A chimeric antigen receptor targeting cd200 and uses thereof

Abstract

The application provides a targeting CD200 chimeric antigen receptor and application, wherein the targeting CD200 chimeric antigen receptor comprises, from N-terminal to C-terminal, a signal peptide, a single-chain antibody ScFv, strepII, CD8 alpha hinge, a CD28 transmembrane region, a CD28 intracellular domain, an intracellular costimulatory domain 4-1BB and a CD3 zeta chain which are sequentially spliced; the single-chain antibody ScFv specifically recognizes a CD200 antigen on the surface of a tumor cell. The chimeric antigen receptor has remarkable effects in the preparation of a drug for treating acute myeloid leukemia.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals and biotechnology, and more particularly to a chimeric antigen receptor targeting CD200 and its applications. Background Technology

[0002] CAR-T cells, short for chimeric antigen receptor T cells, are based on the principle of genetic engineering. The single-chain variable region (Scfv) of an antibody that recognizes a specific tumor antigen is coupled in vitro to the intracellular region of the CD3-ζ chain to form a chimeric protein. This protein is then transfected into cultured patient T cells via gene transduction, causing them to express the chimeric antibody receptor (CAR). After being "reprogrammed," the patient's T cells generate a large number of cytotoxic CAR-T cells capable of specifically targeting tumor cells. Compared to traditional immunotherapy, CAR-T therapy offers significant advantages, including more precise treatment, more accurate targeting, broader tumor killing, and longer-lasting effects.

[0003] Despite the continued success of CAR-T therapy in cancer treatment, the process still faces challenges such as off-target effects due to the high heterogeneity of tumors and limitations imposed by the single target. The high heterogeneity of tumor cells directly limits the effectiveness of CAR-T therapy, while the single target restricts its broad applicability. Therefore, selecting membrane surface markers that are specifically expressed in tumor cells and have broad expression across different tumor types is a crucial step in ensuring the effectiveness of CAR-T therapy.

[0004] CD200 (OX-2) is a cell surface glycoprotein that inhibits alloimmune and autoimmune responses through its receptor CD200R. It is highly expressed primarily in many malignant tumors, including chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML), as well as in cancer stem cells. A major obstacle in treating AML is the elimination of leukemia stem cells (LSCs), these drug-resistant cells with long-term self-renewal capabilities are considered a source of relapse. Patent CN114728048A discloses that CD200 receptor antagonist binding molecules can be used as drugs for treating solid tumors, liquid tumors, or neuroendocrine tumors. Patent CN102906115A discloses that anti-CD200 antibodies can be used as cancer treatment drugs. Neither of these patents discloses that a chimeric antigen receptor targeting CD200 can be used as a drug for treating AML. Summary of the Invention

[0005] In view of this, the present invention proposes a chimeric antigen receptor targeting CD200 as a drug for treating AML.

[0006] The technical solution of the present invention is implemented as follows: First, the present invention provides a CD200-targeting chimeric antigen receptor, which includes a signal peptide, a single-chain antibody ScFv, strepII, CD8αhinge, a CD28 transmembrane region, a CD28 intracellular domain, an intracellular co-stimulatory domain 4-1BB, and a CD3ζ chain sequentially spliced ​​from the N-terminus to the C-terminus; the single-chain antibody ScFv specifically recognizes the CD200 antigen on the surface of tumor cells.

[0007] Based on the above technical solutions, preferably, the amino acid sequence of the single-chain antibody ScFv is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2.

[0008] Based on the above technical solutions, preferably, the amino acid sequence of the CD28 intracellular domain is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4.

[0009] Based on the above technical solutions, the preferred amino acid sequence of the intracellular co-stimulatory domain 4-1BB is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.6.

[0010] Second, the present invention provides an application of a chimeric antigen receptor targeting CD200 in the preparation of a drug for treating acute myeloid leukemia.

[0011] Third, the present invention provides a gene vector for a recombinant chimeric antigen receptor, which uses a BRD-PTK-EF1α vector as a backbone and inserts a lentivirus, retrovirus or transposon vector that encodes the nucleotide sequence of the chimeric antigen receptor.

[0012] Fourth, this invention provides the application of a recombinant chimeric antigen receptor gene vector in the preparation of drugs for treating acute myeloid leukemia.

[0013] Fifth, the present invention provides an immune cell expressing a chimeric antigen receptor, which is obtained by transfecting immune cells with the encoding nucleotide sequence of the chimeric antigen receptor or a recombinant chimeric antigen receptor gene vector; the immune cell is selected from one of the following: umbilical cord blood, peripheral blood, IPSC-derived T cells, NK cells, NKT cells, αβT cells, γδT cells, CD4+T cells, and CD8+T cells.

[0014] Sixth, the present invention provides a method for preparing immune cells expressing chimeric antigen receptors, comprising the following steps: activating the isolated immune cells for 1 day and then infecting the immune cells with a gene vector of recombinant chimeric antigen receptors to obtain immune cells expressing chimeric antigen receptors; performing antitumor activity detection on the immune cells expressing chimeric antigen receptors, wherein the selected cell line or clinical patient sample cells are those that highly or moderately express CD200 protein outside the cell membrane.

[0015] Seventh, the present invention provides the application of immune cells expressing chimeric antigen receptors in the preparation of drugs for treating acute myeloid leukemia.

[0016] The CD200-targeting chimeric antigen receptor and its application of the present invention have the following advantages over the prior art:

[0017] (1) The chimeric antigen receptor targeting CD200 provided by the present invention includes a specific single-chain antibody ScFv, which is used to modify immune cells. The modified immune cells can be used to treat CD200-positive tumors and have a significant tumor-killing effect on acute myeloid leukemia.

[0018] (2) The present invention also provides a method for preparing immune cells expressing chimeric antigen receptors, which involves activating the isolated immune cells for 2-15 days and then infecting them with lentiviruses expressing chimeric antigen receptors. In this way, the original immune cells will not affect the tumor-killing effect of the transfected immune cells expressing chimeric antigen receptors. Furthermore, when performing in vitro functional tests on immune cells expressing chimeric antigen receptors, the selected cell lines are cell lines that highly or moderately express CD200 targets outside the cell membrane. This makes the evaluation of the tumor-killing effect of immune cells expressing chimeric antigen receptors more scientific. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a schematic diagram of the DNA fragment of anti-CD200-CAR in the examples;

[0021] Figure 2 This is a schematic diagram of the plasmid pattern of BRD-PTK-EF1α-anti-CD200-CAR in the example;

[0022] Figure 3A schematic diagram showing the proportion of CAR-positive cells after Lenti3-anti-CD200-CAR transduction into 293T cells;

[0023] Figure 4 This is a schematic diagram showing the CAR-T cell transduction efficiency results after Lenti3-anti-CD200-CAR transduction into T cells;

[0024] Figure 5 This is a schematic diagram showing the detection results of the expression of MOLM-13, K562-CD200, and CD200 on the surface of K562 cells.

[0025] Figure 6 This is a schematic diagram showing the in vitro killing effect of CAR-T cells on the acute myeloid leukemia cell line MOLM-13.

[0026] Figure 7 This is a schematic diagram showing the in vitro killing results of CAR-T cells on the negative cell line K562 and the overexpressing cell line K562-CD200. Detailed Implementation

[0027] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0028] This invention provides a CAR targeting the CD200 chimeric antigen receptor, a gene vector for the recombinant chimeric antigen receptor, and immune cells expressing the chimeric antigen receptor, as well as their applications, as detailed below:

[0029] I. Chimeric antigen receptor (CAR)

[0030] like Figure 1 As shown, the chimeric antigen receptor CAR of the present invention is sequentially spliced ​​from the N-terminus to the C-terminus with a signal peptide, a single-chain antibody ScFv, strepII, CD8 hinge, CD28 transmembrane region, CD28 intracellular domain, intracellular co-stimulatory domain 4-1BB, and CD3ζ chain; the single-chain antibody ScFv can recognize the CD200 antigen on the surface of tumor cells.

[0031] The nucleotide sequence of the signal peptide is shown in SEQ ID NO.7; the amino acid sequence of the single-chain antibody ScFv is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2; the nucleotide sequence of strepII is shown in SEQ ID NO.8; the nucleotide sequence of CD8αhinge is shown in SEQ ID NO.9; the nucleotide sequence of the transmembrane region of CD28 is shown in SEQ ID NO.10; the amino acid sequence of the intracellular domain of CD28 is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4; the amino acid sequence of the intracellular co-stimulatory domain 4-1BB is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.6; the amino acid sequence of the CD3ζ chain is shown in SEQ ID NO.11, and the nucleotide sequence is shown in SEQ ID NO.12.

[0032] II. Gene vectors for recombinant chimeric antigen receptors

[0033] The method for preparing the gene vector of the recombinant chimeric antigen receptor is as follows: using a viral or non-viral expression vector as a backbone, inserting the above-mentioned chimeric antigen receptor encoding nucleotide sequence into a lentivirus, adenovirus, adeno-associated virus, retrovirus, or transposon vector; preferably, using the viral vector BRD-PTK-EF1α as a backbone, inserting the above-mentioned chimeric antigen receptor encoding nucleotide sequence into a lentiviral vector.

[0034] 1. Construction of BRD-PTK-EF1α-anti-CD200-CAR plasmid

[0035] 1.1 Artificially synthesized anti-CD200-CAR fragment, the structure of which is shown in the schematic diagram. Figure 1 As shown.

[0036] 1.2 PCR amplification was performed using the anti-CD200-CAR fragment as a template to obtain the corresponding fragment containing the homologous arm of the BRD-PTK-EF1α vector after digestion with EcoRI and BamHI.

[0037] The nucleotide sequences of the signal peptide (SP) are shown in SEQ ID NO.7, the strepII nucleotide sequence is shown in SEQ ID NO.8, the CD8αhinge nucleotide sequence is shown in SEQ ID NO.9, the CD28TM nucleotide sequence is shown in SEQ ID NO.10, the CD28ICD nucleotide sequence is shown in SEQ ID NO.4, the 4-1BB nucleotide sequence is shown in SEQ ID NO.6, and the CD3ζ nucleotide sequence is shown in SEQ ID NO.12.

[0038] 1.3 The plasmid BRD-PTK-EF1α was double-digested with EcoRI and BamHI restriction endonucleases. The product was subjected to 1.0% agarose gel electrophoresis, and the gel was excised and collected in Eppendorf tubes. The corresponding fragments were recovered using an Axygen agarose gel recovery kit, and the purity and concentration of the product were determined.

[0039] 1.4 The fragment was added to an Eppendorf tube at a 1:2 molar ratio, along with Exnase II ligase (Vazyme) and 5×CE II buffer (homogeneous recombinase), and incubated at 37°C for 0.5 hours. 10 μL of the ligation solution was then added to 100 μL of LDH5α competent cells and incubated on ice for 30 min, followed by heat shock at 42°C for 90 s. After this, 500 μL of 2YT medium was added, and the cells were incubated at 37°C and 220 rpm for 2 hours. Two hours later, the Eppendorf tube was centrifuged at 4000g for 1 min to remove 400 μL of excess liquid. The remaining liquid was plated on LB agar plates and incubated at 37°C for 12 hours. Single colonies were picked from the plates and inoculated into 5 mL of LB liquid medium, and incubated at 37°C and 220 rpm for 12 hours.

[0040] 1.5 Plasmid was extracted using the Axygen miniprep kit to obtain the BRD-PTK-EF1α-anti-CD200-CAR plasmid. After verification by first-generation sequencing at the Wuhan branch of Beijing Qingke Biotechnology Co., Ltd., the *E. coli* DH5α strain containing the BRD-PTK-EF1α-anti-CD200-CAR plasmid was preserved. A complete schematic diagram of the BRD-PTK-EF1α-anti-CD200-CAR plasmid is shown below. Figure 2 As shown.

[0041] 2. Preparation and live droplet detection of BRD-PTK-EF1α-anti-CD200-CAR lentiviral vector

[0042] 2.1 Preparation of Lentiviral Vectors

[0043] E. coli with correct sequencing and containing the BRD-PTK-EF1α-anti-CD200-CAR plasmid were subjected to endotoxin-free large-scale extraction and co-transfected with three lentiviral packaging plasmids (pMDLg / pRRE, pRSV-Rev, and pMD2.G) into 293T cells. After 18-24 hours, fluid was added and the supernatant was collected after 48-72 hours.

[0044] Centrifuge the supernatant at 4000 rpm (or 3000 g) for 30 min, then filter the lentivirus supernatant through a 0.22 μm filter membrane and centrifuge at 30000 g, 4 °C for 2.5–3 h. Remove the supernatant and resuspend the pellet in 1 mL of T cell culture medium. After resuspending, reserve 20 μL for viral activity titer testing. Aliquot the remaining lentivirus concentrate into 100 μL vials, label them, and store at -80 °C for later use.

[0045] 2.2 Detection of lentiviral vector activity titer

[0046] Principle: The anti-strepII antibody is labeled with fluorescein, and the anti-strepII antibody can specifically bind to strepII in CAR. The fluorescence signal detected by flow cytometry indirectly reflects the expression of CAR in 293T cells.

[0047] Method: Introduce 5.0 × 10⁻⁶ mm wire into a 6-well plate. 5 293T cells per well were added, with 0.1 μL, 0.5 μL, and 1 μL of lentiviral concentrate added to each well, and one negative control was included. Cells were incubated at 37°C in a 5% CO2 incubator. After three days, 293T cells were collected using Versene solution (Gibco) and the proportion of CAR-positive 293T cells was determined by flow cytometry. Figure 3 As shown, the activity titer of the BRD-PTK-EF1α-anti-CD200-CAR lentivirus concentrate was calculated to be 2.01 × 10⁻⁶. 8 TU / mL, which can be used for the subsequent preparation of chimeric antigen receptor immune cells.

[0048] The calculation formula is: Activity titer (TU / mL) = (5.0 × 10⁻⁶) / mL 5 ×Positive cell percentage ×1000) / Virus addition amount.

[0049] III. Immune cells with chimeric antigen receptors

[0050] The method for preparing chimeric antigen receptor immune cells is as follows: immune cells are transfected with the above-mentioned chimeric antigen receptor encoding nucleotide sequence or the above-mentioned recombinant chimeric antigen receptor gene vector to obtain chimeric antigen receptor immune cells. The immune cells are selected from umbilical cord blood, peripheral blood or IPSC-derived T cells, NK cells, NKT cells, αβT cells, γδT cells, CD4+T cells, CD8+T cells, preferably peripheral blood-derived T cells, that is, CAR-T cells for treating broad-spectrum tumors with CD200 as the target are obtained. When the single-chain antibody ScFv of the chimeric antigen receptor CAR binds to CD200, the immune cells expressing the chimeric antigen receptor exhibit anti-tumor activity.

[0051] 3.1 Preparation of anti-CD200-CART cells

[0052] 3.1.1 Preparation of CAR-T cell preparations:

[0053] 100 mL of peripheral blood was collected from healthy subjects, and mononuclear cells were isolated using Ficoll lymphocyte separation medium. After counting, CD3-positive cells were sorted using an appropriate amount of CD3 MicroBeads, human (Medini), and counted at 1.0–2.0 × 10⁻⁶ cells / mL. 6 T cell density / mL in complete T cell culture medium (OpTmizer) TM CTS TM T-Cell Expansion Basal Medium, OpTmizer TM CTS T-Cell Expansion Supplement (Invitrogen), 500 IU / mL IL-2 (Shuanglu Pharmaceutical) were cultured in the solution, and the solution was administered at a rate of 10 mg / mL. 6 Add 25 μL of Dynabeads Human T-Activator CD3 / CD28 (Invitrogen) to each cell to activate T cells.

[0054] 24 hours later, Lenti3-anti-CD200-CAR lentiviral vector was added at MOI=5 for transduction, mixed well and incubated in a CO2 incubator. After 4 hours, an appropriate amount of T cell complete culture medium was added for further culture.

[0055] 24 hours after lentivirus transduction, the transduced cells were replaced with fresh complete T cell culture medium, and the viable cell density was adjusted to 1.0-2.0 × 10⁶ cells / year. 6 Continue culturing and expanding the cell culture for 10–20 days at a density of 1.0–2.0 × 10⁶ cells / mL, observing and counting the cells daily, and replenishing the culture medium as needed based on the calculated cell count, always maintaining a cell culture density of 1.0–2.0 × 10⁶ cells / mL. 6 / mL.

[0056] 3.1.2 Detection of anti-CD200-CART cell transduction efficiency

[0057] Take 1.0 × 10 6 After transduction, T cells were incubated with anti-strepII antibody at room temperature for 30 minutes, washed twice with physiological saline, and the anti-strepII-APC fluorescence signal was detected by flow cytometry to measure the APC positive cell ratio.

[0058] The results are as follows Figure 4 As shown, Figure 4This reflects the ratio of anti-CD200-CART cells in the total cells, with an anti-CD200-CART cell transduction efficiency of 29.1%.

[0059] 4. In vitro killing function detection of anti-CD200-CAR-T cells

[0060] The in vitro tumor-killing function of T cells and anti-CD200-CAR T cells was detected by the calcein assay.

[0061] Target cells were selected from the MOLM-13 acute myeloid leukemia cell line, which highly expresses CD200, and the K562 cell line, which overexpresses CD200. The negative target cells in the experimental group were K562 cells. The CD200 expression levels of these three cell types are as follows: Figure 5 As shown.

[0062] Depend on Figure 5 It can be seen that the acute myeloid leukemia cell line MOLM-13, which highly expresses CD200, and the K562 cell line, which overexpresses CD200, have high in vitro killing power.

[0063] Take an appropriate amount of target cells, in 1×10 6 Add Calcein-AM to a final concentration of 25 μM in a 1 mL / mL cell suspension (PBS, 5% fetal bovine serum) and incubate for 30 min. After washing twice at room temperature, resuspend the cells at a concentration of 0.5 × 10⁶ cells / mL. 5 / mL, add 0.5×10 to each well of a 96-well plate. 5 T cells and anti-CD200-CART cells were added at effector-to-target ratios of 25:1, 5:1, and 1:1, respectively, and incubated at 37°C for 2.5–3 hours. After incubation, the supernatant was collected, and the fluorescence intensity of calcein was measured. The percentage of target cell lysis was calculated based on the spontaneous release control and the maximum release control.

[0064] The in vitro killing and lysis results of anti-CD200-CAR T cells against the CD200-overexpressing acute myeloid leukemia cell line MOLM-13 are as follows: Figure 6 As shown, the in vitro killing and lysis results of CD200-non-expressing K562 and CD200-overexpressing cell lines K562-CD200 are as follows: Figure 7 As shown.

[0065] The results showed that anti-CD200-CAR T cells had improved lysis ability against CD200-positive cell lines compared to T cells, but had no lysis ability against the CD200-negative cell line K562, indicating that it specifically recognizes CD200 and suggests that it has a good therapeutic effect on acute myeloid leukemia.

[0066] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A chimeric antigen receptor targeting CD200, characterized in that: The chimeric antigen receptor comprises a signal peptide, a single-chain antibody ScFv, strepII, CD8α hinge, CD28 transmembrane region, CD28 intracellular domain, intracellular co-stimulatory domain 4-1BB, and CD3ζ chain, sequentially spliced ​​from N-terminus to C-terminus; the single-chain antibody ScFv specifically recognizes the CD200 antigen on the surface of tumor cells; The amino acid sequence of the single-chain antibody ScFv is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.

2.

2. The CD200-targeting chimeric antigen receptor as described in claim 1, characterized in that: The amino acid sequence of the CD28 intracellular domain is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.

4.

3. The CD200-targeting chimeric antigen receptor as described in claim 1, characterized in that: The amino acid sequence of the intracellular co-stimulatory domain 4-1BB is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.

6.

4. The use of a CD200 chimeric antigen receptor as described in any one of claims 1-3 in the preparation of a drug for treating acute myeloid leukemia.

5. A gene vector for a recombinant chimeric antigen receptor, characterized in that: Lentiviral, retroviral, or transposon vectors that use the BRD-PTK-EF1α vector as a backbone and insert the encoding nucleotide sequence of the chimeric antigen receptor as described in any one of claims 1-3.

6. The use of the gene vector of the recombinant chimeric antigen receptor as described in claim 5 in the preparation of a drug for treating acute myeloid leukemia.

7. An immune cell expressing a chimeric antigen receptor, characterized in that: The immune cells are obtained by transfecting immune cells with the encoding nucleotide sequence of the chimeric antigen receptor as described in any one of claims 1-3 or the recombinant chimeric antigen receptor gene vector as described in claim 5; the immune cells are selected from one of the following: umbilical cord blood, peripheral blood, IPSC-derived T cells, NK cells, NKT cells, αβT cells, γδT cells, CD4+T cells, and CD8+T cells.

8. The method for preparing immune cells expressing chimeric antigen receptors as described in claim 7, characterized in that, The procedure includes the following steps: activating the isolated immune cells for 1 day and then infecting the immune cells with the gene vector of the recombinant chimeric antigen receptor as described in claim 5 to obtain immune cells expressing the chimeric antigen receptor; and performing antitumor activity detection on the immune cells expressing the chimeric antigen receptor, wherein the selected cell line is one that highly or moderately expresses CD200 protein outside the cell membrane.

9. The use of an immune cell expressing a chimeric antigen receptor as described in claim 7 in the preparation of a drug for treating acute myeloid leukemia.

Images