Small peptides and use thereof in preparation of medicament for treating cancer

Abstract

Small peptides, and a use thereof in the preparation of a medicament for treating cancer. Provided are a group of DSP small peptides, as well as full-length and truncated DDR1 proteins, all of which have the same active structure. Using in vivo tumor experiments, it is demonstrated that this group of DSP small peptides or truncated proteins can increase stem-like CD8+ memory T cells in bone marrow, and enhance the cytotoxic activity of CD8+ effector T cells, ultimately inhibiting the metastasis of lung cancer cells to brain tissue. In vitro, it is demonstrated that they have cytotoxic effects on tumor cells of eight types, including lung cancer, breast cancer, colorectal cancer, melanoma, prostate cancer, liver cancer, pancreatic cancer and bladder cancer.

Description

Small peptides and their use in the preparation of a medicament for treating cancer

[0001] This application claims priority to the application with the application date of September 13, 2024, the application number of “202411289533.6”, and the patent name of “Small peptides and their use in the preparation of a medicament for treating cancer”, the entire content of which is incorporated herein by reference. TECHNICAL FIELD

[0002] The present application relates to the field of pharmaceutical technology, in particular to small peptides and their use in the preparation of a medicament for treating cancer. BACKGROUND

[0003] Cancer is a major disease that threatens the life and health of contemporary human beings, and it is urgent to develop more effective anti-tumor drugs. At present, the research focus of anti-tumor drugs includes:

[0004] 1. Small peptide drugs: they can bind to membrane surface proteins, cause protein-protein interactions, or directly enter cells to mediate changes in intracellular signaling pathways, ultimately exerting anti-cancer function. The affinity, stability, and half-life of small peptides in the body are between small molecule drugs and large molecule drugs such as antibodies. The main advantages are: (1) Small peptides have stronger tissue permeability, can quickly enrich in target organs, and are easily internalized into cells. (2) At the same time, small peptides have lower toxicity and immunogenicity, and are less likely to cause liver and spinal cord damage. (3) From the perspective of drug manufacturing, small peptides are chemically synthesized, the process is simple, quality control is convenient, and the price is lower than that of large molecule drugs. (4) In addition, small peptides are more convenient for chemical modification, such as loading on nanoparticles or cell vesicles, or coupling with radioactive elements or other toxic substances to improve their efficacy.

[0005] 2. Cell-based tumor immunotherapy: including chimeric antigen receptor T cells (CAR-T), and adoptive cell transfer (ACT) of other immune cells. In 2017, CAR-T cells were approved by the FDA for the treatment of hematological malignancies. Clinical trials have shown that even in patients with advanced acute hematological leukemia, CAR-T cell therapy has achieved a complete recovery rate of up to 92%; and in the treatment of difficult large B-cell lymphoma, it has achieved an objective response rate (ORR) of 82%. Although CAR-T cells can effectively treat hematological malignancies, due to their limited ability to enter and survive in tumor tissue, they have not achieved ideal therapeutic effects in solid tumors.

[0006] 3. Antibody-based tumor immunotherapy: This includes targeted therapy using antibodies and their derivatives, such as anti-Her2 / neu monoclonal antibodies widely used in breast cancer, anti-EGFR monoclonal antibodies commonly used in colorectal and head and neck cancers, and the currently highly popular immune checkpoint inhibitors (ICIs). Although PD-1 / PD-L1 inhibitors have become routine treatments for more than 20 indications, they only produce an objective response rate of about 70% in patients with Hodgkin's lymphoma. They have not achieved good responses in other solid tumors such as esophageal cancer, head and neck cancer, melanoma, and non-small cell lung cancer (NSCLC). Furthermore, even patients who initially respond to ICIs often experience disease progression as treatment continues.

[0007] The tumor immunotherapy mentioned in points 2 and 3 above works by enhancing CD8. + T effector cells (CD8) + T, CD8 + T EFF This revolutionary cancer treatment method utilizes CD8+ cell-killing capabilities. Although tumor immunotherapy has been widely used in clinical cancer treatment with significant efficacy, its average effectiveness in treating solid tumors is only 20%. Increasing research has demonstrated that... + T EFF Compared to CD8 + Stem cell memory CD8 + T cells, CD8 + T SCM It possesses stronger and longer-lasting anti-tumor functions. However, currently, there is a lack of drugs targeting CD8. + T SCM Furthermore, research on its regulatory mechanisms is lacking, and there is a greater lack of specific targeting of CD8. + T SCM Clinical anti-tumor drugs.

[0008] Application content

[0009] Therefore, the technical problem to be solved by the present invention is to provide small peptides and their application in the preparation of drugs for treating cancer.

[0010] The small peptide provided by this invention is any one of the following I) to III):

[0011] I) It has the amino acid sequence shown in SEQ ID NO:1;

[0012] II) a small peptide having one or more amino acid residues substituted, deleted, added and / or replaced in the amino acid sequence of I);

[0013] III) a small peptide having at least 80% identity to the amino acid sequence of I) or II).

[0014] In the present application, the amino acid sequence of the small peptide is Ala-Arg-His-Ser-Arg-Leu-Glu-Ser-Ser-Asp-Gly-Asp-Gly-Ala (SEQ ID NO: 1). The small peptide is a core fragment obtained through screening, and experiments have shown that polypeptides containing the fragment can all have the effect of increasing CD8 + T SCM cell number and enhancing CD8 + T cell killing ability, so that polypeptides containing the core fragment as described above have the effect of treating tumors. In some cases, amino acid replacement of individual sites in the small peptide of the present application can change the spatial structure of the small peptide, enhance the stability or bioavailability of the small peptide. In the present application, the same identity of more than 80% means an identity of 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%.

[0015] In some embodiments, the structure of the small peptide is:

[0016] (X1-(X2)m-X3)n:

[0017] wherein:

[0018] X2 has an amino acid sequence as shown in SEQ ID NO: 1;

[0019] X1 consists of 0-15 amino acid residues;

[0020] X3 consists of 0-15 amino acid residues;

[0021] m and n are independently selected from integers from 1 to 5.

[0022] In the present application, X1, X2 and X3 are all amino acids or polypeptides, wherein X1 and X2 or X2 and X3 are connected by an amide bond.

[0023] In the present application, m is 1, 2 or 3; n is 1, 2 or 3. That is, the X2 fragment can be two repeats or three repeats, or the whole X1+(X2)m+X3 is two repeats or three repeats.

[0024] In some embodiments, the number of amino acid residues in X1 is 0, or is 1 to 15, for example, it is 1 to 5, or 5 to 10, or 10 to 15. Specifically, it is 1 to 3, 3 to 6, 7 to 9, 10 to 12, 13 to 15. More specifically, it is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In some specific embodiments, the amino acid residues in X1 include at least one of Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val. Preferably, the amino acid residues in X1 include at least one of Asp, Ser, Thr, Ala or Trp.

[0025] In specific embodiments of the present application, X1 is Ala, Thr-Ala, Ser-Thr-Ala, Asp-Ser-Thr-Ala, Ser-Asp-Ser-Thr-Ala, Trp-Ser-Asp-Ser-Thr-Ala, Ser-Trp-Ser-Asp-Ser-Thr-Ala, Ser-Ser-Trp-Ser-Asp-Ser-Thr-Ala, Ser-Ser-Ser-Trp-Ser-Asp-Ser-Thr-Ala or Ala-Ser-Ser-Ser-Trp-Ser-Asp-Ser-Thr-Ala.

[0026] In some embodiments, the number of amino acid residues in X3 is 0, or is 1 to 15, for example, it is 1 to 5, or 5 to 10, or 10 to 15. Specifically, it is 1 to 3, 3 to 6, 7 to 9, 10 to 12, 13 to 15. More specifically, it is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0027] In some specific embodiments, the amino acid residues in X3 include at least one of Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val. Preferably, the amino acid residues in X3 include at least one of Trp, Cys, Pro, Ala, Gly or Ser.

[0028] In some embodiments of the present application, X3 is Trp, Trp-Cys, Trp-Cys-Pro, Trp-Cys-Pro-Ala, Trp-Cys-Pro-Ala-Gly, Trp-Cys-Pro-Ala-Gly-Pro or Trp-Cys-Pro-Ala-Gly-Ser.

[0029] More specifically, the amino acid sequence of the small peptide of the present application is as shown in any one of SEQ ID NO: 2-6.

[0030] For example, the small peptide of the present application is:

[0031] In the present application, the small peptide is a linear peptide or a cyclic peptide.

[0032] In the cyclic peptide, the N-terminus and / or the C-terminus further comprises at least one Cys, and the two Cys in the small peptide form a disulfide bond.

[0033] In some embodiments, the cyclic peptide is:

[0034] Further, the present application also provides a truncated body of DDR1 protein comprising the small peptide as described above.

[0035] That is, the amino acid sequence of the truncated body of the present application comprises the amino acid sequence of the small peptide as described above. In some embodiments, the DDR1 protein is a human DDR1 protein or a mouse DDR1 protein.

[0036] In the present application, the amino acid sequence of the human DDR1 protein is as shown in SEQ ID NO: 19, and the amino acid sequence of the mouse DDR1 protein is as shown in SEQ ID NO: 20.

[0037] In some embodiments, the length of the truncated body of the present application is 30-913 aa, preferably 50-529 aa. For example, the length of the truncated body is 50-159 aa, or 417-529 aa. Specifically, the length of the truncated body is 50 aa, 60 aa, 69 aa, 70 aa, 80 aa, 159 aa, 417 aa, 444 aa, 527 aa or 529 aa.

[0038] In some embodiments, the truncated body has an amino acid sequence as shown in any one of SEQ ID NO: 7-14, or has an amino acid sequence as shown in any one of SEQ ID NO: 15-18.

[0039] The present application also provides some biomaterials comprising at least one of the following:

[0040] a) nucleic acid encoding said small peptide and / or said truncation;

[0041] b) expression unit comprising a promoter and said nucleic acid of a);

[0042] c) plasmid vector containing said nucleic acid of a) or containing said expression unit of b);

[0043] d) host having said nucleic acid of a) or said expression unit of b) integrated into its genome, or being transformed or transfected with said plasmid vector of c);

[0044] e) derivative comprising a modification and said small peptide and / or truncation; said modification is PEG modification;

[0045] f) fusion protein comprising an antibody Fc fragment and said small peptide and / or said truncation as described above.

[0046] Further, the present application also provides a method for preparing said small peptide and / or said truncation, which comprises:

[0047] synthesizing by chemical method,

[0048] or culturing said host as described above to obtain a product containing said small peptide and / or said truncation.

[0049] The chemical method described in the present application includes but is not limited to liquid phase synthesis, solid phase synthesis and solid-liquid phase synthesis of polypeptide. The steps of synthesis include individual coupling and / or fragment coupling, which are not limited in the present application. The activity of said small peptide or said truncation is not affected by the method of synthesis. The small peptide or truncation prepared by any method of preparing said small peptide or said truncation in the present application can have comparable physiological activity. In the fusion protein described in the present application, the antibody Fc fragment can be located at the C-terminal or N-terminal, which is not limited in the present application.

[0050] Further, the present application also provides the use of DDR1 protein, said small peptide and / or said truncation in the preparation of a medicament for preventing and treating tumor.

[0051] In the present application, said preventing and treating tumor includes: increasing the number of CD8 + T SCM cells in bone marrow, increasing the killing ability of CD8 + T cells in bone marrow, inhibiting tumor cell metastasis and / or prolonging the survival period of patients.

[0052] In the present application, the tumor is at least one of lung cancer, breast cancer, bladder cancer, colorectal cancer, liver cancer, melanoma, pancreatic cancer, prostate cancer, blood cancer, bone cancer, brain cancer, breast cancer, central nervous system cancer, cervical cancer, endometrial cancer, esophageal cancer, gallbladder cancer, gastrointestinal cancer, genital cancer, genitourinary tract cancer, head cancer, kidney cancer, laryngeal cancer, lung cancer, muscle tissue cancer, neck cancer, oral or nasal mucosa cancer, ovarian cancer, skin cancer, spleen cancer, small intestine cancer, large intestine cancer, stomach cancer, testicular cancer and / or thyroid cancer.

[0053] In the present application, the tumor is a human tumor or a mammalian tumor. The mammal is a primate, a canine, a feline and / or a rodent.

[0054] The present application also provides a medicament comprising the small peptide and / or the truncated protein.

[0055] As preferred, the medicament of the present application further comprises at least one of DDR1 protein, immune checkpoint inhibitor, CAR-T cell, CAR-NK cell, PD-1 / PD-L1 inhibitor, CTLA-4 inhibitor, paclitaxel, docetaxel, gemcitabine, vinorelbine, cisplatin, carboplatin, oxaliplatin, etoposide, epirubicin, doxorubicin, oxaliplatin, cyclophosphamide, fluorouracil, methotrexate, mitomycin, tamoxifen, gefitinib, imatinib, bevacizumab and rituximab.

[0056] The present application also provides a method for treating a tumor, comprising administering the medicament as described above.

[0057] The present application provides a group of small peptides and truncated DDR1 proteins, which all have the same active structure. It is proved by in vivo tumor experiments that this group of DSP small peptides or truncated proteins can increase the number of CD8 + Stem cell memory CD8 + T cells, CD8 + T SCM ) in bone marrow and enhance the killing effect of CD8 + Effector CD8 + T cells, CD8 + T EFF ) T cells, ultimately inhibiting the metastasis of lung cancer cells to brain tissue, and in vitro experiments have shown their killing effect on 8 types of tumor cells, including lung cancer, breast cancer, colorectal cancer, melanoma, prostate cancer, liver cancer, pancreatic cancer and bladder cancer. BRIEF DESCRIPTION OF DRAWINGS

[0058] 1Cyclized small peptide CDSP1 inhibits LLC-BrM lung cancer cell metastasis to brain tissue and prolongs the survival of mice in wild-type mice, wherein:

[0059] (A) Representative images of mice live imaging at day 16 after intraventricular injection of LLC-BrM-TL cells; (B) Statistics of lung cancer brain metastasis signals of mice treated with control and different concentrations of cyclized small peptide CDSP1; the signal scale range of live imaging is 0-5.3 (x 10 5 ph / s), n is the number of mice in each group; (C) Survival curves and median survival time of mice in each group;

[0060] Figure 2 shows that cyclized small peptide CDSP2 inhibits LLC-BrM lung cancer cell metastasis to brain tissue and prolongs the survival of mice in wild-type mice, wherein:

[0061] (A) Representative images of mice live imaging at day 16 after intraventricular injection of LLC-BrM-TL cells; (B) Statistics of lung cancer brain metastasis signals of mice treated with control and different concentrations of cyclized small peptide CDSP2; the signal scale range of live imaging is 0-5.3 (x 10 5 ph / s), n is the number of mice in each group; (C) Survival curves and median survival time of mice in each group;

[0062] Figure 3 shows that cyclized small peptide CDSP3 inhibits LLC-BrM lung cancer cell metastasis to brain tissue and prolongs the survival of mice in wild-type mice, wherein:

[0063] (A) Representative images of mice live imaging at day 16 after intraventricular injection of LLC-BrM-TL cells; (B) Statistics of lung cancer brain metastasis signals of mice treated with control and different concentrations of cyclized small peptide CDSP3; the signal scale range of live imaging is 0-5.3 (x 10 5 ph / s), n is the number of mice in each group; (C) Survival curves and median survival time of mice in each group;

[0064] Figure 4 shows that cyclized small peptide CDSP3 inhibits LLC-BrM lung cancer cell metastasis to brain tissue in nude mice, wherein:

[0065] (A) Schematic diagram of cyclized small peptide CDSP3 inhibiting LLC-BrM brain metastasis in nude mice: on day -1, 6 and 13, 1 x 10 4 CD8 + T SCM cells were intravenously injected; on day 0, 3 x 10 5LLC-BrM-TL cells; from day 1, control peptide or different concentrations of cyclic peptide CDSP3 (1, 3, 10 mg / kg) were injected into the orbital vein, and then injected every two days until the mice died; (B) representative pictures of the mice live imaging on day 16; the signal scale of the live imaging was 0-6.4 (x 10 5 ph / s), n was the number of mice in each group; (C) statistics of the brain metastasis signals of the mice administrated with control and different concentrations of cyclic peptide CDSP3;

[0066] Figure 5 shows that cyclic peptide CDSP3 inhibits LLC-BrM-OVA lung cancer cells to the brain tissue and prolongs the survival of mice in OT-I mice, wherein:

[0067] (A) schematic diagram of cyclic peptide CDSP3 inhibiting LLC-BrM-OVA brain metastasis in OT-I mice: on day -35, 1 x 10 6 LLC-BrM-OVA cells were injected into the heart chamber; on day 0, 1 x 10 6 LLC-BrM-OVA cells; from day -1, 10 mg / kg of control peptide or cyclic peptide CDSP3 was injected into the orbital vein, and then injected every two days until the mice died; (B) representative pictures of the mice live imaging on day 16; the signal scale of the live imaging was 0-5.3 (x 10 5 ph / s), n was the number of mice in each group; (C) statistics of the brain metastasis signals of the mice administrated with control peptide and cyclic peptide CDSP3; (D) survival curve and median survival time of the mice in each group;

[0068] Figure 6 shows that a group of DSP peptides can increase the number of CD8 + T SCM cells in the bone marrow of mice, wherein:

[0069] In the mice immunized with LLC-BrM tumor cell lysate, 10 mg / kg of control peptide or linear peptides LDSP1 (A), LDSP2 (B), LDSP3 (C), or cyclic peptides CDSP1 (D), CDSP2 (E), CDSP3 (F) was injected intravenously every other day; LLC-BrM tumor cells were injected into the heart chamber, and the number of CD8 + T SCM cells in the bone marrow of mice was detected after 1 and 3 days; the number of mice in each group was 3;

[0070] Figure 7 shows that cyclic peptide CDSP1 enhances the in vitro killing effect of CD8 + T cells on tumor cells, thereby treating lung cancer, breast cancer, bladder cancer, and colorectal cancer, wherein:

[0071] CD8 + T SCM cells were treated with 30, 100 or 300 nM cyclic peptide CDSP1 for 48 hours, and then separated from CD8 + T cells, which were co-cultured with lung cancer cells LLC (A), breast cancer cells 4T1 (B), bladder cancer cells RT4 (C), or colorectal cancer cells CT26 (D). After 72 hours, the apoptosis of tumor cells was detected. Annexin V + cells were apoptotic cells. The graph on the left shows the statistical analysis of the apoptosis level of tumor cells in each group, and the graph on the right shows the representative flow cytometry analysis.

[0072] Figure 8 shows that cyclic peptide CDSP1 enhances the in vitro killing effect of CD8 + T cells on tumor cells, and further treats hepatocarcinoma, melanoma, pancreatic cancer, and prostate cancer cells.

[0073] CD8 + T SCM cells were treated with 30, 100 or 300 nM cyclic peptide CDSP1 for 48 hours, and then separated from CD8 + T cells, which were co-cultured with hepatocarcinoma cells Hep3B (A), melanoma cells B16 (B), pancreatic cancer cells CFPAC-1 (C), or prostate cancer cells PC-3 (D). After 72 hours, the apoptosis of tumor cells was detected. Annexin V + cells were apoptotic cells. The graph on the left shows the statistical analysis of the apoptosis level of tumor cells in each group, and the graph on the right shows the representative flow cytometry analysis.

[0074] Figure 9 shows that cyclic peptide CDSP2 enhances the in vitro killing effect of CD8 + T cells on tumor cells, and further treats lung cancer, breast cancer, bladder cancer, and colorectal cancer.

[0075] CD8 + T SCM cells were treated with 30, 100 or 300 nM cyclic peptide CDSP2 for 48 hours, and then separated from CD8 + T cells, which were co-cultured with lung cancer cells LLC (A), breast cancer cells 4T1 (B), bladder cancer cells RT4 (C), or colorectal cancer cells CT26 (D). After 72 hours, the apoptosis of tumor cells was detected. Annexin V +The cells are apoptotic cells; the figure shows the statistical analysis of the apoptosis level of tumor cells in each group (left) and a representative flow cytometry analysis (right);

[0076] Figure 10 shows the enhancement of CD8 by the cyclized small peptide CDSP2. + The in vitro killing effect of T cells on tumor cells, thereby treating liver cancer, melanoma, pancreatic cancer, and prostate cancer cells:

[0077] In vitro treatment of CD8 cells specifically targeting tumor cells with the cyclic small peptide CDSP2 at concentrations of 30, 100, or 300 nM. + T SCM Cells, 48 ​​hours later, CD8 were isolated + T cells were co-cultured with Hep3B liver cancer cells (A), B16 melanoma cells (B), CFPAC-1 pancreatic cancer cells (C), or PC-3 prostate cancer cells (D) for 72 hours. Tumor cell apoptosis was then assessed. Flow cytometry analysis revealed the presence of Annexin V. + The cells are apoptotic cells; the figure shows the statistical analysis of the apoptosis level of tumor cells in each group (left) and a representative flow cytometry analysis (right);

[0078] Figure 11 shows the enhancement of CD8 by the cyclized small peptide CDSP3. + The in vitro killing effect of T cells on tumor cells can be used to treat lung cancer, breast cancer, bladder cancer, and colorectal cancer.

[0079] In vitro treatment of CD8 cells specifically targeting tumor cells with cyclic small peptide CDSP3 at concentrations of 30, 100, or 300 nM. + T SCM Cells, 48 ​​hours later, CD8 were isolated + T cells were co-cultured with lung cancer cells LLC (A), breast cancer cells 4T1 (B), bladder cancer cells RT4 (C), or colorectal cancer cells CT26 (D). After 72 hours, tumor cell apoptosis was detected. Flow cytometry analysis showed that Annexin V... + The cells are apoptotic cells; the figure shows the statistical analysis of the apoptosis level of tumor cells in each group (left) and a representative flow cytometry analysis (right);

[0080] Figure 12 shows the enhancement of CD8 by the cyclized small peptide CDSP3. + The in vitro killing effect of T cells on tumor cells, thereby treating liver cancer, melanoma, pancreatic cancer, and prostate cancer cells:

[0081] In vitro treatment of CD8 cells specifically targeting tumor cells with cyclic small peptide CDSP3 at concentrations of 30, 100, or 300 nM. + T SCMcells were isolated and CD8 + T cells were co-cultured with liver cancer cells Hep3B (A), melanoma cells B16 (B), pancreatic cancer cells CFPAC-1 (C), or prostate cancer cells PC-3 (D) for 72 hours, and the apoptosis of tumor cells was detected; Annexin V + The cells were apoptotic cells; the graph shows the statistics of the apoptosis level of tumor cells in each group (left), and the representative flow cytometry graph (right);

[0082] Figure 13 shows that cyclized small peptide CDSP4 enhances the in vitro killing effect of CD8 + T cells on tumor cells, and further treats lung cancer, breast cancer, bladder cancer, and colorectal cancer:

[0083] Specifically target tumor cells CD8 + T SCM cells were isolated and CD8 + T cells were co-cultured with lung cancer cells LLC (A), breast cancer cells 4T1 (B), bladder cancer cells RT4 (C), or colorectal cancer cells CT26 (D) for 72 hours, and the apoptosis of tumor cells was detected; Annexin V + The cells were apoptotic cells; the graph shows the statistics of the apoptosis level of tumor cells in each group (left), and the representative flow cytometry graph (right);

[0084] Figure 14 shows that cyclized small peptide CDSP4 enhances the in vitro killing effect of CD8 + T cells on tumor cells, and further treats liver cancer, melanoma, pancreatic cancer, and prostate cancer cells in vitro killing effect:

[0085] Specifically target tumor cells CD8 + T SCM cells were isolated and CD8 + T cells were co-cultured with liver cancer cells Hep3B (A), melanoma cells B16 (B), pancreatic cancer cells CFPAC-1 (C), or prostate cancer cells PC-3 (D) for 72 hours, and the apoptosis of tumor cells was detected; Annexin V + The cells were apoptotic cells; the graph shows the statistics of the apoptosis level of tumor cells in each group (left), and the representative flow cytometry graph (right);

[0086] Figure 15 shows that cyclized small peptide CDSP5 enhances the in vitro killing effect of CD8 +In vitro killing of tumor cells by T cells, and then treating lung cancer, breast cancer, bladder cancer, and colorectal cancer:

[0087] CD8 T cells that specifically target tumor cells were treated in vitro with 30, 100 or 300 nM concentration of cyclic small peptide CDSP5 + TSCM cells, 48 hours later, CD8 + T cells, and then co-cultured with lung cancer cells LLC (A), breast cancer cells 4T1 (B), bladder cancer cells RT4 (C), or colorectal cancer cells CT26 (D), 72 hours later, the apoptosis of tumor cells was detected; by flow cytometry analysis, Annexin V + cells were apoptotic cells; the graph was the statistics of the apoptosis level of tumor cells in each group (left), and the representative flow cytometry analysis graph (right);

[0088] Figure 16 shows that cyclic small peptide CDSP5 enhances the in vitro killing of tumor cells by CD8 + T cells, and then treating liver cancer, melanoma, pancreatic cancer, and prostate cancer cells:

[0089] CD8 T cells that specifically target tumor cells were treated in vitro with 30, 100 or 300 nM concentration of cyclic small peptide CDSP5 + T SCM cells, 48 hours later, CD8 + T cells, and then co-cultured with liver cancer cells Hep3B (A), melanoma cells B16 (B), pancreatic cancer cells CFPAC-1 (C), or prostate cancer cells PC-3 (D), 72 hours later, the apoptosis of tumor cells was detected; by flow cytometry analysis, Annexin V + cells were apoptotic cells; the graph was the statistics of the apoptosis level of tumor cells in each group (left), and the representative flow cytometry analysis graph (right);

[0090] Figure 17 shows that linear small peptide LDSP1 enhances the in vitro killing of tumor cells by CD8 + T cells, and then treating lung cancer, breast cancer, bladder cancer, and colorectal cancer:

[0091] CD8 T cells that specifically target tumor cells were treated in vitro with 30, 100 or 300 nM concentration of linear small peptide LDSP1 + T SCM cells, 48 hours later, CD8 + T cells, and then co-cultured with lung cancer cells LLC (A), breast cancer cells 4T1 (B), bladder cancer cells RT4 (C), or colorectal cancer cells CT26 (D), 72 hours later, the apoptosis of tumor cells was detected; by flow cytometry analysis, Annexin V+ Apoptotic cells; the graph is the statistics of the apoptosis level of tumor cells in each group (left), and the representative flow cytometry graph (right);

[0092] Figure 18 Linear small peptide LDSP1 enhances CD8 + T cells to kill tumor cells in vitro, and further treat hepatoma, melanoma, pancreatic cancer, and prostate cancer cells in vitro

[0093] Specifically target tumor cells CD8 + T SCM cells, 48 hours later, CD8 + T cells were isolated, and then co-cultured with hepatoma cells Hep3B (A), melanoma cells B16 (B), pancreatic cancer cells CFPAC-1 (C), or prostate cancer cells PC-3 (D), 72 hours later, the apoptosis of tumor cells was detected; by flow cytometry, Annexin V + Apoptotic cells; the graph is the statistics of the apoptosis level of tumor cells in each group (left), and the representative flow cytometry graph (right); make

[0094] Figure 19 Linear small peptide LDSP2 enhances CD8 + T cells to kill tumor cells in vitro, and further treat lung cancer, breast cancer, bladder cancer, and colorectal cancer, wherein:

[0095] Specifically target tumor cells CD8 + T SCM cells, 48 hours later, CD8+ T cells were isolated, and then co-cultured with lung cancer cells LLC (A), breast cancer cells 4T1 (B), bladder cancer cells RT4 (C), or colorectal cancer cells CT26 (D), 72 hours later, the apoptosis of tumor cells was detected; by flow cytometry, Annexin V + Apoptotic cells; the graph is the statistics of the apoptosis level of tumor cells in each group (left), and the representative flow cytometry graph (right);

[0096] Figure 20 Linear small peptide LDSP2 enhances CD8 + T cells to kill tumor cells in vitro, and further treat hepatoma, melanoma, pancreatic cancer, and prostate cancer cells in vitro

[0097] Specifically target tumor cells CD8 + T SCMcells were isolated and CD8 + T cells were co-cultured with hepatoma cells Hep3B (A), melanoma cells B16 (B), pancreatic cancer cells CFPAC-1 (C), or prostate cancer cells PC-3 (D), and the apoptosis of tumor cells was detected after 72 hours; Annexin V + The cells were apoptotic cells; the graph shows the statistics of the apoptosis level of tumor cells in each group (left), and the representative flow cytometry graph (right);

[0098] Figure 21 Linear small peptide LDSP3 enhances the in vitro killing effect of CD8 + T cells on tumor cells, thereby treating lung cancer, breast cancer, bladder cancer, and colorectal cancer:

[0099] Specifically targeted tumor cells CD8 + T SCM cells were isolated and CD8 + T cells were co-cultured with lung cancer cells LLC (A), breast cancer cells 4T1 (B), bladder cancer cells RT4 (C), or colorectal cancer cells CT26 (D), and the apoptosis of tumor cells was detected after 72 hours; Annexin V + The cells were apoptotic cells; the graph shows the statistics of the apoptosis level of tumor cells in each group (left), and the representative flow cytometry graph (right);

[0100] Figure 22 Linear small peptide LDSP3 enhances the in vitro killing effect of CD8 + T cells on tumor cells, thereby treating hepatoma, melanoma, pancreatic cancer, and prostate cancer cells in vitro:

[0101] Specifically targeted tumor cells CD8 + T SCM cells were isolated and CD8 + T cells were co-cultured with hepatoma cells Hep3B (A), melanoma cells B16 (B), pancreatic cancer cells CFPAC-1 (C), or prostate cancer cells PC-3 (D), and the apoptosis of tumor cells was detected after 72 hours; Annexin V + The cells were apoptotic cells; the graph shows the statistics of the apoptosis level of tumor cells in each group (left), and the representative flow cytometry graph (right);

[0102] Figure 23 Linear small peptide LDSP4 enhances the in vitro killing effect of CD8 +The in vitro killing effect of T cells on tumor cells can be used to treat lung cancer, breast cancer, bladder cancer, and colorectal cancer.

[0103] In vitro treatment of CD8 cells specifically targeting tumor cells with the linear small peptide LDSP4 at concentrations of 30, 100, or 300 nM. + T SCM Cells, 48 ​​hours later, CD8 were isolated + T cells were co-cultured with lung cancer cells LLC (A), breast cancer cells 4T1 (B), bladder cancer cells RT4 (C), or colorectal cancer cells CT26 (D). After 72 hours, tumor cell apoptosis was detected. Flow cytometry analysis showed that Annexin V... + The cells are apoptotic cells; the figure shows the statistical analysis of the apoptosis level of tumor cells in each group (left) and a representative flow cytometry analysis (right);

[0104] Figure 24. Linear small peptide LDSP4 enhances CD8 + The in vitro killing effect of T cells on tumor cells, thereby treating liver cancer, melanoma, pancreatic cancer, and prostate cancer cells:

[0105] In vitro treatment of CD8 cells specifically targeting tumor cells with the linear small peptide LDSP4 at concentrations of 30, 100, or 300 nM. + T SCM Cells, 48 ​​hours later, CD8 were isolated + T cells were co-cultured with Hep3B liver cancer cells (A), B16 melanoma cells (B), CFPAC-1 pancreatic cancer cells (C), or PC-3 prostate cancer cells (D) for 72 hours. Tumor cell apoptosis was then assessed. Flow cytometry analysis revealed the presence of Annexin V. + The cells are apoptotic cells; the figure shows the statistical analysis of the apoptosis level of tumor cells in each group (left) and a representative flow cytometry analysis (right);

[0106] Figure 25. Linear small peptide LDSP5 enhances CD8 + The in vitro killing effect of T cells on tumor cells can be used to treat lung cancer, breast cancer, bladder cancer, and colorectal cancer.

[0107] In vitro treatment of CD8 cells specifically targeting tumor cells with the linear small peptide LDSP5 at concentrations of 30, 100, or 300 nM. + T SCM Cells, 48 ​​hours later, CD8 were isolated +T cells, and then co-cultured with lung cancer cells LLC (A), breast cancer cells 4T1 (B), bladder cancer cells RT4 (C), or colorectal cancer cells CT26 (D), after 72 hours, the apoptosis of tumor cells was detected; by flow cytometry analysis, Annexin V + positive cells were apoptotic cells; the graph was the statistics of the apoptosis level of tumor cells in each group (left), and the representative flow cytometry analysis graph (right);

[0108] Figure 26 Linear small peptide LDSP5 enhances the in vitro killing effect of CD8 + T cells on tumor cells, and then treat hepatocarcinoma, melanoma, pancreatic cancer, and prostate cancer cells in vitro:

[0109] Specifically target tumor cells CD8 + T SCM cells in vitro, after 48 hours, CD8 + T cells, and then co-cultured with hepatocarcinoma cells Hep3B (A), melanoma cells B16 (B), pancreatic cancer cells CFPAC-1 (C), or prostate cancer cells PC-3 (D), after 72 hours, the apoptosis of tumor cells was detected; by flow cytometry analysis, Annexin V + positive cells were apoptotic cells; the graph was the statistics of the apoptosis level of tumor cells in each group (left), and the representative flow cytometry analysis graph (right);

[0110] Figure 27 Overexpression of full-length DDR1 or C-terminal deletion and truncation of DDR1 529*, DDR1 444* and the like can inhibit lung cancer cell metastasis in nude mice with adoptive CD8 + T SCM cells:

[0111] (A) Western blotting (left) and flow cytometry (right) were used to detect the expression of full-length DDR1 and C-terminal deletion and truncation of DDR1 529*, DDR1 444* and the like in H460 human lung cancer cell stable lines; (B) On day -1, 1×10 6 CD8 + T SCM cells, 3×10 5 H460 tumor cells were injected into the ventricle on day 0, and then the tumor metastasis signal was continuously monitored; the metastasis signal statistics of mice in each group, and the representative pictures of in vivo imaging on day 0 and day 17; the signal scale range of in vivo imaging was 0-3.1 (×10 4 ph / s), n is the number of mice in each group;

[0112] Figure 28 Overexpression of full-length DDR1 or C-terminal deletion and truncation of DDR1 529*, DDR1 444* and the like can inhibit lung cancer cell metastasis in nude mice with adoptive CD8 +T SCM Overexpression of human full-length DDR1 or C-terminal deletion DDR1 417* in nude mice can inhibit lung cancer cell metastasis:

[0113] (A) Western blotting (left) detected the expression of human full-length DDR1 and C-terminal deletion DDR1 417* in H460 human lung cancer cell stable lines; (B) On day -1, 1 x 10 6 CD8 + T SCM cells, 3 x 10 5 H460 tumor cells, and then continuously monitor tumor metastasis signals; metastasis signal statistics of mice in each group, and representative pictures of in vivo imaging on day 0 and day 17; the signal scale range of in vivo imaging is 0-4.47 (x 10 7 ph / s), n is the number of mice in each group;

[0114] Figure 29 Overexpression of mouse full-length DDR1 or C-terminal deletion DDR1 527* in wild-type mice can inhibit lung cancer cell metastasis:

[0115] (A) Western blotting detected the expression of mouse full-length DDR1 and C-terminal deletion DDR1 527* in LLC-BrM mouse lung cancer cell stable lines; (B) On day -35, 1 x 10 6 LLC-BrM cell lysates were used to immunize mice; on day 0, 1 x 10 6 Control or DDR1-overexpressing LLC-BrM live cells, and then continuously monitor tumor metastasis signals; the statistical graph is the metastasis signal statistics of mice in each group (left), and the representative pictures of in vivo imaging on day 19; the signal scale range of in vivo imaging is 0-1.5 (x 10 4 ph / s), n is the number of mice in each group; (C) Survival curve and median survival time of mice in each group;

[0116] Figure 30 Different lengths of human DDR1 protein enhance the in vitro killing effect of CD8 + T cells on tumor cells, thereby treating cancer:

[0117] (A) Western blotting (left) detected the expression of DDR1 417* (extracellular region of DDR1) in the conditioned medium (CM) of 293FT cells; CD8 + T SCMCells were then co-cultured with H460 human lung cancer cells, and the apoptosis of H460 cells was detected (right); (B) Dot blotting (left) was used to detect the expression of 29-108aa (DSN), 109-187aa (DSC), and 29-187aa (DS domain) in the conditioned medium (CM) of 293FT cells; CD8 + T SCM Cells were then co-cultured with H460 human lung cancer cells, and the apoptosis of H460 cells was detected (right); (C) Dot blotting (left) was used to detect the expression of 29-97aa (DSNN1), 29-88aa (DSNN2), 29-78 (DSNN3), and 29-187aa (DS domain) in the conditioned medium (CM) of 293FT cells; CD8 + T SCM Cells were then co-cultured with H460 human lung cancer cells, and the apoptosis of H460 cells was detected (right); (D) Dot blotting (left) was used to detect the expression of 39-108aa (DSNC1), 49-108aa (DSNC2), and 59-108aa (DSNC3), and 29-187aa (DS domain) in the conditioned medium (CM) of 293FT cells; CD8 + T SCM Cells were then co-cultured with H460 human lung cancer cells, and the apoptosis of H460 cells was detected (right). DETAILED DESCRIPTION

[0118] The present application provides small peptides and their use in the preparation of medicaments for treating cancer. Those skilled in the art can refer to the content of the present application and appropriately modify the process parameters to achieve the desired results. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are considered to be included in the present application. The methods and uses of the present application have been described by preferred embodiments, and those skilled in the art can obviously modify or appropriately change and combine the methods and uses described herein without departing from the content, spirit and scope of the present application, to achieve and apply the present application technology.

[0119] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have meanings that are commonly understood by those of ordinary skill in the art.

[0120] Also, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Specifically, as used herein and in the appended claims, the singular forms "a" (or "an"), "the" and "said" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes a single compound or a combination of two or more compounds.

[0121] In this application, "include," "includes" and "including" are used interchangeably and mean the inclusion of an element, step, operation, or the like to the extent that the context in which they are used permits.

[0122] In this application, "and / or," where used, means "and", "or", "and / or" the inclusive or, "and" the exclusive-or, and "and" the sense used in arithmetic.

[0123] In this application, "amino acid" refers to an organic compound containing both an amino group and a carboxyl group. Amino acids referred to in this application include D-type amino acids and / or L-type amino acids. References to amino acids and their abbreviations herein include:

[0124] Alanine is abbreviated as Ala or A. Valine is abbreviated as Val or V. Leucine is abbreviated as Leu or L. Isoleucine is abbreviated as Ile or I. Phenylalanine is abbreviated as Phe or F. Tryptophan is abbreviated as Trp or W. Methionine is abbreviated as Met or M. Proline is abbreviated as Pro or P. Glycine is abbreviated as Gly or G. Serine is abbreviated as Ser or S. Threonine is abbreviated as Thr or T. Cysteine is abbreviated as Cys or C. Tyrosine is abbreviated as Tyr or Y. Asparagine is abbreviated as Asn or N. Glutamine is abbreviated as Gln or Z or Q. Lysine is abbreviated as Lys or K. Arginine is abbreviated as Arg or R. Aspartic acid is abbreviated as Asp or written as Asx or D. Glutamic acid is abbreviated as Glu or written as Gls or E.

[0125] In the present application, "DDR1 protein" and / or "DDR1" refer to single-pass transmembrane receptor Discoidin Domain Receptor 1, which have the same meaning and can be used interchangeably. Its extracellular domain Discoidin is also referred to as DS in the present application.

[0126] In the present application, "peptide" refers to a compound formed by two or more amino acid molecules linked by peptide bonds (i.e. amide bonds,— CO— NH—). These amino acids can be the same or different. In the present application, "small peptide" and "polypeptide" have the same meaning and can be used interchangeably.

[0127] In the present application, "truncation" or truncated protein, particularly truncated DDR1 protein, refers to a protein produced by removing one or more amino acids from the N-terminus and / or C-terminus of wild-type DDR1 protein, wherein the wild-type DDR1 protein is from human or mouse.

[0128] In the present application, the number of amino acids in a truncation is counted from the N-terminus, for example, DDR1 amino acids 55-72 refers to a polypeptide consisting of 18 amino acids at positions 55-72 of the N-terminus of wild-type DDR1 protein.

[0129] In the present application, "identity" can be calculated by determining the percent "identity" of two amino acid sequences or two nucleic acid sequences by aligning the sequences for optimal comparison purposes (e.g., introducing gaps into one or both of the first and second amino acid sequences or nucleic acid sequences, or adding nucleotides or amino acids to either sequence, for optimal alignment). Subsequently, the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0130] In the present invention, "treatment" refers to a surgical or therapeutic treatment whose purpose is to prevent, slow down (reduce) or arrest an undesired physiological change or pathological condition, such as cancer and tumors, in a subject. Beneficial or desired results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. A subject in need of treatment includes a subject who has a condition or disease, a subject who is predisposed to a condition or disease, or a subject who is intended to prevent a condition or disease. When referring to terms such as slowing down, reducing, diminishing, palliating, alleviating, etc., the meaning also includes elimination, disappearance, non-occurrence, etc.

[0131] In the present invention, "cancer" refers to or describes the physiological condition that is typically characterized by unregulated cell growth in a mammal. This definition includes both benign and malignant cancers.

[0132] In the present invention, "tumor" or "neoplasm" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when referred to herein.

[0133] The materials used in the present invention are all commercially available and can be purchased in the market.

[0134] I. Amino acid sequences of small peptides involved in the present invention:

[0135] 1. Linear DSP small peptides (LDSP), a total of 5:

[0136] LDSP1 (18 aa): corresponding to amino acids 55-72 of human DDR1

[0137] LDSP2 (19 aa): corresponding to amino acids 59-77 of human DDR1

[0138] LDSP3 (20 aa): corresponding to amino acids 60-79 of murine DDR1

[0139] LDSP4 (20 aa): corresponding to amino acids 59-78 of human DDR1

[0140] LDSP5 (30 aa): corresponding to amino acids 49-78 of human DDR1

[0141] 2. Cyclic DS Peptide (CDSP) corresponding to the linear DSP small peptide, totally 5:

[0142] CDSP1: corresponding to the 55-72 amino acids of human DDR1, with an additional Cys at the N-terminus and C-terminus, respectively, and the two Cys are connected by disulfide bond:

[0143] CDSP2: corresponding to the 59-77 amino acids of human DDR1, with an additional Cys at the N-terminus, which is connected to another Cys in the sequence by disulfide bond:

[0144] CDSP3: corresponding to the 60-79 amino acids of mouse DDR1, with an additional Cys at the N-terminus, which is connected to another Cys in the sequence by disulfide bond:

[0145] CDSP4: corresponding to the 59-78 amino acids of human DDR1, with an additional Cys at the N-terminus, which is connected to another Cys in the sequence by disulfide bond:

[0146] CDSP5: corresponding to the 49-78 amino acids of human DDR1, with an additional Cys at the N-terminus, which is connected to another Cys in the sequence by disulfide bond:

[0147] 3. Human full-length DDR1: 1-913aa

[0148] The sequence is shown in SEQ ID NO: 19.

[0149] 4. Human DDR1 529* (C-terminal deletion and truncation): 1-529aa

[0150] The sequence is shown in SEQ ID NO: 15.

[0151] 5. Human DDR1 444* (C-terminal deletion and truncation): 1-444aa

[0152] The sequence is shown in SEQ ID NO: 16.

[0153] 6. Human DDR1 417* (C-terminal deletion and truncation): 1-417aa

[0154] The sequence is shown in SEQ ID NO: 17.

[0155] 7. Mouse full-length DDR1: 1-911aa:

[0156] The sequence is shown as SEQ ID NO: 20.

[0157] 8. Mouse DDR1 527* (C-terminal deletion truncation): 1-527 aa

[0158] The sequence is shown as SEQ ID NO: 18.

[0159] 9. Purified protein of 29-187 aa of human DDR1 (DS domain):

[0160] The sequence is shown as SEQ ID NO: 7.

[0161] 10. Purified protein of 29-108 aa of human DDR1 (DSN):

[0162] The sequence is shown as SEQ ID NO: 8.

[0163] 11. Purified protein of 29-97 aa of human DDR1 (DSNN1):

[0164] The sequence is shown as SEQ ID NO: 9.

[0165] 12. Purified protein of 29-88 aa of human DDR1 (DSNN2):

[0166] The sequence is shown as SEQ ID NO: 10.

[0167] 13. Purified protein of 29-78 aa of human DDR1 (DSNN3):

[0168] The sequence is shown as SEQ ID NO: 11.

[0169] 14. Purified protein of 39-108 aa of human DDR1 (DSNC1):

[0170] The sequence is shown as SEQ ID NO: 12.

[0171] 15. Purified protein of 49-108 aa of human DDR1 (DSNC2):

[0172] The sequence is shown as SEQ ID NO: 13.

[0173] 16. Purified protein of 59-108 aa of human DDR1 (DSNC3):

[0174] The sequence is shown as SEQ ID NO: 14.

[0175] The application is further described below in conjunction with examples:

[0176] Example 1 Preparation of test products

[0177] The small peptides were synthesized by solid phase synthesis and purified by high performance liquid chromatography.

[0178] The truncated peptides were prepared by expression in mammalian cells using genetic recombination.

[0179] Example 2

[0180] Cyclized small peptides CDSP1, CDSP2 and CDSP3 inhibit lung cancer cell metastasis in vivo

[0181] 2.1 Cyclized small peptide CDSP1 inhibits lung cancer cell metastasis to brain tissue and prolongs survival in wild-type mice

[0182] First, a wild-type C57 mouse LLC-BrM lung cancer brain metastasis model was constructed. Wild-type C57 mice were intravenously injected with LLC-BrM-TL lung cancer cell lysate to immunize the mice. After 35 days, CD8+ T cells specific to LLC-BrM-TL lung cancer cells were generated in the mice. In these immunized wild-type mice, LLC-BrM lung cancer cells were injected into the ventricle, and then the control peptide or CDSP1 peptide at 1, 3, or 10 mg / kg was intravenously injected once every other day until the mice died. + T SCM

[0183] In the wild-type C57 mouse LLC-BrM lung cancer brain metastasis model, after treatment with cyclized small peptide CDSP1, lung cancer cell metastasis to brain tissue in the mice was significantly inhibited compared with the control group, and the degree of inhibition increased with increasing CDSP1 peptide concentration (Fig. 1, A, B). After administration of CDSP1 peptide at 10 mg / kg, the median survival of the mice was prolonged by 50% compared with the control group (Fig. 1, C).

[0184] 2.2 Cyclized small peptide CDSP2 inhibits lung cancer cell metastasis to brain tissue and prolongs survival in wild-type mice

[0185] In the wild-type C57 mouse LLC-BrM lung cancer brain metastasis model, after treatment with cyclized small peptide CDSP2, lung cancer cell metastasis to brain tissue in the mice was significantly inhibited compared with the control group, and the degree of inhibition increased with increasing CDSP2 peptide concentration (Fig. 2, A, B). After administration of CDSP2 peptide at 10 mg / kg, the median survival of the mice was prolonged by 27% compared with the control group (Fig. 2, C).

[0186] 2.3.1 Cyclized small peptide CDSP3 inhibits lung cancer cell metastasis to brain tissue and prolongs survival in wild-type mice

[0187] ​In the LLC-BrM lung cancer cell in vivo killing experiment of wild type C57 mice, compared with the control group, the lung cancer cell metastasis to brain tissue of mice was significantly inhibited after the cyclized small peptide CDSP3 treatment, and the inhibition degree was enhanced with the increase of CDSP3 small peptide concentration (Figure 3 B, C). After the administration of 10 mg / kg CDSP3 small peptide, the median survival of mice was prolonged by 50% compared with the control group (Figure 3 D).

[0188] 2.3.2 In the adoptive CD8 + T SCM cell nude mice, cyclized small peptide CDSP3 inhibits lung cancer cell metastasis to brain tissue

[0189] Then, in the adoptive CD8 + T SCM cell lung cancer cell in vivo killing experiment, CD8 + T SCM cells specifically targeting LLC-BrM were injected into immunodeficient nude mice, which can more specifically explore whether cyclized small peptide CDSP3 mediates the inhibition of lung cancer metastasis by exogenously introduced CD8 + T SCM cells (Figure 4 A). The results of this model are similar to the results of the wild type mouse LLC-BrM lung cancer cell in vivo killing experiment, that is, compared with the control group, the lung cancer cell metastasis to brain tissue of mice was significantly inhibited after the administration of cyclized small peptide CDSP3 (Figure 4 B, C).

[0190] 2.3.3 In OT-I mice, cyclized small peptide CDSP3 inhibits lung cancer cell metastasis to brain tissue and prolongs survival

[0191] In immunological studies, OT-I / OVA system is often used to explore the function of CD8 + T cells. The CD8 + T cells in this system can specifically recognize the antigen peptide of OVA protein 257-264 aa. OT-I mice were immunized by injecting LLC-BrM-OVA tumor cell lysate to produce CD8 + T SCM cells specifically targeting OVA in vivo. After the immunization, LLC-BrM-OVA was injected into the ventricle of OT-I mice, and 10 mg / kg of cyclized small peptide CDSP3 was injected once every other day (Figure 5 A). The results showed that in OT-I mice, cyclized small peptide CDSP3 could significantly inhibit the metastasis of LLC-BrM-OVA lung cancer cells to brain tissue (Figure 5 B, C). And the median survival of mice was prolonged by 51% compared with the control group (Figure 5 D).

[0192] II. A group of DSP small peptides increase CD8+ T SCM cells

[0193] In wild type C57 mice immunized with LLC-BrM tumor cell lysate, from day -1, every other day, control small peptides or 10 mg / kg of linear small peptides LDSP1 (18 aa), LDSP2 (19 aa), LDSP3 (20 aa), or the corresponding cyclized small peptides CDSP1, CDSP2, CDSP3 were injected via orbital vein. On day 0, LLC-BrM cells were injected into the heart chamber, and on day 1 and day 3, the number of CD8 + T SCM cells in the bone marrow of the mice was detected. It was found that, 1 day after tumor cell injection, the number of CD8 + T SCM cells in the bone marrow of mice treated with linear small peptides LDSP1, LDSP2, LDSP3, or cyclized small peptides CDSP1, CDSP2, CDSP3 was significantly increased compared to the control group; 3 days later, the number of CD8 + T SCM cells continued to accumulate, and the number of CD8 + T SCM cells was 2-3 times higher than the control group (Figure 6). Thus, it was found that DSP small peptides mainly inhibit the metastasis of lung cancer cells to brain tissue by increasing the number of CD8 + T SCM cells in the bone marrow, thereby treating lung cancer.

[0194] Three, a group of DSP small peptides enhance the in vitro killing effect of CD8 + T cells on tumor cells, thereby treating lung cancer, breast cancer, bladder cancer, colorectal cancer, liver cancer, melanoma, pancreatic cancer, and prostate cancer

[0195] 3.1 Cyclized small peptide CDSP1 enhances the in vitro killing effect of CD8 + T cells on tumor cells, thereby treating lung cancer, breast cancer, bladder cancer, colorectal cancer, liver cancer, melanoma, pancreatic cancer, and prostate cancer

[0196] Cyclized small peptide CDSP1 in vitro treatment of antigen-specific CD8 + T SCM cells, 48 hours later, CD8 + T cells were isolated and co-cultured with lung cancer cells LLC in vitro. 72 hours later, the apoptosis of LLC-BrM cells was detected. The results showed that cyclized small peptide CDSP1 in vitro treatment of CD8 + T SCMAfter the cells, enhanced its lung cancer cell LLC killing effect, and the killing intensity with the concentration of CDSP1 increased (Figure 7, Figure 8). The results are consistent with the results of the cyclization of small peptides CDSP1 in vivo tumor killing experiment (Figure 1), indicating that the cyclization of small peptides CDSP1 can treat lung cancer.

[0197] The above results show that the results of in vitro co-culture killing experiment can directly reflect the results of tumor killing experiment in mice in vivo, therefore, in accordance with the 3R principle of experimental animals, the following are all used in vitro co-culture killing experiment to prove that 10 kinds of small peptides can treat 8 types of cancer:

[0198] 3.2 Cyclization of small peptides CDSP2 enhances CD8 + T cells to kill tumor cells in vitro, and then treat lung cancer, and then treat lung cancer, breast cancer, bladder cancer, colorectal cancer, liver cancer, melanoma, pancreatic cancer, and prostate cancer, the results are shown in Figures 9-10.

[0199] 3.3 Cyclization of small peptides CDSP3 enhances CD8 + T cells to kill tumor cells in vitro, and then treat lung cancer, and then treat lung cancer, breast cancer, bladder cancer, colorectal cancer, liver cancer, melanoma, pancreatic cancer, and prostate cancer, the results are shown in Figures 11-12.

[0200] 3.4 Cyclization of small peptides CDSP4 enhances CD8 + T cells to kill tumor cells in vitro, and then treat lung cancer, and then treat lung cancer, breast cancer, bladder cancer, colorectal cancer, liver cancer, melanoma, pancreatic cancer, and prostate cancer, the results are shown in Figures 13-14.

[0201] 3.5 Cyclization of small peptides CDSP5 enhances CD8 + T cells to kill tumor cells in vitro, and then treat lung cancer, and then treat lung cancer, breast cancer, bladder cancer, colorectal cancer, liver cancer, melanoma, pancreatic cancer, and prostate cancer, the results are shown in Figures 15-16.

[0202] The results show that cyclization of small peptides CDSP1-5 have good anti-tumor effect.

[0203] 3.6 Linear small peptides LDSP1 enhances CD8 + T cells to kill tumor cells in vitro, and then treat lung cancer, and then treat lung cancer, breast cancer, bladder cancer, colorectal cancer, liver cancer, melanoma, pancreatic cancer, and prostate cancer, the results are shown in Figures 17-18.

[0204] 3.7 Linear small peptides LDSP2 enhances CD8 +In vitro killing of tumor cells by T cells, and further treatment of lung cancer, and further treatment of lung cancer, breast cancer, bladder cancer, colorectal cancer, liver cancer, melanoma, pancreatic cancer, and prostate cancer, with results as shown in Figures 19-20.

[0205] 3.8 Linear small peptide LDSP3 enhances CD8 + In vitro killing of tumor cells by T cells, and further treatment of lung cancer, and further treatment of lung cancer, breast cancer, bladder cancer, colorectal cancer, liver cancer, melanoma, pancreatic cancer, and prostate cancer, with results as shown in Figures 21-22.

[0206] 3.9 Linear small peptide LDSP4 enhances CD8 + In vitro killing of tumor cells by T cells, and further treatment of lung cancer, and further treatment of lung cancer, breast cancer, bladder cancer, colorectal cancer, liver cancer, melanoma, pancreatic cancer, and prostate cancer, with results as shown in Figures 23-24.

[0207] 3.10 Linear small peptide LDSP5 enhances CD8 + In vitro killing of tumor cells by T cells, and further treatment of lung cancer, and further treatment of lung cancer, breast cancer, bladder cancer, colorectal cancer, liver cancer, melanoma, pancreatic cancer, and prostate cancer, with results as shown in Figures 25-26.

[0208] The results show that linear small peptides CDSP1-5 all have good anti-tumor effect.

[0209] Four, treatment of lung cancer mice by DDR1 truncation

[0210] In adoptive CD8 + T SCM In nude mice with adoptive CD8 T cells, H460 human lung cancer cells overexpressing human DDR1, or C-terminal deletion truncations DDR1 529*, DDR1 444*, DDR1 417*, can inhibit lung cancer cell metastasis. Results as shown in Figures 27-28.

[0211] Five, DDR1 truncation inhibits lung cancer cell metastasis

[0212] In wild-type mice, LLC mouse lung cancer cells overexpressing mouse DDR1, or C-terminal deletion truncation DDR1 527*, can inhibit lung cancer cell metastasis to brain tissue. Results as shown in Figure 29.

[0213] Six, different lengths of human DDR1 protein can enhance the in vitro killing of lung cancer cells by CD8+ T cells, and further treatment of lung cancer

[0214] In 293FT cells, different lengths of human DDR1 were overexpressed by transient transfection, including 1-417aa (DDR1 extracellular region), 29-187aa (DS domain), 29-108aa (DSN), 29-97aa (DSNN1), 29-88aa (DSNN2), 29-78aa (DSNN3), 39-108aa (DSNC1), 49-108aa (DSNC2), and 59-108aa (DSNC3). The DDR1 extracellular region, because these different lengths of DDR1 all have signal peptides at the N segment, and all do not have transmembrane regions, can be expressed in the conditioned medium outside the cells. Therefore, CD8 + T SCM cells were treated in vitro with the 293FT cell conditioned medium expressing different lengths of DDR1 protein, and then the CD8 + T cells were co-cultured with H460 human lung cancer cells in vitro. After 72 hours, the apoptosis of H460 human lung cancer cells was detected.

[0215] The results showed that 1-417aa (DDR1 extracellular region), 29-187aa (DS domain), 29-108aa (DSN), 29-97aa (DSNN1), 29-88aa (DSNN2), 29-78aa (DSNN3), 39-108aa (DSNC1), 49-108aa (DSNC2), and 59-108aa (DSNC3) proteins can enhance the in vitro killing effect of CD8 + T cells on tumor cells, and then treat cancer. The results are shown in Figure 30.

[0216] In the present application, data analysis: using Two-way ANOVA analysis method, and with Fisher's LSD test as post-test, mean ± S.E.M., statistical analysis of tumor metastasis signals of each group of mice in (B) of Figures 1-3, (C) of Figures 4-5 and (B) of Figures 27-29. Using Log-rank test analysis method, statistical analysis of survival time of each group of mice in (C) of Figures 1-3, (D) of Figure 5 and (C) of Figure 29. Using Two-way ANOVA analysis method, and with Fisher's LSD test as post-test, mean ± S.E.M., statistical analysis of each group of data in Figure 6. Using One-way ANOVA analysis method, and with Fisher's LSD test as post-test, mean ± S.E.M., statistical analysis of tumor cell apoptosis level of each group in Figures 7-26 and (B)-(D) of Figure 30. Using t test analysis method, mean ± S.E.M., statistical analysis of tumor cell apoptosis level of each group in (A) of Figure 30. (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., no significant difference).

[0217] The above is only the preferred embodiment of the present application, it should be noted that for those skilled in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered within the scope of the present application.

Claims

1. A small peptide, which is any one of I) to III) below: I) which has an amino acid sequence as shown in SEQ ID NO: 1; II) a small peptide in which one or more amino acid residues are substituted, deleted, added and / or replaced in the amino acid sequence described in I); III) a small peptide which has at least 80% identity to the amino acid sequence described in I) or II).

2. The small peptide according to claim 1, characterized in that, which has a structure of: (X1-(X2)m-X3)n: wherein: X2 has an amino acid sequence as shown in SEQ ID NO: 1; X1 consists of 0 to 15 amino acid residues; X3 consists of 0 to 15 amino acid residues; m and n are independently selected from an integer of 1 to 5.

3. The small peptide according to claim 2, wherein: m is 1, 2 or 3; and n is 1, 2 or 3; X1 is Ala, Thr-Ala, Ser-Thr-Ala, Asp-Ser-Thr-Ala, Ser-Asp-Ser-Thr-Ala, Trp-Ser-Asp-Ser-Thr-Ala, Ser-Trp-Ser-Asp-Ser-Thr-Ala, Ser-Ser-Trp-Ser-Asp-Ser-Thr-Ala, Ser-Ser-Ser-Trp-Ser-Asp-Ser-Thr-Ala or Ala-Ser-Ser-Ser-Trp-Ser-Asp-Ser-Thr-Ala; X3 is Trp, Trp-Cys, Trp-Cys-Pro, Trp-Cys-Pro-Ala, Trp-Cys-Pro-Ala-Gly, Trp-Cys-Pro-Ala-Gly-Pro or Trp-Cys-Pro-Ala-Gly-Ser.

4. The small peptide according to claim 3, characterized in that, which has an amino acid sequence as shown in any one of SEQ ID NOs: 2 to 6.

5. The small peptide according to any one of claims 2 to 4, characterized in that, which further comprises at least one Cys at the N-terminus and / or C-terminus, and two Cys in the small peptide form a disulfide bond.

6. The small peptide according to claim 5, characterized in that, It is:

7. A truncation of DDR1 protein, which contains the small peptide according to any one of claims 1 to 6.

8. The truncation according to claim 7, characterized in that The DDR1 protein is a human DDR1 protein or a mouse DDR1 protein.

9. The truncation according to claim 7 or 8, characterized in that, which has a length of 50 aa to 529 aa.

10. The truncation according to claim 9, characterized in that which has an amino acid sequence as shown in any one of SEQ ID NOs: 7 to 14 or an amino acid sequence as shown in any one of SEQ ID NOs: 15 to 18.

11. A biological material, which comprises at least one of: a) a nucleic acid encoding the small peptide according to any one of claims 1 to 6 and / or the truncation according to any one of claims 7 to 10; b) an expression unit comprising a promoter and the nucleic acid described in a); c) a plasmid vector containing the nucleic acid described in a) or containing the expression unit described in b); d) a host into the genome of which the nucleic acid described in a) or the expression unit described in b) is integrated, or which is transformed or transfected with the plasmid vector described in c). e) derivatives comprising a modified moiety and the small peptide according to any one of claims 1 to 6 and / or the truncation according to any one of claims 7 to 10; the modified moiety is a PEG modification; f) fusion proteins comprising an antibody Fc fragment and the small peptide according to any one of claims 1 to 6 and / or the truncation according to any one of claims 7 to 10.

12. A method for preparing the small peptide according to any one of claims 1 to 6 and / or the truncation according to any one of claims 7 to 10, comprising: synthesizing by chemical methods, or culturing the host according to claim 11 to obtain a product containing the small peptide and / or the truncation.

13. Use of DDR1 protein, the small peptide according to any one of claims 1 to 6 and / or the truncation according to any one of claims 7 to 10 for the preparation of a medicament for preventing and treating tumors.

14. Use according to claim 13, characterized in that, The prevention and treatment of tumors includes: increasing the number of CD8 + T SCM cells in bone marrow, increasing the killing ability of CD8 + T cells, inhibiting tumor cell metastasis and / or prolonging the survival of patients.

15. The use according to claim 13, characterized in that, The tumors are lung cancer, breast cancer, bladder cancer, colorectal cancer, liver cancer, melanoma, pancreatic cancer and / or prostate cancer.

16. A medicament comprising the small peptide according to any one of claims 1 to 6 and / or the truncation according to any one of claims 7 to 10.

17. The medicament according to claim 16, characterized in that, Also comprising: at least one of DDR1 protein, immune checkpoint inhibitors, CAR-T cells, CAR-NK cells.

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