Cd19-targeting humanized antibody and chimeric antigen receptor, and use thereof

Abstract

The invention provides a humanized antibody targeting CD19, a chimeric antigen receptor and application of the humanized antibody, and the humanized antibody comprises CD19VH and CD19VL which are selected from one of the first group to the eighth group. The chimeric antigen receptor targeting CD19 comprises an extracellular antigen recognition structural domain targeting CD19, a hinge region, a transmembrane region and an intracellular structural domain, the extracellular antigen recognition structural domain targeting CD19 comprises CD19VH and CD19VL, and the CD19VH and the CD19VL are selected from one of the first group to the eighth group.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202411958226.2 (22) Application Date 2024.12.27 (71) Applicant Heyuan Kanghua Pharmaceutical Technology (Beijing) Co., Ltd. Address 102204, Beijing Changping District, Liucun Town, Beiliu Village, Jia 6-587 (72) Inventors Li Zhu, Zheng Yaling, Zhang Chao, Wang Huanyu, Zhang Xuehan, Shi Lin, Wang Yongzeng, Lü Lulu (74) Patent Agency Beijing Beixiang Intellectual Property Agency Co., Ltd. 11285 Patent Attorney Zhang Yong, Zhang Guangyu (51) Int.Cl. C07K 16 / 28 (2006.01) C07K 19 / 00 (2006.01) C12N 15 / 13 (2006.01) C12N 15 / 62 (2006.01) C12N 15 / 867(2006.01) C12N 5 / 10(2006.01) A61K 39 / 395(2006.01) A61K 40 / 11(2025.01) A61K 40 / 15(2025.01) A61K 40 / 31(2025.01) A61K 40 / 42(2025.01) A61P 35 / 00(2006.01) A61P 35 / 02(2006.01) A61P 37 / 02(2006.01) A61P 13 / 12(2006.01) A61P 7 / 06(2006.01) A61P 7 / 04(2006.01) A61P 9 / 14 (2006.01) A61P 17 / 00 (2006.01) A61P 29 / 00 (2006.01) A61P 21 / 04 (2006.01) A61K 47 / 68 (2017.01) (54) Invention Title: A humanized antibody targeting CD19, a chimeric antigen receptor and its uses (57) Abstract: This invention provides a humanized antibody targeting CD19, a chimeric antigen receptor and its uses, wherein the humanized antibody comprises CD19VH and CD19VL, selected from groups 1) to 8). The chimeric antigen receptor targeting CD19 of this invention comprises an extracellular antigen recognition domain, a hinge region, a transmembrane region and an intracellular domain targeting CD19, wherein the extracellular antigen recognition domain targeting CD19 comprises CD19VH and CD19VL, selected from groups 1) to 8). Claims (5 pages), Description (19 pages), Serial List (electronic publication), Drawings (5 pages), CN 122302055 A, 2026.06.30, CN 1 22 30 20 55 A1. A humanized antibody targeting CD19 or an antigen-binding fragment thereof, comprising CD19 VH and CD19 VL, selected from one of the following: 1) the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:1, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:2; or 2) the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:3, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:2; or 3) the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:4, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:5; or 4) the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:4, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:6; or 5) the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:3, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:7; or 6) the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:3, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:7; or 7) the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:4, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:5; or 8) the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:6; or 9) the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:7; or 10) the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:4; or 11) the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:4; or 12) the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:5; or 13) the CD19 VH sequence comprises the amino 1. The CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:8, wherein the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:9; or 7) the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:10, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:11; or 8) the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:8, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:11. 2. The humanized antibody or antigen-binding fragment thereof according to claim 1, wherein the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:4, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:6; or the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:10, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:11. 3. The humanized antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein it is an scFv antibody, an sc(Fv)2 antibody, or a [sc(Fv)2]2 antibody. 4. The humanized antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein it is an scFv antibody as shown in any one of SEQ ID NO:31-38. 5. An antibody drug comprising the humanized antibody or antigen-binding fragment thereof according to any one of claims 1-4.6. An antibody-drug conjugate comprising any one of claims 1-4 of the humanized antibody or its antigen-binding fragment. 7. A chimeric antigen receptor targeting CD19, comprising an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain targeting CD19, wherein the extracellular antigen recognition domain targeting CD19 comprises CD19VH and CD19VL, selected from one of the following: 1) the CD19VH sequence comprises the amino acid sequence shown in SEQ ID NO:1, and the CD19VL sequence comprises the amino acid sequence shown in SEQ ID NO:2; or 2) the CD19VH sequence comprises the amino acid sequence shown in SEQ ID NO:3, and the CD19VL sequence comprises the amino acid sequence shown in SEQ ID NO:2; or 3) the CD19VH sequence comprises the amino acid sequence shown in SEQ ID NO:4, and the CD19VL sequence comprises the amino acid sequence shown in SEQ ID NO:5; or 4) the CD19VH sequence comprises the amino acid sequence shown in SEQ ID NO:4, and the CD19VL sequence comprises the amino acid sequence shown in SEQ ID NO:5. The amino acid sequence shown in SEQ ID NO:6; or 5) the CD19 VH sequence includes the amino acid sequence shown in SEQ ID NO:3, and the CD19 VL sequence includes the amino acid sequence shown in SEQ ID NO:7; or 6) the CD19 VH sequence includes the amino acid sequence shown in SEQ ID NO:8, and the CD19 VL sequence includes the amino acid sequence shown in SEQ ID NO:9; or 7) the CD19 VH sequence includes the amino acid sequence shown in SEQ ID NO:10, and the CD19 VL sequence includes the amino acid sequence shown in SEQ ID NO:11; or 8) the CD19 VH sequence includes the amino acid sequence shown in SEQ ID NO:8, and the CD19 VL sequence includes the amino acid sequence shown in SEQ ID NO:11. 8. The chimeric antigen receptor according to claim 7, wherein the extracellular antigen recognition domain comprises any one of the following structures: amino acid sequence-linking sequence as shown in SEQ ID NO:1-amino acid sequence as shown in SEQ ID NO:2; amino acid sequence-linking sequence as shown in SEQ ID NO:2-amino acid sequence as shown in SEQ ID NO:1; amino acid sequence-linking sequence as shown in SEQ ID NO:3-amino acid sequence as shown in SEQ ID NO:2; amino acid sequence-linking sequence as shown in SEQ ID NO:2-amino acid sequence as shown in SEQ ID NO:2-amino acid sequence as shown in SEQ ID NO:1.The amino acid sequence shown in SEQ ID NO:3, the amino acid sequence shown in SEQ ID NO:4-linked sequence, the amino acid sequence shown in SEQ ID NO:5, the amino acid sequence shown in SEQ ID NO:4-linked sequence, the amino acid sequence shown in SEQ ID NO:6, the amino acid sequence shown in SEQ ID NO:6-linked sequence, the amino acid sequence shown in SEQ ID NO:4, the amino acid sequence shown in SEQ ID NO:3-linked sequence, the amino acid sequence shown in SEQ ID NO:7, the amino acid sequence shown in SEQ ID NO:7-linked sequence, the amino acid sequence shown in SEQ ID NO:3, the amino acid sequence shown in SEQ ID NO:8-linked sequence, the amino acid sequence shown in SEQ ID NO:9, the amino acid sequence shown in SEQ ID NO:9-linked sequence, the amino acid sequence shown in SEQ ID NO:8, the amino acid sequence shown in SEQ ID NO:10-linked sequence, the amino acid sequence shown in SEQ ID NO:11, the amino acid sequence shown in SEQ ID NO:11-linked sequence, the amino acid sequence shown in SEQ ID NO:10, and so on. The amino acid sequence NO:8-linked sequence-amino acid sequence as shown in SEQ ID NO:11, or the amino acid sequence as shown in SEQ ID NO:11-linked sequence-amino acid sequence as shown in SEQ ID NO:8; Optionally, the extracellular antigen recognition domain includes any one of the following structures: amino acid sequence as shown in SEQ ID NO:4-linked sequence-amino acid sequence as shown in SEQ ID NO:6, amino acid sequence as shown in SEQ ID NO:6-linked sequence-amino acid sequence as shown in SEQ ID NO:4, amino acid sequence as shown in SEQ ID NO:10-linked sequence-amino acid sequence as shown in SEQ ID NO:11, or amino acid sequence as shown in SEQ ID NO:11-linked sequence-amino acid sequence as shown in SEQ ID NO:10; Further optionally, the extracellular antigen recognition domain includes any one of the following structures: amino acid sequence as shown in SEQ ID NO:6-linked sequence-amino acid sequence as shown in SEQ ID NO:4, or amino acid sequence as shown in SEQ ID NO:11-linked sequence-amino acid sequence as shown in SEQ ID NO:10. 9. The chimeric antigen receptor according to claim 8, wherein the linker sequence is selected from one or more of the following sequences: SEQ IDNO:23 and SEQ ID NO:24. 10. The chimeric antigen receptor according to claim 9, wherein the extracellular antigen recognition domain comprises: an scFv antibody as shown in any one of SEQ ID NO:31-38. Claims 2 / 5 Page 3 CN 122302055 A 11. The chimeric antigen receptor according to claim 7, wherein the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α; optionally, the amino acid sequence of the hinge region is derived from CD8α; further optionally, the amino acid sequence of the hinge region comprises the amino acid sequence shown in SEQ ID NO:25; and / or the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70; optionally, the amino acid sequence of the transmembrane region is derived from CD8α; further optionally, the amino acid sequence of the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:26. 12. The chimeric antigen receptor according to claim 7, wherein the intracellular domain comprises an intracellular signal transduction region; optionally, the intracellular signal transduction region is derived from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CCD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk; further optionally, the intracellular signal transduction region is derived from CD3ζ; and even more optionally, the amino acid sequence of the intracellular signal transduction region comprises the amino acid sequence shown in SEQ ID NO: 28. 13. The chimeric antigen receptor according to claim 7, wherein the intracellular domain further comprises a co-stimulatory signal transduction region; optionally, the co-stimulatory signal transduction region is derived from one, two, or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, and MyD88; further optionally, the co-stimulatory signal transduction region is derived from 4-1BB; even further optionally, the amino acid sequence of the co-stimulatory signal transduction region comprises the amino acid sequence shown in SEQ ID NO:27. 14. The chimeric antigen receptor according to any one of claims 7-13, further comprising a guide peptide located at the N-terminus of the chimeric antigen receptor amino acid sequence; optionally, the guide peptide is derived from CD8α; further optionally, the amino acid sequence of the guide peptide comprises the amino acid sequence shown in SEQ ID NO:29.15. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a humanized antibody or an antigen-binding fragment thereof as described in any one of claims 1-4, or comprising a nucleotide sequence encoding a chimeric antigen receptor as described in any one of claims 7-14; Optionally, the nucleotide sequence encoding the humanized antibody or an antigen-binding fragment thereof or the nucleotide sequence encoding the chimeric antigen receptor comprises a nucleotide sequence encoding CD19 VH and CD19 VL, selected from a group consisting of: 1) a nucleotide sequence encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:1, as shown in SEQ ID NO:12; and a nucleotide sequence encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:2, as shown in SEQ ID NO:13; and / or 2) a nucleotide sequence encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:3, as shown in SEQ ID NO:14; and a nucleotide sequence encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:2, as shown in SEQ ID NO:13; and / or 3) a nucleotide sequence encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:4. The nucleotide sequence encoding the VH amino acid sequence is shown in SEQ ID NO:15; and the nucleotide sequence encoding the CD19 VL amino acid sequence is shown in SEQ ID NO:16; and / or 4) the nucleotide sequence encoding the CD19 VH amino acid sequence is shown in SEQ ID NO:4, as shown in SEQ ID NO:15; and the nucleotide sequence encoding the CD19 VL amino acid sequence is shown in SEQ ID NO:6, as shown in SEQ ID NO:17; and / or 5) the nucleotide sequence encoding the CD19 VH amino acid sequence is shown in SEQ ID NO:3, as shown in SEQ ID NO:14; and the nucleotide sequence encoding the CD19 VL amino acid sequence is shown in SEQ ID NO:7, as shown in SEQ ID NO:18; and / or the nucleotide sequence encoding the CD19 VH amino acid sequence is shown in SEQ ID NO:8, as shown in SEQ ID NO:19; and the nucleotide sequence encoding the CD19 VL amino acid sequence is shown in SEQ ID NO:9. The nucleotide sequence encoding the VL amino acid sequence, as shown in SEQ ID NO:20; and / or 7) the nucleotide sequence encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:10, as shown in SEQ ID NO:21; and the nucleotide sequence encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:11, as shown in SEQ ID NO:20.16. A vector comprising the isolated nucleic acid molecule of claim 15; optionally, the vector is an expression vector; further optionally, the vector is a viral vector; and even more optionally, the vector is a lentiviral vector. 17. An engineered immune effector cell comprising the humanized antibody or antigen-binding fragment thereof of any one of claims 1-4, the chimeric antigen receptor of any one of claims 7-14, the isolated nucleic acid of claim 15, or the vector of claim 16. 18. The engineered immune effector cells of claim 17, wherein the engineered immune effector cells are selected from one or more of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMCs), pluripotent stem cells, T cells differentiated from pluripotent stem cells, NK cells differentiated from pluripotent stem cells, and embryonic stem cells; optionally, the engineered immune effector cells are T lymphocytes; further optionally, the T lymphocytes are derived from autologous T lymphocytes or allogeneic T lymphocytes. 19. A pharmaceutical composition comprising the engineered immune effector cells of claim 17 or 18 and pharmaceutically acceptable excipients; optionally, the pharmaceutically acceptable excipients include protective agents; optionally, the pharmaceutically acceptable excipients include cell cryopreservation solutions. 20. The pharmaceutical composition of claim 19, wherein the pharmaceutical composition is a cell suspension or cryopreserved cells thereof; or the pharmaceutical composition is an intravenous injection. 21. The use of the humanized antibody or antigen-binding fragment thereof according to any one of claims 1-4, the chimeric antigen receptor according to any one of claims 7-14, the isolated nucleic acid according to claim 15, the vector according to claim 16, or the engineered immune effector cell according to claim 17 in the preparation of a medicament for treating diseases or conditions associated with CD19 expression. 22. The use according to claim 21, wherein the diseases or conditions associated with CD19 expression include non-solid tumors, acute lymphoblastic leukemia, and / or B-cell lymphoma; optionally, the non-solid tumors include leukemia and / or lymphoma; optionally, the acute lymphoblastic leukemia includes adult acute lymphoblastic leukemia and / or childhood acute lymphoblastic leukemia; optionally, the B-cell lymphoma includes non-Hodgkin's lymphoma. 23. The use according to claim 21, wherein the diseases or conditions associated with CD19 expression include refractory diseases.Systemic lupus erythematosus immune thrombocytopenic purpura (SLE-ITP), refractory lupus nephritis (LN), refractory immune hemolytic anemia (AIHA), systemic lupus erythematosus (SLE), autoimmune thrombocytopenic purpura (ITP), antineutrophil cell antibody-associated vasculitis (AAV), dermatomyositis (DM), myasthenia gravis (MG), or antisynthetic antibody syndrome (ASS). Claims 5 / 5 pages 6 CN 122302055 A A humanized antibody targeting CD19, a chimeric antigen receptor, and its uses Technical Field

[0001] This application relates to the field of biomedicine, specifically to a humanized antibody targeting CD19, a chimeric antigen receptor, and its uses. Background Technology

[0002] Cell therapy is an emerging medical technology. Its main principle is to utilize the patient's own immune cells, which are cultured and modified in vitro to give them the ability to specifically kill tumor cells. These modified immune cells are then reinfused into the patient to treat tumors. Among them, chimeric antigen receptor T cell (CAR-T) technology is an important cell therapy technology. It uses genetic engineering to make T cells express a specific antibody, thereby enabling T cells to specifically recognize and kill tumor cells expressing the corresponding antigen. Tumor immunotherapy is a treatment method that uses the body's own immune system to fight tumors. Its main goal is to enhance the body's immune system's ability to recognize and kill tumor cells. Among them, CAR-T cell therapy is an important tumor immunotherapy method. It directly introduces T cells with specific killing capabilities into the patient's body to achieve specific killing of tumor cells. Genetic engineering technology is an important part of modern biotechnology. It achieves genetic modification of organisms by cutting, connecting, and transferring genes. In CAR-T cell therapy, genetic engineering technology is widely used to modify T cells to give them specific anti-tumor capabilities.

[0003] CD19 is a CD molecule (i.e., leukocyte differentiation antigen) expressed by B cells. All B cell lines except plasma cells, malignant B cells, and FDCs (follicular dendritic cells) express this molecule. It is an important membrane antigen involved in B cell proliferation, differentiation, activation, and antibody production, and can also promote BCR signal transduction. CD19 acts as a co-receptor in B cell activation and signal transduction, regulating B cell activation and proliferation, participating in B cell signal transduction, and mediating T cell killing of target cells.

[0004] Currently, most antibodies or CARs targeting CD19 contain murine antibodies, which have high immunogenicity in humans and easily induce human anti-mouse antibodies (HAMA).Antibody reactions can induce rejection in patients, reduce efficacy, and increase relapse rates. Humanized antibodies or humanized CAR-T cells may help reduce immunogenicity, decrease immune rejection in patients, maintain the long-term presence of humanized antibodies or CAR-T cells in patients, and improve long-term treatment efficacy.

[0005] This application provides a humanized antibody targeting CD19, a chimeric antigen receptor, and its uses. Based on existing CD19 murine antibody sequences, the inventors designed eight humanized CD19 antibodies to reduce their heterogeneity. However, during the humanization process, the antibody's functionality (e.g., CAR positivity expression, in vitro killing activity, cytokine release, etc.) may be compromised. Therefore, obtaining humanized antibodies that reduce immunogenicity without sacrificing functionality is of practical significance. The inventors used eight humanized CD19 antibodies as the extracellular antigen recognition domain of CD19 CARs and evaluated the proliferation and function of CAR-T cells using humanized CD19 scFv antibodies through in vitro experiments. From these, they screened out the two humanized CD19 scFv antibodies with the best overall performance.

[0006] The present invention provides a humanized antibody targeting CD19 or an antigen-binding fragment thereof, comprising CD19VH and CD19VL, selected from one of the following:

[0007] 1) The CD19VH sequence comprises the amino acid sequence shown in SEQ ID NO:1, and the CD19VL sequence comprises the amino acid sequence shown in SEQ ID NO:2; or

[0008] 2) The CD19VH sequence comprises the amino acid sequence shown in SEQ ID NO:3, and the CD19VL sequence comprises the amino acid sequence shown in SEQ ID NO:2; or

[0009] 3) The CD19VH sequence comprises the amino acid sequence shown in SEQ ID NO:4, and the CD19VL sequence comprises the amino acid sequence shown in SEQ ID NO:5; or

[0010] 4) The CD19VH sequence comprises the amino acid sequence shown in SEQ ID NO:4, and the CD19VL sequence comprises the amino acid sequence shown in SEQ ID NO:5. The amino acid sequence shown in NO:6; or

[0011] 5) the CD19 VH sequence includes the amino acid sequence shown in SEQ ID NO:3, and the CD19 VL sequence includes the amino acid sequence shown in SEQ ID NO:7; or

[0012] 6) the CD19 VH sequence includes the amino acid sequence shown in SEQ ID NO:8, and the CD19 VL sequence includes the amino acid sequence shown in SEQ ID NO:9; or

[0013] 7) the CD19The VH sequence comprises the amino acid sequence shown in SEQ ID NO:10, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:11; or

[0014] 8) the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:8, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:11.

[0015] In a preferred embodiment of the present invention, the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:4, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:6; or

[0016] the CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:10, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:11.

[0017] In a preferred embodiment of the present invention, the humanized antibody targeting CD19 is an scFv antibody, an sc(Fv)2 antibody, or a [sc(Fv)2]2 antibody.

[0018] In a preferred embodiment of the present invention, the humanized antibody targeting CD19 is an scFv antibody as shown in any of SEQ ID NO:31-38.

[0019] The present invention also provides an antibody drug comprising the above-described humanized antibody or its antigen-binding fragment.

[0020] The present invention also provides an antibody-drug conjugate comprising the above-described humanized antibody or its antigen-binding fragment.

[0021] The present invention also provides a chimeric antigen receptor targeting CD19, comprising an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain targeting CD19, wherein the extracellular antigen recognition domain targeting CD19 comprises CD19 VH and CD19 VL, selected from one of the following:

[0022] 1) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:1, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:2; or

[0023] 2) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:3, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:2; or

[0024] 3) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:4, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:5; or

[0025] 4) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:3. The amino acid sequence shown in NO:4, wherein the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:6; or page 2 / 19, CN of the specification.122302055 A

[0026] 5) The CD19 VH sequence includes the amino acid sequence shown in SEQ ID NO:3, and the CD19 VL sequence includes the amino acid sequence shown in SEQ ID NO:7; or

[0027] 6) The CD19 VH sequence includes the amino acid sequence shown in SEQ ID NO:8, and the CD19 VL sequence includes the amino acid sequence shown in SEQ ID NO:9; or

[0028] 7) The CD19 VH sequence includes the amino acid sequence shown in SEQ ID NO:10, and the CD19 VL sequence includes the amino acid sequence shown in SEQ ID NO:11; or

[0029] 8) The CD19 VH sequence includes the amino acid sequence shown in SEQ ID NO:8, and the CD19 VL sequence includes the amino acid sequence shown in SEQ ID NO:11.

[0030] In a preferred embodiment of the present invention, the extracellular antigen recognition domain comprises any one of the following structures:

[0031] an amino acid sequence-linking sequence as shown in SEQ ID NO:1-an amino acid sequence as shown in SEQ ID NO:2, an amino acid sequence-linking sequence as shown in SEQ ID NO:2-an amino acid sequence as shown in SEQ ID NO:1, an amino acid sequence-linking sequence as shown in SEQ ID NO:3-an amino acid sequence as shown in SEQ ID NO:2, an amino acid sequence-linking sequence as shown in SEQ ID NO:2-an amino acid sequence as shown in SEQ ID NO:3, an amino acid sequence-linking sequence as shown in SEQ ID NO:4-an amino acid sequence as shown in SEQ ID NO:5, an amino acid sequence-linking sequence as shown in SEQ ID NO:5-an amino acid sequence as shown in SEQ ID NO:4, an amino acid sequence-linking sequence as shown in SEQ ID NO:4-an amino acid sequence as shown in SEQ ID NO:4-an amino acid sequence as shown in SEQ ID NO:6, an amino acid sequence-linking sequence as shown in SEQ ID NO:6-an amino acid sequence as shown in SEQ ID NO:4, an amino acid sequence-linking sequence as shown in SEQ ID NO:3-an amino acid sequence as shown in SEQ ID NO:7, and so on. The amino acid sequence shown in NO:7 - linking sequence - amino acid sequence as shown in SEQ ID NO:3, amino acid sequence as shown in SEQ ID NO:8 - linking sequence - amino acid sequence as shown in SEQ ID NO:9, amino acid sequence as shown in SEQ ID NO:9 - linking sequence - amino acid sequence as shown in SEQ ID NO:8, amino acid sequence as shown in SEQ ID NO:10 - linking sequence - as shown in SEQ IDThe amino acid sequence shown in NO:11, the amino acid sequence-linking sequence shown in SEQ ID NO:11-the amino acid sequence shown in SEQ ID NO:10, the amino acid sequence-linking sequence shown in SEQ ID NO:8-the amino acid sequence shown in SEQ ID NO:11, the amino acid sequence-linking sequence shown in SEQ ID NO:11-the amino acid sequence shown in SEQ ID NO:8;

[0032] Optionally, the extracellular antigen recognition domain includes any one of the following structures:

[0033] the amino acid sequence-linking sequence shown in SEQ ID NO:4-the amino acid sequence shown in SEQ ID NO:6, the amino acid sequence-linking sequence shown in SEQ ID NO:6-the amino acid sequence shown in SEQ ID NO:4, the amino acid sequence-linking sequence shown in SEQ ID NO:10-the amino acid sequence shown in SEQ ID NO:11, the amino acid sequence-linking sequence shown in SEQ ID NO:11-the amino acid sequence shown in SEQ ID NO:11-the amino acid sequence shown in SEQ ID NO:10;

[0034] Further optionally, the extracellular antigen recognition domain includes any one of the following structures:

[0035] the amino acid sequence shown in SEQ ID NO:11-the amino acid sequence shown in SEQ ID NO:11-the amino acid sequence shown in SEQ ID NO:11-the amino acid sequence shown in SEQ ID NO:10; The amino acid sequence shown in NO:6 - linker sequence - amino acid sequence shown in SEQ ID NO:4, amino acid sequence shown in SEQ ID NO:11 - linker sequence - amino acid sequence shown in SEQ ID NO:10.

[0036] In the above description, "-" indicates mutual linking, and "-" is directional, indicating a link from the N-terminus to the C-terminus of the amino acid; alternatively, "-" indicates direct linking.

[0037] In a preferred embodiment of the invention, the linker sequence is selected from one or more of the following sequences: SEQ ID NO:23 and SEQ ID NO:24.

[0038] In a preferred embodiment of the invention, the extracellular antigen recognition domain comprises: an scFv antibody as shown in any one of SEQ ID NO:31-38. Specification 3 / 19 Page 9 CN 122302055 A

[0039] In a preferred embodiment of the present invention, the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α; optionally, the amino acid sequence of the hinge region is derived from CD8α; further optionally, the amino acid sequence of the hinge region comprises the amino acid sequence shown in SEQ ID NO:25; and / or

[0040] the transmembrane region is derived from CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152,One or more of OX40 and Fc70; optionally, the amino acid sequence of the transmembrane region is derived from CD8α; further optionally, the amino acid sequence of the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:26.

[0041] In a preferred embodiment of the present invention, the intracellular domain comprises an intracellular signal transduction region; optionally, the intracellular signal transduction region is derived from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CCD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk; further optionally, the intracellular signal transduction region is derived from CD3ζ; even further optionally, the amino acid sequence of the intracellular signal transduction region comprises the amino acid sequence shown in SEQ ID NO:28.

[0042] In a preferred embodiment of the present invention, the intracellular domain further includes a co-stimulatory signal transduction region; optionally, the co-stimulatory signal transduction region is derived from one, two, or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, and MyD88; further optionally, the co-stimulatory signal transduction region is derived from 4-1BB; even further optionally, the amino acid sequence of the co-stimulatory signal transduction region comprises the amino acid sequence shown in SEQ ID NO:27.

[0043] In a preferred embodiment of the present invention, the chimeric antigen receptor further includes a guide peptide located at the N-terminus of the chimeric antigen receptor amino acid sequence; optionally, the guide peptide is derived from CD8α; further optionally, the amino acid sequence of the guide peptide comprises the amino acid sequence shown in SEQ ID NO:29.

[0044] The present invention also provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the above-mentioned humanized antibody or its antigen-binding fragment, or comprising a nucleotide sequence encoding the above-mentioned chimeric antigen receptor;

[0045] Optionally, the nucleotide sequence encoding the humanized antibody or its antigen-binding fragment or the nucleotide sequence encoding the chimeric antigen receptor comprises a nucleotide sequence encoding CD19 VH and CD19 VL, selected from a group of the following:

[0046] 1) a nucleotide sequence encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:1, as shown in SEQ ID NO:12; and a nucleotide sequence encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:2, as shown in SEQ ID NO:13; and / or

[0047] 2) a nucleotide sequence encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:3.The nucleotide sequence encoding the VH amino acid sequence, as shown in SEQ ID NO: 14; and the nucleotide sequence encoding the CD19 VL amino acid sequence, as shown in SEQ ID NO: 2, as shown in SEQ ID NO: 13; and / or

[0048] 3) the nucleotide sequence encoding the CD19 VH amino acid sequence, as shown in SEQ ID NO: 4, as shown in SEQ ID NO: 15; and the nucleotide sequence encoding the CD19 VL amino acid sequence, as shown in SEQ ID NO: 5, as shown in SEQ ID NO: 16; and / or

[0049] 4) the nucleotide sequence encoding the CD19 VH amino acid sequence, as shown in SEQ ID NO: 4, as shown in SEQ ID NO: 15; and the nucleotide sequence encoding the CD19 VL amino acid sequence, as shown in SEQ ID NO: 6, as shown in SEQ ID NO: 17; and / or

[0050] 5) the nucleotide sequence encoding the CD19 VH amino acid sequence, as shown in SEQ ID NO: 3, as shown in SEQ ID NO: 14; and the nucleotide sequence encoding the CD19 VL amino acid sequence, as shown in SEQ ID NO: 2, as shown in SEQ ID NO: 13; and / or

[0048] 4) the nucleotide sequence encoding the CD19 VH amino acid sequence, as shown in SEQ ID NO: 4, as shown in SEQ ID NO: 15; and the nucleotide sequence encoding the CD19 VL amino acid sequence, as shown in SEQ ID NO: 6, as shown in SEQ ID NO: 17; and / or

[0050] 5) the nucleotide sequence encoding the CD19 VH amino acid sequence, as shown in SEQ ID NO: 3, as shown in SEQ ID NO: 14; and the nucleotide sequence encoding the CD19 VL amino acid sequence, as shown in SEQ ID NO: 15; and the nucleotide sequence encoding the CD19 VL amino acid The nucleotide sequence encoding the CD19 VL amino acid sequence shown in NO:7, as shown in SEQ ID NO:18; and / or

[0051] 6) the nucleotide sequence encoding the CD19 VH amino acid sequence shown in SEQ ID NO:8, as shown in SEQ ID NO:19; and the nucleotide sequence encoding the CD19 VL amino acid sequence shown in SEQ ID NO:9, as shown in SEQ ID NO:20; and / or

[0052] 7) the nucleotide sequence encoding the CD19 VH amino acid sequence shown in SEQ ID NO:10, as shown in SEQ ID NO:21; and the nucleotide sequence encoding the CD19 VL amino acid sequence shown in SEQ ID NO:11, as shown in SEQ ID NO:22; and / or

[0053] 8) the nucleotide sequence encoding the CD19 VH amino acid sequence shown in SEQ ID NO:8, as shown in SEQ ID NO:19; and the nucleotide sequence encoding the CD19 VH amino acid sequence shown in SEQ ID NO:11, as shown in SEQ ID NO:22; and / or

[0053] 8) the nucleotide sequence encoding the CD19 VH amino acid sequence shown in SEQ ID NO:8, as shown in SEQ ID NO:19; and the nucleotide sequence encoding the CD19 VH amino acid sequence shown in SEQ ID NO:19; The nucleotide sequence of the CD19 VL amino acid sequence shown in NO:11 is as shown in SEQ ID NO:22.

[0054] The present invention also provides a vector comprising the above-isolated nucleic acid molecule;

[0055] Optionally, the vector is an expression vector;

[0056] Further optionally, the vector is a viral vector;

[0057] Even further optionally, the vector is a lentiviral vector.

[0058] The present invention also provides an engineered immune effector cell comprising the above-mentioned humanized antibody or its antigen-binding fragment, the above-mentioned chimeric antigen receptor, the above-mentioned isolated nucleic acid, or the above-mentioned carrier.

[0059] In a preferred embodiment of the present invention, the engineered immune effector cell is selected from one or more of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMCs), pluripotent stem cells, T cells differentiated from pluripotent stem cells, NK cells differentiated from pluripotent stem cells, and embryonic stem cells;

[0060] Optionally, the engineered immune effector cell is a T lymphocyte;

[0061] Further optionally, the T lymphocyte is derived from autologous T lymphocytes or allogeneic T lymphocytes.

[0062] The present invention also provides a pharmaceutical composition comprising the above-mentioned engineered immune effector cells and pharmaceutically acceptable excipients; pharmaceutically acceptable excipients include one or more of the following: carriers, protectants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and preservatives.

[0063] Optionally, pharmaceutically acceptable excipients include protective agents; alternatively, pharmaceutically acceptable excipients include cell cryopreservation solutions.

[0064] In a preferred embodiment of the invention, the pharmaceutical composition is a cell suspension or cryopreserved cells thereof; or the pharmaceutical composition is an intravenous injection.

[0065] The invention also provides the use of the above-described humanized antibody or its antigen-binding fragment, the above-described chimeric antigen receptor, the above-described isolated nucleic acid, the above-described carrier, or the above-described engineered immune effector cells in the preparation of a medicament for treating diseases or conditions associated with CD19 expression.

[0066] In a preferred embodiment of the present invention, the disease or condition associated with CD19 expression includes non-solid tumors, acute lymphoblastic leukemia, and / or B-cell lymphoma;

[0067] Optionally, the non-solid tumor includes leukemia and / or lymphoma;

[0068] Optionally, the acute lymphoblastic leukemia includes adult acute lymphoblastic leukemia and / or childhood acute lymphoblastic leukemia;

[0069] Optionally, the B-cell lymphoma includes non-Hodgkin lymphoma.

[0070] In a preferred embodiment of the present invention, the diseases or conditions associated with CD19 expression include refractory systemic lupus erythematosus immune thrombocytopenic purpura (SLE-ITP), refractory lupus nephritis (LN), refractory immune hemolytic anemia (AIHA), systemic lupus erythematosus (SLE), autoimmune thrombocytopenic purpura (ITP), antineutrophil cytoplasmic antibody-associated vasculitis (AAV), dermatomyositis (DM), myasthenia gravis (MG), or antisynthetic enzyme antibody syndrome (ASS). Specification 5 / 19 pages 11 CN 122302055 A

[0071] On the other hand, this application also provides a method for treating diseases or conditions related to CD19 expression, the method comprising administering an effective dose of the chimeric antigen receptor, the isolated nucleic acid molecule, the vector, and / or the engineered immune effector cells to a subject who requires treatment for a disease or condition related to CD19 expression.

[0072] In some embodiments, the administration can be performed in different ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, local, or intradermal administration. For example, the administration can be performed by intravenous injection. In some embodiments, an effective dose of the engineered immune effector cells or pharmaceutical composition can be administered to the subject once or in multiple doses over a period of time, such as once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months.

[0073] In some embodiments, the dosage can be different for different indications; the dosage can also be different for patients with different disease severity. The dosage range can be from 1×10⁵ CAR-positive T cells / kg to 1×10⁷ CAR-positive T cells / kg, for example, 1×10⁵ CAR-positive T cells / kg to 1×10⁶ CAR-positive T cells / kg, 1×10⁶ CAR-positive T cells / kg to 1×10⁷ CAR-positive T cells / kg, 0.5×10⁶ CAR-positive T cells / kg, 0.6×10⁶ CAR-positive T cells / kg, 0.7×10⁶ CAR-positive T cells / kg, 0.8×10⁶ CAR-positive T cells / kg, 0.9×10⁶ CAR-positive T cells / kg, 1.0×10⁶ CAR-positive T cells / kg, 1.1×10⁶ CAR-positive T cells / kg, 1.2×10⁶ CAR-positive T cells / kg, 1.3×10⁶ CAR-positive T cells / kg, 1.4×10⁶ CAR-positive T cells / kg, 1.5 ...1×10⁶ CAR-positive T cells / kg, 1.2×10⁶ CAR-positive T cells / kg, 1.3×10⁶ CAR-positive T cells / kg, 1.4×10⁶ CAR-positive T cells / kg, 1.5×10⁶ CAR-positive T cells / kg, 1.6 × 10⁶ CAR-positive T cells / kg, 1.7 × 10⁶ CAR-positive T cells / kg, 1.8 × 10⁶ CAR-positive T cells / kg, 1.9 × 10⁶ CAR-positive T cells / kg, 2.0 × 10⁶ CAR-positive T cells / kg.

[0074] In some embodiments, the subject may include humans and non-human animals. For example, the subject may include, but is not limited to, mice, rats, cats, dogs, horses, pigs, cattle, sheep, rabbits, or monkeys.

[0075] The present invention also provides the above-described humanized antibody or its antigen-binding fragment, the above-described chimeric antigen receptor, the above-described isolated nucleic acid, the above-described vector, or the above-described engineered immune effector cells for treating diseases or conditions related to CD19 expression.

[0076] In a preferred embodiment of the present invention, the disease or condition associated with CD19 expression includes non-solid tumors, acute lymphoblastic leukemia, and / or B-cell lymphoma;

[0077] Optionally, the non-solid tumor includes leukemia and / or lymphoma;

[0078] Optionally, the acute lymphoblastic leukemia includes adult acute lymphoblastic leukemia and / or pediatric acute lymphoblastic leukemia;

[0079] Optionally, the B-cell lymphoma includes non-Hodgkin lymphoma.

[0080] In a preferred embodiment of the present invention, the diseases or conditions associated with CD19 expression include refractory systemic lupus erythematosus immune thrombocytopenic purpura (SLE-ITP), refractory lupus nephritis (LN), refractory immune hemolytic anemia (AIHA), systemic lupus erythematosus (SLE), autoimmune thrombocytopenic purpura (ITP), antineutrophil cytoplasmic antibody-associated vasculitis (AAV), dermatomyositis (DM), myasthenia gravis (MG), or antisynthetic antibody syndrome (ASS).

[0081] Compared with the prior art, the humanized antibody and chimeric antigen receptor targeting CD19 of the present invention have the following beneficial effects:

[0082] The humanized CD19 antibody or humanized CAR-T of the present invention has comparable efficacy to the CD19 murine antibody or murine CD19 CAR-T in terms of killing activity against tumor cells and amplification fold after multiple rounds of stimulation. Furthermore, the humanized CD19 antibody or humanized CAR-T of the present invention has higher IL-2 release and IFN-γ release than the CD19 murine antibody or murine CD19 CAR-T. In addition, the humanized CD19 antibody or humanized CAR-T of the present invention has lower immunogenicity than the CD19 murine antibody or murine CD19 CAR-T, thereby having better safety. Specification 6 / 19 pages 12 CN 122302055 A Description of Drawings

[0083] Figure 1: Graph of CD19-CAR positivity rate in cells at different time points.

[0084] Figure 2: Statistical graph of CD19-CAR MFI at different time points.

[0085] Figure 3: In vitro killing activity against positive target cells Nalm6.

[0086] Figure 4: Detection of cytokine release during killing of target cells Nalm6.

[0087] Figure 5: CAR-T cell expansion fold during multiple rounds of stimulation of target cells Nalm6.

[0088] Figure 6: Scoring results of CD19 humanized sequences using three biological computer evaluation tools (T20 score, Hscore, QASis).

[0089] Figure 7: Immunogenicity risk prediction results of CD19 humanized sequences using two immunogenicity prediction tools (AlphaMHC v2, CD4 T cell Immunogenicity prediction tool).

[0090] In Figures 1-2, each bar represents CNCT19, Q11, Q14, Q23, Q33, Q54, GS32, GS21, and GS22 from left to right. In Figures 3-4, each bar represents UTD (untransduced CAR T cells), CNCT19, Q11, Q14, Q33, Q54, GS32, GS21, and GS22 from left to right. In Figure 5, each bar represents CNCT19, Q11, Q14, Q33, Q54, GS32, GS21, and GS22 from left to right. Detailed Description

[0091] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0092] The invention is further described below: In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology-related terms and laboratory procedures used in this article are all widely used terms and routine procedures in their respective fields. Meanwhile, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0093] In this application, the term "Chimeric Antigen Receptor" (CAR) is a core component of CAR cell therapy drugs, which may include an extracellular antigen recognition domain (e.g., a portion that binds to tumor-associated antigens (TAA)), a hinge region, a transmembrane region, and an intracellular domain. CAR-T (Chimeric Antigen Receptor T) cell immunotherapy is considered one of the most promising means of conquering tumors. CAR-T cells utilize genetic modification to enable T cells to express CAR proteins, which are capable of recognizing intact proteins on the membrane surface without relying on antigen presentation, thereby activating and functionally affecting T cells.

[0094] In this application, the term "extracellular antigen recognition domain" refers to an antigen recognition domain (ARD). CAR cell therapy products (such as CAR-T cells) can specifically recognize and / or bind to target antigens expressed by tumor cells because of their extracellular antigen recognition domains. To date, these antigen recognition domains have evolved from single-chain variable fragments (scFv) of antibodies, or from receptor-ligand interactions, TCR mimics, and variable lymphocyte receptors (VLRs).Come. To date, the most common source is the scFv segment of the antibody. The scFv includes the variable region of the antibody heavy chain and the variable region of the light chain, which are connected by a peptide chain, such as the 18-amino acid linker sequence GSTGSGSGKPGSGEGSTKG. Common CDR partitioning rules for antibodies include Kabat, AbM, Chothia, Contact, and IMGT. These rules are well known to those skilled in the art. When applying a website that executes these rules, simply input the VH and VL sequences and select the corresponding rule to obtain CDR sequences according to different rules. Those skilled in the art should understand that the scope of protection of this application covers the combination of CDR sequences obtained by analyzing different rules using the specification on page 7 / 19 of CN 122302055 A.

[0095] In this application, the term "hinge region" refers to the linker segment that acts between the extracellular antigen recognition domain and the transmembrane domain. This region allows the CAR to recognize the antigen by giving the antigen recognition domain a certain range of motion. The hinge region currently used is mainly derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α. In addition, a typical hinge region also contains residues that participate in CAR dimerization, which helps enhance antigen sensitivity.

[0096] In this application, "transmembrane region" refers to a transmembrane domain connecting the intracellular and extracellular components of the CAR structure. Different transmembrane domains can affect CAR expression and stability to some extent, but do not directly participate in signal transduction; however, they can enhance downstream signal transduction through interaction. The transmembrane region may be derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70.

[0097] In this application, the term "intracellular domain" includes intracellular signal transduction regions and may also include co-stimulatory signal transduction regions.

[0098] In this application, the term "intracellular signal transduction region" refers to the activation of at least one normal effector function of an immune effector cell responsible for expressing CAR. The intracellular signal transduction region may originate from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CCD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk.

[0099] In this application, the term "co-stimulatory signal transduction region" is used because, in addition to stimulation by antigen-specific signals, many immune effector cells require co-stimulation to promote cell proliferation, differentiation, and survival, as well as to activate the effector functions of cells. In some embodiments, the CAR may further include one or more co-stimulatory signal transduction regions, wherein co-stimulation...The excitation signal transduction region can be derived from one, two, or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, and MyD88.

[0100] In this application, the term "scFv" has the conventional meaning in the art, referring to a single-chain variable fragment (scFv), which is an antibody formed by linking the antibody heavy chain variable fragment and the light chain variable fragment through a short peptide (linker).

[0101] In this application, terms such as "Sc(Fv)2" and "[Sc(Fv)2]2" that are not specifically explained also have the conventional meaning in the art.

[0102] In this application, one of the key factors determining the efficacy of CAR-immune effector cell therapy is the selection of tumor target antigens. However, the selection of antigen targets is not necessarily singular. Therefore, the extracellular antigen recognition domain may also include an extracellular antigen recognition domain targeting any of the following targets (e.g., scFv antibodies): CD5, CD20, CD22, CD33, CD123, CLL1, BCMA, CD138, CS1. For example, in dual-target CAR-T products, the extracellular antigen recognition domain includes scFv sequences targeting two targets. scFv antibodies targeting a single target include a heavy chain variable region (VH) and a light chain variable region (VL), which are linked together by a linker sequence. scFv antibodies targeting two or more targets include VH and VL regions targeting different targets, which are also directly or indirectly linked by a linker sequence. The arrangement can be any of the following forms: target 1VL-target 1VH-target 2VL-target 2VH, target 2VL-target 2VH-target 1VL-target 1VH, target 1VL-target 2VL-target 2VH-target 1VH, target 2VL-target 1VL-target 1VH, target 2VL-target 1VL-target 1VH-target 2VH, where "-" indicates a linker sequence.

[0103] In this application, the term "isolated" generally refers to something obtained artificially from its natural state. If a substance or component appears in nature as "separated," it may be due to a change in its natural environment, the separation of the substance from its natural environment, or both. For example, a certain unseparated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide isolated from this natural state is called separated. The term "separated" does not exclude the possibility of obtaining it from its natural state through artificial means and then processing it artificially or synthetically. (See page 8 / 19 of the specification, CN 122302055 A)The substances formed may contain other impurities that do not affect the activity of the substance.

[0104] In this application, the term "guide peptide" refers to a short peptide preceding the extracellular antigen recognition domain (such as the scFv sequence), which guides the recombinant protein synthesized in the cell to be exported to the extracellular space. Commonly used guide peptides include human CD8α signal peptide or human GM-CSF receptor α signal peptide.

[0105] In this application, the term "HI19a antibody" refers to an existing murine antibody. The HI19a antibody can specifically bind to the extracellular domain of CD19 on the surface of human B cells and has high affinity. If scFv derived from murine antibodies is used directly in CAR-T drugs, it may increase the risk of immune infection. In order to reduce the immunogenicity of scFv derived from murine antibodies and improve the safety of treatment, it can be achieved by humanizing scFv derived from murine antibodies. However, during the humanization process, the binding affinity, specificity, and other functions of the antibody to a specific antigen may be lost. Therefore, obtaining humanized antibodies with reduced immunogenicity but without loss of function is of practical significance.

[0106] In this application, the term "humanized antibody" also refers to a humanized antibody. The methods of humanization are known (e.g., WO96 / 02576). The purpose of humanization is to basically retain the affinity and specificity of the parent antibody while reducing its heterologity. For example, in the case where the CDR is obtained from a mouse antibody, primer 25 can be synthesized (the corresponding primer can be obtained by referring to the method described in WO98 / 13388) and used to link the CDR of the mouse antibody to the framework region (FR) of the human antibody.

[0107] In this application, the term "linking sequence" generally refers to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length that links any structure / region of the chimeric antigen receptor of the present invention together. The linker sequence can consist of different amino acid residues (such as glycine and serine) so that adjacent protein domains can move freely relative to each other. Longer linker sequences can be used when it is desirable to ensure that two adjacent domains do not interfere with each other spatially.

[0108] In this application, the term "isolated nucleic acid molecule" generally refers to an isolated form of nucleotide, deoxyribonucleotide, or ribonucleotide of any length, which may be isolated from its natural environment or an analogue synthesized artificially.

[0109] In this application, gene transduction / transfection methods for CAR gene transduction / transfection and target gene expression mainly include viral and non-viral methods. For example: through γ-retroviral vectors, lentiviral vectors, adenovirus-associated viral vectors, plasmid DNA-dependent vectors, transposon-dependent gene transfer, and mRNA-mediated gene transduction.

[0110] The term "vector" generally refers to a vector into which a polynucleotide encoding a protein can be inserted to achieve protein expression.A vector is a nucleic acid delivery vehicle. Vectors can transform, transduce, or transfect host cells, enabling the expression of their carried genetic material elements within the host cells. Examples of vectors include: plasmids; phage particles; Cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain replication initiation sites. Vectors may also include components that facilitate their entry into cells, such as viral particles, liposomes, or protein coats, but are not limited to these substances. The term "transposon" refers to a discontinuous DNA segment with the ability to migrate between chromosomal loci and carry genetic information, such as the Sleeping Beauty SB system and the PB system derived from lepidopteran insects. In some embodiments, electroporation can also be used to transduce mRNA into T cells.

[0111] In this application, the term "immune effector cell" generally refers to a cell that participates in an immune response, such as promoting an immune effector response. Immune effector cells may be selected from one or more of the following groups: T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMCs), pluripotent stem cells, T lymphocytes differentiated from pluripotent stem cells, NK cells differentiated from pluripotent stem cells, and embryonic stem cells.

[0112] In this application, the term "pharmaceutical composition" generally refers to a pharmaceutical composition suitable for administration to a patient, which may contain the immune effector cells described in this application, and may also contain one or more pharmaceutically acceptable excipients, such as: carriers, protectants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, preservatives, or one or more of these. In some embodiments, pharmaceutically acceptable excipients include protectants, such as: cell cryopreservation solutions. In some embodiments, the pharmaceutical composition of this application is a cell suspension or its cryopreserved cells.

[0113] In this application, the term "antibody drug" has the conventional meaning in the art, that is, a drug with an antibody substance as its active ingredient, such as: monoclonal antibody drugs, bispecific antibody drugs.

[0114] In this application, the term "antibody drug conjugate" has the conventional meaning in the art, that is, Antibodydrug conjugate, abbreviated as ADC. It is a small molecule drug with biological activity linked to a monoclonal antibody through a chemical chain, and the monoclonal antibody acts as a carrier to target and transport the small molecule drug to the target cells.

[0115] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to mice, rats, cats, dogs, rabbits, horses, pigs, cattle, sheep, or monkeys.

[0116] In this application, the term "comprising" generally means including the explicitly specified features, but does not exclude other elements.

[0117] In this application, the term "about" generally means a range of fluctuation acceptable to those skilled in the art above or below a specified value, such as variation within ±0.5% to 10%, for example, variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0118] Not intended to be limited by any theory, the embodiments described below are merely for illustrating the chimeric antigen receptor, engineered immune effector cells, preparation methods, and uses of this application, and are not intended to limit the scope of the invention. The embodiments do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods for inserting genes encoding proteins into such vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those skilled in the art and have been described in numerous publications, including Sambrook, J., Fritsch, E.F., and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press.

[0119] The murine antibody and humanized antibody scFv of the present invention

[0120] >HI19a(CNCT19)scFv

[0121] DIVLTQSPKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKPLIYSATYRNSGVPDRFTGSGSG TDFTLTITNVQSKDLADYFCQQYNRYPYTSGGGTKLEIKRGGGGSGGGGSGGGGSQVQLQQSGAELVRPGSSVKIS CKASGYAFSSYWMNWVKQRPGQGLEWIGQIYPGDGDTNYNGKFKGQATLTADKSSSTAYMQLSGLTSEDSAVYFCA RKTISSVVDFYFDYWGQGTTLTVSS(SEQ ID NO:30)

[0122] >Q11 scFv

[0123] DIQLTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSATYRNSGVPSRFSGSGSG TDFTLTISSLQPEDFATYFCQQYNRYPYTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSC KASGYAFSSYWMNWVRQAPGQGLEWMGQIYPGDGDTNYNGKFKGRVTMTRDTSTSTAYMELSSLRSEDTAVYFCAR KTISSVVDFYFDYWGQGTTVTVSS(SEQ ID NO:31)

[0124] Q11 scFv contains Q11 VH and Q11 VL, with the structure: Q11 VL-linking sequence (GGGGSGGGGSGGGGS)-Q11 VH, wherein the amino acid sequence of Q11 VH is as follows:

[0125] QVQLVQSGAEVKKPGASVKVSCKASGYAFSSYWMNWVRQAPGQGLEW MGQIYPGDGDTNYNGKFKGR Instruction manual 10 / 19 pages 16 CN 122302055 A VTMTRDTSTSTAYMELSSLRSEDTAVYFCARK TISSVVDFYFDYWGQGTTVTVSS (SEQ ID NO:1)

[0126] The nucleotide sequence encoding Q11 VH is as follows:

[0127] CAAGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCTCCGTGAAGGTGAGCTGCAA AGCCTCCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAGACAAGCCCCCGGCCAAGGCCTGGAGTGGATG GGGCAGATCTACCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAGAGTCACCATGACAAGAGACA CAAGCACAAGCACCGCCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTTCTGCGCTAGAAA GACAATCAGCAGCGTGGTGGACTTCTACTTCGACTACTGGGGCCAAGGCACCACCGTGACCGTGAGCAGC (SEQ ID NO: 12) The amino acid sequence of Q11 VL is as follows:

[0129] DIQLTQSPSSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSA TYRNSGVPSRFSGSGS GTDFTLTISSLQPEDFATYFCQQYNRYPYTFGGGTKLE IK (SEQ ID NO: 2)

[0130] The nucleotide sequence encoding Q11 VL is as follows:

[0131] GACATTCAGCTGACACAGAGCCCTAGCAGCCTGAGCGCCTCCGTGGGCGACAGAGTGACCATCACATG CAAGGCCTCCCAAAACGTGGGCACCAACGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCCCCTGATC TACAGCGCCACCTACAGAAACAGCGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGA CCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTTCTGTCAGCAGTACAACAGATACCCCTACACATTTGG CGGGGGCACAAAGCTGGAGATTAAG (SEQ ID NO: 13)

[0132] >Q14 scFv

[0133] DIQLTQSPSSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSATYRNSGVPSRFSGSGSG TDFTLTISSLQPEDFATYFCQQYNRYPYTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSC KASGYAFSSYWMNWVRQAPGQGLEWMGQIYPGDGDTNYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYFCAR KTISSVVDFYFDYWGQGTTVTVSS(SEQ ID NO:32)

[0134] Q14 scFv contains Q14 VH and Q14 VL, and the structure is: Q14 VL-linking sequence (GGGGSGGGGSGGGGS)-Q14 VH, where the amino acid sequence of Q14 VH is as follows:

[0135] QVQLVQSGAEVKKPGSSVKVSCKASGYAFSSYWMNWVRQAPGQGLEW MGQIYPGDGDTNYNGKFKGR VTITADKSTSTAYMELSSLRSEDTAVYFCARKTISSVVDFYFDYWGQGTTVTVSS (SEQ ID NO: 3)

[0136] The nucleotide sequence encoding Q14 VH is as follows:

[0137] CAAGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCTCCTCCGTGAAGGTGAGCTGCAA AGCCTCCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAGACAAGCCCCCGGCCAAGGCCTGGAGTGGATG GGGCAGATCTACCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAGAGTCACCATCACAGCAGACA AAAGCACAAGCACCGCCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTTCTGCGCTAGAAA GACAATCAGCAGCGTGGTGGACTTCTACTTCGACTACTGGGGCCAAGGCACCACCGTGACCGTGAGCAGC (SEQ ID NO: 14)

[0138] The amino acid sequence of Q14 VL is as follows:

[0139] DIQLTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSA TYRNSGVPSRFSGSGS GTDFTLTISSLQPEDFATYFCQQYNRYPYTFGGGTKLE IK (SEQ ID NO:2)

[0140] The nucleotide sequence encoding Q14 VL is as follows:

[0141] GACATTCAGCTGACACAGAGCCCTAGCAGCCTGAGCGCCTCCGTGGGCGACAGAGTGACCATCACATG CAAGGCCTCCCAAAACGTGGGCACCAACGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCCCCTGATC Specification 11 / 19 pages 17 CN 122302055 A TACAGCGCCACCTACAGAAACAGCGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGA CCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTTCTGTCAGCAGTACAACAGATACCCCTACACATTTGGCGGGGGCACAAAGCTGGAGATTAAG(SEQ ID NO: 13)

[0142] >Q23 scFv

[0143] DIQLTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKSPKPLIYSATYRNSGVPSRFSGSGSG TDFTLTISSLQPKDFATYFCQQYNRYPYTSGGGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLKISC KASGYAFSSYWMNWVRQMPGKGLEWMGQIYPGDGDTNYNGKFKGQVTLSADKSISTAYLQWSSLKASDTAMYFCAR KTISSVVDFYFDYWGQGTTVTVSS(SEQ ID NO: 33)

[0144] Q23 scFv comprises Q23 VH and Q23 VL, and has a structure of: Q23 VL-linker sequence (GGGGSGGGGSGGGGS)-Q23 VH, wherein the amino acid sequence of Q23 VH is shown below:

[0145] EVQLVQSGAEVKKPGESLKISCKASGYAFSSYWMNWVRQMPGKGLEWM GQIYPGDGDTNYNGKFKGQ VTLSADKSISTAYLQWSSLKASDTAMYFCARKTI SSVVDFYFDYWGQGTTVTVSS(SEQ ID NO: 4)

[0146] The nucleotide sequence encoding Q23 VH is shown below:

[0147] GAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGAGAGCCTGAAGATCAGCTGCAA AGCTTCCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAGACAGATGCCCGGCAAGGGCCTGGAGTGGATG GGGCAGATCTACCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCCAAGTGACCCTGAGCGCCGACA AGAGCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCCGACACCGCCATGTACTTCTGCGCTAGAAA GACCATCAGCAGCGTGGTGGACTTCTACTTCGACTACTGGGGCCAAGGCACCACCGTGACCGTGAGCAGC(SEQ ID NO: 15)

[0148] Q23The amino acid sequence of VL is as follows:

[0149] DIQLTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKSPKPLIYSA TYRNSGVPSRFSGSGS GTDFTLTISSLQPKDFATYFCQQYNRYPYTSGGGTKLE IK (SEQ ID NO:5)

[0150] The nucleotide sequence encoding Q23 VL is as follows:

[0151] GACATTCAGCTGACACAGAGCCCTAGCAGCCTGAGCGCCTCCGTGGGCGACAGAGTGACCATCACCTG CAAGGCCTCCCAAAACGTGGGCACCAACGTGGCCTGGTATCAGCAGAAGCCCGGCAAGAGCCCCAAGCCCCTGATC TACAGCGCCACCTACAGAAACAGCGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGA CCATCAGCTCCCTGCAGCCCAAGGACTTCGCCACCTACTTCTGTCAGCAGTACAACAGATACCCCTACACATCCGG GGGCGGCACCAAGCTTGGAAATCAAG(SEQ ID NO:16)

[0152] >Q33 scFv

[0153] EIVMTQSPATLSVSPGERATLSCKASQNVGTNVAWYQQKPGQAPRPLIYSATYRNSGIPARFSGSGSG TEFTLTISSLQSEDFAVYFCQQYNRYPYTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLKISC KASGYAFSSYWMNWVRQMPGKGLEWMGQIYPGDGDTNYNGKFKGQVTLSADKSISTAYLQWSSLKASDTAMYFCAR KTISSVVDFYFDYWGQGTTVTVSS(SEQ ID NO:34)

[0154] Q33 scFv contains Q33 VH and Q33 VL, with the structure: Q33 VL-linking sequence (GGGGSGGGGSGGGGS)-Q33 VH, wherein the amino acid sequence of Q33 VH is as follows:

[0155] EVQLVQSGAEvkkPGeSlKISCKASGYAFSSYWMNWVrQmPGkGLEWmGQI YPGDGDTNYNGKFKGQvTLsADKSiSTAYlQwSsLkasDtAmYFCARKTISSVVDF YFDYWGQGTTVTVSS (SEQ ID NO: 4)

[0156] The nucleotide sequence encoding Q33 VH is as follows:

[0157] GAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGAGAGCCTGAAGATCAGCTGCAA Instructions 12 / 19 Page 18 CN 122302055 A AGCTTCCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAGACAGATGCCCGGCAAGGGCCTGGAGTGGATG GGGCAGATCTACCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCCAAGTGACCCTGAGCGCCGACA AGAGCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCCGACACCGCCATGTACTTCTGCGCTAGAAA GACCATCAGCAGCGTGGTGGACTTCTACTTCGACTACTGGGGCCAAGGCACCACCGTGACCGTGAGCAGC (SEQ ID NO:15)

[0158] The amino acid sequence of Q33 VL is as follows:

[0159] EIVMTQSPATLSVSPGERATLSCKASQNVGTNVAWYQQKPGQAPRPLIYS ATYRNSGIPARFSGSGS GTEFTLTISSLQSEDFAVYFCQQYNRYPYTfGGGTKLE IK (SEQ ID NO:6)

[0160] The nucleotide sequence encoding Q33 VL is as follows:

[0161] GAAATCGTGATGACCCAGTCCCCTGCTACACTGAGCGTGTCCCCAGGCGAGCGGGCCACACTGTCTTG CAAGGCCTCCCAAAACGTGGGCACCAACGTGGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGGCCCCTGATC TACAGCGCCACCTACAGAAACAGCGGCATCCCTGCCAGATTCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAGCCTGCAGTCCGAGGACTTCGCCGTCTACTTCTGTCAGCAGTACAACAGATACCCCTACACATTCGG CGGGGGGACCAAGCTGGAGATCAAA(SEQ ID NO: 17)

[0162] >Q54 scFv

[0163] DIQLTQSPSFLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSATYRNSGVPSRFSGSGSG TEFTLTISSLQPEDFATYFCQQYNRYPYTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLvQSGAEvkkPGSSVKvSC KASGYAFSSYWMNWVrQaPGQGLEWmGQIYPGDGDTNYNGKFKGrvTiTADKStSTAYMeLSsLrSEDtAVYFCAR KTISSVVDFYFDYWGQGTTVTVSS(SEQ ID NO: 35)

[0164] Q54 scFv comprises Q54 VH and Q54 VL, with a structure of: Q54 VL-linker sequence (GGGGSGGGGSGGGGS)- Q54 VH, wherein the amino acid sequence of Q54 VH is shown as follows:

[0165] QVQLvQSGAEvkkPGSSVKvSCKASGYAFSSYWMNWVrQaPGQGLEWmGQ IYPGDGDTNYNGKFKGr vTiTADKStSTAYMeLSsLrSEDtAVYFCARKTISSVVDF YFDYWGQGTTVTVSS(SEQ ID NO: 3)

[0166] The nucleotide sequence encoding Q54 VH is shown as follows:

[0167] CAAGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCTCCTCCGTGAAGGTGAGCTGCAA AGCCTCCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAGACAAGCCCCCGGCCAAGGCCTGGAGTGGATG GGGCAGATCTACCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAGAGTCACCATCACAGCAGACA AAAGCACAAGCACCGCCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTTCTGCGCTAGAAAGACAATCAGCAGCGTGGTGGACTTCTACTTCGACTACTGGGGCCAAGGCACCACCGTGACCGTGAGCAGC (SEQ ID NO:14)

[0168] The amino acid sequence of Q54 VL is as follows:

[0169] DIQLTQSPSFLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSA TYRNSGVPSRFSGSGS GTEFTLTISSLQPEDFATYFCQQYNRYPYTFGGGTKVE IK (SEQ ID NO:7)

[0170] The nucleotide sequence encoding Q54 VL is as follows:

[0171] GACATCCAACTGACCCAGAGCCCCTCCTTCCTGAGCGCCTCTGTCGGCGATAGAGTGACAATCACCTG TAAGGCCAGCCAGAACGTGGGCACCAATGTGGCCTGGTACCAGCAGAAACCAGGCAAGGCTCCTAAGCCTCTGATC TACTCCGCTACATATCGGAACAGCGGCGTGCCTTCGAGATTTTCTGGCAGCGGCCTGGAACCGAGTTCACCCTGA CCATCTCCTCTCTGCAGCCTGAGGACTTCGCCACCTACTTCTGCCAGCAGTACAACAGATACCCCTACACCTTCGG AGGCGGCACCAAGGTGGAGATTAAG(SEQ ID NO:18) Instructions 13 / 19 Page 19 CN 122302055 A GS32 scFv DIQLTQSPSFLSAVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSATYRNSGVPSRFSGSGSG TEFTLTISSLQPEDFADYFCQQYNRYPYTFGGGTKVEIKRGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVS CKASGYAFSSYWMNWVRQAPGQGLEWIGQIYPGDGDTNYNGKFKGRATITADKSTSTAYMELSSLRSEDTAVYFCA RKTISSVVDFYFDYWGQGTLVTVSS(SEQ ID NO:36)

[0174] GS 3 2 sc F v includes GS 3 2 VH and GS 3 2 VL, with the structure: GS 3 2VL-linking sequence (GGGGSGGGGSGGGGS)-GS32 VH, wherein the amino acid sequence of GS32 VH is as follows:

[0175] QVQLVQSGAEVKKPGSSVKVSCKASGYAFSSYWMNWVRQAPGQGLEWI GQIYPGDGDTNYNGKFKGR ATITADKSTSTAYMELSSLRSEDTAVYFCARKTIS SVVDFYFDYWGQGTLVTVSS (SEQ ID NO:8)

[0176] The nucleotide sequence encoding GS32 VH is as follows:

[0177] CAAGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCAGCAGCGTGAAGGTGAGCTGTAA AGCTTCCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAGACAAGCCCCCGGCCAAGGCCTGGAGTGGATC GGGCAGATCTACCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCAGAGCCACCATCACCGCCGACA AGAGCACAAGCACCGCCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTTCTGCGCTAGAAA GACCATCAGCAGCGTGGTGGACTTCTACTTCGACTACTGGGGCCAAGGCACCCTGGTGACCGTGAGCAGC(SEQ ID NO:19)

[0178] The amino acid sequence of GS32 VL is as follows:

[0179] DIQLTQSPSFLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSA TYRNSGVPSRFSGSGS GTEFTLTISSLQPEDFADYFCQQYNRYPYTFGGGTKVE IKR(SEQ ID NO:9)

[0180] The nucleotide sequence encoding GS32 VL is as follows:

[0181] GACATTCAGCTGACACAGAGCCCTAGCTTCCTGAGCGCCTCCGTGGGCGACAGAGTGACCATCACCTG CAAGGCTTCCCAAAACGTGGGCACCAACGTGGCCTGGTATCAGCAGAAACCCGGCAAGGCCCCCAAGCCCTGATCTACAGCGCCACCTACAGAAACAGCGGCGTGCCTAGCAGATTCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGA CAATCAGCAGCCTGCAGCCCGAGGACTTCGCCGACTACTTCTGTCAGCAGTACAACAGATACCCCTACACCTTCGG CGGGGGCACCAAGGTGGAGATCAAGCGG(SEQ ID NO:20)

[0182] >GS21 scFv

[0183] DIQLTQSPSFLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSATYRNSGVPSRFSGSGSG TEFTLTISSLQPEDFATYFCQQYNRYPYTSGGGTKVEIKRGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVS CKASGYAFSSYWMNWVRQAPGQGLEWMGQIYPGDGDTNYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCA RKTISSVVDFYFDYWGQGTLVTVSS (SEQ ID NO:37)

[0184] GS 2 1 sc F v contains GS 2 1 VH and GS 2 1 VL, with the structure: GS 2 1 VL - linker sequence (GGGGSGGGGSGGGGS) - GS21 VH, wherein the amino acid sequence of GS21 VH is as follows:

[0185] QVQLVQSGAEVKKPGSSVKVSCKASGYAFSSYWMNWVRQAPGQGLEW MGQIYPGDGDTNYNGKFKGR VTITADKSTSTAYMELSSLRSEDTAVYYCARK TISSVVDFYFDYWGQGTLVTVSS (SEQ ID NO:37) NO:10)

[0186] The nucleotide sequence encoding GS21 VH is shown below:

[0187] CAAGTGCAGCTGGTCCAGAGCGGCGCCGAGGTGAAGAAACCTGGCTCTAGCGTGAAAGTGTCCTGCAA GGCCTCTGGCTACGCCTTTTCCAGCTATTGGATGAACTGGGTGCGGCAGGCTCCTGGCCAGGGCCTGGAATGGATGGGCCAGATCTACCCCGGAGATGGCGATACCAACTACAATGGCAAGTTCAAGGGCAGAGTGACCATCACCGCTGACAAGTCTACAAGCACAGCCTACATGGAGCTGTCCAGCCTGCGGAGCGAGGACACCGCCGTGTACTACTGTGCCAGAAA Description 14 / 19 page 20 CN 122302055 A GACCATCTCCTCTGTGGTGGACTTCTACTTCGACTACTGGGGACAGGGCACGCTGGTGACCGTGTCCTCT(SEQ ID NO: 21)

[0188] The amino acid sequence of GS21 VL is as follows:

[0189] DIQLTQSPSFLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSA TYRNSGVPSRFSGSGS GTEFTLTISSLQPEDFATYFCQQYNRYPYTSGGGTKVE IKR(SEQ ID NO: 11)

[0190] The nucleotide sequence encoding GS21 VL is as follows:

[0191] GACATCCAACTGACCCAGAGCCCCTCCTTCCTGAGCGCCTCTGTCGGCGATAGAGTGACAATCACCTG TAAGGCCAGCCAGAACGTGGGCACCAATGTGGCCTGGTACCAGCAGAAACCAGGCAAGGCTCCTAAGCCTCTGATC TACTCCGCTACATATCGGAACAGCGGCGTGCCTTCGAGATTTTCTGGCAGCGGCTCTGGAACCGAGTTCACCCTGA CCATCTCCTCTCTGCAGCCTGAGGACTTCGCCACCTACTTCTGCCAGCAGTACAACAGATACCCCTACACCTCCGG AGGCGGCACCAAGGTGGAAATCAAG(SEQ ID NO: 22)

[0192] >GS22 scFv

[0193] DIQLTQSPSFLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSATYRNSGVPSRFSGSGSG TEFTLTISSLQPEDFATYFCQQYNRYPYTSGGGTKVEIKRGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYAFSSYWMNWVRQAPGQGLEWIGQIYPGDGDTNYNGKFKGRATITADKSTSTAYMELSSLRSEDTAVYFCA RKTISSVVDFYFDYWGQGTLVTVSS (SEQ ID NO:38)

[0194] GS 2 2 sc F v contains GS 2 2 VH and GS 2 2 VL, with the structure: GS 2 2 VL - linker sequence (GGGGSGGGGSGGGGS) - GS22 VH, wherein the amino acid sequence of GS22 VH is as follows:

[0195] QVQLVQSGAEVKKPGSSVKVSCKASGYAFSSYWMNWVRQAPGQGLEWI GQIYPGDGDTNYNGKFKGR ATITADKSTSTAYMELSSLRSEDTAVYFCARKTIS SVVDFYFDYWGQGTLVTVSS (SEQ ID NO:8)

[0196] The nucleotide sequence encoding GS22 VH is shown below:

[0197] CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGATCTAGCGTGAAAGTGTCCTGCAA GGCCTCCGGCTACGCCTTCAGCTCCTATTGGATGAACTGGGTGCGGCAGGCTCCTGGACAAGGCCTGGAATGGATC GGCCAGATCTACCCTGGCGATGGCGATACCAACTACAATGGCAAGTTCAAGGGCAGAGCCACCATCACCGCCGACA AGTCCACATCTACCGCCTACATGGAGCTGAGCAGCCTGAGATCCGAGGACACCGCTGTGTACTTTTGTGCCAGAAA GACCATCAGCTCCGTGGTGGACTTCTACTTCGACTACTGGGCCAGGGCACCCTGGTCACAGTGTCTTCT(SEQ ID NO:19)

[0198] The amino acid sequence of GS22 VL is shown below:

[0199] DIQLTQSPSFLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKPLIYSA TYRNSGVPSRFSGSGS GTEFTLTISSLQPEDFATYFCQQYNRYPYTSGGGTKVE IKR (SEQ ID NO:11)

[0200] The nucleotide sequence encoding GS22 VL is shown below:GACATCCAACTGACCCAGAGCCCCTCCTTCCTGAGCGCCTCTGTCGGCGATAGAGTGACAATCACCTG TAAGGCCAGCCAGAACGTGGGCACCAATGTGGCCTGGTACCAGCAGAAACCAGGCAAGGCTCCTAAGCCTCTGATC TACTCCGCTACATATCGGAACAGCGGCGTGCCTTCGAGATTTTCTGGCAGCGGCCTCTGGAACCGAGTTCACCCTGA CCATCTCCTCTCTGCAGCCTGAGGACTTCGCCACCTACTTCTGCCAGCAGTACAACAGATACCCCTACACCTCCGG AGGCGGCACCAAGGTGGAAATCAAGCGG (SEQ ID NO: 22)

[0202] The first connection sequence:

[0203] GGGGSGGGGSGGGGS (SEQ ID NO: 23)

[0204] The second connection sequence: Instructions 15 / 19 Page 21 CN 122302055 A

[0205] GGGGS (SEQ ID NO:24)

[0206] Amino acid sequence of CD8 hinge region:

[0207] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO:25)

[0208] Amino acid sequence of CD8 transmembrane region:

[0209] IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:26)

[0210] Amino acid sequence of 4-1BB intracellular signal transduction region:

[0211] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK PRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKD TYDALHMQALPPR (SEQ ID NO:27)

[0212] The amino acid sequence of the CD3ζ co-stimulatory signal transduction region:

[0213] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:28)

[0214] The amino acid sequence of the CD8α guide peptide:

[0215] MALPVTALLL PLALLLHAARP (SEQ ID NO:29)

[0216] Example 1, Preparation of humanized CD19 CAR-T cells

[0217] I. Humanization Screening of Anti-CD19 Mouse Antibodies

[0218] 1. Construction and Lentiviral Packaging of Humanized Anti-CD19-CAR

[0219] The mouse antibody HI19a (named CNCT19 in this patent, the amino acid sequence of its scFv is shown in SEQ ID NO:30) was designed for humanization. The humanized antibody sequence numbers are Q11 (the amino acid sequence of its scFv is shown in SEQ ID NO:31, the amino acid sequence of VH is shown in SEQ ID NO:1, and the amino acid sequence of VL is shown in SEQ ID NO:2), Q14 (the amino acid sequence of its scFv is shown in SEQ ID NO:32, the amino acid sequence of VH is shown in SEQ ID NO:3, and the amino acid sequence of VL is shown in SEQ ID NO:2), Q23 (the amino acid sequence of its scFv is shown in SEQ ID NO:33, the amino acid sequence of VH is shown in SEQ ID NO:4, and the amino acid sequence of VL is shown in SEQ ID NO:5), Q33 (the amino acid sequence of its scFv is shown in SEQ ID NO:31, and the amino acid sequence of VH is shown in SEQ ID NO:4, and the amino acid sequence of VL is shown in SEQ ID NO:5), and Q33 (the amino acid sequence of its scFv is shown in SEQ ID NO:31, and the amino acid sequence of VL is shown in SEQ ID NO:32, and the amino acid sequence of VL is shown in SEQ ID NO:32), Q23 (the amino acid sequence of its scFv is shown in SEQ ID NO:33, and the amino acid sequence of VH is shown in SEQ ID NO:4, and the amino acid sequence of VL is shown in SEQ ID NO:5), and Q33 (the amino acid sequence of its scFv is shown in SEQ ID NO:32, and the amino acid sequence of VL is shown in SEQ ID NO:3 NO:34, the amino acid sequence of VH is shown in SEQ ID NO:4, the amino acid sequence of VL is shown in SEQ ID NO:6), Q54 (the amino acid sequence of its scFv is shown in SEQ ID NO:35, the amino acid sequence of VH is shown in SEQ ID NO:3, the amino acid sequence of VL is shown in SEQ ID NO:7), GS32 (the amino acid sequence of its scFv is shown in SEQ ID NO:36, the amino acid sequence of VH is shown in SEQ ID NO:8, the amino acid sequence of VL is shown in SEQ ID NO:9), GS21 (the amino acid sequence of its scFv is shown in SEQ ID NO:37, the amino acid sequence of VH is shown in SEQ ID NO:10, the amino acid sequence of VL is shown in SEQ ID NO:11), GS22 (the amino acid sequence of its scFv is shown in SEQ ID NO:38, the amino acid sequence of VH is shown in SEQ ID NO:8, the amino acid sequence of VL is shown in SEQ ID NO:11). Different anti-CD19 humanized scFvs were used to construct CAR genes with CD8α guide chain signal peptide (as shown in SEQ ID NO:29), CD8 hinge region (as shown in SEQ ID NO:25), CD8 transmembrane region (as shown in SEQ ID NO:26), 4-1BB intracellular domain (as shown in SEQ ID NO:27), and CD3ζ (as shown in SEQ ID NO:28). Different anti-CD19 CAR genes were then constructed into different lentiviral master plasmids (manufacturer: SBI, catalog numbers: CD500-CD800, e.g., ...).The CAR expression vector was obtained by conventional resistance modification as described in Example 1 of WO2021 / 121227. The main plasmid and three packaging plasmids (pMD2.G (purchased from Biovector, product number Biovector012259), pMDLg / pRRE (purchased from Biovector, product number Biovector012251), and pRSV-Rev (purchased from Biovector, product number Biovector012253)) were mixed and co-transfected into 293T cells. Lentiviral viruses containing different anti-CD19-CARs were collected for infecting T cells.

[0220] 2. Preparation of different humanized anti-CD19 CAR-T cells

[0221] The T cell culture medium was Optimizer basal medium supplemented with OpTmizer amplification additive, ISR and GlutaMAX (commercially available), and cytokines IL-7 (10 ng / mL) and IL-15 (5 ng / mL) were added. Instructions for Use, Pages 16 / 19, 22 CN 122302055 A

[0222] Day 1: PBMC cell resuscitation;

[0223] Day 0: T cells in PBMCs are sorted using CD4 and CD8 magnetic beads, and T cells are activated using Thermo CD3 / CD28 activation magnetic beads.

[0224] Day 1: T cells are infected with the corresponding humanized anti-CD19-CAR lentivirus in the CNCT19, Q11, Q14, Q23, Q33, Q54, GS32, GS21, and GS22 groups.

[0225] T cell culture medium was added every 2-3 days according to the culture status. Cell viability and CD19-CAR positivity were detected by sampling and counting. After 9 days of culture, CAR-T cells (humanized Q11 CAR-T cells, Q14 CAR-T cells, Q23 CAR-T cells, Q33 CAR-T cells, Q54 CAR-T cells, GS32 CAR-T cells, GS21 CAR-T cells, GS22 CAR-T cells and one type of mouse-derived CNCT19 CAR-T cell) were harvested and subjected to in vitro functional testing.

[0226] After humanizing the CNCT19 sequence, Q11, Q14, Q23, Q33, Q54, GS32, GS21 and GS22 were selected for a series of experimental functional verifications. The experimental results are shown in Figures 1-5.

[0227] 1. Detection of CAR-T cell positivity rate

[0228] Eight types of humanized CAR-T cells and one type of mouse-derived CNCT19 CAR-T cells obtained in Example 1 were mixed with CD19 antigen-conjugated PE fluorescein and incubated at room temperature in the dark for 15 minutes; the supernatant was removed by centrifugation, and the CAR-T cells were compared with CD19 antigen-conjugated PE fluorescein on different days after transduction (Day 3, Day 4, Day 5, Day 6, Day 7, Day 8, Day 9, Day 10, Day 11, Day 12, Day 13, Day 14, Day 15, Day 16, Day 17, Day 18, Day 19 ...On Day 6 and Day 9, the CAR molecule positivity ratio of various cells was detected by flow cytometry. The results are shown in Figure 1 and Figure 2: except for the poor CAR positivity rate of Q23 structure (Q23 CAR-T cell), the expression of other structures was normal; except for the low MFI (mean fluorescence intensity) of CAR expression in Q23 structure (Q23 CAR-T cell), the expression of other structures was normal.

[0229] 2. In vitro killing of CAR-T cells

[0230] 1. Target cell plate

[0231] 1.1 Target cell preparation:

[0232] 1.1.1 The target cells are in good logarithmic growth phase with a cell viability of more than 85%.

[0233] 1.1.2 Count the target cells according to the standard cell counting procedure. Take 2×10⁶ cells of target cells into a 15ml centrifuge tube, centrifuge at 300g for 8min, remove the supernatant, and resuspend the target cells in TO medium (X-Vivo medium + 5% inactivated FBS + 1% GlutaMax) until the cell density is 2×10⁵ cells / ml.

[0234] 1.1.3 Carefully transfer the diluted target cells into a sterile sample loading tray and add the target cells into a black, transparent, flat-bottomed 96-well plate using a multi-channel pipette, 50μL / well.

[0235] 1.2 Effector cell plating

[0236] 1.2.1 Effector cell preparation: Calculate the cell quantities according to the effector-to-target ratios of 9:1, 3:1, and 1:1, respectively, to be 9×10⁴, 3×10⁴, and 1×10⁴ cells. Count the effector cells, take the required number of CAR-T cell samples into a 15ml centrifuge tube, centrifuge at 300g for 8min, and discard the supernatant.

[0237] 1.2.2 Resuspend the CAR-T cells in TO medium (X-Vivo medium + 5% inactivated FBS + 1% GlutaMax), and adjust the effector cell density according to the effector-to-target ratio.

[0238] 1.2.3 Add effector cells to the well plates at effector-to-target ratios of 9:1, 3:1, and 1:1, 50μL / well.

[0239] 1.3 Target cell control group setup

[0240] Add 50μL of target cells and 50μL of TO medium (X-Vivo medium + 5% inactivated FBS + 1% GlutaMax) to the wells of the target cell control group, so that the total culture volume of the target cell control group is consistent with the total culture volume of the co-culture experimental group.

[0241] 2. Detection of luc value

[0242] 2.1 Cell co-culture: Place the 96-well plate with the sample added in a carbon dioxide incubator and co-culture for 4 hours.

[0243] 2.2 Addition of detection reagent: Melt steadyglo luciferase (commercially available) at 4°C or room temperature in the dark. Dilute steadyglo required for the experiment (page 17 / 19 of the instruction manual, 23 CN 122302055 A) 3 times with PBS, mix well, add 50 μL to each well, and place in a 96-well plate shaker to mix.1. Shake at 100 rpm for 15 min.

[0244] 2.3 Detection on the instrument: Turn on the multi-functional microplate reader and computer software, put the shaken 96-well plate into the multi-functional microplate reader, and detect the luc fluorescence intensity. Take out the 96-well plate and turn off the instrument and computer.

[0245] 3. Calculation of cell killing activity

[0246] After obtaining the luc values ​​of all wells, calculate the killing activity according to the following formula

[0247] Cell killing activity = (average luc value of individual target cell group - luc value of co-culture well) / average luc value of individual target cell group × 100%

[0248] The detection results are shown in Figure 3: There is no significant difference in the killing activity of CAR-T cells against target cell Nalm6 in all groups.

[0249] 3. Cytokine Detection

[0250] 1. CAR-T cells and target cells were mixed at an effector-to-target ratio of 1:1.

[0251] 2. Incubated in carbon dioxide medium for 24 hours.

[0252] 3. After incubation, the mixture was centrifuged at 500g for 10 minutes, and the supernatant was collected.

[0253] 4. The cytokine concentration in the supernatant was detected using the "Human Th1 Panel (5-Plex) with Filter Plate V02" kit.

[0254] The detection results are shown in Figures 4A and 4B: The IL-2 release of CAR-T cells from humanized structures Q11, Q14, Q33, Q54, GS32, and GS21 was higher than that of mouse-derived CNCT19. The IFN-γ release of CAR-T cells from humanized structures Q11, Q33, GS32, and GS21 was higher than that of mouse-derived CNCT19. This experimental result shows that the humanization modification of the murine antibody in this application alters the antibody's ability to bind to the antigen, thereby making the humanized CAR-T cells of this application superior to the murine CNCT19 structure in terms of IL-2 release and IFN-γ release.

[0255] 4. Multiple rounds of stimulation

[0256] 1. CAR-T cells and target cells are mixed at an effector-to-target ratio of 1:1 and a cell number of 0.5×106:0.5×106

[0257] 2. Every 2 days, the number of CAR-T cells is detected, and 0.5×106 CAR-T cells are taken and 0.5×106 target cells are added

[0258] 3. After 4 rounds of target cell stimulation, the fold increase of CAR-T cells is calculated.

[0259] The detection results are shown in Figure 5: After 4 rounds of target cell stimulation, the fold increase of CAR-T cells in humanized structures Q11, Q33, and GS21 is consistent with that in murine structure CNCT19.

[0260] In addition, three biocomputing tools, T20 score, HS score, and QASis, were used to analyze the VH and VL chains of CD19.Humanization scores from the three tools showed a convergence, with Q11, Q14, Q23, Q33, Q54, GS32, GS21, and GS22 showing higher humanization scores compared to murine CNCT19 (see Figure 6). Two immunogenicity prediction tools, Alpha MHC v2 and CD4 T cell Immunogenicity Prediction Tool, were used to predict the immunogenicity of humanized antibodies. The prediction results were largely consistent, showing that Q11, Q14, Q23, Q33, Q54, and GS21 had the lowest immunogenicity risk (see Figure 7).

[0261] The immunogenicity of antibody drugs may trigger an anti-drug immune response, thereby inducing the production of anti-drug antibodies (ADA). Antibody immunogenicity evaluation is mainly performed using anti-drug antibody (ADA) analysis. CAR-T immunogenicity causes an anti-CAR immune response, which in turn destroys and eliminates CAR-T cells, contributing to CAR-T therapy failure. The scFvs used in CAR-T contain mouse-derived sequences and are an important factor in generating anti-CAR immune responses. Humanizing mouse-derived scFvs to avoid anti-CAR immune responses associated with such mouse-derived scFvs is an important strategy. However, whether the antibody functional reduction or loss can be avoided during the modification process is the key to the success or failure of this strategy. Specification 18 / 19 pages 24 CN 122302055 A

[0262] Based on the in vitro functional data of different humanized CD19 CAR-Ts, combined with antibody humanization degree scores and antibody immunogenicity prediction data, the Q33 and GS21 sequences have good CAR positive rate expression, high in vitro killing activity and cytokine release, and low immunogenicity. Instruction manual 19 / 19 pages 25 CN 122302055 A Figure 1 Figure 2 Figure 3 Instruction manual Figure 1 / 5 pages 26 CN 122302055 A Figure 4A Figure 4B Instruction manual Figure 2 / 5 pages 27 CN 122302055 A Figure 5 Instruction manual Figure 3 / 5 pages 28 CN 122302055 A Figure 6 Instruction manual Figure 4 / 5 pages 29 CN 122302055 A Figure 7 Instruction manual Figure 5 / 5 pages 30 CN 122302055 A Abstract The present invention provides a CD19-targeting humanized antibody and chimeric antigen receptor, and theuse thereof. The humanized antibody contains CD19 VH and CD19 VL which are selected from one of groups 1) to 8). The CDl9-targeting chimeric antigen receptor of the present invention contains a CD19-targeting extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain, wherein the CD19-targeting extracellular antigen recognition domain contains CD19 VH and CD19 VL which are selected from one of groups 1) to 8). D ay 3 D ay 6 D ay 9 0 20 40 60 80 C A R + % CNCT19 Q11 Q14 Q23 Q33 Q54 GS32 GS21 GS22 Figure 1

Claims

1. A humanized antibody targeting CD19 or an antigen-binding fragment thereof, comprising CD19 VH and CD19VL, selected from one of the following: 1) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:1, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:2; or 2) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:3, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:2; or 3) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:4, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:5; or 4) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:4, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:6; or 5) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:3, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:7; or 6) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:8, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:9; or 7) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:10, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:11; or 8) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:8, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:

11.

2. The humanized antibody or its antigen-binding fragment according to claim 1, wherein, The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:4, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:6; or The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:10, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:

11.

3. The humanized antibody or its antigen-binding fragment according to claim 1 or 2, wherein it is an scFv antibody, an sc(Fv)2 antibody or a [sc(Fv)2]2 antibody.

4. The humanized antibody or its antigen-binding fragment according to claim 1 or 2, wherein it is an scFv antibody as shown in any of SEQ ID NO:31-38.

5. An antibody drug comprising the humanized antibody or antigen-binding fragment thereof as described in any one of claims 1-4.

6. An antibody-drug conjugate comprising any one of claims 1-4, or an antigen-binding fragment thereof.

7. A chimeric antigen receptor targeting CD19, comprising an extracellular antigen recognition domain targeting CD19, a hinge region, a transmembrane region, and an intracellular domain, wherein the extracellular antigen recognition domain targeting CD19 comprises CD19VH and CD19VL, selected from one of the following: 1) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:1, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:2; or 2) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:3, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:2; or 3) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:4, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:5; or 4) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:4, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:6; or 5) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:3, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:7; or 6) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:8, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:9; or 7) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:10, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:11; or 8) The CD19 VH sequence comprises the amino acid sequence shown in SEQ ID NO:8, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:

11.

8. The chimeric antigen receptor according to claim 7, wherein, The extracellular antigen recognition domain includes any one of the following structures: The amino acid sequence shown in SEQ ID NO:1-linked sequence-amino acid sequence shown in SEQ ID NO:2, amino acid sequence shown in SEQ ID NO:2-linked sequence-amino acid sequence shown in SEQ ID NO:1, amino acid sequence shown in SEQ ID NO:3-linked sequence-amino acid sequence shown in SEQ ID NO:2, amino acid sequence shown in SEQ ID NO:2-linked sequence-amino acid sequence shown in SEQ ID NO:3, amino acid sequence shown in SEQ ID NO:4-linked sequence-amino acid sequence shown in SEQ ID NO:5, amino acid sequence shown in SEQ ID NO:5-linked sequence-amino acid sequence shown in SEQ ID NO:4, amino acid sequence shown in SEQ ID NO:4-linked sequence-amino acid sequence shown in SEQ ID NO:6, amino acid sequence shown in SEQ ID NO:6-linked sequence-amino acid sequence shown in SEQ ID NO:4, amino acid sequence shown in SEQ ID NO:3-linked sequence-amino acid sequence shown in SEQ ID NO:7, amino acid sequence shown in SEQ ID NO:7-linked sequence-amino acid sequence shown in SEQ ID NO:3, amino acid sequence shown in SEQ ID NO:8-linked sequence-amino acid sequence shown in SEQ ID NO:1, amino acid sequence shown in SEQ ID NO:2, amino acid sequence shown in SEQ ID NO:2-linked sequence-amino acid sequence shown in SEQ ID NO:3, amino acid sequence shown in SEQ ID NO:8, amino acid sequence shown in SEQ ID NO:1, amino acid sequence shown in SEQ ID NO:2, amino acid sequence shown in SEQ ID NO:3, amino acid sequence shown in SEQ ID NO:4, amino acid sequence shown in SEQ ID NO:4-linked sequence-amino acid sequence shown in SEQ ID NO:7, amino acid sequence shown in SEQ ID NO:7-linked sequence-amino acid sequence shown in SEQ ID NO:3, amino acid sequence shown in SEQ ID NO:8, amino acid sequence shown in SEQ ID NO:1, amino acid sequence shown in SEQ ID NO:2, amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in NO:9, the amino acid sequence shown in SEQ ID NO:9 - linking sequence - the amino acid sequence shown in SEQ ID NO:8, the amino acid sequence shown in SEQ ID NO:10 - linking sequence - the amino acid sequence shown in SEQ ID NO:11, the amino acid sequence shown in SEQ ID NO:11 - linking sequence - the amino acid sequence shown in SEQ ID NO:10, the amino acid sequence shown in SEQ ID NO:8 - linking sequence - the amino acid sequence shown in SEQ ID NO:11, the amino acid sequence shown in SEQ ID NO:11 - linking sequence - the amino acid sequence shown in SEQ ID NO:8; Optionally, the extracellular antigen recognition domain includes any one of the following structures: Amino acid sequence as shown in SEQ ID NO:4 - linking sequence - amino acid sequence as shown in SEQ ID NO:6, amino acid sequence as shown in SEQ ID NO:6 - linking sequence - amino acid sequence as shown in SEQ ID NO:4, amino acid sequence as shown in SEQ ID NO:10 - linking sequence - amino acid sequence as shown in SEQ ID NO:11, amino acid sequence as shown in SEQ ID NO:11 - linking sequence - amino acid sequence as shown in SEQ ID NO:10; Further, optionally, the extracellular antigen recognition domain includes any one of the following structures: Amino acid sequence as shown in SEQ ID NO:6 - linker sequence - amino acid sequence as shown in SEQ ID NO:4, amino acid sequence as shown in SEQ ID NO:11 - linker sequence - amino acid sequence as shown in SEQ ID NO:

10.

9. The chimeric antigen receptor according to claim 8, wherein, The connection sequence is selected from one or more of the following sequences: SEQ ID NO:23 and SEQ ID NO:

24.

10. The chimeric antigen receptor according to claim 9, wherein, The extracellular antigen recognition domain includes: scFv antibodies as shown in any of SEQ ID NO:31-38.

11. The chimeric antigen receptor according to claim 7, wherein, The hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α; optionally, the amino acid sequence of the hinge region is derived from CD8α; further optionally, the amino acid sequence of the hinge region comprises the amino acid sequence shown in SEQ ID NO:25; and / or The transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70; optionally, the amino acid sequence of the transmembrane region is derived from CD8α; further optionally, the amino acid sequence of the transmembrane region comprises the amino acid sequence shown in SEQ ID NO:

26.

12. The chimeric antigen receptor according to claim 7, wherein, The intracellular domain includes an intracellular signal transduction region; optionally, the intracellular signal transduction region is derived from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CCD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk; further optionally, the intracellular signal transduction region is derived from CD3ζ; even further optionally, the amino acid sequence of the intracellular signal transduction region comprises the amino acid sequence shown in SEQ ID NO:

28.

13. The chimeric antigen receptor according to claim 7, wherein, The intracellular domain further includes a co-stimulatory signal transduction region; optionally, the co-stimulatory signal transduction region is derived from one, two, or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, and MyD88; further optionally, the co-stimulatory signal transduction region is derived from 4-1BB; even further optionally, the amino acid sequence of the co-stimulatory signal transduction region comprises the amino acid sequence shown in SEQ ID NO:

27.

14. The chimeric antigen receptor according to any one of claims 7-13, further comprising a guide peptide located at the N-terminus of the amino acid sequence of the chimeric antigen receptor; optionally, the guide peptide is derived from CD8α; further optionally, the amino acid sequence of the guide peptide comprises the amino acid sequence shown in SEQ ID NO:

29.

15. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a humanized antibody or an antigen-binding fragment thereof as described in any one of claims 1-4, or comprising a nucleotide sequence encoding a chimeric antigen receptor as described in any one of claims 7-14; Optionally, the nucleotide sequence encoding the humanized antibody or its antigen-binding fragment, or the nucleotide sequence encoding the chimeric antigen receptor, comprises nucleotide sequences encoding CD19 VH and CD19 VL, selected from a group of: 1) A nucleotide sequence encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:1, as shown in SEQ ID NO:12; and a nucleotide sequence encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:2, as shown in SEQ ID NO:13; and / or 2) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:3, as shown in SEQ ID NO:14; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:2, as shown in SEQ ID NO:13; and / or 3) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:4, as shown in SEQ ID NO:15; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:5, as shown in SEQ ID NO:16; and / or 4) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:4, as shown in SEQ ID NO:15; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:6, as shown in SEQ ID NO:17; and / or 5) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:3, as shown in SEQ ID NO:14; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:7, as shown in SEQ ID NO:18; and / or 6) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:8, as shown in SEQ ID NO:19; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:9, as shown in SEQ ID NO:20; and / or 7) Nucleotide sequences encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:10, as shown in SEQ ID NO:21; and nucleotide sequences encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:11, as shown in SEQ ID NO:22; and / or 8) A nucleotide sequence encoding the CD19 VH amino acid sequence as shown in SEQ ID NO:8, as shown in SEQ ID NO:19; and a nucleotide sequence encoding the CD19 VL amino acid sequence as shown in SEQ ID NO:11, as shown in SEQ ID NO:

22.

16. A vector comprising the isolated nucleic acid molecule of claim 15; Optionally, the carrier is an expression carrier; Alternatively, the vector may be a viral vector; Alternatively, the vector may be a lentiviral vector.

17. An engineered immune effector cell comprising the humanized antibody or antigen-binding fragment thereof as described in any one of claims 1-4, the chimeric antigen receptor as described in any one of claims 7-14, the isolated nucleic acid as described in claim 15, or the vector as described in claim 16.

18. The engineered immune effector cells according to claim 17, wherein, The engineered immune effector cells are selected from one or more of the following: T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMCs), pluripotent stem cells, T cells differentiated from pluripotent stem cells, NK cells differentiated from pluripotent stem cells, and embryonic stem cells. Optionally, the engineered immune effector cells are T lymphocytes; Alternatively, the T lymphocytes may be derived from autologous T lymphocytes or allogeneic T lymphocytes.

19. A pharmaceutical composition comprising engineered immune effector cells as described in claim 17 or 18 and a pharmaceutically acceptable excipient; optionally, the pharmaceutically acceptable excipient comprises a protective agent; optionally, the pharmaceutically acceptable excipient comprises a cell cryopreservation solution.

20. The pharmaceutical composition according to claim 19, wherein, The pharmaceutical composition is a cell suspension or frozen cells thereof; or the pharmaceutical composition is an intravenous injection.

21. Use in the preparation of a medicament for the treatment of diseases or conditions associated with CD19 expression, the humanized antibody or antigen-binding fragment thereof as described in any one of claims 1-4, the chimeric antigen receptor as described in any one of claims 7-14, the isolated nucleic acid as described in claim 15, the vector as described in claim 16, or the engineered immune effector cell as described in claim 17.

22. The use according to claim 21, wherein, The diseases or conditions associated with CD19 expression include non-solid tumors, acute lymphoblastic leukemia, and / or B-cell lymphoma. Optionally, the non-solid tumors include leukemia and / or lymphoma; Optionally, the acute lymphoblastic leukemia includes adult acute lymphoblastic leukemia and / or childhood acute lymphoblastic leukemia; Optionally, the B-cell lymphoma includes non-Hodgkin lymphoma.

23. The use according to claim 21, wherein, The diseases or conditions associated with CD19 expression include refractory systemic lupus erythematosus immune thrombocytopenic purpura (SLE-ITP), refractory lupus nephritis (LN), refractory immune hemolytic anemia (AIHA), systemic lupus erythematosus (SLE), autoimmune thrombocytopenic purpura (ITP), antineutrophil cytoplasmic antibody-associated vasculitis (AAV), dermatomyositis (DM), myasthenia gravis (MG), or antisynthetic antibody syndrome (ASS).

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