, the product was transformed into competent cells. After sequencing verification confirmed the sequence was correct, the mutant gene was cloned into a prokaryotic expression vector (such as pET-28a). This vector contains an N-terminal His tag and a protease cleavage site, thus constructing a recombinant expression plasmid.

[0039] 2. Induction of expression: Transform the correctly sequenced recombinant plasmid into E. coli BL21(DE3) competent cells. Incubate in LB medium at 37°C until the OD600 reaches 0.6-0.8, then add IPTG (final concentration 0.5 mM) to induce expression for 4-6 hours. Collect the bacterial cells by centrifugation.

[0040] 3. Inclusion Body Denaturation and Renaturation: IFN-γ exists primarily as inclusion bodies in *E. coli*. After sonication, the bacterial cells are centrifuged to collect the precipitate (inclusion bodies). The inclusion bodies are washed with washing buffer (containing Triton X-100) to remove impurities. Subsequently, the inclusion bodies are dissolved using denaturing buffer containing 8M urea. The denatured protein solution is slowly added to refolding buffer (containing arginine and redox pairs) via dialysis or dilution, allowing the protein to refold and form an active dimer.

[0041] 4. Purification: The refolded protein solution was passed through a Ni-NTA affinity chromatography column, captured using a His tag, and eluted using an imidazole gradient. The eluent was dialyzed to replace the buffer, yielding soluble IFN-γ mutant protein with a purity greater than 90%.

[0042] Results: It should be noted that under the preparation conditions of this embodiment, both the mutant L103G and the control mutant D63A of the present invention could be successfully refolded and yielded soluble proteins. However, for the control mutant F15A, under the same expression and refolding conditions, the protein mainly existed in the form of misfolded inclusion bodies and was extremely difficult to refold and dissolve, resulting in insufficient soluble samples that met the experimental requirements. Therefore, it was not included in subsequent biological activity assays.

[0043] This result shows that L103G can maintain the stability of the overall protein structure while changing surface amino acids, which is not possible at all predicted sites (such as F15A).

[0044] Example 3 Validation of the biological activity of mutant L103G To verify whether the L103G mutant retains its function of activating immune cells while achieving antibody escape, this embodiment tested its ability to induce HLA-DR expression in THP-1 cells.

[0045] 1. Experimental Methods Cell treatment: THP-1 mononuclear cells were seeded in culture plates and stimulated for 24 hours with wild-type IFN-γ, the mutant L103G of this invention, and the control mutant D63A at a final concentration of 20 ng / mL. An untreated group was set up as a negative control.

[0046] Note: A concentration of 20 ng / mL was confirmed as the linear reaction range in preliminary experiments. The control mutant F15A was not included in this functional experiment because it was shown in Example 2 to be unable to fold correctly and was insoluble.

[0047] Staining and detection: Cells were collected and surface stained with FITC-labeled anti-HLA-DR antibody, then incubated at 4°C in the dark for 30 minutes. The fluorescence intensity of the FITC channels was detected using flow cytometry.

[0048] 2. Results Analysis (see...) Figure 4 ) Histogram analysis: The peaks of wild-type IFN-γ and mutant groups were significantly shifted to the right (in the direction of high fluorescence intensity), indicating that all three could effectively upregulate the expression of HLA-DR on the cell surface.

[0049] MFI Statistical Analysis: (1) Wild-type human IFN-γ (WT): MFI value is 2867.

[0050] (2) Mutant 2 of the present invention (L103G): MFI value is 2856.

[0051] (3) Control mutant 5 (D63A): MFI value is 2582.

[0052] Data comparison: Calculations show that the activity of mutant L103G is approximately 100% of that of the wild type. This indicates that although mutant L103G has undergone mutations at key sites to evade antibodies, it still retains most of its receptor binding and immune activation capabilities.

[0053] Example 4 ELISA competitive inhibition detection method based on plasma from AIGA patients This embodiment establishes a modified competitive ELISA method to evaluate the neutralization escape ability of mutant proteins in the plasma environment of patients with anti-IFN-γ autoantibody syndrome (AIGA). This method combines an AIGA plasma pre-incubation step with a standardized double-antibody sandwich ELISA assay procedure.

[0054] 1. Reagents and Materials Test kit: BD OptEIA™ Human IFN-γ ELISA Set (Cat. No. 555142).

[0055] The accompanying reagent set, BD OptEIA™ Reagent Set B (Cat. No. 550534), includes coating buffer, Assay Diluent, wash buffer concentrate, TMB substrate, and stop solution.

[0056] Sample: Plasma from a clinically diagnosed AIGA patient (Patient ID: CSG).

[0057] Note: This patient sample was selected because previous screening showed that it represents a typical group sensitive to a specific epitope.

[0058] Proteins to be tested: wild-type human IFN-γ protein, the mutant L103G of this invention and the control mutant D63A.

[0059] 2. Solution preparation Coating Buffer: 0.1 M sodium carbonate buffer, pH 9.5. Use the components provided in Reagent Set B or prepare your own (1.26 g NaHCO3, 0.64 g Na2CO3, bring to 1 L).

[0060] Diluent / Blocking Buffer (Assay Diluent): PBS (pH 7.0) containing 10% fetal bovine serum (FBS).

[0061] Wash Buffer: PBS (pH 7.0) containing 0.05% Tween-20. Dilute the 20X concentrate with deionized water to a 1X working solution.

[0062] Working Detector: Prepare within 15 minutes of use. Add the biotinylated detection antibody and enzyme conjugate (SAv-HRP) to the Assay Diluent in the proportions specified in the instructions and mix well.

[0063] 3. Experimental Procedure Step A: Pretreatment of standards and plasma samples.

[0064] Preparation of IFN-γ working solutions: After reconstituted the lyophilized standard, dilute it with Assay Diluent to prepare working solutions of wild type, mutant L103G and control mutant D63A with a concentration of 2000 pg / mL.

[0065] Plasma serial dilution: Assay Diluent was used to serially dilute patient CSG plasma. The initial dilution was set to 1:50, followed by successive serial dilutions, selecting an appropriate gradient range (e.g., 1:3200 to 1:409600) for testing.

[0066] Step B: Neutralization reaction (pre-incubation).

[0067] Collect 50 μL of CSG plasma from patients at different dilution levels.

[0068] Add 50 μL of IFN-γ protein working solution (WT, L103G, or control D63A) at a concentration of 2000 pg / mL. Set up 3 replicates for each concentration point, and repeat the experiment independently at least 3 times. Data are expressed as Mean ± SD.

[0069] After shaking and mixing, incubate at room temperature (20-25℃) for 1 hour.

[0070] Explanation of the principle: In this step, if the plasma contains an auto-neutralizing antibody that can recognize the IFN-γ, the two will combine to form a complex; if the mutant (such as L103G) achieves epitope escape, it remains in a free state.

[0071] Step C: Double antibody sandwich ELISA detection.

[0072] Coating: Dilute the capture antibody with coating buffer at the recommended ratio and add it to a 96-well microplate (100 μL / well). Seal the plate and incubate overnight at 4°C.

[0073] Wash 1: Aspirate the liquid from the wells and add ≥ 300 μL of washing buffer to each well for 3 washes. After the last wash, pat dry on absorbent paper.

[0074] Block: Add ≥ 200 μL of Assay Diluent to each well and incubate at room temperature for 1 hour.

[0075] Washing 2: Absorb the liquid and wash 3 times.

[0076] Incubation: Add the pre-incubated "plasma-protein mixture" from step B to the wells (100 μL / well). Seal the plate and incubate at room temperature for 2 hours.

[0077] Washing 3: Absorb the liquid and wash 5 times.

[0078] Detecting antibody binding: Add 100 μL of the prepared working detector to each well. Seal the plate and incubate at room temperature for 1 hour.

[0079] Washing 4 (Critical Step): Absorb the liquid and wash 7 times. Note: In this step, soak for 30 seconds to 1 minute after each addition of washing solution to thoroughly remove non-specific binders.

[0080] Color development: Add 100 μL of TMB substrate mixture to each well. Incubate at room temperature in the dark for 30 minutes.

[0081] Stop and reading: Add 50 μL of stop solution to each well; the solution will change from blue to yellow. Measure the absorbance at 450 nm using a microplate reader within 30 minutes.

[0082] 4. Data processing: The inhibition rate at different plasma concentrations was calculated using the following formula:

[0083] in: OD 实验组 : Well readings after pre-incubation with added plasma; OD 最大信号 Readings of positive control wells without plasma (with only Assay Diluent + IFN-γ added), (0% inhibition); OD 背景 : Blank control well reading (no plasma, IFN-γ, only Assay Diluent added).

[0084] Example 5 Evaluation of the neutralization escape effect of mutant L103G I. Using the method of Example 4, the neutralization escape performance of the mutant of the present invention in CSG plasma of AIGA patients was evaluated.

[0085] Patient's CSG test results (see appendix) Figure 5 The results showed a significant pattern of difference: Wild-type human IFN-γ: The inhibition rate is extremely high, indicating that the patient's autoantibodies can efficiently recognize and neutralize wild-type IFN-γ.

[0086] The L103G mutant of this invention exhibits a neutralization curve significantly lower than that of the wild-type. Particularly under high plasma concentrations, L103G is neutralized to a significantly lower degree than the wild-type. This demonstrates that for this patient, leucine at position 103 is a key epitope for autoantibody recognition, and mutating it to glycine (L103G) successfully disrupts the antigen-antibody binding interface.

[0087] The control mutant D63A showed that its inhibition curve was almost identical to that of the wild type, indicating that the mutation at this site failed to achieve effective escape.

[0088] Conclusion: Experimental data show that the antibody profile in AIGA patients is specific. For the CSG patient population, the mutant L103G of this invention exhibits the best escape effect while retaining 100% of its biological activity, demonstrating extremely high drug development potential.

[0089] II. Summary.

[0090] In summary, the mutant L103G provided by this invention effectively solves the technical problem in existing technologies where exogenous IFN-γ is easily neutralized by autoantibodies from AIGA patients. This study, through systematic screening and validation, confirms that the mutant L103G has significant and unexpected technical advantages compared to other predicted sites: Excellent drug-like properties and structural stability (compared to F15A): The predicted mutant F15A designed at the same time forms insoluble inclusion bodies and cannot meet the drug-like properties requirements. In contrast, the mutant L103G of this invention exhibits good solubility and structural stability in prokaryotic expression systems.

[0091] Excellent preservation of biological activity: Experiments have shown that the biological activity of the L103G mutant (MFI 2526) is almost identical to that of the wild type (MFI 2521), proving that the mutation does not affect the receptor binding domain and is superior to some mutants with impaired activity.

[0092] Specific neutralization escape capability (compared to D63A): Although the control mutant D63A retained activity, it failed to escape neutralization. In stark contrast, the mutant L103G successfully achieved significant escape from autoantibodies against AIGA patients.

[0093] Therefore, the mutant L103G of this invention simultaneously meets the three key technical indicators of "structural stability", "100% biological activity retention" and "antibody neutralization escape", providing a novel candidate drug for personalized precision treatment of AIGA.

[0094] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0095] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. An L103G site-modified anti-IFN-γ autoantibody escape mutant, characterized in that, The 103rd amino acid of the L103G site-modified anti-IFN-γ autoantibody escape mutant is glycine.

2. The L103G site-modified anti-IFN-γ autoantibody escape mutant according to claim 1, characterized in that, The L103G site-modified anti-IFN-γ autoantibody escape mutant is an IFN-γ mutant. The remaining amino acid sequence of the L103G site-modified anti-IFN-γ autoantibody escape mutant, except for the 103rd amino acid, has at least 95% sequence identity with the amino acid sequence of wild-type IFN-γ, and the amino acid sequence of wild-type IFN-γ is shown in SEQ ID NO:

1.

3. The L103G site-modified anti-IFN-γ autoantibody escape mutant according to claim 2, characterized in that, The amino acid sequence of the L103G site-modified anti-IFN-γ autoantibody escape mutant, except for the amino acid at position 103, is identical to that of wild-type IFN-γ. The amino acid sequence of the L103G site-modified anti-IFN-γ autoantibody escape mutant is shown in SEQ ID NO:

2.

4. An expression gene encoding an anti-IFN-γ autoantibody escape mutant modified at the L103G site as described in any one of claims 1-3.

5. An expression carrier, characterized in that, Includes the expressed gene as described in claim 4.

6. A recombinant cell, characterized in that, Includes the expression vector as described in claim 5.

7. A method for preparing the L103G site-modified anti-IFN-γ autoantibody escape mutant according to any one of claims 1-3, characterized in that, The process includes the following steps: inserting the expression gene described in claim 4 into an initial vector to construct an expression vector; transforming the expression vector into a host cell; inducing expression; and obtaining the L103G site-modified anti-IFN-γ autoantibody escape mutant.

8. A drug, characterized in that, It includes a pharmaceutically active ingredient, said pharmaceutically active ingredient including an anti-IFN-γ autoantibody escape mutant modified at the L103G site according to any one of claims 1-3.

9. A reagent kit, characterized in that, The invention comprises a solid-phase carrier, a detection reagent, and an anti-IFN-γ autoantibody escape mutant modified with the L103G site according to any one of claims 1-3, wherein the L103G site modified anti-IFN-γ autoantibody escape mutant is coated on the solid-phase carrier, the L103G site modified anti-IFN-γ autoantibody escape mutant is used to capture anti-IFN-γ autoantibodies in a sample, and the detection reagent is used to detect the captured anti-IFN-γ autoantibodies.

10. The use of the L103G site modified anti-IFN-γ autoantibody escape mutant according to any one of claims 1-3 in a drug or kit.

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