WO2021129559A1 - T cell receptor fusion protein and use thereof - Google Patents

Abstract

The present invention provides a recombinant T cell receptor (TCR) fusion protein, which comprises (a) an antigen binding region binding to a surface antigen; (b) a constant region of a TCRγ chain; and (c) a constant region of the TCRδ chain. The present invention further provides a protein complex containing the TCR fusion protein, a nucleic acid containing a coding sequence of the TCR fusion protein, a vector, a cell and a pharmaceutical composition containing the nucleic acid, and use thereof in treatment of diseases.

Description

T cell receptor fusion protein and its use Technical field

The present invention relates to T Cell Receptor (TRC) fusion protein, its encoding nucleic acid molecule and their use.

Background technique

In recent years, the development and achievements of Chimeric Antigen Receptor (CAR)-T cell therapy technology in the field of tumor immunotherapy are obvious to all. However, CAR-T cells have always had many problems in clinical application, especially Cytokine Release Syndrome (CRS) and neurotoxicity caused by CAR-T treatment. CRS is caused by CAR-T off-target or tumor-free T cells attacking normal tissues or T cells killing tumors in large quantities in a short time. Since CAR-T cells have strong affinity for antigens and signal transduction functions, when they bind to related antigens, they will release a large amount of cytokines, causing inflammation and causing CRS. The main manifestations of CRS include high fever, hypotension, shock and so on. At present, the mechanism of neurotoxicity caused by CAR-T cells is not clear, but symptoms such as cerebral edema, increased intracranial pressure, epilepsy, and changes in consciousness caused by CAR-T cells have been observed, and it has been reported that some patients have been caused by this. Kind of neurotoxicity died.

In addition to CAR-T cell therapy, TCR-T cell therapy is also being vigorously developed and promoted, and has achieved good results in clinical trials. TCR is a characteristic mark on the surface of all T cells, including two types: the αβ type composed of two chains of α and β, and the γδ type composed of two chains of γ and δ. Each T cell expresses only one type of TCR, resulting in two T cell populations: αβT cells and γδT cells. In humans, most mature T cells are αβ T cells, and only 1%-5% are γδ T cells. In T cells, when the extracellular domain of the αβ chain or the γδ chain recognizes the antigen, the TCR will non-covalently bind to the CD3 complex to form the TCR-CD3 complex, and then conduct signal transduction and T cell activation. Among them, γδ T cells are a subgroup that regulates and initiates an immune response against infection. For example, there is a type of γδ T cell in peripheral blood, and the Vγ9Vδ2 TCR molecule expressed by it (that is, the TCR composed of the 9th fragment of the γ chain V gene and the No. 2 fragment of the δ chain V gene) can directly recognize the phosphoantigen. Then the activation signal is transmitted through TCR/CD3 to exercise its killing function. At the same time, these cells also secrete cytokines TNF-α and TNF-γ, and participate in the activation of helper T cells Th1.

At present, TCR-T cell therapy generally involves genetic modification of the α chain and β chain of the TCR, and then introducing the modified TCR into T cells to produce tumor antigen-specific T cells, which enhances the effect of TCR on tumor-associated antigens (Tumor Associated Antigen, TAA), thereby enhancing the recognition of tumor cells by T cells, and ultimately killing tumor cells. However, the ability of this TCR to recognize specific antigens will be restricted by the Major Histocompatibility Complex (MHC), and the introduced modified αβ chain and the αβ chain in the endogenous TCR Mismatches may be formed between them, thereby inducing harmful recognition of self-antigens, leading to graft-versus-host disease.

The present invention provides a new T cell receptor fusion protein, which comprises the constant regions of the TCR γ chain and the δ chain and an antigen binding region that specifically binds to cell surface antigens connected thereto. This new fusion protein can release much less pro-inflammatory cytokines than CAR while maintaining the lethality comparable to traditional CAR. The release level of these cytokines is comparable to that of CRS and adoptive CAR-T therapy. Dose limiting toxicity is related.

Summary of the invention

The first subject of the present invention is a recombinant T cell receptor (TCR) fusion protein, which includes an antigen binding region that binds to a surface antigen, a constant region of the TCR γ chain, and a constant region of the TCR δ chain.

In one embodiment, the antigen binding region is directly connected to the constant region of the TCR γ chain and/or the constant region of the TCR δ chain. In another embodiment, the antigen binding region is connected to the constant region of the TCR γ chain and/or the constant region of the TCR δ chain through a connecting peptide. In this embodiment, the connecting peptide is selected from (G4S)n, CD8 and IgG4, where n is an integer of 1-4.

In one embodiment, the TCR fusion protein of the present invention does not include the variable region of the TCR gamma chain and/or the variable region of the TCR delta chain. In another embodiment, the TCR fusion protein of the present invention includes the variable region of the TCR γ chain and/or the variable region of the TCR δ chain, and preferably includes both the variable region of the TCR γ chain and the variable region of the TCR δ chain. . In a specific embodiment, the constant region of the TCR γ chain is selected from SEQ ID NO: 14, 32, or a functional variant with at least 85% sequence identity therewith; wherein the constant region of the TCR δ chain It is selected from SEQ ID NO: 16, 34, or a functional variant with at least 85% sequence identity thereto. When the TCR fusion protein of the present invention further includes the variable region of the TCR γ chain and the variable region of the TCR δ chain (that is, when the TCR fusion protein includes a complete TCR γ chain and a δ chain, the TCR γ chain is selected from SEQ ID NO: 18, or a functional variant with at least 85% sequence identity therewith; the TCR δ chain is selected from SEQ ID NO: 20, or a functional variant with at least 85% sequence identity with it.

In one embodiment, the TCR fusion protein of the present invention contains one or two antigen binding regions that bind the same or different antigens. Specifically, the antigen binding region is selected from scFv, VH domain, VL domain, single domain antibody, Nanobody, antigen binding ligand (such as PD1, PDL1, PDL2, TGFβ, APRIL and NKG2D), recombinant fibronectin structure Domain, anticalin and DARPIN.

In one embodiment, the antigen binding region contained in the TCR fusion protein of the present invention binds to an antigen selected from the group consisting of TSHR, CD19, CD123, CD22, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33 , EGFRvIII, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-1 Ra, PSCA , PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, Folate receptor α, ERBB2 (Her2/neu), MUC1, EGFR, NCAM, Claudin18.2, Prostase, PAP, ELF2M, Ephrin B2 IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, Fucosyl GMl, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY- ESO-1, LAGE-la, MAGE-A1, legumin, HPV E6, E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos related Antigen 1, p53, p53 mutant, prostate specific protein, survivin and telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG( TMPRSS2ETS fusion gene), NA17, PAX3, androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES 1, LCK, AKAP-4, SSX2, RAGE-1 , Human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5 , IGLL1 and their Meaning combination. Preferably, the surface antigen is selected from: CD19, CD20, CD22, BAFF-R, CD33, EGFRvIII, BCMA, GPRC5D, PSMA, ROR1, FAP, ERBB2 (Her2/neu), MUC1, EGFR, CAIX, WT1, NY -ESO-1, CD79a, CD79b, GPC3, Claudin 18.2.

In one embodiment, the antigen binding region is a monoclonal antibody, polyclonal antibody, recombinant antibody, human antibody, humanized antibody, murine antibody, chimeric antibody and functional fragments thereof. In a specific embodiment, the antigen binding region is selected from the heavy chain variable region or light chain variable region of an anti-CD19 scFv, an anti-CD19 antibody, preferably, the antigen binding region is selected from SEQ ID NO: 4 , 6, 8, 10, 12, or functional variants with at least 95% sequence identity with which retain surface antigen binding activity.

In one embodiment, the TCR fusion protein of the present invention further includes a transmembrane domain. Specifically, the transmembrane domain is selected from the transmembrane domains of the following proteins: TCRγ chain, TCRδ chain, CD3ζ subunit, CD3εTCR subunit, CD3γTCR subunit, CD3δTCR subunit, CD45, CD4, CD5, CD8, CD9 , CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and their functional fragments. Preferably, the transmembrane domain is selected from the transmembrane domains of the following proteins: TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit and functional fragments thereof.

In one embodiment, the TCR fusion protein of the present invention further includes a costimulatory domain. Specifically, the costimulatory domain is a functional signaling domain obtained from the following proteins: TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, CD66d, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD -L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM and ZAP70.

In one embodiment, the TCR fusion protein of the present invention further comprises a signal peptide and/or 2A peptide.

In one embodiment, the present invention also provides a protein complex comprising the fusion protein of the present invention and at least one endogenous CD3 subunit or endogenous CD3 complex. Preferably, the endogenous CD3 subunit is selected from the group consisting of CD3ζ subunit, CD3ε subunit, CD3γ subunit and CD3δ subunit; the endogenous CD3 complex is composed of CD3ζ subunit, CD3ε subunit, CD3γ subunit And the complex formed by the CD3δ subunit.

The second subject of the present invention is a nucleic acid comprising a sequence encoding the TCR fusion protein of the present invention, a vector comprising the nucleic acid, the system of the present invention, a cell comprising the nucleic acid or the vector or system, and the TCR fusion protein , The nucleic acid, the vector, the system or the pharmaceutical composition of the cell.

In one embodiment, the invention provides a nucleic acid comprising a sequence encoding the TCR fusion protein of the invention. Preferably, the nucleic acid is DNA or RNA, more preferably mRNA.

In one embodiment, the present invention provides a vector comprising the aforementioned nucleic acid. Specifically, the vector is selected from linear nucleic acid molecules, plasmids, retroviruses, lentiviruses, adenoviruses, vaccinia virus, Rous sarcoma virus (RSV), polyoma virus and adeno-associated virus (AAV), bacteriophages, bacteriophages Granules, cosmids or artificial chromosomes. In some embodiments, the vector also includes a starter, selection marker, restriction enzyme cleavage site, promoter, polyadenylic acid tail (polyA), 3'UTR, 5'UTR, enhanced Element, terminator, insulator, operon, selectable marker, reporter gene, targeting sequence and/or protein purification tag. In a specific embodiment, the vector is an in vitro transcribed vector.

In one embodiment, the present invention also provides a cell comprising the aforementioned nucleic acid or vector or system. Preferably, the cells are immune cells, such as T cells, macrophages, dendritic cells, monocytes, NK cells or NKT cells, more preferably T cells or natural killer cells, even more preferably CD4+/CD8+ double positive T cells, CD4+ helper T cells, CD8+ T cells, tumor infiltrating cells, memory T cells, naive T cells, natural killer (NK) cells, natural killer T (NKT) cells, γδ-T cells, αβ-T cells. In a specific embodiment, the cell contains one or more TCR fusion proteins or protein complexes of the present invention. In another specific embodiment, the cell lacks TCR α chain and/or TCR β chain.

In one embodiment, the present invention also provides a vector system, which includes: (a) a first nucleic acid sequence encoding the constant region of the TCR gamma chain; (b) a second nucleic acid sequence encoding the constant region of the TCR delta chain; The first nucleic acid sequence and/or the second nucleic acid sequence are operably linked to the third nucleic acid sequence encoding the antigen binding region, and the first nucleic acid sequence and the second nucleic acid sequence are located in the same vector or different vectors. The meaning of the carrier is as defined above.

In one embodiment, the present invention also provides a pharmaceutical composition comprising the TCR fusion protein, nucleic acid, vector, system, cell or cell population of the present invention and pharmaceutically acceptable excipients. In a specific embodiment, the pharmaceutically acceptable excipient is selected from one or more of the following: fillers, binders, disintegrants, coating agents, adsorbents, anti-adhesive agents , Glidants, antioxidants, flavors, colorants, sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, Isotonic agents, absorption delay agents, stabilizers and tonicity modifiers. In a specific embodiment, the pharmaceutical composition is administered topically, intraarterially, intramuscularly, subcutaneously, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual, or intranasal Route administration. In a specific embodiment, the pharmaceutical composition is an ointment, cream, transdermal patch, gel, powder, tablet, solution, aerosol, granule, pill, suspension, In the form of emulsion, capsule, syrup, liquid, elixir, extract, tincture or liquid extract extract.

The third subject of the present invention relates to a method for preparing modified immune cells, including introducing the TCR fusion protein, protein complex, nucleic acid or vector of the present invention into the immune cells, preferably T cells, macrophages, and dendritic cells , Monocytes, NK cells or NKT cells, more preferably T cells, NK cells or NKT cells.

The fourth subject of the present invention relates to a method of treating a subject suffering from a disease related to surface antigen expression.

In one embodiment, the above method includes administering to the mammal an effective amount of the TCR fusion protein, protein complex, nucleic acid, vector, cell or pharmaceutical composition of the present invention. In another embodiment, the above-mentioned method includes the following steps: (a) providing a sample of the subject, the sample containing immune cells; (b) combining the TCR fusion protein, protein complex, nucleic acid, and carrier of the present invention in vitro , Cells and/or pharmaceutical compositions are introduced into the immune cells to obtain modified immune cells, and (c) administering the modified immune cells to the subject. Preferably, the immune cells are autologous or allogeneic cells, preferably T cells, macrophages, dendritic cells, monocytes, NK cells or NKT cells, more preferably T cells, NK cells or NKT cells.

In one embodiment, the subject is a human, non-human primate, mouse, rat, dog, cat, horse, or cow.

In one embodiment, the disease related to surface antigen expression is selected from: blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer, peritoneal cancer, Cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma (GBM ), liver cancer, hepatocellular tumor, intraepithelial tumor, kidney cancer, laryngeal cancer, leukemia, liver tumor, lung cancer (such as small cell lung cancer, non-small cell lung cancer, glandular lung cancer and squamous lung cancer), lymphoma (including Hodge Gold lymphoma and non-Hodgkin lymphoma), melanoma, myeloma, neuroblastoma, oral cancer (such as lips, tongue, mouth and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, Rhabdomyosarcoma, rectal cancer, cancer of the respiratory system, salivary gland cancer, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system cancer, vulvar cancer and other cancers and sarcomas , And B-cell lymphoma (including low-grade/follicular non-Hodgkin’s lymphoma (NHL), small lymphocytic (SL) NHL, intermediate/follicular NHL, intermediate-level diffuse NHL, high-grade immune cells NHL, high-grade lymphoblastic NHL, high-grade small non-cracked cell NHL, large mass disease NHL), mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom macroglobulinemia, chronic lymphocytic leukemia (CLL), Acute lymphocytic leukemia (ALL), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), B-cell young lymphocytic leukemia, blastic plasmacytoid dendritic cell tumor , Burkitt’s lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, chronic myelogenous leukemia (CML), malignant lymphoproliferative disease, MALT lymphoma, hairy cell leukemia, marginal zone lymphoma, multiple Myeloma, myelodysplasia, plasmablastic lymphoma, pre-leukemia, plasmacytoid dendritic cell tumor, and post-transplant lymphoproliferative disorder (PTLD).

Detailed description of the invention

Unless otherwise specified, the meanings of all scientific and technical terms used herein are the same as those commonly understood by those of ordinary skill in the art to which the present invention belongs.

TCR fusion protein

The first subject of the present invention is a recombinant T cell receptor (TCR) fusion protein, which includes an antigen binding region that binds to a surface antigen, a constant region of the TCR γ chain, and a constant region of the TCR δ chain.

As used herein, the term "TCR fusion protein" refers to an isolated recombinant protein comprising an antigen binding region and TCR gamma and delta chains or parts of their constant regions. Generally speaking, when correctly expressed, the TCR fusion protein of the present invention can bind to the surface antigen on the target cell and interact with the endogenous CD3 component in the target cell to form a TCR fusion protein-CD3 complex.

"T cell receptor" or "TCR" are used interchangeably herein, and refer to a specific receptor on the surface of T cells, whose main function is to recognize antigens and activate T cells. TCR is a heterodimer fixed on the cell membrane and consists of two different subunits connected by disulfide bonds. According to the different subunits, TCR is divided into two types: αβ type and γδ type. Among them, about 95% of TCRs are composed of α subunits and β subunits, and about 5% of TCRs are composed of γ subunits and δ subunits. TCRα, TCRβ, TCRγ, and TCRδ subunits are encoded by the TCRA, TCRB, TCRG and TCRD loci, respectively.

TCR and CD3 molecules (including CD3δ/ε dimer, CD3γ/ε dimer, and CD3ζ/ζ) form a TCR-CD3 complex (see Figure 2). Therefore, as used herein, the term "CD3 subunit" refers to the CD3δ, CD3ε, CD3γ, and CD3ζ subunits, which together make up the CD3 complex. There are many immunoreceptor tyrosine activation motifs (Immunoreceptor Tyrosine-based Activation Motifs, ITAM) in the intracellular region of CD3. When the TCR is activated by binding to the antigen, the tyrosine in these ITAM sequences is phosphorylated, thus realizing the signal transmission across the membrane, and finally activating T cells.

Each subunit of TCR contains two extracellular domains: variable region (V region) and constant region (C region). The variable region of TCR is responsible for recognizing the antigen, and the constant region is responsible for anchoring the chain in the transmembrane region of the plasma membrane. The variable region of TCR includes a framework region and three Complementary Determining Regions (CDR): CDR1, CDR2 and CDR3, among which CDR3 is the main determinant of antigen recognition and specificity. Therefore, the variable region of the TCR of the present invention includes a complete TCR variable region, functional fragments thereof such as one or more of CDR1, CDR2, and CDR3, or functional variants thereof. The constant/variable regions of the TCR gamma chain and delta chain can be derived from humans or other species, such as mice. For example, the constant region/variable region of the human TCR γ chain or δ chain can be replaced with a murine constant region/variable region to form a "chimeric constant region/variable region", or a murine constant region/variable region can be completely used. The variable region replaces the constant region/variable region of the human TCR gamma chain and delta chain. In one embodiment, the constant region of the TCR γ chain is selected from SEQ ID NO: 14 and SEQ ID NO: 32 and functional variants thereof; the constant region of the TCR δ chain is selected from SEQ ID NO: 16 and SEQ ID NO: 34 and Its functional variant.

In one embodiment, the TCR fusion protein of the present invention does not include the variable region of the TCR gamma chain and/or the variable region of the TCR delta chain. In another embodiment, the TCR fusion protein of the present invention includes the variable region of the TCR γ chain and/or the variable region of the TCR δ chain, and preferably includes both the variable region of the TCR γ chain and the variable region of the TCR δ chain. . In another embodiment, when the variable region is included, the complete TCR gamma chain is selected from SEQ ID NO: 18 and functional variants thereof; the complete TCR gamma chain is selected from SEQ ID NO: 20 and functional variants thereof.

In one embodiment, the antigen binding region is directly connected to the constant region of the TCR γ chain and/or the constant region of the TCR δ chain. In another embodiment, the antigen binding region is connected to the constant region of the TCR γ chain and/or the constant region of the TCR δ chain through a connecting peptide.

As used herein, the term "connector peptide" refers to a peptide linker composed of amino acids that connects the antigen binding region and the TCR constant region, and its length is generally 10-120 amino acids, preferably 10-100, 10-80, 10-60, More preferably 10-50 amino acids. The connecting peptides that can be used in the present invention are well known to those skilled in the art.

In one embodiment, the connecting peptide is (G4S)n, where n is an integer from 1-4. In other embodiments, the connecting peptide is a CD8 or IgG connecting peptide, preferably a CD8α, IgG1 or IgG4 connecting peptide.

In one embodiment, the TCR fusion protein of the present invention further includes a signal peptide.

As used herein, the term "signal peptide" refers to a short peptide chain (5-30 amino acids in length) that guides the transfer of newly synthesized proteins to the secretory pathway. The signal peptide is generally located at the N-terminus of the peptide chain, which can guide the transmembrane transfer (localization) of the protein and is responsible for guiding the protein into the subcellular organelles of the cell. The present invention can use signal peptides well known to those skilled in the art, such as membrane protein signal peptides, such as CD8α signal peptide, CD33 signal peptide, CD4 signal peptide; and cell secretion factor signal peptides, such as IL-2 signal peptide, CCL19 signal peptide.

In one embodiment, the TCR fusion protein of the present invention further includes 2A peptide.

As used herein, the terms "2A sequence", "2A peptide" or "2A viral peptide" are used interchangeably and belong to the cis-hydrolase action element (CHYSEls), which was originally found in foot-and-mouth disease virus (FMDV). The average length of the 2A peptide is 18-22 amino acids. During protein translation, the 2A peptide can be broken from the C-terminus of the last two amino acids of itself through ribosome jumping. Specifically, the peptide chain binding group between glycine and proline is damaged at position 2A, which can trigger ribosome jumping and start translation from the second codon, thereby making two proteins in one transcription unit Independent expression. This 2A peptide-mediated cleavage is widespread in eukaryotic animal cells. Using the higher shearing efficiency of 2A peptide and the ability to promote balanced expression of upstream and downstream genes can improve the expression efficiency of heterologous polyproteins (such as cell surface receptors, cytokines, immunoglobulins, etc.). Conventional 2A peptides include: P2A, T2A, E2A, F2A, etc. For example, in the present invention, when the constant regions of the TCR γ chain and the δ chain are located on the same expression vector, the 2A peptide may be located between the transcription unit containing the TCR γ chain constant region and the transcription unit containing the TCR δ chain constant region, so that These two transcription units can be expressed independently without affecting each other.

In one embodiment, the TCR fusion protein of the present invention contains one or two antigen binding regions that bind the same or different antigens.

In one embodiment, the antigen binding region contained in the TCR fusion protein of the present invention binds to one or more surface antigens selected from the group consisting of TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-1 Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, Folate receptor α, ERBB2 (Her2/neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor , CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, Fucosyl GMl, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1/CD248, TEM7R, CLDN6, GPRC5D , CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE -la, MAGE-A1, legumin, HPV E6, E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostate-specific protein, survivin and telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2ETS fusion gene), NA17, PAX3, androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reversal Recordase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1 and any of them combination. In a preferred embodiment, the surface antigen is selected from: CD19, CD20, CD22, BAFF-R, CD33, EGFRvIII, BCMA, GPRC5D, PSMA, ROR1, FAP, ERBB2 (Her2/neu), MUC1, EGFR, CAIX, WT1, NY-ESO-1, CD79a, CD79b, GPC3, Claudin 18.2.

As used herein, "antigen binding region" refers to any structure or functional variant thereof that can bind to an antigen, including but not limited to monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric Antibodies and their functional fragments. For example, the antigen binding region includes but is not limited to single chain antibody (Single Chain Antibody Fragment, scFv) and its fragments (for example, heavy chain variable domain (VH), light chain variable domain (VL)), single domain Antibodies, nanobodies, antigen-binding ligands, and alternative scaffolds known in the art that can be used as antigen-binding regions, such as recombinant fibronectin domain, anticalin, DARPIN, etc. In the present invention, the antigen binding region may be monovalent or divalent.

A "single chain antibody" or "scFv" is an antibody in which the variable region of the heavy chain (VH) of the antibody and the variable region of the light chain (VL) are connected by a linker. The optimal length and/or amino acid composition of the linker can be selected. The length of the linker will significantly affect the folding and interaction of the variable region of scFv. In fact, if a shorter linker (for example, between 5-10 amino acids) is used, intra-chain folding can be prevented. For the selection of the size and composition of the linker, see, for example, Hollinger et al., 1993 Proc Natl Acad. Sci. USA 90: 6444-6448; U.S. Patent Application Publication Nos. 2005/0100543, 2005/0175606, 2007/0014794; and PCT Publication Nos. WO2006/020258 and WO2007/024715, the entire contents of which are incorporated herein by reference.

"Single domain antibody" refers to an antibody that naturally lacks the light chain. The antibody contains only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, also known as "heavy chain antibodies." The separately cloned and expressed VHH structure has structural stability and antigen binding activity equivalent to that of the original heavy chain antibody. It is the smallest unit known to bind the target antigen, also known as Nanobody (Nb) .

The antigen binding region of the TCR fusion protein of the present invention may also include a natural or synthetic ligand that specifically recognizes and binds to the target antigen. For example, ligands that can be used in the present invention include but are not limited to PD1, PDL1, PDL2, TGFβ, APRIL, NKG2D, and the like.

The term "functional variant" or "functional fragment" refers to a variant that essentially contains the amino acid sequence of the parent but contains at least one amino acid modification (ie substitution, deletion or insertion) compared to the parent amino acid sequence, provided that all The variant retains the biological activity of the parent amino acid sequence. For example, the above-mentioned amino acid modifications can be introduced or present in the variable region and/or constant region and/or antigen binding region of the TCR fusion protein, and can be used to adjust binding strength and specificity, post-translational processing (e.g., glycosylation) , Thermodynamic stability, solubility, surface expression or TCR assembly properties. In one embodiment, the amino acid modification is preferably a conservative modification.

As used herein, the term "conservative modification" refers to an amino acid modification that does not significantly affect or change the binding characteristics of an antibody or antibody fragment containing the amino acid sequence. These conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the TCR fusion protein 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 having a similar side chain. Amino acid residue families with similar side chains have been defined in the art, including 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), non-polar side chains (e.g. alanine, valine) Acid, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g. threonine, valine, isoleucine) And aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine). Conservative modifications can be selected, for example, based on polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or similarity in the amphipathic properties of the residues involved.

Therefore, a "functional variant" or "functional fragment" has at least 75%, preferably at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84% of the parent amino acid sequence. %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, And retain the biological activity of the parent amino acid, such as binding activity.

As used herein, the term "sequence identity" refers to the degree to which two (nucleotide or amino acid) sequences have the same residue at the same position in the alignment, and is usually expressed as a percentage. Preferably, identity is determined over the overall length of the sequences being compared. Therefore, two copies with exactly the same sequence have 100% identity. Those skilled in the art will recognize that some algorithms can be used to determine sequence identity using standard parameters, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147: 195-197) and ClustalW.

In one embodiment, the invention encompasses modifications to the amino acid sequence of the starting antibody or fragment (e.g., scFv) that result in functionally equivalent molecules. For example, the VH or VL of the antigen binding region (such as scFv) contained in the TCR fusion protein can be modified to maintain at least about 75%, 76%, 77%, 78%, 79%, 80%, and the parent VH or VL framework. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% sequence identity. In order to produce a functionally equivalent molecule, the present invention covers the modification of the complete TCR fusion protein, for example, the modification in one or more amino acid sequences of various domains of the TCR fusion protein. The TCR fusion protein can be modified to maintain at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86% with the parent TCR fusion protein , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

Therefore, in one embodiment, the TCR fusion protein of the present invention comprises the heavy chain variable region or the light chain variable region of an anti-CD19 scFv, an anti-CD19 antibody. Preferably, the antigen binding region contained in the TCR fusion protein of the present invention is selected from SEQ ID NO: 4, 6, 8, 10, 12 and functional fragments thereof, wherein the functional fragment is the same as SEQ ID NO: 4, 6 , 8, 10, 12 have at least 95%, 96%, 97%, 98% or 99% sequence identity of the amino acid sequence of the heavy chain variable region or light chain variable region of an anti-CD19 scFv or anti-CD19 antibody.

In one embodiment, the constant region of the TCR γ chain of the present invention is selected from SEQ ID NO: 14, 32, or a functional variant having at least 85% sequence identity therewith; wherein the constant region of the TCR δ chain is selected From SEQ ID NO: 16, 34, or a functional variant with at least 85% sequence identity thereto. In one embodiment, when the variable regions of the TCR γ chain and the δ chain are included, the TCR γ chain of the present invention is selected from SEQ ID NO: 18, or a functional variant having at least 85% sequence identity therewith; The TCR δ chain is selected from SEQ ID NO: 20, or a functional variant with at least 85% sequence identity thereto.

In one embodiment, the TCR fusion protein of the present invention further includes a transmembrane domain. In this embodiment, in general, the transmembrane domain and the constant region contained in the TCR fusion protein are derived from the same genomic sequence, that is, the transmembrane domain of the TCR γ chain or the δ chain. However, TCR fusion proteins can also be designed to contain a transmembrane domain heterologous to the constant region. The transmembrane domain may include one or more additional amino acids adjacent to the transmembrane region, for example, one or more amino acids associated with the extracellular region of the protein that is the source of the transmembrane protein (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 15 amino acids) and/or one or more additional amino acids associated with the intracellular region of the protein that is the source of the transmembrane protein (e.g., intracellular region) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 15 amino acids). In one embodiment, the transmembrane domains may be selected or modified by amino acid substitutions to prevent such domains from binding to the transmembrane domains of the same or different surface membrane proteins, for example, to interact with other members of the receptor complex. The interaction is minimized. In one embodiment, the transmembrane domain is capable of homodimerization with another TCR fusion protein on the surface of the T cell expressing the TCR fusion protein. In another embodiment, the amino acid sequence of the transmembrane domain may be modified to minimize the interaction with the binding domain of the natural binding partner present in the same TCR fusion protein.

The transmembrane domain can be derived from a natural source or a recombinant source. Where the source is a natural source, the domain can be derived from any membrane-bound or transmembrane protein. In one embodiment, when the TCR fusion protein binds to the target antigen, the transmembrane domain is capable of signal transduction. The transmembrane domain particularly suitable for use in the present invention may at least include, for example, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22 , CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and their functional fragments.

In some cases, the transmembrane domain can be connected to the constant region of the TCR fusion protein by a hinge, such as a hinge from a human protein. For example, in one embodiment, the hinge may be a human immunoglobulin (Ig) hinge, such as an IgG4 hinge, or a CD8a hinge.

In one embodiment, the TCR fusion protein of the present invention may also include a costimulatory domain.

The term "costimulatory domain" refers to a homologous binding partner that specifically binds to a costimulatory ligand on T cells, thereby mediating the costimulatory response of T cells. The costimulatory domain may be an intracellular functional signaling domain derived from a costimulatory molecule, which may include the entire intracellular part of the molecule from which the domain is derived, or the entire natural intracellular signaling domain, or its function Fragment. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for efficient immune response. The proliferation of lymphocytes requires not only antigen binding, but also signals from costimulatory molecules. Co-stimulatory molecules include, but are not limited to, Class 1 MHC molecules, BTLA and Toll ligand receptors, as well as OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278) and 4-1BB (CD137). Costimulatory molecules can be represented by the following protein families: TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), and activated NK cell receptor. Examples of these molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C , SLAMF7, NKp80, CD160, B7-H3, and ligands that specifically bind to CD83. Therefore, in one embodiment, the costimulatory domain present in the TCR fusion protein of the present invention is a signal transduction domain from a protein selected from the group consisting of: TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22 , CD79a, CD79b, CD66d, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM and ZAP70. The term "4-1BB" refers to a member of the TNFR superfamily with the amino acid sequence as provided in GenBank accession number AAA62478.2 or equivalent residues from non-human species, such as mouse, rodent, monkey, ape, etc.; And "4-1BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank accession number AAA62478.2, or equivalent residues from non-human species, such as mouse, rodent, monkey, ape, etc.

In one embodiment, the present invention also provides a protein complex comprising the TCR fusion protein of the present invention and at least one endogenous CD3 subunit or endogenous CD3 complex. Preferably, the endogenous CD3 subunit is selected from the group consisting of CD3ζ subunit, CD3ε subunit, CD3γ subunit and CD3δ subunit; the endogenous CD3 complex is composed of CD3ζ subunit, CD3ε subunit, CD3γ subunit And the complex formed by the CD3δ subunit.

Nucleic Acid

The present invention also provides a nucleic acid comprising a sequence encoding the TCR fusion protein of the present invention.

As used herein, the term "nucleic acid" includes sequences of polyribonucleotides and polydeoxyribonucleotides, such as modified or unmodified RNA or DNA, each in a single-stranded and/or double-stranded form of linear or Cyclic, or their mixtures (including hybrid molecules). Therefore, the nucleic acid according to the present invention includes DNA (such as dsDNA, ssDNA, cDNA), RNA (such as dsRNA, ssRNA, mRNA, ivtRNA), combinations or derivatives thereof (such as PNA). Preferably, the nucleic acid is DNA or RNA, more preferably mRNA.

Nucleic acids may contain conventional phosphodiester bonds or unconventional bonds (such as amide bonds, such as those found in peptide nucleic acids (PNA)). The nucleic acid of the present invention may also contain one or more modified bases, such as, for example, trityl bases and unusual bases (such as inosine). Other modifications are also conceivable, including chemical, enzymatic or metabolic modifications, as long as the binding molecule of the present invention can be expressed from a polynucleotide. The nucleic acid can be provided in an isolated form. In one embodiment, the nucleic acid may also include regulatory sequences, such as transcription control elements (including promoters, enhancers, operators, repressors, and transcription termination signals), ribosome binding sites, introns, and the like.

The nucleic acid sequence of the present invention can be codon-optimized for optimal expression in desired host cells (eg, human lymphocytes); or for expression in bacteria, yeast, or insect cells. Codon optimization refers to the replacement of codons that are generally rare in the highly expressed genes of a given species in the target sequence with codons that are generally common in the highly expressed genes of such species, and the codons before and after the replacement Code the same amino acid. Therefore, the choice of the best codon depends on the codon usage preference of the host genome.

Carrier or carrier system

The present invention also provides a vector comprising one or more nucleic acids as described in the present invention.

The present invention also provides a vector system comprising (a) a first nucleic acid sequence encoding the constant region of the TCR gamma chain; (b) a second nucleic acid sequence encoding the constant region of the TCR delta chain; wherein the first nucleic acid sequence And/or the second nucleic acid sequence is operably linked to the third nucleic acid sequence encoding the antigen binding region, and the first nucleic acid sequence and the second nucleic acid sequence are located in the same vector or in different vectors.

As used herein, the term "operably linked" refers to a functional connection relationship between two nucleic acid sequences, especially on the same polynucleotide molecule. For example, when the first nucleic acid sequence and the second nucleic acid sequence are in a functional relationship, the first nucleic acid sequence and the second nucleic acid sequence are operably linked. For example, if the promoter affects the transcription or expression of the coding sequence, the promoter is operably linked to the coding sequence. The operably linked DNA sequences can be adjacent to each other and, for example, are in the same reading frame when two protein coding regions need to be joined.

As used herein, the term "vector" is a nucleic acid molecule used as a vehicle for transferring (exogenous) genetic material into a host cell, where the nucleic acid molecule can be replicated and/or expressed, for example.

Vectors generally include targeting vectors and expression vectors. A "targeting vector" is a medium that delivers an isolated nucleic acid to the inside of a cell by, for example, homologous recombination or a hybrid recombinase using a sequence at a specific targeting site. An "expression vector" is a vector used for the transcription of heterologous nucleic acid sequences (such as those encoding the TCR fusion protein of the present invention) in a suitable host cell and the translation of their mRNA. Suitable vectors that can be used in the present invention are known in the art, and many are commercially available. In one embodiment, the vector of the present invention includes, but is not limited to, linear nucleic acid molecules (e.g. DNA or RNA), plasmids, viruses (e.g. retrovirus, lentivirus, adenovirus, vaccinia virus, Rous sarcoma virus (RSV, multiple Oncovirus and adeno-associated virus (AAV), etc.), phage, phagemid, cosmid and artificial chromosome (including BAC and YAC). The vector itself is usually a nucleotide sequence, usually a DNA sequence containing an insert (transgene) And the larger sequence as the "backbone" of the vector. The engineered vector usually also contains a starting point for autonomous replication in the host cell (if stable expression of the polynucleotide is required), a selection marker and a restriction enzyme cleavage site (such as a multiple cloning site) , MCS). The vector may additionally include a promoter, polyadenylic acid tail (polyA), 3'UTR, enhancer, terminator, insulator, operon, selectable marker, reporter gene, targeting sequence and/or protein purification Elements such as tags, etc. In a specific embodiment, the vector is an in vitro transcribed vector.

Host cell

The present invention also provides a host cell, which comprises the TCR fusion protein, nucleic acid or vector of the present invention.

As used herein, the term "host cell" refers to a cell that can be or has been a recipient of the nucleic acid or vector described herein and/or express (and optionally secrete) the TCR fusion protein of the present invention. Generally speaking, host cells include prokaryotic or eukaryotic cells, and also include, but are not limited to, bacteria, yeast cells, fungal cells, plant cells, and animal cells, such as insect cells and mammalian cells. Such as mouse, rat, macaque or human cells.

In one embodiment, the host cell is an immune cell, such as T cells, macrophages, dendritic cells, monocytes, NK cells, and/or NKT cells. Preferably, the host cell is a T cell. In the present invention, T cells capable of expressing the TCR fusion protein of the present invention are also referred to as TRUE-T (T cell receptor fusion engager-T) cells. The T cell may be any T cell, such as a T cell cultured in vitro, such as a primary T cell, or a T cell derived from a T cell line cultured in vitro, such as Jurkat, SupT1, etc., or a T cell obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. T cells can also be concentrated or purified. T cells can be any type of T cells and can be at any stage of development, including but not limited to CD4+/CD8+ double positive T cells, CD4+ helper T cells (such as Th1 and Th2 cells), CD8+ T cells (such as cytotoxicity) T cells), tumor infiltrating cells, memory T cells, naive T cells, γδ-T cells, αβ-T cells, etc. In a preferred embodiment, the host cell is a human T cell. Various techniques known to those skilled in the art, such as Ficoll isolation, can be used to obtain T cells from the blood of the subject.

The polynucleotides and/or vectors of the present invention can be introduced into host cells using conventional methods known in the art (such as by transduction, transfection, transformation, etc.). "Transfection" is the process of introducing nucleic acid molecules or polynucleotides (including vectors) into target cells. One example is RNA transfection, the process of introducing RNA (such as in vitro transcribed RNA, ivtRNA) into host cells. The term is mainly used for non-viral methods in eukaryotic cells. The term "transduction" is generally used to describe virus-mediated transfer of nucleic acid molecules or polynucleotides. Transfection of animal cells usually involves opening transient holes or "holes" in the cell membrane to allow uptake of material. Transfection can be performed using calcium phosphate, by electroporation, by cell extrusion, or by mixing cationic lipids with materials to produce liposomes that fuse with cell membranes and deposit their cargoes inside. Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate/DNA co-precipitation), microinjection, and electroporation. perforation. The term "transformation" is used to describe the non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria and non-animal eukaryotic cells (including plant cells). Therefore, transformation is a genetic modification of bacteria or non-animal eukaryotic cells, which is produced by the direct uptake of the cell membrane from its surroundings and subsequent incorporation of exogenous genetic material (nucleic acid molecules). Conversion can be achieved by manual means. In order for transformation to occur, the cell or bacteria must be in a competent state. For prokaryotic transformation, techniques can include heat shock-mediated uptake, bacterial protoplast fusion with intact cells, microinjection, and electroporation. Techniques for plant transformation include Agrobacterium-mediated transfer (such as by A. tumefaciens), rapidly advanced tungsten or gold microprojectiles, electroporation, microinjection, and polyethylene glycol mediation. Guided intake.

In a preferred embodiment, the host cell lacks TCR α chain and/or TCR β chain. The inventors unexpectedly discovered that, compared with wild-type T cells, the TRUE-T cells of the present invention can express TCR fusion proteins more efficiently in T cells lacking TCR α chain and/or β chain, and achieve the same effect as traditional CAR-T. The cell has comparable tumor killing effect, while significantly reducing the release of cytokines, thereby reducing the risk of developing CRS. In addition, since the TRUE-T cell of the present invention expresses γδ type TCR, it can also avoid mismatches with endogenous TCRα chain and/or TCR β chain, thereby reducing the occurrence of graft-versus-host disease.

The technology of knocking out TCR α chain and/or TCR β chain in cells is well known to those skilled in the art, such as CRISPR system, TALEN system, zinc finger nuclease system, base editor, RNAi technology, antisense? Morpholino (Morpholino) and other technologies are used to knock out TCRα chains and/or TCRβ chains in T cells.

Pharmaceutical composition

In one embodiment, the present invention also provides a pharmaceutical composition comprising the TCR fusion protein, nucleic acid, vector, system or host cell of the present invention as an active agent, and one or more pharmaceutically acceptable Excipients. Therefore, the present invention also covers the use of the TCR fusion protein, nucleic acid, vector, system or host cell in the preparation of pharmaceutical compositions or medicines.

As used herein, the term "pharmaceutically acceptable excipient" refers to pharmacologically and/or physiologically compatible with the subject and the active ingredient (that is, capable of eliciting the desired therapeutic effect without causing any undesirable effects). The carriers and/or excipients for the desired local or systemic effects) are well-known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coating agents, adsorbents, anti-adherents, glidants, antioxidants, flavoring agents, coloring agents, Sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delaying agents, stabilizers and tonicity regulators . It is known to those skilled in the art to select suitable excipients to prepare the desired pharmaceutical composition of the present invention. Exemplary excipients used in the pharmaceutical composition of the present invention include saline, buffered saline, dextrose, and water. In general, the choice of suitable excipients depends inter alia on the active agent used, the disease to be treated, and the desired dosage form of the pharmaceutical composition.

The pharmaceutical composition according to the present invention can be applied to various routes of administration. Usually, administration is accomplished parenterally. Parenteral delivery methods include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual, or intranasal administration.

The pharmaceutical composition according to the present invention can also be prepared into various forms, such as solid, liquid, gaseous or freeze-dried forms, especially ointments, creams, transdermal patches, gels, powders, tablets, and solutions. Formulations, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, liquids, elixirs, extracts, tinctures or liquid extracts, or are particularly suitable for the desired administration The form of the method. The processes known in the present invention for the production of drugs may include, for example, conventional mixing, dissolving, granulating, sugar coating, grinding, emulsifying, encapsulating, embedding or freeze-drying processes. A pharmaceutical composition comprising, for example, the host cell or TCR fusion protein, nucleic acid or carrier described herein is usually provided in liquid form, and preferably comprises a pharmaceutically acceptable buffer.

The pharmaceutical composition according to the present invention can also be administered in combination with one or more other agents suitable for the treatment and/or prevention of the disease to be treated. Preferable examples of agents suitable for the combination include known anticancer drugs such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide , Gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate glucuronate, Austria Auristatin E (auristatin E), vincristine and doxorubicin; peptide cytotoxins, such as ricin, diphtheria toxin, pseudomonas bacterial exotoxin A, DNase and RNase; radionuclides, such as iodine 131, rhenium 186, indium 111, iridium 90, bismuth 210 and 213, actinium 225 and astatine 213; prodrugs, such as antibody-directed enzyme prodrugs; immunostimulants, such as IL-2, chemokines such as IL-8, Platelet factor 4, melanoma growth stimulating protein, etc.; antibodies or fragments thereof, such as anti-CD3 antibody or fragments, complement activators, heterogeneous protein domains, homologous protein domains, virus/bacterial protein domains and virus/bacterial peptides .

Method for preparing modified immune cells

The present invention also provides a method for preparing modified immune cells, which includes introducing the nucleic acid or vector of the present invention into immune cells so that the immune cells express the TCR fusion protein or protein complex of the present invention.

In one embodiment, the immune cells are human immune cells, more preferably human T cells, macrophages, dendritic cells, monocytes, NK cells and/or NKT cells.

Methods of introducing nucleic acids or vectors into immune cells and expressing them are known in the art. For example, the nucleic acid or vector can be introduced into T cells by physical methods, such as calcium phosphate precipitation method, lipofection method, particle bombardment method, microinjection method, electroporation method, etc. Alternatively, chemical methods can also be used, such as through colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and lipids The body introduces the nucleic acid or vector. In addition, biological methods can also be used to introduce nucleic acids or vectors. For example, viral vectors, especially retroviral vectors, have become the most common method for inserting genes into mammalian, such as human cells. Other viral vectors can be derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus and adeno-associated virus.

After the nucleic acid or vector is introduced into the immune cells, those skilled in the art can amplify and activate the obtained immune cells by conventional techniques.

treatment

The present invention also provides a method of providing anti-tumor immunity to a subject or a method of preventing or treating a disease in a subject, comprising administering to the subject an effective amount of a TCR fusion protein expressing the present invention cell.

In one embodiment, an effective amount of the TCR fusion protein, nucleic acid, vector, vector system, host cell and/or pharmaceutical composition of the present invention is directly administered to the subject.

In another embodiment, the treatment method of the present invention is ex vivo treatment. Specifically, the method includes the following steps: (a) providing a sample of the subject, the sample containing immune cells; (b) in vitro combining the TCR fusion protein, protein complex, nucleic acid, carrier, carrier system, The cells and/or the pharmaceutical composition are introduced into the immune cells to obtain modified immune cells, and (c) administering the modified immune cells to a subject in need thereof. Preferably, the immune cells provided in step (a) are "effector cells" and are advantageously selected from T cells, NK cells and/or NKT cells; and the immune cells can be used in step by conventional methods known in the art. (a') is obtained from a sample of a subject (especially a blood sample). However, other immune cells capable of expressing the TCR fusion protein of the present invention and exerting the desired biological effect function as described herein can also be used. In addition, the immune cells that are usually selected are compatible with the immune system of the subject, i.e. it is preferred that the immune cells do not elicit an immunogenic response. For example, "universal acceptor cells" can be used, that is, universally compatible lymphocytes that can grow and expand in vitro that perform the desired biological effect function. The use of such cells will not require obtaining and/or providing the subject's own lymphocytes. The in vitro introduction of step (c) can be carried out by introducing the nucleic acid or vector described herein into immune cells via electroporation or by infecting immune cells with a viral vector, the viral vector being the aforementioned lentiviral vector, adenoma Viral vector, adeno-associated virus vector or retroviral vector. Other conceivable methods include the use of transfection reagents (such as liposomes) or transient RNA transfection. The transfer of antigen-specific TCR genes into (primary) T cells via, for example, (retro) viral vectors or transient RNA transfection is a promising tool for generating tumor-associated antigen-specific T cells. T cells are reintroduced into the donor, where these T cells specifically target and destroy tumor cells that express the antigen.

In one embodiment, the host cell or immune cell is an autologous or allogeneic cell, preferably T cell, macrophage, dendritic cell, monocyte, NK cell and/or NKT cell, more preferably T cell Cells, most preferably human T cells.

As used herein, the term "autologous" refers to any material derived from an individual that will later be reintroduced into that same individual.

As used herein, the term "allogeneic" refers to any material derived from a different animal or a different patient of the same species as the individual into which the material is introduced. When the genes at one or more loci are different, two or more individuals are considered to be allogeneic to each other. In some cases, the genetic differences of allogeneic materials from individual individuals of the same species may be sufficient for antigenic interaction to occur.

As used herein, the term "subject" is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects representing animal models of cancer. Preferably, the subject is a human.

In one embodiment, the disease is a disease related to the expression of a surface antigen bound to an antigen binding region. For example, the diseases include, but are not limited to: blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon And rectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma (GBM), liver cancer, hepatocellular tumor, Intraepithelial tumor, kidney cancer, laryngeal cancer, leukemia, liver tumor, lung cancer (such as small cell lung cancer, non-small cell lung cancer, glandular lung cancer, and squamous lung cancer), lymphoma (including Hodgkin's lymphoma and non-Hodgkin's Lymphoma), melanoma, myeloma, neuroblastoma, oral cancer (such as lip, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory system Cancer, salivary gland cancer, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary system cancer, vulvar cancer and other cancers and sarcomas, and B-cell lymphoma (including Low-grade/follicular non-Hodgkin’s lymphoma (NHL), small lymphocytic (SL) NHL, intermediate/follicular NHL, intermediate diffuse NHL, high-grade immunocytoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cracked cell NHL, large mass disease (NHL), mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), B-cell young lymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt’s lymphoma, Diffuse large B-cell lymphoma, follicular lymphoma, chronic myelogenous leukemia (CML), malignant lymphoproliferative disease, MALT lymphoma, hairy cell leukemia, marginal zone lymphoma, multiple myeloma, myelodysplasia, Plasmablastic lymphoma, pre-leukemia, plasmacytoid dendritic cell tumor, and post-transplant lymphoproliferative disorder (PTLD); and other diseases related to surface antigen expression. Preferably, the disease that can be treated with the TCR fusion protein, nucleic acid, vector, host cell or pharmaceutical composition of the present invention is selected from leukemia, lymphoma, multiple myeloma, brain glioma, pancreatic cancer, gastric cancer and the like.

In one embodiment, the method further comprises administering one or more additional chemotherapeutic agents, biological agents, drugs, or treatments to the subject. In this embodiment, the chemotherapeutic agent, biological agent, drug or treatment is selected from radiotherapy, surgery, antibody agents and/or small molecules and any combination thereof.

Hereinafter, the present invention will be described in detail with reference to the drawings and examples. It should be noted that those skilled in the art should understand that the drawings and embodiments of the present invention are for illustrative purposes only, and should not constitute any limitation on the present invention. If there is no contradiction, the embodiments in the application and the features in the embodiments can be combined with each other.

Description of the drawings

Fig. 1 shows various exemplary embodiments of the natural γδ type TCR structure and the TCR fusion protein of the present invention. A. Natural γδ type TCR structure, including γ chain and δ chain, each chain contains a variable region (V region) and a constant region (C region); B. The two antigen binding regions scFv are separated by connecting peptides Connected to the γ chain and the δ chain; C. The two antigen binding regions scFv are respectively connected to the constant regions of the γ chain and the δ chain through a connecting peptide (that is, the variable regions of the γ chain and the δ chain are knocked out); D. One The antigen binding region scFv is connected to the constant region of the γ chain through a connecting peptide; E. One antigen binding region scFv is connected to the constant region of the δ chain through a connecting peptide; F. Two antigen binding regions scFv are directly connected to the γ chain and the δ chain respectively. The constant region is connected; G and H. The variable region of the heavy chain and the variable region of the light chain of the antibody are respectively connected to the constant region of the γ chain and the δ chain through a connecting peptide; I. The variable region of the heavy chain and the variable light chain of the antibody The region is directly connected to the constant regions of the γ chain and the δ chain; J. Two different antigen binding regions scFv are connected to the constant regions of the γ chain and the δ chain respectively through a connecting peptide.

Figure 2 shows the structure of the natural γδTCR-CD3 complex and the TCR fusion protein-CD3 complex of the present invention. The natural γδTCR-CD3 complex includes two CD3ε polypeptides, one CD3γ polypeptide, one CD3δ polypeptide, two CD3ζ polypeptides, one TCR γ subunit and one TCR δ subunit. The horizontal gray bar represents the cell membrane. When the TCR fusion protein of the present invention is introduced into T cells, it competes with the natural TCR to bind to CD3 to form a modified TCR fusion protein-CD3 complex.

Figure 3 shows the efficiency of preparing TCRα/TCRβ double knockout T cells. A. T cells electrotransfected with Cas9 protein and two sgRNAs (TRAC sgRNA and TRBC sgRNA) (ie, TCRα/TCRβ double knockout T cells); B. T cells not transfected with Cas9 protein and sgRNA.

Figure 4 shows the average fluorescence intensity MFI after transduction of TCRα/TCRβ double knockout T cells and wild-type T cells with the TCR fusion protein of the present invention. Mock: Blank electrotransfected T cells.

Figure 5 shows the expression of scFv after transduction of wild-type T cells with scFv-TRG or scFv-TRD alone. NT: Untransduced wild-type T cells.

Figure 6 shows the expression levels of scFv after TCRα/TCRβ double knockout T cells are transduced with TCR fusion proteins containing different linking peptides or without linking peptides. NT: TCRα/TCRβ double knockout T cells without TCR fusion protein transduction. Two-way ANOVA was used for analysis, and T test was used for statistical analysis. * Indicates that the P value is less than 0.05, and ** indicates that the P value is less than 0.01, both reaching a significant level. ns means there is no significant difference.

Figure 7 shows the killing effect of TRUE-T cells and traditional CAR-T cells on target cells. NT: T cells not transduced with TCR fusion protein.

Figure 8 shows the IL2 (A) and IFN-γ (B) release levels of TRUE-T cells and traditional CAR-T cells. NT: T cells not transduced with TCR fusion protein. Two-way ANOVA was used for analysis, and T test was used for statistical analysis. * Indicates that the P value is less than 0.05, and ** indicates that the P value is less than 0.01, both reaching a significant level.

Figure 9 shows the structure of the pLv-BHAm vector.

Figure 10 shows the amount of scFv after transduction of wild-type T cells (Mock) and TCRα/TCRβ double knockout T (dKO) with murine TCR fusion protein. NT: Wild-type T cells that have not been transduced with TCR fusion protein.

Detailed ways

The sequences used in the following examples are summarized in Table 1 below.

Table 1. Sequences used in the present invention

SEQ ID NO description SEQ ID NO: 1 TRAC sgRNA SEQ ID NO: 2 TRBC sgRNA SEQ ID NO: 3 Nucleotide sequence of the heavy chain variable region (VH) of anti-CD19 scFv SEQ ID NO: 4 Anti-CD19 scFv heavy chain variable region (VH) amino acid sequence SEQ ID NO: 5 The nucleotide sequence of the light chain variable region (VL) of anti-CD19 scFv SEQ ID NO: 6 The amino acid sequence of the light chain variable region (VL) of anti-CD19 scFv SEQ ID NO: 7 Nucleotide sequence of anti-CD19 Fab light chain (VL-CL) SEQ ID NO: 8 Amino acid sequence of anti-CD19 Fab light chain (VL-CL) SEQ ID NO: 9 Nucleotide sequence of anti-CD19 Fab heavy chain (VH-CH1) SEQ ID NO: 10 Amino acid sequence of anti-CD19 Fab heavy chain (VH-CH1) SEQ ID NO: 11 Nucleotide sequence of anti-CD19 scFv SEQ ID NO: 12 Amino acid sequence of anti-CD19 scFv SEQ ID NO: 13 The nucleotide sequence of the constant region of human TCRγ chain (hTRGC) SEQ ID NO: 14 Amino acid sequence of the constant region of human TCRγ chain (hTRGC) SEQ ID NO: 15 The nucleotide sequence of the constant region (hTRDC) of the human TCRδ chain SEQ ID NO: 16 Amino acid sequence of the constant region (hTRDC) of human TCRδ chain SEQ ID NO: 17 Nucleotide sequence of human TCR gamma chain (hTRG) SEQ ID NO: 18 Amino acid sequence of human TCRγ chain (hTRG) SEQ ID NO: 19 Nucleotide sequence of human TCRδ chain (hTRD) SEQ ID NO: 20 Amino acid sequence of human TCRδ chain (hTRD) SEQ ID NO: 21 Connecting peptide (G4S) 3 nucleotide sequence SEQ ID NO: 22 Amino acid sequence of connecting peptide (G4S) 3 SEQ ID NO: 23 The nucleotide sequence of connecting peptide CD8α

SEQ ID NO: 24 Amino acid sequence of connecting peptide CD8α SEQ ID NO: 25 The nucleotide sequence of the connecting peptide IgG4 SEQ ID NO: 26 Amino acid sequence of connecting peptide IgG4 SEQ ID NO: 27 Nucleotide sequence of Cas9 SEQ ID NO: 28 Amino acid sequence of Cas9 SEQ ID NO: 29 Nucleotide sequence of anti-CD19 scFv-CAR SEQ ID NO: 30 Amino acid sequence of anti-CD19 scFv-CAR SEQ ID NO: 31 Nucleotide sequence of the constant region of mouse TCRγ chain (mTRGC) SEQ ID NO: 32 Amino acid sequence of the constant region of mouse TCRγ chain (mTRGC) SEQ ID NO: 33 Nucleotide sequence of the constant region (mTRDC) of the mouse TCRδ chain SEQ ID NO: 34 Amino acid sequence of the constant region (mTRDC) of the mouse TCRδ chain SEQ ID NO: 35 Nucleotide sequence of B2m signal peptide SEQ ID NO: 36 Amino acid sequence of B2m signal peptide SEQ ID NO: 37 Nucleotide sequence of CD8α signal peptide SEQ ID NO: 38 Amino acid sequence of CD8α signal peptide SEQ ID NO: 39 Nucleotide sequence of F2A peptide SEQ ID NO: 40 Amino acid sequence of F2A peptide

Example 1. Preparation of TCRα/TCRβ double knockout T cells

The Cas9 protein (sequence shown in SEQ ID NO: 28) used in the present invention was purchased from Thermo (Cat. No. A36499).

The T cells used in all the examples of the present invention are Ficoll-PaqueTM PREMIUM (GE Healthcare, article number 17-5442-02) using leukocyte separation to isolate primary human CD4+CD8+ T cells from healthy donors.

The T cells were stimulated with DynaBeads CD3/CD28 CTSTM (Gibco, catalog number 40203D), and cultured at 37°C and 5% CO2 for 3 days. Then, using a BTX Agile Pulse Max electroporator (Harvard Apparatus BTX), 10ug Cas9 protein and 10ug sgRNA (5ug TRAC sgRNA+5ug TRBC sgRNA) were electrotransfected into activated T cells at 400V and 0.7ms to obtain TCRα/ TCRβ double knockout T cells. T cells not transfected with Cas9 protein and sgRNA were used as controls. Immediately after electrotransfection, the T cells were placed in 1ml of pre-warmed medium and cultured in the presence of IL-2 (300IU/ml) at 37°C and 5% CO2. After 11 days, the APC Mouse Anti-Human CD3 (BD Pharmingen, catalog number 555335) antibody was used to detect the expression of TCR/CD3 by flow cytometry to detect the knockout efficiency of TCRα/TCRβ. The results are shown in Figure 3.

It can be seen that compared with the untransfected control T cells (Figure 3B), the TCR/CD3 expression in the T cells transfected with Cas9 protein and sgRNA was significantly reduced (Figure 3A), indicating the double knockout efficiency of TCRα/TCRβ Achieve more than 90%.

Example 2. Preparation of anti-CD19 scFv-CAR T cells

Synthesize the sequence encoding the CD8α signal peptide (SEQ ID NO: 37), the nucleic acid encoding the anti-CD19 scFv-CAR (including anti-CD19 scFv, CD8α hinge region, CD8α transmembrane region, 4-1BB intracellular region, CD3ζ intracellular region) Sequence (SEQ ID NO: 29), clone it into the pGEM-T Easy vector (Promega, catalog number A1360), and confirm the correct insertion of the target sequence by sequencing.

T7 Ultra Kit试剂盒(Invitrogen，货号AM1345)制备mRNA，并用Fastpure cell/Tissue total RNA isolation kit试剂盒(Vazyme，货号RC101-01)进行纯化，获得纯化的mRNA。 Then, the mRNA is prepared: the expression vector prepared above is digested with SpeI enzyme, and the linearized vector is obtained after purification and recovery. Then, according to the manufacturer’s recommendations, use the linearized vector as a template and use mMESSAGE

T7 Ultra Kit (Invitrogen, article number AM1345) was used to prepare mRNA, and the Fastpure cell/Tissue total RNA isolation kit (Vazyme, article number RC101-01) was used for purification to obtain purified mRNA.

Finally, perform electrotransfection: use a BTX Agile Pulse Max electroporator (Harvard Apparatus BTX) to electrotransfect the 10ug purified mRNA prepared above into activated T cells at 400V and 0.7ms to obtain anti-CD19scFv-CAR T cells .

Example 3. Preparation of TRUE-T cells

(1) Construction of the TCR fusion protein expression vector of the present invention

The heavy chain variable region (SEQ ID NO: 3) and light chain variable region (SEQ ID NO: 3) of the anti-CD19 scFv carrying B2m signal peptide (SEQ ID NO: 35) or CD8α signal peptide (SEQ ID NO: 37) will be synthesized. ID NO: 5) or complete scFv (SEQ ID NO: 11) coding sequence; connecting peptide (CD8α, IgG4 or (G4S)3) or heavy chain/light chain constant region (CL or CH1) coding sequence; TCR γ The constant region of the chain (SEQ ID NO: 13), the constant region of the TCR δ chain (SEQ ID NO: 15), the complete TCR γ chain (SEQ ID NO: 17) or the complete TCR δ chain (SEQ ID NO: 19) The coding sequence of) was sequentially cloned into the pGEM-TEasy vector (Promega, catalog number A1360) to obtain the TCR fusion protein expression vector of the present invention, and the correct insertion of the target sequence was confirmed by sequencing. The prepared expression vector is shown in Table 2 below.

Table 2. The expression vector prepared in Example 3

(2) Preparation of mRNA

T7 Ultra Kit试剂盒(Invitrogen，货号AM1345)制备mRNA，并用Fastpure cell/Tissue total RNA isolation kit试剂盒(Vazyme，货号RC101-01)进行纯化，获得纯化的mRNA。 The expression vector prepared in step 1 was digested with SpeI enzyme, and the linearized vector was obtained after purification and recovery. Then, according to the manufacturer’s recommendations, use the linearized vector as a template and use mMESSAGE

T7 Ultra Kit (Invitrogen, article number AM1345) was used to prepare mRNA, and the Fastpure cell/Tissue total RNA isolation kit (Vazyme, article number RC101-01) was used for purification to obtain purified mRNA.

(3) Prepare TRUE-T cells and detect the expression of scFv

According to the electrotransfection method described in Example 2, 10ug of purified mRNA (scFv-hTRG+scFv-hTRD) obtained from vectors 11 and 12 was simultaneously transfected into TCRα/TCRβ double knockout T cells by electroporation Or wild-type T cells to obtain TRUE-T cells. At the same time, TCRα/TCRβ double knockout T cells or wild-type T cells (Mock) that were not transfected by electroporation were used as negative controls.

Using Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG, F(ab') ₂ Fragment Specific (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, catalog number 115-065-072) as the primary antibody, APC Streptavidin (BD Pharmingen, catalog number 554067) or PE Streptavidin (BD Pharmingen, catalog number 554061) is used as a secondary antibody to detect the expression level of scFv in TRUE-T cells by flow cytometry (only when the transfected TCR fusion protein is expressed correctly , Can detect the expression of the complete scFv), and use FlowJo software to calculate the MFI value according to the detection result. The result is shown in Figure 4.

It can be seen that the expression of scFv in TCRα/TCRβ double knockout T cells transfected with scFv-TRG+scFv TRD is much higher than that in wild-type T cells, indicating that the endogenous TCR α and TCR β subunits will interact with The TCRγ and TCRδ subunits in the exogenous TCR fusion protein compete to bind to the CD3 component, resulting in the inhibition of the expression of the TCR fusion protein.

In addition, in order to verify whether the TRG or TRD subunit alone is sufficient for the correct expression of scFv, the purified mRNA of the vector 11 or 12 alone (ie, scFv-TRG or scFv-TRD) was electrotransfected into wild-type T cells in the same manner , And used Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG, F(ab') ₂ Fragment Specific (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, catalog number 115-065-072) as the primary antibody , APC Streptavidin (BD Pharmingen, article number 554067) or PE Streptavidin (BD Pharmingen, article number 554061) was used as a secondary antibody, and the expression level of scFv was detected by flow cytometry. The results are shown in Figure 5.

It can be seen that neither scFv-TRG nor scFv-TRD can be effectively expressed by electrotransfection alone, indicating that the correct expression of TRUE-T depends on the simultaneous expression of γ and δ subunits, while excluding scFv-TRG or scFv-TRD and scFv-TRD. The mismatch combination of TCRα or TCRβ subunits leads to the possibility of GvHD.

Therefore, in order to efficiently express the TCR fusion protein and avoid causing graft-versus-host disease, the following experiments all use TCRα/TCRβ double knockout T cells to prepare the TRUE-T cells of the present invention.

According to the following pairing method, the corresponding purified mRNA was transfected into TCRα/TCRβ double knockout T cells by electroporation to obtain TRUE-T cells of the present invention: VL-CL-TRGC+VH-CH1-TRDC; VL- (G4S)3-TRGC+VH-(G4S)3-TRDC; VL-CD8α-TRGC+VH-CD8α-TRDC; VL-IgG4-TRGC+VH-IgG4-TRDC; VL-TRGC+VH-TRDC; scFv- TRG+scFv-TRD; scFv-TRGC+TRDC. At the same time, anti-CD19scFv-CAR T cells were used as a positive control.

Using Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG, F(ab') ₂ Fragment Specific (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, catalog number 115-065-072) as the primary antibody, APC Streptavidin (BD Pharmingen, catalog number 554067) or PE Streptavidin (BD Pharmingen, catalog number 554061) was used as a secondary antibody to detect the above TRUE-T cells, scFv-CAR T cells (positive control) and untransfected TCRα by flow cytometry /TCRβ double knockout T cells (NT, negative control) scFv expression level, the results are shown in Figure 6.

It can be seen that the expression level of scFv in TRUE-T cells of the present invention is basically the same as that of scFv-CAR T cells, and there is no significant difference, while the scFv in cells carrying VL-CL-TRGC+VH-CH1-TRDCT The expression level of scFv-CAR T cells was significantly lower than that of control scFv-CAR T cells.

At the same time, it can also be seen that the expression level of scFv in transfected T cells with or without the connecting peptide is significantly higher than that in transfected T cells containing the heavy chain/light chain constant region (CL/CH1) of CD19 scFv. This may be because the steric hindrance formed by the length of CL/CH affects the folding of CD19 scFv, which in turn affects its cell surface expression level.

Example 4. The killing effect of TRUE-T cells

When T cells kill target cells, the number of target cells will decrease. After co-cultivating T cells with target cells that can express luciferase, the number of target cells decreases, and the secreted luciferase also decreases. Luciferase can catalyze the conversion of luciferin to oxidized luciferin, and during this oxidation process, bioluminescence will be produced, and the intensity of this luminescence will depend on the level of luciferase expressed by the target cell. Therefore, the detected fluorescence intensity can reflect the killing ability of T cells to target cells.

In order to detect the killing ability of TRUE-T cells on target cells, firstly Nalm6 target cells carrying the fluorescein gene were plated into a 96-well plate at a rate of 1× ^{10 4 /well, and then an effective target ratio of 4:1 (ie effector T cells and} Target cell ratio) Spread TRUE-T, untransfected T cells (negative control) and anti-CD19 scFv-CAR T cells (positive control) into a 96-well plate for co-cultivation, and use a microplate reader after 16-18 hours Determine the fluorescence value. According to the calculation formula: (average fluorescence of target cells-average fluorescence of samples)/average fluorescence of target cells×100%, the killing efficiency is calculated, and the result is shown in FIG. 7.

It can be seen that compared with NT, it carries VL-(G4S)3-TRGC+VH-(G4S)3-TRDC; VL-CD8α-TRGC +VH-CD8α-TRDC; VL-IgG4-TRGC+VH-IgG4- TRDC; VL-TRGC+VH-TRDC; scFv-TRG+scFv-TRD TRUE-T cells can effectively kill target cells, and the killing effect is not lower than that of scFv-CAR T cells.

Example 5. Cytokine release of TRUE-T cells

When T cells kill target cells, the decrease in the number of target cells will also release the cytokines IL2 and IFN-γ. According to the following steps, enzyme-linked immunosorbent assay (ELISA) is used to determine the release levels of cytokines IL2 and IFN-γ when TRUE-T cells kill target cells.

(1) Collection of cell co-culture supernatant

Spread the target cell Nalm6 and non-target cell K562 on a 96-well plate at 1x10 ⁵ /well, and then place TRUE-T cells, untransfected T cells (negative control) and anti-CD19 scFv-CAR at a ratio of 1:1 T cells (positive control) were co-cultured with target cells and non-target cells, and the supernatant was collected 18-24 hours later.

(2) ELISA to detect the secretion of IL2 and IFNγ in the supernatant

ELISA was used to determine the secretion of cytokines IL2 and IFNγ in the supernatant. Use the capture antibody Purified anti-human IL-2 Antibody (Biolegend, catalog number 500302) and Purified anti-human IFN-γ Antibody (Biolegend, catalog number 506502) to coat 96-well plates and incubate overnight at 4°C, then remove the supernatant. Add 250 μL of PBST (1XPBS containing 0.1% Tween) solution containing 2% BSA (sigma, article number V900933-1kg), and incubate at 37° C. for 2 hours. After removing the supernatant, 250 μL PBST (1XPBS containing 0.1% Tween) was added and washed 3 times. Then add 50μL of cell co-culture supernatant or standard to each well, and incubate at 37°C for 1 hour. Remove the supernatant, then add 250 μL PBST (1XPBS containing 0.1% Tween), and wash 3 times. Then add 50μL of the detection antibody Biotin anti-human IL-2 Antibody (Biolegend, catalog number 517605) and Anti-Interferon gamma antibody [MD-1] (Biotin) (abcam, catalog number ab25017) to each well, and incubate at 37°C for 1 hour Afterwards, the supernatant was discarded, 250 μL PBST (1XPBS containing 0.1% Tween) was added, and washed 5 times. HRP Streptavidin (Biolegend, catalog number 405210) was added, and after incubating at 37°C for 30 minutes, 50 μL of TMB substrate solution was added to each well. The reaction was allowed to occur in the dark at room temperature for 30 minutes, and then 50 μL of 1 mol/L H ₂ SO ₄ was added to each well to stop the reaction. Within 30 minutes of stopping the reaction, use a microplate reader to detect the absorbance at 450 nm, and calculate the cytokine content according to the standard curve (drawn from the reading and concentration of the standard).

It can be seen that it carries VL-(G4S)3-TRGC+VH-(G4S)3-TRDC; VL-CD8α-TRGC+VH-CD8α-TRDC; VL-IgG4-TRGC+VH-IgG4-TRDC; VL-TRGC +VH-TRDC; scFv-TRG+scFv-TRD TRUE-T cells when killing target cells, the release level of cytokines IL2 and IFN-γ is far lower than the release of anti-CD19 scFv-CAR T cells when killing target cells Level, which can effectively reduce CRS.

Example 6. Construction of TRUE-T cells using lentiviral vectors

(1) TRUE-T lentivirus packaging

Synthesize the following coding sequences: complete CD19 scFv sequence (SEQ ID NO: 11), (G4S) 3 connecting peptide (SEQ ID NO: 21), hTGC (SEQ ID NO: 13), 2A peptide (SEQ ID NO: 39), hTRDC (SEQ ID NO: 15), and cloned it into the pLv-BHAm lentiviral vector (Figure 9) to obtain the TRUE-T plasmid. Prepare the mTRUE-T plasmid at the same time, which contains mTRGC (SEQ ID NO: 31) and mTRDC (SEQ ID NO: 33), and other elements are the same as the TRUE-T plasmid.

Add 3ml Opti-MEM (Gibco, article number 31985-070) to the sterile tube to dilute the above two plasmids, and then add the packaging vector psPAX2( at the ratio of plasmid: virus packaging vector: virus envelope vector = 4:2:1 Addgene, article number 12260) and envelope vector pMD2.G (Addgene, article number 12259). Then, add 120ul X-treme GENE HP DNA transfection reagent (Roche, catalog number 0636236601), mix immediately, incubate at room temperature for 15 minutes, and then add the plasmid/vector/transfection reagent mixture dropwise to the 293T cell culture flask . The virus was collected at 24 hours and 48 hours, and after combining them, ultracentrifugation (25000g, 4°C, 2.5 hours) was used to obtain concentrated TRUE-T or mTRUE-T lentivirus.

(2) Preparation of TRUE-T cells

T cells were activated with DynaBeads CD3/CD28 CTSTM (Gibco, catalog number 40203D), and cultured at 37°C and 5% CO2 for 1 day. Then, add concentrated TRUE-T or mTRUE-T lentivirus, continue to culture for 3 days, use BTX Agile Pulse Max (Harvard Apparatus BTX), 400V, 0.7ms 10ug Cas9 protein and 10ug sgRNA (5ug TRAC sgRNA+ 5ug TRBC sgRNA) was electrotransfected into activated T cells to obtain TCRα/TCRβ double knockout TRUE-T cells (TRUE-T-dKO) and mTRUE-T cells (mTRUE-T-dKO). Viruses without Cas9+sgRNA electrotransfection infect T cells (ie, TRUE-T-Mock and mTRUE-T-Mock) as controls.

The T cells were placed in 1ml of pre-warmed medium and cultured in the presence of IL-2 (300IU/ml) at 37°C and 5% CO2. After 11 days, use Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG, F(ab') ₂ Fragment Specific (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, item number 115-065-072) as The primary antibody, APC Streptavidin (BD Pharmingen, article number 554067) or PE Streptavidin (BD Pharmingen, article number 554061) was used as the secondary antibody, and the expression level of the above scFv was detected by flow cytometry. The results are shown in FIG. 10.

It can be seen that the expression level of scFv in mTRUE-T cells is comparable to that in TRUE-T cells, and similarly, the expression level in TCRα/TCRβ double knockout T cells is higher than that in wild-type T cells.

In summary, the inventors found that TRUE-T cells containing the TCR fusion protein of the present invention can maintain a target cell killing effect comparable to that of traditional CART cells, while also significantly reducing the release level of cytokines, thereby effectively reducing CRS. risk. Moreover, the inventors also unexpectedly discovered that in TRUE-T cells lacking TCRα/β chains, the expression level of scFv is much higher than that of wild-type T cells, that is, the killing effect on target cells is better.

It should be noted that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Those skilled in the art understand that any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (47)

A recombinant T cell receptor (TCR) fusion protein, including: (a) The antigen binding region that binds to the surface antigen; (b) The constant region of the TCRγ chain; and (c) The constant region of the TCRδ chain. The fusion protein according to claim 1, characterized in that it further comprises the variable region of the TCRγ chain and/or the variable region of the TCRδ chain. The fusion protein of any one of claims 1-2, wherein the constant region and/or variable region are derived from human, murine or human-mouse chimera. The fusion protein according to claim 1, characterized in that it does not include the variable region of the TCRγ chain and/or the variable region of the TCRδ chain. The fusion protein of any one of claims 1 to 4, wherein the antigen binding region is directly connected to the constant region of the TCRγ chain and/or the constant region of the TCRδ chain. The fusion protein of any one of claims 1 to 4, wherein the antigen binding region is connected to the constant region of the TCRγ chain and/or the constant region of the TCRδ chain through a connecting peptide. The fusion protein of claim 6, wherein the connecting peptide is selected from (G4S)n, CD8 and IgG4, wherein n is an integer of 1-4. The fusion protein of any one of claims 1-7, wherein the antigen binding region binds the same or different surface antigens. The fusion protein of any one of claims 1-7, wherein the surface antigen is selected from: TSHR, CD19, CD123, CD22, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-1 Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, Folate receptor α, ERBB2 (Her2/neu), MUC1, EGFR, NCAM, Claudin18.2, Prostase, PAP, ELF2M, Ephrin B2, IGF-I Receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, Fucosyl GMl, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1/CD248, TEM7R, CLDN6 , GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1 , LAGE-la, MAGE-A1, legume protein, HPV E6, E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostate-specific protein, survivin and telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2ETS fusion gene ), NA17, PAX3, androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES 1, LCK, AKAP-4, SSX2, RAGE-1, human Granzyme reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1 and Any combination of them. The fusion protein of claim 9, wherein the surface antigen is selected from: CD19, CD20, CD22, BAFF-R, CD33, EGFRvIII, BCMA, GPRC5D, PSMA, ROR1, FAP, ERBB2 (Her2/neu), MUC1, EGFR, CAIX, WT1, NY-ESO-1, CD79a, CD79b, GPC3, Claudin 18.2 and any combination thereof. The fusion protein of any one of claims 1-10, wherein the antigen binding region is selected from the group consisting of scFv, VH domain, VL domain, single domain antibody, nanobody, antigen binding ligand, recombinant fibronectin structure Domain, anticalin and DARPIN. The fusion protein of claim 11, wherein the antigen binding region is a monoclonal antibody, polyclonal antibody, recombinant antibody, human antibody, humanized antibody, murine antibody, chimeric antibody and functional fragments thereof. The fusion protein of claim 11, wherein the antigen binding ligand is selected from: PD1, PDL1, PDL2, TGFβ, APRIL and NKG2D. The fusion protein of any one of claims 1-13, wherein the antigen binding region is selected from the heavy chain variable region or the light chain variable region of an anti-CD19 scFv, an anti-CD19 antibody, preferably, the antigen binding region is selected from From SEQ ID NO: 4, 6, 8, 10, 12, or a functional variant with at least 95% sequence identity with which retains surface antigen binding activity. The fusion protein of any one of claims 1-14, wherein the constant region of the TCRγ chain is selected from SEQ ID NO: 14, 32, or a functional variant with at least 85% sequence identity thereto; wherein the The constant region of the TCRδ chain is selected from SEQ ID NO: 16, 34, or a functional variant with at least 85% sequence identity thereto. The fusion protein of claim 2, wherein the TCRγ chain is selected from SEQ ID NO: 18, or a functional variant with at least 85% sequence identity therewith; wherein the variable region of the TCRδ chain is selected from SEQ ID NO: 20, or a functional variant with at least 85% sequence identity to it. The fusion protein of any one of claims 1-6, wherein the fusion protein further comprises a transmembrane domain. The fusion protein of claim 17, wherein the transmembrane domain is selected from the transmembrane domains of the following proteins: TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and their functional fragments. The fusion protein of any one of claims 1-18, characterized in that it further comprises a costimulatory domain. The fusion protein of claim 19, wherein the costimulatory domain is a functional signaling domain obtained from the following proteins: TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, CD66d , CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, ZAP70, 41BB and their functional fragments. The fusion protein of any one of claims 1-20, which further comprises a signal peptide. The fusion protein of any one of claims 1-21, which is characterized in that it further comprises a 2A peptide. A protein complex comprising the fusion protein of any one of claims 1-22 and at least one endogenous CD3 subunit or endogenous CD3 complex. A nucleic acid comprising: (a) a sequence encoding the fusion protein of any one of claims 1-22. The nucleic acid of claim 24, wherein the nucleic acid is DNA or RNA. A vector comprising the nucleic acid of any one of claims 1-25. The vector of claim 26, wherein the vector further comprises a starter that replicates autonomously in the host cell, a selection marker, a restriction enzyme cleavage site, a promoter, a polyadenylic acid tail (polyA), 3'UTR, 5'UTR, enhancer, terminator, insulator, operon, selectable marker, reporter gene, targeting sequence and/or protein purification tag. The vector of claim 26 or 27, wherein the vector is a linear nucleic acid molecule, plasmid, retrovirus, lentivirus, adenovirus, vaccinia virus, Rous sarcoma virus (RSV), polyoma virus, and adeno-associated virus ( AAV), phage, phagemid, cosmid or artificial chromosome. A carrier system, including: (a) The first nucleic acid sequence encoding the constant region of the TCRγ chain; (b) The second nucleic acid sequence encoding the constant region of the TCRδ chain; The first nucleic acid sequence and/or the second nucleic acid sequence are operably linked to the third nucleic acid sequence encoding the antigen binding region, and the first nucleic acid sequence and the second nucleic acid sequence are located in the same vector or different vectors. A cell comprising the fusion protein according to any one of claims 1-22, the protein complex according to claim 23, the nucleic acid according to any one of claims 24-25, and any one of claims 26-28 The carrier or the system of claim 29. The cell of claim 30, wherein the cell is an immune cell. The cell of claim 31, wherein the immune cells are T cells, macrophages, dendritic cells, monocytes, NK cells, or NKT cells. . The cell of claim 32, wherein the T cell is selected from the group consisting of CD4+/CD8+ double positive T cells, CD4+ helper T cells, CD8+ T cells, tumor infiltrating cells, memory T cells, naive T cells, γδ-T cells and αβ-T cells. The cell of claim 32 or 33, wherein the T cell lacks TCR alpha chain and/or TCR beta chain. A pharmaceutical composition comprising the fusion protein according to any one of claims 1-22, the protein complex according to claim 23, the nucleic acid according to any one of claims 24-25, and any one of claims 26-28 One of the carrier, the system of claim 29, or the cell of any one of claims 30-34, and one or more pharmaceutically acceptable excipients. The pharmaceutical composition of claim 35, wherein the pharmaceutically acceptable excipient is selected from one or more of the following: fillers, binders, disintegrants, coating agents, adsorbents, anti-sticking agents Adjuvants, glidants, antioxidants, flavors, colorants, sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coatings Agents, isotonic agents, absorption delay agents, stabilizers and tonicity modifiers. The pharmaceutical composition of claim 35 or 36, wherein the pharmaceutical composition is administered locally, intraarterially, intramuscularly, subcutaneously, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, Administration is intravaginal, sublingual or intranasal route. The pharmaceutical composition of claim 35 or 36, wherein the pharmaceutical composition is an ointment, cream, transdermal patch, gel, powder, tablet, solution, aerosol, granule In the form of medicine, pill, suspension, emulsion, capsule, syrup, liquid, elixir, medicinal extract, tincture or liquid medicinal extract. The composition of any one of claims 35-38, wherein the pharmaceutical composition is administered in combination with one or more agents selected from the group consisting of: cisplatin, maytansine derivatives, and rapamycin (rachelmycin), calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II (sorfimer sodium photofrin II) ), Temozolomide, Topotecan, Trimetreate glucuronate, Auristatin E, Vincristine, Adriamycin, Ricin, Diphtheria toxin, Pseudomonas bacteria Toxin A, DNase, RNase, iodine 131, rhenium 186, indium 111, iridium 90, bismuth 210 and 213, actinium 225, astatine 213, antibody-directed enzyme prodrugs, IL-2, IL-8, platelet factor 4 , Melanoma growth stimulating proteins, antibodies or fragments thereof, complement activators, heterogeneous protein domains, homologous protein domains, viral/bacterial protein domains and viral/bacterial peptides. A method for preparing modified immune cells, comprising combining the fusion protein according to any one of claims 1-22, the protein complex according to claim 23, the nucleic acid according to any one of claims 24-25, and The vector of any one of claims 26-28 or the system of claim 29 is introduced into the immune cells. The method of claim 40, wherein the immune cells are T cells, macrophages, dendritic cells, monocytes, NK cells, or NKT cells. A method for treating a subject suffering from a disease related to surface antigen expression, comprising administering to the subject an effective amount of the fusion protein according to any one of claims 1 to 22, or the fusion protein according to claim 23 Protein complex, the nucleic acid of any one of claims 24-25, the vector of any one of claims 26-28, the system of claim 29, the cell of any one of claims 30-34 Or the pharmaceutical composition of any one of claims 35-39. The method of claim 42, wherein the method comprises the following steps: (a) providing a sample of the subject, the sample comprising immune cells; (b) applying the method of any one of claims 1-22 in vitro The fusion protein, the protein complex of claim 23, the nucleic acid of any one of claims 24-25, the vector of any one of claims 26-28, or the system of claim 29 introduces the immunity Cells, obtaining modified immune cells, and (c) administering the modified immune cells to the subject. The method of claim 43, wherein the immune cells are autologous or allogeneic T cells, NK cells, or NKT cells. The method of any one of claims 42-44, wherein the subject is a human, non-human primate, mouse, rat, dog, cat, horse, or cow. The method of any one of claims 42-45, wherein the disease related to surface antigen expression is selected from the group consisting of: blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS Cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, glioblastoma (GBM), liver cancer, hepatocellular tumor, intraepithelial tumor, kidney cancer, laryngeal cancer, leukemia, liver tumor, lung cancer, lymphoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, Prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, cancer of the respiratory system, salivary gland cancer, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, malignant tumors of the urinary system , Vulvar cancer and other cancers and sarcomas, as well as B-cell lymphoma, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom macroglobulinemia, chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), B -Cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), B-cell young lymphocytic leukemia, blastic plasmacytoid dendritic cell tumor, Burkitt’s lymphoma, diffuse Large B-cell lymphoma, follicular lymphoma, chronic myelogenous leukemia (CML), malignant lymphoproliferative disease, MALT lymphoma, hairy cell leukemia, marginal zone lymphoma, multiple myeloma, myelodysplasia, plasma Blastic lymphoma, pre-leukemia, plasmacytoid dendritic cell tumor, and post-transplant lymphoproliferative disorder (PTLD). The method of claim 46, wherein the lung cancer is selected from the group consisting of small cell lung cancer, non-small cell lung cancer, glandular lung cancer, and squamous lung cancer; and the lymphoma is selected from the group consisting of Hodgkin's lymphoma and non-Hodgkin's lymphoma; The B-cell lymphoma is selected from the group consisting of low-grade/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate/follicular NHL, intermediate diffuse NHL, high-grade immunoblasts NHL, high-grade lymphoblastic NHL, high-grade small non-cracked cell NHL, and mass NHL.

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