CN108295244B - Polypeptides for treating breast tumors - Google Patents
Polypeptides for treating breast tumors Download PDFInfo
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- CN108295244B CN108295244B CN201710026136.3A CN201710026136A CN108295244B CN 108295244 B CN108295244 B CN 108295244B CN 201710026136 A CN201710026136 A CN 201710026136A CN 108295244 B CN108295244 B CN 108295244B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
Abstract
The present application provides polypeptides for the treatment of breast tumors, particularly triple negative breast cancers. Specifically, the polypeptide or the pharmaceutical composition thereof is a fragment of endostatin with the N-terminal length of 45 amino acid residues or less and at least contains amino acid residues 1-20 of the N-terminal, wherein the amino acid residues 2 and 18 of the endostatin are respectively shown in the text, and optionally mutations in 17 th and 20-22 of the endostatin exist in the text.
Description
Technical Field
The application belongs to the field of tumor treatment, and particularly relates to a polypeptide for treating breast tumors.
Background
Breast cancer is a malignancy that occurs in the epithelial tissue of the mammary gland, 99% of which occur in females. Breast is not an important organ for maintaining vital activities of human body, and in situ breast cancer is not fatal; however, the breast cancer cells lose the characteristics of normal cells, so that the connection between the cells is loose and easy to fall off. Once the cancer cells fall off, the free cancer cells can spread throughout the body along with blood or lymph fluid to form metastasis, thereby endangering life. Breast cancer is now a common tumor that threatens female physical and mental health. Global breast cancer incidence has been on the rise since the end of the 70 s of the 20 th century. 1 woman in the united states suffers from breast cancer in the life of 8 women. China is not a high-incidence country of breast cancer, but is not optimistic, and the rate of increase of the incidence of breast cancer in China is 1-2 percent higher in recent years. 2009 breast cancer incidence data published by the national cancer center and health disease prevention control agency 2012 showed that: the incidence of breast cancer in the tumor-registered region of the country is located at position 1 of female malignant tumors. Treatment of breast cancer mainly includes surgery, radiation therapy, chemotherapy, estrogen endocrine therapy and targeted therapy for HER-2.
Triple-negative breast cancer (TNBC) is a special type of breast cancer, and the estrogen receptor (estrogen receptor, ER), the progestogen receptor (progesterone receptor, PR) and the human EGF receptor 2 (human epidermal growth factor receptor, HER-2) of tumor cells are all negative breast cancer types accounting for 10% -20% of all pathological types of breast cancer. Because of the lack of hormone receptor and HER-2, the tumor is insensitive to hormone endocrine treatment and HER-2 targeting treatment, so that the triple-negative breast cancer is the most dangerous breast cancer, has strong invasiveness, is easy to transfer and extremely poor prognosis, the survival time of the patients after diagnosis is usually not more than 20 months, and the survival rate of 5 years is less than 15%.
The triple-negative breast cancer is not clinically treated currently, and is generally treated conventionally according to the breast cancer with poor prognosis, and the standard therapy is novel auxiliary chemotherapy containing anthracyclines or taxol after operation. Since there are multiple subtypes of estrogen receptor, progestin receptor or HER2 receptor in TNBC patients, even with the same treatment regimen, there is a large difference in response levels among patients. No targeted drug for treating TNBC is currently approved in the united states.
Endostatin is an endogenous angiogenesis inhibitor isolated and purified from cultured mouse endothelial cell tumor (EOMA) supernatant of 1997O' Reilly et al, is a 20kd molecular weight protein, and is derived from hydrolysate of XVIII type collagen. Experiments show that endostatin plays an inhibitory role on vascular endothelium and tumor cells. Because of the difficulty factors such as difficult renaturation of recombinant endostatin, entreMed corporation in U.S. has abandoned the clinical study of recombinant endostatin, and it is not possible to prepare endostatin with higher in vitro activity in large quantities.
The binding site of zinc ions in endostatin sequence, which consists of three histidine residues at 1 st, 3 rd and 11 th positions and aspartic acid residue at 76 th position, is critical to the activity of endostatin binding with zinc ions. Polypeptides derived from the N-terminus of endostatin have been reported to have some vascular endothelial cell and tumor cell inhibiting activity (Cancer Res.2005;65 (9): 3656-63, U.S. Pat. No. 3,262B 2). However, the above experiments also showed that the polypeptide containing amino acids 1-25 from the N-terminal from human endostatin source could not significantly inhibit the growth of human tumors inoculated on mouse animal models, and that the activity of endostatin derived peptides was to be improved.
Disclosure of Invention
The application provides application of polypeptide in preparing a medicament for treating breast tumor, wherein the polypeptide is a fragment of endostatin with the N-terminal length of less than 45 amino acid residues and at least contains amino acid residues at the 1 st to 20 th positions at the N-terminal, and the amino acid residues at the 2 nd and 18 th positions at the N-terminal of the endostatin are respectively selected from the following groups:
and optionally, the endostatin has amino acid residue at position 17 at the N-terminus of S, A, L, I or T, and/or amino acid residue at position 20 of S or T, and/or amino acid residue at position 21 of G, A, L, I or V if containing amino acid residue at position 21 and/or 22, and/or amino acid residue at position 22 of G, A, L, I or V;
preferably, the amino acid sequence of the endostatin is shown as SEQ ID NO. 1.
In one or more embodiments, the polypeptide comprises at least the amino acid sequence of SEQ ID NO:38 amino acid residues 1-22, and amino acid residues 2 and 18 are as described above.
In one or more embodiments, the polypeptide comprises at least the amino acid sequence of SEQ ID NO:38 amino acid residues 1-25, and amino acid residues 2 and 18 are as described above.
In one or more embodiments, the polypeptide comprises at least the amino acid sequence of SEQ ID NO:38, preferably at least amino acid residues 1-22 of SEQ ID NO:38 amino acid residues 1-25, and amino acid residue 2 is T, amino acid residue 18 is N, G, K, M, F, S or T, and amino acids 17, 20, 21, and 22 are as described above.
In one or more embodiments, the polypeptide comprises at least the amino acid sequence of SEQ ID NO:38, preferably at least amino acid residues 1-22 of SEQ ID NO:38 amino acid residues 1-25, and 18 amino acid residue is N, 2 amino acid residue is T, and 17, 20, 21, and 22 amino acids are as described above.
In one or more embodiments, the polypeptide comprises at least the amino acid sequence of SEQ ID NO:38, preferably at least amino acid residues 1-22 of SEQ ID NO:38 amino acid residues 1-25, and 18 amino acid residue S, 2 amino acid residue E, H, L, T, W or V, and 17, 20, 21 and 22 amino acids are as described above.
In one or more embodiments, the amino acid sequence of the polypeptide is as set forth in any one of SEQ ID NOs 4, 5, 6, 7, 27-30, 39, 41 and 47-49.
In one or more embodiments, the polypeptide consists of SEQ ID NO 38, wherein the amino acid residue at position 2 is T, the amino acid residue at position 18 is N or S, and the amino acids at positions 17, 20, 21 and 22 are as described previously.
In one or more embodiments, the polypeptide is selected from the group consisting of the amino acid sequences consisting of amino acid residues 1 to 39, 38, 37, 36, 34, 33, 32, 31, 29, 28, 27 or 26 of SEQ ID NO. 4, and the amino acid sequences consisting of amino acid residues 1 to 39, 38, 37, 36, 35, 34, 33, 32, 31, 29, 28, 27, 26 or 25 of SEQ ID NO. 39.
In one or more embodiments, the amino acid residue at position 1 of the N-terminus of the polypeptide is histidine, which is formylated, acetylated, propionylated or butyrylated, and the amino acid at position 1 of the C-terminus may be modified by PEG, cholesterol or amidation.
In one or more embodiments, the polypeptide is selected from the group consisting of:
HTHRDFQPVLHLVALNSSLSGGMRGIRGAD；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGAD；
HTHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH 2 ；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH 2 ；
Ac-HTHRDFQPVLHLVALNSSLSGGMRG-NH 2 ；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGADFQCFQ-NH 2 ；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGADFQCFQQARAV-NH 2 ；
HTHRDFQPVLHLVALNSNLSGGMRGIRGAD；
Ac-HTHRDFQPVLHLVALNSNLSGGMRGIRGAD；
HTHRDFQPVLHLVALNSNLSGGMRGIRGAD-NH 2 ；
Ac-HTHRDFQPVLHLVALNSNLSGGMRGIRGAD-NH 2 ；
Ac-HTHRDFQPVLHLVALNASLSGGMRGIRGAD-NH 2 ；
Ac-HTHRDFQPVLHLVALNSSLTGGMRGIRGAD-NH 2 ；
Ac-HTHRDFQPVLHLVALNASLTGGMRGIRGAD-NH 2 ；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRG-NH 2 ；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRG；
HTHRDFQPVLHLVALNSSLSGGMRGIRG-NH 2 ；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGA-NH 2 ；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGA；
HTHRDFQPVLHLVALNSSLSGGMRGIRGA-NH 2 ；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGADF-NH 2 ；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGADF；
HTHRDFQPVLHLVALNSSLSGGMRGIRGADF-NH 2 ；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGADFQ-NH 2 ；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGADFQ; and
HTHRDFQPVLHLVALNSSLSGGMRGIRGADFQ-NH 2 ；
wherein Ac is an acetyl modification, NH 2 Is an amidation modification.
In one or more embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
In one or more embodiments, the breast tumor is selected from the group consisting of triple negative breast cancer, both negative breast cancer, one negative breast cancer, and all three positive breast cancers, wherein HER2, estrogen receptor, and progestin receptor are all negative.
In one or more embodiments, the breast tumor is selected from the group consisting of a breast epithelial tumor, a breast mesenchymal tumor, a breast fibroepithelial tumor, a nipple tumor, and a male breast cancer.
In one or more embodiments, the breast epithelial tumor is selected from the group consisting of a micro invasive carcinoma, an invasive breast carcinoma, an epithelial-myoepithelial tumor, a precursor lesion, an intraductal proliferative lesion, and a papillary lesion.
In one or more embodiments, the breast mesenchymal tumor package is selected from myofibroblastic tumor, liposarcoma, and hemangiosarcoma.
In one or more embodiments, the mammary fibroepithelial tumor is a phylloma or a hamartoma.
The application also provides application of the polypeptide or the pharmaceutical composition thereof in preparing a medicament for improving the therapeutic effect of the chemotherapeutic medicament. In one or more embodiments, the chemotherapeutic agent is one or more of cisplatin, carboplatin, oxaliplatin, cyclophosphamide, methotrexate, fluorouracil, doxorubicin, taxol, mitoxantrone, hiloda, robusta, epirubicin, gemcitabine, and the like.
The application also provides application of the polypeptide or the pharmaceutical composition thereof in preparing a medicament for improving the therapeutic effect of endocrine treatment medicaments. In one or more embodiments, the endocrine therapeutic agent is one or more of megestrol, ai Luomei new, tamoxifen, rilidine, letrozole, no Lei Deku, and the like.
The application also provides application of the polypeptide or the pharmaceutical composition thereof in preparing a medicament for improving therapeutic effect of the targeted therapeutic medicament. In one or more embodiments, the targeted therapeutic is an antibody and a tyrosine kinase inhibitor, wherein the antibody is herceptin, pertuzumab, bevacizumab, keyruda mab, durvalumab, or the like; the tyrosine kinase inhibitor is one or more of lapatinib, nolatinib, sunitinib and the like.
The application also provides application of the polypeptide or the pharmaceutical composition thereof in preparing a medicament for improving the therapeutic effect of the immunotherapy medicament. In one or more embodiments, the immunotherapeutic agent is one or more of an anti-PD-1 antibody, an anti-PD-L1 antibody, a CAR-T cell, and the like.
Drawings
FIGS. 1a and 1b show the HPLC and MASS profiles, respectively, of polypeptide P1.
FIGS. 1c and 1d show the HPLC and MASS profiles, respectively, of polypeptide P2.
FIGS. 1e and 1f show the HPLC and MASS profiles, respectively, of polypeptide P2T2S 18.
FIGS. 1g and 1h show the HPLC and MASS profiles, respectively, of polypeptide P2T2N 18.
FIG. 2 shows the biological activity of P1, P2, P3, P4 polypeptides, endostatin, endostar on HUVEC inhibition.
Figures 3a and 3b show the HPLC and MASS profiles, respectively, of the polypeptide P2T2s18Δ1.
Figures 3c and 3d show the HPLC and MASS profiles, respectively, of the polypeptide P2T2s18Δ2.
FIGS. 3e and 3f show HPLC and MASS profiles, respectively, of the polypeptide P2T2S 18. Delta.3.
FIG. 4 shows the biological activity of a portion of the polypeptide on HUVEC inhibition.
FIGS. 5a and 5b show the inhibition of BT-483 by several polypeptides.
FIG. 6a shows the inhibition of BT-483, MDA-MB-453 and MDA-MB-468 by several polypeptides.
FIG. 6b shows a photomicrograph of the inhibition of MDA-MB-453 cells by P2 and P2T2S18 at various concentrations.
FIG. 7 shows the inhibition of SK-BR-3, T-47D and BT-474 by P2 and P2T2S 18-29.
FIGS. 8a and 8b show HPLC and MASS profiles, respectively, of the polypeptide P2T2S 18-20.
FIGS. 8c and 8d show HPLC and MASS profiles, respectively, of the polypeptide P2T2S 18-25.
FIGS. 8e and 8f show HPLC and MASS profiles, respectively, of the polypeptide P2T2N 18-35.
FIGS. 8g and 8h show HPLC and MASS profiles, respectively, of the polypeptide P2T2N 18-40.
FIGS. 8i and 8j show HPLC and MASS profiles, respectively, of the polypeptide P2T2N 18-45.
FIG. 9 shows inhibition of HUVEC growth by the polypeptide in vitro. In the figure, each curve represents the cell viability of P2T2-15, P2T2S18-45, P2T2S18-40, P2T2S18-20, P2T2S18-25, P2T2S18-35 and P2T2S18 in order from top to bottom, based on the cell viability at a concentration of 180. Mu.M.
FIG. 10 shows inhibition of HUVEC growth by the polypeptide in vitro.
FIGS. 11a and 11b show HPLC and MASS profiles, respectively, of the polypeptide P2T2S 18-29.
FIG. 12 shows the inhibition of HUVEC by polypeptide P2T2S 18-29. The curves represent Endostar, endostatin, P, P2T2S18-29 and P2T2S18 in order from top to bottom, in terms of cell viability at 2.5 mg/ml.
Detailed Description
The application relates to treatment of breast tumors, in particular to treatment of breast tumors using polypeptides as follows: a fragment of endostatin having less than 45 amino acid residues N-terminal, comprising at least amino acid residues 1-20 of endostatin N-terminal, and wherein:
(1) Residue corresponding to amino acid 2 of the N-terminal of endostatin is A, R, N, D, Q, E, H, I, L, K, M, F, P, T, W, Y or V; and
(2) Residue corresponding to amino acid 18 of the N-terminal end of endostatin is A, R, N, D, C, E, G, H, I, L, K, M, F, S, T, W, Y or V;
and the polypeptide has an inhibition rate of HUVEC at the same concentration of at least 15%, preferably at least 20% higher than its corresponding sequence without mutation; or the polypeptide is compared with its corresponding sequence without mutation 50 IC with the concentration of the latter 50 One half of the concentration, preferably the former IC 50 IC with the concentration of the latter 50 One fifth of the concentration, further preferably the former IC 50 IC with the concentration of the latter 50 One tenth of the concentration.
The endostatin is preferably human endostatin. An example of recombinant human vascular endothelial chalone is shown in SEQ ID NO. 1. Preferably, the amino acid sequence of the application at least comprises amino acid residues 1-20 of the N-terminal of endostatin as shown in SEQ ID NO. 1, and the amino acids at positions 2 and 18 are as described herein.
Preferably, the residue of the polypeptide corresponding to amino acid 2 at the N-terminal end of endostatin is D, L, T, W or Y. Preferably, the residue of the polypeptide corresponding to amino acid 18 at the N-terminus of endostatin is N, E, K, M, S, T or V. More preferably, the residue of the polypeptide corresponding to amino acid 2 at the N-terminal end of endostatin is D, T, W or Y. More preferably, the polypeptide corresponds to residue N, S or V of amino acid 18 of endostatin.
In certain embodiments, the polypeptide corresponds to amino acid residue 2 and amino acid residue 18 of endostatin, respectively, in the following combination:
| amino acid 2 | Amino acid 18 |
| A | M |
| R | I |
| N | K |
| D | E. M, T or Y |
| Q | A or H |
| E | S or V |
| H | A or S |
| L | R, E or S |
| K | V |
| M | L or W |
| F | T |
| P | C or V |
| T | N, G, K, M, F, S or T |
| W | C. E, I, K, S or Y |
| Y | R, H, W or V |
| V | D or S |
Alternatively, in certain embodiments, the polypeptide corresponds to the amino acid residue at position 2 and the amino acid residue at position 18 of endostatin, respectively, in combination of:
| amino acid 18 | Amino acid 2 |
| A | Q or H |
| R | L or Y |
| N | T |
| D | V |
| C | P or W |
| E | D. L or W |
| G | T |
| H | Q or Y |
| I | R or W |
| L | M |
| K | N, T or W |
| M | A. D or T |
| F | T |
| S | E. H, L, T, W or V |
| T | D. F or T |
| W | M or Y |
| Y | D or W |
| V | E. K, P or Y |
It is understood that a "fragment" refers to a contiguous portion of the full-length sequence. For example, the polypeptides herein are preferably sequences consisting of amino acid residues 1 to 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 of the N-terminal endostatin and the amino acid residues described herein at positions 2 and 18. In other words, the polypeptide of the present application is 20-45 amino acid residues in length, starting from amino acid residue 1 at the N-terminus of endostatin. More preferably, the polypeptide of the application is 25-40 amino acid residues in length, starting from the 1 st amino acid residue at the N-terminus.
In certain embodiments, the fragment may optionally be any, any three, or all four of the amino acid residues at positions 17, 20, 21, and 22 in addition to the amino acid residues described herein at positions 2 and 18:
amino acid residue 17: s, A, L, I, V or T;
amino acid residue 20: s or T;
amino acid residue 21: G. a, L, I or V;
amino acid residue 22: G. a, L, I or V.
Thus, in certain embodiments, a polypeptide of the application that is a fragment within 45 amino acid residues of the N-terminus of endostatin comprises at least the amino acid sequence of SEQ ID NO:38, preferably at least amino acid residues 1-22 of SEQ ID NO:38 from amino acid residues 1-25, and amino acid residues 2 and 18 as described herein, and any, any three, or all four of positions 17, 20-22 are as described above. Further, such polypeptides are 25-40 amino acid residues in length.
In certain embodiments, the polypeptide of the application, which is a fragment within 45 amino acid residues of the N-terminus of endostatin, comprises at least the amino acid sequence of SEQ ID NO:38, preferably at least amino acid residues 1-22 of SEQ ID NO:38 amino acid residues 1-25, and amino acid residue 2 is T, amino acid residue 18 is N, G, K, M, F, S or T (more preferably N or S), optionally amino acids 17, 20, 21 and 22 as previously described. Further, such polypeptides are 25-40 amino acid residues in length.
In certain embodiments, the polypeptide of the application, which is a fragment within 45 amino acid residues of the N-terminus of endostatin, comprises at least the amino acid sequence of SEQ ID NO:38, preferably at least amino acid residues 1-22 of SEQ ID NO:38 amino acid residues 1-25, and 18 amino acid residue is N, 2 amino acid residue is T, optionally 17, 20, 21 and 22 amino acids are as previously described. Further, such polypeptides are 25-40 amino acid residues in length.
In certain embodiments, the polypeptide of the application, which is a fragment within 45 amino acid residues of the N-terminus of endostatin, comprises at least the amino acid sequence of SEQ ID NO:38, preferably at least amino acid residues 1-22 of SEQ ID NO:38 amino acid residues 1-25, and 18 amino acid residue S, 2 amino acid residue E, H, L, T, W or V, optionally, 17, 20, 21 and 22 amino acids as described above. Further, such polypeptides are 25-40 amino acid residues in length.
The amino acid sequence of the preferred polypeptide of the present application is shown in any one of SEQ ID NOs 4, 5, 6, 7, 27-30, 39, 41 and 47-49. The polypeptides of the application also include amino acid sequences consisting of amino acid residues 1 to 39, 38, 37, 36, 34, 33, 32, 31, 29, 28, 27 or 26 of SEQ ID NO. 4 and amino acid sequences consisting of amino acid residues 1 to 39, 38, 37, 36, 35, 34, 33, 32, 31, 29, 28, 27, 26 or 25 of SEQ ID NO. 39.
The amino acid residue at the 1 st position of the N-terminal of the polypeptide is histidine, the histidine can be formylated, acetylated, propionylated or butyrylated, and the amino acid at the 1 st position of the C-terminal of the polypeptide can be modified by PEG, cholesterol or amidation.
Preferably, histidine at amino acid residue 1 at N-terminus of the polypeptide of the present application is acetylated, and amino acid at C-terminus thereof is amidated.
It will be appreciated that in gene cloning operations, it is often necessary to design suitable cleavage sites, which tend to introduce one or more unrelated residues at the end of the expressed amino acid sequence, without affecting the activity of the sequence of interest. As another example, to construct a fusion protein, facilitate expression of a recombinant protein, obtain a recombinant protein that is automatically secreted outside of a host cell, or facilitate purification of a recombinant protein, it is often desirable to add some amino acid to the N-terminus, C-terminus, or other suitable region within the recombinant protein, including, for example, but not limited to, a suitable linker peptide, signal peptide, leader peptide, terminal extension, and the like. The amino-or carboxy-terminal end of the amino acid sequences of the application may also contain one or more polypeptide fragments as protein tags. Any suitable label may be used with the present application. For example, the tag may be FLAG, HA, HA1, c-Myc, poly-His, poly-Arg, strep-TagII, AU1, EE, T7,4A6, ε, B, gE, and Ty1. These tags can be used to purify proteins. Examples of tags used include Poly-Arg, such as RRRRR (SEQ ID NO: 42); poly-His 2-10 (typically 6), such as HHHHH (SEQ ID NO: 43); FLAG, DYKDDDDK (SEQ ID NO: 44); strep-TagII, WSHPQFEK (SEQ ID NO: 45); and C-myc, WQKLISEEDL (SEQ ID NO: 46).
Thus, the application also includes polypeptides comprising or consisting of the tag sequences and fragments described above.
The amino acid sequences of the application may be the products of chemical synthesis or recombinant polypeptides produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacterial, yeast, filamentous fungi, higher plant, insect and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptides of the application may be glycosylated or may be non-glycosylated.
For example, the amino acid sequences of the present application can be synthesized using polypeptide chemical synthesis methods well known in the art. The chemical synthesis method of the polypeptide includes a solid phase synthesis method and a liquid phase synthesis method, wherein the solid phase synthesis method is commonly used. Solid phase synthesis methods include, but are not limited to, fmoc and tBOC. Typically, using resins as insoluble solid supports, amino acids are attached to the peptide chain, typically one by one, from the C-terminus (carboxy-terminus) to the N-terminus (amino-terminus), each amino acid attachment cycle consisting of the following three steps of reaction: 1) Deprotection: the protected amino acid must be deprotected with a deprotecting solvent to remove the protecting group for the amino group; 2) Activating: the carboxyl group of the amino acid to be linked is activated by an activator; and 3) coupling: the activated carboxyl group reacts with the naked amino group of the previous amino acid to form a peptide bond. The cycle is repeated until the peptide chain is extended to the desired length. Finally, the cleavage solution is used to cleave the connection between the peptide chain and the solid support, thus obtaining the required amino acid sequence. The chemical synthesis described above may be performed on a programmed automated polypeptide synthesizer, such as, but not limited to, the Tribute two-channel polypeptide synthesizer from Protein Technologies, the UV Online Monitor system from CS Bio, the Focus XC three-channel synthesizer from Aapptec, and the like.
The application also includes polynucleotides encoding the polypeptides of the application. For example, SEQ ID NO. 30 shows the coding sequence of SEQ ID NO. 1; SEQ ID NO. 31 shows the coding sequence of SEQ ID NO. 3; SEQ ID NO. 32 shows the coding sequence of SEQ ID NO. 4; SEQ ID NO. 33 shows the coding sequence of SEQ ID NO. 5; SEQ ID NO. 34 shows the coding sequence of SEQ ID NO. 6; SEQ ID NO. 35 shows the coding sequence of SEQ ID NO. 7; SEQ ID NO. 36 shows the coding sequence of SEQ ID NO. 8; SEQ ID NO. 37 shows the coding sequence of SEQ ID NO. 9; SEQ ID NO. 40 shows the coding sequence of SEQ ID NO. 39.
In certain embodiments, the polynucleotide sequence is selected from the group consisting of the bases 1 to 117, 114, 111, 108, 102, 99, 96, 93, 87, 84, 81, or 78 of SEQ ID NO. 32. In certain embodiments, the polynucleotide sequence is selected from the group consisting of the bases 1 to 117, 114, 111, 108, 105, 102, 99, 96, 93, 87, 84, 81, 78, or 75 of SEQ ID NO. 40.
The polynucleotides of the application may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical to the DNA sequence described above or degenerate variants. As used herein, "degenerate variant" refers to a nucleic acid sequence that encodes an amino acid sequence of the present application, but differs from the sequence shown as SEQ ID NO. 31, etc.
The term "polynucleotide encoding a polypeptide" may include polynucleotides encoding the polypeptide, or may include additional coding and/or non-coding sequences.
The polypeptides and polynucleotides of the application are preferably provided in isolated form, and more preferably purified to homogeneity.
The nucleotide sequence of the present application can be usually obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. For the PCR amplification method, primers can be designed according to the nucleotide sequences disclosed in the present application, particularly the open reading frame sequences, and amplified to obtain the relevant sequences using a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.
At present, it is already possible to obtain the DNA sequences encoding the amino acid sequences according to the application entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art.
The application also relates to vectors comprising the polynucleotides of the application, host cells genetically engineered with the vectors of the application, and methods of producing the polypeptides of the application by recombinant techniques. Preferably, the vector of the application is an expression vector.
The polynucleotide sequences of the application may be used to express or produce the polypeptides of the application by conventional recombinant DNA techniques. Generally, there are the following steps:
(1) Transforming or transducing a suitable host cell with a polynucleotide of the application or a degenerate variant thereof, or with a recombinant expression vector comprising the polynucleotide;
(2) A host cell cultured in a suitable medium;
(3) Separating and purifying the protein from the culture medium or the cells.
The polynucleotide sequences of the application may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. Any plasmid or vector may be used as long as it is replicable and stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translational control elements. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
Methods well known to those skilled in the art can be used to construct expression vectors containing the nucleic acid sequences of the application and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The nucleic acid sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.coli; a lambda phage PL promoter; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, LTRs from retroviruses, and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or viruses thereof.
In addition, the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.
Vectors comprising the appropriate DNA sequences as described above, as well as appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.
The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; filamentous fungal cells, or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast, filamentous fungi, plant cells; insect cells of Drosophila S2 or Sf 9; CHO, COS, 293 cells, or Bowes melanoma cells.
When the polynucleotide of the present application is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 base pairs, that act on a promoter to increase the transcription of a gene.
It will be clear to a person of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.
Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which can take up DNA, can be obtained after the exponential growth phase and then treated with CaCl 2 The process is carried out using procedures well known in the art. Another approach is to use MgCl 2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.
The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present application. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.
The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods. Various methods for producing polypeptides by recombinant techniques are known in the art.
The application also provides a pharmaceutical composition comprising the polypeptide of the application and a pharmaceutically acceptable carrier.
The pharmaceutical compositions may contain a therapeutically or prophylactically effective amount of a polypeptide of the application. An "effective amount" means an amount of an ingredient sufficient to produce the desired reaction. The specific effective amount depends on a variety of factors such as the particular condition to be treated, the physical condition of the patient (e.g., patient weight, age, or sex), the duration of the treatment, the co-administered therapy (if any), and the specific formulation used. An "effective amount" also means that the toxicity or negative effects of the polypeptides of the application are less than the positive efficacy associated therewith.
Pharmaceutically acceptable carriers are generally safe, nontoxic, and may broadly include any of the known substances used in the pharmaceutical industry for preparing pharmaceutical compositions, such as fillers, diluents, coagulants, binders, lubricants, glidants, stabilizers, colorants, wetting agents, disintegrants, and the like. The mode of administration of the pharmaceutical composition is primarily considered in the selection of excipients suitable for delivery of the synthetic peptide, and those skilled in the art are familiar with this technology.
The polypeptide is present in the pharmaceutical composition of the application in an amount of about 0.01 to 1000. Mu.M.
The above pharmaceutical compositions may be prepared according to known pharmaceutical procedures, such as those described in detail in Remington pharmaceutical sciences (Remington's Pharmaceutical Sciences) (17 th edition, alfonoso R.Gennaro, mitsubishi (Mack Publishing Company), iston, pa., 1985).
The pharmaceutical compositions of the present application may be in a variety of suitable dosage forms including, but not limited to, tablets, capsules, injections, and the like.
The pharmaceutical compositions of the present application may also contain other known chemotherapeutic agents, particularly those known to treat breast cancer, including but not limited to cisplatin, carboplatin, oxaliplatin, cyclophosphamide, methotrexate, fluorouracil, doxorubicin, taxol, taxotere, mitoxantrone, hilder, valance, epirubicin, gemcitabine, and the like.
Other known endocrine treatment agents, particularly those known to treat breast cancer, may also be included in the pharmaceutical compositions of the present application, including, but not limited to, megestrol, ai Luomei novel, tamoxifen, rilidine, letrozole, nor Lei Deku, and the like.
The pharmaceutical composition of the application may also contain other known targeted therapeutic drugs, particularly targeted therapeutic drugs known to be used for treating breast cancer, including but not limited to antibodies and tyrosine kinase inhibitors, wherein the antibodies include but are not limited to herceptin, pertuzumab, bevacizumab, keytruuda monoclonal antibody, durvalumab and the like; tyrosine kinase inhibitors include, but are not limited to, lapatinib, noratinib, sunitinib, and the like.
Other known immunotherapeutic agents, particularly those known to treat breast cancer, may also be included in the pharmaceutical compositions of the application, including but not limited to anti-PD-1 antibodies, anti-PD-L1 antibodies, CAR-T cells, and the like.
The polypeptides and pharmaceutical compositions of the application are useful for treating or preventing various diseases for which endostatin is known to treat or prevent, and for alleviating or alleviating various symptoms that endostatin is known to alleviate or alleviate.
For example, the polypeptide of the application or a pharmaceutical composition thereof may be administered to a subject in need thereof for the treatment of breast tumors. The subject may be a mammal, in particular a human.
Breast tumors include, but are not limited to, breast epithelial tumors, breast mesenchymal tumors, fibroepithelial tumors, nipple tumors, male breast cancers. In certain embodiments, the breast tumor is a malignancy, i.e., breast cancer. Breast epithelial tumors include, but are not limited to, micro invasive carcinoma, invasive breast carcinoma, epithelial-myoepithelial tumors, precursor lesions, intraductal proliferative lesions, papillary lesions, and the like. Breast mesenchymal tumors include, but are not limited to, myofibroblastic tumors, liposarcoma, hemangiosarcoma, and the like. The mammary gland fibrous epithelial tumor is leaf tumor or hamartoma, etc. In certain embodiments, the breast cancer is a triple negative breast cancer.
Accordingly, the present application also provides a method of treating a breast tumor, particularly breast cancer, comprising administering to a subject in need thereof a polypeptide of the present application or a pharmaceutical composition thereof.
The polypeptide or the pharmaceutical composition thereof can also improve the curative effects of chemotherapeutic drugs (such as chemotherapeutic drugs for treating breast cancer, especially triple-negative breast cancer), endocrine therapeutic drugs, molecular targeting drugs and immunotherapy. Accordingly, the present application also provides a method of increasing the efficacy of a chemotherapeutic, endocrine therapeutic, molecularly targeted or immunotherapeutic agent, the method comprising administering to a subject in need thereof a polypeptide of the application or a pharmaceutical composition thereof, either before, simultaneously with or after administration of the chemotherapeutic, endocrine therapeutic, molecularly targeted or immunotherapeutic agent.
In the methods and uses of the application, the amount of polypeptide contained in the administered polypeptide or pharmaceutical composition should be a therapeutically effective amount. The breast tumor may be any of the breast tumors described previously. The method of administration is not particularly limited, and may be, for example, oral, subcutaneous, intravenous, or the like. Chemotherapeutic agents, endocrine therapeutic agents, molecular targeted agents, and immunotherapeutics include various chemotherapeutic agents, endocrine therapeutic agents, molecular targeted agents, and various immunotherapeutic agents for treating tumors, particularly breast cancer, including but not limited to the chemotherapy, molecular targeted, and immunotherapeutic agents described previously.
The application also provides application of the polypeptide or the pharmaceutical composition in preparing medicines for improving the curative effects of chemotherapeutic medicines (such as chemotherapeutic medicines for treating breast cancer, especially triple-negative breast cancer), endocrine therapeutic medicines, molecular targeting medicines or immunotherapeutic medicines.
The application also provides a polypeptide for use as a medicament, as described in the preceding aspects or embodiments of the application. The application also provides polypeptides for treating various breast tumors (further triple negative breast cancer) as described above or for improving the efficacy of a chemotherapeutic agent (e.g., a chemotherapeutic agent for treating breast cancer, particularly triple negative breast cancer), an endocrine therapeutic agent, a targeted therapeutic agent or an immunotherapeutic agent, as described in the foregoing aspects or embodiments of the application.
It is to be understood that the various aspects, embodiments, and features of the specific examples described herein may be combined arbitrarily to form preferred embodiments. For example, each of the polypeptides described herein may be used to treat any of the listed diseases.
Examples
The application will be further illustrated with reference to specific examples. The practice of the present application will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, recombinant DNA technology and immunology, which are known to those of skill in the art. These techniques are fully explained in the literature. See, e.g., peptides: chemistry and biology, scientific press, n.luer, h.d. Gu Kubu g, liu Keliang, he Junlin, etc.; basic immunology (Fundamental Virology), second edition, volumes I and II (b.n. fields and d.m. knipe); manual of Experimental immunology (Handbook of Experimental Immunology), volumes I-IV (D.M. Weir and C.C. Blackwell, blackwell Scientific Publications); e.Cright on, protein: structure and molecular Properties (Proteins: structures and Molecular properties) (W.H. Freeman and Company, 1993); l. lehninger, biochemistry (world Publishers, inc. Latest edition); sambrook et al, molecular cloning: laboratory Manual (Molecular Cloning: a Laboratory Manual), second edition, 1989; methods in enzymology (Methods in Engymology) (S.Colowick and N.Kaplan, academic Press, inc.). Furthermore, it should be understood that the term "comprising" in the present application also includes "consisting of … …". As used herein, amino acid sequence numbers, i.e. "SEQ ID NOS: 1-29, 38, 39, 41 and 47-49", refer only to the amino acid sequence itself, excluding N-terminal modifications and C-terminal modifications.
Embodiment one: preparation and modification of polypeptides
The N-terminal modification can be added last, starting with 0.25mM resin, according to the Fmoc protocol standard for polypeptide synthesis, and extending from the carboxy-terminal to the amino-terminal residue by residue according to the following table sequence. After the peptide synthesis is finished, the peptide is cut by a cutting fluid, the resin is filtered by a G6 glass sand funnel, the filtrate is vacuumized, and the C terminal of the polypeptide can be further amidated. The polypeptide product is dissolved in deionized water,purifying by using a C18 column of an Explorer 100 type medium pressure liquid chromatograph, and collecting main peaks step by step. Target peak collection samples were identified by Agilent 1100 reverse phase high pressure liquid chromatography Phenomenex C18 analytical column purity, LCQ Advantage type mass spectrometer molecular weight. The collected solution obtained by the purification of the medium-pressure liquid chromatography is freeze-dried and dissolved in PBS to form polypeptide storage solution, and the polypeptide storage solution is filtered and sterilized by 0.20 mu M and frozen at-80 ℃. HPLC purity identification and MASS MASS spectrum molecular weight identification are shown in FIG. 1.
| Polypeptide numbering | Sequence numbering | Sequence (from N-terminal to C-terminal) |
| P1 | SEQ ID NO:2 | HSHRDFQPVLHLVALNSPLSGGMRGIRGAD |
| P2 | SEQ ID NO:2 | Ac-HSHRDFQPVLHLVALNSPLSGGMRGIRGAD-NH 2 |
| P2T2S18 | SEQ ID NO:6 | Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH 2 |
| P2T2N18 | SEQ ID NO:9 | Ac-HTHRDFQPVLHLVALNSNLSGGMRGIRGAD-NH 2 |
Embodiment two: isolation and culture of Human Umbilical Vein Endothelial Cells (HUVECs)
Preparing umbilical cord preservation solution: 150ml PBS+3 working concentration of double antibody (cyan/chain); preparing a complete medium: 80ml M199+20ml FBS+1ml ECGS+1ml 100X diabody+1 ml heparin solution (0.5% W/V) +1ml 200mM glutamine; preparing a separation instrument: 1 surgical bending disc, 4-5 vascular clamps, 2 surgical scissors and glass culture dish with the diameter of about 10 cm; configuration of type I collagenase: configured to be 1% (W/V).
Taking 20cm of umbilical cord near the fetal end, washing, ligating the two ends, and placing in 150ml of umbilical cord preservation solution; placing in a refrigerator at 4 ℃ for preservation and digestion within 6 hours; checking umbilical cord, removing damaged part, washing umbilical vein completely, injecting 10ml collagenase solution, transferring to 37 ℃ incubator for digestion for 15 min; taking out umbilical cord, collecting digestive juice, washing with PBS, centrifuging, culturing in suspension, and changing liquid after 24 hr to remove cells incapable of adhering to wall.
Embodiment III: inhibition of Human Umbilical Vein Endothelial Cells (HUVECs) and tumor cells by polypeptides
The MTT assay was used to examine the growth inhibitory effect on cells. The principle is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT (3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazolium bromide) into water-insoluble blue-violet crystal Formazan (Formazan) and deposit in cells, while dead cells have no function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and the light absorption value of the formazan can be measured at the wavelength of 490/570nm by an enzyme-linked immunosorbent assay, so that the number of living cells can be indirectly reflected. The amount of MTT crystals formed is proportional to the number of cells over a range of cell numbers.
HUVEC or tumor cells in logarithmic growth phase, discarding culture supernatant, washing with PBS for 1 time, adding 1ml 0.25% pancreatin (4deg.C), digesting for 2min at 37deg.C, adding culture supernatant for neutralization, blowing cells into suspension, and centrifuging for 3min at 1000 rpm. The supernatant was discarded and resuspended in 5ml of medium. According to 3X 10 4 Per ml, 48 well plate, 500 ul/well. 5% CO2, incubated at 37℃for 24 hours. The supernatant of the cultured cells was discarded, and a medium containing the polypeptide (Zn was contained in the medium) 2+ The concentration was 17.39. Mu. Mol/L), and the culture was continued for 48 hours. The supernatant was carefully discarded from each well and gently rinsed 1 time with 450 ul/well PBS. 450ul MTT medium was added to each well and culture was continued for 4h. The culture supernatant was carefully discarded, 450 ul/well of dimethyl sulfoxide was added, and the mixture was placed on a shaking table and shaken at a low speed for 10min under light-shielding conditions. 150ul of the supernatant was transferred to a 96-well ELISA plate, and absorbance values of each well were measured on an ELISA detector OD490nm and 570 nm.
Embodiment four: influence of polypeptide N-terminal and C-terminal modifications on its Activity
The procedure as shown in example one synthesizes a polypeptide of the sequence shown in the following Table as SEQ ID NO. 2, with or without N-terminal and/or C-terminal modifications, wherein Ac is an acetylation modification, NH 2 Is an amidation modification. The HPLC purity and MASS MASS spectrum molecular weight are identified.
| Polypeptide numbering | Sequence (from N-terminal to C-terminal) |
| P1 | HSHRDFQPVLHLVALNSPLSGGMRGIRGAD |
| P2 | Ac-HSHRDFQPVLHLVALNSPLSGGMRGIRGAD-NH 2 |
| P3 | Ac-HSHRDFQPVLHLVALNSPLSGGMRGIRGAD |
| P4 | HSHRDFQPVLHLVALNSPLSGGMRGIRGAD-NH 2 |
Recombinant human endostatin (SEQ ID NO: 1) is commercially available (e.g., genetex accession number GTX65524, bioVision product number 4799-1000, shanghai Bosch Biotechnology Co., ltd. Accession number E2296-05, wuhan Bode Biotechnology Co., ltd. Accession number BP4153, etc.). Recombinant human endostatin drug Endostar (SEQ ID NO: 10) has been purchased from medical institutions.
The biological activity of P1, P2, P3, P4 polypeptides, endostatin, endostar against HUVEC inhibition was measured under the conditions of the assay of 1mg/ml polypeptide concentration and 5mg/ml recombinant human endostatin concentration (approximately equimolar concentrations of polypeptide and endostatin) as shown in example III, and the measurement results are shown in FIG. 2.
Fifth embodiment: research on structure-activity relationship of polypeptides
The polypeptides having the sequences shown in the following table were synthesized as in example one and their HPLC purity identification and MASS MASS spectral molecular weight were determined. The biological activity of the above polypeptides against HUVEC inhibition was measured at a concentration of 1mg/ml as described in example III, and the measurement results are shown in FIG. 4.
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Based on the vascular endothelial chalone structure (PDB database structure number 1 BNL) disclosed in the literature (EMBO J.1998Mar 16;17 (6): 1656-1664), the above polypeptides were subjected to homologous modeling using INSIGHT II software to obtain a dominant conformation of the P2 polypeptide. And then the highest occupied orbital (HOMO) energy and the lowest unoccupied orbital (LUMO) energy of the P2 polypeptide are calculated by adopting a fully activated spatial self-consistent field (CASSCF). Based on the method, an original algorithm of polypeptide amino acid 2-dimensional collaborative iterative space-position field algorithm (2-D synergistic iterative algorithms in spacial point field,2-D SIASPF) is adopted, amino acids in the P2 polypeptide are subjected to pairwise combination simulation iterative replacement, electron density variance accumulation caused by polypeptide Zn ion binding activity domains (1H, 3H and 11H) is calculated and replaced, biological activity corresponding to an actual test amino acid combination is combined, the biological activity synergistic relationship between two sites of each amino acid of the P2 is scored, and the result shows that the synergistic effect of amino acid at the 2 nd site and the amino acid at the 18 th site in the polypeptide on the biological activity is highest.
Example six: influence of polypeptide amino acid substitutions on its Activity
Construction of bit 2P2 peptide combinatorial peptide library of amino acid 18. Polypeptide with the sequence shown in the following table is synthesized by using an AAPPTEC company Apex396 full-automatic high-throughput polypeptide synthesizer, wherein X 1 And X 3 Is any natural amino acid (see table below), X 2 And X 4 Is S, X 5 And X 6 G.
| Polypeptide numbering | Sequence (from N-terminal to C-terminal) |
| P2X2X18 | Ac-HX 1 HRDFQPVLHLVALNX 2 X 3 LX 4 X 5 X 6 MRGIRGAD-NH 2 (SEQ ID NO:38) |
The biological activity of the above polypeptides against HUVEC inhibition was determined as described in example three at a concentration of 1mg/ml and the results are given in the following table.
In the table, lower case a, b, c, d, e, f, g, h, i and j represent respectively:
a: cell activity 0-10%; b: cell activity 11-20%; c: cell activity 21-30%; d: cell activity 31-40%; e: cell activity 41-50%; f: cell activity 51-60%; g: cell activity 61-70%; h: cell activity 71-80%; i: cell activity 81-90%; j: the cell activity is 91-100%.
Embodiment seven: in vitro growth inhibition of human triple negative breast cancer cell BT-483 by polypeptide
According to the method described in example III, each polypeptide was tested for inhibition of in vitro growth of human triple negative breast cancer cells BT-483 by the polypeptides shown in the following table at equimolar concentrations (150. Mu.M concentration of P2T2S18 polypeptide corresponds to about 0.5 mg/ml), and the results of the test are shown in FIG. 5a and the following table.
The results show that the Endological and P2 have little effect on BT-483 cells at 150uM concentration. The inhibition effect of P2T2S18, P2T2N18 and P2T2S18-29 on BT-483 cells is obvious, the activity of tumor cells is less than 15%, and the tumor cells are almost completely killed. P2T2S18-20, P2T2S18-25, P2T2S18-35, P2T2S18-40, P2T2S18-45 show different degrees of tumor inhibition.
| Polypeptide numbering | Sequence numbering | Cell viability (%) |
| P2T2S18-45 | SEQ ID NO:3 | 44 |
| P2T2S18-40 | SEQ ID NO:4 | 39 |
| P2T2S18-35 | SEQ ID NO:5 | 26 |
| P2T2S18 | SEQ ID NO:6 | 14 |
| P2T2S18-29 | SEQ ID NO:41 | 9 |
| P2T2S18-25 | SEQ ID NO:7 | 17 |
| P2T2S18-20 | SEQ ID NO:8 | 28 |
| P2T2-15 | SEQ ID NO:24 | 86 |
| P2T2N18 | SEQ ID NO:9 | 20 |
| P2 | SEQ ID NO:2 | 99 |
| endostar | SEQ ID NO:10 | 93 |
Example eight: in vitro growth inhibition of human triple negative breast cancer cell BT-483 by polypeptide
The polypeptides listed below were synthesized as in example one, with HPLC purity identification and MASS MASS spectral molecular weight identification shown in FIGS. 1 and 3a-3f. The inhibition of BT-483 was tested as described in example three, and the results are shown in FIG. 5b.
Example nine: in vitro growth inhibition of polypeptides on multiple human triple negative breast cancer cells
According to the method described in example III, each of the polypeptides was tested for inhibition of growth in vitro of the P2 (SEQ ID NO: 2), P2T2S18 (SEQ ID NO: 6), P2T2N18 (SEQ ID NO: 9) and P2T2S18-29 (SEQ ID NO: 41) polypeptides against human triple negative breast cancer cells BT-483, MDA-MB-453 and MDA-MB-468 at equal gradient concentrations, as shown in FIGS. 6a, 6b and the following tables.
The result shows that at 0.5mg/ml, the P2 has little inhibition effect on various human triple negative breast cancer cells, and the cell survival rate is more than 90%; and the P2T2S18, the P2T2N18 and the P2T2S18-29 can play an obvious inhibiting role on a plurality of human triple negative breast cancer cells. P2T2S18, P2T2N18 and P2T2S18-29 almost completely kill human triple negative breast cancer cells at 2.5mg/ml, and the survival rate of partial tumor cells is less than 10%. Accordingly, the IC50 concentration of P2 on human triple negative breast cancer cells is significantly higher than that of P2T2S18, P2T2N18 and P2T2S18-29, even up to 17-fold. The micrograph shows (FIG. 6 b) that the respective concentrations of P2 show no effect on MDA-MB-453 cells. Whereas the P2T2S18 polypeptide causes a significant change in cell morphology, most cells are in a dead state at a concentration of 0.7 mg/ml; as the concentration of polypeptide increases, MDA-MB-453 fuses with each other to form a bolus. It can be seen that there is a substantial difference in the effect of P2 and P2T2S18 on MDA-MB-453 cells.
Example ten: in vitro growth inhibition of polypeptides on multiple human breast cancer cells
According to the method described in example III, each polypeptide was tested for the inhibition of the growth of human breast cancer cells SK-BR-3, T-47D and BT-474 in vitro by P2 (SEQ ID NO: 2) and P2T2S18-29 (SEQ ID NO: 41) at equal gradient concentrations, the expression and test results of each cell surface receptor being shown in FIG. 7 and the following table.
The result shows that at 0.5mg/ml, the P2 has little inhibition effect on human breast cancer cells, and the cell survival rate is more than 80 percent; the P2T2S18-29 can also play a significant role in inhibiting non-triple negative breast cancer cells, the cell survival rate of the non-triple negative breast cancer cells is less than 50% at 0.5mg/ml, and the cell survival rate is not obviously related to the expression of surface receptors. Accordingly, the IC50 concentration of P2 on human triple negative breast cancer cells is significantly higher than that of P2T2S18-29, even up to 17-fold.
Example eleven: establishment of breast cancer in-vivo tumor model
Taking out logarithmic phase human breast cancer tumor cells with good in vitro culture condition, subcutaneously inoculating nude mice with 5×10 cells 6 Culture cell suspension of tumor cells 100ul. After 15 days, well-grown solid tumors were harvested, aseptically cut into uniform small pieces of about 3mm size, and inoculated subcutaneously in the right axilla of each nude mouse with a trocar. Animals with oversized and undersized tumors were eliminated by regrouping them according to tumor size 10-14 days after inoculation, with average tumor volumes per group being substantially consistent. Each group was given a test drug according to the test protocol. The major (a) and minor (b) diameters of the tumor mass were measured 2 times per week. Animals are sacrificed after the test is finished, tumor mass is dissected, the tumor is weighed, and the pictures are taken. Tumor volume tv=1/2×a×b 2 The method comprises the steps of carrying out a first treatment on the surface of the Tumor relative volume rtv=vt/Vo, vo is the tumor volume measured at the time of caging (i.e. 1 day prior to dosing), vt is the tumor volume at each measurement. Tumor inhibition (%) = (1-T/C) ×100%, where T is the average tumor volume of the treatment group and C is the average tumor volume of the negative control group.
Embodiment twelve: inhibition of human breast cancer cell growth by polypeptide
The procedure as in example one synthesizes polypeptides having the sequences shown in the following table:
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in a method as shown in example eleven, an in vivo tumor model of human breast cancer was established, the following 6 groups of 6 animals per group were tested except for 9 animals in the negative control group, wherein the dosages of the polypeptide and endostatin were approximately equimolar, and the endostar group used the marketed drug Endostar (SEQ ID NO: 10):
1) Negative control group (normal saline, sc subcutaneously, 2 times/day, 21 days of continuous administration);
2) Cyclophosphamide CTX (30 mg/kg, ip,1 time/day, 7 days of continuous administration);
3) Group P2 (15 mg/kg/time, sc,2 times/day, 21 days of continuous administration);
4) Group P2T2S18 (15 mg/kg/time, sc,2 times/day, 21 days of continuous administration);
5) Group P2T2S18-29 (15 mg/kg/sc, 2 times/day, 21 days of continuous administration);
6) endostar (50 mg/kg/sc, 2 times/day, 21 days of continuous dosing).
Embodiment thirteen: inhibition of HUVEC in vitro growth by polypeptide
The polypeptides having the sequences shown in the following table were synthesized and subjected to HPLC purity identification and MASS spectrometry molecular weight identification as described in example one.
| Polypeptide numbering | Sequence numbering | Sequence (from N-terminal to C-terminal) |
| P2 | SEQ ID NO:2 | Ac-HSHRDFQPVLHLVALNSPLSGGMRGIRGAD-NH 2 |
| P2T2S18 | SEQ ID NO:6 | Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH 2 |
| P2T2N18 | SEQ ID NO:9 | Ac-HTHRDFQPVLHLVALNSNLSGGMRGIRGAD-NH 2 |
| P2S18 | SEQ ID NO:25 | Ac-HSHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH 2 |
| P2N18 | SEQ ID NO:26 | Ac-HSHRDFQPVLHLVALNSNLSGGMRGIRGAD-NH 2 |
| P2T2 | SEQ ID NO:11 | Ac-HTHRDFQPVLHLVALNSPLSGGMRGIRGAD-NH 2 |
The sequence of the recombinant vascular endothelial chalone (endostatin) is shown as SEQ ID NO. 1, and the Endostar (endostar) of the marketed medicine is shown as SEQ ID NO. 10.
Inhibition of HUVEC was tested as described in example three, with the results set forth in the following table. The results show that the biological activity of P2T2S18 and P2T2N18 is obviously higher than that of P2, and the I thereofC 50 The concentration was reduced by about 10-fold compared to P2. The biological activity of the polypeptide P2T2 carrying single point mutation of amino acid at position 2 and the biological activity of the polypeptide P2N18 and P2S18 carrying single point mutation of amino acid at position 18 are lower than that of P2, so that the high biological activity of the polypeptide P2T2S18 and the polypeptide P2T2N18 is generated by unexpected synergistic effect caused by common mutation of amino acid at position 2 and amino acid at position 18, and the high biological activity structure of the polypeptide P2T2S18 and the polypeptide P2T2N18 which are related to the application are difficult to obtain by a conventional single point mutation scanning method.
Fourteen examples: inhibition of HUVEC in vitro growth by polypeptide
The polypeptides having the sequences shown in the following table were synthesized by the method described in example one, and HPLC purity identification and MASS MASS spectral molecular weight identification are shown in FIGS. 1 and 8a-8j.
The polypeptides were tested for their inhibitory activity against HUVEC as described in example three. Each polypeptide was tested at equimolar concentrations, where the concentration of 300uM of the P2T2S18 polypeptide corresponds to about 1mg/ml. The results are shown in FIG. 9 and the following table, which show that the C-terminal end of P2T2S18 is still biologically active after shortening or lengthening within a certain range.
| Polypeptide numbering | Sequence numbering | Cell viability (%) |
| P2T2S18-45 | SEQ ID NO:3 | 80 |
| P2T2S18-40 | SEQ ID NO:4 | 49 |
| P2T2S18-35 | SEQ ID NO:5 | 15 |
| P2T2S18 | SEQ ID NO:6 | 2 |
| P2T2S18-25 | SEQ ID NO:7 | 31 |
| P2T2S18-20 | SEQ ID NO:8 | 52 |
| P2T2-15 | SEQ ID NO:24 | 95 |
Example fifteen: inhibition of tumor cell and HUVEC in vitro growth by polypeptide
The polypeptides having the sequences shown in the following Table were synthesized as in example one, and the inhibition of HUVEC was tested at a polypeptide concentration of 0.1mg/ml as in example three, and the results are shown in FIG. 10.
| Polypeptide numbering | Sequence numbering | Sequence (from N-terminal to C-terminal) |
| P2T2S18 | SEQ ID NO:6 | Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH 2 |
| P2T2A17S18 | SEQ ID NO:27 | Ac-HTHRDFQPVLHLVALNASLSGGMRGIRGAD-NH 2 |
| P2T2S18T20 | SEQ ID NO:28 | Ac-HTHRDFQPVLHLVALNSSLTGGMRGIRGAD-NH 2 |
| P2T2A17S18T20 | SEQ ID NO:29 | Ac-HTHRDFQPVLHLVALNASLTGGMRGIRGAD-NH 2 |
Example sixteen: inhibition of HUVEC in vitro growth by polypeptide
The polypeptides having the sequences shown in the following table were synthesized by the method described in example one, and HPLC purity identification and MASS MASS spectral molecular weight identification are shown in FIGS. 11a and 11b. Inhibition of HUVEC was tested as described in example three. The measurement results are shown in FIG. 12. The results show that P2T2S18 and P2T2S18-29 are similar in biological activity and are both significantly higher than P2.
| Polypeptide numbering | Sequence numbering | Sequence (from N-terminal to C-terminal) |
| P2T2S18-29 | SEQ ID NO:41 | Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGA-NH 2 |
The particular embodiments described above are illustrative only and not limiting. The scope of the application is to be defined by the appended claims. Those skilled in the art will appreciate that various modifications and changes can be made to the technical solution of the present application without departing from the spirit and scope thereof, and such modifications and changes are still included in the scope of the present application.
Sequence listing
<110> Shanghai Jibei pharmaceutical technology Co., ltd
<120> polypeptide for treating breast tumor
<130> 170293
<160> 49
<170> PatentIn version 3.3
<210> 1
<211> 183
<212> PRT
<213> Homo sapiens (Homo sapiens)
<400> 1
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln
20 25 30
Cys Phe Gln Gln Ala Arg Ala Val Gly Leu Ala Gly Thr Phe Arg Ala
35 40 45
Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser Ile Val Arg Arg Ala
50 55 60
Asp Arg Ala Ala Val Pro Ile Val Asn Leu Lys Asp Glu Leu Leu Phe
65 70 75 80
Pro Ser Trp Glu Ala Leu Phe Ser Gly Ser Glu Gly Pro Leu Lys Pro
85 90 95
Gly Ala Arg Ile Phe Ser Phe Asp Gly Lys Asp Val Leu Arg His Pro
100 105 110
Thr Trp Pro Gln Lys Ser Val Trp His Gly Ser Asp Pro Asn Gly Arg
115 120 125
Arg Leu Thr Glu Ser Tyr Cys Glu Thr Trp Arg Thr Glu Ala Pro Ser
130 135 140
Ala Thr Gly Gln Ala Ser Ser Leu Leu Gly Gly Arg Leu Leu Gly Gln
145 150 155 160
Ser Ala Ala Ser Cys His His Ala Tyr Ile Val Leu Cys Ile Glu Asn
165 170 175
Ser Phe Met Thr Ala Ser Lys
180
<210> 2
<211> 30
<212> PRT
<213> artificial sequence
<220>
<223> fragments of human endostatin
<400> 2
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 3
<211> 45
<212> PRT
<213> artificial sequence
<220>
<223> mutants of fragments of human endostatin
<400> 3
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Ser Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln
20 25 30
Cys Phe Gln Gln Ala Arg Ala Val Gly Leu Ala Gly Thr
35 40 45
<210> 4
<211> 40
<212> PRT
<213> artificial sequence
<220>
<223> mutants of fragments of human endostatin
<400> 4
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Ser Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln
20 25 30
Cys Phe Gln Gln Ala Arg Ala Val
35 40
<210> 5
<211> 35
<212> PRT
<213> artificial sequence
<220>
<223> mutants of fragments of human endostatin
<400> 5
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Ser Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln
20 25 30
Cys Phe Gln
35
<210> 6
<211> 30
<212> PRT
<213> artificial sequence
<220>
<223> mutants of fragments of human endostatin
<400> 6
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Ser Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 7
<211> 25
<212> PRT
<213> artificial sequence
<220>
<223> mutants of fragments of human endostatin
<400> 7
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Ser Leu Ser Gly Gly Met Arg Gly
20 25
<210> 8
<211> 20
<212> PRT
<213> artificial sequence
<220>
<223> mutants of fragments of human endostatin
<400> 8
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Ser Leu Ser
20
<210> 9
<211> 30
<212> PRT
<213> artificial sequence
<220>
<223> mutants of fragments of human endostatin
<400> 9
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Asn Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 10
<211> 192
<212> PRT
<213> artificial sequence
<220>
<223> Endosis of recombinant human vascular endothelial chalone drug
<400> 10
Met Gly Gly Ser His His His His His His Ser His Arg Asp Phe Gln
1 5 10 15
Pro Val Leu His Leu Val Ala Leu Asn Ala Pro Leu Ser Gly Gly Met
20 25 30
Arg Gly Ile Arg Gly Ala Asp Phe Gln Cys Phe Gln Gln Ala Arg Ala
35 40 45
Val Gly Leu Ala Gly Thr Phe Arg Ala Phe Leu Ser Ser Arg Leu Gln
50 55 60
Asp Leu Tyr Ser Ile Val Arg Arg Ala Asp Arg Ala Ala Val Pro Ile
65 70 75 80
Val Asn Leu Lys Asp Glu Leu Leu Phe Pro Ser Trp Glu Ala Leu Phe
85 90 95
Ser Gly Ser Glu Gly Pro Leu Lys Pro Gly Ala Arg Ile Phe Ser Phe
100 105 110
Asp Gly Lys Asp Val Leu Arg His Pro Thr Trp Pro Gln Lys Ser Val
115 120 125
Trp His Gly Ser Asp Pro Asn Gly Arg Arg Leu Thr Glu Ser Tyr Cys
130 135 140
Glu Thr Trp Arg Thr Glu Ala Pro Ser Ala Thr Gly Gln Ala Ser Ser
145 150 155 160
Leu Leu Gly Gly Arg Leu Leu Gly Gln Ser Ala Ala Ser Cys His His
165 170 175
Ala Tyr Ile Val Leu Cys Ile Glu Asn Ser Phe Met Thr Ala Ser Lys
180 185 190
<210> 11
<211> 30
<212> PRT
<213> artificial sequence
<220>
<223> mutants of fragments of human endostatin
<400> 11
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 12
<211> 30
<212> PRT
<213> artificial sequence
<220>
<223> mutants of fragments of human endostatin
<400> 12
His Ala His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 13
<211> 30
<212> PRT
<213> artificial sequence
<220>
<223> mutants of fragments of human endostatin
<400> 13
His Glu His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 14
<211> 30
<212> PRT
<213> artificial sequence
<220>
<223> mutants of fragments of human endostatin
<400> 14
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Ala Asn
1 5 10 15
Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 15
<211> 30
<212> PRT
<213> artificial sequence
<220>
<223> mutants of fragments of human endostatin
<400> 15
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Ala
1 5 10 15
Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 16
<211> 30
<212> PRT
<213> artificial sequence
<220>
<223> mutants of fragments of human endostatin
<400> 16
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Thr Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 17
<211> 30
<212> PRT
<213> artificial sequence
<220>
<223> mutants of fragments of human endostatin
<400> 17
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ala Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 18
<211> 30
<212> PRT
<213> artificial sequence
<220>
<223> mutants of fragments of human endostatin
<400> 18
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Ala Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 19
<211> 30
<212> PRT
<213> artificial sequence
<220>
<223> mutants of fragments of human endostatin
<400> 19
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Pro Ala Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 20
<211> 30
<212> PRT
<213> artificial sequence
<220>
<223> mutants of fragments of human endostatin
<400> 20
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Pro Leu Ala Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 21
<211> 30
<212> PRT
<213> artificial sequence
<220>
<223> mutants of fragments of human endostatin
<400> 21
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Pro Leu Ser Ala Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 22
<211> 30
<212> PRT
<213> artificial sequence
<220>
<223> mutants of fragments of human endostatin
<400> 22
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Pro Leu Ser Gly Ala Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 23
<211> 30
<212> PRT
<213> artificial sequence
<220>
<223> mutants of fragments of human endostatin
<400> 23
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Pro Leu Ser Gly Gly Met Arg Gly Asp Arg Gly Ala Asp
20 25 30
<210> 24
<211> 17
<212> PRT
<213> artificial sequence
<220>
<223> mutants of fragments of human endostatin
<400> 24
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser
<210> 25
<211> 30
<212> PRT
<213> artificial sequence
<220>
<223> mutants of fragments of human endostatin
<400> 25
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Ser Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 26
<211> 30
<212> PRT
<213> artificial sequence
<220>
<223> mutants of fragments of human endostatin
<400> 26
His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Asn Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 27
<211> 30
<212> PRT
<213> artificial sequence
<220>
<223> mutants of fragments of human endostatin
<400> 27
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ala Ser Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 28
<211> 30
<212> PRT
<213> artificial sequence
<220>
<223> mutants of fragments of human endostatin
<400> 28
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Ser Leu Thr Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 29
<211> 30
<212> PRT
<213> artificial sequence
<220>
<223> mutants of fragments of human endostatin
<400> 29
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ala Ser Leu Thr Gly Gly Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 30
<211> 549
<212> DNA
<213> Homo sapiens (Homo sapiens)
<400> 30
cacagccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag ccccctgtca 60
ggcggcatgc ggggcatccg cggggccgac ttccagtgct tccagcaggc gcgggccgtg 120
gggctggcgg gcaccttccg cgccttcctg tcctcgcgcc tgcaggacct gtacagcatc 180
gtgcgccgtg ccgaccgcgc agccgtgccc atcgtcaacc tcaaggacga gctgctgttt 240
cccagctggg aggctctgtt ctcaggctct gagggtccgc tgaagcccgg ggcacgcatc 300
ttctcctttg acggcaagga cgtcctgagg caccccacct ggccccagaa gagcgtgtgg 360
catggctcgg accccaacgg gcgcaggctg accgagagct actgtgagac gtggcggacg 420
gaggctccct cggccacggg ccaggcctcc tcgctgctgg ggggcaggct cctggggcag 480
agtgccgcga gctgccatca cgcctacatc gtgctctgca ttgagaacag cttcatgact 540
gcctccaag 549
<210> 31
<211> 135
<212> DNA
<213> artificial sequence
<220>
<223> coding sequences of mutants of human endostatin fragments
<400> 31
cacacccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag cagcctgtca 60
ggcggcatgc ggggcatccg cggggccgac ttccagtgct tccagcaggc gcgggccgtg 120
gggctggcgg gcacc 135
<210> 32
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> coding sequences of mutants of human endostatin fragments
<400> 32
cacacccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag cagcctgtca 60
ggcggcatgc ggggcatccg cggggccgac ttccagtgct tccagcaggc gcgggccgtg 120
<210> 33
<211> 105
<212> DNA
<213> artificial sequence
<220>
<223> coding sequences of mutants of human endostatin fragments
<400> 33
cacacccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag cagcctgtca 60
ggcggcatgc ggggcatccg cggggccgac ttccagtgct tccag 105
<210> 34
<211> 90
<212> DNA
<213> artificial sequence
<220>
<223> coding sequences of mutants of human endostatin fragments
<400> 34
cacacccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag cagcctgtca 60
ggcggcatgc ggggcatccg cggggccgac 90
<210> 35
<211> 75
<212> DNA
<213> artificial sequence
<220>
<223> coding sequences of mutants of human endostatin fragments
<400> 35
cacacccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag cagcctgtca 60
ggcggcatgc ggggc 75
<210> 36
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> coding sequences of mutants of human endostatin fragments
<400> 36
cacacccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag cagcctgtca 60
<210> 37
<211> 90
<212> DNA
<213> artificial sequence
<220>
<223> coding sequences of mutants of human endostatin fragments
<400> 37
cacacccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag caacctgtca 60
ggcggcatgc ggggcatccg cggggccgac 90
<210> 38
<211> 30
<212> PRT
<213> artificial sequence
<220>
<221> MISC_FEATURE
<222> (2)..(2)
<223> Xaa is any amino acid
<220>
<221> MISC_FEATURE
<222> (17)..(17)
<223> Xaa is S, A, L, I, V or T
<220>
<221> MISC_FEATURE
<222> (18)..(18)
<223> Xaa is any amino acid
<220>
<221> MISC_FEATURE
<222> (20)..(20)
<223> Xaa is S or T
<220>
<221> MISC_FEATURE
<222> (21)..(21)
<223> Xaa is G, A, L, I or V
<220>
<221> MISC_FEATURE
<222> (22)..(22)
<223> Xaa is G, A, L, I or V
<400> 38
His Xaa His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Xaa Xaa Leu Xaa Xaa Xaa Met Arg Gly Ile Arg Gly Ala Asp
20 25 30
<210> 39
<211> 40
<212> PRT
<213> artificial sequence
<220>
<223> mutants of human endostatin fragments
<400> 39
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Asn Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln
20 25 30
Cys Phe Gln Gln Ala Arg Ala Val
35 40
<210> 40
<211> 120
<212> DNA
<213> artificial sequence
<220>
<223> coding sequences of mutants of human endostatin fragments
<400> 40
cacacccacc gcgacttcca gccggtgctc cacctggttg cgctcaacag caacctgtca 60
ggcggcatgc ggggcatccg cggggccgac ttccagtgct tccagcaggc gcgggccgtg 120
<210> 41
<211> 29
<212> PRT
<213> artificial sequence
<220>
<223> mutants of human endostatin fragments
<400> 41
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Ser Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala
20 25
<210> 42
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> Tag
<400> 42
Arg Arg Arg Arg Arg
1 5
<210> 43
<211> 6
<212> PRT
<213> artificial sequence
<220>
<223> Tag
<400> 43
His His His His His His
1 5
<210> 44
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> Tag
<400> 44
Asp Tyr Lys Asp Asp Asp Asp Lys
1 5
<210> 45
<211> 8
<212> PRT
<213> artificial sequence
<220>
<223> Tag
<400> 45
Trp Ser His Pro Gln Phe Glu Lys
1 5
<210> 46
<211> 10
<212> PRT
<213> artificial sequence
<220>
<223> Tag
<400> 46
Trp Gln Lys Leu Ile Ser Glu Glu Asp Leu
1 5 10
<210> 47
<211> 28
<212> PRT
<213> artificial sequence
<220>
<223> mutants of human endostatin fragments
<400> 47
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Ser Leu Ser Gly Gly Met Arg Gly Ile Arg Gly
20 25
<210> 48
<211> 31
<212> PRT
<213> artificial sequence
<220>
<223> mutants of human endostatin fragments
<400> 48
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Ser Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe
20 25 30
<210> 49
<211> 32
<212> PRT
<213> artificial sequence
<220>
<223> mutants of human endostatin fragments
<400> 49
His Thr His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn
1 5 10 15
Ser Ser Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln
20 25 30
Claims (15)
1. Use of a polypeptide or a pharmaceutical composition thereof in the manufacture of a medicament for the treatment of a breast tumor, wherein the polypeptide is a fragment of endostatin having an N-terminal length of 45 amino acid residues or less and comprising at least amino acid residues 1-20 of the N-terminal, and wherein the amino acid residue 2 of the polypeptide is T, the amino acid residue 18 is S or N; wherein the amino acid sequence of the endostatin is shown as SEQ ID NO. 1.
2. The use according to claim 1, wherein the amino acid at position 17 of the polypeptide is S or a.
3. The use of claim 1, wherein the amino acid residue at position 20 of the polypeptide is S or T.
4. The use according to claim 1, wherein the polypeptide comprises at least the amino acid sequence of SEQ ID NO:38, wherein the amino acid residue at position 1-22 is T, the amino acid at position 17 is S or A, the amino acid residue at position 18 is S or N, the amino acid residue at position 20 is S or T, the amino acid residue at position 21 is G, and the amino acid residue at position 22 is G.
5. The use according to claim 4, wherein the polypeptide comprises at least the amino acid sequence of SEQ ID NO:38 amino acid residues 1-25.
6. The use according to claim 1, wherein the amino acid sequence of the polypeptide is as set forth in any one of SEQ ID NOs 3, 4, 5, 6, 7, 8, 27-29, 39, 41 and 47-49.
7. The use according to claim 1, wherein the polypeptide consists of SEQ ID NO 38, wherein the amino acid residue at position 2 is T, the amino acid at position 17 is S or A, the amino acid residue at position 18 is N or S, the amino acid residue at position 20 is S or T, the amino acid residue at position 21 is G, and the amino acid residue at position 22 is G.
8. The use according to claim 1, wherein the polypeptide is selected from the group consisting of amino acid sequences of amino acid residues 1 to 39, 38, 37, 36, 34, 33, 32, 31, 29, 28, 27 or 26 of SEQ ID NO. 4.
9. The use according to claim 1, wherein the polypeptide is selected from the group consisting of amino acid sequences of amino acid residues 1 to 39, 38, 37, 36, 35, 34, 33, 32, 31, 29, 28, 27, 26 or 25 of SEQ ID NO. 39.
10. The use according to any one of claims 1 to 9, wherein the amino acid residue at position 1 of the N-terminal end of the polypeptide is histidine which is formylated, acetylated, propionylated or butyrylated and the amino acid at position 1 of the C-terminal end is PEG, cholesterol or amidated.
11. The use according to claim 1, wherein the polypeptide is selected from the group consisting of:
HTHRDFQPVLHLVALNSSLSGGMRGIRGAD；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGAD；
HTHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH 2 ；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGAD-NH 2 ；
Ac-HTHRDFQPVLHLVALNSSLSGGMRG-NH 2 ；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGADFQCFQ-NH 2 ；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGADFQCFQQARAV-NH 2 ；
HTHRDFQPVLHLVALNSNLSGGMRGIRGAD；
Ac-HTHRDFQPVLHLVALNSNLSGGMRGIRGAD；
HTHRDFQPVLHLVALNSNLSGGMRGIRGAD-NH 2 ；
Ac-HTHRDFQPVLHLVALNSNLSGGMRGIRGAD-NH 2 ；
Ac-HTHRDFQPVLHLVALNASLSGGMRGIRGAD-NH 2 ；
Ac-HTHRDFQPVLHLVALNSSLTGGMRGIRGAD-NH 2 ；
Ac-HTHRDFQPVLHLVALNASLTGGMRGIRGAD-NH 2 ；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRG-NH 2 ；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRG；
HTHRDFQPVLHLVALNSSLSGGMRGIRG-NH 2 ；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGA-NH 2 ；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGA；
HTHRDFQPVLHLVALNSSLSGGMRGIRGA-NH 2 ；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGADF-NH 2 ；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGADF；
HTHRDFQPVLHLVALNSSLSGGMRGIRGADF-NH 2 ；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGADFQ-NH 2 ；
Ac-HTHRDFQPVLHLVALNSSLSGGMRGIRGADFQ; and
HTHRDFQPVLHLVALNSSLSGGMRGIRGADFQ-NH 2 ；
wherein Ac is an acetyl modification, NH 2 Is an amidation modification.
12. The use according to any one of claims 1 to 9 and 11, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
13. The use according to any one of claims 1-9 and 11, wherein the breast tumor is selected from the group consisting of triple negative breast cancer, both negative breast cancer, one negative breast cancer and all three positive breast cancers, wherein HER2, estrogen receptor, and progestin receptor are all negative.
14. The use according to any one of claims 1-9 and 11, wherein the breast tumor is selected from the group consisting of a breast epithelial tumor, a breast mesenchymal tumor, a breast fibroepithelial tumor, a nipple tumor and a male breast cancer.
15. The use according to claim 14, wherein,
the breast epithelial tumor is selected from the group consisting of micro invasive carcinoma, invasive breast cancer, epithelial-myoepithelial tumor, precursor lesions, intraductal proliferative lesions, and papillary lesions;
the breast mesenchymal tumor package is selected from myofibroblastic tumor, liposarcoma and angiosarcoma;
The mammary gland fibrous epithelial tumor is phylloma or hamartoma.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710026136.3A CN108295244B (en) | 2017-01-13 | 2017-01-13 | Polypeptides for treating breast tumors |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710026136.3A CN108295244B (en) | 2017-01-13 | 2017-01-13 | Polypeptides for treating breast tumors |
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| CN114931634B (en) * | 2022-03-18 | 2023-03-17 | 广州达博生物制品有限公司 | Combined treatment method and pharmaceutical application of E10A and PD1 monoclonal antibody to tumors |
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| CN1875104A (en) * | 2003-08-29 | 2006-12-06 | 儿童医学中心公司 | Anti-angiogenic peptides from the N-terminus of endostatin |
| CN102924578A (en) * | 2011-08-09 | 2013-02-13 | 哈药集团技术中心 | Anti-tumor polypeptide, preparation method and anti-tumor applications thereof |
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| CN1875104A (en) * | 2003-08-29 | 2006-12-06 | 儿童医学中心公司 | Anti-angiogenic peptides from the N-terminus of endostatin |
| CN102924578A (en) * | 2011-08-09 | 2013-02-13 | 哈药集团技术中心 | Anti-tumor polypeptide, preparation method and anti-tumor applications thereof |
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