Bispecific chimeric antigen receptor targeting CD38 and CLL1 and application thereof

Abstract

The invention belongs to the field of immune cell therapy, and particularly relates to a CD38 and CLL1 targeted bispecific chimeric antigen receptor, and the chimeric antigen receptor fusion protein at least comprises: i) an antigen binding domain for specifically recognizing CLL1 and CD38 from an N terminal to a C terminal; ii) a transmembrane domain; iii) at least one co-stimulatory domain; iv) a signal transduction domain; the dual-targeting CAR T cell prepared by using the chimeric antigen receptor shows high-specificity in-vitro cytotoxicity to CLL1 positive and / or CD38 positive target cells.

Description

Technical Field

[0001] The present invention belongs to the field of immunocyte therapy, and specifically, relates to a bispecific chimeric antigen receptor targeting CD38 and CLL1 and its application. Background Art

[0002] Acute myeloid leukemia (AML) is a cancer of myeloid blood cells, 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, greatly increasing the difficulty of AML treatment. Commonly used treatments for AML include chemotherapy and hematopoietic stem cell transplantation. However, since AML is most common in the elderly, most patients cannot tolerate intensive chemotherapy, and at the same time, the donor sources for stem cell transplantation are few. Therefore, the survival rate of patients is extremely poor and the recurrence rate is high. There is a need to seek new treatment means for treatment.

[0003] A chimeric antigen receptor (CAR) is an artificial receptor that mimics the function of the T cell receptor, which fuses the antigen recognition fragment (or antigen corresponding ligand) of an antibody and 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 the activation, proliferation, and subsequent tumor killing of CAR-T cells.

[0004] The CD38 antigen is a type II transmembrane glycoprotein with a size of 45 kDa, which can catalyze the synthesis and degradation of cyclic adenosine diphosphate ribose. CD38 is also related to the regulation of apoptosis and cell proliferation and has important adhesion properties. The high expression of CD38 in most AML blasts makes it a potential target for AML treatment.

[0005] C-type lectin-like-1 (CLL1) is a glycoprotein receptor and a member of the large family of C-type lectin-like receptors involved in immune regulation. CLL1 is mainly expressed in hematopoietic cells, mainly in innate immune cells including monocytes, DCs, and granulocytes and bone marrow progenitor cells. CLL-1 is also found in acute myeloid leukemia (AML) blasts, leukemia stem cells (such as 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] Due to the lack of suitable target surface antigens, CAR-T cells have not been able to make a breakthrough in the treatment of AML for a long time. Because the targeted AML antigens are often co-expressed on healthy hematopoietic stem / progenitor cells (HSPCs), resulting in the loss of all myeloid progeny cells. Creative solutions are being sought to overcome these obstacles, making CART therapy a viable option for AML patients. Summary of the Invention

[0007] 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. The chimeric antigen receptor fusion protein comprises at least, from the N-terminus to the C-terminus:

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

[0010] ii) A transmembrane domain;

[0011] iii) At least one co-stimulatory domain;

[0012] iv) A signal transduction domain.

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

[0014] Preferably, the above antigen domain includes single-domain antibodies; others that can also be selected include camel Ig, IgNAR, Fab fragments, Fab' fragments, F(ab')z fragments, F(ab')3 fragments, Fv, single-chain antibodies (such as scFv, di-scFv, (scFv)z), minibodies, bispecific antibodies, trispecific antibodies, tetravalent antibodies, disulfide-stabilized Fv proteins ("dsFv"), chimeric antibodies, humanized antibodies, single-domain antibodies, bispecific antibodies or multispecific antibodies, binding ligands or protein domains.

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

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

[0017] i) VHH1-VHH2; or

[0018] ii) VHH2 - VHH1.

[0019] Preferably, the single - domain antibody in this application is a heavy - chain single - domain antibody.

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

[0021] As another preferred embodiment, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH1 further include amino acid sequences having 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%, 99% identity with SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 11 respectively; or have one or several amino acid substitutions, deletions, or additions (such as 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 this application, the amino acid sequence of CDR1 of VHH1 is as shown in SEQ ID NO: 7; the CDR2 of VHH1 includes an amino acid sequence as shown in SEQ ID NO: 9; the amino acid sequence of CDR3 of VHH1 is as shown in SEQ ID NO: 12.

[0023] As another preferred embodiment, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH1 further include amino acid sequences having 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%, 99% identity with SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO: 12 respectively; or have one or several amino acid substitutions, deletions, or additions (such as 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.

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

[0025] As another preferred embodiment, the amino acid sequences of CDR1, CDR2, and CDR3 of VHH1 further include amino acid sequences having 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%, 99% identity with SEQ ID NO:7, SEQ ID NO:10, and SEQ ID NO:13 respectively; or having one or several amino acid substitutions, deletions or additions (such as 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared with the sequence from which it is derived. Preferably, the substitution is a conservative substitution.

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

[0027] As another preferred embodiment, the amino acid sequence of VHH1 further includes amino acid sequences having 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%, 99% identity with SEQ ID NO:1-3 respectively; or having one or several amino acid substitutions, deletions or additions (such as 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared with the sequence from which it is derived. Preferably, the substitution is a conservative substitution.

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

[0029] As another preferred embodiment, the amino acid sequences of CDR1, CDR2, and CDR3 of the VHH2 further include amino acid sequences having 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%, 99% identity to SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 17, respectively; or having one or several amino acid substitutions, deletions, or additions (such as 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived, and preferably, the substitution is a conservative substitution.

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

[0031] As another preferred embodiment, the amino acid sequences of CDR1, CDR2, and CDR3 of the VHH2 further include amino acid sequences having 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%, 99% identity to SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 18, respectively; or having one or several amino acid substitutions, deletions, or additions (such as 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived, and preferably, the substitution is a conservative substitution.

[0032] Preferably, the VHH2 includes 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 the VHH2 further includes an amino acid sequence having 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%, 99% identity to SEQ ID NO: 4 or 5; or having one or several amino acid substitutions, deletions, or additions (such as 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions) compared to the sequence from which it is derived, and preferably, the substitution is a conservative substitution.

[0034] Preferably, the transmembrane domain includes a molecule selected from the following: CD8α, CD28, IgG1, IgG4, 4-1BB, PD-1, CD34, OX40, CD3ε, IL-2 receptor, IL-7 receptor.

[0035] More preferably, the transmembrane domain is selected as CD8α or CD28. The transmembrane domain of CD8α includes the amino acid sequence shown in SEQ ID NO: 19 or consists thereof; the transmembrane domain of CD28 includes the amino acid sequence shown in SEQ ID NO: 20 or consists thereof.

[0036] As another preferred embodiment, the amino acid sequence of the transmembrane domain further includes amino acid sequences having 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%, 99% identity with SEQ ID NO: 19 or 20 respectively; or having one or several amino acid substitutions, deletions or additions (such as 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared with the sequence from which it is derived. Preferably, the substitution is a conservative substitution.

[0037] Preferably, the intracellular signaling domain includes 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. The cytoplasmic signaling sequence of CD3ζ 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 further includes an amino acid sequence having 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%, 99% identity to SEQ ID NO: 21; or having one or several amino acid substitutions, deletions or additions (such as 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, preferably, the substitution is a conservative substitution.

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

[0041] Preferably, the above co-stimulatory signal domain includes molecules selected from the following: 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, or a combination thereof.

[0042] More preferably, the co-stimulatory signal domain is preferably 4-1BB or CD28. The co-stimulatory signal structure of 4-1BB includes the amino acid sequence shown in SEQ ID NO: 22, or is composed of it; the co-stimulatory signal structure of CD28 includes the amino acid sequence shown in SEQ ID NO: 23, or is composed of it.

[0043] As another preferred embodiment, the amino acid sequence of the co-stimulatory signal structure further includes an amino acid sequence having 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%, 99% identity to SEQ ID NO: 22 or 23 respectively; or having one or several amino acid substitutions, deletions or additions (such as 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived, preferably, the substitution is a conservative substitution.

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

[0045] Preferably, the hinge region is preferably CD8α. 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 further includes an amino acid sequence having 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%, 99% identity to SEQ ID NO: 24; or has one or several amino acid substitutions, deletions or additions (such as 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived. Preferably, the substitution is a conservative substitution.

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

[0048] Preferably, the signal peptide includes a molecule selected from the following: 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, GM-CSF.

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

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

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

[0052] In the formula,

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

[0054] L is a 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 linker peptide;

[0058] F is either absent or a Flag Tag sequence;

[0059] H is the hinge region;

[0060] TM is the transmembrane domain;

[0061] C is the co-stimulatory domain;

[0062] CD3ζ is the cytoplasmic signaling sequence derived from CD3ζ, including its wild-type, mutants, or modified forms.

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

[0064] L-VHH1-L1-VHH2-H-TM-C-CD3ζ

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

[0066] L is the CD8a signal peptide molecule;

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

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

[0069] L1 is a linker peptide;

[0070] H is the hinge region of CD8a;

[0071] TM is the transmembrane domain of CD8a;

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

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

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

[0075] L-VHH1-L1-VHH2-F-H-TM-C-CD3ζ

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

[0077] L is the CD8a signal peptide molecule;

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

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

[0080] L1 is a linker 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 co-stimulatory domain of CD28;

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

[0086] Preferably, the above-mentioned linker peptide is selected from linkers with the following amino acid sequences: SGG, GGS, SGGS, SSGGS, GGGG, SGGGG, GGGGS, SGGGGS, GGGGGG, SGGGGGS, SGGGGG, GSGGGGS, GGGGGGGGS, SGGGGGGG, SGGGGGGGS, SGGGGSGGGGS or GGGGSGGGGSGGGGS.

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

[0088] As another preferred embodiment, the amino acid sequence of the Flag tag further includes an amino acid sequence having 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%, 99% identity with SEQ ID NO: 27; or having one or several amino acid substitutions, deletions or additions (such as 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared with the sequence from which it is derived. Preferably, the substitution is a conservative substitution.

[0089] The second aspect of the present invention discloses a nucleic acid molecule, which is a nucleotide sequence encoding the above-mentioned 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, and the nucleotide sequence of C9B5 is shown by SEQ ID NO: 30.

[0091] The nucleic acid molecule further includes the nucleotide sequence shown in any one of SEQ ID NO: 28-29, or 31-51.

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

[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, a retroviral vector.

[0095] The fourth aspect of the present invention discloses engineered immune cells, which include and / or express the chimeric antigen receptor described above, the nucleic acid molecule, or the recombinant expression vector described above.

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

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

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

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

[0100] As another preferred embodiment, compared with the cells in a comparable population that have undergone ex vivo amplification for 1 week or more weeks, the engineered immune cells prepared by the FAST-CAR process are observed to have stronger ex vivo amplification and continuous killing ability.

[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 preferably be T cells and NK cells.

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

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

[0105] Preferably, the drug composition further includes a pharmaceutically acceptable carrier and adjuvant.

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

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

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

[0109] (3) An additional agent for treating diseases associated with CD38 and CLL1.

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

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

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

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

[0114] (3) The dual-target CAR T cells produced by the FAST process of the present invention have a younger phenotype than those produced by the traditional process and show more persistent tumor-killing ability and amplification ability in in vitro experiments;

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

[0116] Figure 1 It is a schematic diagram of the surface antigen expression of the target cells described in Example 1;

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

[0118] Figure 3 It is the positive rate of CAR of the Dual-CAR T cells detected by antigen in Example 3;

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

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

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

[0122] Figure 7 It is a schematic diagram of the multiple-round killing results of Dual-CAR T on HL60 cells in Example 5;

[0123] Figure 8 It is a schematic diagram of the multiple-round killing results of Dual-CAR T on KG1 cells in Example 5;

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

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

[0126] Figure 11 It is a schematic diagram of the killing results of CAR-NK92 on tumor cells in Example 9;

[0127] Figure 12 It is a schematic diagram of the multiple-round killing results of CAR-NK92 in Example 10;

[0128] Figure 13Schematic diagram of the changes in CD38 expression, CAR expression, expansion, and viability after the resuscitation of F Dual-CAR T in Example 12;

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

[0130] Figure 15 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 Schematic diagram of the in vitro multi-round killing results of F Dual-CAR T and C Dual-CAR T cells in Example 14;

[0132] Figure 17 Schematic diagram of the in vivo pharmacodynamic experiment of F Dual-CAR T and C Dual-CAR T cells in Example 15. Detailed implementation manners

[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 do not 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, easily obtained, or prepared in the market.

[0135] As used herein, the term "antigen" refers to a molecule or a 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 immunoprotein (e.g., an antibody). An antigen can also refer to a molecule or a fragment thereof that can be used in an animal to generate an antibody that can bind to the antigen. In some cases, an antigen can bind to a substrate (e.g., a cell membrane). Alternatively, an antigen may not bind to a substrate (e.g., a secreted molecule, such as a secreted polypeptide).

[0136] The term "antibody" (Ab) shall include, without limitation, immunoglobulins that specifically bind 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 L 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), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged in the following order from the amino terminus to the 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 in this text, amino acid names are identified by a single English letter in the international common usage, and their corresponding three-letter abbreviations of 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), Val (V).

[0138] The term "complementarity-determining region" or "CDR" refers to the hypervariable regions of the heavy and light chains of an immunoglobulin, 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 editions). This application uses the Kabat numbering system to define CDRs. There are three heavy chain CDRs and three light chain CDRs. Here, depending on the context, the terms "CDR" and "CDRs" are used to refer to regions that contain one or more or even all of the major amino acid residues that contribute to the binding affinity of an antibody for the antigen or epitope it recognizes. In another specific embodiment, the CDR region or CDR refers to the hypervariable regions of the heavy and light chains of an immunoglobulin as defined by IMGT.

[0139] Preparation of Antibodies

[0140] The antibodies of the present invention can be prepared by various methods known in the art, for example, obtained by genetic engineering recombinant techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention are obtained by chemical synthesis or PCR amplification. The obtained DNA molecules are inserted into an expression vector and then transfected into host cells. Then, the transfected host cells are 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 hydrolysis of the intact antibody molecule (see Morimoto et al., J. Biochem. Biophys. Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81 (1985)). Additionally, these antigen-binding fragments can also be directly produced 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 directly obtained from host cells; Fab' fragments can be chemically conjugated to form F(ab')2 fragments (Carter et al., Bio / Technology, 10: 163-167 (1992)). Additionally, Fv, Fab or F(ab')2 fragments can also be directly isolated from the culture broth of recombinant host cells. Those of ordinary skill in the art are fully aware of other techniques for preparing these antigen-binding fragments.

[0142] Conservative substitution

[0143] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the expected properties of a protein / polypeptide containing 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 an amino acid residue with another amino acid residue having a similar side chain, e.g., a substitution with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., having a similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having 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), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to substitute 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 comprises an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain comprises a target-specific binding element (also referred to as an antigen-binding domain) and a hinge region. The intracellular domain comprises a co-stimulatory signaling region and a CD3ζ chain portion.

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

[0147] For the hinge region and the transmembrane region (transmembrane domain), the CAR can be designed to comprise a transmembrane domain fused to the extracellular domain of the CAR. In some examples, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding such a domain to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0148] Between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR, a linker can be incorporated. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that serves to link the transmembrane domain to the extracellular or cytoplasmic domain of a polypeptide chain. The linker can comprise from 0 to 300 amino acids, preferably from 2 to 100 amino acids and most preferably from 3 to 50 amino acids.

[0149] The co-stimulatory signaling region refers to a part of the intracellular domain that includes the co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules required for the effective response of lymphocytes 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 with the hinge region and transmembrane region, and the intracellular domain from one or more of the co-stimulatory molecule and the CD3ζ chain.

[0151] In another preferred example, the antigen-binding domain is fused with the intracellular domain combined with the CD8a hinge region and CD8a transmembrane region, the 4-1BB signaling domain and the CD3ζ signaling domain.

[0152] In another preferred example, the antigen-binding domain is fused with the intracellular domain combined with the CD8a hinge region and CD28 transmembrane region, the CD28 signaling domain and the CD3ζ signaling domain.

[0153] Bispecific CAR targeting CD38 and CLL1

[0154] Bispecific means that the same CAR can specifically bind and immunologically recognize two different antigens, and an immune response can be generated when the CAR binds to any one of the antigens.

[0155] In another preferred example, 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 example, the present invention provides a bispecific chimeric antigen receptor against CD38 and CLL1 antigens. The CAR structural components targeting CD38 and CLL1 simultaneously may include a signal peptide, a single-domain antibody against CD38, a single-domain antibody against CLL1, a hinge region, a transmembrane region, and an intracellular T cell signaling region.

[0158] Bispecific CAR-T has a wider treatment range. CAR-immune cells targeting CD38 and CLL1 simultaneously can reduce the possibility of antigen escape caused by downregulation or deletion of a single surface antigen. In addition, the bispecific chimeric antigen receptor targeting the CD38 and CLL1 target combination shows high-specific in vitro cytotoxicity.

[0159] Natural killer cell (NK cell)

[0160] NK cells are important immune cells in the body. Morphologically, NK cells belong to large granular lymphocytes, originating from the bone marrow. They are the third major type of lymphocytes besides 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, mainly distributed in peripheral blood, liver and spleen. In the human body, the main characteristic of NK cells is the CD3-CD56+ lymphocyte population. Among them, in the blood, it is mainly the CD16+CD56dim subtype (According to the difference in the expression density of CD56 molecules on the cells, NK cells are divided into two subsets: CD56dim and CD56bright; CD56dim accounts for more than 90% of NK cells, mainly for cytotoxic effects, expressing medium-affinity interleukin-2 receptor (IL-2R), and having stronger killing activity; CD56bright can produce a large number of cytokines, mainly playing an immunomodulatory role, and highly expressing IL-2R).

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

[0162] As used herein, the terms "CAR-T cells", "CAR-T", "CAR-T cells of the present invention". Chimeric antigen receptor T cells (CAR-T cells) are formed by coupling the antigen-binding portion of an antibody that can recognize a certain tumor antigen with the intracellular portion of CD3-ζ chain or FcεRIγ in vitro into a chimeric protein, and then transfecting the patient's T cells by gene transduction method to make them express 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) refers to genetically modifying NK cells with chimeric antigen receptor (CAR) to endow NK cells with the ability to target and recognize tumor cells, and then injecting them into the human body after in vitro expansion to achieve the effect of tumor treatment. Currently, there are mainly five sources of NK cells used clinically: human peripheral blood (PB), umbilical cord blood (UCB), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), and NK-92 cell line.

[0164] The NK-92 cell line is the most widely studied cell line in CAR-NK at present. It was isolated from a 50-year-old male patient with non-Hodgkin's lymphoma in 1992 and its growth is IL-2 dependent. Compared with primary NK cells, the greatest advantage of the NK-92 cell line is that the expression of inhibitory receptors (such as KIR) on its surface is very low. The absence of inhibitory receptor signals makes its killing ability against various tumors superior to that of primary NK cells or other killer cells activated by cytokines. In addition, NK-92 also has certain potential in the treatment of solid tumors. An important reason for the poor effect of CAR-T in solid tumors is that tumor cells highly express PD-L1, which binds to the inhibitory molecule PD-1 on the surface of T cells, thereby inhibiting their killing activity. The absence of inhibitory receptors on the surface of NK-92 enables it to avoid the interference of similar inhibitory signals. However, NK-92 also has some obvious disadvantages, such as potential tumorigenicity and EBV susceptibility, etc. Therefore, NK-92 must be irradiated before it can be used.

[0165] FAST-CAR platform

[0166] The "FAST process" described herein is an engineered immune cell preparation process.

[0167] In the traditional CAR-T production process, T cells of patients are first activated with CD3 and / or CD28 antibodies, and then transduced with viral vectors to express one or more CARs. These modified CAR-T cells are amplified in vitro before being injected back into the human body, and the overall production process usually takes 1 to 6 weeks.

[0168] The FasTCAR platform can use the XLenti vector from lentivirus with high quality and high gene transduction efficiency to simultaneously activate and transduce resting T cells. After transduction, one or more CARs are integrated into the T cell genome and stably expressed. Based on our preclinical studies, the transduced T cells have high amplification and tumor cell clearance activities, eliminating the need for the in vitro cell amplification step and can be directly formulated and used in patients. Based on such innovation, the FasTCAR technology can transform the activation, transduction, and amplification steps into a single "simultaneous 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 within 72 hours.

[0169] vector

[0170] Nucleic acid sequences encoding the desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening a library from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by directly isolating it from cells and tissues containing the gene using standard techniques. Optionally, the gene of interest can be produced synthetically.

[0171] The invention also provides vectors into which the expression cassette of the invention is inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncoviruses such as murine leukemia virus because they can transduce non-proliferating cells such as hepatocytes. They also have the advantage of low immunogenicity.

[0172] Briefly, generally the expression cassette or nucleic acid sequence of the invention is operably linked to a promoter and incorporated into an expression vector. The vector is suitable for replication and integration in eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

[0173] The expression constructs of the 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, 5,589,466, which are hereby incorporated by reference in their entirety. In another embodiment, the invention provides gene therapy vectors.

[0174] The nucleic acid can be encapsulated 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. Particular vectors of interest include expression vectors, replication vectors, probe production vectors, and sequencing vectors.

[0175] Furthermore, the expression vector can be provided to cells in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include but are not limited to retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, suitable vectors contain an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (e.g., WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0176] Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to target cells in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0177] Additional promoter elements, such as enhancers, can modulate the frequency of transcription initiation. Generally, these are located in the region 30 - 110 bp 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 promoter function is maintained when the elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by up to 50 bp before activity starts to decline. Depending on the promoter, individual elements can act cooperatively 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 capable of driving high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences can 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 leukosis virus promoter, 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 use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of inducible promoters provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or turn off the expression when it is not desired. Examples of inducible promoters include but are not limited to metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0179] To evaluate the expression of a CAR polypeptide or a portion thereof, the expression vector introduced into the cell may also contain either or both of a selectable marker gene and a reporter gene to facilitate the identification and selection of expressing cells from a population of cells sought to be transfected or infected by the viral vector. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.

[0180] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Typically, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property such as enzyme activity. After the DNA has been introduced into the recipient cell, the expression of the reporter gene is assayed at an appropriate time. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are known in the art and can be prepared using known techniques or obtained commercially. Generally, constructs having at least 5 flanking regions that display the highest level of reporter gene expression are identified as promoters. Such promoter regions can be ligated to a reporter gene and used to evaluate the ability of a reagent to modulate promoter-driven transcription.

[0181] Methods for introducing genes into cells and for expressing genes in cells are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell by any method in the art, such as mammalian, bacterial, yeast, or insect cells. For example, the expression vector can be transferred into the host cell 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 comprising a vector and / or exogenous nucleic acid are 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, e.g., human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. See, e.g., 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 an in vitro and in vivo delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0185] In the case of using non-viral delivery systems, an exemplary delivery vehicle is a liposome. Lipid formulations are contemplated for introducing nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid can be associated with lipids. Nucleic acids associated with lipids can be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes via a linking molecule associated with both the liposome and the oligonucleotide, entrapped within liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, included as a suspension within lipids, included within micelles or complexed with micelles, or otherwise associated with lipids. The lipids, lipid / DNA, or lipid / expression vector associated with the composition are not limited to any particular structure in solution. For example, they can be present in bilayer structures, as micelles, or have a "collapsed" structure. They can also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances, which can be naturally occurring or synthetic lipids. For example, lipids include lipid droplets that occur naturally in the cytoplasm and such compounds that contain long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0186] Pharmaceutical compositions

[0187] The isolated nucleic acid molecules, vectors, host cells, engineered immune cells or immune cell compositions of the present invention can be formulated into any dosage forms known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, plasters, lozenges, suppositories, injections (including injection solutions, 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 compositions of the present invention should be sterile and stable under the conditions of production and storage. A preferred dosage form is injection. Such injections can be sterile injection solutions. In addition, sterile injection solutions can be prepared as sterile lyophilized powders (for example, by vacuum drying or freeze-drying) for ease of 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 (for example, 0.9% (w / v) NaCl), glucose solution (for example, 5% glucose), solution containing surfactant (for example, 0.01% polysorbate 20), pH buffer solution (for example, phosphate buffer solution), Ringer's solution and any combination thereof.

[0188] The isolated nucleic acid molecules, vectors, host cells, engineered immune cells or immune cell compositions of the present invention can be administered by any suitable method known in the art, including but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracisternal, inguinal, intravesical, local (such as powders, ointments or drops), or nasal routes. However, for many therapeutic uses, the preferred route / mode of administration is parenteral administration (such as intravenous injection or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art should understand that the route and / or mode of administration will vary according to the intended purpose. In certain embodiments, the isolated nucleic acid molecules, nucleic acid constructs, vectors, host cells, engineered immune cells or immune cell compositions of the present invention are administered by intravenous injection or bolus injection.

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

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

[0191] Therapeutic applications

[0192] Accordingly, the present invention also provides a method of stimulating a T cell-mediated immune response against a target cell population or tissue in a mammal, which comprises the step of administering to the mammal the CAR-T cells of the present invention.

[0193] In one embodiment, the present invention includes a class of cell therapies in which autologous T cells of a patient (or NK cells from a heterologous donor source) are isolated, activated and genetically engineered to produce CAR-T and CAR-NK cells, which are then infused into the patient. This approach has a very low probability of graft-versus-host disease, and the antigen is recognized by T cells or NK cells in an MHC-independent manner. In addition, one type of CAR-T or CAR-NK can treat all cancers expressing the antigen. Unlike antibody therapies, CAR-T and CAR-NK cells can replicate in vivo, resulting in 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 persist for a prolonged period of time. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy procedure, wherein the antigen-binding domain in the CAR induces a specific immune response of the CAR-modified immune cells against antigen-expressing target cells. For example, CAR-T and CAR-NK cells against CD38 and / or CLL1 elicit a specific immune response against cells expressing CD38 and / or CLL1.

[0195] Although the data disclosed herein specifically disclose lentiviral vectors comprising a CD38 and / or CLL1 antigen recognition and binding domain, a hinge and transmembrane region, a 4-1BB or CD28 co-stimulatory domain, and a CD3ζ intracellular signaling domain, the invention is to be construed as encompassing any number of variations to each of the components of the construct.

[0196] The CAR-modified T cells of the invention can also be used as a type of vaccine 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 before the cells are administered into the mammal: i) expanding the cells, ii) introducing a nucleic acid encoding the CAR into the cells, and / or iii) cryopreserving the cells.

[0198] Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (preferably human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing the CAR disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient can be human, and the CAR-modified cells can be autologous with respect to the recipient. Optionally, the cells can be allogeneic, syngeneic, or xenogeneic with respect to the recipient.

[0199] In addition to using cell-based vaccines for ex vivo immunization, the invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

[0200] The invention provides a method of treating a tumor, which comprises administering to a subject in need thereof a therapeutically effective amount of the CAR-modified T cells of the invention.

[0201] The CAR-modified T cells of the 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 populations. Briefly, the pharmaceutical compositions of the invention can comprise a population of target cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can include buffers such as neutral buffered saline, sulfate buffered saline, and the like; 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 invention are preferably formulated for intravenous administration.

[0202] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease - although appropriate doses 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 all cultured in RPMI 1640 medium; 293T and wild-type, and Hela cells expressing CD38, expressing CLL1, and simultaneously expressing CLL1 and CD38 (Hela-WT, Hela-CD38, Hela-CLL1, Hela-CLL1-CD38) were cultured in DMEM medium. All of the above media were supplemented with 10% (v / v) fetal bovine serum and 100 U / ml penicillin and streptomycin, 2 mM L-glutamine, and 1 mM sodium pyruvate. The cells were cultured at 37 °C, 5% CO 2 and saturated humidity.

[0205] Among them, the Hela cells expressing CD38 were a stable transfected cell line obtained by transferring the CD38 antigen into Hela cells through a lentiviral vector, and could specifically express the CD38 protein molecule; the Hela cells expressing CLL1 were a stable transfected cell line obtained by transferring the CLL1 antigen into Hela cells through a lentiviral vector, and could specifically express the CLL1 protein molecule; the Hela cells simultaneously expressing CLL1 and CD38 were a stable transfected cell line obtained by transferring the CD38 antigen into the Hela cells expressing CLL1 again through a lentiviral vector, and could specifically express the CLL1 and CD38 protein molecules. Molm13-LucR cells were a stable transfected cell line obtained by screening after being infected with a lentivirus expressing firefly luciferase-RFP (T2A-linked). KG1-LucG cells, HL60-LucG cells, U937-LucG cells, THP1-LucG cells and K562-LucG cells were stable transfected cell lines obtained by screening after being infected with a lentivirus expressing firefly luciferase-GFP (T2A-linked). Molm13-CLL1-LucR cells were a stable transfected cell line obtained by transferring the CLL1 antigen into Molm13-LucR cells through a lentiviral vector, and could specifically express the CLL1 protein molecule.

[0206] The expression of the target cell surface antigen used was as Figure 1As shown, 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 cells negative for both CLL1 and CD38, Molm13 is a cell positive only for CD38, and HL60, KG1, U937, THP1, and Molm13-CLL1 are cells positive for both CLL1 and CD38.

[0207] Example 2: Construction of Dual CAR Vector and Preparation of Virus

[0208] The structure of the CAR (Dual CAR) that simultaneously targets CLL1 and CD38 is as Figure 2 shown, and 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 co-stimulatory signal region, and a CD3z signal region. The Dual CAR gene is placed under the promoter of EF1α (EF-1α) to form a Dual CAR expression vector. The Dual CAR expression vectors are numbered C7B5, C7B8, C9B5, C9B8, B5C7, B5C9, B8C7, B8C9, C7B3, C9B3, B3C7, and B3C9 according to the composition of the VHH sequences.

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

[0210]

[0211]

[0212] Inoculate 2.5×10 6 293T cells in a 150 cm 2 culture dish with DMEM medium containing 10% FBS, and culture the cells overnight at 37°C, 5% CO 2 and saturated humidity before transfection.

[0213] On the second day, the helper plasmid and the Dual CAR expression vector were added to a centrifuge tube containing 13.8 mL of Opti MEM medium, and then 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 added to obtain the transfection medium. For transfection, after removing the 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 medium was collected and centrifuged at 3000 g at 4 °C for 15 min. The supernatant was further centrifuged at 27000 g at 4 °C for 2 hours. The precipitate was collected and resuspended in 400 μL of pre-cooled X-VIVO medium to obtain the Dual CAR lentivirus suspension, which was kept overnight at 4 °C. On the next day, the virus suspension was aliquoted for further use.

[0214] Example 3: Preparation of Dual-CAR T cells

[0215] All T cells were cultured using X-VIVO medium. Pan T cells were incubated with CD3 / CD28 Dynabeads at a ratio of 1:1 (CD3 / CD28 Dynabeads: T cells) and 300 IU / mL IL2 to activate the cells. After 2 days, the activated cells were electroporated to knockout the CD38 gene and then virus transfected to prepare Dual-CAR T.

[0216] The specific operation is as follows: 0.25 nmol of gRNA targeting the CD38 gene and 16.5 μg of Cas9 protein were mixed evenly and incubated at 37 °C for 15 min to prepare RNP; during the incubation, 18 μL of supplement buffer was added to 82 μL of Nucleofector Solution to prepare 100 μL of lonza P3 electroporation buffer; at the same time, the CD3 / CD28 Dynabeads were removed using a magnetic column, and 1×10 7 activated T cells were taken out, and the cell pellet was collected after centrifugation at 400 g for 5 min; after the incubation, 1×10 7 T cell pellets were resuspended in 100 μL of lonza P3 electroporation buffer and then added to the RNP and mixed evenly. Subsequently, it was transferred to an electroporation cuvette and placed in a lonza 2B electroporator, and electroporation was performed using the FI-115 program. The electroporated cells were incubated overnight with 300 IU / mL IL2.

[0217] On the second day after electroporation, at 37 °C at 1×10 6T cells were transfected with Dual CAR virus at a cell density of cells / mL. The transfected cells were continuously cultured and expanded. During the expansion process and cryopreservation, the CAR positive rate of Dual-CAR T cells was detected using CLL1 and CD38 antigens. Half of the culture medium was changed every 2 - 3 days. Dual-CAR T cells were harvested on the 8th day after removing CD3 / CD28 Dynabeads.

[0218] Figure 3 It shows the detection of the CAR positive rate of Dual-CAR T cells using antigens. The expression of CLL1 CAR and CD38 CAR could be detected on the surface of T cells after virus transfection using both CLL1 antigen and CD38 antigen.

[0219] Example 4: In vitro killing of Dual-CAR T cells

[0220] An in vitro killing experiment was performed on the obtained Dual-CAR T cells. RTCA killing analysis was performed using the Hela overexpression cell line overexpressing CLL1 and CD38, or detection was performed using luciferase-labeled tumor target cells.

[0221] By transferring the luciferase gene into target cells, stable transfected cell lines (HL60, Molm13, U937, and K562) were obtained after cloning and screening. During the experiment, luciferin substrate was added, and the reaction of luciferase with luciferin could produce fluorescence. By detecting the intensity of the fluorescence, the activity of luciferase could be measured, and the survival ratio of the cells was detected to obtain the killing effect of CAR-T cells.

[0222] The results showed that after co-culturing CAR-T cells with each target cell (CLL1 / CD38 double positive, CLL1 single positive, CD38 single positive), the target cells would lyse, indicating that Dual-CAR T had a killing effect on CLL1 / CD38 double positive, CLL1 single positive, and CD38 single positive cells.

[0223] The specific results are as Figure 4 shown. Bispecific CAR-T had a significant killing effect on single positive CLL1 positive target cells (Hela-CLL1) or single positive CD38 positive target cells (Hela-CD38), and also had a significant killing effect on the CLL1 and CD38 double positive target cell Hela-CLL1-CD38. It shows that the bispecific CAR-T cells combined with CLL1 and CD38 had a killing effect on single target and double target cells. And Dual-CAR T had no killing effect on the Hela target cell that was negative for both CLL1 and CD38.

[0224] Figure 5It was shown that Dual-CAR T could significantly kill tumor target cells HL60, U937, and Molm13 expressing CD38 and CLL1, while having no killing effect on CLL1- and CD38-double negative cells K562.

[0225] Example 5: Multi-round killing of Dual-CAR T cells in vitro

[0226] Resuscitate Dual-CAR T on the first day and incubate it at 37°C at a cell density of 1×10 6 cells / mL together with 300 IU / mL IL2. On the second day, take out 1×10 5 cell samples and use CLL1 and CD38 antigens to detect the proportion of DualCAR-positive cells.

[0227] Take out 2×10 5 CAR-positive Dual-CAR T cells according to the CAR positive rate of the cells and co-culture them with 6×10 5 HL60-LucG and KG1-LucG cells respectively for multi-round killing analysis (E:T = 1:3). On the third day of each round, take out some co-cultured cells for analysis by flow cytometry. The analysis process is shown in Figure 6 , and the specific method is described as follows: Circle out the 7AAD-negative live cell population from the main cell population, and then distinguish T cells (GFP-negative) from target cells (GFP-positive) through the GFP signal. Use CLL1 and CD38 antigens to label and analyze the CAR positive rate in the T cell population. According to the results of flow cytometry and cell counting, take out the same number of Dual-CAR T cells and supplement tumor cells to a fixed effector-to-target ratio (E:T is 1:3) for the next round of killing experiment.

[0228] The results of multi-round killing against HL60 target cells are as Figure 7 shown. Figure 7 A shows the amplification multiple of residual target cells (HL60) at the end of each round of Dual-CAR T cells; Figure 7 B shows the change in the CAR positive rate of Dual-CAR T cells during multi-round killing against HL60; Figure 7 C shows the total amplification multiple of CAR T cells of Dual-CAR T cells during multi-round killing against HL60.

[0229] From Figure 7 A, it can be observed that C7B3 and C9B5 have a stronger inhibitory ability on HL60 cells than other Dual-CAR T; at the end of the third round, most of the Dual-CAR T could no longer inhibit HL60 cells and the CAR positive rate began to decline ( Figure 7 B); fromFigure 7 In C, it can be observed that in multiple rounds of killing against HL60, the amplification of C7B3 is significantly better than that of other Dual-CAR Ts.

[0230] The results of multiple rounds of killing against KG1 target cells are as Figure 8 shown. Figure 8 A shows the amplification multiple of residual target cells (KG1) at the end of each round of Dual-CAR T cells; Figure 8 B shows the change in the CAR positive rate of Dual-CAR T cells during multiple rounds of killing against KG1; Figure 8 C shows the total amplification multiple of CAR T cells of Dual-CAR T cells during multiple rounds of killing against KG1.

[0231] At the end of the third round, all Dual-CAR T cells could not inhibit KG1 cells well, but the inhibitory ability of C9B5 and C9B8 against KG1 cells was stronger than that of other Dual-CAR Ts ( Figure 8 A). From Figure 8 C, it can be observed that in multiple rounds of killing against KG1, the amplification of C9B5 and C9B8 is better than that of other Dual-CAR T cells.

[0232] The results of multiple rounds of killing against THP1 target cells are as Figure 9 shown. Figure 9 A shows the amplification multiple of residual target cells (THP1) at the end of each round of Dual-CAR T cells; Figure 9 B shows the change in the CAR positive rate of Dual-CAR T cells during multiple rounds of killing against THP1; Figure 9 C shows the total amplification multiple of CAR T cells of Dual-CAR T cells during multiple rounds of killing against THP1.

[0233] At the end of the fifth round, all Dual-CAR T cells could not inhibit THP1 cells well, but the ability of C7B5, C9B5 and C9B8 to maintain CAR expression was stronger than that of other Dual-CAR Ts ( Figure 9 B). From Figure 9 C, it can be observed that in multiple rounds of killing against THP1, the amplification of C9B8, C7B5 and C9B5 is better than that of other Dual-CAR T cells.

[0234] Example 6: NK cells and culture method

[0235] The complete medium for NK92 cells is Alpha MEM supplemented with 12.5% FBS, 12.5% Horse Serum, 1% Pen / strep, 1% sodium pyruvate, 1% L-glutamine, 100U IL2. The complete medium for HL60, Molm13, KG-1, THP-1, and K562 cells is RPMI1640 supplemented with 10% FBS, 1% Pen / strep, 1% sodium pyruvate, 1% L-glutamine. The cells are cultured at 37 °C, 5% CO 2 and saturated humidity.

[0236] Example 7: Packaging and transfection of CAR-NK lentivirus

[0237] Resuscitate 293T and seed it in one 150 cm 2 culture dish, and place it in the CO 2 incubator for 72 h. After two passages, seed it in a 150 cm 2 culture dish for transfection. Mix the four-plasmid system composed of the lentiviral expression vector, the helper plasmid gag / pol, Rev, and VSV-G with the PEI transfection reagent, then add it to a certain volume of serum-free DMEM, mix well and let it stand for 15 min; add the above mixture to the 150 cm 2 culture dish seeded with 293T cells, mix gently, and culture it in a 37 °C, 5% CO 2 cell incubator for 6 h. After 6 h, replace the fresh medium and continue to culture. Collect the lentiviral culture supernatant for infection after 48 and 72 h. Transfer the harvested supernatant to a centrifuge tube, centrifuge at 4000 rpm for 10 min to remove cell debris. Transfer all the centrifuged LVV supernatant to a 0.45 μm filter for clarification, and then transfer the filtrate to a new centrifuge tube. Add the clarified lentiviral supernatant to an ultracentrifuge tube and balance it on a balance. Place the balanced ultracentrifuge tube in a hanging cup, place the hanging cup in the corresponding position of the rotor, and then put them together into an ultracentrifuge for centrifugation. The centrifugation temperature is 4 °C, the rotation speed is 100000 g, the centrifugation time is 90 min, and the acceleration and deceleration speeds are set to the maximum. After ultracentrifugation, discard the supernatant, add 0.5 mL of medium to each tube, and resuspend it at 2 - 8 °C for 2 h. Add the harvested virus to 1e6 NK92 and mix it in a 24-well plate, and place it in the CO 2 incubator for 72 h.

[0238] Example 8: Flow cytometry detection and sorting of CAR-NK

[0239] Take out the cell suspension, wash it three times with DPBS, centrifuge at 300g for 5 min, discard the supernatant, add the antibody mixture (anti-FLAG-APC, added to DPBS at 1:100), mix well and place at 4°C for 30 min of staining, wash three times with DPBS, centrifuge at 300g for 5 min. After resuspending with DPBS, it is used for flow cytometry detection and flow sorting. During sorting, gate the PE-positive cells and collect them in a 5-ml flow tube. After sorting, centrifuge at 300g for 5 min, discard the supernatant, resuspend the cells with pre-warmed medium, and place at 37°C, 5% CO 2 cell incubator.

[0240] For the sorted CAR-NK92 cells, the expression of CAR was detected by flow cytometry through anti-FLAG-APC antibody staining.

[0241] The results of flow cytometry detection are as Figure 10 shown. After sorting, the positive rate of CAR in each CAR-NK92 cell reached over 98%.

[0242] Example 9: CAR-NK killing experiment

[0243] The killing ability was detected using luciferase-labeled tumor target cells. By transferring the luciferase gene into the target cells, stable cell lines expressing the luciferase gene of Molm13, HL60, THP-1, and KG1 were obtained. During the experiment, add the luciferin substrate, and the luciferase reacts with luciferin to produce fluorescence. By detecting the intensity of the fluorescence, the activity of luciferase can be measured, and by detecting the survival ratio of the cells, the killing effect of each effector cell can be obtained.

[0244] The results are as Figure 11 shown. All CAR-NK92 showed more obvious killing on each target cell than NK92, suggesting the killing effect mediated by CAR.

[0245] Example 10: CAR-NK multi-round killing experiment

[0246] One day before the experiment, take the fluorescent target cell THP1, resuspend and count it, adjust the cell density to 8×10 4 / ml, and seed 100 μl per well in 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. Start the experiment in the Cellcyte, and take pictures every 4 hours through the bright field and fluorescence channels. Every two days is one round. After one round of killing, remove part of the supernatant, transfer it to a new plate seeded with target cells, and put it into the Cellcyte to start a new round of killing. The KillingIndex is calculated by the reduction in the number of fluorescent cells after adjusting for the zero time point and the error of normal cell growth.

[0247] The results of multiple rounds of killing experiments are as Figure 12 shown. All CAR-NK92 cells showed stronger killing ability than NK92 cells, indicating that CAR-mediated killing was continuously functioning.

[0248] Example 11: Preparation of Fast Dual-CAR T cells

[0249] Peripheral blood was collected from healthy donors, and PBMC was separated by centrifugation at 500 - 600 g for 20 - 30 minutes using a density gradient centrifuge. Magnetic beads conjugated with CD28 antibody and CD3 antibody (CD3 / CD28 Dynabeads) were used to sort and enrich T cells. The T cells conjugated with CD3 / CD28 Dynabeads were further incubated with 300 IU / mL IL2 to activate the cells. Meanwhile, at 37 °C, the cells were transfected overnight with Dual-CAR virus at a cell density of 0.1 - 10×10 6 cells / mL, and after washing with saline buffer the next day, they were directly cryopreserved. FAST Dual-CAR T cells could be obtained without further amplification. During this process, the cells were activated, and these cells were also called F Dual-CAR T cells.

[0250] Example 12: Phenotype of Fast Dual-CAR T cells

[0251] Since there is CD38 protein on the surface of T cells, the expression of CD38 CAR will lead to autologous killing, resulting in the clearance of T cells expressing CD38. To analyze whether severe autologous killing would occur in Dual-CAR T produced by the FAST process, while performing CAR positive calibration after the resuscitation of F Dual-CAR T cells, the cell viability, amplification, and CD38 expression on the cell surface were also monitored.

[0252] After resuscitation, the F Dual-CAR T cells were cultured at a cell density of 1×10 6 cells / mL at 37 °C in the presence of 300 IU / mL IL2. The cells were counted at D2, D3, D5, and D8 after resuscitation, and 1×10 5 cell samples were taken to detect the proportion of Dual-CAR positive cells using CLL1 and CD38 antigens, and at the same time, the expression of CD38 on the surface of T cells was analyzed using an antibody targeting CD38.

[0253] The data of F Dual-CAR T cells after resuscitation are as Figure 13 shown. Figure 13 A shows CD38 on the surface of T cells +Percentage, F-C9B5 has a CD38 similar to that of F-NT + Ratio, F-B8C9 in the pre-recovery stage CD38 + The proportion of cells is relatively small, but it recovers to a level similar to that of F-NT on the fifth day after recovery, while CD38 cannot be detected on the cell surface of F-C7B3 + Cells. The positive rate of CAR in each group of F Dual-CAR T gradually increases after recovery ( Figure 13 B). F-C9B5 has a cell expansion and viability similar to that of F-NT, while the expansion and viability of F-B8C9 and F-C7B3 are relatively low ( Figure 13 C, B), combined with CD38 + Percentage speculation may be due to fratricide resulting in reduced cell viability and amplification multiple.

[0254] On the third day after the recovery of F Dual-CAR T, CD45RO and CCR7 were used to analyze the cell differentiation phenotypes of F Dual-CAR T and C Dual-CART (traditional production process) cells. Stem memory-like T cells (Tscm) were defined as CD45RO - CCR7 + , central memory T cells (Tcm) were defined as CD45RO + CCR7 + , effector memory T cells (Tem) were defined as CD45RO + CCR7 － , terminally differentiated effector T cells (Teff) were defined as CD45RO － CCR7 － . As Figure 14 shown, F-C9B5 cells have a relatively large proportion of young Tscm and Tcm phenotypes, while C-C9B5 cells are mostly in the late-stage differentiated Tem and Teff phenotypes.

[0255] Example 13: In vitro killing experiment of Fast Dual-CAR T cells and C Dual-CAR T cells

[0256] On the first day, Fast Dual-CAR T was recovered and cultured at 37°C at 1×10 6Cells were incubated at a cell density of 1×10 cells / mL and 300 IU / mL IL2. On the second day, C Dual-CAR T cells were resuscitated and incubated at a cell density of 1×10 cells / mL and 300 IU / mL IL2 at 37°C. On the third day, they were co-cultured with K562, HL60, Molm13, and THP1 expressing firefly luciferase respectively, and the killing effect was detected 4 hours and 24 hours after co-culture respectively. The results are as Figure 15 shown, F-C9B5 and C-C9B5 have similar effects on killing target cells.

[0257] Example 14: Multi-round killing of F Dual-CAR T cells and C Dual-CAR T cells in vitro

[0258] On the first day, F CAR T cells were resuscitated and incubated at a cell density of 1×10 6 cells / mL and 300 IU / mL IL2 at 37°C. On the second day, C CAR T cells were resuscitated and incubated at a cell density of 1×10 6 cells / mL and 300 IU / mL IL2 at 37°C. On the third day, 2×10 5 CAR-positive F Dual-CAR T cells and C Dual-CAR T cells were taken out and co-cultured with 1×10 6 HL60, Molm13, and THP1 cells (E:T was 1:5) respectively for multi-round killing analysis. On the third day of each round, 1 / 6 of the co-cultured cells were taken out and directly added with 1×10 6 corresponding target cells for the next round of killing experiment. At the same time, part of the co-cultured cells were counted and the proportion of residual target cells and CAR + cell proportion were analyzed by flow cytometry. Combining the counting results, the cumulative amplification multiple of CAR T cells could be calculated.

[0259] The results of multi-round killing are as Figure 16 shown, regardless of the target cells, F-C9B5 can obtain a more persistent ability to kill target cells in vitro than C-C9B5 ( Figure 16 B) and a greater amplification multiple ( Figure 16 A).

[0260] Example 15: Comparison of the in vivo efficacy of F Dual-CAR T cells and C Dual-CAR T cells in mice

[0261] 4- to 6-week-old NOG-dKO mice were selected and injected with 2×10 6Molm13-CLL1-LucR cells. After three days, the tumor burden was detected by in vivo imaging of small animals. On the same day, the groups were divided and F-C9B5 cells and C-C9B5 cells were injected respectively. After T cell treatment, the tumor burden of mice was evaluated by in vivo imaging of small animals twice a week.

[0262] The results are as Figure 17 shown. Compared with the NT control group, the tumor growth of mice injected with CAR T cells was inhibited; F-C9B5 cells had a dose-related pharmacodynamic effect and could obtain a more persistent tumor inhibitory effect than C-C9B5 at a lower dose. The reinfusion of CAR T cells in each group had no significant effect on the body weight of mice.

[0263] At this point, those skilled in the art should recognize that although multiple exemplary embodiments of the present invention have been shown and described in detail herein, many other variations or modifications that conform to the principles of the present invention can still be directly determined or derived from the content disclosed in 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 determined to cover all these other variations or modifications.

Claims

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

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

3. The chimeric antigen receptor according to claim 2, wherein the antigen domain comprises a single-domain antibody.

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

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

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

11.

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

12.

8. The chimeric antigen receptor according to claim 5, wherein the amino acid sequence of CDR1 of VHH1 is as shown in SEQ ID NO:7; the CDR2 of VHH1 comprises an amino acid sequence as shown in SEQ ID NO:10; 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 6-8, wherein VHH1 comprises the amino acid sequence shown in SEQ ID NO:1-3, or consists of the same.

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

17.

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

18.

12. The chimeric antigen receptor according to claim 10 or 11, wherein VHH2 comprises the amino acid sequences shown in SEQ ID NO:4 and SEQ ID NO:5, or consists of the same.

13. 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 the amino acid sequence shown in SEQ ID NO:19, or consists of the same; the transmembrane domain of CD28 comprises the amino acid sequence shown in SEQ ID NO:20, or consists of the same.

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

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

17. The chimeric antigen receptor according to claim 16, wherein the cytoplasmic signaling sequence of CD3ζ comprises the amino acid sequence shown in SEQ ID NO:21, or consists of the same.

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

19. The chimeric antigen receptor according to claim 18, wherein the co-stimulatory signal domain comprises a molecule selected from the following: 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, CD226, or a combination thereof.

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

21. The chimeric antigen receptor according to claim 20, wherein the co-stimulatory signal structure of 4-1BB comprises the amino acid sequence shown in SEQ ID NO:22, or consists of the same; the co-stimulatory signal structure of CD28 comprises the amino acid sequence shown in SEQ ID NO:23, or consists of the same.

22. The chimeric antigen receptor according to claim 21, wherein the chimeric antigen receptor further comprises a hinge region.

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

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

24.

25. The chimeric antigen receptor according to claim 24, wherein the chimeric antigen receptor further comprises a signal peptide; the signal peptide is selected from the following molecules: 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, GM-CSF, or a combination thereof.

26. 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 the amino acid sequence shown in SEQ ID NO: 25 or consists of the same.

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

29. The chimeric antigen receptor according to claim 27, wherein its specific structure is: L-VHH1-L1-VHH2-F-H-TM-C-CD3ζ Each "-" is independently a linker peptide or a peptide bond; L is a 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 linker peptide; F is a Flag tag sequence (Flag Tag); H is the hinge region of CD8a; TM is the transmembrane domain of CD28; C is the co-stimulatory domain of CD28; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

30. The chimeric antigen receptor according to any one of claims 28-29, wherein the linker peptide is selected from linkers having the following amino acid sequences: SGG, GGS, SGGS, SSGGS, GGGG, SGGGG, GGGGS, SGGGGS, GGGGGG, SGGGGGG, GSGGGGS, GGGGGGGG, SGGGGGGGG, SGGGGGGG, SGGGGSGGGGS, or GGGGSGGGGSGGGGS.

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

26.

32. The chimeric antigen receptor according to claim 29, wherein the fusion protein expression tag Flag tag is as shown 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 6-12.

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

30.

35. The nucleic acid molecule according to claim 33, wherein the nucleic acid molecule further comprises the nucleotide sequence shown in any one of SEQ ID NO: 28-29, or 31-51.

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

37. The recombinant vector according to claim 36, characterized in that 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 according to claim 37, wherein the viral vector comprises a lentiviral vector, an adenoviral vector, a retroviral vector.

39. Engineered immune cells, which comprise the chimeric antigen receptor according to any one of claims 6-12, or the nucleic acid molecule according to any one of claims 33-35, or the recombinant expression vector according to any one of claims 36-38.

40. The engineered immune cells according to claim 39, wherein the immune cells are prepared by the FAST-CAR process.

41. The engineered immune cells according to claim 40, wherein the FAST CAR converts the activation, transduction and amplification steps into a single "synchronous activation-transduction" step, and the engineered immune cells undergo ex vivo amplification for less than 72 hours.

42. The engineered immune cells according to claim 41, which can reduce the effect of fratricide caused by the recognition of the CD38 antigen on the surface of immune cells by CD38 CAR during the production process, or the effect of fratricide caused by the recognition of the corresponding target antigen on the surface of immune cells by other CARs, such as the apoptosis of T cells caused by the recognition of the CD70 antigen on the surface of T cells by CD70 CAR; the immune cells can be applied to any CAR-targeted antigen and different tumor markers.

43. The engineered immune cells according to claim 41, wherein the cell phenotype of the immune cells is younger compared to the cells in a comparable population that has undergone ex vivo amplification for one week or more.

44. The engineered immune cells according to claim 43, wherein the young feature is that the proportions of T n / scm (CD45RO - , CCR7 + ) and Tcm (CD45RO + , CCR7 + ) are higher.

45. The engineered immune cells as claimed in claim 41 show stronger in vitro expansion and sustained killing ability compared to cells in a comparable population that have undergone ex vivo expansion for 1 week or more.

46. The engineered immune cells as claimed in claim 39, wherein the immune cells comprise T cells, NK cells, iNKT cells, CTL cells, monocytes, macrophages, dendritic cells, and / or NKT cells.

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

48. Use of the chimeric antigen receptor as claimed in any one of claims 6 - 12, the nucleic acid molecule as claimed in any one of claims 33 - 35, the recombinant expression vector as claimed in claims 36 - 38, or the engineered immune cells as claimed in any one of claims 39 - 47 in the preparation of a medicament for treating tumors.

49. The use as claimed in claim 48, wherein the tumor is a tumor expressing CD38 and / or CLL1.

50. The use as claimed in claim 49, wherein the tumor is acute myeloid leukemia.

51. The use as claimed in claim 48, wherein the medicament further comprises a pharmaceutically acceptable carrier and adjuvant.

52. Use of the chimeric antigen receptor as claimed in any one of claims 6 - 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 comprising CAR nucleic acid or CAR polypeptide; (2) An agent that ameliorates one or more side effects associated with the administration of cells comprising CAR nucleic acid or CAR polypeptide; (3) An additional agent 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 selected from surgery, chemotherapy, radiotherapy, immunotherapy, gene therapy, DNA therapy, RNA therapy, nanotherapy, virus therapy, adjuvant therapy, and any combination thereof.

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