CD19-targeting humanized antibody and chimeric antigen receptor, and use thereof

Abstract

Provided are a CD19-targeting humanized antibody and chimeric antigen receptor, and the use thereof. The humanized antibody contains CD19 VH and CD19 VL which are selected from one of groups 1) to 8). The CD19-targeting chimeric antigen receptor 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).

Description

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 their uses. Background Technology

[0002] Cell therapy is an emerging medical technology. Its main principle is to utilize the patient's own immune cells, culturing and modifying them in vitro to give them the specific ability to kill tumor cells. These modified immune cells are then reinfused into the patient to treat tumors. Chimeric antigen receptor T-cell (CAR-T) technology is an important cell therapy technique. It uses genetic engineering to make T cells express a specific antibody, enabling them to specifically recognize and kill tumor cells expressing the corresponding antigen. Tumor immunotherapy is a treatment method that utilizes 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. CAR-T cell therapy is an important tumor immunotherapy method that directly introduces T cells with specific killing capabilities into the patient, thereby achieving specific killing of tumor cells. Genetic engineering technology is an important component of modern biotechnology. It achieves genetic modification of organisms through operations such as gene cutting, ligation, and transfer. In CAR-T cell therapy, genetic engineering technology is widely used to modify T cells, giving them specific anti-tumor capabilities.

[0003] CD19 is a CD molecule (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 also promotes BCR signaling. 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 CD19-targeting antibodies or CARs contain murine antibodies, which have high immunogenicity in humans and easily induce human anti-mouse antibody (HAMA) reactions, leading to rejection, reduced efficacy, and increased relapse rate. Humanized antibodies or humanized CAR-T cells may help reduce immunogenicity, decrease immune rejection, maintain the long-term presence of humanized antibodies or CAR-T cells in the patient, and improve long-term treatment efficacy. Summary of the Invention

[0005] This application provides a humanized antibody targeting CD19, a chimeric antigen receptor, and their uses. Based on existing CD19 murine antibody sequences, the inventors designed eight humanized CD19 antibodies to reduce their heterology. However, during the humanization process, the antibody's functionality (e.g., CAR positivity expression, in vitro cytotoxic activity, cytokine release, etc.) may be compromised. Therefore, obtaining humanized antibodies with reduced immunogenicity but undiminished functionality is of practical significance. The inventors used the eight humanized CD19 antibodies as the extracellular antigen recognition domain of CD19 CARs and evaluated the proliferation and functionality of CAR-T cells using humanized CD19 scFv antibodies in vitro, selecting the two humanized CD19 scFv antibodies with the best overall performance.

[0006] This invention provides a humanized antibody targeting CD19 or its antigen-binding fragment thereof, comprising CD19VH and CD19 VL, selected from one of the following:

[0007] 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

[0008] 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

[0009] 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

[0010] 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

[0011] 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

[0012] 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

[0013] 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

[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:4, and the CD19 VL sequence comprises the amino acid sequence shown in SEQ ID NO:6; or

[0026] 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

[0027] 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

[0028] 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

[0029] 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.

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

[0031] 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-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; the amino acid sequence shown in SEQ ID NO:8-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:8;

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

[0033] 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;

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

[0035] 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.

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

[0037] In a preferred embodiment of the invention, the connection 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 present invention, the extracellular antigen recognition domain comprises: an scFv antibody as shown in any of SEQ ID NO:31-38.

[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 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.

[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; and even more 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 comprises 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.

[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 nucleotide sequences encoding CD19 VH and CD19 VL, selected from a group of:

[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) 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

[0048] 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

[0049] 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

[0050] 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

[0051] 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

[0052] 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

[0053] 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.

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

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

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

[0057] Alternatively, the vector may be a lentiviral vector.

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

[0059] In a preferred embodiment of the present invention, the engineered immune effector cells are selected from one or more of T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMC cells), 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 cells are T lymphocytes;

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

[0062] The present invention also provides a pharmaceutical composition comprising the above-described engineered immune effector cells and pharmaceutically acceptable excipients; the 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 cryoprotectants; alternatively, pharmaceutically acceptable excipients include cell cryopreservation solutions.

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

[0065] The present invention also provides the use of the above-mentioned humanized antibody or its antigen-binding fragment, the above-mentioned chimeric antigen receptor, the above-mentioned isolated nucleic acid, the above-mentioned vector or the above-mentioned engineered immune effector cells in the preparation of a drug for treating diseases or conditions related to CD19 expression.

[0066] In a preferred embodiment of the present invention, the diseases or conditions associated with CD19 expression include non-solid tumors, acute lymphoblastic leukemia, and / or B-cell lymphoma.

[0067] Optionally, the non-solid tumors include 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 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).

[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 in need of treatment for diseases or conditions related to CD19 expression.

[0072] In some embodiments, the administration can be performed in various ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, local, or intradermal administration. For example, administration can be given to the subject via intravenous injection. In some embodiments, an effective dose of engineered immune effector cells or pharmaceutical composition can be administered to the subject in a single dose 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 implementation schemes, the dosage may vary depending on the indication; the dosage may also vary depending on the severity of the patient's condition. The dosage range may be 1×10⁻⁶. 5 CAR-positive T cells / kg to 1×10 7 CAR-positive T cells / kg, for example, 1×10 5 CAR-positive T cells / kg to 1×10 6 CAR-positive T cells / kg, 1×10 6 CAR-positive T cells / kg to 1×10 7 CAR-positive T cells / kg, 0.5×10 6 CAR-positive T cells / kg, 0.6×10 6 CAR-positive T cells / kg, 0.7×106 CAR-positive T cells / kg, 0.8×10 6 CAR-positive T cells / kg, 0.9 × 10 6 CAR-positive T cells / kg, 1.0×10 6 CAR-positive T cells / kg, 1.1×10 6 CAR-positive T cells / kg, 1.2 × 10 6 CAR-positive T cells / kg, 1.3×10 6 CAR-positive T cells / kg, 1.4 × 10 6 CAR-positive T cells / kg, 1.5 × 10 6 CAR-positive T cells / kg, 1.6 × 10 6 CAR-positive T cells / kg, 1.7 × 10 6 CAR-positive T cells / kg, 1.8 × 10 6 CAR-positive T cells / kg, 1.9 × 10 6 CAR-positive T cells / kg, 2.0 × 10 6 CAR-positive T cells / kg.

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

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

[0076] In a preferred embodiment of the present invention, the diseases or conditions associated with CD19 expression include non-solid tumors, acute lymphoblastic leukemia, and / or B-cell lymphoma.

[0077] Optionally, the non-solid tumors include leukemia and / or lymphoma;

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

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

[0080] In a preferred embodiment of the 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 exhibits comparable efficacy to mouse CD19 antibodies or mouse CD19 CAR-T in terms of tumor cell killing activity and fold expansion after multiple rounds of stimulation. Furthermore, the humanized CD19 antibody or humanized CAR-T of the present invention demonstrates higher IL-2 and IFN-γ release than mouse CD19 antibodies or mouse CD19 CAR-T. Additionally, the humanized CD19 antibody or humanized CAR-T of the present invention exhibits lower immunogenicity compared to mouse CD19 antibodies or mouse CD19 CAR-T, thereby providing better safety. Attached Figure Description

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

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

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

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

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

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

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

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

[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 following further describes this application: 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 terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein 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 (TAAs)), 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 approaches to combating cancer. CAR-T cells utilize genetic modification to enable T cells to express CAR proteins. These CAR proteins are capable of recognizing intact proteins on the cell membrane surface without antigen presentation, thereby activating and functionally affecting T cells.

[0094] In this application, the term "extracellular antigen recognition domain" refers to the antigen recognition domain (ARD). CAR cell therapy products (such as CAR-T cells) rely on extracellular antigen recognition domains to specifically recognize and / or bind to target antigens expressed by tumor cells. To date, antigen recognition domains are derived from the single-chain variable fragment (scFv) of antibodies, or from receptor-ligand interactions, TCR mimics, and variable lymphocyte receptors (VLRs). The most common source to date is the scFv segment of antibodies, which includes both heavy and light chain variable regions linked by a peptide chain, such as the 18-amino acid linker sequence GSTGSGSGKPGSGEGSTKG. In antibody analysis, common CDR (Continuous Derivative) rules include Kabat, AbM, Chothia, Contact, and IMGT. These rules are well-known to those skilled in the art. When applying websites that execute these rules, simply inputting the VH and VL sequences and selecting the corresponding rule will yield CDR sequences based on different rules. Those skilled in the art should understand that the scope of this application covers combinations of CDR sequences obtained through analysis using different rules.

[0095] In this application, the term "hinge region" refers to the connecting segment that acts between the extracellular antigen recognition domain and the transmembrane domain. This region allows the CAR to recognize the antigen by providing a certain range of motion to the antigen recognition domain. Currently used hinge regions are mainly derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α. In addition, typical hinge regions also contain residues that participate in CAR dimerization, which helps to 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 interactions. 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 antigen-specific signal stimulation, many immune effector cells require co-stimulation to promote cell proliferation, differentiation, and survival, as well as to activate effector functions. In some embodiments, the CAR may further include one or more co-stimulatory signal transduction regions, wherein the co-stimulatory signal transduction regions may 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 and refers to a single chain variable fragment (scFv), which is an antibody composed of antibody heavy chain variable fragments and light chain variable fragments linked by 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 their conventional meanings 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 can also include extracellular antigen recognition domains targeting any of the following targets (e.g., scFv antibodies): CD5, CD20, CD22, CD33, CD123, CLL1, BCMA, CD138, and 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 "separated" generally refers to substances obtained artificially from their natural state. If a substance or component is found in nature as a "separated" substance, 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 separated from this natural state is called a separated substance. The term "separated" does not exclude substances obtained artificially from their natural state and then synthesized, nor does it exclude the presence of other impurities that do not affect the substance's activity.

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

[0105] In this application, the term "HI19a antibody" refers to an existing murine antibody that specifically binds to the extracellular domain of CD19 on the surface of human B cells and exhibits high affinity. Directly using murine antibody-derived scFv in CAR-T drugs may increase the risk of immune responses. To reduce the immunogenicity of murine antibody-derived scFv and improve treatment safety, it can be achieved by humanizing the murine antibody-derived scFv. However, during the humanization process, the antibody's binding affinity, specificity, and other functionalities may be compromised. Therefore, obtaining humanized antibodies with reduced immunogenicity but without loss of functionality is of practical significance.

[0106] In this application, the term "humanized antibody" also refers to a humanized antibody. Methods for humanization are known (e.g., WO96 / 02576). The purpose of humanization is to reduce heterology while essentially preserving the affinity and specificity of the parent antibody. For example, when the CDR is derived from a mouse antibody, primer 25 (the corresponding primer can be obtained by referring to the method described in WO98 / 13388) can be synthesized and used to link the CDR of the mouse antibody to the frame 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 together any structure / region of the chimeric antigen receptor of the present invention. The linking sequence may consist of different amino acid residues (such as glycine and serine) so that adjacent protein domains can move freely relative to each other. Longer linking sequences may 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, the gene transduction / transfection methods for CAR gene transduction / transfection and target gene expression mainly include viral and non-viral methods. These include: gamma 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 nucleic acid delivery vehicle that inserts a polynucleotide encoding a protein into itself, thereby enabling the protein to be expressed. Vectors can transform, transduce, or transfect host cells, allowing the genetic material they carry to be expressed within the host cell. Examples of vectors include: plasmids; phage particles; Cos plasmids; 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 segment of DNA capable of migrating between chromosomal loci and carrying 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, and preservatives. 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 that uses antibody substances as active ingredients, such as monoclonal antibody drugs and bispecific antibody drugs.

[0114] In this application, the term "antibody-drug conjugate" has its conventional meaning in the art, abbreviated as ADC. It refers to a small molecule drug with biological activity linked to a monoclonal antibody via a chemical chain, with the monoclonal antibody acting as a carrier to target and deliver the small molecule drug to 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 refers to a range of fluctuations acceptable to a person skilled in the art above or below a specified value, such as a variation within ±0.5% to 10%, for example, a 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 a specified value.

[0118] Not intended to be limited by any theory, the embodiments described below are merely illustrative of 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 mouse-derived antibody and humanized antibody scFv of the present invention

[0120] >HI19a(CNCT19)scFv

[0121] >Q11 scFv

[0122] Q11 scFv contains Q11 VH and Q11 VL, with the structure: Q11 VL-linking sequence (GGGGSGGGGSGGGGS)-Q11 VH, where the amino acid sequence of Q11 VH is shown below:

[0123] The nucleotide sequence encoding Q11 VH is shown below:

[0124] The amino acid sequence of Q11 VL is shown below:

[0125] The nucleotide sequence encoding Q11 VL is shown below:

[0126] >Q14 scFv

[0127] Q14 scFv contains Q14 VH and Q14 VL, with the structure: Q14 VL-linking sequence (GGGGSGGGGSGGGGS)-Q14 VH, where the amino acid sequence of Q14 VH is shown below:

[0128] The nucleotide sequence encoding Q14 VH is shown below:

[0129] The amino acid sequence of Q14 VL is shown below:

[0130] The nucleotide sequence encoding Q14 VL is shown below:

[0131] >Q23 scFv

[0132] Q23 scFv contains Q23 VH and Q23 VL, with the structure: Q23 VL-linking sequence (GGGGSGGGGSGGGGS)-Q23 VH, where the amino acid sequence of Q23 VH is shown below:

[0133] The nucleotide sequence encoding Q23 VH is shown below:

[0134] The amino acid sequence of Q23 VL is shown below:

[0135] The nucleotide sequence encoding Q23 VL is shown below:

[0136] >Q33 scFv

[0137] Q33 scFv contains Q33 VH and Q33 VL, with the structure: Q33 VL-linking sequence (GGGGSGGGGSGGGGS)-Q33 VH, where the amino acid sequence of Q33 VH is shown below:

[0138] The nucleotide sequence encoding Q33 VH is shown below:

[0139] The amino acid sequence of Q33 VL is shown below:

[0140] The nucleotide sequence encoding Q33 VL is shown below:

[0141] >Q54 scFv

[0142] Q54 scFv contains Q54 VH and Q54 VL, with the structure: Q54 VL-linking sequence (GGGGSGGGGSGGGGS)-Q54 VH, where the amino acid sequence of Q54 VH is shown below:

[0143] The nucleotide sequence encoding Q54 VH is shown below:

[0144] The amino acid sequence of Q54 VL is shown below:

[0145] The nucleotide sequence encoding Q54 VL is shown below:

[0146] >GS32 scFv

[0147] GS32 scFv contains GS32 VH and GS32 VL, with the structure: GS32 VL-linking sequence (GGGGSGGGGSGGGGS)-GS32 VH, where the amino acid sequence of GS32 VH is shown below:

[0148] The nucleotide sequence encoding GS32 VH is shown below:

[0149] The amino acid sequence of GS32 VL is shown below:

[0150] The nucleotide sequence encoding GS32 VL is shown below:

[0151] >GS21 scFv

[0152] GS21 scFv contains GS21 VH and GS21 VL, with the structure: GS21 VL-linker sequence (GGGGSGGGGSGGGGS)-GS21 VH, where the amino acid sequence of GS21 VH is shown below:

[0153] The nucleotide sequence encoding GS21 VH is shown below:

[0154] The amino acid sequence of GS21 VL is shown below:

[0155] The nucleotide sequence encoding GS21 VL is shown below:

[0156] >GS22 scFv

[0157] GS22 scFv contains GS22 VH and GS22 VL, with the structure: GS22 VL-linking sequence (GGGGSGGGGSGGGGS)-GS22 VH, where the amino acid sequence of GS22 VH is shown below:

[0158] The nucleotide sequence encoding GS22 VH is shown below:

[0159] The amino acid sequence of GS22 VL is shown below:

[0160] The nucleotide sequence encoding GS22 VL is shown below:

[0161] First type of connection sequence:

[0162] The second type of connection sequence:

[0163] The amino acid sequence of the CD8 hinge region:

[0164] The amino acid sequence of the CD8 transmembrane region:

[0165] The amino acid sequence of the 4-1BB intracellular signal transduction region:

[0166] The amino acid sequence of the CD3ζ co-stimulatory signal transduction region:

[0167] The amino acid sequence of the CD8α guide peptide:

[0168] Example 1: Preparation of humanized CD19 CAR-T cells

[0169] I. Humanization Screening of Anti-CD19 Mutual Antibodies

[0170] 1. Construction and lentiviral packaging of humanized anti-CD19-CAR

[0171] Humanization was performed on the murine antibody HI19a (named CNCT19 in this patent, with its scFv amino acid sequence as shown in SEQ ID NO:30). The humanized antibody sequences are numbered as follows: Q11 (with scFv amino acid sequence as shown in SEQ ID NO:31, VH amino acid sequence as shown in SEQ ID NO:1, and VL amino acid sequence as shown in SEQ ID NO:2), Q14 (with scFv amino acid sequence as shown in SEQ ID NO:32, VH amino acid sequence as shown in SEQ ID NO:3, and VL amino acid sequence as shown in SEQ ID NO:2), Q23 (with scFv amino acid sequence as shown in SEQ ID NO:33, VH amino acid sequence as shown in SEQ ID NO:4, and VL amino acid sequence as shown in SEQ ID NO:5), Q33 (with scFv amino acid sequence as shown in SEQ ID NO:34, VH amino acid sequence as shown in SEQ ID NO:4, and VL amino acid sequence as shown in SEQ ID NO:6), and Q54 (with scFv amino acid sequence as shown in SEQ ID NO:30). 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 combined 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). CAR genes were constructed by combining different anti-CD19 CAR genes into different lentiviral master plasmids (manufacturer: SBI, catalog number: CD500-CD800, as described in Example 1 of WO2021 / 121227 for routine resistance modification) to obtain CAR expression vectors. The master plasmids were co-transfected with 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)) into 293T cells. Lentiviral viruses containing different anti-CD19-CARs were collected for infecting T cells.

[0172] 2. Preparation of different humanized anti-CD19 CAR-T cells

[0173] The T cell culture medium is 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).

[0174] PBMC cell resuscitation was performed on day -1.

[0175] On day 0, T cells in PBMCs were sorted using CD4 and CD8 magnetic beads, and T cells were activated using Thermo CD3 / CD28 activation magnetic beads.

[0176] On day 1, T cells were infected with the corresponding humanized anti-CD19-CAR lentivirus in the CNCT19, Q11, Q14, Q23, Q33, Q54, GS32, GS21, and GS22 groups.

[0177] T cell culture medium was added every 2-3 days according to the culture progress. Cell viability and CD19-CAR positivity were measured 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 murine CNCT19 CAR-T cell) were harvested and subjected to in vitro functional tests.

[0178] 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.

[0179] 1. Detection of CAR-positive proportion in CAR-T cells

[0180] 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 molecule positivity rate of each cell type was detected by flow cytometry at different days after transduction (Day 3, Day 6, and Day 9). The detection results are shown in Figures 1 and 2: except for the poor CAR positivity rate of Q23 structure (Q23 CAR-T cells), the expression of other structures was normal; except for the low MFI (mean fluorescence intensity) of CAR expression in Q23 structure (Q23 CAR-T cells), the expression of other structures was normal.

[0181] 2. In vitro killing effect of CAR-T cells

[0182] 1. Target cell plate

[0183] 1.1 Target cell preparation:

[0184] 1.1.1 The target cells are in good logarithmic growth phase with a cell viability of over 85%.

[0185] 1.1.2 Count the target cells according to standard cell counting procedures, and take 2 × 10⁻⁶ cells. 6 Transfer the target cells to a 15ml centrifuge tube, centrifuge at 300g for 8 minutes, remove the supernatant, and resuspend the target cells in T0 medium (X-Vivo medium + 5% inactivated FBS + 1% GlutaMax) until the cell density is 2×10⁻⁶ cells / mL. 5 per ml.

[0186] 1.1.3 Carefully transfer the diluted target cells into a sterile sample loading tray, and use a pipette to add the target cells into a black, transparent, flat-bottomed 96-well plate at a rate of 50 μL / well.

[0187] 1.2 Effector cell plate

[0188] 1.2.1 Effector cell preparation: Based on effector-to-target ratios of 9:1, 3:1, and 1:1, the cell dosage was calculated to be 9 × 10⁻⁶ cells / year. 4 1, 3×10 4 1×10 4 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.

[0189] 1.2.2 Resuspend CAR-T cells in T0 medium (X-Vivo medium + 5% inactivated FBS + 1% GlutaMax) and adjust the effector cell density according to the effector-target ratio.

[0190] 1.2.3 Add effector cells to the well plates at ratios of 9:1, 3:1, and 1:1, 50 μL / well.

[0191] 1.3 Setting up the target cell control group

[0192] Add 50 μL of target cells and 50 μL of T0 medium (X-Vivo medium + 5% inactivated FBS + 1% GlutaMax) to the wells of the target cell control group to make the total culture volume of the target cell control group consistent with the total culture volume of the co-culture experimental group.

[0193] 2. Detect luc value

[0194] 2.1 Cell co-culture: Place the 96-well plate with the added samples in a carbon dioxide incubator and co-culture for 4 hours.

[0195] 2.2 Adding the detection reagent: Melt steadyglo luciferase (commercially available) at 4°C or room temperature in the dark. Dilute the steadyglo required for the experiment with PBS 3 times and mix well. Add 50 μL to each well and place on a 96-well plate shaker. Shake at 100 rpm for 15 min.

[0196] 2.3 Instrumental Detection: Turn on the multi-mode microplate reader and computer software. Place the shaken 96-well plate into the multi-mode microplate reader and detect the luciferase fluorescence intensity. Remove the 96-well plate and turn off the instrument and computer.

[0197] 3. Calculation of cell killing activity

[0198] After obtaining the luc values ​​of all wells, calculate the cytotoxic activity using the following formula.

[0199] Cell-killing activity = (average luc value of target cells alone - luc value of co-culture wells) / average luc value of target cells alone × 100%

[0200] The test results are shown in Figure 3: there was no significant difference in the killing activity of CAR-T cells against target cells Nalm6 in all groups.

[0201] 3. Cytokine detection

[0202] 1. CAR-T cells and target cells are mixed at an effector-to-target ratio of 1:1.

[0203] 2. Incubate in carbon dioxide medium for 24 hours

[0204] 3. After incubation, centrifuge at 500g for 10 minutes and collect the supernatant.

[0205] 4. Use the Human Th1 Panel (5-Plex) with Filter Plate V02 kit to detect the cytokine concentration in the supernatant.

[0206] The test results are shown in Figures 4A and 4B: IL-2 release from CAR-T cells in the humanized structures Q11, Q14, Q33, Q54, GS32, and GS21 was higher than that from the murine CNCT19. IFN-γ release from CAR-T cells in the humanized structures Q11, Q33, GS32, and GS21 was also higher than that from the murine CNCT19. These results indicate that the humanization modification of the murine antibody in this application alters the antibody's binding ability to the antigen, thereby enabling the humanized CAR-T cells of this application to exhibit superior IL-2 and IFN-γ release compared to the murine CNCT19.

[0207] 4. Multiple rounds of stimulation

[0208] 1. CAR-T cells and target cells were prepared at an effector-to-target ratio of 1:1, with a cell count of 0.5 × 10⁻⁶. 6 0.5×10 6 Mix

[0209] 2. Detect the number of CAR-T cells every two days, and at the same time take 0.5 × 10⁻⁶ cells. 6 CAR-T cells, added 0.5×10 6 target cells

[0210] 3. After four rounds of target cell stimulation, the expansion fold of CAR-T cells was calculated.

[0211] The test results are shown in Figure 5: After four rounds of target cell stimulation, the CAR-T amplification fold of humanized structures Q11, Q33, and GS21 was consistent with that of mouse structure CNCT19.

[0212] In addition, three biocomputation tools—T20 score, HS score, and QASis—were used to score the humanization of the VH and VL chains of CD19. The scores from the three tools were consistent, 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 the 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).

[0213] The immunogenicity of antibody drugs may trigger an anti-drug immune response, thereby inducing the production of anti-drug antibodies (ADAs). The evaluation of antibody immunogenicity primarily utilizes ADA analysis. The immunogenicity of CAR-T cells induces 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 therapy contain murine sequences, a crucial factor in generating anti-CAR immune responses. Humanizing murine scFvs to circumvent these murine-derived anti-CAR immune responses is a vital strategy; however, the success of this strategy hinges on whether antibody functional degradation or loss can be avoided during the modification process.

[0214] Based on in vitro functional data of different humanized CD19 CAR-T cells, combined with antibody humanization score and antibody immunogenicity prediction data, the Q33 and GS21 sequences showed good CAR positivity, high in vitro killing activity and cytokine release, and low immunogenicity.

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 antigen-binding fragment thereof of 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 of the humanized antibody or its antigen-binding fragment.

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 of 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-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; the amino acid sequence shown in SEQ ID NO:8-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: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 of 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 of 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 of 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 of 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 of 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; Further, optionally, the vector is 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 cell of 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; Further, optionally, the T lymphocytes are 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 of 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 of 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 of 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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