CAR-T cell targeting SLC7A11

Abstract

The invention relates to a CAR-T (Chimeric Antigen Receptor-T) cell targeting SLC7A11.

Description

Technical Field

[0001] This invention relates to CAR-T cells targeting SLC7A11. Background Technology

[0002] Cancer is a major disease threatening human life and health, ranking second only to cardiovascular disease as the leading cause of death. Current treatments for cancer include surgery, chemotherapy, targeted therapy, immune checkpoint inhibitor therapy, and chimeric antigen receptor-T-cell (CAR-T) therapy, which have made significant progress in breast cancer, lung cancer, and melanoma. However, for breast cancer, colorectal cancer, lung cancer, and pancreatic cancer with distant metastases or those initially unresectable, treatment needs remain highly unmet. For these patients, treatment options and strategies, including large molecule targeted drugs such as monoclonal antibodies targeting novel targets and the combination of these monoclonal antibodies with traditional chemotherapy and immunotherapy drugs, offer new choices and strategies to address this enormous unmet clinical need.

[0003] SLC7A11 (Solute Carrier Family 7 Member 11, also known as xCT) is the 11th member of the 7th family of solute transporters, belonging to the cysteine / glutamate antitransporter group. It primarily participates in the transport of cysteine ​​and glutamate on the cell membrane. The human SLC7A11 gene is located on chromosome 4q28.3, has 14 exons, and encodes a protein composed of 501 amino acids, including 12 transmembrane domains. Its N-terminus and C-terminus are both located in the cytoplasm. SLC7A11, as the light chain subunit, together with SLC3A2, as the heavy chain subunit, constitutes the cysteine / glutamate antitransporter (Xc-system), but SLC7A11 performs the main transport function. Recent domestic and international studies have shown that SLC7A11 is highly expressed in various solid malignant tumors, such as colon cancer, rectal cancer, breast cancer, pancreatic cancer, ovarian cancer, and glioma, and is closely related to drug resistance in malignant tumors. SLC7A11 mediates cysteine ​​transport and regulates intracellular cysteine ​​concentration and glutathione (GSH) levels. GSH has antioxidant and detoxification functions; the sulfhydryl group on cysteine ​​is its active group (hence often abbreviated as G-SH), which readily binds to certain drugs and toxins, thus enabling its detoxification effect. Recent studies have found that downregulation of SLC7A11 can indirectly inhibit GPX4 activity by inhibiting the cysteine ​​metabolic pathway, leading to decreased intracellular cysteine ​​levels and depletion of GSH biosynthesis, thereby causing lipid peroxide accumulation and ultimately inducing ferroptosis. In previous studies, small molecule inhibitors of SLC7A11, including Erastin, IKE, and SAS, have been found to have significant inhibitory effects on various tumor cell lines that highly express this gene.

[0004] However, due to the multiple transmembrane nature of SLC7A11, the extracellular segments of this protein are short (the lengths of the six extracellular segments are 10, 13, 24, 31, 2, and 6 amino acids, respectively), making the preparation of monoclonal antibodies difficult. Currently, there is a lack of effective antibodies (especially humanized antibodies) and chimeric antigen receptors (and related cells) targeting human SLC7A11 in this field.

[0005] This invention obtained a humanized anti-human SLC7A11 monoclonal antibody by humanization based on a murine anti-human SLC7A11 monoclonal antibody, and measured the anti-tumor effects of CAR-T cells constructed with the above antibody on human colon cancer, pancreatic cancer and breast cancer cell lines in vivo and in vitro. Summary of the Invention

[0006] On one hand, the present invention provides a murine antibody or a humanized antibody, which comprises at least one, at least two, or all three of the following: CDR-L1 as shown in SEQ ID NO: 1, CDR-L2 as shown in SEQ ID NO: 2, and CDR-L3 as shown in SEQ ID NO: 3.

[0007] On one hand, the present invention provides a murine antibody or a humanized antibody, which comprises at least one, at least two, or all three of the following: CDR-H1 as shown in SEQ ID NO: 4, CDR-H2 as shown in SEQ ID NO: 5, and CDR-H3 as shown in SEQ ID NO: 6.

[0008] On one hand, the present invention provides a murine antibody or a humanized antibody, which comprises at least one, at least two, at least three, at least four, at least five, or all six of the following: CDR-L1 as shown in SEQ ID NO: 1, CDR-L2 as shown in SEQ ID NO: 2, CDR-L3 as shown in SEQ ID NO: 3, CDR-H1 as shown in SEQ ID NO: 4, CDR-H2 as shown in SEQ ID NO: 5, and CDR-H3 as shown in SEQ ID NO: 6.

[0009] In one embodiment, the murine antibody of the present invention comprises at least one, at least two, at least three, or all four of FR-L1, FR-L2, FR-L3, and FR-L4 contained in VL as shown in SEQ ID NO: 15.

[0010] In one embodiment, the murine antibody of the present invention comprises at least one, at least two, at least three, or all four of FR-H1, FR-H2, FR-H3, and FR-H4 contained in VH as shown in SEQ ID NO: 16.

[0011] In one embodiment, the murine antibody of the present invention comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or all eight of the following: FR-L1, FR-L2, FR-L3, and FR-L4 contained in VL as shown in SEQ ID NO: 15, and FR-H1, FR-H2, FR-H3, and FR-H4 contained in VH as shown in SEQ ID NO: 16.

[0012] In one embodiment, the murine antibody of the present invention comprises VL as shown in SEQ ID NO: 15, and / or VH as shown in SEQ ID NO: 16.

[0013] In one embodiment, the humanized antibody of the present invention comprises: a VL frame region derived from V gene IGKV2-28*014 and J gene IGKJ2*01, and / or a VH frame region derived from V gene IGHV4-59*01 and J gene IGHJ6*01.

[0014] In one embodiment, the humanized antibody of the present invention comprises at least one, at least two, at least three, or all four of FR-L1 as shown in SEQ ID NO: 7, FR-L2 as shown in SEQ ID NO: 8, FR-L3 as shown in SEQ ID NO: 9, and FR-L4 as shown in SEQ ID NO: 10.

[0015] In one embodiment, the humanized antibody of the present invention comprises at least one, at least two, at least three, or all four of FR-H1 as shown in SEQ ID NO: 11, FR-H2 as shown in SEQ ID NO: 12, FR-H3 as shown in SEQ ID NO: 13, and FR-H4 as shown in SEQ ID NO: 14.

[0016] In one embodiment, the humanized antibody of the present invention comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or all eight of the following: FR-L1 as shown in SEQ ID NO: 7, FR-L2 as shown in SEQ ID NO: 8, FR-L3 as shown in SEQ ID NO: 9, FR-L4 as shown in SEQ ID NO: 10, FR-H1 as shown in SEQ ID NO: 11, FR-H2 as shown in SEQ ID NO: 12, FR-H3 as shown in SEQ ID NO: 13, and FR-H4 as shown in SEQ ID NO: 14.

[0017] In one embodiment, the humanized antibody of the present invention comprises: VL as shown in SEQ ID NO: 17, and / or VH as shown in SEQ ID NO: 18.

[0018] In one embodiment, the humanized antibody of the present invention (e.g., a humanized antibody comprising VL as shown in SEQ ID NO: 17 and VH as shown in SEQ ID NO: 18) comprises amino acid substitutions at one or more positions selected from the group consisting of:

[0019] (1) L7, L8, L9, L11, L15, L17, L18, L64, L74 and L100 in VL; and / or

[0020] (2) H1, H16, H17, H25, H40, H43, H44, H48, H67, H68, H70, H71, H79, H81, H82a, H84, H85, H89, H108 and H112 in VH.

[0021] In one embodiment, the amino acid substitution is:

[0022] (1) S7A, P8A, L9F, L11N, P15L, E17T, P18S, G64S, K74R and Q100G in VL; and / or

[0023] (2) Q1D, E16Q, T17S, S25T, P40F, K43N, G44K, I48M, V67I, T68S, S70T, V71R, S79F, K81Q, S82aN, A84T, A85E, V89T, T108L and S112A in VH,

[0024] In one embodiment, the humanized antibody of the present invention (e.g., a humanized antibody comprising VL as shown in SEQ ID NO: 17 and VH as shown in SEQ ID NO: 18) comprises one or more amino acid substitutions selected from the group consisting of:

[0025] (1) K74R in VL; and / or

[0026] (2) T17S, S25T, K43N, I48M, V67I, S70T, V71R, S79F, A84T and V89T in VH.

[0027] In one embodiment, the humanized antibody of the present invention (e.g., a humanized antibody comprising VL as shown in SEQ ID NO: 17 and VH as shown in SEQ ID NO: 18) comprises the following amino acid substitutions:

[0028] (1) K74R in VL; and

[0029] (2) T17S, S25T, K43N, I48M, V67I, S70T, V71R, S79F, A84T and V89T in VH.

[0030] In one embodiment, the humanized antibody of the present invention comprises: VL as shown in SEQ ID NO: 19, and / or VH as shown in SEQ ID NO: 20.

[0031] In one embodiment, the murine antibody and humanized antibody of the present invention are antibody fragments, such as antigen-binding fragments, such as scFv, Fab, Fab', F(ab')2, scFab, xFab (i.e., cross-Fab, wherein either the variable domain or constant region of the light chain and the heavy chain is exchanged) or Fd.

[0032] In one embodiment, the murine antibody and humanized antibody of the present invention are scFv. In one embodiment, VL and VH are linked via a peptide linker. In one embodiment, VL is at the N-terminus and VH is at the C-terminus. In one embodiment, VH is at the N-terminus and VL is at the C-terminus.

[0033] In one embodiment, the murine antibody and humanized antibody of the present invention are scFab. In one embodiment, the Fab light chain and the Fab heavy chain are linked via a peptide linker. In one embodiment, the Fab light chain is at the N-terminus and the Fab heavy chain is at the C-terminus. In one embodiment, the Fab heavy chain is at the N-terminus and the Fab light chain is at the C-terminus.

[0034] In one embodiment, a peptide linker refers to an oligopeptide or polypeptide region with a length of about 2 to 100, for example, about 5 to 50, for example, about 10 to 25, for example, about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In one embodiment, the linker may include or be composed of flexible residues (such as glycine and serine) that allow adjacent protein domains to move freely relative to each other. Longer linkers may be used when it is desirable to ensure that two adjacent domains do not spatially interfere with each other. In one embodiment, the peptide linker is (GS4). n (S4G) n (GS4) n G or G (S4G) n , where n is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the peptide linker is as shown in SEQ ID NO: 30.

[0035] In one embodiment, the murine antibody and humanized antibody of the present invention are full-length antibodies. In one embodiment, the humanized antibody of the present invention has a κ or λ light chain. In one embodiment, the humanized antibody of the present invention has an IgA, IgD, IgE, IgG, or IgM heavy chain. In one embodiment, the humanized antibody of the present invention has an IgG1, IgG2, IgG, or IgG4 heavy chain.

[0036] In one embodiment, the humanized antibody of the present invention comprises a constant region of the human κ light chain as shown in SEQ ID NO: 21.

[0037] In one embodiment, the humanized antibody of the present invention comprises a human IgG1 heavy chain constant region as shown in SEQ ID NO: 22. In one embodiment, the humanized antibody of the present invention comprises a human IgG2 heavy chain constant region as shown in SEQ ID NO: 23.

[0038] In one embodiment, the murine antibody and humanized antibody of the present invention have one or more of the following characteristics:

[0039] (1) Combined with SLC7A11;

[0040] (2) Binding to the SLC7A11 / SLC3A2 complex;

[0041] (3) Inhibits the proliferation of cancer cells in vitro, such as colon cancer, breast cancer, or pancreatic cancer;

[0042] (4) Inducing apoptosis in cancer cells in vitro, such as colon cancer, breast cancer, or pancreatic cancer;

[0043] (5) Inhibits CD4+ T cell proliferation;

[0044] (6) Inhibits the differentiation of CD4+ T cells into Treg cells;

[0045] (7) Inhibits tumor growth in vivo, such as in colon cancer; and / or

[0046] (8) Eliminate tumors in the body, such as colon cancer.

[0047] On one hand, the present invention provides a chimeric antigen receptor polypeptide comprising: an optional signal peptide, an extracellular antigen-binding domain, a hinge region, a transmembrane region, an intracellular signal domain, and an optional co-stimulatory domain.

[0048] In one embodiment, the signal peptide is derived from human CD8α, as shown in a further embodiment, such as SEQ ID NO: 28. In one embodiment, the signal peptide is derived from CSF2Rα or IL-2. In one embodiment, the signal peptide is derived from the molecular origin of other components of the CAR described herein.

[0049] In one embodiment, the extracellular antigen-binding domain binds to SLC7A11. In one embodiment, the extracellular antigen-binding domain is a murine or humanized antibody of the present invention, particularly scFv.

[0050] In one embodiment, the hinge region is derived from human CD8α, and in a further embodiment, as shown in SEQ ID NO: 24. In one embodiment, the hinge region is derived from CD8β, CD4, CD28, and CD7.

[0051] In one embodiment, the transmembrane region is derived from human CD8α. In a further embodiment, as shown in SEQ ID NO: 25, the transmembrane region is derived from the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8β, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), 4-1BBL, GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2Rβ, IL2Rγ, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITG AX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD1 50, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D or NKG2C.

[0052] In one embodiment, the intracellular signaling domain is derived from human CD3ζ, and in a further embodiment, as shown in SEQ ID NO: 27. In one embodiment, the intracellular signaling domain is derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3θ, CD3δ, CD3ε, CD22, CD79a, CD79b, or CD66d.

[0053] In one embodiment, the co-stimulatory domain is derived from human 4-1BB, and in a further embodiment, as shown in SEQ ID NO: 26. In one embodiment, the co-stimulatory domain is derived from one or more (e.g., two) of MHC class I molecules, BTLA, Toll-like receptors, CD27, CD28, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. In one implementation, the co-stimulatory domain is derived from one or more (e.g., two) of CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM, and ZAP70.

[0054] On the other hand, the present invention provides a nucleic acid that encodes the humanized antibody or chimeric antigen receptor polypeptide of the present invention.

[0055] On the other hand, the present invention provides a vector comprising the nucleic acid of the present invention. In one embodiment, the vector is a cloning vector or an expression vector.

[0056] On the other hand, the present invention provides a cell containing the nucleic acid or vector of the present invention, or expressing the humanized antibody or chimeric antigen receptor polypeptide of the present invention.

[0057] On the other hand, the present invention provides a cell that displays the chimeric antigen receptor polypeptide of the present invention on its cell membrane.

[0058] On the other hand, the present invention provides a method for generating humanized antibodies or chimeric antigen receptor peptides, comprising culturing the cells of the present invention under conditions suitable for the expression of humanized antibodies or chimeric antigen receptor peptides, thereby generating the humanized antibodies or chimeric antigen receptor peptides. In one embodiment, the method further includes recovering the humanized antibodies or chimeric antigen receptor peptides. In one embodiment, the method further includes purifying the humanized antibodies or chimeric antigen receptor peptides.

[0059] On the other hand, the present invention provides a humanized antibody or chimeric antigen receptor polypeptide, which is generated by the method of the present invention.

[0060] On the other hand, the present invention provides a method for generating cells in which a chimeric antigen receptor polypeptide is integrated on the cell membrane, comprising culturing the cells of the present invention under conditions suitable for the integration of the chimeric antigen receptor polypeptide on the cell membrane, thereby enabling the integration of the chimeric antigen receptor polypeptide on the cell.

[0061] On the other hand, the present invention provides a cell in which a chimeric antigen receptor polypeptide is integrated on the cell membrane, which is generated by the method of the present invention.

[0062] In one embodiment, the cell is a prokaryotic or eukaryotic cell. In one embodiment, the cell is a bacterial, fungal, or mammalian cell. In one embodiment, the cell is an *Escherichia coli* cell. In one embodiment, the cell is a *Saccharomyces cerevisiae* cell. In one embodiment, the cell is a CHO cell. In one embodiment, the cell is an immune cell. In one embodiment, the cell is a lymphocyte, such as B cells and T cells; a natural killer cell, such as NK cells or NKT cells; or a myeloid cell, such as a monocyte, macrophage, eosinophil, mast cell, basophil, or granulocyte. In one embodiment, the cell is a T cell, NK cell, or macrophage. In one embodiment, the cell is a T effector (Teff) cell.

[0063] On the other hand, the present invention provides a composition comprising the humanized antibody, chimeric antigen receptor polypeptide, nucleic acid, vector, or cell of the present invention. In one embodiment, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0064] On the other hand, the present invention provides humanized antibodies, chimeric antigen receptor peptides, nucleic acids, vectors, cells or compositions thereof, which are used as pharmaceuticals.

[0065] On the other hand, the present invention provides humanized antibodies, chimeric antigen receptor peptides, nucleic acids, vectors, cells or compositions thereof for treating cancer (in subjects) or delaying cancer progression.

[0066] On the other hand, the present invention provides humanized antibodies, chimeric antigen receptor peptides, nucleic acids, vectors, cells or compositions thereof for use in the preparation of medicaments for treating (in subjects) cancer or delaying cancer progression.

[0067] On the other hand, the present invention provides a method for treating cancer (in a subject) or delaying cancer progression, comprising administering to a subject suffering from cancer a humanized antibody, chimeric antigen receptor polypeptide, nucleic acid, vector, cell or composition of the present invention.

[0068] On the other hand, the present invention provides the use of the humanized antibodies, chimeric antigen receptor peptides, nucleic acids, carriers, cells or compositions of the present invention in the preparation of medicaments for treating (in subjects) cancer or delaying cancer progression.

[0069] On the other hand, the present invention provides the use of the humanized antibodies, chimeric antigen receptor peptides, nucleic acids, vectors, cells or compositions of the present invention for treating cancer (in subjects) or delaying cancer progression.

[0070] In one implementation, the cancer is colon cancer, breast cancer, or pancreatic cancer. In one implementation, the subject is a human being. Attached Figure Description

[0071] Figure 1 This shows a sequence alignment of the light chain variable domain of the humanized antibody and the mouse antibody of the present invention.

[0072] Figure 2 This shows a sequence alignment of the heavy chain variable domain of the humanized antibody and the mouse antibody of the present invention.

[0073] Figure 3 This demonstrates the effect of the humanized antibody of the present invention on the cell activity of tumor cells (**, P<0.01; ***, P<0.001).

[0074] Figure 4 This demonstrates the effect of the humanized antibody of the present invention on apoptosis of tumor cells (***, P<0.001).

[0075] Figure 5 This demonstrates the effect of the humanized antibody of the present invention on T cells (**, P<0.01).

[0076] Figure 6 This demonstrates the effect of the humanized antibody of the present invention on tumor volume in mice (***, P<0.001).

[0077] Figure 7 This demonstrates the effect of the humanized antibody of the present invention on tumor weight in mice (***, P<0.001).

[0078] Figure 8 This shows the flow cytometry results of the humanized CAR-T cells of the present invention.

[0079] Figure 9 The results of qPCR for humanized CAR-T cells of this invention are shown (****, P<0.0001).

[0080] Figure 10 This demonstrates the effect of the humanized CAR-T cells of the present invention on the activity of DLD1 colon cancer cells (****, P<0.0001).

[0081] Figure 11 This demonstrates the effect of the humanized CAR-T cells of the present invention on the activity of HT29 colon cancer cells (***, P<0.001; ****, P<0.0001).

[0082] Figure 12 The curve showing the tumor volume change in a single mouse in the control group is displayed.

[0083] Figure 13 The curve showing the tumor volume change in a single mouse in the experimental group is displayed.

[0084] Figure 14 This demonstrates the effect of the humanized CAR-T cells of the present invention on tumor volume in mice (**, P<0.01).

[0085] Figure 15 This demonstrates the effect of the humanized CAR-T cells of this invention on the body weight of mice.

[0086] Figure 16 This demonstrates the effect of the humanized CAR-T cells of this invention on tumor weight in mice (**, P<0.01).

[0087] Figure 17 This shows the flow cytometry results of the mouse-derived CAR-T cells of the present invention.

[0088] Figure 18 The results of qPCR for the murine CAR-T cells of this invention are shown (****, P<0.0001).

[0089] Figure 19 This demonstrates the effect of the murine CAR-T cells of the present invention on the lysis of DLD1 colon cancer cells (***, P<0.001).

[0090] Figure 20 This demonstrates the effect of the murine CAR-T cells of the present invention on the lysis of HT29 colon cancer cells (***, P<0.001).

[0091] Figure 21This demonstrates the effect of the murine CAR-T cells of the present invention on tumor volume in mice (**, P<0.01).

[0092] Figure 22 This invention demonstrates the effect of the mouse-derived CAR-T cells of the present invention on the body weight of mice. Detailed Implementation

[0093] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Specific details not specified in the examples are performed under conventional conditions in the art or under conditions recommended by the manufacturer.

[0094] Example 1: Humanized antibody against SLC7A11

[0095] 1. Humanization of antibodies

[0096] Murine antibodies can trigger a strong immune response in the human body, producing human anti-mouse antibodies (HAMA), which affects the safety and therapeutic efficacy of murine antibodies in clinical applications. Antibody humanization allows non-human antibodies to have sequences similar to human antibodies, thereby evading recognition by the immune system and avoiding the induction of a HAMA response.

[0097] Compared with parental non-human antibodies, humanized antibodies generated through CDR transplantation may exhibit alterations in properties such as activity and affinity. To effectively maintain the activity and affinity of parental non-human antibodies, potential reversion mutation sites are selected based on antibody structure modeling. The amino acids at these sites in the humanized antibodies generated through CDR transplantation are then reverted to the amino acids at the corresponding sites in the non-human antibodies. The selection principles for reversion mutation sites are as follows: (1) amino acid residues located at the VH-VL interface that have a significant impact on the conformation of VH and VL; (2) amino acid residues in the FR that are close to the CDR, directly interact with the CDR, and provide support for the CDR loop; (3) considering immunogenicity, amino acid residues embedded within the protein should be selected as much as possible.

[0098] Humanization and sequence optimization of the mouse monoclonal antibody BDF12-M (see CN115490769A) were performed using CDR substitution and computer-aided structural simulation, as detailed below.

[0099] (1) Determination of CDR

[0100] The CDR of antibodies has several definitions in this field, which are summarized in Table 1.

[0101] Table 1: Definition of Antibody CDR

[0102] Loop Kabat AbM Chothia Contact IMGT L1 L24-L34 L24-L34 L24-L34 L30-L36 L27-L32 L2 L50-L56 L50-L56 L50-L56 L46-L55 L50-L51 L3 L89-L97 L89-L97 L89-L97 L89-L96 L89-L97 H1 H31-H35B H26-H35B H26-H32..H34 H30-H35B H26-H35B H2 H50-H65 H50-H58 H52-H56 H47-H58 H51-H56 H3 H95-H102 H95-H102 H95-H102 H93-H101 H93-H102

[0103] This invention uses the Kabat definition of antibody CDR. The CDR of BDF12-M is shown in Table 2.

[0104] Table 2: CDR of murine antibodies

[0105] CDR sequence SEQ ID NO CDR-L1 RSSESLLHTNGITYLY 1 CDR-L2 QMSNLAS 2 CDR-L3 AQNLQLPYT 3 CDR-H1 SDYAWN 4 CDR-H2 YISYSGTISYNPSLTS 5 CDR-H3 WRFDGAWFSY 6

[0106] (2) Porting of CDR

[0107] The heavy and light chain variable domain sequences of BDF12-M were queried in the human antibody germline database IGKV. Human antibody sequences with the highest homology were selected to provide the frame regions (FRs), namely the V gene IGKV2-28*014 and the J gene IGKJ2*01 for the light chain, and the V gene IGHV4-59*01 and the J gene IGHJ6*01 for the heavy chain. The complementarity-determining region (CDR) of the murine antibody BDF12-M, as defined by Kabat, was transplanted onto the selected human antibody frame regions (FRs), forming primary humanized antibody heavy and light chain variable domains composed of the murine CDR and the human FR.

[0108] Human FRs used for transplanting mouse CDRs are shown in Table 3.

[0109] Table 3: Human FRs used for mouse-derived CDR transplantation

[0110] FR sequence SEQ ID NO FR-L1 DIVMTQSPLSLPVTPGEPASISC 7 FR-L2 WYLQKPGQSPQLLIY 8 FR-L3 GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC 9 FR-L4 FGQGTKLEIK 10 FR-H1 QVQLQESGPGLVKPSETLSLTCTVSGYSIT 11 FR-H2 WIRQPPGKGLEWIG 12 FR-H3 RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR 13 FR-H4 WGQGTTVTVSS 14

[0111] (3) Reversion mutation

[0112] Sequence alignment of the light chain variable domain of the primary humanized antibody generated via CDR transplantation with the mouse antibody BDF12-M showed that... Figure 1 Sequence alignment showed that BDF12-M and IGKV2-28*01 had high homology in the light chain variable domain frame region, differing by only 10 amino acids (see Table 4), mainly concentrated in FR1, while FR2 was completely identical. A 3D structural model of the murine antibody was established using computer-aided simulation, and reversion mutation sites were selected according to the principles described above. One amino acid (L74) in the light chain variable domain of the primary humanized antibody was reverted to the corresponding amino acid in the murine antibody, resulting in the optimized light chain variable domain sequence of the humanized antibody, named BDF12-L-7-1 (see Table 4).

[0113] Table 4: Sequence alignment of light chain variable domains between murine and humanized antibodies (positions of light chain variable domains are based on Kabat numbering).

[0114]

[0115] Similarly, sequence alignment of the heavy chain variable domain of the primary humanized antibody generated via CDR transplantation with the mouse antibody BDF12-M showed... Figure 2 Sequence alignment revealed that BDF12-M differs from IGHV4-59*01 by 20 amino acids in the heavy chain variable domain frame region (see Table 5). A 3D structural model of the murine antibody was constructed using computer-aided simulation, and reversion mutation sites were selected according to the principles described above. Ten amino acids in the heavy chain variable domain of the primary humanized antibody were reverted to the corresponding amino acids in the murine antibody, resulting in the optimized heavy chain variable domain sequence of the humanized antibody, named BDF12-H-17-2 (see Table 5).

[0116] Table 5: Sequence alignment of heavy chain variable domains between murine and humanized antibodies (heavy chain variable domain positions are based on Kabat numbering).

[0117]

[0118] Mouse antibody light and heavy chain variable domain amino acid sequences:

[0119] Light chain variable domain, BDF12-LM:

[0120] DIVMTQAAFSNPVTLGTSASISCRSSESLLHTNGITYLYWYLQKPGQSPQL

[0121] LIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLQLPY

[0122] TFGGGTKLEIK

[0123] (SEQ ID NO: 15)

[0124] Heavy chain variable domain, BDF12-HM:

[0125] DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMG

[0126] YISYSGTISYNPSLTSRISITRDTSKNQFFLQLNSVTTEDTATYYCARWRFD

[0127] GAWFSYWGQGTLVTVSA

[0128] (SEQ ID NO: 16)

[0129] The light and heavy chain variable domain amino acid sequences of humanized antibodies generated by CDR transplantation:

[0130] Light chain variable domain, BDF12-LG:

[0131] DIVMTQSPLSLPVTPGEPASISCRSSESLLHTNGITYLYWYLQKPGQSPQLL

[0132] IYQMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLQLPYT

[0133] FGQGTKLEIK

[0134] (SEQ ID NO: 17)

[0135] Heavy chain variable domain, BDF12-HG:

[0136] QVQLQESGPGLVKPSETLSLTCTVSGYSITSDYAWNWIRQPPGKGLEWIG

[0137] YISYSGTISYNPSLTSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARWRFD

[0138] GAWFSYWGQGTTVTVSS

[0139] (SEQ ID NO: 18)

[0140] The reversal mutation of the humanized antibody light and heavy chain variable domain amino acid sequences:

[0141] Light chain variable domain, BDF12-L-7-1:

[0142] DIVMTQSPLSLPVTPGEPASISCRSSESLLHTNGITYLYWYLQKPGQSPQLL

[0143] IYQMSNLASGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCAQNLQLPYT

[0144] FGQGTKLEIK

[0145] (SEQ ID NO: 19)

[0146] Heavy chain variable domain, BDF12-H-17-2:

[0147] QVQLQESGPGLVKPSESLSLTCTVTGYSITSDYAWNWIRQPPGNGLEWMG

[0148] YISYSGTISYNPSLTSRITITRDTSKNQFFLKLSSVTTADTATYYCARWRFD

[0149] GAWFSYWGQGTTVTVSS

[0150] (SEQ ID NO: 20)

[0151] (4) Construction of full-length antibody

[0152] The final humanized VH sequence BDF12-H-17-2 was combined with the constant region sequences of human heavy chain IgG1 and IgG2, respectively. The optimized humanized VL sequence BDF12-L-7-1 was combined with the constant region sequence of human light chain κ to obtain the full-length humanized antibody sequences, which were named BDF12-H17-2L7-1 (IgG1 / κ) and BDF12-H17-2L7-2 (IgG2 / κ), respectively.

[0153] Human light chain κ constant region sequence:

[0154] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG

[0155] NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK

[0156] SFNRGEC

[0157] (SEQ ID NO: 21)

[0158] Human heavy chain IgG1 constant region sequence:

[0159] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV

[0160] HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPK

[0161] SCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE

[0162] DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG

[0163] KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTC

[0164] LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW

[0165] QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0166] (SEQ ID NO: 22)

[0167] Human heavy chain IgG2 constant region sequence:

[0168] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH

[0169] TFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERK

[0170] CCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV

[0171] QFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKC

[0172] KVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF

[0173] YPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN

[0174] VFSCSVMHEALHNHYTQKSLSLSPGK

[0175] (SEQ ID NO: 23)

[0176] 2. Binding activity of the humanized antibody

[0177] The SLC3A2 (Uniprot, P08195-2; Sinopharm, 0.5 mg / mL) used as antigen and its complex with SLC7A11 (Uniprot, Q9UPY5; Sinopharm, 0.22 mg / mL) (the above antigens were dissolved in HEPES, Thermo Fisher, 15630130) were diluted to 5 μg / mL with diluent (0.1% BSA-PBST; BSA: Sigma-Aldrich, A3311; PBST: Thermo Fisher, 28352) and used to coat 96-well ELISA plates (Nex, 514201), 100 μL / well, overnight at 4°C. The following day, block with blocking buffer (2% BSA-PBST), 300 μL / well, at room temperature for 1 hour; wash twice with washing buffer (PBST pH 7.4, ThermoFisher, 28352), 300 μL / well each time; dilute the mouse antibody and humanized antibody used as primary antibodies to 1 μg / mL with diluent, add 100 μL of the corresponding primary antibody dilution to each well, and incubate at room temperature for 2 hours; wash three times with washing buffer, 300 μL / well each time; rabbit anti-mouse IgG used as secondary antibody... F(ab)2 / HRP (Jackson, 315035047), goat anti-mouse IgG Fc / HRP (Jackson, 115035071), and goat anti-human IgG (H+L) / HRP (Jackson, 109-036-088) were diluted to the working concentration. 100 μL of the corresponding secondary antibody dilution buffer was added to each well, and the mixture was incubated at room temperature for 1 hour. The wells were washed 3 times, 300 μL each time. 200 μL of chromogenic solution A and chromogenic solution B (Beyotime, P0209) were mixed at a 1:1 ratio and added to each well. The mixture was incubated at room temperature in the dark for 20 minutes. 50 μL of stop solution (Lianke Biotechnology, E0301) was added to each well, and the mixture was mixed. The OD value was immediately measured at 450 nm using a BioTek Synergy H2 microplate reader.

[0178] Table 6 summarizes the binding of murine antibodies to antigens. Table 7 summarizes the binding of humanized antibodies to antigens. ELISA results show that murine antibodies have weak binding abilities to both SLC3A2 and the SLC7A11 / SLC3A2 complex. The final humanized antibody obtained through CDR transplantation and reversion mutation showed improved binding to both SLC3A2 and the SLC7A11 / SLC3A2 complex compared to the murine antibody.

[0179] Table 6: Binding of murine antibodies to antigens (OD) 450nm )

[0180]

[0181] Table 7: Binding of humanized antibodies to antigens (OD)450nm )

[0182]

[0183] 3. In vitro antitumor activity of humanized antibodies

[0184] The inhibitory effect of the full-length humanized antibody on cancer cells was determined using the CCK8 (DOJINDO, CK04) cell viability assay kit. Colon cancer cell lines DLD1 (ATCC, CCL-221) and LOVO (ATCC, CCL-229), breast cancer cell lines MCF7 (ATCC, HTB-22) and MDA-MB-231 (ATCC, CRM-HTB-26), and pancreatic cancer cell lines PANC-1 (ATCC, CRL-1469) and MIA-PaCa-2 (ATCC, CRL-1420) were resuspended in DMEM complete medium (Gibco, 11995065) and seeded into 96-well plates (Korning, 3599), 100 μL per well, 1×10⁻⁶. 4 Cells were cultured at 37°C in a 5% CO2 incubator for 24 hours. Cancer cell viability was then measured using a kit as a baseline. 1 μL of the corresponding full-length humanized antibody (final concentration 100 μg / mL) was added to each well of the experimental groups (IgG1 and IgG2 groups), while an equal volume of PBS (Gibco, 10010023) was added to the control group. Cancer cell viability was measured every 12 hours, and cell proliferation curves were plotted. Figure 3 The results are shown in Table 8. The results indicate that the full-length humanized antibody can significantly inhibit the growth of colon cancer, breast cancer, and pancreatic cancer cells.

[0185] Table 8: Inhibition of cancer cells by humanized antibodies

[0186] Inhibition rate (%) LOVO DLD1 MDA-MB-231 MCF7 PANC1 MIA PaCa-2 IgG1 group 38.46 40.37 24.43 37.57 44.91 33.05 IgG2 group 28.84 49.64 33.90 31.08 32.84 49.91

[0187] The killing effect of the full-length humanized antibody on cancer cells was determined using the Annexin-V and PI apoptosis kit (Biolegend, 640932). Colon cancer cell lines DLD1 (ATCC, CCL-221) and LOVO (ATCC, CCL-229), breast cancer cell lines MCF7 (ATCC, HTB-22) and MDA-MB-231 (ATCC, CRM-HTB-26), and pancreatic cancer cell lines PANC-1 (ATCC, CRL-1469) and MIA-PaCa-2 (ATCC, CRL-1420) were resuspended in DMEM complete medium (Gibco, 11995065) and seeded into 6-well plates (Korning, 3516), 1 mL per well, 3 × 10⁻⁶. 5Cells were cultured at 37°C in a 5% CO2 incubator for 24 hours. The experimental groups (IgG1 and IgG2 groups) were treated with 10 μL of their respective full-length humanized antibodies (final concentration 100 μg / mL), while the control group received an equal volume of PBS. After 48 hours of further culture, the apoptosis rate of cancer cells was measured using a kit. The results showed... Figure 4 See Table 9. Compared with the control group, the experimental groups all had a higher apoptosis rate, indicating that the full-length humanized antibody can significantly kill tumor cells.

[0188] Table 9: Induction of apoptosis in cancer cells by humanized antibodies

[0189] Apoptosis rate (%) LOVO DLD1 MDA-MB-231 MCF7 PANC1 MIA PaCa-2 control group 9.05 3.89 6.39 5.24 7.86 11.14 IgG1 group 31.17 15.51 22.86 15.85 23.53 29.36 IgG2 group 29.34 14.42 22.46 16.41 23.25 31.33

[0190] 4. In vitro inhibitory effect of humanized antibodies on CD4 T cells

[0191] With informed consent, fresh peripheral blood was obtained from healthy volunteers. Human lymphocyte separation medium (Dayou, 7111012) was added, and peripheral blood mononuclear cells (PBMCs) were separated by centrifugation at 800g for 30 minutes. Human CD4+ was used. + T-cell magnetic beads (Stemcell, 17852) sorting CD4 + T cells were resuspended in T cell culture medium (Stemcell, 10981) to 10... 6 Cells were cultured at 37°C in a 5% CO2 incubator, with IL-2 (10 ng / mL, Stemcell, 78036) and CD3 / CD28 (25 μL / mL, Stemcell, 10971) added. After 48 hours of culture, CD4+ was... + T cells were seeded in 24-well plates (Corning, 3524), 1 ml per well, 1×10⁶ cells / well. 6 Cells were cultured for 48 hours. Experimental groups (IgG1 and IgG2 groups) received 10 μL of the corresponding full-length humanized antibody (final concentration 100 μg / mL), while the control group received an equal volume of PBS (Gibco, 10010023). After culturing in a cell incubator for 48 hours, CD4+ was detected using flow cytometry (Biolegend, 320114 and 302604). + Changes in the number of T cells and their conversion to Treg (FOXP3) + CD25 + The differentiation status of cells. The results showed... Figure 5 The results indicate that the full-length humanized antibody can significantly inhibit the proliferation of CD4+ T cells (inhibition rate: 13.22% in IgG1 group and 23.02% in IgG2 group) and the differentiation into Tregs (Treg ratio: 10.94% in control group, 8.51% in IgG1 group and 8.16% in IgG2 group).

[0192] 5. In vivo antitumor activity of humanized antibodies

[0193] A subcutaneous tumor model was constructed using humanized immunodeficient mice (huHSC-NCG, GemPharmatech, T037620). 1×10 6 Personal colon cancer cells (DLD1) were resuspended in 200 μL PBS (Gibco, 10010023) and injected subcutaneously into the left abdomen of mice. One week later, drug administration began. The experimental group received intraperitoneal injections of full-length humanized antibodies IgG1 and IgG2 (10 mg / kg, 1 mg / mL, prepared in PBS), while the control group received an equal volume of PBS intraperitoneally, every two days. Tumor volume was measured every three days. Tumor volume = (long axis × wide axis) 2 Mice were sacrificed after 2-3 weeks, and tumor weight was measured. The tumor volume change curve and the endpoint tumor weight are shown on [the graphs]. Figure 6 and Figure 7 The results are summarized in Table 10. The results show that the full-length humanized antibody can significantly inhibit the growth of colon cancer tumors in vivo.

[0194] Table 10: Effects of humanized antibodies on tumor mass and volume

[0195] control group IgG1 group IgG2 group Tumor mass (g) 0.98 0.27 0.52 <![CDATA[Tumor volume (mm 3 )]]> 804.16 195.05 409.31

[0196] Example 2: CAR-T cells based on humanized antibodies

[0197] 1. CAR Design

[0198] A representative CAR has an ABD-Hin-TM-CS-IS structure. Here, ABD is the extracellular antigen-binding domain, a scFv specifically targeting SLC7A11, with a VL-(G4S)3-VH structure, based on the humanized antibody BDF12-H17-2L7-1 of this invention; Hin is the hinge region, derived from human CD8α; TM is the transmembrane region, derived from human CD8α; IS is the intracellular signaling domain, derived from human CD3ζ; and CS is the co-stimulatory domain, derived from human 4-1BB. It also contains an N-terminal signal peptide, derived from human CD8α.

[0199] Human CD8α signal peptide:

[0200] MALPVTALLLPLALLLHAARP (SEQ ID NO: 28);

[0201] VL (BDF12-L-7-1) combined with SLC7A11:

[0202] DIVMTQSPLSLPVTPGEPASISCRSSESLLHTNGITYLYWYLQKPGQSPQLL

[0203] IYQMSNLASGVPDRFSGSGSGTDFTLRISRVEAEDVGVYYCAQNLQLPYT FGQGTKLEIK (SEQ IDNO: 19);

[0204] SCFV connector:

[0205] GGGGSGGGGSGGGGS (SEQ ID NO: 30)

[0206] VH (BDF12-H-17-2) combined with SLC7A11:

[0207] QVQLQESGPGLVKPSESLSLTCTVTGYSITSDYAWNWIRQPPGNGLEWMG

[0208] YISYSGTISYNPSLTSRITITRDTSKNQFFLKLSSVTTADTATYYCARWRFDGAWFSYWGQGTTVTVSS (SEQ ID NO: 20);

[0209] Human CD8α hinge region:

[0210] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY (SEQ ID NO: 24);

[0211] Human CD8α transmembrane region:

[0212] IWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 25);

[0213] Human 4-1BB costimulatory domain:

[0214] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 26);

[0215] Intracellular signaling domain of human CD3ζ:

[0216] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK

[0217] PRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT KDTYDALHMQALPPR (SEQ ID NO: 27).

[0218] The full-length amino acid sequence of a representative CAR:

[0219] MALPVTALLLPLALLLHAARPDIVMTQSPLSLPVTPGEPASISCRSSESLLH

[0220] TNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSGTDFTLRISR

[0221] VEAEDVGVYYCAQNLQLPYTFGQGTKLEIKGGGGSGGGGSGGGGSQVQ

[0222] LQESGPGLVKPSESLSLTCTVTGYSITSDYAWNWIRQPPGNGLEWMGYIS

[0223] YSGTISYNPSLTSRITITRDTSKNQFFLKLSSVTTADTATYYCARWRFDGA

[0224] WFSYWGQGTTVTVSSTTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH

[0225] TRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRP

[0226] VQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNL

[0227] GRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 29).

[0228] In the above sequence, positions 1-21 are the human CD8α signal peptide; positions 22-267 are the scFv sequence that binds to SLC7A11; positions 268-314 are the human CD8α hinge region; positions 315-336 are the human CD8α transmembrane region; positions 337-378 are the human 4-1BB co-stimulatory domain; and positions 379-490 are the human CD3ζ intracellular signaling domain. The mature sequence of a representative CAR is positions 22-490 of the full-length sequence.

[0229] 2. Preparation of CAR-T cells

[0230] The coding sequence of the representative CAR was inserted into the lentiviral vector GV401 (GeneChem), named pLV-hCAR(SLC7A11). The pLV-hCAR(SLC7A11) was packaged into virus in HEK293T cells (Cell Bank of the Chinese Academy of Sciences) using the pHelper 1.0 vector (containing gag, pol and rev genes; Addgene, 12260, psPAX2) and the pHelper 2.0 vector (containing the VSVG gene; Addgene, 12259, pMD2.G).

[0231] Fresh peripheral blood was obtained from healthy volunteers under informed consent, and human lymphocyte separation medium (Dayou, 7111012) was added. Peripheral blood mononuclear cells (PBMCs) were separated by centrifugation at 800g for 30 minutes, and T cells were sorted using human T cell magnetic beads (Biolengend, 480022). After sorting, T cells were seeded in a 24-well plate (Corning, 3524) at a density of 1×10 6 cells / mL using T cell medium (Stemcell, 10981), and IL-2 (10 ng / ml, Stemcell, 78036) and CD3 / CD28 (25 μL / mL, Stemcell, 10971) were added simultaneously, and the cells were cultured in a 37 °C, 5% CO2 cell culture incubator. After 48 hours of culture, the T cells were infected with the virus (moi = 50) and expanded in vitro.

[0232] Flow cytometry results showed that the virus transfection efficiency reached more than 90% (see Figure 8 ).

[0233] RNA was extracted, reverse transcribed, and the overexpression of CAR was identified by qPCR. T cells not transfected with the virus (UTD) were used as the control group, and GAPDH was used as the internal reference. The results showed that the CAR was overexpressed 732-fold in virus-transfected T cells, indicating successful overexpression (see Figure 9 ). \n

[0234] 3. In vitro tumor killing efficacy of CAR-T cells

[0235] The killing efficacy of the CAR-T cells of the present invention against the human colon cancer cell lines DLD1 (ATCC, CCL-221) and HT29 (ATCC, HTB-38) was examined using a CCK8 kit (DOJINDO, CK04). One day before the experiment, 1×10 4DLD1 and HT29 cells were resuspended in 100 μL of DMDM ​​(Gibco, 11995065). The next day, after tumor cells adhered, CAR-T cells were co-cultured with the previously seeded DLD1 cells (effect-to-target ratio 1:1) or HT29 cells (effect-to-target ratio 2:1) in 100 μL of RIPM 1640 medium (Gibco, 11875119) at 37°C in a 5% CO2 cell culture incubator. After 24 hours of co-culture, cancer cell viability was measured using a kit. Controls included untreated T cells (CON), untransfected T cells (UTD), and T cells transfected with EGFP-carrying virus (MOCK). Results (n=4) showed that the cytotoxic efficacy of the CAR-T group was significantly higher than that of the three control groups (see...). Figure 10 and Figure 11 The viability of DLD1 cells co-cultured with CAR-T cells was 19%, the target cell viability in the UTD group was 83%, and the target cell viability in the MOCK group was 82%. The viability of HT29 cells co-cultured with CAR-T cells was 34%, the target cell viability in the UTD group was 68%, and the target cell viability in the MOCK group was 66%.

[0236] 4. In vivo tumor-killing efficacy of CAR-T cells

[0237] A subcutaneous tumor model was established using NCG mice (GemPharmatech, T001475) (approximately 10 mm was transplanted subcutaneously into the lateral ventral wall of the mice). 3 (fresh colon cancer tissue from patients). When the average subcutaneous tumor volume in the experimental and control groups increased to 150 mm... 3 At approximately 10:00 PM, cells were infused via the tail vein. The experimental group received 8 × 10⁸ cells resuspended in 200 μL PBS. 6 8 × 10⁸ CAR-T cells were infused into the MOCK control group and resuspended in 200 μL PBS. 6 T cells transfected with control virus were used. Tumor volume and mouse weight were measured every 3 days for 30 days. Tumor volume = (long axis × wide axis) 2 ) / 2. Mice were sacrificed 30 days later and their tumors were weighed (n=6). The results showed that Figure 16 The tumor volume change curves of individual mice in the experimental and control groups are shown in [the figures]. Figure 12 and Figure 13 The tumor volume and mouse body weight changes in the experimental and control groups are shown in the curves. Figure 14 and Figure 15 (n=6). By day 30, the average tumor volume in the control group was 932.56 mm. 3 The average tumor volume in the experimental group was 161.59 mm. 3In the experimental group, tumors disappeared in 3 mice. The average tumor weight in the control group was 1.10g, while the average tumor weight in the experimental group was 0.14g. The results indicate that tumor growth in the CAR-T group was significantly inhibited compared to the control group, and there was no significant difference in body weight between the CAR-T group and the control group.

[0238] In summary, the CAR-T cells constructed based on the humanized anti-SLC7A11 monoclonal antibody of this invention have good in vivo and in vitro killing effects, while also exhibiting good safety.

[0239] Example 3: CAR-T cells based on murine antibodies

[0240] 1. CAR Design

[0241] A representative CAR has an ABD-Hin-TM-CS-IS structure. Here, ABD is the extracellular antigen-binding domain, a scFv specifically targeting SLC7A11, with a VH-(G4S)3-VL structure, based on the murine antibody BDF12-M of this invention; Hin is the hinge region, derived from human CD8α; TM is the transmembrane region, derived from human CD8α; IS is the intracellular signaling domain, derived from human CD3ζ; CS is the co-stimulatory domain, derived from human 4-1BB. It also contains an N-terminal signal peptide, derived from human CD8α.

[0242] The full-length amino acid sequence of a representative CAR:

[0243] MALPVTALLLPLALLLHAARPDVQLQESGPGLVKPSQSLSLTCTVTGYSIT

[0244] SDYAWNWIRQFPGNKLEWMGYISYSGTISYNPSLTSRISITRDTSKNQFFL

[0245] QLNSVTTEDTATYYCARWRFDGAWFSYWGQGTLVTVSAGGGGSGGGGS

[0246] GGGGSDIVMTQAAFSNPVTLGTSASISCRSSESLLHTNGITYLYWYLQKPG

[0247] QSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQN

[0248] LQLPYTFGGGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVH

[0249] TRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRP

[0250] VQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNL

[0251] GRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 31).

[0252] In the above sequence, positions 1 - 21 are the human CD8α signal peptide; positions 22 - 267 are the scFv based on the murine antibody BDF12 - M; positions 268 - 314 are the human CD8α hinge region; positions 315 - 336 are the human CD8α transmembrane region; positions 337 - 378 are the human 4 - 1BB co - stimulatory domain; positions 379 - 490 are the human CD3ζ intracellular signaling domain. The mature sequence of the representative CAR is positions 22 - 490 of the full - length sequence.

[0253] 2. Preparation of CAR - T cells

[0254] The coding sequence of the representative CAR was inserted into the lentiviral vector GV401 (GeneChem), named pLV - mCAR(SLC7A11). The pLV - mCAR(SLC7A11) was packaged into virus in HEK293T cells (Cell Bank of the Chinese Academy of Sciences) using the pHelper 1.0 vector (containing gag, pol, and rev genes; Addgene, 12260, psPAX2) and the pHelper 2.0 vector (containing the VSVG gene; Addgene, 12259, pMD2.G).

[0255] Fresh peripheral blood was obtained from healthy volunteers under informed consent, added with human lymphocyte separation medium (Dayou, 7111012), and peripheral blood mononuclear cells (PBMC) were separated by centrifugation at 800g for 30 minutes. T cells were sorted using human T cell magnetic beads (Biolengend, 480022). After sorting, T cell medium (Stemcell, 10981) was used in 24 - well plates (Corning, 3524) at 1×10 6T cells were seeded at a density of cells / mL, supplemented with IL-2 (10 ng / mL, Stemcell, 78036) and CD3 / CD28 (25 μL / mL, Stemcell, 10971), and cultured in a 37°C, 5% CO2 cell incubator. After 48 hours of culture, the T cells were infected with a virus (moi=50) and expanded in vitro.

[0256] Flow cytometry results showed that the viral transfection efficiency reached over 90% (see...). Figure 17 ).

[0257] RNA was extracted, reverse transcribed, and CAR expression was identified by qPCR. Untransfected T cells (UTDs) were used as the control group, and GAPDH was used as an internal control. The results showed that virus-transfected T cells overexpressed CAR by 6209-fold, indicating successful overexpression (see [link to relevant documentation]). Figure 18 ).

[0258] 3. In vitro tumor-killing efficacy of CAR-T cells

[0259] The killing efficacy of the CAR-T cells of this invention against the colon cancer cell lines DLD1 (ATCC, CCL-221) and HT29 (ATCC, HTB-38) was tested using an LDH kit (DOJINDO, CK12). One day prior to the experiment, 1×10⁶ cells were seeded into 96-well plates (Corning, 3599). 4 DLD1 and HT29 cells were resuspended in 100 μL of DMDM ​​(Gibco, 11995065). The next day, after tumor cells adhered, CAR-T cells were co-cultured with the previously seeded DLD1 cells (effect-to-target ratio 1:1) or HT29 cells (effect-to-target ratio 2:1) in 100 μL of RIPM 1640 medium (Gibco, 11875119) at 37°C in a 5% CO2 cell culture incubator. The lysis rate of cancer cells was measured using a kit after 6, 12, and 24 hours of co-culture. Untransfected T cells (UTD) and T cells transfected with a virus carrying the EGFP gene (MOCK) served as controls. Results (n=3) showed that after 24 hours of co-culture, the cytotoxic efficacy of the CAR-T group was significantly higher than that of the two control groups (see...). Figure 19 and Figure 20 After co-culturing with CAR-T cells, the DLD1 lysis rate was 81.32%, the target cell lysis rate in the UTD group was 8.15%, and the target cell lysis rate in the MOCK group was 10.29%. After co-culturing with CAR-T cells, the HT29 lysis rate was 85.61%, the target cell lysis rate in the UTD group was 17.61%, and the target cell lysis rate in the MOCK group was 26.68%.

[0260] The killing efficacy of the CAR-T cells of this invention against the colon cancer cell line HT29 was tested using Annexin-V and the PI apoptosis kit (Biolegend, 640932). One day prior to the experiment, 5 × 10⁶ cells were seeded in 48-well plates (Corning, 3548). 4 HT29 cells were resuspended in 250 μL of DMEM (Gibco, 11995065). The next day, after the tumor cells adhered, 1×10⁻⁶ cells were added to the culture dish. 5 CAR-T cells and HT29 cells seeded the previous day (effect-to-target ratio 2:1) were co-cultured in 250 μL RIPM 1640 medium (Gibco, 11875119) at 37°C in a 5% CO2 cell culture incubator for 20 hours. Untransfected T cells (UTD) and T cells transfected with the virus carrying the EGFP gene (MOCK) were used as controls, and no T cells (CON) were used as background. The results (n=4) showed that the cytotoxic efficacy of the CAR-T group was significantly stronger than that of the two control groups (see Table 11).

[0261] Table 11: Apoptosis induction of target cells by murine CAR-T cells (%)

[0262] CON CAR-T UTD MOCK HT29 6 84.825 21.54 21.628

[0263] 4. In vivo tumor-killing efficacy of CAR-T cells

[0264] A subcutaneous tumor model was established using NCG mice (GemPharmatech, T001475) (3 × 10⁶ cells were subcutaneously injected into the lateral ventral wall of the mice). 6 DLD1 cells resuspended in 200 μL PBS (Gibco, 10010023). When the average subcutaneous tumor volume in the experimental and control groups increased to 100 mm... 3 At approximately 10:00 PM, cells were infused via the tail vein. The experimental group received 6 × 10⁶ cells resuspended in 200 μL PBS. 6 CAR-T cells, UTD control group, 6×10⁶ cells resuspended in 200 μL PBS 6 Untransfected T cells. Tumor volume and mouse body weight were measured every 2 days (n=10), and the observation period was 30 days. Tumor volume = (long axis × wide axis) 2 ) / 2. By day 30, the average tumor volume in the control group was 370.77 mm. 3 The average tumor volume in the experimental group was 131.47 mm. 3 The curves showing changes in tumor volume and mouse body weight are shown in Figure 1. Figure 21 and Figure 22The results showed that tumor growth in the CAR-T group was significantly inhibited compared to the control group, and there was no significant difference in body weight between the CAR-T group and the control group.

[0265] In summary, the CAR-T cells constructed based on the murine anti-SLC7A11 monoclonal antibody of this invention have good in vivo and in vitro killing effects, while also exhibiting good safety.

Claims

1. A chimeric antigen receptor polypeptide comprising: Extracellular antigen-binding domain, which is the antibody that binds to SLC7A11; The hinge region, optionally, is derived from human CD8α, and further optionally, is as shown in SEQ ID NO: 24; Transmembrane region, optionally derived from human CD8α, further optionally as shown in SEQ ID NO: 25; and Intracellular signaling domain, optionally derived from human CD3ζ, and further optionally as shown in SEQ ID NO:

27. Optional, it also includes: a costimulatory domain, optional, derived from human 4-1BB, and further optional, As shown in SEQ ID NO: 26, Optionally, it further comprises: a signal peptide, optionally derived from human CD8α, and further optionally, as shown in SEQ ID NO:

28. in The antibody comprises CDR-L1 as shown in SEQ ID NO:1, CDR-L2 as shown in SEQ ID NO:2, CDR-L3 as shown in SEQ ID NO:3, CDR-H1 as shown in SEQ ID NO:4, CDR-H2 as shown in SEQ ID NO:5, and CDR-H3 as shown in SEQ ID NO:

6.

2. The chimeric antigen receptor polypeptide according to claim 1, wherein the antibody comprises: (1) VL as shown in SEQ ID NO: 15 and / or VH as shown in SEQ ID NO: 16; or (2) VL as shown in SEQ ID NO: 19 and / or VH as shown in SEQ ID NO:

20.

3. The chimeric antigen receptor polypeptide according to claim 1 or 2, wherein the antibody is scFv, optionally, VL and VH are linked via a peptide linker, further optionally, the peptide linker is as shown in SEQ ID NO: 30, and / or, optionally, VL is at the N-terminus and VH is at the C-terminus, or VH is at the N-terminus and VL is at the C-terminus.

4. A nucleic acid encoding the chimeric antigen receptor polypeptide of any one of claims 1 to 3.

5. A vector comprising the nucleic acid of claim 4.

6. A cell comprising the nucleic acid of claim 4 or the vector of claim 5, optionally, the cell being a prokaryotic cell or a eukaryotic cell, further optionally, the cell being an Escherichia coli cell or a mammalian cell.

7. A cell displaying the chimeric antigen receptor polypeptide of any one of claims 1 to 3 on its cell membrane.

8. A method for generating cells in which a chimeric antigen receptor polypeptide is integrated on a cell membrane, comprising culturing the cells of claim 6 under conditions suitable for the integration of the chimeric antigen receptor polypeptide on a cell membrane, such that the chimeric antigen receptor polypeptide is integrated on the cell.

9. A cell generated by the method of claim 8, optionally, said cell being an immune cell, and further optionally, said cell being a T cell.

10. A composition comprising the nucleic acid of claim 4, the vector of claim 5, or the cell of any one of claims 6, 7, and 9.

11. The nucleic acid of claim 4, the vector of claim 5, the cell of any one of claims 6, 7 and 9, or the composition of claim 10, used as a medicine, optionally for treating cancer or delaying cancer progression, further optionally, said cancer being colon cancer, breast cancer, or pancreatic cancer.

12. A method of treating cancer or delaying cancer progression, comprising administering to a subject suffering from cancer the nucleic acid of claim 4, the vector of claim 5, the cell of any one of claims 6, 7 and 9, or the composition of claim 10, optionally, the cancer being colon cancer, breast cancer, or pancreatic cancer.

13. Use of the nucleic acid of claim 4, the vector of claim 5, the cell of any one of claims 6, 7 and 8, or the composition of claim 10 in the preparation of a medicament for treating cancer or delaying cancer progression, optionally, wherein the cancer is colon cancer, breast cancer, or pancreatic cancer.

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