CN116836291B - Anti-idiotype antibody of anti-CD 47-CLDN18.2 bispecific antibody, preparation method and application thereof - Google Patents
Anti-idiotype antibody of anti-CD 47-CLDN18.2 bispecific antibody, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an anti-idiotype antibody of an anti-CD 47-CLDN18.2 bispecific antibody, a preparation method and application thereof. The invention develops an anti-idiotype antibody for identifying the anti-CD 47-CLDN18.2 double antibody, can be applied to quantitative detection of the anti-CD 47-CLDN18.2 double antibody in preclinical research, and is an important reagent for analysis of Pharmacokinetics (PK) and immunogenicity (ADA). Provides a powerful tool for biological analysis of clinical tests, and has wide application prospect and great market value.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to an anti-idiotype antibody of an anti-CD 47-CLDN18.2 bispecific antibody, a preparation method and application thereof.
Background
CD47, also known as an integrin-associated protein, belongs to the immunoglobulin superfamily, and is widely found on the surface of a variety of normal cells and is also overexpressed in tumor tissue of a variety of tumor types (hematological tumors, solid tumors). CD47 ligands include sirpa (signal regulatory protein α), TSP-1, integrins, etc., with the most reported sirpa being mainly expressed on the cell surface of macrophages, dendritic cells, etc. The CD47 is connected with SIRPalpha on the surface of the macrophage to cause the intracellular ITIM of the SIRPalpha to be phosphorylated, and a series of cascade reactions are generated to inhibit the phagocytosis of the macrophage, thereby avoiding the accidental injury of normal cells and also causing tumor cells expressing the CD47 to generate immune escape. Therefore, the development of the medicine targeting CD47 can block the immune inhibition signal mediated by CD47/SIRP alpha in tumor tissues, thereby playing an anti-tumor role.
CLDN18.2 belongs to members of the family of compact junction proteins (CLDNs), and is mainly expressed in epithelial cells of an organism, and abnormal CLDN proteins can cause structural damage and functional impairment of the epithelial cells, lower intercellular adhesion and lost polarity, cause normal differentiation of cells to be lost and invasive, cause occurrence and development of various tumors, and have a certain relationship with invasion and metastasis of tumors. CLDN18 is one of the members of the CLDN protein family, including CLDN18.1 and CLDN18.2 subtypes, with the expression of CLDN18.1 and CLDN18.2 being tissue specific in humans, CLDN18.1 being predominantly expressed in lung tissue and CLDN18.2 being predominantly expressed on gastric mucosa, small intestinal epithelial cells of gastrointestinal tissue. The CLDN18.2 molecules are highly expressed in the primary gastric cancer and various cancer types after metastasis, and in addition, the activated expression of the CLDN18.2 is observed in pancreatic cancer, esophageal cancer, colon cancer and ovarian cancer, and the high expression characteristic of the CLDN18.2 tumor makes the CLDN18.2 tumor become a tumor treatment target with great potential. WO2016165762A1 discloses an anti-CLDN 18.2 antibody, IMAB362 (Zolbetuximab), which shows significantly longer survival than standard chemotherapy in clinical secondary trials of gastric cancer (13.2 versus 8.4 months), with a more pronounced advantage in CLDN18.2 highly expressed patients.
Conventional targeted or immunotherapeutic drugs can only be used to inhibit one target or class of targets, so that some unique combined drug schemes are created; the biggest characteristic of the double antibody is that the medicine can inhibit two or two kinds of targets at the same time, and the effect of the combined treatment of the two medicines can be achieved by using the force of one medicine. As in WO2021003082A1, anti-CLDN 18.2 and CD47 bispecific antibodies are disclosed which do not substantially bind human erythrocytes. CN115991784A discloses an anti-CD 47-CLDN18.2 specific antibody NA3S-H1, the double antibody has high affinity with CLDN18.2, and the medicine can specifically bind to CLDN18.2 positive tumor cells; has low affinity with CD47, but can still block the CD47/SIRP alpha signal path so as to relieve the immune inhibition mediated by CD47 in tumor. In non-clinical studies, the diabody showed dose-dependent anti-tumor effects in human gastric cancer, human pancreatic cancer and human colon cancer mouse tumor models over-expressing CLDN18.2, and was significantly superior to CD47 mab (Hu 5F 9) and CLDN18.2 mab (IMAB 362) currently in clinical triple development.
However, when the anti-CD 47-CLDN18.2 double-antibody is used as an exogenous protein, the anti-antibody can be generated after being injected into a body, so that the drug effect is reduced, even the body is initiated to generate autoimmune reaction, and the life and health are threatened. Therefore, it is important to conduct pharmacokinetic, immunogenicity, etc. analyses of animals and humans before antibody drugs are put into production and marketed. In the development and detection of antibody drugs, a particular class of antibodies is of interest. The method can be used as an important reference for detecting the anti-drug antibody, is used for evaluating the immunogenicity of the drug, can also be used as a detection reagent for detecting the content of the antibody drug in human or animal serum, and can be used for analyzing the pharmacokinetics of the antibody drug. Such antibodies are anti-idiotype antibodies.
In summary, the development of an anti-idiotype antibody for recognizing the anti-CD 47-CLDN18.2 double antibody can be applied to quantitative detection of the anti-CD 47-CLDN18.2 double antibody drug, and has important significance for application of the anti-CD 47-CLDN18.2 double antibody drug.
Disclosure of Invention
Aiming at the defects and actual demands of the prior art, the invention provides an anti-idiotype antibody of an anti-CD 47-CLDN18.2 bispecific antibody, a preparation method and application thereof, and the invention develops the anti-idiotype antibody for identifying the anti-CD 47-CLDN18.2 bispecific antibody, can be applied to quantitative detection of anti-CD 47-CLDN18.2 diabodies in preclinical research, is an important reagent for analysis of Pharmacokinetics (PK) and immunogenicity (ADA), and provides a powerful tool for biological analysis of clinical experiments.
In order to achieve the above purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides an anti-idiotype antibody to a CD47-CLDN18.2 bispecific antibody comprising a first anti-idiotype antibody and/or a second anti-idiotype antibody; the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of the first anti-idiotype antibody respectively comprise sequences shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain respectively comprise sequences shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6; the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of the second anti-idiotype antibody comprise the sequences shown in SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain comprise the sequences shown in SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12, respectively.
The anti-idiotype antibody capable of effectively recognizing the anti-CD 47-CLDN18.2 specific antibody NA3S-H1 is developed, can be specifically combined with the anti-CD 47-CLDN18.2 double antibody, can be used for quantitative analysis of the anti-CD 47-CLDN18.2 antibody by a sandwich ELISA method, monitors and evaluates the metabolic process of the anti-CD 47-CLDN18.2 double antibody and the like, provides a powerful tool for biological analysis of clinical experiments, and has wide application prospect and huge market value.
SEQ ID NO.1：GYYMH。
SEQ ID NO.2：YINCYNGATSYNQNFKG。
SEQ ID NO.3：WEAYYGNYDY。
SEQ ID NO.4：RASKSVSTSGYSYMH。
SEQ ID NO.5：LVSNLES。
SEQ ID NO.6：QHIRELTR。
SEQ ID NO.7：SYTMH。
SEQ ID NO.8：YINPSSGYTNYNQKFKD。
SEQ ID NO.9：LHYYGY。
SEQ ID NO.10：QSIGTS。
SEQ ID NO.11：YAS。
SEQ ID NO.12：QQSNSWPNT。
Preferably, the amino acid sequence of the heavy chain of the first anti-idiotype antibody comprises the sequence shown in SEQ ID NO.13, and the amino acid sequence of the light chain comprises the sequence shown in SEQ ID NO. 14; the amino acid sequence of the heavy chain of the second anti-idiotype antibody comprises the sequence shown in SEQ ID NO.15, and the amino acid sequence of the light chain comprises the sequence shown in SEQ ID NO. 16.
SEQ ID NO.13：
EVQLQQSGPELVKTGASVKISCKASGYSFTGYYMHWVKQSHGKSLEWIGYINCYNGATSYNQNFKGKATFTVDTSSSTAYMQFNSLTSEDSAVYYCARWEAYYGNYDYWGQGTTLTVSS。
SEQ ID NO.14：
DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWEIK。
SEQ ID NO.15：
QVQLQQSGAELARPGASVKMSCKASGYTFTSYTMHWVKQRPGQGLEWIGYINPSSGYTNYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSAVYSCATLHYYGYWGQGTTLTVSS。
SEQ ID NO.16：
DILLTQSPAILSVSPGERVSFSCRASQSIGTSTHWYQQRKNGSPRLLIKYASESISGIPSRFSGSGSGTEFTLSINSVESEDIADYYCQQSNSWPNTLGGGTKLEIK。
In the invention, two anti-idiotype antibodies of the anti-CD 47-CLDN18.2 bispecific antibody are developed and respectively named as 30D12D3B5 (SEQ ID NO.13 and SEQ ID NO. 14) and 15E5G6D1 (SEQ ID NO.15 and SEQ ID NO. 16), wherein the 30D12D3B5 is combined with the anti-CD 47 end of the diabody, and the 15E5G6D1 is combined with the anti-CLDN 18.2 end of the diabody, and the two antibodies can be in the form of single preparations or combined as a combined preparation.
In a second aspect, the invention provides a nucleic acid molecule encoding an anti-idiotype antibody of the anti-CD 47-CLDN18.2 bispecific antibody of the first aspect.
In a third aspect, the present invention provides an expression vector comprising a nucleic acid molecule according to the second aspect.
In a fourth aspect, the invention provides a recombinant cell comprising a nucleic acid molecule according to the second aspect.
In a fifth aspect, the invention provides the use of an anti-idiotype antibody of an anti-CD 47-CLDN18.2 bispecific antibody according to the first aspect for the preparation of a product for detecting an anti-CD 47-CLDN18.2 bispecific antibody.
In a sixth aspect, the invention provides a kit for detecting an anti-CD 47-CLDN18.2 bispecific antibody comprising an anti-idiotype antibody of the anti-CD 47-CLDN18.2 bispecific antibody of the first aspect.
In a seventh aspect, the invention provides the use of an anti-idiotype antibody of an anti-CD 47-CLDN18.2 bispecific antibody according to the first aspect for detecting an anti-CD 47-CLDN18.2 bispecific antibody.
In an eighth aspect, the invention provides a method of detecting an anti-CD 47-CLDN18.2 bispecific antibody, the method comprising:
detection was performed using an anti-idiotype antibody of the anti-CD 47-CLDN18.2 bispecific antibody of the first aspect based on an enzyme-linked immunosorbent assay (ELISA).
Preferably, the method for detecting the anti-CD 47-CLDN18.2 bispecific antibody is a sandwich ELISA method, and specifically comprises the following steps:
coating the anti-idiotype antibody to ELISA plate reaction holes with the concentration of 1-3 mug/mL, standing at 20-30 ℃ for 0.5-3 h, discarding liquid in the holes, and then cleaning with PBS; adopting PBS to carry out 10-time serial dilution on the humanized antibody of the anti-human CD47-CLDN18.2, standing for 0.5-3 h at 20-30 ℃, discarding the liquid in the hole, and then washing with PBS; adding an anti-idiotype antibody to be detected and paired, wherein the concentration is 1.0-3 mug/mL, standing for 1-3 h at 20-30 ℃, discarding the liquid in the hole, and then cleaning with PBS; adding strepitavidin-HRP, standing at 20-30 ℃ for 10-40 min, discarding liquid in the holes, and then washing with PBS; adding a reaction substrate, performing light-shielding color development at 20-30 ℃ for 10-15 min, adding a stop solution, and detecting the light absorption value by using an enzyme-labeled instrument.
Compared with the prior art, the invention has the following beneficial effects:
the anti-idiotype antibody for screening and preparing the anti-CD 47-CLDN18.2 double antibody can be specifically and efficiently combined with the anti-CD 47-CLDN18.2 double antibody, has no cross reaction with other IgG1 type antibodies, can determine the serum level of the antibody given in an early test, is favorable for planning a future treatment method and is used for determining the Pharmacokinetics (PK), immunogenicity (ADA) and the like of the CD47-CLDN18.2 double antibody, provides a powerful tool for biological analysis of clinical tests, and has wide application prospect and huge market value.
Drawings
FIG. 1A is a graph showing the results of specific binding between anti-idiotype antibodies (15G 11E11A12, 29A1C6B 10) at the anti-CD 47-CDN18.2 double anti-CD 47 end and CD47-CLDN18.2 double antibodies;
FIG. 1B is a graph showing the results of specific binding between anti-idiotype antibodies (6E 7C8G4, 26C1E8A 6) at the anti-CD 47-CDN18.2 double anti-CD 47 end and CD47-CLDN18.2 double antibodies;
FIG. 1C is a graph showing the results of specific binding between anti-idiotype antibodies (19A 2C8D6, 9F6D6E 7) at the anti-CD 47 end of anti-CD 47-CDN18.2 dual anti-CD 47 and CD47-CLDN18.2 dual anti-antibodies;
FIG. 1D is a graph showing the results of specific binding between anti-idiotype antibodies (27A 11B4B10, 1E9B7A 8) at the anti-CD 47-CDN18.2 dual anti-CD 47 end and CD47-CLDN18.2 dual antibodies;
FIG. 1E is a graph showing the results of specific binding between anti-idiotype antibodies (23F 4D9B2, 30D12D3B 5) at the anti-CD 47-CDN18.2 dual anti-CD 47 end and CD47-CLDN18.2 dual antibodies;
FIG. 2 is a graph showing the results of specific binding between anti-idiotype antibody (1G 1B6F12, 1D2F4B 11) at the end of anti-CD 47-CDN18.2 dual anti-CLDN 18.2 and CDLN18.2 mab and CD47-CLDN18.2 dual anti-antibody;
FIG. 3 is a graph showing the results of specific binding between anti-idiotype antibody (15E 5G6D1, 3C3F10B 5) at the end of anti-CD 47-CDN18.2 dual anti-CLDN 18.2 and CDLN18.2 monoclonal antibody, CD47-CLDN18.2 dual antibody;
FIG. 4 is a graph showing the results of specific binding between anti-idiotype antibody (20E 12E8H 10) at the end of anti-CD 47-CDN18.2 dual anti-CLDN 18.2 and CDLN18.2 mab, CD47-CLDN18.2 dual antibody;
FIG. 5 is a graph showing the blocking activity of anti-idiotype antibodies at the anti-CD 47-CDN18.2 dual anti-CLDN 18.2 end.
Detailed Description
The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase through regular channels, with no manufacturer noted.
Example 1
This example screens for the preparation of anti-idiotype antibodies against the CD47 end.
(1) Immunization and cell fusion
The animals were digested with 5 Balb/C females and 5C 57 females, anti-CD 47-CLDN18.2 bispecific antibody (disclosed in CN 115991784A) and digested with enzyme to F (ab ') 2, 50. Mu.g of F (ab ') 2 in complete Freund's adjuvant (1:1, v: v) were injected subcutaneously into the animals on day 0, serum from immunized animals was detected by subcutaneous injection of incomplete Freund's adjuvant containing 25. Mu. g F (ab ') 2 on day 14, blood was collected on day 21 and ELISA, serum from immunized animals was detected by subcutaneous injection of incomplete Freund's adjuvant containing 25. Mu. g F (ab ') 2 on day 28, blood was collected on day 35 and ELISA. When the mice were able to reach the level of immune response against the immunogen (OD >1.0, titer 1:8,000), spleens of the mice were taken under sterile conditions and prepared into spleen cell suspensions, which were fused with myeloma cells SP2/0 2 times by electrofusion, and all cells fused each time were plated in 96-well plates.
(2) Monoclonal antibody selection
The supernatant of the fused cells was screened by ELISA, and the supernatant positive for the cleavage product F (ab') 2 was selected. In the case of the maternal clone selection, the supernatant of all maternal clone cells positive for the CD47-CLDN18.2 diabody was screened by indirect ELISA, while the negative screening was performed with human total IgG and IgG1 isotype control antibodies.
(3) Cloning hybridomas by limiting dilution
Positive master clones were subcloned by limiting dilution to ensure that these positive master clones were derived from individual master clone cells, respectively. Positive master clone cells were transferred to 24-well plates for expansion, and supernatants were collected for each expansion clone. 10 positive parent clones were obtained in the subcloning stage, with clone numbers 15G11E11A12, 29A1C6B10, 6E7C8G4, 26C1E8A6, 19A2C8D6, 9F6D6E7, 27A11B4B10, 1E9B7A8, 23F4D9B2, 30D12D3B5, respectively, and were stable in growth. Wherein 23F4D9B2 and 30D12D3B5 are non-blocking and non-competing, and the others are blocking.
(4) Antibody production
Performing expansion culture on the hybridoma cells, taking a mouse with the age of about 6 weeks,intraperitoneal injection of 0.5. 0.5 mL paraffin oil, 2×10 after one week 6 The abdomen of the mice is obviously enlarged 7 days after injection, ascites is collected, supernatant is centrifugally taken, monoclonal antibody ascites is obtained, and an anti-idiotype antibody is purified by using a protein A affinity chromatography method.
Example 2
This example uses an indirect ELISA method to detect the specific binding of an anti-idiotype antibody to the CD47-CLDN18.2 diabody.
Anti-idiotype antibodies with clone numbers 15G11E11a12, 29A1C6B10, 6E7C8G4, 26C1E8A6, 19A2C8D6, 9F6D6E7, 27a11B4B10, 1E9B7A8, 23F4D9B2 and 30D12D3B5 were validated in this experiment. Respectively adopting humanized antibody, humanized IgG antibody and humanized IgG1 antibody of anti-human CD47-CLDN18.2 double antibody to coat the ELISA reaction holes, wherein the addition amount of each hole is 100 mu L, the concentration is 1.0 mu g/mL, standing for 1. 1 h at 25 ℃, discarding liquid in the holes, and then washing with PBS; diluting the anti-idiotype antibody by 3 times serial with PBS, standing at 25deg.C for 1 h, discarding the liquid in the hole, and cleaning with PBS; adding peroxidase-labeled goat anti-mouse IgG antibody, standing at 25deg.C for 30 min, removing liquid in the hole, and cleaning with PBS; adding a reaction substrate, developing at 25 ℃ in dark for 10-15 min, adding a stop solution, and detecting the light absorption value by using an enzyme-labeled instrument.
As can be seen from table 1 and fig. 1A-1E, 15G11E11A12, 29A1C6B10, 6E7C8G4, 26C1E8A6, 19A2C8D6, 9F6D6E7, 27a11B4B10, 1E9B7A8, 23F4D9B2 and 30D12D3B5 anti-idiotype antibodies bind only to humanized antibodies against human CD47-CLDN18.2, and do not bind to human IgG antibodies and human IgG1 antibodies, indicating that anti-idiotype antibodies can specifically bind to CD47-CLDN18.2 diabodies.
TABLE 1
Note that: a, a double antibody against human CD47/CLDN 18.2; b, human IgG; c is Human IgG1.
Example 3
The blocking ELISA method is used in this example to detect the blocking activity of anti-idiotype antibodies.
15G11E11A12, 29A1C6B10, 6E7C8G4, 26C1E8A6, 19A2C8D6, 9F6D6E7, 27A11B4B10, 1E9B7A8, 23F4D9B2, and 30D12D3B5 anti-idiotype antibodies were validated in this experiment. Coating a human CD47 antigen containing a His tag into ELISA reaction holes, wherein the addition amount of each hole is 100 mu L, the concentration is 0.5 mu g/mL, standing at 25 ℃ for 1-h, discarding liquid in the holes, and then washing with PBS; adding the anti-idiotype antibody, wherein the addition amount of each hole is 50 mu L, the concentration is 10 mu g/mL, standing at 25 ℃ for 1 to h, discarding the liquid in the holes, and then washing with PBS; adding humanized antibody of anti-human CD47-CLDN18.2 double antibody, adding 50 mu L of the humanized antibody into each hole, keeping the concentration at 1000 ng/mL, standing at 25 ℃ for 1 h, discarding liquid in the hole, and washing with PBS; adding a mouse anti-human IgG Fc fragment enzyme conjugate IgG-HRP, standing at 25 ℃ for 30 min, removing liquid in the hole, and washing with PBS; adding a reaction substrate, developing at 25deg.C in dark for 13 min, adding a stop solution, detecting light absorption value with an enzyme-labeled instrument, and blocking rate= (1-sample OD) 450 Value/blocking Buffer average OD 450 Value) x 100%.
As can be seen from Table 2, the anti-idiotype antibody blocking rates of 15G11E11A12, 29A1C6B10, 6E7C8G4, 26C1E8A6, 19A2C8D6, 9F6D6E7, 27A11B4B10, 1E9B7A8, 23F4D9B2 and 30D12D3B5 were 81.27%, 93.27%, 92.61%, 70.76, 85.75%, 92.31%, 92.88%, 86.81%, -6.58% and 15.8%, respectively, indicating that 15G11E11A12, 29A1C6B10, 6E7C8G4, 26C1E8A6, 19A2C8D6, 9F6D6E7, 27A11B4B10, 1E9B7A8 were effective in blocking binding of the anti-CD 47-DN 18.2 diabody to the CD47 antigen.
TABLE 2
Example 4
This example prepares an anti-idiotype antibody against the CLDN18.2 end.
(1) Immunization and cell fusion
The anti-CD 47-CLDN18.2 bispecific antibody cleaves Fc by pharmaceutical enzymes and the cleavage product VHH-conjugated KLH carrier protein serves as an immunogen. 10 mice, 5 Balb/C mice and 5C 57 mice were routinely immunized with VHH+KLH. Animals were subcutaneously injected with 50. Mu.g of immunogen in complete Freund's adjuvant (1:1, v: v), animals were immunized by subcutaneous injection of incomplete Freund's adjuvant containing 25. Mu.g of immunogen on day 14, blood was collected on day 21 and serum from the immunized animals was detected by ELISA, and animals were immunized by subcutaneous injection of incomplete Freund's adjuvant containing 25. Mu.g of immunogen on day 28, blood was collected on day 35 and serum from the immunized animals was detected by ELISA. When the mice were able to reach the level of immune response against the immunogen (OD >1.0, titer 1:8,000), spleens of the mice were taken under sterile conditions and prepared into spleen cell suspensions, which were fused with myeloma cells SP2/0 2 times by electrofusion, and all cells fused each time were plated in 96-well plates.
(2) Monoclonal antibody selection
The supernatant of the fused cells was screened by ELISA, and the supernatant positive for the anti-VHH-Fc full-length antibody drug was selected. In the case of the parental screening, all positive parental cell supernatants against the BC007 diabodies were screened by indirect ELISA, while the anti-screening was performed with human total IgG and IgG1 isotype control antibodies.
(3) Cloning hybridomas by limiting dilution
Positive master clones were subcloned by limiting dilution to ensure that these positive master clones were derived from individual master clone cells, respectively. Positive master clone cells were transferred to 24-well plates for expansion, and supernatants were collected for each expansion clone. In the subcloning stage, 5 positive parent clones were obtained, with clone numbers 1G1B6F12, 1D2F4B11, 15E5G6D1, 3C3F10B5, and 20E12E8H10, respectively, and all grew stably.
(4) Antibody production and pairing
Amplifying and culturing hybridoma cells, taking about 6 weeks old mice, and abdominal cavity0.5. 0.5 mL paraffin oil was injected and 2X 10 was intraperitoneally injected after one week 6 The abdomen of the mice is obviously enlarged 7 days after injection, ascites is collected, supernatant is centrifugally taken, monoclonal antibody ascites is obtained, protein A affinity chromatography is used for purifying anti-idiotype antibody, pairing experiment based on sandwich ELISA is carried out on the antibody, and the pairing detection effect is confirmed.
Example 5
In this example, an indirect ELISA method was used to detect the specific binding of anti-idiotype antibodies to both the CLDN18.2 mab and the CD47-CLDN18.2 mab.
Anti-idiotype antibodies with clone numbers 1G1B6F12, 1D2F4B11, 15E5G6D1, 3C3F10B5, and 20E12E8H10 were validated in this experiment. Respectively adopting humanized antibodies of anti-human CLDN18.2 monoclonal antibodies and anti-human CD47-CLDN18.2 monoclonal antibodies, humanized IgG antibodies and humanized IgG1 antibodies to coat ELISA reaction holes, wherein the addition amount of each hole is 100 mu L, the concentration is 1.0 mu g/mL, standing at 25 ℃ for 1 h, discarding liquid in the holes, and then washing with PBS; diluting the anti-idiotype antibody by 3 times serial with PBS, standing at 25deg.C for 1 h, discarding the liquid in the hole, and cleaning with PBS; adding peroxidase-labeled goat anti-mouse IgG antibody, standing at 25deg.C for 30 min, removing liquid in the hole, and cleaning with PBS; adding a reaction substrate, developing for 13 min at 25 ℃ in a dark place, adding a stop solution, and detecting the light absorption value by using an enzyme-labeled instrument.
As can be seen from table 3 and fig. 2-4, the 1G1B6F12, 1D2F4B11, 15E5G6D1, 3C3F10B5, and 20E12E8H10 anti-idiotype antibodies bind only to the anti-human CLDN18.2 mab and the anti-human CD47-CLDN18.2 mab, and do not bind to the human IgG antibody and the human IgG1 antibody, indicating that the anti-idiotype antibodies can specifically bind to both CLDN18.2 and CD47-CLDN18.2 mab.
TABLE 3 Table 3
Example 6
This example uses a blocking FACS method to detect the blocking activity of anti-idiotype antibodies.
1G1B6F12, 1D2F4B11, 15E5G6D1, 3C3F10B5 and 20E12E8H10 anti-idiotype antibodies were validated in this experiment. Respectively adopting fusion serum as positive control, mouse IgG as isotype control, PBS as negative control, and adding corresponding anti-unique antibody into sample hole; the medicine adopts an anti-CLDN 18.2 antibody (working concentration is 0.1 ug/mL). 25. Incubation of the mu L anti-idiotype antibody and 25 mu L medicine at 4 ℃ for 40 min, centrifugation, washing with 150 mu L PBS after removing liquid in the hole, and centrifugation for 3 min at 300 g, and repeating for 2 times. CLDN18.2-HEK293 cells were used as the test cell line followed by incubation for 40 min with 1×10 cells per tube 5 And/50 μl/tube. Incubation was completed by rinsing with 150 μl PBS, centrifuging for 3 min at 300 g, and repeating 2 times. Alexa Fluor 647 marked goat anti-human IgG is added as a secondary antibody, incubated for 40 min at 4 ℃, and detected on the machine.
As shown in FIG. 5, the anti-idiotype antibodies 1G1B6F12, 1D2F4B11, 15E5G6D1, 3C3F10B5, and 20E12E8H10 blocked binding of the anti-CLDN 18.2 antibody to the over-expressed CLDN18.2-HEK293 cells.
Example 7
This example uses a sandwich ELISA method to detect pairing of anti-idiotype antibodies.
Idiotype antibodies derived from examples 1 and 4 were taken and validated in this experiment. Coating the anti-idiotype antibody to ELISA reaction holes with the concentration of 2.0 mug/mL, standing at 25 ℃ for 1 to h, discarding liquid in the holes, and then washing with PBS; the humanized antibody of the anti-human CD47-CLDN18.2 is serially diluted 10 times by adopting PBS, and is kept stand at 25 ℃ for 1 h, and after the liquid in the hole is removed, the humanized antibody is washed by PBS; adding an anti-uniqueness antibody at the other end, wherein the concentration is 1.0 mug/mL, standing at 25 ℃ for 1 h, discarding the liquid in the hole, and then washing with PBS; adding strepitavidin-HRP, standing at 25deg.C for 30 min, discarding the liquid in the hole, and cleaning with PBS; adding a reaction substrate, developing for 14 min at 25 ℃ in a dark place, adding a stop solution, and detecting the light absorption value by using an enzyme-labeled instrument.
From the pairing results of tables 4 and 5, it was found that 15E5G6D1 and 1G1B6F12 were the best paired anti-idiotype antibodies. 30D12D3B5 and 15E5G6D1 are a group of non-competitive coatings that can be used to check total drug content.
Coating an anti-idiotype antibody to ELISA reaction holes with the concentration of 2.0 mug/mL, standing at 25 ℃ for 1 to h, discarding liquid in the holes, and then washing with PBS; the humanized antibody of the anti-human CD47-CLDN18.2 is serially diluted 10 times by adopting PBS, and is kept stand at 25 ℃ for 1 h, and after the liquid in the hole is removed, the humanized antibody is washed by PBS; adding an anti-uniqueness antibody at the CLDN18.2 end of the anti-CD 47-CLDN18.2 double antibody obtained in the example 6, wherein the concentration is 1.0 mug/mL, standing at 25 ℃ for 1 h, discarding liquid in a hole, and then washing with PBS; adding strepitavidin-HRP, standing at 25deg.C for 30 min, discarding the liquid in the hole, and cleaning with PBS; adding a reaction substrate, developing for 12 min at 25 ℃ in a dark place, adding a stop solution, and detecting the light absorption value by using an enzyme-labeled instrument.
From the pairing results of table 4 (coated CD47 end idiotype antibody) and table 5 (coated CLDN18.2 end idiotype antibody), it was shown that the combination of multiple strains of idiotype antibodies, including 1E9B7A8, 15G11E11a12, 30D12D3B5, involved in CD47 end idiotype antibodies, including 1G1B6F12, 15E5G6D1, where 30D12D3B5 and 15E5G6D1 are a non-competitive coated set, could be used in drug generation studies to detect drug content in blood without being affected by free antigen, for subsequent sequencing.
TABLE 4 Table 4
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TABLE 5
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In summary, the anti-idiotype antibody capable of effectively recognizing the anti-CD 47-CLDN18.2 specific antibody NA3S-H1 is developed, can be specifically combined with the anti-CD 47-CLDN18.2 double antibody, can be used for quantitatively analyzing the anti-CD 47-CLDN18.2 antibody by a sandwich ELISA method, monitors and evaluates the metabolic process of the anti-CD 47-CLDN18.2 double antibody and the like, provides a powerful tool for biological analysis of clinical experiments, and has wide application prospect and huge market value.
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
Claims (9)
1. An anti-idiotype antibody against a CD47-CLDN18.2 bispecific antibody, characterized in that said anti-idiotype antibody comprises a first anti-idiotype antibody and/or a second anti-idiotype antibody;
the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of the first anti-idiotype antibody are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain are respectively shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6;
the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of the second anti-idiotype antibody are shown as SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9 respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain are shown as SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12 respectively.
2. The anti-idiotype antibody of anti-CD 47-CLDN18.2 bispecific antibody of claim 1, wherein the amino acid sequence of the heavy chain of said first anti-idiotype antibody comprises the sequence shown in SEQ ID No.13 and the amino acid sequence of the light chain comprises the sequence shown in SEQ ID No. 14;
the amino acid sequence of the heavy chain of the second anti-idiotype antibody comprises the sequence shown in SEQ ID NO.15, and the amino acid sequence of the light chain comprises the sequence shown in SEQ ID NO. 16.
3. A nucleic acid molecule encoding an anti-idiotype antibody of the anti-CD 47-CLDN18.2 bispecific antibody of claim 1 or 2.
4. An expression vector comprising the nucleic acid molecule of claim 3.
5. A recombinant cell comprising the nucleic acid molecule of claim 3.
6. Use of an anti-idiotype antibody of an anti-CD 47-CLDN18.2 bispecific antibody according to claim 1 or 2 for the preparation of a product for detecting an anti-CD 47-CLDN18.2 bispecific antibody.
7. A kit for detecting an anti-CD 47-CLDN18.2 bispecific antibody comprising an anti-idiotype antibody of an anti-CD 47-CLDN18.2 bispecific antibody of claim 1 or 2.
8. Use of an anti-idiotype antibody of an anti-CD 47-CLDN18.2 bispecific antibody according to claim 1 or 2 for detecting an anti-CD 47-CLDN18.2 bispecific antibody.
9. A method of detecting an anti-CD 47-CLDN18.2 bispecific antibody, comprising:
detection based on an enzyme-linked immunosorbent assay using an anti-idiotype antibody of the anti-CD 47-CLDN18.2 bispecific antibody of claim 1 or 2.
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