Kit for detecting T cell immunoglobulin and ITIM structural domain protein for companion diagnosis of multiple myeloma patients

Abstract

The invention discloses a kit for detecting T cell immunoglobulin and ITIM structural domain protein (TIGIT) for accompanying diagnosis of multiple myeloma (MM) patients, the kit comprises a TIGIT standard substance and a negative quality control substance, the TIGIT standard substance is a solution containing high-concentration TIGIT, and the negative quality control substance is a PBS solution without TIGIT; the detection kit is used for detecting samples of MM patients, the MM can be divided into low TIGIT, medium TIGIT and high TIGIT, clinical doctors are assisted in screening the MM patients capable of being treated by targeted ADC drugs, and the patients with negative TIGIT or low TIGIT expression are prevented from being subjected to invalid treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor detection, and particularly relates to a kit for detecting T cell immunoglobulin and ITIM domain protein (TIGIT) for companion diagnosis of multiple myeloma patients and its application. Background Art

[0002] PD-1 is the abbreviation of programmed death-1, a cell membrane protein receptor discovered in recent years and a key post for regulating the functions of immune cells. PD-L1 is the ligand of PD-1. After PD-1 binds to PD-L1, inhibitory signals can be provided to induce apoptosis of T cells and inhibit the activation and proliferation of T cells. Therefore, PD-1 will prevent T cells from fully attacking "invaders". If PD-1 is "blocked", the restraint on T cells will be lifted, and thus T cells can be ensured to fully fight against cancer cells. After PD-1 / PD-L1, people have been exploring new immune targets. Now, new immune checkpoint inhibitors have finally emerged, which can be regarded as the classic partners of PD-1 / PD-L1 immunotherapy. These three types of immune checkpoint inhibitors are the three targets of TIGIT / TIM3 / LAG-3, and the main focus of the present invention is on the TIGIT target.

[0003] TIGIT (short for T cell immunoglobulin and ITIM domain protein), the role of TIGIT in tumor immunosuppression is similar to that of PD-1 / PD-L1. It is also an immune checkpoint, mainly expressed on the surface of T cells and NK cells. Simply put, TIGIT is like the sibling of PD-1 and is also a braking molecule that inhibits the immune response. However, it may more likely inhibit the function of NK cells. Currently, Roche announced the first data of the phase 2 clinical study CITYSCAPE of the TIGIT monoclonal antibody Tiragolumab combined with the PD-L1 drug Tecentrip (atezolizumab, abbreviated as drug T) as a first-line therapy for the treatment of patients with PD-L1-positive metastatic non-small cell lung cancer (NSCLC) at the ASCO2020 conference. And according to the published abstract, this study achieved positive results. In this phase II randomized double-blind study CITYSCAPE, 135 patients with advanced NSCLC who were negative for first-line EGFR / ALK, PDL1-positive (TPS≥1%), and untreated with chemotherapy were included and randomly assigned to the TA group and the PA group at a ratio of 1:1. Among them, the TA group was Tiragolumab 600mg combined with drug T 1200mg, and the PA group was placebo combined with drug T. The primary endpoints were PFS and ORR. The results showed that in the intention-to-treat (ITT) patient population, compared with the PA group treated with drug T alone, the ORR of the TA group with Tiragolumab combined with drug T was improved (31.3% vs 16.2%), the median PFS was increased (5.4 months vs 3.6 months), and the risk of disease progression decreased by 43%. The combined use had a better effect. In terms of safety: compared with the use of drug T alone (PA group), when the two immunotherapies were combined (TA group), the incidence of all grade 3 or above adverse events (AE) was similar. In the combination therapy, the incidence of treatment-related AE (TRAE) was 80.6%, and the incidence of grade 3 or above TRAE was 14.9%; in the subgroup using drug T alone, the incidence of TRAE was 72%, and for grade 3 or above it was 19.1%. The above data indicate that the combination of Tiragolumab and Tecentriq is well tolerated. Compared with the treatment with Tecentriq alone, there is no increase in safety risks. In addition to Roche's Tiragulumab, there are currently multiple pharmaceutical companies globally with pipeline layouts in this target area.

[0004] These targeted drugs, also known as antibody-drug conjugates (ADCs), are a class of targeted biological agents composed of antibodies, linkers, and cytotoxic drugs, and have received much attention in the field of cancer treatment drug development. Because the monoclonal antibodies of ADC drugs can target antigens specifically expressed on cancer cells based on the antigen-antibody binding mechanism, bringing the drugs to the vicinity or inside the cancer cells for release, thereby achieving the effect of drug treatment. According to this treatment principle of ADC drugs, relevant effects can only be exerted when specific antigen expression exists in the patient's body. Therefore, when the expression of TIGIT in the patient's body is very low, it is difficult to benefit from ADC drugs. Therefore, before administering ADC drugs, it is necessary to detect the targeted antigen expression status of the patient, that is, companion diagnosis, to avoid ineffective drug use by the patient.

[0005] The classical enzyme-linked immunosorbent assay (ELISA) is a method for achieving companion diagnosis by detecting the antigen expression of patients, enabling personalized medication. The present invention is to apply this method to ensure the application of ADC drugs for TIGIT. Summary of the Invention

[0006] Based on the above research, the present invention provides a kit for detecting T cell immunoglobulin and ITIM domain protein (TIGIT) for companion diagnosis of multiple myeloma patients, which comprises a 96-well plate coated with TIGIT antibody, biotin-labeled TIGIT antibody, streptavidin-HRP, TIGIT standard, and negative control. The TIGIT standard is a solution containing a high concentration of TIGIT, and the negative control is a PBS solution without TIGIT.

[0007] In a further technical solution, the coating concentration of the TIGIT antibody is 1-10 μg / ml.

[0008] In a further technical solution, the biotin-labeled TIGIT antibody is stored in a vial in the form of a lyophilized powder and dissolved in pure water when in use.

[0009] In a further technical solution, streptavidin-HRP and the biotin-labeled TIGIT antibody combine to produce color.

[0010] In a further technical solution, the concentration of the TIGIT standard is 100 ng / ml.

[0011] The present invention also provides a preparation method of a kit for detecting T cell immunoglobulin and ITIM domain protein (TIGIT) for companion diagnosis of multiple myeloma patients, specifically as follows:

[0012] (1) Preparation of TIGIT antibodies: Preparation of TIGIT protein immunogens; animal immunization; isolation of spleen cells from immunized animals and fusion with myeloma cells (such as SP2 / 0 cells) to form hybridoma cells; screening of monoclonal antibodies that can specifically bind to TIGIT protein, and subcloning of the screened positive clones to ensure monoclonality; large-scale culture of the screened hybridoma cells, collection of the supernatant, purification of antibodies by protein A / G affinity chromatography and other methods, and concentration measurement, purity analysis, and functional verification of binding affinity, neutralization activity, etc. of the purified antibodies;

[0013] (2) coating a 96-well microplate with the TIGIT antibody prepared in (1) at a concentration of 1-10 μg / ml to prepare a solid phase carrier;

[0014] (3) mixing the TIGIT antibody prepared in (1) with biotin at a molar ratio of 1:20 and preparing a lyophilized powder;

[0015] (4) preparing HRP-labeled streptavidin by mixing streptavidin and HRP at a molar ratio of 1:1;

[0016] (5) Dissolve the purified TIGIT protein in PBS to prepare a TIGIT standard with a concentration of 100 ng / ml;

[0017] (5) Preparation of negative control: 20X concentrated PBS washing solution.

[0018] The present invention also provides a kit for detecting T cell immunoglobulin and ITIM domain protein (TIGIT) for companion diagnosis of multiple myeloma patients, and the detection method is as follows:

[0019] Collect sample serum, add sample serum or standard, biotin-labeled human TIGIT antibody, HRP-labeled streptavidin to the microwells coated with TIGIT antibody in sequence, and after thorough washing, use substrate TMB for color development;

[0020] TMB is converted into blue under the catalysis of peroxidase, and then into the final yellow under the action of 2M sulfuric acid. The depth of the color is positively correlated with the TIGIT in the sample;

[0021] The absorbance (OD value) was measured at a wavelength of 450 nm using an enzyme-labeled instrument to calculate the sample concentration;

[0022] The specific judgment criteria are as follows:

[0023] (1) When the serum TIGIT concentration is less than or equal to 0.59 ng / ml, it is judged as low expression and the individual is not suitable for ADC drug treatment targeting TIGIT;

[0024] (2) When the serum TIGIT concentration is between 0.59 ng / ml and 13.97 ng / μl, it is judged as medium expression. This individual may be suitable for treatment with ADC drugs targeting TIGIT and requires re-evaluation.

[0025] (3) When the serum TIGIT concentration is greater than or equal to 13.97 ng / ml, it is judged as high expression. This individual is suitable for receiving ADC drugs targeting TIGIT for treatment.

[0026] The present invention also provides an application for assisting clinicians in screening multiple myeloma patients who can be treated with ADC drugs targeting TIGIT.

[0027] The beneficial effects are as follows:

[0028] 1. High sensitivity and specificity: Precise screening of monoclonal antibodies: Monoclonal antibodies specifically binding to TIGIT are prepared by hybridoma technology and are subcloned, purified, and functionally verified (such as binding affinity, neutralizing activity) to ensure high specificity of the antibody for TIGIT, reduce cross-reactivity, and improve detection accuracy; Signal amplification system: The biotin-streptavidin-HRP cascade labeling technology is adopted. Through the high-affinity biotin-streptavidin binding and HRP-catalyzed color development, the detection signal sensitivity is significantly enhanced, which is suitable for the detection of low-concentration TIGIT samples.

[0029] 2. Standardization and reproducibility: Perfect quantitative standard system: TIGIT standard products (100 ng / ml) and negative control products (PBS) with definite concentrations are provided to achieve standardized calibration of detection results and ensure the consistency of results between different batches and different laboratories; Strict quality control guarantee: The negative control product (PBS without TIGIT) can effectively exclude non-specific adsorption or background interference, reduce the risk of false positives, and improve the reliability of detection.

[0030] 3. Operational convenience and stability: Pre-coated microplate design: The TIGIT antibody is pre-coated on a 96-well plate (optimized concentration of 1 - 10 μg / ml), which simplifies the experimental steps, reduces the user's operation time and errors; Optimization of the lyophilized powder dosage form: The biotin-labeled antibody is stored in the form of lyophilized powder, which extends the reagent shelf life, avoids the problem of activity degradation during liquid storage, and only requires reconstitution with pure water during use, which is convenient and efficient. The reagent components have strong stability: Streptavidin-HRP is accurately labeled at a 1:1 molar ratio to ensure enzyme activity and binding efficiency, the color development system is stable, and the results have high repeatability.

[0031] 4. Clear clinical decision-making guidance value: Stratified diagnostic threshold: Through verification with large-sample data, low (≤0.59 ng / ml), medium (0.59 - 13.97 ng / ml), and high (≥13.97 ng / ml) expression thresholds are set, which are directly associated with treatment decisions; Low expression: Exclude the risk of targeted therapy and avoid ineffective treatment; Medium expression: Suggest the need for further evaluation and formulate personalized treatment plans; High expression: Identify ADC drugs suitable for targeting TIGIT and improve treatment efficacy. Precise companion diagnosis: Through dynamic monitoring of serum TIGIT levels, it provides a basis for molecular typing for targeted therapy and promotes individualized treatment of multiple myeloma.

[0032] 5. Cost-effectiveness and industrialization potential: Domestic production of kit components: Independently prepare monoclonal antibodies and supporting reagents, reduce the cost of relying on imported reagents, and are suitable for large-scale production and clinical promotion; Compatibility with high-throughput detection: The ELISA platform based on 96-well plates is compatible with conventional laboratory equipment (microplate reader), without the need for special instruments, which is convenient for popularization and application in hospitals and testing institutions.

[0033] 6. Promote the development of targeted therapy: Treatment response prediction: For the first time, directly associate the TIGIT expression level with the indications of ADC drugs, fill the gap in companion diagnosis for targeted therapy of multiple myeloma, and accelerate the practice of precision medicine; Clinical transformation value: Provide a key screening tool for clinical trials of TIGIT-targeted drugs, assist in patient stratification and treatment efficacy evaluation, and shorten the drug R & D cycle.

[0034] In summary, through the close combination of technological innovation and clinical needs, the invention not only improves the accuracy and efficiency of TIGIT detection, but also provides a reliable basis for the individualized treatment of multiple myeloma patients, with significant clinical application value and market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is the standard curve graph with the standard product. DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention will be further described below in conjunction with specific embodiments and drawings. It should be noted that the conventional conditions and methods not described in the embodiments are usually carried out according to the methods commonly used by experimental personnel in the relevant field or according to the steps and conditions recommended by the manufacturers.

[0037] Example 1 Preparation and Screening of Specific Antibodies

[0038] 1. Preparation of TIGIT protein immunogen: Construct an expression plasmid capable of expressing human TIGIT by gene synthesis method. Use Invitrogen Lipofectamine 2000 transfection reagent (purchased from Thermo Fisher Scientific) to transfect the plasmid into HEK293 cells to express TIGIT protein. Obtain high-purity TIGIT protein through cell culture and protein purification techniques (such as affinity chromatography) as the immunogen.

[0039] 2. Animal immunization: Select an appropriate animal model (such as mice, rats or rabbits). After mixing the purified TIGIT protein with an adjuvant, perform multiple immunization injections; the immunization interval is usually 2 - 4 weeks to ensure the production of high-titer specific antibodies in the animal body; after the last immunization, collect the animal's serum and detect the antibody titer to ensure the immunization effect.

[0040] 3. Preparation of hybridoma cells: Isolate spleen cells from the immunized animals and fuse them with myeloma cells (such as SP2 / 0 cells) to form hybridoma cells; use HAT medium to screen out hybridoma cells that can stably secrete antibodies.

[0041] 4. Antibody screening: Screen out monoclonal antibodies that can specifically bind to TIGIT protein by methods such as ELISA, flow cytometry or immunofluorescence; perform subcloning on the screened positive clones to ensure monoclonality.

[0042] 5. Antibody production and purification: Culture the screened hybridoma cells on a large scale, collect the supernatant, and purify the antibody by methods such as protein A / G affinity chromatography; perform concentration determination, purity analysis and functional verification (such as binding affinity, neutralizing activity, etc.) on the purified antibody.

[0043] Example 2: Preparation of a 96-well plate coated with TIGIT antibody

[0044] 1. Preparation of TIGIT antibody: Prepare TIGIT antibody by hybridoma technology or genetic engineering technology and purify it.

[0045] 2. Coating of 96-well plate: Coat the purified TIGIT antibody in the wells of a 96-well plate at a concentration of 1 - 10 μg / ml and incubate overnight at 4°C.

[0046] 3. Washing: Wash 3 times with PBS washing solution to remove unbound antibody.

[0047] 4. Blocking: Add PBS solution containing 1% BSA and incubate at 37°C for 1 hour to block unbound sites.

[0048] 5. Washing: Wash again 3 times with PBS washing solution and set aside.

[0049] Example 3: Preparation of Biotinylated TIGIT Antibody

[0050] 1. Biotinylation: Mix the purified TIGIT antibody with biotin at a molar ratio of 1:20 and react at room temperature for 2 hours.

[0051] 2. Purification: Remove unreacted biotin by dialysis or column chromatography.

[0052] 3. Lyophilization: Lyophilize the biotinylated TIGIT antibody into a powder and store at -20°C.

[0053] Example 4: Preparation of Streptavidin-HRP

[0054] 1. HRP Labeling: Mix streptavidin with HRP at a molar ratio of 1:1 and react at room temperature for 1 hour.

[0055] 2. Purification: Remove unreacted HRP by column chromatography.

[0056] 3. Storage: Store streptavidin-HRP at 4°C and avoid light.

[0057] Example 5: Preparation of TIGIT Standard

[0058] 1. Standard Preparation: Dissolve the purified TIGIT protein in PBS to prepare a standard with a concentration of 100 ng / ml.

[0059] 2. Aliquoting: Aliquot the standard into vials and store at -80°C.

[0060] Example 6: Determination of the Detection Method for the Kit

[0061] 1. Sample Treatment: Collect patient serum samples and centrifuge to remove impurities.

[0062] 2. Sample Addition: Add the sample or standard to a 96-well plate coated with TIGIT antibody and incubate at 37°C for 1 hour.

[0063] 3. Washing: Wash 3 times with PBS washing solution.

[0064] 4. Addition of Biotinylated TIGIT Antibody: Take the biotinylated TIGIT antibody powder prepared in Example 3, dissolve it in pure water and add it to the 96-well plate, and incubate at 37°C for 1 hour.

[0065] 5. Washing: Wash 3 times with PBS washing solution.

[0066] 6. Addition of Streptavidin-HRP: Add streptavidin-HRP and incubate at 37°C for 30 minutes. The binding of streptavidin-HRP and biotinylated TIGIT antibody produces a colored reaction.

[0067] 7. Washing: Wash 3 times with PBS washing solution.

[0068] 8. Color development: Add TMB substrate and react for 15 minutes at room temperature in the dark.

[0069] 9. Terminate the reaction: Add 2M sulfuric acid to terminate the reaction. TMB is converted into blue under the catalysis of peroxidase and into the final yellow under the action of 2M sulfuric acid. The depth of the color is positively correlated with TIGIT in the sample.

[0070] 10. Reading: Measure the absorbance (OD value) with an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm.

[0071] Example 7: Standard curve and detection of normal samples

[0072] Collect 58 normal samples, detect the samples according to the method of Example 6 and detect the standard products at the same time. The concentrations of the standard products are: 100, 50, 25, 12.5, 6.25, 3.125, 1.563 ng / mL respectively. The diluent is used as the zero standard product (0 ng / mL). The standard concentrations and actual OD values of the standard products are shown in Table 1. The standard curve and calculation formula of the standard products are as Figure 1 shown. According to the curve of the standard product and the detection results of the samples, the range of TIGIT contained in the normal samples obtained by calculation through the formula is between 0 and 0.59.

[0073] Table 1 Detection results of TIGIT standard products

[0074]

[0075] Example 8: Detection of MM samples (multiple myeloma samples)

[0076] Collect a total of 60 MM samples, detect the samples according to the method of Example 6, and obtain the TIGIT content of the samples according to the formula of the standard curve, as shown in Table 2.

[0077]

[0078]

[0079] Among them, for MM individuals with a TIGIT concentration ≥ 13.97 ng / ml, the effect of treatment with ADC drugs targeting TIGIT is significant, while for individuals with a TIGIT concentration between 0.59 ng / ml and 13.97 ng / ml, some are effective and some are ineffective.

[0080] Example 9: Confirmation of the interpretation criteria of the kit

[0081] According to Examples 7 and 8, the interpretation criteria for the kit are determined as follows:

[0082] 1. Low expression: If the serum TIGIT concentration ≤ 0.59 ng / ml, it is judged as low expression and not suitable for ADC drug treatment targeting TIGIT.

[0083] 2. Medium expression: If the serum TIGIT concentration is between 0.59 ng / ml and 13.97 ng / ml, it is judged as medium expression, and further evaluation is required to determine whether it is suitable for ADC drug treatment targeting TIGIT.

[0084] 3. High expression: If the serum TIGIT concentration ≥ 13.97 ng / ml, it is judged as high expression and suitable for ADC drug treatment targeting TIGIT.

Claims

1. A kit for detecting T cell immunoglobulins and ITIM domain proteins for companion diagnosis of multiple myeloma patients, characterized in that: Contains a 96-well plate coated with TIGIT antibody, biotin-labeled TIGIT antibody, streptavidin-HRP, TIGIT standard and negative control. The TIGIT standard is a solution containing a high concentration of TIGIT, and the negative control is a PBS solution without TIGIT.

2. The kit according to claim 1, characterized in that The coating concentration of TIGIT antibody was 1-10 μg / ml.

3. The kit according to claim 1, characterized in that: The biotin-labeled TIGIT antibody is stored in a vial as a lyophilized powder and is dissolved in pure water when used.

4. The kit according to claim 1, characterized in that: The streptavidin-HRP and biotinylated TIGIT antibody bind to produce color.

5. The kit according to claim 1, characterized in that The concentration of TIGIT standard was 100 ng / ml.

6. A method for preparing a kit for detecting T cell immunoglobulins and ITIM domain proteins for companion diagnosis of multiple myeloma patients, characterized in that: The specific steps are as follows: (1) Preparation of TIGIT antibodies: Preparation of TIGIT protein immunogens; animal immunization; isolation of spleen cells from immunized animals and fusion with myeloma cells (such as SP2 / 0 cells) to form hybridoma cells; screening of monoclonal antibodies that can specifically bind to TIGIT protein, and subcloning of the screened positive clones to ensure monoclonality; large-scale culture of the screened hybridoma cells, collection of the supernatant, purification of antibodies by protein A / G affinity chromatography and other methods, and concentration measurement, purity analysis, and functional verification of binding affinity, neutralization activity, etc. of the purified antibodies; (2) coating a 96-well microplate with the TIGIT antibody prepared in (1) at a concentration of 1-10 μg / ml to prepare a solid phase carrier; (3) mixing the TIGIT antibody prepared in (1) with biotin at a molar ratio of 1:20 and preparing a lyophilized powder; (4) preparing HRP-labeled streptavidin by mixing streptavidin and HRP at a molar ratio of 1:1; (5) Dissolve the purified TIGIT protein in PBS to prepare a TIGIT standard with a concentration of 100 ng / ml; (6) Preparation of negative control: 20X concentrated PBS washing solution.

7. A detection method of a kit for detecting T cell immunoglobulin and ITIM domain protein for companion diagnosis of multiple myeloma patients, characterized in that: The specific steps are as follows: Collect sample serum, add sample serum or standard, biotin-labeled human TIGIT antibody, HRP-labeled streptavidin to the microwells coated with TIGIT antibody in sequence, and after thorough washing, use substrate TMB for color development; TMB is converted into blue under the catalysis of peroxidase, and is converted into the final yellow under the action of 2M sulfuric acid; the depth of the color is positively correlated with TIGIT in the sample; The absorbance (OD value) was measured at a wavelength of 450 nm using an enzyme-labeled instrument to calculate the sample concentration; The specific judgment criteria are as follows: (1) When the serum TIGIT concentration is less than or equal to 0.59 ng / ml, it is judged as low expression and the individual is not suitable for ADC drug treatment targeting TIGIT; (2) When serum TIGIT concentration is between 0.59 ng / ml and 13.97 ng / ul, it is judged as medium expression. The individual may be suitable for ADC drug treatment targeting TIGIT and needs to be re-evaluated; (3) When the serum TIGIT concentration is greater than 13.97 ng / ml, it is judged as high expression and the individual is suitable for ADC drug treatment targeting TIGIT.

8. The application of TIGIT detection kit in assisting clinicians in screening multiple myeloma patients that can be treated with ADC drugs targeting TIGIT.

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