Engineered immune cell for overexpressing integrin as well as preparation and application of engineered immune cell

Abstract

The invention relates to an engineered immune cell for overexpressing integrin as well as preparation and application of the engineered immune cell. The engineered immune cell expresses integrin beta1 or integrin beta3. The expression of the integrin beta1 or the integrin beta3 can improve the in-vitro migration ability of the T cells, enhance the in-vitro tumor killing ability of the T cells, improve the in-vivo anti-tumor ability of the T cells and improve the infiltration proportion in T cell tumors. The T cell overexpressing the integrin beta1 or the integrin beta3 provided by the invention can enhance the infiltration capacity of the T cell and improve the effector function of the T cell so as to promote the anti-tumor capacity of the T cell, and is suitable for clinical treatment of any adoptive immunotherapy, including clinical treatment of tumors, infection and autoimmune diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an engineered immune cell overexpressing integrin, and the preparation and application thereof. Background Art

[0002] Cancer is one of the leading causes of death worldwide, severely threatening human health and life. Traditional cancer treatments, including surgery, chemotherapy, and radiotherapy, can kill tumor cells, but they are limited in scope, prone to developing drug resistance, and exhibit significant toxic side effects. Following these therapies, the emergence of tumor immunotherapy has ushered in a third revolution in cancer treatment. Unlike traditional chemotherapy and radiotherapy, immunotherapy primarily activates the body's anti-tumor immune response to eliminate tumor cells, providing long-term protection against cancer recurrence. It also offers minimal toxicity and is considered one of the most promising cancer treatments.

[0003] Among them, adoptive T cell therapies (ACT) are a type of therapy that involves isolating the body's immune cells, inducing, modifying, and expanding them in vitro, and then returning the obtained effector cells with specificity and high cytotoxicity to the patient's body to kill tumors. Currently, there are three mature adoptive T cell therapies, including tumor-infiltrating lymphocytes (TILs), T cell receptor engineered T cell therapy (TCR-engineeredT cells, TCR-T), and chimeric antigen receptor T cell therapy (CAR-T). Currently, adoptive T cell therapy has shown good efficacy in a variety of hematological tumors such as leukemia, multiple myeloma, and malignant lymphoma, such as Kymria, Yescarta, Tecartus, and Breyanzi approved by the US FDA. However, to date, adoptive T cell therapy for most solid tumors still faces huge challenges.

[0004] Among them, how to relieve the inhibitory effect of the complex tumor microenvironment on lymphocytes in solid tumors is the main challenge in the current application of adoptive immunotherapy in solid tumors. Unlike blood tumors, at the boundaries of most solid tumors, that is, the invasion front, there are a large number of cross-linked extracellular matrix proteins, chemokines, growth factors and other protein components, as well as a large number of tumor-associated fibroblasts (CAFs) and other interstitial cell components. It is precisely because tumor cells are wrapped in the extracellular matrix that the infiltration of immune cells into tumor tissues is restricted. However, the existing methods of transforming immune cells have the defects of being unable to effectively penetrate the extracellular matrix, producing certain toxic side effects, limited delivery effect in solid tumors, and insufficient infiltration ability into solid tumor cells.

[0005] Studies have shown that the infiltration rate of lymphocytes in the tumor tissue of cancer patients before immunotherapy is positively correlated with the response rate of the patient to immunotherapy. This suggests that improving the tumor infiltration ability of lymphocytes may be one of the key factors to improve the response rate of cancer patients to cellular immunotherapy. There are currently a variety of transformation strategies that target the tumor microenvironment and enhance the infiltration ability of T cells, but these methods still have certain defects. First, the transformation of chemokine receptors such as CXCR1, CXCR2 and CXCR4 can enable T cells to better respond to chemokines secreted in tumors, thereby promoting the recruitment of cells to tumor sites. However, recent studies have shown that the expression of CXCR4 in T cells will also promote T cells to migrate out of the tumor site through the peripheral lymphatic vessels enriched with CXCL12, promoting tumor growth; at the same time, CAR-T cells targeting fibroblast activation protein (Fibroblast Activation Protein, FAP) enhance the migration ability of T cells by clearing the interstitial components around solid tumors, but FAP-CAR-T cells also target FAP. + Bone marrow stromal cells cause severe cachexia and bone toxicity. Similar to FAP-CAR-T cells, CAR-T cells expressing heparanase can enhance T cell infiltration and anti-tumor activity by degrading heparan sulfate proteoglycans in the extracellular matrix (ECM). However, intravenous injection of ECM-degrading enzymes increases the risk of thromboembolism. Therefore, further research is needed in this area to develop engineered immune cells with strong infiltration capabilities and minimal side effects.

[0006] Integrin β1 (ITGB1) and integrin β3 (ITGB3) play a crucial role in T cell migration. T cell infiltration into tumors occurs in two main phases: transendothelial migration and crawling within the tumor's interstitial matrix. Adhesion molecules such as integrin β1 and integrin β3 not only mediate T cell rolling on the endothelium, promoting T cell adhesion to endothelial cells and passage through the endothelium and basement membrane into the interstitial matrix, but also regulate T cell motility within the interstitial matrix by regulating the formation of T cell pseudopodia. Therefore, integrin β1 and integrin β3 play a crucial role in T cell migration. Summary of the Invention

[0007] Problems to be solved by the invention

[0008] Based on the above problems existing in the prior art, the main purpose of the present invention is to transform T cells using integrin β1 and integrin β3 to promote the infiltration and tumor killing ability of T cells in solid tumors.

[0009] Solutions for solving problems

[0010] The present invention provides an engineered immune cell, wherein the engineered immune cell expresses integrin.

[0011] Preferably, the immune cells are selected from lymphocytes and / or their modified products.

[0012] More preferably, the lymphocytes are selected from T cells and / or monocytes.

[0013] Further preferably, the T cells are selected from one or more of αβT cells, NKT cells or γδT cells.

[0014] Preferably, the integrin is selected from one or more of integrin α1, integrin α2, integrin α3, integrin α4, integrin α6, integrin α7, integrin β1, integrin β2, integrin β3, integrin β6, integrin β7 or integrin β8.

[0015] More preferably, the integrin is selected from integrin β1 and / or integrin β3.

[0016] Preferably, the integrin β1 is the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0017] More preferably, the integrin β1 is a sequence having one or more conservative modifications compared to the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2, wherein the conservative modifications include amino acid substitution, deletion or insertion.

[0018] Further preferably, the integrin β1 is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0019] Preferably, the nucleotide sequence of the gene encoding the integrin β1 is the nucleotide sequence shown in SEQ ID NO.5 or SEQ ID NO.6 or a degenerate sequence thereof.

[0020] More preferably, the nucleotide sequence of the gene encoding the integrin β1 is a sequence having one or more conservative modifications compared to the nucleotide sequence shown in SEQ ID NO.5 or SEQ ID NO.6, wherein the conservative modification includes substitution, deletion or insertion of nucleotide derivatives.

[0021] Further preferably, the nucleotide sequence of the gene encoding the integrin β1 has the same or similar function as the protein encoded by SEQ ID NO. 5 or SEQ ID NO. 6.

[0022] Preferably, the integrin β3 has an amino acid sequence as shown in SEQ ID NO.3 or SEQ ID NO.4.

[0023] More preferably, the integrin β3 is a sequence having one or more conservative modifications compared to the amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4, wherein the conservative modifications include amino acid substitution, deletion or insertion.

[0024] Further preferably, the integrin β3 is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4.

[0025] Preferably, the nucleotide sequence of the gene encoding the integrin β3 is the nucleotide sequence shown in SEQ ID NO. 7 or SEQ ID NO. 8 or a degenerate sequence thereof.

[0026] More preferably, the nucleotide sequence of the gene encoding the integrin β3 is a sequence having one or more conservative modifications compared to the nucleotide sequence shown in SEQ ID NO.7 or SEQ ID NO.8, wherein the conservative modifications include substitution, deletion or insertion of nucleotide derivatives.

[0027] Further preferably, the nucleotide sequence of the gene encoding the integrin β3 has the same or similar function as the protein encoded by SEQ ID NO.7 or SEQ ID NO.8.

[0028] The present invention also provides an expression vector, which expresses one or more of integrin β1 or integrin β3 or their modified variants.

[0029] Preferably, the expression vector is a viral vector and / or a non-viral vector.

[0030] More preferably, the viral vector is a lentiviral vector and / or a retroviral vector.

[0031] The present invention also provides an engineered immune cell, wherein the engineered immune cell co-expresses the integrin and the tumor cell targeting receptor.

[0032] Preferably, the immune cells are selected from lymphocytes and / or their modified products.

[0033] More preferably, the lymphocytes are selected from T cells and / or monocytes.

[0034] Further preferably, the T cells are selected from one or more of αβT cells, NKT cells or γδT cells.

[0035] Preferably, the integrin is selected from one or more of integrin α1, integrin α2, integrin α3, integrin α4, integrin α6, integrin α7, integrin β1, integrin β2, integrin β3, integrin β6, integrin β7 or integrin β8.

[0036] More preferably, the integrin is selected from integrin β1 and / or integrin β3.

[0037] Preferably, the tumor cell targeting receptor is selected from chimeric antigen receptor CAR, T cell receptor TCR and / or other targeting receptors.

[0038] Preferably, the antigen binding region of the CAR is selected from the single chain variable region of a monoclonal antibody, a tumor antigen binding receptor or a ligand.

[0039] Preferably, the tumor antigen targeted by the CAR is selected from one or more of CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, AFP, CEA, PSCA, GD2, NKG2D, BCMA, EGFR, Her2, EGFRvⅢ, CD171, FAP, IL13Rα2, VEGFR1, VEGFR2, GPC-3, Mesothelin, claudin 18.2, EpCAM, MUC1, MUC16, EPHA2, EPHA3, CD133 or PSMA.

[0040] Preferably, the TCR is selected from human TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, mouse TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain or the products after replacement, deletion or insertion thereof.

[0041] Preferably, the tumor antigen targeted by the TCR is selected from one or more of HBV, HPV E6, P53, MART-1, gp100, NYESO-1, MAGE-A3, MAGE-A4, WT1, CEA, Thyroglobulin or Tyrosinase.

[0042] The present invention also provides a co-expression vector, characterized in that the co-expression vector simultaneously expresses:

[0043] (1) One of CAR or TCR;

[0044] (2) one of integrin β1 or integrin β3 or their modified variants.

[0045] Preferably, the co-expression vector simultaneously expresses CAR and integrin β1, or simultaneously expresses CAR and a modified variant of integrin β1, or simultaneously expresses CAR and integrin β3, or simultaneously expresses CAR and a modified variant of integrin β3, or simultaneously expresses TCR and integrin β1, or simultaneously expresses TCR and a modified variant of integrin β1, or simultaneously expresses TCR and integrin β3, or simultaneously expresses TCR and a modified variant of integrin β3.

[0046] Preferably, the co-expression vector is a viral vector and / or a non-viral vector.

[0047] More preferably, the viral vector is a lentiviral vector and / or a retroviral vector.

[0048] Preferably, the co-expression vector is constructed by 2A peptide.

[0049] More preferably, the 2A peptide is selected from one or more of P2A, T2A or F2A.

[0050] More preferably, the 2A peptide is P2A.

[0051] Preferably, the P2A is the amino acid sequence shown in SEQ ID NO.9.

[0052] More preferably, the P2A is a sequence having one or more conservative modifications compared to the amino acid sequence shown in SEQ ID NO. 9, wherein the conservative modifications include amino acid substitution, deletion or insertion.

[0053] Further preferably, the P2A is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO.9.

[0054] Preferably, the nucleotide sequence of the gene encoding the P2A is the nucleotide sequence shown in SEQ ID NO. 10 or a degenerate sequence thereof.

[0055] More preferably, the nucleotide sequence of the gene encoding the P2A is a sequence having one or more conservative modifications compared to the nucleotide sequence shown in SEQ ID NO. 10, wherein the conservative modifications include substitution, deletion or insertion of nucleotide derivatives.

[0056] Further preferably, the nucleotide sequence of the gene encoding the P2A has the same or similar function as the protein encoded by SEQ ID NO.10.

[0057] Preferably, the T2A is the amino acid sequence shown in SEQ ID NO.11.

[0058] More preferably, the T2A is a sequence having one or more conservative modifications compared to the amino acid sequence shown in SEQ ID NO. 11, wherein the conservative modifications include amino acid substitution, deletion or insertion.

[0059] Further preferably, the T2A is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO.11.

[0060] Preferably, the nucleotide sequence of the gene encoding the T2A is the nucleotide sequence shown in SEQ ID NO. 12 or a degenerate sequence thereof.

[0061] More preferably, the nucleotide sequence of the gene encoding the T2A is a sequence having one or more conservative modifications compared to the nucleotide sequence shown in SEQ ID NO. 12, wherein the conservative modifications include substitution, deletion or insertion of nucleotide derivatives.

[0062] Further preferably, the nucleotide sequence of the gene encoding the T2A has the same or similar function as the protein encoded by SEQ ID NO.12.

[0063] Preferably, the F2A is the amino acid sequence shown in SEQ ID NO.13.

[0064] More preferably, the F2A is a sequence having one or more conservative modifications compared to the amino acid sequence shown in SEQ ID NO. 13, wherein the conservative modifications include amino acid substitution, deletion or insertion.

[0065] Further preferably, the F2A is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO. 13.

[0066] Preferably, the nucleotide sequence of the gene encoding the F2A is the nucleotide sequence shown in SEQ ID NO. 14 or a degenerate sequence thereof.

[0067] More preferably, the nucleotide sequence of the gene encoding the F2A is a sequence having one or more conservative modifications compared to the nucleotide sequence shown in SEQ ID NO. 14, wherein the conservative modifications include substitution, deletion or insertion of nucleotide derivatives.

[0068] Further preferably, the nucleotide sequence of the gene encoding the F2A has the same or similar function as the protein encoded by SEQ ID NO.14.

[0069] The present invention also provides a host cell expressing the expression vector or the co-expression vector, wherein the host cell is an immune cell.

[0070] Preferably, the immune cells are selected from lymphocytes and / or their modified products.

[0071] More preferably, the lymphocytes are selected from T cells and / or monocytes.

[0072] Further preferably, the T cells are selected from one or more of αβT cells, NKT cells or γδT cells.

[0073] The present invention also provides a method for constructing the engineered immune cells, comprising the following steps:

[0074] (1) Collecting and activating host cells;

[0075] (2) adding the expression vector or co-expression vector to the activated host cells for the first infection;

[0076] (3) After the first infection, the expression vector or co-expression vector is added for a second infection to obtain host cells expressing integrin.

[0077] Preferably, the host cell is an immune cell.

[0078] More preferably, the immune cells are selected from lymphocytes and / or their modified products.

[0079] Further preferably, the lymphocytes are selected from T cells and / or monocytes.

[0080] Most preferably, the T cells are selected from one or more of αβT cells, NKT cells or γδT cells.

[0081] Preferably, the expression vector is an expression vector that expresses integrin β1 or integrin β3 or a modified variant thereof.

[0082] Preferably, the co-expression vector is a co-expression vector that expresses (1) CAR or TCR and (2) integrin β1 or integrin β3 or a modified variant thereof.

[0083] The present invention also provides a pharmaceutical composition, which includes the engineered immune cell, or the expression vector, or the co-expression vector, or the host cell, or the engineered immune cell constructed according to the method, and a pharmaceutically acceptable carrier or excipient.

[0084] The present invention also provides a kit comprising the engineered immune cell, or the expression vector, or the co-expression vector, or the host cell, or the engineered immune cell constructed according to the method, or the pharmaceutical composition, and instructions for use.

[0085] The present invention also provides a use of the engineered immune cell, or the expression vector, or the co-expression vector, or the host cell, or the engineered immune cell constructed according to the method, or the pharmaceutical composition, or the kit in preparing a drug for treating solid tumor-related diseases.

[0086] Preferably, the solid tumor-related disease is selected from one or more of lung cancer, liver cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, kidney cancer, lymphoma, esophageal cancer, bile duct cancer, ovarian cancer, gastric cancer, pancreatic cancer, head and neck cancer, synovial sarcoma, angiosarcoma, osteosarcoma, multiple myeloma, glioma, thyroid cancer, endometrial cancer, neuroblastoma, glioblastoma, rhabdomyosarcoma, melanoma, prostate cancer, cervical cancer, soft tissue sarcoma, urothelial carcinoma, bile duct cancer, esophageal cancer, mesothelioma and melanoma.

[0087] More preferably, the solid tumor-related disease is selected from one or more of lung cancer, liver cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, kidney cancer, lymphoma, ovarian cancer, gastric cancer, pancreatic cancer, multiple myeloma, glioma, prostate cancer, cervical cancer, bile duct cancer, esophageal cancer, and melanoma.

[0088] Effects of the Invention

[0089] 1. The present invention provides a T cell overexpressing Integrinβ1 / Integrinβ3 and its application. The expression of Integrinβ1 / Integrinβ3 can improve the migration ability of T cells;

[0090] 2. The integrin β1 / integrin β3-overexpressing T cells obtained by the present invention have significantly higher cytokine expression in vitro than unmodified T cells and have stronger tumor killing ability in vitro;

[0091] 3. The present invention discovered that overexpressing integrin β1 / integrin β3 can significantly enhance the anti-tumor function of T cells in vivo and prolong the survival of tumor-bearing mice. Furthermore, the proportion of T cells modified with integrin β1 / integrin β3 infiltrating into tumors is significantly increased, and they produce more cytokines.

[0092] In summary, the present invention found that Integrinβ1 / Integrinβ3 can enhance the infiltration ability of T cells and improve their effector function, thereby promoting their anti-tumor ability, and is suitable for any clinical adoptive immunotherapy, including the clinical treatment of tumors, infections and autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1 This is an example diagram of the MSGV-Thy1.1-P2A-Integrinβ3 plasmid.

[0094] Figure 2 To detect the in vitro infiltration ratio of T cells overexpressing Integrinβ1 / Integrinβ3.

[0095] Figure 3To detect the in vitro infiltration function of OT-1T cells and human T cells overexpressing Integrinβ1 / Integrinβ3.

[0096] Figure 4 To detect the in vivo effector function of OT-1T cells overexpressing Integrinβ1 / Integrinβ3.

[0097] Figure 5 To detect the in vivo anti-tumor function of OT-1T cells overexpressing Integrinβ1 / Integrinβ3 and human T cells overexpressing Integrinβ1. DETAILED DESCRIPTION

[0098] To make the technical solutions and beneficial effects of the present invention more clearly understood, the following detailed description is given by way of specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly illustrate the details of the local features. Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application belongs.

[0099] As used herein, the singular forms "a," "an" or "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0100] As used herein, the term "about" or "approximately" means that a particular value is within an acceptable error range as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" can mean within 3 or more standard deviations. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude of a value, within 5-fold of a value, or within 2-fold of a value.

[0101] As used herein, the term "administering" includes any route of introducing or delivering an agent to a subject to perform its intended function. Administration can be performed by any suitable route, including but not limited to intravenous, intramuscular, intraperitoneal, subcutaneous, or other suitable routes as described herein.

[0102] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally occurring amino acid (e.g., an amino acid analog). The terms encompass amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0103] As used herein, the term "amino acid" refers to naturally occurring amino acids and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), as well as pyrolysine and selenocysteine. Amino acid analogs refer to reagents having the same basic chemical structure as naturally occurring amino acids (i.e., α-carbon bound to hydrogen, carboxyl group, amino group, and R group), such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (such as norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. In some embodiments, the amino acids forming the polypeptide are in the D form. In some embodiments, the amino acids forming the polypeptide are in the L form.

[0104] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides are referred to by their commonly accepted single-letter codes.

[0105] As used herein, the term "percent homology" or "sequence identity" refers to the percent identity between two amino acid sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions × 100), taking into account the number of gaps and the length of each gap, which need to be introduced for optimal alignment of the two sequences. Mathematical algorithms can be used to compare sequences and determine the percent identity between two sequences.

[0106] The percent homology between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 444-453 (1970)), which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), can be used using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6 to determine the percent homology between two amino acid sequences.

[0107] As used herein, the term "substantially homologous" or "substantially identical" refers to a polypeptide or nucleic acid molecule that exhibits at least 50% or greater homology or identity relative to a reference amino acid sequence (e.g., any amino acid sequence described herein) or nucleic acid sequence (e.g., any nucleic acid sequence described herein). For example, such sequences are at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99% homologous or identical at the amino acid level or nucleic acid relative to the sequence used for comparison (e.g., a wild-type or native sequence). In some embodiments, a substantially homologous or substantially identical polypeptide contains an amino acid substitution, insertion, or deletion of one or more amino acids relative to the sequence used for comparison. In some embodiments, a substantially homologous or substantially identical polypeptide contains one or more non-natural amino acids or amino acid analogs (including D-amino acids and retro-inverso amino groups) in place of a homologous sequence.

[0108] As used herein, the term "analog" refers to a structurally related polypeptide or nucleic acid molecule that has the function of a reference polypeptide or nucleic acid molecule.

[0109] As used herein, the term "antibody" refers not only to complete antibody molecules, but also to antibody molecule fragments that retain immunogen binding ability. Such fragments are also well known in the art and are generally used both in vitro and in vivo. Therefore, "antibody" refers not only to complete immunoglobulin molecules, but also to the well-known active fragments F(ab')2 and Fab. F(ab')2 and Fab fragments lacking the Fc fragment of a complete antibody are cleared faster from the circulation and can be less than the nonspecific tissue binding of a complete antibody (Wahl et al., J.Nucl.Med.24:316-325 (1983)). Antibodies can include whole natural antibodies, monoclonal antibodies, human antibodies, humanized antibodies, camelization (camelised) antibodies, multispecific antibodies, bispecific antibodies, chimeric antibodies, Fab, Fab', single chain V region fragments (scFv), single domain antibodies (e.g., nanobodies and single domain camelid antibodies), VNAR fragments, bispecific T cell engager (BiTE) antibodies, miniantibodies, disulfide bonded Fv (sdFv) and anti-idiotypic (anti-Id) antibodies, intracellular antibodies, fusion polypeptides, unconventional antibodies and the antigen binding fragment of any of the above antibodies. Specifically, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen binding sites. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2).

[0110] Antibodies and antibody fragments can be derived in whole or in part from mammals (e.g., humans, non-human primates, goats, guinea pigs, hamsters, horses, mice, rats, rabbits, and sheep) or non-mammalian antibody-producing animals (e.g., chickens, ducks, geese, snakes, and uromorph amphibians). Antibodies and antibody fragments can be produced in vivo or in vitro, such as from yeast or phage (e.g., as single antibodies or antibody fragments or as part of an antibody library).

[0111] As used herein, the term "F(ab)" refers to a fragment of an antibody structure that binds to an antigen but is monovalent and lacks the Fc portion, e.g., papain digestion of an antibody produces two F(ab) fragments and one Fc fragment (e.g., a heavy (H) chain constant region; an Fc region that does not bind to an antigen).

[0112] As used herein, the term "F(ab')2" refers to an antibody fragment produced by pepsin digestion of an intact IgG antibody, wherein the fragment has two antigen-binding (ab') (divalent) regions, wherein each (ab') region comprises two separate amino acid chains (a portion of a heavy (H) chain and a light (L) chain connected by an SS bond for antigen binding) and wherein the remaining H chain portions are linked together. The "F(ab')2" fragment can be divided into two separate Fab' fragments.

[0113] As used herein, the term "antigen" refers to a molecule to which an antibody can selectively bind. The target antigen can be a protein (e.g., an antigenic peptide), a carbohydrate, a nucleic acid, a lipid, a hapten, or other naturally occurring or synthetic compound. Antigens can also be administered to an animal subject to generate an immune response in the subject.

[0114] As used herein, a "host cell" is a cell that is used to receive, maintain, propagate, and amplify a vector. A host cell can also be used to express a polypeptide encoded by a vector. When the host cell divides, the nucleic acid contained in the vector is replicated, thereby amplifying the nucleic acid.

[0115] As used herein, the term "immune cell" refers to any cell that plays a role in a subject's immune response. Immune cells are derived from the hematopoietic system and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells (such as monocytes, macrophages, dendritic cells, eosinophils, neutrophils, mast cells, basophils, and granulocytes).

[0116] As used herein, the term "lymphocyte" refers to all immature, mature, undifferentiated and differentiated leukocyte populations derived from lymphoid progenitor cells, including tissue-specific and specialized types, and encompasses B cells, T cells, NKT cells and NK cells by way of non-limiting example. In some embodiments, lymphocytes include all B cell lineages, including pre-B cells, progenitor B cells, early progenitor B cells, late progenitor B cells, large pre-B cells, small pre-B cells, immature B cells, mature B cells, plasma B cells, memory B cells, B-1 cells, B-2 cells and anergic AN1 / T3 cell populations.

[0117] As used herein, the term "engineered immune cell" refers to an immune cell that has been genetically modified.

[0118] As used herein, the term "conservative modifications" or "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies or antibody fragments of the present invention by standard techniques known in the art (such as site-directed mutagenesis and PCR-mediated mutagenesis). Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar 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). Therefore, one or more amino acid residues within the CAR of the present invention can be replaced by amino acid residues from the same side chain family, and the changed CAR can be tested using functional assays as described herein.

[0119] As used herein, the term "disease" refers to any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include neoplasia or infection of cells by pathogens.

[0120] As used herein, the term "tumor" or "solid tumor" refers to a disease characterized by pathological proliferation of cells or tissues and their subsequent migration or invasion to other tissues or organs. Tumor growth is typically uncontrolled and progressive and occurs under conditions that do not cause normal cell proliferation or would cause normal cell proliferation to cease. Tumors can affect a variety of cell types, tissues, or organs, including but not limited to organs selected from the group consisting of the bladder, colon, bone, brain, breast, cartilage, glial cells, esophagus, fallopian tubes, gallbladder, heart, intestine, kidney, liver, lung, lymph nodes, nervous tissue, ovary, pleura, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testicle, thymus, thyroid, trachea, genitourinary tract, ureters, urethra, uterus, and vagina, or tissues or cell types thereof. Tumors include cancers such as sarcomas, epithelial cancers, or plasmacytomas (malignant tumors of plasma cells).

[0121] As used herein, the term "effective amount" refers to the amount of a medicament sufficient to achieve a beneficial or desired result after administration. The amount of the medicament administered to the subject can depend on the characteristics of the individual (such as overall health, age, sex, weight), the effective concentration of the engineered immune cells administered, and the tolerance to the drug. Those skilled in the art will be able to determine the appropriate dosage and administer it to the subject based on these and other factors.

[0122] As used herein, the term "sample" refers to a clinical sample obtained from a subject. In certain embodiments, the sample is obtained from a biological source (i.e., a "biological sample"), such as a tissue, body fluid, or microorganism collected from a subject. Sample sources include, but are not limited to, mucus, sputum, bronchoalveolar lavage fluid (BAL), bronchial washings (BW), whole blood, body fluids, cerebrospinal fluid (CSF), urine, plasma, serum, or tissue.

[0123] As used herein, the terms "subject," "individual," or "patient" are used interchangeably and refer to an individual organism, vertebrate, or mammal and may include humans, non-human primates, rodents, etc. (e.g., a subject to be the recipient of a particular medical intervention or from which cells are harvested). In certain embodiments, the individual, patient, or subject is a human.

[0124] As used herein, the term "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression in eukaryotic cells can include splicing of mRNA. The expression level of a gene can be determined by measuring the amount of mRNA or protein in a cell or tissue sample. On the one hand, the expression level of a gene from a sample can be directly compared to the expression level of the gene from a control or reference sample. On the other hand, the expression level of a gene from a sample can be directly compared to the expression level of the gene from the same sample after administering a composition disclosed herein.

[0125] As used herein, the terms "isolated" or "purified" refer to a material that is free to varying degrees from the components that normally accompany it as found in its native state. "Isolated" refers to a degree of separation from the original source or environment. "Purified" refers to a degree of separation that is greater than isolated. A "purified" protein is sufficiently free of other materials so that any impurities do not substantially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or polypeptide of the presently disclosed subject matter is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA technology or substantially free of chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques (e.g., polyacrylamide gel electrophoresis or high performance liquid chromatography). The term "purified" can mean that a nucleic acid or protein produces essentially one band in an electrophoretic gel. For proteins that can undergo modification (e.g., phosphorylation or glycosylation), different modifications can produce different isolated proteins, which can be purified individually.

[0126] As used herein, the term "integrin" or "Integrin" is a heterodimer composed of an α chain and a β chain, which together form a complete receptor in the plasma membrane. In mammals, the integrin family consists of 18 α subunits and 8 β subunits, which combine to form 24 pairs of different αβ integrin molecules. Structurally, the αβ integrin subunit is a type 1 transmembrane protein, and each subunit is typically composed of a long and large extracellular domain, a transmembrane helical domain, and a short unstructured cytoplasmic tail. The αβ pairing of integrin subunits determines the specificity of the integrin for a specific ligand, promotes the formation of intracellular adhesion complexes, and regulates downstream signal transduction. Integrins are essential for T cell recirculation, migration to tumor tissue, and antigen recognition.

[0127] As used herein, the term "T cell" refers to a cell having a TCR on its surface or a TCR that has been genetically edited or modified. In the present invention, "T cell" includes T cells including CD4+T cells, CD8+T cells, γδT cells, TCR-T cells and CAR-T cells.

[0128] As used herein, the term "T cell receptor" or "TCR" refers to a heterodimeric membrane protein on the surface of T cells that specifically recognizes antigens and mediates immune responses. TCR includes two receptors, αβ and γδ, but approximately 95% of human peripheral blood T lymphocytes express αβ chain receptors. αβTCR mediates T cell immune function by binding to the major histocompatibility complex (MHC) on antigen presenting cells (APC) and activating the TCR downstream signaling pathway. TCR-T cells can not only recognize specific antigens on the surface of tumor cells, but also recognize antigens within the cells, which enables TCR-T cells to recognize a wider spectrum of antigens. Specifically, the T cell receptor (TCR) used in the present invention is IG4 TCR. The IG4 TCR antigen recognition region is Vα and Vβ targeting the NY-ESO-1 antigen, and the constant region is Cα and Cβ of mouse origin.

[0129] As used herein, the term "chimeric antigen receptor" or "CAR" consists of an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain. The extracellular domain is a single-chain variable fragment (scFv) that can specifically recognize tumor surface antigens. The first-generation CAR structure contains an antigen recognition domain scFv and an intracellular CD3ζ activation domain. Due to the lack of co-stimulatory signals, they exhibit limited proliferation and anti-tumor capabilities. The second-generation CAR adds co-stimulatory domains such as CD28, 4-1BB, OX40, or ICOS to the first-generation CAR structure, giving it better proliferation capacity and the ability to release more cytokines. Currently commercialized CAR-T products are mainly based on the second-generation CAR structure. The third-generation CAR contains two different co-stimulatory molecules to enhance the killing ability of CAR-T cells. The fourth-generation CAR, also known as TRUCK or armored CAR, is further modified to secrete cytokines or express suicide genes such as IL-7, IL-12, IL-15, IL-21, and iCaspase-9. Specifically, the chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain and an intracellular domain, which is a second generation CAR. The extracellular domain is a scFv targeting CD19 antigen, and the intracellular domain includes CD3ζ and 41BB costimulatory signal transduction region. Costimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens, and the costimulatory signal transduction region refers to the intracellular domain of costimulatory molecules.

[0130] As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to the portion of the intracellular domain of the CAR comprising a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for the effective activation and function of T lymphocytes after binding to an antigen. Examples of such costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS (CD278), LFA-1, CD3, CD2, CD7, LIGHT, NKD2C, B7-H2, and a ligand that specifically binds to CD83. The inclusion of one or more costimulatory signaling domains can enhance the efficacy and amplification of T cells expressing CAR receptors. Intracellular signaling and costimulatory signaling domains can be connected in series to the carboxyl terminus of the transmembrane domain in any order.

[0131] As used herein, the term "CD3" consists of five peptide chains: the gamma chain, the delta chain, the epsilon chain, the zeta chain, and the eta chain, all of which are transmembrane proteins. The transmembrane region of the CD3 molecule connects to the transmembrane regions of the two peptide chains of the TCR via a salt bridge, forming a TCR-CD3 complex that participates in antigen recognition by T cells. The activation signal generated by TCR recognition of antigens is transduced by CD3 into T cells, primarily used to label thymocytes, T lymphocytes, and T cell lymphomas.

[0132] As used herein, the term "CD28" is a co-stimulatory molecule that participates in the reception of the second signal and plays an important role in the activation of T cells. It binds to the B7 molecule on APCs (antigen presenting cells), mediates T cell co-stimulation, and promotes their survival, proliferation, and cytokine production.

[0133] As used herein, the term "CD28 antibody" refers to a monoclonal antibody that can bind to the CD28 molecule and activate T cells.

[0134] As used herein, the term "vector" is a replicable nucleic acid that, when transformed into an appropriate host cell, can express one or more heterologous proteins from the replicable nucleic acid. Reference to a vector includes those vectors into which a nucleic acid encoding a polypeptide or its fragment can be introduced typically by restriction digestion and connection. Reference to a vector also includes those vectors containing nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells to amplify nucleic acids or express / display polypeptides encoded by nucleic acids. Vectors typically remain in an episomal state, but can be designed to achieve integration of a gene or a portion thereof into a genomic chromosome. Also contemplated are vectors as artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes. The selection and use of such vectors are well known to those skilled in the art. Vectors also include "viral vectors" (virus vector or viral vector). Viral vectors are engineered viruses that are operably connected to exogenous genes to transfer exogenous genes (as a vehicle or shuttle) to cells.

[0135] As used herein, the term "expression vector" includes vectors capable of expressing DNA operably linked to regulatory sequences (such as promoter regions) that can achieve expression of such DNA fragments. Such other fragments may include promoter and terminator sequences and may optionally include one or more origins of replication, one or more selective markers, enhancers, polyadenylation signals, etc. Expression vectors are typically derived from plasmids or viral DNA, or may contain both elements. Therefore, expression vectors refer to recombinant DNA or RNA constructs, such as plasmids, phages, recombinant viruses or other vectors, that cause expression of cloned DNA after introduction into a suitable host cell. Suitable expression vectors are well known to those skilled in the art and are included in those replicable in eukaryotic and / or prokaryotic cells and those that maintain episomal states or are integrated into the host cell genome.

[0136] As used herein, the term "Matrigel" refers to a soluble extracellular matrix extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in extracellular matrix proteins, including laminin, type IV collagenase, heparin sulfate proteoglycan, nestin, and a large number of growth factors.

[0137] As used herein, the term "tumor fibroblasts" or "CAFs" refers to cells that are transformed from resident fibroblasts or stellate cells in tissues into CAFs under the stimulation of growth factors. CAFs can not only inhibit the function of immune cells by secreting a variety of cytokines or metabolites, thereby promoting tumor development, invasion, and metastasis, but also have the ability to shape the extratumoral matrix and form a drug or therapeutic immune cell penetration barrier to prevent drugs and immune cells from penetrating deep into tumor tissues, thereby reducing the effectiveness of tumor treatment.

[0138] As used herein, the term "MC38 cells" refers to a colon adenocarcinoma cell line derived from C57BL / 6 mice, which grows adherently and has a fibroblastic morphology.

[0139] As used herein, the term "B16 cells" or "mouse melanoma cells" refers to malignant melanoma, a highly malignant tumor derived from melanocytes. It primarily occurs in the skin, but can also be found in mucous membranes and internal organs, accounting for approximately 3% of all tumors. It is caused by excessive proliferation of abnormal melanocytes and is highly malignant, accounting for the vast majority of deaths from skin tumors.

[0140] As used herein, the term "cytokine" refers to a class of small molecule proteins with a wide range of biological activities that are synthesized and secreted by immune cells (such as monocytes, macrophages, T cells, B cells, NK cells, etc.) and certain non-immune cells (endothelial cells, epidermal cells, fibroblasts, etc.) upon stimulation. The cytokines may have multiple functions such as regulating innate immunity and adaptive immunity, hematopoiesis, cell growth, APSC multipotent cells, and damaged tissue repair. In the present invention, the cytokines may include interleukins, interferons, tumor necrosis factor superfamily, colony stimulating factors, chemokines, and growth factors.

[0141] As used herein, the term "interleukin" or "interleukin" refers to a secretory protein or signaling molecule that promotes the development and differentiation of T and / or B lymphocytes and / or hematopoietic cells. Interleukins can be synthesized by helper CD4 T lymphocytes, monocytes, macrophages, and endothelial cells. As used herein, interleukin 2 (IL-2) is a cytokine that is primarily synthesized by T cells and plays an important role in the body's immune response and antiviral infection, and can be used in clinical research and tumor treatment.

[0142] As used herein, the term "control" is an alternative sample used in an experiment for comparison purposes, and a control can be "positive" or "negative."

[0143] The present invention provides an engineered immune cell, wherein the engineered immune cell expresses integrin.

[0144] In certain embodiments, the immune cells are selected from lymphocytes and / or modified products thereof.

[0145] In certain embodiments, the immune cell is a lymphocyte.

[0146] In certain embodiments, the immune cells are products of lymphocyte modification.

[0147] In certain embodiments, the lymphocytes are selected from T cells and / or monocytes.

[0148] In certain embodiments, the lymphocytes are T cells.

[0149] In certain embodiments, the lymphocytes are monocytes.

[0150] In certain embodiments, the T cells are selected from one or more of αβ T cells, NK cells, or γδ T cells.

[0151] In certain embodiments, the T cells are αβ T cells.

[0152] In certain embodiments, the T cells are NKT cells.

[0153] In certain embodiments, the T cells are γδ T cells.

[0154] In certain embodiments, the integrin is selected from one or more of integrin α1, integrin α2, integrin α3, integrin α4, integrin α6, integrin α7, integrin β1, integrin β2, integrin β3, integrin β6, integrin β7 or integrin β8.

[0155] In certain embodiments, the integrin is selected from integrin β1 and / or integrin β3.

[0156] In certain embodiments, the integrin is integrin β1.

[0157] In certain embodiments, the integrin is integrin β3.

[0158] In certain embodiments, the integrin β1 has an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.2.

[0159] In certain embodiments, the integrin β1 has an amino acid sequence as shown in SEQ ID NO.1.

[0160] In certain embodiments, the integrin β1 has an amino acid sequence as shown in SEQ ID NO.2.

[0161] In certain embodiments, the integrin β1 is a sequence having one or more conservative modifications compared to the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0162] In certain embodiments, the conservative modifications include amino acid substitutions, deletions or insertions.

[0163] In certain embodiments, the integrin β1 is a sequence having one or more amino acids substituted, deleted or inserted compared to the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0164] In certain embodiments, the integrin β1 is a sequence having one or more amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0165] In certain embodiments, the integrin β1 is a sequence having one or more amino acids deleted compared to the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0166] In certain embodiments, the integrin β1 is a sequence having one or more amino acids inserted compared to the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0167] In certain embodiments, the integrin β1 is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0168] In certain embodiments, the nucleotide sequence of the gene encoding the integrin β1 is the nucleotide sequence shown in SEQ ID NO. 5 or SEQ ID NO. 6 or a degenerate sequence thereof.

[0169] In certain embodiments, the nucleotide sequence of the gene encoding the integrin β1 is a sequence having one or more conservative modifications compared to the nucleotide sequence shown in SEQ ID NO. 5 or SEQ ID NO. 6.

[0170] In certain embodiments, the conservative modification comprises substitution, deletion or insertion of a nucleotide derivative.

[0171] In certain embodiments, the nucleotide sequence of the gene encoding the integrin β1 is a sequence having one or more nucleotide derivatives substituted, deleted or inserted compared to the nucleotide sequence shown in SEQ ID NO. 5 or SEQ ID NO. 6.

[0172] In certain embodiments, the nucleotide sequence of the gene encoding the integrin β1 is a sequence having one or more nucleotide derivatives substituted compared to the nucleotide sequence shown in SEQ ID NO. 5 or SEQ ID NO. 6.

[0173] In certain embodiments, the nucleotide sequence of the gene encoding the integrin β1 is a sequence having one or more nucleotide derivatives deleted compared to the nucleotide sequence shown in SEQ ID NO.5 or SEQ ID NO.6.

[0174] In certain embodiments, the nucleotide sequence of the gene encoding the integrin β1 is a sequence having one or more nucleotide derivatives inserted therein compared to the nucleotide sequence shown in SEQ ID NO. 5 or SEQ ID NO. 6.

[0175] In certain embodiments, the nucleotide sequence of the gene encoding the integrin β1 has the same or similar function as the protein encoded by SEQ ID NO. 5 or SEQ ID NO. 6.

[0176] In certain embodiments, the integrin β3 has an amino acid sequence as shown in SEQ ID NO.3 or SEQ ID NO.4.

[0177] In certain embodiments, the integrin β3 is a sequence having one or more conservative modifications compared to the amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4.

[0178] In certain embodiments, the conservative modifications include amino acid substitutions, deletions or insertions.

[0179] In certain embodiments, the integrin β3 is a sequence having one or more amino acids substituted, deleted or inserted compared to the amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4.

[0180] In certain embodiments, the integrin β3 is a sequence having one or more amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4.

[0181] In certain embodiments, the integrin β3 is a sequence having one or more amino acids deleted compared to the amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4.

[0182] In certain embodiments, the integrin β3 is a sequence having one or more amino acids inserted compared to the amino acid sequence shown in SEQ ID NO. 3 or SEQ ID NO. 4.

[0183] In certain embodiments, the integrin β3 is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4.

[0184] In certain embodiments, the nucleotide sequence of the gene encoding the integrin β3 is the nucleotide sequence shown in SEQ ID NO. 7 or SEQ ID NO. 8 or a degenerate sequence thereof.

[0185] In certain embodiments, the nucleotide sequence of the gene encoding the integrin β3 is a sequence having one or more conservative modifications compared to the nucleotide sequence shown in SEQ ID NO. 7 or SEQ ID NO. 8.

[0186] In certain embodiments, the conservative modification comprises substitution, deletion or insertion of a nucleotide derivative.

[0187] In certain embodiments, the nucleotide sequence of the gene encoding the integrin β3 is a sequence having one or more nucleotide derivatives substituted, deleted or inserted compared to the nucleotide sequence shown in SEQ ID NO. 7 or SEQ ID NO. 8.

[0188] In certain embodiments, the nucleotide sequence of the gene encoding the integrin β3 is a sequence having one or more nucleotide derivatives substituted compared to the nucleotide sequence shown in SEQ ID NO. 7 or SEQ ID NO. 8.

[0189] In certain embodiments, the nucleotide sequence of the gene encoding the integrin β3 is a sequence having one or more nucleotide derivatives deleted compared to the nucleotide sequence shown in SEQ ID NO. 7 or SEQ ID NO. 8.

[0190] In certain embodiments, the nucleotide sequence of the gene encoding integrin β3 is a sequence having one or more nucleotide derivatives inserted therein compared to the nucleotide sequence shown in SEQ ID NO. 7 or SEQ ID NO. 8.

[0191] In certain embodiments, the nucleotide sequence of the gene encoding the integrin β3 has the same or similar function as the protein encoded by SEQ ID NO. 7 or SEQ ID NO. 8.

[0192] The present invention also provides an expression vector, which expresses one or more of integrin β1 or integrin β3 or their modified variants.

[0193] In certain embodiments, the expression vector expresses integrin β1.

[0194] In certain embodiments, the expression vector expresses integrin β3.

[0195] In certain embodiments, the expression vector expresses an engineered variant of integrin β1.

[0196] In certain embodiments, the expression vector expresses an engineered variant of integrin β3.

[0197] In certain embodiments, the expression vector is a viral vector and / or a non-viral vector.

[0198] In certain embodiments, the expression vector is a viral vector.

[0199] In certain embodiments, the expression vector is a non-viral vector.

[0200] In certain embodiments, the viral vector is a lentiviral vector and / or a retroviral vector.

[0201] In certain embodiments, the viral vector is a lentiviral vector.

[0202] In certain embodiments, the viral vector is a retroviral vector.

[0203] The present invention also provides an engineered immune cell, wherein the engineered immune cell co-expresses the integrin and the tumor cell targeting receptor.

[0204] In certain embodiments, the co-expression is that the engineered immune cells simultaneously express the integrin and the tumor cell targeting receptor.

[0205] In certain embodiments, the immune cells are selected from lymphocytes and / or modified products thereof.

[0206] In certain embodiments, the immune cell is a lymphocyte.

[0207] In certain embodiments, the immune cells are products of lymphocyte modification.

[0208] In certain embodiments, the lymphocytes are selected from T cells and / or monocytes.

[0209] In certain embodiments, the lymphocytes are T cells.

[0210] In certain embodiments, the lymphocytes are monocytes.

[0211] In certain embodiments, the T cells are selected from one or more of αβ T cells, NKT cells, or γδ T cells.

[0212] In certain embodiments, the T cells are αβ T cells.

[0213] In certain embodiments, the T cells are NKT cells.

[0214] In certain embodiments, the T cells are γδ T cells.

[0215] In certain embodiments, the integrin is selected from one or more of integrin β1, integrin β2, integrin β3, integrin β6, integrin β7, or integrin β8.

[0216] In certain embodiments, the integrin is selected from integrin β1 and / or integrin β3.

[0217] In certain embodiments, the integrin is integrin β1.

[0218] In certain embodiments, the integrin is integrin β3.

[0219] In certain embodiments, the tumor cell targeting receptor is selected from a chimeric antigen receptor CAR, a T cell receptor TCR and / or other targeting receptors.

[0220] In certain embodiments, the tumor cell targeting receptor is a chimeric antigen receptor CAR.

[0221] In certain embodiments, the tumor cell targeting receptor is a T cell receptor TCR.

[0222] In certain embodiments, the antigen binding region of the CAR is selected from a single chain variable region of a monoclonal antibody, a tumor antigen binding receptor or a ligand.

[0223] In certain embodiments, the tumor antigen targeted by the CAR is selected from one or more of CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, AFP, CEA, PSCA, GD2, NKG2D, BCMA, EGFR, Her2, EGFRvⅢ, CD171, FAP, IL13Rα2, VEGFR1, VEGFR2, GPC-3, Mesothelin, claudin 18.2, EpCAM, MUC1, MUC16, EPHA2, EPHA3, CD133 or PSMA.

[0224] In certain embodiments, the TCR is selected from human TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, mouse TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, or products thereof after replacement, deletion, or insertion.

[0225] In certain embodiments, the tumor antigen targeted by the TCR is selected from one or more of HBV, HPV E6, P53, MART-1, gp100, NYESO-1, MAGE-A3, MAGE-A4, WT1, CEA, Thyroglobulin or Tyrosinase.

[0226] The present invention also provides a co-expression vector, which simultaneously expresses:

[0227] (1) One of CAR or TCR;

[0228] (2) one of integrin β1 or integrin β3 or their modified variants.

[0229] In certain embodiments, the co-expression vector simultaneously expresses CAR and integrin β1.

[0230] In certain embodiments, the co-expression vector simultaneously expresses CAR and an engineered variant of integrin β1.

[0231] In certain embodiments, the co-expression vector simultaneously expresses CAR and integrin β3.

[0232] In certain embodiments, the co-expression vector simultaneously expresses CAR and an engineered variant of integrin β3.

[0233] In certain embodiments, the co-expression vector simultaneously expresses TCR and integrin β1.

[0234] In certain embodiments, the co-expression vector simultaneously expresses TCR and an engineered variant of integrin β1.

[0235] In certain embodiments, the co-expression vector simultaneously expresses TCR and integrin β3.

[0236] In certain embodiments, the co-expression vector simultaneously expresses the engineered variants of TCR and integrin β3.

[0237] In certain embodiments, the co-expression vector is a viral vector and / or a non-viral vector.

[0238] In certain embodiments, the co-expression vector is a viral vector.

[0239] In certain embodiments, the co-expression vector is a non-viral vector.

[0240] In certain embodiments, the viral vector is a lentiviral vector and / or a retroviral vector.

[0241] In certain embodiments, the viral vector is a lentiviral vector.

[0242] In certain embodiments, the viral vector is a retroviral vector.

[0243] In certain embodiments, the co-expression vector is constructed from a 2A peptide.

[0244] In certain embodiments, the 2A peptide is selected from one or more of P2A, T2A, or FA2.

[0245] In certain embodiments, the 2A peptide is P2A.

[0246] In certain embodiments, the 2A peptide is T2A.

[0247] In certain embodiments, the 2A peptide is FA2.

[0248] In certain embodiments, the 2A peptides are P2A and T2A.

[0249] In certain embodiments, the 2A peptides are P2A and F2A.

[0250] In certain embodiments, the 2A peptides are T2A and F2A.

[0251] In certain embodiments, the 2A peptide is P2A, T2A, and F2A.

[0252] In certain embodiments, the P2A is the amino acid sequence shown in SEQ ID NO.9.

[0253] In certain embodiments, the P2A is a sequence having one or more conservative modifications compared to the amino acid sequence shown in SEQ ID NO.9.

[0254] In certain embodiments, the conservative modifications include amino acid substitutions, deletions or insertions.

[0255] In certain embodiments, the P2A is a sequence having one or more amino acids substituted, deleted or inserted compared to the amino acid sequence shown in SEQ ID NO.9.

[0256] In certain embodiments, the P2A is a sequence having one or more amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO.9.

[0257] In certain embodiments, the P2A is a sequence having one or more amino acids deleted compared to the amino acid sequence shown in SEQ ID NO.9.

[0258] In certain embodiments, the P2A is a sequence having one or more amino acids inserted compared to the amino acid sequence shown in SEQ ID NO.9.

[0259] In certain embodiments, the P2A is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO.9.

[0260] In certain embodiments, the nucleotide sequence of the gene encoding the P2A is the nucleotide sequence shown in SEQ ID NO. 10 or a degenerate sequence thereof.

[0261] In certain embodiments, the nucleotide sequence of the gene encoding the P2A is a sequence having one or more conservative modifications compared to the nucleotide sequence shown in SEQ ID NO.10.

[0262] In certain embodiments, the conservative modification comprises substitution, deletion or insertion of a nucleotide derivative.

[0263] In certain embodiments, the nucleotide sequence of the gene encoding the P2A is a sequence having one or more nucleotide derivatives substituted, deleted or inserted compared to the nucleotide sequence shown in SEQ ID NO. 10.

[0264] In certain embodiments, the nucleotide sequence of the gene encoding the P2A is a sequence having one or more nucleotide derivatives substituted compared to the nucleotide sequence shown in SEQ ID NO. 10.

[0265] In certain embodiments, the nucleotide sequence of the gene encoding the P2A is a sequence having one or more nucleotide derivatives deleted compared to the nucleotide sequence shown in SEQ ID NO.10.

[0266] In certain embodiments, the nucleotide sequence of the gene encoding the P2A is a sequence having one or more nucleotide derivatives inserted therein compared to the nucleotide sequence shown in SEQ ID NO. 10.

[0267] In certain embodiments, the nucleotide sequence of the gene encoding the P2A has the same or similar function as the protein encoded by SEQ ID NO.10.

[0268] In certain embodiments, the T2A is the amino acid sequence shown in SEQ ID NO.11.

[0269] In certain embodiments, the T2A is a sequence having one or more conservative modifications compared to the amino acid sequence shown in SEQ ID NO.11.

[0270] In certain embodiments, the conservative modifications include amino acid substitutions, deletions or insertions.

[0271] In certain embodiments, the T2A is a sequence having one or more amino acids substituted, deleted or inserted compared to the amino acid sequence shown in SEQ ID NO.11.

[0272] In certain embodiments, the T2A is a sequence having one or more amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO.11.

[0273] In certain embodiments, the T2A is a sequence having one or more amino acids deleted compared to the amino acid sequence shown in SEQ ID NO.11.

[0274] In certain embodiments, the T2A is a sequence having one or more amino acids inserted compared to the amino acid sequence shown in SEQ ID NO.11.

[0275] In certain embodiments, the T2A is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO.11.

[0276] In certain embodiments, the nucleotide sequence of the gene encoding the T2A is the nucleotide sequence shown in SEQ ID NO. 12 or a degenerate sequence thereof.

[0277] In certain embodiments, the nucleotide sequence of the gene encoding the T2A is a sequence having one or more conservative modifications compared to the nucleotide sequence shown in SEQ ID NO.12.

[0278] In certain embodiments, the conservative modification comprises substitution, deletion or insertion of a nucleotide derivative.

[0279] In certain embodiments, the nucleotide sequence of the gene encoding the T2A is a sequence having one or more nucleotide derivatives substituted, deleted or inserted compared to the nucleotide sequence shown in SEQ ID NO. 12.

[0280] In certain embodiments, the nucleotide sequence of the gene encoding the T2A is a sequence having one or more nucleotide derivatives substituted compared to the nucleotide sequence shown in SEQ ID NO. 12.

[0281] In certain embodiments, the nucleotide sequence of the gene encoding the T2A is a sequence having one or more nucleotide derivatives deleted compared to the nucleotide sequence shown in SEQ ID NO.12.

[0282] In certain embodiments, the nucleotide sequence of the gene encoding the T2A is a sequence having one or more nucleotide derivatives inserted therein compared to the nucleotide sequence shown in SEQ ID NO. 12.

[0283] In certain embodiments, the nucleotide sequence of the gene encoding the T2A has the same or similar function as the protein encoded by SEQ ID NO.12.

[0284] In certain embodiments, the F2A is the amino acid sequence shown in SEQ ID NO.13.

[0285] In certain embodiments, the F2A is a sequence having one or more conservative modifications compared to the amino acid sequence shown in SEQ ID NO.13.

[0286] In certain embodiments, the conservative modifications include amino acid substitutions, deletions or insertions.

[0287] In certain embodiments, the F2A is a sequence having one or more amino acids substituted, deleted or inserted compared to the amino acid sequence shown in SEQ ID NO.13.

[0288] In certain embodiments, the F2A is a sequence having one or more amino acid substitutions compared to the amino acid sequence shown in SEQ ID NO.13.

[0289] In certain embodiments, the F2A is a sequence having one or more amino acids deleted compared to the amino acid sequence shown in SEQ ID NO.13.

[0290] In certain embodiments, the F2A is a sequence having one or more amino acids inserted compared to the amino acid sequence shown in SEQ ID NO.13.

[0291] In certain embodiments, the F2A is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO. 13.

[0292] In certain embodiments, the nucleotide sequence of the gene encoding the F2A is the nucleotide sequence shown in SEQ ID NO. 14 or a degenerate sequence thereof.

[0293] In certain embodiments, the nucleotide sequence of the gene encoding the F2A is a sequence having one or more conservative modifications compared to the nucleotide sequence shown in SEQ ID NO.14.

[0294] In certain embodiments, the conservative modification comprises substitution, deletion or insertion of a nucleotide derivative.

[0295] In certain embodiments, the nucleotide sequence of the gene encoding the F2A is a sequence having one or more nucleotide derivatives substituted, deleted or inserted compared to the nucleotide sequence shown in SEQ ID NO. 14.

[0296] In certain embodiments, the nucleotide sequence of the gene encoding the F2A is a sequence having one or more nucleotide derivatives substituted compared to the nucleotide sequence shown in SEQ ID NO. 14.

[0297] In certain embodiments, the nucleotide sequence of the gene encoding the F2A is a sequence having one or more nucleotide derivatives deleted compared to the nucleotide sequence shown in SEQ ID NO. 14.

[0298] In certain embodiments, the nucleotide sequence of the gene encoding the F2A is a sequence having one or more nucleotide derivatives inserted therein compared to the nucleotide sequence shown in SEQ ID NO. 14.

[0299] In certain embodiments, the nucleotide sequence of the gene encoding the F2A has the same or similar function as the protein encoded by SEQ ID NO.14.

[0300] The present invention also provides a host cell expressing the expression vector or the co-expression vector, wherein the host cell is an immune cell.

[0301] In certain embodiments, the immune cells are selected from lymphocytes and / or modified products thereof.

[0302] In certain embodiments, the immune cell is a lymphocyte.

[0303] In certain embodiments, the immune cells are products of lymphocyte modification.

[0304] In certain embodiments, the lymphocytes are selected from T cells and / or monocytes.

[0305] In certain embodiments, the lymphocytes are T cells.

[0306] In certain embodiments, the lymphocytes are monocytes.

[0307] In certain embodiments, the T cells are selected from one or more of αβ T cells, NKT cells, or γδ T cells.

[0308] In certain embodiments, the T cells are αβ T cells.

[0309] In certain embodiments, the T cells are NKT cells.

[0310] In certain embodiments, the T cells are γδ T cells.

[0311] The present invention also provides a method for constructing the engineered immune cells, comprising the following steps:

[0312] (1) Collecting and activating host cells;

[0313] (2) adding the expression vector or co-expression vector to the activated host cells for the first infection;

[0314] (3) After the first infection, the expression vector or co-expression vector is added for a second infection to obtain host cells expressing integrin.

[0315] In certain embodiments, the activation is performed in a 24-well plate coated with CD3 antibody by adding CD28 antibody and mouse IL-2.

[0316] In certain embodiments, the first infection is performed by adding integrin β3 virus to a retronectin-coated 24-well low-adhesion plate, and then adding the cell suspension.

[0317] In certain embodiments, the cell suspension has a cell density of 2×10 6 pieces / mL.

[0318] In certain embodiments, the second infection is performed after the first infection is completed.

[0319] In certain embodiments, the second infection is performed 24 hours after the end of the first infection.

[0320] In certain embodiments, the host cell is an immune cell.

[0321] In certain embodiments, the immune cells are selected from lymphocytes and / or engineered products thereof.

[0322] In certain embodiments, the immune cell is a lymphocyte.

[0323] In certain embodiments, the immune cells are products of lymphocyte modification.

[0324] In certain embodiments, the lymphocytes are selected from T cells and / or monocytes.

[0325] In certain embodiments, the lymphocytes are T cells.

[0326] In certain embodiments, the lymphocytes are monocytes.

[0327] In certain embodiments, the T cells are selected from one or more of αβ T cells, NKT cells, or γδ T cells.

[0328] In certain embodiments, the T cells are αβ T cells.

[0329] In certain embodiments, the T cells are NKT cells.

[0330] In certain embodiments, the T cells are γδ T cells.

[0331] In certain embodiments, the expression vector is an expression vector that expresses integrin β1 or integrin β3 or an engineered variant thereof.

[0332] In certain embodiments, the expression vector is an expression vector for expressing integrin β1.

[0333] In certain embodiments, the expression vector is an expression vector that expresses an engineered variant of integrin β1.

[0334] In certain embodiments, the expression vector is an expression vector for expressing integrin β3.

[0335] In certain embodiments, the expression vector is an expression vector that expresses an engineered variant of integrin β1.

[0336] In certain embodiments, the co-expression vector is a co-expression vector that expresses (1) CAR or TCR and (2) integrin β1 or integrin β3 or an engineered variant thereof.

[0337] In certain embodiments, the co-expression vector simultaneously expresses CAR and integrin β1.

[0338] In certain embodiments, the co-expression vector simultaneously expresses CAR and an engineered variant of integrin β1.

[0339] In certain embodiments, the co-expression vector simultaneously expresses CAR and integrin β3.

[0340] In certain embodiments, the co-expression vector simultaneously expresses CAR and an engineered variant of integrin β3.

[0341] In certain embodiments, the co-expression vector simultaneously expresses TCR and integrin β1.

[0342] In certain embodiments, the co-expression vector simultaneously expresses TCR and an engineered variant of integrin β1.

[0343] In certain embodiments, the co-expression vector simultaneously expresses TCR and integrin β3.

[0344] In certain embodiments, the co-expression vector simultaneously expresses the engineered variants of TCR and integrin β3.

[0345] The present invention also provides a pharmaceutical composition, which includes the engineered immune cell, or the expression vector, or the co-expression vector, or the host cell, or the engineered immune cell constructed according to the method, and a pharmaceutically acceptable carrier or excipient.

[0346] The present invention also provides a kit comprising the engineered immune cell, or the expression vector, or the co-expression vector, or the host cell, or the engineered immune cell constructed according to the method, or the pharmaceutical composition, and instructions for use.

[0347] The present invention also provides a use of the engineered immune cell, or the expression vector, or the co-expression vector, or the host cell, or the engineered immune cell constructed according to the method, or the pharmaceutical composition, or the kit in preparing a drug for treating solid tumor-related diseases.

[0348] In certain embodiments, the solid tumor-related disease is selected from one or more of lung cancer, liver cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, kidney cancer, lymphoma, esophageal cancer, bile duct cancer, ovarian cancer, gastric cancer, pancreatic cancer, head and neck cancer, synovial sarcoma, angiosarcoma, osteosarcoma, multiple myeloma, glioma, thyroid cancer, endometrial cancer, neuroblastoma, glioblastoma, rhabdomyosarcoma, melanoma, prostate cancer, cervical cancer, soft tissue sarcoma, urothelial carcinoma, bile duct cancer, esophageal cancer, mesothelioma and melanoma.

[0349] In certain embodiments, the solid tumor-related disease is selected from one or more of lung cancer, liver cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, kidney cancer, lymphoma, ovarian cancer, gastric cancer, pancreatic cancer, multiple myeloma, glioma, prostate cancer, cervical cancer, bile duct cancer, esophageal cancer, and melanoma.

[0350] Experimental Materials

[0351] C57BL / 6 mice were purchased from Beijing Weitonglihua Company, 6-8 weeks old, female, 18-20 g. All animal husbandry was in accordance with standard experimental animal husbandry requirements.

[0352] CD3 Antibody: Ultra-LEAF TM Purified anti-mouse CD3ε Antibody and InVivoMAb anti-human CD3, purchased from Biolegend, were used to activate primary cells.

[0353] CD28 antibody: Ultra-LEAF TM Purified anti-mouse CD28 Antibody and InVivoMAb anti-human CD28 were purchased from Biolegend and used to activate primary cells.

[0354] Mouse IL-2: Recombinant murine IL-2, purchased from PeproTech, is used to culture T cells and promote T cell proliferation.

[0355] Integrinβ1 / Integrinβ3: Mouse and human integrin β1 and β3 amplified from cDNA of mouse OT-1 T cells or human T cells.

[0356] DPBS: Dulbecco's Phosphate Buffered Saline, purchased from Thermo, can be obtained using conventional methods in the art or through commercial channels.

[0357] Lymphocyte separation medium: Ficoll-Paque PLUS, a ready-to-use sterile culture medium purchased from Cytiva, is used for high-yield isolation of human lymphocytes from peripheral blood.

[0358] Human IL-2: Recombinant human IL-2, purchased from PeproTech, is used to culture T cells and promote T cell proliferation.

[0359] Matrigel solution: Matrigel, purchased from Corning, was used for transwell infiltration experiments and in vivo inoculation of tumor cells.

[0360] RPMI 1640 basal medium: used to culture primary T cells and B16 tumor cells.

[0361] DMEM basal medium: used to culture MC38 tumor cells, CAF cells and 293T cells.

[0362] MC38 cells: A mouse colon cancer cell line, preserved in our laboratory for a long time, was used to prepare 3D spheres in this invention.

[0363] CAF cells: tumor fibroblasts, isolated by our laboratory staff, are used to prepare 3D spheres in this invention.

[0364] CFSE: carboxyfluorescein diacetate succinimidyl ester, purchased from Biolegend, was used to detect cell proliferation.

[0365] 4% paraformaldehyde fixative: purchased from Formazan, used to fix cells.

[0366] B16-OVA tumor cells: A cell line obtained by stably expressing the full-length OVA antigen on B16 tumor cells (mouse melanoma tumor cells).

[0367] Percoll separation medium: Percoll medium, a low viscosity, non-toxic medium suitable for density gradient centrifugation of cells, viruses and subcellular particles.

[0368] PMA solution: phorbol ester 12-myristate 13-acetate, used to stimulate T cells, enhance TCR signaling, and promote cytokine secretion.

[0369] BFA: Brefeldin A Solution (1,000X), Brefeldin A, purchased from Biolegend, is an intracellular protein blocking inhibitor.

[0370] Example 1: Preparation of Integrinβ1 / Integrinβ3 Virus

[0371] (1) Construction of MSGV-Thy1.1-P2A-Integrinβ1 / MSGV-Thy1.1-P2A-Integrinβ3 plasmids

[0372] Mouse Itgβ3 and human ITGβ3 genes were amplified from primary cells, and the target fragments were inserted into the MSGV retroviral vector by enzyme digestion and ligation using P2A peptide, T2A peptide, or F2A peptide, thereby obtaining the MSGV-Thy1.1-P2A-Integrinβ1 / MSGV-Thy1.1-P2A-Integrinβ3 target plasmids. The MSGV-Thy1.1-P2A-Integrinβ3 plasmid example is shown in the figure. Figure 1 shown.

[0373] The amino acid sequence of the P2A peptide is shown in SEQ ID NO.9, the amino acid sequence of the T2A peptide is shown in SEQ ID NO.11, the amino acid sequence of the F2A peptide is shown in SEQ ID NO.13, the nucleotide sequence encoding the P2A peptide is shown in SEQ ID NO.10, the nucleotide sequence encoding the T2A peptide is shown in SEQ ID NO.12, and the nucleotide sequence encoding the F2A peptide is shown in SEQ ID NO.14.

[0374] (2) MSGV-Thy1.1-P2A-Integrinβ1 / MSGV-Thy1.1-P2A-Integrinβ3 Retroviral Packaging

[0375] Collect well-growing 293T cells, add an appropriate number of cells to a 6cm culture dish, plate the cells, shake well, and culture in a 37°C incubator. When the cell density reaches 70-80%, replace the medium with antibiotic-free DMEM complete medium and place it in a 37°C incubator for transfection. Add 500μL of OPTI-MEM medium to a 1.5mL EP tube, and then add the PCL-eco packaging plasmid, MSGV-Thy1.1-P2A-Integrinβ1 / MSGV-Thy1.1-P2A-Integrinβ3 target plasmid, and transfection reagent in sequence. Prepare plasmid droplets and add them to 293T cells. After 48 hours, harvest the virus to obtain the Integrinβ1 / Integrinβ3 virus.

[0376] Example 2: Detection of in vitro infiltration function of OT-1 T cells and human T cells overexpressing Integrin β1 / Integrin β3

[0377] (1) Activation and infection of OT-1T cells

[0378] The spleen of OT-1 transgenic mice was isolated, ground, and red blood cells were lysed. The filtrate was filtered through a nylon wool column, and T cells were collected. The obtained T cells were expressed as 1×10 6 Each well of a 24-well plate coated with CD3 antibody was activated by adding CD28 antibody and mouse IL-2.

[0379] OT-1T cells were collected after activation for 24 h, and the cell density was adjusted to 2×10 6 Add 500 μL of murine integrin β1 / β3 virus to a retronectin-coated 24-well low-adhesion plate, followed by 500 μL of cell suspension. Centrifuge at 2500 rpm for 90 minutes at 30°C for primary infection. 24 hours after infection, aspirate 500 μL of cell supernatant and add 500 μL of virus for secondary infection to obtain OT-1T cells overexpressing integrin β1 / β3.

[0380] The amino acid sequence of mouse Integrin β1 is shown in SEQ ID NO.2, the amino acid sequence of mouse Integrin β3 is shown in SEQ ID NO.4, the nucleotide sequence encoding mouse Integrin β1 is shown in SEQ ID NO.6, and the nucleotide sequence encoding mouse Integrin β3 is shown in SEQ ID NO.8.

[0381] (2) Activation and infection of human T cells

[0382] An equal volume of DPBS or saline is added to the peripheral blood collected from the vein (1:1), diluted, and slowly added to a flow cytometry tube containing lymphocyte separation fluid (Ficoll or Histopaque-1077). Centrifuge at 800g for 20 minutes, aspirate the cells above the lymphocyte separation fluid, transfer to a new centrifuge tube, add 1640 culture medium, centrifuge and discard the supernatant, retaining the cell pellet at the bottom of the centrifuge tube to obtain peripheral blood mononuclear cells.

[0383] The peripheral blood mononuclear cells were collected at a rate of 1×10 6 Each well was added to a 24-well plate coated with CD3 antibody, and CD28 antibody and 300 U / ml human IL-2 were added for activation to obtain human T cells.

[0384] After 48 h of T cell activation, 1×10 6Human Integrinβ1 / Integrinβ3 virus was added to T cells at a volume ratio of 1:1, and the cells were centrifuged at 2500 rpm for 90 minutes. After further culture for 24 hours, the medium was replaced to obtain human T cells overexpressing Integrinβ1 / Integrinβ3.

[0385] The amino acid sequence of human Integrin β1 is shown in SEQ ID NO.1, and the amino acid sequence of human Integrin β3 is shown in SEQ ID NO.3; the nucleotide sequence encoding human Integrin β1 is shown in SEQ ID NO.5, and the nucleotide sequence encoding human Integrin β3 is shown in SEQ ID NO.7.

[0386] (3) Transwell infiltration assay

[0387] 130 μL of diluted 130 μg / mL Matrigel solution was added to a 5 μm Transwell chamber and placed in a 37°C incubator for 2 h. OT-1 T cells and human T cells overexpressing Integrinβ3 were collected and the cell density of each group was adjusted to 2×10 6 After the Matrigel solidifies, add 650 μL of RPMI 1640 basal medium containing 5% FBS to the lower chamber. Add 100 μL of diluted T cells to each chamber and culture in a 37°C incubator.

[0388] After 24 h of culture, the cells infiltrating into the lower chamber were collected and counted, and then the infiltration ratio was calculated according to the following formula. Figure 2 As shown in A / C / E / G, compared with the control group (vector), the infiltration ratio of mouse OT-1T cells (mITGβ1 and mITGβ3) and human T cells (hITGβ1 and hITGβ3) overexpressing Integrinβ1 / Integrinβ3 was higher, indicating that Integrinβ1 / Integrinβ3 modification can enhance the penetration ability of T cells.

[0389]

[0390] (4) 3D Spheroids Infiltration Experiment

[0391] The cell density of MC38 cells and CAFs was adjusted to 4.6×10 4 75 μL of each cell type was placed in a 96-well U-bottom low-adsorption plate, pipetted evenly, centrifuged, and cultured for about 24 hours. After pelleting, OT-1T cells and human T cells were stained with CFSE and the cell density was adjusted to 1×10 cells / mL using normal culture medium. 6Select well-shaped pellets, label them, and then aspirate 100 μL of culture medium from the U-bottom plate. Then, add 100 μL of stained OT-1 T cells and human T cells to the U-bottom plate and continue culturing in a 37°C incubator for 24 h.

[0392] Microscope observation: After 24 hours, the pellet was transferred to a 96-well U-bottom plate containing 100 μL of fixative solution and placed in a confocal microplate for photography. Figure 2 As shown in B / D / F / H, compared with the control group (vector), the infiltration ratio of OT-1T cells (mITGβ1 and mITGβ3) and human T cells (hITGβ1 and hITGβ3) overexpressing Integrinβ1 / Integrinβ3 in the spheres was significantly increased, indicating that overexpression of Integrinβ1 / Integrinβ3 can enhance the infiltration ability of T cells into 3D spheres.

[0393] Example 3: Detection of the Intratumor Infiltration Ratio of OT-1 T Cells Overexpressing Integrin β1 / Integrin β3 and Human T Cells Overexpressing Integrin β1

[0394] B16-OVA tumor cells (cell lines obtained by stably expressing full-length OVA antigen on B16 tumor cells) were collected and counted by centrifugation. The cell suspension was evenly mixed with matrigel at a ratio of 1:1 on ice and the cell density was adjusted to 3 × 10 6 C57BL / 6N mice were subcutaneously inoculated with 75 μL of tumor cells. When the tumor volume of the tumor-bearing mice reached 100 mm 3 Around 14 days, the tumor-bearing mice were randomly divided into groups and irradiated (5 Gy) one day before adoptive transfer. OT-1T cells were activated and infected according to the method of Example 1 and cultured normally until the 6th day. The cells were collected and centrifuged with Ficoll (to remove dead cells) to adjust the cell density to 15×10 6 Each mouse was injected with 200 μL of cell suspension into the tail vein. After 6 days of adoptive therapy, the mice were sacrificed and the tumors were weighed. After collagenase digestion, single cell suspension was prepared and finally detected by flow cytometry. Figure 3 As shown in AC, compared with the control group (vector), overexpression of Integrinβ1 / Integrinβ3 (mITGβ1 and mITGβ3) can significantly increase the infiltration ratio of OT-1T cells in solid tumors.

[0395] At the same time, NSG mice were subcutaneously inoculated with 3×10 6HEPG2-CD19 tumor cells (cell lines obtained by stably expressing the full-length CD19 antigen on HEPG2 tumor cells). When the tumor volume of the tumor-bearing mice reached 100mm 3 Around 14 days, the tumor-bearing mice were randomly divided into groups and irradiated (2 Gy) one day before adoptive transfer. Human T cells were activated and infected according to the method of Example 1 and cultured normally until the 12th day. The cells were collected and centrifuged with Ficoll (to remove dead cells) to adjust the cell density to 30×10 6 Each mouse was injected with 200 μL of cell suspension into the tail vein. After 6 days of adoptive therapy, the mice were sacrificed, the tumors were isolated and weighed. After collagenase digestion, single cell suspension was prepared and finally detected by flow cytometry. Figure 3 As shown in D, compared with the control group (vector), overexpression of Integrinβ1 (hITGβ1) can significantly increase the infiltration ratio of human T cells in solid tumors.

[0396] Example 4: Detection of effector function of OT-1 T cells overexpressing Integrin β1 / Integrin β3 in vivo

[0397] As described in Example 2, B16-OVA tumor-bearing mice were treated with adoptive T cell therapy for 12-16 days. The mice were then sacrificed and the tumor tissues were removed. The tissues were ground, filtered, and centrifuged, and then subjected to density gradient centrifugation using Percoll separation medium to obtain lymphocytes. After stimulation with PMA (containing BFA) solution for 5 hours, the cytokine production of T cells was detected by flow cytometry. The results are shown in Figure 2. Figure 4 As shown in AC, compared with the control group (vector), Integrinβ1 / Integrinβ3 (mITGβ1 and mITGβ3) engineered OT-1T cells can produce more cytokines.

[0398] Example 5: In vivo anti-tumor function detection of OT-1 T cells overexpressing Integrin β1 / Integrin β3 and human T cells overexpressing Integrin β1

[0399] As described in Example 2, the length and width of the tumors of the tumor-bearing mice were measured every 2 days starting from the day of adoptive T cell transfer, and the tumor volume was calculated according to the following formula: 3 The mice were killed at 4 hr and the tumor volume and survival period curves were plotted. Figure 5As shown in AD, the tumor volume of mice bearing tumors overexpressing Integrinβ1 / Integrinβ3 (mITGβ1 and mITGβ3) and human T cells (hITGβ1) was significantly reduced compared with the control group (vector).

[0400]

[0401] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations of the claims. Various modifications and variations may be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments may be arbitrarily combined to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments merely illustrate several implementations of the present invention and do not limit the scope of protection of the patent of this invention.

Claims

1. An engineered immune cell, characterized in that: The engineered immune cells express integrins.

2. The engineered immune cell according to claim 1, characterized in that The immune cells are selected from lymphocytes and / or their modified products; Preferably, the lymphocytes are selected from T cells and / or monocytes; More preferably, the T cells are selected from one or more of αβT cells, NKT cells or γδT cells.

3. The engineered immune cell according to claim 1, characterized in that The integrin is selected from one or more of integrin α1, integrin α2, integrin α3, integrin α4, integrin α6, integrin α7, integrin β1, integrin β2, integrin β3, integrin β6, integrin β7 or integrin β8; Preferably, the integrin is selected from integrin β1 and / or integrin β3.

4. The engineered immune cell according to claim 3, characterized in that The integrin β1 is the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2; Preferably, the integrin β1 is a sequence having one or more conservative modifications compared to the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2, wherein the conservative modification includes amino acid substitution, deletion or insertion; More preferably, the integrin β1 is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.

2.

5. The engineered immune cell according to claim 4, characterized in that The nucleotide sequence of the gene encoding the integrin β1 is the nucleotide sequence shown in SEQ ID NO.5 or SEQ ID NO.6 or a degenerate sequence thereof; Preferably, the nucleotide sequence of the gene encoding the integrin β1 is a sequence having one or more conservative modifications compared to the nucleotide sequence shown in SEQ ID NO.5 or SEQ ID NO.6, wherein the conservative modification includes substitution, deletion or insertion of a nucleotide derivative; More preferably, the nucleotide sequence of the gene encoding the integrin β1 has the same or similar function as the protein encoded by SEQ ID NO.5 or SEQ ID NO.

6.

6. The engineered immune cell according to claim 3, characterized in that The integrin β3 is the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4; Preferably, the integrin β3 is a sequence having one or more conservative modifications compared to the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.4, wherein the conservative modification includes amino acid substitution, deletion or insertion; More preferably, the integrin β3 is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO.3 or SEQ ID NO.

4.

7. The engineered immune cell according to claim 6, characterized in that The nucleotide sequence of the gene encoding the integrin β3 is the nucleotide sequence shown in SEQ ID NO.7 or SEQ ID NO.8 or a degenerate sequence thereof; Preferably, the nucleotide sequence of the gene encoding the integrin β3 is a sequence having one or more conservative modifications compared to the nucleotide sequence shown in SEQ ID NO.7 or SEQ ID NO.8, wherein the conservative modification includes substitution, deletion or insertion of a nucleotide derivative; More preferably, the nucleotide sequence of the gene encoding the integrin β3 has the same or similar function as the protein encoded by SEQ ID NO.7 or SEQ ID NO.

8.

8. An expression vector, characterized in that The expression vector expresses one or more of integrin β1 or integrin β3 or their modified variants.

9. The expression vector according to claim 8, characterized in that The expression vector is a viral vector and / or a non-viral vector; preferably, the viral vector is a lentiviral vector and / or a retroviral vector.

10. An engineered immune cell, characterized in that: The engineered immune cells co-express the integrin and the tumor cell targeting receptor.

11. The engineered immune cell according to claim 10, characterized in that: The immune cells are selected from lymphocytes and / or their modified products; Preferably, the lymphocytes are selected from T cells and / or monocytes; More preferably, the T cells are selected from one or more of αβT cells, NKT cells or γδT cells.

12. The engineered immune cell according to claim 10, characterized in that The integrin is selected from one or more of integrin α1, integrin α2, integrin α3, integrin α4, integrin α6, integrin α7, integrin β1, integrin β2, integrin β3, integrin β6, integrin β7 or integrin β8; Preferably, the integrin is selected from integrin β1 and / or integrin β3.

13. The engineered immune cell according to claim 10, characterized in that The tumor cell targeting receptor is selected from a chimeric antigen receptor CAR, a T cell receptor TCR and / or other targeting receptors.

14. The engineered immune cell according to claim 13, characterized in that The antigen binding region of the CAR is selected from the single chain variable region of a monoclonal antibody, a tumor antigen binding receptor or a ligand.

15. The engineered immune cell according to claim 14, characterized in that: The tumor antigen targeted by the CAR is selected from one or more of CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, AFP, CEA, PSCA, GD2, NKG2D, BCMA, EGFR, Her2, EGFRvⅢ, CD171, FAP, IL13Rα2, VEGFR1, VEGFR2, GPC-3, Mesothelin, claudin18.2, EpCAM, MUC1, MUC16, EPHA2, EPHA3, CD133 or PSMA.

16. The engineered immune cell according to claim 13, characterized in that: The TCR is selected from human TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, mouse TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain or the products after replacement, deletion or insertion thereof.

17. The engineered immune cell according to claim 16, characterized in that The tumor antigen targeted by the TCR is selected from one or more of HBV, HPVE6, P53, MART-1, gp100, NYESO-1, MAGE-A3, MAGE-A4, WT1, CEA, Thyroglobulin or Tyrosinase.

18. A co-expression vector, characterized in that: The co-expression vector simultaneously expresses: (1) One of CAR or TCR; (2) one of integrin β1 or integrin β3 or their modified variants; Preferably, the co-expression vector simultaneously expresses CAR and integrin β1, or simultaneously expresses CAR and a modified variant of integrin β1, or simultaneously expresses CAR and integrin β3, or simultaneously expresses CAR and a modified variant of integrin β3, or simultaneously expresses TCR and integrin β1, or simultaneously expresses TCR and a modified variant of integrin β1, or simultaneously expresses TCR and integrin β3, or simultaneously expresses TCR and a modified variant of integrin β3.

19. The co-expression vector according to claim 18, characterized in that The co-expression vector is a viral vector and / or a non-viral vector; preferably, the viral vector is a lentiviral vector and / or a retroviral vector.

20. The co-expression vector according to claim 19, characterized in that The co-expression vector is constructed by 2A peptide; Preferably, the 2A peptide is selected from one or more of P2A, T2A or F2A; More preferably, the 2A peptide is P2A.

21. The co-expression vector according to claim 20, characterized in that: The P2A is the amino acid sequence shown in SEQ ID NO.9; Preferably, the P2A is a sequence having one or more conservative modifications compared to the amino acid sequence shown in SEQ ID NO.9, wherein the conservative modifications include substitution, deletion or insertion of amino acids; More preferably, the P2A is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO.

9.

22. The co-expression vector according to claim 21, characterized in that The nucleotide sequence of the gene encoding the P2A is the nucleotide sequence shown in SEQ ID NO.10 or its degenerate sequence; Preferably, the nucleotide sequence of the gene encoding the P2A is a sequence having one or more conservative modifications compared to the nucleotide sequence shown in SEQ ID NO.10, wherein the conservative modification includes substitution, deletion or insertion of a nucleotide derivative; More preferably, the nucleotide sequence of the gene encoding the P2A has the same or similar function as the protein encoded by SEQ ID NO.

10.

23. The co-expression vector according to claim 20, characterized in that The T2A is the amino acid sequence shown in SEQ ID NO.11; Preferably, the T2A is a sequence having one or more conservative modifications compared to the amino acid sequence shown in SEQ ID NO.11, wherein the conservative modification includes amino acid substitution, deletion or insertion; More preferably, the T2A is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO.

11.

24. The co-expression vector according to claim 23, characterized in that The nucleotide sequence of the gene encoding the T2A is the nucleotide sequence shown in SEQ ID NO.12 or a degenerate sequence thereof; Preferably, the nucleotide sequence of the gene encoding the T2A is a sequence having one or more conservative modifications compared to the nucleotide sequence shown in SEQ ID NO.12, wherein the conservative modification includes substitution, deletion or insertion of a nucleotide derivative; More preferably, the nucleotide sequence of the gene encoding the T2A has the same or similar function as the protein encoded by SEQ ID NO.

12.

25. The co-expression vector according to claim 20, characterized in that The F2A is the amino acid sequence shown in SEQ ID NO.13; Preferably, the F2A is a sequence having one or more conservative modifications compared to the amino acid sequence shown in SEQ ID NO.13, wherein the conservative modification includes amino acid substitution, deletion or insertion; More preferably, the F2A is a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO.

13.

26. The co-expression vector according to claim 25, characterized in that The nucleotide sequence of the gene encoding the F2A is the nucleotide sequence shown in SEQ ID NO.14 or its degenerate sequence; Preferably, the nucleotide sequence of the gene encoding the F2A is a sequence having one or more conservative modifications compared to the nucleotide sequence shown in SEQ ID NO.14, wherein the conservative modification includes substitution, deletion or insertion of a nucleotide derivative; More preferably, the nucleotide sequence of the gene encoding the F2A has the same or similar function as the protein encoded by SEQ ID NO.

14.

27. A host cell expressing the expression vector according to any one of claims 8 to 9, or expressing the co-expression vector according to any one of claims 18 to 26, characterized in that: The host cell is an immune cell; Preferably, the immune cells are selected from lymphocytes and / or their modified products; More preferably, the lymphocytes are selected from T cells and / or monocytes; Further preferably, the T cells are selected from one or more of αβT cells, NKT cells or γδT cells.

28. A method for constructing an engineered immune cell according to any one of claims 1 to 7 or an engineered immune cell according to any one of claims 10 to 17, characterized in that: The method comprises the following steps: (1) Collecting and activating host cells; (2) adding the expression vector or co-expression vector to the activated host cells for the first infection; (3) After the first infection is completed, an expression vector or a co-expression vector is added for a second infection to obtain host cells expressing integrins.

29. The method according to claim 28, characterized in that The host cell is an immune cell; preferably, the immune cell is selected from lymphocytes and / or their modified products; more preferably, the lymphocyte is selected from T cells and / or monocytes; further preferably, the T cell is selected from one or more of αβT cells, NKT cells or γδT cells; Preferably, the expression vector is an expression vector expressing integrin β1 or integrin β3 or a modified variant thereof; Preferably, the co-expression vector is a co-expression vector that expresses (1) CAR or TCR and (2) integrin β1 or integrin β3 or a modified variant thereof.

30. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises an engineered immune cell according to any one of claims 1-7, or an expression vector according to any one of claims 8-9, or an engineered immune cell according to any one of claims 10-17, or a co-expression vector according to any one of claims 18-26, or a host cell according to claim 27, or an engineered immune cell constructed according to the method of any one of claims 28-29, and a pharmaceutically acceptable carrier or excipient.

31. A kit, characterized in that The kit comprises an engineered immune cell according to any one of claims 1-7, or an expression vector according to any one of claims 8-9, or an engineered immune cell according to any one of claims 10-17, or a co-expression vector according to any one of claims 18-26, or a host cell according to claim 27, or an engineered immune cell constructed according to the method of any one of claims 28-29, or a pharmaceutical composition according to claim 30, and instructions for use.

32. Use of the engineered immune cell according to any one of claims 1-7, or the expression vector according to any one of claims 8-9, or the engineered immune cell according to any one of claims 10-17, or the co-expression vector according to any one of claims 18-26, or the host cell according to claim 27, or the engineered immune cell constructed by the method according to any one of claims 28-29, or the pharmaceutical composition according to claim 30, or the kit according to claim 31 in the preparation of a medicament for treating solid tumor-related diseases.

33. The use according to claim 32, characterized in that The solid tumor-related disease is selected from one or more of lung cancer, liver cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, kidney cancer, lymphoma, esophageal cancer, bile duct cancer, ovarian cancer, gastric cancer, pancreatic cancer, head and neck cancer, synovial sarcoma, angiosarcoma, osteosarcoma, multiple myeloma, glioma, thyroid cancer, endometrial cancer, neuroblastoma, glioblastoma, rhabdomyosarcoma, melanoma, prostate cancer, cervical cancer, soft tissue sarcoma, urothelial carcinoma, bile duct cancer, esophageal cancer, mesothelioma and melanoma; Preferably, the solid tumor-related disease is selected from one or more of lung cancer, liver cancer, breast cancer, colorectal cancer, nasopharyngeal cancer, kidney cancer, lymphoma, ovarian cancer, gastric cancer, pancreatic cancer, multiple myeloma, glioma, prostate cancer, cervical cancer, bile duct cancer, esophageal cancer, and melanoma.

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