A preparation method and application of CD7-CAR-T cells

Abstract

The present invention provides a method for preparing CD7-CAR-T cells and their application. Specifically, the present invention provides a method for preparing CD7-CAR-T cells, adjusting the transfection sequence and transfection time of a lentivirus expressing a CD7 blocking molecule and a lentivirus expressing a CD7-CAR, thereby improving the transfection efficiency and enhancing the cytotoxicity of CAR-T cells. The present invention also provides CD7-CAR-T cells prepared by the method and their application.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to a method for preparing CD7-CAR-T cells and applications thereof. Background Art

[0002] T-cell-derived malignancies represent a typical type of hematological malignancy, with high recurrence and mortality rates in both children and adults, and there is currently no very effective treatment. T-cell acute lymphoblastic leukemia (T-ALL) is a highly heterogeneous hematological malignancy, accounting for approximately 25% of adult acute lymphoblastic leukemia cases and about 15% of childhood acute lymphoblastic leukemia cases. The clinical symptoms of T-ALL mainly include infection, anemia, fever, abnormal bleeding, etc. T-ALL progresses rapidly and can quickly infiltrate tissues and organs such as lymph nodes, liver, spleen, central nervous system and testicles in a short period of time. If there is no timely and effective treatment, the patient will die soon after the onset of the disease. At present, the treatment strategies for T-cell acute lymphoblastic leukemia (T-ALL) mainly include intensive chemotherapy, allogeneic hematopoietic stem cell transplantation (allo-HSCT), antiviral therapy and molecular targeted therapy. However, intensive chemotherapy usually cannot prevent relapse of the disease after treatment. For those patients who relapse after initial treatment, the remission rate of salvage chemotherapy is only about 20-40%. In addition, although hematopoietic stem cell transplantation may be the only curative option at present, it is also accompanied by life-threatening conditions such as graft-versus-host disease.

[0003] In recent years, chimeric antigen receptor T cell (CAR-T) therapy has shown very significant clinical efficacy. As a new adoptive immunotherapy technology, it has been used to treat a variety of solid tumors and blood tumors, among which the most significant efficacy is the treatment of B-cell-derived lymphocytic leukemia and lymphoma. CAR-T therapy targets and eliminates malignant tumors in a major histocompatibility complex-independent manner by genetically modifying the patient's T lymphocytes. One of the keys to the effective application of this technology is to select a suitable target to construct the corresponding CAR molecule. The best target antigen should be expressed only in tumor cells, not in normal cells, or it can also be expressed on cells that have a clinical response plan after a temporary loss of normal cells.

[0004] The CD7 molecule is highly expressed on most T-cell hematological tumor cells. CD7 is a transmembrane glycoprotein expressed by T, NK cells and their precursor cells, which stimulates T cell activation after binding to its ligand K12 / SECTM1. However, the CD7 molecule does not seem to play a key role in T cell development or function, because the destruction of the CD7 molecule in the progenitor cells of mouse T cells does not affect normal T cell development and homeostasis, and has no effect on T cell effector function. Importantly, CD7 has been used as an antibody target for the treatment of T-cell lymphoma, and patients have not experienced serious adverse reactions after targeting CD7. Therefore, the CD7 molecule may be a very suitable therapeutic target for T-cell hematological tumors. However, the targeted treatment of T-cell acute lymphoblastic leukemia with CD7-CAR-T cells still faces great problems, because both normal effector T cells and T-cell tumors express the CD7 antigen, which will lead to the fratricide of CD7-CAR-T cells, and CD7-CAR-T cells are difficult to prepare successfully in vitro.

[0005] Therefore, there is an urgent need in the art to develop new methods for preparing CD7-CAR-T cells. Summary of the invention

[0006] The object of the present invention is to provide a preparation method and application of CD7-CAR-T cells.

[0007] Specifically, the present invention provides a method for preparing CD7-CAR-T cells based on humanized CD7 nanobody sequences.

[0008] The present invention also provides the in vitro killing results of CD7-CART cells constructed with PA007CD and PA107CD vectors on CD7-positive tumor cells.

[0009] In a first aspect of the present invention, a method for preparing CD7-CAR-T cells is provided, comprising the steps of:

[0010] (i) providing a sample to be processed containing T cells,

[0011] (ii) sorting and activating T cells contained in the sample to obtain activated T cells,

[0012] (iii) introducing a first viral vector for expressing a CD7 blocking molecule into the activated T cells, thereby obtaining CD7 blocked T cells, and

[0013] (iv) introducing a second viral vector for expressing CD7-CAR into the CD7-blocked T cells to obtain the CD7-CAR-T cells,

[0014] Wherein, after the T cells are co-incubated with the activator for 12-36 hours, preferably 18-30 hours, and more preferably 22-26 hours, the first viral vector for expressing the CD7 blocking molecule is introduced into the activated T cells.

[0015] In another preferred embodiment, the interval between the introduction of the first viral vector and the introduction of the second viral vector is 36-60 hours, preferably, 42-54 hours, and more preferably, 46-50 hours.

[0016] In another preferred embodiment, in step (iii), the steps are included:

[0017] (a) co-culturing the first viral vector for expressing the CD7 blocking molecule with the activated T cells under conditions suitable for transfection for 36-60 hours, preferably 42-54 hours, more preferably 46-50 hours, thereby obtaining a first transfection mixture;

[0018] (b) removing the first viral vector in the first transfection mixture to obtain CD7-blocked T cells.

[0019] In another preferred embodiment, step (b) further comprises the step of removing the activator added in step (ii).

[0020] In another preferred embodiment, in step (b), the first viral vector and the activator in the first transfection mixture are removed.

[0021] In another preferred embodiment, in step (iv), the steps are included:

[0022] (c) co-culturing the second viral vector for expressing CD7-CAR with the CD7-blocked T cells under conditions suitable for transfection for 12-36 hours, preferably 18-30 hours, and more preferably 22-26 hours, to obtain a second transfection mixture;

[0023] (d) removing the second viral vector in the second transfection mixture to obtain the CD7-CAR-T cells.

[0024] In another preferred embodiment, the first viral vector and the second viral vector are lentiviral vectors.

[0025] In another preferred embodiment, the co-cultivation is carried out at 36-38°C.

[0026] In another preferred embodiment, in step (a), the number ratio of the first viral vector to T cells (multiplicity of infection) is 1:0.05-0.4.

[0027] In another preferred embodiment, in step (c), the number ratio of the second viral vector to T cells (multiplicity of infection) is 1:0.5-4.

[0028] In another preferred embodiment, the T cells are washed with a buffer to remove the viral vector.

[0029] In another preferred embodiment, the sample is peripheral blood mononuclear cells.

[0030] In another preferred embodiment, in step (ii), an activator is used to perform the separation and activation.

[0031] In another preferred embodiment, the activator is magnetic beads targeting CD3 and CD28.

[0032] In another preferred embodiment, the magnetic beads are Transact (which is easy to remove and degrade and has good activation effect). Transact is the CD3 and CD28 targeting magnetic beads used in the examples.

[0033] In another preferred embodiment, the ratio of T cells to activator is 1×10 6 Pieces: 5-20ul.

[0034] In another preferred embodiment, the CD3 / CD28-targeting magnetic beads are incubated with T cells for 12-36 hours, preferably 18-30 hours, and more preferably 22-26 hours, and then the first viral vector for expressing the CD7 blocking molecule is introduced into the activated T cells.

[0035] In another preferred embodiment, after step (iv), the method further comprises the following steps:

[0036] (v) expanding and culturing the CD7-CAR-T cells under conditions suitable for T cell expansion.

[0037] In another preferred embodiment, the antigen binding domain of the CD7-CAR includes one or more CD7 nanobodies.

[0038] In another preferred example, the amino acid sequence of the CD7 nanobody is as shown in SEQ ID NO:13.

[0039] In another preferred embodiment, the CAR further comprises a transmembrane region derived from ICOS and / or an intracellular co-stimulatory region derived from ICOS.

[0040] In another preferred embodiment, the CAR comprises an intracellular co-stimulatory region derived from ICOS and an intracellular co-stimulatory region derived from 4-1BB.

[0041] In another preferred embodiment, the structure of the CAR is shown in Formula I below:

[0042] L-VHH-H-TM-C-CD3ζ(I)

[0043] In the formula,

[0044] Each "-" is independently a connecting peptide or a peptide bond;

[0045] L is the signal peptide sequence;

[0046] VHH is an antigen-binding domain containing one or two CD7 nanobodies;

[0047] H is the hinge region;

[0048] TM is the transmembrane domain;

[0049] C is a co-stimulatory signal molecule;

[0050] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

[0051] In another preferred embodiment, the structure of the CAR is as follows Figure 1 shown.

[0052] In another preferred embodiment, the L is a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

[0053] In another preferred embodiment, the L is a signal peptide derived from CD8.

[0054] In another preferred embodiment, the amino acid sequence of L is shown in SEQ ID NO:4.

[0055] In another preferred embodiment, the VHH comprises two CD7 nanobodies. Preferably, the two CD7 nanobodies are connected by a connecting peptide. More preferably, the amino acid sequence of the connecting peptide is as shown in SEQ ID NO:8.

[0056] In another preferred example, the nucleotide sequence of the connecting peptide is shown in SEQ ID NO:7.

[0057] In another preferred embodiment, the CD7 nanobody includes humanized nanobodies and camel-derived nanobodies, preferably humanized nanobodies.

[0058] In another preferred embodiment, the VHH is a humanized CD7 nanobody.

[0059] In another preferred embodiment, the H is a hinge region derived from a protein selected from the group consisting of Fc, CD8, CD28, CD137, or a combination thereof.

[0060] In another preferred embodiment, the H is an Fc fragment, and preferably, the Fc fragment is a human IgG4 Fc fragment.

[0061] In another preferred embodiment, the amino acid sequence of the Fc fragment is shown in SEQ ID NO:14.

[0062] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the following group: ICOS, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0063] In another preferred embodiment, the TM is a transmembrane region derived from ICOS.

[0064] In another preferred example, the amino acid sequence of the ICOS-derived transmembrane region is shown in SEQ ID NO:15.

[0065] In another preferred embodiment, C is a co-stimulatory signal molecule of a protein selected from the following group: ICOS, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.

[0066] In another preferred embodiment, the C is a co-stimulatory signal molecule derived from ICOS and / or 4-1BB.

[0067] In another preferred example, the amino acid sequence of the ICOS-derived co-stimulatory signal molecule is shown in SEQ ID NO:16.

[0068] In another preferred example, the amino acid sequence of the co-stimulatory signal molecule derived from 4-1BB is shown in SEQ ID NO:17.

[0069] In another preferred example, the amino acid sequence of CD3ζ is shown in SEQ ID NO:18.

[0070] In another preferred embodiment, the CAR (preferably the C-terminus) also includes a cell suicide element.

[0071] In another preferred embodiment, the CAR is connected to the cell suicide element via a self-cleavage element.

[0072] In another preferred embodiment, the cell suicide element is connected to the CD3ζ of the CAR via T2A.

[0073] In another preferred embodiment, the cell suicide element is selected from the following group: HSV-TK, iCasp9, ΔCD20, mTMPK, ΔCD19, RQR8, EGFRt, or a combination thereof.

[0074] In another preferred embodiment, the cell suicide element is tEGFR.

[0075] In another preferred example, the amino acid sequence of the tEGFR is shown in SEQ ID NO:20.

[0076] In another preferred example, the amino acid sequence of T2A is shown in SEQ ID NO:19.

[0077] In another preferred embodiment, the CAR is PA107CD CAR (i.e., the CD7 CAR named PA3-17 in the Chinese patent application with application number 201911150678.7).

[0078] In another preferred example, the amino acid sequence of the CAR is shown in SEQ ID NO:12.

[0079] In another preferred embodiment, the CD7 blocking molecule includes one or more CD7 nanobodies and an endoplasmic reticulum retention sequence.

[0080] In another preferred embodiment, the structure of the CD7 blocking molecule is shown in the following formula II:

[0081] L'-VHH'-ER(II)

[0082] In the formula,

[0083] Each "-" is independently a connecting peptide or a peptide bond;

[0084] L' is the signal peptide sequence;

[0085] VHH' is the binding region containing two CD7 nanobodies;

[0086] ER stands for endoplasmic reticulum retention sequence.

[0087] In another preferred embodiment, the amino acid sequence of L' is shown in SEQ ID NO:4.

[0088] In another preferred embodiment, the nucleotide sequence of L' is shown in SEQ ID NO:3.

[0089] In another preferred embodiment, the two CD7 nanobodies of the VHH' are connected by a connecting peptide, and more preferably, the connecting peptide is as shown in SEQ ID NO:8.

[0090] In another preferred example, the amino acid sequence of the VHH' is shown in SEQ ID NO:6.

[0091] In another preferred example, the nucleotide sequence of the VHH' is shown in SEQ ID NO:5.

[0092] In another preferred embodiment, the VHH' is a camel-derived nanobody.

[0093] In another preferred example, the amino acid sequence of the ER is shown in SEQ ID NO:10.

[0094] In another preferred example, the nucleotide sequence of the ER is shown in SEQ ID NO:9.

[0095] In another preferred example, the amino acid sequence of the CD7 blocking molecule is as shown in SEQ ID NO:2.

[0096] In another preferred embodiment, the nucleotide sequence of the CD7 blocking molecule is as shown in SEQ ID NO:1.

[0097] In the second aspect of the present invention, a CD7-CAR-T cell prepared by the method described in the first aspect of the present invention is provided.

[0098] In another preferred embodiment, at least 80%, preferably 90%, more preferably 95%, and optimally 98% of the endogenous CD7 expression in the CD7-CAR-T cells is blocked.

[0099] In the third aspect of the present invention, a preparation is provided, which contains the CD7-CAR-T cells described in the second aspect of the present invention and a pharmaceutically acceptable carrier.

[0100] In another preferred embodiment, the preparation is a liquid preparation.

[0101] In another preferred embodiment, the preparation is in the form of an injection.

[0102] In another preferred embodiment, the concentration of the engineered immune cells in the preparation is 1×10 3 -1×10 8 cells / ml, preferably 1×10 4 -1×10 7 cells / ml.

[0103] In the fourth aspect of the present invention, a method for treating a disease is provided, comprising administering an appropriate amount of the CD7-CAR-T cells described in the second aspect of the present invention or the preparation described in the third aspect of the present invention to a subject in need of treatment.

[0104] In another preferred embodiment, the disease is cancer or tumor.

[0105] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] Figure 1 The vector structure diagram of the CD7-CAR and CD blocking molecules of the present invention is shown.

[0107] Figure 2 A flow chart for preparing the CD7-CART of the present invention is shown.

[0108] Figure 3 Flow cytometry is shown for detecting the expression of CD7 molecules on the surface of T cells (using the CD7 antibody of the FITC channel to detect CD7 molecule expression) and the transfection efficiency of CD7-CART (using the CD3 antibody of the PE channel to detect T cell expression and the FC antibody of the APC channel to detect CAR expression).

[0109] Figure 4 The cytotoxicity of CD7-CAR-T cells against 293T cells overexpressing CD7 was shown.

[0110] Figure 5 The cytotoxicity of CD7-CAR-T cells against acute lymphoblastic leukemia cells (CEM) that naturally express CD7 was shown.

[0111] Figure 6 The cytokine secretion after incubation of CD7-CAR-T cells with 293T-CD7 cells is shown.

[0112] Figure 7 The cytokine secretion after incubation of CD7-CAR-T cells with CEM cells is shown. DETAILED DESCRIPTION

[0113] After extensive and in-depth research, the inventors unexpectedly discovered for the first time a method for preparing and using CD7-CAR-T cells. Specifically, the method of the present invention improves the transfection efficiency and enhances the cytotoxicity of CAR-T cells by adjusting the transfection sequence and transfection time of the lentivirus expressing the CD7 blocking molecule and the lentivirus expressing the CD7-CAR. On this basis, the present invention was completed.

[0114] Specifically, the present invention is to provide a method for preparing CD7-CAR-T cells based on humanized CD7 nano antibody sequences. Since the preparation process of CD7-CAR-T cells involves the transfection of two lentiviruses, one is the PA007CD virus that anchors the CD7 antigen expressed on the surface of T cells in the endoplasmic reticulum or Golgi apparatus of the cell, and the other is the PA107CD lentivirus that can express CD7-CAR on the surface of T cells. The inventors found that the order of use of the two lentiviruses, the time interval of transfection and other factors will directly affect the quality of the final CART cells. The present invention describes a method for preparing CD7-CAR-T by transfecting PA007CD lentivirus 24 hours after T cell sorting and activation, washing and removing PA007CD lentivirus and activator after 48 hours, and then washing and removing PA107CD lentivirus again after 24 hours of transfection. The CD7-CAR-T cells prepared using this method have a higher CAR transfection efficiency and better cell viability. In vitro functional experiments have shown that the CD7-CAR-T cells prepared in this application can specifically kill CD7-positive target cells and secrete higher specific cytokines.

[0115] the term

[0116] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0117] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0118] The term "administering" refers to the physical introduction of the product of the invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion.

[0119] The term "antibody" (Ab) shall include, but is not limited to, immunoglobulins that specifically bind to an antigen and comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementary determining regions (CDRs), which are interspersed with more conservative regions called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

[0120] It should be understood that the amino acid names herein are identified by internationally accepted single-letter English letters, and the corresponding three-letter abbreviations of the amino acid names are: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), and Val (V).

[0121] The sequence table of the present application provides the sequences of CD7 blocking molecules (PA007CD), CD7-CAR (PA107CD) and their elements, and the meanings represented by each sequence are shown in Table 1 below.

[0122] Table 1 Names of the sequences in the sequence listing

[0123] sequence name SEQ ID NO:1 CD7 blocking molecule base sequence SEQ ID NO:2 CD7 blocking molecule amino acid sequence SEQ ID NO:3 sp (signal peptide) base sequence SEQ ID NO:4 sp (signal peptide) amino acid sequence SEQ ID NO:5 Base sequence of CD7 nanobody (VHH6) SEQ ID NO:6 Amino acid sequence of CD7 nanobody (VHH6) SEQ ID NO:7 (G4S) 4 base sequence SEQ ID NO:8 (G4S)4 amino acid sequence SEQ ID NO:9 ER base sequence SEQ ID NO:10 ER amino acid sequence SEQ ID NO:11 PA107CD base sequence SEQ ID NO:12 PA107CD amino acid sequence SEQ ID NO:13 HuVHH6 (humanized sequence of CD7 nanobody) amino acid sequence SEQ ID NO:14 Fc (human IgG4 Fc sequence, hinge region) amino acid sequence SEQ ID NO:15 ICOS-tran (ICOS transmembrane region sequence) amino acid sequence SEQ ID NO:16 ICOS (ICOS intracellular domain sequence) amino acid sequence SEQ ID NO:17 41BB (41BB intracellular region sequence) amino acid sequence SEQ ID NO:18 CD3ζ amino acid sequence SEQ ID NO:19 T2A amino acid sequence SEQ ID NO:20 tEGFR (truncated EGFR sequence) amino acid sequence

[0124] Chimeric Antigen Receptor (CAR)

[0125] The design of CARs has gone through the following process: The first generation of CAR has only one intracellular signal component CD3ζ or FcγRI molecule. Since there is only one activation domain in the cell, it can only cause short-term T cell proliferation and less cytokine secretion, but cannot provide long-term T cell proliferation signals and sustained anti-tumor effects in vivo, so it has not achieved good clinical efficacy. The second generation of CARs introduces a co-stimulatory molecule such as CD28, 4-1BB, OX40, and ICOS based on the original structure. Compared with the first generation of CARs, the function is greatly improved, further enhancing the persistence of CAR-T cells and the ability to kill tumor cells. On the basis of the second generation of CARs, some new immune co-stimulatory molecules such as CD27 and CD134 are connected in series, and developed into the third and fourth generation of CARs.

[0126] The chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen binding domain). The intracellular domain includes a costimulatory signaling region and a ζ chain portion. The costimulatory signaling region refers to a portion of the intracellular domain including a costimulatory molecule. Costimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens, rather than antigen receptors or their ligands.

[0127] Between the extracellular domain and the transmembrane domain of CAR, or between the cytoplasmic domain and the transmembrane domain of CAR, a joint may be incorporated. As used herein, the term "joint" generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane domain to the extracellular domain or cytoplasmic domain of a polypeptide chain. The joint may include 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

[0128] In a preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention includes an antigen binding domain targeting CD7. When the CAR of the present invention is expressed in T cells, it is possible to perform antigen recognition based on antigen binding specificity. When it binds to its associated antigen, it affects tumor cells, causing tumor cells not to grow, to be caused to die or otherwise affected, and causes the patient's tumor load to be reduced or eliminated. The antigen binding domain is preferably fused with one or more intracellular domains from a costimulatory molecule and a ζ chain. Preferably, the antigen binding domain is fused with an intracellular domain of a combination of ICOS and / or 4-1BB signaling domains and CD3ζ signaling domains.

[0129] As used herein, "antigen binding domain" refers to a Fab fragment, Fab' fragment, F(ab')2 fragment, or a single Fv fragment having antigen binding activity. As a preferred embodiment of the present invention, the antigen binding domain comprises one or more nanobodies that specifically recognize CD7.

[0130] For hinge region and transmembrane region (transmembrane domain), CAR can be designed to include a transmembrane domain fused to the extracellular domain of CAR. In one embodiment, a transmembrane domain naturally associated with one of the domains in CAR is used. In some examples, a transmembrane domain can be selected, or modified by amino acid replacement to avoid binding such a domain to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complex.

[0131] The intracellular domain in the CAR of the present invention includes the signaling domain of ICOS and / or 4-1BB and the signaling domain of CD3ζ.

[0132] The present invention also provides CAR-T cells targeting CD7. Unless otherwise specified, the CD7 expression on the surface of the CAR-T cells of the present invention is blocked, preferably, blocked by the CD7 blocking molecules described in the second aspect of the present invention.

[0133] Suicide gene switch

[0134] In order to further control adverse effects such as non-tumor targeting and cytokine release syndrome of CAR-T cells, the CART cells in the present invention are all equipped with a suicide gene switch, which can effectively eliminate CAR-T cells in the body under the action of exogenous drugs and block unknown or uncontrollable long-term toxicity to ensure the safety of patients.

[0135] The suicide gene switch used in the present invention can be HSV-TK, iCasp9, CD20, mTMPK, etc. In comparison, HSV-TK, iCasp9 and CD20 have the same ability to clear T cells, but iCasp9 and CD20 are cleared more quickly, while HSV-TK is cleared more slowly.

[0136] The iCasp9 suicide gene contains the FKBP12-F36V domain, which can be connected to caspase 9 through a flexible linker, which does not contain a recruitment domain. FKBP12-F36V contains an FKBP domain with phenylalanine replacing valine at the 36th amino acid residue. It has high selectivity and subnanomolar affinity and can bind to dimerization synthetic ligands, such as the otherwise inert small molecule AP1903. When small molecules are added, they can promote their dimerization, thereby inducing cell apoptosis, while having no effect on normal cells that do not carry the suicide gene.

[0137] Carrier

[0138] The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening a library from a cell expressing the gene, by obtaining the gene from a known vector comprising the gene, or by directly isolating from cells and tissues comprising the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically.

[0139] The present invention also provides vectors into which the expression cassette of the present invention is inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia viruses because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.

[0140] In brief summary, the expression cassette or nucleic acid sequence of the present invention is usually operably connected to a promoter and incorporated into an expression vector. The vector is suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

[0141] The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0142] The nucleic acid can be cloned into many types of vectors. For example, the nucleic acid can be cloned into such vectors, which include but are not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors.

[0143] Further, expression vector can be provided to cell in the form of viral vector. Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and slow viruses. Generally, suitable vectors are included in at least one organism that works on the origin of replication, promoter sequence, convenient restriction enzyme sites and one or more selectable markers (for example, WO01 / 96584; WO01 / 29058; and U.S. Patent number 6,326,193).

[0144] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to a subject's cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0145] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. Typically, these are located in the 30-110bp region upstream of the start site, although recently it has been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that when an element is inverted or moved relative to another, the promoter function is maintained. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50bp before activity begins to decline. Depending on the promoter, it is shown that individual elements can work cooperatively or independently to initiate transcription.

[0146] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive any polynucleotide sequence operably connected thereto to express at a high level. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr (Epstein-Barr) virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the present invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered to be part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0147] In order to evaluate the expression of CAR polypeptides or parts thereof, the expression vector introduced into the cell may also include any one or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a cell population seeking to be transfected or infected by a viral vector. In other aspects, selectable markers may be carried on a single DNA segment and used for co-transfection procedures. Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences so that they can be expressed in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

[0148] Reporter gene is used to identify the cells of potential transfection and to evaluate the functionality of regulatory sequences. Generally, reporter gene is the following gene: it is not present in or expressed by a receptor organism or tissue, and it encodes a polypeptide whose expression is clearly indicated by some easily detectable properties such as enzymatic activity. After DNA has been introduced into the receptor cell, the expression of the reporter gene is measured at the appropriate time. Suitable reporter gene can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein (for example, Ui-Tei et al., 2000FEBS Letters479:79-82). Suitable expression systems are well known and can be prepared using known techniques or commercially available. Generally, the construct with a minimum of 5 flanking regions showing the highest level of reporter gene expression is identified as a promoter. Such a promoter region can be connected to a reporter gene and used to evaluate the ability of a reagent to regulate promoter-driven transcription.

[0149] Methods for introducing genes into cells and expressing genes into cells are known in the art. In the context of expression vectors, vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical or biological means.

[0150] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0151] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used methods for inserting genes into mammalian cells such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0152] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0153] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. Consider using lipid preparations to introduce nucleic acid into a host cell (in vitro, ex vivo or in vivo). On the other hand, the nucleic acid can be associated with a lipid. Nucleic acids associated with lipids can be encapsulated in the aqueous interior of the liposome, dispersed in the lipid bilayer of the liposome, attached to the liposome through a connecting molecule associated with both the liposome and the oligonucleotide, trapped in the liposome, compounded with the liposome, dispersed in a solution comprising lipids, mixed with lipids, combined with lipids, included in lipids as a suspension, included in micelles or compounded with micelles, or otherwise associated with lipids. The lipids, lipid / DNA or lipid / expression vectors associated with the composition are not limited to any specific structure in the solution. For example, they can be present in a bilayer structure, as micelles or have a "collapsed (collapsed)" structure. They can also be simply dispersed in a solution, possibly forming aggregates of size or shape inhomogeneity. Lipid is a fatty substance, which can be a naturally occurring or synthetic lipid. For example, lipids include fat droplets that occur naturally in the cytoplasm as well as compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0154] In a preferred embodiment of the present invention, the vector is a lentiviral vector.

[0155] preparation

[0156] The present invention provides a preparation containing the CAR-T cells described in the first aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the preparation is a liquid preparation. Preferably, the preparation is an injection. Preferably, the concentration of the CAR-T cells in the preparation is 1×10 3 -1×10 8 cells / ml, preferably 1×10 4 -1×10 7 cells / ml.

[0157] In one embodiment, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, etc.; a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or an amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The formulation of the present invention is preferably formulated for intravenous administration.

[0158] Therapeutic applications

[0159] The present invention includes therapeutic applications of cells (e.g., T cells) transduced with a lentiviral vector (LV) encoding the expression cassette of the present invention. The transduced T cells can target the tumor cell marker CD7, synergistically activate T cells, and induce a T cell immune response, thereby significantly improving its efficiency in killing tumor cells.

[0160] Therefore, the present invention also provides a method for stimulating a T cell-mediated immune response to a target cell population or tissue in a mammal, comprising the following steps: administering the CAR-T cell of the present invention to the mammal.

[0161] In one embodiment, the present invention includes a type of cell therapy that separates the patient's autologous T cells (or allogeneic donors), activates and genetically modifies them to produce CAR-T cells, which are then injected into the same patient. This approach has a very low probability of graft-versus-host disease, and antigens are recognized by T cells in an MHC-free manner. In addition, one CAR-T can treat all cancers that express the antigen. Unlike antibody therapy, CAR-T cells can replicate in vivo, producing long-term persistence that can lead to sustained tumor control.

[0162] In one embodiment, the CAR-T cells of the present invention can undergo robust in vivo T cell expansion and can sustain an extended amount of time. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy step, in which the CAR-modified T cells induce an immune response specific to the antigen binding domain in the CAR. For example, anti-CD7 CAR-T cells cause a specific immune response against cells expressing CD7.

[0163] Although the data disclosed herein specifically discloses a lentiviral vector comprising a CD7 nanobody, an Fc fragment, a transmembrane region, and an ICOS intracellular region, 4-1BB and a CD3ζ signaling domain, the present invention should be construed to include any number of changes to each of the components of the construct.

[0164] Treatable cancers include tumors that are not vascularized or substantially not vascularized, and vascularized tumors.Cancers may include non-solid tumors (such as hematological tumors, such as leukemia and lymphoma) or may include solid tumors.The types of cancers treated with the CAR of the present invention include, but are not limited to, cancer, blastoma and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, cancers, and melanomas.Also include adult tumors / cancers and childhood tumors / cancers.

[0165] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, myelo-monocytic, monocytic, and erythroleukemias), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphomas, Hodgkin's disease, non-Hodgkin's lymphomas (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0166] Solid tumors are abnormal masses of tissue that usually do not contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell types that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcomas, myxosarcoma, liposarcoma mesothelioma, lymphoid malignancies, pancreatic cancer, ovarian cancer.

[0167] The CAR-modified T cells of the present invention can also be used as a type of vaccine for ex vivo immunization and / or in vivo therapy of mammals. Preferably, the mammal is a human.

[0168] For ex vivo immunization, at least one of the following occurs in vitro prior to administering the cells into a mammal: i) expanding the cells, ii) introducing a nucleic acid encoding a CAR into the cells, and / or iii) cryopreserving the cells.

[0169] Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from mammals (preferably humans) and genetically modified (i.e., in vitro transduction or transfection) with vectors expressing CAR disclosed herein. CAR-modified cells can be administered to a mammalian recipient to provide therapeutic benefits. The mammalian recipient can be human, and the CAR-modified cells can be autologous relative to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic relative to the recipient.

[0170] In addition to the use of cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

[0171] The present invention provides a method for treating a tumor, comprising administering a therapeutically effective amount of the CAR-modified T cells of the present invention to a subject in need thereof.

[0172] The CAR-modified T cells of the present invention may be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as IL-2, IL-17 or other cytokines or cell groups. Simply put, the pharmaceutical composition of the present invention may include a target cell group as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such a composition may include a buffer such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The composition of the present invention is preferably formulated for intravenous administration.

[0173] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease-although the appropriate dosage can be determined by clinical trials.

[0174] When an "immunologically effective amount", "anti-tumor effective amount", "tumor-suppressive effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, who takes into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. It can be generally stated that the pharmaceutical composition comprising the T cells described herein can be administered in an amount of 10 4 Up to 10 9 The dosage is preferably 10 cells / kg body weight. 5 Up to 10 6 The T cell composition can also be administered at these doses multiple times. The cells can be administered by using well-known injection techniques in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dose and treatment regimen for a specific patient can be easily determined by a medical technician by monitoring the patient's disease signs and adjusting the treatment accordingly.

[0175] Administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous (iv) injection or intraperitoneally. In one embodiment, the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by iv injection. The composition of the T cell can be injected directly into the tumor, lymph node or infection site.

[0176] In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art to expand T cells to therapeutic levels are administered to patients in combination with any number of related treatment forms (e.g., before, simultaneously or after), including but not limited to treatment with the following agents: agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or efavirenz treatment for psoriasis patients or other treatments for PML patients. In further embodiments, the T cells of the present invention can be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunotherapeutic agents. In further embodiments, the cell compositions of the present invention are administered to patients in combination with bone marrow transplantation, using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide (e.g., before, simultaneously or after). For example, in one embodiment, the subject can undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives an infusion of the expanded immune cells of the invention following transplantation. In an additional embodiment, the expanded cells are administered prior to or after surgery.

[0177] The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The dosage ratio for human administration can be implemented according to practices accepted in the art. Typically, 1×10 6 to 1×10 10 The modified T cells of the present invention (e.g., CAR-T20 cells) are administered to the patient, for example, by intravenous infusion.

[0178] The main advantages of the present invention include:

[0179] (a) The CD7-CAR-T cells prepared by the method of the present invention have higher CAR transfection efficiency and better cell viability.

[0180] (b) The CD7-CAR-T cells prepared by the method of the present invention can specifically kill CD7-positive target cells and secrete higher specific cytokines.

[0181] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0182] Example 1

[0183] Preparation of CD7-CAR and CD7 blocking molecules

[0184] The CD7 blocking molecule is constructed by conventional techniques in the art, and the structure is as follows Figure 1 The amino acid sequence is shown in SEQ ID NO: 2. The CD7 blocking molecule is inserted into a lentiviral vector, and then a CD7 blocking lentivirus, namely PA007CD, is prepared by lentiviral packaging technology.

[0185] CD7-CAR is constructed using conventional techniques in the art, and the structure is as follows: Figure 1 The amino acid sequence is shown in SEQ ID NO: 12. The above CD7-CAR is inserted into a lentiviral vector, and then a CD7-CAR lentivirus, namely PA107CD, is prepared by lentiviral packaging technology.

[0186] Example 2

[0187] Preparation of CD7-CAR-T cells

[0188] Since more than 90% of normal human T cells express CD7 antigen, the CD7 antigen on the surface of T cells needs to be blocked inside the cell during the preparation of CD7-CAR-T cells. Otherwise, suicide will occur due to CD7-CAR-T recognizing its own antigen CD7.

[0189] T cells were transfected with CD7 blocking lentivirus (PA007CD), so that the CD7 expressed by T cells was blocked in the endoplasmic reticulum or Golgi apparatus and could not be presented on the surface of T cells. Then CD7-CAR lentivirus (PA107CD) was used to transfect T cells to prepare CD7-CAR-T cells.

[0190] In this embodiment, a single blood sample from a donor was used, and Ficoll was used to obtain mononuclear cells (PBMC) by density gradient centrifugation. Then, CD4 / CD8 dual magnetic beads were used to enrich T cells from the mononuclear cells. After obtaining the T cells, an activator (Transact) was used to activate the T cells. On the second day after activation (24 hours after activation), an appropriate number of PA007CD viruses was selected according to the previous verification results for transfection of T cells. The specific ratio was MOI (multiplicity of infection) = 0.05-0.4. Under this MOI condition, T cell expression The CD7 molecule can be completely blocked and has little effect on the later PA107CD virus transfection. After 48 hours of PA007CD lentivirus transfection, the residual activator and PA007CD lentivirus are removed by centrifugation and then PA107CD lentivirus is added for transfection. The amount of PA107CD used in transfection is also selected according to the previous verification results. The specific ratio is MOI (multiplicity of infection) = 0.5-4. The selection of this MOI ensures the transfection efficiency. After 24 hours of transfection, the residual PA107CD lentivirus is removed by centrifugation and the expansion culture of CAR-T cells is started. The preparation flow chart is as follows Figure 2 shown.

[0191] Example 3

[0192] Expression of CD7 molecules on the surface of T cells and transfection efficiency of CD7-CART

[0193] In the preparation process of CD7-CAR-T cells, the expression rate of CD7 on the surface of T cells and the proportion of CAR-positive cells (transfection efficiency) are important indicators for evaluating CD7-CAR-T cells. After transfecting the PA007CD virus, the expression of CD7 molecules on the surface of T cells needs to be blocked, and then transfecting PA107CD can successfully prepare CD7-CAR-T cells. The three batches of CD7-CAR-T prepared by this method, the cell CD7 molecule expression and transfection efficiency statistics at the time of sampling are shown in Table 2.

[0194] Table 2 Cell surface CD7 molecule expression and transfection efficiency

[0195] Cell lot number LZ012019122101 ZL012019123001 ZZY12020010701 CD7 molecule expression 0.20 1.05 0.99 Transfection efficiency 37.80 18.86 11.56

[0196] Flow chart Figure 3 shown.

[0197] The results showed that after transfection with the PA007CD virus, the CD7 molecules on the surface of T cells were almost completely blocked, with an expression level of about 1%. At this time, transfection with the PA107CD lentivirus to prepare CD7-CAR-T reduced the suicide phenomenon of cells, effectively ensured the viability of CD7 CAR-T cells, and significantly improved the success rate of cell preparation.

[0198] Example 4

[0199] Cytotoxicity of CD7-CAR-T cells against 293T cells overexpressing CD7

[0200] An important method to detect the biological efficacy of CD7-CAR-T cells is to detect the killing ability of CAR-T cells on target cells after co-incubation of CD7-CAR-T cells with CD7-positive target cells.

[0201] CD7-overexpressing 293T cells (293T-CD7) were constructed as target cells to evaluate the in vitro killing function of CD7-CAR-T cells. Using LZ012019122101, ZL012019123001, and ZZY12020010701 prepared in Example 2, three groups of CD7-CAR-T were used for killing experiments, with effect-target ratios of 20:1, 10:1, and 5:1, respectively. 293T cells are CD7-negative target cells.

[0202] The results are as follows Figure 4 As shown, it can be seen from the killing results of the following three groups of cells that CD7-CAR-T cells have a significant specific killing effect on 293T-CD7 cells (CD7 positive), but have no specific killing effect on 293T cells (CD7 negative), indicating that the CD7-CAR-T cells prepared in Example 2 can produce specific cytotoxicity to 293T-CD7 cells overexpressing CD7 positive in vitro, but have no specific cytotoxicity to CD7 negative 293T cells.

[0203] Example 5

[0204] Cytotoxicity of CD7-CAR-T cells against acute lymphoblastic leukemia cells (CEM) that naturally express CD7

[0205] The CD7-CAR-T cells prepared in Example 2 were used to perform an in vitro killing experiment on acute lymphoblastic leukemia cells (CEM), and the effector-target ratios were 1:1, 1:10, 1:50, and 1:100, respectively. T cells were control effector cells.

[0206] The results are as follows Figure 5 As shown, compared with the control T cells, CD7-CART cells have a significant specific killing effect on CEM, indicating that the CD7-CART cells prepared in Example 2 have significant cytotoxicity to CD7-positive CEM cells in vitro.

[0207] Example 6

[0208] Cytokine secretion after incubation of CD7-CAR-T cells and 293T-CD7 cells

[0209] The release of cytokines after incubation of CD7-CAR-T cells with 293T-CD7 cells was analyzed. The killing supernatant of the group in which the effector cells and target cells were co-incubated with an effector-target ratio of 1:1 was selected to detect the secretion of cytokines IL-2, IFN-γ, and Granzyme B.

[0210] The results are as follows Figure 6 As shown, there is basically no cytokine production after CD7-CAR-T cells are co-incubated with 293T cells, while there is a significant increase in cytokine release after CD7-CAR-T cells are incubated with 293T-CD7 cells, indicating that the prepared CD7-CAR-T cells can specifically activate CD7-positive 293T-CD7 cells.

[0211] Example 7

[0212] Cytokine secretion after incubation of CD7-CAR-T cells and CEM cells

[0213] The release of cytokines after co-incubation of CD7-CAR-T cells with CEM cells was further analyzed. The effector-target ratios of CD7-CAR-T cells and target cells were 1:10, 1:50, and 1:100, respectively. The T cell group served as the control group, and the secretion of cytokines IL-2, IFN-γ, and Granzyme B was detected.

[0214] The results are as follows Figure 7 As shown in the figure, there is basically no cytokine production after T cells are co-incubated with target cells, while there is a significant increase in cytokine release after CD7-CAR-T cells are incubated with CEM cells. This further proves that the prepared CD7-CAR-T cells can be specifically activated by CD7-positive tumor cells.

[0215] Summary: The main content of the present invention is to successfully prepare CD7-CAR-T cells with high transfection efficiency and good cell activity using a new transfection method, and through a series of in vitro functional experiments, it is proved that the prepared CD7-CAR-T cells have significant specific killing effects on CD7-positive tumor cells.

[0216] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application. Sequence Listing <110> Boshengji Pharmaceutical Technology (Suzhou) Co., Ltd. Bosheng Jianke Cell Technology Co., Ltd. <120> Individual CD7-CAR-T activation was performed in a manner <130> P2020-0474 <160> 20 <170> SIPOSequenceList 1.0 <210> 1 <211> 933 <212> DNA <213> Artificial Sequence <400> 1 atgctgctgc tggtgacctc tctgctgctc tgcgaactgc ctcacccagc ctttctgctg atccccatgg acgtgcagct gcaggaaagc ggaggagc tggtgcaggc aggaggatct 180. ctgaggctgt cttgcgcagt gtccggatac ccctacagca gctactgcat gggttggttc agacaggccc caggaaagga gagagagcta tcgatagcga cggaagaacc agatacgccg acagcgtga gggcaggttc accatcagcc aggacaacgc caagacacc ctgtacctgc agatgaaccg gatgaagccc gaggacaccg ccatgtacta ttgcgccgct 360 cgcttcggac ctatgggttg cgtggatctg agcaccctga gctttggcca ttggggacag 420 ggacccagg tcaccgtgtc catcactgga ggaggagga ggaggaggagg 480 rich gcgaggagg aggatctatg gacgtgcagc tgcaggagag cggagga 540 ctggtgcagg caggaggatc tctgagactg tcttgcgccg tgtccggata cccttacagc 600 agctactgca tgggctggtt caggcaggct ccaggaaagg agagagaggg agtggcagcc 660 atcgatagcg acggcagaac cagatacgcc gatagcgtga agggcaggtt caccatcagc 720 caggacaacg ccaagaacac cctgtacctg cagatgaacc ggatgaagcc cgaggatacc 780 gccatgtact attgcgccgc caggttcgga cctatgggtt gcgtggacct gagcacactg 840 agcttcggcc attggggaca gggaacacag gtcaccgtgt ccatcaccga gcagaagctg 900 atcagcgagg aggacctgaa ggacgagctg tga 933 <210> 2 <211> 310 <212> PRT <213> Artificial Sequence <400> 2 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Met Asp Val Gln Leu Gln Glu Ser Gly Gly 20 25 30 Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser 35 40 45 Gly Tyr Pro Tyr Ser Ser Tyr Cys Met Gly Trp Phe Arg Gln Ala Pro 50 55 60 Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Asp Ser Asp Gly Arg Thr 65 70 75 80 Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn 85 90 95 Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Arg Met Lys Pro Glu Asp 100 105 110 Thr Ala Met Tyr Tyr Cys Ala Ala Arg Phe Gly Pro Met Gly Cys Val 115 120 125 Asp Leu Ser Thr Leu Ser Phe Gly His Trp Gly Gln Gly Thr Gln Val 130 135 140 Thr Val Ser Ile Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 145 150 155 160 Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Asp Val Gln Leu Gln Glu 165 170 175 Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys 180 185 190 Ala Val Ser Gly Tyr Pro Tyr Ser Ser Tyr Cys Met Gly Trp Phe Arg 195 200 205 Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Asp Ser Asp 210 215 220 Gly Arg Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 225 230 235 240 Gln Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Arg Met Lys 245 250 255 Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Arg Phe Gly Pro Met 260 265 270 Gly Cys Val Asp Leu Ser Thr Leu Ser Phe Gly His Trp Gly Gln Gly 275 280 285 Thr Gln Val Thr Val Ser Ile Thr Glu Gln Lys Leu Ile Ser Glu Glu 290 295 300 Asp Leu Lys Asp Glu Leu 305 310 <210> 3 <211> 66 <212> DNA <213> Artificial Sequence <400> 3 atgctgctgc tggtgacctc tctgctgctc tgcgaactgc ctcacccagc ctttctgctg 60 atcccc 66 <210> 4 <211> 22 <212> PRT <213> Artificial Sequence <400> 4 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro 20 <210> 5 <211> 381 <212> DNA <213> Artificial Sequence <400> 5 atggacgtgc agctgcagga aagcggagga ggactggtgc aggcaggagg atctctgagg 60 ctgtcttgcg cagtgtccgg atacccctac agcagctact gcatgggttg gttcagacag 120 gccccaggaa aggagagaga gggagtggcc gctatcgata gcgacggaag aaccagatac 180 gccgacagcg tgaagggcag gttcaccatc agccaggaca acgccaagaa caccctgtac 240 ctgcagatga accggatgaa gcccgaggac accgccatgt actattgcgc cgctcgcttc 300 ggacctatgg gttgcgtgga tctgagcacc ctgagctttg gccattgggg acagggaacc 360 caggtcaccg tgtccatcac t 381 <210> 6 <211> 127 <212> PRT <213> Artificial Sequence <400> 6 Met Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Pro Tyr Ser Ser 20 25 30 Tyr Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly 35 40 45 Val Ala Ala Ile Asp Ser Asp Gly Arg Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Arg Met Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Ala Arg Phe Gly Pro Met Gly Cys Val Asp Leu Ser Thr Leu Ser 100 105 110 Phe Gly His Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ile Thr 115 120 125 <210> 7 <211> 60 <212> DNA <213> Artificial Sequence <400> 7 ggaggaggag gaagcggagg aggaggatct ggaggaggag gaagcggagg aggaggatct 60 <210> 8 <211> 20 <212> PRT <213> Artificial Sequence <400> 8 Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 9 <211> 42 <212> DNA <213> Artificial Sequence <400> 9 gagcagaagc tgatcagcga gaggacctg aaggacgagc tg <210> 10 <211> 14 <212> PRT <213> Artificial Sequence <400> 10 Glu Gln Light Leu Ile Ser Glu Glu Asp Leu Light Asp Glu Leu 1 5 10 <210> 11 <211> 1770 <212> DNA <213> Artificial Sequence <400> 11 atgctgctgc tggtgacctc tctgctgctc tgcgaactgc ctcacccagc ctttctgctg atccccatgc aggtgcagct ggtggaagc ggaggagc tggtgcagcc aggaggatct 120 ctgagactgt cttgcgccgc cagcggctac ccctacagca gctactgcat gggttggttc 180 agacaggccc caggacaggg actggaagca gtggcagcca tcgatagcga cggcagaaca 240 cgctacgccg attccgtgaa gggcagattc accatcagcc gggacaacag caagaacacc 300 ctgtacctgc agatgaacag cctgagggcc gaggataccg cagtgtacta ttgcgccgcc 360 cgctttggac ctatgggttg cgtggacctg agcacactga gcttcggcca ttggggacag 420 ggaacactgg tgacagtgtc cagcgagagc aaatacggcc ctccttgccc tccttgccca 480 gccccagaat ttgagggagg acctagcgtg ttcctgttcc ctcccaagcc caaggacacc 540 ctgatgatca gccggacccc agaagtcacc tgcgtggtgg tggacgtgtc tcaggaagac 600 cccgaggtgc agttcaattg gtacgtggac ggcgtggaag tgcacaacgc caagaccaag 660 cccagagagg agcagttcca gagcacctac agagtggtgt ccgtgctgac cgtgctgcat 720 caggattggc tgaacggcaa ggagtacaag tgcaaggtgt ccaacaaggg cctgcctagc 780 agcatcgaga agaccatcag caaggccaag ggccagccta gagagcctca ggtgtacaca 840 ctgccccctt ctcaggagga gatgaccaag aaccaggtgt ccctgacttg cctcgtgaag 900 ggcttctacc ccagcgatat tgccgtggag tgggagtcta acggccagcc cgagaacaac 960 tacaagacca cccctcccgt gctggatagc gacggctctt tcttcctgta cagccggctg 1020 acagtggaca aaagtcgctg gcaggagggc aacgtgttca gttgcagcgt gatgcacgag 1080 gccctgcaca accactacac ccagaagagc ctgtctctgt ctctcggcaa gtggctccct 1140 atcggttgcg ccgcctttgt cgtcgtctgt atcctcggct gcatcctcat ctgttggctc 1200 accaagaaga agtacagcag cagcgtgcac gaccccaacg gcgagtacat gttcatgcgg 1260 gccgtcaaca ccgccaagaa gagcagactg accgacgtga ccctgaagag aggcaggaag 1320 aagctgctgt acatcttcaa gcagcccttc atgcggccag tgcagacaac ccaggaggaa 1380 gacggctgct cttgcagatt ccccgaggaa gaagagggcg gttgcgagct gagagtgaag 1440 ttcagcagaa gcgccgacgc tccagcctat cagcagggcc agaaccagct gtacaacgag 1500 ctgaacctgg gcaggagaga ggagtacgac gtgctggaca agaggagagg cagagaccca 1560 gagatgggcg gcaagcctag acggaagaac cctcaggagg gcctgtacaa cgaactgcag 1620 aaggacaaga tggccgaggc ctacagcgag atcggcatga agggcgagag gagaagaggc 1680 aagggacacg acggactgta ccagggactg agcacagcca ccaaggacac atacgacgcc 1740 ctgcacatgc aggctctgcc tcctagatga 1770 <210> 12 <211> 589 <212> PRT <213> Artificial Sequence <400> 12 Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro 1 5 10 15 Ala Phe Leu Leu Ile Pro Met Gln Val Gln Leu Val Glu Ser Gly Gly 20 25 30 Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser 35 40 45 Gly Tyr Pro Tyr Ser Ser Tyr Cys Met Gly Trp Phe Arg Gln Ala Pro 50 55 60 Gly Gln Gly Leu Glu Ala Val Ala Ala Ile Asp Ser Asp Gly Arg Thr 65 70 75 80 Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 85 90 95 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Ala Arg Phe Gly Pro Met Gly Cys Val 115 120 125 Asp Leu Ser Thr Leu Ser Phe Gly His Trp Gly Gln Gly Thr Leu Val 130 135 140 Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro 145 150 155 160 Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220 Gln Phe Gln Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 260 265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 305 310 315 320 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 340 345 350 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser Leu Gly Lys Trp Leu Pro Ile Gly Cys Ala 370 375 380 Ala Phe Val Val Val Cys Ile Leu Gly Cys Ile Leu Ile Cys Trp Leu 385 390 395 400 Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr 405 410 415 Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp 420 425 430 Val Thr Leu Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 435 440 445 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 450 455 460 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 465 470 475 480 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 485 490 495 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 500 505 510 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 515 520 525 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 530 535 540 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 545 550 555 560 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 565 570 575 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 580 585 <210> 13 <211> 126 <212> PRT <213> Artificial Sequence <400> 13 Met Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Pro Tyr Ser Ser 20 25 30 Tyr Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Gln Gly Leu Glu Ala 35 40 45 Val Ala Ala Ile Asp Ser Asp Gly Arg Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Arg Phe Gly Pro Met Gly Cys Val Asp Leu Ser Thr Leu Ser 100 105 110 Phe Gly His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 14 <211> 229 <212> PRT <213> Artificial Sequence <400> 14 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225 <210> 15 <211> 23 <212> PRT <213> Artificial Sequence <400> 15 Trp Leu Pro Ile Gly Cys Ala Ala Phe Val Val Val Cys Ile Leu Gly 1 5 10 15 Cys Ile Leu Ile Cys Trp Leu 20 <210> 16 <211> 35 <212> PRT <213> Artificial Sequence <400> 16 Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn Gly Glu Tyr 1 5 10 15 Met Phe Met Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp 20 25 30 Val Thr Leu 35 <210> 17 <211> 42 <212> PRT <213> Artificial Sequence <400> 17 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 18 <211> 112 <212> PRT <213> Artificial Sequence <400> 18 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 19 <211> 18 <212> PRT <213> Artificial Sequence <400> 19 Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro 1 5 10 15 Gly Pro <210> 20 <211> 352 <212> PRT <213> Artificial Sequence <400> 20 Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile 1 5 10 15 Ser Arg Lys Val Cys Asn Gly Ile Gly Ile Gly Glu Phe Lys Asp Ser 20 25 30 Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser 35 40 45 Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser 50 55 60 Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu Lys 65 70 75 80 Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile Gln Ala Trp Pro Glu 85 90 95 Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu Glu Ile Ile Arg Gly 100 105 110 Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala Val Val Ser Leu Asn 115 120 125 Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu Ile Ser Asp Gly Asp 130 135 140 Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn 145 150 155 160 Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser 165 170 175 Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala 180 185 190 Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp Cys Val 195 200 205 Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys Val Asp Lys Cys Asn 210 215 220 Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu Asn Ser Glu Cys Ile 225 230 235 240 Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met Asn Ile Thr Cys Thr 245 250 255 Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala His Tyr Ile Asp Gly 260 265 270 Pro His Cys Val Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn 275 280 285 Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys 290 295 300 His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly Cys 305 310 315 320 Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala Thr Gly Met Val Gly 325 330 335 Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly Ile Gly Leu Phe Met 340 345 350

Claims

1. A method for preparing CD7-CAR-T cells, characterized in that: Includes steps: (i) providing a sample containing T cells to be processed, (ii) sorting and activating T cells contained in the sample to obtain activated T cells, (iii) introducing a first viral vector for expressing a CD7 blocking molecule into the activated T cells, thereby obtaining CD7 blocked T cells, and (iv) introducing a second viral vector for expressing CD7-CAR into the CD7-blocked T cells to obtain the CD7-CAR-T cells, Wherein, after the T cells are co-incubated with the activator for 22-26 hours, the first viral vector for expressing the CD7 blocking molecule is introduced into the activated T cells; In step (iii), the steps include: (a) co-culturing the first viral vector for expressing the CD7 blocking molecule with the activated T cells for 46-50 hours under conditions suitable for transfection, wherein the ratio of the first viral vector to the T cells is 1:0.05-0.4, thereby obtaining a first transfection mixture; (b) removing the first viral vector in the first transfection mixture to obtain CD7-blocked T cells; In step (iv), the steps include: (c) co-culturing the second viral vector for expressing CD7-CAR with the CD7-blocked T cells for 22-26 hours under conditions suitable for transfection, wherein the ratio of the second viral vector to the T cells is 1:0.5-4, thereby obtaining a second transfection mixture; (d) removing the second viral vector in the second transfection mixture to obtain the CD7-CAR-T cells.

2. The method according to claim 1, characterized in that The sample is peripheral blood mononuclear cells.

3. The method according to claim 1, characterized in that In step (b), the method further comprises removing the activator added in step (ii).

4. The method according to claim 1, characterized in that The first viral vector and the second viral vector are lentiviral vectors.

5. The method according to claim 1, characterized in that The co-cultivation is carried out at 36-38°C.

6. The method according to claim 1, characterized in that In step (ii), the sorting and activation are performed using an activator, which is a magnetic bead targeting CD3 and CD28.

7. The method according to claim 1, characterized in that After step (iv), the method further comprises the steps of: (v) expanding and culturing the CD7-CAR-T cells under conditions suitable for T cell expansion.

8. The method according to claim 1, characterized in that The CD7 blocking molecule includes one or more CD7 nanobodies and an endoplasmic reticulum retention sequence.

9. The method according to claim 1, characterized in that The antigen binding domain of the CD7-CAR includes one or more CD7 nanobodies.

10. The method according to claim 8 or 9, characterized in that The amino acid sequence of the CD7 nanobody is shown in SEQ ID NO:

13.

11. The method according to claim 1, wherein: The amino acid sequence of the CAR is shown in SEQ ID NO:

12.

12. The method according to claim 1, wherein: The amino acid sequence of the CD7 blocking molecule is shown in SEQ ID NO: 2.

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