A specific chimeric antigen receptor targeting CD3L1, immune cell and application thereof

Abstract

The present invention discloses a specific chimeric antigen receptor targeting CD3L1, an immune cell and an application thereof, and belongs to the field of biological genetic engineering; the structure of the specific chimeric antigen receptor contains an antigen binding domain targeting CD3L1; the antigen binding domain targeting CD3L1 is the extracellular segment of the CD3E receptor, and can specifically bind to CD3L1 in a ligand manner. It is beneficial to the phenomenon that tumor tissues highly express CD3L1, while normal tissues do not express (rarely express) CD3L1. By using the extracellular segment of the natural receptor (CD3E) of CD3L1 as one of the structural components of the specific chimeric antigen receptor, the targeting efficiency of the antigen receptor to the tumor tissue can be significantly improved, the probability of the occurrence of the tumor immune escape phenomenon is reduced, and the immune effect of the antigen receptor on the tumor tissue is greatly improved, which is beneficial to the large-scale promotion and application of the specific chimeric antigen receptor in the treatment of tumor diseases.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, primarily to the field of tumor immunotherapy biomedical technology, specifically to a specific chimeric antigen receptor targeting CD3L1, immune cells, and their applications. Background Art

[0002] In the research of novel cancer treatment methods, tumor immunotherapy has received increasing attention. Studies on the tumor microenvironment and anti-tumor immunity have provided a new research foundation for tumor immunotherapy, which includes adoptive immunotherapy and targeted drug therapy. Rosenberg et al. first proposed the concept of adoptive immunotherapy, which refers to the in vitro culture, expansion, and activation of the patient's own immune cells, such as T lymphocytes and natural killer cells (NK cells), combined with growth factors, and then infused back into the patient, enabling these immune effector cells to kill tumor cells, thereby achieving the goal of treating tumors. Although this therapy has achieved good results in the treatment of some tumors, including melanoma, it is not effective for most malignant tumors, mainly due to the limitation of the major histocompatibility complex, which greatly reduces the effectiveness of T lymphocytes in directly recognizing and killing tumors. Against this background, chimeric antigen receptor (CAR) modified T lymphocytes, with their high efficiency, specificity, persistence, and killing activity, have come into view. The basic structure of a CAR includes a tumor-associated antigen-binding domain (usually derived from the scFV segment of the antigen-binding region of a monoclonal antibody), an extracellular hinge region, a transmembrane region, and an intracellular immune receptor tyrosine-based activation motif (ITAM). This structure can overcome MHC constraints and can specifically target tumor cells through the extracellular expressed antigen-binding domain, thereby exerting highly efficient and durable anti-tumor activity.

[0003] CAR-T cells have undergone four generations of structural development. First-generation CARs have a simple structure and lack a co-stimulatory signaling domain, resulting in incomplete T cell activation and an inability to expand in vivo to meet clinical needs, leading to unsatisfactory therapeutic effects. Second-generation CARs added a co-stimulatory domain (CD28 or 4-1BB), significantly increasing the duration of CAR-T cell activity. Third-generation CARs added two co-stimulatory domains, resulting in stronger CAR-T cell activity in vivo; however, some studies suggest that third-generation CARs may lower the threshold for T cell response to stimulation, inducing more severe cytokine release syndrome. Fourth-generation CAR-T cells, also known as TRUCK T cells, contain an activated T cell nuclear factor transcription element, enabling CAR-T cells to secrete specific cytokines within the tumor infiltration area to modify the tumor microenvironment and promote the participation of other immune cells in the immune response. In the preparation of chimeric antigen receptor immune cells, target selection is crucial; broad-spectrum and relatively specific targets are ideal.

[0004] CD3L1 is a ligand of CD3E. CD3L1 on the surface of tumor cells binds to CD3E on T cells to inhibit T cell activation. CD3L1 is not expressed in most normal tissues, but is highly expressed in many tumor tissues. Xu Jie et al. first proposed that inhibiting the interaction between CD3L1 and CD3E activates T cells to kill tumors.

[0005] Although CAR-T cell antibodies have a certain therapeutic effect in blocking the binding of CD3L1 and CD3E in tumor treatment, their poor targeting ability leads to poor effectiveness, which seriously affects the large-scale promotion and application of CAR-T cells in tumor immunotherapy. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing specific chimeric antigen receptors, which have poor targeting effects on tumors and lead to prominent tumor immune escape phenomena. This invention proposes a specific chimeric antigen receptor targeting CD3L1, immune cells, and their applications.

[0007] To achieve the above objectives, the present invention provides a specific chimeric antigen receptor targeting CD3L1, wherein the antigen receptor contains an antigen-binding domain targeting CD3L1; the antigen-binding domain targeting CD3L1 is an extracellular segment of the CD3E receptor, which can specifically bind to CD3L1 antibodies in a ligand manner.

[0008] This invention discloses a specific chimeric antigen receptor targeting CD3L1, which is beneficial for the phenomenon that tumor tissues highly express CD3L1 while normal tissues do not express (or express very little) CD3L1. By incorporating the extracellular segment of the natural receptor of CD3L1 (CD3E) as one of the structural components of the specific chimeric antigen receptor, the targeting efficiency of the antigen receptor to tumor tissues can be significantly improved, the probability of tumor immune escape can be reduced, and the immune effect of the antigen receptor on tumor tissues can be greatly enhanced. This is conducive to the large-scale application of specific chimeric antigen receptors in the treatment of tumor diseases.

[0009] Preferably, the amino acid sequence of the antigen-binding domain targeting CD3L1 is as shown in SEQ ID NO:1; SEQ ID NO:1 is: DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGG DEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD; or the antigen-binding domain targeting CD3L1 may also be a polypeptide molecule structure that maintains a high degree of identity with the amino acid sequence of SEQ ID NO:1 and has the same function.

[0010] Preferably, the structure of the antigen receptor further includes a signal peptide, a hinge region, a transmembrane domain, a co-stimulatory signaling molecule, and an intracellular signal transduction sequence; the signal peptide, hinge region, transmembrane domain, co-stimulatory signaling molecule, and intracellular signal transduction sequence are connected by linker peptides or peptide bonds; by embedding multiple polypeptide functional segments into the antigen receptor, the targeting effect and expression of the antigen receptor can be significantly improved, which is beneficial for overexpression in immune cells.

[0011] Preferably, the amino acid sequence of the signal peptide is SEQ ID NO:4: MALPVTALLLPLALLLHAARP.

[0012] Preferably, the amino acid sequence of the hinge region is SEQ ID NO:5: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP.

[0013] Preferably, the amino acid sequence of the transmembrane domain is SEQ ID NO:6: FWVLVVVGGVLACYSLLVTVAFIIFWV.

[0014] Preferably, the co-stimulatory signaling molecule is at least one selected from CD27, CD28, 4-1BB, CD11c, ITGB1, OX40, CD30, CD40, ICOS, CD11b, and ITGAX; more preferably, the co-stimulatory signaling molecule is CD28 and / or 4-1BB; even more preferably, the co-stimulatory signaling molecule is 4-1BB; wherein the amino acid sequence of the 4-1BB is SEQ ID NO:7: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0015] Preferably, the amino acid sequence of the intracellular signal transduction sequence is SEQ ID NO:8: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0016] Preferably, the amino acid sequence of the antigen receptor is as shown in SEQ ID NO:2, wherein SEQ ID NO:2 NO:2 is: MALPVTALLLPLALLLHAARPDYKDDDDKDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDGSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVL ACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR; or the amino acid sequence of the antigen receptor can also be the same as SEQ ID The NO:2 amino acid sequence maintains a high degree of identity and has the same function in the polypeptide molecular structure.

[0017] Furthermore, the specific chimeric antigen receptor targeting CD3L1 of the present invention is obtained through artificial synthesis or genetic engineering transcription encoding.

[0018] Furthermore, the present invention also provides a nucleic acid molecule that encodes the aforementioned specific chimeric antigen receptor targeting CD3L1.

[0019]

[0020] Furthermore, the present invention also provides a carrier containing the above-mentioned nucleic acid molecule that can encode a specific chimeric antigen receptor targeting CD3L1.

[0021] Furthermore, the present invention also provides a host cell containing the above-mentioned vector or chromosome in which an exogenous nucleic acid molecule or expressing the above-mentioned specific chimeric antigen receptor targeting CD3L1 is integrated.

[0022] Furthermore, the present invention also provides an immune cell containing the above-mentioned vector or chromosome in which an exogenous nucleic acid molecule is integrated or which expresses the above-mentioned specific chimeric antigen receptor targeting CD3L1.

[0023] Furthermore, the present invention also provides a method for preparing the above-mentioned immune cells, comprising the following steps: transducing the above-mentioned nucleic acid molecules or the above-mentioned vector into NK92, T and / or NK cells to obtain the immune cells.

[0024] Furthermore, the present invention also provides a pharmaceutical composition comprising the above-described specific chimeric antigen receptor targeting CD3L1, the above-described nucleic acid molecule, the above-described carrier, the above-described host cell, and / or the above-described immune cell, as well as a pharmaceutically acceptable carrier, diluent, or excipient.

[0025] Furthermore, the present invention also provides the application of the above-mentioned CD3L1-targeting chimeric antigen receptor, the above-mentioned nucleic acid molecule, the above-mentioned carrier, the above-mentioned host cell, and / or the above-mentioned immune cell in the preparation of an oncology drug; the oncology drug can be used to prevent and / or treat tumor diseases; the tumor diseases include, but are not limited to, liver cancer, gastric cancer, pancreatic cancer, or combinations thereof.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] This invention, which targets CD3L1 with a specific chimeric antigen receptor, significantly improves the targeting efficiency of the antigen receptor to tumor tissues by incorporating the extracellular segment of the natural CD3L1 receptor (CD3E) as one of the structural components of the specific chimeric antigen receptor. This reduces the probability of tumor immune escape and greatly enhances the immune effect of the antigen receptor on tumor tissues, which is conducive to the large-scale application of specific chimeric antigen receptors in the treatment of tumor diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a bar chart showing the results of the in vitro tumor cell (hepatocellular carcinoma cell line Huh7-CD3L1) killing experiment in Experiment Example 1 of the present invention;

[0029] Figure 2 This is a bar chart showing the results of the in vitro tumor cell (gastric cancer cell line MKN-45-CD3L1) killing experiment in Experiment Example 1 of the present invention;

[0030] Figure 3 This is a bar chart showing the results of the inhibitory effect on mouse tumor cells (hepatocellular carcinoma cell line Huh7-CD3L1) in Experiment Example 2 of the present invention.

[0031] Figure 4 This is a bar chart showing the results of the inhibitory effect on tumor cells (gastric cancer cell line MKN-45-CD3L1) in mice in Experiment Example 2 of this invention. DETAILED DESCRIPTION

[0032] The present invention will now be described in further detail with reference to specific embodiments. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the reagents, biological materials, and detection kits used in the experiments are all commercially available unless otherwise specified.

[0034] Example 1:

[0035] A specific chimeric antigen receptor targeting CD3L1 has the structure S-CD3E-CD28hinge-CD28TM-4-1BB-CD3ζ, where CD3E is the antigen-binding domain targeting CD3L1 (the extracellular segment of the CD3E receptor, which can specifically bind to CD3L1 in a receptor-ligand manner); "-" represents a linker peptide or peptide bond; S represents a signal peptide; CD28hinge is the hinge region; CD28TM is the transmembrane domain; 4-1BB is a co-stimulatory signaling molecule; and CD3ζ is an intracellular signal transduction sequence.

[0036] The specific amino acid sequences of each segment in the antigen receptor are as follows:

[0037]

[0038] The amino acid sequence of the specific chimeric antigen receptor targeting CD3L1 is as follows: MALPVTALLLPLALLLHAARPDYKDDDDKDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDGSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0039]

[0040] The gene fragment (nucleic acid molecule) of the chimeric antigen receptor was introduced into the PCDH vector, and then the vector was introduced into Stab3 competent cells. Plasmids were extracted using a plasmid extraction kit (purchased from Qingke Company). After sequencing to confirm whether the target gene sequence was correct, the positive clone plasmids were synthesized into lentiviral vectors.

[0041] NK92 cells were cultured in NK92-specific medium at 37°C and 5% CO2, and the cells were collected by centrifugation. The cells were then resuspended in fresh medium and the cell density was adjusted to 1×10⁻⁶. 6 Cells / mL, lentiviral vector was added to 24-well culture plates at MOI=10, mixed well, and incubated at 37℃ in a 5% CO2 incubator. After 48h-96h, the culture medium was replaced with fresh medium. The transduction efficiency of CAR-NK92 cells was determined by PCR, and positive NK92 cells were screened out, which are immune cells (CAR-NK92) that can express the above-mentioned specific chimeric antigen receptor targeting CD3L1.

[0042] Experimental Example 1: CAR-NK92 Cells

[0043] In vitro tumor cell killing experiment

[0044] To verify the tumor-killing effect of CAR-NK92 cells, the human liver cancer cell line Huh7-CD3L1 and the gastric cancer cell line MKN-45-CD3L1 were selected as experimental subjects in this invention; and blank group (PBS saline buffer only), control group NK92, and CARNC-NK92 group (CARNC does not contain the extracellular CD3E component, but the rest is the same as CAR) were set up. The specific experimental methods are as follows:

[0045] (1) Tumor cell culture

[0046] Huh7-CD3L1 and MKN-45-CD3L1 cells were removed and revived in liquid nitrogen at a concentration of 1×10⁻⁶. 6 Cells / mL were seeded in RPMI 1640 complete medium (containing 10% FBS) and cultured aseptically in a 37°C, 5% CO2 cell culture incubator. Cells were counted and passaged every 48 hours to observe cell status. When cell viability reached 80% or higher, subsequent experiments were performed.

[0047] (2) Co-culture of CAR-NK92 cells

[0048] CAR-NK92 cells and the aforementioned tumor cells were harvested separately, washed three times with sterile PBS, and then mixed at a 2:1 ratio to adjust the cell density to 1×10⁻⁶. 6Inoculate 1 / mL of the sample into 24-well plates and incubate at 37°C in a 5% CO2 incubator for 24 hours. Use sterile PBS solution as a blank control.

[0049] (3) LDH method for detecting tumor killing efficiency of different CAR-NK92 cells

[0050] After CAR-NK92 cells were co-cultured with tumor cells for 24 hours, LDH was measured according to the Promega instructions.

[0051] The results are as follows Figure 2 As shown, the NK92 cells provided in this invention can kill hepatocellular carcinoma cells, but surprisingly, CAR-NK92 targeting CD3L1 is even more effective at killing cells.

[0052] The results are as follows Figure 3 As shown, the NK92 cells provided in this invention can kill gastric cancer cells, but surprisingly, CAR-NK92 targeting CD3L1 is even more effective at killing cells.

[0053] Experimental Example 2: Inhibitory effect of CAR-NK92 cells on tumors in vivo

[0054] (1) Animal model preparation and drug administration

[0055] Tumor cells were resuscitated and cultured using the same method as in Experiment 1. Twenty-four healthy BALB / c nude mice of similar weight, aged 6-8 weeks, were used. Huh7-CD3L1 was diluted to 1×10⁻⁶. 6 Cells / mL, 200 μL of the above cell solution was injected into the axilla of nude mice (subcutaneous tumor construction), and the tumor volume was increased to 100 mm². 3 The left and right positions indicate successful model establishment. The experimental animals were randomly divided into 4 groups (n=6 per group): NK92 group, CARNC-NK92 group, CAR-NK92 and PBS blank group, and injected with 1×10⁻⁶ mg / L of each group. 6 Cells were collected, with an equal volume of physiological saline as a control group. The same method was used for the gastric cancer cell line MKN-45-CD3L1.

[0056] (2) Tumor volume detection

[0057] The first CAR-NK92 injection was recorded as week 0. Tumor volume was measured every 5 days using the following method: the long diameter (L) and short diameter (W) of the tumor were measured with calipers, and the volume was calculated using the formula V = (W / L). 2 Calculate the tumor volume using (×L) / 2.

[0058] like Figure 3As shown, the CAR-NK92 cells provided in this invention can significantly inhibit the increase in Huh7-CD3L1 tumor volume and inhibit the tumor growth process. In in vivo experiments, CAR-NK92 cells can exert a long-lasting and efficient tumor-suppressive effect.

[0059] like Figure 4 As shown, the CAR-NK92 cells provided in this invention can significantly inhibit the increase in MKN-45-CD3L1 tumor volume and inhibit the tumor growth process. In in vivo experiments, CAR-NK92 cells can exert a long-lasting and efficient tumor-suppressive effect.

Claims

1. A specific chimeric antigen receptor targeting CD3L1, characterized in that: The structure of the specific chimeric antigen receptor contains an antigen binding domain targeting CD3L1; the antigen binding domain targeting CD3L1 is the extracellular segment of the CD3E receptor, and can specifically bind to CD3L1 in a ligand manner.

2. The specific chimeric antigen receptor according to claim 1, characterized in that The structure of the specific chimeric antigen receptor also includes a signal peptide, a hinge region, a transmembrane domain, a co-stimulatory signal molecule and an intracellular signal transduction sequence; the signal peptide, the antigen binding domain targeting CD3L1, the hinge region, the transmembrane domain, the co-stimulatory signal molecule and the intracellular signal transduction sequence are connected in sequence through a connecting peptide or a peptide bond.

3. The specific chimeric antigen receptor according to claim 2, characterized in that The co-stimulatory signal molecule is at least one of CD27, CD28, 4-1BB, CD11c, ITGB1, OX40, CD30, CD40, ICOS, CD11b, and ITGAX.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the specific chimeric antigen receptor targeting CD3L1 according to any one of claims 1 to 3.

5. A carrier, characterized in that The vector contains the nucleic acid molecule encoding the specific chimeric antigen receptor targeting CD3L1 according to claim 4.

6. A host cell, characterized in that The host cell contains the vector according to claim 5 or a chromosome in which the exogenous nucleic acid molecule according to claim 4 is integrated, or expresses the specific chimeric antigen receptor targeting CD3L1 according to any one of claims 1 to 3.

7. An immune cell, characterized in that The immune cell contains the vector according to claim 5 or a chromosome in which the exogenous nucleic acid molecule according to claim 4 is integrated, or expresses the specific chimeric antigen receptor targeting CD3L1 according to any one of claims 1 to 3.

8. A method for preparing immune cells according to claim 7, characterized in that: The method comprises the following steps: transducing the nucleic acid molecule according to claim 4 or the vector according to claim 5 into NK92, T and / or NK cells, thereby obtaining the immune cells.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the specific chimeric antigen receptor targeting CD3L1 as described in any one of claims 1 to 3, the nucleic acid molecule as described in claim 4, the vector as described in claim 5, the host cell as described in claim 6, and / or the immune cell as described in claim 7, and a pharmaceutically acceptable carrier, diluent or excipient.

10. Use of a specific chimeric antigen receptor targeting CD3L1 according to any one of claims 1 to 3, a nucleic acid molecule according to claim 4, a vector according to claim 5, a host cell according to claim 6, and / or an immune cell according to claim 7 in the preparation of an anticancer drug.

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