S84g site-modified anti-ifn-gamma autoantibody escape mutants and uses thereof

Abstract

The application relates to an S84G site modified anti-IFN-gamma autoantibody escape mutant and application thereof, and relates to the technical field of biological medicine and immunotherapy. The mutant is an IFN-gamma mutant, the 84th amino acid of which is glycine; the mutant has a significant neutralization escape function, can effectively avoid the attack of autoantibodies, and retains biological activity. For a part of patient groups who are not sensitive to other mutation sites, the S84G site modified anti-IFN-gamma autoantibody escape mutant has unique and superior escape characteristics, and can effectively fill the treatment blind area.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and immunotherapy, and to an S84G site modified anti-IFN-γ autoantibody escape mutant 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. This disease is prevalent in Southeast Asian populations. Patients produce high titers of anti-IFN-γ autoantibodies, which specifically bind to and neutralize IFN-γ in the body, blocking the IFN-γ / JAK / STAT1 signaling pathway. This makes patients extremely susceptible to intracellular pathogens (such as *Cladosporium marneffei*, nontuberculous mycobacteria, and Salmonella), clinically manifesting as recurrent, refractory disseminated infections.

[0003] Currently, the clinical treatment of AIGA faces significant challenges. Traditional anti-infective therapies often fail to eradicate the pathogen, while treatments targeting autoantibodies (such as using rituximab to clear B cells or plasma exchange) suffer from high relapse rates and significant side effects. For immune reconstitution, directly supplementing with exogenous wild-type IFN-γ (such as recombinant human IFN-γ-1b) is usually ineffective because the exogenous protein is rapidly recognized and neutralized by existing high-titer autoantibodies after entering the patient's body, thus failing to exert its biological effects.

[0004] Therefore, there is an urgent need to develop a novel IFN-γ molecule that possesses two core characteristics: first, it must be able to alter key epitopes recognized by its own antibodies, thereby escaping neutralization; second, it must retain the ability to bind to the IFN-γ receptor (IFNGR1) to maintain normal immune activation. This invention is based on this requirement and is achieved by modifying specific regions of the IFN-γ molecule. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides an S84G site-modified anti-IFN-γ autoantibody escape mutant. This mutant is an IFN-γ mutant with significant neutralizing escape capabilities, effectively evading the attack of anti-IFN-γ autoantibodies while retaining biological activity. For some patient groups insensitive to other mutation sites, the S84G site-modified anti-IFN-γ autoantibody escape mutant possesses unique and superior escape characteristics, effectively filling treatment blind spots and solving 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 S84G site modified anti-IFN-γ autoantibody escape mutant, wherein the 84th amino acid of the anti-IFN-γ autoantibody escape mutant is glycine.

[0007] The aforementioned anti-IFN-γ autoantibody escape mutants retain the activity of binding to IFN-γ receptor 1 (IFNGR1), and compared with wild-type IFN-γ, the binding affinity or degree of neutralization of autoantibodies derived from the plasma of AIGA patients is reduced.

[0008] This invention first uses computer-aided structural simulation to predict the B-cell antigen epitopes and receptor-binding interface on the surface of IFN-γ, and screens a series of amino acid residues located in highly immunogenic regions but not at key receptor-binding sites for mutation. Then, multiple potential sites are experimentally verified, confirming that a mutant with glycine at amino acid position 84 exhibits significant neutralization escape ability in the plasma of AIGA patients, especially in patient populations with specific antibody profiles, where its escape effect is particularly excellent. Furthermore, this mutant retains its biological activity binding to the IFN-γ receptor, suggesting that this mutant has important application value in personalized precision medicine.

[0009] It should be noted that, due to 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 S84G 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.

[0010] In one embodiment, the anti-IFN-γ autoantibody escape mutant is an IFN-γ mutant, and the remaining amino acid sequence of the anti-IFN-γ autoantibody escape mutant, except for amino acid position 84, has at least 95% sequence identity with the amino acid sequence of wild-type IFN-γ, the amino acid sequence of which is shown in SEQ ID NO:1.

[0011] In one embodiment, the amino acid sequence of the anti-IFN-γ autoantibody escape mutant, except for the 84th amino acid, is identical to that of wild-type IFN-γ, and the amino acid sequence of the anti-IFN-γ autoantibody escape mutant is shown in SEQ ID NO:2.

[0012] The present invention also provides an expression gene encoding the escape mutant of the anti-IFN-γ autoantibody.

[0013] The present invention also provides an expression vector comprising the aforementioned expression gene.

[0014] The present invention also provides a recombinant cell comprising the aforementioned expression vector.

[0015] In one embodiment, the method for preparing the recombinant cells includes: inserting the expression gene into an initial vector to construct an expression vector, and transforming the expression vector into a host cell to obtain recombinant cells.

[0016] In one embodiment, the host cell is a prokaryotic cell or a eukaryotic cell.

[0017] In one embodiment, the prokaryotic cells include Escherichia coli.

[0018] The present invention also provides a method for preparing the 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 anti-IFN-γ autoantibody escape mutant.

[0019] The present invention also provides a drug comprising a pharmaceutically active ingredient, said pharmaceutically active ingredient comprising the aforementioned anti-IFN-γ autoantibody escape mutant.

[0020] Understandably, the aforementioned anti-IFN-γ autoantibody escape mutants can serve as active ingredients in immune reconstitution drugs. By utilizing their dual characteristics of neutralization escape and preservation of biological activity, they can, on the one hand, evade the recognition of high-titer anti-IFN-γ autoantibodies in the patient's body, avoiding neutralization and inactivation; on the other hand, they can still effectively bind to the IFN-γ receptor (IFNGR1), maintaining the patient's normal immune activation state, thereby achieving immune reconstitution in AIGA patients.

[0021] In one embodiment, the drug further includes pharmaceutically acceptable excipients.

[0022] The present invention also provides a kit comprising a solid-phase carrier, a detection reagent, and the aforementioned anti-IFN-γ autoantibody escape mutant, wherein the anti-IFN-γ autoantibody escape mutant is coated on the solid-phase carrier, the 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.

[0023] The above kit determines the suitability (or clinical sensitivity) of the patient providing the sample for the anti-IFN-γ autoantibody escape mutant by detecting the binding signal between the anti-IFN-γ autoantibody and the escape mutant, and comparing it with the binding signal of wild-type IFN-γ. The kit can be used to detect the absence of a binding epitope between the anti-IFN-γ autoantibody and the mutant, or to screen target patient groups for whom the anti-IFN-γ autoantibody cannot effectively neutralize the mutant.

[0024] The present invention also provides the application of the anti-IFN-γ autoantibody escape mutant in a kit for detecting the specificity of binding of the anti-IFN-γ autoantibody to the anti-IFN-γ autoantibody escape mutant and / or screening sensitive patients.

[0025] Understandably, the aforementioned sensitive patients refer to those whose anti-IFN-γ autoantibodies, upon testing, cannot effectively recognize or bind to the anti-IFN-γ autoantibody escape mutant, or whose anti-IFN-γ autoantibody escape mutant can effectively evade the neutralizing effect of their anti-IFN-γ autoantibodies. In other words, the anti-IFN-γ autoantibodies in sensitive patients do not bind to or bind minimally to the anti-IFN-γ autoantibody escape mutant.

[0026] Compared with the prior art, the present invention has the following beneficial effects: This invention discloses an S84G site-modified anti-IFN-γ autoantibody escape mutant and its application. This mutant has the following advantages: (1) Significant neutralization escape function and specificity: Compared with wild-type IFN-γ, the anti-IFN-γ autoantibody escape mutant modified by the S84G site of the present invention can effectively avoid the attack of anti-IFN-γ autoantibodies and retain biological activity. For some patient groups that are not sensitive to other mutation sites, the anti-IFN-γ autoantibody escape mutant modified by the S84G site has unique and superior escape characteristics, which can effectively fill the treatment blind spot.

[0027] (2) Preservation of structure and function: The S84G site modified anti-IFN-γ autoantibody escape mutant of the present invention can be expressed in the prokaryotic system and refolded into a soluble dimer, thus maintaining the basic structural characteristics of cytokines and overcoming the technical defects of similar designs (such as F15A) that are prone to forming inclusion bodies and difficult to refold.

[0028] (3) Diagnostic typing tool: Using the anti-IFN-γ autoantibody escape mutant modified by the S84G site of the present invention as a probe, the recognition of the antibody in the body of AIGA patients at this specific site can be detected, thereby screening out the patient group sensitive to this mutant and guiding the precise clinical medication. Attached Figure Description

[0029] Figure 1 This is a comparative analysis diagram of IFN-γ antigen epitope prediction and receptor binding interface in this invention; Figure 2 This diagram illustrates the mutation sites, with yellow arrows indicating the mutation sites designed in this invention. S84G is the key site with neutralization escape function verified in this invention; F15A and D63A are predicted sites designed concurrently (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 S84G 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 This diagram illustrates the effect of flow cytometry on the induction of HLA-DR expression in THP-1 cells by mutant S84G. The first histogram from left to right shows the shift in HLA-DR fluorescence intensity on the cell surface after treatment with mutant S84G. The second table summarizes the mean fluorescence intensity (MFI) for each group. Figure 5 The graph shows the neutralization and inhibition curves of XSZ plasma from AIGA patients against wild-type IFN-γ, the mutant S84G of this invention, and the control mutant D63A. Detailed Implementation

[0030] 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.

[0031] 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.

[0032] 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.

[0033] Example 1 Prediction of IFN-γ neutralizing epitopes and design of S84G mutants.

[0034] For ease of description, the proteins and illustrations involved in this embodiment and subsequent embodiments are numbered and explained as follows: Mutant of this invention: Mutant 1 (S84G) Control mutants: Mutant 3 (F15A); Mutant 5 (D63A) Wild-type human IFN-γ: WT 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.

[0035] 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.

[0036] The prediction results show that the region of amino acid residues 85-93 (NKKKRDDFE) and the adjacent region (including the 84th position) have extremely high antigenicity scores.

[0037] 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.

[0038] Site Identification: Based on the above predictions, this invention focused on designing the S84G site, located in a highly immunogenic region, for mutation verification. Simultaneously, to verify the accuracy of the prediction strategy and as a control, two other predicted sites, F15A and D63A, were selected. Subsequent experiments confirmed that only the S84G mutant successfully achieved the design goal (both retaining activity and escaping neutralization). The specific mutation design is as follows (site distribution is shown in the figure). Figure 2 (as shown) (1) S84G (preferred in this invention): located in the interhelical junction region, predicted to be a strong antigenic site, and its spatial position is conducive to maintaining protein folding. (2) F15A (Control 1): Located at the N-terminal helix, it is a potential epitope (later confirmed to have poor drug-like properties). (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).

[0039] Sequence of mature wild-type human IFN-γ protein: QDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRG (SEQ IDNO: 1).

[0040] Sequence of mutant 1 (S84G): QDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNGNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRG (SEQ ID NO: 2).

[0041] Sequence of mutant 3 (F15A): QDPYVKEAENLKKYANAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRG (SEQ ID NO: 3).

[0042] Sequence of mutant 5 (D63A): QDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDAQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRG (SEQ IDNO: 4).

[0043] Example 2 Expression and purification of the anti-IFN-γ autoantibody escape mutant S84G.

[0044] This embodiment describes the preparation process of the recombinant mutant protein. The flowchart is shown below. Figure 3 .

[0045] 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) Construction of the S84G mutant (in this invention, the 84th serine residue is mutated to glycine): S84G-F (forward): GTGAAGTTCTTCAACGGCAACAAGAAGAAGC (SEQ ID NO: 5) S84G-R (reverse):GCTTCTTCTTGTTGCCGTTGAAGAACTTCAC (SEQ ID NO: 6) Note: For parallel comparison, control mutants D63A and F15A were also constructed in this embodiment. The construction method was the same as above. Mutant D63A was created 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).

[0046] 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.

[0047] 2. Induction of Expression: The correctly sequenced recombinant plasmid was transformed into *E. coli* BL21(DE3) competent cells. The cells were cultured in LB medium at 37°C until the OD600 reached 0.6-0.8. IPTG (final concentration 0.5 mM) was then added to induce expression for 4-6 hours. The cells were collected by centrifugation.

[0048] 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 in 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.

[0049] 4. The purified and refolded protein solution was passed through a Ni-NTA affinity chromatography column, captured using a His tag, and eluted using an imidazole gradient. After dialysis to replace the buffer, soluble IFN-γ mutant protein with a purity greater than 90% was obtained.

[0050] Results: It should be noted that under the preparation conditions of this embodiment, both the mutant S84G 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 meeting experimental requirements. Therefore, it was not included in subsequent biological activity assays. This result indicates that S84G can maintain the stability of the overall protein structure while altering surface amino acids, a capability not achieved at all predicted sites (such as F15A). Although this embodiment uses an inclusion body refolding process, those skilled in the art can also attempt to achieve soluble expression through low-temperature induction or co-expression of chaperone proteins, which also falls within the scope of the preparation method of this invention.

[0051] Example 3 Validation of the biological activity of mutant S84G.

[0052] To verify whether the S84G 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.

[0053] 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 S84G 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.

[0054] Note: A concentration of 20 ng / mL was confirmed as the linear response range in preliminary experiments. Meanwhile, 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.

[0055] 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.

[0056] 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.

[0057] MFI Statistical Analysis: (1) Wild-type human IFN-γ (WT): MFI value is 2867.

[0058] (2) Mutant 1 of the present invention (S84G): MFI value is 2808.

[0059] (3) Control mutant 5 (D63A): MFI value is 2582.

[0060] Data comparison: Calculations show that the activity of mutant S84G is approximately 98% of that of the wild type. This indicates that although mutant S84G has undergone mutations at key sites to evade antibodies, it still retains the vast majority of its receptor binding and immune activation capabilities.

[0061] Example 4 I. ELISA competitive inhibition detection method based on plasma from AIGA patients.

[0062] 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.

[0063] 1. Reagents and Materials Test kit: BD OptEIA™ Human IFN-γ ELISA Set (Cat. No. 555142).

[0064] 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.

[0065] Sample: Plasma from a clinically diagnosed AIGA patient (Patient ID: XSZ).

[0066] Note: This patient sample was selected because previous screening showed that it represents a typical group sensitive to a specific epitope.

[0067] Proteins to be tested: wild-type human IFN-γ protein, the mutant S84G of this invention and the control mutant D63A.

[0068] 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).

[0069] Diluent / Blocking Buffer (Assay Diluent): PBS (pH 7.0) containing 10% fetal bovine serum (FBS).

[0070] Wash Buffer: PBS (pH 7.0) containing 0.05% Tween-20. Dilute the 20X concentrate with deionized water to a 1X working solution.

[0071] 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.

[0072] 3. Experimental Procedure Step A: Pretreatment of standards and plasma samples.

[0073] Preparation of IFN-γ working solutions: After reconstituted the lyophilized standards, dilute them with Assay Diluent to prepare working solutions of wild type, mutant S84G and control mutant D63A with a concentration of 2000 pg / mL.

[0074] Plasma serial dilution: Assay Diluent was used to serially dilute the plasma of patients with XSZ. The initial dilution was set to 1:50, followed by continuous serial dilutions, selecting an appropriate gradient range (e.g., 1:3200 to 1:409600) for testing.

[0075] Step B: Neutralization reaction (pre-incubation).

[0076] Collect 50 μL of XSZ plasma from patients at different dilution gradients.

[0077] Add 50 μL of IFN-γ protein working solution (WT, S84G, 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.

[0078] After shaking and mixing, incubate at room temperature (20-25℃) for 1 hour.

[0079] Explanation of the principle: In this step, if the plasma contains a self-neutralizing antibody that can recognize the IFN-γ, the two will combine to form a complex; if the mutant (such as S84G) achieves epitope escape, it remains in a free state.

[0080] Step C: Double antibody sandwich ELISA detection.

[0081] 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.

[0082] 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.

[0083] Block: Add ≥ 200 μL of Assay Diluent to each well and incubate at room temperature for 1 hour.

[0084] Washing 2: Absorb the liquid and wash 3 times.

[0085] 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.

[0086] Washing 3: Absorb the liquid and wash 5 times.

[0087] 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.

[0088] 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.

[0089] Color development: Add 100 μL of TMB substrate mixture to each well. Incubate at room temperature in the dark for 30 minutes.

[0090] 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.

[0091] 4. Data processing: The inhibition rate at different plasma concentrations was calculated using the following formula:

[0092] in: OD 实验组 : Well readings after pre-incubation with added plasma; OD 最大信号 Readings of the 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).

[0093] II. Evaluation of the neutralization escape effect of mutant S84G.

[0094] Using the above methods, the neutralization escape performance of the mutant of the present invention in XSZ plasma of AIGA patients was evaluated.

[0095] Patient's XSZ test results (see) Figure 5 The results showed a significant pattern of difference: Wild-type human IFN-γ: The inhibition rate remained high (close to 100%), indicating that the patient's autoantibodies could efficiently recognize and neutralize wild-type IFN-γ.

[0096] The S84G mutant of this invention exhibits a neutralization curve significantly lower than that of the wild type. At the same plasma concentration, the degree of neutralization of S84G is drastically reduced. This demonstrates that, for this patient, serine at position 84 is a key epitope for autoantibody recognition, and mutating it to glycine (S84G) successfully disrupts the antigen-antibody binding interface, thereby achieving immune escape.

[0097] 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.

[0098] Conclusion: Experimental data show that the antibody profile in AIGA patients is specific. For patient groups such as XSZ who are sensitive to the S84G site, the S84G mutant of this invention exhibits the best escape effect and has the potential to be used as a precision medicine.

[0099] III. Conclusion.

[0100] In summary, the mutant S84G 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 S84G has significant and unexpected technical advantages compared to other predicted sites: 1. Excellent druggability and structural stability (compared to F15AA): Experimental results show that high antigenicity scores predicted by computers alone cannot guarantee the expressibility of mutants. The concurrently designed predicted mutant F15AA formed insoluble inclusion bodies due to impaired protein folding characteristics, failing to meet druggability requirements. In contrast, the mutant S84G of this invention exhibits good solubility and structural stability in prokaryotic expression systems and retains the biological activity of inducing HLA-DR expression on the cell surface (Example 3), demonstrating that it maintains the correct three-dimensional spatial structure.

[0101] 2. Specific Neutralization Escape Function (Compared to D63A): In further functional validation, although the control mutant D63A retained its biological activity, it failed to escape recognition by autoantibodies in AIGA patients (XSZ), confirming that simple surface charge or local amino acid alterations do not necessarily lead to neutralization escape. In stark contrast, the mutant S84G successfully achieved significant escape from AIGA patient autoantibodies while retaining immunogenicity (Example 4).

[0102] 3. Conclusion: The mutant S84G of this invention overcomes the common technical defects of "poor solubility" or "ineffective escape" in similar designs, while simultaneously meeting the three key technical indicators of "structural stability," "preservation of biological activity," and "antibody neutralization escape." The acquisition of this mutant can be easily predicted by those skilled in the art through conventional methods, possessing outstanding substantive characteristics and significant clinical application value, providing a novel candidate drug for personalized precision treatment of AIGA.

[0103] 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.

[0104] 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 S84G site-modified anti-IFN-γ autoantibody escape mutant, characterized in that, The 84th amino acid of this anti-IFN-γ autoantibody escape mutant is glycine.

2. The anti-IFN-γ autoantibody escape mutant according to claim 1, characterized in that, The anti-IFN-γ autoantibody escape mutant is an IFN-γ mutant. The remaining amino acid sequence of the anti-IFN-γ autoantibody escape mutant, except for amino acid position 84, 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 anti-IFN-γ autoantibody escape mutant according to claim 1, characterized in that, The amino acid sequence of the anti-IFN-γ autoantibody escape mutant, except for amino acid position 84, is identical to that of wild-type IFN-γ. The amino acid sequence of the 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 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. The method for preparing the 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 as 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 anti-IFN-γ autoantibody escape mutant.

8. A drug, characterized in that, It includes a pharmaceutically active ingredient, said pharmaceutically active ingredient including any one of the anti-IFN-γ autoantibody escape mutants according to 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 as described in any one of claims 1-3, wherein the anti-IFN-γ autoantibody escape mutant is coated on the solid-phase carrier, the 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 anti-IFN-γ autoantibody escape mutant according to any one of claims 1-3 in a pharmaceutical or kit.

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