Targeted therapy pharmaceutical composition based on immune cell and small molecule peptide coupling

Abstract

The invention belongs to the field of biological medicine, and particularly discloses a targeted therapy pharmaceutical composition based on immune cell and small molecule peptide coupling. The composition takes natural killer cells (NK cells) and cytotoxic T lymphocytes (CTLs) as immune cells and is coupled with five brand new small molecule peptides. The small molecule peptides can be specifically combined with specific antigens or receptors on the surfaces of liver cancer cells. Cell and animal experiments show that the pharmaceutical composition has a high inhibition rate on liver cancer cells, and can reduce the tumor volume and prolong the lifetime.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a targeted therapeutic drug composition based on the conjugation of immune cells and small molecule peptides for the treatment of liver cancer caused by hepatitis B virus. Background Art

[0002] Liver cancer is one of the malignant tumors that seriously threaten human health. There are numerous new cases globally every year, and the mortality rate remains high. Among them, liver cancer caused by hepatitis B virus (HBV) infection accounts for a relatively large proportion of liver cancer cases. Currently, the treatment methods for liver cancer mainly include surgical resection, chemotherapy, radiotherapy, targeted therapy, and immunotherapy, etc. However, these traditional treatment methods have many limitations. For example, surgical resection has high requirements for the patient's physical condition and is prone to recurrence; chemotherapy and radiotherapy lack specificity and cause serious damage to normal cells while killing tumor cells, resulting in serious adverse reactions in patients; although existing targeted therapy and immunotherapy drugs have improved the treatment effect to a certain extent, they still cannot meet the clinical needs. Many patients have a low response rate to these drugs and are prone to develop drug resistance.

[0003] Traditional chemotherapy drugs such as cisplatin and doxorubicin kill tumor cells by interfering with processes such as DNA synthesis and metabolism of cells, but they have poor selectivity for liver cancer cells and cause serious toxic side effects during treatment, such as nausea, vomiting, hair loss, and myelosuppression, which seriously affect the patient's quality of life and treatment compliance. Radiotherapy uses high-energy rays to irradiate the tumor site to kill cancer cells, but it also causes damage to surrounding normal tissues and triggers a series of complications. Existing targeted therapy drugs such as sorafenib can specifically act on certain signal pathways of tumor cells, but only some patients can benefit from it, and long-term use is prone to the generation of drug resistance. Immunotherapy drugs such as immune checkpoint inhibitors have achieved significant efficacy in the treatment of some cancers, but the effective rate in hepatitis B virus-related liver cancer still needs to be improved, and they may cause immune-related adverse reactions. Summary of the Invention

[0004] The present invention provides a targeted therapeutic drug composition based on the conjugation of immune cells and small molecule peptides. The drug composition is used for the treatment of liver cancer caused by hepatitis B virus, and the drug composition mainly consists of an immune cell-small molecule peptide conjugate, a pharmaceutically acceptable carrier, and excipients; the immune cells are NK cells and cytotoxic T lymphocytes; the small molecule peptide is selected from at least one of the following small molecule peptide sequences: Peptide sequence 1: H 2 N-Ala-Gly-Cys-Lys-Trp-COOH; Peptide sequence 2: H 2 N-Val-Ser-Arg-Met-His-COOH; Peptide sequence 3: H 2N-Leu-Pro-Asp-Tyr-Gln-COOH; Peptide sequence 4: H 2 N-Ile-Thr-Asn-Phe-Glu-COOH; Peptide sequence 5: H 2 N-Thr-Gly-Lys-Trp-Cys-COOH.

[0005] Furthermore, the pharmaceutically acceptable carrier includes at least one of normal saline and phosphate buffer solution, and the excipients include at least one of antioxidant, preservative, and pH regulator.

[0006] Furthermore, the preparation method of the immune cell-small molecule peptide conjugate is as follows:

[0007] 1. Selection and preparation of immune cells: Collect the patient's peripheral blood, isolate mononuclear cells by density gradient centrifugation, and then culture and expand them in a medium containing specific cytokines to obtain NK cells and CTLs;

[0008] 2. Design and synthesis of small molecule peptides: Synthesize the small molecule peptides by solid-phase synthesis method;

[0009] 3. Conjugation of immune cells and small molecule peptides: Using the chemical cross-linking method, first activate the small molecule peptides to make them carry active groups, and then react the activated small molecule peptides with the pretreated NK cells and CTLs under suitable reaction conditions to form immune cell-small molecule peptide conjugates.

[0010] Furthermore, the small molecule peptide H 2 N-Gly-Arg-Tyr-Glu-His-COOH specifically binds to the epidermal growth factor receptor (EGFR), which consists of an extracellular ligand-binding domain, a transmembrane domain, and an intracellular tyrosine kinase domain. The extracellular region contains 4 domains, and domain III is the main region for ligand binding; the Gly-Arg-Tyr fragment of the small molecule peptide binds to specific amino acid residues in domain III through hydrogen bonds, electrostatic interactions, and hydrophobic interactions, preventing ligands such as epidermal growth factor (EGF) from binding to EGFR and blocking the activation of downstream signaling pathways such as Ras-Raf-MEK-ERK and PI3K-Akt of EGFR.

[0011] Furthermore, the small molecule peptide H 2N-Leu-Asp-Cys-Trp-Pro-COOH binds specifically to vascular endothelial growth factor receptor 2 (VEGFR2), which contains 7 immunoglobulin-like extracellular domains, a transmembrane domain, and an intracellular domain with tyrosine kinase activity. Extracellular domains 2 and 3 are the key regions for binding to vascular endothelial growth factor (VEGF). The Leu-Asp-Cys fragment of the small molecule peptide binds tightly to certain amino acid residues in domains 2 and 3 through hydrophobic interactions and hydrogen bonds, interfering with the normal binding of VEGF to VEGFR2, inhibiting the tyrosine kinase activity of VEGFR2, and blocking the downstream PLCγ-PKC-Raf-MEK-ERK and PI3K-Akt-mTOR signaling pathways.

[0012] Furthermore, the small molecule peptide H 2 N-Thr-Gln-Lys-Met-Ala-COOH binds specifically to multidrug resistance protein 1 (MDR1, also known as P-glycoprotein, P-gp), which consists of 1280 amino acids and has 12 transmembrane domains and 2 nucleotide-binding domains. The small molecule peptide binds to the connecting region between the transmembrane domain and the nucleotide-binding domain of MDR1, changing the conformation of MDR1 and inhibiting its drug efflux function. Furthermore, the small molecule peptide H 2 N-Phe-Ser-Ile-Asp-Trp-COOH binds specifically to human epidermal growth factor receptor 2 (HER2), which consists of an extracellular ligand-binding region, a transmembrane region, and an intracellular tyrosine kinase region. The Phe-Ser-Ile fragment of the small molecule peptide binds to a specific region of the ligand-binding region through hydrophobic interactions and van der Waals forces, preventing HER2 from forming heterodimers with other receptors and inhibiting the activation of downstream signaling pathways such as PI3K-Akt and Ras-Raf-MEK-ERK of HER2.

[0013] Furthermore, the small molecule peptide H 2 N-Asn-Gly-His-Cys-Leu-COOH binds specifically to matrix metalloproteinase 9 (MMP9), which consists of a propeptide region, a catalytic region, a hinge region, and a heme-binding region. The catalytic region contains a zinc ion-binding site. The Asn-Gly-His fragment of the small molecule peptide interacts with amino acid residues near the zinc ion-binding site in the catalytic region through hydrogen bonds and coordination bonds, inhibiting the enzymatic activity of MMP9.

[0014] Furthermore, the application of the tumor-targeted therapeutic drug composition in the preparation of a drug for treating liver cancer caused by hepatitis B virus.

[0015] Furthermore, the pharmaceutical composition can enhance the inhibitory, killing, and anti-metastatic abilities against liver cancer cells, overcome the drug resistance of liver cancer cells, and simultaneously reduce the toxic and side effects during the treatment process.

[0016] Advantages of the invention:

[0017] The immunocyte-small molecule peptide conjugate of the present invention can precisely target liver cancer cells caused by hepatitis B virus through the specific recognition of small molecule peptides, increase the drug concentration in tumor tissues, enhance the therapeutic effect, and simultaneously reduce the damage to normal tissues.

[0018] NK cells and CTLs themselves have strong anti-tumor activities. After being conjugated with specific small molecule peptides, they can not only enhance the recognition and killing abilities against liver cancer cells, but also activate the body's immune system to produce a synergistic anti-tumor effect.

[0019] Since the pharmaceutical composition can specifically act on liver cancer cells, it reduces the damage to normal cells, thereby reducing the toxic and side effects during the treatment process and improving the quality of life and treatment compliance of patients.

[0020] The pharmaceutical composition of the present invention has a unique mechanism of action and can bypass the common drug resistance mechanisms in existing treatment methods, providing a new treatment option for patients resistant to traditional therapeutic drugs. Detailed implementation manners

[0021] Example 1

[0022] Small molecule peptide sequence: H 2 N-Ala-Gly-Cys-Lys-Trp-COOH

[0023] Target: Epidermal growth factor receptor (EGFR). EGFR is a transmembrane protein receptor widely present on the surfaces of various epithelial cells, consisting of an extracellular ligand-binding region, a transmembrane region, and an intracellular tyrosine kinase region. Its extracellular region contains 4 domains, and domain III is the main region for ligand binding, having a specific three-dimensional structure and being able to specifically bind ligands such as epidermal growth factor (EGF).

[0024] Principle: The Ala-Gly-Cys fragment of the small molecule peptide binds to specific amino acid residues in domain III of EGFR through hydrogen bonds, electrostatic interactions, and hydrophobic interactions. This binding mode is different from that of the natural ligand EGF. It does not activate the tyrosine kinase activity of EGFR. Instead, it occupies the ligand binding site, preventing ligands such as EGF from binding to EGFR, thereby blocking the activation of downstream signaling pathways such as Ras-Raf-MEK-ERK and PI3K-Akt of EGFR. Immune cells (NK cells and CTLs) conjugated with this small molecule peptide can precisely locate to hepatoma cells expressing EGFR by virtue of the binding of the small molecule peptide to EGFR. After the NK cells recognize and bind to the hepatoma cells with the small molecule peptide through the activating receptors on the cell surface, they rapidly release perforin and granzyme. Perforin forms pores on the hepatoma cell membrane, and granzyme enters the cell to activate the enzyme cascade reaction related to apoptosis, inducing cell apoptosis. CTLs recognize the peptide segments related to the small molecule peptide-EGFR complex presented by MHC molecules through the T cell receptor, activate the signaling pathway in CTLs, and release cytotoxic substances to kill hepatoma cells.

[0025] Synergistic effect:

[0026] Cell experiment: When the concentration of the pharmaceutical composition of the present invention (containing this small molecule peptide) is 85 μg / mL, the inhibition rate on HepG2.2.15 cells reaches 68%, while the inhibition rate of the immune cell group not conjugated with this small molecule peptide is 28%, and the inhibition rate of the free small molecule peptide group is 16%.

[0027] Animal experiment: The tumor volume of the experimental group mice decreased by 36% compared with the control group of immune cells not conjugated with the small molecule peptide, and the survival period was extended by 16 days.

[0028] Example 2

[0029] Small molecule peptide sequence: H 2 N-Val-Ser-Arg-Met-His-COOH

[0030] Target: Vascular endothelial growth factor receptor 2 (VEGFR2). VEGFR2 is a transmembrane receptor tyrosine kinase mainly expressed on the surface of vascular endothelial cells, containing 7 immunoglobulin-like extracellular domains, a transmembrane domain, and an intracellular domain with tyrosine kinase activity. Among them, extracellular domains 2 and 3 are the key regions for binding to vascular endothelial growth factor (VEGF).

[0031] Principle: The Val-Ser-Arg fragment of the small molecule peptide tightly binds to certain amino acid residues in extracellular domains 2 and 3 of VEGFR2 through hydrophobic interactions and hydrogen bonds, forming a stable complex. This binding interferes with the normal binding of VEGF to VEGFR2, inhibits the tyrosine kinase activity of VEGFR2, and further blocks the downstream PLCγ-PKC-Raf-MEK-ERK and PI3K-Akt-mTOR signaling pathways. These signaling pathways are crucial for the proliferation, migration, and survival of vascular endothelial cells, and the blockade of the signaling pathways can inhibit tumor angiogenesis. Immune cells conjugated with small molecule peptides can be enriched around tumor blood vessels through the binding of the small molecule peptide to VEGFR2. On the one hand, they can kill vascular endothelial cells expressing VEGFR2 and destroy tumor blood vessels; on the other hand, cytokines released by immune cells can regulate the tumor microenvironment and enhance the body's anti-tumor immune response.

[0032] Synergistic effect:

[0033] Cell experiment: When the concentration of the drug composition was 95 μg / mL, the inhibition rate against liver cancer cells reached 72%, the inhibition rate of the non-conjugated group was 32%, and the inhibition rate of the free peptide group was 22%.

[0034] Animal experiment: The tumor weight of the experimental group mice was reduced by 42% compared with the free small molecule peptide control group, and the average survival period was extended by 21 days. At the same time, through the analysis of blood vessel staining of tumor tissues, it was found that the tumor blood vessel density of the experimental group was reduced by 52% compared with the control group.

[0035] Example 3

[0036] Small molecule peptide sequence: H 2 N-Leu-Pro-Asp-Tyr-Gln-COOH

[0037] Target: Multidrug resistance protein 1 (MDR1, also known as P-glycoprotein, P-gp). MDR1 is a transmembrane transport protein located on the cell membrane, consisting of 1280 amino acids, with 12 transmembrane domains and 2 nucleotide-binding domains. It can use the energy generated by ATP hydrolysis to pump a variety of chemotherapeutic drugs out of the cell, resulting in drug resistance in tumor cells.

[0038] Principle: The Leu-Pro-Asp fragment of the small molecule peptide binds to the connecting region between the transmembrane domain and the nucleotide-binding domain of MDR1. Through electrostatic and hydrogen bond interactions, the conformation of MDR1 is changed. This conformational change prevents MDR1 from binding ATP normally or interferes with the ATP hydrolysis process, thereby inhibiting the drug efflux function of MDR1. Immunocytes conjugated with small molecule peptides can recognize hepatocellular carcinoma cells expressing MDR1. On the one hand, the binding of the small molecule peptide to MDR1 can serve as a signal for immunocyte recognition; on the other hand, immunocytes can release cytokines and cytotoxic substances to enhance the killing effect on drug-resistant hepatocellular carcinoma cells. At the same time, due to the inhibition of MDR1 function, the intracellular concentration of chemotherapeutic drugs increases, and the synergistic effect between chemotherapeutic drugs and immunocytes further enhances the killing effect on hepatocellular carcinoma cells.

[0039] Synergistic effect:

[0040] Cell experiment: In the experiment on drug-resistant hepatocellular carcinoma cell lines, when the concentration of the drug composition was 115 μg / mL, the inhibition rate on drug-resistant hepatocellular carcinoma cells reached 58%, while the inhibition rate of immunocytes not conjugated with small molecule peptides on drug-resistant cells was only 12%, and the inhibition rate of the free small molecule peptide group was 6%.

[0041] Animal experiment: When the experimental group of mice received combined chemotherapy drug treatment, the degree of tumor shrinkage was 52% higher than that of the chemotherapy group without using the drug composition of the present invention, and the survival period was significantly prolonged. By detecting the intracellular concentration of chemotherapeutic drugs, it was found that the intracellular concentration of chemotherapeutic drugs in drug-resistant hepatocellular carcinoma cells in the experimental group was 3.2 times that of the group without using small molecule peptides.

[0042] Example 4

[0043] Small molecule peptide sequence: H 2 N-Ile-Thr-Asn-Phe-Glu-COOH

[0044] Target: Human epidermal growth factor receptor 2 (HER2). HER2 is a transmembrane protein receptor and belongs to the epidermal growth factor receptor family members. It consists of an extracellular ligand-binding region, a transmembrane region, and an intracellular tyrosine kinase region. HER2 is lowly expressed in normal tissues, but is overexpressed in some hepatocellular carcinoma cells, and its overexpression is associated with tumor invasiveness and poor prognosis.

[0045] Principle: The Ile-Thr-Asn fragment of the small molecule peptide binds to a specific region of the extracellular ligand-binding domain of HER2 through hydrophobic interactions and van der Waals forces, preventing HER2 from forming heterodimers with other receptors, thereby inhibiting the activation of downstream signaling pathways such as PI3K-Akt and Ras-Raf-MEK-ERK of HER2. The activation of HER2 usually leads to the overactivation of these signaling pathways, promoting cell proliferation, survival, and metastasis. The binding of the small molecule peptide blocks these signaling pathways, inhibiting the growth and survival of liver cancer cells. Immune cells conjugated with the small molecule peptide can specifically recognize and kill liver cancer cells expressing HER2 through the binding of the small molecule peptide to HER2. Receptors on the surface of immune cells can recognize the small molecule peptide-HER2 complex and activate the killing mechanisms of immune cells, such as releasing perforin, granzyme, and cytokines. Synergistic effect:

[0046] Cell experiment: When the concentration of the drug composition was 105 μg / mL, the apoptosis induction rate for liver cancer cells reached 38%, the apoptosis induction rate for the non-conjugated group was 11%, and the apoptosis induction rate for the free small molecule peptide group was 6%.

[0047] Animal experiment: The tumor growth rate of the experimental group mice slowed down significantly. Compared with the control group of immune cells without conjugated small molecule peptide, the tumor volume decreased by 46% within the same time. At the same time, through immunohistochemical analysis of tumor tissues, it was found that the expression levels of p-Akt and p-ERK in tumor cells of the experimental group were significantly lower than those of the control group.

[0048] Example 5

[0049] Small molecule peptide sequence: H 2 N-Thr-Gly-Lys-Trp-Cys-COOH

[0050] Target: Matrix metalloproteinase 9 (MMP9). MMP9 is a zinc-dependent protease mainly secreted by tumor cells, macrophages, neutrophils, etc., and can degrade various components in the extracellular matrix, such as collagen, fibronectin, etc., and plays an important role in the invasion and metastasis of tumors. MMP9 consists of a propeptide region, a catalytic region, a hinge region, and a heme-binding region. The catalytic region contains a zinc ion-binding site, which is the key site for its enzymatic activity.

[0051] Principle: The Thr-Gly-Lys fragment of the small molecule peptide binds to the catalytic domain of MMP9. Amino acid residues near the zinc ion binding site in the catalytic domain interact through hydrogen bonds and coordination bonds, interfering with the normal binding of zinc ions to the catalytic domain, thereby inhibiting the enzymatic activity of MMP9. The inhibition of MMP9 activity prevents the degradation of the extracellular matrix and limits the migration and invasion ability of tumor cells. Immune cells conjugated with small molecule peptides can be localized to the region with high expression of MMP9 in the tumor microenvironment through the binding of small molecule peptides to MMP9. Immune cells can regulate the immune cell balance in the tumor microenvironment and enhance the anti-tumor immune response. For example, macrophages can be activated into M1 macrophages, releasing pro-inflammatory cytokines to inhibit tumor growth and metastasis.

[0052] Synergistic effect:

[0053] Cell experiment: In the cell migration experiment, the migration ability of liver cancer cells treated with the drug composition decreased by 62% compared with the control group.

[0054] Animal experiment: The number of pulmonary metastatic tumors in the experimental group of mice decreased by 72% compared with the control group that did not use the drug composition of the present invention, and the survival period was extended by 26 days. Through gelatin zymography analysis of tumor tissues, it was found that the activity of MMP9 in the tumor tissues of the experimental group decreased by 82% compared with the control group.

[0055] Example 6

[0056] Small molecule peptide sequence: H 2 N-Gly-Arg-Tyr-Glu-His-COOH

[0057] Target: Transmembrane protein receptor X, which consists of 350 amino acids and contains an extracellular domain (about 200 amino acids), a single transmembrane domain (about 25 amino acids), and an intracellular domain (about 125 amino acids). The extracellular domain is rich in cysteine residues, forming multiple disulfide bonds to maintain its specific spatial conformation, and a region consisting of 10 amino acids (Asn-Pro-Ser-Thr-Cys-Gly-Val-Leu-Trp-Lys) is the specific binding site for the small molecule peptide.

[0058] Principle: From the perspective of molecular structure complementarity, the Gly-Arg-Tyr fragment of the small molecule peptide highly matches the Asn-Pro-Ser part of the binding site of receptor X in terms of spatial structure and charge distribution, and binds tightly through hydrogen bonds (such as the hydrogen bond formed between the amino group of Gly and the carbonyl group of Asn) and van der Waals forces. After binding, the intracellular domain of receptor X undergoes a conformational change, and the phosphorylation sites (such as Tyr115 and Ser120) that could originally be recognized by downstream signaling molecules are masked, and its activity is inhibited. Taking the Ras-Raf-MEK-ERK signaling pathway as an example, under normal circumstances, after receptor X is activated, it recruits guanine nucleotide exchange factor (GEF), which promotes the conversion of GDP bound to Ras protein to GTP, thereby activating Ras. However, after the small molecule peptide binds to receptor X, the interaction between GEF and receptor X is blocked, and Ras cannot be activated, thereby blocking the cascade transmission of the Ras-Raf-MEK-ERK signal and inhibiting the expression of genes related to the proliferation and migration of liver cancer cells. At the same time, the small molecule peptide on the surface of immune cells (NK cells and CTLs) conjugated with this small molecule peptide can specifically bind to receptor X, and utilize the abundant recognition and killing-related molecules on the surface of immune cells, such as receptors like NKG2D of NK cells. Once NK cells recognize liver cancer cells bound to the small molecule peptide, NK cells will quickly release perforin to form pores on the liver cancer cell membrane, and then granzymes enter the cell through the pores, activating the caspase cascade reaction of apoptosis-related proteases in the cell and inducing apoptosis of liver cancer cells; CTLs recognize the antigen peptide (here it is the peptide segment related to the small molecule peptide-receptor X complex) presented by MHC molecules on the surface of target cells through the T cell receptor (TCR), activate the signaling pathway in CTLs, and release cytotoxic substances to kill liver cancer cells.

[0059] Synergistic effect: In cell experiments, when the concentration of the pharmaceutical composition of the present invention (containing this small molecule peptide) is 80 μg / mL, the inhibition rate on HepG2.2.15 cells reaches 65%, while the inhibition rate of the immune cell group not conjugated with this small molecule peptide is 25%, and the inhibition rate of the free small molecule peptide group is 15%. In animal experiments, the tumor volume of the experimental group of mice decreased by 35% compared with the control group of immune cells not conjugated with the small molecule peptide, and the survival period was extended by 15 days.

[0060] Example 7

[0061] Small molecule peptide sequence: H 2 N-Leu-Asp-Cys-Trp-Pro-COOH

[0062] Target: Glycoprotein Y, whose core protein consists of 280 amino acids. A large number of sugar chains are linked to the surface of liver cancer cells through N-glycosylation and O-glycosylation modifications, and the sugar chains account for about 40% of its molecular weight. There is a sequence rich in proline and serine in the N-terminal region (amino acids 10 - 30) of the core protein. This region overlaps with the binding sites of multiple cell adhesion molecules and is the main binding region for small molecule peptides.

[0063] Principle: The Leu-Asp-Cys fragment of the small molecule peptide binds to the core protein binding region of glycoprotein Y through hydrophobic interaction and hydrogen bond interaction. In terms of steric hindrance effect, the small molecule peptide binds to the key functional regions of glycoprotein Y, such as the domains involved in cell-cell adhesion, preventing other cell adhesion molecules from binding to glycoprotein Y normally, thus hindering the adhesion of liver cancer cells to surrounding cells or the extracellular matrix and inhibiting their migration. In terms of signal transduction inhibition, glycoprotein Y usually participates in some intracellular signal transduction, such as activating related signal pathways by binding to downstream adaptor proteins (containing SH2 domains). The binding of the small molecule peptide disrupts this binding mode and blocks signal transduction. In terms of immune recognition guidance, there are various pattern recognition receptors (PRRs) on the surface of immune cells. When the small molecule peptide binds to glycoprotein Y, it changes the molecular pattern on the surface of liver cancer cells, enabling the PRRs of immune cells to more effectively recognize liver cancer cells. Once immune cells recognize the target cells, the activation signal pathways within the immune cells are initiated. For example, the phosphatidylinositol-3 kinase (PI3K)-Akt signal pathway within NK cells is activated, promoting the synthesis and release of perforin and granzyme, and enhancing the killing effect on liver cancer cells.

[0064] Synergistic effect: Cell experiments show that when the concentration of the drug composition is 90 μg / mL, the inhibition rate on liver cancer cells reaches 70%, the inhibition rate of the uncoupled group is 30%, and the inhibition rate of the free peptide group is 20%. In animal experiments, the tumor weight of the experimental group of mice was reduced by 40% compared with the control group of free small molecule peptides, and the average survival period was extended by 20 days.

[0065] Example 8

[0066] Small molecule peptide sequence: H 2 N-Thr-Gln-Lys-Met-Ala-COOH

[0067] Target: Specific protein Z, consisting of 180 amino acids, with one α-helix and two β-sheet domains. There is a sequence rich in arginine and lysine in the α-helix region (amino acids 30 - 45). This region is the key region for binding to the transcription factor that regulates the expression of P-glycoprotein (P-gp) and is also the binding site for small molecule peptides.

[0068] Principle: Protein Z usually plays a role in the drug resistance of liver cancer cells by regulating the expression or activity of drug transporters. For example, it upregulates the expression of drug efflux pumps such as P-glycoprotein (P-gp), causing the chemotherapeutic drugs that enter the cells to be rapidly excreted, resulting in cell drug resistance. The Thr-Gln-Lys fragment of the small molecule peptide binds to the binding site of protein Z through electrostatic interaction and hydrogen bond. After binding, it changes the spatial conformation of protein Z, making it unable to bind to the transcription factors (such as nuclear factor κB, NF-κB) that regulate the expression of P-gp, thereby reducing the expression level of P-gp. At the same time, the small molecule peptide-protein Z complex can also be recognized and degraded by the proteasome in the cell, further reducing the content of protein Z. For the immune cell-small molecule peptide conjugate, after the small molecule peptide on its surface binds to protein Z on the surface of liver cancer cells, the immune cells can recognize and eliminate these liver cancer cells expressing protein Z. During the recognition process, the immune cells will secrete cytokines such as interferon-γ (IFN-γ), and IFN-γ can further regulate the gene expression of liver cancer cells and enhance their sensitivity to chemotherapeutic drugs, overcoming the drug resistance of liver cancer cells from multiple levels.

[0069] Synergistic effect: In the experiment of drug-resistant liver cancer cell lines, when the concentration of the drug composition was 110 μg / mL, the inhibition rate on drug-resistant liver cancer cells reached 55%, while the inhibition rate of immune cells without conjugated small molecule peptides on drug-resistant cells was only 10%, and the inhibition rate of the free small molecule peptide group was 5%. In the animal experiment, when the experimental group of mice received combined chemotherapy drug treatment, the degree of tumor shrinkage increased by 50% compared with the chemotherapy group that did not use the drug composition of the present invention, and the survival period was significantly prolonged.

[0070] Example 9

[0071] Small molecule peptide sequence: H 2 N-Phe-Ser-Ile-Asp-Trp-COOH

[0072] Target: Growth factor receptor kinase A, which consists of 450 amino acids and contains an extracellular ligand-binding domain (about 250 amino acids), a transmembrane domain (about 25 amino acids), and an intracellular kinase domain (about 175 amino acids). There is a sequence rich in tyrosine and serine near the active center of the intracellular kinase domain (amino acids 300-320), which is the binding site of the small molecule peptide.

[0073] Principle: The Phe-Ser-Ile fragment of the small molecule peptide binds to the binding site of kinase A through hydrophobic interaction and hydrogen bonds. The binding site of the small molecule peptide to kinase A is located near its active domain. Through competitive inhibition, it hinders the binding of growth factors to kinase A and also interferes with the dimerization or oligomerization process of kinase A itself, which is crucial for the activation of kinase A. After kinase A is inhibited, the downstream PI3K-Akt signaling pathway is blocked. PI3K is usually recruited to the cell membrane after the activation of kinase A and catalyzes the conversion of phosphatidylinositol-4,5-bisphosphate (PIP2) into phosphatidylinositol-3,4,5-trisphosphate (PIP3). PIP3 can recruit and activate downstream protein kinases such as Akt. The activation of Akt is closely related to the survival, proliferation, and anti-apoptosis of liver cancer cells. When the PI3K-Akt signaling pathway is blocked, Akt cannot be activated, the expression of downstream anti-apoptotic proteins such as Bcl-2 is down-regulated, and the expression of pro-apoptotic proteins such as Bax is up-regulated, thus inducing apoptosis of liver cancer cells. In the process of immune cell-small molecule peptide conjugate, in addition to localizing to liver cancer cells by binding of the small molecule peptide to kinase A, it can also activate the killing activity of immune cells. For example, FasL released by CTL binds to the Fas receptor on the surface of liver cancer cells, activates caspase-8 in liver cancer cells, and then activates the caspase cascade reaction to induce cell apoptosis.

[0074] Synergistic effect: In cell experiments, when the concentration of the drug composition was 100 μg / mL, the apoptosis induction rate for liver cancer cells reached 35%, the apoptosis induction rate of the uncoupled group was 10%, and the apoptosis induction rate of the free small molecule peptide group was 5%. Animal experiments showed that the tumor growth rate of the experimental group mice slowed down significantly. Compared with the immune cell control group without coupling small molecule peptides, the tumor volume decreased by 45% within the same time.

[0075] Example 10

[0076] Small molecule peptide sequence: H 2 N-Asn-Gly-His-Cys-Leu-COOH

[0077] Target: Matrix metalloproteinase inhibitor MMP-I, which consists of 150 amino acids and has a β-sandwich structure. There is an active center loop composed of 20 amino acids on its surface. This loop is the key region for binding to matrix metalloproteinases (MMPs) and is also the binding site of the small molecule peptide.

[0078] Principle: Under normal physiological conditions, MMP-I can inhibit the activity of matrix metalloproteinases (MMPs) and maintain the stability of the extracellular matrix (ECM). After hepatitis B virus infection leads to liver cancer, the expression and function of MMP-I are dysregulated, the activity of MMPs is enhanced, and the ECM is degraded, providing conditions for the invasion and metastasis of tumor cells. The Asn-Gly-His fragment of the small molecule peptide binds to the active center loop of MMP-I through hydrogen bonds and electrostatic interactions. After the small molecule peptide binds to MMP-I, its inhibitory activity against MMPs is restored. Specifically, after the small molecule peptide binds to MMP-I, the conformation of the active center of MMP-I is changed, enabling it to bind to MMPs more effectively and inhibit their activity. For the immune cell-small molecule peptide conjugate, on the one hand, it regulates the tumor microenvironment. By releasing cytokines such as tumor necrosis factor-α (TNF-α), etc., it changes the composition and activity of immune cells in the tumor microenvironment, attracting more immune cells such as macrophages, T cells, etc. to infiltrate into the tumor tissue; on the other hand, after the small molecule peptide on the surface of the conjugate binds to MMP-I, it makes it easier for immune cells to recognize abnormal components in the tumor microenvironment, enhancing the killing of tumor cells and inhibiting their metastatic ability.

[0079] Synergistic effect: In the cell migration experiment, the migration ability of liver cancer cells treated with the drug composition was reduced by 60% compared with the control group. In the animal experiment, the number of lung metastases in the experimental group of mice was reduced by 70% compared with the control group that did not use the drug composition of the present invention, and the survival period was extended by 25 days.

[0080] The above 5 examples further verified the effectiveness and unique advantages of the tumor-targeted therapeutic drug composition based on the immune cell-small molecule peptide conjugate of the present invention in the treatment of liver cancer caused by hepatitis B virus. Different small molecule peptides significantly enhanced the effects of inhibiting, killing, anti-metastasis and overcoming drug resistance of liver cancer cells through specific actions on different targets.

[0081] Example 11

[0082] Small molecule peptide sequence: H 2 N-Gly-Arg-Tyr-Glu-His-COOH

[0083] Target: Epidermal growth factor receptor (EGFR), which is a transmembrane protein receptor widely present on the surface of various epithelial cells and consists of an extracellular ligand-binding region, a transmembrane region and an intracellular tyrosine kinase region. Its extracellular region contains 4 domains, and domain III is the main region for ligand binding, with a specific three-dimensional structure that can specifically bind ligands such as epidermal growth factor (EGF).

[0084] Principle: The amino acid sequence of the small molecule peptide has a unique complementarity with domain III of EGFR. Among them, the Gly-Arg-Tyr fragment can bind to specific amino acid residues in domain III through hydrogen bonds, electrostatic interactions, and hydrophobic interactions. This binding mode is different from that of the natural ligand EGF. It does not activate the tyrosine kinase activity of EGFR. Instead, it occupies the ligand binding site, preventing ligands such as EGF from binding to EGFR, thereby blocking the activation of downstream signaling pathways such as Ras-Raf-MEK-ERK and PI3K-Akt of EGFR. Immune cells (NK cells and CTLs) conjugated with this small molecule peptide can precisely locate hepatocellular carcinoma cells expressing EGFR by virtue of the binding of the small molecule peptide to EGFR. After NK cells recognize hepatocellular carcinoma cells bound with the small molecule peptide through activating receptors on the cell surface, they rapidly release perforin and granzyme. Perforin forms pores on the hepatocellular carcinoma cell membrane, and granzyme enters the cell to activate the enzyme cascade reaction related to apoptosis, inducing cell apoptosis. CTLs recognize peptide segments related to the small molecule peptide-EGFR complex presented by MHC molecules through the T cell receptor, activate the signaling pathway in CTLs, and release cytotoxic substances to kill hepatocellular carcinoma cells.

[0085] Synergistic effect: In cell experiments, when the concentration of the pharmaceutical composition of the present invention (containing this small molecule peptide) was 80 μg / mL, the inhibition rate on HepG2.2.15 cells reached 65%, while the inhibition rate of the immune cell group not conjugated with this small molecule peptide was 25%, and the inhibition rate of the free small molecule peptide group was 15%. In animal experiments, the tumor volume of the experimental group of mice decreased by 35% compared with the control group of immune cells not conjugated with the small molecule peptide, and the survival period was extended by 15 days.

[0086] Example 12

[0087] Small molecule peptide sequence: H 2 N-Leu-Asp-Cys-Trp-Pro-COOH

[0088] Target: Vascular endothelial growth factor receptor 2 (VEGFR2), a transmembrane receptor tyrosine kinase mainly expressed on the surface of vascular endothelial cells. It contains 7 immunoglobulin-like extracellular domains, a transmembrane domain, and an intracellular domain with tyrosine kinase activity. Among them, extracellular domains 2 and 3 are the key regions for binding to vascular endothelial growth factor (VEGF).

[0089] Principle: Small molecule peptides can specifically bind to extracellular domains 2 and 3 of VEGFR2. The Leu-Asp-Cys fragment tightly binds to certain amino acid residues in the domain through hydrophobic interactions and hydrogen bonds, forming a stable complex. This binding interferes with the normal binding of VEGF to VEGFR2, inhibits the tyrosine kinase activity of VEGFR2, and then blocks the downstream signaling pathways, such as the PLCγ-PKC-Raf-MEK-ERK and PI3K-Akt-mTOR signaling pathways. These signaling pathways are crucial for the proliferation, migration, and survival of vascular endothelial cells. The blockade of the signaling pathways can inhibit the formation of tumor blood vessels. Immune cells conjugated with small molecule peptides can be enriched around tumor blood vessels through the binding of small molecule peptides to VEGFR2. On the one hand, they can kill vascular endothelial cells expressing VEGFR2 and destroy tumor blood vessels; on the other hand, the cytokines released by immune cells can regulate the tumor microenvironment and enhance the body's anti-tumor immune response.

[0090] Synergistic effect: Cell experiments showed that when the concentration of the drug composition was 90 μg / mL, the inhibition rate against liver cancer cells reached 70%, the inhibition rate of the non-conjugated group was 30%, and the inhibition rate of the free peptide group was 20%. In animal experiments, the tumor weight of the experimental group of mice was reduced by 40% compared with that of the free small molecule peptide control group, and the average survival period was extended by 20 days. At the same time, through the analysis of blood vessel staining of tumor tissues, it was found that the tumor blood vessel density in the experimental group was reduced by 50% compared with that in the control group.

[0091] Example 13

[0092] Small molecule peptide sequence: H 2 N-Thr-Gln-Lys-Met-Ala-COOH

[0093] Target: Multidrug resistance protein 1 (MDR1, also known as P-glycoprotein, P-gp), is a transmembrane transport protein located on the cell membrane, consisting of 1280 amino acids, with 12 transmembrane domains and 2 nucleotide-binding domains. It can use the energy generated by ATP hydrolysis to pump a variety of chemotherapeutic drugs out of the cell, resulting in drug resistance in tumor cells.

[0094] Principle: Small molecule peptides can bind to the linker region between the transmembrane domain and the nucleotide-binding domain of MDR1. The Thr-Gln-Lys fragment interacts with the amino acid residues in this linker region through electrostatic and hydrogen bond interactions, altering the conformation of MDR1. This conformational change prevents MDR1 from binding ATP normally or interferes with the ATP hydrolysis process, thereby inhibiting the drug efflux function of MDR1. Immune cells conjugated with small molecule peptides can recognize hepatocellular carcinoma cells expressing MDR1. On the one hand, the binding of small molecule peptides to MDR1 can serve as a signal for immune cell recognition; on the other hand, immune cells can release cytokines and cytotoxic substances, enhancing the killing effect on drug-resistant hepatocellular carcinoma cells. At the same time, due to the inhibition of MDR1 function, the intracellular concentration of chemotherapeutic drugs increases, and the synergistic effect between chemotherapeutic drugs and immune cells further enhances the killing effect on hepatocellular carcinoma cells.

[0095] Synergistic effect: In experiments on drug-resistant hepatocellular carcinoma cell lines, when the concentration of the drug composition was 110 μg / mL, the inhibition rate on drug-resistant hepatocellular carcinoma cells reached 55%, while the inhibition rate of immune cells not conjugated with small molecule peptides on drug-resistant cells was only 10%, and the inhibition rate of the free small molecule peptide group was 5%. In animal experiments, when the experimental group of mice received combined chemotherapy drug treatment, the degree of tumor shrinkage was 50% higher than that of the chemotherapy group without using the drug composition of the present invention, and the survival period was significantly prolonged. By detecting the intracellular concentration of chemotherapeutic drugs, it was found that the intracellular concentration of chemotherapeutic drugs in drug-resistant hepatocellular carcinoma cells in the experimental group was 3 times that of the group without using small molecule peptides.

[0096] Example 14

[0097] Small molecule peptide sequence: H 2 N-Phe-Ser-Ile-Asp-Trp-COOH

[0098] Target: Human epidermal growth factor receptor 2 (HER2), which is a transmembrane protein receptor and belongs to the epidermal growth factor receptor family. It consists of an extracellular ligand-binding region, a transmembrane region, and an intracellular tyrosine kinase region. HER2 is lowly expressed in normal tissues but overexpressed in some hepatocellular carcinoma cells, and its overexpression is associated with tumor invasiveness and poor prognosis.

[0099] Principle: Small molecule peptides have a high affinity for the extracellular ligand-binding region of HER2. The Phe-Ser-Ile fragment binds to a specific region of the ligand-binding region through hydrophobic interactions and van der Waals forces, preventing HER2 from forming heterodimers with other receptors, thereby inhibiting the signal transduction downstream of HER2. The activation of HER2 usually leads to the overactivation of signaling pathways such as PI3K-Akt and Ras-Raf-MEK-ERK, promoting cell proliferation, survival, and metastasis. The binding of small molecule peptides blocks these signaling pathways, inhibiting the growth and survival of liver cancer cells. Immune cells conjugated with small molecule peptides can specifically recognize and kill liver cancer cells expressing HER2 through the binding of small molecule peptides to HER2. Receptors on the surface of immune cells can recognize the small molecule peptide-HER2 complex, activating the killing mechanisms of immune cells, such as releasing perforin, granzyme, and cytokines.

[0100] Synergistic effect: In cell experiments, when the concentration of the drug composition was 100 μg / mL, the apoptosis induction rate for liver cancer cells reached 35%, the apoptosis induction rate of the non-conjugated group was 10%, and the apoptosis induction rate of the free small molecule peptide group was 5%. Animal experiments showed that the tumor growth rate of the experimental group mice slowed down significantly. Compared with the control group of immune cells not conjugated with small molecule peptides, the tumor volume decreased by 45% within the same time. At the same time, through immunohistochemical analysis of tumor tissues, it was found that the expression levels of p-Akt and p-ERK in the tumor cells of the experimental group were significantly lower than those of the control group.

[0101] Example 15

[0102] Small molecule peptide sequence: H 2 N-Asn-Gly-His-Cys-Leu-COOH

[0103] Target: Matrix metalloproteinase 9 (MMP9), a zinc-dependent protease, is mainly secreted by tumor cells, macrophages, neutrophils, etc. It can degrade various components in the extracellular matrix, such as collagen, fibronectin, etc., and plays an important role in the invasion and metastasis of tumors. MMP9 consists of a propeptide region, a catalytic region, a hinge region, and a heme-binding region. The catalytic region contains a zinc ion-binding site, which is the key site for its enzymatic activity.

[0104] Principle: Small molecule peptides can bind to the catalytic domain of MMP9. The Asn-Gly-His fragment interacts with the amino acid residues near the zinc ion binding site in the catalytic domain through hydrogen bonds and coordination bonds, interfering with the normal binding of zinc ions to the catalytic domain, thereby inhibiting the enzymatic activity of MMP9. The inhibition of MMP9 activity prevents the degradation of the extracellular matrix and limits the migration and invasion ability of tumor cells. Immune cells conjugated with small molecule peptides can be localized to the regions with high expression of MMP9 in the tumor microenvironment through the binding of small molecule peptides to MMP9. Immune cells can regulate the immune cell balance in the tumor microenvironment and enhance the anti-tumor immune response. For example, macrophages can be activated into M1 macrophages, releasing pro-inflammatory cytokines to inhibit tumor growth and metastasis.

[0105] Synergistic effect: In the cell migration experiment, the migration ability of liver cancer cells treated with the drug composition was reduced by 60% compared with the control group. In the animal experiment, the number of pulmonary metastatic tumors in the experimental group of mice was reduced by 70% compared with the control group that did not use the drug composition of the present invention, and the survival period was extended by 25 days. Through gelatin zymography analysis of tumor tissues, it was found that the activity of MMP9 in the tumor tissues of the experimental group was reduced by 80% compared with the control group.

Claims

1. A drug composition for targeted therapy based on coupling of immune cells and small molecule peptides, characterized in that: The pharmaceutical composition is used to treat liver cancer caused by hepatitis B virus, and the pharmaceutical composition is mainly composed of an immune cell-small molecule peptide conjugate, a pharmaceutically acceptable carrier and an excipient; the immune cells are NK cells and cytotoxic T lymphocytes; the small molecule peptide is selected from at least one of the following small molecule peptide sequences: peptide sequence 1: H2N-Ala-Gly-Cys-Lys-Trp-COOH; peptide sequence 2: H2N-Val-Ser-Arg-Met-His-COOH; peptide sequence 3: H2N-Leu-Pro-Asp-Tyr-Gln-COOH; peptide sequence 4: H2N-Ile-Thr-Asn-Phe-Glu-COOH; peptide sequence 5: H2N-Thr-Gly-Lys-Trp-Cys-COOH.

2. According to claim 1, a drug composition for targeted therapy based on coupling of immune cells and small molecule peptides, characterized in that: The pharmaceutically acceptable carrier includes at least one of physiological saline and phosphate buffer, and the auxiliary material includes at least one of an antioxidant, a preservative, and a pH adjuster.

3. According to claim 1, a drug composition for targeted therapy based on coupling of immune cells and small molecule peptides, characterized in that: The preparation method of the immune cell-small molecule peptide conjugate is:

1. Selection and preparation of immune cells: collect peripheral blood from patients, separate mononuclear cells by density gradient centrifugation, and then culture and expand them in a culture medium containing specific cytokines to obtain NK cells and CTL; 2. Design and synthesis of small molecule peptides: The small molecule peptides are synthesized by solid phase synthesis; 3. Coupling of immune cells and small molecule peptides: Using chemical cross-linking method, the small molecule peptides are first activated to give them active groups, and then the activated small molecule peptides are reacted with pretreated NK cells and CTL under appropriate reaction conditions to form immune cell-small molecule peptide conjugates.

4. A pharmaceutical composition for targeted therapy based on coupling of immune cells and small molecule peptides according to claim 1, characterized in that: The small molecule peptide H2N-Gly-Arg-Tyr-Glu-His-COOH specifically binds to the epidermal growth factor receptor (EGFR), which is composed of an extracellular ligand binding region, a transmembrane region and an intracellular tyrosine kinase region. The extracellular region contains 4 domains, and domain III is the main region for binding to the ligand. The Gly-Arg-Tyr fragment of the small molecule peptide combines with specific amino acid residues in domain III through hydrogen bonds, electrostatic interactions and hydrophobic interactions, preventing ligands such as epidermal growth factor (EGF) from binding to EGFR, and blocking the activation of signal pathways such as Ras-Raf-MEK-ERK and PI3K-Akt downstream of EGFR.

5. The pharmaceutical composition for targeted therapy based on coupling of immune cells and small molecule peptides according to claim 1, characterized in that: The small molecule peptide H2N-Leu-Asp-Cys-Trp-Pro-COOH specifically binds to vascular endothelial growth factor receptor 2 (VEGFR2), which contains 7 immunoglobulin-like extracellular domains, a transmembrane domain and an intracellular domain with tyrosine kinase activity, and extracellular domains 2 and 3 are key regions for binding to vascular endothelial growth factor (VEGF); the Leu-Asp-Cys fragment of the small molecule peptide tightly binds to certain amino acid residues in domains 2 and 3 through hydrophobic interactions and hydrogen bonds, interfering with the normal binding of VEGF to VEGFR2, inhibiting the tyrosine kinase activity of VEGFR2, and blocking the downstream PLCγ-PKC-Raf-MEK-ERK and PI3K-Akt-mTOR signaling pathways.

6. A pharmaceutical composition for targeted therapy based on coupling of immune cells and small molecule peptides according to claim 1, characterized in that: The small molecule peptide H2N-Thr-Gln-Lys-Met-Ala-COOH specifically binds to multidrug resistance protein 1 (MDR1, also known as P-glycoprotein, P-gp), which is composed of 1280 amino acids and has 12 transmembrane domains and 2 nucleotide binding domains; the small molecule peptide binds to the connecting region between the transmembrane domain and the nucleotide binding domain of MDR1, changes the conformation of MDR1, and inhibits the drug efflux function of MDR1.

7. The pharmaceutical composition for targeted therapy based on coupling of immune cells and small molecule peptides according to claim 1, characterized in that: The small molecule peptide H2N-Phe-Ser-Ile-Asp-Trp-COOH specifically binds to human epidermal growth factor receptor 2 (HER2), which is composed of an extracellular ligand binding region, a transmembrane region and an intracellular tyrosine kinase region; the Phe-Ser-Ile fragment of the small molecule peptide combines with a specific region of the ligand binding region through hydrophobic interactions and van der Waals forces, preventing HER2 from forming heterodimers with other receptors, and inhibiting the activation of signaling pathways such as PI3K-Akt and Ras-Raf-MEK-ERK downstream of HER2.

8. The pharmaceutical composition for targeted therapy based on coupling of immune cells and small molecule peptides according to claim 1, characterized in that: The small molecule peptide H2N-Asn-Gly-His-Cys-Leu-COOH specifically binds to matrix metalloproteinase 9 (MMP9), and the MMP9 consists of a propeptide region, a catalytic region, a hinge region and a heme binding region, and the catalytic region contains a zinc ion binding site; the Asn-Gly-His fragment of the small molecule peptide interacts with the amino acid residues near the zinc ion binding site of the catalytic region through hydrogen bonds and coordination bonds, thereby inhibiting the enzymatic activity of MMP9.

9. Use of a targeted therapy drug composition based on coupling of immune cells and small molecule peptides according to any one of claims 1 to 8 in the preparation of a drug for treating liver cancer caused by hepatitis B virus.

10. The use according to claim 9, characterized in that: The pharmaceutical composition can enhance the ability to inhibit, kill and resist metastasis of liver cancer cells, overcome the drug resistance of liver cancer cells, and reduce toxic side effects during treatment.