Bispecific chimeric antigen receptors targeting cd38 and cll1 and uses thereof

Abstract

A bispecific chimeric antigen receptor targeting CD38 and CLL1 is provided, wherein the chimeric antigen receptor comprises, from the N-terminus to the C-terminus, at least: i) an antigen-binding domain specifically recognizing CLL1 and CD38; ii) a transmembrane domain; iii) at least one co-stimulatory domain; and iv) a signal transduction domain. Bispecific CAR-T cells prepared using the above chimeric antigen receptor exhibit high specific in vitro cytotoxicity against CLL1-positive and / or CD38-positive target cells.

Description

A bispecific chimeric antigen receptor targeting CD38 and CLL1 and its application Technical Field

[0001] The present invention belongs to the field of immune cell therapy, and in particular, relates to a bispecific chimeric antigen receptor targeting CD38 and CLL1 and applications thereof. Background Art

[0002] Acute myeloid leukemia (AML) is a cancer of the myeloid lineage, characterized by the clonal expansion of malignant hematopoietic cells that accumulate in the bone marrow and blood and interfere with normal blood cells. The biological and clinical complexity of AML stems from its multiple subtypes, which greatly increases the difficulty of AML treatment. Common treatments for AML include chemotherapy and hematopoietic stem cell transplantation. However, since AML is most common in the elderly, most patients are unable to undergo intensive chemotherapy, and there are few sources of stem cell transplant donors. As a result, patient survival is extremely poor and relapse rates are high. New treatments are needed to treat it.

[0003] Chimeric antigen receptors (CARs) are artificial receptors that mimic the function of T cell receptors. They fuse the antigen recognition fragment of an antibody (or its corresponding ligand) with the downstream signaling domain of a T cell. Under the action of this chimeric receptor, T cells are specifically targeted to tumor-associated or specific antigens expressed on the surface of tumor cells, inducing CAR-T cell activation, proliferation, and subsequent tumor killing.

[0004] The CD38 antigen is a 45kDa type II transmembrane glycoprotein that catalyzes the synthesis and degradation of cyclic adenosine diphosphate ribose. CD38 is also involved in the regulation of apoptosis and cell proliferation and has important adhesion properties. Most AML progenitor cells have high CD38 expression, making it a potential target for AML treatment.

[0005] C-type lectin-like-1 (CLL1) is a member of the large family of glycoprotein receptors and C-type lectin-like receptors involved in immune regulation. CLL1 is primarily expressed in hematopoietic cells, primarily in innate immune cells and myeloid progenitor cells, including monocytes, DCs, and granulocytes. CLL-1 is also found in acute myeloid leukemia (AML) blasts, leukemic stem cells (e.g., CD34+ / CD38-), and a small fraction of hematopoietic progenitor cells (CD34+ / CD38+ or CD34+ / CD33+), but is not expressed on normal hematopoietic stem cells (CD34+ / CD38- or CD34+ / CD33-).

[0006] CAR-T cells have been slow to achieve breakthroughs in the treatment of AML due to a lack of suitable target surface antigens. This is because the targeted AML antigens are often co-expressed on healthy hematopoietic stem / progenitor cells (HSPCs), leading to the loss of all myeloid progenitor cells. Creative solutions are being sought to overcome these obstacles and make CAR-T therapy a viable option for AML patients. Summary of the Invention

[0007] In order to solve the above-mentioned problems, the present invention provides the following technical solutions:

[0008] The first aspect of the present invention discloses a bispecific chimeric antigen receptor targeting CD38 and CLL1, wherein the chimeric antigen receptor fusion protein comprises at least:

[0009] i) an antigen binding domain that specifically binds to CD38 and / or CLL1;

[0010] ii) transmembrane domain;

[0011] iii) at least one costimulatory domain;

[0012] iv) Signal transduction domain.

[0013] Preferably, the antigen-binding domain is monovalent or multivalent, that is, the antigen-binding domain contains a single antigenic determinant or multiple antigenic determinants and can specifically bind to a single antigenic epitope or multiple antigenic epitopes.

[0014] Preferably, the above-mentioned antigenic domain comprises a single domain antibody; others may also be selected, such as camel Ig, IgNAR, Fab fragment, Fab' fragment, F(ab')z fragment, F(ab')3 fragment, Fv, single-chain antibody (e.g., scFv, di-scFv, (scFv)z), miniantibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, disulfide-stabilized Fv protein ("dsFv"), chimeric antibodies, humanized antibodies, single domain antibodies, bispecific antibodies or multispecific antibodies, binding ligands or protein domains.

[0015] Preferably, the antigenic domain has two single domain antibodies, for example, VHH1, which represents a single domain antibody of a first antigen binding domain, and VHH2, which represents a single domain antibody of a second antigen binding domain; the first antigen binding domain targets and binds to CD38, and the second antigen binding domain targets and binds to CLL1.

[0016] The first antigen-binding domain and the second antigen-binding domain are arranged from amino terminus to carboxyl terminus in a pattern selected from one of the following groups:

[0017] i) VHH1-VHH2; or

[0018] ii) VHH2-VHH1.

[0019] The present application preferably uses a heavy chain single domain antibody as the single domain antibody.

[0020] As a preferred embodiment of the present application, the amino acid sequence of the CDR1 of the VHH1 is shown as SEQ ID NO: 6; the amino acid sequence of the CDR2 of the VHH1 is shown as SEQ ID NO: 8; and the amino acid sequence of the CDR3 of the VHH1 is shown as SEQ ID NO: 11.

[0021] As another preferred embodiment, the CDR1, CDR2, and CDR3 amino acid sequences of the VHH1 further include amino acid sequences that are at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 11, respectively; or have one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the sequences from which they are derived. Preferably, the substitutions are conservative substitutions.

[0022] As a preferred embodiment of the present application, the amino acid sequence of the CDR1 of the VHH1 is shown as SEQ ID NO: 7; the amino acid sequence of the CDR2 of the VHH1 is shown as SEQ ID NO: 9; and the amino acid sequence of the CDR3 of the VHH1 is shown as SEQ ID NO: 12.

[0023] As another preferred embodiment, the CDR1, CDR2, and CDR3 amino acid sequences of the VHH1 further include an amino acid sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 12, respectively; or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived. Preferably, the substitutions are conservative substitutions.

[0024] As a preferred embodiment of the present application, the amino acid sequence of the CDR1 of the VHH1 is shown as SEQ ID NO: 7; the amino acid sequence of the CDR2 of the VHH1 is shown as SEQ ID NO: 10; and the amino acid sequence of the CDR3 of the VHH1 is shown as SEQ ID NO: 13.

[0025] As another preferred embodiment, the CDR1, CDR2, and CDR3 amino acid sequences of the VHH1 further include amino acid sequences that are at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7, SEQ ID NO: 10, or SEQ ID NO: 13, respectively; or have one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the sequences from which they are derived. Preferably, the substitutions are conservative substitutions.

[0026] Preferably, the VHH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 1-3.

[0027] As another preferred embodiment, the amino acid sequence of VHH1 also includes an amino acid sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1-3, respectively; or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived. Preferably, the substitutions are conservative substitutions.

[0028] Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 of the above-mentioned VHH2 are shown as SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 17, respectively.

[0029] As another preferred embodiment, the CDR1, CDR2, and CDR3 amino acid sequences of the VHH2 further include amino acid sequences that are at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 17, respectively; or have one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the sequences from which they are derived. Preferably, the substitutions are conservative substitutions.

[0030] Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 of the above-mentioned VHH2 are shown as SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18, respectively.

[0031] As another preferred embodiment, the CDR1, CDR2, and CDR3 amino acid sequences of the VHH2 further include amino acid sequences that are at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14, SEQ ID NO: 16, or SEQ ID NO: 18, respectively; or have one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the sequences from which they are derived. Preferably, the substitutions are conservative substitutions.

[0032] Preferably, the VHH2 comprises the amino acid sequence shown in SEQ ID NO: 4 or 5, or consists of the same.

[0033] As another preferred embodiment, the amino acid sequence of VHH2 also includes an amino acid sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4 or 5, respectively; or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived. Preferably, the substitutions are conservative substitutions.

[0034] Preferably, the transmembrane domain comprises a molecule selected from the group consisting of CD8α, CD28, IgG1, IgG4, 4-1BB, PD-1, CD34, OX40, CD3ε, IL-2 receptor, and IL-7 receptor.

[0035] More preferably, the transmembrane domain is CD8α or CD28, wherein the transmembrane domain of CD8α comprises or consists of the amino acid sequence shown in SEQ ID NO: 19; and the transmembrane domain of CD28 comprises or consists of the amino acid sequence shown in SEQ ID NO: 20.

[0036] As another preferred embodiment, the amino acid sequence of the transmembrane domain also includes an amino acid sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19 or 20, respectively; or has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived. Preferably, the substitutions are conservative substitutions.

[0037] Preferably, the intracellular signaling domain comprises one or a combination of the following molecules: CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, CD66d.

[0038] More preferably, the intracellular signaling domain is preferably the cytoplasmic signaling sequence of CD3ζ, which has the amino acid sequence shown in SEQ ID NO: 21.

[0039] As another preferred embodiment, the amino acid sequence of the cytoplasmic signaling sequence of CD3ζ also includes an amino acid sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 21; or it has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived. Preferably, the substitutions are conservative substitutions.

[0040] As another preferred embodiment, the above-mentioned CD3ζ cytoplasmic signal transduction sequence also includes its wild type, or its mutant / modified form.

[0041] Preferably, the above-mentioned co-stimulatory signaling domain includes one or a combination of the following molecules: 4-1BB (CD137), CD27, CD19, CD4, CD28, ICOS (CD278), CD8α, CD82β, BAFFR, HVEM, LIGHT, KIRDS2, SLAMF7, NKp30, NKp46, CD40, CDS, ICAM-1, B7-H3, OX40, DR3, GITR, CD30, TIM1, CD2, CD7, CD226.

[0042] More preferably, the costimulatory signaling domain is preferably 4-1BB or CD28. The costimulatory signaling structure of 4-1BB comprises or consists of the amino acid sequence shown in SEQ ID NO: 22; the costimulatory signaling structure of CD28 comprises or consists of the amino acid sequence shown in SEQ ID NO: 23.

[0043] As another preferred embodiment, the amino acid sequence of the co-stimulatory signal structure also includes an amino acid sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 22 or 23, respectively; or it has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived. Preferably, the substitutions are conservative substitutions.

[0044] The chimeric antigen receptor further includes a hinge region.

[0045] Preferably, the hinge region is CD8α, and the hinge region of CD8α has the amino acid sequence shown in SEQ ID NO: 24.

[0046] As another preferred embodiment, the amino acid sequence of the hinge region also includes an amino acid sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 24; or it has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived. Preferably, the substitutions are conservative substitutions.

[0047] The chimeric antigen receptor further includes a signal peptide.

[0048] Preferably, the signal peptide comprises a molecule selected from the group consisting of the α and β chains of the T cell receptor, CD3ζ, CD3ε, CD4, CD5, CD8, CD9, CD28, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, GITR, and GM-CSF.

[0049] More preferably, the signal peptide is CD8α. The signal peptide of CD8α has the amino acid sequence shown in SEQ ID NO: 25.

[0050] The specific structure is obtained by connecting the above signal peptide, antigen binding domain, hinge region, transmembrane domain, co-stimulatory signal domain and signal transduction sequence in series:

[0051] L-VHH1-L1-VHH2-FH-TM-C-CD3ζ(Ia)

[0052] Where,

[0053] Each "-" is independently a connecting peptide or a peptide bond;

[0054] L is the signal peptide sequence;

[0055] VHH1 is an antigen-binding domain that specifically binds to CD38 or CLL1;

[0056] VHH2 is an antigen-binding domain that specifically binds to CLL1 or CD38;

[0057] L1 is a connecting peptide;

[0058] F is none or Flag tag sequence (Flag Tag);

[0059] H is the hinge region;

[0060] TM is the transmembrane domain;

[0061] C is the costimulatory signaling domain;

[0062] CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ, and also includes its wild type, or mutants / modified forms thereof.

[0063] Preferably, the specific structure of CAR-T is:

[0064] L-VHH1-L1-VHH2-H-TM-C-CD3ζ

[0065] Each "-" is independently a connecting peptide or a peptide bond;

[0066] L is the CD8a signal peptide molecule;

[0067] VHH1 is an antigen-binding domain that specifically binds to CD38 or CLL1;

[0068] VHH2 is an antigen-binding domain that specifically binds to CLL1 or CD38;

[0069] L1 is a connecting peptide;

[0070] H is the hinge region of CD8a;

[0071] TM is the transmembrane domain of CD8a;

[0072] C is the 4-1BB costimulatory domain;

[0073] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

[0074] Preferably, the specific structure of CAR-NK is:

[0075] L-VHH1-L1-VHH2-FH-TM-C-CD3ζ

[0076] Each "-" is independently a connecting peptide or a peptide bond;

[0077] L is the CD8a signal peptide molecule;

[0078] VHH1 is an antigen-binding domain that specifically binds to CD38 or CLL1;

[0079] VHH2 is an antigen-binding domain that specifically binds to CLL1 or CD38;

[0080] L1 is a connecting peptide;

[0081] F is the Flag tag sequence (Flag Tag);

[0082] H is the hinge region of CD8a;

[0083] TM is the transmembrane domain of CD28;

[0084] C is the costimulatory domain of CD28;

[0085] CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

[0086] Preferably, the above-mentioned connecting peptide is selected from the linker of the following amino acid sequence: SGG, GGS, SGGS, SSGGS, GGGG, SGGGG, GGGGS, SGGGGGS, SGGGGG, GSGGGGGS, GGGGGGGS, SGGGGGGG, SGGGGGGG, SGGGGSGGGGGGS or GGGGSGGGGSGGGGS.

[0087] Preferably, the fusion protein expression tag is a Flag tag, represented by the amino acid sequence of SEQ ID NO: 27.

[0088] As another preferred embodiment, the amino acid sequence of the Flag tag also includes an amino acid sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 27; or it has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived. Preferably, the substitutions are conservative substitutions.

[0089] The second aspect of the present invention discloses a nucleic acid molecule, which is a nucleotide sequence encoding the chimeric antigen receptor.

[0090] Preferably, the nucleic acid molecule C9B5 encoding the amino acids shown in SEQ ID NO: 2 and SEQ ID NO: 5 is preferred, and the nucleotide sequence of C9B5 is shown in SEQ ID NO: 30.

[0091] The nucleic acid molecule further comprises a nucleotide sequence shown in any one of SEQ ID NOs: 28-29, or 31-51.

[0092] The third aspect of the present invention discloses a recombinant vector, which contains the chimeric antigen receptor or the nucleic acid molecule.

[0093] Preferably, the recombinant vector includes a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR / Cas9 vector or a viral vector.

[0094] More preferably, the viral vector includes a lentiviral vector, an adenoviral vector, or a retroviral vector.

[0095] The fourth aspect of the present invention discloses an engineered immune cell, wherein the engineered immune cell comprises and / or expresses the chimeric antigen receptor, the nucleic acid molecule, or the recombinant expression vector.

[0096] Preferably, the immune cells are prepared using the FAST-CAR process.

[0097] More preferably, the FAST CAR converts the activation, transduction, and expansion steps into a single "synchronous activation-transduction" step, wherein the engineered immune cells undergo ex vivo expansion in less than 72 hours.

[0098] As another preferred embodiment, the engineered immune cells prepared using the FAST-CAR process can reduce the fratricide caused by CD38CAR recognizing the CD38 antigen on the surface of immune cells during the production process; or the fratricide caused by other CARs recognizing the corresponding target antigens on the surface of immune cells (for example, CD70CAR recognizes the CD70 antigen on the surface of T cells). At the same time, the immune cells can be applied to any CAR targeting antigens and different tumor markers.

[0099] As another preferred embodiment, the engineered immune cells prepared using the FAST-CAR process are observed to have a younger phenotype than cells in a comparable population that have undergone ex vivo expansion for 1 week or more. n / scm (CD45RO - , CCR7 + ) and Tcm(CD45RO + , CCR7 + ) accounts for a higher proportion.

[0100] As another preferred embodiment, engineered immune cells prepared using the FAST-CAR process were observed to have stronger in vitro expansion and sustained killing capabilities compared to cells in a comparable population that underwent ex vivo expansion for one week or more.

[0101] Preferably, the immune cells include T cells, NK cells, iNKT cells, CTL cells, monocytes, macrophages, dendritic cells and / or NKT cells.

[0102] More preferably, the immune cells may be T cells and NK cells.

[0103] A fifth aspect of the present invention discloses the use of the chimeric antigen receptor, nucleic acid molecule, recombinant expression vector, or engineered immune cell described above in the preparation of a drug or pharmaceutical composition for treating tumors. The tumor may be a cancer. Preferably, the tumor is a tumor or cancer that expresses CD38 and CLL1.

[0104] More preferably, the tumor is acute myeloid leukemia.

[0105] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and adjuvant.

[0106] Use of the chimeric antigen receptor described above in combination with one or more of the following in the preparation of a pharmaceutical combination for treating and / or preventing cancer:

[0107] (1) an agent that increases the efficacy of cells containing a CAR nucleic acid or CAR polypeptide;

[0108] (2) an agent that ameliorates one or more side effects associated with administration of cells containing a CAR nucleic acid or CAR polypeptide;

[0109] (3) Additional agents for treating diseases associated with CD38 and CLL1.

[0110] Preferably, the treatment and / or prevention also includes use in combination with a second therapy selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, virotherapy, adjuvant therapy and any combination thereof.

[0111] The present invention has the following significant advantages and effects compared to the prior art:

[0112] (1) The dual-targeted CAR T cells of the present invention show highly specific in vitro cytotoxicity against CLL1-positive and / or CD38-positive target cells.

[0113] (2) The dual-targeted CAR T cells produced by the present invention using the FAST process are not affected by CD38-CAR-based fratricide;

[0114] (3) The dual-targeted CAR T cells produced by the FAST process in the present invention have a younger phenotype than those produced by traditional processes and exhibit more sustained tumor killing and expansion capabilities in in vitro experiments;

[0115] (4) The dual-targeted CAR T cells produced by the FAST process in the present invention can effectively inhibit the growth of CD38- and CLL1-positive tumors in vivo at lower doses and show long-lasting anti-tumor efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0116] FIG1 is a schematic diagram of target cell surface antigen expression described in Example 1;

[0117] FIG2 is a schematic diagram of the structure of the Dual-CAR described in Example 2;

[0118] Figure 3 shows the CAR positivity rate of Dual-CAR T cells detected using antigens in Example 3;

[0119] Figure 4 is a schematic diagram of the killing results of Dual-CAR T against Hela-WT and CLL1 and CD38 overexpressing Hela cells in Example 4;

[0120] Figure 5 is a schematic diagram of the killing results of target cells expressing CLL1 and CD38 by Dual-CAR T in Example 4;

[0121] Figure 6 is a schematic diagram of the flow cytometry analysis process of multiple rounds of CAR-T cell killing in vitro in Example 5;

[0122] FIG7 is a schematic diagram showing the results of multiple rounds of killing of HL60 cells by Dual-CAR T in Example 5;

[0123] FIG8 is a schematic diagram showing the results of multiple rounds of killing of KG1 cells by Dual-CAR T in Example 5;

[0124] Figure 9 is a schematic diagram of the results of multiple rounds of killing of THP1 cells by Dual-CAR T in Example 5;

[0125] Figure 10 is a schematic diagram of the CAR positive rate after CAR-NK92 sorting in Example 8;

[0126] FIG11 is a schematic diagram showing the results of CAR-NK92 killing tumor cells in Example 9;

[0127] FIG12 is a schematic diagram of the multiple rounds of killing results of CAR-NK92 in Example 10;

[0128] Figure 13 is a schematic diagram of the changes in CD38 expression, CAR expression, expansion, and activity of F Dual-CAR T cells after recovery in Example 12;

[0129] Figure 14 is a schematic diagram comparing the differentiation phenotypes of F Dual-CAR T and C Dual-CAR T cells in Example 12;

[0130] Figure 15 is a schematic diagram of the in vitro killing results of F Dual-CAR T and C Dual-CAR T cells in Example 13;

[0131] Figure 16 is a schematic diagram of the results of multiple rounds of killing by F Dual-CAR T and C Dual-CAR T cells in vitro in Example 14;

[0132] Figure 17 is a diagram showing the in vivo efficacy experiment of F Dual-CAR T and C Dual-CAR T cells in mice in Example 15. DETAILED DESCRIPTION

[0133] The present invention will be further described below through specific examples. It should be understood that the following examples are only for illustrating the present invention and are not intended to limit the content of the invention.

[0134] The raw materials and equipment used in the examples are well known to those skilled in the art and can be purchased or easily obtained or prepared on the market.

[0135] As used herein, the term "antigen" refers to a molecule or fragment thereof that can be bound by a selective binding agent. For example, an antigen can be a ligand that can be bound by a selective binding agent such as a receptor. As another example, an antigen can be an antigen molecule that can be bound by a selective binding agent such as an immune protein (e.g., an antibody). Antigen can also refer to a molecule or fragment thereof that can be used in an animal to produce an antibody that can bind to the antigen. In some cases, an antigen can be bound to a substrate (e.g., a cell membrane). Alternatively, an antigen may not be bound to a substrate (e.g., a secreted molecule, such as a secreted polypeptide).

[0136] The term "antibody" (Ab) shall include, but is not limited to, immunoglobulins that specifically bind to an antigen and comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen.

[0137] 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).

[0138] The term "complementarity determining region" or "CDR" refers to the hypervariable regions of the heavy and light chains of immunoglobulins as defined by Kabat et al. (Kabat et al., Sequences of proteins of immunological interest, 5th Ed. US Department of Health and Human Services, NIH, 1991, and later versions). The Kabat numbering system is used herein to define CDRs. There are three heavy chain CDRs and three light chain CDRs. Herein, the terms "CDR" and "CDRs" are used to refer to a region comprising one or more, or even all, of the major amino acid residues that contribute to the binding affinity of an antibody to its recognized antigen or epitope. In another specific embodiment, the CDR region or CDRs refers to the hypervariable regions of the heavy and light chains of immunoglobulins as defined by IMGT.

[0139] Antibody preparation

[0140] The antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombinant technology. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the antibodies of the present invention.

[0141] The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). Alternatively, these antigen-binding fragments can be produced directly by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000)). For example, Fab' fragments can be obtained directly from host cells; Fab' fragments can be chemically coupled to form F(ab')z fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). In addition, Fv, Fab, or F(ab')z fragments can also be directly isolated from recombinant host cell culture medium. Other techniques for preparing such antigen-binding fragments are well known to those of ordinary skill in the art.

[0142] Conservative substitution

[0143] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions of residues physically or functionally similar to corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family.

[0144] Chimeric Antigen Receptor (CAR)

[0145] 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) and a hinge region. The intracellular domain includes a costimulatory signaling region and a CD3ζ chain portion.

[0146] A target-specific binding element (also called an antigen-binding domain) refers to an element capable of antigen recognition based on antigen-binding specificity.

[0147] 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 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.

[0148] 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.

[0149] The co-stimulatory signaling region refers to the portion of the intracellular domain that includes co-stimulatory molecules. Co-stimulatory molecules are cell surface molecules that are required for lymphocytes to effectively respond to antigens, rather than antigen receptors or their ligands.

[0150] When the CAR of the present invention is expressed in T cells, the antigen binding domain is fused to the hinge and transmembrane regions, the intracellular domains from one or more of the co-stimulatory molecules and the CD3 zeta chain.

[0151] In another preferred embodiment, the antigen binding domain is fused to the intracellular domain of a combination of the CD8a hinge region and the CD8a transmembrane region, the 4-1BB signaling domain, and the CD3ζ signaling domain.

[0152] In another preferred embodiment, the antigen binding domain is fused to the intracellular domain of a combination of the CD8a hinge region, the CD28 transmembrane region, the CD28 signaling domain, and the CD3ζ signaling domain.

[0153] Bispecific CAR targeting CD38 and CLL1

[0154] Bispecificity means that the same CAR can specifically bind to and immune-recognize two different antigens, and CAR can produce an immune response by binding to either antigen.

[0155] In another preferred embodiment, the bispecific CAR targeting CD38 and CLL1 is as described in the first aspect of the present invention.

[0156] 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 CD38 and CLL1.

[0157] In another preferred embodiment, the present invention provides a bispecific chimeric antigen receptor for CD38 and CLL1 antigens. The structural components of the CAR that simultaneously targets CD38 and CLL1 may include a signal peptide, an anti-CD38 single-domain antibody, an anti-CLL1 single-domain antibody, a hinge region, a transmembrane region, and an intracellular T cell signaling region.

[0158] Dual-targeted CAR-T cells offer a broader therapeutic scope. CAR-immune cells that simultaneously target both CD38 and CLL1 can reduce the possibility of antigen escape caused by downregulation or loss of a single surface antigen. Furthermore, bispecific chimeric antigen receptors targeting the combination of CD38 and CLL1 have demonstrated highly specific in vitro cytotoxicity.

[0159] Natural killer cells (NK cells)

[0160] NK cells are important immune cells in the body. Morphologically, NK cells belong to large granular lymphocytes and originate from the bone marrow. They are the third largest type of lymphocytes after T cells and B cells, accounting for about 15% of all immune cells (white blood cell count) in the blood. They are core cells of the innate immune system and are mainly distributed in peripheral blood, liver and spleen. The main characteristics of NK cells in the human body are CD3-CD56+ lymphocyte populations, of which the main subtype in the blood is CD16+CD56dim (based on the difference in the expression density of CD56 molecules on cells, NK cells are divided into two subpopulations, CD56dim and CD56bright; CD56dim accounts for more than 90% of NK cells, mainly for cytotoxic effects, expresses the moderate-affinity IL-2 receptor (IL-2R), and has stronger killing activity; CD56bright can produce a large amount of cytokines, mainly plays an immunomodulatory role, and highly expresses IL-2R).

[0161] Chimeric antigen receptor T cells (CAR-T cells)

[0162] As used herein, the terms "CAR-T cells," "CAR-T," and "CAR-T cells of the present invention" refer to chimeric antigen receptor T cells (CAR-T cells). A chimeric antigen receptor T cell (CAR-T cell) is a chimeric protein that is formed by coupling the antigen-binding portion of an antibody that recognizes a tumor antigen with the intracellular portion of the CD3-ζ chain or FcεRIγ in vitro. The chimeric protein is then transfected into the patient's T cells through gene transduction to express the chimeric antigen receptor (CAR). After the patient's T cells are "recoded," a large number of tumor-specific CAR-T cells are generated. The basic principle is to use the patient's own immune cells to eliminate cancer cells.

[0163] CAR-NK adoptive cell therapy (ACT) involves genetically modifying NK cells with a chimeric antigen receptor (CAR), giving them the ability to target and recognize tumor cells. After expansion in vitro, they are then injected into the human body to achieve the desired therapeutic effect. Currently, NK cells used clinically come from five main sources: human peripheral blood (PB), umbilical cord blood (UCB), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), and the NK-92 cell line.

[0164] The NK-92 cell line is currently the most extensively studied cell line in CAR-NK therapy. It was isolated in 1992 from a 50-year-old male patient with non-Hodgkin's lymphoma and exhibits IL-2-dependent growth. Compared to primary NK cells, the NK-92 cell line's greatest advantage lies in its low expression of inhibitory receptors (such as KIR) on its surface. This lack of inhibitory receptor signaling enhances its ability to kill a variety of tumors compared to primary NK cells or other cytokine-activated killer cells. NK-92 also has potential in the treatment of solid tumors. A key reason for the poor efficacy of CAR-T in solid tumors is the high expression of PD-L1 on tumor cells, which binds to the inhibitory molecule PD-1 on the surface of T cells, thereby inhibiting their cytotoxic activity. The lack of inhibitory receptors on the NK-92 cell line prevents interference from these inhibitory signals. However, NK-92 also has significant drawbacks, such as potential tumorigenicity and susceptibility to Epstein-Barr virus. Therefore, NK-92 must be irradiated before use.

[0165] FAST-CAR Platform

[0166] The "FAST process" described in this article is a process for preparing engineered immune cells.

[0167] In the traditional CAR-T production process, the patient's T cells are first activated using CD3 and / or CD28 antibodies, and then transduced with a viral vector to express one or more CARs. These modified CAR-T cells are then expanded in vitro before being injected back into the body. The entire production process usually takes 1 to 6 weeks.

[0168] The FasTCAR platform can simultaneously activate and transduce resting T cells using XLenti vectors, which are high-quality and exhibit high gene transduction efficiency and are derived from lentiviruses. After transduction, one or more CARs will be integrated into the T cell genome and stably expressed. Based on our preclinical studies, transduced T cells have high expansion and tumor cell clearance activity, eliminating the need for in vitro cell expansion steps and can be directly dosed and given to patients. Based on such innovations, FasTCAR technology can transform the activation, transduction, and amplification steps into a single "synchronous activation-transduction" step. This technology can significantly shorten the production time of autologous CAR-T cells from the industry standard of 1 to 6 weeks to completion within 72 hours.

[0169] carrier

[0170] 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.

[0171] The present invention also provides vectors into which the 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.

[0172] 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.

[0173] 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.

[0174] The nucleic acid can be packaged into many types of vectors. For example, the nucleic acid can be cloned into vectors including, but 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.

[0175] 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).

[0176] 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.

[0177] 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.

[0178] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive any polynucleotide sequence operably connected thereto for high-level expression. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr (Epstein-Barr) virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the present invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered to be part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0179] 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.

[0180] Reporter gene is used to identify the cells of potential transfection and to evaluate the functionality of regulatory sequences. Generally, reporter gene is following gene: it is not present in or is expressed by receptor organism or tissue, and its coded polypeptide, the expression of this polypeptide is clearly represented by some easily detectable properties such as enzymatic activity. After DNA has been introduced into receptor cells, the expression of reporter gene is measured under the appropriate time. Suitable reporter gene can comprise the gene (for example, Ui-Tei etc., 2000FEBS Letters479:79-82) of coding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein. Suitable expression system is well known and can utilize known technology to prepare or obtain commercially. Generally, the construct with minimum 5 flanking regions showing the highest level of reporter gene expression is identified as promoter. Such promoter region can be connected to reporter gene and be used to evaluate the ability of reagent regulating promoter-driven transcription.

[0181] 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.

[0182] 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). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0183] 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.

[0184] 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).

[0185] 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.

[0186] Pharmaceutical composition

[0187] The isolated nucleic acid molecules, vectors, host cells, modified immune cells or immune cell compositions of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The pharmaceutical composition of the present invention should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such an injection can be a sterile injection solution. In addition, the sterile injection solution can be prepared as a sterile lyophilized powder (for example, by vacuum drying or freeze drying) for storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCI), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0188] The nucleic acid molecules of the present invention, vectors, host cells, the immune cells of the present invention or immune cell compositions can be used by any suitable method known in the art, including but not limited to oral, oral, sublingual, eyeball, local, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, inguinal, intravesical, local (such as, powder, ointment or drops), or nasal route. However, for many therapeutic uses, preferred route of administration / mode is parenteral administration (such as intravenous injection or push injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). It will be understood by those skilled in the art that route of administration and / or mode will change according to the intended purpose. In certain embodiments, the nucleic acid molecules of the present invention, nucleic acid construct, vectors, host cells, the immune cells of the present invention or immune cell compositions are given by intravenous injection or push injection.

[0189] The pharmaceutical compositions of the present invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of an isolated nucleic acid molecule, nucleic acid construct, vector, host cell, modified immune cell of the present invention, or immune cell composition. A "prophylactically effective amount" refers to an amount sufficient to prevent, prevent, or delay the onset of a disease. A "therapeutically effective amount" refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. The therapeutically effective amount of an isolated nucleic acid molecule, nucleic acid construct, vector, host cell, modified immune cell of the present invention, or immune cell composition may vary depending on factors such as the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition, such as age, weight, and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0190] In the present invention, the dosage regimen can be adjusted to obtain the best desired response (e.g., therapeutic or preventive response). For example, the dosage can be a single dose, multiple doses can be administered over a period of time, or the dosage can be proportionally reduced or increased according to the urgency of the treatment situation.

[0191] Therapeutic applications

[0192] 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.

[0193] In one embodiment, the present invention includes a type of cell therapy that separates the patient's autologous T cells (or NK cells from a heterologous donor), activates and genetically modifies them to produce CAR-T or CAR-NK cells, which are then injected into the patient. This approach has a very low probability of developing graft-versus-host disease, and the antigen is recognized by T cells or NK cells in an MHC-independent manner. In addition, one CAR-T or CAR-NK cell can treat all cancers that express the antigen. Unlike antibody therapy, CAR-T and CAR-NK cells can replicate in vivo, producing long-term persistence that can lead to sustained tumor control.

[0194] In one embodiment, the CAR-T cells of the present invention can undergo robust in vivo T cell expansion and can sustain an extended period of time. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy step, wherein the antigen binding domain in the CAR induces a specific immune response of the CAR-modified immune cells to the antigen-expressing target cells. For example, anti-CD38 and / or CLL1 CAR-T, CAR-NK cells induce a specific immune response against cells expressing CD38 and / or CLL1.

[0195] Although the data disclosed herein specifically disclose lentiviral vectors comprising CD38 and / or CLL1 antigen recognition binding domains, hinge and transmembrane regions, 4-1BB or CD28 costimulatory domains, and CD3ζ intracellular signaling domains, the present invention should be construed to include any number of variations to each of the construct components.

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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.

[0200] The present invention provides a method for treating tumors, comprising administering to a subject in need thereof a therapeutically effective amount of the CAR-modified T cells of the present invention.

[0201] 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.

[0202] 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.

[0203] Example 1: Cell culture and construction

[0204] KG1-LucG cells, HL60-LucG cells, U937-LucG cells, THP1-LucG cells, Molm13-LucR cells, Molm13-CLL1-LucR cells, and K562-LucG cells were cultured in RPMI 1640 medium. 293T and wild-type cells, as well as Hela cells expressing CD38, CLL1, and both CLL1 and CD38 (Hela-WT, Hela-CD38, Hela-CLL1, and Hela-CLL1-CD38), were cultured in DMEM medium. All of the above media were supplemented with 10% (v / v) fetal bovine serum, 100 U / ml penicillin and streptomycin, 2 mM L-glutamine, and 1 mM sodium pyruvate. Cells were cultured at 37°C, 5% CO2, and saturated humidity.

[0205] Hela cells expressing CD38 are stably transfected cells obtained by transfecting CD38 antigens into Hela cells using a lentiviral vector, specifically expressing the CD38 protein. Hela cells expressing CLL1 are stably transfected cells obtained by transfecting CLL1 antigens into Hela cells using a lentiviral vector, specifically expressing the CLL1 protein. Hela cells expressing both CLL1 and CD38 are stably transfected cells obtained by transfecting CLL1-expressing Hela cells with the CD38 antigen via a lentiviral vector, specifically expressing both CLL1 and CD38. Molm13-LucR cells are stably transfected cells obtained by transfecting with a lentivirus expressing firefly luciferase-RFP (T2A linker). KG1-LucG cells, HL60-LucG cells, U937-LucG cells, THP1-LucG cells, and K562-LucG cells are stably transfected cells obtained by transfecting with a lentivirus expressing firefly luciferase-GFP (T2A linker). Molm13-CLL1-LucR cells are a stable cell line obtained by transferring CLL1 antigen into Molm13-LucR cells via a lentiviral vector, and can specifically express CLL1 protein molecules.

[0206] The expression of target cell surface antigens is shown in Figure 1. Hela-CD38 is a cell overexpressing CD38, Hela-CLL1 is a cell overexpressing CLL1, Hela-CLL1-CD38 is a cell overexpressing CLL1 and CD38, Hela-WT and K562 are double-negative cells for CLL1 and CD38, Molm13 is a single-positive cell for CD38, and HL60, KG1, U937, THP1, and Molm13-CLL1 are double-positive cells for CLL1 and CD38.

[0207] Example 2: Dual CAR vector construction and virus preparation

[0208] The structure of a CAR (Dual CAR) that simultaneously targets CLL1 and CD38 is shown in Figure 2. It consists of a CD8α signal peptide, a VHH that recognizes CD38 (or CLL1), a G4S sequence, a VHH that recognizes CLL1 (or CD38), a Flag tag, a hinge and transmembrane region, a costimulatory signaling region, and a CD3z signaling region. The Dual CAR gene is placed under the EF1α (EF-1α) promoter to form a Dual CAR expression vector. Dual CAR expression vectors are numbered C7B5, C7B8, C9B5, C9B8, B5C7, B5C9, B8C7, B8C9, C7B3, C9B3, B3C7, and B3C9, respectively, based on the composition of the VHH sequence.

[0209] Among them, B3, B5, B8, C7, and C9 represent single domain antibodies having the B3 alpaca antibody amino acid sequence (SEQ ID NO: 1), B5 alpaca antibody amino acid sequence (SEQ ID NO: 2), B8 alpaca antibody amino acid sequence (SEQ ID NO: 3), C7 alpaca antibody amino acid sequence (SEQ ID NO: 4), and C9 alpaca antibody amino acid sequence (SEQ ID NO: 5), respectively.

[0210] 2.5×10 6 293T cells were seeded in 150 cm 2 The cells were cultured in 10% FBS-containing DMEM medium in a culture dish at 37°C, 5% CO2 and saturated humidity overnight before transfection.

[0211] The next day, the auxiliary plasmid and Dual CAR expression vector were added to a centrifuge tube containing 13.8 mL of Opti MEM medium, and 80 μg of PEI was added to the tube to obtain a mixture. The mixture was allowed to stand at room temperature for 20 minutes, and then 12 mL of Opti MEM medium was supplemented to obtain the transfection medium. For transfection, after removing the culture medium, the 293T cells were incubated with the transfection medium for 4-6 hours, and then the transfection medium was replaced with 20 mL of DMEM medium containing 2% FBS. After 72 hours, the culture medium was collected and centrifuged at 3000 g and 4 ° C for 15 minutes. The supernatant was further centrifuged at 27000 g and 4 ° C for 2 hours. The precipitate was collected and resuspended with 400 μL of pre-cooled X-VIVO medium to obtain the Dual CAR lentiviral suspension and kept at 4 ° C overnight. The next day, the viral suspension was aliquoted for further use.

[0212] Example 3: Dual-CAR T cell preparation

[0213] T cells were cultured in X-VIVO medium. Pan T cells were activated by incubating with CD3 / CD28 Dynabeads at a 1:1 ratio (CD3 / CD28 Dynabeads: T cells) and 300 IU / mL IL-2. Two days later, the activated cells were electroporated to knock out the CD38 gene and then virally transfected to generate Dual-CAR T cells.

[0214] The specific operation is as follows: 0.25 nmol of CD38-targeting gRNA and 16.5 μg of Cas9 protein were mixed evenly and incubated at 37°C for 15 min to prepare RNP; during the incubation period, 18 μL of supplement buffer was added to 82 μL of Nucleofector Solution to prepare 100 μL of Ionza P3 electroporation buffer; at the same time, the CD3 / CD28 Dynabeads were removed using a magnetic column, and 1×10 7 After centrifugation at 400 g for 5 min, the cell pellet was collected. After incubation, 1×10 7 The T cell pellet was added to the RNP and mixed evenly. The cells were then transferred to an electroporation cup and placed in a Lonza 2B electroporator. The electroporation was performed using the FI-115 program. The electroporated cells were incubated with 300 IU / mL IL-2 overnight.

[0215] On the second day after electroporation, 1 × 10 6T cells were transfected with Dual CAR virus at a cell density of 10 cells / mL. The transfected cells were cultured and expanded. CLL1 and CD38 antigens were used to detect the CAR positivity of Dual-CAR T cells during the expansion process and when frozen. Half of the culture medium was replaced every 2-3 days. Dual-CAR T cells were harvested on the 8th day after removing the CD3 / CD28 Dynabeads.

[0216] Figure 3 shows the CAR positivity rate of Dual-CAR T cells using antigen detection. The expression of CLL1CAR and CD38CAR can be detected simultaneously on the surface of virally transfected T cells using CLL1 antigen and CD38 antigen.

[0217] Example 4: Dual-CAR T cell killing in vitro

[0218] The obtained Dual-CAR T cells were subjected to in vitro cytotoxicity assays. RTCA cytotoxicity assays were performed using Hela cell lines overexpressing CLL1 and CD38, or luciferase-labeled tumor target cells were used for detection.

[0219] By transferring the luciferase gene into target cells and cloning and screening, stable cell lines (HL60, Molm13, U937, and K562) are obtained. During the experiment, the luciferin substrate is added, and the luciferase reacts with the luciferin to produce fluorescence. The intensity of the fluorescence can be used to determine the activity of the luciferase, and the cell survival rate can be measured to determine the killing effect of the CAR-T cells.

[0220] The results showed that after co-culturing CAR-T cells with various target cells (CLL1 / CD38 double positive, CLL1 single positive, CD38 single positive), the target cells were lysed, indicating that Dual-CAR T has a killing effect on CLL1 / CD38 double positive, CLL1 single positive, and CD38 single positive cells.

[0221] The specific results are shown in Figure 4. The bispecific CAR-T cells significantly kill single-positive CLL1-positive target cells (Hela-CLL1) or single-positive CD38-positive target cells (Hela-CD38), and also significantly kill the target cells Hela-CLL1-CD38 that are both positive for both CLL1 and CD38. This shows that the bispecific CAR-T cells combined with CLL1 and CD38 have a killing effect on both single and dual target cells. In addition, Dual-CAR T has no killing effect on the target cells Hela that are both negative for CLL1 and CD38.

[0222] Figure 5 shows that Dual-CAR T can significantly kill tumor target cells HL60, U937 and Molm13 that express CD38 and CLL1, while having no killing effect on CLL1 and CD38 double-negative cells K562.

[0223] Example 5: Dual-CAR T cells kill multiple rounds in vitro

[0224] On the first day, Dual-CAR T cells were revived and incubated at 37°C at a concentration of 1 × 10 6 The cells were cultured at a density of 10 cells / mL and 300 IU / mL IL2. On the second day, 1×10 5 The proportion of Dual CAR-positive cells in each cell sample was detected using CLL1 and CD38 antigens.

[0225] According to the CAR positive rate of cells, 2×10 5 CAR-positive Dual-CAR T cells were respectively 5 HL60-LucG and KG1-LucG cells were co-cultured (E:T = 1:3) for multiple rounds of killing analysis. On the third day of each round, a portion of the co-cultured cells was removed for flow cytometry analysis. The analysis process is shown in Figure 6. The specific method is described as follows: 7AAD-negative live cell populations were circled from the main cell population, and then T cells (GFP-negative) and target cells (GFP-positive) were distinguished by GFP signal. CLL1 and CD38 antigen markers were used to analyze the CAR positivity rate in the T cell population. Based on the flow cytometry and cell count results, the same number of Dual-CAR T cells were removed and supplemented with tumor cells to a fixed effector-target ratio (E:T of 1:3) for the next round of killing experiments.

[0226] The results of multiple rounds of killing against HL60 target cells are shown in Figure 7. Figure 7A shows the expansion fold of residual target cells (HL60) at the end of each round of Dual-CAR T cells; Figure 7B shows the change in CAR positivity rate during multiple rounds of killing against HL60 by Dual-CAR T cells; Figure 7C shows the total expansion fold of CAR T cells during multiple rounds of killing against HL60 by Dual-CAR T cells.

[0227] As can be seen from Figure 7A, C7B3 and C9B5 have stronger inhibitory abilities against HL60 cells than other Dual-CAR T cells. At the end of the third round, most Dual-CAR T cells were no longer able to inhibit HL60 cells, and the CAR positivity rate began to decline (Figure 7B). As can be seen from Figure 7C, in multiple rounds of killing against HL60 cells, the expansion of C7B3 was significantly better than that of other Dual-CAR T cells.

[0228] The results of multiple rounds of killing against KG1 target cells are shown in Figure 8. Figure 8A shows the expansion fold of residual target cells (KG1) at the end of each round of Dual-CAR T cells; Figure 8B shows the change in CAR positivity during multiple rounds of killing against KG1 by Dual-CAR T cells; Figure 8C shows the total expansion fold of CAR T cells during multiple rounds of killing against KG1 by Dual-CAR T cells.

[0229] At the end of the third round, all Dual-CAR T cells were unable to inhibit KG1 cells well, but C9B5 and C9B8 had stronger inhibitory abilities against KG1 cells than other Dual-CAR T cells (Figure 8A). Figure 8C shows that in multiple rounds of killing against KG1, the expansion of C9B5 and C9B8 was better than that of other Dual-CAR T cells.

[0230] The results of multiple rounds of killing against THP1 target cells are shown in Figure 9. Figure 9A shows the expansion fold of residual target cells (THP1) at the end of each round of Dual-CAR T cells; Figure 9B shows the change in CAR positivity during multiple rounds of killing against THP1 by Dual-CAR T cells; Figure 9C shows the total expansion fold of CAR T cells during multiple rounds of killing against THP1 by Dual-CAR T cells.

[0231] At the end of the fifth round, all Dual-CAR T cells were unable to inhibit THP1 cells well, but C7B5, C9B5, and C9B8 were more capable of maintaining CAR expression than other Dual-CAR T cells (Figure 9B). As can be seen from Figure 9C, in multiple rounds of killing against THP1, the expansion of C9B8, C7B5, and C9B5 was better than that of other Dual-CAR T cells.

[0232] Example 6: NK cells and culture methods

[0233] The complete culture medium for NK92 cells is AlphaMEM supplemented with 12.5% ​​FBS, 12.5% ​​Horse Serum, 1% Pen / Strep, 1% sodium pyruvate, 1% L-glutamine, and 100 U IL2. The complete culture medium for HL60, Molm13, KG-1, THP-1, and K562 cells is RPMI1640 supplemented with 10% FBS, 1% Pen / Strep, 1% sodium pyruvate, and 1% L-glutamine. Cells were cultured at 37°C, 5% CO2, and saturated humidity.

[0234] Example 7: CAR-NK lentiviral packaging and transfection

[0235] Resuscitation 293T inoculated one 150cm 2 The culture dish was placed in a CO2 incubator and cultured for 72 hours. After two passages, the culture dish was inoculated in a 150 cm 2 Culture dish for transfection. Mix the four-plasmid system consisting of lentiviral expression vector, auxiliary plasmids gag / pol, Rev, and VSV-G with PEI transfection reagent, add to a certain volume of serum-free DMEM, mix well and let stand for 15 minutes; add the above mixture to a 150cm dish with 293T cells. 2 Place the culture dish in a 37°C, 5% CO2 incubator for 6 hours. After 6 hours, replace the culture medium with fresh medium and continue culturing. After 48 and 72 hours, collect the lentiviral culture supernatant for infection. Transfer the harvested supernatant to a centrifuge tube and centrifuge at 4000 rpm for 10 minutes to remove cell debris. Transfer all centrifuged LVV supernatants to a 0.45μm filter for filtration and clarification, and transfer the filtrate to a new centrifuge tube. Add the clarified lentiviral supernatant to an ultracentrifuge tube and balance it on a scale. Place the balanced ultracentrifuge tube in a hanging cup, position the hanging cup in the corresponding position of the rotor, and then place the tube in an ultracentrifuge for centrifugation at 4°C, 100,000g, and 90 minutes, with the ascent and descent speeds set to the highest setting. After ultracentrifugation, discard the supernatant, add 0.5mL of culture medium to each tube, and resuspend at 2-8°C for 2 hours. The harvested virus was added to 1e6NK92 and mixed in a 24-well plate, and then cultured in a CO2 incubator for 72 h.

[0236] Example 8: CAR-NK cell flow cytometry and sorting

[0237] Remove the cell suspension, wash three times with DPBS, centrifuge at 300g for 5 minutes, discard the supernatant, add the antibody cocktail (anti-FLAG-APC, 1:100 in DPBS), mix well, incubate at 4°C, stain for 30 minutes, wash three times with DPBS, and centrifuge at 300g for 5 minutes. Resuspend in DPBS and use for flow cytometry and flow sorting. During sorting, circle PE-positive cells and collect them in a 5ml flow cytometer. After sorting, centrifuge at 300g for 5 minutes, discard the supernatant, resuspend the cells in prewarmed culture medium, and incubate at 37°C, 5% CO2 in a cell culture incubator.

[0238] The sorted CAR-NK92 cells were stained with anti-FLAG-APC antibody, and the expression of CAR was detected by flow cytometry.

[0239] The results of flow cytometry are shown in FIG10 . The CAR positivity rate of each CAR-NK92 cell after sorting reached more than 98%.

[0240] Example 9: CAR-NK killing experiment

[0241] Luciferase-labeled tumor target cells are used to test their killing ability. By transferring the luciferase gene into target cells, stably expressing Molm13, HL60, THP-1, and KG1 cell lines were generated. During the experiment, the addition of a luciferin substrate causes the luciferase to react with the luciferin to produce fluorescence. The intensity of this fluorescence determines the luciferase activity, and the cell survival rate determines the killing effect of each effector cell.

[0242] The results are shown in Figure 11. All CAR-NK92s showed more obvious killing than NK92 on each target cell, suggesting a CAR-mediated killing effect.

[0243] Example 10: CAR-NK multi-round killing experiment

[0244] One day before the experiment, THP1 cells with fluorescent target were resuspended and counted, and the cell density was adjusted to 8×10 4 / ml, 100ul per well of a 96-well plate. On the day of the experiment, take the corresponding number of NK92 and CAR-NK92 cells and add them to the wells. Place them in Cellcyte to start the experiment, and take pictures every 4 hours using bright field and fluorescence channels. Every two days is a round. After a round of killing, remove part of the supernatant, transfer it to a new plate with target cells, and place it in Cellcyte to start a new round of killing. The killing index is calculated by adjusting the zero-crossing time and the normal cell growth error, and then the reduction in the number of fluorescent cells is used to calculate the killing.

[0245] The results of multiple rounds of killing experiments are shown in Figure 12. Each CAR-NK92 cell showed a stronger killing ability than NK92 cells, indicating that CAR-mediated killing is continuing to play a role.

[0246] Example 11: Fast Dual-CAR T cell preparation

[0247] Peripheral blood was collected from healthy donors and PBMCs were isolated by density gradient centrifugation at 500-600 g for 20-30 minutes. Magnetic beads conjugated to CD28 and CD3 antibodies (CD3 / CD28 Dynabeads) were used to sort and enrich T cells. T cells bound to CD3 / CD28 Dynabeads were further incubated with 300 IU / mL IL-2 to activate the cells. Simultaneously, 0.1-10 × 10 6Cells are transfected with the Dual-CAR virus overnight at a density of 10 cells / mL. The cells are washed with saline buffer the next day and then directly frozen. FAST Dual-CAR T cells are obtained without further expansion. During this process, the cells become activated and are also referred to as F Dual-CAR T cells.

[0248] Example 12: Fast Dual-CAR T cell phenotype

[0249] Because CD38 protein is present on the surface of T cells, expression of CD38CAR can lead to fratricide, resulting in the elimination of CD38-expressing T cells. To analyze whether Dual-CAR T cells produced by the FAST process would experience severe fratricide, CAR positivity was tested after recovery of F Dual-CAR T cells, while also monitoring cell viability, proliferation, and cell surface CD38 expression.

[0250] After thawing, F Dual-CAR T cells were cultured at 37°C in the presence of 300 IU / mL IL-2 at a concentration of 1×10 6 The cells were cultured at a density of 10 cells / mL. The cells were counted on D2, D3, D5, and D8 after recovery, and 1×10 5 The proportion of Dual-CAR positive cells was detected using CLL1 and CD38 antigens in each cell sample, and the expression of CD38 on the surface of T cells was analyzed using an antibody targeting CD38.

[0251] The data of F Dual-CAR T cells after recovery are shown in Figure 13. Figure 13A shows CD38 on the surface of T cells. + Percentage, F-C9B5 has CD38 similar to F-NT + Ratio, F-B8C9 in the early stage of recovery CD38 + The percentage of cells was low, but it recovered to a level similar to that of F-NT on the fifth day after recovery, while CD38 could not be detected on the cell surface of F-C7B3. + The CAR positive rate of F Dual-CAR T cells in each group gradually increased after recovery (Figure 13B). F-C9B5 had similar cell proliferation and viability to F-NT, while F-B8C9 and F-C7B3 had lower proliferation and viability (Figure 13C, B). + The percentage may be due to cannibalism, which leads to reduced cell viability and expansion times.

[0252] On the third day after F Dual-CAR T recovery, CD45RO and CCR7 were used to analyze the differentiation phenotype of F Dual-CAR T and C Dual-CAR T (traditional production process). Stem memory-like T cells (Tscm) were defined as CD45RO - CCR7 + Central memory T cells (Tcm) are defined as CD45RO + CCR7 + , effector memory T cells (Tem) are defined as CD45RO + CCR7 － Terminally differentiated effector T cells (Teff) are defined as CD45RO － CCR7 － As shown in FIG14 , F-C9B5 cells have a large proportion of young Tscm and Tcm phenotypes, while C-C9B5 cells are mostly in the late differentiation stage of Tem and Teff phenotypes.

[0253] Example 13: In vitro killing experiment of Fast Dual-CAR T cells and C Dual-CAR T cells

[0254] On the first day, Fast Dual-CAR T cells were revived and incubated at 37°C at a concentration of 1×10 6 C-C9B5 cells were cultured at a cell density of 1 × 106 cells / mL and 300 IU / mL IL2. On the second day, C Dual-CAR T cells were revived and incubated at 37°C at a cell density of 1 × 106 cells / mL and 300 IU / mL IL2. On the third day, they were co-cultured with K562, HL60, Molm13, and THP1 cells expressing firefly luciferase, and the killing effect was measured after 4 and 24 hours of co-culture. As shown in Figure 15, F-C9B5 and C-C9B5 had similar killing effects on target cells.

[0255] Example 14: Multiple rounds of killing by F Dual-CAR T cells and C Dual-CAR T cells in vitro

[0256] On the first day, F CAR T cells were revived and incubated at 37°C at a concentration of 1 × 10 6 The cells were cultured at a density of 1×10 cells / mL and 300 IU / mL IL2. The C CAR T cells were revived on the next day and incubated at 37°C at a density of 1×10 6 The cells were cultured at a density of 10 cells / mL and 300 IU / mL IL2. On the third day, 2 × 105 CAR-positive F Dual-CAR T cells and C Dual-CAR T cells were respectively mixed with 1×10 6 HL60, Molm13 and THP1 cells (E:T ratio was 1:5) were co-cultured for multiple rounds of killing analysis. On the third day of each round, 1 / 6 of the co-cultured cells were removed and directly added with 1×10 6 The corresponding target cells were used for the next round of killing experiments. At the same time, some co-cultured cells were taken out for counting and the residual target cell ratio and CAR were analyzed by flow cytometry. + The cell ratio can be combined with the counting results to calculate the cumulative expansion times of CAR T cells.

[0257] The results of multiple rounds of killing are shown in FIG16 . Regardless of the target cells, F-C9B5 can achieve a more sustained in vitro killing ability of target cells ( FIG16B ) and a greater expansion multiple ( FIG16A ) than C-C9B5.

[0258] Example 15: Comparison of the in vivo efficacy of F Dual-CAR T cells and C Dual-CAR T cells in mice

[0259] 4-6 week old NOG-dKO mice were selected and injected with 2×10 6 Molm13-CLL1-LucR cells. Three days later, tumor burden was assessed by small animal live imaging. The mice were divided into groups and injected with F-C9B5 cells and C-C9B5 cells on the same day. After T cell treatment, tumor burden in the mice was assessed by small animal live imaging twice a week.

[0260] The results, as shown in Figure 17, show that tumor growth in mice injected with CAR T cells was suppressed compared to the NT control group. F-C9B5 cells exhibited dose-dependent efficacy and achieved a more sustained tumor suppression effect than C-C9B5 at a lower dose. Infusion of CAR T cells in each group did not significantly affect the body weight of the mice.

[0261] At this point, those skilled in the art will recognize that, although a number of exemplary embodiments of the present invention have been shown and described in detail herein, many other variations or modifications consistent with the principles of the present invention may be directly determined or derived from the disclosure of the present invention without departing from the spirit and scope of the present invention. Therefore, the scope of the present invention should be understood and deemed to cover all such other variations or modifications.

Claims

1. A bispecific chimeric antigen receptor targeting CD38 and CLL1, wherein the chimeric antigen receptor fusion protein comprises from the N-terminus to the C-terminus at least: i) antigen binding domain that specifically recognizes CLL1 and CD38; ii) transmembrane domain; iii) at least one co-stimulatory domain; iv) Signal transduction domain.

2. The chimeric antigen receptor of claim 1, wherein the antigen binding domain is monovalent or multivalent.

3. The chimeric antigen receptor of claim 2, wherein the antigenic domain comprises a single domain antibody.

4. The chimeric antigen receptor according to claim 3, wherein the single domain antibody has been humanized.

5. The chimeric antigen receptor of claim 4, wherein the antigen binding domain comprises two single domain antibodies VHH1 and VHH2, wherein VHH1 represents a single domain antibody of a first antigen binding domain, and VHH2 represents a single domain antibody of a second antigen binding domain; The first antigen binding domain targets CD38, and the second antigen binding domain targets CLL1; The first antigen-binding domain and the second antigen-binding domain are arranged from the amino terminus to the carboxyl terminus in a pattern selected from one of the following groups: i) VHH1-VHH2; or ii) VHH2-VHH1.

6. The chimeric antigen receptor of claim 5, wherein the amino acid sequence of CDR1 of VHH1 is as shown in SEQ ID NO:6; the amino acid sequence of CDR2 of VHH1 is as shown in SEQ ID NO:8; and the amino acid sequence of CDR3 of VHH1 is as shown in SEQ ID NO:

11.

7. The chimeric antigen receptor of claim 5, wherein the amino acid sequence of CDR1 of VHH1 is as shown in SEQ ID NO:7; the amino acid sequence of CDR2 of VHH1 is as shown in SEQ ID NO:9; and the amino acid sequence of CDR3 of VHH1 is as shown in SEQ ID NO:

12.

8. The chimeric antigen receptor of claim 5, wherein the amino acid sequence of CDR1 of VHH1 is as shown in SEQ ID NO:7; the amino acid sequence of CDR2 of VHH1 is as shown in SEQ ID NO:10; and the amino acid sequence of CDR3 of VHH1 is as shown in SEQ ID NO:

13.

9. The chimeric antigen receptor according to any one of claims 6 to 8, wherein the VHH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 1-3.

10. The chimeric antigen receptor of claim 5, wherein the CDR1 amino acid sequence of the VHH2 is shown in SEQ ID NO: 14; the CDR2 amino acid sequence of the VHH2 is shown in SEQ ID NO: 15; and the CDR3 amino acid sequence of the VHH2 is shown in SEQ ID NO:

17.

11. The chimeric antigen receptor of claim 5, wherein the CDR1 amino acid sequence of the VHH2 is shown as SEQ ID NO: 14; the CDR2 amino acid sequence of the VHH2 is shown as SEQ ID NO: 16; and the CDR3 amino acid sequence of the VHH2 is shown as SEQ ID NO:

18.

12. The chimeric antigen receptor according to claim 10 or 11, wherein the VHH2 comprises or consists of the amino acid sequences shown in SEQ ID NO: 4 and SEQ ID NO:

5. The chimeric antigen receptor according to claim 1 , wherein the transmembrane domain is CD8α or CD28.

14. The chimeric antigen receptor according to claim 13, wherein the transmembrane domain of CD8α comprises or consists of the amino acid sequence shown in SEQ ID NO: 19; and the transmembrane domain of CD28 comprises or consists of the amino acid sequence shown in SEQ ID NO:

20.

15. The chimeric antigen receptor of claim 1, wherein the intracellular signaling domain comprises a molecule selected from the group consisting of CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, FcεRI, DAP10, DAP12, and CD66d, or a combination thereof.

16. The chimeric antigen receptor according to claim 15, wherein the intracellular signaling domain is preferably a cytoplasmic signaling sequence of CD3ζ.

17. The chimeric antigen receptor according to claim 16, wherein the cytoplasmic signaling sequence of CD3ζ comprises or consists of the amino acid sequence shown in SEQ ID NO:

21.

18. The chimeric antigen receptor according to claim 16, wherein the cytoplasmic signaling sequence of CD3ζ further comprises its wild type, or a mutant / modified form thereof.

19. The chimeric antigen receptor of claim 18, wherein the co-stimulatory signaling domain comprises a molecule selected from the group consisting of 4-1BB (CD137), CD27, CD19, CD4, CD28, ICOS (CD278), CD82β, BAFFR, HVEM, LIGHT, KIRDS2, SLAMF7, NKp30, NKp46, CD40, ICAM-1, B7-H3, OX40, DR3, GITR, CD30, TIM1, CD2, CD7, and CD226, or a combination thereof.

20. The chimeric antigen receptor of claim 19, wherein the co-stimulatory signaling domain is preferably 4-1BB or CD28.

21. The chimeric antigen receptor of claim 20, wherein the costimulatory signal structure of 4-1BB comprises or consists of the amino acid sequence shown in SEQ ID NO: 22; and the costimulatory signal structure of CD28 comprises or consists of the amino acid sequence shown in SEQ ID NO:

23.

22. The chimeric antigen receptor of claim 21, further comprising a hinge region.

23. The chimeric antigen receptor according to claim 22, wherein the hinge region is preferably CD8α.

24. The chimeric antigen receptor of claim 23, wherein the hinge region of CD8α has the amino acid sequence shown in SEQ ID NO:

24.

25. The chimeric antigen receptor of claim 24, further comprising a signal peptide; The signal peptide is selected from the following molecules: one or a combination of the α chain and β chain of the T cell receptor, CD3ζ, CD3ε, CD4, CD5, CD8, CD9, CD28, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, GITR, and GM-CSF. The chimeric antigen receptor according to claim 25 , wherein the signal peptide is preferably CD8α.

27. The chimeric antigen receptor according to claim 26, wherein the signal peptide of CD8α comprises or consists of the amino acid sequence shown in SEQ ID NO:

25.

28. The chimeric antigen receptor according to claim 27, wherein the specific structure is: L-VHH1-L1-VHH2-H-TM-C-CD3ζ Each "-" is independently a connecting peptide or a peptide bond; L is the CD8a signal peptide molecule; VHH1 is an antigen binding domain that specifically binds to CD38 or CLL1; VHH2 is an antigen binding domain that specifically binds to CLL1 or CD38; L1 is a connecting peptide; H is the hinge region of CD8a; TM is the transmembrane domain of CD8a; C is the 4-1BB co-stimulatory domain; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

29. The chimeric antigen receptor according to claim 27, wherein the specific structure is: L-VHH1-L1-VHH2-FH-TM-C-CD3ζ Each "-" is independently a connecting peptide or a peptide bond; L is the CD8a signal peptide molecule; VHH1 is an antigen binding domain that specifically binds to CD38 or CLL1; VHH2 is an antigen binding domain that specifically binds to CLL1 or CD38; L1 is a connecting peptide; F is the Flag tag sequence (Flag Tag); H is the hinge region of CD8a; TM is the transmembrane domain of CD28; C is the costimulatory domain of CD28; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

30. The chimeric antigen receptor of any one of claims 28-29, wherein the connecting peptide is selected from a linker of the following amino acid sequence: SGG, GGS, SGGS, SSGGS, GGGG, SGGGG, GGGGS, SGGGGGS, SGGGGG, GSGGGGGS, GGGGGGS, SGGGGGG, GSGGGGGGS, GGGGGGG, SGGGGGGGGS, SGGGGGGGGS, SGGGGSGGGGGGS or GGGGSGGGGSGGGGS.

31. The chimeric antigen receptor according to claim 30, wherein the connecting peptide is preferably GGGGSGGGGSGGGGS, as represented by the amino acid sequence of SEQ ID NO:

26.

32. The chimeric antigen receptor of claim 29, wherein the fusion protein expression tag Flag tag is represented by the amino acid sequence of SEQ ID NO:

27.

33. A nucleic acid molecule, which is a nucleotide sequence encoding the chimeric antigen receptor according to any one of claims 6 to 12.

34. The nucleic acid molecule according to claim 33, preferably a nucleic acid molecule C9B5 encoding the amino acids shown in SEQ ID NO:2 and SEQ ID NO:5, wherein the nucleotide sequence of C9B5 is shown in SEQ ID NO:

30.

35. The nucleic acid molecule of claim 33, further comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 28-29, or 31-51.

36. A recombinant vector comprising the chimeric antigen receptor according to any one of claims 6 to 12, or the nucleic acid molecule according to any one of claims 33 to 35.

37. The recombinant vector according to claim 36, wherein The recombinant vector includes a DNA vector, an RNA vector, a plasmid, a transposon vector, a liposome, a CRISPR / Cas9 vector or a viral vector.

38. The recombinant vector of claim 37, wherein the viral vector comprises a lentiviral vector, an adenoviral vector, or a retroviral vector.

39. An engineered immune cell comprising the chimeric antigen receptor of any one of claims 6-12, or the nucleic acid molecule of any one of claims 33-35, or the recombinant expression vector of any one of claims 36-38.

40. The engineered immune cell of claim 39, wherein the immune cell is prepared using the FAST-CAR process.

41. An engineered immune cell as described in claim 40, wherein the FAST CAR converts the activation, transduction and expansion steps into a single "synchronous activation-transduction" step, wherein the engineered immune cell undergoes ex vivo expansion for less than 72 hours.

42. The engineered immune cell as described in claim 41 can reduce the effects of fratricide caused by CD38 CAR recognizing CD38 antigen on the surface of immune cells during the production process, or the effects of fratricide caused by other CAR recognizing corresponding target antigens on the surface of immune cells, such as T cell apoptosis caused by CD70 CAR recognizing CD70 antigen on the surface of T cells; The immune cells can be applied to any CAR targeting antigen and different tumor markers.

43. The engineered immune cell of claim 41, which has a younger cellular phenotype than cells in a comparable population that have undergone ex vivo expansion for one week or more.

44. The engineered immune cell of claim 43, wherein the youthfulness is characterized by T cells in the immune cell n / scm (CD45RO - , CCR7 + ) and Tcm(CD45RO + , CCR7 + ) accounts for a higher proportion.

45. The engineered immune cells of claim 41 are observed to have greater in vitro expansion and sustained killing capabilities than cells in a comparable population that have undergone ex vivo expansion for one or more weeks.

46. ​​The engineered immune cell of claim 39, comprising a T cell, a NK cell, an iNKT cell, a CTL cell, a monocyte, a macrophage, a dendritic cell and / or a NKT cell.

47. The engineered immune cells of claim 46, wherein the immune cells are preferably T cells and NK cells.

48. Use of the chimeric antigen receptor according to any one of claims 6 to 12, the nucleic acid molecule according to any one of claims 33 to 35, the recombinant expression vector according to any one of claims 36 to 38, or the engineered immune cell according to any one of claims 39 to 47 in the preparation of a drug for treating a tumor.

49. The use according to claim 48, wherein the tumor is a tumor expressing CD38 and / or CLL1.

50. The use according to claim 49, wherein the tumor is acute myeloid leukemia.

51. The use according to claim 48, wherein the medicine further comprises a pharmaceutically acceptable carrier and adjuvant.

52. Use of the chimeric antigen receptor according to any one of claims 6 to 12 in combination with one or more of the following in the preparation of a pharmaceutical combination for treating and / or preventing cancer: (1) an agent that increases the efficacy of cells containing a CAR nucleic acid or a CAR polypeptide; (2) an agent that ameliorates one or more side effects associated with administration of cells containing a CAR nucleic acid or a CAR polypeptide; (3) Additional agents for treating diseases associated with CD38 and CLL1.

53. The use as claimed in claim 52, wherein the treatment and / or prevention further comprises use in combination with a second therapy, wherein the second therapy is selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, virotherapy, adjuvant therapy and any combination thereof.

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