Anti-MUC1 single-domain antibody chimeric antigen receptor and application thereof

Abstract

The invention relates to an anti-MUC1 single-domain antibody chimeric antigen receptor and application thereof, and particularly provides a chimeric antigen receptor, and an antigen binding domain of the chimeric antigen receptor comprises an MUC1 binding molecule containing an anti-MUC1 single-domain antibody. The immune cell containing the chimeric antigen receptor disclosed by the invention has a relatively high cell killing effect.

Description

Technical Field

[0001] The present invention relates to the field of chimeric antigen receptor therapy, and in particular to an anti-MUC1 single domain antibody chimeric antigen receptor and applications thereof. Background Art

[0002] Over the past decade, the engineering of personalized immune responses to cancer has gone from theory to reality. Chimeric antigen receptor (CAR) T cell therapy, made from the patient's own immune cells, has brought a turnaround for certain types of aggressive leukemia and other blood cancers. This therapy uses genetic engineering technology to splice the variable region gene sequence of antibodies that can recognize tumor antigen molecules with the intracellular region sequence of T lymphocyte immune receptors, and then introduces these fusion gene sequences into the T lymphocyte genome through a lentiviral vector system or a transposon transposase system. In this way, T cells can express CAR, recognize and attack specific antigens, and enhance their ability to kill tumor cells. In some cases, CAR-T cells can even cure people whose cancer has relapsed after multiple other treatments. However, CAR-T cells have not yet achieved the effect of allowing most people to survive for a long time. In addition, although CAR-T therapy has achieved some success in treating blood cancers, there is still a long way to go in the treatment of solid tumors (such as pancreatic cancer, lung cancer or colorectal cancer).

[0003] Selecting the right cancer cell antigen is the key to designing an effective CAR T cell approach and avoiding side effects. Among various cancer-related antigens, mucin 1 (MUC1) is a potential target. MUC1 is expressed in a variety of healthy epithelial tissues and is glycosylated to form a mucus gel that provides protection for epithelial cells. Abnormally glycosylated MUC1 is found in most epithelial cell cancers and has attracted much attention from scientists as a carcinogenic molecule. Tumor-associated MUC1 is different from MUC1 expressed by normal cells. Due to the loss of Core 2 transferase and abnormal activation of sialyltransferase, most of the MUC1 expressed by tumors is hypoglycosylated or has truncated sugar chains, which exposes some new antibody binding epitopes. CAR-T designed for this tumor-specific antigen epitope is often considered to have fewer off-target effects. Summary of the invention

[0004] In a first aspect, the present invention provides a chimeric antigen receptor comprising an optional signal peptide sequence, a MUC1 binding molecule comprising a MUC1 single domain antibody, a hinge region, a transmembrane region, and an intracellular region.

[0005] In one or more embodiments, the intracellular region includes an intracellular co-stimulatory domain and / or an intracellular signaling domain.

[0006] In one or more embodiments, the complementarity determining region CDR of the MUC1 single domain antibody comprises CDR1, CDR2 and CDR3, wherein CDR1 comprises the sequence shown in SEQ ID NO: 1, CDR2 comprises the sequence shown in SEQ ID NO: 2, and CDR3 comprises the sequence shown in SEQ ID NO: 3, wherein,

[0007] SEQ ID NO: 1 is GX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 , where X 1 is P, F, L, A or R, X 2 H, T, S or F, X 3 L, F, S, G, T or A, X 4 is E, R, D, L or N, X 5 L, R, Y, N, D, Q, I or E, X 6 is Y, H, L, E or I, X 7 A, Y, D, M, E or T, X 8 is Y, E or None, X 9 is Y or None, X 10 is N or None,

[0008] SEQ ID NO: 2 is IX 1 X 2 X 3 X 4 X 5 X 6 X 7 , where X 1 S, N or R, X 2 is E, T, G, R or W, X 3 is S, N, R, I, F or Y, X 4 is G, D or N, X 5 D, G, S, A, E, T or none, X 6 D, T, S, R, P, L, W or none, X 7 is T, I or None,

[0009] SEQ ID NO:3 is

[0010] X 1 X 2 X 3 X 4 X5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 , where X 1 A, T or N, X 2 A or T, X 3 is C, I, E, G or V, X 4 T, G, R, V or D, X 5 is F, Y, T, I, A, N or P, X 6 Y, P, R, G, L or A, X 7 G, S, F, R, L or H, X 8 is S, Q, I, T, W, K or Y, X 9 T, L, F, P, S or none, X 10 F, S, G, C, T or None, X 11 R, Y, T, L, S, C or none, X 12 E, D, C, F, Y or None, X 13 M, Y, V, L or none, X 14 G, P, H, A, T or none, X 15 Y, S, Q, R, W, V or none, X 16 N, R, T, M, P or None, X 17 F, E, A, Q or none, X 18 A, G, Y or none, X 19 S, D, R or none, X 20 Is Y or None.

[0011] In one or more embodiments, the CDR1 of the MUC1 single domain antibody comprises a sequence as shown in any one of SEQ ID NOs: 4-13, CDR2 comprises a sequence as shown in any one of SEQ ID NOs: 14-24, and CDR3 comprises a sequence as shown in any one of SEQ ID NOs: 25-35.

[0012] Preferably, the single-domain antibody contains CDR1, CDR2 and CDR3 shown in SEQ ID NO of any one of the following groups a1 to a11:

[0013]

[0014]

[0015] In one or more embodiments, the FR region of the MUCl single domain antibody comprises the FR region of any one VHH selected from SEQ ID NOs: 36-46.

[0016] In one or more embodiments, the sequence of the MUCl single domain antibody is as shown in any one of SEQ ID NOs:36-46, or has at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of any one of SEQ ID NOs:36-46.

[0017] In one or more embodiments, the MUCl binding molecule is a monovalent or multivalent single domain antibody, a multispecific single domain antibody, a heavy chain antibody or an antigen binding fragment thereof, an antibody or an antigen binding fragment thereof comprising one, two or more of the single domain antibodies.

[0018] In one or more embodiments, the chimeric antigen receptor contains, from N-terminus to C-terminus, a signal peptide, a MUCl binding molecule including a MUCl single domain antibody, a hinge region, a transmembrane region, an intracellular co-stimulatory domain, and an intracellular signaling domain.

[0019] In one or more embodiments, the signal peptide is a CD8 signal peptide, a CD28 signal peptide, a CD4 signal peptide or a light chain signal peptide; more preferably, it is a CD8 signal peptide; preferably, the amino acid sequence of the CD8 signal peptide is as shown in SEQ ID NO:47.

[0020] In one or more embodiments, the hinge region is selected from a CD8 hinge region, an IgD hinge region, an IgG1FcCH2CH3 hinge region and an IgG4 Fc CH2CH3 hinge region; preferably, the hinge region is a CD8 hinge region; more preferably, the amino acid sequence of the CD8 hinge region is as shown in SEQ ID NO:48.

[0021] In one or more embodiments, the transmembrane region is one of the CD28 transmembrane region, CD8 transmembrane region, CD3ζ transmembrane region, CD134 transmembrane region, CD137 transmembrane region, ICOS transmembrane region and DAP10 transmembrane region; preferably it is the CD8 transmembrane region, and more preferably its amino acid sequence is as shown in SEQ ID NO:49.

[0022] In one or more embodiments, the intracellular co-stimulatory domain includes the intracellular domain of a co-stimulatory signal molecule, including CD28, CD134 / OX40, 4-1BB, lymphocyte-specific protein tyrosine kinase, inducible T cell co-stimulatory factor (ICOS) and DNAX activation protein 10; preferably, the intracellular co-stimulatory domain is the intracellular domain of 4-1BB; preferably, the amino acid sequence of the 4-1BB intracellular domain is as shown in SEQ ID NO: 50.

[0023] In one or more embodiments, the intracellular signaling domain is a CD3ζ intracellular signaling domain or a FcεRIγ intracellular signaling domain; preferably, it is a CD3ζ intracellular signaling domain, and preferably, the amino acid sequence of the CD3ζ intracellular signaling domain is as shown in SEQ ID NO:51.

[0024] In one or more embodiments, the chimeric antigen receptor contains, from N-terminus to C-terminus, a CD8 signal peptide, an anti-MUC1 nanobody, a CD8 hinge region, a CD8 transmembrane region, a 4-1BB co-stimulatory domain, and a CD3ζ intracellular signaling domain.

[0025] In one or more embodiments, the amino acid sequence of the chimeric antigen receptor is shown in any one of SEQ ID NOs: 52-62.

[0026] The present invention also provides a nucleic acid molecule comprising a sequence selected from the group consisting of:

[0027] (1) a coding sequence of a chimeric antigen receptor as described in any embodiment of the present invention;

[0028] (2) the complementary sequence of (1);

[0029] (3) A 5-50 bp fragment of any of the sequences in (1) or (2).

[0030] In one or more embodiments, the fragment is a primer.

[0031] In one or more embodiments, the coding sequence is DNA or RNA.

[0032] The second aspect of the present invention also provides a nucleic acid construct, which comprises the nucleic acid molecule described in any embodiment of the present invention.

[0033] In one or more embodiments, the nucleic acid construct further comprises a coding sequence for a transposase, which is used to transpose the coding sequence of the chimeric antigen receptor, such as integrating the coding sequence or expression cassette of the transposed chimeric antigen receptor into the genome.

[0034] In one or more embodiments, the coding sequence for the transposase is DNA or RNA.

[0035] In one or more embodiments, the nucleic acid construct is a vector, such as a cloning vector, an expression vector, or an integrating vector.

[0036] In one or more embodiments, the expression vector is a constitutive expression vector. Preferably, the constitutive expression vector is a transposable vector.

[0037] In one or more embodiments, the coding sequence of the chimeric antigen receptor is located between the recognition sequences of the transposase.

[0038] In one or more embodiments, the transposase is PiggyBac transposase (PBase).

[0039] In one or more embodiments, the transposase is as described in CN105154473A.

[0040] In one or more embodiments, the nucleic acid construct comprises an expression cassette for CAR. In one or more embodiments, the nucleic acid construct further comprises an expression cassette for transposase. The one or two expression cassettes are contained in one or more vectors.

[0041] In one or more embodiments, the nucleic acid construct contains an expression cassette of the chimeric antigen receptor and an expression cassette of the transposase; or the nucleic acid construct is an expression cassette, wherein the coding sequence of the chimeric antigen receptor and the coding sequence of the transposase are within the expression cassette.

[0042] In one or more embodiments, the nucleic acid construct is one or more vectors, each vector comprising one or two expression cassettes as described in any of the embodiments herein.

[0043] In one or more embodiments, the nucleic acid construct comprises:

[0044] A vector comprising (a) an expression cassette for a CAR and optionally (b) an expression cassette for a transposase;

[0045] The two vectors contain the expression cassette of CAR and the expression cassette of transposase respectively.

[0046] On the other hand, the present invention also provides a combination of coding sequences, comprising the coding sequence of the chimeric antigen receptor according to any embodiment of the present invention, and further comprising the coding sequence of a transposase.

[0047] In one or more embodiments, any one of the coding sequences of the combination of coding sequences can be freely selected as DNA or RNA. For example, both coding sequences are DNA, or both coding sequences are RNA, or any one of the two is DNA.

[0048] In one or more embodiments, the combination of coding sequences includes a coding sequence for a chimeric antigen receptor and a coding sequence for a transposase, and both coding sequences are DNA.

[0049] In one or more embodiments, the combination of coding sequences includes a coding sequence of a chimeric antigen receptor and a coding sequence of a transposase, and the coding sequence of the chimeric antigen receptor is DNA, and the coding sequence of the transposase is RNA, more preferably mRNA.

[0050] In one or more embodiments, in the combination of the coding sequences, the coding sequences may be located in the same nucleic acid molecule or in different nucleic acid molecules. For example, the coding sequence of the chimeric antigen receptor and the coding sequence of the transposase are located in the same nucleic acid molecule; or the coding sequence of the chimeric antigen receptor and the coding sequence of the transposase are located in two nucleic acid molecules respectively.

[0051] Another aspect of the present invention provides a host cell, wherein:

[0052] (1) comprising and / or expressing a chimeric antigen receptor as described in any of the embodiments herein, and / or

[0053] (2) comprising the nucleic acid molecules and / or nucleic acid constructs described herein, and / or

[0054] (3) comprising the coding sequence combination described herein.

[0055] In one or more embodiments, the cell is an immune cell, preferably a T cell.

[0056] In one or more embodiments, the cell contains: a coding sequence for a chimeric antigen receptor as described in any of the embodiments herein.

[0057] Another aspect of the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and:

[0058] (1) the chimeric antigen receptor and / or its coding sequence as described in any embodiment herein, optionally further comprising a transposase and / or its coding sequence, or

[0059] (2) A nucleic acid molecule, a nucleic acid construct, a combination of coding sequences or a cell as described in any embodiment herein.

[0060] In one or more embodiments, the pharmaceutical composition is used to treat cancer.

[0061] In one or more embodiments, the cancer comprises a MUCl -mediated cancer.

[0062] In one or more embodiments, the cancer comprises breast cancer, kidney cancer, ovarian cancer, gastric cancer, pancreatic cancer, lung cancer, colon cancer, osteosarcoma, adenocarcinoma, bladder cancer, colorectal cancer, cervical cancer, head and neck cancer, fallopian tube cancer, multiple myeloma, bile duct cancer, gallbladder cancer, esophageal cancer, prostate cancer, or glioblastoma.

[0063] Another aspect of the present invention provides the use of a reagent in preparing activated immune cells (such as T cells), wherein the reagent comprises:

[0064] (1) the chimeric antigen receptor and / or its coding sequence as described in any embodiment herein, optionally further comprising a transposase and / or its coding sequence, or

[0065] (2) A nucleic acid molecule, a nucleic acid construct, a combination of coding sequences or a cell as described in any embodiment herein.

[0066] Another aspect of the present invention provides the use of a reagent in the preparation of a drug for treating cancer, wherein the reagent comprises:

[0067] (1) the chimeric antigen receptor and / or its coding sequence as described in any embodiment herein, optionally further comprising a transposase and / or its coding sequence, or

[0068] (2) A nucleic acid molecule, a nucleic acid construct, a combination of coding sequences or a cell as described in any embodiment herein.

[0069] The present invention also provides a method for treating or preventing cancer, comprising administering a therapeutically effective amount of the cell or pharmaceutical composition according to any embodiment of the present invention to a patient in need thereof.

[0070] In one or more embodiments, the cancer comprises a MUC1-mediated cancer. The cancer comprises breast cancer, kidney cancer, ovarian cancer, gastric cancer, pancreatic cancer, lung cancer, colon cancer, osteosarcoma, adenocarcinoma, bladder cancer, colorectal cancer, cervical cancer, head and neck cancer, fallopian tube cancer, multiple myeloma, bile duct cancer, gallbladder cancer, esophageal cancer, prostate cancer or glioblastoma.

[0071] In one or more embodiments, the therapeutically effective amount is 1-100*10 6 cells / mouse, preferably 5*10 6 cells / mouse.

[0072] In one or more embodiments, the cells or pharmaceutical compositions are administered by intravenous injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 : CAR-T cell proliferation multiples.

[0074] Figure 2: Cell viability during CAR-T cell proliferation.

[0075] Figure 3 : CAR-T cell positivity rate on day 9.

[0076] Figure 4 : The ratio of CD4 and CD8 CAR-T cells on day 9.

[0077] Figure 5 : The time required for CAR-T cells in each group to kill 50% of MUC1-high-expressing tumor cells MB468 under different effector-target ratios.

[0078] Figure 6 :48h tumor lysis rate of each group of CAR-T on HCT116 tumor cells with extremely low expression of MUC1.

[0079] Figure 7 : CAR positivity rate of two donors after each round of co-culture.

[0080] Figure 8 : At the end of the fifth round, the theoretical proliferation multiples of the CAR-positive T cells of the two donors compared to the number of CAR-positive cells initially inoculated.

[0081] Fig. 9 : IFN-γ secretion of CAR-T in each group in the first three rounds of co-culture experiment. DETAILED DESCRIPTION

[0082] The present invention provides a chimeric antigen receptor (CAR) targeting MUC1. The CAR contains an optional signal peptide sequence, a MUC1 binding molecule containing a MUC1 single domain antibody sequence, a hinge region, a transmembrane region, an intracellular costimulatory domain and an intracellular signaling domain.

[0083] Herein, the "binding molecule" of an antigen is a protein that specifically binds to an antigen, including but not limited to, antibodies, antigen-binding fragments of antibodies, heavy chain antibodies, nanobodies, minibodies, affibodies, target binding regions of receptors, cell adhesion molecules, ligands, enzymes, cytokines, and chemokines. Herein, the term "antibody" includes monoclonal antibodies (including full-length antibodies, which have an immunoglobulin Fc region), antibody compositions with multiple epitope specificities, multispecific antibodies (e.g., bispecific antibodies), double antibodies and single-chain molecules, as well as antibody fragments, especially antigen-binding fragments, e.g., Fab, F(ab')2 and Fv). Herein, the terms "immunoglobulin" (Ig) and "antibody" are used interchangeably.

[0084] The "heavy chain antibody" described herein is an antibody derived from a camelid or cartilaginous fish. Compared with the above-mentioned 4-chain antibody, the heavy chain antibody lacks the light chain and the heavy chain constant region 1 (CH1), and only contains 2 heavy chains composed of variable regions (VHH) and other constant regions, and the variable regions are connected to the constant regions by a structure similar to the hinge region. Each heavy chain of the camelid heavy chain antibody contains 1 variable region (VHH) and 2 constant regions (CH2 and CH3), and each heavy chain of the cartilaginous fish heavy chain antibody contains 1 variable region and 5 constant regions (CH1-CH5). The antigen-binding fragment of the heavy chain antibody includes VHH and a single-chain heavy chain antibody. By fusion with the constant region of human IgG Fc, the heavy chain antibody can have CH2 and CH3 of human IgG Fc. Exemplarily, the heavy chain constant region comprises the sequence shown in SEQ ID NO:63.

[0085] As used herein, the terms "single domain antibody", "heavy chain variable region domain of heavy chain antibody", "VHH", "nanobody", and "single variable domain" are used interchangeably and refer to a single domain polypeptide or protein that specifically recognizes and binds to an antigen. A single domain antibody is a variable region of a heavy chain antibody. Typically, a single domain antibody contains three CDRs and four FRs. A single domain antibody is the smallest functional antigen-binding fragment. Usually, an antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody consisting of only one heavy chain variable region.

[0086] A binding molecule comprising two or more single domain antibodies is a multivalent single domain antibody; a binding molecule comprising two or more single domain antibodies of different specificities is a multispecific single domain antibody. A multivalent single domain antibody or a multispecific single domain antibody connects multiple single domain antibodies via a linker. The linker is generally composed of 1-15 amino acids selected from G and S.

[0087] In this article, heavy chain antibodies and antibodies are intended to distinguish different combinations of antibodies. Due to the similarity between the two structures, the following structural description of antibodies is also applicable to heavy chain antibodies in addition to light chains.

[0088] The "variable region" or "variable domain" of an antibody refers to the amino terminal domain of the heavy or light chain of an antibody. The variable domains of the heavy and light chains may be referred to as "VH" and "VL", respectively. These domains are usually the most variable parts of an antibody (relative to other antibodies of the same type) and contain the antigen binding site.

[0089] The term "variable" refers to the situation that certain segments in the variable domain vary widely in the antibody sequence. The variable domain mediates antigen binding and defines the specificity of a particular antibody to its specific antigen. However, the variability is not evenly distributed over all the amino acids spanned by the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) (in both the light chain and heavy chain variable domains), namely, HCDR1, HCDR2, HCDR3 (heavy chain antibodies can be referred to as CDR1, CDR2, CDR3) of the heavy chain variable region and LCDR1, LCDR2 and LCDR3 of the light chain variable region. The more highly conserved parts of the variable domain are called framework regions (FRs). The variable domains of natural heavy and light chains each contain four FR regions (FR1, FR2, FR3 and FR4), most of which adopt a β-folded conformation, connected by three HVRs that form a loop connection and in some cases form a part of the β-folded structure. The HVR in each chain is held together in close proximity by the FR region and contributes to the formation of the antigen binding site of the antibody together with the HVR of the other chain. Typically, the structure of the light chain variable region is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, and the structure of the heavy chain variable region is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. The constant domain is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions, such as the participation of the antibody in antibody-dependent cell-mediated cytotoxicity.

[0090] "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates the binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells), or binding to the first component (C1q) of the classical complement system. In IgG, IgA, and IgD antibody isotypes, the Fc region is composed of two identical protein fragments, the CH2 domain and the CH3 domain from the two heavy chains of the antibody; the Fc region of IgM and IgE contains three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as the sequence segment from the amino acid residue at position C226 or P230 of the heavy chain to the carboxyl terminus, where the numbering is according to the EU index, as in Kabat. Each heavy chain of a camelid heavy chain antibody comprises one variable region (VHH) and two constant regions (CH2 and CH3). By fusing with the constant region of human IgG Fc, the heavy chain antibody can have the CH2 and CH3 of human IgG Fc.

[0091] "Antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding and / or variable regions of an intact antibody. Antibody fragments are preferably antigen binding fragments of antibodies. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; scFv-Fc fragments; multispecific antibodies formed from antibody fragments; and any fragments that should be able to increase half-life by chemical modification or by incorporation into liposomes. Digestion of antibodies with papain produces two identical antigen binding fragments called "Fab" fragments, and a residual "Fc" fragment, the name of which reflects its ability to crystallize easily. Fab fragments consist of an intact light chain and heavy chain variable domain (VH) and one heavy chain first constant domain (CH1). Each Fab fragment is monovalent in terms of antigen binding, i.e., it has a single antigen binding site. Pepsin treatment of antibodies produces a larger F(ab')2 fragment, which is roughly equivalent to two Fab fragments linked by disulfide bonds, with different antigen binding activities and still able to cross-link antigen. Fab' fragments differ from Fab fragments by the addition of some additional residues at the carboxyl terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. F(ab')2 antibody fragments were originally generated as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known. The Fc fragment contains the carboxyl terminal portions of the two heavy chains held together by disulfide bonds. The effector functions of antibodies are determined by sequences in the Fc region, which is also the region recognized by Fc receptors (FcR) found on certain types of cells. Antigen binding fragments of heavy chain antibodies include VHH and single-chain heavy chain antibodies.

[0092] Antibodies herein also include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

[0093] A "humanized" form of a non-human (e.g., mouse) antibody refers to a chimeric antibody that minimally contains sequences derived from non-human immunoglobulins. Therefore, a "humanized antibody" generally refers to a non-human antibody in which the variable domain framework region is exchanged with sequences found in human antibodies. Typically in a humanized antibody, the entire antibody (except for the CDRs) is encoded by a polynucleotide of human origin or is identical to such an antibody (except for the CDRs). The CDRs (some or all of which are encoded by nucleic acids derived from non-human organisms) are transplanted into the β-folded framework of the variable region of a human antibody to produce an antibody whose specificity is determined by the transplanted CDRs. Methods for producing such antibodies are well known in the art, for example, using mice with a genetically engineered immune system.

[0094] "Human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and / or produced using any of the techniques disclosed herein for producing human antibodies. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. The binding molecules described in any embodiment of the present invention are chimeric antibodies or fully human antibodies; preferably fully human antibodies.

[0095] The term "immune cell" refers to a cell involved in or associated with an immune response, examples of which include, but are not limited to, lymphocytes, dendritic cells, monocytes / macrophages, granulocytes, mast cells, etc. Preferably, the immune cell is, for example, a T cell, a NK cell, a NKT cell, a tumor-infiltrating lymphocyte, and a macrophage. In certain embodiments, the immune cell of the present invention includes a T cell, such as a CD4+T cell and / or a CD8+T cell.

[0096] The "MUC1 binding molecule" described herein comprises a MUC1 single domain antibody, wherein the complementarity determining region CDR of the MUC1 single domain antibody comprises CDR1, CDR2 and CDR3, wherein CDR1 comprises the sequence shown in SEQ ID NO:1, CDR2 comprises the sequence shown in SEQ ID NO:2, and CDR3 comprises the sequence shown in SEQ ID NO:3, wherein,

[0097] SEQ ID NO: 1 is GX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 , where X 1 is P, F, L, A or R, X 2 H, T, S or F, X 3L, F, S, G, T or A, X 4 is E, R, D, L or N, X 5 L, R, Y, N, D, Q, I or E, X 6 is Y, H, L, E or I, X 7 A, Y, D, M, E or T, X 8 is Y, E or None, X 9 is Y or None, X 10 is N or None,

[0098] SEQ ID NO: 2 is IX 1 X 2 X 3 X 4 X 5 X 6 X 7 , where X 1 S, N or R, X 2 is E, T, G, R or W, X 3 is S, N, R, I, F or Y, X 4 is G, D or N, X 5 D, G, S, A, E, T or none, X 6 D, T, S, R, P, L, W or none, X 7 is T, I or None,

[0099] SEQ ID NO:3 is

[0100] X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 , where X 1 A, T or N, X 2 A or T, X 3 is C, I, E, G or V, X 4 T, G, R, V or D, X 5 is F, Y, T, I, A, N or P, X 6 Y, P, R, G, L or A, X7 G, S, F, R, L or H, X 8 is S, Q, I, T, W, K or Y, X 9 T, L, F, P, S or none, X 10 F, S, G, C, T or None, X 11 R, Y, T, L, S, C or none, X 12 E, D, C, F, Y or None, X 13 M, Y, V, L or none, X 14 G, P, H, A, T or none, X 15 Y, S, Q, R, W, V or none, X 16 N, R, T, M, P or None, X 17 F, E, A, Q or none, X 18 A, G, Y or none, X 19 S, D, R or none, X 20 Is Y or None.

[0101] In one or more embodiments, the CDR1 of the MUC1 single domain antibody comprises a sequence as shown in any one of SEQ ID NOs: 4-13, CDR2 comprises a sequence as shown in any one of SEQ ID NOs: 14-24, and CDR3 comprises a sequence as shown in any one of SEQ ID NOs: 25-35.

[0102] Preferably, the single-domain antibody contains CDR1, CDR2 and CDR3 shown in SEQ ID NO of any one of the following groups a1 to a11:

[0103] Group CDR1 CDR2 CDR3 a1 4 14 25 a2 5 15 26 a3 6 16 27 a4 7 17 28 a5 8 18 29 a6 9 19 30 a7 10 20 31 a8 10 21 32 a9 11 22 33 a10 12 23 34 a11 13 24 35 .

[0104] In one or more embodiments, the FR region of the MUCl single domain antibody comprises the FR region of any one VHH selected from SEQ ID NOs: 36-46.

[0105] In one or more embodiments, the MUCl single domain antibody VHH is as shown in any one of SEQ ID NOs:36-46, or has at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of any one of SEQ ID NOs:36-46.

[0106] The MUC1 binding molecules described herein are monovalent or multivalent single domain antibodies, multispecific single domain antibodies, heavy chain antibodies or antigen binding fragments thereof, antibodies or antigen binding fragments thereof comprising one, two or more MUC1 single domain antibodies described herein. The multivalent single domain antibody or multispecific single domain antibody connects multiple single domain antibodies via a linker. The linker consists of 1-15 amino acids selected from G and S. The heavy chain antibody is a camel heavy chain antibody or a cartilaginous fish heavy chain antibody. The antigen binding fragment of the heavy chain antibody is a single chain heavy chain antibody. The heavy chain antibody further comprises a heavy chain constant region. In one or more embodiments, the heavy chain constant region is a constant region of a camel heavy chain antibody, comprising CH2 and CH3. In one or more embodiments, the CH2 and CH3 are CH2 and CH3 of human IgG Fc, such as CH2 and CH3 of IgG4.

[0107] The optional signal peptide on CAR can be selected as needed. For example, a CD8 signal peptide, a CD28 signal peptide, a CD4 signal peptide or a light chain signal peptide, the sequence of which is within the knowledge of those skilled in the art. The CD8 signal peptide suitable for the present invention can be various human CD8 signal peptide sequences commonly used in CAR in the art. In certain embodiments, the amino acid sequence of the human CD8 signal peptide can be as shown in SEQ ID NO:47.

[0108] The hinge region of CAR is selected from CD8α hinge region, IgD hinge region, IgG1 Fc CH2CH3 hinge region or IgG4 FcCH2CH3 hinge region, and its sequence is within the knowledge of those skilled in the art. The CD8 hinge region suitable for the present invention can be various human CD8 hinge region sequences commonly used in CAR in the art. In certain embodiments, the amino acid sequence of the human CD8 hinge region can be as shown in SEQ ID NO:48.

[0109] The transmembrane region of CAR is selected from one of the CD28 transmembrane region, the CD8 transmembrane region, the CD3ζ transmembrane region, the CD134 transmembrane region, the CD137 transmembrane region, the ICOS transmembrane region and the DAP10 transmembrane region; preferably the CD8 transmembrane region, the sequence of which is within the knowledge of those skilled in the art. The human CD8 transmembrane region suitable for the present invention can be various human CD8 transmembrane region sequences commonly used in CAR in the art. In certain embodiments, the amino acid sequence of the human CD8 transmembrane region is as shown in SEQ ID NO:49.

[0110] Suitable intracellular costimulatory domains can be selected as needed, including intracellular domains with costimulatory signal molecules, such as CD28, CD134 / OX40, CD137 / 4-1BB, lymphocyte-specific protein tyrosine kinase, inducible T cell costimulatory factor (ICOS) and DNAX activation protein 10 intracellular domains. The human 4-1BB costimulatory domains suitable for the present invention can be various human 4-1BB costimulatory domain sequences commonly used in CAR in the art. In certain embodiments, the amino acid sequence of the 4-1BB costimulatory domain is as shown in SEQ ID NO:50.

[0111] Similarly, the intracellular signaling domain of CAR can be selected as needed, including but not limited to CD3ζ intracellular signaling domain or FcεRIγ intracellular signaling domain. The CD3ζ intracellular signaling domain suitable for the present invention can be various CD3ζ intracellular signaling domains for CAR known in the art. In certain embodiments, the amino acid sequence of the CD3ζ intracellular signaling domain is shown in SEQ ID NO:51.

[0112] The above-mentioned parts forming the chimeric antigen receptor of the present invention, such as CD8 signal peptide, anti-MUC1 single domain antibody, CD8 hinge region, CD8 transmembrane region, 4-1BB co-stimulatory domain, CD3ζ intracellular signal domain, etc., can be directly connected to each other, or can be connected through a linker sequence. The linker sequence can be a linker sequence suitable for antibodies known in the art, such as a linker sequence containing G and S. Generally, the linker contains one or more motifs repeated before and after. For example, the motif can be GGGS, GGGGS, SSSSG, GSGSA and GGSGG. Preferably, the motif is adjacent in the linker sequence, and no amino acid residues are inserted between the repeats. The linker sequence can be composed of 1, 2, 3, 4 or 5 repeating motifs. The length of the linker can be 3 to 25 amino acid residues, such as 3 to 15, 5 to 15, 10 to 20 amino acid residues. In certain embodiments, the linker sequence is a polyglycine linker sequence. The number of glycine residues in the linker sequence is not particularly limited, and is generally 2 to 20, such as 2 to 15, 2 to 10, or 2 to 8. In addition to glycine and serine, the linker may also contain other known amino acid residues, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), glutamine (Q), etc. In certain embodiments, the linker sequence is (GGGGS)n, where n is an integer of 1 to 5.

[0113] In an exemplary embodiment, CAR contains CD8 signal peptide, anti-MUC1 nanobody, CD8 hinge region, CD8 transmembrane region, 4-1BB costimulatory domain, CD3ζ intracellular signal domain from N-terminus to C-terminus. In a specific embodiment, an exemplary CAR with the above structure is shown in any one of SEQ ID NO: 52-62.

[0114] It should be understood that in gene cloning operations, it is often necessary to design suitable restriction sites, which will inevitably introduce one or more irrelevant residues at the end of the expressed amino acid sequence, which does not affect the activity of the target sequence. In order to construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside the host cell, or facilitate the purification of recombinant proteins, it is often necessary to add some amino acids to the N-terminus, C-terminus or other suitable regions within the protein of the recombinant protein, for example, including but not limited to suitable linker peptides, signal peptides, leader peptides, terminal extensions, etc. Therefore, the amino or carboxyl terminus of the CAR of the present invention may also contain one or more polypeptide fragments as protein tags. Any suitable tag can be used herein. For example, the tag may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE and Ty1. These tags can be used to purify proteins.

[0115] The present invention also includes mutants of CAR described in any embodiment. These mutants include: an amino acid sequence having at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity with the CAR and retaining the biological activity (such as activated T cells) of the CAR. The sequence identity between the two aligned sequences can be calculated using, for example, NCBI's BLASTp.

[0116] Mutants also include: amino acid sequences having one or more mutations (insertions, deletions or substitutions) in the amino acid sequence of the CAR described in any embodiment, while still retaining the biological activity of the CAR. The several mutations generally refer to within 1-10, such as 1-8, 1-5 or 1-3. The substitution is preferably a conservative substitution. For example, in the art, when conservative substitutions are made with amino acids with similar or similar properties, the function of the protein or polypeptide is generally not changed. "Amino acids with similar or similar properties" include, for example, families of amino acid residues with similar side chains, including amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, replacing one or several sites in the polypeptide of the present invention with another amino acid residue from the same side chain class will not substantially affect its activity.

[0117] The present invention includes polynucleotide sequences encoding the fusion proteins of the present invention. The polynucleotide sequences of the present invention may be in the form of DNA or RNA. The DNA forms include cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The present invention also includes degenerate variants of the polynucleotide sequences encoding the fusion proteins, i.e., nucleotide sequences encoding the same amino acid sequence but with different nucleotide sequences.

[0118] The polynucleotide sequences described herein can generally be obtained by PCR amplification. Specifically, primers can be designed based on the nucleotide sequences disclosed herein, especially the open reading frame sequences, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art can be used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified in each amplification together in the correct order.

[0119] The present invention also relates to nucleic acid constructs, and the term "nucleic acid construct" or "polynucleotide construct" refers to one or more single-stranded or double-stranded nucleic acid molecules that are separated from naturally occurring genes or modified to contain nucleic acid fragments in a manner that does not exist in nature. The term "nucleic acid molecule" mainly refers to a physical nucleic acid molecule, and the term "nucleic acid sequence" mainly refers to a nucleotide sequence on a nucleic acid molecule, but the two terms are interchangeable, especially with respect to nucleic acid molecules, or nucleic acid sequences, that can encode proteins or protein domains. The nucleic acid construct contains the polynucleotide sequences described herein, and one or more regulatory sequences operably connected to these sequences. The polynucleotide sequences described in the present invention can be manipulated in a variety of ways to ensure the expression of the CAR. Before the nucleic acid construct is inserted into the vector, the nucleic acid construct can be manipulated according to the different or required expression vectors. The technology of using recombinant DNA methods to change polynucleotide sequences is known in the art.

[0120] The regulatory sequence can be a suitable promoter sequence. The promoter sequence is usually operably connected to the coding sequence of the protein to be expressed. The promoter can be any nucleotide sequence that shows transcriptional activity in the selected host cell, including mutant, truncated and hybrid promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell. The regulatory sequence can also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably connected to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used in the present invention. The regulatory sequence can also be a suitable leader sequence, a non-translated region of an mRNA that is important for host cell translation. The leader sequence is operably connected to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used in the present invention.

[0121] In certain embodiments, the nucleic acid construct is a vector. The term "vector" can transfer a gene sequence to a target cell. Generally, "vector construct", "expression vector" and "gene transfer vector" mean any nucleic acid construct that can direct the expression of a gene of interest and can transfer a gene sequence to a target cell, which can be achieved by genome integration of the whole or part of the vector, or by transient or heritable maintenance of the vector as an extrachromosomal element. Therefore, the term includes cloning vectors, expression vectors and integration vectors. Usually, the expression of the polynucleotide sequence of the present invention is achieved by operably connecting the polynucleotide sequence of the present invention to a promoter, and incorporating the construct into an expression vector. The vector may be suitable for replication and integration of eukaryotic cells. A typical cloning vector comprises transcription and translation terminators, initiation sequences and promoters that can be used to regulate the expression of the desired nucleic acid sequence. The nucleic acid construct may be one or more vectors, each of which comprises one or two or three expression cassettes described in any embodiment of this article.

[0122] The polynucleotide sequence of the present invention can be cloned into many types of vectors. For example, it can be cloned into plasmids, phagemids, phage derivatives, animal viruses and cosmids. Further, the vector is an expression vector. The expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and slow viruses.

[0123] Typically, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (eg, WO 01 / 96584; WO 01 / 29058; and US Pat. No. 6,326,193).

[0124] The nucleic acid construct may be a cloning vector or an expression vector. The expression vector is preferably a constitutive expression vector, such as a transposition vector (or "transposon vector"). In certain embodiments, the transposon vector is a eukaryotic expression vector containing a transposition element selected from piggybac, sleeping beauty, frog prince, Tn5 or Ty. This type of transposon vector contains the 5' inverted terminal repeat sequence (5'ITR) of the corresponding transposon and the 3' inverted terminal repeat sequence (3'ITR) of the corresponding transposon. Therefore, in some embodiments of the present invention, the nucleic acid construct further comprises a coding sequence of a transposase, which is used to transpose the coding sequence of a chimeric antigen receptor, for example, integrating the coding sequence or expression cassette of a chimeric antigen receptor into the genome. The transposase may be any transposase known in the art that is suitable for human cells, for example, a transposase from a piggybac, sleeping beauty, frog prince, Tn5 or Ty transposition system. When using transposases from different transposition systems, the sequences of 5'ITR and 3'ITR in the vector are also changed accordingly to sequences compatible with the transposition system, which can be easily determined by those skilled in the art. The expression frame of the CAR or antibody of the present invention is contained between 5'ITR and 3'ITR, including the corresponding promoter sequence, the coding sequence of CAR or antibody, and the polyA tailing signal sequence. An exemplary transposase is a PB enzyme. In certain embodiments, the 5' reverse terminal repeat sequence of the transposon is shown in SEQ ID NO:3 of CN111206043A (the contents of which are incorporated herein by reference). In certain embodiments, the 3' reverse terminal repeat sequence of the transposon is shown in SEQ ID NO:7 of CN111206043A. In certain embodiments, the piggybac transposase is a transposase containing a c-myc nuclear localization signal coding sequence. In certain embodiments, the coding sequence of the piggybac transposase is shown in SEQ ID NO:2 of CN111206043A. Exemplary vector backbones are pNB338B and pS338B described in CN111206043A and CN114729361A.

[0125] Therefore, in one or more embodiments, the nucleic acid construct comprises the coding sequence of CAR and transposase. In one or more embodiments, the nucleic acid construct contains the expression cassette of the chimeric antigen receptor and the expression cassette of the transposase. The two expression cassettes are contained in one or two vectors. Alternatively, the nucleic acid construct is an expression cassette, wherein the coding sequence of the chimeric antigen receptor and the coding sequence of the transposase are in the expression cassette.

[0126] In some cases, the nucleic acid construct described herein comprises a CAR and a transposase coding sequence. The nucleic acid construct may contain two expression cassettes, i.e., an expression cassette of the chimeric antigen receptor and an expression cassette of the transposase; the two expression cassettes may be contained in one or two vectors.

[0127] In one or more embodiments, the nucleic acid construct comprises: a vector comprising (a) an expression cassette for CAR and optionally (b) an expression cassette for a transposase; or two vectors comprising an expression cassette for CAR and an expression cassette for a transposase, respectively.

[0128] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence that can drive any polynucleotide sequence operably connected thereto to express at a high level. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). Another example of a suitable promoter is the promoter driven by IFNγ. However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia 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, it may also be considered to use an inducible promoter. The use of an inducible promoter provides a molecular switch that is capable of turning on the expression of a polynucleotide sequence operably linked to the inducible promoter when expression is desired, and turning off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0129] In order to evaluate the expression of CAR polypeptides or parts thereof, the expression vector introduced into the cell may also include any one or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a cell population seeking to be transfected or infected by a viral vector. In other aspects, selectable markers may be carried on a single DNA segment and used for co-transfection procedures. Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences so that they can be expressed in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

[0130] Reporter genes are used to identify cells of potential transfection and to evaluate the functionality of regulatory sequences. After DNA has been introduced into recipient cells, the expression of reporter genes is measured at the appropriate time. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein genes. Suitable expression systems are known and can utilize known techniques to prepare or commercially obtain.

[0131] Methods for introducing genes into cells and expressing genes into cells are known in the art. Vectors can be easily introduced into host cells, for example, mammalian, bacterial, yeast or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical or biological means.

[0132] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. 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.

[0133] T cells suitable for the present invention can be various types of T cells from various sources. For example, T cells can be derived from PBMCs of patients with B cell malignancies. In certain embodiments, after obtaining T cells, they can be first stimulated and activated with an appropriate amount (e.g., 30 to 80 ng / ml, such as 50 ng / ml) of CD3 antibody, and then cultured in a medium containing an appropriate amount (e.g., 30 to 80 IU / ml, such as 50 IU / ml) of IL2 for standby use.

[0134] Therefore, in certain embodiments, the present invention provides a genetically modified T cell, which contains a polynucleotide sequence as described herein, or contains a nucleic acid construct as described herein, or is prepared using the method described herein, or stably expresses CAR as described herein. Cells described herein (e.g., T cells) may also include or express a transposase or its coding sequence.

[0135] The CAR-T cells of the present invention can undergo robust in vivo T cell expansion and persist at high levels in the blood and bone marrow for an extended amount of time, and form specific memory T cells. Without wishing to be bound by any particular theory, after encountering and subsequently eliminating target cells expressing alternative antigens, the CAR-T cells of the present invention can differentiate into a central memory-like state in vivo.

[0136] The present invention also includes a class of cell therapies in which T cells are genetically modified to express the CAR described herein, and the CAR-T cells are infused into a recipient in need thereof. The infused cells are capable of killing the recipient's tumor cells. Unlike antibody therapies, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

[0137] The anti-tumor immune response caused by CAR-T cells can be an active or passive immune response. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy procedure, in which the CAR-T cells induce an immune response specific to the antigen-binding portion in the CAR.

[0138] Therefore, the disease that can be treated with the CAR, its coding sequence, nucleic acid construct, expression vector and CAR-T cell of the present invention is preferably MUC1-mediated cancer.

[0139] Specifically, herein, "MUC1-mediated cancer" especially includes various types of breast cancer, kidney cancer, ovarian cancer, gastric cancer, pancreatic cancer, lung cancer, colon cancer, osteosarcoma, adenocarcinoma, bladder cancer, colorectal cancer, cervical cancer, head and neck cancer, fallopian tube cancer, multiple myeloma, bile duct cancer, gallbladder cancer, esophageal cancer, prostate cancer or glioblastoma.

[0140] The CAR-modified T cells of the present invention may be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as related cytokines or cell groups. Simply put, the pharmaceutical composition of the present invention may include CAR-T cells as described herein, combined with one or more pharmaceutically or physiologically acceptable excipients (e.g., carriers, diluents, or excipients). Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0141] 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 such factors as the patient's condition, and the type and severity of the patient's disease.

[0142] When an "immunologically effective amount", "anti-tumor effective amount", "tumor-suppressive effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, who takes into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. It can be generally stated that the pharmaceutical composition comprising the T cells described herein can be administered in an amount of 10 4 Up to 10 9The dosage is preferably 10 cells / kg body weight. 5 Up to 10 6 The T cell composition can also be administered multiple times at these doses. The cells can be administered using well-known infusion techniques in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a particular patient can be easily determined by a medical technician by monitoring the patient's disease signs and adjusting the treatment accordingly.

[0143] Administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous injection or intraperitoneally. In one embodiment, the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by intravenous injection. The composition of the T cell can be directly injected into a tumor, lymph node or infection site.

[0144] In some embodiments of the present invention, the CAR-T cells or compositions thereof of the present invention may be combined with other therapies known in the art. The therapies include, but are not limited to, chemotherapy, radiotherapy, and immunosuppressants. For example, the treatment may be combined with radiotherapy or chemotherapy agents known in the art for treating MUC1-mediated diseases.

[0145] Herein, "anti-tumor effect" refers to a biological effect which can be represented by a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with cancer.

[0146] "Patient", "subject", "individual" and the like are used interchangeably herein and refer to a living organism, such as a mammal, that can elicit an immune response. Examples include, but are not limited to, humans, dogs, cats, mice, rats, and transgenic species thereof.

[0147] The present invention is further described in detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention should never be construed as being limited to the following examples, but should be construed as including any and all variations that become apparent due to the teachings provided herein. The methods and reagents used in the examples, unless otherwise stated, are conventional methods and reagents in the art.

[0148] Example

[0149] Example 1, MUC1 CAR-T cell plasmid construction

[0150] The chimeric antigen receptors involved in Example 1 are shown in Table 1, wherein the chimeric antigen receptors 1A2, 1C4, EFP-B02, EFP-B03, 1D2-2, AI-120, AI-121, AI-181, AI-201, AI-212, and A12-6-LST-D5-2 contain the MUC1 single domain antibody provided by the present invention, and the chimeric antigen receptor BMK is derived from CN109715670A, and the sequence of the antigen binding domain of BMK is shown in SEQ ID NO:64.

[0151] Table 1: Chimeric antigen receptor sequences involved in Example 1

[0152]

[0153]

[0154]

[0155] MUC1 CAR-T cells were constructed by using MUC1 CAR and BMK CAR derived from single-domain antibodies of different sequences and then transferring the genes into PBMCs from healthy subjects by electroporation. The in vitro performance of CAR-T cells was verified, including proliferation ability, CAR positivity rate, cell phenotype, and in vitro killing ability of target cells.

[0156] Experimental instruments: ordinary PCR instrument (Jena, TRIO), electrophoresis instrument (Bio-Rad, 1645050), electrophoresis tank (Bio-Rad, 1704483), desktop microcentrifuge (Thermo, Pico17), metal bath heating module (Thermo, DCD), electric constant temperature water bath (Shanghai Yiheng, DK-8D), automatic gel imager (Tianneng Tanon, 2500)

[0157] Experimental reagents

[0158] Reagent name Manufacturer MUC1 single domain antibody Self-produced PCR tubes AXYGEN 1.5mL EP tube AXYGEN Restriction enzymes BioLabs DNA ligase TaKaRa PCR enzymes TOYOBO Homologous recombination enzyme BioLabs Agarose TIANGEN Agarose Gel Extraction Kit ZYMO RESEARCH TRYPTONE OXOID YEAST EXTRACT OXOID NaCl Shanghai Test Plasmid Extraction Kit TIANGEN Plasmid Maxi-Pull Kit MACHEREY-NAGEL Kana Bioengineering

[0159] Experimental methods

[0160] According to the sequences of MUC1 single domain antibody and BMK, the full-length nucleic acid sequence and primers were synthesized, a large number of target fragments were obtained by PCR, the PC23S vector was double-digested (EcoR1 and Sal1) to obtain the vector fragment, and the target fragment and the vector fragment were connected by DNA ligase to obtain a plasmid. The constructed vector was sequenced, and the plasmid was extracted in large quantities after the sequence was correct. The PC23S vector sequence is shown in SEQ ID NO:65.

[0161] Example 2: Preparation of CAR-T cells targeting MUC1

[0162] Mock-T and MUC1 CAR-T were prepared by electroporation.

[0163] Experimental instruments: electroporator (Lonza 4D), automated cell counter (Cellomter K2), microscope (Jiangnan XD-202), biological safety cabinet (Thermo Fisher Scientific), refrigerator (Qingdao Haier), carbon dioxide incubator (Thermo), desktop low-speed centrifuge (Xiangyi), Pico17 desktop microcentrifuge (Thermo).

[0164] PBMCs were revived using AIM-V medium (containing 2% FBS), inoculated into culture flasks at 5E6 / mL, and placed in an incubator (37°C; 5% CO 2 ) overnight. 6-well plates or culture flasks were coated with 5ug / mL anti-CD3 and anti-CD28 at 4°C overnight. PBMCs placed overnight were inoculated into the coated 6-well plates or culture flasks and stimulated for 48h. Cells were collected and electroporated using a Lonza 4D electroporator. The electroporation process was optimized according to the electroporator manual. 5×10 6 to 7×10 6 For PBMC cells, 6ug of the above-prepared plasmid was added to the cell suspension, and 20ug of PB mRNA (the amino acid sequence of the PB enzyme is shown in SEQ ID NO:66, and the nucleotide sequence of the template plasmid of the PB enzyme mRNA is shown in SEQ ID NO:67) and 200IU of Rnase inhibitor were added to the electroporation system, and electroporation was performed using the EO-115 program. After electroporation, the cells were allowed to stand at room temperature for 10 minutes. After that, the PBMCs were transferred to a 6-well plate containing 2mL of AIM-V (containing 2% FBS and 100IU / mL IL-2) culture medium (need to be preheated to 37°C in advance), placed in an incubator (37°C; 5% CO2) and cultured for 24h-48h, and then the culture medium was supplemented to 4mL. The first subculture was performed on the 3rd day after electroporation, the second subculture was performed on the 6th day, and the CAR-T cell preparation was completed on the 9th day. During the subculture process, PBMCs were cultured in AIM-V medium containing 2% FBS and 100 IU / ml IL-2, and the cell density was 5×10 5 / ml. During and after the CAR-T cell preparation process, flow cytometry was used to detect the cell surface CAR positivity rate and other surface markers.

[0165] Example 3, in vitro proliferation, positive rate and cell phenotype detection of MUC1 CAR-T cells

[0166] The purpose of this experiment is to evaluate the quality of the prepared mock-T, BMK CAR-T cells, and MUC1 CAR-T cells by proliferation and positive rate.

[0167] Experimental instruments: automated cell counter (Cellomter K2), microscope (Jiangnan XD-202), biological safety cabinet (Thermo Fisher Scientific), refrigerator (Qingdao Haier), carbon dioxide incubator (Thermo), desktop low-speed centrifuge (Xiangyi), flow cytometer (Beckman Coulter CytoFLEX S Flow Cytometer) Pico17 desktop microcentrifuge (Thermo).

[0168] Experimental reagents

[0169] Reagent name Manufacturer AIM-V medium GIBCO FBS GIBCO DPBS Hyclone Recombinant human interleukin-2 for injection Shandong Quangang Pharmaceutical Co., Ltd. Serum-free cell freezing medium New Saimei 0.4% Trypan blue dye Gibco Biotinylated MUC1 antigen Jiangsu Huakang Biotechnology Co., Ltd. PE Streptavidin BD

[0170] Donor cell information

[0171] Donor Number supplier Product form Z0020 Aoneng PBMCs Z0313 Aoneng PBMCs

[0172] Experimental methods

[0173] 1) CAR-T cell culture and passaging

[0174] By counting cells and passing them at equal density, the proliferation times of each group of cells were calculated and the cell proliferation curve was drawn. The passaging density was 5×10 5 Cells were passaged and counted on Day 3, 6, and 9; the IL-2 concentration was 100 U / mL. See the experimental procedure for details.

[0175] 2) Detection of the proportion of MUC1 CAR-T positive cells

[0176] The CAR positive rate of cells during proliferation was detected by flow cytometry. See the experimental procedure for details.

[0177] 3) MUC1 CAR-T cell phenotype detection

[0178] The phenotype of the CART cells we prepared was evaluated by detecting the CD3 / CD4 / CD8 and TCM / TEM of MUC1 CART using flow cytometry.

[0179] Experimental instruments: automated cell counter (Invitrogen CountessⅡFL Cellomter K2), microscope (Jiangnan XD-202), Pico17 desktop microcentrifuge (Thermo), flow cytometer (Beckman Coulter CytoFLEX S Flow Cytometer)

[0180] Experimental reagents

[0181]

[0182]

[0183] Experimental Results

[0184] CAR-T cell proliferation, viability, positive rate and phenotype Figure 1-4 As shown. Figure 1 , Figure 2 As shown in the figure, the total proliferation of T cells in group 1C4 on D9 was about 15 times higher than that on D0, and the cell viability was maintained above 90%. Figure 3 As shown, the positive rate of group 1C4 was higher. Figure 4 It shows that the proportion of CD4 and CD8 in the preparation of D9 CAR T, the proportion of CD8+T cells in the 1C4 group is significantly higher than that of CD4+T cells.

[0185] Example 4, MUC1 CAR-T cell killing experiment

[0186] The killing effect of CAR-T cells on tumor cells was detected using the xCELLigence RTCA device (Roche AppliedScience, Canada), and the detection process was carried out according to the device operating instructions. 10,000 target cells (MB468, HCT116) were resuspended in 50ul culture medium and inoculated into the microplate of the RTCA device. The tumor cells were cultured on the RTCA device for about 24 hours. When the cell index (CI, indicating cell growth) reached about 1.5, different numbers of effector cells (CAR-T cells) were resuspended in 50ul culture medium according to different effect-target ratios (effector cells: target cells), mixed with tumor cells, and continued to be cultured on the RTCA device for 48h-96h. The cell killing curve was recorded by the RTCA device, and the data was analyzed using RTCAPro 2.3.0 software.

[0187] Figure 5The figure shows the time required for CAR T cells in the 1C4 group to kill 50% of MB468 MUC1-high-expressing tumor cells under different effector-target ratios, i.e., the KT50 value. CAR T cells in the 1C4 group were able to reach KT50 within 24 hours at an effector-target ratio of 1:4. As the effector-target ratio decreased, the killing ability of each group also decreased accordingly.

[0188] Figure 6 The 48h tumor lysis rate of 1C4 group CAR T for HCT116 tumor cells with extremely low MUC1 expression. The results showed that 1C4 group CAR T had no killing ability for tumor cells with low MUC1 expression, proving that the CAR-T in this patent has low non-target specific killing and high safety.

[0189] Example 5, Continuous Proliferation Experiment of MUC1 CAR-T Cells

[0190] MB468 tumor cells were inoculated into six-well plates one day in advance (600,000 tumor cells per well). After overnight incubation at 37°C, the tumor cells were treated with mitomycin C (25ug / mL) for 4h to inhibit their growth. The tumor cells were washed 5 times with PBS. CAR-positive T cells were inoculated at a 1:1 effector-target ratio, and one round was performed every 5 days. After one round, the number of cells was counted using a cell counter, and the CAR positivity rate was detected using a flow cytometer. The number of CAR-positive cells was calculated, and a new round of tumor co-culture experiments was started at a 1:1 effector-target ratio. This was repeated until the end of the fifth round. At the same time, 24h after the start of each round of co-culture experiments, that is, 24h after CAR-T was inoculated into the tumor cell plate, part of the supernatant was collected, and the Cytokine Beads Assay detection kit was used to detect the level of CAR-T cell factor release in each group.

[0191] Figure 7 The CAR positivity rate of the two donors after each round of co-culture is shown. The CAR positivity rate of the 1C4 group gradually increased in the first two rounds of co-culture, and a large difference began to appear after the third round, and 1C4 was still able to maintain a high level.

[0192] Figure 8 The figure shows the theoretical proliferation times of CAR-positive T cells from two donors at the end of the fifth round compared to the number of CAR-positive cells inoculated at the beginning. The proliferation times of the two donor 1C4 groups were higher.

[0193] Fig. 9 The results show the IFN-γ secretion of CAR T in the 1C4 group in the first three rounds of the co-culture experiment. Both donors showed that the cytokine release of CAR T in the 1C4 group was high in the second round of the experiment, and began to decrease in the third round, which was consistent with the results of the change in CAR positivity rate.

[0194] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation modes of the present invention. For ordinary technicians in the relevant field, various changes, modifications, substitutions and modifications made on the basis of the present invention without departing from the principles and purpose of the present invention are still within the scope of protection of the present invention.

Claims

1. A chimeric antigen receptor, the antigen binding domain of which comprises a MUC1 binding molecule comprising a MUC1 single domain antibody, The complementary determining region CDR of the MUC1 single domain antibody comprises CDR1, CDR2 and CDR3, wherein CDR1 comprises the sequence shown in SEQ ID NO: 1, CDR2 comprises the sequence shown in SEQ ID NO: 2, and CDR3 comprises the sequence shown in SEQ ID NO: 3, wherein, SEQ ID NO: 1 is GX1X2X3X4X5X6X7X8X9X 10 , wherein X1 is P, F, L, A or R, X2 is H, T, S or F, X3 is L, F, S, G, T or A, X4 is E, R, D, L or N, X5 is L, R, Y, N, D, Q, I or E, X6 is Y, H, L, E or I, X7 is A, Y, D, M, E or T, X8 is Y, E or none, X9 is Y or none, X 10 is N or None, SEQ ID NO:2 is IX1X2X3X4X5X6X7, wherein X1 is S, N or R, X2 is E, T, G, R or W, X3 is S, N, R, I, F or Y, X4 is G, D or N, X5 is D, G, S, A, E, T or none, X6 is D, T, S, R, P, L, W or none, X7 is T, I or none, SEQ ID NO:3 is X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 , wherein X1 is A, T or N, X2 is A or T, X3 is C, I, E, G or V, X4 is T, G, R, V or D, X5 is F, Y, T, I, A, N or P, X6 is Y, P, R, G, L or A, X7 is G, S, F, R, L or H, X8 is S, Q, I, T, W, K or Y, X9 is T, L, F, P, S or none, X 10 F, S, G, C, T or None, X 11 R, Y, T, L, S, C or none, X 12 E, D, C, F, Y or None, X 13 M, Y, V, L or none, X 14 G, P, H, A, T or none, X 15 Y, S, Q, R, W, V or none, X 16 N, R, T, M, P or None, X 17 F, E, A, Q or none, X 18 A, G, Y or none, X 19 S, D, R or none, X 20 Is Y or None.

2. The chimeric antigen receptor according to claim 1, wherein The CDR1 of the MUC1 single domain antibody comprises a sequence shown in any one of SEQ ID NOs: 4-13, the CDR2 comprises a sequence shown in any one of SEQ ID NOs: 14-24, and the CDR3 comprises a sequence shown in any one of SEQ ID NOs: 25-35, Preferably, the single-domain antibody contains CDR1, CDR2 and CDR3 shown in SEQ ID NO of any one of the following groups a1 to a11:

3. The chimeric antigen receptor according to claim 1 or 2, characterized in that The FR region of the MUC1 single-domain antibody comprises the FR region of any VHH selected from SEQ ID NO: 36-46, and / or The MUC1 single domain antibody VHH is as shown in any one of SEQ ID NOs: 36-46, or has at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence of any one of SEQ ID NOs: 36-46, and / or The MUC1 binding molecule is a monovalent or multivalent single domain antibody, a multispecific single domain antibody, a heavy chain antibody or an antigen binding fragment thereof, an antibody or an antigen binding fragment thereof comprising one, two or more of the single domain antibodies.

4. The chimeric antigen receptor according to claim 3, wherein The chimeric antigen receptor comprises: an optional signal peptide sequence, a MUC1 binding molecule comprising a MUC1 single domain antibody, a hinge region, a transmembrane region, and an intracellular region; Preferably, the chimeric antigen receptor also has one or more characteristics selected from the following: The signal peptide includes a CD8 signal peptide, a CD28 signal peptide, a CD4 signal peptide or a light chain signal peptide, The hinge region includes a CD8 hinge region, an IgD hinge region, an IgG1 Fc CH2CH3 hinge region or an IgG4 Fc CH2CH3 hinge region, The transmembrane region includes a CD28 transmembrane region, a CD8 transmembrane region, a CD3ζ transmembrane region, a CD134 transmembrane region, a CD137 transmembrane region, an ICOS transmembrane region or a DAP10 transmembrane region, The intracellular region includes an intracellular co-stimulatory domain and / or an intracellular signaling domain. The intracellular co-stimulatory domain includes the intracellular domain of CD28, CD134 / OX40, CD137 / 4-1BB, lymphocyte-specific protein tyrosine kinase, inducible T cell co-stimulator (ICOS) or DNAX activation protein 10, The intracellular signaling domain includes a CD3ζ intracellular signaling domain or a FcεRIγ intracellular signaling domain.

5. A nucleic acid molecule comprising a sequence selected from the group consisting of: (1) The coding sequence of the chimeric antigen receptor according to any one of claims 1 to 4, (2) the complementary sequence of (1), and (3) A 5-50 bp fragment of any sequence in (1) or (2), Preferably, The fragment is a primer, and / or The coding sequence is DNA or RNA.

6. A nucleic acid construct comprising the nucleic acid molecule according to claim 5, Preferably, the nucleic acid construct comprises a coding sequence for a transposase and comprises one or more features selected from the following: (1) The transposase is used to transpose the coding sequence of the chimeric antigen receptor, (2) The coding sequence of the transposase is DNA or RNA, (3) the nucleic acid construct is a cloning vector, an expression vector or an integration vector, (4) The transposase is PiggyBac transposase.

7. A combination of coding sequences, comprising the coding sequence of the chimeric antigen receptor according to any one of claims 1 to 4, and further comprising a coding sequence of a transposase, preferably, the coding sequence is DNA or RNA, and the transposase is PiggyBac transposase.

8. A cell, wherein: (1) comprising and / or expressing the chimeric antigen receptor according to any one of claims 1 to 4, and / or (2) comprising the nucleic acid molecule according to claim 5 and / or the nucleic acid construct according to claim 6, and / or (3) comprising the coding sequence combination of claim 7; Preferably, the cell is a T cell.

9. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and: (1) (i) the chimeric antigen receptor and / or its coding sequence according to any one of claims 1 to 4, optionally further comprising (ii) a transposase and / or its coding sequence, or (2) The nucleic acid molecule according to claim 5, the nucleic acid construct according to claim 6, the combination of coding sequences according to claim 7, or the cell according to claim 8.

10. Use of a reagent in preparing activated immune cells, the reagent comprising: (1) (i) the chimeric antigen receptor and / or its coding sequence according to any one of claims 1 to 4, optionally further comprising (ii) a transposase and / or its coding sequence, or (2) A combination of the nucleic acid molecule according to claim 5, the nucleic acid construct according to claim 6 or the coding sequence according to claim 7.

11. Use of a reagent in the preparation of a drug for treating cancer, the reagent comprising: (1) (i) the chimeric antigen receptor and / or its coding sequence according to any one of claims 1 to 4, optionally further comprising (ii) a transposase and / or its coding sequence, or (2) the nucleic acid molecule according to claim 5, the nucleic acid construct according to claim 6, the combination of coding sequences according to claim 7, or the cell according to claim 8, Preferably, the cancer comprises a MUC1-mediated cancer, More preferably, the cancer is selected from breast cancer, kidney cancer, ovarian cancer, gastric cancer, pancreatic cancer, lung cancer, colon cancer, osteosarcoma, adenocarcinoma, bladder cancer, colorectal cancer, cervical cancer, head and neck cancer, fallopian tube cancer, multiple myeloma, bile duct cancer, gallbladder cancer, esophageal cancer, prostate cancer or glioblastoma.

Images