Single-chain antibodies, fusion proteins targeting cd43, and immune cells expressing the same and uses thereof

Abstract

The application discloses a single-chain antibody targeting CD43, and constructs a fusion protein and immune cells expressing the same on the basis of the single-chain antibody, and further discloses the use of the single-chain antibody, the fusion protein or the immune cells in treating tumors or cancers. The CAR-T cell CAR-T-43 targeting CD43 has high safety and effectiveness.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the field of cell therapy technology, and particularly to a single-chain antibody targeting CD43, a fusion protein, immune cells constructed based thereon, and their applications. Background Technology

[0002] T-cell acute lymphoblastic leukemia (T-ALL) is a malignant hematologic disorder caused by early T-cell progenitor cells. It accounts for approximately 15% of childhood ALL cases and 25% of adult ALL cases, with a male incidence rate twice that of females. Compared to B-ALL, T-ALL has a higher relapse rate, poorer remission rate, and lower long-term survival. Chemotherapy alone is ineffective and prone to relapse. With improvements in treatment regimens, including improved methotrexate (MTX) dosage, the prognosis for childhood T-ALL has become close to that of B-ALL, with a 5-year progression-free survival rate of 80%. The prognosis for adult T-ALL is worse than for children; approximately 50% of adult patients relapse about one year after treatment remission, and the remission rate with repeat chemotherapy is only 30%-45%, ultimately resulting in only 40% of patients achieving long-term survival.

[0003] CD43 is a cell surface protein expressed in various blood cells except erythrocytes. Studies have shown that monoclonal antibodies targeting CD43 and antibody-drug conjugates targeting toxic effectors have demonstrated preliminary anti-tumor effects in mouse models. CAR-T-43 targets a glycosylation-associated mCD43 epitope on CD43. Previous studies have shown that this epitope is not expressed on mature blood cells, hematopoietic stem cells, and other normal cells, except on CD4 and CD8 double-positive T cells. However, it is highly expressed on acute T-cell leukemia cells (T-ALL) and T-lymphoblastic lymphoma (T-LBL) cells, making it a promising tumor-specific antigenic epitope for therapeutic applications.

[0004] The chimeric antigen receptor (CAR) T-cell modification method originated from Carl June's use of Anti-CD19 CAR-T to treat Emily, a clinically cured child with B-cell acute lymphoblastic leukemia. CAR-T therapy involves extracting peripheral blood from the patient, genetically modifying it in vitro to enable T cells to express CAR and activate them to a certain level, before reinfusing them into the patient. The modified T cells then target, expand, and efficiently kill tumor cells. Currently, CAR-T cell therapy targeting CD5 or CD7 has shown some efficacy in clinical patients with relapsed / refractory T-cell acute lymphoblastic disease / lymphoma. However, these targets face several obstacles. Because normal T cells also express a small amount of CD5 or CD7, two potential problems arise: 1) During the preparation process, CAR-T cells may kill CAR-T cells expressing CD5 or CD7, leading to cannibalism and CAR-T cell preparation failure; 2) The targeted CAR-T cells reinfused into the body may kill normal T cells, resulting in incomplete T cell development and ultimately causing immunodeficiency in patients, increasing the risk of infection with exogenous viruses and bacteria. Although gene editing technology can prevent the expression of CD5 or CD7 on normal T cells in the patient's peripheral blood, these gene editing technologies increase the complexity of the CAR-T cell preparation process and pose a serious off-target risk.

[0005] TCR-T therapy is based on the T cell receptor (TCR). The TCR is the identity characteristic of T cells, and based on the type of TCR, T cells can be divided into αβ T cells and γδ T cells. During development, T precursor cells undergo VDJ rearrangement of the TCRγ and TCRδ chains. If the rearrangement is successful, they develop into γδ T cells; if the rearrangement fails, the precursor cells undergo VDJ recombination of the TCRα and TCRβ chains, subsequently developing into αβ T cells. αβ T cells account for 90%-95% of peripheral blood T cells, while γδ T cells account for 5%-10%. These two types of T cells recognize antigens in an MHC-restricted and MHC-unrestricted manner, respectively, playing important roles in the immunity against pathogens and tumors.

[0006] The T-cell receptor (TCR) complex molecule contains multiple chains. The TCRα and TCRβ chains (or TCRγ and TCRδ chains) are responsible for recognizing MHC-peptide molecules, while the other six CD3 subunits bind to the TCRα / β chains (or TCRγ / δ chains), functioning as signal transduction agents. The natural TCR complex contains 10 ITAM signaling sequences, theoretically capable of transmitting stronger signals than CARs. Utilizing the signal transduction function of the natural TCR, it is possible to construct a novel receptor to alleviate T-cell dysfunction, enabling it to better exert its anti-solid tumor effects. The extracellular region of the TCR is very similar to the Fab domain of an antibody; therefore, the variable region sequence of the TCR can be replaced with the variable region sequence of an antibody, resulting in a synthetic T-cell receptor antigen receptor that possesses both the specificity of an antibody and the superior signal transduction function of the natural TCR, mediating T-cell activation. Summary of the Invention

[0007] Based on research on CD43, this invention aims to provide an immune cell that targets CD43 for the treatment of diseases expressing CD43, such as acute T-cell leukemia and T-cell lymphoma.

[0008] This invention first provides a single-chain antibody targeting CD43, which is sequentially linked from the N-terminus to the C-terminus with the following structure: VL-Linker-VH or VH-Linker-VL; wherein, the amino acid sequence of VL is SEQ ID NO:1, and the amino acid sequence of VH is SEQ ID NO:2; the linker is selected from peptides with amino acid sequences of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5; and when the structure of the single-chain antibody is VH-linker-VL, the linker is selected from peptides with amino acid sequences of SEQ ID NO:4 and SEQ ID NO:5.

[0009] In one embodiment of the present invention, the amino acid sequence of the single-chain antibody is SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:49.

[0010] The present invention provides a nucleic acid molecule encoding the above-mentioned single-chain antibody, wherein the nucleotide sequence is selected from SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:49.

[0011] Another aspect of the present invention provides a fusion protein comprising a light chain variable region peptide of an antibody targeting CD43, a heavy chain variable region peptide of a single-chain antibody targeting CD43, and a transmembrane functional region; wherein the amino acid sequence of the light chain variable region peptide is SEQ ID NO:1, and the amino acid sequence of the heavy chain variable region peptide is SEQ ID NO:2; wherein,

[0012] 1) The transmembrane functional region is a chimeric antigen receptor region comprising a hinge region, a transmembrane domain, a co-stimulatory domain, and an intracellular signal transduction domain. The light chain variable region peptide and the heavy chain variable region peptide are linked via a linker to form a single-chain antibody. The single-chain antibody is sequentially tandemly linked with the transmembrane domain, the co-stimulatory domain, and the intracellular signal transduction domain to form a fusion protein. Preferably, the C-terminus of the intracellular signal transduction domain is linked to a co-expressed protein via a first linker peptide. The co-expressed protein is OX40 protein or c-jun protein. The amino acid sequence of the OX40 protein is SEQ ID NO:17, and the amino acid sequence of the c-jun protein is SEQ ID NO:18. Alternatively,

[0013] 2) The transmembrane functional region includes a constant region of the TCRα subunit and a constant region of the TCRβ subunit; a first peptide chain obtained by fusing the variable region of the antibody heavy chain with the first subunit; and a second peptide chain obtained by fusing the variable region of the antibody light chain with the second subunit; wherein when the first subunit is a constant region of the TCRα chain, the second subunit is a constant region of the TCRβ chain; and when the first subunit is a constant region of the TCRβ chain, the second subunit is a constant region of the TCRα chain; preferably, it further includes a first signal peptide, a second signal peptide, and a second linker peptide, wherein the first signal peptide is linked to the N-terminus of the first peptide chain, the second linker peptide is linked to the C-terminus of the first peptide chain, the N-terminus of the second signal peptide is linked to the C-terminus of the second linker peptide, and the C-terminus of the second signal peptide is linked to the N-terminus of the second peptide chain; or,

[0014] 3) The transmembrane functional region includes the CD3ε subunit. The light chain variable region peptide and the heavy chain variable region peptide are linked via a linker to form a single-chain antibody. The single-chain antibody is fused to the N-terminus of the CD3ε subunit via a third linker peptide.

[0015] In one embodiment of the present invention, the single-chain antibody is the single-chain antibody described above.

[0016] In one embodiment of the invention, each Linker is independently selected from peptides with amino acid sequences of SEQ ID NO:3, 4, and 5 each time it appears; and / or, the amino acid sequence of the hinge region is as shown in SEQ ID NO:6 or 7; and / or, the amino acid sequence of the transmembrane domain is as shown in SEQ ID NO:8; and / or, the amino acid sequence of the co-stimulatory domain is as shown in SEQ ID NO:9, 10, or 11; and / or, the amino acid sequence of the intracellular signal transduction domain is as shown in SEQ ID NO:12, 13, or 14; and / or, the first signal peptide is a CD8α signal peptide, and the amino acid sequence of the CD8α signal peptide is as shown in SEQ ID NO:15; and / or, the second signal peptide is CD8α signal peptide 2, and its amino acid sequence is SEQ ID NO:50; and / or, the amino acid sequence of the TCRβ chain constant region is SEQ ID NO:19; and / or, the amino acid sequence of the TCRα chain constant region is SEQ ID NO:50. NO:20; and / or, the first linker, the second linker, and the third linker are each independently selected from peptides with amino acid sequences of SEQ ID NO:3, 4, and 5; and / or, the amino acid sequence of the CD3ε subunit is SEQ ID NO:21.

[0017] In one embodiment of the present invention, the amino acid sequence of the fusion protein is SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:32.

[0018] The present invention further provides the application of the above-mentioned single-chain antibody or fusion protein in the preparation of CD43-targeting immune T cells.

[0019] Another aspect of the present invention provides a nucleic acid molecule encoding the above-described fusion protein; preferably, it is a nucleic acid molecule selected from those with nucleotide sequences of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 or SEQ ID NO:45.

[0020] The present invention also provides a vector comprising the above-described nucleic acid molecules; the vector is selected from one or more of DNA vectors, RNA vectors, plasmids, lentiviral vectors, adenovirus vectors, and retroviral vectors.

[0021] The present invention further provides engineered immune cells, wherein the immune cells contain the above-described vector, or have an exogenous nucleic acid molecule integrated into the chromosome, or express the above-described fusion protein.

[0022] Another aspect of the present invention provides a formulation comprising the above-described single-chain antibody, fusion protein, or engineered immune cell, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0023] Furthermore, the present invention also discloses the use of the above-mentioned fusion protein or engineered immune cells in the preparation of drugs or formulations for treating cancer or tumors.

[0024] In one embodiment of the invention, the cancer or tumor is selected from adenoid cystic carcinoma (ACC), diffuse large B-cell lymphoma (DLBCL), myeloid sarcoma, blastic plasmacytoid dendritic cell neoplasm (BPDCN), T-cell lymphoma, and T-cellacute lymphoblastic leukemia (T-ALL).

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. This invention successfully constructed CAR-T cells targeting CD43 and verified that the CAR-T 43 cells provided by this invention have high safety and efficacy. Since CD43 is basically not expressed on the surface of normal T cells, it is not necessary to block CD43 expression when preparing CAR-T cells, making the CAR-T cell preparation process simpler.

[0027] 2. The CAR-T-43 provided by this invention can specifically target tumor cells while almost completely avoiding attacks on normal T cells. This prevents severe immunodeficiency and facilitates the rapid reconstruction of the patient's own immune system, preventing fatal complications such as severe infections. The CAR-T-43 prepared by this invention does not kill normal T cells but exhibits strong killing activity against T-ALL cell lines. Attached Figure Description

[0028] Figure 1 The transduction efficiency detection results of CD43-targeted CAR-T cells prepared in Example 5 of this invention are shown in the figure.

[0029] Figure 2 The transduction efficiency detection results of CD43-targeted CAR-T cells prepared in Example 6 of this invention are shown in the figure.

[0030] Figure 3 Figure 1 shows the flow cytometry results of CD43 expression in healthy human PBMCs, T-ALL patient PBMCs, tumor cell lines MOLT-4 and CCRF-CEM.

[0031] Figure 4 The killing effect of CD43-targeting CAR-T cells prepared according to Example 5 of the present invention on the CD43-expressing tumor cell line CCRF-CEM after 70 hours;

[0032] Figure 5 Line graph showing the 70-hour killing effect of CD43-targeting CAR-T cells prepared according to Example 6 of the present invention on the CD43-expressing tumor cell line CCRF-CEM;

[0033] Figure 6 Line graph showing the 20-hour killing effect of CD43-targeted CAR-T cells on CD43-expressing tumor cell line MOLT-4 and normal human T cells;

[0034] Figure 7 The figure shows the 20-hour killing effect of CD43-targeted CAR-T cells and CD7-targeted CAR-T cells on the CCRF-CEM cell line. Detailed Implementation

[0035] The following detailed explanation and description of the present invention, in conjunction with specific embodiments, does not limit the scope of protection of the present invention.

[0036] General Technology

[0037] Unless otherwise indicated, the practice of this invention will employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the scope of the art. This technique is well explained in the literature, including, for example: *Molecular Cloning: A Laboratory Manual, 2nd Edition* (Sambrook et al., 1989), Cold Spring Harbor Press; *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Methods in Molecular Biology*, Humana Press; *Cell Biology: A Laboratory Notebook* (edited by J.E. Cell, 1998), Academic Press; *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Introduction to Cell and Tissue Culture* (edited by J.M. Pather and P.E. Roberts, 1998), Plenum Press; *Cell and Tissue Culture: Laboratory Procedures* (edited by A. Doyle, J.B. Griffiths, and D.G. Newell, 1993-1998), J. Wiley and Sons; *Methods in Enzymology* (Academic Press, Inc.); and *Handbook of Experimental... Immunology (edited by DMWeir and CC Blackwell); GeneTransfer Vectors for Mammalian Cells (edited by JMMiller and MPCalos, 1987); Current Protocols in Molecular Biology (edited by FMAusubel et al., 1987); PCR: The PolymeraseChainReaction, (edited by Mullis et al., 1994); Current Protocols in Immunology (JEColigan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: a practical approach (edited by D. Catty., IRL Press, 1988-1989); Monoclonalantibodies: a practical approach (edited by P. Shepherd and C. Dean, Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (edited by M. Zanetti and JD Capra, Harwood Academic Publishers, 1995).

[0038] In some cases, for clarity and / or ease of reference, terms with their commonly understood meanings are defined herein. The invention will now be described in detail by way of the following definitions and examples. All patents and publications mentioned herein (including all sequences disclosed in such patents and publications) are expressly incorporated by reference.

[0039] Unless otherwise stated, when this invention relates to percentages between liquids, the percentage is volume / volume percentage; when this invention relates to percentages between liquids and solids, the percentage is volume / weight percentage; when this invention relates to percentages between solids and liquids, the percentage is weight / volume percentage; the remainder is weight / weight percentage.

[0040] definition

[0041] Generally, unless otherwise defined, all terms, symbols and other scientific terms or terminology used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains.

[0042] As used herein, the numerical range includes the numbers that define the range.

[0043] The terms “approximately,” “close to,” etc., when preceding a list of values ​​or ranges, independently refer to each individual value in that list or range as if each individual value immediately followed the term. The term implies that the value referred to by the term is exactly the same as, close to, or similar to. For example, in some embodiments, “approximately” or “close to” a particular value may indicate 99%, 95%, or 90% of that value.

[0044] As used in this article, nucleic acids are written from left to right in a 5' to 3' direction; amino acid sequences are written from left to right in an orientation from amino (N-terminus) to carboxyl (C-terminus).

[0045] The terms “polypeptide,” “oligopeptide,” “peptide,” and “protein” are used interchangeably herein to refer to an amino acid chain of any length (e.g., 10-100 amino acids). The chain may be linear or branched, may contain modified amino acids, and / or may be broken by non-amino acid components. The term also includes amino acid chains that have been modified naturally or through intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation to a labeled component. The definition also includes, for example, analogs containing one or more amino acids (including, for example, non-natural amino acids), and other modified polypeptides known in the art. It should be understood that polypeptides can exist as single chains or associated chains. Preferably, mammalian polypeptides (polypeptides originally derived from mammalian organisms) are used, more preferably those polypeptides secreted directly into the culture medium.

[0046] The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains (two heavy (H) chains and two light (L) chains) linked by disulfide bonds, as well as their polymers (e.g., IgM). Each heavy chain contains a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region. The "variable region" of an antibody refers to the variable region of either the antibody light chain alone or in combination with the variable region of the antibody heavy chain.

[0047] The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are contiguously linked via a short, flexible polypeptide linker and are capable of being expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it originates. Unless otherwise stated, as described herein, scFv may have V L and V H For example, relative to the N-terminus and C-terminus of the peptide, scFv may contain VL-linker-VH or VH-linker-VL.

[0048] As used herein, the terms "linker," "connector," and "flexible peptide linker" refer to peptide linkers composed of amino acid residues, either alone or in combination, such as glycine and / or serine, to link heavy chain variable regions and light chain variable regions together. In one embodiment, the flexible peptide linker is a Gly / Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Ser). n Or it can be written as (G4S) n, where n is a positive integer equal to or greater than 1. For example, n = 1, n = 2, n = 3, n = 4, n = 5, n = 6, n = 7, n = 8, n = 9, and n = 10.

[0049] The term "antigen" or "Ag" refers to a molecule that can be specifically bound by an antibody or otherwise elicit an immune response. This immune response may involve the production of antibodies, the activation of specific immune cells, or both.

[0050] The term "stimulus" refers to a primary response induced by the binding of a stimulating domain or stimulating molecule (e.g., the TCR / CD3 complex) to its homologous ligand, thereby mediating signal transduction events, such as, but not limited to, signal transduction via the TCR / CD3 complex. Stimulus can mediate alterations in the expression of certain molecules and / or reorganization of cytoskeleton structures.

[0051] The terms "stimulatory molecule" or "stimulatory domain" refer to a molecule or portion thereof expressed by T cells that provides primary cytoplasmic signal transduction. A sequence that stimulates the primary activation of the TCR complex targets at least some aspect of the T cell signaling pathway. On one hand, primary signaling is initiated, for example, by the binding of the TCR / CD3 complex to a peptide-loaded MHC molecule, and this leads to the mediation of T cell responses, including but not limited to proliferation, activation, differentiation, etc. Primary cytoplasmic signal transduction sequences that act in a stimulatory manner (also called "primary signal transduction domains") may contain signal transduction motifs known as immune receptor tyrosine-based activation motifs or "ITAMs."

[0052] As used in this article, the term "intracellular signal transduction domain" refers to the intracellular portion of a molecule. Intracellular signal transduction domains generate signals that promote the immune effector functions of cells containing TFP (e.g., TFP-expressing T cells). Examples of immune effector functions, such as in TFP-expressing T cells, include cytolytic activity and T helper cell activity, including cytokine secretion.

[0053] Primary intracellular signal transduction domains may contain ITAMs (“immunoreceptor tyrosine-based activation motifs”). Examples of ITAMs containing primary cytoplasmic signal transduction sequences include, but are not limited to, those derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d, as well as DAP10 and DAP12.

[0054] The term "co-stimulatory molecule" refers to a homologous binding partner on a T cell that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response in T cells, such as, but not limited to, proliferation. Co-stimulatory molecules are cell surface molecules required for an effective immune response, other than antigen receptors or their ligands. Co-stimulatory molecules can be derived from the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, KG2C, SLAMF7, NKp80, CD160, B7-H3, and ligands that specifically bind to CD83. Intracellular signal transduction domains may contain the entire intracellular portion of the molecules from which they are derived, or the entire native intracellular signal transduction domain or a functional fragment thereof.

[0055] The term "4-1BB" refers to a member of the TNFR superfamily, whose amino acid sequence is provided by GenBank Acc. No. AAA62478.2, or equivalent residues from non-human species such as mice, rodents, monkeys, apes, etc.; the "4-1BB co-stimulatory domain" is defined as amino acid residues 214-255 of GenBank Acc. No. AAA62478.2, or equivalent residues from non-human species such as rodents, monkeys, and apes.

[0056] The term "antitumor effect" refers to biological effects that can be manifested in a variety of ways, including but not limited to, reductions in tumor volume, number of tumor cells, metastasis, increased life expectancy, reduced tumor cell proliferation, reduced tumor cell survival, or improvement of various physiological symptoms associated with cancer.

[0057] As used herein, “treatment” or “curing” refers to any successful indication of treating or improving a disease or condition. Treatment may include, for example, reducing, delaying, or alleviating the severity of one or more symptoms of a disease or condition, or may include reducing the frequency of the occurrence of symptoms of a disease, defect, condition, or adverse condition.

[0058] Reagents and materials

[0059] Restriction endonucleases such as XbaI, BamHI, AgeI, and XhoI were all purchased from New England Biolabs.

[0060] T4 DNA ligase was purchased from New England Biolabs;

[0061] pPVLV4, pMDLg / pRRE(K), pRSV-Rev(K), and pMD.G(K) were all purchased from Pharosvaccine Inc., Gyeonggi, Republic of Korea.

[0062] 293T cells were purchased from ATCC (American Type Culture Collection);

[0063] opti-MEM medium was purchased from Gibco, catalog number 51985034;

[0064] PEI MAX was purchased from Polysciences Incorporate;

[0065] Ficoll, purchased from Cytiva

[0066] X-VIVO15 serum-free culture medium was purchased from Lonza, catalog number: 04-418Q;

[0067] Sample purchased from Mixer Thermo Fisher Scientific;

[0068] Dynabeads were purchased from Thermo Fisher Scientific;

[0069] Polybrene was purchased from Yisheng Biotechnology.

[0070] PBS was purchased from Gibco;

[0071] FBS was purchased from Gibco;

[0072] RPMI-1640 was purchased from Gibco;

[0073] CCRF-CEM cell line was purchased from Zhongyuan Union Cell & Gene Engineering Co., Ltd.

[0074] The MOLT-4 cell line was purchased from Zhongyuan Union Cell & Gene Engineering Co., Ltd.

[0075] CFSE fluorescent dye was purchased from AAT Bioquest;

[0076] Unless otherwise stated, all reagents, instruments and materials involved in this invention are commercially available.

[0077] Example 1: Construction and expression of scFV antibody targeting CD43

[0078] The single-chain antibody targeting CD43 provided by this invention consists of a light chain sequence of a single-chain antibody targeting CD43, a linker, and a heavy chain sequence of a single-chain antibody targeting CD43. This invention constructs scFVs with the structure VH-linker-VL or VL-linker-VH, as shown in Table 1. The amino acid sequence of the single-chain antibody light chain VL is shown in SEQ ID NO:1; the amino acid sequence of the single-chain antibody heavy chain VH targeting CD43 is shown in SEQ ID NO:2; and the amino acid sequence of the single-chain antibody hinge linker targeting CD43 is shown in any one of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.

[0079] Table 1. Structure and sequence of scFV antibodies

[0080] name structure Linker sequence scFV amino acid sequence ScFV01 VH-linker-VL SEQ ID NO:3 SEQ ID NO:46 ScFV02 VL-linker-VH SEQ ID NO:3 SEQ ID NO:47 ScFV03 VL-linker-VH SEQ ID NO:4 SEQ ID NO:48 ScFV04 VL-linker-VH SEQ ID NO:5 SEQ ID NO:49

[0081] Example 2: Construction of a chimeric antigen receptor or T-cell receptor targeting CD43

[0082] This invention combines the aforementioned scFV to design a chimeric antigen receptor with the structural formula SP-scFv-H-TM-CR-ST. SP is a signal peptide that helps the CAR protein fold correctly and locate on the cell membrane. It is located at the N-terminus of the CAR (chimeric antigen receptor) and is typically implemented as the CD8 signal peptide (amino acid sequence SEQ ID NO:15) in this invention. scFv is a single-chain variable fragment (scFv). In the embodiments of this invention, it is implemented as the single-chain antibody designed in Example 1, and in the comparative example, it is implemented as an anti-CD7 scFV and its chimeric antigen receptor (amino acid sequence SEQ ID NO:33). H is a hinge region located between the extracellular domain and the transmembrane domain, connecting the scFv and the transmembrane domain. In this invention, it is implemented as the CD28 Hinge (amino acid sequence SEQ ID NO:6) or the IgG4 Hinge (amino acid sequence SEQ ID NO:33). NO:7); TM is the transmembrane domain connecting the extracellular and intracellular regions of the CAR and anchoring the entire CAR structure to the T cell membrane. In this invention, it is implemented as the CD28 transmembrane domain (CD28 TM, amino acid sequence SEQ ID NO:8); CR is the co-stimulation region, which in this invention is implemented as the CD28 co-stimulation domain (amino acid sequence SEQ ID NO:10), a CD28 co-stimulation domain mutant (mutCD28 CR, amino acid sequence SEQ ID NO:11), or a 4-1BB co-stimulation domain (4-1BB CR, amino acid sequence SEQ ID NO:10); ST is the signal transduction domain, which in this invention is implemented as the CD3ζ chain (amino acid sequence SEQ ID NO:12) or a modified CD3ζ chain, for example, a CD3ζ chain mutant with K mutated to R in the sequence (mutCD3ζ chain, amino acid sequence SEQ ID NO:12). NO:13), or CD3ζ-TIR (the amino acid sequence of the TIR domain is SEQ ID NO:14) fused with the TLR2-TIR intracellular signaling domain; some embodiments of the present invention also include co-expressed proteins linked by a linker peptide after the ST portion, such as OX-40 (amino acid sequence is SEQ ID NO:18) or c-jun (amino acid sequence is SEQ ID NO:19).

[0083] Based on research on T-cell antigen receptors, this invention also designs a novel fusion protein structure, linking the VL region of the antibody to the β-chain-C region of the TCR subunit, and linking the VH region of the antibody to the α-chain-C region of the TCR subunit. The structure of this fusion protein, from the N-terminus to the C-terminus, is as follows:

[0084] CD8αSP+VL+mutCβ+P2A+CD8αSP+VH+mutCα.

[0085] Among them, CD8αSP is the signal peptide, and its amino acid sequence is SEQ ID NO:15; VL is the variable region peptide of the antibody light chain, and its amino acid sequence is SEQ ID NO:1; VH is the variable region peptide of the antibody heavy chain, and its amino acid sequence is SEQ ID NO:2; mutCβ is the constant region peptide of the TCRβ chain, and its amino acid sequence is SEQ ID NO:19; mutCα is the constant region peptide of the TCRα chain, and its amino acid sequence is SEQ ID NO:20.

[0086] Another fusion protein designed in this invention links scFV to the CD3ε group via a linker, with the structure scFV+linker+CD3ε. After expression in cells, this fusion protein can autonomously assemble with other TCR subunits to form a complete TCR group, expressing scFV on the cell surface. In this invention, the structure is implemented as scFV02+(G4S)3+CD3ε; the amino acid sequence of CD3ε is SEQ ID NO:21. The specific structure is shown in Table 2.

[0087] Table 2. Structure of the fusion protein

[0088]

[0089] Comparative CD7 CAR-T sequence

[0090] This invention designs a chimeric antigen receptor targeting CD7, the amino acid sequence of which is SEQ ID NO:33.

[0091] Example 3: Construction of recombinant lentiviral plasmid targeting CD43 chimeric antigen receptor

[0092] 1. Expression gene synthesis

[0093] Based on the fusion proteins of Example 2 and the comparative example, Genscript Biotech Inc. was commissioned to synthesize a cloning vector containing the gene capable of expressing the corresponding fusion protein. The expression gene of the fusion protein is shown in Table 3.

[0094] Table 3. Genes encoding the fusion protein

[0095] name Fusion protein sequence gene sequence Fusion protein 1 (CD43 CAR 01) SEQ ID NO:22 SEQ ID NO:34 Fusion protein 2 (CD43CAR 02) SEQ ID NO:23 SEQ ID NO:35 Fusion protein 3 (CD43CAR 03) SEQ ID NO:24 SEQ ID NO:36 Fusion protein 4 (CD43CAR 04) SEQ ID NO:25 SEQ ID NO:37 Fusion protein 5 (CD43CAR 05) SEQ ID NO:26 SEQ ID NO:38 Fusion protein 6 (CD43CAR 06) SEQ ID NO:27 SEQ ID NO:39 Fusion protein 7 (CD43CAR 07) SEQ ID NO:28 SEQ ID NO:40 Fusion protein 8 (CD43CAR 08) SEQ ID NO:29 SEQ ID NO:41 Fusion protein 9 (CD43CAR 09) SEQ ID NO:30 SEQ ID NO:42 Fusion protein 10 (CD43CAR 10) SEQ ID NO:31 SEQ ID NO:43 Fusion protein 11 (CD43CAR 11) SEQ ID NO:32 SEQ ID NO:44 Comparative Example 12 (CD7 CAR) SEQ ID NO:33 SEQ ID NO:45

[0096] 2. Construction of lentiviral vectors

[0097] 1) The synthesized cloning vector was digested with enzymes to obtain the coding genes of each fusion protein, and appropriate restriction endonucleases were selected for digestion based on each gene sequence.

[0098] Specifically, this invention is implemented as follows:

[0099] Cloning vectors containing the coding genes for fusion protein 1 or fusion protein 2 were digested with XbaI and BamHI, respectively; cloning vectors containing the coding genes for fusion protein 3, fusion protein 4, or fusion protein 5 were digested with AgeI and XhoI, respectively; and cloning vectors containing the coding genes for fusion protein 6, fusion protein 7, fusion protein 8, fusion protein 9, fusion protein 10, or fusion protein 11 were digested with XbaI and XhoI, respectively.

[0100] 2) The target fragment was harvested from the gel, and then the coding gene was ligated into the corresponding pPVLV4 vector using T4 DNA ligase. The vector was then transformed into competent cells. The next day, single clones were picked and cultured in antibiotic-free LB liquid medium for 12 hours in a shaker for plasmid extraction. The concentration and purity of the plasmid were detected, and the correct clones were identified by enzyme digestion. The clones were then sent for DNA sequencing verification. The shuttle plasmid pPVLV4 identified by sequencing was used for subsequent experiments.

[0101] Example 4: Recombinant Lentiviral Virus Targeting CD43 Chimeric Antigen Receptor

[0102] (1) Cell plating

[0103] 293T cells were revived and cultured in DMEM medium containing 10% FBS at a depth of 225 cm. 2 In cell culture flasks, when the cell confluence reaches 85-95%, 293T cells are digested with recombinant trypsin. The digested cells are then plated in 225 cm⁻¹ plates. 2 Cells were cultured in cell culture flasks at 37°C in a 5% CO2 incubator.

[0104] (2) Plasmid transfection and virus packaging

[0105] Once the 293T cells reach a confluence of 60-80%, plasmid transfection will be performed.

[0106] Preparation of plasmid transfection solution: The shuttle plasmid pPVLV4 and three helper plasmids pMDLg / pRRE(K), pRSV-Rev(K), and pMD.G(K) were added at a mass ratio of 4:2:2:1. The plasmid mixture was prepared by adding the above plasmids to 2.5 ml of opti-MEM medium and mixing thoroughly. PEI MAX was added to opti-MEM medium at a volume ratio of 1 mg / ml (1:(15-20)) to prepare a PEI MAX mixture. The PEI MAX mixture and plasmid mixture were thoroughly mixed at a 1:1 ratio to form a PEI-DNA mixture, and incubated at room temperature for 20 min. The PEI-DNA mixture was then added to cell culture flasks to form the plasmid transfection solution. The cell culture flasks were incubated in a CO2 incubator for 18 ± 2 h.

[0107] (3) Change the fluid

[0108] Discard the transfection medium in the cell culture flask from step 2), and add 15-25 mL each of opti-MEM + 2% FBS fresh complete medium.

[0109] (4) Harvesting and preservation of lentiviral vectors

[0110] Collect the virus after 45-72 hours of culture: Collect the supernatant from the cell culture flask into a centrifuge tube, add 20-25 mL of Opti-MEM + 2% FBS fresh complete culture medium, and tighten the cap.

[0111] (5) After collecting the supernatant, centrifuge at 4000g and 4℃ for 15min. Filter the supernatant through a 0.45μM filter, transfer it to a new centrifuge tube, add 6× lentivirus concentrate at a ratio of 1:5, mix well, wrap with sealing film, and place in a 4℃ refrigerator to stand.

[0112] (6) Lentiviral centrifugation and aliquoting

[0113] After the virus has been allowed to stand for 1-2 days, centrifuge at a maximum speed of 4000g and 4℃ for 30 minutes, then discard the supernatant. Resuspend the virus in 100uL of serum-free DMEM medium in each tube, aliquot into 40-50uL portions, and store at -80℃.

[0114] Example 5: Construction and transduction efficiency detection of CD43-targeted T cells

[0115] 1. PBMC resuscitation and T cell sorting and activation

[0116] (1) Separation of PBMC

[0117] Transfer the volunteer's blood into centrifuge tubes, add an appropriate amount of sodium chloride to dilute the blood, and use Ficoll to separate PBMCs using density gradient centrifugation. Separate the PBMC cells, and bring the volume of the separated PBMC cells to 50 ml with X-VIVO15 serum-free medium (Lonza), mix well, and centrifuge at 526 g for 5 min; remove the supernatant, resuspend the cells in cell cryopreservation solution, and cryopreserve.

[0118] (2) The recovery of PBMC

[0119] Preheat the X-VIVO15 medium in the centrifuge tubes in a 37°C water bath. Remove the frozen PBMCs from liquid nitrogen and thaw them rapidly in a 37°C water bath. Transfer the cells to the preheated X-VIVO15 medium and mix well. Centrifuge at 526g for 5 min, discard the supernatant, resuspend the cells in X-VIVO15 medium, mix well, and then sample for cell counting.

[0120] (3) Adhesion removal of mononuclear cells

[0121] Add an appropriate amount of X-VIVO15 medium to the cell suspension from step (2) above, and adjust the PBMC density to 2 × 10⁻⁶. 6 Cells were counted at a density of 10 cells / ml. The cell suspension was transferred to a cell culture flask and incubated in a CO2 incubator (37℃, 5% CO2) for 30 min. The suspended cells were collected in centrifuge tubes, centrifuged at 400g for 10 min, the supernatant was discarded, and the cell pellet was resuspended in X-VIVO15 medium (X-200) containing 200 IU / mL IL-2. The cells were mixed well, and 0.5 ml was taken for cell count and phenotypic analysis.

[0122] (4) Sorting of T cells

[0123] CD3 was adjusted using X-VIVO15 medium containing 200 IU / mL IL-2. + T cell density reached 2 × 10⁶ 7 -4×10 7 Dynabeads CD3 / CD28 and T cells were mixed evenly at a ratio of (2-5):1. Centrifuge at 2 rpm / min at room temperature for 30 minutes to 1 hour using a sample mixer. After centrifugation, place the centrifuge tubes on a Magent and let them stand for about 2 minutes, then discard the supernatant.

[0124] (5) T cell activation

[0125] The mixture of Dynabeads and cells was resuspended in X-VIVO15 medium containing 200 IU / mL IL-2 to achieve a T cell density of 5 × 10⁶ cells / mL. 6Cells / ml were transferred to containers for culture and incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0126] 2. Lentiviral transduction of T cells

[0127] After activation, the cells were removed from the incubator and observed under a microscope. The cells were then mixed and transferred to 15ml centrifuge tubes for sampling to determine cell count and viability. Based on the cell count results, the T cell density was adjusted to 2×10⁻⁶ cells / mL. 6 The cells were divided into two groups: a non-transduced T cell group (NC) and a recombinant immune T cell group (which were transduced with fusion proteins 1, 2, 4 and 5 respectively to obtain CD43 CAR-T 01 cells, CD43 CAR-T 03 cells, CD43 CAR-T 04 cells and CD43 CAR-T 05 cells). No lentivirus was added to the NC group. Each recombinant immune T cell group was given an appropriate volume of the corresponding lentivirus according to the lentivirus MOI of 3. Polybrene was added to both the NC group and the T cell group to a final concentration of 8 μg / ml.

[0128] Add the appropriate volume of cells, virus solution, and polybrene (Yisheng Biotechnology) to a cell culture plate, label the cell culture plate according to the number, and mix thoroughly. Incubate at 37°C in a 5% CO2 incubator for 24 hours to obtain lentivirus-transduced T cells.

[0129] 3. Elution of T cells after lentiviral transduction

[0130] Remove cells from the incubator and observe them under a microscope. Collect cells and centrifuge at 526g for 5 min. Adjust the cell density to 5 × 10⁶ cells / mL with X-VIVO15 medium containing 200 IU / mL IL-2. 5 The cells were incubated at 37°C in a 5% CO2 incubator for 48 hours.

[0131] 4. Detection of CD43-targeted chimeric antigen receptor T cell transduction efficiency

[0132] Cells were removed from the incubator, and the cells in the culture flask were mixed. Approximately 0.5 ml was taken for cell count and viability analysis. The same number of cells were taken from both the non-transduced T cell group (NC) and the recombinant immune T cell group for flow cytometry analysis. The results are as follows: Figure 1 As shown. The remaining cell suspension was adjusted to a cell density of 5 × 10⁶ cells using X-VIVO15 medium containing 200 IU / mL IL-2. 5 Cells / mL were incubated at 37°C in a 5% CO2 incubator for 2-3 days for in vitro killing analysis.

[0133] Flow cytometry analysis of CAR-T cell transduction efficiency:

[0134] (1) Mix the sample, take the cell suspension and add it to the labeled EP tube, remove the beads, add 2 mL of PBS (1X) to each tube, mix well, centrifuge at 1500 rpm for 3 min, discard the supernatant, and disperse the cells by shaking.

[0135] (2) Add 50 μL PBS to resuspend the cells, perform instrumental analysis, and collect 20,000 cells from the cell gate;

[0136] Flow cytometry analysis of recombinant immune T cell transduction efficiency: Figure 1 The results showed that the constructed recombinant immune T cells had high transduction efficiency, indicating that the recombinant immune T cells were successfully prepared.

[0137] Example 6: Construction and Transduction Efficiency Detection of Recombinant CD43-Targeting Immune T Cells

[0138] CD43 CAR-T 01 cells constructed using fusion proteins 1, 2, 6, 7, 8, 9, 10, and 11 were transduced using the same procedures as in Example 5, and non-transduced T cell group (NC) and recombinant immune T cell group were set up.

[0139] Flow cytometry analysis of recombinant immune T cell transduction efficiency: results are as follows Figure 2 As shown, the recombinant immune T cells constructed in this embodiment have high transduction efficiency, indicating that the recombinant immune T cells were successfully prepared.

[0140] Experiment 1: Expression of CD43 in T-ALL patients and tumor cells

[0141] The expression of CD43 in healthy human PBMCs, T-ALL patient PBMCs, human acute lymphoblastic leukemia cells MOLT-4, and CCRF-CEM was detected by flow cytometry.

[0142] The results are as follows Figure 3 As shown, CD43 is expressed at low levels in normal cells, but at high levels in T-ALL patients, tumor cells MOLT-4, and tumor cells CCRF-CEM.

[0143] Experiment 2: Validation of in vitro killing effect of recombinant immune T cells targeting CD43

[0144] 1. Effector cells

[0145] The recombinant immune T cells (CD43 CAR-T 01 cells, CD43 CAR-T 03 cells, CD43 CAR-T 04 cells, and CD43 CAR-T 05 cells) prepared in Example 5 above were used as effector cells. According to the proportion of CAR positive cells, the effector cells were prepared into cell suspensions of different concentrations according to the effector-target ratio using RPMI-1640 medium containing 10% FBS.

[0146] 2. Target cells

[0147] CCRF-CEM and MOLT-4 cells (both purchased from Zhongyuan Xiehe) were used as target cells. The target cells were incubated with 0.5 μM CFSE fluorescent dye at 37±1℃ and 5±1% CO2 for 25-40 min in the dark. After incubation, the cells were washed with RPMI-1640 medium, and finally adjusted to a target cell density of 1×10⁶ cells / cells using RPMI-1640 medium containing 10% FBS. 6 cells / ml.

[0148] 3. Cell plating

[0149] Target cells were seeded at 100 μl / well in 96-well cell culture plates, with a target cell count of 1 × 10⁶ cells / well. 5 Cells / well: A target cell control group was set up and supplemented with 100 μl of RPMI-1640 medium containing 10% FBS; effector cells with different effector-to-target ratios were added to the corresponding wells at 100 μl / well. The 96-well plates were incubated at 37±1℃ and 5±1% CO2 for 20-72 hours.

[0150] 4. Kill efficiency test

[0151] Cells were cultured in 96-well plates at 37±1℃ and 5±1% CO2, and all cells were transferred to Eppendorf tubes. Cells were resuspended in 1 ml PBS in each Eppendorf tube, centrifuged, and the supernatant discarded. 50 μl of PBS containing 2.5 μl of 7-AAD was added to each Eppendorf tube. All samples were incubated at 4℃ in the dark for 25 min. After incubation, samples were removed and flow cytometry was used to determine the cell killing rate.

[0152] 5. Test Results

[0153] Experimental success condition: The self-killing efficiency of the target cell blank well is less than 20%.

[0154] The killing efficiency was calculated based on the flow cytometry results using the following formula: Killing efficiency (%) = 7 - AAD+% (transducer group) - 7 - AAD+% (target cells).

[0155] Among them, 7-AAD+% (transduction group) is the positive cell population of the transduction group, and 7-AAD+% (target cells) is the positive cell population of the target cells.

[0156] Kill efficiency results are as follows Figure 4 As shown in the figure. The results indicate that the recombinant immune T cells targeting CD43 prepared based on the fusion protein constructed in Example 5 can effectively kill the CD43-expressing tumor cell line CCRF-CEM.

[0157] Experiment 3: Validation of in vitro killing effect of recombinant immune T cells targeting CD43

[0158] 1. Effector cells

[0159] The CD43 CAR-T 01 cells, CD43 CAR-T 02 cells, CD43 CAR-T 06 cells, CD43 CAR-T 07 cells, CD43 CAR-T 08 cells, CD43 CAR-T 09 cells, CD43 CAR-T 10 cells, and CD43 CAR-T 11 cells prepared in Example 6 above were used as effector cells. According to the proportion of positive cells, the effector cells were prepared into cell suspensions of different concentrations according to the effector-target ratio using RPMI-1640 medium containing 10% FBS.

[0160] 2. Target cells

[0161] CCRF-CEM and MOLT-4 cells (both purchased from Zhongyuan Xiehe) were used as target cells. The target cells were incubated with 0.5 μM CFSE fluorescent dye at 37±1℃ and 5±1% CO2 for 25-40 min in the dark. After incubation, the cells were washed with RPMI-1640 medium, and finally adjusted to a target cell density of 1×10⁶ cells / cells using RPMI-1640 medium containing 10% FBS. 6 cells / ml.

[0162] The experimental methods and procedures for verifying lethality are consistent with those in Experiment Example 2.

[0163] Kill efficiency results are as follows Figure 5 As shown in the figure. The results showed that recombinant immune T cells targeting CD43 based on the fusion protein CAR structure constructed in Example 6 could effectively kill tumor cells expressing CD43.

[0164] Experiment 4: Validation of in vitro killing effect of CD43 chimeric antigen receptor T cells

[0165] Comparative Example 1 compares the in vitro killing effects of recombinant CD43-containing immune T cells on tumor cells that highly express CD43 and normal human T cells that do not express CD43, as follows:

[0166] 1. Effector cells

[0167] The CD43 CAR-T 01 cells prepared above were used as effector cells. Based on the proportion of positive cells, the effector cells were prepared into cell suspensions of different concentrations according to the effector-target ratio using RPMI-1640 medium containing 10% FBS.

[0168] 2. Target cells

[0169] The target cells were the CD43-expressing tumor cell line MOLT-4 and normal human T cells. The target cells were incubated with 0.5 μM CFSE fluorescent dye at 37±1℃ and 5±1% CO2 for 25-40 min in the dark. After incubation, the cells were washed with RPMI-1640 medium and finally adjusted to a target cell density of 1×10⁶ cells / cells using RPMI-1640 medium containing 10% FBS. 6 cells / ml.

[0170] 3. Cell plating

[0171] Target cells were seeded at 100 μl / well in the corresponding wells of a 96-well cell culture plate, with a target cell count of 1 × 10⁶ cells / well. 5 Cells / well: A target cell control group was set up and supplemented with 100 μl of RPMI-1640 medium containing 10% FBS; effector cells with different effector-to-target ratios were added to the corresponding wells at 100 μl / well. The 96-well plate was incubated at 37±1℃ and 5±1% CO2 for 20 hours.

[0172] 4. Kill Efficiency Detection

[0173] From a culture condition of 37±1℃ and 5±1% CO2, remove 96-well round-bottom cell culture plates and transfer all cells to 1.5ml Eppendorf tubes. Resuspend cells in 1ml PBS in each Eppendorf tube, centrifuge, and discard the supernatant. Add 50μl of PBS containing 2.5μl of 7-AAD to each Eppendorf tube. Incubate all samples at 4℃ in the dark for 25min. After incubation, remove the samples and perform flow cytometry to determine the cell killing rate.

[0174] 5. Test Results

[0175] Test results as follows Figure 6 As shown. From Figure 6 The CD43 CAR-T cells of this invention show a comparison of their killing effects on tumor cells and normal human T cells. Left image: CD43 CAR-T cells have a strong killing effect on MOLT-4 tumor cells that highly express CD43; Right image: They have no killing effect on normal T cells that do not express CD43.

[0176] CD43 CAR-T cells (CD43 CAR-T 01 cells) exhibited a strong killing effect on the MOLT-4 tumor cell line, which highly expresses CD43, but showed no killing effect on healthy donor T cells that do not express CD43. These results indicate that targeting CD43 CAR-T cells (CD43CAR-T 01 cells) has high safety.

[0177] Experiment 5: Validation of in vitro killing effect of CD43 chimeric antigen receptor T cells

[0178] In this experimental example, the in vitro killing effects of CD43-targeting T cells prepared in the above embodiments of the present invention are compared with those of CD7 CAR-T cells, as detailed below:

[0179] (1) Effector cells

[0180] The CD43 CAR-T 01 cells and CD7 CAR-T cells prepared above were used as effector cells. According to the proportion of CAR-positive cells, the effector cells were prepared into cell suspensions of different concentrations according to the effector-target ratio using RPMI-1640 medium containing 10% FBS.

[0181] (2) Target cells

[0182] CCRF-CEM cells were used as target cells. The target cells were incubated with 0.5 μM CFSE fluorescent dye at 37±1℃ and 5±1% CO2 for 25-40 min in the dark. After incubation, the cells were washed with RPMI-1640 medium and finally adjusted to a target cell density of 1×10⁶ cells / cells using RPMI-1640 medium containing 10% FBS. 6 cells / ml.

[0183] (3) Cell plating

[0184] Target cells were seeded at 100 μl / well in the corresponding wells of a 96-well cell culture plate, with a target cell count of 1 × 10⁶ cells / well. 5 Cells / well: A target cell control group was set up and supplemented with 100 μl of RPMI-1640 medium containing 10% FBS; effector cells with different effector-to-target ratios were added to the corresponding wells at 100 μl / well. The 96-well plate was incubated at 37±1℃ and 5±1% CO2 for 20 hours.

[0185] (4) Kill efficiency test

[0186] From a culture condition of 37±1℃ and 5±1% CO2, remove 96-well round-bottom cell culture plates and transfer all cells to 1.5ml Eppendorf tubes. Resuspend cells in 1ml PBS in each Eppendorf tube, centrifuge, and discard the supernatant. Add 50μl of PBS containing 2.5μl of 7-aminoactinomycin D (7-AAD) to each Eppendorf tube. Incubate all samples at 4℃ in the dark for 25min. After incubation, remove samples and analyze the cell killing rate using flow cytometry.

[0187] (5) Test results

[0188] Test results as follows Figure 7 As shown. From Figure 7 The results show the 20-hour killing effect of CD43 CAR-T cells (CD43CAR-T 01 cells) and CD7 CAR-T cells on the CCRF-CEM cell line. At multiple effector-to-target ratios, CD43 CAR-T cells (CD43 CAR-T 01 cells) and CD7 CAR-T cells exhibited consistent killing activity and strong killing effect on the CCRF-CEM cell line.

[0189] Figure 7 Results of 20-hour killing of CCRF-CEM cell line by CD43-targeted CAR-T cells and CD7-targeted CAR-T cells. At multiple effector-to-target ratios, CD43 CAR-T cells (CD43 CAR-T 01 cells) showed consistent killing activity with the control group targeting CD7 CAR-T cells, and exhibited strong killing activity against the CCRF-CEM cell line.

[0190] The above description of specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of protection of the claims of the present invention.

[0191] sequence list

[0192] SEQ ID NO:1VL

[0193]

[0194] SEQ ID NO:2VH

[0195]

[0196] SEQ ID NO:3linker1

[0197]

[0198] SEQ ID NO:4linker2

[0199]

[0200] SEQ ID NO:5linker3

[0201]

[0202] SEQ ID NO:6hinge1(CD28 hinge)

[0203]

[0204] SEQ ID NO:7hinge2(IgG4 hinge)

[0205]

[0206] SEQ ID NO:8CD28 TM

[0207]

[0208] SEQ ID NO:9CD28 co-stimulatory domain

[0209] SEQ ID NO:10 4-1BB co-stimulatory domain

[0210] SEQ ID NO:11mutCD28 co-stimulatory domain

[0211] SEQ ID NO:12ST1

[0212]

[0213] SEQ ID NO:13ST2

[0214]

[0215] SEQ ID NO:14TIR

[0216]

[0217] SEQ ID NO:15CD8a SP

[0218]

[0219] SEQ ID NO:16 Linker peptide

[0220] SEQ ID NO:17 Co-expressed gene OX40 amino acids

[0221] SEQ ID NO:18 Co-expressed gene C-JUN amino acids

[0222] SEQ ID NO:19 β-chain constant region

[0223] SEQ ID NO:20α chain constant region

[0224] SEQ ID NO:21CD3ε amino acid sequence

[0225] SEQ ID NO:22 Fusion protein 1 (Chimeric antigen receptor 1)

[0226]

[0227] SEQ ID NO:23 Fusion protein 2 (Chimeric antigen receptor 2)

[0228]

[0229] SEQ ID NO:24 Fusion protein 3 (Chimeric antigen receptor 3)

[0230]

[0231] SEQ ID NO:25 Fusion protein 4 (Chimeric antigen receptor 4)

[0232]

[0233] SEQ ID NO:26 Fusion protein 5 (Chimeric antigen receptor 5)

[0234]

[0235] SEQ ID NO:27 Fusion protein 6 (Chimeric antigen receptor 6)

[0236]

[0237] SEQ ID NO:28 Fusion protein 7 (Chimeric antigen receptor 7)

[0238]

[0239] SEQ ID NO:29 Fusion protein 8 (Chimeric antigen receptor 8)

[0240]

[0241] SEQ ID NO:30 Fusion protein 9 (Chimeric antigen receptor 9)

[0242]

[0243] SEQ ID NO:31 Fusion protein 10 (STAR)

[0244]

[0245] SEQ ID NO:32 Fusion protein 11 (ε-TRuC)

[0246]

[0247] SEQ ID NO:33CD7 fusion protein

[0248] SEQ ID NO:34 Fusion protein 1 gene

[0249]

[0250] SEQ ID NO:35 Fusion protein 2 gene

[0251] SEQ ID NO:36 Fusion protein 3 gene

[0252]

[0253] SEQ ID NO:37 Fusion Protein 4 Gene

[0254]

[0255] SEQ ID NO:38 Fusion protein 5 gene

[0256] SEQ ID NO:39 Fusion protein 6 gene

[0257]

[0258] SEQ ID NO:40 Fusion protein 7 gene

[0259] SEQ ID NO:41 Fusion protein 8 gene

[0260]

[0261] SEQ ID NO:42 Fusion protein 9 gene

[0262] SEQ ID NO:43 Fusion protein 10 gene

[0263]

[0264] SEQ ID NO:44 Fusion protein 11 gene

[0265] SEQ ID NO:45CD7 fusion protein gene

[0266]

[0267] SEQ ID NO:46ScFV01

[0268]

[0269] SEQ ID NO:47ScFV02

[0270]

[0271] SEQ ID NO:48ScFV03

[0272]

[0273] SEQ ID NO:49ScFV04

[0274]

[0275] SEQ ID NO:50CD8α signal peptide 2

[0276]

Claims

1. A single-chain antibody targeting CD43, wherein the following structures are sequentially linked from the N-terminus to the C-terminus: VL-Linker-VH or VH-Linker-VL; wherein, The amino acid sequence of VL is SEQ ID NO:1, and the amino acid sequence of VH is SEQ ID NO:2; The linker is selected from peptides with amino acid sequences of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5; Furthermore, when the structure of the single-chain antibody is VH-linker-VL, the linker is selected from peptides with amino acid sequences SEQ ID NO:4 and SEQ ID NO:

5.

2. The single-chain antibody according to claim 1 or 2, wherein the amino acid sequence is SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48 or SEQ ID NO:

49.

3. Nucleic acid molecules encoding the above-mentioned single-chain antibodies.

4. A fusion protein comprising a light chain variable region peptide of an antibody targeting CD43, a heavy chain variable region peptide of a single-chain antibody targeting CD43, and a transmembrane functional region; wherein the amino acid sequence of the light chain variable region peptide is SEQ ID NO:1, and the amino acid sequence of the heavy chain variable region peptide is SEQ ID NO:2; wherein, 1) The transmembrane functional region is a chimeric antigen receptor region comprising a hinge region, a transmembrane domain, a co-stimulatory domain, and an intracellular signal transduction domain. The light chain variable region peptide and the heavy chain variable region peptide are linked via a linker to form a single-chain antibody. The single-chain antibody is sequentially tandemly linked with the transmembrane domain, the co-stimulatory domain, and the intracellular signal transduction domain to form a fusion protein. Preferably, the C-terminus of the intracellular signal transduction domain is linked to a co-expressed protein via a first linker peptide. The co-expressed protein is OX40 protein or c-jun protein. The amino acid sequence of the OX40 protein is SEQ ID NO:17, and the amino acid sequence of the c-jun protein is SEQ ID NO:

18. Alternatively, 2) The transmembrane functional region includes a constant region of the TCRα subunit and a constant region of the TCRβ subunit; a first peptide chain obtained by fusing the variable region of the antibody heavy chain with the first subunit; and a second peptide chain obtained by fusing the variable region of the antibody light chain with the second subunit; wherein when the first subunit is a constant region of the TCRα chain, the second subunit is a constant region of the TCRβ chain; and when the first subunit is a constant region of the TCRβ chain, the second subunit is a constant region of the TCRα chain; preferably, it further includes a first signal peptide, a second signal peptide, and a second linker peptide, wherein the first signal peptide is linked to the N-terminus of the first peptide chain, the second linker peptide is linked to the C-terminus of the first peptide chain, the N-terminus of the second signal peptide is linked to the C-terminus of the second linker peptide, and the C-terminus of the second signal peptide is linked to the N-terminus of the second peptide chain; or, 3) The transmembrane functional region includes the CD3ε subunit. The light chain variable region peptide and the heavy chain variable region peptide are linked via a linker to form a single-chain antibody. The single-chain antibody is fused to the N-terminus of the CD3ε subunit via a third linker peptide.

5. The fusion protein of claim 4, wherein, The single-chain antibody is the single-chain antibody according to claim 1 or 2.

6. The fusion protein as described in claim 4 or 5, wherein, Each linker is independently selected from peptides with amino acid sequences SEQ ID NO:3, 4, and 5 each time it appears; and / or, The amino acid sequence of the hinge region is as shown in SEQ ID NO:6 or 7; and / or, The amino acid sequence of the transmembrane domain is shown in SEQ ID NO:8; and / or, The amino acid sequence of the co-stimulatory domain is shown in SEQ ID NO: 9, 10 or 11; and / or, The amino acid sequence of the intracellular signal transduction domain is shown in SEQ ID NO:12, 13, or 14; and / or, the first signal peptide is a CD8α signal peptide, the amino acid sequence of which is shown in SEQ ID NO:15; and / or, The second signal peptide is CD8α signal peptide 2, whose amino acid sequence is SEQ ID NO:50; and / or, The amino acid sequence of the constant region of the TCRβ chain is SEQ ID NO:19; and / or, The amino acid sequence of the constant region of the TCRα chain is SEQ ID NO:20; and / or, The first linker peptide, the second linker peptide, and the third linker peptide are each independently selected from peptides with amino acid sequences of SEQ ID NO: 3, 4, and 5; and / or, The amino acid sequence of the CD3ε subunit is SEQ ID NO:

21.

7. The fusion protein according to any one of claims 4-6, wherein the amino acid sequence is SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 or SEQ ID NO:

32.

8. The use of the single-chain antibody as described in claim 1 or 2, or the fusion protein as described in any one of claims 4-6, in the preparation of CD43-targeting immune T cells.

9. A nucleic acid molecule encoding a fusion protein as described in any one of claims 4-6; preferably, selected from nucleic acid molecules with nucleotide sequences of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 or SEQ ID NO:

45.

10. A vector comprising a nucleic acid molecule as described in claim 3 or 9; said vector being selected from one or more of DNA vectors, RNA vectors, plasmids, lentiviral vectors, adenovirus vectors, and retroviral vectors.

11. Engineered immune cells, wherein the immune cells contain the vector of claim 10, or have an exogenous nucleic acid molecule of claim 9 integrated into a chromosome, or express a fusion protein of any one of claims 4-6.

12. A formulation comprising a single-chain antibody according to any one of claims 1 or 2, a fusion protein according to any one of claims 4-6, or an engineered immune cell according to claim 11, and a pharmaceutically acceptable carrier, diluent, or excipient.

13. Use of a fusion protein according to any one of claims 4-6, or an engineered immune cell according to claim 11, in the preparation of a medicament or formulation for treating cancer or tumors.

14. The use as described in claim 13, wherein, The cancer or tumor mentioned is selected from adenoid cystic carcinoma (ACC), diffuse large B-cell lymphoma (DLBCL), myeloid sarcoma, blastic plasmacytoid dendritic cell neoplasm (BPDCN), T-cell lymphoma, and T-cellacute lymphoblastic leukemia (T-ALL).

Images