CN110850067B

CN110850067B - Chimeric antigen receptor affinity detection method

Info

Publication number: CN110850067B
Application number: CN201810951161.7A
Authority: CN
Inventors: 王海鹰; 张双双
Original assignee: Shanghai Hengrun Dasheng Biotechnology Co ltd
Current assignee: Shanghai Hengrun Dasheng Biotechnology Co ltd
Priority date: 2018-08-21
Filing date: 2018-08-21
Publication date: 2023-08-15
Anticipated expiration: 2038-08-21
Also published as: CN110850067A

Abstract

The invention discloses a BCMAAR affinity detection method, which mainly comprises the following steps: 1) BCMA IgG binding affinity to BCMA positive cell lines; 2) BCMA IgG binding affinity ELISA assay to BCMA. The method can effectively measure the affinity of the BCMAAR antibody, and has the advantages of safety, high sensitivity, good repeatability, stable and reliable detection result and the like.

Description

Chimeric antigen receptor affinity detection method

Technical Field

The invention belongs to the field of cell therapy, and particularly relates to a BCMA CAR affinity detection method.

Background

Chimeric antigen receptor (Chimeric Antigen Receptor-T cell, CAR-T) T cells refer to T cells that, after genetic modification, recognize a specific antigen of interest in an MHC non-limiting manner and continue to activate expansion. The annual meeting of the international cell therapy association in 2012 indicates that biological immune cell therapy has become a fourth means for treating tumors outside surgery, radiotherapy and chemotherapy, and is becoming an essential means for future tumor treatment. CAR-T cell feedback therapy is the most clearly effective form of immunotherapy in current tumor therapy. A large number of researches show that the CAR-T cells can effectively recognize tumor antigens, cause specific anti-tumor immune response and obviously improve the survival condition of patients.

Chimeric Antigen Receptors (CARs) are the core component of CAR-T, conferring to T cells the ability to recognize tumor antigens in an HLA-independent manner, which enables CAR engineered T cells to recognize a broader range of targets than native T cell surface receptor TCRs. The basic design of a CAR includes a Tumor Associated Antigen (TAA) binding region (typically derived from the scFV segment of a monoclonal antibody antigen binding region), an extracellular hinge region, a transmembrane region and an intracellular signaling region. The choice of antigen of interest is a critical determinant of the specificity, effectiveness of the CAR and safety of the genetically engineered T cells themselves.

CAR-T activates an activation signal by specific binding to a tumor antigen, thereby generating a targeted killing function on tumor cells. This specific binding depends on the single chain variable region (scFv) of the antibody in the CAR structure that specifically binds to the antigen. Anti-human BCMA T cell injection uses murine anti-human BCMA scFv, wherein the light chain variable region and heavy chain variable region sequences are derived from BCMA antibody clone No. c11d 5.3. Therefore, the strength of the binding affinity of the monoclonal antibody to the antigen is an important factor affecting the anti-tumor activity of the BCMA CAR-T cells.

Multiple Myeloma (MM) is a malignant tumor characterized by clonal proliferation of bone marrow malignant plasma cells, which is characterized by a massive proliferation of clonal plasma cells, resulting in myelohematopoietic suppression and osteolytic disease. Despite the use of traditional chemotherapy and hematopoietic stem cell transplantation and targeted drug therapy for myeloma, MM is still not completely cured.

MM originates from plasma cells in the bone marrow, which are cells that develop into the final functional stage of B lymphocytes, and thus generally fall into the category of B lymphomas. However, MM abnormal plasma cells hardly express CD19, and most B cell marker proteins are expressed very little or even not, so CAR-T treatment against MM needs to find new targets.

One candidate antigen for immunotherapy of MM is the B cell maturation antigen (B Cell Maturation Antigen, BCMA). BCMA RNA is commonly detected in MM cells and BCMA protein is detectable on the plasma cell surface of patients with multiple myeloma. BCMA is a member of the Tumor Necrosis Factor Receptor (TNFR) superfamily that binds B cell activating factor (BAFF) and proliferation-inducing ligand (APRIL). BCMA is reported to be expressed in normal cells primarily by plasma cells and a portion of mature B cells, but not in most B cells and other organs. Therefore, the anti-human BCMA T cell injection adopts a CAR structure specific to the BCMA antigen, is used for targeting and identifying and destroying BCMA-expressing cells, and is intended for BCMA positive relapsed/refractory multiple myeloma.

Antibody affinity is one of the important parameters in determining the nature of an antibody, and it is indicative of the degree of binding firmness between the binding site of an antibody and an epitope, and is often expressed as the dissociation equilibrium constant KD, which refers to the concentration of antibody required to bind half of the antigen molecules after equilibrium has been reached. The affinity of the antibody and antigen can provide basis for selecting monoclonal antibodies with different purposes, and the high-affinity antibody is useful for quantitative detection, diagnosis and targeted treatment of diseases, and can improve the positive detection rate or the drug effect of therapeutic antibodies; the antibody with the same affinity is used for affinity purification, and is not easy to adsorb due to the too low affinity and not easy to elute due to the too high affinity. Therefore, to select antibodies of different affinities according to the purpose of the application, it is necessary to detect the affinity of the antibodies first.

The CAR structure of BCMACR adopts C11D5.3scFv, so that the C11D5.3scFv has high affinity to target antigen, and the BCMACR has higher affinity to target antigen. The invention establishes a detection method of the affinity of BCMAAR to a target antigen. The experimental method is based on flow cytometry for relevant project detection.

Disclosure of Invention

The invention aims to provide a method for detecting BCMAAR affinity, which has the advantages of safety, high sensitivity, good repeatability, stable and reliable detection result and the like.

The technical scheme adopted for solving the technical problems is as follows:

(1) Binding affinity of BCMA IgG to BCMA positive cell lines

Expressed and purified Anti-human BCMA IgG (clone number: C11D 5.3) was prepared by the same company, set to an initial concentration of 100ug/ml, and then diluted in a 1:3 gradient at 7 spots, with 3X 10, respectively ⁵ The human BCMA positive cell lines MM.1S and H929 were incubated together, and then the BCMA IgG bound to the target cells was detected by using the flow antibody PE-anti human IgG-Fc, and the fluorescence intensity was detected by flow and the binding affinity was judged.

(2) BCMA IgG and BCMA binding affinity ELISA detection

The ELISA plate is coated with 2 mug/ml Human BCMA protein, free protein is washed after 1h, then 8 concentration gradients are diluted by self-made purified Anti-Human BCMA IgG 2ug/ml 1:3 of the company, the mixture is incubated for 1h at 37 ℃, excessive antibody is washed, and then secondary antibody Biotin Goat Anti mouse F (ab) is sequentially used ₂ And three antibodies strepitavidin-HRP are respectively incubated for 1h at 37 ℃, finally developed, and OD450/620 values are read on an enzyme labeling instrument.

Drawings

FIG. 1 MM.1S and BCMA IgG binding EC50 results

FIG. 2H 929 results of EC50 binding to BCMA IgG

FIG. 3 ELISA results of detection of BCMA IgG binding EC50

Detailed Description

The present invention is described in further detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Accordingly, the present invention should in no way be construed as being limited to the following examples, but rather should be construed to include any and all variations that become apparent from the teachings provided herein. The methods and reagents used in the examples are, unless otherwise indicated, conventional in the art.

Example 1: binding affinity of BCMA IgG to BCMA positive cell lines

The method comprises the following specific steps: expressed and purified Anti-human BCMA IgG (clone number: C11D 5.3) was prepared by the same company, set to an initial concentration of 100ug/ml, and then diluted in a 1:3 gradient at 7 spots, with 3X 10, respectively ⁵ The human BCMA positive cell lines mm.1s and H929 of (see table 1 for specific groupings) were co-incubated, followed by detection of BCMA IgG bound to target cells by the flow antibody PE-anti human IgG-Fc, fluorescence intensity was detected by flow and binding affinity was judged.

TABLE 1 binding affinity assay packets for BCMA IgG and BCMA positive cell lines

The results of the examples are shown in fig. 1 and fig. 2. Wherein MM.1S binds to BCMAIgG with an EC50 of 37nM and H929 reaches 57nM.

Example 2: BCMA IgG and BCMA binding affinity ELISA detection

The method comprises the following specific steps: the ELISA plate is coated with 2 mug/ml Human BCMA protein, free protein is washed after 1h, then 8 concentration gradients are diluted by self-made purified Anti-Human BCMA IgG 2ug/ml 1:3 of the company, the mixture is incubated for 1h at 37 ℃, excessive antibody is washed, and then secondary antibody Biotin Goat Anti mouse F (ab) is sequentially used ₂ And three antibodies strepitavidin-HRP are respectively incubated for 1h at 37 ℃, finally developed, and OD450/620 values are read on an enzyme labeling instrument.

The results of this example are shown in FIGS. 1 and 2. The binding affinity was measured by ELISA using an excess of Human BCMA-Fc protein coated plate, followed by incubation with an initial concentration of 2ug/ml, a 1:3 gradient of 8 concentrations of Anti-Human BCMA IgG, and an EC50 value of 1.2 nM.

Claims

1. A method of detecting BCMA CAR affinity comprising the steps of:

(1) Binding affinity of BCMA IgG to BCMA positive cell lines

The method comprises the following specific steps: the initial concentration was set to 100ug/ml with self-made expressed and purified Anti-human BCMA IgG clone number C11D5.3, and then 7 concentration gradients were diluted 1:3, respectively, in equal amounts to 3X 10 ⁵ Incubating the human BCMA positive cell lines MM.1S and H929 together, detecting BCMA IgG bound with target cells by using a flow antibody PE-anti human IgG-Fc, detecting fluorescence intensity by using a flow cytometer, and judging binding affinity;

(2) BCMA IgG and BCMA binding affinity ELISA detection

The method comprises the following specific steps: the ELISA plate is coated with 2 mug/ml Human BCMA protein, free protein is washed off after 1h, then 8 concentration gradients are diluted by self-made purified Anti-Human BCMA IgG 2ug/ml 1:3, incubated for 1h at 37 ℃, superfluous antibody is washed off, and then secondary antibody Biotin Goat Anti mouse F (ab) is sequentially used ₂ And three antibodies strepitavidin-HRP are respectively incubated for 1h at 37 ℃, finally developed, and OD450/620 values are read on an enzyme labeling instrument.