Humanized Anti-CD19 single-chain variable region fragment and use thereof

Abstract

Provided is a new humanized anti-CD19 antibody. A humanized CAR-T cell is constructed on the basis of the antibody. The constructed humanized CAR-T cell has a killing effect and a tumor removing ability which are comparable to those of a CAR-T cell constructed using FMC63, and avoids safety risks such as an immune response caused by murine scFv. Therefore, the humanized CAR-T cell is more suitable for clinical treatment.

Description

Humanized anti-CD19 single-chain variable region fragment and its use Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to a humanized anti-CD19 single-chain variable region fragment and uses thereof. Background Art

[0002] Adoptive cellular immunotherapy (ACCT) has achieved remarkable success in recent years. With the US Food and Drug Administration's approval of KYMRIAH (Novartis Pharmaceuticals Corporation), YESCARTA and TECARTUS (Kite Pharma, Inc.), BREYANZI (Juno Therapeutics, Inc.), ABECMA (Celgene Corporation), and CARVYKTI (Janssen Biotech, Inc.), ACCT has become a new and reliable therapeutic strategy for the treatment of relapsed and refractory hematologic malignancies, including large B-cell lymphoma, mantle cell lymphoma, and multiple myeloma. These FDA-approved ACCTs all engineer autologous T cells to express chimeric antigen receptors (CARs), which target specific tumor antigens and stimulate and activate the adopted T cells to kill tumors, achieving anti-tumor effects. The ACC structure consists of an extracellular antigen-binding domain connected to an intracellular secondary signaling domain and primary signaling domain via a hinge and transmembrane region. The extracellular antigen-binding domains of KYMRIAH, YESCARTA, TECARTUS, and BREYANZI are all mouse-derived single-chain variant fragments of anti-CD19; ABECMA's is a mouse-derived single-chain variant fragment of anti-BCMA; and CARVYKTI's is a single-domain antibody derived from alpaca. However, these non-human protein sequences, when exposed outside of T cells, increase the immunogenicity of these agents, potentially eliciting an immune response in the human body. This shortens the effectiveness of these adoptive T cell immunotherapies and raises safety concerns.

[0003] CAR-T cells with mouse-derived scFvs can induce humoral and cellular immune responses, including excessive expression of HAMA (human anti-mouse antibodies) and proliferation of CD8 T cells targeting the mouse scFv. This results in a short CAR-T half-life, poor efficacy, and safety concerns. If patients have previously received treatment with antibodies containing mouse sequences (such as Rituximab, infliximab, and Aciximab), the potential for anti-drug antibodies (ADA), whether related to or unrelated to the disease being treated by the CAR-T, increases the risk of mouse-derived CAR-T therapy. CAR-T cells based on non-mouse and non-human extracellular antigen-binding domains face the same challenges as mouse-derived scFv-based CAR-T cells.

[0004] Among patients with acute lymphoblastic leukemia who had relapsed or were unresponsive to CD19-CAR-T cells containing mouse scFvs, 64% (7 / 11) achieved complete remission within one month after receiving CD19-CAR-T cells containing human scFvs. A clinical study using a humanized scFv-based CD19-CAR-T cell with 25% residual mouse sequence showed that eight of 10 patients with relapsed or refractory acute lymphoblastic leukemia achieved complete remission for more than six months. HAMA levels peaked between three weeks and three months after infusion, and then declined sharply after three months. Compared to CD19-CAR-T cells based on mouse scFvs, which still fluctuated three months after infusion, this CAR-T cell-induced HAMA exhibited a significantly lower immunogenicity. In vitro studies have shown that humanized scFv-based CD19-CAR-T cells have a weaker ability to induce IL-6 release from monocytes than mouse scFv-based CD19-CAR-T cells, thus reducing the risk of CRS in clinical use. In 2021, Myers et al. reported the treatment outcomes of 72 patients with relapsed or refractory B-cell acute lymphoblastic leukemia and two patients with B-cell lymphoblastic lymphoma treated with humanized scFv-based CD19-CAR-T cells. Among the 41 patients who had not previously received mouse CAR-T therapy, the one-month complete remission rate was 98%, while the one-month complete remission rate reached 64% in the 33 patients who had previously received mouse CAR-T therapy but had relapsed or had no response. The 12- and 24-month relapse-free survival rates for patients who had not previously received mouse CAR-T therapy were 84% and 74%, respectively, compared to 73% and 58% for patients who had previously received mouse CAR-T therapy. These studies have shown that humanized scFv-based CAR-T has significant advantages in efficacy and safety compared to mouse scFv-based CAR-T. Even patients who have received unsuccessful mouse CAR-T treatment may have a chance of being cured with humanized CAR-T.

[0005] Therefore, there is still a great demand for humanized CAR-T in this field.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to provide a series of anti-CD19 single-chain variable region fragments with a degree of humanization greater than 90% (93.83%-99.56%), which are integrated into chimeric antigen receptors to prepare chimeric antigen receptor T cells, and have good in vivo and in vitro anti-tumor (especially B cell lymphoma) functions.

[0008] In a first aspect, the present invention provides a humanized anti-CD19 antibody, comprising an antibody heavy chain variable region as shown in any one of SEQ ID NOs: 1-4, and an antibody light chain variable region as shown in any one of SEQ ID NOs: 5-7.

[0009] In another preferred embodiment, the antibody comprises an antibody light chain variable region and an antibody heavy chain variable region selected from the following groups:

[0010] In another preferred embodiment, the antibody comprises the antibody heavy chain variable region shown in SEQ ID NO: 1, and the antibody light chain variable region shown in SEQ ID NO: 5; or

[0011] The antibody comprises the antibody heavy chain variable region shown in SEQ ID NO: 2, and the antibody light chain variable region shown in SEQ ID NO: 6; or

[0012] The antibody comprises an antibody heavy chain variable region as shown in SEQ ID NO: 3, and an antibody light chain variable region as shown in SEQ ID NO: 6.

[0013] In another preferred embodiment, the antibody comprises the antibody heavy chain variable region shown in SEQ ID NO: 1, and the antibody light chain variable region shown in SEQ ID NO: 5.

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

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

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

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

[0018] In another preferred embodiment, the structure of the antibody is shown in the following formula A or formula B: H -V L (A); V L -V H (B)

[0019] Where V H is the antibody heavy chain variable region; V L is the antibody light chain variable region; "-" is a connecting peptide or peptide bond; preferably, it is a structure of formula B.

[0020] In another preferred embodiment, the connecting peptide is 1-5 consecutive sequences shown in SEQ ID NO: 11 (GGGGS), SEQ ID NO: 12 (GGGS), SEQ ID NO: 13 (SSSSG), SEQ ID NO: 14 (GSGSA), or SEQ ID NO: 15 (GGSGG), preferably 1-4, and more preferably 3-4.

[0021] In another preferred embodiment, the amino acid sequence of the antibody is shown in any one of SEQ ID NOs: 8-10.

[0022] In a second aspect of the present invention, there is provided an antigen binding receptor (CAR) targeting CD19, wherein the antigen binding domain of the CAR comprises the humanized anti-CD19 antibody described in the first aspect of the present invention.

[0023] In another preferred embodiment, the structure of the CAR is shown in the following formula I: L-scFv-H-TM-C-CS (I)

[0024] Where,

[0025] Each "-" is independently a connecting peptide or a peptide bond;

[0026] L is none or a signal peptide sequence;

[0027] scFv is the antigen-binding domain targeting CD19;

[0028] H is none or hinge region;

[0029] TM is the transmembrane domain;

[0030] C is a co-stimulatory signal molecule;

[0031] CS is an intracellular signaling sequence derived from CD3ζ.

[0032] In another preferred embodiment, the scFv has a structure as shown in Formula A or Formula B: H -V L (A); V L -V H (B)

[0033] Where V H is the heavy chain variable region of the humanized anti-CD19 antibody; V L is the light chain variable region of the humanized anti-CD19 antibody; "-" is a connecting peptide or peptide bond; preferably, it is a structure of formula B.

[0034] In another preferred embodiment, the connecting peptide is 1-5 consecutive sequences shown in SEQ ID NO: 11 (GGGGS), SEQ ID NO: 12 (GGGS), SEQ ID NO: 13 (SSSSG), SEQ ID NO: 14 (GSGSA), or SEQ ID NO: 15 (GGSGG), preferably 1-4, and more preferably 3-4.

[0035] In another preferred embodiment, the V H The amino acid sequence of V is shown in SEQ ID NO: 1. l The amino acid sequence is shown in SEQ ID NO: 5; or

[0036] The V H The amino acid sequence of V is shown in SEQ ID NO: 2. l The amino acid sequence is shown in SEQ ID NO: 6; or

[0037] The V H The amino acid sequence of V is shown in SEQ ID NO: 3. l The amino acid sequence is shown in SEQ ID NO:6.

[0038] In another preferred embodiment, the scFv amino acid sequence is shown in any one of SEQ ID NOs: 8-10.

[0039] In another preferred embodiment, the L is a signal peptide of a protein selected from the group consisting of CD8α, CD28, GM-CSF, IL-12, CD4, CD137, or a combination thereof.

[0040] In another preferred embodiment, the L is a signal peptide derived from CD8α.

[0041] In another preferred embodiment, the L has an amino acid sequence as shown in SEQ ID NO: 16.

[0042] In another preferred embodiment, the H is the hinge region of a protein selected from the group consisting of CD8α, CD28, CD4, CD7, CD152, CD137, PD-1, or a combination thereof.

[0043] In another preferred embodiment, the H is independently a hinge region derived from CD8.

[0044] In another preferred embodiment, the H has an amino acid sequence as shown in SEQ ID NO: 17.

[0045] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the following group: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD27, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, TCRα / β / γ / δ chain, PD-1 or a combination thereof.

[0046] In another preferred embodiment, the TMs are each independently a transmembrane region derived from CD28.

[0047] In another preferred embodiment, the CD8-derived transmembrane region has an amino acid sequence as shown in SEQ ID NO: 18.

[0048] In another preferred embodiment, the C is a co-stimulatory signal molecule of a protein selected from the following group: CD28, CD2, CD7, CD27, CD30, CD40, CD70, OX40 (CD134), 4-1BB (CD137), CD154 (CD40L), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.

[0049] In another preferred embodiment, the C is a costimulatory signal molecule derived from CD28.

[0050] In another preferred embodiment, the CD28-derived costimulatory signal molecule has an amino acid sequence as shown in SEQ ID NO: 19 or 20.

[0051] In another preferred embodiment, the CS is derived from the intracellular signaling region of CD3ζ or FcγR.

[0052] In another preferred embodiment, the CS is derived from the intracellular signaling region of CD3ζ, which has an amino acid sequence as shown in SEQ ID NO: 21.

[0053] In another preferred example, the CAR has an amino acid sequence as shown in any one of SEQ ID NOs: 22-25.

[0054] The third aspect of the present invention provides a nucleic acid molecule encoding the humanized anti-CD19 antibody as described in the first aspect of the present invention or the CD19-targeting CAR as described in the second aspect of the present invention.

[0055] The fourth aspect of the present invention provides a vector, wherein the vector contains the nucleic acid molecule described in the third aspect of the present invention.

[0056] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, or a combination thereof.

[0057] In another preferred embodiment, the vector is a lentiviral vector.

[0058] In the fifth aspect of the present invention, a host cell is provided, which contains the vector as described in the fourth aspect of the present invention, or an exogenous nucleic acid molecule as described in the third aspect of the present invention integrated into the chromosome, or expresses the humanized anti-CD19 antibody as described in the first aspect of the present invention or the CD19-targeting CAR as described in the second aspect of the present invention.

[0059] The sixth aspect of the present invention provides an engineered immune cell, which contains the vector as described in the fourth aspect of the present invention, or an exogenous nucleic acid molecule as described in the third aspect of the present invention integrated into the chromosome, or expresses the humanized anti-CD19 antibody as described in the first aspect of the present invention or the CD19-targeting CAR as described in the second aspect of the present invention.

[0060] In another preferred embodiment, the engineered immune cells are selected from the following group:

[0061] (i) chimeric antigen receptor T cells (CAR-T cells); or

[0062] (ii) Chimeric antigen receptor NK cells (CAR-NK cells).

[0063] In another preferred embodiment, the engineered immune cells include universal CAR-T cells.

[0064] The seventh aspect of the present invention provides an immunoconjugate, comprising:

[0065] (a) the humanized anti-CD19 antibody according to the first aspect of the present invention;

[0066] (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0067] In another preferred embodiment, the coupling moiety is a drug or a toxin.

[0068] In another preferred embodiment, the coupling moiety is a detectable label.

[0069] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (for example, cisplatin) or any form of nanoparticles, etc.

[0070] In an eighth aspect, the present invention provides a kit comprising the humanized anti-CD19 antibody according to the first aspect of the present invention or the immunoconjugate according to the seventh aspect of the present invention.

[0071] In another preferred embodiment, the kit further comprises an instruction manual, which indicates that the kit is used to detect CD19 molecules.

[0072] The ninth aspect of the present invention provides a preparation containing the humanized anti-CD19 antibody as described in the first aspect of the present invention, or the immunoconjugate as described in the seventh aspect of the present invention, or the engineered immune cell as described in the sixth aspect of the present invention, and a pharmaceutically acceptable carrier.

[0073] In another preferred embodiment, the preparation is a liquid preparation.

[0074] In another preferred embodiment, the preparation is in the form of an injection.

[0075] In another preferred embodiment, the concentration of the engineered immune cells in the preparation is 1×10 3 -1×10 8 cells / ml, preferably 1×10 4 -1×10 7 cells / ml.

[0076] The tenth aspect of the present invention provides the use of the humanized anti-CD19 antibody as described in the first aspect of the present invention, or the engineered immune cell as described in the sixth aspect of the present invention, or the immunoconjugate as described in the seventh aspect of the present invention for (1) preparing a drug or preparation for treating cancer or tumors; (2) preparing a reagent or kit for detecting CD19 molecules.

[0077] In another preferred embodiment, the tumor or cancer is a tumor or cancer that highly expresses CD19.

[0078] In another preferred embodiment, the tumor or cancer is selected from the following group: hematological tumors, solid tumors, or a combination thereof.

[0079] In another preferred embodiment, the blood tumor is selected from the group consisting of lymphoma, multiple myeloma (MM), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), or a combination thereof.

[0080] In another preferred embodiment, the solid tumor is selected from the following group: gastric cancer, gastric cancer peritoneal metastasis, liver cancer, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer, testicular cancer, colorectal cancer, urinary tract tumor, thyroid cancer, or a combination thereof.

[0081] In another preferred embodiment, the cancer or tumor is lymphoma.

[0082] In another preferred embodiment, the cancer is B-cell lymphoma.

[0083] The eleventh aspect of the present invention provides a method for preparing the engineered immune cell according to the sixth aspect of the present invention, comprising the following steps:

[0084] The nucleic acid molecule described in the third aspect of the present invention or the vector described in the first aspect of the present invention is transduced into immune cells, and the immune cells are cultured under conditions suitable for antibody or CAR expression to obtain the engineered immune cells.

[0085] In another preferred embodiment, the immune cells are T cells or NK cells.

[0086] A twelfth aspect of the present invention provides a method for detecting CD19 protein in a sample, the method comprising the steps of:

[0087] (1) contacting a sample with the humanized anti-CD19 antibody according to the first aspect of the present invention or the immunoconjugate according to the seventh aspect of the present invention;

[0088] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of CD19 protein in the sample.

[0089] In another preferred embodiment, the method is an in vitro method.

[0090] In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

[0091] The thirteenth aspect of the present invention provides a method for treating cancer or tumors, comprising administering to a subject in need thereof a therapeutically effective amount of the humanized anti-CD19 antibody as described in the first aspect of the present invention, or the immunoconjugate as described in the seventh aspect of the present invention, or the engineered immune cell as described in the sixth aspect of the present invention, or the preparation as described in the ninth aspect of the present invention.

[0092] In another preferred embodiment, the subject includes a human or a non-human mammal.

[0093] In another preferred embodiment, the tumor or cancer is a tumor or cancer that highly expresses CD19.

[0094] In another preferred embodiment, the tumor or cancer is selected from the following group: hematological tumors, solid tumors, or a combination thereof.

[0095] In another preferred embodiment, the blood tumor is selected from the group consisting of lymphoma, multiple myeloma (MM), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), or a combination thereof.

[0096] In another preferred embodiment, the solid tumor is selected from the following group: gastric cancer, gastric cancer peritoneal metastasis, liver cancer, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymphoma, nasopharyngeal carcinoma, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer, testicular cancer, colorectal cancer, urinary tract tumor, thyroid cancer, or a combination thereof.

[0097] In another preferred embodiment, the cancer or tumor is lymphoma.

[0098] In another preferred embodiment, the cancer is B-cell lymphoma.

[0099] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1: V L3 V H4 Schematic diagram of CARs of different designs assembled with combined scFvs;

[0101] In the figure, SP: CD8α signal peptide; V L1 : Antibody light chain variable region 1; V L3 : Antibody light chain variable region 3; L: single linker (ie, GGGGS); V H1 : Antibody heavy chain variable region 1; VH2 : Antibody heavy chain variable region 2; V H4 : antibody heavy chain variable region 4; H: CD8α hinge region; TM: CD8α transmembrane domain; CD28: CD28 intracellular domain; CD28Δ: CD28 intracellular region with Lck binding site mutation; CD3ζ: CD3zeta intracellular signaling domain.

[0102] Figure 2: V L1 V H1 、V L1 V H2 、V L3 V H4 The combined scFv assembly of different designed CARs was expressed in T cells using a retroviral system.

[0103] Figure 3: V L1 V H1 、V L1 V H2 、V L3 V H4 Statistical results of T cell expression of CARs of different designs assembled with combined scFvs.

[0104] FIG4 : Comparison of the yields of the CAR-T cells of the present invention and FMC63 CAR-T cells.

[0105] Figure 5: V L1 V H1 、V L1 V H2 、V L3 V H4 Target cell killing ability of CAR-T with different designs assembled by combinatorial scFv.

[0106] Figure 6: The ability of humanized CD19 CAR-T to kill CD19-positive target cells in vitro is comparable to that of murine FMC63 CD19 CAR-T.

[0107] Figure 7: In vivo efficacy test results of humanized CD19 CAR-T. DETAILED DESCRIPTION

[0108] After extensive and in-depth research, the inventors have, for the first time, constructed a novel humanized anti-CD19 antibody, derived from the humanized FMC63 antibody. The present invention also provides CAR-T cells constructed based on this humanized antibody and related applications. Compared to CAR-T cells constructed using FMC63, the CAR-T cells constructed in the present invention exhibit similar killing efficacy and tumor clearance capabilities while avoiding the safety risks associated with immune responses caused by murine scFvs. This work was completed on this basis.

[0109] the term

[0110] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0111] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0112] The terms "administering" or "administering" refer to the physical introduction of the product of the invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion.

[0113] It should be understood that the amino acid names herein are identified by internationally accepted single-letter English letters, and the corresponding three-letter abbreviations of the amino acid names are: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), and Val (V).

[0114] Antibody

[0115] As used herein, the terms "antibody" or "immunoglobulin" are heterotetrameric glycoproteins of approximately 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0116] As used herein, the term "variable" refers to certain parts of the variable region in an antibody that are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are generally in a β-pleated configuration and are connected by three CDRs that form a connecting loop, and in some cases can form a partial β-pleated structure. The CDRs in each chain are closely together through the FR region and form the antigen-binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume 1, pages 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cytotoxicity of the antibody.

[0117] The "light chains" of vertebrate antibodies (immunoglobulins) can be assigned to one of two distinct classes, called kappa and lambda, based on the amino acid sequence of their constant regions. Immunoglobulins can be divided into different classes based on the amino acid sequence of their heavy chain constant regions. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known in the art.

[0118] Generally, an antibody's antigen-binding properties are described by three specific regions located in the variable regions of the heavy and light chains, known as the variable regions (CDRs). These regions are divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, spatially close to each other through the β-sheet formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antibody's antigen-binding site. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0119] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0120] In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human parts, can be obtained by standard DNA recombinant techniques, and they are all useful antibodies. A chimeric antibody is a molecule in which different parts are derived from different animal species, such as a chimeric antibody having a variable region from a mouse monoclonal antibody and a constant region from a human immunoglobulin (see, for example, U.S. Patent No. 4,816,567 and U.S. Patent No. 4,816,397, which are hereby incorporated by reference in their entirety). A humanized antibody refers to an antibody molecule derived from a non-human species, having one or more complementary determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Patent No. 5,585,089, which is hereby incorporated by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombinant techniques well known in the art.

[0121] In the present invention, the antibodies can be monospecific, bispecific, trispecific, or more multispecific.

[0122] In the present invention, the antibodies of the present invention also include conservative variants thereof, which refer to polypeptides in which no more than 10, preferably no more than 8, more preferably no more than 5, and most preferably no more than 3 amino acids are replaced with amino acids having similar or similar properties, compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by making amino acid substitutions according to Table A.

[0123] Table A

[0124] Chimeric Antigen Receptor (CAR)

[0125] The chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen binding domain). The intracellular domain includes a costimulatory signaling region and a ζ chain portion. The costimulatory signaling region refers to a portion of the intracellular domain including costimulatory molecules. Costimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens, rather than antigen receptors or their ligands.

[0126] Between the extracellular domain and the transmembrane domain of CAR, or between the cytoplasmic domain and the transmembrane domain of CAR, a linker can be incorporated. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that acts to connect the transmembrane domain to the extracellular domain or cytoplasmic domain of a polypeptide chain. The linker may include 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

[0127] In a preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention includes an antigen binding domain targeting CD19. When the CAR of the present invention is expressed in T cells, it is possible to perform antigen recognition based on antigen binding specificity. When it is combined with its associated antigen, it affects tumor cells, causing tumor cells to not grow, be prompted to die or otherwise be affected, and causes the patient's tumor load to shrink or be eliminated. The antigen binding domain is preferably fused with one or more intracellular domains from a costimulatory molecule and a ζ chain. Preferably, the antigen binding domain is fused with the intracellular domain of a CD28 signaling domain (including wild type and variants thereof, such as CD28 intracellular regions of Lck binding site mutations) and a combination of CD3ζ signaling domains.

[0128] As used herein, "antigen binding domain" and "single-chain antibody fragment" refer to Fab fragments, Fab' fragments, F(ab')2 fragments, or single Fv fragments with antigen binding activity. Fv antibodies contain the variable region of the heavy chain and the variable region of the light chain of the antibody, but no constant region, and are the smallest antibody fragments with all antigen binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains, and are capable of forming the structure required for antigen binding. The antigen binding domain is usually scFv (single-chain variable fragment). The size of scFv is generally 1 / 6 of a complete antibody. A single-chain antibody is preferably an amino acid chain sequence encoded by a nucleotide chain. As a preferred embodiment of the present invention, the antigen binding domain comprises an antibody that specifically recognizes CD19, preferably a single-chain antibody. Preferably, the scFv targeting CD19 of the present invention comprises an antibody heavy chain variable region shown in any one of SEQ ID NOs: 1-4, and an antibody light chain variable region shown in any one of SEQ ID NOs: 5-7.

[0129] For hinge region and transmembrane region (transmembrane domain), CAR can be designed to include a transmembrane domain fused to the extracellular domain of CAR. In one embodiment, a transmembrane domain naturally associated with one of the domains in CAR is used. In some examples, a transmembrane domain can be selected, or modified by amino acid replacement to avoid such a domain being bound to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complex.

[0130] Chimeric antigen receptor T cells (CAR-T cells)

[0131] As used herein, the terms "CAR-T cells," "CAR-T," and "CAR-T cells of the present invention" include the CAR-T cells of the sixth aspect of the present invention. The CAR-T cells of the present invention can be used to treat tumors that overexpress CD19, such as multiple myeloma and lymphoma.

[0132] CAR-T cells have the following advantages over other T-cell-based treatments: (1) The action of CAR-T cells is not restricted by MHC; (2) Since many tumor cells express the same tumor antigens, once the CAR gene construction for a certain tumor antigen is completed, it can be widely used; (3) CAR can utilize both tumor protein antigens and glycolipid non-protein antigens, expanding the target range of tumor antigens; (4) The use of the patient's own cells reduces the risk of rejection; (5) CAR-T cells have immune memory function and can survive in the body for a long time.

[0133] Chimeric antigen receptor NK cells (CAR-NK cells)

[0134] As used herein, the terms "CAR-NK cells," "CAR-NK," and "CAR-NK cells of the present invention" all refer to the CAR-NK cells encompassed by the sixth aspect of the present invention. The CAR-NK cells of the present invention can be used to treat tumors that overexpress CD19, such as multiple myeloma and lymphoma.

[0135] Natural killer (NK) cells are a major type of immune effector cell that protects the body from viral infection and tumor cell invasion through non-antigen-specific pathways. Engineered (genetically modified) NK cells may acquire new functions, including the ability to specifically recognize tumor antigens and possess enhanced anti-tumor cytotoxicity.

[0136] Compared with autologous CAR-T cells, CAR-NK cells have the following advantages: (1) they directly kill tumor cells by releasing perforin and granzymes, but have no killing effect on normal cells in the body; (2) they release very small amounts of cytokines, thereby reducing the risk of cytokine storms; (3) they are extremely easy to expand in vitro and develop into "ready-made" products. Other than that, it is similar to CAR-T cell therapy.

[0137] Nucleic acid molecules and vectors

[0138] The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by directly isolating from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically.

[0139] The present invention also provides vectors into which the nucleic acid sequences or expression cassettes of the present invention are inserted. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses, such as murine leukemia viruses, because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.

[0140] In brief summary, the expression cassette or nucleic acid sequence of the present invention is generally operably linked to a promoter and incorporated into an expression vector. Such vectors are suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that can be used to regulate expression of the desired nucleic acid sequence.

[0141] The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entireties. In another embodiment, the present invention provides gene therapy vectors.

[0142] Nucleic acids can be cloned into many types of vectors. For example, nucleic acids can be cloned into such vectors, which include but are not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors and sequencing vectors.

[0143] Further, expression vector can be provided to cell in the form of viral vector.Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. The virus that can be used as a vector includes but is not limited to retrovirus, adenovirus, adeno-associated virus, herpes virus and slow virus. Generally, suitable vectors are included in at least one organism and work in the origin of replication, promoter sequence, convenient restriction enzyme site and one or more selectable markers (for example, WO01 / 96584; WO01 / 29058; and U.S. Patent number 6,326,193).

[0144] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject's cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0145] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. Typically, these are located in the 30-110 bp region upstream of the start site, although recently it has been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so that when an element is inverted or moved relative to another, promoter function is maintained. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50 bp before activity begins to decline. Depending on the promoter, it has been shown that individual elements can work together or independently to initiate transcription.

[0146] In order to evaluate the expression of the CAR polypeptide or its portion, the expression vector introduced into the cell may also include any one or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a cell population that is sought to be transfected or infected by a viral vector. In other aspects, selectable markers can be carried on a single DNA segment and used for co-transfection procedures. Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

[0147] Methods for introducing genes into cells and expressing genes in cells are known in the art. In the context of expression vectors, the vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0148] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0149] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, for example, human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0150] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0151] In the case of using non-viral delivery system, exemplary delivery means is liposome. Consider using lipid preparation, to introduce nucleic acid into host cell (in vitro, in vitro (ex vivo) or in vivo). On the other hand, this nucleic acid can be associated with lipid. The nucleic acid associated with lipid can be encapsulated in the aqueous interior of liposome, dispersed in the lipid bilayer of liposome, attached to liposome through the connecting molecule associated with liposome and oligonucleotide, trapped in liposome, compounded with liposome, dispersed in the solution comprising lipid, mixed with lipid, united with lipid, included in lipid as suspension, included in micelle or with micelle compound, or otherwise associated with lipid. The lipid, lipid / DNA or lipid / expression vector associated with composition are not limited to any specific structure in solution. For example, they can be present in bilayer structure, as micelle or have " collapsed (collapsed) " structure. They can also be simply dispersed in solution, may form aggregates of size or shape inhomogeneity. Lipid is a fatty substance, which can be a naturally occurring or synthetic lipid. For example, lipids include fat droplets that occur naturally in the cytoplasm as well as compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0152] preparation

[0153] The present invention provides a preparation comprising the humanized anti-CD19 antibody described in the first aspect of the present invention, or the immunoconjugate described in the seventh aspect of the present invention, or the engineered immune cell described in the sixth aspect of the present invention, and a pharmaceutically acceptable carrier. In a preferred embodiment of the present invention, the preparation comprises the CAR-T cells described in the sixth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the preparation is a liquid preparation. Preferably, the preparation is an injection. Preferably, the concentration of the CAR-T cells in the preparation is 1×103 -1×10 8 cells / ml, preferably 1×10 4 -1×10 7 cells / ml.

[0154] In one embodiment, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, or the like; a carbohydrate such as glucose, mannose, sucrose, or dextran, mannitol; a protein; a polypeptide or amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The formulation of the present invention is preferably formulated for intravenous administration.

[0155] Therapeutic applications

[0156] The present invention includes therapeutic applications of cells (e.g., T cells) transduced with a retrovirus encoding an expression cassette of the present invention. The transduced T cells can target the tumor cell marker CD19, activate the T cells, elicit a T cell immune response, and thereby kill the tumor cells.

[0157] Therefore, the present invention also provides a method for stimulating a T cell-mediated immune response to a target cell population or tissue in a mammal, comprising the steps of administering the CAR-T cells of the present invention to the mammal.

[0158] In one embodiment, the present invention comprises a type of cell therapy in which a patient's own T cells (or those of an allogeneic donor) are isolated, activated, and genetically modified to produce CAR-T cells, which are then infused back into the same patient. This approach significantly reduces the risk of graft-versus-host disease, as antigens are recognized by T cells in an MHC-free manner. Furthermore, a single CAR-T cell can treat all cancers expressing that antigen. Unlike antibody therapies, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

[0159] In one embodiment, the CAR-T cells of the present invention can undergo robust in vivo T cell expansion and can sustain for an extended period of time. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy procedure, wherein the CAR-modified T cells induce an immune response specific to the antigen binding domain in the CAR. For example, anti-CD19 CAR-T cells induce a specific immune response against cells expressing CD19.

[0160] Although the data disclosed herein specifically disclose lentiviral vectors comprising an anti-CD19 scFv, hinge and transmembrane regions, and CD28 and CD3ζ signaling domains, the invention should be construed to encompass any number of variations in each of the construct's components.

[0161] Treatable cancers include tumors that are not vascularized or substantially not yet vascularized, and vascularized tumors. Cancer may include non-solid tumors (such as hematological tumors, such as leukemia and lymphoma) or may include solid tumors. The types of cancer treated with the CAR of the present invention include but are not limited to cancer, blastoma and sarcoma, and certain leukemia or lymphoma, benign and malignant tumors and malignant tumors, such as sarcoma, cancer and melanoma. Also included are adult tumors / cancers and childhood tumors / cancers.

[0162] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, granulocytic-monocytic, monocytic, and erythroleukemias), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphomas, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0163] Solid tumors are abnormal masses of tissue that usually do not contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcomas, myxosarcoma, liposarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer, ovarian cancer, etc.

[0164] The CAR-modified T cells of the present invention can also be used as a vaccine type for ex vivo immunization and / or in vivo therapy of mammals. Preferably, the mammal is a human.

[0165] For ex vivo immunization, at least one of the following occurs in vitro prior to administering the cells into a mammal: i) expanding the cells, ii) introducing a nucleic acid encoding a CAR into the cells, and / or iii) cryopreserving the cells.

[0166] In vitro procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from mammals (preferably humans) and genetically modified (i.e., in vitro transduction or transfection) with a vector expressing the CAR disclosed herein. CAR-modified cells can be administered to a mammalian recipient to provide therapeutic benefits. The mammalian recipient can be a human, and the CAR-modified cells can be autologous relative to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic relative to the recipient.

[0167] In addition to the use of cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

[0168] The present invention provides a method for treating tumors, comprising administering a therapeutically effective amount of CAR-modified T cells of the present invention to a subject in need thereof.

[0169] The CAR-modified T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as IL-2, IL-17 or other cytokines or cell groups. Briefly, the pharmaceutical composition of the present invention may include a target cell group as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0170] The pharmaceutical compositions of the present invention can be administered in a manner appropriate to the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease, although appropriate dosages can be determined by clinical trials.

[0171] When an "immunologically effective amount," "anti-tumor effective amount," "tumor-suppressive effective amount," or "therapeutically effective amount" is indicated, the precise amount of the composition of the present invention to be administered can be determined by a physician, who takes into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. It can generally be stated that a pharmaceutical composition comprising the T cells described herein can be administered in an amount of 10 4 to 10 9 The dose of cells / kg body weight is preferably 10 5 to 10 6 The T cell composition can be administered at a dose of 10 cells / kg body weight (including all integer values ​​within those ranges). The T cell composition can also be administered multiple times at these doses. The cells can be administered using infusion techniques well known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dose and treatment regimen for a specific patient can be easily determined by a person skilled in the art of medicine by monitoring the patient's disease signs and adjusting the treatment accordingly.

[0172] Administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous (iv) injection or intraperitoneally. In one embodiment, the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by iv injection. The composition of the T cell can be injected directly into the tumor, lymph node or infection site.

[0173] In certain embodiments of the present invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in combination with (e.g., before, simultaneously, or after) any number of related treatment modalities, including, but not limited to, treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or natalizumab treatment for MS patients, or efavirenz treatment for psoriasis patients, or other treatments for PML patients. In further embodiments, the T cells of the present invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell compositions of the present invention are administered to a patient in combination with (e.g., before, simultaneously, or after) bone marrow transplantation, chemotherapy agents such as fludarabine, external beam radiation therapy (XRT), or cyclophosphamide. For example, in one embodiment, a subject may undergo standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In some embodiments, the subject receives an infusion of the expanded immune cells of the invention following transplantation. In an additional embodiment, the expanded cells are administered before or after surgery.

[0174] The dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. Dosage ratios for human administration may be implemented according to practices accepted in the art. Typically, 1×10 6 to 1×10 10 The modified T cells of the present invention are administered to the patient, for example, by intravenous infusion.

[0175] The main advantages of the present invention include:

[0176] The CAR-T cells constructed using the humanized antibody scFv sequence designed and screened in the present invention are significantly superior to the CAR-T cells prepared based on FMC63 in terms of positive rate and total CAR-T cell yield. At the same time, they have similar in vivo and in vitro tumor killing abilities as the CAR-T constructed with FMC63, and can effectively avoid the humoral and cellular immune responses caused by the CAR-T constructed with mouse scFv, thereby effectively improving the safety and efficacy of the drug.

[0177] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0178] Example 1 Humanized Design

[0179] The FMC63 sequence was compared with a database of known human antibodies to identify the germline genes with the highest homology: IGHV4 for the heavy chain and IGKV1 for the light chain. Their framework regions were selected (using AbM to define CDRs and framework regions). Then, using computer prediction and simulation, mouse amino acids in the FMC63 antibody framework region that are important for antigen binding were retained through back mutation. Finally, the antigen-determining CDRs were replaced with the corresponding CDR sequences in the FMC63 antibody. The following humanized sequence was obtained:

[0180] Humanized anti-CD19 V H4 Sequence (SEQ ID NO:1):

[0181] Humanized anti-CD19 V H1 Sequence (SEQ ID NO:2):

[0182] Humanized anti-CD19 V H2 Sequence (SEQ ID NO:3):

[0183] Humanized anti-CD19 V H3 Sequence (SEQ ID NO:4):

[0184] Humanized anti-CD19 V L3Sequence (SEQ ID NO:5):

[0185] Humanized anti-CD19 V L1 Sequence (SEQ ID NO:6):

[0186] Humanized anti-CD19 V L2 Sequence (SEQ ID NO:7):

[0187] Example 2 Construction of CAR-T cells containing humanized scFv

[0188] Furthermore, based on the heavy chain and light chain variable region sequences in Example 1, scFv comprising the above variable regions was constructed:

[0189] >V L3 V H4 scFv (SEQ ID NO: 8)

[0190] >V L1 V H1 scFv (SEQ ID NO: 9)

[0191] >V L1 V H2 scFv (SEQ ID NO: 10)

[0192] V L3 and V H4 The scFv composed of different CARs was constructed, in which V L3 and V H4 The intermediate linker is 3 repeated GGGGS (SEQ ID NO: 11) sequences; the hinge region (SEQ ID NO: 17) and the transmembrane region (SEQ ID NO: 18) are selected from CD8 alpha; the second signaling domain is the wild-type CD28 intracellular region (SEQ ID NO: 19) or the CD28 intracellular region with a Lck binding site mutation (CD28Δ) (SEQ ID NO: 20); the first signaling domain is selected from the intracellular region of CD3 zeta (SEQ ID NO: 21); and the signal peptide is selected from CD8 alpha (SEQ ID NO: 16).

[0193] >CD8 alpha signal peptide (SEQ ID NO: 16)

[0194] Hinge region (SEQ ID NO: 17)

[0195] >Mold penetration region (SEQ ID NO: 18)

[0196] >Wild-type CD28 intracellular region (SEQ ID NO: 19)

[0197] >CD28 intracellular region with Lck binding site mutation (CD28Δ) (SEQ ID NO: 20)

[0198] >Intracellular region of CD3 zeta (SEQ ID NO: 21)

[0199] The constructed CARs were named CAR3 (the full-length amino acid sequence is shown in SEQ ID NO: 22), CAR24 (the full-length amino acid sequence is shown in SEQ ID NO: 23), CAR1 (the full-length amino acid sequence is shown in SEQ ID NO: 24), and CAR2 (the full-length amino acid sequence is shown in SEQ ID NO: 25).

[0200] >CAR3 (SEQ ID NO: 22)

[0201] >CAR24 (SEQ ID NO: 23)

[0202] >CAR1 (SEQ ID NO: 24)

[0203] >CAR2 (SEQ ID NO: 25)

[0204] After the four CARs shown in Figure 1 were constructed into retroviral vectors, the CAR expression plasmid, viral packaging plasmid, and viral envelope protein expression plasmid were introduced into 293T cells at a ratio of 4:3:3 using PEI transfection to produce retroviral particles. The supernatants were harvested on the first and second days after transfection and mixed into a viral harvest solution. After two days of activation of primary T cells with TransAct (Mi ltenyi Biotec), the T cells were infected with the viral harvest solution. One day after infection, the virus was removed and the T cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and interleukin 2 (IL-2, 400 IU / mL). After another 4-7 days of culture, the CAR positivity rate was detected. The CAR positivity rate was labeled with FITC-CD19-Fc protein and stained with 7AAD to exclude dead cells.

[0205] The results are shown in Figure 2, which shows that CAR1, CAR2, CAR3, and CAR24 are all normally expressed on the surface of T cells and can be recognized by the CD19 protein. This shows that the designed CAR and the vector and expression system carrying this CAR can be used to prepare CAR-T cells targeting CD19. As shown in the statistical results of Figure 3, compared with FMC63-CAR, CAR1, CAR2, CAR3, and CAR24 all expressed a higher CAR positive rate (2.5-2.8 times), and the FITC median fluorescence intensity (MFI) was also 1.6-2.1 times higher. As shown in Figure 4, the CAR-T yields after 4 days, 8 days, and 12 days of culture after infection were compared. The yield of CAR-T cells based on the humanized scFv of the present invention was 1.7-2.7 times that of FMC63 CAR-T cells.

[0206] Example 3 Verification of the in vitro killing ability of CAR-T cells containing humanized scFv

[0207] The in vitro killing abilities of the four CAR-T cells prepared in Example 2 were detected.

[0208] CD19 surface antigen negative K562 human chronic myeloid leukemia cells and CD19 surface antigen positive Daudi B lymphoma cells were used as target cells. TM K562 cells and Daudi cells were labeled with CAR-T cells using Blue (Invitrogen) for 30 minutes. After washing off the dye, the cells were mixed with CAR-T cells at an effector-target ratio (E:T) of 0.3, 1, and 3, and co-cultured with X-VIVO15 medium containing 10% fetal bovine serum. Ctrl-T is an empty electroporated T cell; its cell number in the killing system is equivalent to the group with the lowest positive rate and the largest number of total T cells among the four groups of CAR-T. CAR-T targeting CEA (based on BW431 / 26scFv) is another control group. After 16 to 24 hours, the death rate of K562 cells and Daudi cells was detected by flow cytometry after staining with 7AAD / Annexin V-FITC. Circle the CellTracker TM After counting blue positive cells, draw a four-quadrant graph with Annexin V-FITC as the X-axis and 7AAD as the Y-axis to get the proportion of each quadrant. The percentage of dead cells is 7AAD. + Annexin V - , 7AAD + Annexin V + , 7AAD - Annexin V +The sum of the three quadrants.

[0209] The results are shown in Figure 5, which show that CAR-T cells expressing CAR1, CAR2, and CAR3 effectively kill CD19 antigen-positive Daudi B lymphoma cells, but have no killing effect on CD19 antigen-negative K562 cells. This result demonstrates that the scFv-based CAR of the present invention can be used to prepare CAR-T cells, and has good in vitro killing ability and is specific for CD19 antigen-positive tumor cells.

[0210] Furthermore, the in vitro killing ability of the humanized anti-CD19 CAR-T (CAR24) of the present invention and the murine FMC63 anti-CD19 CAR-T was compared. Compared with the murine FMC63 anti-CD19 CAR-T in terms of structure, the humanized anti-CD19 CAR-T (CAR24) and the murine FMC63 anti-CD19 CAR-T differ only in the scFv region, and the rest of the structures are the same. Using Daudi B lymphoma cells as target cells, CAR-T targeting CEA (based on BW431 / 26scFv) was used as the control group. First, CellTracker TM K562 cells and Daudi cells were labeled with CAR-T cells using 7AAD / Annexin V-FITC staining for 30 minutes. After washing, the cells were mixed with CAR-T cells at an effector-target ratio (E:T) of 0.3, 1, and 3, and co-cultured in X-VIVO15 medium containing 10% fetal bovine serum. After 24 hours, the cell death rate of K562 cells and Daudi cells was determined by flow cytometry after staining with 7AAD / Annexin V-FITC. TM After counting blue positive cells, draw a four-quadrant graph with Annexin V-FITC as the X-axis and 7AAD as the Y-axis to get the proportion of each quadrant. The percentage of dead cells is 7AAD. + Annexin V - , 7AAD + Annexin V + , 7AAD - Annexin V + The sum of the three quadrants.

[0211] The results are shown in Figure 6, which show that the in vitro killing ability of the CAR-T (CAR24) based on the humanized scFv of the present invention is comparable to that of the CAR-T based on FMC63, and both specifically kill CD19 antigen-positive tumor cells.

[0212] Example 4 Verification of the In Vivo Tumor Suppression Ability of Humanized Anti-CD19 CAR-T

[0213] NOG immunodeficient mice were intravenously inoculated with 5×10 6Seven days after inoculation of Daudi B lymphoma cells carrying the luciferase gene (Figure 7, left) or five days after inoculation (Figure 7, right), mice were infused with the humanized anti-CD19 CAR-T (CAR24) prepared by the present invention. The buffer group received injections of the formulation buffer. Luciferase activity in the mice was measured every 3-4 days after CAR-T injection. Tumor burden was expressed as whole-body bioluminescence (photos / s).

[0214] Experimental results showed that humanized CD19 CAR-T exhibited good therapeutic effects on B lymphoma (Figure 7).

[0215] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A humanized anti-CD19 antibody, characterized in that: The antibody comprises an antibody heavy chain variable region as shown in any one of SEQ ID NOs: 1-4, and an antibody light chain variable region as shown in any one of SEQ ID NOs: 5-7.

2. The antibody according to claim 1, characterized in that The antibody comprises an antibody heavy chain variable region as shown in SEQ ID NO: 1, and an antibody light chain variable region as shown in SEQ ID NO: 5; or The antibody comprises an antibody heavy chain variable region as shown in SEQ ID NO: 2, and an antibody light chain variable region as shown in SEQ ID NO: 6; or The antibody comprises an antibody heavy chain variable region as shown in SEQ ID NO:3, and an antibody light chain variable region as shown in SEQ ID NO:

6.

3. The antibody according to claim 1, characterized in that The structure of the antibody is shown in the following formula A or formula B: V H -V L (A);V L -V H (B) Where V H V is the variable region of the antibody heavy chain; L is the antibody light chain variable region; "-" is a connecting peptide or peptide bond; preferably, it is a structure of formula B.

4. The antibody according to any one of claims 1 to 3, characterized in that The amino acid sequence of the antibody is shown in any one of SEQ ID NOs: 8-10.

5. A CD19-targeting antigen binding receptor (CAR), characterized in that: The antigen binding domain of the CAR comprises the humanized anti-CD19 antibody of claim 1.

6. The CAR according to claim 5, wherein: The structure of the CAR is shown in Formula I below: L-scFv-H-TM-C-CS (I) In the formula, Each "-" is independently a connecting peptide or a peptide bond; L is none or a signal peptide sequence; scFv is the antigen binding domain targeting CD19; H is none or hinge region; TM is the transmembrane domain; C is a co-stimulatory signal molecule; CS is an intracellular signaling sequence derived from CD3ζ.

7. The CAR according to claim 6, wherein: The scFv has a structure as shown in Formula A or Formula B: V H -V L (A);V L -V H (B) Where V H is the heavy chain variable region of the humanized anti-CD19 antibody; V L is the light chain variable region of the humanized anti-CD19 antibody; "-" is a connecting peptide or peptide bond; preferably, it is a structure of formula B.

8. A nucleic acid molecule encoding the humanized anti-CD19 antibody according to claim 1 or the CD19-targeting CAR according to claim 5.

9. A vector comprising the nucleic acid molecule according to claim 8.

10. A host cell, comprising the vector of claim 9, or an exogenous nucleic acid molecule of claim 8 integrated into the chromosome, or expressing the humanized anti-CD19 antibody of claim 1 or the CD19-targeting CAR of claim 5.

11. An engineered immune cell, comprising the vector of claim 9, or an exogenous nucleic acid molecule of claim 8 integrated into the chromosome, or expressing the humanized anti-CD19 antibody of claim 1 or the CD19-targeting CAR of claim 5.

12. An immunoconjugate, characterized in that: The immunoconjugate comprises: (a) the humanized anti-CD19 antibody according to claim 1; (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

13. A kit comprising the humanized anti-CD19 antibody according to claim 1 or the immunoconjugate according to claim 12.

14. A preparation comprising the humanized anti-CD19 antibody of claim 1, or the immunoconjugate of claim 12, or the engineered immune cell of claim 11, and a pharmaceutically acceptable carrier.

15. Use of the humanized anti-CD19 antibody according to claim 1, or the engineered immune cell according to claim 11, or the immunoconjugate according to claim 12 for (1) preparing a drug or preparation for treating cancer or tumors; (2) preparing a reagent or kit for detecting CD19 molecules.

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