Humanized cd19 antibodies and uses thereof

Abstract

The application provides a humanized CD19 antibody and a chimeric antigen receptor, an immune cell and an application thereof. The humanized CD19 antibody of the application is based on the FMC63 chimeric antibody and is humanized. The application also provides a CAR-T cell and a double CAR-T cell based on the humanized antibody and related applications thereof. Compared with the CAR-T cell based on the FMC63, the CAR-T cell constructed in the application has higher killing effect and tumor clearance capacity.

Description

Technical Field

[0001] This invention relates to the field of immunotherapy, and more specifically to a humanized CD19 antibody and its applications. Background Technology

[0002] Antibodies are protective proteins produced by the body in response to antigen stimulation, secreted by plasma cells into bodily fluids such as the blood. Antibodies can specifically bind to antigens, neutralizing toxins and preventing pathogen invasion. Based on this antigen-specific binding characteristic, antibody drugs targeting disease-specific biological targets can be developed for disease treatment. Antibody drugs have already been applied in the fields of anti-tumor therapy and autoimmune disease treatment, and are playing an increasingly important role in antiviral and bacterial infections, cardiovascular and cerebrovascular diseases, diabetes, and rare disease treatment. They represent the class of drugs with the highest compound annual growth rate among current biopharmaceuticals.

[0003] CD19 is a cluster differentiation antigen and an important membrane antigen involved in B cell proliferation, differentiation, activation, and antibody production. The vast majority of B-cell malignancies highly express CD19 on their surface. Multiple centers have independently achieved unprecedented success in treating relapsed and refractory B-cell malignancies using chimeric antigen receptor (CAR)-modified T cells that target CD19. Immunotherapy, spearheaded by CAR-T therapy, has brought hope of a "cure for cancer" to countless patients.

[0004] However, current immunotherapy still suffers from problems such as high relapse rates and low safety, and there is a need in this field to develop safer and more effective immunotherapy methods. Summary of the Invention

[0005] The purpose of this invention is to provide a humanized CD19 antibody and its application.

[0006] In a first aspect of the invention, a humanized CD19 antibody is provided, the antibody comprising a variable region of the antibody light chain shown in any of SEQ ID NO:1-7 and a variable region of the antibody heavy chain shown in any of SEQ ID NO:8-17.

[0007] In another preferred embodiment, the antibody includes the antibody light chain variable region shown in SEQ ID NO:5 or 6, and the antibody heavy chain variable region shown in any of SEQ ID NO:15, 16 or 17.

[0008] In another preferred embodiment, the antibody includes a variable region of the antibody light chain and a variable region of the antibody heavy chain selected from the group consisting of:

[0009]

[0010]

[0011] In another preferred embodiment, the antibody comprises the antibody light chain variable region shown in SEQ ID NO:5 and the antibody heavy chain variable region shown in SEQ ID NO:16; or,

[0012] The antibody includes the antibody light chain variable region shown in SEQ ID NO:5 and the antibody heavy chain variable region shown in SEQ ID NO:17; or,

[0013] The antibody includes the antibody light chain variable region shown in SEQ ID NO:6 and the antibody heavy chain variable region shown in SEQ ID NO:15.

[0014] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0015] In another preferred embodiment, the antibody is a full-length antibody protein or an antigen-binding fragment.

[0016] In another preferred embodiment, the antibody is a bispecific antibody or a multispecific antibody.

[0017] In another preferred embodiment, the antibody further comprises a linker peptide located between the heavy chain variable region and the light chain variable region.

[0018] In another preferred embodiment, the antibody is represented by formula A or formula B as follows:

[0019] V H -V L (A); V L -V H (B)

[0020] In the formula, V H V is the variable region of the antibody heavy chain; L This refers to the variable region of the antibody light chain; "-" represents a linking peptide or peptide bond.

[0021] The preferred structure is structure B.

[0022] In another preferred embodiment, the linker peptide is 1-4 consecutive sequences as shown in SEQ ID NO:22 (GGGGS), more preferably 1-4, and more preferably 3-4.

[0023] In another preferred embodiment, the connection order of each segment in the light chain variable region is: humanized VL FR1-VLCDR1-humanized VL FR2-VL CDR2-humanized VL FR3-VL CDR3-humanized VL FR4;

[0024] In another preferred embodiment, the connection order of each segment in the heavy chain variable region is: humanized VH FR1- VHCDR1- humanized VH FR2- VH CDR2- humanized VH FR3- VH CDR3- humanized VH FR4.

[0025] In a second aspect of the invention, a chimeric antigen receptor (CAR) targeting CD19 is provided, wherein the antigen-binding domain of the CAR is the humanized CD19 antibody described in the first aspect of the invention.

[0026] In another preferred embodiment, the structure of the CAR is shown in Equation I:

[0027] L-scFv-H-TM-C-CD3ζ(I)

[0028] In the formula,

[0029] Each "-" independently represents a linking peptide or peptide bond;

[0030] L represents the absence of a signal peptide sequence;

[0031] scFv is an antigen-binding domain that targets CD19;

[0032] H represents the area with no hinge or no connection.

[0033] TM represents a transmembrane domain;

[0034] C is a co-stimulatory signaling molecule;

[0035] CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ.

[0036] In a third aspect of the invention, a bispecific CAR is provided, the bispecific CAR targeting CD19 and a first target.

[0037] Wherein, the antigen-binding domain targeting CD19 in the bispecific CAR is the humanized CD19 antibody described in the first aspect of the present invention;

[0038] Furthermore, the first target is selected from the following group:

[0039] CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, D151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2 / HER2, ErbB3, HER-2, HER3, ErbB4 / HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetylGD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, T GFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, PSCA, HVEM, MAGE-A, MSLN, N Y-ESO-1, PSMA, RANK, RORl, TNFRSF4, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, MUC16, TCRα, TCRβ, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, Notch-1-4, APRIL, CS1, MAGE3, Claudin18.2, Folate receptor α, Folate receptor β, GPC2, CD70, BAFF-R, TROP-2, or combinations thereof.

[0040] In another preferred embodiment, the first target is BCMA, and the antigen-binding domain (scFv) targeting BCMA in the bispecific CAR includes the antibody heavy chain variable region shown in SEQ ID NO:21 and the antibody light chain variable region shown in SEQ ID NO:20.

[0041] In another preferred embodiment, the bispecific CAR simultaneously includes an antigen-binding domain targeting a first target and an antigen-binding domain targeting CD19.

[0042] In another preferred embodiment, the structure of the bispecific CAR is shown in Formula II:

[0043] L-scFv1-I-scFv2-H-TM-C-CD3ζ(II)

[0044] In the formula,

[0045] Each "-" independently represents a linking peptide or peptide bond;

[0046] L represents the absence of a signal peptide sequence;

[0047] I represents a flexible joint;

[0048] H represents the area with no hinge or no connection.

[0049] TM represents a transmembrane domain;

[0050] C is a co-stimulatory signaling molecule;

[0051] CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ;

[0052] Of the two, scFv1 and scFv2, one is an antigen-binding domain that targets the first target, and the other is an antigen-binding domain that targets CD19.

[0053] In another preferred embodiment, scFv1 and scFv2 can be independent, connected in series, or in a loop structure.

[0054] In another preferred embodiment, scFv1 is an antigen-binding domain targeting a first target, and scFv2 is an antigen-binding domain targeting CD19.

[0055] In another preferred embodiment, scFv1 is an antigen-binding domain targeting CD19, and scFv2 is an antigen-binding domain targeting a first target.

[0056] In another preferred embodiment, the sequence of the flexible joint I comprises 1-6, preferably 3-5 consecutive sequences as shown in SEQ ID NO:22 (GGGGS).

[0057] In another preferred embodiment, the structure of the antigen-binding domain targeting the first target is shown in formula C or formula D as follows:

[0058] V L1 -V H1 (C); V H1 -V L1 (D)

[0059] Among them, V L1 For the variable region of the light chain of the antibody against the first target; V H1 This represents the variable region of the heavy chain of the antibody against the first target; "-" indicates a linking peptide or peptide bond.

[0060] In another preferred embodiment, the structure of the antigen-binding domain targeting BCMA is shown in formula C or formula D below:

[0061] V L1 -V H1 (C); V H1 -V L1 (D)

[0062] Among them, V L1 For the variable region of the light chain of the anti-BCMA antibody; V H1 This is the variable region of the heavy chain of the anti-BCMA antibody; "-" indicates a linking peptide or peptide bond.

[0063] The preferred structure is the D-type structure.

[0064] In another preferred embodiment, the structure of the CD19-targeting antigen-binding domain is shown in formula A or formula B as follows:

[0065] V H -V L , (A); V L -V H (B)

[0066] In the formula, V H V is the variable region of the antibody heavy chain; L "-" represents the variable region of the antibody light chain; "-" represents a linking peptide or peptide bond.

[0067] In another preferred embodiment, the scFv1 and / or scFv2 are murine, human, human-mice chimeric, or fully humanized single-chain antibody variable region fragments, preferably humanized single-chain antibody variable region fragments.

[0068] In another preferred embodiment, the structure of the bispecific CAR is shown in formula III or III':

[0069] LV L3 -scFv3-V H3 -H-TM-C-CD3ζ (III)

[0070] LV H3 -scFv3-V L3 -H1-TM-C-CD3ζ (III')

[0071] In the formula,

[0072] Each "-" independently represents a linking peptide or peptide bond;

[0073] Components L, H, TM, C, and CD3ζ are as described above;

[0074] scFv3 is an antigen-binding domain targeting CD19, V H3 For the variable region of the heavy chain of the antibody against the first target, and V L3The variable region of the light chain of the antibody against the first target; or scFv3 is the antigen-binding domain targeting the first target, V H3 It is the variable region of the heavy chain of the anti-CD19 antibody, and V L3 This is the variable region of the light chain of the anti-CD19 antibody.

[0075] In another preferred embodiment, scFv3 is an antigen-binding domain targeting BCMA, V H3 It is the variable region of the heavy chain of the anti-CD19 antibody, and V L3 This is the variable region of the light chain of the anti-CD19 antibody.

[0076] In another preferred embodiment, the antigen-binding domain targeting BCMA includes the antibody heavy chain variable region shown in SEQ ID NO:21 and the antibody light chain variable region shown in SEQ ID NO:20.

[0077] In another preferred embodiment, the structure of the bispecific CAR is shown in Formula III below.

[0078] In another preferred embodiment, L is a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

[0079] In another preferred embodiment, the L is a signal peptide derived from CD8.

[0080] In another preferred embodiment, the L has an amino acid sequence as shown in SEQ ID NO:23.

[0081] In another preferred embodiment, H is a hinge region of a protein selected from the group consisting of CD8, CD28, CD137, or a combination thereof.

[0082] In another preferred embodiment, each of the H's is an independent hinge region derived from CD8.

[0083] In another preferred embodiment, H has an amino acid sequence as shown in SEQ ID NO:24.

[0084] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0085] In another preferred embodiment, each of the TMs is an independent transmembrane region derived from CD8 or CD28.

[0086] In another preferred embodiment, the transmembrane region derived from CD8 has an amino acid sequence as shown in SEQ ID NO:25.

[0087] In another preferred embodiment, the transmembrane region derived from CD28 has an amino acid sequence as shown in SEQ ID NO:26.

[0088] In another preferred embodiment, C is a co-stimulatory signaling molecule selected from the group consisting of: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or combinations thereof.

[0089] In another preferred embodiment, C is a co-stimulatory signaling molecule derived from CD28 and / or 4-1BB.

[0090] In another preferred embodiment, the 4-1BB-derived co-stimulatory signaling molecule has an amino acid sequence as shown in SEQ ID NO:27.

[0091] In another preferred embodiment, the CD28-derived co-stimulatory signaling molecule has an amino acid sequence as shown in SEQ ID NO:28.

[0092] In another preferred embodiment, the CD3ζ has an amino acid sequence as shown in SEQ ID NO:29.

[0093] In another preferred embodiment, the CAR (preferably C-terminus or N-terminus) further includes a cell suicide element.

[0094] In another preferred embodiment, the cell suicide element is connected to the L or CD3ζ of the CAR or the bispecific CAR via T2A.

[0095] In a fourth aspect of the invention, a nucleic acid molecule is provided, the nucleic acid molecule encoding the humanized CD19 antibody of the first aspect of the invention, the CAR of the second aspect of the invention, or the bispecific CAR of the third aspect of the invention.

[0096] In a fifth aspect of the invention, a carrier is provided, said carrier containing the nucleic acid molecule described in the fourth aspect of the invention.

[0097] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmids, lentiviral vectors, adenovirus vectors, retroviral vectors, transposons, or combinations thereof.

[0098] In another preferred embodiment, the vector is a lentiviral vector.

[0099] In a sixth aspect of the invention, a host cell is provided, said host cell containing the vector described in the fifth aspect of the invention, or having an exogenous nucleic acid molecule described in the fourth aspect of the invention integrated into the chromosome, or expressing the humanized CD19 antibody described in the first aspect of the invention, the CAR described in the second aspect of the invention, or the bispecific CAR described in the third aspect of the invention.

[0100] In a sixth aspect of the invention, an engineered immune cell is provided, the immune cell containing the vector described in the fourth aspect of the invention, or having an exogenous nucleic acid molecule described in the third aspect of the invention integrated into the chromosome, or expressing the humanized CD19 antibody described in the first aspect of the invention, the CAR described in the second aspect of the invention, or the bispecific CAR described in the third aspect of the invention.

[0101] In another preferred embodiment, the immune cells have one or more characteristics selected from the group consisting of:

[0102] (a) The PD-1 gene expression of the immune cells was silenced;

[0103] (b) The immune cells are T cells, and the TCR gene expression of the T cells is silenced; and

[0104] (c) The immune cells express exogenous cell suicide elements;

[0105] (d) The immune cells express or secrete PD-1 antibody, PD-L1 antibody, CD47 antibody, Tim3 antibody, Lag3 antibody, Tigit antibody, OX40 antibody, ICOS antibody, IL7, CXCL19, IL21, IL15, IL2, IL18, or combinations thereof; and

[0106] (e) The cytokine-related signaling pathways of the immune cells are enhanced, wherein the cytokines are selected from the group consisting of IL7, CXCL19, IL21, IL15, IL2, IL18, or combinations thereof.

[0107] In another preferred embodiment, the engineered immune cells are selected from the group consisting of:

[0108] (i) Chimeric antigen receptor T cells (CAR-T cells); or

[0109] (ii) Chimeric antigen receptor NK cells (CAR-NK cells).

[0110] In another preferred embodiment, the immune cells express exogenous cell suicide elements.

[0111] In another preferred embodiment, the CAR is co-expressed with the cell suicide element in the immune cells.

[0112] In another preferred embodiment, the CAR is connected to the cell suicide element via a self-cleaving element.

[0113] In another preferred embodiment, the cell suicide element is located at the N-terminus or C-terminus of the CAR.

[0114] In another preferred embodiment, the self-cutting element comprises a 2A sequence or an IRES sequence, preferably P2A and T2A.

[0115] In another preferred embodiment, the cell suicide element is selected from the group consisting of HSV-TK, iCasp9, ΔCD20, mTMPK, ΔCD19, RQR8, EGFRt, or combinations thereof.

[0116] In another preferred embodiment, the structure of the cell suicide element is shown in Formula IV:

[0117] L2-DF (IV)

[0118] In the formula,

[0119] Each "-" independently represents a linking peptide or peptide bond;

[0120] L2 is an optional signal peptide sequence;

[0121] D is a suicide switch element;

[0122] F represents a transmembrane element.

[0123] In another preferred embodiment, the signal peptide is a signal peptide derived from GM-CSFR.

[0124] In another preferred embodiment, the cell suicide element is selected from the group consisting of: truncated epidermal growth factor receptor (EGFRt), truncated CD19 (CD19t) gene, induced caspase 9 gene (iCasp9), HSV-TK, ΔCD20, mTMPK, or combinations thereof.

[0125] In another preferred embodiment, the cell suicide element is EGFRt.

[0126] In another preferred embodiment, the engineered immune cells include universal CAR-T cells.

[0127] In another preferred embodiment, the TRAC and B2M genes of the universal CAR-T cells are knocked out.

[0128] In an eighth aspect of the invention, an engineered immune cell is provided, the immune cell comprising an exogenous first expression cassette and a second expression cassette, wherein the first expression cassette is used to express a first CAR targeting a first target, and the second expression cassette is used to express a second CAR targeting CD19.

[0129] Alternatively, the immune cells may express the first CAR targeting the first target and the second CAR targeting CD19;

[0130] Wherein, the antigen-binding domain (scFv) targeting CD19 in the second CAR is the humanized CD19 antibody described in the first aspect of the present invention;

[0131] Furthermore, the first target is selected from the following group:

[0132] CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, D151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2 / HER2, ErbB3, HER-2, HER3, ErbB4 / HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetylGD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, T GFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, PSCA, HVEM, MAGE-A, MSLN, N Y-ESO-1, PSMA, RANK, RORl, TNFRSF4, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, MUC16, TCRα, TCRβ, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, Notch-1-4, APRIL, CS1, MAGE3, Claudin18.2, Folate receptor α, Folate receptor β, GPC2, CD70, BAFF-R, TROP-2, or combinations thereof.

[0133] In another preferred embodiment, the first target is BCMA, and the antigen-binding domain (scFv) targeting BCMA in the first CAR includes the antibody heavy chain variable region shown in SEQ ID NO:21 and the antibody light chain variable region shown in SEQ ID NO:20.

[0134] In another preferred embodiment, the second CAR is the CAR described in the second aspect of the present invention.

[0135] In another preferred embodiment, the first CAR and the second CAR are located on the cell membrane of the immune cell.

[0136] In another preferred embodiment, the immune cells express a first CAR targeting BCMA and a second CAR targeting CD19 on their cell membranes.

[0137] In another preferred embodiment, the first expression box and the second expression box are located on the same or different carriers.

[0138] In another preferred embodiment, the first expression box and the second expression box are located on the same carrier.

[0139] In another preferred embodiment, the structure of the first CAR is shown in equation V:

[0140] L-scFv1'-H-TM-C-CD3ζ (V)

[0141] In the formula,

[0142] Each "-" independently represents a linking peptide or peptide bond;

[0143] Components L, H, TM, C, and CD3ζ are as described above;

[0144] scFv1' is an antigen-binding domain that targets BCMA.

[0145] In another preferred embodiment, the first CAR and the second CAR are linked by a 2A peptide.

[0146] In another preferred embodiment, the sequence of the 2A peptide is shown in SEQ ID NO:30.

[0147] In another preferred embodiment, the immune cells also include a cell suicide element.

[0148] In another preferred embodiment, the cell suicide element is connected (or tandemly) with the bispecific CAR via T2A.

[0149] In another preferred embodiment, the cell suicide element is connected to the first CAR and / or the second CAR via a T2A.

[0150] In another preferred embodiment, the PD1 gene expression of the immune cells is silenced.

[0151] In another preferred embodiment, "PD-1 gene expression is silenced" means that the PD-1 gene is not expressed or is expressed at low levels.

[0152] In another preferred embodiment, "low expression" refers to the ratio of the expression level G1 of the PD-1 gene on immune cells to the expression level G0 of the PD-1 gene on normal immune cells, i.e., G1 / G0≤0.5, preferably G1 / G0≤0.3, more preferably ≤0.2, even more preferably ≤0.1, and most preferably 0.

[0153] In another preferred embodiment, "low expression" refers to the ratio of the expression level G1 of the PD-1 gene in the CAR-T cells to the expression level G0 of the PD-1 gene in normal T cells, i.e., G1 / G0≤0.5, preferably G1 / G0≤0.3, more preferably ≤0.2, even more preferably ≤0.1, and most preferably 0.

[0154] In a ninth aspect of the invention, a formulation is provided comprising the humanized CD19 antibody as described in the first aspect of the invention, or the engineered immune cells as described in the seventh or eighth aspect of the invention, and a pharmaceutically acceptable carrier, diluent, or excipient.

[0155] In another preferred embodiment, the formulation is a liquid formulation.

[0156] In another preferred embodiment, the dosage form of the preparation is an injection.

[0157] In another preferred embodiment, the concentration of the engineered immune cells in the formulation is 1 × 10⁻⁶. 3 -1×10 8 Cells / ml, preferably 1×10⁻⁶ 4 -1×10 7 Cells / ml

[0158] In a tenth aspect of the invention, the use of the humanized CD19 antibody described in the first aspect of the invention, or the engineered immune cells described in the seventh or eighth aspect of the invention, is provided for the preparation of a medicament or formulation for the prevention and / or treatment of cancer or tumors.

[0159] In another preferred embodiment, the tumor is selected from the group consisting of hematologic malignancies, solid tumors, or combinations thereof.

[0160] In another preferred embodiment, the hematologic malignancy is selected from the group consisting of: acute myeloid leukemia (AML), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or combinations thereof.

[0161] In another preferred embodiment, the solid tumor is selected from the group consisting of: gastric cancer, peritoneal metastasis of gastric cancer, liver cancer, kidney tumor, lung cancer, small bowel cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), glioma, endometrial cancer, testicular cancer, colorectal cancer, urinary tract tumor, thyroid cancer, or combinations thereof.

[0162] In another preferred embodiment, the cancer or tumor is multiple myeloma.

[0163] In another preferred embodiment, the cancer or tumor is lymphoma.

[0164] In another preferred embodiment, the lymphoma is selected from the group consisting of: Hodgkin lymphoma (HL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), Burkitt lymphoma (BL), and other complex B-cell non-Hodgkin lymphomas.

[0165] In another aspect of the present invention, a method for preparing engineered immune cells is provided, wherein the engineered immune cells express the humanized CD19 antibody described in the first aspect of the present invention, the CAR described in the second aspect of the present invention, or the bispecific CAR described in the third aspect of the present invention, comprising the following steps:

[0166] The engineered immune cells are obtained by introducing the nucleic acid molecule described in the fourth aspect of the present invention or the vector described in the fifth aspect of the present invention into immune cells.

[0167] In another preferred embodiment, the immune cells are T cells or NK cells.

[0168] In an eleventh aspect of the present invention, a method for preparing engineered immune cells is provided, comprising the following steps:

[0169] (1) Provide an immune cell to be modified; and

[0170] (2) Introducing a first expression cassette for expressing a first CAR targeting a first target into the immune cells; and

[0171] (3) The second expression cassette for expressing a second CAR targeting CD19 is introduced into the immune cells to obtain the engineered immune cells.

[0172] Wherein, the antigen-binding domain (scFv) targeting CD19 in the second CAR is the humanized CD19 antibody described in the first aspect of the present invention;

[0173] Furthermore, the first target is selected from the following group:

[0174] CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, C D81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2 / HER2, ErbB3, HER-2, HER3, ErbB4 / HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, PSCA, HVEM, MAGE-A, MSLN, N Y-ESO-1, PSMA, RANK, RORl, TNFRSF4, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, MUC16, TCRα, TCRβ, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, Notch-1-4, APRIL, CS1, MAGE3, Claudin 18.2. Folate receptor α, Folate receptor β, GPC2, CD70, BAFF-R, TROP-2, or combinations thereof.

[0175] In another preferred embodiment, step (2) may be performed before, after, simultaneously with, or alternately with step (3).

[0176] In another preferred embodiment, if the immune cells to be modified in step (1) have already expressed the first CAR or the second CAR, then step (2) or step (3) can be omitted.

[0177] In a twelfth aspect of the invention, a kit is provided for preparing engineered immune cells as described in the seventh or eighth aspect of the invention, and the kit contains a container and a nucleic acid molecule as described in the fourth aspect of the invention, or a carrier as described in the fifth aspect of the invention, located within the container.

[0178] In a thirteenth aspect of the invention, a kit is provided for preparing engineered immune cells as described in the seventh or eighth aspect of the invention, and the kit includes a container, and the following are located within the container:

[0179] (1) A first nucleic acid sequence, the first nucleic acid sequence containing a first expression cassette, the first expression cassette being used to express a first CAR targeting a first target; and

[0180] (2) A second nucleic acid sequence, wherein the second nucleic acid sequence contains a second expression cassette, the second expression cassette being used to express the second CAR targeting CD19;

[0181] Wherein, the antigen-binding domain (scFv) targeting CD19 in the second CAR is the humanized CD19 antibody described in the first aspect of the present invention;

[0182] Furthermore, the first target is selected from the following group:

[0183] CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, D151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2 / HER2, ErbB3, HER-2, HER3, ErbB4 / HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetylGD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, T GFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, PSCA, HVEM, MAGE-A, MSLN, N Y-ESO-1, PSMA, RANK, RORl, TNFRSF4, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, MUC16, TCRα, TCRβ, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, Notch-1-4, APRIL, CS1, MAGE3, Claudin18.2, Folate receptor α, Folate receptor β, GPC2, CD70, BAFF-R, TROP-2, or combinations thereof.

[0184] In another preferred embodiment, the first and second nucleic acid sequences are located in the same or different containers.

[0185] In another preferred embodiment, the first and second nucleic acid sequences are located in the same expression vector.

[0186] In a fourteenth aspect of the invention, the use of engineered immune cells as described in the seventh or eighth aspect of the invention is provided for the prevention and / or treatment of cancer or tumors.

[0187] In another preferred embodiment, the cancer or tumor is multiple myeloma, hematologic malignancy, or lymphoma.

[0188] In a fifteenth aspect of the invention, a method for treating a disease is provided, comprising administering an appropriate amount of the cells described in the seventh or eighth aspect of the invention, or the preparation described in the sixth aspect of the invention, to a subject requiring treatment.

[0189] In another preferred embodiment, the disease is cancer or a tumor.

[0190] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0191] Figure 1 The CAR expression of FMC63 and humanized FMC63 antibody scFv modified CAR-T cells was shown.

[0192] Figure 2 The figure shows the killing effect of CAR-T cells modified with FMC63 and humanized FMC63 antibody scFv on Hela-CD19 cells (RTCA method). Each number in the figure represents a CAR-T cell constructed using the corresponding antibody, and the corresponding antibody structures are shown in Table 1.

[0193] Figure 3 The comparison of the killing ability of CAR-T cells modified with FMC63 and humanized FMC63 antibody scFv against Nalm6-luc cells at different effector-to-target ratios is shown (Luciferase assay). Each number in the figure represents a CAR-T cell constructed using the corresponding antibody, and the corresponding antibody structure is shown in Table 1.

[0194] Figure 4 The binding ability of FMC63 and humanized FMC63 antibodies to CD19-positive SU-DHL-10 and Raji cells is shown (FACS assay). In the figure, FMC63 represents the chimeric antibody FMC63.

[0195] Figure 5 The results show the positive rates of different flow cytometry assays for FMC63 and CAR-T cell surface CAR molecules modified with humanized FMC63 antibody scFv.

[0196] Figure 6 The study demonstrated the killing ability of FMC63 and humanized FMC63 antibody scFv-modified CAR-T cells against Hela-CD19 target cells under different effector-to-target ratios (RTCA method, where Index 80 represents the number of hours required for 80% of the target cells to be killed).

[0197] Figure 7This study compares the killing ability of FMC63 and humanized FMC63 antibody-modified CAR-T cells against Nalm6-luc and Raji-luc target cells under different effector-to-target ratios.

[0198] Figure 8 The study showed the release of IFNγ during the killing of Nalm6-luc and Raji-luc target cells by CAR-T cells modified with FMC63 and humanized FMC63 antibody scFv under different effector-to-target ratios.

[0199] Figure 9 The study compared the in vivo efficacy of FMC63 and humanized FMC63 antibody scFv-modified CAR-T cells against Raji-induced NOG mice.

[0200] Figure 10 The results showed that CAR-T cells modified with FMC63 and the humanized FMC63 antibody scFv(H9L5) did not kill negative target cells (Luciferase assay).

[0201] Figure 11 This study compares the tumor clearance capabilities of FMC63-modified bispecific CAR-T cells and humanized FMC63 antibody scFv (H9L5) in Raji-induced NOG mice.

[0202] Figure 12 A shows the structure of the dual CAR constructed in Example 15.

[0203] Figure 12 B shows the structure of the mouse-derived dual CAR constructed in Example 16.

[0204] Figure 12 C shows the structure of the humanized dual CAR constructed in Example 16.

[0205] Figure 13 The comparison of the killing capacity of humanized dual CAR-T cells and mouse dual CAR-T cells is shown. In the figure, NT represents the negative control, L represents mouse dual CAR-T cells, and HL represents humanized dual CAR-T cells.

[0206] Figure 14 This figure shows the production of universal CAR-T cells from humanized dual CAR-T cells. In the figure, NT represents the negative control, and HL-DKO represents double knockout universal humanized dual CAR-T cells.

[0207] Figure 15This figure shows a comparison of the killing effects of humanized dual CAR-T cells against conventional CAR-T and universal CAR-T cells. In the figure, NT represents the negative control, HL-DKO represents double knockout universal humanized dual CAR-T cells, and HL represents humanized dual CAR-T cells.

[0208] In the attached diagram, LmHn or HnLm (m, n are positive integers) represent CAR-T cells constructed using humanized antibodies containing VLm and VHn. In the specific structure of the CAR, VL comes first, followed by VH. Except... Figure 4 In addition, FMC63 in each figure represents CAR-T cells constructed using FMC63. NT in each figure represents untreated T cells. Detailed Implementation

[0209] Through extensive and in-depth research, the inventors have for the first time constructed a novel humanized CD19 antibody, which is obtained by humanization modification based on an FMC63 chimeric antibody. This invention also provides CAR-T cells and dual CAR-T cells constructed based on this humanized antibody, as well as their related applications. Compared with CAR-T cells constructed using FMC63, the CAR-T cells and dual CAR-T cells constructed in this invention exhibit higher killing efficacy and tumor clearance capacity. This invention was completed based on these findings.

[0210] the term

[0211] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0212] The term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0213] The term “giving” means the physical introduction of the product of the present invention into a subject using any of the various methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion.

[0214] It should be understood that the amino acid names in this article adopt the internationally accepted single-letter identifiers, and the corresponding three-letter abbreviations of the amino acid names are: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), I1e (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), Val (V).

[0215] CD19

[0216] CD19 is a transmembrane protein on the surface of B cells, closely related to B cell activation, signal transduction, and growth regulation. CD19 is expressed on the surface of almost all B cells, and CAR-T cells targeting CD19 have shown significant efficacy in the treatment of leukemia and lymphoma. CD19 can also be used to treat multiple myeloma.

[0217] B cell maturation antigen (BCMA)

[0218] BCMA is a transmembrane protein expressed on the surface of mature B lymphocytes, specifically plasmablasts and plasma cells. Multiple myeloma is caused by the abnormal proliferation and invasion of the bone marrow by plasma cells. Studies have shown that BCMA is expressed on multiple myeloma cells. CAR-T cell therapy targeting BCMA has been shown to specifically kill myeloma cells. However, some patients still experience relapse after receiving BCMA-targeted CAR-T cell therapy. For these relapsed patients, it is necessary to find a target different from BCMA to continue treatment.

[0219] Antibody

[0220] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0221] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are tightly packed together by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0222] Vertebrate antibodies (immunoglobulins) can be classified into two distinct classes (denoted as κ and λ) based on the amino acid sequence of their constant region. Immunoglobulins can be further classified into different types based on the amino acid sequence of their heavy chain constant region. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are respectively called α, δ, ε, γ, and μ. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known to those skilled in the art.

[0223] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs). These regions are divided into four frame regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, and are spatially close to each other through β-sheets formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0224] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0225] In this invention, antibodies include mouse, chimeric, humanized, or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including both human and non-human portions, can be obtained using standard DNA recombination techniques and are all useful antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as a chimeric antibody having a variable region derived from a mouse monoclonal antibody and a constant region derived from a human immunoglobulin (see, for example, U.S. Patents 4,816,567 and 4,816,397, which are incorporated herein by reference in their entirety). A humanized antibody is an antibody molecule derived from a non-human species, having one or more complementarity-determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Patent 5,585,089, which is incorporated herein by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombination techniques well known in the art.

[0226] In this invention, the antibody can be monospecific, bispecific, trispecific, or more multiple specific.

[0227] In this invention, the antibody also includes its conserved variants, which are polypeptides formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of this invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0228] Table A

[0229] Ala(A) Val; Leu; Ile Val Arg(R) Lys;Gln;Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg;Gln;Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe

[0230] Val(V) Ile; Leu; Met; Phe; Ala Leu

[0231] Chimeric antigen receptor (CAR)

[0232] The chimeric antigen receptor (CAR) of this invention comprises an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen-binding domain). The intracellular domain includes a co-stimulatory signaling region and a ζ-chain portion. The co-stimulatory signaling region refers to a portion of the intracellular domain containing a co-stimulatory molecule. The co-stimulatory molecule is a cell surface molecule required for an effective lymphocyte response to an antigen, rather than an antigen receptor or its ligands.

[0233] A linker may be incorporated between the extracellular and transmembrane domains of the CAR, or between the cytoplasmic and transmembrane domains of the CAR. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular or cytoplasmic domain of the polypeptide chain. Linkers may comprise 0-300 amino acids, preferably 2 to 100 amino acids, and most preferably 3 to 50 amino acids.

[0234] In a preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention includes an antigen-binding domain targeting CD19. When expressed in T cells, the CAR of the present invention is capable of antigen recognition based on antigen-binding specificity. When it binds to its associated antigen, it affects tumor cells, causing them to stop growing, be induced to die, or otherwise be affected, leading to a reduction or elimination of the patient's tumor burden. The antigen-binding domain is preferably fused with an intracellular domain derived from one or more of the co-stimulatory molecule and the ζ chain. Preferably, the antigen-binding domain is fused with an intracellular domain combining a 4-1BB signaling domain and a CD3ζ signaling domain.

[0235] As used herein, "antigen-binding domain" and "single-chain antibody fragment" refer to Fab fragments, Fab' fragments, F(ab')2 fragments, or single Fv fragments with antigen-binding activity. Fv antibodies contain variable regions of the antibody heavy chain and light chain, but no constant region, and are the smallest antibody fragments possessing all antigen-binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding. The antigen-binding domain is typically scFv (single-chain variable fragment). The size of an scFv is generally 1 / 6 of a complete antibody. A single-chain antibody is preferably a single amino acid chain sequence encoded by a single nucleotide chain. As a preferred embodiment of the invention, the antigen-binding domain contains an antibody that specifically recognizes CD19; optionally, the antigen-binding domain also contains an antibody that specifically recognizes BCMA, preferably a single-chain antibody.

[0236] For the hinge region and transmembrane region (transmembrane domain), the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one implementation, a transmembrane domain naturally associated with one of the domains in the CAR is used. In some examples, the transmembrane domain can be selected, or modified by amino acid substitution, to avoid binding such a domain to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0237] The intracellular domains in the CAR of this invention include the 4-1BB signal transduction domain and the CD3ζ signal transduction domain.

[0238] Preferably, the CAR of the present invention also includes a cell suicide element.

[0239] Preferably, the CD19-targeting scFv of the present invention includes the antibody light chain variable region shown in any of SEQ ID NO:1-7 and the antibody heavy chain variable region shown in any of SEQ ID NO:8-17.

[0240] Bispecific CAR

[0241] CD19 is a 95kDa glycoprotein expressed on the surface of pre-B cells and mature B cells. It is closely related to the transmembrane signaling pathway of Ca++ in B cells and plays a regulatory role in B cell proliferation and differentiation. CD19 is mainly expressed in normal B cells and cancerous B cells, exhibiting high tissue specificity, making it a promising target for antibody or CAR-T immunotherapy. However, during immunotherapy, the loss of the CD19 epitope on B cells frequently occurs, leading to unresponsiveness or relapse in patients.

[0242] Bispecificity means that the same CAR can specifically bind to and be recognized by the immune system for two different antigens. The CAR can generate an immune response by binding to either antigen.

[0243] This invention provides a bispecific CAR that targets CD19 and another tumor target, as described in the third aspect of this invention.

[0244] In another preferred embodiment, the bispecific CAR targets CD19 and BCMA.

[0245] In a preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention includes an antigen-binding domain targeting CD19 and BCMA, including an anti-CD19 scFv and an anti-BCMA scFv.

[0246] In another preferred embodiment, the present invention provides a bispecific chimeric antigen receptor targeting both CD19 and BCMA antigens. The CAR structural components simultaneously targeting CD19 and BCMA may include a signal peptide, an anti-CD19 scFv, an anti-BCMA scFv, a hinge region, a transmembrane region, and an intracellular T cell signaling region, wherein the CD19 scFv and the BCMA scFv are linked by a short peptide (G4S)xN. The CAR structure simultaneously targeting CD19 and BCMA is as described in the third aspect of the present invention.

[0247] In another preferred embodiment, the CD19 and BCMA bispecific CAR of the present invention is a single structure comprising scFvs that resist both CD19 and BCMA. The CAR comprises CD19 scFvs and BCMA scFvs, and the ordering and hinge of the CD19 scFvs and BCMA scFvs are the main factors influencing its function.

[0248] This invention utilizes a CAR that bidirectionally targets CD19 and BCMA. Compared to CARs targeting a single antigen, it exhibits significantly enhanced affinity and increased immune cell activity, demonstrating a synergistic effect. Furthermore, due to the heterogeneous expression levels of CD19 and BCMA in tumor cells, the dual-targeting CAR-T therapy has a broader therapeutic scope. Simultaneously targeting CD19 and BCMA with CAR-immune cells can reduce the possibility of antigen escape caused by downregulation or absence of a single surface antigen.

[0249] In a preferred embodiment of the present invention, the present invention utilizes humanized CD19 scFv to construct a bispecific CAR, which can further improve its killing effect and tumor clearance ability.

[0250] Chimeric antigen receptor T cells (CAR-T cells)

[0251] As used herein, the terms “CAR-T cell”, “CAR-T”, and “CAR-T cell of the present invention” include the CAR-T cells included in the third aspect of the present invention.

[0252] CAR-T cells have the following advantages over other T-cell-based therapies: (1) The action of CAR-T cells is not restricted by MHC; (2) Given that many tumor cells express the same tumor antigens, once the CAR gene targeting a certain tumor antigen is constructed, it can be widely used; (3) CAR can utilize both tumor protein antigens and glycolipid non-protein antigens, thus expanding the target range of tumor antigens; (4) Using the patient's own cells reduces the risk of rejection; (5) CAR-T cells have immune memory function and can survive in the body for a long time.

[0253] The present invention provides bispecific CAR-T cells comprising a CAR targeting CD19 and a CAR targeting another tumor target, as described in the eighth aspect of the present invention.

[0254] In another preferred embodiment, the other tumor target is BCMA.

[0255] In a preferred embodiment of the present invention, bispecific CAR-T cells are constructed using humanized CD19 scFv, which can further enhance their killing effect and tumor clearance ability.

[0256] Chimeric antigen receptor NK cells (CAR-NK cells)

[0257] As used herein, the terms "CAR-NK cell," "CAR-NK," and "CAR-NK cell of the present invention" all refer to the CAR-NK cell included in the third aspect of the present invention. The CAR-NK cell of the present invention can be used to treat tumors with high CD19 expression, such as multiple myeloma and lymphoma.

[0258] Natural killer (NK) cells are a major type of immune effector cells that protect the body from viral infections and tumor cell invasion through non-antigen-specific pathways. Engineered (genetically modified) NK cells may acquire new functions, including the ability to specifically recognize tumor antigens and enhanced anti-tumor cytotoxicity.

[0259] Compared with autologous CAR-T cells, CAR-NK cells have the following advantages, such as: (1) they directly kill tumor cells by releasing perforin and granzymes, without killing normal cells in the body; (2) they release very little cytokine, thus reducing the risk of cytokine storm; and (3) they are very easy to expand in vitro and develop into "ready-made" products. In addition, they are similar to CAR-T cell therapy.

[0260] Suicide Gene Switch

[0261] To further control adverse effects such as non-tumor targeting of CAR-T cells and cytokine release syndrome, the CAR-T cells in this invention all carry suicide gene switches. Under the action of exogenous drugs, they can effectively eliminate CAR-T cells in the body and block unknown or uncontrollable long-term toxicity, thereby ensuring the safety of patients.

[0262] The suicide switch used in this invention can be the herpes simplex virus thymidine kinase (HSV-TK), inducible caspase 9 (iCasp9), CD20, mutated human thymidylate kinase (mTMPK), etc. Comparatively, HSV-TK, iCasp9, and CD20 have equivalent clearance abilities against CAR-cells, but iCasp9 and CD20 clear cells more rapidly, while HSV-TK clears cells more slowly.

[0263] The iCasp9 suicide switch contains the FKBP12-F36V domain, which connects to cysteine-aspartic protease 9 via a flexible linker. The latter lacks a recruitment domain. FKBP12-F36V contains an FKBP domain where phenylalanine replaces valine at the 36th amino acid residue. It exhibits high selectivity and sub-nanomolar affinity, enabling it to bind dimerizing ligands such as other inert small molecules like AP1903. Upon addition of these small molecules, dimerization is induced, thereby inducing apoptosis, while having no effect on normal cells lacking the suicide switch.

[0264] The inducible safety switch caspase9 (iCasp9) uses human caspase9 fused with FK506 binding protein (FKBP), which can be induced to form a dimer with a chemical inducer (AP1903 / Rimiducid, Bellicum Pharmaceutical), leading to apoptosis in cells expressing the fusion protein.

[0265] Although CD19 and BCMA are highly expressed in tumor cells, they are also expressed in normal B cells. The engineered immune cells of this invention will attack normal B cells in vivo.

[0266] Controlling the safety of CAR cells has always been a pressing issue. Adding a safety switch to CAR cells is the safest way to terminate their activity. Inducible iCasp9 safety switches can control CAR cell clearance in cases of severe toxicity (CRS / neurotoxicity) or after a patient achieves long-term remission.

[0267] carrier

[0268] The nucleic acid sequence encoding the desired molecule can be obtained using recombination methods known in the art, such as, for example, by screening a library from a cell expressing the gene, by obtaining the gene from a vector known to contain the gene, or by directly isolating the gene from cells and tissues containing the gene using standard techniques. Optionally, the gene of interest can be synthesized.

[0269] This invention also provides vectors in which the expression cassette of this invention is inserted. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses, such as murine leukemia viruses, because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.

[0270] In short, the expression cassette or nucleic acid sequence of this invention is typically operatively linked to a promoter and incorporated into an expression vector. This vector is suitable for replication and integration into eukaryotic cells. A typical cloning vector contains transcription and translation terminators, an initial sequence, and a promoter that can be used to regulate the expression of the desired nucleic acid sequence.

[0271] The expression constructs of the present invention can also be used with standard gene delivery protocols for nucleic acid immunotherapy and gene therapy. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the present invention provides a gene therapy vector.

[0272] This nucleic acid can be cloned into many types of vectors. For example, it can be cloned into vectors including, but not limited to, plasmids, phage particles, phage derivatives, animal viruses, and granules. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0273] Furthermore, the expression vector can be provided to cells in the form of a viral vector. Viral vector technology is well known in the art and has been described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Typically, a suitable vector contains at least one origin of replication functioning in an organism, a promoter sequence, a convenient restriction enzyme site, and one or more optional markers (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0274] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to target cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0275] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. These are typically located in a 30–110 bp region upstream of the start site, although recent studies have shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible to maintain promoter function when an element is inverted or moved relative to another. In the thymidine kinase (TK) promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, individual elements can function cooperatively or independently to initiate transcription.

[0276] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the early promoter of simian virus 40 (SV40), mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Russ's sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Furthermore, the invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered as part of the invention. The use of inducible promoters provides a molecular switch that can turn on the expression of a polynucleotide sequence operatively linked to the inducible promoter when such expression is desired, or turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0277] To assess the expression of CAR peptides or portions thereof, the expression vector introduced into cells may also contain either or both of an optional marker gene or a reporter gene to facilitate the identification and selection of expressing cells from a population of cells seeking transfection or infection via a viral vector. Alternatively, the optional marker may be carried on a separate DNA segment and used in co-transfection procedures. Both the optional marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful optional markers include, for example, antibiotic resistance genes such as neo.

[0278] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Typically, a reporter gene is a gene that is either absent from or expressed by the recipient organism or tissue, and that encodes a polypeptide whose expression is clearly indicated by readily detectable properties such as enzyme activity. After DNA has been introduced into the recipient cells, reporter gene expression is measured at an appropriate time. Suitable reporter genes may include those encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well-known and can be prepared using known techniques or are commercially available. Typically, a construct with at least five flanking regions exhibiting the highest level of reporter gene expression is identified as a promoter. Such promoter regions can be ligated into reporter genes and used to evaluate the ability of reagents to regulate promoter-driven transcription.

[0279] Methods for introducing genes into cells and expressing genes into cells are known in the art. Within the scope of expression vectors, the vector can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0280] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, and so on. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0281] Biological approaches to introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0282] Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, and beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in both in vitro and in vivo is the liposome (e.g., an artificial membrane capsule).

[0283] In the case of using a non-viral delivery system, an exemplary delivery tool is a liposome. Consider using a lipid formulation to introduce nucleic acid into host cells (in vitro, ex vivo, or in vivo). Alternatively, the nucleic acid may be associated with a lipid. Lipid-associated nucleic acid can be encapsulated within the aqueous interior of a liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linker molecule associated with both the liposome and the oligonucleotide, trapped within the liposome, complexed with the liposome, dispersed in a solution containing lipids, mixed with lipids, conjugated with lipids, contained in lipids as a suspension, contained in or complexed with micelles, or otherwise associated with lipids. The lipids, lipid / DNA, or lipid / expression vector associated with the composition are not limited to any specific structure in solution. For example, they may be present in a bilayer structure, as micelles, or have a “collapsed” structure. They may also be simply dispersed in solution, possibly forming aggregates of varying sizes or shapes. Lipids are fatty substances and can be naturally occurring or synthetic lipids. For example, lipids include fat droplets, which occur naturally in the cytoplasm and in compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols and aldehydes.

[0284] In a preferred embodiment of the present invention, the carrier is a lentivirus carrier.

[0285] preparation

[0286] This invention provides a formulation containing CAR-T cells as described in the first aspect of this invention, and a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injectable formulation. Preferably, the concentration of the CAR-T cells in the formulation is 1 × 10⁻⁶. 3 -1×10 8 Cells / ml, more optimal 1×10 4 -1×10 7 Cells / ml

[0287] In one embodiment, the formulation may include buffer solutions such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The formulations of the present invention are preferably formulated for intravenous administration.

[0288] Therapeutic applications

[0289] This invention includes therapeutic applications using cells (e.g., T cells) transduced with a lentiviral vector (LV) encoding the expression cassette of this invention. The transduced T cells can target the tumor cell marker CD19 or CD19 and BCMA, synergistically activating T cells and evoking a T cell immune response, thereby significantly enhancing their efficiency in killing tumor cells.

[0290] Therefore, the present invention also provides a method for stimulating a T-cell-mediated immune response against a target cell population or tissue of a mammal, comprising the step of administering the CAR-T cells of the present invention to a mammal.

[0291] In one embodiment, the present invention includes a type of cell therapy in which the patient's own T cells (or those from a heterologous donor) are isolated, activated, and genetically modified to produce CAR-T cells, which are then injected into the same patient. This method results in an extremely low probability of graft-versus-host disease, and the antigen is recognized by the T cells in an MHC-free manner. Furthermore, a single CAR-T cell can treat all cancers expressing that antigen. Unlike antibody therapy, CAR-T cells can replicate in vivo, producing long-lasting, durable antibodies that lead to sustained tumor control.

[0292] In one embodiment, the CAR-T cells of the present invention can undergo robust in vivo T cell expansion and sustain for an extended period of time. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step, wherein CAR-modified T cells induce an immune response specific to the antigen-binding domain in the CAR. For example, anti-BCMA and / or CD19 CAR-T cells elicit a specific immune response against cells expressing BCMA and / or CD19.

[0293] Although the data disclosed herein specifically disclose lentiviral vectors including anti-CD19 scFv or CD19 and BCMA scFv, hinge and transmembrane regions, and 4-1BB / CD28 and CD3ζ signaling domains, the invention should be construed as including any number of variations in each of the construct components.

[0294] Treatable cancers include tumors that are not vascularized or are substantially not vascularized, as well as vascularized tumors. Cancers may include non-solid tumors (such as hematologic malignancies, such as leukemia and lymphoma) or may include solid tumors. Types of cancers treatable with the CAR of this invention include, but are not limited to, carcinomas, germ cell tumors, and sarcomas, and certain leukemias or lymphomas, benign and malignant tumors, and malignant tumors such as sarcomas, carcinomas, and melanomas. Adult tumors / cancers and childhood tumors / cancers are also included.

[0295] Hematologic cancers are cancers of the blood or bone marrow. Examples of hematologic (or blood-borne) cancers include leukemia, including acute leukemia (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, granulocytic, monocytic, and erythroleukemia), chronic leukemia (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (painless and high-grade forms), multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease, myelodysplastic syndromes, hairy cell leukemia, and spinal dysplasia.

[0296] Solid tumors are abnormal masses of tissue that do not typically contain cysts or fluid-filled areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell types that form them (such as sarcoma, carcinoma, and lymphoma). Examples of solid tumors such as sarcoma and carcinoma include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, malignant lymphoma, pancreatic cancer, and ovarian cancer.

[0297] The CAR-modified T cells of the present invention can also be used as a type of vaccine for in vitro immunization and / or in vivo therapy in mammals. Preferably, the mammal is human.

[0298] For in vitro immunization, at least one of the following occurs in vitro before the cells are administered into a mammal: i) cell expansion, ii) introduction of nucleic acid encoding CAR into the cells, and / or iii) cryopreservation of the cells.

[0299] In vitro procedures are well known in the art and are discussed more fully below. Simply put, cells are isolated from a mammal (preferably human) and genetically modified (i.e., transduced or transfected in vitro) using a vector expressing a CAR disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide therapeutic benefit. The mammalian recipient can be human, and the CAR-modified cells can be autologous relative to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic relative to the recipient.

[0300] In addition to using cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against antigens in a patient.

[0301] The present invention provides a method for treating tumors, comprising administering a therapeutically effective amount of the CAR-modified T cells of the present invention to a subject in need of the treatment.

[0302] The CAR-modified T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or other components such as IL-2, IL-17, or other cytokines or cell populations. In short, the pharmaceutical compositions of the present invention may comprise target cell populations as described herein, combined with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may comprise buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0303] The pharmaceutical compositions of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease—although the appropriate dosage can be determined by clinical trials.

[0304] When referring to "immunologically effective amount," "antitumor effective amount," "tumor-suppressive effective amount," or "therapeutic amount," the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and disease condition. It can generally be indicated that a pharmaceutical composition including T cells described herein can be administered in doses of 10... 4 Up to 10 9 A dose of cells / kg body weight, preferably 10. 5 Up to 10 6 The T-cell composition can be administered at a dose of cells per kg of body weight (including all integer values ​​within those ranges). These doses can also be administered multiple times. The cells can be administered using infusion techniques known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a specific patient can be readily determined by a physician skilled in the medical field by monitoring the patient's disease signs and thus adjusting the treatment accordingly.

[0305] The application of the target composition can be performed in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered to patients subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, intravenously (iv), or intraperitoneally. In one embodiment, the T-cell composition of the present invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T-cell composition of the present invention is preferably administered by intravenous injection. The T-cell composition can be injected directly into the tumor, lymph node, or site of infection.

[0306] In some embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in combination with any number of relevant treatment modalities (e.g., before, simultaneously with, or after), including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or nastatinumab treatment for MS patients or erfaizumab treatment for psoriasis patients or other treatments for PML patients. In further embodiments, the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell composition of the invention is administered to a patient in combination with bone marrow transplantation, chemotherapy agents such as fludarabine, external beam radiotherapy (XRT), or cyclophosphamide (e.g., before, simultaneously with, or after). For example, in one embodiment, the subject may undergo standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives an injection of the expanded immune cells of the present invention after transplantation. In an additional embodiment, the expanded cells are administered before or after surgery.

[0307] The dosage of the above treatments administered to patients will vary depending on the precise nature of the condition being treated and the recipient of the treatment. The dosage ratios administered to individuals can be implemented according to accepted practices in the field. Typically, 1 × 10⁻⁶ ppm can be administered per treatment or per course of treatment. 6 One to 1×10 10 The modified T cells of this invention (e.g., CAR-T20 cells) are administered to the patient via, for example, intravenous infusion.

[0308] The main advantages of this invention include:

[0309] (a) The humanized antibodies screened have similar affinity.

[0310] (b) CAR-T cells constructed from the selected humanized antibody scFv sequence exhibited higher killing capacity than CAR-T cells constructed from FMC63.

[0311] (c) Bispecific CAR-T cells constructed from the selected humanized antibody scFv sequence exhibited higher killing ability against CD19 target cells than bispecific CAR-T cells constructed from FMC63.

[0312] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0313] Example 1: Design of Humanized Antibodies

[0314] Using the FMC63 chimeric antibody against human CD19 antigen as the parent antibody, humanized antibodies against human CD19 antigen were designed by humanizing the backbone region based on the amino acid sequences of its heavy and light chain variable regions.

[0315] Using software, seven humanized light chain variable regions (VL1-VL7, amino acid sequences as shown in SEQ ID NO: 1-7) and ten humanized heavy chain variable regions (VH1-VH10, amino acid sequences as shown in SEQ ID NO: 8-17) were designed and obtained. Combining these light and heavy chain variable regions yielded multiple humanized antibody sequences containing different heavy and light chain variable regions. In subsequent embodiments, the affinity of the humanized antibodies was further tested.

[0316] Example 2: Affinity activities of different antibodies as determined by Biacore:

[0317] Affinity tests were performed on some of the antibodies in Example 1. The composition of the heavy chain variable region and light chain variable region of the antibody to be tested is shown in Table 1.

[0318] The target antibody was expressed in HEK293 cells and purified using Protein A. Surface plasmon resonance (SPR) assays were performed, and antibody immobilization was achieved using the Fc capture method.

[0319] Table 1. Affinity test results of humanized antibodies

[0320] FMC63 FMC63 1.29E-04 4.28E-09 6 VH2+VL3 3.95E-04 4.47E-08 12 VH4+VL3 4.37E-04 5.61E-08 5 VH2+VL2 5.26E-04 6.68E-08 4 VH2+VL1 5.68E-04 6.98E-08 10 VH4+VL1 5.84E-04 8.10E-08 11 VH4+VL2 6.05E-04 8.92E-08 13 VH5+VL1 6.37E-04 9.97E-08 8 VH3+VL2 6.38E-04 1.09E-07 7 VH3+VL1 6.88E-04 1.11E-07 15 VH5+VL3 1.43E-03 1.42E-08 9 VH3+VL3 1.59E-03 1.58E-08 14 VH5+VL2 1.80E-03 1.48E-08 16 VH6+VL4 4.93E-03 2.10E-05 17 negative control 7.15E-03 3.39E-07 2 VH1+VL2 7.38E-03 7.95E-09 1 VH1+VL1 8.08E-03 5.76E-08 3 VH1+VL3 7.94E-02 4.07E-08

[0321] The results showed that the humanized test antibodies VH2+VL3, VH4+VL3, and VH2+VL2 exhibited relatively high affinity. In the table, FMC63 is a chimeric antibody containing a human Fc fragment, and the negative control represents the affinity assay without a capture antibody.

[0322] Example 3: Preparation and expression of CAR-T cells

[0323] Mononuclear cells were isolated from donor blood, and T cells were enriched using a density gradient centrifugation method with Histopaque-1077 (Sigma-Aldrich) (EasySep Human T Cell Enrichment Kit, Stemcell Technologies). T cells were activated and expanded using magnetic beads coupled with anti-CD3 / anti-CD28. X-vivo15 (300 IU / ml rhIL2) was used as the culture medium. All cells were cultured in a 37°C, 5% CO2 incubator.

[0324] CD19-targeting CAR-T cells were constructed using some of the antibodies designed in Example 1. Lentiviral cells were packaged, transfected into T cells, and the expression of CAR on the surface of CAR-T cells was measured by flow cytometry.

[0325] The results are as follows Figure 1 As shown, CAR expression can be detected on the surface of CAR-T cells constructed using each of the test antibodies.

[0326] Example 4: CAR-T cell killing assay

[0327] The killing power of the CAR-T cells constructed in Example 3 was detected by real-time cell analysis (RTCA) using HeLa cells that stably express CD19.

[0328] HeLa-CD19 target cells were cultured overnight, and then effector cells (FMC63 CAR-T cells, humanized CD19 CAR-T cells prepared in Example 3, and untransfected T cells) were mixed with HeLa-CD19 target cells at a ratio of 1:1. The killing effector cells on target cells was detected by RTAC.

[0329] The results are as follows Figure 2As shown, the NT control group (untransfected T cell control group) and the culture medium control group (blank control group) did not kill Hela-CD19 cells, while the CAR-T cells constructed from humanized scFv showed significant killing ability against target cells. Compared with the killing ability of CAR-T cells constructed using FMC63 (FMC63 CAR-T), cells 11# (L3H1), 1# (L1H1), 6# (L2H1), and 16# (L4H6) showed significantly improved killing ability relative to CD19.

[0330] Example 5: Detection of CAR-T cell killing of target cells Nalm6 by Luciferase method

[0331] The cytotoxic ability of tumor target cells labeled with luciferase was detected. The luciferase gene was transferred into target cells, and a stable cell line, Nalm6-Luc, was obtained after cloning and selection. During the experiment, a luciferin substrate was added, and the luciferase reacted with luciferin to produce fluorescence. The activity of luciferase was determined by detecting the intensity of the fluorescence, and the cell survival rate was measured to obtain the cytotoxic effect of each CAR-T cell.

[0332] The results are as follows Figure 3 As shown, CAR-T cells constructed from humanized scFv exhibited significantly higher killing ability against target cells than FMC63-CAR-T cells.

[0333] Example 6 Affinity activity of different antibodies as determined by Biacore

[0334] Affinity tests were performed on some of the antibodies in Example 1. The composition of the heavy chain variable region and light chain variable region of the antibody to be tested is shown in Table 2.

[0335] The target antibody was expressed in HEK293 cells and purified using Protein A. Surface plasmon resonance (SPR) assays were performed, and antibody immobilization was achieved using the Fc capture method.

[0336] Table 2. Affinity test results of humanized antibodies and chimeric antibodies

[0337] FMC63 1.83E+05 2.38E-04 1.30E-09 80.2 VH7+VL6 1.30E+05 5.92E-04 4.57E-09 93.1 VH8+VL6 1.17E+05 3.83E-04 3.26E-09 78.3 VH8+VL5 1.55E+05 6.02E-04 3.87E-09 91.2 VH9+VL5 1.09E+05 1.86E-04 1.70E-09 81 VH10+VL5 1.18E+05 1.82E-04 1.55E-09 87 VH4+VL6 1.29E+05 1.21E-03 9.31E-09 106.8 VH2+VL6 1.79E+05 1.24E-03 6.93E-09 96.9 VH2+VL7 1.81E+05 1.16E-03 6.39E-09 96.4

[0338] The results showed that the affinity of each test antibody was slightly lower than that of the chimeric antibody. Among them, the humanized test antibodies VH9+VL5, VH10+VL5, and VH8+VL6 had relatively high affinity, which was not significantly different from that of the chimeric antibody FMC63.

[0339] Example 7: Detection of antibody binding to target cells

[0340] The binding of some of the antibodies in Example 1 to the surface of CD19 positive target cells SU-DHL-10 and Raji was detected.

[0341] CD19-positive SU-DHL-10 or Raji cell lines were used to bind target cells with humanized antibodies at different concentrations. After binding, the cells were stained with secondary antibodies (fluorescently labeled anti-Fc antibodies). After washing away residual antibodies, the positive rate was analyzed by flow cytometry, and the binding curves of antibodies to cell surface antigens were plotted.

[0342] The results are as follows Figure 4 As shown, all the antibodies tested could bind to CD19-positive target cells, and their binding ability was not significantly different from that of FMC63.

[0343] Example 8: Preparation and expression of CAR-T cells

[0344] Using some of the antibodies designed in Example 1, humanized CAR-T cells were constructed using a method similar to that in Example 3.

[0345] The expression of CAR was detected using different detection methods (protein L, anti-FMC63 antibody, CD19 antigen), and the expression rates were compared.

[0346] The results are as follows Figure 5 As shown, different levels of CAR expression can be detected on the surface of CAR-T cells constructed using each of the test antibodies.

[0347] Example 10: Detection of CAR-T cell killing by RTCA method

[0348] Using a method similar to that in Example 4, the killing effect of the CAR-T cells constructed in Example 8 on target cells Hela-CD19 was detected by real-time cell analysis (RTCA).

[0349] The results are as follows Figure 6 As shown, CAR-T cells constructed from humanized scFv exhibited significant killing ability against target cells, and some humanized CAR-T cells showed superior killing ability compared to FMC63 CAR-T cells.

[0350] Example 11 Detection of CAR-T cell killing by Luciferase method

[0351] Using a method similar to that in Example 5, the killing effect of the CAR-T cells constructed in Example 8 on target cells Nalm6 and Raji was detected.

[0352] The results are as follows Figure 7As shown, CAR-T cells constructed from humanized scFv exhibited significantly higher killing ability against target cells than FMC63-CAR-T cells.

[0353] Example 12: Release of IFNγ during CAR-T cell killing of target cells Nalm6 and Raji

[0354] After the target cell killing experiment in Example 8 was completed, the final supernatant was taken and the cytokine IFNγ was measured by ELISA.

[0355] The results are as follows Figure 8 As shown, CAR-T cells constructed from partially humanized scFv produced high levels of INF-γ release in target cells Nalm6 and Raji.

[0356] Example 13: Detection of in vivo cytotoxic activity

[0357] NOG mice aged 6-12 weeks were selected and subcutaneously injected with 3×10 5 Raji cells. Six days later, tumor graft burden was assessed, and mice were divided into groups with comparable tumor burdens. One day after grouping, each group was injected with the CAR-T cells constructed in Example 8. Tumor volume burden in mice was evaluated after CAR-T treatment. Each mouse received an intraperitoneal injection of 3 mg d-luciferin (Perkin Elmer Life Sciences), and four minutes later, images were taken using the Xenogen IVIS Imaging System (Perkin Elmer Life Sciences) with a 30-second exposure. The bioluminescent signal was calculated based on the emitted photon quantity, which was normalized using exposure time and surface area to obtain the final photon quantity / s / cm². 2 / Spherical angle (p / s / cm) 2 / sr).

[0358] The results showed that CAR-T cells modified with humanized CD19 antibodies VH9+VL5, VH10+VL5, and VH8+VL6 could eliminate tumors in Raji cell-induced subcutaneous mouse models and prolong the survival of these mice. Among them, CAR-T cells modified with humanized CD19 antibody VH9+VL5 showed the best effect. Figure 9 As shown, it can more effectively eliminate tumors in Raji cell-induced model mice, and its anti-tumor effect is superior to that of FMC63-modified CAR-T cells.

[0359] Example 14 Non-specific detection of CAR-T cells

[0360] The non-specific killing effect of CAR-T cells constructed in Example 8 was detected using the Lucierase method. Raji-KO19 and Nalm6-KO19 cells with CD19 knockout, as well as K562 and CCRF cells that do not express CD19 on their cell surface, were used as target cells to detect the killing effect of CAR-T cells on these cells.

[0361] The results are as follows Figure 10 As shown, CAR-T cells modified with humanized CD19 antibody did not exhibit non-specific killing and did not kill cells that do not express CD19.

[0362] Example 15 Humanized dual CAR in animals

[0363] Dual CAR-T cells targeting both BCMA and CD19 were constructed using humanized scFv and FMC63, and BCMA antibody, respectively, and their in vivo cytotoxic activity was assessed. The structure of the dual CAR is shown below. Figure 12 As shown in A. The heavy chain variable region of BCMA scFv is shown in SEQ ID NO:21, and the light chain variable region is shown in SEQ ID NO:20.

[0364] NOG mice aged 6-12 weeks were selected and subcutaneously injected with 3×10 5 Raji cells. Six days later, tumor graft burden was assessed, and mice were divided into groups with comparable tumor burdens. One day after grouping, each group was injected with the prepared dual CAR-T cells. Tumor volume burden was evaluated after CAR-T treatment. Each mouse received an intraperitoneal injection of 3 mg d-luciferin (Perkin Elmer Life Sciences), and four minutes later, images were taken using the Xenogen IVIS Imaging System (Perkin Elmer Life Sciences) with a 30-second exposure. The bioluminescent signal was calculated based on the emitted photon quantity, which was normalized using exposure time and surface area to obtain the final photon quantity / s / cm². 2 / Spherical angle (p / s / cm) 2 / sr).

[0365] The results showed that bispecific CAR-T cells modified with humanized CD19 antibodies VH9+VL5, VH10+VL5, and VH8+VL6 all exhibited stronger ability to eliminate tumors in Raji cell-induced mouse models than FMC63-modified bispecific CAR-T cells (Dual CAR-T), with essentially comparable antitumor effects. Among them, the bispecific CAR-T cells modified with humanized CD19 antibody VH9+VL5 showed the best effect. Figure 11 As shown, this demonstrates its significant anti-tumor efficacy.

[0366] Example 16: Comparison of the killing power of humanized dual CAR-T and mouse-derived dual CAR-T.

[0367] Bispecific CAR-T cells targeting both BCMA and CD19 were constructed using humanized CD19 scFv (VH9+VL5) and FMC63, respectively, and BCMA antibody. The structure of the bispecific CAR is shown below. Figure 12 As shown in B and 12C. The heavy chain variable region of BCMA scFv is shown in SEQ ID NO:21, and the light chain variable region is shown in SEQ ID NO:20.

[0368] Using a method similar to that in Example 5, the killing ability of the constructed bispecific CAR-T cells was detected by using luciferase-labeled tumor target cells.

[0369] The results are as follows Figure 13 As shown, the humanized scFv-constructed dual CAR-T cells exhibited significantly higher killing ability against target cells than the murine dual CAR-T cells.

[0370] Example 17: Production and Expression Testing of Humanized Universal Dual CAR-T

[0371] Using the humanized dual CAR-T cells and mouse dual CAR-T cells constructed in Example 16, and by knocking out the TRAC and B2M genes of T cells, humanized universal CAR-T cells were constructed.

[0372] Using a method similar to that in Example 3, different detection methods (anti-FMC63 antibody, CD19 antigen) were used to detect CAR expression and determine the expression rate. Anti-B2M and CD3 antibodies were used to determine the knockout efficiency.

[0373] The results are as follows Figure 14 As shown, the TRAC and B2M genes of universal CAR-T cells are knocked out, while CAR cells can express BCMA and humanized CD19.

[0374] Example 18 Killing ability of humanized universal dual CAR-T cells and non-universal CAR-T cells

[0375] Using a method similar to that in Example 5, the killing ability of the dual CAR-T cells constructed in Example 17 was tested using luciferase-labeled tumor target cells.

[0376] The results are as follows Figure 15 As shown, universal dual CAR-T cells exhibit similar killing ability to non-universal CAR-T cells.

[0377] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims. sequence list <110> Gerniscin Biotech (Shanghai) Co., Ltd. <120> Humanized CD19 Antibody and Its Application <130> P202-1937 <150> CN202010374769.5 <151> 2020-05-06 <160> 30 <170> PatentIn version 3.5 <210> 1 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Humanized light chain variable region L1 <400> 1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 2 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Humanized light chain variable region L2 <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 3 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Humanized light chain variable region L3 <400> 3 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 4 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Humanized light chain variable region L4 <400> 4 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 5 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Humanized light chain variable region L5 <400> 5 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 6 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Humanized light chain variable region L6 <400> 6 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gly Ala Val Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 7 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Humanized light chain variable region L7 <400> 7 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 8 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Humanized heavy chain variable region H1 <400> 8 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 9 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Humanized heavy chain variable region H2 <400> 9 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 10 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Humanized heavy chain variable region H3 <400> 10 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 11 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Humanized heavy chain variable region H4 <400> 11 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 12 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Humanized heavy chain variable region H5 <400> 12 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 13 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Humanized heavy chain variable region H6 <400> 13 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ala Ala Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 14 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Humanized heavy chain variable region H7 <400> 14 Gln Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 15 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Humanized heavy chain variable region H8 <400> 15 Gln Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 16 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Humanized heavy chain variable region H9 <400> 16 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 17 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Humanized heavy chain variable region H10 <400> 17 Gln Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 18 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> FMC63 chimeric antibody light chain variable region <400> 18 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr 100 105 <210> 19 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> FMC63 chimeric antibody heavy chain variable region <400> 19 Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30 Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95 Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 20 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> BCMA scFv light chain variable region <400> 20 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Leu Ala Ser Glu Asp Ile Ser Asp Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Val 35 40 45 Tyr Thr Thr Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Thr Tyr Lys Phe Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 <210> 21 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> BCMA scFv heavy chain variable region <400> 21 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Asn Ser Gly Tyr Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Ala Thr Met Thr Ala Asp Lys Ser Ile Asn Thr Ala Tyr 65 70 75 80 Val Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys 85 90 95 Thr Arg Tyr Met Trp Glu Arg Val Thr Gly Phe Phe Asp Phe Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> twenty two <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Linking peptides <400> twenty two Gly Gly Gly Gly Ser 1 5 <210> twenty three <211> twenty one <212> PRT <213> Artificial Sequence <220> <223> CD8 signal peptide <400> twenty three Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> twenty four <211> 45 <212> PRT <213> Artificial Sequence <220> <223> CD8 hinge area <400> twenty four Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 25 <211> twenty four <212> PRT <213> Artificial Sequence <220> <223> CD8 transmembrane region <400> 25 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys 20 <210> 26 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> CD28 transmembrane region <400> 26 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25 <210> 27 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> 41BB signal area <400> 27 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 28 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> CD28 signal area <400> 28 Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr 1 5 10 15 Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30 Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 <210> 29 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> CD3z signaling region <400> 29 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 30 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Self-cleaving 2A peptide <400> 30 Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val 1 5 10 15 Glu Glu Asn Pro 20

Claims

1. A humanized CD19 antibody, said antibody comprising: (a) Antibody light chain variable region (VL), which is shown in the amino acid sequence of SEQ ID NO:5; and (b) Antibody heavy chain variable region (VH), which is shown in the amino acid sequence of SEQ ID NO:

16.

2. A chimeric antigen receptor (CAR) targeting CD19, wherein the antigen-binding domain (scFv) of the CAR is the humanized CD19 antibody of claim 1.

3. A bispecific CAR, characterized in that, The bispecific CAR targets CD19 and the first target. Wherein, the antigen-binding domain targeting CD19 in the bispecific CAR is the humanized CD19 antibody as described in claim 1; Furthermore, the first target is selected from the following group: CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, C EACAM6, c-Met, EGFRvIII, ErbB2 / HER2, ErbB3, HER-2, HER3, ErbB4 / HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, PSCA, HVEM, MAGE-A, MSLN, N Y-ESO-1, PSMA, RANK, RORl, TNFRSF4, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, MUC16, TCRα, TCRβ, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, Notch-1-4, APRIL, CS1, MAGE3, Claudin 18.2, Folate receptorα, Folate receptorβ, GPC2, CD70, BAFF-R, TROP-2, or combinations thereof.

4. The bispecific CAR as described in claim 3, characterized in that, The structure of the bispecific CAR is shown in formula III or III' below: L-V L3 -scFv3-V H3 -H-TM-C-CD3ζ (III) L-V H3 -scFv3-V L3 -H1-TM-C-CD3ζ (III’) In the formula, Each "-" independently represents a linking peptide or peptide bond; L represents the absence of a signal peptide sequence; H represents the hinge area; TM represents a transmembrane domain; C is a co-stimulatory signaling molecule; CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ; scFv3 is an antigen-binding domain targeting CD19, V H3 For the variable region of the heavy chain of the antibody against the first target, and V L3 The variable region of the light chain of the antibody against the first target; Alternatively, scFv3 may be an antigen-binding domain targeting the first target, V H3 It is the variable region of the heavy chain of the anti-CD19 antibody, and V L3 This is the variable region of the light chain of the anti-CD19 antibody.

5. The bispecific CAR as described in claim 3, characterized in that, The structure of the bispecific CAR is shown in Formula II below: L-scFv1-I-scFv2-H-TM-C-CD3ζ (II) In the formula, Each "-" independently represents a linking peptide or peptide bond; L represents the absence of a signal peptide sequence; I represents a flexible joint; H represents the hinge area; TM represents a transmembrane domain; C is a co-stimulatory signaling molecule; CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ; Of the two, scFv1 and scFv2, one is an antigen-binding domain that targets the first target, and the other is an antigen-binding domain that targets CD19.

6. The bispecific CAR as described in claim 3, characterized in that, The bispecific CAR includes a first CAR targeting a first target and a second CAR targeting CD19, wherein the first CAR and the second CAR are connected by a self-cleaving element.

7. A polynucleotide, characterized in that, The polynucleotide encodes the humanized CD19 antibody of claim 1, the chimeric antigen receptor targeting CD19 of claim 2, or the bispecific CAR of claim 3.

8. An engineered immune cell, characterized in that, The immune cell chromosomes of the aforementioned immune cells are integrated with exogenous nucleic acid molecules as described in claim 7, or express humanized CD19 antibody as described in claim 1, chimeric antigen receptor targeting CD19 as described in claim 2, or bispecific CAR as described in claim 3.

9. The engineered immune cells as described in claim 8, characterized in that, The engineered immune cells include universal CAR-T cells.

10. The engineered immune cells as described in claim 9, characterized in that, The TRAC and B2M genes of the universal CAR-T cells were knocked out.

11. An engineered immune cell, characterized in that, The immune cells contain an exogenous first expression cassette and a second expression cassette, wherein the first expression cassette is used to express a first CAR targeting a first target, and the second expression cassette is used to express a second CAR targeting CD19; Alternatively, the immune cells may express the first CAR targeting the first target and the second CAR targeting CD19; Wherein, the antigen-binding domain (scFv) targeting CD19 in the second CAR is the humanized CD19 antibody as described in claim 1; Furthermore, the first target is selected from the following group: CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, C EACAM6, c-Met, EGFRvIII, ErbB2 / HER2, ErbB3, HER-2, HER3, ErbB4 / HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, TGFBR2, TGFBR1, IL6R, gp130, Lewis, TNFR1, TNFR2, PD1, PD-L1, PD-L2, PSCA, HVEM, MAGE-A, MSLN, N Y-ESO-1, PSMA, RANK, RORl, TNFRSF4, TWEAK-R, LTPR, LIFRP, LRP5, MUC1, MUC16, TCRα, TCRβ, TLR7, TLR9, PTCH1, WT-1, Robol, Frizzled, OX40, Notch-1-4, APRIL, CS1, MAGE3, Claudin 18.2, Folate receptorα, Folate receptorβ, GPC2, CD70, BAFF-R, TROP-2, or combinations thereof.

12. A formulation, characterized in that, The formulation contains the humanized CD19 antibody of claim 1, or the engineered immune cells of claim 8 or 11, and a pharmaceutically acceptable carrier.

13. A formulation, characterized in that, The formulation contains the humanized CD19 antibody of claim 1, or the engineered immune cells of claim 8 or 11, and a diluent.

14. A formulation, characterized in that, The formulation contains the humanized CD19 antibody of claim 1, or the engineered immune cells of claim 8 or 11, and excipients.

15. The use of the humanized CD19 antibody of claim 1, the chimeric antigen receptor targeting CD19 of claim 2, the bispecific CAR of claim 3, or the engineered immune cells of claim 8 or 11, characterized in that, Used to prepare drugs or preparations for the prevention and / or treatment of lymphoma.

Images