Anti-tumor fusion protein and its preparation and use

Abstract

The present invention provides an anti-tumor fusion protein and its preparation and use. Specifically, the fusion protein of the present invention contains a CPP element, optionally a linking element, and the SH2 domain of SHP2 or SHP1 or an active fragment thereof, and the fusion protein obtained by the present invention has excellent anti-tumor activity.

Description

[Technical Field]

[0001] The present invention relates to the fields of biology and medicine, and more particularly to anti-tumor fusion proteins and their preparation and use. [Background technology]

[0002] Immune checkpoint inhibitors have become an important target in tumor immunotherapy. PD-1 / PD-L1-targeting immune checkpoint inhibitors are a new generation of anti-cancer immunotherapy that harness the body's own immune system to fight malignant tumors and substantially improve overall patient survival. However, numerous studies have reported that only 20-30% of patients respond to these inhibitors, and some patients develop drug resistance after receiving drug treatment. Given the low response rates associated with monotherapy, the development of novel immune checkpoint inhibitors and the combination of different immune checkpoint drugs are currently offering novel approaches to improving patient response rates.

[0003] However, they have not been able to completely solve the problems of low response rates and drug resistance in clinical settings, and the combination of multiple therapies poses challenges in terms of timing, dosage optimization, pharmacoeconomics, etc. Therefore, universal immune checkpoint blockades (UICBs), which are designed to target multiple immune checkpoints in common, could be an important breakthrough in resolving these issues.

[0004] Therefore, there is a strong demand in this field for the development of more effective anti-tumor fusion proteins. DISCLOSURE OF THE INVENTION [Problem to be solved by the invention]

[0005] An object of the present invention is to provide more effective anti-tumor fusion proteins. [Means for solving the problem]

[0006] In a first aspect, the present invention provides a fusion protein having a structure represented by Formula I or II from the N-terminus to the C-terminus.

number

number

[0007] In another preferred embodiment, Z1 and Z2 are linked in a head-to-head, head-to-tail, tail-to-head, or tail-to-tail manner.

[0008] In another preferred embodiment, the "head" is the N-terminus of a polypeptide or a fragment thereof, in particular the N-terminus of a wild-type polypeptide or a fragment thereof.

[0009] In another preferred embodiment, the "tail" is the C-terminus of the polypeptide or fragment thereof, particularly the C-terminus of the wild-type polypeptide or fragment thereof.

[0010] In another preferred embodiment, Z1 and Z2 are D- or L-amino acids.

[0011] In another preferred embodiment, when Z1 and Z2 are linked in a head-to-head configuration, Z1 and Z2 are L- or D-amino acids.

[0012] In another preferred embodiment, when Z1 and Z2 are linked tail-to-tail, Z1 and Z2 are L- or D-amino acids.

[0013] In another preferred embodiment, the CPP element is of human or non-human mammalian origin.

[0014] In another preferred embodiment, the CPP elements include wild-type and mutant types.

[0015] In another preferred embodiment, the CPP element comprises a full-length, mature form of a CPP, or an active fragment thereof, preferably 5-30 amino acids in length.

[0016] In another preferred embodiment, the CPP element is the TAT membrane-permeable peptide.

[0017] In another preferred embodiment, the CPP element is selected from the group consisting of polyarginine (R5-9), MPG, CADY, pVEC, penetratin, Kaposi's fibroblast growth factor peptide, VP22, or a combination thereof.

[0018] In another preferred embodiment, the sequence of the CPP element is set forth in any of SEQ ID NOs: 1, 15-21.

[0019] In another preferred embodiment, L consists of glycine and serine.

[0020] In another preferred embodiment, L is -(Gly-Ser) n -, wherein n is 1-5, preferably 1-3.

[0021] In another preferred embodiment, L is -(Gly-Ser-Ser-Ser-Ser) n -, wherein n is 1-5, preferably 1-3.

[0022] In another preferred embodiment, L contains 1-6 prolines, preferably 1-3 prolines.

[0023] In another preferred embodiment, the Z2 is of human or non-human mammalian origin.

[0024] In another preferred embodiment, the Z2 includes a wild type and a mutant type.

[0025] In another preferred embodiment, Z2 comprises the full-length, mature form of the SH2 domain of SHP2 or SHP1, or an active fragment thereof.

[0026] In another preferred embodiment, Z2 is selected from the group consisting of an N-terminal SH2 domain, a C-terminal SH2 domain, or a combination thereof.

[0027] In another preferred embodiment, Z2 further comprises an ITIM motif binding region of a C-terminal SH2 domain.

[0028] In another preferred embodiment, Z2 is the N-terminal SH2 domain of SHP2, having amino acids 1-99 or 9-51 of SEQ ID NO:2, preferably containing amino acids 9, 28, 49, and 51 of SEQ ID NO:2, and is 43-99 amino acids in length.

[0029] In another preferred embodiment, Z2 is the C-terminal SH2 domain of SHP2, having amino acids 27-39 or 29-37 of SEQ ID NO:3, preferably containing amino acids 29, 31, 36, and 37 of SEQ ID NO:3, and is 9-108 or 13-108 amino acids in length.

[0030] In another preferred embodiment, Z2 is the N-terminal SH2 domain of SHP1, having amino acids 9-51 of SEQ ID NO:12, preferably containing amino acids 9, 28, 49, and 51 of SEQ ID NO:12, and being 43-99 amino acids in length.

[0031] In another preferred embodiment, Z2 is the C-terminal SH2 domain of SHP1, which comprises amino acids 29-37 of SEQ ID NO:13, preferably amino acids 29, 31, 36, and 37 of SEQ ID NO:13, and is 9-107 amino acids in length.

[0032] In another preferred embodiment, Z2 has the structure of formula III from the N-terminus to the C-terminus.

number

[0033] In another preferred embodiment, element A is the N-terminal SH2 domain of SHP2, comprising positions 1-99 or 9-51 of SEQ ID NO:2, preferably positions 9, 28, 49 and 51 of SEQ ID NO:2, and is 43-99 amino acids in length.

[0034] In another preferred embodiment, element B is the C-terminal SH2 domain of SHP2, has the sequence set forth in SEQ ID NO: 3, and contains positions 27-39 or 29-37 of SEQ ID NO: 3, preferably positions 29, 31, 36, and 37 of SEQ ID NO: 3, and is 9-108 or 13-108 amino acids in length.

[0035] In another preferred embodiment, element A is the N-terminal SH2 domain of SHP1, which comprises amino acids 9-51 of SEQ ID NO:12, preferably amino acids 9, 28, 49, and 51 of SEQ ID NO:12, and is 43-99 amino acids in length.

[0036] In another preferred embodiment, element B is the C-terminal SH2 domain of SHP1, comprising amino acids 29-37 of SEQ ID NO:13, preferably containing amino acids 29, 31, 36, and 37 of SEQ ID NO:13, and having a length of 9-107 amino acids.

[0037] In another preferred example, the length of L' is 1 to 20 aa, preferably 3 to 15 aa, more preferably 5 to 10 aa.

[0038] In another preferred embodiment, the amino acid sequence of L' is selected from the following group: (1) a polypeptide having an amino acid sequence represented by SEQ ID NO: 10; (2) A polypeptide derived from the polypeptide of the amino acid sequence shown in SEQ ID NO: 10, which has undergone substitution, deletion or addition of one or more, preferably 1 to 20, more preferably 1 to 15, even more preferably 1 to 10, even more preferably 1 to 8, even more preferably 1 to 3, and most preferably 1 amino acid residue, and which has the function of the polypeptide described in (1).

[0039] In another preferred embodiment, Z2 has the sequence shown in SEQ ID NO: 11 or 14.

[0040] In another preferred embodiment, the sequence of Z2 is set forth in SEQ ID NO: 11 or 14.

[0041] In another preferred embodiment, the length of the peptide linker is 0-10 amino acids, preferably 0-5 amino acids.

[0042] In another preferred embodiment, the fusion protein is selected from the group consisting of: (A) a polypeptide having an amino acid sequence set forth in any one of SEQ ID NOs: 4-8; (B) a polypeptide having 80% or more homology (preferably 90% or more homology, preferably 95% or more homology, most preferably 97% or more, e.g., 98% or more, 99% or more homology) to the amino acid sequence shown in any one of SEQ ID NOs: 4-8 and having tumor-suppressing activity; (C) A derived polypeptide having an amino acid sequence represented by any one of SEQ ID NOs: 4-8, with 1-5 amino acid residues substituted, deleted or added, and which retains tumor-suppressing activity.

[0043] In another preferred embodiment, the amino acid sequence of the fusion protein is set forth in any of SEQ ID NOs: 4-8.

[0044] In a second aspect of the invention, there is provided an isolated polynucleotide that encodes a fusion protein according to the first aspect of the invention.

[0045] In another preferred embodiment, the polynucleotide further contains auxiliary elements selected from the group consisting of a signal peptide, a secretory peptide, a tag sequence (e.g., 6His), or a combination thereof, next to the ORF of the mutant protein or fusion protein.

[0046] In another preferred embodiment, the polynucleotide is selected from the group consisting of a DNA sequence, an RNA sequence, or a combination thereof.

[0047] In a third aspect of the present invention, there is provided a vector comprising a polynucleotide according to the second aspect of the present invention.

[0048] In another preferred embodiment, the vector comprises one or more promoters operably linked to the nucleic acid sequence, an enhancer, a transcription termination signal, a polyadenylation sequence, an origin of replication, a selectable marker, a nucleic acid restriction site, and / or a homologous recombination site.

[0049] In another preferred embodiment, the vector includes a plasmid and a viral vector.

[0050] In another preferred embodiment, the viral vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpesvirus, SV40, poxvirus, or a combination thereof.

[0051] In another preferred embodiment, the vector includes an expression vector, a shuttle vector, and an integration vector.

[0052] In a fourth aspect of the invention, there is provided a host cell which comprises a vector according to the third aspect of the invention or has incorporated into its genome a polynucleotide according to the second aspect of the invention.

[0053] In another preferred embodiment, the host cell is a eukaryotic cell, such as a yeast cell, a plant cell or a mammalian cell (including human and non-human mammals).

[0054] In another preferred embodiment, the host cell is a prokaryotic cell, such as E. coli.

[0055] In another preferred embodiment, the yeast cells are from one or more yeasts selected from the group consisting of Pichia yeast, Kluyveromyces yeast, or a combination thereof, and preferably, the yeast cells include Kluyveromyces yeast, more preferably Kluyveromyces marxianus and / or Kluyveromyces lactis.

[0056] In another preferred embodiment, the host cell is selected from the group consisting of E. coli, wheat germ cells, insect cells, SF9, Hela, HEK293, CHO, yeast cells, or combinations thereof.

[0057] In a fifth aspect of the present invention, there is provided a method of producing a fusion protein according to the first aspect of the present invention, the method comprising the steps of: expressing the fusion protein by culturing a host cell according to the fourth aspect of the invention under conditions suitable for expression; and / or isolating said fusion protein.

[0058] In a sixth aspect of the present invention, there is provided a pharmaceutical composition comprising the fusion protein according to the first aspect of the present invention and a pharmaceutically acceptable carrier therefor.

[0059] In another preferred embodiment, the pharmaceutical composition further comprises other drugs for suppressing tumor activity.

[0060] In another preferred embodiment, the other drug for suppressing tumor activity is a PD-1 antibody, a PD-L1 antibody, a HER2 monoclonal antibody, a BTLA antibody, a CTLA-4 antibody, a CD47 antibody, an NKG2A antibody, an NKTR-214, a GDF-15 antibody, a LILRB4 antibody, an LAIR1 antibody, a Tim-3 antibody, a Lag-3 antibody, a Tight antibody, CD160 antibody, KLRG-1 antibody, GP49B antibody, CD31 antibody, CD38 antibody, Lair-1 antibody, CD200 / CD200R antibody, Catumaxomab, Blinatumomab, EGFR monoclonal antibody, CD20 monoclonal antibody, VEGF / VEGFR monoclonal antibody, Licartin, Zevalin, Bexxar, Mylotarg, Kadcyla, Aflibercept, Conbercept, Apalutamide (ARN-509), Rova-T, TNFα, IFNγ, IL-2, Tβ4, small molecule inhibitors targeting EGFR, taxol (PTX), docetaxel (TXT), cisplatin (DDP), carboplatin (CBP), oxaliplatin, nedaplatin, cyclophosphamide (CTX), ifosfamide (IFO), doxorubicin (ADM), pirarbucin (THP), epirubicin (EPI), fluorouracil (5-Fu), gemcitabine (GEM), vinorelbine (NVB), pemetrexed (PEM), irinotecan (CPT-11), etoposide (VP-16), capecitabine (Xeloda), leuprolide acetate, goserelin acetate, or a combination thereof.

[0061] In another preferred embodiment, the PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, cemiplimab, toripalimab, sintilimab, camrelizumab, tislelizumab, or a combination thereof.

[0062] In another preferred embodiment, the PD-L1 antibody is selected from the group consisting of atezolizumab, durvalumab, avelumab, or a combination thereof.

[0063] In another preferred embodiment, the HER2 monoclonal antibody is selected from the group consisting of trastuzumab, pertuzumab, T-DM1, or a combination thereof.

[0064] In another preferred embodiment, the CTLA-4 antibody comprises ipilimumab.

[0065] In another preferred embodiment, the EGFR monoclonal antibody includes Necitumumab, Panitumumab, Nimotuzumab, and Cetuximab.

[0066] In another preferred embodiment, the CD20 monoclonal antibody is selected from the group consisting of rituximab, ibritumomab, tositumomab, ofatumumab, ocrelizumab, atezolizumab, or a combination thereof.

[0067] In another preferred embodiment, the VEGF / VEGFR monoclonal antibody is selected from the group consisting of bevacizumab, ramucirumab, ranibizumab, or a combination thereof.

[0068] In another preferred embodiment, the small molecule inhibitor targeting EGFR is selected from the group consisting of erlotinib, gefitinib, gefitinib, gefitinib (including afatinib maleate), dacotinib, osimertinib, nazartinib, neratinib (including neratinib maleate), sorafenib, apatinib (e.g., Aitan, apatinib mesylate), imatinib, sunitinib, dasatinib, lapatinib, pazopanib, crizotinib, vandetanib, regorafenib, axitinib, ponatinib, neratinib, zanubrutinib, anlotinib, ceritinib, fruquintinib, pyrotinib, lenvatinib, or a combination thereof.

[0069] In a seventh aspect of the present invention, there is provided the use of a fusion protein according to the first aspect of the invention, a polynucleotide according to the second aspect of the invention, a vector according to the third aspect of the invention, or a host cell according to the fourth aspect of the invention in the manufacture of a composition or formulation for treating or preventing tumors.

[0070] In another preferred embodiment, the composition or formulation is further used for one or more purposes selected from the following group: (a) Enhancement of the killing activity of T cells against tumor cells; (b) inhibition of tumor growth; (c) inhibition of T cell apoptosis; (d) increased levels of T cell IL-2 secretion; (e) Enhancement of the killing activity of NK cells against tumor cells; (f) increased expression levels of CD107a on NK cells; (g) Increased secretion of perforin and granzymes from NK cells; (h) Increased secretion of IFN-γ and TNF-α by NK cells; (i) Enhancement of macrophage phagocytic function; (j) Enhancement of the phagocytic killing activity of macrophages against tumor cells; (k) Increased secretion of NO, TNF-α and IL-1β by macrophages.

[0071] In another preferred embodiment, the composition is a pharmaceutical composition.

[0072] In another preferred embodiment, the tumor is selected from the group consisting of breast cancer, colon cancer, lung cancer, colorectal cancer, gastric cancer, esophageal cancer, pancreatic adenocarcinoma, ovarian cancer, prostate cancer, kidney cancer, liver cancer, brain cancer, melanoma, multiple myeloma, leukemia, lymphoma, head and neck tumors, thyroid cancer, or a combination thereof.

[0073] In another preferred embodiment, the tumor cells are derived from one or more tumors selected from the group consisting of breast cancer, colon cancer, lung cancer, colorectal cancer, gastric cancer, esophageal cancer, pancreatic adenocarcinoma, ovarian cancer, prostate cancer, kidney cancer, liver cancer, brain cancer, melanoma, multiple myeloma, leukemia, lymphoma, head and neck tumor, thyroid cancer, or a combination thereof.

[0074] In an eighth aspect of the present invention, there is provided a method for non-therapeutic inhibition of tumor growth ex vivo, the method comprising the step of inhibiting tumor growth by culturing tumor cells in the presence of a fusion protein according to the first aspect of the present invention.

[0075] In another preferred embodiment, the tumor cells are cells cultured in vitro. In a ninth aspect of the invention, there is provided a method of treating a tumor, comprising administering to a subject in need thereof a fusion protein according to the first aspect of the invention.

[0076] In another preferred embodiment, the fusion protein is applied in the form of a monomer and / or a dimer.

[0077] In another preferred embodiment, the subject is a human.

[0078] Of course, it is understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (e.g., in the Examples) can be combined with each other to form new or preferred technical solutions, which will not be described here one by one due to space limitations. [Effects of the Invention]

[0079] The main advantages of the present invention are as follows:

[0080] (a) In the present invention, for the first time, a fusion protein has been discovered, which contains a CCP protein, optionally a linking element, and the SH2 domain of SHP2 or SHP1 or an active fragment thereof, and the fusion protein obtained in the present invention has excellent tumoricidal activity.

[0081] (b) The fusion protein of the present invention can also significantly enhance the killing activity of T cells against tumor cells, and / or inhibit tumor growth, and / or inhibit T cell apoptosis, and / or increase the level of IL-2 secretion by T cells.

[0082] (c) The fusion protein of the present invention can also enhance the killing activity of NK cells against tumor cells, increase the CD107a expression level of NK cells, increase the secretion of perforin and granzymes by NK cells, and increase the secretion of IFN-γ and TNF-α by NK cells.

[0083] (d) The fusion protein of the present invention can also enhance the phagocytic function of macrophages, enhance the phagocytic killing action of macrophages against tumor cells, and enhance the secretion of NO, TNF-α, and IL-1β by macrophages.

[0084] (e) In this invention, for the first time, we have designed and produced fusion proteins and mimetic polypeptides of the N-SH2 and C-SH2 domains of SHP2 or SHP1, respectively, capable of binding to ITIMs, using genetic engineering and solid-phase polypeptide synthesis technology, with the aim of blocking the binding of the N-SH2 and C-SH2 domains of SHP2 or SHP1 to ITIMs in immune cells. To enable the fusion proteins and polypeptides to enter cells, the membrane-permeable peptide TAT was fused to the N-terminus of the fusion protein and the C-terminus of the polypeptide. The HIV-1 transactivator protein (TAT) allows efficient and rapid intracellular delivery of polypeptides and proteins without affecting the normal structure and function of cells. Once inside cells, the fusion proteins and polypeptides compete with SHP2 or SHP1 normally expressed in T cells / NK cells / macrophages to bind to the ITIMs in the intracellular domains of inhibitory receptors, thereby maintaining SHP2 or SHP1 in an inactive state within immune cells and eliminating the inhibitory effect of immune inhibitory receptors.

[0085] (f) The fusion protein of the present invention has a broad range of antitumor activity. BEST MODE FOR CARRYING OUT THE INVENTION

[0086] After extensive research, the present inventors unexpectedly discovered a fusion protein containing a CPP element, optionally a linking element, and the SH2 domain of SHP2 or SHP1 or an active fragment thereof, which has excellent tumor-killing activity and can significantly enhance the killing activity of T cells against tumor cells. Based on this, the present invention was completed.

[0087] <CPPエレメント> The cell membrane is the main barrier between cells and the extracellular environment. This natural barrier makes it difficult for many biopolymers to penetrate the cell membrane and enter cells, posing a major obstacle to current targeted drug delivery. Cell penetration peptides (CPPs) are polypeptides consisting of 5–30 amino acid residues that have the ability to penetrate cell membranes and can carry biopolymers, such as proteins and nucleic acids, to enter cells. CPP-mediated delivery of biopolymers into cells is safer and more efficient than conventional methods and is currently the most successful delivery system for direct biopolymer transport both in vivo and in vivo. Initial CPP studies demonstrated that arginine-rich polypeptide (TAT), a member of the reverse transcription activator protein (RTP) of the human immunodeficiency virus (HIV-1), can effectively penetrate the cell membrane and activate the transcription of the corresponding viral promoter. TAT can efficiently deliver drugs into cells. The membrane permeability function of TAT was precisely identified as being located in an 11-amino acid core region (aa47-aa57), which was named the TAT-protein transduction domain (TAT-PTD) and successfully applied to the intracellular transport of various heterologous proteins.

[0088] CPPs are divided into three types based on their chemical properties: (1) cationic CPPs are rich in arginine and lysine residues and have a strong positive charge at physiological pH, mainly including TAT, R9, and hLF. (2) Amphipathic peptides, in which the positive charge is provided by lysine residues, contain hydrophobic and hydrophilic domains, and their amphipathic character is determined by both their primary and secondary structures. These CPPs mainly include MPG, CADY, pVEC, and SAP. (3) Hydrophobic CPPs have a low net charge and are primarily composed of nonpolar amino acids. For example, Kaposi's fibroblast growth factor (K-FGF) peptide is included.

[0089] In one preferred embodiment, the CPP element of the present invention is the TAT membrane-permeable peptide.

[0090] In one preferred embodiment, the CPP element of the present invention comprises polyarginine (R5-9), MPG (a fusion protein domain of the gp41 protein of human immunodeficiency virus type 1 and a nuclear localization sequence region of the large T antigen of simian vacuolar virus, amino acid sequence: GLAFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 15)), CADY (a second amphipathic peptide consisting of 20 amino acids containing a tryptophan residue and an arginine residue, amino acid sequence: GLWRALWRLLRSLWRLLWKA (SEQ ID NO: 16)-CyA), pVEC (mouse vascular endothelial cell-derived cadherin, amino acid sequence: L LIILRRRIRKQAHAHSK (SEQ ID NO: 17)), penetratin (the positively charged sequence 43-58 of the third alpha helix in the Drosophila antennapedia protein, amino acid sequence: RQIKIWFQNRRMKWKK (SEQ ID NO: 18)), Kaposi's fibroblast growth factor peptide (K-FGF, amino acid sequence: AAVLLPVLLAAP (SEQ ID NO: 19)), VP22 (the intermediate layer protein of herpes simplex virus type 1, amino acid sequence: DAATATRGRSAASRPTERPRAPARSASRPRRVD (SEQ ID NO: 20)), or a combination thereof.

[0091] <Immunoreceptor tyrosine-dependent inhibitory motif (ITIM)> Currently, many immune checkpoint inhibitor receptors (e.g., PD-1, BTLA, KIR, CD31, and SIRPα) are known. Although their extracellular domains differ, their intracellular domains all contain one or more immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Tyrosine phosphorylation of ITIMs recruits the signaling molecules protein tyrosine phosphatase 1 or 2 (SH2 domain-containing inositol phosphatase-1, SHP1; SH2 domain-containing inositol phosphatase-2, SHP2), generating inhibitory signals that lead to the loss of effector T cell function and / or apoptosis. Therefore, ITIMs are a common domain that mediates the inhibitory effects of many immune checkpoints and are important targets for designing versatile immune checkpoint drugs. SHP1 and SHP2 are highly homologous and recruit to similar binding sites, but the distinct functions of SHP1 and SHP2 phosphatases remain unclear.

[0092] <shp1> Protein tyrosine phosphatase 1 (SH2 domain-containing protein tyrosine phosphates, SHP1) is primarily expressed in human hematopoietic cells and is a critical negative regulator of tyrosine phosphorylation levels in lymphocyte signaling pathways. SHP1 contains two SH2 domains, N-SH2 (3-101) and C-SH2 (109-215), a catalytic (PTP) domain (272-514), a proline-rich tail, and a tyrosine phosphorylation tail.

[0093] <shp2> SHP2 is a protein tyrosine phosphatase encoded by the non-receptor type 11 (PTPN11) gene. It contains two SH2 domains, N-SH2 (5-103) and C-SH2 (111-218), a catalytic (PTP) domain (276-523), a proline-rich tail, and a tyrosine phosphorylation site. The PTPN11 gene contains 16 exons, generates a widely expressed 7-kb transcript, and contains a 1.779-bp open reading frame encoding a 593-amino acid protein. The human and mouse SHP2 gene sequences share 100% homology.

[0094] The N-SH2 domain of inactive SHP2 / SHP1 makes extensive contact with the catalytic domain PTP through charge-charge interactions, and a portion of the SH2 domain (NXGDY / F motif) inserts into the catalytic cleft, blocking substrate entry into the active site and resulting in catalytic inactivity. Binding of the N-SH2 domain to a ligand containing a phosphotyrosine residue leads to the conversion of an allosteric switch in the SH2 domain from its inactive state to its active state. This conformational change in the N-SH2 domain of SHP2 / SHP1 disrupts the interaction between the SH2 domain and the phosphatase domain, eliminating its own inhibition and allowing substrate entry.

[0095] In this study, we aimed to block the binding of the N-SH2 and C-SH2 domains of SHP2 / SHP1 to ITIMs in immune cells. Using genetic engineering and solid-phase polypeptide synthesis, we designed and produced fusion proteins and mimetic polypeptides of the N-SH2 and C-SH2 domains of SHP2 / SHP1, respectively, that can bind to ITIMs. To facilitate cell entry, the membrane-permeable peptide TAT was fused to the N-terminus of the fusion protein and the C-terminus of the polypeptide. The HIV-1 transactivator protein (TAT) allows efficient and rapid intracellular delivery of polypeptides and proteins without affecting the normal structure and function of cells. Upon entry into cells, the fusion proteins and polypeptides compete with SHP2 / SHP1 normally expressed in T cells to bind to ITIMs in the intracellular domains of inhibitory receptors, thereby maintaining SHP2 / SHP1 in an inactive state within immune cells and eliminating the inhibitory effects of immune inhibitory receptors. This is the concept behind the research and development of a versatile immune checkpoint inhibitor designed to target ITIMs.

[0096] <Fusion protein> As used herein, the term "fusion protein of the invention" or "polypeptide" refers to any fusion protein according to the first aspect of the invention.

[0097] In another preferred embodiment, the structure of the fusion protein is represented by Z1-L-Z2 (I) or Z2-L-Z1 (II), where B is a CCP protein, L is nothing or a linking element, and Z2 is the SH2 domain of SHP2 or SHP1 or an active fragment thereof.

[0098] In another preferred embodiment, the fusion protein has an amino acid sequence set forth in any of SEQ ID NOs: 4-8.

[0099] As used herein, the term "fusion protein" further encompasses variants of the fusion protein (sequences represented by any of SEQ ID NOS: 4-8) that have the above-described activity. These variants include, but are not limited to, the deletion, insertion, and / or substitution of one to three (usually one to two, more preferably one) amino acids, and the addition or deletion of one or more (usually up to three, preferably up to two, more preferably up to one) amino acids at the C-terminus and / or N-terminus. For example, substitution with amino acids with close or similar functions generally does not alter the function of the protein. Furthermore, the addition or deletion of one or more amino acids at the C-terminus and / or N-terminus generally does not alter the structure and function of the protein. The term also encompasses the polypeptides of the present invention in both monomeric and multimeric forms. The term further encompasses linear and nonlinear polypeptides (e.g., cyclic peptides).

[0100] The present invention further encompasses active fragments, derivatives, and analogs of the above-described fusion proteins. As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that essentially maintain the function or activity of the fusion proteins of the present invention. Fragments, derivatives, and analogs of the polypeptides of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) have been substituted, or (ii) polypeptides in which one or more amino acid residues have been substituted, or (iii) polypeptides in which the antigenic peptide is fused to another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides in which an additional amino acid sequence has been fused to the polypeptide (e.g., a fusion protein fused to a leader sequence, secretory sequence, or tag sequence such as 6xHis). Based on the disclosure herein, these fragments, derivatives, and analogs are within the scope known to those skilled in the art.

[0101] One preferred type of active derivative is a polypeptide in which, compared to the amino acid sequence of Formula I or Formula II, three or fewer, preferably two or fewer, and more preferably one or fewer amino acids are substituted with amino acids of similar or similar properties. These conservatively mutated polypeptides are preferably generated by amino acid substitutions as shown in Table A. [Table 1]

[0102] The present invention also provides analogs of the fusion proteins of the present invention. These analogs may differ from the polypeptides set forth in any of SEQ ID NOS: 4-8 in amino acid sequence, in modifications that do not affect sequence, or in both. Analogs further include analogs containing residues other than naturally occurring L-amino acids (e.g., D-amino acids) and analogs containing unnatural or synthetic amino acids (e.g., β- and γ-amino acids). Of course, the polypeptides of the present invention are not limited to the representative polypeptides listed above.

[0103] Modified forms (which usually do not alter the primary structure) include chemically derivatized forms of polypeptides, either in vivo or in vitro, such as acetylation or carboxylation. Modifications also include polypeptides that have undergone glycosylation, e.g., glycosylation modifications during polypeptide synthesis and processing or during further processing. Such modifications are accomplished by exposing the polypeptide to glycosylating enzymes (e.g., mammalian glycosylating or deglycosylating enzymes). Modified forms further include sequences with phosphorylated amino acid residues (e.g., tyrosine phosphate, serine phosphate, threonine phosphate). Furthermore, modified polypeptides include polypeptides with improved resistance to proteolysis or improved solubility.

[0104] <Expression vectors and host cells> The present invention also relates to vectors comprising polynucleotides of the invention and host cells produced by genetic engineering using the vectors of the invention or the coding sequences of the fusion proteins of the invention, as well as methods for producing the polypeptides of the invention by recombinant techniques.

[0105] The polynucleotide sequences of the present invention can be used to express or produce recombinant fusion proteins by conventional recombinant DNA techniques, which generally involve the following steps: (1) A polynucleotide (or a variant) encoding the fusion protein of the present invention, or a recombinant expression vector containing this polynucleotide, is used to transform or transduce a suitable host cell. (2) culturing the host cells in an appropriate medium; (3) Isolating and purifying the protein from the medium or cells.

[0106] In the present invention, the polynucleotide sequence encoding the fusion protein may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian viruses such as adenoviruses and retroviruses, or other vectors well known in the art. Any plasmid or vector can be used as long as it can stably replicate in the host. One important feature of an expression vector is that it usually contains an origin of replication, a promoter, a marker gene, and a translation control element.

[0107] Methods well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding the fusion protein of the present invention and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be effectively linked to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of such promoters include eukaryotic promoters, including the lac or trp promoter of E. coli, the PL promoter of phage lambda, the CMV immediate-early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, retroviral LTRs, and other known regulatable gene promoters expressed in prokaryotic or eukaryotic cells or their viruses. The expression vector may further contain a ribosome binding site for translation initiation and a transcription terminator.

[0108] Expression vectors also preferably contain one or more selectable marker genes to provide a trait for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E. coli.

[0109] A vector containing the appropriate DNA sequence as described above and an appropriate promoter or control sequence can be transformed into an appropriate host cell so as to express the protein.

[0110] Host cells may be prokaryotic cells (e.g., Escherichia coli), lower eukaryotic cells, or higher eukaryotic cells, such as yeast cells, plant cells, or mammalian cells (including human and non-human mammalian cells). Representative examples include Escherichia coli, wheat germ cells, insect cells, SF9, Hela, HEK293, CHO, and yeast cells. In one preferred embodiment of the present invention, yeast cells (e.g., Pichia yeast, Kluyveromyces yeast, or a combination thereof, preferably, the yeast cells include Kluyveromyces yeast, more preferably Kluyveromyces marxianus and / or Kluyveromyces lactis) are selected as host cells.

[0111] When the polynucleotide of the present invention is expressed in higher eukaryotic cells, transcription can be enhanced by inserting an enhancer sequence into the vector. Enhancers are DNA cis elements, typically about 10 to 300 bp long, that act on promoters to enhance gene transcription. Examples include the SV40 enhancer, located 100 to 270 bp long on the late side of the replication origin, the polyoma enhancer located on the late side of the replication origin, and adenovirus enhancers.

[0112] Those skilled in the art will know how to select appropriate vectors, promoters, enhancers and host cells.

[0113] When the host is a prokaryotic cell, such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, a procedure well known in the art. Another method uses MgCl2. Transformation can also be performed by electroporation, if desired. When the host is a eukaryotic organism, DNA transfection methods such as calcium phosphate precipitation, microinjection, conventional mechanical methods such as electroporation, and lipofection can be used.

[0114] The resulting transformant can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Conventional culture media may be selected depending on the host cells used. The cells are cultured under conditions suitable for the growth of the host cells. Once the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (e.g., temperature shift or chemical induction), and the cells are further cultured.

[0115] The recombinant polypeptides in the above methods can be expressed intracellularly or at the cell membrane, or can be secreted extracellularly. If necessary, the recombinant proteins can be isolated and purified using various isolation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatments, treatment with protein precipitants (salting out), centrifugation, osmotic shock, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0116] <Peptide linker> The present invention provides fusion proteins that optionally contain a peptide linker. The activity of the protein is affected by the size and complexity of the peptide linker. Typically, the peptide linker must be long and flexible enough to allow the two proteins it links to have sufficient spatial freedom to perform their functions. At the same time, the peptide linker must be free from the formation of α-helices or β-sheets to avoid affecting the stability of the fusion protein.

[0117] The length of the connecting peptide is generally 0-10 amino acids, preferably 0-5 amino acids.

[0118] <Drug Composition> Furthermore, the present invention provides a pharmaceutical composition. In a preferred embodiment, the composition is a pharmaceutical composition containing the above-mentioned fusion protein and a pharmaceutically acceptable carrier, diluent, stabilizer and / or thickener, and can be prepared in the form of a lyophilized powder, tablet, capsule, syrup, solution or suspension.

[0119] "Pharmaceutically acceptable carrier or excipient" refers to one or more compatible solid or liquid fillers or gel substances that are suitable for human administration and must have sufficient purity and sufficiently low toxicity. "Compatibility" means that each component in the composition can be mixed with the active ingredient of the present invention and with each other without significantly reducing the effectiveness of the active ingredient.

[0120] The composition may be liquid or solid, for example a powder, gel or paste. Suitably, the composition is a liquid, preferably an injectable liquid. Suitable excipients are known to those skilled in the art.

[0121] Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyhydric alcohols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., Tween®), wetting agents (e.g., sodium dodecyl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free distilled water, etc.

[0122] The compositions may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions and emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous or non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0123] Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier, and the pH value is usually about 5 to 8, preferably about 6 to 8, depending on the properties of the formulated substances and the disease to be treated. The formulated pharmaceutical composition can be administered by any conventional route, including, but not limited to, intraperitoneal, intravenous, or topical administration. The pharmaceutical composition is used for (a) treating or preventing tumors, (b) enhancing the killing activity of T cells against tumor cells, (c) inhibiting tumor growth, (d) inhibiting apoptosis of T cells, and (e) increasing the level of IL-2 secretion by T cells. (a) Enhanced killing activity of NK cells against tumor cells, (b) Increased CD107a expression level of NK cells, (c) Increased secretion of perforin and granzymes by NK cells, (d) Increased secretion of IFN-γ and TNF-α by NK cells, (a) Enhanced phagocytic function of M cells, (b) Enhanced phagocytic killing activity of M cells against tumor cells, (c) Enhanced secretion of NO, TNF-α and IL-1β by M cells.

[0124] The present invention will be further described below with reference to specific examples. It should be understood that these examples are used only to illustrate the present invention and do not limit the scope of the present invention. Experimental methods for which specific conditions are not specified in the following examples generally follow conventional conditions, such as those described in Sambrook et al., "Molecular Cloning: A Laboratory Manual" (New York: Cold Spring Harbor Laboratory Press, 1989), or manufacturer recommendations. Unless otherwise specified, percentages and parts are by weight.

[0125] Unless otherwise stated, all reagents and materials used in the examples of the present invention are commercially available. Example 1

[0126] Example 1: Recombinant TAT-N-SH2 fusion protein and its production method

[0127] The recombinant TAT-N-SH2 fusion protein of the present invention is a TAT membrane-permeable peptide linked to N-SH2 via glycine and serine (TAT-N-SH2), and its linkage form is TAT-Gly-Ser-N-SH2, with the amino acid sequence shown in SEQ ID NO: 4.

[0128] The nucleotide sequence of the recombinant TAT-N-SH2 fusion protein is a nucleotide sequence in which the TAT membrane-permeable peptide and the N-terminal SH2 domain of SHP2 are linked via a BamHI enzyme cleavage site, with an NdeI enzyme cleavage site at the 5' end and a termination codon TGA and a SalI enzyme cleavage site at the 3' end. The ligation configuration is NdeI enzyme cleavage site-TAT gene-BamHI enzyme cleavage site-N-SH2 gene-TGA-SalI enzyme cleavage site, and the nucleotide sequence is set forth in SEQ ID NO: 9.

[0129] The nucleotide sequence of the recombinant TAT-N-SH2 fusion protein is constructed in the expression vector or recombinant plasmid pET-22b(+)-TAT-N-SH2, which is ligated to the plasmid pET-22b(+) at the NdeI and SalI enzyme sites via double enzyme cleavage and ligation.

[0130] The method for producing the recombinant TAT-N-SH2 fusion protein comprises the following steps.

[0131] 1. Construction of the recombinant TAT-N-SH2 fusion protein gene expression vector or recombinant plasmid pET-22b(+)-TAT-N-SH2

[0132] 1) Design and synthesis of TAT-N-SH2 The amino acid sequences of TAT and N-SH2 were converted into gene sequences using codons suitable for E. coli. The TAT and N-SH2 gene sequences were linked together at a BamHI site, and an NdeI site was added to the 5' end and a stop codon TGA and a SalI site to the 3' end to synthesize the TAT-N-SH2 gene, the nucleotide sequence of which is shown below. [ka]

[0133] The synthesis of the TAT-N-SH2 gene was outsourced to another company.

[0134] 2) Identification of the expression vector pET-22b(+)-TAT-N-SH2 BL21 cells were transformed with the recombinant plasmid pET-22b(+)-TAT-N-SH2 and cultured for 12 hours at 37°C. A single clone colony was selected and inoculated into LB medium containing 100 mg / L ampicillin. The colony was then cultured at 37°C in a shaker. The bacteria were then harvested and the plasmid was extracted. The plasmid was double-cleaved with NdeI and SalI and identified by agarose gel electrophoresis. The expected insert fragment of 342 bp was obtained (Figure 1). DNA sequencing (Figure 2) confirmed the sequence of the recombinant plasmid pET-22b(+)-TAT-N-SH2, demonstrating the successful construction of the recombinant plasmid.

[0135] The recombinant plasmid pET-22b(+)TAT-SHP2-N-SH2 obtained was double digested with Nde I and Sal I enzymes and then subjected to agarose gel electrophoresis. The red arrow indicates the target fragment.

[0136] 2. Expression of Recombinant TAT-N-SH2 Fusion Protein

[0137] Competent E. coli BL21 cells were transformed with the recombinant plasmid pET-22b(+)-TAT-N-SH2 and cultured for 12 hours in a 37°C incubator. Colonies with uniform margins and good growth were selected and inoculated into LB medium containing 100 mg / L ampicillin and cultured overnight in a shaker at 37°C. The next day, the colonies were inoculated at a 1:100 ratio into fresh LB medium containing 100 mg / L ampicillin and cultured at 37°C until the bacterial density reached an OD600 of 0.4-0.6. After induction with 0.01-0.1 mM IPTG for 4 hours, the cells were harvested by spinning at 12,000 rpm for 20 minutes and stored at -20°C.

[0138] 3. Purification of Recombinant TAT-N-SH2 Fusion Protein

[0139] 1) The bacterial solution was weighed and mixed with PB decomposition buffer (pH 6.5) at a ratio of 1:7. The mixture was stirred in a refrigerator at 4°C until uniformly dissolved. The mixture was then sonicated in an ice bath for 20 minutes at 300 W output, 5 seconds running time, and 10 seconds interval time. The mixture was then centrifuged at 12,000 rpm at 4°C for 20 minutes, and the supernatant was collected.

[0140] 2) 25 mL of bacterial lysis supernatant was transferred to a pre-prepared dialysis bag and dialyzed overnight with 30-fold volume of equilibrated solution A (20 mM pH 6.5, PB) as the external solution. The mixture was then centrifuged at 12,000 rpm for 20 min at 4°C. The supernatant was collected and passed through a 0.22 μm filter, and the volume was recorded. A pre-prepared 25 mL SP-cation exchange chromatography column was used. The column was equilibrated with equilibrated solution A until the baseline stabilized, zero-calibrated, and loaded at a flow rate of 1 mL / min. The flow-through peak was collected. Peak collection was stopped once the baseline stabilized. Linear elution was performed from 0 to 100% with equilibrated solution A and eluent solution B (20 mM pH (5.4, 6.5, 7.4) PB, 1 M NaCl) at a flow rate of 1.5 mL / min, and elution peak 4 was collected. The target protein elution peak was then dialyzed against 30-fold volume of PBS. After dialysis, the mixture was filtered through a 0.22 μm filter membrane to remove bacteria, and the target protein, TAT-N-SH2 fusion protein, was obtained.

[0141] 4. Identification of the Inducible Expression and Purification Results of Recombinant TAT-N-SH2 Fusion Protein

[0142] 1) The expression level of the target protein was analyzed by SDS-PAGE (polyacrylamide gel electrophoresis) (shown in Figure 3). Compared with the uninduced group, the induced group showed a specific protein band, the size of which matched the theoretical molecular weight (12.870 KD).

[0143] After lysing the bacteria with ultrasound, the supernatant and precipitate were collected and analyzed by SDS-PAGE to see if the target protein was expressed in the supernatant. As shown in Figure 4, the target protein was present in both the bacterial lysis supernatant and the bacterial lysis precipitate, but the amount of the bacterial lysis precipitate was negligible compared to the bacterial lysis supernatant. Therefore, in subsequent experiments, the bacterial lysis supernatant was used as the raw material for precise separation.

[0144] The supernatant after bacterial digestion and crude extraction was dialyzed and centrifuged, and then further separated using an SP-cation exchange chromatography column. Peaks were collected at different detection wavelengths, and the target protein was only detected at a wavelength of 215 nm. A total of one pass-through peak and four elution peaks were obtained, which were SP peak 1, SP peak 2, SP peak 3, and SP peak 4 (shown in Figure 5). Analysis of each peak by SDS-PAGE revealed that the peak containing the target protein was SP4 (shown in Figure 6).

[0145] 5. Identification of the Specificity and Purity of Recombinant TAT-N-SH2 Fusion Protein

[0146] Qualitative detection of the target protein by Western blot revealed that the rabbit anti-human SHP2 polyclonal antibody was able to specifically bind to the target protein (shown in Figure 7), demonstrating that the purified product was the target protein.

[0147] The purified fusion protein was analyzed by HPLC. A 20 μL sample was taken and run for 20 min at a flow rate of 0.8 mL / min at a detection wavelength of 280 nm. The purity of the sample was determined from the area of ​​the main peak. The result showed that the purity of the fusion protein was 95.92% (Figure 8). Example 2

[0148] <Example 2: Fusion polypeptides of four types of TAT membrane-penetrating peptides and the ITIM motif-binding region of the C-terminal SH2 domain of SHP2>

[0149] 1. A fusion polypeptide of the TAT membrane-permeable peptide and the ITIM motif-binding region of the C-terminal SH2 domain of SHP2, in which the TAT membrane-permeable peptide and the ITIM motif-binding region of C-SH2 are linked by two prolines (TAT-C-SH2), the linkage form being TAT-Phe-Phe-C-SH2, and the amino acid sequence being as follows: YGRKKRRQRRR-PP-VRESQSHPGDFVL (SEQ ID NO: 5)

[0150] We commissioned a third party to synthesize the TAT-C-SH2 polypeptide (designated L1) using all L-amino acids. Using the sequence of polypeptide L1 as a reference, we synthesized the reverse sequence of L1, the L1 sequence using D-amino acids, and the L1 reverse sequence using D-amino acids, designated L2, D1, and D2, respectively. Their amino acid sequences are as follows:

[0151] 1) Reverse L-type polypeptide (L2): LVFDGPHSQSERVPPRRRQRRKKRGY (SEQ ID NO: 6) 2) D-type polypeptide (D1): YGRKKRRQRRRPPVRESQSHPGDFVL (SEQ ID NO: 7) 3) Reverse D-type polypeptide (D1): LVFDGPHSQSERVPPRRRQRRKKRGY (SEQ ID NO: 8) All chemical synthesis was outsourced to other companies.

[0152] 2. Polypeptide Identification The purity of the synthesized polypeptides was analyzed by HPLC. A 20 μL sample was taken and loaded. The run time was 20 min, the flow rate was 1.0 mL / min, the detection wavelength was 220 nm, and the purity of the sample was determined from the area of ​​the main peak. The results showed that the purity of the L1 polypeptide was 96.74% (Figure 9A), the L2 polypeptide was 98.1% (Figure 9B), the D1 polypeptide was 99.27% ​​(Figure 9C), and the D2 polypeptide was 96.66% (Figure 9D).

[0153] The molecular weights of the synthesized polypeptides were determined by mass spectrometry. The results showed that the molecular weights of the L1 polypeptide were 3206 (Figure 10A), the L2 polypeptide was 3206.67 (Figure 10B), the D1 polypeptide was 3206.63 (Figure 10C), and the D2 polypeptide was 3206.79 (Figure 10D), which were consistent with the theoretical molecular weights.

[0154] The results indicated that the polypeptide was successfully synthesized and could be used for further in vitro and in vivo experimental studies. Example 3

[0155] Example 3: Cooperation of recombinant fusion protein and polypeptide of membrane-permeable peptide TAT in T cell entry

[0156] Entry of recombinant TAT-N-SH2 fusion proteins and polypeptides into T cells is a prerequisite for binding to the ITIM (intracellular domain) of inhibitory receptors on the cell membrane and exerting their effects. The membrane-permeable peptide TAT is a positively charged short peptide that can facilitate membrane permeation of many substances and their biological functions. To determine whether fusion proteins and polypeptides containing the TAT membrane-permeable sequence can enter T cells, the TAT-N-SH2 fusion protein, L1 polypeptide, and control peptide were FITC-labeled and their entry into T cells was observed using a confocal microscope at 0, 0.5, 4, 24, 72, 96, 120, and 144 h (Figure 11). The results showed that the fusion protein containing the TAT membrane-permeable sequence significantly entered T cells at 24 h, gradually decreasing by 120 h (Figure 11c). In contrast, the L1 polypeptide containing the TAT membrane-permeable sequence significantly entered T cells at 0.5 h, gradually decreasing by 72 h (Figure 11b). The control peptide without the TAT membrane-permeable sequence was unable to enter T cells (Figure 11a). This result indicates that the TAT membrane-permeable sequence can facilitate the entry of the fusion protein and polypeptide into target cells, and that the polypeptide and fusion protein have different times to enter T cells, which may be related to their molecular weight. Example 4

[0157] Example 4: Effects of recombinant fusion proteins and polypeptides on T cell function

[0158] 1. Effects of recombinant TAT-N-SH2 fusion protein and L1 (TAT-SHP2-C-SH2) polypeptide on T cell proliferation

[0159] To determine whether the recombinant TAT-N-SH2 fusion protein and L1(TAT-SHP2-C-SH2) polypeptide enhanced T cell proliferation, T cells were stimulated with 80 μg / mL of TAT-SHP2-N-SH2 fusion protein and TAT-SHP2-C-SH2 polypeptide, respectively, for 3 days, and then cell proliferation was detected using CFSE. The proliferation rate of T cells in the control group was 62.83% (Figure 12a), the proliferation rate of T cells in the recombinant TAT-N-SH2 fusion protein group was 61.05% (Figure 12b), and the proliferation rate of T cells in the L1(TAT-SHP2-C-SH2) polypeptide group was 60.33% (Figure 12c). The statistical results showed that compared with the control group, the recombinant TAT-N-SH2 fusion protein and L1 polypeptide did not significantly enhance T cell proliferation (Figure 12d), indicating that the recombinant TAT-N-SH2 fusion protein and L1 (TAT-SHP2-C-SH2) polypeptide did not affect T cell proliferation.

[0160] 2. Effects of recombinant TAT-N-SH2 fusion protein and L1 (TAT-SHP2-C-SH2) polypeptide on T cell apoptosis

[0161] After 3 days of stimulation with recombinant TAT-N-SH2 fusion protein and L1 polypeptide, T cells were stimulated with the recombinant TAT-N-SH2 fusion protein and L1 polypeptide, and the apoptosis status of T cells in each treatment group was monitored by flow cytometry. Figure 13 (a-d) shows the effect of polypeptides on T cell apoptosis monitored by Annexin V-FITC. Cells were resuspended and treated with SHP2-NC (untreated group) or SHP2-N-SH2 (80 μg / mL) or TAT-SHP2-C-SH2 polypeptide (80 μg / mL) for each group. Then, cells were cultured in EASY-T-primed culture plates (24-well plates were coated with 50 ng of PD-L1 antibody at 37°C 3 h before adding the cell mixture) for 3 days. Cells were harvested, stained with FITC and PI, and monitored by flow cytometry. Figure 13 (e) shows the percentage of apoptotic cells measured by flow cytometry compared with the control group; *p<0.05 and **p<0.01. As described above, compared with the control group, the apoptosis of T cells was significantly reduced in the groups treated with the recombinant TAT-N-SH2 fusion protein and L1 polypeptide.

[0162] 3. Effects of recombinant TAT-N-SH2 fusion protein and L1 (TAT-SHP2-C-SH2) polypeptide on the phosphorylation levels of downstream signaling molecules mediated by T cells

[0163] T cells were collected after 3 days of stimulation with recombinant TAT-N-SH2 fusion protein and L1 (TAT-SHP2-C-SH2) polypeptide, and changes in the phosphorylation levels of ERK, AKT, and JNK molecules were detected. Compared with the untreated group, there were no significant changes in the expression levels of ERK, AKT, and JNK. However, the TAT-SHP2-N-SH2 fusion protein-stimulated group showed the most significant changes in the expression levels of p-ERK and p-AKT, and the TAT-SHP2-C-SH2 polypeptide-stimulated group showed the most significant changes in the expression level of p-AKT (Figure 14 ad).

[0164] 4. Effects of recombinant TAT-N-SH2 fusion protein and L1 (TAT-SHP2-C-SH2) polypeptide on the cytokine IL-2 secreted by T cells

[0165] T cells were harvested after 3 days of stimulation with the recombinant TAT-N-SH2 fusion protein and L1 polypeptide, and the levels of the cytokine IL-2 secreted were measured. The results showed that the levels of IL-2 secreted by T cells stimulated with the TAT-SHP2-N-SH2 fusion protein and SHP2-C-SH polypeptide were significantly higher than those in the control group (Figure 15). Example 5

[0166] Example 5: Study of the direct killing ability of recombinant TAT-N-SH2 fusion protein and L1 (TAT-SHP2-C-SH2) polypeptide against tumor cells

[0167] To determine whether the recombinant TAT-N-SH2 fusion protein and L1 polypeptide exerted a direct killing effect on tumor cells, different concentrations (20 μg / mL, 40 μg / mL, and 80 μg / mL) of the recombinant TAT-N-SH2 fusion protein and L1 polypeptide were incubated with MDA-MB-231 and SW480 tumor cells, and their effects on tumor cells were monitored in real time using a zenCell owl live-cell dynamic imaging analysis system. The experimental results showed that the recombinant TAT-N-SH2 fusion protein and L1 polypeptide had no direct toxic effect on MDA-MB-231 and SW480 tumor cells compared with the untreated group. However, these results suggest that the recombinant TAT-N-SH2 fusion protein and L1 polypeptide exerted their killing effect on tumor cells by affecting T cells (Figure 16). Example 6

[0168] Example 6: Study on the enhancement of the killing ability of T cells against tumor cells in vitro by recombinant fusion proteins and polypeptides

[0169] 1. Effect of recombinant TAT-N-SH2 fusion protein on T cell killing of breast cancer cells MDA-MB-231 and colon cancer cells SW480

[0170] Three concentrations of TAT-SHP2-N-SH2 fusion protein (20 μg / mL, 40 μg / mL, and 80 μg / mL) were used to stimulate human T cells, and their killing activity against tumor cells MDA-MB-231 and SW480 was detected using a CytoTox 96 non-radioactive cytotoxicity assay at an effector cell to target cell ratio of 30:1. Experimental results showed that when the target cells were breast cancer cell line MDA-MB-231, the TAT-SHP2-N-SH2 fusion protein concentrations at 40 μg / mL and 80 μg / mL significantly increased killing compared to the control group (p=0.0379 and p=0.0367, respectively). When the target cells were colon cancer cell line SW480, all three concentrations of protein significantly increased killing compared to the untreated group (Figure 17, Table 1). The TAT-SHP2-N-SH2 fusion protein was shown to enhance the killing of tumor cells by T cells, and to have a better killing effect on colon cancer cells than on breast cancer cells. [Table 2]

[0171] 2. Effect of L1 (TAT-SHP2-C-SH2) polypeptide on T cell killing of breast cancer cells MDA-MB-231 and colon cancer cells SW480

[0172] CytoTox 96 non-radioactive cytotoxicity assay was used to detect the killing activity of human T cells against tumor cells MDA-MB-231 and SW480 after 4 days of stimulation with TAT-SHP2-C-SH2 (20 μg / mL, 40 μg / mL, 80 μg / mL). At an effector cell to target cell ratio of 30:1 and at TAT-SHP2-C-SH2 polypeptide concentrations of 40 μg / mL and 80 μg / mL, respectively, the killing ability of T cells stimulated with the TAT-SHP2-C-SH2 polypeptide was significantly enhanced compared to the control group (Figure 18, Table 2). [Table 3]

[0173] 3. Effect of L1, L2, D1, and D2 polypeptides on T cell killing of lung cancer cell H460

[0174] Using the CytoTox 96 non-radioactive cytotoxicity assay, human T cells were stimulated with L1, L2, D1, and D2 polypeptides (40 μg / mL) for 4 days and then their killing activity against lung cancer cells H460 was detected. At an effector cell to target cell ratio of 20:1, T cells stimulated with L1, L2, D1, and D2 polypeptides showed significantly improved killing ability against lung cancer cells H460 compared to the untreated group and the control peptide, with the D2 polypeptide having the strongest effect (Figure 19, Table 3). [Table 4] Example 7

[0175] Example 7: Study of the in vivo antitumor activity of recombinant fusion proteins and polypeptides

[0176] 1. Detection of the effect of recombinant TAT-N-SH2 fusion protein on mouse colon cancer

[0177] A subcutaneously xenografted MC38 colon cancer mouse model was established, and αPD-1 antibody was used as a positive control at a dose of 50 μg / mouse. TAT-SHP2-N-SH2 fusion protein was administered intraperitoneally at concentrations of 1.25 μg / mouse (N1.25), 2.5 μg / mouse (N2.5), 5 μg / mouse (N5), and 10 μg / mouse (N10). After eight doses, tumor-bearing mice were sacrificed and tumors were weighed. Tumor weights in the TAT-SHP2-N-SH2 fusion protein group were significantly lower than those in the control group, and in the N5 group were lower than those in the αPD-1 antibody group (Figure 20, ab). The tumor inhibition rate in the N5 group was the highest, reaching 80.9% (Table 4). These results suggest that a dose of 5 μg / mouse of TAT-SHP2-N-SH2 fusion protein had the best colon cancer tumor inhibition effect. [Table 5]

[0178] 2. Tumor suppression effect of L1 (TAT-SHP2-C-SH2) polypeptide on mouse colon cancer

[0179] The effect of TAT-SHP2-C-SH2 polypeptide on tumor growth in mice was evaluated using a constructed MC38 colon cancer mouse subcutaneous tumor-bearing model. TAT-SHP2-C-SH2 polypeptide was administered intraperitoneally at doses of 1.25 μg / mouse (C1.25), 2.5 μg / mouse (C2.5), 5 μg / mouse (C5), and 10 μg / mouse (C10). Simultaneously, αPD-1 antibody (αPD-1) was administered at a dose of 50 μg / mouse as a positive control drug. In addition, TAT-SHP2-N-SH2 (5 μg / mouse) and combination groups (TAT-SHP2-N-SH2 fusion protein and TAT-SHP2C-SH2 polypeptide, 5 μg / mouse each) were added. Compared with the control group, the TAT-SHP2-C-SH2 polypeptide group, αPD-1 antibody group, and TAT-SHP2 The tumor weight was significantly reduced in the TAT-SHP2-C-SH2 fusion protein group and the combination group. The combination group showed the best tumor suppression effect, followed by the N5 group. The TAT-SHP2-C-SH2 polypeptide at a dose of 5 μg / mouse slowed tumor growth (Figure 21, ab). The combination group showed the best tumor suppression rate, followed by the N5 group (Table 5). These results indicate that each dose group had an inhibitory effect on tumor growth in mice, with the combination group showing the best effect. [Table 6]

[0180] 3. Detection of the tumor-suppressive effect of recombinant TAT-N-SH2 fusion protein and L1 (TAT-SHP2-C-SH2) polypeptide on mouse breast cancer

[0181] Furthermore, the effects of the TAT-SHP2SHP2-N-SH2 fusion protein and the TAT-SHP2SHP2-C-SH2 polypeptide on tumor growth in mice were evaluated using a subcutaneous tumor-bearing mouse model of EMT breast cancer. The TAT-SHP2SHP2-C-SH2 polypeptide and the TAT-SHP2SHP2-N-SH2 fusion protein were administered intraperitoneally at 5 μg / mouse (C5), 10 μg / mouse (C10), and 15 μg / mouse (C15), respectively, and 2 μg / mouse (N2), 5 μg / mouse (N5), 10 μg / mouse (N10), and 15 μg / mouse (N15), respectively. Using αPD-1 antibody (αPD-1) at a dose of 5 μg / mouse as a positive control, the results showed that tumor volumes in the TAT-SHP2SHP2-C-SH2 polypeptide, αPD-1 antibody, and TAT-SHP2SHP2-N-SH2 fusion protein treatment groups were significantly smaller than those in the control group. The TAT-SHP2SHP2-N-SH2 fusion protein (5 μg / mouse) and TAT-SHP2SHP2 or SHP1-C-SH2 polypeptide (5 μg / mouse) groups had the lowest tumor weights and the best tumor inhibition rates (Figure 22, ab, Table 6). The results showed that each dose group inhibited tumor growth in mice, with the fusion protein and polypeptide showing the best effects at 5 μg / mouse, consistent with their colon cancer tumor inhibition effects. [Table 7]

[0182] 4. Tumor-suppressive effects of recombinant TAT-N-SH2 fusion protein and L1 (TAT-SHP2-C-SH2) polypeptide on transplanted tumors in nude mice

[0183] 1) Tumor-suppressive effects of recombinant TAT-N-SH2 fusion protein and L1 (TAT-SHP2-C-SH2) polypeptide on MC38 colon cancer-bearing nude mice

[0184] To rule out the direct tumor-inhibitory effects of the recombinant TAT-N-SH2 fusion protein and L1(TAT-SHP2-C-SH2) polypeptide, immunodeficient nude mice were inoculated with MC38 colon cancer tumors. Tumors were formed 14 days later, and treatment was initiated. Tumor volumes were measured once 4 days later. After 30 days, the mice were sacrificed and dissected for tumor weight measurement. The tumor volumes, tumor weights, and tumor inhibition rates of the recombinant TAT-N-SH2 fusion protein and L1(TAT-SHP2-C-SH2) polypeptide were not statistically significantly different from those of the control group (Figure 23, a-c, Table 7). These results indicate that the TAT-SHP2-N-SH2 fusion protein and TAT-SHP2-C-SH2 polypeptide do not have a direct tumor-inhibitory effect on colon cancer. [Table 8]

[0185] 2) Tumor-suppressive effects of recombinant TAT-N-SH2 fusion protein and L1 (TAT-SHP2-C-SH2) polypeptide on EMT-6 breast cancer-bearing nude mice

[0186] Simultaneously, immunocompromised nude mice were inoculated with EMT-6 breast cancer tumors. After 5 days, tumors formed. The mice were randomly divided into three groups of six mice each and administered the same doses as above, once every other day for a total of eight doses. After 4 days, tumor volumes were measured and growth curves were plotted. After 21 days, the mice were sacrificed and weighed. Compared with the control group, there were no significant differences in tumor volume, tumor weight, or tumor inhibition rate between the recombinant TAT-N-SH2 fusion protein and L1(TAT-SHP2-C-SH2) polypeptide-treated groups. These results suggest that the recombinant TAT-N-SH2 fusion protein and L1(TAT-SHP2-C-SH2) polypeptide cannot directly inhibit tumor cells, but exert their inhibitory effect on breast cancer tumor growth via immune cells (Figure 24, a-c, Table 8). [Table 9]

[0187] 5. Tumor-suppressive effects of recombinant TAT-N-SH2 fusion protein and L1 (TAT-SHP2-C-SH2) polypeptide on breast cancer xenograft tumors in PD-L1 knockout mice

[0188] Because the recombinant TAT-N-SH2 fusion protein and L1 (TAT-SHP2-C-SH2) polypeptide target the ITIM motif of the inhibitory receptor, they offer a broader therapeutic range than PD-1 monoclonal antibodies, which simply block the PD-1 inhibitory receptor. In theory, they could treat tumors that are resistant to PD-1 antibody therapy. Therefore, we constructed a PD-L1 stable knockout breast cancer EMT-6 cell line (PD-L1-KO-EMT6) and inoculated it into the mammary fat pad of mice. We intraperitoneally injected 5 μg / mouse of the recombinant TAT-N-SH2 fusion protein (N5) and the TAT-SHP2-C-SH2 polypeptide (C5), respectively, with 50 μg / mouse of αPD-1 antibody (αPD-1) as a control. The results showed that both N5 and C5 significantly inhibited the growth of PD-L1-KO-EMT6 tumors, but treatment with PD-1 antibody was ineffective (Figure 25, a-c, Table 9). [Table 10]

[0189] 6. Tumor suppression effects of L1, L2, D1, and D2 polypeptides on lung cancer in mice

[0190] Furthermore, the effects of L1, L2, D1, and D2 polypeptides on tumor growth in mice were evaluated using a constructed mouse model of Lewis lung carcinoma xenografts. L1, L2, D1, and D2 polypeptides were administered intraperitoneally at 5 μg / mouse. A control peptide (R peptide) was administered at 5 μg / mouse as a negative control, and saline was administered as a blank control (NC) once every other day for a total of eight doses. Results showed that L1, L2, D1, and D2 polypeptides all significantly inhibited tumor growth, with the D2 group showing the highest tumor inhibition rate (P<0.001), consistent with the results of the in vitro killing experiments (Figure 26, a-c, Table 10). [Table 11]

[0191] All documents related to the present invention are incorporated herein by reference as if each document were individually incorporated by reference. After reading the above content of the present invention, it should be understood that those skilled in the art can make various changes and modifications to the present invention, and that equivalents thereof are within the scope of the claims of the present invention. [Brief explanation of the drawings]

[0192] [Figure 1] FIG. 1 shows the results of identification by double enzyme digestion of pET-22b(+)TAT-SHP2-N-SH2. [Figure 2] FIG. 2 shows the sequencing results of the recombinant plasmid pET-22b(+)TAT-SHP2-N-SH2. [Figure 3] Figure 3 shows the expression status of the target protein detected by SDS-PAGE. 1 indicates the marker, 2 indicates uninduced, 3 indicates IPTG-induced 1, and 4 indicates IPTG-induced 2. The red arrow indicates the target protein. [Figure 4] Figure 4 shows the expression status of the target protein detected by SDS-PAGE. Note: 1 is uninduced, 2 is induced, 3 is bacterial digestion supernatant, and 4 is digestion precipitate. [Figure 5] Figure 5 shows the separation of the target protein from impurity proteins by cation exchange chromatography. Note: SP1-SP4 are elution peaks, of which SP4 is the peak containing the target protein. [Figure 6] Figure 6 shows the purification efficiency of TAT-SHP2-N-SH2 detected by SDS-PAGE. Note: 1 is the pass-through, 2 is SP1, 3 is SP2, 4 is SP3, and 5 is SP4 (target protein peak). [Figure 7] Figure 7 shows the identification of the target protein of SHP2-N-SH2 by Western blot. 1 is the recombinant bacteria not induced with IPTG, and 2 is the recombinant bacteria induced with 1 mM IPTG. [Figure 8] FIG. 8 shows the results of HPLC purification chromatography of the fusion protein. [Figure 9] 9 shows the results of HPLC purification chromatography of polypeptides, where A is the L1 polypeptide, B is the L2 polypeptide, C is the D1 polypeptide, and D is the D2 polypeptide. [Figure 10] 10 shows the molecular weights of the polypeptides detected by mass spectrometry: A is the L1 polypeptide, B is the L2 polypeptide, C is the D1 polypeptide, and D is the D2 polypeptide. [Figure 11] Figure 11 shows the localization of the fusion protein and polypeptide in T cells as detected by laser confocal microscopy. a) The ability of a control peptide (20 μg / mL) lacking the TAT membrane-permeable sequence to enter cells as observed by confocal microscopy; b) SHP2-C-SH2 with the TAT sequence; c) SHP2-N-SH2 with the TAT sequence; and c) the time of action at 0, 0.5, 4, 24, 72, 96, 120, and 144 h (25 μm (×60)). [Figure 12] Figure 12 shows the effects of recombinant TAT-N-SH2 fusion protein and L1 polypeptide on T cell proliferation. (a) CFSE assay for the effects of each treatment group on T cell proliferation. (d) Flow cytometry analysis showed that the T cell proliferation rate was statistically compared with the control group. p>0.05 indicates no statistical significance. [Figure 13] Figure 13 shows the effects of recombinant TAT-N-SH2 fusion protein and L1 polypeptide on T cell apoptosis. a) Annexin V-FITC assay was used to detect the effects of polypeptides on T cell apoptosis. Cells were resuspended and treated with SHP2-NC (untreated group) or SHP2-N-SH2 (80μg / mL) or TAT-SHP2-C-SH2 polypeptide (80μg / mL) for each group. Then, cells were cultured in EASY-T-stimulated culture plates (24-well plates were coated with 50ng PD-L1 antibody at 37℃ 3 hours before adding the cell mixture) for 3 days. Cells were harvested, stained with FITC and PI, and detected by flow cytometry. e) The percentage of apoptotic cells was calculated from the flow cytometry results and compared with the control group. *p<0.05 or **p<0.01. [Figure 14] Figure 14 shows the effects of fusion proteins and polypeptides on the phosphorylation levels of downstream single molecules mediated by T cell proliferation. a) TAT-SHP2-N-SH2 fusion protein treatment group; c) TAT-SHP2-C-SH2 polypeptide treatment group; b) and d) Western blot analysis of T cell-mediated phosphorylation of JNK, AKT, and ERK proteins using grayscale analysis with β-actin as an internal reference. Compared to the negative control, unpaired t-test showed **p<0.01, NS indicates statistically significant difference. [Figure 15] Figure 15 shows the detection of the cytokine IL-2 secreted by T cells. *p<0.05 and ****p<0.0001 compared to control by unpaired t-test. [Figure 16] FIG. 16 shows the results of detecting direct tumor killing activity. [Figure 17] Figure 17 shows the killing effect of T cells stimulated with the TAT-SHP2-N-SH2 fusion protein against different tumor cell lines. Compared to the untreated group, *p<0.05, **p<0.01, or ***p<0.001; p>0.05 is not statistically significant. [Figure 18] Figure 18 shows the killing effect of T cells stimulated with the TAT-SHP2-C-SH2 polypeptide against different tumor cell lines. Compared to the untreated group, *p<0.05, **p<0.01, or ***p<0.001; p>0.05 is not statistically significant. [Figure 19] Figure 19 shows the killing effect of T cells stimulated with L1, L2, D1, and D2 polypeptides on lung cancer cells H460. Compared to the control group, *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. Compared to the D2 group, #P<0.05, ##P<0.01, and ###P<0.001. [Figure 20] Figure 20 shows the tumor-suppressing effect of TAT-SHP2-N-SH2 fusion protein on colon cancer tumor-bearing mice. a) Tumor weights of mice treated with different doses after 30 days of anatomical analysis; b) Tumor size; *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 compared with the control group by unpaired t-test. [Figure 21] Figure 21 shows the tumor-suppressing effect of the TAT-SHP2-C-SH2 polypeptide on colon cancer tumor-bearing mice. a) Tumor weights of mice in different dose groups after 30 days of anatomical analysis; b) Tumor size; **p<0.01, ***p<0.001, and ****p<0.0001 compared with the control group by unpaired t-test. [Figure 22] Figure 22 shows the tumor-suppressing effects of the TAT-SHP2-N-SH2 fusion protein and the TAT-SHP2-C-SH2 polypeptide on breast cancer tumor-bearing mice. a) Tumor weights of mice from different treatment groups after 21 days of necropsy; b) Tumor size; *p<0.05, **p<0.01, ***p<0.001 compared with the control group by unpaired t-test. [Figure 23] Figure 23 shows the tumor-suppressing effect of recombinant TAT-N-SH2 fusion protein and L1 polypeptide on colon cancer-bearing nude mice. a) Tumor weight of mice after 30 days of necropsy; b) Tumor size; c) Tumor growth curve; compared with the control group by unpaired t-test; NS: not statistically significant. [Figure 24] Figure 24 shows the tumor-suppressing effect of recombinant TAT-N-SH2 fusion protein and L1 polypeptide on breast cancer in nude mice. a) Tumor weight of mice after 21 days of necropsy; b) Tumor size; c) Tumor growth curve; NS: no statistically significant difference. [Figure 25] Figure 25 shows the tumor-suppressive effects of recombinant TAT-N-SH2 fusion protein and L1 polypeptide on PD-L1 knockout breast cancer tumor-bearing mice. a) Tumor weight of mice after 23 days of necropsy; b) Tumor size; c) Tumor growth curve; *P<0.05, **P<0.01, ***P<0.001 compared to the control group by unpaired t-test. [Figure 26] Figure 26 shows the tumor-suppressing effect of L1, L2, D1, and D2 polypeptides on lung cancer in mice. a. Tumor weight of mice after 21 days of necropsy; b. Tumor size; c. Tumor growth curve; *P<0.05, **P<0.01, ***P<0.001 compared with the control group by unpaired t-test.

Claims

1. A fusion protein consisting of the amino acid sequence of any one of SEQ ID NOs: 5, 6, 7, and 8.

2. An isolated polynucleotide, characterized in that it encodes the fusion protein of claim 1.

3. A vector comprising the polynucleotide of claim 2.

4. A host cell characterized by containing the vector according to claim 3 or having the polynucleotide according to claim 2 integrated into its genome.

5. 10. A method for producing the fusion protein of claim 1, comprising the steps of:

5. Expressing the fusion protein by culturing the host cell of claim 4 under conditions suitable for expression; and / or isolating said fusion protein.

6. A pharmaceutical composition comprising the fusion protein of claim 1 and a pharmaceutically acceptable carrier thereof.

7. Use of the fusion protein of claim 1, the polynucleotide of claim 2, the vector of claim 3, or the host cell of claim 4, characterized in that they are used for the manufacture of a composition or formulation for treating or preventing tumors.

8. 8. The use according to claim 7, The use wherein the tumor is selected from the group consisting of breast cancer, colon cancer, lung cancer, colorectal cancer, gastric cancer, esophageal cancer, pancreatic adenocarcinoma, ovarian cancer, prostate cancer, kidney cancer, liver cancer, brain cancer, melanoma, multiple myeloma, leukemia, lymphoma, head and neck tumors, thyroid cancer, or a combination thereof.

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