CAR-T cells targeting CLL1, and their manufacturing method and use.

Abstract

This invention relates to CAR-T cells targeting CLL1, as well as a method for producing and using the same. Specifically, it relates to CAR-T cells containing a chimeric antigen receptor, the chimeric antigen receptor comprising a single-domain antibody, a hinge region, a transmembrane region, and an intracellular signaling region, with the amino acid sequence of the single-domain antibody being at positions 22-150 of SEQ ID No. 1. Experiments have demonstrated that CLL1-VHH-16 CAR-T cells effectively secrete the T cell-specific effector molecule IFN-γ, efficiently and specifically killing CLL1-positive target cells, exhibiting excellent in vivo tumor-killing activity, significantly inhibiting tumor cell proliferation in mice, and significantly extending the survival time of mice. CLL1-VHH-16 CAR-T cells possess excellent antitumor capabilities and can be used in immunotherapy for CLL1-targeted diseases, offering potential for a wide range of clinical applications.

Description

[Technical Field]

[0001] This invention belongs to the field of cell immunotherapy, and more specifically relates to CAR-T cells targeting CLL1, as well as methods for producing and using the same. [Background technology]

[0002] Acute myeloid leukemia (AML) is a malignant disease of the hematopoietic stem / progenitor cells in the bone marrow. For a long time, anthracycline and cytarabine chemotherapy has been used to treat mild cases, and hematopoietic stem cell transplantation has been used to treat intermediate- and high-risk cases. Due to the heterogeneity of acute myeloid leukemia, treating AML patients remains challenging. Recently, researchers have discovered that immunotherapy targeting CLL1 is effective in treating AML. C-type lectin-like molecule 1 (CLL1), also known as C-type lectin domain family 12 member A (CLEC12A), is a type II transmembrane glycoprotein that plays an important role in immunomodulation as an inhibitory receptor. CLL1 is present in peripheral blood, bone marrow cells, and most AML cells, and is found in many AML cells. + CD38 - CD34 is expressed in stem cells, but is normal. + CD38 - Because it is not expressed in stem cells, CLL1 is a potential target in the treatment and diagnosis of AML due to its unique expression pattern. Furthermore, CLL1 is also expressed in cells of myelodysplastic syndromes (MDS) and chronic myeloid leukemia (CML, chronic granulocytic leukemia).

[0003] Conventional radiotherapy, chemotherapy, and hematopoietic stem cell transplantation have low efficacy rates in treating AML, but the advent of CAR-T cell therapy has made it possible to cure AML. CAR-T stands for Chimeric Antigen Receptor T-Cell Immunotherapy. Chimeric antigen receptor technology uses genetic engineering to modify immune cells and express exogenous antitumor genes (CAR genes), giving immune cells such as lymphocytes the ability to recognize surface antigens of tumor cells. This allows them to specifically recognize and kill tumor cells without being restricted by the major histocompatibility complex (MHC). The structure of CAR mainly consists of an extracellular domain that recognizes surface antigens of tumor cells and an intracellular signaling domain. The extracellular domain is used to specifically recognize specific proteins (antigens) on the tumor surface, while the intracellular domain, which includes a co-stimulatory molecule domain, is used to initiate an immune response by lymphocytes after the specific proteins (antigens) on the tumor surface are recognized, exerting a cytotoxic effect on specific target cells and killing the target cells (tumor cells). Currently, CAR-T technology is being applied in clinical medicine, and numerous clinical successes have been reported worldwide. CAR-T cell therapy has shown promising efficacy in hematological malignancies such as B-cell tumors. However, the antigenic properties of bone marrow cells are fundamentally different from those of B cells, and CAR-T damage to normal bone marrow cells leads to bone marrow failure. Because there is no corresponding replacement therapy, progress in CAR-T therapy for AML has been very slow. Many challenges remain for the success of CAR-T technology in AML. Therefore, further thorough research and development of novel and effective CAR-T cells and improvements in the efficacy of CAR-T cells have important theoretical and clinical application value for immunotherapy of AML. [Overview of the Initiative]

[0004] One of the objects of the present invention is to provide CLL1-targeting CAR-T cells for use in the treatment of acute myeloid leukemia (AML). The technical problems to be solved are not limited to the technical subjects described herein, and those skilled in the art will be able to clearly understand other technical subjects not described herein through the following description.

[0005] To achieve the above objective, the present invention provides CAR-T cells containing a chimeric antigen receptor. The chimeric antigen receptor may include a single-domain antibody, a hinge region, a transmembrane region, and an intracellular signaling region, and the amino acid sequence of the single-domain antibody may be from position 22 to 150 of SEQ ID No. 1.

[0006] CAR-T cells (CAR-expressing T cells or CAR-modified T cells) may be CAR-T cells that target CLL1, and CAR-T cells express chimeric antigen receptors.

[0007] Furthermore, the intracellular signaling region may also include the co-stimulatory domain 4-1BB and the activation domain CD3ζ, the amino acid sequence of 4-1BB may be at positions 220-266 of SEQ ID No. 1, and the amino acid sequence of CD3ζ may be at positions 267-378 of SEQ ID No. 1.

[0008] Furthermore, the hinge region may be the CD8a hinge region whose amino acid sequence is at positions 151-195 of SEQ ID No. 1, and the transmembrane region may be the CD8a transmembrane region whose amino acid sequence is at positions 196-219 of SEQ ID No. 1.

[0009] Furthermore, the chimeric antigen receptor, named CLL1-VHH-16 CAR, may contain one of the following: A1) A protein having the amino acid sequence shown at positions 22-378 of SEQ ID No. 1, A2) A protein having 80% or more identity with the protein described in A1) and having the same function, in which amino acid residues are substituted and / or deleted and / or added in the amino acid sequence shown at positions 22-378 of SEQ ID No. 1. A3) A fusion protein having the same function as A1), with a tag or signal peptide added to the N-terminus and / or C-terminus of A1) or A2).

[0010] Furthermore, the amino acid sequence of the signal peptide described in A3) may also be positions 1-21 of SEQ ID No. 1.

[0011] Furthermore, the chimeric antigen receptor may include one of the following: E1) A protein having the amino acid sequence shown in SEQ ID No. 1, E2) A protein having 80% or more identity with the protein described in E1) and having the same function, in which amino acid residues are substituted and / or deleted and / or added in the amino acid sequence shown in SEQ ID No. 1. E3) A fusion protein having the same function as E1), with tags added to the N-terminus and / or C-terminus of E1) or E2).

[0012] The tags described herein include, but are not limited to, GST (glutathione S-transferase) tagged proteins, His6 tagged proteins (His-tag), MBP (maltose-binding protein) tagged proteins, Flag tagged proteins, SUMO tagged proteins, HA tagged proteins, Myc tagged proteins, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tagged proteins.

[0013] A person skilled in the art can easily mutate the nucleotide sequence encoding the chimeric antigen receptor of the present invention using known methods such as the directed evolution method or the site-directed mutagenesis method. Nucleotides having 75% or more identity with the nucleotide sequence of the chimeric antigen receptor of the present invention that have been artificially modified are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode a chimeric antigen receptor and have the function of the chimeric antigen receptor.

[0014] Any of the chimeric antigen receptors described herein is within the scope of protection of the present invention.

[0015] The chimeric antigen receptor may sequentially include a signal peptide, a single-domain antibody, a hinge region, a transmembrane region, a co-stimulatory domain, and an activation domain from the N-terminus to the C-terminus.

[0016] The present invention further provides a biological material. The biological material may be any one of the following: B1) A nucleic acid molecule encoding any of the chimeric antigen receptors described herein, B2) An expression cassette containing the nucleic acid molecule described in B1), B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2), B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

[0017] In the above biological material, the nucleic acid molecule described in B1) may be any one of the following: C1) A DNA molecule whose nucleotide sequence or coding sequence is at positions 64 to 1134 of SEQ ID No. 2, C2) A DNA molecule having 75% or more identity with the nucleotide sequence defined in C1) and having the same function, C3) A DNA molecule whose nucleotide sequence or coding sequence is SEQ ID No. 2, A DNA molecule having at least 75% identity with the nucleotide sequence defined by C4)C3) and having the same function.

[0018] In one embodiment of the present invention, the chimeric antigen receptor is named CLL1-VHH-16 CAR and, from the N-terminus to the C-terminus, in the order of a signal peptide (Signal), a single-domain antibody (CLL1-VHH-16), a CD8a hinge region (Hinge), a CD8a transmembrane region, a co-stimulatory domain 4-1BB, and an activation domain CD3ζ (Figure 1). Among these, the single-domain antibody is the single-domain antibody CLL1-VHH-16 that specifically binds to the CLL1 protein, and the single-domain antibody CLL1-VHH-16 is the extracellular antigen-binding region of the CAR and plays a role in recognizing and binding to the tumor surface antigen CLL1. A signal peptide (Signal) for inducing the extracellular translocation of the peptide chain of the antigen-binding region is added in front of (N-terminus) the antigen-binding region so as to recognize the tumor surface antigen CLL1. The CD8a hinge region (Hinge) is the peptide chain of the CAR located extracellularly and connects the extracellular antigen-binding region and the CD8a transmembrane region. The CD8a transmembrane region is used to fix the single-domain antibody (CLL1-VHH-16) to the cell membrane. The intracellular signal region includes a co-stimulatory domain 4-1BB and an activation domain CD3ζ, synergistically stimulates T cells, activates intracellular signals, enables continuous proliferation and cytokine release of T cells, enhances the anti-tumor ability of T cells, and also plays the signal transduction function of T cells and is used to promote a stronger and more persistent T cell response.

[0019] The amino acid sequence of CLL1-VHH-16 CAR is shown in SEQ ID NO.1. Positions 1-21 of SEQ ID NO.1 represent the amino acid sequence of the signal peptide, positions 22-150 represent the amino acid sequence of the single-domain antibody (CLL1-VHH-16), positions 151-195 represent the amino acid sequence of the CD8a hinge region, positions 196-219 represent the amino acid sequence of the CD8a transmembrane region, positions 220-266 represent the amino acid sequence of the costimulatory domain 4-1BB, and positions 267-378 represent the amino acid sequence of the activation domain CD3ζ.

[0020] The gene encoding CLL1-VHH-16 CAR is the CLL1-VHH-16 CAR gene, and its nucleotide sequence is shown in SEQ ID NO. 2. Positions 1-63 of SEQ ID NO. 2 are the nucleotide sequence of the signal peptide, positions 64-450 are the nucleotide sequence of the single-domain antibody (CLL1-VHH-16), positions 451-585 are the nucleotide sequence of the CD8a hinge region, positions 586-657 are the nucleotide sequence of the CD8a transmembrane region, positions 658-798 are the nucleotide sequence of the costimulatory domain 4-1BB, and positions 799-1134 are the nucleotide sequence of the activation domain CD3ζ.

[0021] A 75% or higher degree of identity may also be 80%, 85%, 90%, or 95% or higher.

[0022] Identity of 80% or more may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Identity of 85% or more may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. Identity of 90% or more may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. A 95% or higher level of identity may be at least 95%, 96%, 97%, 98%, or 99%.

[0023] In this specification, identity refers to the identity of an amino acid sequence or nucleotide sequence. Amino acid sequence identity can be measured using homology search sites on the internet, such as the BLAST page on the NCBI homepage. For example, using the advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the Matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, the identity value (%) can be obtained by searching for and calculating amino acid sequence identity.

[0024] The vectors described herein mean vectors that can introduce, amplify, and express foreign DNA or a target gene into host cells. The vectors may be cloning vectors or expression vectors, and include, but are not limited to, plasmids, phages (such as λ phages and M13 filamentous phages), cosmids, and viral vectors (such as baculovirus vectors, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, and herpesviruses (such as herpes simplex virus)). In one or more embodiments of the present invention, the vector is the pUC57 vector and / or the retroviral vector MP71.

[0025] The microorganisms described herein may be bacteria, yeasts, algae, or fungi. Here, bacteria may include, but are not limited to, genera such as Escherichia sp., Erwinia sp., Agrobacterium sp., Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., and Bacillus sp. Yeasts may include, but are not limited to, budding yeasts such as Saccharomyces sp., Pichia sp., Yarrowia sp., Candida sp., fission yeasts such as Schizosaccharomyces sp., Hansenula sp., and killer yeasts such as Kluyreromyces sp. Algae may include, but are not limited to, genera such as Fucus sp., Achnanthes sp., Amphiprora sp., Amphora sp., Ankistrodesmus sp., Asteromonas sp., and Boekelovia sp. Fungi may include, but are not limited to, genera such as Fusarium sp., Rhizoctonia sp., Verticillium sp., Penicillium sp., Aspergillus sp., and Cephalosporium sp. In one or more embodiments of the present invention, the microorganism is Escherichia coli DH5α.

[0026] The recombinant vectors described herein mean recombinant vector DNA molecules constructed by ligating a foreign target gene to a vector in vitro, and can be constructed by any suitable method, as long as the constructed recombinant vector delivers the foreign target gene to a receptor cell and imparts the ability to replicate, integrate, amplify and / or express the foreign target gene in the receptor cell. In one or more embodiments of the present invention, the recombinant vector is the recombinant retroviral vector MP71-CLL1-VHH-16 CAR.

[0027] The recombinant retroviral vector MP71-CLL1-VHH-16 CAR is a recombinant expression vector obtained by replacing the fragment (small fragment) between the NotI and EcoRI recognition sites of the retroviral vector MP71 with a DNA fragment having the nucleotide sequence shown in SEQ ID No. 2 of the sequence listing, without altering the other nucleotide sequences of the retroviral vector MP71.

[0028] The recombinant microorganisms described herein refer to microorganisms whose function has been altered by manipulating or modifying the genes of the target microorganism. Examples include recombinant microorganisms obtained by introducing an exogenous target gene or recombinant vector into the target microorganism, or recombinant microorganisms obtained by directly editing the endogenous genes of the target microorganism. In one or more embodiments of the present invention, the recombinant microorganism is a recombinant bacterium containing the DNA molecule shown in SEQ ID No. 2, obtained by introducing the recombinant retroviral vector MP71-CLL1-VHH-16 CAR into Escherichia coli DH5α.

[0029] The CAR-T cells described herein are recombinant cells obtained by introducing a constructed chimeric antigen receptor (CAR) gene into T cells using an in vitro recombination method in genetic engineering and stably expressing it. Furthermore, the CAR-T cells described herein may also be CLL1-VHH-16 CAR-T cells obtained by introducing the CLL1-VHH-16 CAR gene (SEQ ID No. 2) into T cells and stably expressing it.

[0030] Specifically, CLL1-VHH-16 CAR-T cells may be recombinant cells obtained by packaging the recombinant retroviral vector MP71-CLL1-VHH-16 CAR with packaging cells to obtain a recombinant retrovirus, and then introducing the recombinant retrovirus into T cells. The packaging cells may be Phoenix Ecotropic (ECO) cells and PG13 cells.

[0031] CLL1-VHH-16 CAR-T cells contain and / or express the CLL1-VHH-16 CAR gene (SEQ ID No. 2).

[0032] The CLL1-VHH-16 CAR-T cells described herein have at least one of the following characteristics: F1) Secretion of the specific effector molecule IFN-γ, F2) Specific killing of CLL1-positive tumor cells, F3) Inhibition of proliferation of CLL1-positive tumor cells.

[0033] The present invention further provides the use of the chimeric antigen receptor CLL1-VHH-16 CAR in the production of CLL1-VHH-16 CAR-T cells.

[0034] The present invention further provides pharmaceutical compositions comprising any of the CAR-T cells described herein.

[0035] The pharmaceutical composition is used for the prevention or treatment of CLL1 target-related diseases.

[0036] Furthermore, the pharmaceutical composition may contain one or more pharmaceutically acceptable carriers.

[0037] Furthermore, as is well known to those skilled in the art, pharmaceutically acceptable carriers may include excipients, stabilizers, suspending agents, or diluents.

[0038] The present invention further provides any one of the following uses of any CAR-T cell, chimeric antigen receptor, biomaterial, or pharmaceutical composition described herein: D1) Use in the manufacture of pharmaceuticals for the prevention or treatment of tumors, or use in the prevention or treatment of tumors, D2) Use in the manufacture of pharmaceuticals for the prevention or treatment of CLL1 target-related diseases, or use in the prevention or treatment of CLL1 target-related diseases.

[0039] Furthermore, in the above use, the CLL1 target-related disease may be a CLL1-positive cancer (a cancer that expresses CLL1).

[0040] Furthermore, CLL1-positive cancer may also be acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), or chronic myeloid leukemia (CML, chronic granulocytic leukemia).

[0041] The present invention further provides a method for producing any of the CAR-T cells described herein. This method may include introducing any of the nucleic acid molecules described herein into T cells and stably expressing them to obtain CAR-T cells.

[0042] Furthermore, this method may include the following steps: (1) The CLL1-VHH-16 CAR gene (SEQ ID No. 2) is constructed between the NotI and EcoRI recognition sites of the retroviral vector MP71 to obtain the recombinant retroviral vector MP71-CLL1-VHH-16 CAR. (2) The recombinant retrovirus vector MP71-CLL1-VHH-16 CAR is introduced into packaging cells and packaged to obtain a recombinant retrovirus. (3) A step in which recombinant retrovirus is used to infect human T cells to obtain recombinant cells called CLL1-VHH-16 CAR-T cells.

[0043] Furthermore, the packaging cells may also be Phoenix Ecotropic (ECO) cells and PG13 cells.

[0044] The present invention further provides a method for preventing or treating CLL1 target-related diseases. This method may include administering any of the CAR-T cells or pharmaceutical compositions described herein to a subject suffering from a CLL1 target-related disease.

[0045] Furthermore, CLL1 target-related tumors may also be CLL1-positive cancers.

[0046] Furthermore, CLL1-positive cancer may also be acute myeloid leukemia, myelodysplastic syndrome, or chronic myeloid leukemia.

[0047] CLL1 as described herein is a C-type lectin-like molecule 1, also known as C-type lectin domain family 12 member A (CLEC12A), and is a type II transmembrane glycoprotein that plays an important role in immunomodulation as an inhibitory receptor. CLL1 is present in peripheral blood, bone marrow cells, and most acute myeloid leukemia (AML) cells, and is found in many AML CD34 + CD38 - It is expressed in stem cells, but normal CD + CD38 - Because it is not expressed in stem cells, CLL1, with its unique expression pattern, is a potential target in the treatment and diagnosis of AML.

[0048] This invention designs a novel CLL1-targeting CAR-T cell (CLL1-VHH-16 CAR-T cell) with a unique structure. Experiments have demonstrated that the CLL1-VHH-16 CAR-T cells of this invention effectively secrete the T cell-specific effector molecule IFN-γ, efficiently and specifically killing CLL1-positive target cells, exhibiting excellent in vivo tumor-killing activity. This not only significantly inhibits tumor cell proliferation in mice but also significantly extends the survival time of mice. The CLL1-VHH-16 CAR-T cells of this invention possess excellent antitumor capabilities and can be used in immunotherapy for CLL1-targeted diseases (such as acute myeloid leukemia, myelodysplastic syndrome, or chronic myeloid leukemia), offering potential for a wide range of clinical applications. [Brief explanation of the drawing]

[0049] [Figure 1] This is a schematic diagram of the CLL1-VHH-16 CAR molecular structure. [Figure 2] This figure shows the detection results of CAR expression in CLL1-VHH-16 CAR-T cells. [Figure 3] This figure shows the results of detecting cellular function of IFN-γ secretion by CLL1-VHH-16 CAR-T cells. [Figure 4] This figure shows the results of detecting cytotoxicity in CLL1-VHH-16 CAR-T cells. [Figure 5] This figure shows the results of detecting the in vivo tumor-killing activity of CLL1-VHH-16 CAR-T cells. [Modes for carrying out the invention]

[0050] The present invention will be described in detail below with reference to specific embodiments, but the embodiments shown are for illustrative purposes only and do not limit the scope of the present invention. The embodiments provided below can be used by those skilled in the art as a guide for further improvements, but do not limit the present invention in any way.

[0051] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were carried out in accordance with the techniques or conditions described in the literature in the art, or in accordance with the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0052] The retroviral vector MP71 used in the following examples is described in the following literature: Engels B, Cam H, et al. Retroviral Vectors for High-Level Transgene Expression in TLymphocytes[J]. Human Gene Therapy, 2003, 14(12):1155-1168. This biomaterial is available from the applicant and shall be used solely for the purpose of repeating the experiments of the present invention and not for any other purpose.

[0053] The human peripheral blood mononuclear cells (PBMCs) in the following examples were collected from the venous blood of healthy volunteers.

[0054] The U937 cells (CLL1-positive cells) in the following examples are acute myeloid leukemia cells and are a product of Beijing Baiou Boyei Biotechnology Co., Ltd. (catalog number bio-73180).

[0055] Example 1: Structure and sequence of a chimeric antigen receptor (CAR) The chimeric antigen receptor (CAR) designed and constructed in this example is named CLL1-VHH-16 CAR, and from the N-terminus to the C-terminus, it consists of a signal peptide, a single-domain antibody (CLL1-VHH-16), a CD8a hinge region, a CD8a transmembrane region, a costimulatory domain 4-1BB, and an activating domain CD3ζ (Figure 1).

[0056] Of these, the single-domain antibody is CLL1-VHH-16, which specifically binds to the CLL1 protein. The single-domain antibody CLL1-VHH-16 is the extracellular antigen-binding domain of the CAR, and plays a role in recognizing and binding to the tumor surface antigen CLL1. A signal peptide is added to the front (N-terminus) of the antigen-binding domain to induce the extracellular translocation of the peptide chain of the antigen-binding domain to recognize the tumor surface antigen CLL1. The CD8a hinge domain is the extracellular peptide chain of the CAR, and it links the extracellular antigen-binding domain and the CD8a transmembrane domain. The CD8a transmembrane domain is used to immobilize the single-domain antibody (CLL1-VHH-16) to the cell membrane. The intracellular signaling region, comprising the co-stimulatory domain 4-1BB and the activating domain CD3ζ, synergistically stimulates T cells, activates intracellular signaling, enables sustained T cell proliferation and cytokine release, enhances the antitumor capacity of T cells, and exerts T cell signaling function, thereby promoting a stronger and more sustained T cell response.

[0057] The amino acid sequence of the designed CLL1-VHH-16 CAR is shown in SEQ ID NO.1. Positions 1-21 of SEQ ID NO.1 represent the amino acid sequence of the signal peptide, positions 22-150 represent the amino acid sequence of the single-domain antibody (CLL1-VHH-16), positions 151-195 represent the amino acid sequence of the CD8a hinge region, positions 196-219 represent the amino acid sequence of the CD8a transmembrane region, positions 220-266 represent the amino acid sequence of the costimulatory domain 4-1BB, and positions 267-378 represent the amino acid sequence of the activation domain CD3ζ.

[0058] The gene encoding CLL1-VHH-16 CAR is the CLL1-VHH-16 CAR gene, and its nucleotide sequence is shown in SEQ ID NO. 2. Positions 1-63 of SEQ ID NO. 2 are the nucleotide sequence of the signal peptide, positions 64-450 are the nucleotide sequence of the single-domain antibody (CLL1-VHH-16), positions 451-585 are the nucleotide sequence of the CD8a hinge region, positions 586-657 are the nucleotide sequence of the CD8a transmembrane region, positions 658-798 are the nucleotide sequence of the costimulatory domain 4-1BB, and positions 799-1134 are the nucleotide sequence of the activation domain CD3ζ.

[0059] Example 2: Production of CLL1-VHH-16 CAR-T cells The CAR-T cells produced in this example were stably expressed by introducing the CLL1-VHH-16 CAR gene (SEQ ID No. 2) from Example 1 into T cells, thereby obtaining T cells expressing the CLL1-VHH-16 CAR gene (named CLL1-VHH-16 CAR-T cells). The specific procedure is as follows.

[0060] 2-1. Construction of recombinant retroviral vectors (1) The recombinant vector pUC57-CLL1-VHH-16 CAR was double-digested with NotI (NEB) and EcoRI (NEB), and the target gene fragment was isolated and recovered. The recombinant vector pUC57-CLL1-VHH-16 CAR was obtained by cloning the CLL1-VHH-16 CAR gene (SEQ ID No. 2) into the pUC57 vector, and was synthesized and supplied by ▲Chin▼ Biotechnology Co., Ltd. (2) The retrovirus vector MP71 was double digested with NotI and EcoRI, and the large vector fragments were isolated and recovered. (3) The above target gene fragment and the large vector fragment were ligated using T4 ligase (NEB) to obtain a recombinant retroviral vector MP71-CLL1-VHH-16 CAR carrying the CLL1-VHH-16 CAR gene. (4) The recombinant retroviral vector MP71-CLL1-VHH-16 CAR was transformed into competent Escherichia coli DH5α, and the plasmid was extracted and purified using a plasmid purification kit from Qiagen to obtain the MP71-CLL1-VHH-16 CAR plasmid.

[0061] The recombinant retroviral vector MP71-CLL1-VHH-16 CAR (i.e., the MP71-CLL1-VHH-16 CAR plasmid) is a recombinant expression vector obtained by replacing the fragment (small fragment) between the NotI and EcoRI recognition sites of the retroviral vector MP71 with a DNA fragment having the nucleotide sequence shown in SEQ ID No. 2 of the sequence listing, without changing the other nucleotide sequences of the retroviral vector MP71.

[0062] 2-2, Packaging of Retrovirus The MP71-CLL1-VHH-16 CAR plasmid produced in Procedure 2-1 was introduced into packaging cells for packaging to complete virus assembly and obtain retrovirus. The specific procedure for virus packaging is as follows. a) Day 1: Phoenix Ecotropic (ECO) cells must have a passage number less than 20 and not be fully confluent. Seed at a cell density of 0.6×10 6 / ml, add 10 mL of DMEM medium to a 10 cm dish, mix the cells well, and culture overnight at 37°C. b) Day 2: Transfection was performed when the ECO cells reached approximately 90% confluence (usually about 14-18 hours after seeding). 12.5 μg of plasmid, 250 μL of 1.25 M CaCl2, and 1 mL of H2O were prepared to make a total volume of 1.25 mL. An equal volume of 2 × HBS was added to a separate test tube and the plasmid complex was vortexed for 20 seconds while adding the plasmid complex. The mixture was gently added along the edge of the ECO dish and incubated at 37°C for 4 hours. The medium was removed, washed once with PBS, and then preheated fresh medium was added again. c) Day 4: The supernatant was collected 48 hours after transfection, filtered through a 0.45 μm filter to obtain the retrovirus solution, aliquoted, and stored at -80°C. d) 1.2 mL of 15 μg / mL Retronectin coating solution was added to each well of an NTC 6-well plate, and the plates were incubated overnight at 4°C. e) Carefully aspirate and remove the blocking solution, wash with 2 mL / well of PBS, add 5 mL of the above virus solution to each well, centrifuge at 32°C and 2000 × g for 2 hours, and discard the unbound virus supernatant. f) PG13 cells in the logarithmic growth phase were rinsed once with 10 mL of PBS, 1 mL of 0.25% recombinant trypsin was added, and the cells were allowed to stand at room temperature for 2-3 minutes. g) Add 5 ml of complete medium containing 10% FBS to stop digestion, and centrifuge at 1500 rpm for 5 minutes. h) Discard the supernatant and reduce the cell density to 0.5 × 10⁶ in complete medium. 5 Prepare the solution to cells / mL and add 3 mL / well to an NTC 6-well plate coated with the above virus, and the final cell count should be 1.5 × 10⁶. 5 The cells were divided into wells. i) Centrifuged at 1000 rpm for 1 minute, then incubated at 37°C and 5% CO2 for 48 hours. j) After 1-2 passaging cycles, the cultures were transferred to T175 culture flasks and incubated in DEME medium containing 12% FBS for 2 days. k) Replace with DEME medium containing fresh 12% FBS, incubate for 48 hours, collect the supernatant, filter through a 0.45 μm filter to obtain the retrovirus solution, aliquot and store at -80°C.

[0063] 2-3. Retroviral infection of human T cells a) Thaw frozen healthy human peripheral blood PBMCs and use RPMI-1640 medium containing 10% fetal bovine serum (FBS) to increase the cell density to 1 × 10⁶ 6 ~2×10 6 The concentration was adjusted to cells / mL. b) PBMCs were collected with Ficoll isolation solution (Tianjin, China), CD3-positive T cells were separated using magnetic beads, and clinical-grade Dynabeads Human T Expander CD3 / CD28 magnetic beads (Invitrogen) were added in a ratio of magnetic beads:CD3-positive cells = 3:1 to activate the T cells. c) Two days after T cell activation, 1.2 mL of RetroNectin (TAKARA), diluted to a final concentration of 15 μg / mL in PBS, was coated into each well of a non-tissue culture plate in a 6-well plate. The plates were incubated overnight at 4°C in the dark. d) Two days after T cell activation culture, the coated 6-well plate was removed, the coating solution was aspirated and removed, and the plate was washed once with PBS. e) Add 5-6 mL of the retrovirus solution prepared in step 2-2 to each well and centrifuge at 32°C and 2000 × g for 2 hours. Add 3 mL of fresh complete medium containing hIL-2 (500 U / mL) to each well and incubate for 1 day. f) After cell infection, observe the cell density daily and set the T cell density to approximately 5 × 10⁻⁶. 5 T cell culture medium containing 100 U / mL of IL-2 was added periodically to maintain the concentration at / mL and promote cell proliferation. g) CAR-T cells infected with the retrovirus prepared in step 2-2 were obtained and named CLL1-VHH-16 CAR-T cells (i.e., T cells expressing the CLL1-VHH-16 CAR gene having the nucleotide sequence of SEQ ID No. 2).

[0064] The constructed CLL1-VHH-16 CAR-T cells and CTR T cells (T cells that have not undergone viral transfection, as a control) were cultured at 37°C in RPMI-1640 medium containing 10% fetal bovine serum (FBS), and the date was recorded as day 0. After 10 days of culture, various functional tests were performed.

[0065] Example 3: Detection of CAR expression in CLL1-VHH-16 CAR-T cells The constructed CLL1-VHH-16 CAR-T cells contain the CLL1-VHH-16 CAR gene with the nucleotide sequence of SEQ ID No. 2 and express the chimeric antigen receptor CLL1-VHH-16 CAR with the amino acid sequence of SEQ ID No. 1. The procedure for detecting CAR expression is as follows. 1. The cells were centrifuged (1500 rpm × 5 min), the supernatant was discarded, and 200 μL of FACS buffer (1 × PBS containing 0.1% NaN3 and 2% FBS) was added to each well of a 96-well round-bottom plate to resuspend the cells. The cells were then centrifuged at 1500 rpm for 5 minutes. 2. Add 60 μL of the prepared fluorescently labeled anti-human CD4-APC Cy7 / CLL1-FITC to each well, resuspend and mix, and incubate at 4°C for 30 minutes. 3. Add 200 μL of FACS buffer to each well and centrifuge at 1500 rpm for 5 minutes. 4. The supernatant was discarded, the cells were resuspended in 400 μL of FACS buffer and transferred to a flow cytometer tube, and the cells were read using a flow cytometer (BDCanto-II) to analyze the proportion of CLL1 antibody in CLL1-VHH-16 CAR-T cells.

[0066] The detection results are shown in Figure 2. The fact that CLL1-VHH-16 CAR-T cells can successfully express the CLL1 antibody (i.e., the single-domain antibody CLL1-VHH-16) indicates that the design anticipates the expression of CAR molecules in CLL1-VHH-16 CAR-T cells.

[0067] Example 4: Functional detection of specific effector molecule IFN-γ secretion by CLL1-VHH-16 CAR-T cells 1. The cell density of the test cells, CLL1-VHH-16 CAR-T cells and CTR T cells (T cells that have not undergone viral transfection, as a control), was set to 2 × 10⁻⁶. 6 The solution was adjusted to cells / mL, 100 μL was added to a 96-well U-bottom plate, U937 cells were added in a ratio of test cells:target cells = 1:1, Brefeldin A (Med Chem Express, HY-16592) at a final concentration of 5 μg / mL was added to each well, and the mixture was incubated in a 37°C incubator for 6 hours.

[0068] CTR+U937:1×10 6 In T cells (CTR T cells) that have not undergone viral transfection at a rate of cells / mL / well, 1 × 10⁶ 6 U937 cells were added at a concentration of cells / mL. CLL1-VHH-16 CAR-T+U937:1×10 6 To CLL1-VHH-16 CAR-T cells at a rate of cells / mL / well, 1 × 10⁶ 6 U937 cells were added at a concentration of cells / mL.

[0069] 2. After incubation, flow cytometry staining is performed, and the procedure is as follows: (1) The cells were centrifuged (1500 rpm × 5 min), the supernatant was discarded, 200 μL of FACS buffer (1 × PBS containing 0.1% NaN3 and 2% FBS) was added to each well and the cells were resuspended. The cells were then centrifuged at 1500 rpm for 5 minutes, and this procedure was repeated twice. (2) Add 60 μL of the prepared fluorescently labeled CLL1(rp)-PE to each well, resuspend and mix, and incubate in the dark at room temperature for 10 minutes. (3) Add 200 μL of FACS buffer to each well, and centrifuge at 1500 rpm for 5 minutes, then discard the supernatant. (4) Add 150 μL of Cytofix / Cytoperm (BD, catalog number 55472) to each well, resuspend and mix, and incubate in the dark at room temperature for 15 minutes. (5) Centrifuge at 1500 rpm for 5 minutes, discard the supernatant, add 200 μL of Perm / Wash buffer (BD, catalog number 554723) to each well, resuspend and mix, centrifuge at 1500 rpm for 5 minutes, and wash twice by centrifugation. (6) Diluted APC-labeled anti-human IFN-γ (Biolegend, catalog number 506510) was added to 20 μL of each well, resuspended and mixed, and incubated in the dark at room temperature for 20 minutes. (7) 200 μL of Perm buffer was added to each well and the cells were centrifuged at 1500 rpm for 5 minutes. After discarding the supernatant, the cells were resuspended in 400 μL of FACS buffer and transferred to flow cytometer tubes. The cells were read using a flow cytometer (BD Canto-II), and the proportion of the functional effector molecule IFN-γ in CLL1-VHH-16 CAR-T cells and control cells was analyzed.

[0070] The detection results are shown in Figure 3. After co-culturing with U937 target cells, CLL1-VHH-16 CAR-T cells can effectively secrete IFN-γ, a T cell-specific effector molecule.

[0071] Example 5: Detection of cytotoxicity of CLL1-VHH-16 CAR-T cells 1. The cell density of effector cells, CLL1-VHH-16 CAR-T cells and CTR T cells (T cells that have not undergone viral transfection, as a control) was set to 2 × 10⁻⁶. 5 The solution was adjusted to cells / mL, 100 μL was added to a 96-well U-bottom plate, and D-firefly luciferin sodium salt (Yeasen Biotechnology, catalog number 40901ES08, storage concentration 100 mg / mL) was added to a final concentration of 100 μg / mL, with 3 wells duplicated.

[0072] 2. Different effectors: Target cell U937-luc under target ratios (9:1, 3:1, 1:1, 1:3) (Under the 1:1 condition, the cell density of target cells is 2 × 10⁻¹⁶). 4 Each cell type was added and cultured at 37°C for 16 hours.

[0073] 3. Fluorescence values ​​were measured using a TECAN spark microplate reader, and the specific cytotoxicity of CLL1-VHH-16 CAR-T cells was calculated using the average value of three overlapping wells. Specific lysis%=100-100×(Eexp-Emin) / (Tmax-Tmin) Eexp: RLU value when effector cells and target cells are co-cultured. Emin: The RLU value of natural death of effector cells when no cells are present. Tmax: The RLU value of natural death of target cells in the absence of effector cells. Tmin: RLU value under conditions of maximum lethality.

[0074] The detection results are shown in Figure 4. CLL1-VHH-16 CAR-T cells can efficiently and specifically kill CLL1-positive target cells (U937 cells), while control CTR T cells did not show any killing effect against CLL1-positive target cells.

[0075] Example 6: Detection of in vivo tumor-killing activity of CLL1-VHH-16 CAR-T cells 1. Each of 8 female NSG mice (Shanghai Southern Model Biotechnology Co., Ltd., catalog number NM-NSG-001, 8-10 weeks old) was given 2.0 × 10⁶ 6 U937-Luc-GFP cells were inoculated into the tail vein. 2. Six days after vaccination, the mice were randomly divided into two groups, and each mouse received 4.0 × 10⁶ 6 CLL1-VHH-16 CAR-T cells or CTR T cells (T cells that have not undergone viral transfection, as a control) were injected via tail vein and recorded as day 0. Tumor luminescence signals were captured using the Photo Acquisition small animal imaging system on days 3, 7, 14, 21, 28, 35, and 42.

[0076] The detection results are shown in Table 1 and Figure 5. Compared to the CTR control group, CLL1-VHH-16 CAR-T cells not only significantly inhibited tumor cell proliferation in mice but also significantly extended the survival time of the mice, with tumor tissue completely disappearing from the mice by day 35 and day 42.

[0077] TIFF2026515021000001.tif110170

[0078] The present invention has been described in detail above. Those skilled in the art will be able to implement the present invention over a wider range of parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without performing unnecessary experiments. While the present invention has shown specific examples, it should be understood that further improvements can be made to the present invention. In short, based on the principles of the present invention, this application is intended to include any modifications, uses, or improvements to the present invention, including those that deviate from the scope disclosed herein and are made using prior art known in the art. Some basic features may be applied within the scope of the appended claims below. [Industrial applicability]

[0079] The novel CLL1-targeting CAR-T cells (CLL1-VHH-16 CAR-T cells) designed in this invention effectively secrete the T cell-specific effector molecule IFN-γ, efficiently and specifically killing CLL1-positive target cells and exhibiting excellent in vivo tumor-killing activity. This not only significantly inhibits tumor cell proliferation in mice but also significantly extends the survival time of mice. The CLL1-VHH-16 CAR-T cells of this invention possess excellent antitumor capabilities and can be used in immunotherapy for CLL1-targeted diseases (such as acute myeloid leukemia, myelodysplastic syndrome, or chronic myeloid leukemia), offering potential for a wide range of clinical applications.

Claims

1. A CAR-T cell, characterized in that the CAR-T cell contains a chimeric antigen receptor, the chimeric antigen receptor comprises a single-domain antibody, a hinge region, a transmembrane region, and an intracellular signaling region, and the amino acid sequence of the single-domain antibody is at positions 22 to 150 of SEQ ID No.

1.

2. The CAR-T cell according to claim 1, characterized in that the intracellular signaling region comprises a co-stimulatory domain 4-1BB and an activation domain CD3ζ, the amino acid sequence of 4-1BB is at positions 220-266 of SEQ ID No. 1, and the amino acid sequence of CD3ζ is at positions 267-378 of SEQ ID No.

1.

3. The CAR-T cell according to claim 1 or 2, characterized in that the hinge region is a CD8a hinge region whose amino acid sequence is at positions 151 to 195 of SEQ ID No. 1, and the transmembrane region is a CD8a transmembrane region whose amino acid sequence is at positions 196 to 219 of SEQ ID No.

1.

4. The CAR-T cell according to any one of claims 1 to 3, characterized in that the chimeric antigen receptor comprises one of the following: A1) A protein having the amino acid sequence shown at positions 22-378 of SEQ ID No. 1, A2) A protein having 80% or more identity with the protein described in A1) and having the same function, wherein amino acid residues are substituted and / or deleted and / or added in the amino acid sequence shown at positions 22 to 378 of SEQ ID No.

1. A3) A fusion protein having the same function as A1), with a tag or signal peptide added to the N-terminus and / or C-terminus of A1) or A2).

5. The CAR-T cell according to claim 4, characterized in that the amino acid sequence of the signal peptide described in A3) is at positions 1 to 21 of SEQ ID No.

1.

6. A chimeric antigen receptor according to any one of claims 1 to 5.

7. A biomaterial characterized by being one of the following: B1) A nucleic acid molecule encoding the chimeric antigen receptor according to claim 6, B2) Expression cassette containing the nucleic acid molecule described in B1), B3) Recombinant vectors containing nucleic acid molecules as described in B1), or recombinant vectors containing expression cassettes as described in B2), B4) Recombinant microorganisms containing the nucleic acid molecule described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3).

8. The biomaterial according to claim 7, characterized in that the nucleic acid molecule described in B1) is one of the following: C1) A DNA molecule whose nucleotide sequence or coding sequence is at positions 64-1134 of SEQ ID No.

2. A DNA molecule having 75% or more identity with the nucleotide sequence defined in C2) and C1) and having the same function, C3) DNA molecules whose nucleotide sequence or coding sequence is SEQ ID No. 2, A DNA molecule that has 75% or more identity with the nucleotide sequence defined in C4) and C3) and has the same function.

9. A pharmaceutical composition characterized by comprising CAR-T cells as described in any one of claims 1 to 5.

10. Any one of the following uses of the CAR-T cell according to any one of claims 1 to 5, the chimeric antigen receptor according to claim 6, the biomaterial according to claim 7 or 8, or the pharmaceutical composition according to claim 9: D1) Use in the manufacture of pharmaceuticals for the prevention or treatment of tumors, or use in the prevention or treatment of tumors, D2) Use in the manufacture of pharmaceuticals for the prevention or treatment of CLL1 target-related diseases, or use in the prevention or treatment of CLL1 target-related diseases.

11. The use according to claim 10, characterized in that the CLL1 target-related disease is a CLL1-positive cancer.

12. The use according to claim 11, characterized in that the CLL1-positive cancer is acute myeloid leukemia, myelodysplastic syndrome, or chronic myeloid leukemia.

13. A method for producing CAR-T cells according to any one of claims 1 to 5, characterized by comprising introducing a nucleic acid molecule according to claim 7 or 8 into T cells and stably expressing it to obtain the CAR-T cells.

14. A method for preventing or treating CLL1 target-related disease, characterized by comprising administering CAR-T cells according to any one of claims 1 to 5 or the pharmaceutical composition according to claim 9 to a subject suffering from CLL1 target-related disease.

15. The method according to claim 14, characterized in that the CLL1 target-related disease is a CLL1-positive cancer.

16. The method according to claim 15, characterized in that the CLL1-positive cancer is acute myeloid leukemia, myelodysplastic syndrome, or chronic myeloid leukemia.

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