Method for evaluating drug effect of rituximab on primary membranous nephropathy

Abstract

The invention discloses a method for evaluating the drug effect of rituximab on primary membranous nephropathy. According to the method, an SNP (Single Nucleotide Polymorphism) site rs396991 of FCGR3A is mutated into AC or CC from wild type AA, and the 158th amino acid coded by the SNP site rs396991 is mutated into phenylalanine from valine; according to the invention, an iMLDR typing technology is adopted to detect an FGCR3A base mutation site, if rs396991 is AA type, the rs396991 is considered to be effective to an RTX drug, and if the rs396991 is detected to be AC type or CC type, the rs396991 is considered to be insensitive or ineffective to an RTX reaction; therefore, the rituximab can be used for effectively evaluating the drug effect of the rituximab on the primary membranous nephropathy.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and more specifically, to a method for evaluating the drug effect of rituximab on primary membranous nephropathy. Background Art

[0002] Primary membranous nephropathy (PMN) is a common histopathological type of adult nephrotic syndrome. Its typical feature is the thickening of the glomerular basement membrane caused by subepithelial immune complex deposition (mainly IgG and complement C3). Patients present with clinical manifestations such as severe edema, massive proteinuria, hypoproteinemia, and hyperlipidemia, and are extremely prone to complications such as acute infection, deep vein thrombosis, acute kidney injury, and protein metabolism disorders, which seriously endanger the life and health of patients. The natural course of PMN varies greatly among individuals. Approximately 20% of patients can achieve spontaneous remission, and 30%-40% of patients progress to end-stage renal disease (ESRD) 5-10 years after the onset of the disease, endangering the lives of patients and causing serious health problems and economic burdens.

[0003] In the past decade, with the discovery of anti-phospholipase A2 antibody and thrombospondin type 1 domain-containing 7A antibody, PMN has been considered an autoimmune disease targeting podocytes. The pathogenic role of B cells that produce autoantibodies in PMN has been gradually recognized, which provides strong evidence for the treatment of PMN with rituximab (RTX). RTX is a human-mouse chimeric anti-CD20 antibody with natural IgG1-Fc, which can crosslink Fcγ receptors (FcγRs) expressed on B cells, thereby triggering or inhibiting effector functions and playing an important role in antibody-dependent cell-mediated cytotoxicity (ADCC), immune regulation, endocytosis, phagocytosis, and the release of inflammatory factors and antigen presentation. The FCGR gene encodes Fcγ receptors, and its single nucleotide polymorphisms (SNPs) are closely related to many phenotypic differences in the human body, susceptibility to drug efficacy, or diseases.

[0004] Compared with traditional therapeutic drugs, RTX can significantly reduce the proteinuria level in patients, effectively prolong the remission time, reduce the recurrence risk of the disease, and has relatively mild adverse reactions and no nephrotoxicity. However, the clinical use of RTX has certain limitations. According to statistics, about 20%-40% of PMN patients are ineffective in RTX treatment. Therefore, there is an urgent need to find a method to detect the drug effect of rituximab (RTX) on primary membranous nephropathy. Summary of the Invention

[0005] The purpose of the present invention is to solve the deficiencies existing in the prior art and propose a method for efficiently detecting the drug effectiveness of rituximab (RTX) on primary membranous nephropathy.

[0006] The technical solution of the present invention is as follows: A method for evaluating the drug effect of rituximab on primary membranous nephropathy, taking the SNP locus rs396991 of FCGR3A mutated from wild-type AA to AC or CC, and the 158th amino acid encoded by it mutated from valine to phenylalanine as the evaluation basis.

[0007] The method for evaluating the drug effect of rituximab on primary membranous nephropathy specifically includes the following steps:

[0008] 1) Collect 1 ml of fasting peripheral venous blood from the patient in the early morning, extract peripheral blood DNA using the Flexigene DNA kit, and store it at a temperature of -80 °C for analysis.

[0009] 2) Take 1 μl of the DNA sample for 1% agarose electrophoresis to check the quality of the sample and estimate the concentration, and then dilute the sample to the working concentration of 5-10 ng / μl according to the estimated concentration.

[0010] 3) Design and synthesize PCR specific amplification primers with rs396991; among them, the nucleotide sequence of the F primer is as shown in SEQ ID NO.1: 5’-CCCAACTCAACTTCCCAGTGTGAT-3’; the nucleotide sequence of the R primer is as shown in SEQ ID NO.2: 5’-CAGAATGGCAAAGGCAGGAAGT-3’; use the iMLDR genotyping technology to detect the FGCR3A base mutation site. If rs396991 is of the AA type, it is considered effective for RTX drugs. If rs396991 is detected as AC type or CC type, it is considered insensitive or ineffective to RTX.

[0011] The beneficial effects of the present invention are as follows: Research has confirmed that in patients with rheumatoid arthritis and systemic lupus erythematosus receiving RTX treatment, the FCGR3A gene is involved in rituximab-induced ADCC, and the homozygosity and increased copy number of FCGR3A rs396991 are closely related to the complete clearance of B cells and the improvement of clinical symptoms. Based on this, the present invention selects the SNP loci rs1801274 and rs396991 of FCGR2A and FCGR3A, and analyzes their pharmacodynamic relationship with RTX treatment of PMN. The results show that when the rs396991 locus mutates from the wild-type AA to AC or CC, the 158th amino acid encoded by it mutates from valine to phenylalanine, which is related to RTX resistance. Therefore, it can be used to effectively evaluate the drug effect of rituximab on primary membranous nephropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 iMLDR in the embodiment of the present invention TM Schematic diagram of SNP genotyping. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0013] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments.

[0014] Screening of FCGR3A gene loci related to RTX efficacy

[0015] 1. Selection of research subjects

[0016] (1) Inclusion criteria

[0017] 1) Aged 18 - 85 years old; 2) Diagnosed as PMN by renal biopsy, and positive for serum PLA2R antibody (i.e., ELISA test value ≥ 20 RU / ml); 3) Proteinuria within the range of nephropathy (24-hour urinary protein quantification > 3.5 g or urinary protein / creatinine > 3.5) accompanied by hypoproteinemia and / or dyslipidemia; 4) Stable condition, without oliguria, abnormal heart function, etc.; 5) Blood CD20 B cell lymphocyte count > 100 cells / μl.

[0018] (2) Exclusion criteria

[0019] 1) Secondary MN, such as hepatitis B-related glomerulonephritis, lupus nephritis, malignant tumors, etc.; 2) Patients with any active infections, severe heart and liver diseases, infectious diseases and other wasting diseases, and psychiatric patients; 3) CD4+ and CD8+ T lymphocyte counts < 200 cells / μl; 4) Patients treated with combined hormones and / or other immunosuppressive agents.

[0020] (3) Treatment plan

[0021] Before RTX administration, promethazine (0.5 - 1.0 mg / kg) and dexamethasone (0.1 - 0.3 mg / kg) were intravenously injected, and electrocardiogram and blood pressure monitoring were given. RTX was diluted in 9 g / l normal saline to prepare a 1 g / l RTX solution. The initial intravenous drip rate was 50 mg / h, and then increased by 50 mg / h every 30 min, up to a maximum of 400 mg / l. The usage method of RTX: 375 mg / (m 2 *week), for 4 consecutive weeks.

[0022] (4) Efficacy evaluation

[0023] The patients were observed and followed up at 0, 3, and 6 months. The main observation index was the remission of nephropathy, and the secondary observation indexes included adverse events and renal outcomes. Complete remission was defined as urine protein / creatinine < 0.3 g / g, serum albumin > 35 g / l, and creatinine stable or increased by < 30% compared with the baseline value; partial remission was defined as urine protein / creatinine < 3.5 g / g and decreased by more than 50% compared with the peak value, while the serum albumin level increased, and creatinine was stable or increased by < 30% compared with the baseline value. Patients with complete remission or partial remission were classified into the effective group, and the remaining study subjects were included in the drug-resistant group.

[0024] 2. Evaluation steps

[0025] (1) Collect the general information and clinical data of the study subjects: age, gender, drug treatment situation, serum albumin, serum creatinine, urine protein / creatinine, 24-hour urine protein quantification, and PLA2R antibody titer.

[0026] (2) After determining the inclusion, collect the fasting peripheral blood specimens of the study subjects in the early morning: After obtaining the written informed consent of the patients, 1 ml of peripheral venous blood was collected, and the peripheral blood DNA was extracted using the Flexigene DNA kit and stored at -80 °C for analysis.

[0027] (3) iMLDR technology typing of gene SNP loci

[0028] The multiplex SNP typing technology is a multiplex SNP typing technology improved based on the traditional ligation reaction. Compared with the traditional ligation reaction technology, the multiplex SNP typing technology improves the accuracy and typing success rate. Its working principle is as Figure 1 shown:

[0029] 1) The target SNP locus region is first amplified in one system by multiplex PCR reaction.

[0030] 2) The amplified product is purified by exonuclease and shrimp alkaline phosphatase (ExoI / SAP) and used as the template for the subsequent ligation reaction.

[0031] 3) In a ligation reaction, each site contains two 5'-end allele-specific probes (with two allele-specific bases at their 3'-ends respectively) and a fluorescence-labeled specific probe for the 3'-end site immediately following. The ligation products are distinguished by capillary electrophoresis on an ABI 3730XL, and the raw data files are analyzed using GeneMapper 4.1 software (Applied biosystems).

[0032] (4) Statistical processing

[0033] 1) Clinical information

[0034] Statistical analysis was performed using SPSS 26.0 software. Measurement data conforming to a normal distribution were expressed as mean ± standard deviation. The independent samples t-test was used for comparison between two groups, and one-way analysis of variance was used for comparison among multiple groups. Non-normal distribution data were expressed as median and quartiles, and the rank sum test was used for comparison between groups; the chi-square test was used for constituent ratios. P < 0.05 was considered statistically significant.

[0035] 2) Association analysis of the SPN locus of the FCGR3A gene

[0036] Before performing the association analysis, SNP loci need to be filtered by the following conditions: 1. SNP genotyping rate ≥ 95%; 2. Minor allele frequency ≥ 1%; 3. The allele frequencies of SNP loci conform to the Hardy-Weinberg equilibrium law; 4. Remove duplicate loci.

[0037] Plink v1.90 software was used for gene locus-based association analysis. Taking individual loci as units, chi-square analysis, Fisher's exact test, and logistic regression analysis were performed according to four hypothetical genetic models: Dominant (the allele with a low frequency is dominant), Recessive (the allele with a low frequency is recessive), Genotype (codominant, with homozygous normal as the reference), and Allele (allele model).

[0038] A total of 132 PMN patients receiving RTX treatment were included in this example. According to the treatment effect, they were divided into an effective group (n = 88) and a drug-resistant group (n = 44). The iMLDR genotyping technique was used to detect the FGCR3A mutation sites, and the correlation between the genotypes of SNP loci rs1801274 and rs396991 of FCGR2A / 3A and RTX drug resistance was analyzed in detail. The results showed that if the rs396991 single nucleotide polymorphism was of the AA type, the RTX drug was effective for primary membranous nephropathy. If rs396991 was detected as the AC type or CC type, RTX was ineffective or insensitive to primary membranous nephropathy. The analysis results are shown in Table 1.

[0039]

[0040] Taking the SNP locus rs396991 of FCGR3A mutated from wild-type AA to AC or CC, and the 158th amino acid encoded by it mutated from valine to phenylalanine as the evaluation basis, the rituximab drug effects of 5 primary membranous nephropathy patients in Examples 1-5 were evaluated according to the following steps:

[0041] 1) Collect 1 ml of fasting peripheral venous blood from the patient in the early morning, extract peripheral blood DNA using the Flexigene DNA kit, and store it at -80 °C for analysis;

[0042] 2) Take 1 μl of the DNA sample for 1% agarose electrophoresis to check the quality of the sample and estimate the concentration, and then dilute the sample to the working concentration of 5-10 ng / μl according to the estimated concentration;

[0043] 3) Design and synthesize PCR specific amplification primers with rs396991; among them, the nucleotide sequence of the F primer is as shown in SEQ ID NO.1: 5'-CCCAACTCAACTTCCCAGTGTGAT-3'; the nucleotide sequence of the R primer is as shown in SEQ ID NO.2: 5'-CAGAATGGCAAAGGCAGGAAGT-3'; use the iMLDR genotyping technology to detect the FGCR3A mutation site. The detection results of Example 1 were AA type, Example 2 was AC type, Example 3 was AA type, Example 4 was CC type, and Example 5 was AC type. According to the results, if rs396991 is AA type, it is considered effective for RTX drug, and if rs396991 is detected as AC type or CC type, it is considered insensitive or ineffective to RTX. 5 groups of patients received RTX treatment, and were clinically observed and followed up at 0, 3, and 6 months to observe the treatment conditions, blood pressure, serum albumin, serum creatinine, urine protein / creatinine, and 24-hour urine protein quantification. Complete remission was defined as urine protein / creatinine <0.3 g / g, serum albumin >35 g / l, and creatinine stable or increased <30% compared with the baseline value; partial remission was defined as urine protein / creatinine

[0044] <3.5 g / g and decreased by more than 50% compared with the peak value, and at the same time the serum albumin level increased, and the creatinine was stable or increased <30% compared with the baseline value. Patients with complete remission or partial remission were classified as effective, and the rest were regarded as drug-resistant. The clinical evaluation results are as follows:

[0045] Example 1 (AA type):

[0046] 0 months 3 months 6 months Albumin (g / L) 32.7 37.2 38.8 Serum creatinine (μmol / L) 53.8 56.6 56.8 Urinary protein / creatinine (mg / g) 1.49 0.1 0.04 24-hour urinary protein (mg / d) 3515 1052.9 358.8

[0047] Example 2 (AC type):

[0048] 0 months 3 months 6 months Albumin (g / L) 27.7 27.2 33.4 Serum creatinine (μmol / L) 69.7 74.2 61.0 Urinary protein / creatinine (mg / g) 3.72 3.66 3.81 24-hour urinary protein (mg / d) 13267.2 7323.3 10000.3

[0049] Example 3 (AA type):

[0050] 0 months 3 months 6 months Albumin (g / L) 30.7 28.1 35.2 Serum creatinine (μmol / L) 50.6 87.2 76.9 Urinary protein / creatinine (mg / g) 3.59 2.17 0.27 24-hour urinary protein (mg / d) 2491.5 1100 806.8

[0051] Example 4 (CC type):

[0052] 0 months 3 months 6 months Albumin (g / L) 32.3 26.3 26.5 Serum creatinine (μmol / L) 225.1 267.1 183.8 Urinary protein / creatinine (mg / g) 5.67 3.18 3.46 24-hour urinary protein (mg / d) 3461 1770 1620

[0053] Example 5 (AC type):

[0054] 0 months 3 months 6 months Albumin (g / L) 20.3 30.3 26.6 Serum creatinine (μmol / L) 72.2 82.6 71.9 Urinary protein / creatinine (mg / g) 2.22 2.81 2.7 24-hour urinary protein (mg / d) 4075 890.2 12051

[0055] It can be seen that in the 3rd and 6th months after the 1st and 3rd patients were treated with the standard dose of rituximab, their blood albumin levels were found to have increased, and both the urine protein / creatinine and the 24-hour urine protein quantification were significantly reduced. However, in the 3rd and 6th months after the 2nd, 4th, and 5th patients were treated with the same dose of rituximab, their blood albumin levels did not increase significantly or were even lower than before treatment, and the urine protein / creatinine and the 24-hour urine protein quantification did not decrease or even increased compared with before treatment. Therefore, the RTX drug is effective for the 1st and 3rd patients and is insensitive or ineffective for the 2nd, 4th, and 5th patients.

[0056] The above are only the characteristic implementation examples of the present invention and do not constitute any limitation to the protection scope of the present invention. Any technical solutions formed by equivalent exchange or equivalent substitution fall within the scope of the protection of the present invention.

Claims

1. A method for evaluating the drug effect of rituximab on primary membranous nephropathy, characterized in that: The SNP site rs396991 of FCGR3A mutated from the wild-type AA to AC or CC, and the 158th amino acid encoded by it mutated from valine to phenylalanine was used as the evaluation basis.

2. The method for evaluating the drug effect of rituximab on primary membranous nephropathy according to claim 1 specifically includes the following steps: 1) Collect 1 ml of fasting peripheral venous blood from the patient in the early morning, extract peripheral blood DNA using the Flexigene DNA kit, and store it at -80 °C for analysis; 2) Take 1 μl of the DNA sample for 1% agarose electrophoresis to check the quality of the sample and estimate the concentration, and then dilute the sample to the working concentration of 5 - 10 ng / μl according to the estimated concentration; 3) Design and synthesize PCR specific amplification primers with rs396991; among them, the nucleotide sequence of the F primer is as shown in SEQ ID NO.1: 5’-CCCAACTCAACTTCCCAGTGTGAT -3’; the nucleotide sequence of the R primer is as shown in SEQ ID NO.2: 5’-CAGAATGGCAAAGGCAGGAAGT -3’; use the iMLDR typing technology to detect the FGCR3A base mutation site. If rs396991 is of the AA type, it is considered effective for the RTX drug. If rs396991 is detected as AC type or CC type, it is considered insensitive or ineffective to RTX.

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