Anti-idiotypic antibody and its applications

Abstract

The present invention discloses an anti-idiotypic antibody and its application. The provided anti-idiotypic antibody is a monoclonal antibody that can specifically bind to the corresponding PD-L1 antibody drug, and the content of the antibody drug in the human body can be reflected by the anti-idiotypic antibody. The anti-idiotypic antibody of the present invention is prepared by hybridoma technology. The ADA kit using this antibody can quantitatively detect the concentration level of the PD-L1 antibody drug in vivo and has a significant role in pharmacokinetic studies.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically refers to an anti-idiotypic antibody and its application. Background Art

[0002] PD-L1 is a protein that allows some cells to escape the attack of the immune system. PD-L1 extends from the surface of cancer cells and interacts with a protein called PD-1 on important immune system cells, T cells. This coupling - known as an immune checkpoint - instructs T cells to leave tumor cells. Checkpoint inhibitor drugs prevent the occurrence of the encounter between PD-1 / PD-L1. In the absence of the "stop" signal sent by the PD-L1 protein, T cells can continue to attack tumor cells. PD-L1 Antibody is a non-conjugated, rabbit-derived, anti-PD-L1 monoclonal antibody with a molecular weight of approximately 33 kDa. In the prior art, there are already marketed drugs acting on the target PD-1, and different anti-PD-1 / PD-L1 monoclonal antibody drugs vary in terms of stability, affinity, Fc segment characteristics, dosage, indications, immunogenicity, and pharmacokinetics. Anti-idiotypic antibodies play an important role in pharmacokinetic studies and immunogenicity detection during the research and development of such antibody drugs.

[0003] An anti-idiotypic antibody refers to an antibody that can specifically recognize the variable region of another antibody and produce specific binding. It has a very wide application in drug development. It can be used as an important reference for the immunogenicity analysis of antibody drugs, and can also specifically detect the level of antibody drugs in vivo, serving as an important reagent for pharmacokinetic studies. It can also be used as a standard product to detect the level of anti-antibodies against antibody drugs in vivo. Summary of the Invention

[0004] 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.

[0005] The anti-idiotypic antibodies provided by the present invention correspond to the cell line names 1B3, 6C1, 8C8, 8H9, 7B8.

[0006] Among them, 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;

[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 6C1 are shown in SEQ ID No.3 and SEQ ID No.4 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 8C8 are shown in SEQ ID No.5 and SEQ ID No.6 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 8H9 are shown in SEQ ID No.7 and SEQ ID No.8 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 7B8 are shown in SEQ ID No.9 and SEQ ID No.10 respectively.

[0011] The heavy chain constant regions of the above antibodies are all the same, and their amino acid sequences are shown in SEQ ID No.11, and the light chain constant regions are also the same, and their amino acid sequences are shown in SEQ ID No.12.

[0012] The present invention also discloses the use of the above anti-idiotype antibody in the preparation of a reagent / kits for detecting anti-PD-L1 antibody in a biological sample.

[0013] For example, an immunoassay kit for specifically detecting anti-PD-L1 antibody in a biological sample, which comprises: (a) the above anti-idiotype antibody; (b) a detectable antibody that binds to the anti-PD-L1 antibody.

[0014] The beneficial effects of the present invention are as follows: The provided anti-idiotype antibody is a monoclonal antibody that can specifically bind to the corresponding PD-L1 antibody drug, and the content of the antibody drug in the human body can be reflected by the anti-idiotype antibody; the anti-idiotype antibody of the present invention is prepared by hybridoma technology, and the ADA kit using this antibody can quantitatively detect the concentration level of the PD-L1 antibody drug in the body, and has a significant effect in pharmacokinetic studies. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is for the FC excision experiment of the antibody drug.

[0016] Figure 2 It is the detection result of the antibody concentration of different antibody strain numbers. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention will be described in detail below in conjunction with the embodiments and the drawings. The following embodiments are implemented on the premise of the technical solution of the present invention, and the detailed implementation manners and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0018] Embodiment

[0019] 1.1 Experimental animals

[0020] Ten Balb / C mice aged 5-8 weeks.

[0021] 1.2 Preparation of F(ab)2 antigen

[0022] The PD-L1 monoclonal antibody drug was enzymatically cleaved and 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 No. 13 and 14, respectively.

[0023] 1.3 Immunization methods

[0024] The first main injection uses Freund's complete adjuvant, and the subsequent booster injections use Freund's incomplete adjuvant, which are fully mixed with an equal volume of antigen before injection. The immunization method is multiple injections at the back. See the immunization cycle for details. Figure 1 .

[0025] Operation Date Dose Adjuvant Primary injection 2023.3.31 100 μg / animal Complete Freund's adjuvant First boost 2023.4.7 50 μg / animal Incomplete Freund's adjuvant Second boost 2023.4.21 50 μg / animal Incomplete Freund's adjuvant Third boost 2023.5.6 50 μg / animal Incomplete Freund's adjuvant Collect small serum sample 2023.5.15 / / Fourth boost 2023.5.22 50 μg / animal Incomplete Freund's adjuvant

[0026] 1.4 Serum titer detection

[0027] Initial screening: three indirect methods + Capture Elisa + Bridge Elisa

[0028] Table 1 Absorbance values of immunogens after enzyme digestion

[0029]

[0030]

[0031] Table 2 Absorbance values of negative screening original determination

[0032]

[0033] Table 3 Absorbance values of human IgG determination

[0034]

[0035]

[0036] Table 4 Absorbance values of Capture Elisa and Bridge Elisa initial screening

[0037]

[0038] After the initial screening, mouse-1R 2# and mouse-2O 9# showed the best results, so we conducted a retest:

[0039] Table 5 Absorbance values of serum titer determination

[0040]

[0041]

[0042]

[0043] Table 6 Absorbance values for determining the strength of serum reaction

[0044]

[0045] Experimental summary: After verification by experimental results, mouse No. 9 showed stronger reactivity, and it was decided to fuse mouse No. 9.

[0046] 1.5 Fusion and screening

[0047] (1) Preparation of myeloma cells

[0048] One week before fusion, resuscitate SP2 / 0 cells and culture them normally until the logarithmic phase.

[0049] (2) Preparation of spleen cells

[0050] Select the mice to be fused, sacrifice them by cervical dislocation on the day of fusion, take out the spleens, and collect spleen cells according to the standard procedure and count them.

[0051] (3) Cell fusion

[0052] Mix myeloma cells and spleen cells at a ratio of 1:3 - 1:10, perform cell fusion operation according to the standard procedure, and then culture them with HAT DMEM complete medium. Hybridoma cells can be seen 3 days after fusion, change to 1 / 2 HAT complete medium on the 7th day, and change to 1 / 2 HT medium on the 8th day. Screening detection starts about 10 days after fusion.

[0053] Results of cell fusion: After fusion, culture with HAT selective medium and observe under the microscope. Multiple growing hybridoma cells are seen, proving that the fusion operation is successful.

[0054] (4) Fusion screening

[0055] Aspirate 100 μL of cell supernatant per well for indirect ELISA detection. According to the ELISA results, judge the positive wells. Use a single-channel pipette to pick and test the positive wells detected on the whole plate for a second recheck to further confirm the positive wells.

[0056] (5) Subcloning

[0057] Perform two rounds of subcloning on the cells in the positive wells after re-screening. (Because the positive well cell lines obtained from the first subcloning are still unstable and may contain multiple hybridoma cells. It is generally believed that the hybridoma cells are single cell lines after the second subcloning and are determined to be positive).

[0058] Subclone the cells in the positive wells of the first subclone by limited dilution into multiple wells, add HT DMEM medium for culture, observe under a microscope after about 7 days, detect the wells with clone growth by indirect ELISA, and select the wells with high OD values as positive wells; pick the cells from the positive wells for the second subclone, detect the stable positive hybridoma cell lines, which serve as the cells for the final preparation of monoclonal antibodies, and expand the culture.

[0059] (6) Identification of monoclonal antibody subtypes

[0060] Use the monoclonal antibody subtype identification kit from Southern Biotech, USA, to measure the subtypes of each supernatant. Prepare the strip plates coated with immunogenic proteins, 50 ng / well. Collect 600 μL of supernatant from each clone and add 100 μL / well to the corresponding enzyme-labeled wells of 6 proteins. Incubate at 37 °C for 1 h, wash three times with PBST. Add the diluted typing secondary antibodies against IgM, IgA, IgG1, IgG2a, IgG2b, and IgG3 to the 6 wells, incubate at 37 °C for 1 h, wash three times with PBST, and develop color with TMB. The subtype of the identification secondary antibody corresponding to the well with a signal reaction is the subtype of the antibody.

[0061] (7) After two rounds of subcloning and rechecking, the following positive cell lines are determined:

[0062] Table 7 Absorbance values for F(ab)2 screening determination

[0063]

[0064] Table 8 Absorbance values for negative screening determination

[0065]

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

[0067]

[0068]

[0069]

[0070]

[0071]

[0072] 1.6 Sequencing of hybridoma cell lines

[0073] 1.6.1 Experimental procedure

[0074] 1) Culture hybridoma cells

[0075] Culture the revived hybridoma cell line. When the cell count expands to approximately 1×10 7 , centrifuge at 1000 rpm for 5 min to collect the cells.

[0076] 2) Extract cell RNA

[0077] Under the environment of a laminar flow hood, add 1 ml of Trizol reagent to the centrifuged cells, let it stand for 5 min, add 2 mL of chloroform, shake vigorously for 15 sec, let it stand at room temperature for 3 min, centrifuge at 12000 rpm for 15 min, transfer the upper aqueous layer to a new EP tube, add 0.5 mL of isopropanol, let it stand at room temperature for 10 min. Centrifuge at 12000 rpm for 10 min. Discard the supernatant, add 1 mL of 75% ethanol, centrifuge at 7500 rpm for 5 min, dry the precipitate, and add 50 μL of double-distilled water. Identify the purity and quantify by agarose gel electrophoresis, and store at -70 °C for later use.

[0078] 3) Reverse transcribe to prepare cDNA

[0079] 1 μL of total cell RNA, 6 μL of RNase Free ddH2O, 0.5 μL of oligodT Primer, 0.5 μL of PRIME Script RT 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.

[0080] 4) Amplify cDNA

[0081] Use the mouse IgG VH VL primer library designed by our company to amplify the above cDNA respectively. 10 μL of 5x PrimeStar Buffer, 4 μL of dNTP, 1 μL of cDNA, 1 μL of upstream primer, 1 μL of downstream primer, 0.5 μL of PrimeSTAR, make up the volume to 50 μL with water. Perform PCR reaction according to the following reaction conditions: incubate at 94 °C for 5 min, denature at 94 °C for 45 s, anneal at 63 °C for 45 s, extend at 72 °C for 1 min, after 30 cycles, extend at 72 °C for 10 min.

[0082] 5) Agarose gel electrophoresis and gel extraction

[0083] Perform agarose gel electrophoresis on the above PCR products, observe the electrophoresis results, and send the amplified products with a molecular weight of 250 - 350 bp for sequencing.

[0084]

[0085] 1.7 Development of the kit

[0086] Perform a primary screening experiment on the anti-idiotypic antibody:

[0087] Table 10 Primary Screening of Anti-idiotypic Antibodies

[0088]

[0089] In this experiment, the OD values of the anti-idiotypic antibody detection of strain number 1B3 had no gradient and did not meet the requirements; the gradients of other strain numbers were normal, and the next standard curve detection experiment could be carried out.

[0090] Table 11 Standard Curve Detection of Anti-idiotypic Antibodies

[0091]

[0092] Table 11 Standard Curve Detection of Anti-idiotypic Antibodies

[0093]

[0094] The detection results of the antibody concentrations of different antibody strain numbers are shown in Figure 2 .

[0095] During this screening process, the detection linear range of the anti-idiotypic antibody of strain number 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 detection linear range of the anti-idiotypic antibody of strain number 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 detection linear range of the anti-idiotypic antibody of strain number 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 detection linear range of the anti-idiotypic antibody of strain number 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; according to the three factors of the small detection linear range, the correlation coefficient R, and the coating concentration, the optimal one should be the anti-idiotypic antibody of strain number 7B8. Using the anti-idiotypic antibody of strain number 7B8 and combining with the antibody concentration standard curve, the antibody concentration level produced in the human body against the antibody drug can be quantitatively analyzed.

Claims

1. An anti-idiotypic antibody against a PD-L1 antibody, characterized in that: The anti-idiotypic 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. 9 and SEQ ID No. 10, respectively.

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

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