CLL1-car-t cell, and preparation method therefor and use thereof

Abstract

Disclosed in the present invention are a CLL1-CAR-T cell, and a preparation method therefor and the use thereof. The CLL1-CAR-T cell contains a chimeric antigen receptor, wherein the chimeric antigen receptor comprises a single domain antibody, a hinge region, a transmembrane region and an intracellular signaling region, and the single domain antibody has an amino acid sequence of positions 22-150 of SEQ ID No. 1. The CLL1-VHH-1 CAR-T cell of the present invention can secrete T cell specific effector molecule IFN-γ to specifically kill CLL1+ target cells, inhibit the proliferation of tumor cells in mice, prolong the survival time of mice, and can be used for immunotherapy of diseases associated with CLL1 target (such as acute myelogenous leukemia).

Description

CLL1-CAR-T cells and preparation methods and applications thereof Technical Field

[0001] The present invention belongs to the field of cellular immunotherapy technology, and specifically relates to CLL1-CAR-T cells and their preparation methods and applications. Background Art

[0002] Acute myeloid leukemia (AML) is a malignant disease of myeloid hematopoietic stem / progenitor cells characterized by abnormal proliferation of primitive and immature myeloid cells in the bone marrow and peripheral blood. Clinical manifestations include anemia, bleeding, infection and fever, organ infiltration, and metabolic abnormalities. Most cases present with severe disease and a dismal prognosis, often leading to life-threatening consequences if left untreated. Anthracycline and cytarabine chemotherapy have long been used to treat mild cases, while hematopoietic stem cell transplantation has been used for intermediate- and high-risk patients. The heterogeneity of AML makes the treatment of AML patients a challenge. In recent years, researchers have discovered that immune-targeted therapies targeting CLL1 are highly 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 immune regulation as an inhibitory receptor. CLL1 is present in myeloid cells in peripheral blood and bone marrow, as well as in most AML cells, and is also expressed on the CD34 + CD38 - On stem cells, normal human CD34 + CD38 - Stem cells do not express CLL1, making it a potential target for the treatment and diagnosis of AML due to its unique expression pattern. Furthermore, CLL1 is also expressed on cells of myelodysplastic syndrome (MDS) and chronic myeloid leukemia (CML).

[0003] Traditional radiotherapy, chemotherapy, and hematopoietic stem cell transplantation have low efficacy rates for treating AML. However, the emergence of CAR-T cell therapy has made a cure possible. CAR-T stands for Chimeric Antigen Receptor T-Cell Immunotherapy. Chimeric antigen receptor (CAR) technology uses genetic engineering to modify immune cells to express exogenous anti-tumor genes (CAR genes). This allows lymphocytes and other immune cells to recognize tumor cell surface antigens and specifically recognize and kill tumor cells without the restriction of the major histocompatibility complex (MHC). The CAR structure primarily consists of an extracellular domain that specifically recognizes tumor cell surface antigens and an intracellular signaling domain. The extracellular domain is responsible for specifically recognizing tumor surface proteins (antigens), while the intracellular domain contains a costimulatory molecule domain that, upon recognition of tumor surface proteins (antigens), triggers a lymphocyte immune response, exerting cytotoxic effects against specific target cells, thereby killing them. Currently, CAR-T cell therapy has achieved encouraging therapeutic effects in hematological tumors such as B cells. However, the antigenic properties of the myeloid lineage are fundamentally different from those of the B lineage. The killing of normal myeloid cells by CAR-T can lead to bone marrow failure and there is a lack of corresponding alternative treatments. This has led to very slow progress in CAR-T treatment of AML. The successful application of CAR-T technology in acute myeloid leukemia (AML) still faces many challenges. Therefore, in-depth research and development of new and effective CAR-T cells and improving the efficacy of CAR-T cells have important theoretical significance and clinical application value for immune cell therapy of AML. SUMMARY OF THE INVENTION

[0004] One objective of the present invention is to provide CLL1-targeted CAR-T cells (CLL1-CAR-T cells) for the treatment of acute myeloid leukemia (AML). The technical problems to be solved are not limited to the technical subject matter described herein. Other technical subjects not described herein will be readily apparent to those skilled in the art from the following description.

[0005] To achieve the above objectives, the present invention first provides CLL1-CAR-T cells, which contain a chimeric antigen receptor (CAR). The chimeric antigen receptor may include a single-domain antibody, a hinge region, a transmembrane region and an intracellular signaling region. The amino acid sequence of the single-domain antibody may be positions 22-150 of SEQ ID No. 1.

[0006] The CLL1-CAR-T cell (i.e., a T cell expressing CLL1-CAR or a CLL1-CAR-modified T cell) may be a CAR-T cell targeting CLL1 (C-type lectin-like molecule 1), and the CLL1-CAR-T cell expresses the chimeric antigen receptor.

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

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

[0009] Furthermore, the CLL1-CAR-T cell may further include a signal peptide, and the amino acid sequence of the signal peptide may be positions 1-21 of SEQ ID No.1.

[0010] Furthermore, the chimeric antigen receptor may be named CLL1-VHH-1 CAR, and may include any of the following:

[0011] A1) the amino acid sequence is SEQ ID No. 1 or the protein shown in positions 22-378 of SEQ ID No. 1;

[0012] A2) a protein having at least 80% identity with the protein of A1) and having the same function as the protein of A1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence of SEQ ID No. 1 or positions 22-378 of SEQ ID No. 1;

[0013] A3) A fusion protein having the same function as A1) is obtained by connecting a tag or signal peptide to the N-terminus and / or C-terminus of A1) or A2).

[0014] Furthermore, the connection in A3) is achieved through a peptide bond.

[0015] The tags described herein include, but are not limited to, GST (glutathione sulfhydryltransferase) tag protein, His-tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag-tagged protein, SUMO-tagged protein, HA-tagged protein, Myc-tagged protein, GFP (green fluorescent protein), CFP (cyan fluorescent protein), YFP (yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein), or AviTag-tagged protein.

[0016] Those skilled in the art can readily mutate the nucleotide sequence encoding the chimeric antigen receptor of the present invention using known methods, such as directed evolution or point mutagenesis. Artificially modified nucleotide sequences that share 75% or greater identity with the nucleotide sequence of the chimeric antigen receptor of the present invention are derived from and are equivalent to the nucleotide sequence of the present invention, as long as they encode the chimeric antigen receptor and possess the function of the chimeric antigen receptor.

[0017] Any chimeric antigen receptor described herein is also within the scope of protection of the present invention.

[0018] The chimeric antigen receptor may comprise, from N-terminus to C-terminus, the signal peptide, the single domain antibody, the hinge region, the transmembrane region, the costimulatory domain and the activation domain.

[0019] The present invention also provides a biomaterial, which may be any of the following:

[0020] B1) a nucleic acid molecule encoding any chimeric antigen receptor described herein;

[0021] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0022] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0023] 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).

[0024] Among them, the expression cassette described in B2), the recombinant vector described in B3), and the recombinant microorganism described in B4) can all express a nucleic acid molecule encoding any chimeric antigen receptor described herein.

[0025] In the above-mentioned biological material, the nucleic acid molecule in B1) can be any of the following:

[0026] C1) The nucleotide sequence or coding sequence is the DNA molecule shown in positions 64 to 1134 of SEQ ID No. 2;

[0027] C2) A DNA molecule that has 75% or more identity with the nucleotide sequence defined in C1) and has the same function;

[0028] C3) a DNA molecule whose nucleotide sequence or coding sequence is SEQ ID No. 2;

[0029] C4) A DNA molecule having 75% or more identity with the nucleotide sequence defined in C3) and having the same function.

[0030] The name of the DNA molecule shown in SEQ ID No. 2 can be CLL1-VHH-1 CAR gene, and the amino acid sequence encoded by it is the chimeric antigen receptor CLL1-VHH-1 CAR shown in SEQ ID No. 1.

[0031] In one embodiment of the present invention, the chimeric antigen receptor is named CLL1-VHH-1 CAR, and from N-terminus to C-terminus are signal peptide (Signal), single domain antibody (CLL1-VHH-1), CD8a hinge region (Hinge), CD8a transmembrane region, co-stimulatory domain 4-1BB and activation domain CD3ζ ( Figure 1 ). Among them, the single-domain antibody CLL1-VHH-1 is a single-domain antibody that specifically binds to the CLL1 protein. It is the extracellular antigen binding region of CAR, responsible for recognizing and binding to the tumor surface antigen CLL1; the N-terminus of the antigen binding region carries a signal peptide (Signal), which is used to guide the peptide chain of the antigen binding region to transfer to the extracellular space, thereby recognizing the tumor cell surface antigen CLL1; the CD8a hinge region (Hinge) is a peptide chain located outside the cell of CAR, connecting the extracellular antigen binding region and the CD8a transmembrane region; the CD8a transmembrane region is used to anchor the single-domain antibody CLL1-VHH-1 to the cell membrane; the intracellular signal region includes the co-stimulatory domain 4-1BB and the activation domain CD3ζ, which are used to synergistically stimulate T cells, activate intracellular signals, enable T cells to continue to proliferate and release cytokines, improve the anti-tumor ability of T cells, and at the same time exert the T cell signal transduction function to promote a stronger and more lasting T cell response.

[0032] The amino acid sequence of the CLL1-VHH-1 CAR is shown in SEQ ID No. 1 (378 aa), where positions 1-21 of SEQ ID No. 1 are the amino acid sequence of the signal peptide (Signal); positions 22-150 of SEQ ID No. 1 are the amino acid sequence of the single-domain antibody CLL1-VHH-1; positions 151-195 of SEQ ID No. 1 are the amino acid sequence of the CD8a hinge region (Hinge); positions 196-219 of SEQ ID No. 1 are the amino acid sequence of the CD8a transmembrane region; positions 220-266 of SEQ ID No. 1 are the amino acid sequence of the costimulatory domain 4-1BB; and positions 267-378 of SEQ ID No. 1 are the amino acid sequence of the activation domain CD3ζ.

[0033] The encoding gene of the CLL1-VHH-1 CAR is the CLL1-VHH-1 CAR gene, and its nucleotide sequence is shown in SEQ ID No. 2 (1134 bp). Positions 1-63 of SEQ ID No. 2 are the nucleotide sequence of the signal peptide (Signal); positions 64-450 of SEQ ID No. 2 are the nucleotide sequence of the single-domain antibody CLL1-VHH-1; positions 451-585 of SEQ ID No. 2 are the nucleotide sequence of the CD8a hinge region (Hinge); positions 586-657 of SEQ ID No. 2 are the nucleotide sequence of the CD8a transmembrane region; positions 658-798 of SEQ ID No. 2 are the nucleotide sequence of the costimulatory domain 4-1BB; and positions 799-1134 of SEQ ID No. 2 are the nucleotide sequence of the activation domain CD3ζ.

[0034] The aforementioned 75% or greater identity may be 80%, 85%, 90% or 95% or greater identity.

[0035] The 80% or greater identity 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. The 85% or greater identity may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 90% or greater identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 95% or greater identity may be at least 95%, 96%, 97%, 98%, or 99% identity.

[0036] As used herein, identity refers to amino acid sequence or nucleotide sequence identity. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, amino acid sequence identity can be calculated using Advanced BLAST 2.1 by using blastp as the program, setting the Expect value to 10, 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 then be obtained.

[0037] The vectors described herein are those capable of transporting exogenous DNA or a gene of interest into host cells for amplification and expression. These vectors may be cloning vectors or expression vectors, including but not limited to plasmids, bacteriophages (e.g., lambda phage or M13 filamentous phage), cosmids (i.e., cosmids), and viral vectors (e.g., baculovirus vectors, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, or herpes viruses (e.g., herpes simplex virus)). In one or more embodiments of the present invention, the vectors are pUC57 vectors and / or retroviral vectors MP71.

[0038] The microorganisms described herein may be bacteria, yeast, algae, or fungi. The bacteria may be from, but are not limited to, Escherichia sp., Erwinia sp., Agrobacterium sp., Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., or Bacillus sp. The yeast may be from, but are not limited to, Saccharomyces sp., Pichia sp., Yarrowia sp., Candida sp., Schizosaccharomyces sp., Hansenula sp., or Kluyreromyces sp. The algae may be from, but are not limited to, Fucus sp., Achnanthes sp., Amphiprora sp., Amphora sp., Ankistrodesmus sp., Asteromonas sp., or Boekelovia sp. The fungi may be from, but are not limited to, Fusarium sp., Rhizoctonia sp., Verticillium sp., Penicillium sp., Aspergillus sp., or Cephalosporium sp. In one or more embodiments of the present invention, the microorganism is Escherichia coli DH5α.

[0039] The recombinant vector described herein refers to a recombinant vector DNA molecule constructed by connecting an exogenous target gene to a vector in vitro. It can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the exogenous target gene into the recipient cell and provide the exogenous target gene with the ability to replicate, integrate, amplify and / or express in the recipient cell. In one or more embodiments of the present invention, the recombinant vector is a recombinant retroviral vector MP71-CLL1-VHH-1 CAR.

[0040] The recombinant retroviral vector MP71-CLL1-VHH-1 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 with a nucleotide sequence of SEQ ID No. 2 in the sequence list, while keeping the other nucleotide sequences of the retroviral vector MP71 unchanged.

[0041] The recombinant microorganism described herein refers to a microorganism whose genes are manipulated and modified to obtain a functionally altered microorganism, such as a recombinant microorganism obtained by introducing an exogenous target gene or recombinant vector into the target microorganism, or a recombinant microorganism 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 obtained by introducing the recombinant retroviral vector MP71-CLL1-VHH-1 CAR into Escherichia coli DH5α, which contains the DNA molecule shown in SEQ ID No. 2.

[0042] The CLL1-CAR-T cells described herein are recombinant cells obtained by stably expressing a constructed chimeric antigen receptor (CAR) gene by genetic engineering in vitro recombination. Furthermore, the CLL1-CAR-T cells described herein may be CLL1-VHH-1 CAR-T cells obtained by stably expressing the CLL1-VHH-1 CAR gene (SEQ ID No. 2) by transfecting the T cells.

[0043] Specifically, the CLL1-VHH-1 CAR-T cells can be recombinant cells obtained by packaging the recombinant retroviral vector MP71-CLL1-VHH-1 CAR using packaging cells to obtain a recombinant retrovirus, and then introducing the recombinant retrovirus into T cells. The packaging cells can be Phoenix Ecotropic (ECO) cells and PG13 cells.

[0044] The CLL1-VHH-1 CAR-T cells contain and / or express the CLL1-VHH-1 CAR gene (SEQ ID No. 2).

[0045] The CLL1-VHH-1 CAR-T cells described herein have at least one of the following characteristics:

[0046] F1) secretes the specific effector molecule IFN-γ;

[0047] F2) Specific killing of CLL1 + Tumor cells;

[0048] F3) Inhibition of CLL1 + Tumor cell proliferation.

[0049] The present invention also provides the use of the chimeric antigen receptor CLL1-VHH-1 CAR in preparing the CLL1-VHH-1 CAR-T cell.

[0050] The present invention also provides a pharmaceutical composition, which may contain any of the CLL1-CAR-T cells described herein.

[0051] The pharmaceutical composition can be used to prevent or treat CLL1 target-related diseases.

[0052] Furthermore, the pharmaceutical composition may further include one or more pharmaceutically acceptable carriers.

[0053] Furthermore, the pharmaceutically acceptable carrier is selected from excipients, stabilizers, suspending agents and diluents, etc., which are well known to those skilled in the art.

[0054] The present invention also provides any of the following uses of any of the CLL1-CAR-T cells, the chimeric antigen receptors, the biomaterials, or the pharmaceutical compositions described herein:

[0055] D1) Use in the preparation of a medicament for preventing or treating tumors, or use in preventing or treating tumors;

[0056] D2) Use in the preparation of a medicament for preventing or treating a CLL1 target-related disease, or use in preventing or treating a CLL1 target-related disease.

[0057] In the above application, the CLL1 target-related disease may be CLL1-positive cancer (i.e., cancer expressing CLL1).

[0058] Furthermore, the CLL1-positive cancer may be acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) or chronic myeloid leukemia (CML).

[0059] The present invention also provides a method for preparing any of the CLL1-CAR-T cells described herein, which may include: transferring any of the nucleic acid molecules described herein into T cells for stable expression to obtain the CLL1-CAR-T cells.

[0060] Furthermore, the method may include the following steps:

[0061] (1) The CLL1-VHH-1 CAR gene (SEQ ID No. 2) was constructed between the NotI and EcoRI recognition sites of the retroviral vector MP71 to obtain the recombinant retroviral vector MP71-CLL1-VHH-1 CAR;

[0062] (2) introducing the recombinant retroviral vector MP71-CLL1-VHH-1 CAR into packaging cells for packaging to obtain a recombinant retrovirus;

[0063] (3) Infecting human T cells with the recombinant retrovirus to obtain recombinant cells, namely CLL1-VHH-1 CAR-T cells.

[0064] Furthermore, the packaging cells may be Phoenix Ecotropic (ECO) cells and PG13 cells.

[0065] The present invention also provides a method for preventing or treating a CLL1 target-related disease, which may include administering any of the CLL1-CAR-T cells or pharmaceutical compositions described herein to a subject suffering from a CLL1 target-related disease.

[0066] Furthermore, the CLL1 target-related disease may be CLL1-positive cancer.

[0067] Furthermore, the CLL1-positive cancer may be acute myeloid leukemia, myelodysplastic syndrome or chronic myeloid leukemia.

[0068] CLL1, described in this article, is a type II transmembrane glycoprotein that acts as an inhibitory receptor and plays an important role in immune regulation. CLL1 is present in myeloid cells in peripheral blood and bone marrow, as well as in most acute myeloid leukemia (AML) cells, and is also expressed on the CD34 + CD38 - On stem cells, normal human CD34 + CD38 - Stem cells do not express CLL1. Due to its special expression pattern, CLL1 has become a potential target for AML treatment and diagnosis.

[0069] The present invention first designed a chimeric antigen receptor (SEQ ID No. 1) for constructing CLL1-CAR-T cells, the structure of which is shown in Figure 1. Furthermore, a CLL1-targeting CAR-T cell (CLL1-VHH-1 CAR-T cell) with a novel structure was prepared. Experimental results showed that the CLL1-VHH-1 CAR-T cell of the present invention can well secrete the T cell-specific effector molecule IFN-γ, effectively and specifically kill CLL1. + The CLL1-VHH-1 CAR-T cells of the present invention exhibit excellent in vivo tumoricidal activity, significantly inhibiting tumor cell proliferation in mice and significantly prolonging their survival. Tumor tissue in the mice was almost completely eliminated on days 28 and 35 after injection. The CLL1-VHH-1 CAR-T cells of the present invention have excellent anti-tumor activity and can be used for immunotherapy of CLL1-targeted diseases (such as acute myeloid leukemia, myelodysplastic syndrome, or chronic myeloid leukemia), with broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 is a schematic diagram of the molecular structure of CLL1-VHH-1 CAR.

[0071] Figure 2 shows the detection of CAR expression in CLL1-VHH-1 CAR-T cells.

[0072] Figure 3 shows the cell function detection of IFN-γ secretion by CLL1-VHH-1 CAR-T cells.

[0073] Figure 4 shows the cytotoxicity assay of CLL1-VHH-1 CAR-T cells.

[0074] FIG5 is the in vivo tumoricidal activity detection of CLL1-VHH-1 CAR-T cells. Modes for Carrying Out the Invention

[0075] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0076] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0077] 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 T Lymphocytes [J]. Human Gene Therapy, 2003, 14(12):1155-1168. The public can obtain this biological material from the applicant. This biological material is only used to repeat the experiments of the present invention and cannot be used for other purposes.

[0078] The human peripheral blood mononuclear cells (PBMCs) in the following examples were derived from the venous blood of healthy volunteers.

[0079] The U937 cells (CLL1-positive cells) in the following examples are acute myeloid leukemia cells, products of Beijing Biobo Biotechnology Co., Ltd., catalog number bio-73180.

[0080] Example 1. Design of Chimeric Antigen Receptor (CAR) for Construction of CLL1-CAR-T Cells

[0081] The chimeric antigen receptor (CAR) designed in this example for constructing CLL1-CAR-T cells is named CLL1-VHH-1 CAR, which consists of a signal peptide (Signal), a single-domain antibody (CLL1-VHH-1), a CD8a hinge region (Hinge), a CD8a transmembrane region, a costimulatory domain 4-1BB, and an activation domain CD3ζ from the N-terminus to the C-terminus (the structure is shown in Figure 1). Among them, the single-domain antibody CLL1-VHH-1 is a single-domain antibody that specifically binds to the CLL1 protein. It is the extracellular antigen binding region of CAR, responsible for recognizing and binding to the tumor surface antigen CLL1; the N-terminus of the antigen binding region carries a signal peptide (Signal), which is used to guide the peptide chain of the antigen binding region to transfer to the extracellular space, thereby recognizing the tumor cell surface antigen CLL1; the CD8a hinge region (Hinge) is a peptide chain located outside the cell of CAR, connecting the extracellular antigen binding region and the CD8a transmembrane region; the CD8a transmembrane region is used to anchor the single-domain antibody CLL1-VHH-1 to the cell membrane; the intracellular signal region includes the co-stimulatory domain 4-1BB and the activation domain CD3ζ, which are used to synergistically stimulate T cells, activate intracellular signals, enable T cells to continue to proliferate and release cytokines, improve the anti-tumor ability of T cells, and at the same time exert the T cell signal transduction function to promote a stronger and more lasting T cell response.

[0082] The designed CLL1-VHH-1 CAR amino acid sequence is shown in SEQ ID No. 1 (378 aa), where positions 1-21 of SEQ ID No. 1 are the amino acid sequence of the signal peptide (Signal); positions 22-150 of SEQ ID No. 1 are the amino acid sequence of the single-domain antibody CLL1-VHH-1; positions 151-195 of SEQ ID No. 1 are the amino acid sequence of the CD8a hinge region (Hinge); positions 196-219 of SEQ ID No. 1 are the amino acid sequence of the CD8a transmembrane region; positions 220-266 of SEQ ID No. 1 are the amino acid sequence of the costimulatory domain 4-1BB; and positions 267-378 of SEQ ID No. 1 are the amino acid sequence of the activation domain CD3ζ.

[0083] The gene encoding CLL1-VHH-1 CAR is the CLL1-VHH-1 CAR gene, and its nucleotide sequence is shown in SEQ ID No. 2 (1134 bp). Positions 1-63 of SEQ ID No. 2 are the nucleotide sequence of the signal peptide (Signal); positions 64-450 of SEQ ID No. 2 are the nucleotide sequence of the single-domain antibody CLL1-VHH-1; positions 451-585 of SEQ ID No. 2 are the nucleotide sequence of the CD8a hinge region (Hinge); positions 586-657 of SEQ ID No. 2 are the nucleotide sequence of the CD8a transmembrane region; positions 658-798 of SEQ ID No. 2 are the nucleotide sequence of the costimulatory domain 4-1BB; and positions 799-1134 of SEQ ID No. 2 are the nucleotide sequence of the activation domain CD3ζ.

[0084] Example 2. Preparation of CLL1-VHH-1 CAR-T cells

[0085] The CLL1-CAR-T cells constructed using the CLL1-VHH-1 CAR designed in Example 1 are named CLL1-VHH-1 CAR-T cells. Specifically, the CLL1-VHH-1 CAR gene (SEQ ID No. 2) in Example 1 is transferred into T cells for stable expression to obtain T cells expressing the CLL1-VHH-1 CAR gene, namely CLL1-VHH-1 CAR-T cells, in the following steps:

[0086] 2-1. Construction of recombinant retroviral vector

[0087] (1) The recombinant vector pUC57-CLL1-VHH-1 CAR was double-digested with NotI (NEB) and EcoRI (NEB), and the target gene fragment was recovered by gel excision. The recombinant vector pUC57-CLL1-VHH-1 CAR was synthesized and provided by Qingke Biotechnology Co., Ltd. by cloning the CLL1-VHH-1 CAR gene (SEQ ID No. 2) into the pUC57 vector.

[0088] (2) The retroviral vector MP71 was double-digested with NotI and EcoRI, and the large vector fragment was recovered by gel cutting.

[0089] (3) Use T4 ligase (NEB) to connect the target gene fragment and the large vector fragment to obtain the recombinant retroviral vector MP71-CLL1-VHH-1 CAR carrying the CLL1-VHH-1 CAR gene.

[0090] (4) The recombinant retroviral vector MP71-CLL1-VHH-1 CAR was transformed into competent Escherichia coli DH5α, and the plasmid was extracted and purified using the Qiagen plasmid purification kit to obtain the MP71-CLL1-VHH-1 CAR plasmid.

[0091] The recombinant retroviral vector MP71-CLL1-VHH-1 CAR (i.e., MP71-CLL1-VHH-1 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 a nucleotide sequence of SEQ ID No. 2 in the sequence listing, while keeping the other nucleotide sequences of the retroviral vector MP71 unchanged.

[0092] 2-2. Retroviral packaging

[0093] The MP71-CLL1-VHH-1 CAR plasmid prepared in step 2-1 is introduced into packaging cells for packaging to complete viral assembly and obtain retrovirus. The specific steps for viral packaging are as follows:

[0094] a) Day 1: Phoenix Ecotropic (ECO) cells are less than 20 generations old and not overgrown. 6 / ml cell density, add 10 mL of DMEM medium to a 10 cm dish, mix the cells thoroughly, and culture at 37°C overnight;

[0095] b) Day 2: Transfect ECO cells when they reach approximately 90% confluency (usually about 14-18 hours after plating). Prepare 12.5 μg of plasmid, 250 μL of 1.25 M CaCl₂, and 1 mL of H₂O for a total volume of 1.25 mL. In a separate tube, add an equal volume of 2× HBS to the plasmid complex and vortex for 20 seconds. Gently add the mixture along the side of the ECO dish. Incubate at 37°C for 4 hours. Remove the culture medium, wash once with PBS, and replace with fresh prewarmed culture medium.

[0096] c) Day 4: 48 hours after transfection, collect the supernatant and filter through a 0.45 μm filter to obtain the retroviral solution. Aliquot and store at -80°C.

[0097] d) Add 1.2 mL of 15 μg / mL Retronectin coating solution to each well of a 6-well NTC plate and incubate at 4°C overnight.

[0098] e) Carefully aspirate the blocking solution, add 2 mL / well PBS for washing, add 5 mL of the above virus solution to each well, centrifuge at 2000 × g at 32°C for 2 h, and discard the unbound virus supernatant.

[0099] f) Rinse logarithmic phase PG13 cells once with 10 mL of PBS, add 1 mL of 0.25% recombinant trypsin, and incubate at room temperature for 2-3 minutes.

[0100] g) Add 5 ml of complete medium containing 10% FBS to terminate the digestion and centrifuge at 1500 rpm for 5 min.

[0101] h) Discard the supernatant and adjust the cell density to 0.5 × 10 5 cell / mL, and add 3 mL / well to the virus-coated NTC 6-well plate to make the final cell count 1.5×10 5 cell / hole;

[0102] i) Centrifuge at 1000 rpm for 1 min and incubate at 37°C, 5% CO2 for 48 h;

[0103] j) After passage 1-2, transfer the cells to T175 culture flasks and culture in DEME medium containing 12% FBS for 2 days.

[0104] k) Replace with fresh DEME medium containing 12% FBS and continue culturing for 48 h. Collect the supernatant and filter through a 0.45 μm filter to obtain the retroviral solution. Aliquot and store at -80°C.

[0105] 2-3. Retroviral infection of human T cells

[0106] a) Thaw frozen healthy human peripheral blood PBMCs and adjust the cell density to 1 × 10 cells / mL using RPMI-1640 medium containing 10% fetal bovine serum (FBS). 6 -2×10 6 cells / mL.

[0107] b) PBMCs were collected using Ficoll separation medium (Tianjin Haoyang), and CD3 + T cells, by magnetic beads: CD3 + Clinical-grade Dynabeads Human T Expander CD3 / CD28 magnetic beads (Invitrogen) were added to activate T cells at a ratio of 3:1.

[0108] c) The day after T cell activation, coat non-tissue-treated culture plates with RetroNectin (TAKARA) diluted in PBS to a final concentration of 15 μg / mL, using 1.2 mL per well of a 6-well plate. Protect from light and incubate at 4°C overnight until ready to use.

[0109] d) After two days of T cell activation culture, remove the coated 6-well plate, discard the coating solution, and wash the plate once with PBS.

[0110] e) Add 5-6 mL of the retroviral solution prepared in step 2-2 to each well and centrifuge at 32°C, 2000 × g for 2 h. Add 3 mL of fresh complete culture medium containing 500 U / mL hIL-2 (human interleukin-2) to each well and continue incubation for 1 day.

[0111] f) After cell infection, the cell density was observed daily and T cell culture medium containing 100 U / mL IL-2 was added as appropriate to maintain the T cell density at 5×10 5 / mL, which is convenient for cell expansion.

[0112] g) CAR-T cells infected with the retrovirus prepared in step 2-2 are obtained, i.e., T cells expressing the CLL1-VHH-1 CAR gene with the nucleotide sequence of SEQ ID No. 2, and are named CLL1-VHH-1 CAR-T cells.

[0113] The constructed CLL1-VHH-1 CAR-T cells and CTR T cells (i.e., T cells without virus transfection, used as a control) were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) at 37°C and recorded as day 0. Various functional tests were performed on day 10.

[0114] Example 3. Detection of CAR Expression in CLL1-VHH-1 CAR-T Cells

[0115] The constructed CLL1-VHH-1 CAR-T cells express the chimeric antigen receptor CLL1-VHH-1 CAR with the amino acid sequence of SEQ ID No. 1, and the CAR expression detection steps are as follows:

[0116] 1. After centrifugation (1500 rpm × 5 min), discard the supernatant and resuspend the cells in 200 μL FACS buffer (1× PBS containing 0.1% NaN3 and 2% FBS) per well of a 96-well round-bottom plate. Centrifuge at 1500 rpm for 5 min.

[0117] 2. Add 60 μL of prepared fluorescent-labeled anti-human CD4-APC Cy7 / CLL1-FITC to each well, resuspend and mix thoroughly, and incubate at 4°C for 30 min.

[0118] 3. Add 200 μL FACS buffer to each well and centrifuge at 1500 rpm for 5 min.

[0119] 4. Discard the supernatant, resuspend the cells in 400 μL FACS buffer, transfer them to a flow cytometer, read the cells using a flow cytometer (BD Canto-II), and analyze the percentage of CLL1 antibodies in CLL1-VHH-1 CAR-T cells.

[0120] The test results are shown in Figure 2. CLL1-VHH-1 CAR-T cells can well express CLL1 antibodies (i.e., single-domain antibodies CLL1-VHH-1), indicating that the expression of CAR molecules in CLL1-VHH-1 CAR-T cells meets the expected design.

[0121] Example 4. Functional detection of CLL1-VHH-1 CAR-T cells secreting specific effector molecule IFN-γ

[0122] 1. The cell density of the test cells CLL1-VHH-1 CAR-T cells and CTR T cells (i.e., T cells without virus transfection, as a control) was adjusted to 2×10 6 100 μL of the solution was added to a 96-well U-bottom plate, and U937 cells were added at a ratio of test cells: target cells = 1:1. Brefeldin A (Med Chem Express, HY-16592) was added to each well at a final concentration of 5 μg / mL, and the cells were incubated in a 37°C incubator for 6 hours.

[0123] CTR+U937: 1 mL per well, 1 × 10 6 1×10 T cells without virus transfection (CTR T cells) were added in 1 mL. 6U937 cells;

[0124] CLL1-VHH-1 CAR-T+U937: 1×10 per well, 1 mL 6 1×10 CLL1-VHH-1 CAR-T cells were added to 1 mL. 6 U937 cells;

[0125] 2. After incubation, perform flow cytometry staining. The steps are as follows:

[0126] (1) After centrifugation (1500 rpm × 5 min), discard the supernatant and add 200 μL FACS buffer (1× PBS containing 0.1% NaN3 and 2% FBS) to each well to resuspend the cells. Centrifuge at 1500 rpm for 5 min. Repeat this step twice.

[0127] (2) Add 60 μL of the prepared fluorescently labeled CLL1(rp)-PE to each well, resuspend and mix thoroughly, and incubate at room temperature for 10 min in the dark.

[0128] (3) Add 200 μL FACS buffer to each well, centrifuge at 1500 rpm for 5 min, and discard the supernatant;

[0129] (4) Add 150 μL of Cytofix / Cytoperm (BD Biosciences, Cat. No. 55472) to each well, resuspend and mix, and incubate at room temperature for 15 min in the dark.

[0130] (5) Centrifuge at 1500 rpm for 5 min, discard the supernatant, add 200 μL Perm / Wash buffer (BD, Cat. No. 554723) to each well, resuspend and mix, centrifuge at 1500 rpm for 5 min, and wash twice;

[0131] (6) Add 20 μL of diluted APC-labeled anti-human IFN-γ (Biolegend, Cat. No. 506510) to each well, resuspend and mix, and incubate at room temperature for 20 min in the dark.

[0132] (7) Add 200 μL of Perm buffer to each well and centrifuge at 1500 rpm for 5 min. After discarding the supernatant, resuspend the cells in 400 μL of FACS buffer and transfer them to a flow cytometer. Read the cells using a flow cytometer (BD Canto-II) to analyze the percentage of functional effector molecule IFN-γ in CLL1-VHH-1 CAR-T cells and control cells (CTR T cells).

[0133] The test results are shown in Figure 3. After co-culture with U937 target cells, CLL1-VHH-1 CAR-T cells were able to secrete the T cell-specific effector molecule IFN-γ well.

[0134] Example 5. Cytotoxicity Detection of CLL1-VHH-1 CAR-T Cells

[0135] 1. Adjust the cell density of effector cells CLL1-VHH-1 CAR-T cells and CTR T cells (i.e., T cells without virus transfection, as a control) to 2×10 5 100 μL was taken and added to a 96-well U-bottom plate; D-firefly luciferin sodium salt (Yeasen Biotechnology, catalog number 40901ES08, stock concentration 100 mg / mL) was added to a final concentration of 100 μg / mL, and repeated for 3 wells;

[0136] 2. Add target cells U937-luc at different effector-target ratios (9:1, 3:1, 1:1, 1:3) (the cell density of target cells under 1:1 condition is 2×10 4 cells) and cultured at 37°C for 16 h.

[0137] 3. Measure the fluorescence value using a TECAN spark microplate reader and take the average value of three replicate wells to calculate the specific cytotoxicity of CLL1-VHH-1 CAR-T cells:

[0138] Specific lysis %=100-100×(Eexp-Emin) / (Tmax-Tmin)

[0139] Eexp: RLU value when effector cells and target cells are co-cultured;

[0140] Emin: RLU value of spontaneous death of effector cells in the absence of cells;

[0141] Tmax: RLU value of spontaneous death of target cells in the absence of effector cells;

[0142] Tmin: RLU value under conditions of maximum killing rate.

[0143] The test results are shown in Figure 4. CLL1-VHH-1 CAR-T cells can effectively and specifically kill CLL1 + target cells (U937 cells), while control CTR T cells were + No killing effect on target cells.

[0144] Example 6. Detection of in vivo tumoricidal activity of CLL1-VHH-1 CAR-T cells

[0145] 1. Eight female NSG mice (Shanghai Model Organisms Science Co., Ltd., catalog number NM-NSG-001, 8-10 weeks old) were inoculated with 2.0 × 10 6 U937-Luc-GFP cells;

[0146] 2. Six days after inoculation, the rats were randomly divided into two groups and each group was injected with 4.0×10 6 CLL1-VHH-1 CAR-T or CTR T (i.e., T cells without virus transfection, as control) cells were injected into the CAR-T cells and recorded as D0 day;

[0147] 3. Use Photo Acquisition small animal imager to capture tumor luminescence signals on D7, D14, D21, D28, and D35.

[0148] The test results are shown in Table 1 and Figure 5. Compared with the CTR control group, CLL1-VHH-1 CAR-T cells can not only significantly inhibit the proliferation of tumor cells in mice, but also significantly prolong the survival time of mice. By D28 and D35 days, the tumor tissue in the mice had almost completely disappeared.

[0149]

[0150] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims. Industrial Applicability

[0151] The CLL1-targeting CAR-T cells (CLL1-VHH-1 CAR-T cells) designed in this invention with a novel structure can effectively secrete the T cell-specific effector molecule IFN-γ and effectively kill CLL1. +The CLL1-VHH-1 CAR-T cells of the present invention have excellent anti-tumor activity against target cells and exhibit excellent in vivo tumoricidal activity, not only significantly inhibiting tumor cell proliferation in mice but also significantly prolonging their survival. The CLL1-VHH-1 CAR-T cells of the present invention have excellent anti-tumor activity and can be used for immunotherapy of CLL1-targeted diseases (such as acute myeloid leukemia, myelodysplastic syndrome, or chronic myeloid leukemia), with broad clinical application prospects.

Claims

1. CLL1-CAR-T cells, characterized in that The CLL1-CAR-T cell contains a chimeric antigen receptor, which includes a single-domain antibody, a hinge region, a transmembrane region and an intracellular signal region. The amino acid sequence of the single-domain antibody is positions 22-150 of SEQ ID No.

1.

2. The CLL1-CAR-T cell according to claim 1, characterized in that The intracellular signaling region includes the co-stimulatory domain 4-1BB and the activation domain CD3ζ, the amino acid sequence of the 4-1BB is positions 220-266 of SEQ ID No.1, and the amino acid sequence of the CD3ζ is positions 267-378 of SEQ ID No.

1.

3. The CLL1-CAR-T cell according to claim 1 or 2, characterized in that The hinge region is the CD8a hinge region whose amino acid sequence is the 151st to 195th positions of SEQ ID No.1, and the transmembrane region is the CD8a transmembrane region whose amino acid sequence is the 196th to 219th positions of SEQ ID No.

1.

4. The CLL1-CAR-T cell according to any one of claims 1 to 3, characterized in that The chimeric antigen receptor includes any of the following: A1) the amino acid sequence is SEQ ID No.1 or the protein shown in positions 22-378 of SEQ ID No.1; A2) a protein having more than 80% identity with the protein shown in A1) and having the same function as the protein shown in A1) obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in SEQ ID No.1 or positions 22 to 378 of SEQ ID No.1; A3) A fusion protein having the same function as A1) is obtained by connecting a tag or a signal peptide to the N-terminus and / or C-terminus of A1) or A2).

5. The chimeric antigen receptor according to any one of claims 1 to 4.

6. Biomaterial, characterized in that The biological material is any of the following: B1) a nucleic acid molecule encoding the chimeric antigen receptor according to claim 5; 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).

7. The biomaterial according to claim 6, characterized in that B1) The nucleic acid molecule is any of the following: C1) The nucleotide sequence or coding sequence is the DNA molecule shown in positions 64 to 1134 of SEQ ID No. 2; C2) A DNA molecule that has 75% or more identity to the nucleotide sequence defined in C1) and has the same function; C3) a DNA molecule whose nucleotide sequence or coding sequence is SEQ ID No. 2; C4) A DNA molecule having 75% or more identity with the nucleotide sequence defined in C3) and having the same function.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the CLL1-CAR-T cells described in any one of claims 1-4.

9. Any of the following uses of the CLL1-CAR-T cell according to any one of claims 1 to 4, the chimeric antigen receptor according to claim 5, the biomaterial according to claim 6 or 7, or the pharmaceutical composition according to claim 8: D1) Use in the preparation of a medicament for preventing or treating tumors, or use in preventing or treating tumors; D2) Use in the preparation of a drug for preventing or treating a CLL1 target-related disease, or use in the prevention or treatment of a CLL1 target-related disease.

10. The use according to claim 9, characterized in that: The CLL1 target-related disease is CLL1-positive cancer.

11. The use according to claim 10, characterized in that: The CLL1-positive cancer is acute myeloid leukemia, myelodysplastic syndrome or chronic myeloid leukemia.

12. A method for preparing the CLL1-CAR-T cell according to any one of claims 1 to 4, characterized in that: The method comprises: transferring the nucleic acid molecule described in claim 6 or 7 into T cells for stable expression to obtain the CLL1-CAR-T cells.

13. A method for preventing or treating a CLL1 target-related disease, characterized in that: The method comprises administering the CLL1-CAR-T cell of any one of claims 1 to 4 or the pharmaceutical composition of claim 8 to a subject suffering from a CLL1 target-related disease.

14. The method according to claim 13, characterized in that The CLL1 target-related disease is CLL1-positive cancer.

15. The method according to claim 14, characterized in that The CLL1-positive cancer is acute myeloid leukemia, myelodysplastic syndrome or chronic myeloid leukemia.

Images