Anti-idiotype antibodies and uses

Abstract

The invention discloses an anti-idiotypic antibody and application, the provided anti-idiotypic antibody is a monoclonal antibody capable of being specifically combined with a corresponding PD-L1 antibody drug, and the content of the antibody drug in a human body is reflected through the anti-idiotypic antibody; the anti-idiotype antibody disclosed by the invention is prepared by adopting a hybridoma technology, and an ADA kit adopting the antibody can quantitatively detect the concentration level of a PD-L1 antibody drug in vivo, and has a remarkable effect in pharmacokinetic research.

Description

[0001] This application is a divisional application of the invention patent application entitled "Anti-idiotypic Antibodies and Applications" and application number 202410863041.7 submitted by the applicant on June 29, 2024. Technical Field

[0002] The present invention relates to the field of biotechnology, in particular to an anti-idiotypic antibody and its application. Background Art

[0003] PD-L1 is a protein that allows some cells to escape attack by the immune system. PD-L1 extends from the surface of cancer cells and interacts with a protein called PD-1 on T cells, a key immune system cell. This coupling—known as an immune checkpoint—instructs T cells to leave tumor cells. Checkpoint inhibitors prevent the PD-1 / PD-L1 interaction from occurring. Without receiving the "stop" signal from the PD-L1 protein, T cells can continue to attack tumor cells. The PD-L1 antibody is an unconjugated, approximately 33 kDa, rabbit-derived, anti-PD-L1 monoclonal antibody. There are already marketed drugs targeting the PD-1 target. Different anti-PD-1 / PD-L1 monoclonal antibodies vary in stability, affinity, Fc region characteristics, dosing, indications, immunogenicity, and pharmacokinetics. Anti-idiotypic antibodies play an important role in pharmacokinetic studies and immunogenicity testing during the development of these antibody drugs.

[0004] Anti-idiotypic antibodies are antibodies that can specifically recognize and bind to the variable region of another antibody. They are widely used in drug development, serving as an important reference for immunogenicity analysis of antibody drugs. They can also specifically detect antibody drug levels in the body, serve as important reagents for pharmacokinetic studies, and serve as standards for detecting the level of anti-antibodies produced in vivo against antibody drugs. Summary of the Invention

[0005] The present invention provides an anti-idiotypic antibody, an anti-drug antibody (ADA) kit and its application for anti-PD-L1 monoclonal antibody drugs. The anti-idiotypic antibody of the present invention is prepared using hybridoma technology.

[0006] The anti-idiotypic antibodies provided by the present invention correspond to the cell line names 1B3, 6C1, 8C8, 8H9, and 7B8.

[0007] The amino acid sequences of the heavy chain variable region of the heavy chain and the light chain variable region of the light chain of 1B3 are shown in SEQ ID No. 1 and SEQ ID No. 2, respectively;

[0008] The amino acid sequences of the heavy chain variable region of the heavy chain and the light chain variable region of the light chain of 6C1 are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively;

[0009] The amino acid sequences of the heavy chain variable region of the heavy chain and the light chain variable region of the light chain of 8C8 are shown in SEQ ID No. 5 and SEQ ID No. 6, respectively;

[0010] The amino acid sequences of the heavy chain variable region of the heavy chain and the light chain variable region of the light chain of 8H9 are shown in SEQ ID No. 7 and SEQ ID No. 8, respectively;

[0011] The amino acid sequences of the heavy chain variable region of the heavy chain and the light chain variable region of the light chain of 7B8 are shown in SEQ ID No. 9 and SEQ ID No. 10, respectively.

[0012] The heavy chain constant regions of the above antibodies are all identical, and their amino acid sequences are shown in SEQ ID No. 11. The light chain constant regions are also identical, and their amino acid sequences are shown in SEQ ID No. 12.

[0013] The present invention also discloses the use of the above-mentioned anti-idiotypic antibody in preparing a reagent / kit for detecting anti-PD-L1 antibodies in a biological sample.

[0014] For example, an immunoassay kit for specifically detecting anti-PD-L1 antibodies in a biological sample comprises: (a) the anti-idiotypic antibody described above; and (b) a detectable antibody that binds to the anti-PD-L1 antibody.

[0015] The beneficial effects of the present invention are as follows: the anti-idiotypic antibody provided is a monoclonal antibody that can specifically bind to the corresponding PD-L1 antibody drug, and the anti-idiotypic antibody is used to reflect the antibody drug content in the human body; the anti-idiotypic antibody of the present invention is prepared using hybridoma technology, and the ADA kit using the antibody can quantitatively detect the concentration level of the PD-L1 antibody drug in the body, which has a significant effect in pharmacokinetic studies. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is an antibody drug FC removal experiment.

[0017] Figure 2 The following are the antibody concentration test results of different antibody strains. DETAILED DESCRIPTION

[0018] The present invention is described in detail below in conjunction with the embodiments and drawings. The following embodiments are implemented on the premise of the technical solution of the present invention, and provide detailed implementation methods and specific operating processes, but the protection scope of the present invention is not limited to the following embodiments.

[0019] Example

[0020] 1.1 Experimental Animals

[0021] Ten Balb / C mice aged 5-8 weeks.

[0022] 1.2 Preparation of F(ab)2 Antigen

[0023] The PD-L1 monoclonal antibody drug was enzymatically cleaved and the F(ab) was purified. The amino acid sequences of the heavy chain and light chain of the PD-L1 monoclonal antibody drug are shown in SEQ ID Nos. 13 and 14, respectively.

[0024] 1.3 Immunization methods

[0025] The first main injection uses Freund's complete adjuvant, and subsequent booster injections use Freund's incomplete adjuvant. Both are fully mixed with equal volumes of antigen before injection. The immunization method is multiple injections at the back. See the immunization cycle for details. Figure 1 .

[0026] operate date dose adjuvant Main injection 2023.3.31 100 μg / mouse Freund's complete adjuvant First reinforcement 2023.4.7 50 μg / mouse Freund's incomplete adjuvant Second reinforcement 2023.4.21 50 μg / mouse Freund's incomplete adjuvant The third reinforcement 2023.5.6 50 μg / mouse Freund's incomplete adjuvant Take a serum sample 2023.5.15 / / The fourth reinforcement 2023.5.22 50 μg / mouse Freund's incomplete adjuvant

[0027] 1.4 Serum titer detection

[0028] Initial screening: three indirect tests + Capture Elisa + Bridge Elisa

[0029] Table 1 Absorbance values ​​of immunogens after enzyme digestion

[0030]

[0031]

[0032] Table 2 Absorbance values ​​of negative screening original determination

[0033]

[0034] Table 3 Absorbance values ​​of human IgG determination

[0035]

[0036]

[0037] Table 4 Absorbance values ​​of Capture Elisa and Bridge Elisa primary screening

[0038]

[0039] The initial screening showed that mouse-1R 2# and mouse-2O 9# had the best results, so retesting was performed:

[0040] Table 5 Absorbance values ​​of serum titer determination

[0041]

[0042]

[0043] Table 6 Absorbance values ​​for determination of serum reaction strength

[0044]

[0045] Experimental summary: After verification by experimental results, 9# mice were more responsive, so it was decided to fuse 9# mice.

[0046] 1.5 Fusion and Screening

[0047] (1) Myeloma cell preparation

[0048] One week before fusion, SP2 / 0 cells were revived and cultured normally to the logarithmic phase.

[0049] (2) Preparation of spleen cells

[0050] The mice to be fused were selected and sacrificed by cervical dislocation on the day of fusion. The spleens were removed and spleen cells were collected and counted according to standard procedures.

[0051] (3) Cell fusion

[0052] Myeloma cells and spleen cells were mixed at a ratio of 1:3-1:10 and cell fusion was performed according to standard procedures. Hybridoma cells were then cultured in HAT complete DMEM medium. Hybridoma cells were visible 3 days after fusion. On the 7th day, the medium was replaced with 1 / 2 HAT complete medium, and on the 8th day, the medium was replaced with 1 / 2 HT medium. Screening began approximately 10 days after fusion.

[0053] Cell fusion results: After fusion, the cells were cultured in HAT selective medium and observed under a microscope. Multiple growing hybridoma cells were seen, proving that the fusion operation was successful.

[0054] (4) Fusion screening

[0055] Pipette 100 μL of cell supernatant per well for indirect ELISA testing. Determine the positive wells based on the ELISA results. Use a single-channel pipette to inspect the entire plate for positive wells and perform a second retest to further confirm the positive wells.

[0056] (5) Subcloning

[0057] The cells from the rescreened positive wells were subcloned twice. (Because the cell lines from the positive wells obtained in the first subcloning are not stable and may contain multiple hybridoma cells, it is generally believed that the hybridoma cells after the second subcloning are a single cell line and are confirmed to be positive).

[0058] For the first subclone, cells from the limiting dilution positive wells were transferred to multiple wells and cultured with HT DMEM medium. After about 7 days, the cells were observed under a microscope and the wells with cloned growth were detected by indirect ELISA. The wells with high OD values ​​were selected as positive wells. The cells from the positive wells were picked for the second subcloning. Stable positive hybridoma cell lines were detected and used as the cells for the final preparation of monoclonal antibodies and expanded in culture.

[0059] (6) Monoclonal antibody subtype identification

[0060] The subtype of each supernatant was determined using a Southern Biotech monoclonal antibody subtyping kit. Prepare a strip coated with the immunogenic protein at 50 ng / well. Collect 600 μL of supernatant from each clone and add 100 μL / well to six enzyme-labeled wells containing the corresponding protein. Incubate at 37°C for 1 hour, wash three times with PBST, and then add diluted secondary antibodies against IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 to each of the six wells. Incubate at 37°C for 1 hour, wash three times with PBST, and develop with TMB. Wells showing a signal corresponding to the subtype of the subtype identified by the secondary antibody are the subtypes of the antibody.

[0061] (7) After two rounds of subcloning and retesting, the following positive cell lines were identified:

[0062] Table 7 Absorbance values ​​of F(ab)2 screening assay

[0063]

[0064] Table 8 Absorbance values ​​determined by negative screening

[0065]

[0066] Table 9 F(ab)2-negative screening difference

[0067]

[0068]

[0069] (8) Final positive cell line number and subtype:

[0070]

[0071] 1.6 Hybridoma cell line sequencing

[0072] 1.6.1 Experimental Procedure

[0073] 1) Cultivation of hybridoma cells

[0074] After reviving the hybridoma cell line, culture it until the cell number is expanded to about 1×10 7The cells were collected by centrifugation at 1000 rpm for 5 min.

[0075] 2) Extraction of cellular RNA

[0076] Under a clean bench, add 1 mL of Trizol reagent to the centrifuged cells, let stand for 5 minutes, add 2 mL of chloroform, shake vigorously for 15 seconds, let stand at room temperature for 3 minutes, and spin at 12,000 rpm for 15 minutes. Transfer the upper aqueous layer to a new EP tube, add 0.5 mL of isopropanol, and let stand at room temperature for 10 minutes. Spin at 12,000 rpm for 10 minutes. Discard the supernatant, add 1 mL of 75% ethanol, and spin at 7,500 rpm for 5 minutes. Dry the pellet and add 50 μL of double-distilled water. Assess purity and quantify by agarose gel electrophoresis and store at -70°C until needed.

[0077] 3) Reverse transcription to prepare cDNA

[0078] 1 μL of total cell RNA, 6 μL of RNase-Free ddH2O, 0.5 μL of oligodT Primer, 0.5 μL of PRIME ScriptRT Enzyme Mix I, 2 μL of 5x Prime Script Buffer, mix well, incubate at 37°C for 15 min, and then at 85°C for 5 s.

[0079] 4) Amplification of cDNA

[0080] The above cDNAs were amplified using the mouse IgG VH and VL primer libraries designed by our company. 10 μL of 5x PrimeStar Buffer, 4 μL of dNTPs, 1 μL of cDNA, 1 μL of upstream primer, 1 μL of downstream primer, and 0.5 μL of PrimeSTAR were added to 50 μL of water. PCR reactions were performed using the following conditions: incubation at 94°C for 5 minutes, denaturation at 94°C for 45 seconds, annealing at 63°C for 45 seconds, extension at 72°C for 1 minute, and 30 cycles followed by extension at 72°C for 10 minutes.

[0081] 5) Agarose gel electrophoresis and gel recovery

[0082] The above PCR products were subjected to agarose gel electrophoresis, the electrophoresis results were observed, and the amplified products with a molecular weight of 250-350 bp were sent for sequencing.

[0083]

[0084] 1.7 Development of the kit

[0085] Perform initial screening tests for anti-idiotypic antibodies:

[0086] Table 10 Primary screening of anti-idiotypic antibodies

[0087]

[0088] In this experiment, the OD value of the anti-idiotypic antibody test of strain 1B3 had no gradient, which did not meet the requirements; the gradients of other strains were normal, and the next step of the standard curve detection experiment could be carried out.

[0089] Table 11 Standard curve detection of anti-idiotypic antibodies

[0090]

[0091] Table 11 Standard curve detection of anti-idiotypic antibodies

[0092]

[0093]

[0094] The antibody concentration test results of different antibody strains are shown in Figure 2 .

[0095] During this screening process, the linear range of the anti-idiotypic antibody detection of strain 6C1 was 0-1500 ng / mL, the correlation coefficient R2 was 0.9703, and the optimal coating concentration of the antibody drug was 1 μg / mL; the linear range of the anti-idiotypic antibody detection of strain 8C8 was 0-750 ng / mL, the correlation coefficient R2 was 0.9892, and the optimal coating concentration of the antibody drug was 0.8 μg / mL; the linear range of the anti-idiotypic antibody detection of strain 8H9 was 0-750 ng / mL, the correlation coefficient R2 was 0.9905, and the optimal coating concentration of the antibody drug was 1 μg / mL; the linear range of the anti-idiotypic antibody detection of strain 7B8 was 0-750 ng / mL, the correlation coefficient R2 was 0.9944, and the optimal coating concentration of the antibody drug was 0.8 μg / mL; based on the three factors of small linear range of detection, correlation coefficient R and coating concentration, the anti-idiotypic antibody of strain 7B8 should be the best. Using the anti-idiotypic antibody strain 7B8, combined with the antibody concentration standard curve, the antibody concentration level produced in the human body against antibody drugs can be quantitatively analyzed.

Claims

1. An anti-idiotypic antibody against PD-L1 antibody, characterized in that: The anti-idiosyncratic antibody comprises a heavy chain and a light chain; The amino acid sequences of the heavy chain variable region of the heavy chain and the light chain variable region of the light chain are shown in SEQ ID No. 7 and SEQ ID No. 8, respectively.

2. The anti-idiotypic antibody against PD-L1 antibody according to claim 1, characterized in that: The heavy chain further comprises a heavy chain constant region, the amino acid sequence of which is shown in SEQ ID No.11, and the light chain further comprises a light chain constant region, the amino acid sequence of which is shown in SEQ ID No.

12.

3. Use of the anti-idiotypic antibody against PD-L1 antibody according to claim 1 in the preparation of a reagent / kit for detecting anti-PD-L1 antibodies in a biological sample.

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