CN113252894B

CN113252894B - Method for detecting scFv affinity of CAR-T cell

Info

Publication number: CN113252894B
Application number: CN202110764864.0A
Authority: CN
Inventors: 何霆; 齐菲菲; 鲁薪安; 刘光华; 胡雪莲; 许文平
Original assignee: Shandong Jinsai Biotechnology Co ltd; Beijing Yimiao Shenzhou Pharmaceutical Technology Co ltd
Current assignee: Shandong Jinsai Biotechnology Co ltd; Beijing Yimiao Shenzhou Pharmaceutical Technology Co ltd
Priority date: 2021-07-07
Filing date: 2021-07-07
Publication date: 2021-11-09
Anticipated expiration: 2041-07-07
Also published as: CN113252894A

Abstract

The invention relates to a method for detecting scFv affinity of CAR-T cells, which is characterized in that the CAR-T cells and a target antigen⁺After the cells are mixed, the cells are incubated together, and the CAR-T cell scFv and the target antigen are detected by a flow method⁺Affinity of the cell. The invention adopts a flow method to directly detect the scFv of the CAR-T cell and the target antigen⁺The affinity of the cells accurately reflects the CAR-T cells to the target antigen⁺The recognition effect of the cells.

Description

Method for detecting scFv affinity of CAR-T cell

Technical Field

The invention relates to the technical field of cell therapy, in particular to a method for detecting the scFv affinity of CAR-T cells.

Background

At present, the main method for detecting the affinity of a target protein (such as CAR-T cell scFv) to a corresponding target antigen is SPR (surface plasmon resonance) technology, and the main operation process is as follows: firstly, an antigen recognized by an antibody to be detected is coupled on the surface of a sensing chip, then the antibody to be detected is injected, the antibody-antigen binding process is directly monitored through the surface of the chip, and data such as a reaction kinetic constant, affinity and the like are provided by utilizing analysis software. The prior patent (application publication No. CN 111351936A) discloses a CD19-CAR affinity detection method, which specifically comprises the following steps: firstly, a stable cell strain with high expression of CD19 is constructed, then the affinities of CD19 antibodies (derived from scFv sequences in CD19 CAR-T) with different concentrations and CD19 positive cells are respectively detected, and the affinities of CD19 proteins with different concentrations and CD19 CAR-T cells are simultaneously detected.

The conventional method for detecting the scFv affinity of the CAR-T cell is separated from the CAR-T cell and directly detects the binding capacity of the scFv protein to a target antigen, namely the scFv protein is separated and purified from the CAR-T cell, then the affinity of the scFv protein to the target antigen is detected, the steric hindrance effect generated when the CAR-T cell is combined with the target antigen cell (such as a tumor cell) is ignored, the affinity of the CAR-T cell to the target antigen cell cannot be truly reflected, and further the accurate detection of the affinity of the CAR-T cell to the corresponding target antigen cell cannot be realized⁺The affinity of the cell, affects the progress of subsequent CAR-T cell product development. There is a need for a method that can accurately detect the affinity of CAR-T cell scFv.

Disclosure of Invention

In view of the above, the present invention provides a method for detecting the affinity of CAR-T cell scFv, which directly detects CAR-T cell scFv and target antigen by flow method⁺The affinity of the cells accurately reflects the CAR-T cells to the target antigen⁺The recognition effect of the cells.

In view of the above, the first aspect of the present invention provides a method for detecting scFv affinity of CAR-T cells by contacting the CAR-T cells with a target antigen⁺Cells are mixed and incubated together, and CAR-T fine is detected by a flow methodCellular scFv and target antigen⁺Affinity of the cell.

In a preferred embodiment of the invention, the CAR-T cell scFv detected by flow assay is conjugated to a target antigen⁺Binding of the cell to the presentation form to judge CAR-T cell scFv to target antigen⁺The magnitude of the affinity of the cell.

In a preferred embodiment of the invention, the CAR-T cell scFv is judged to be associated with the target antigen⁺The method for the affinity of the cell comprises the following steps:

(1) if the CD3 appears in the flow cytometry detection^-Target antigens^-Cells and CD3⁺Target antigens^-Cells, then determining CAR-T cell scFv and target antigen⁺The cells have strong affinity;

(2) if the CD3 appears in the flow cytometry detection^-Target antigens⁺Cell, CD3⁺Target antigens⁺Cells and CD3⁺Target antigens^-Cells, then determining CAR-T cell scFv and target antigen⁺The cells have relatively strong affinity;

(3) if the CD3 appears in the flow cytometry detection⁺Target antigens^-Cells and CD3^-Target antigens⁺Cells, then determining CAR-T cell scFv and target antigen⁺The affinity of the cells is weak or no specific affinity.

In a preferred embodiment of the invention, the CAR-T cell scFv is detected by flow assay as being linked to the target antigen⁺The binding of the cells is in the form of (1) case, CAR-T cell scFv to the target antigen⁺The affinity of the cells was calculated as follows:

；

wherein n = CAR-T cell and target antigen⁺Time of co-incubation after cell mixing, 0 hour target antigen⁺Proportion of cells and nth hour target antigen⁺The proportion of cells was determined by flow assay.

In a preferred embodiment of the invention, the assay is performed if the flow methodDetected CAR-T cell scFv and target antigen⁺The binding of the cells is in the form of (2) case, CAR-T cell scFv to the target antigen⁺The affinity of the cells was calculated as follows:

；

wherein n = CAR-T cell and target antigen⁺Time of co-incubation after cell mixing, n hour CD3⁺Target antigens⁺Proportion of cells and 0 h CD3⁺Target antigens⁺The proportion of cells was determined by flow assay.

In a preferred embodiment of the invention, the CAR-T cells are conjugated to a target antigen⁺After mixing the cells, the mixture is heated at 35-38 ℃ and 3-8% CO₂Co-incubating;

preferably, the CAR-T cell is conjugated to a target antigen⁺After mixing the cells, at 37 ℃ and 5% CO₂Co-incubating;

and/or, the CAR-T cell is associated with a target antigen⁺The time for incubating the cells together after mixing is 1-5 h;

preferably, the CAR-T cells and the target antigen + cells are mixed and then incubated together for 1.5-2.5 h.

In a preferred embodiment of the invention, the CAR-T cells are conjugated to a target antigen⁺The mixing ratio of the cells is 1: 1-5: 1.

In a preferred embodiment of the invention, the target antigen is PSMA, CD19, CD20, CD22, CD30, CD33, CD138, EGFRv III, ErbB, FAP, GD2, HER2, κ -IgLC or BCMA.

Based on the same inventive concept, the second aspect of the invention provides a method for screening the scFv of CAR-T cell, and the scFv of CAR-T cell and the target antigen are obtained by the method⁺Affinity of cells, screening for target antigens based on the affinity obtained⁺The cells have a strong affinity CAR-T cell scFv.

Based on the same inventive concept, the third aspect of the present invention provides a method for detecting protein affinityExpressing the test protein in the cell, and then mixing the cell expressing the test protein with the target antigen⁺Mixing and incubating cells, and detecting the protein to be detected and the target antigen by a flow method⁺Affinity of the cell.

Drawings

FIG. 1 shows CAR-T cell scFv with target antigen⁺A binding pattern of cells;

FIG. 2 is a diagram of a flow-based detection method for a Daudi cell and a target antigen CD19 of a patient tumor cell, wherein the left diagram is a diagram of a flow-based detection method for a Daudi cell target antigen CD19, and the right diagram is a diagram of a flow-based detection method for a patient tumor cell target antigen CD 19;

FIG. 3 is a flow chart of the detection of the affinity of T cells and CD19 CAR-T cell scFv for Daudi cells;

FIG. 4 is a comparison of the affinity assay and killing effect of T cells and CD19 CAR-T cell scFv to Daudi cells;

FIG. 5 is a flow chart of the assay of T cells and CD19 CAR-T cell scFv with patient CD19⁺Affinity of tumor cells;

FIG. 6 is T cell and CD19 CAR-T cell scFv vs. patient CD19⁺Comparing the affinity detection results of the tumor cells;

FIG. 7 is a diagram of a flow-based detection method for a Daudi cell and a target antigen CD22 of a patient's tumor cell, wherein the left diagram is a diagram of a flow-based detection method for a Daudi cell and the right diagram is a diagram of a flow-based detection method for a patient's tumor cell and a target antigen CD22 of a patient's tumor cell and a target antigen CD22 of a patient's tumor cell;

FIG. 8 is a flow chart of the detection of the affinity of T cells and CD22 CAR-T cell scFv for Daudi cells;

FIG. 9 is a comparison of the affinity assay and killing effect of T cells and CD22 CAR-T cell scFv to Daudi cells;

FIG. 10 is a flow chart of the assay of T cells and CD22 CAR-T cells with patient CD22⁺Affinity of tumor cells;

FIG. 11 is a T cell and CD22 CAR-T cell scFv vs. patient CD22⁺Comparing the affinity detection result of the tumor cells with the killing effect;

FIG. 12 shows the detection of the target antigens BCMA of NCI H929 cells and RPMI8226 by flow method;

figure 13 is a flow assay to detect the affinity of T cells and BCMA CAR-T cell scFv to RPMI8226 cells;

figure 14 is a comparison of affinity assay results and killing effect of T cells and BCMA CAR-T cells with RPMI8226 cells;

FIG. 15 is a flow chart that measures the affinity of T cells and BCMA CAR-T cell scFv to NCI H929 cells;

FIG. 16 is a comparison of the affinity assay and killing effect of T cells and BCMA CAR-T with NCI H929 cells;

FIG. 17 is a flow assay to detect the affinity of T cells, 01-BCMA CAR-T cells and 02-BCMA CAR-T cell scFv to NCI H929 cells;

FIG. 18 is a comparison of the results of affinity assays for T cells, 01-BCMA CAR-T cells and 02-BCMA CAR-T cell scFv and NCI H929 cells;

FIG. 19 is a comparison of the killing effect of T cells, 01-BCMA CAR-T cells and 02-BCMA CAR-T cell scFv to NCI H929 cells.

Detailed Description

It should be noted that technical terms or scientific terms used in the embodiments of the present specification should have a general meaning as understood by those skilled in the art, unless otherwise defined.

The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagents used in the following examples are all commercially available products unless otherwise specified.

The term "scFv" as used herein refers to an antibody fragment that comprises a variable region of heavy chain (V) linked by a linker_H) And the light chain variable region (V)_L) The linker associates the two domains to ultimately form the antigen binding site. The size of the scFv is typically 1/6 for a whole antibody. The scFv is preferably an amino acid sequence encoded by one nucleotide chain. The scFv for use in the present invention can be prepared by using conventional techniques known in the art, such as amino acid deletion, insertion, substitution, addition, and/or recombination and/or other modification methods, either alone or in combinationFurther modifying. Methods for introducing such modifications into the DNA sequence of an antibody based on its amino acid sequence are well known to those skilled in the art (see, e.g., Sambrook molecular cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N. Y.). The modification is preferably carried out at the nucleic acid level. The scFv described above may also include derivatives thereof.

The term "target antigen" as used herein refers to any cell surface antigen that can be targeted by an antibody or fragment thereof. In one embodiment, the target antigen is a cell surface-expressed specific protein molecule specifically recognized by an scFv of a CAR-T cell expressed on the target antigen⁺Cell lines and primary target antigens of patients⁺A tumor cell. For example, the target antigen is leukocyte differentiation antigen CD19, leukocyte differentiation antigen CD22, or B Cell Maturation Antigen (BCMA).

The term "target antigen" as used herein⁺Cell "or" target cell "are used interchangeably and refer to a cell that expresses a target antigen on its cell surface, e.g., a target antigen⁺Cell lines (Daudi cells, RPMI8226 cells and NCI H929 cells) or target antigens⁺A tumor cell.

The term "CAR-T cell scFv" as used herein refers to a scFv secreted by a CAR-T cell, and it is an object of the present invention to detect the affinity of the scFv secreted by the CAR-T cell for a target antigen expressed on the surface of a target cell. CAR-T is any cell that recognizes a specific target antigen, and the key structure is the scFv domain that recognizes the target antigen, linked to the CD8 or CD28 transmembrane region by the CD8 hinge region, followed by the CD28 or 4-1BB costimulatory domain and the CD3 zeta domain, to form a complete CAR structure. The present invention is not limited to CAR-T cells or CAR structures, and affinity detection can be performed using the detection method of the present invention as long as CAR-T cells or CAR structures that secrete expression scFv can be detected. Alternatively, the nucleotide sequences of the scFv domain, CD8 hinge region, CD28 transmembrane region, and CD3 zeta domain etc. may be referred to the sequences as disclosed in the invention patent application 20151032458.

The term "specific binding" or "specifically binds" as used herein refers to a non-random binding reaction between two molecules, such as a binding reaction between an antibody and an antigen.

In this context, the CD3 molecule is a membrane surface molecule of a T cell or CAR-T cell, it being noted that T cells may also bind to a target antigen on the surface of a target cell, but that in the presence of both T cells and CAR-T cells, the target antigen on the surface of the target cell preferentially binds to the CAR-T cells. Based on this, the cells to which the present invention relates are explained as follows:

(1) “CD3⁺target antigens^-Cell "refers to a T cell or CAR-T cell having a CD3 molecule (denoted as CD 3) on the membrane surface⁺) But does not express the target antigen on the cell surface (expressed as target antigen)^-) (ii) a Alternatively, the CAR-T cell recognizes a target antigen, absorbs the target antigen, and internalizes the antigen into the cell.

(2) “CD3^-Target antigens⁺A cell "refers to a target cell (e.g., a tumor cell) that expresses a target antigen (denoted as a target antigen) on the surface of the cell⁺) Or part of the target antigen is recognized and abstracted by CAR-T cells, but the cell's membrane surface is free of CD3 molecule (denoted as CD 3)^-）。

(3) “CD3⁺Target antigens⁺Cell "refers to a CAR-T cell having a CD3 molecule (designated as CD 3) on the membrane surface⁺) And the cell forms a complex after binding to a portion of the target antigen on the surface of the target cell or successfully captures the target antigen but has not internalized (expressed as target antigen)⁺）。

(4) “CD3^-Target antigens^-"refers to a target cell (e.g., a tumor cell) whose surface has all of its target antigen recognized and captured by CAR-T cells (denoted as target antigen)^-) And the cell has a membrane surface free of CD3 molecules (designated as CD 3)^-）。

A Chimeric Antigen Receptor (CAR-T) T cell is a T cell after genetic modification, and the CAR protein is over-expressed in the T cell in a lentivirus integration mode, namely the CAR-T cell. Chimeric Antigen Receptors (CARs) are fusion proteins consisting of a Single-Chain antibody Variable Fragment (scFv), a hinge region, a transmembrane domain, and an intracellular signaling domain (costimulatory domain and T cell activation domain). Single chain antibodies (scFv) for CAR-T cells are generally derived from monoclonal antibodies, with scFv having an affinity for the target antigen that is several orders of magnitude higher than TCRs (T cell antigen receptors). In addition, CAR-T cells can bypass Major Histocompatibility Complex (MHC) restriction to recognize intact cell surface proteins.

In recent years, CAR-T cell therapy has been rapidly developed in the treatment of hematological malignancies, and is one of the currently effective ways of treating malignant tumors, and has also been rapidly developed in solid tumors such as liver cancer, which is an emerging direction of future tumor immunotherapy.

Currently, Tumor Associated Antigen (TAA) is a main research target of CAR-T cell therapy, and TAA refers to an Antigen molecule that can be expressed in both Tumor cells and normal cells, i.e., is not specific to Tumor cells, and normal cells can express low levels but have higher expression levels in Tumor cells. Thus, to ensure the efficacy and safety of CAR-T cell therapy, CAR-T cells need to specifically recognize tumor cells without a lethal effect on normal tissue cells.

The key component of the CAR-T cell for killing the tumor cell is the scFv structural domain, and the scFv can induce the CAR-T cell to specifically kill the tumor cell after recognizing the target antigen on the surface of the tumor cell, so that the aim of treating malignant tumor is fulfilled. Therefore, the strength of the affinity of scFv to antigen is a key factor affecting the anti-tumor activity of CAR-T cells, and the affinity of scFv to target antigen-positive cells directly determines the safety and efficacy of CAR-T cell therapy.

As described in the background section of the present specification, although the methods in the prior art can effectively detect the affinity of the scFv protein to the target antigen, the methods individually express and purify the scFv protein using a protein expression system, and then detect the affinity of the scFv protein to the target antigen, but ignore the steric hindrance effect generated when the CAR-T cell is bound to the target antigen cell (e.g., tumor cell), and cannot truly reflect the affinity between the CAR-T cell and the target antigen cell, and further cannot accurately detect the affinity of the CAR-T cell to the corresponding target antigen cell. There is a need for a method that can accurately detect the affinity of CAR-T cell scFv.

In order to solve the problems of the prior art, the invention provides a method for detecting scFv affinity of CAR-T cells in a first aspect, wherein the CAR-T cells are contacted with a target antigen⁺After the cells are mixed, the cells are incubated together, and the CAR-T cell scFv and the target antigen are detected by a flow method⁺Affinity of the cell.

The invention utilizes the CAR-T cell and tumor cell co-incubation mode to reduce the real state of cell contact, considers the steric effect between the CAR-T cell and the tumor cell, and truly reflects the affinity between the CAR-T cell and the tumor cell.

In addition, the existing SPR technology needs special reagent consumables and instruments, is high in price, high in research and development cost, long in experimental period and complex in operation, and samples are easy to degrade in the sample treatment process, so that the test success rate is greatly reduced. The invention adopts a flow method to detect the scFv of the CAR-T cell and a target antigen⁺The affinity of the cells greatly reduces the research and development cost and greatly shortens the research and development period.

In a preferred embodiment of the invention, the CAR-T cell scFv detected by flow assay is conjugated to a target antigen⁺Binding of the cell to the presentation form to judge CAR-T cell scFv to target antigen⁺The magnitude of the affinity of the cell. Specifically, as shown in fig. 1, three cell types initially exist, respectively: CAR-T cells (expressing scFv), tumor cells (expressing target antigen), and T cells, wherein after 2 hours of co-incubation of the CAR-T cells mixed with the target antigen tumor cells, the scFv of the CAR-T cells specifically binds to the target antigen of the tumor cells. According to the size of scFv affinity, CAR-T cell binding to tumor cells appears in three forms:

(1) if the CAR-T cell scFv has strong affinity with the tumor cell target antigen, after 2 hours of incubation, two groups of cells are presented in flow cytometry detection, namely CD3^-Target antigens^-(tumor cells), CD3⁺Target antigens^-(T or CAR-T cells). The cause of this occurs in two ways: first, the tumor cell surface target antigen is rapidly bound by the scFv of the CAR-T cell, blocking the binding of the relevant target antigen flow antibody to the target antigen, and thus presented as CD3^-Target antigens^-(ii) a Secondly, after the tumor cell target antigen is combined with the CAR-T cell, the cell gnawing effect is generated, namely the scFv of the CAR-T cell recognizes the target antigen, absorbs the target antigen and internalizes the target antigen into the CAR-T cell, and the result is shown as CD3⁺Target antigens^-。

(2) If the CAR-T cell scFv has relatively strong affinity with the tumor cell target antigen, namely the affinity is moderate, after 2 hours of incubation, three groups of cells are presented in flow cytometry detection, namely CD3^-Target antigens⁺(tumor cells), CD3⁺Target antigens⁺(CAR-T cells or CAR-T and tumor cell complexes that successfully capture the target antigen) and CD3⁺Target antigens^-(T or CAR-T cells). Part of the tumor cells are still CD3^-Target antigens⁺However, the mean fluorescence intensity was reduced compared to the tumor cells before co-incubation, because: part of the target antigen on the surface of the tumor cell is recognized and abstracted by the CAR-T cell; CD3⁺Target antigens⁺There are two forms of cells, CAR-T cell binding to part of the target antigen of the tumor cell to form a complex and CAR-T cell successfully capturing the target antigen; CD3⁺Target antigens^-Is a T cell or a CAR-T cell that successfully internalizes a target antigen.

(3) If the affinity of scFv of the CAR-T cell to the target antigen of the tumor cell is weak or no specific affinity, the CAR-T cell cannot be combined with the tumor cell, and after 2 hours of incubation, the CAR-T cell appears to be two groups of cells, namely CD3⁺Target antigens^-(T or CAR-T cell) and CD3^-Target antigens⁺(tumor cells).

To summarize: CAR-T cells and target antigens⁺After the cells are incubated together, the three clustering phenomena can occur. Among them, CD3^-Target antigens^-(tumor cell)、CD3⁺Target antigens^-(CAR-T cells) is an ideal phenomenon of cell clustering, i.e. CAR-T cell scFv has strong affinity for the target antigen; if the above-mentioned clusters do not occur, CD3 needs to be analyzed⁺Target antigens^-(T or CAR-T cells), CD3^-Target antigens⁺(tumor cells) and CD3⁺Target antigens⁺(CAR-T cells binding to part of the target antigen of the tumor cell to form a complex and CAR-T cells successfully capturing the target antigen, respectively); the CAR-T cells with the two clustering phenomena can obviously improve the target antigen⁺Cell killing effect of cells. If CD3 is detected⁺Target antigens^-(T or CAR-T cells), CD3^-Target antigens⁺(tumor cells) cell clustering, which means that CAR-T cell scFv has a weak affinity or no specific affinity and cannot induce significant cell killing effect.

In addition, the prior art generally uses binding affinities K_D(concentration of antibody required to bind half of the antigen molecule) to represent CAR-T cell scFv to target antigen⁺Affinity of the cells, which can be determined using any conventional method known in the art, including but not limited to surface plasmon resonance methods, microscale thermophoresis, high performance liquid chromatography-mass spectrometry, and flow cytometry (e.g., FACS) methods, K_DSmaller values represent stronger affinities. The inventors of the present invention provide different methods of affinity calculation based on the different forms of binding presentation of CAR-T cells to tumor cells. The method specifically comprises the following steps: if CAR-T cell scFv binds to the target antigen⁺The cells have a strong affinity, which is calculated as follows:

；

If CAR-T cell scFv andtarget antigens⁺The cells have a relatively strong affinity, which is calculated as follows:

；

Alternatively, the method for detecting the scFv affinity of the CAR-T cell comprises the following steps: CAR-T cells and target antigens⁺Mixing the cells at a certain ratio, and adding 5% CO at 37 deg.C₂The cells were incubated in the incubator for 2 hours. Cells were harvested after 2 hours and then flow antibodies against CD3 and target antigen were used to target CAR-T and target antigen, respectively⁺Staining the cells, detecting the target antigen by flow cytometry⁺Cell Down-Regulation or CD3⁺Target antigens⁺The ratio of cell up-regulation, calculation of CAR-T cells to target antigen⁺Affinity of the cell.

The method for detecting the scFv affinity of the CAR-T cell is suitable for detecting the affinity of most CAR-T cells and target antigens, and therefore, the type of the target antigens is not limited by the invention. Preferably, the target antigen is PSMA (prostate-specific membrane antigen), leukocyte differentiation antigen CD19, leukocyte differentiation antigen CD20, leukocyte differentiation antigen CD22, leukocyte differentiation antigen CD30, leukocyte differentiation antigen CD33, leukocyte differentiation antigen CD138, EGFRv III (epidermal growth factor receptor type III), ErbB (tyrosine kinase receptors), FAP (fibroblast activation protein ), GD2 (disialoglycoside, disialoganglioside), 2 (human epidermal growth factor receptor 2), κ -IgLC (human immunoglobulin light chain, human immunoglobulin light chain immunoglobulin light antibody)Or BCMA (B Cell organization Antigen, BCMA), more preferably the target Antigen is CD19, CD22, or BCMA. The CD19 antigen exists on the cell surface of lymphoma and acute and chronic lymphocytic leukemia, and the CD19 CAR-T cell which is designed by taking the CD19 antigen as a target achieves great success in treating the lymphoma and the acute and chronic lymphocytic leukemia. The CD22 antigen is a transmembrane protein and a member of the immunoglobulin superfamily, is restricted to be expressed on the surfaces of mature B cells and most B cell non-Hodgkin's lymphoma (NHL) and is closely related to the development, differentiation and function of the B cells. CD22 CAR-T cells targeted at CD22 antigen can be used to treat relapsed/refractory B-cell acute lymphoblastic leukemia. B-cell maturation antigens are members of the tumor necrosis factor receptor (TNF) superfamily, which bind to B-cell activating factor (BAFF) and proliferation-inducing ligand (APRIL). BCMA CAR-T cells targeted to BCMA antigens can be used to treat relapsed or refractory multiple myeloma. The detection method can detect CD19 CAR-T cell scFv and CD19⁺Cells, CD22 CAR-T cell scFv and CD22⁺Cells and BCMA CAR-T cell scFv with BCMA⁺Affinity of cells, screening for target antigens based on affinity data⁺The cells have stronger affinity of the CAR-T cell scFv, namely the CAR-T cell scFv meeting the clinical requirement is screened.

Based on the same invention concept, the invention also provides a method for detecting protein affinity, which expresses the protein to be detected in cells, and then expresses the cells expressing the protein to be detected and a target antigen⁺Mixing and incubating cells, and detecting the protein to be detected and the target antigen by a flow method⁺Affinity of the cell. Any test protein (expressed in any cell) to be detected and the target antigen⁺Cell affinity assays can be performed by the detection methods of the invention and are therefore within the scope of the invention.

The strength of the affinity of scFv to antigen is a key factor affecting the anti-tumor activity of CAR-T cells, and the affinity of scFv to target antigen-positive cells directly determines the safety and effectiveness of CAR-T cell therapy. A third aspect of the invention provides a method of screening for CAR-T cells scFv, obtaining CAR-T cell scFv and target antigen by the above method⁺Affinity of cells, screening for target antigens based on the affinity obtained⁺The cells have a strong affinity CAR-T cell scFv. The affinity of the CAR-T cell to a target antigen positive cell line and a patient target antigen positive tumor cell is rapidly detected by a flow method, the pharmacological activity of the CAR-T cell in a patient body can be effectively evaluated, and the success rate of drug development is improved.

The technical solution provided by the present invention is further described with reference to specific embodiments. The following examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

It should be noted that T-Daudi-1 and T-Daudi-2 in Table 1 are only representative of two duplicate wells in the test, i.e., two parallel tests performed on the same sample, and have no other special meaning. The remaining tables, "… … -1" and "… … -2" also represent only two duplicate wells in the assay.

Example 1 CD19 CAR-T cells with CD19⁺Affinity assay for cells

The CD19 CAR-T cells used in this example were prepared in accordance with examples 1 to 3 of the patent literature (title of the invention: chimeric antigen receptor-modified T cells and use thereof, application publication No. CN 105177031A). The Daudi cells (human Burkitt's lymphoma cells) and the patient tumor cells were subjected to flow-based detection of the target antigen CD19, and it was found that the Daudi cells were CD19 positive cells (FIG. 2, left) and the patient CD19 positive tumor cells accounted for 1.92% of the total mononuclear cells (FIG. 2, right), and the two cells were used as affinity detection of the subsequent CD19 CAR-T cell scFv.

And (3) an affinity detection method: mixing CD19 CAR-T cell with Daudi cell and patient CD19 positive tumor cell at a ratio of 3:1, and adding 5% CO at 37 deg.C₂Incubating in a cell incubator for 2 hours, collecting cells after 2 hours, and respectively carrying out treatment on CAR-T cells and target antigens by using a flow antibody (APC (allophycocyanin) fluorescein labeled CD3 antibody) for resisting CD3 and a flow antibody (FITC (isothiocyanic acid) fluorescein labeled CD19 protein antibody) for resisting target antigens⁺Staining cells (Daudi cells or patient CD19 positive tumor cells), detecting CAR-T cell scFv and target antigen by flow method⁺Affinity of the cell.

The killing efficiency detection method comprises the following steps:

the first step is as follows: labelling of target cells with Calcein-AM

1) Calcein-AM (Life Technology, Shanghai) was diluted to 1mg/mL with DMSO;

2) the target cells (Daudi cells or patient CD19 positive tumor cells) were resuspended at 1X 10 with physiological saline⁶Density per mL;

3) adding Calcein-AM of 15 μ M at 37 deg.C and 5% CO₂Culturing for 30min, and mixing gently every 10 min;

4) centrifuging at 1500rpm for 5 min, removing supernatant, resuspending with complete culture medium, and repeating twice;

the second step is that: killing target cells using effector cells

1) Resuspending the labeled target cells according to the density of 5000-;

2) the fluorescence intensity (tetherelase) of the supernatant was measured in 3 replicates with T cells as control cells, while 100. mu.L of effector cells were added at the appropriate ET (effective target ratio) ratio (5: 1); designing a separate group A (6 in parallel), only target cells, and detecting their spontaneous apoptotic cleavage fluorescence intensity (spontaneous release); a separate group B (6 replicates) was designed, with only target cells +2% Triton X-100, and their maximal lytic fluorescence intensity (maximum release) was measured;

3)37℃、5%CO₂after culturing for 6h, centrifuging, taking 75 mu L of supernatant, and transferring the supernatant to a new culture plate;

4) the sample was detected using a spectramax diagnostic-scanning microplate fluorometer (interaction filter: 485. + -. 9 nm; band-pass filter 530 + -9 nm); the percentage of cell lysis was calculated according to the following formula: [ (test release-sponge release)/(maximum release-sponge release) ]. times.100.

The results of the affinity assay and the results of the killing efficiency assay are shown in FIGS. 3-6 and tables 1-2. The results showed that the cells were in phase with T cellsIn contrast, the affinity of CD19 CAR-T cell scFv to Daudi cells was significantly enhanced (fig. 3 and table 1), and the affinity assay results were consistent with the killing effect (fig. 4). CD19 CAR-T cell scFv vs patient CD19 compared to T cells⁺The affinity of tumor cells was significantly enhanced (fig. 5 and table 2), and CD19 CAR-T cells were specific for patient CD19⁺The killing power of the target cells was strong (FIG. 6), which is consistent with CD19⁺The results of the cell line (Daudi) were consistent.

TABLE 1

TABLE 2

Flow-detected CD19 CAR-T cells and Daudi cells or patient CD19⁺The target cells have strong affinity, and the calculation formula of the affinity in table 1 and table 2 is:

；

for example, the calculation of CD19 CAR-T-Daudi-1 in Table 1 is: (24.55-0.86)/24.55 × 100% = 96.49%; the CD19 CAR-T-patient tumor cell-1 in table 2 was calculated as: (0.86-0.06)/0.86 × 100% = 93.02%.

Example 2 CD22 CAR-T cell scFv with CD22⁺Affinity assay for cells

The method of making CD22 CAR-T cells used in this example was the same as the method of making CD19 CAR-T cells in example 1, except that the nucleotide and amino acid sequences of the scFv differed. scFv sequences reference the patents "Mutated anti-CD22 antibodies with increased affinity to CD22-expressing leukamia cells", patent Nos.: US8809502B 2. The Daudi cells (human Burkitt's lymphoma cells) and patient tumor cells were used for detecting the target antigen CD22 by a flow method, wherein the Daudi cells are CD22 positive cells, and the proportion of patient CD22 positive tumor cells to total mononuclear cells is 47.82% (FIG. 7), so that the affinity detection test of CD22 CAR-T cell scFv was performed on the two cells.

And (3) an affinity detection method: mixing CD22 CAR-T cells with Daudi cells and patient CD22 positive tumor cells at a ratio of 3:1, respectively, and adding 5% CO at 37 deg.C₂Incubating in a cell incubator for 2 hours, collecting cells after 2 hours, and respectively carrying out treatment on CAR-T cells and target antigens by using a flow antibody (APC (allophycocyanin) fluorescein labeled CD3 antibody) for resisting CD3 and a flow antibody (PE (phycoerythrin) fluorescein labeled CD22 protein antibody) for resisting target antigens⁺Staining cells (Daudi cells or patient CD22 positive tumor cells), detecting CAR-T cell scFv and target antigen by flow method⁺Affinity of the cell. The killing efficiency was measured in the same manner as in example 1.

The results of the affinity assay and the results of the killing efficiency assay are shown in FIGS. 8-11 and Table 3. The data indicate that the affinity of CD22 CAR-T cell scFv to Daudi cells was significantly enhanced compared to T cells (fig. 8 and table 3), and the affinity assay results are consistent with the killing effect (fig. 9). Compared with T cells, the affinity of CD22 CAR-T cell scFv to patient tumor cells was significantly enhanced (fig. 10), and the affinity assay result was consistent with the killing effect (fig. 11).

TABLE 3

Flow-detected CD22 CAR-T cells and Daudi cells or patient CD19⁺The target cells have strong affinity, and the affinity calculation formula in table 3 is:

；

for example, the CD22 CAR-T-Daudi-1 in Table 3 is calculated as: (24.95-0.35)/24.95 × 100% = 98.59%.

Example 3 BCMA CAR-T cell scFv and different target antigen densities BCMA⁺Affinity assay for cells

The method for producing BCMA CAR-T cells used in this example was carried out in reference to examples 1 to 5 in the patent literature (title of the invention: an anti-BCMA antibody and use thereof, application publication No. CN 112321713A). When the target antigen BCMA assay was performed on RPMI8226 cells (human multiple myeloma cells) and NCI H929 cells (human myeloma cells) by a flow method, both BCMA-positive cells were detected, but the BCMA expression density of NCI H929 was significantly higher than that of RPMI8226 (fig. 12).

And (3) an affinity detection method: mixing BCMA CAR-T cells with RPMI8226 cells and NCI H929 cells at a ratio of 3:1, respectively, and adding 5% CO at 37 deg.C₂Incubating in a cell incubator for 2 hours, collecting cells after 2 hours, and respectively carrying out CAR-T cells and target antigen on the cells by using a flow antibody (FITC (isothiocyanate) fluorescein labeled CD3 antibody) for resisting CD3 and a flow antibody (APC (allophycocyanin) fluorescein labeled BCMA protein antibody) for resisting target antigen⁺Staining cells (RPMI 8226 cells or NCI H929 cells), and detecting scFv of CAR-T cells and target antigen by flow method⁺Affinity of the cell.

The killing efficiency detection method comprises the following steps: resuspend 2X 10 with 1mL physiological saline⁶NCI H929 target cells (ATCC), 5. mu.L of Calcein-AM (concentration 1. mu.g/. mu.L, ThermoFisher, USA) was added, gently mixed, and then placed in a 37 ℃ water bath to incubate for 30min to label the target cells, washed twice with 10mL of physiological saline, excess dye was removed, and cells were resuspended and counted in 1mL of physiological saline and diluted to 1X 10⁶one/mL. To a 48-well cell culture plate (Corning Incorporated, Corning, NY, USA) was added 1X 10 per well⁵Adding the labeled NCI H929 into T cells and BCMA CAR-T cells at 37 deg.C and 5% CO according to E: T (effective target ratio) =5:1₂And (5) incubating for 6h in the cell incubator, and taking culture supernatant for detection. The fluorescence value of the cell supernatant was measured with a fluorescence microplate reader (Varioscan Lux, ThermoFisher) (excitation wavelength: 495 nm, emission wavelength: 515 nm).

The results of the affinity assay and the results of the killing efficiency assay are shown in FIGS. 13-16 and tables 4-5. Data from affinity assays of BCMA CAR-T with low target antigen density cells RPMI8226 showed no enhancing effect of BCMA CAR-T cell scFv affinity with RPMI8226 cells compared to T cells (fig. 13 and table 4), while BCMA CAR-T cells did not increase killing effect on RPMI8226 (fig. 14); thus, CAR-T cell scFv affinity is consistent with cell killing effects.

TABLE 4

Data from affinity assays of BCMA CAR-T cells with the high target antigen density cell line NCI H929 show that the affinity of BCMA CAR-T cell scFv to NCI H929 cells was significantly enhanced compared to T cells (fig. 15 and table 5), while the killing effect of BCMA CAR-T on NCI H929 cells was significantly enhanced (fig. 16). Therefore, the affinity detection result is consistent with the rule of killing effect.

TABLE 5

The BCMA CAR-T cells detected by the flow method have relatively strong affinity with NCI H929 cells with high BCMA expression density, but have weak or no specific affinity for RPMI8226 cells with low BCMA expression density. Thus, CAR-T cells are directed to the target antigen⁺The affinity of the cell is influenced by the density of expression of the target antigen in the target cell: target antigens⁺The higher the density of target antigen expression in the cell, the stronger the CAR-T cell scFv has affinity for it, and conversely, the weaker the CAR-T cell has affinity for it.

The affinity calculation formulas in tables 4 and 5 are:

for example, the calculation of BCMA CAR-T-RPMI8226-1 in Table 4 is: 2.70% -0.40% = 2.30%; the calculation of BCMA CAR-T-NCI H929-1 in Table 5 is: 38.52% -0.69% = 37.83%.

Example 4 different affinity scFv derived BCMA CAR-T cells with BCMA⁺Affinity assay for cells (NCI H929)

The CAR against BCMA was constructed with different scfvs and affinity and killing assays were performed on the target cells NCI H929 with the corresponding CAR-T cells (01-BCMA CAR-T cells and 02-BCMA CAR-T cells). The 01-BCMA CAR-T cells and the 02-BCMA CAR-T cells used in this example were prepared in the same manner, were all prepared by the preparation method in example 3, and specifically recognized BCMA target antigen, but had different affinities due to differences in the amino acid sequences of the scFv. Wherein, the scFv amino acid sequences of the 01-BCMA CAR-T cell and the 02-BCMA CAR-T cell are respectively shown as SEQ ID NO. 1 and SEQ ID NO. 2.

And (3) an affinity detection method: mixing 01-BCMA CAR-T cell and 02-BCMA CAR-T cell with NCI H929 cell at 3:1 ratio, respectively, and adding 5% CO at 37 deg.C₂Incubating in a cell incubator for 2 hours, collecting cells after 2 hours, and respectively subjecting 01-BCMA CAR-T, 02-BCMA CAR-T cells and target antigen to flow antibody (FITC (isothiocyanic acid) fluorescein labeled CD3 antibody) and flow antibody (APC (allophycocyanin) fluorescein labeled BCMA protein antibody) for resisting CD3⁺Staining cells (NCI H929 cells), detecting CAR-T cell scFv and target antigen by flow method⁺Affinity of the cell. The killing efficiency was measured in the same manner as in example 3.

The results of the affinity assay and the killing efficiency assay are shown in FIGS. 17-19. The results showed that the affinity of both scFv of 01-BCMA CAR-T cells and 02-BCMA CAR-T cells to NCI H929 cells was significantly enhanced compared to T cells (fig. 17), but the affinity of scFv of 02-BCMA CAR-T cells was significantly higher than that of 01-BCMA CAR-T cells (fig. 18); meanwhile, consistent with the rule of affinity detection, the killing effect of 01-BCMA CAR-T cells and 02-BCMA CAR-T cells on NCI H929 cells was significantly enhanced compared to T cells, and the killing effect of 02-BCMA CAR-T cells was significantly higher than that of 01-BCMA CAR-T cells (fig. 19).

To sum up the aboveCAR-T cell to target antigen⁺The affinity of a cell is influenced by two factors: (1) CAR-T cell scFv amino acid sequence, i.e. the spatial structure of the scFv protein; (2) target antigens⁺The density of target antigen expression in the cell, the higher the density of target antigen expression, the stronger the CAR-T cell scFv has affinity for it, and vice versa, the weaker the CAR-T cell has affinity for it.

Sequence listing

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Claims

1. A method for detecting scFv affinity of CAR-T cells, wherein the CAR-T cells are contacted with a target antigen⁺After the cells are mixed, the cells are incubated together, and the CAR-T cell scFv and the target antigen are detected by a flow method⁺The affinity of the cell; CAR-T cell scFv and target antigen detected by flow method⁺Binding of the cell to the presentation form to judge CAR-T cell scFv to target antigen⁺The magnitude of the affinity of the cell;

determination of CAR-T cell scFv from target antigen⁺The method for the affinity of the cell comprises the following steps:

(3) if the CD3 appears in the flow cytometry detection⁺Target antigens^-Cells and CD3^-Target antigens⁺Cells, then determining CAR-T cell scFv and target antigen⁺Weak or no specific affinity for the cell;

CAR-T cell scFv and target antigen⁺The binding of the cells takes on the three different grades, and the affinity is (1) > (2) > (3);

wherein the "target antigen⁺Cell "refers to a cell that expresses a target antigen on its cell surface;

“CD3⁺target antigens^-Cell "refers to a T cell or CAR-T cell that has a CD3 molecule on its membrane surface, but does not express a target antigen on its cell surface; alternatively, it refers to a CAR-T cell that recognizes a target antigen, absorbs the target antigen, and internalizes the antigen into the cell;

“CD3^-target antigens⁺By cell "is meant a target cell that expresses on its surface a target antigen or a portion of a target antigen that is recognized and captured by a CAR-T cell, but that has a membrane surface free of CD3 molecules;

“CD3⁺target antigens⁺By cell "is meant a CAR-T cell that has a CD3 molecule on its membrane surface and which, upon binding to a portion of the target antigen on the surface of the target cell, forms a complex or successfully captures the target antigen but has not been internalized;

“CD3^-target antigens^-By cell "is meant a target cell whose surface is recognized and abstracted by CAR-T cells for all of the target antigen and whose membrane surface is free of CD3 molecules.

2. The method of claim 1, wherein the CAR-T cell scFv is bound to the target antigen if detected by flow cytometry⁺The binding of the cells is in the form of (1) case, CAR-T cell scFv to the target antigen⁺The affinity of the cells was calculated as follows:

；

3. The method of claim 1, wherein the CAR-T cell scFv is bound to the target antigen if detected by flow cytometry⁺The binding of the cells is in the form of (2) case, CAR-T cell scFv to the target antigen⁺The affinity of the cells was calculated as follows:

；

4. The method of claim 1, wherein the CAR-T cell is conjugated to a target antigen⁺After mixing the cells, the mixture is heated at 35-38 ℃ and 3-8% CO₂And (4) co-incubation.

5. The method of claim 4, wherein the CAR-T cells are conjugated to a target antigen⁺After mixing the cells, at 37 ℃ and 5% CO₂And (4) co-incubation.

6. The method of claim 1, wherein the CAR-T cell is conjugated to a target antigen⁺The time for incubating the cells together after mixing is 1-5 h.

7. The method of claim 6, wherein the CAR-T cell is conjugated to a target antigen⁺CellsThe time of incubation is 1.5-2.5 h after mixing.

8. The method of claim 1, wherein the CAR-T cell is conjugated to a target antigen⁺The mixing ratio of the cells is 1: 1-5: 1.

9. The method of claim 1, wherein said target antigen is PSMA, CD19, CD20, CD22, CD30, CD33, CD138, EGFRv III, ErbB, FAP, GD2, HER2, kappa-IgLC, or BCMA.

10. A method of screening for a CAR-T cell scFv, wherein the CAR-T cell scFv is obtained by the method of any one of claims 1-9 in combination with a target antigen⁺Affinity data of the cells, screening for target antigens based on the obtained affinity data⁺The cells have a strong affinity CAR-T cell scFv.