Polypeptide and application thereof
阅读说明：本技术 一种多肽及其应用 (Polypeptide and application thereof ) 是由 何明亮 陈缨 周方行 于 2018-06-27 设计创作，主要内容包括：本发明提供一种多肽及其应用。该多肽包括带穿膜肽的线性多肽(如SEQ ID NO：1至SEQ ID NO：5所示)、无穿膜肽的环状多肽(如SEQ ID NO：6至SEQ ID NO：8所示)和突变体多肽(SEQ ID NO：9、SEQ ID NO：11至SEQ ID NO：22所示)中的一种或多种。本发明的多肽缩短了在先申请中TAP21的肽段,并且能够保持原有活性并且具有更高的活性,降低了工艺的难度和合成成本；其在细胞以及动物模型中具有良好的抗肿瘤性能,且效果明显优于已有多肽产品,其在抑制肿瘤或阻断肿瘤细胞增殖中的应用为抗肿瘤治疗提供了良好的前景。(The invention provides a polypeptide and application thereof. The polypeptide comprises one or more of linear polypeptide (shown as SEQ ID NO: 1 to SEQ ID NO: 5) with a cell-penetrating peptide, cyclic polypeptide (shown as SEQ ID NO: 6 to SEQ ID NO: 8) without the cell-penetrating peptide and mutant polypeptide (shown as SEQ ID NO: 9, SEQ ID NO: 11 to SEQ ID NO: 22). The polypeptide shortens the peptide segment of TAP21 in the prior application, can keep the original activity and has higher activity, and reduces the difficulty of the process and the synthesis cost; the polypeptide has good anti-tumor performance in cells and animal models, the effect is obviously better than that of the existing polypeptide products, and the application of the polypeptide in inhibiting tumors or blocking tumor cell proliferation provides good prospects for anti-tumor treatment.)
1. A polypeptide comprising one or more of a linear polypeptide with a cell-penetrating peptide, a cyclic polypeptide without a cell-penetrating peptide, and a mutant polypeptide;
the amino acid sequence of the linear polypeptide with the cell-penetrating peptide comprises one or more of the following amino acid sequences:
RKKRRQRRRLFPYPTYAAVHRHPFL (shown in SEQ ID NO: 1);
RKKRRQRRRLFPYPTYASVHRHPFL (shown in SEQ ID NO: 2);
RKKRRQRRRLFPYPTYAAAVHRHPFL (shown in SEQ ID NO: 3);
RKKRRQRRRLFPYPTYAASAAVHRHPFL (shown in SEQ ID NO: 4);
RKKRRQRRRLFPYPTYAAVHRHPF (shown in SEQ ID NO: 5);
the amino acid sequence of the cyclic polypeptide without the cell-penetrating peptide comprises one or more of the following amino acid sequences:
LFPYPYTYAAVHRHP (shown in SEQ ID NO: 6);
LFPYPTY (shown as SEQ ID NO: 7);
RGDFLFPYPTYR (shown in SEQ ID NO: 8);
the amino acid sequence of the mutant polypeptide includes one or more of the following amino acid sequences:
LFPAAAT (shown as SEQ ID NO: 9);
RKKRRQRRRLFPYPYTYAAVHRHPFL (shown in SEQ ID NO: 11);
RKKRRQRRRLFPYPYTYAASHRHPFL (shown in SEQ ID NO: 12);
RKKRRQRRRFPYPYTYAASHRHPFL (shown in SEQ ID NO: 13);
RKKRRQRRRPYPYTYAASHRHPFL (shown in SEQ ID NO: 14);
RKKRRQRRRLFPYPYTAASHRHPFL (shown in SEQ ID NO: 15);
RKKRRQRRRLFPYPYAASHRHPFL (shown in SEQ ID NO: 16);
RKKRRQRRRFPYPYTAASHRHPFL (shown in SEQ ID NO: 17);
RKKRRQRRRLFPYPYTYAASHRHPF (shown in SEQ ID NO: 18);
RKKRRQRRRLFPYPYTYAASHRHP (shown in SEQ ID NO: 19);
RKKRRQRRRFPYPYTYAASHRHPF (shown in SEQ ID NO: 20);
RKKRRQRRRPYPYTYAASHRHP (shown in SEQ ID NO: 21);
PYPYTYAASHRHP (shown in SEQ ID NO: 22).
2. The polypeptide of claim 1, wherein the polypeptide is synthesized by using D-form amino acids according to the amino acid sequence of the polypeptide.
3. The polypeptide of claim 1, wherein the polypeptide is synthesized using beta-type amino acids based on its amino acid sequence.
4. A modified polypeptide which is modified by N-terminal acetylation and C-terminal amidation of the polypeptide of any one of claims 1 to 3.
5. A pharmaceutical composition for treating a tumor comprising the polypeptide of any one of claims 1-3, the modified polypeptide of claim 4, and an excipient thereof.
6. Use of a polypeptide according to any one of claims 1-3, a modified polypeptide according to claim 4 for the manufacture of a medicament, polypeptide medicament or combination medicament for the treatment of a tumor or cancer in an individual.
7. The use according to claim 6, wherein the tumor or cancer is selected from lung cancer, nasopharyngeal cancer, laryngeal cancer, gastric cancer, liver cancer, esophageal cancer, intestinal cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, leukemia, lymphoma, hemangioma, bone cancer, cervical cancer, cancer of the sub-organs, ovarian cancer, adipose cancer, breast cancer, brain tumor, squamous carcinoma, skin cancer, thyroid cancer, lip cancer, melanoma, tongue cancer, thymus cancer and brain or central nervous system cancer.
8. Use according to claim 7, wherein the tumor or cancer is selected from liver cancer, gastric cancer, colorectal cancer, breast cancer or lung cancer.
9. The use of claim 6, comprising administering to the individual an effective amount of said drug, said polypeptide drug, or said combination.
10. The use of claim 9, wherein the effective amount ranges from 0.1nM to 0.1M.
Technical Field
The invention belongs to the technical field of anti-tumor, and particularly relates to a target-specific anti-tumor polypeptide and application thereof.
Background
Cancer has become an important public health problem worldwide, and there are nearly 1400 million new cases worldwide in 2012, and it is expected that the number of new cases will increase by about 70% in the next 20 years. Over the past decade, the cost of cancer treatment has also increased, but there is still a high mortality rate for several cancer types, e.g., liver and uterine cancer. In addition, the incidence and mortality of cancer and tumors in China are also rising all the time and are one of the major public health problems in China. For example, liver cancer is a common cancer with high mortality, and the commonly used drugs for treating liver cancer include doxorubicin (adriamycin), cissplatin (cisplatin), 5-Fluorouracil and the like, which kill tumor cells through apoptosis, but up to 72% of patients carry multidrug resistance genes, thus causing treatment failure and relapse. The latest oral drug Sorafenib (Sorafenib) on the market in 2005 is an inhibitor of multi-target multi-kinase, and compared with placebo treatment, Sorafenib only prolongs the overall survival rate of patients with late-stage liver cancer by about 3 months, and the treatment scheme is not ideal. Most current treatments employ chemotherapy and/or radiation therapy, immunotherapy, targeted therapy or hormone therapy in combination with surgery to treat tumors and cancers, but still cause side effects/problems that affect healthy tissues or organs. The lack of precise treatment of many tumors and/or cancers is still present, and the search for effective, low-toxicity, anti-drug resistance and inexpensive anti-tumor drugs is a major issue to be solved.
Polypeptide drugs are considered to have the advantages of no toxicity or low toxic and side effects and high selectivity, and the method for treating tumors by using the polypeptide is more and more popular. Currently in clinical trials, more than about 140 polypeptides are in clinical trials. Over 500 polypeptide drugs are in preclinical research. The global polypeptide drug market size was $ 141 billion in 2011 and increased at a rate of 15% to 20% per year, predicted to $ 254 billion in 2018, where the innovative polypeptide drug market would increase from $ 86 billion in 2011 to $ 170 billion in 2018. A research proves that the polypeptide drug interacts with the virus envelope glycoprotein to prevent the conformational change of the virus envelope glycoprotein, thereby blocking the fusion of virus envelope host cells, inhibiting the replication of virus and achieving the aim of preventing and treating HIV infection. In the aspect of tumor polypeptide drugs, more than 20 polypeptides, small molecules, oligoribonucleotides or antibodies form a polypeptide coupler in clinical tests, and the kang Zheng medicine research (Shenzhen) Limited company has two anticancer polypeptide drugs in clinical or preclinical research. The commercial compound annual growth rate of polypeptide medicines in China reaches 20.7 percent, and reaches 195 billion yuan in 2009. Moreover, only more than ten kinds of polypeptide drugs are sold in China, and the tumor polypeptide drugs are only clinically researched and are not on the market, so that the space for developing new polypeptide drugs is huge.
Transcription factors of the T-box family are highly conserved in evolution and play a key role in embryonic development, and deletion or mutation of the transcription factors can cause ulna lesion of human beings and cause various organ diseases. In 1999I first cloned TBX2 and TBX3 and found sequences 90% of which were homologous to the DNA binding domain and the transcription repression domain. The TBX2/TBX3 gene is highly expressed in tissues of various malignant tumors such as liver cancer, breast cancer, ovarian cancer and the like, leads to epithelial-mesenchymal transition (EMT), promotes the growth, invasion and metastasis of tumors, and reduces the sensitivity of antitumor drugs polyploid and cissplatin, and the high expression of TBX2/TBX3 is negatively related to the 5-year survival rate. TBX2/TBX3 binds to and inhibits the transcription of the promoter of p19ARF, p21CIP, and promotes malignant transformation of tumor cells by cooperating with protooncogene Myc, Ras and the like. Which is mediated primarily by the C-terminal repressor domain in Tbx3, involves the amino acid 567-623 sequence, as well as the N-and C-terminal repressor domains in
Based on the biological properties of Tbx3 in cancer development, it is possible to find the major domain of Tbx3 as a polypeptide that targets cancer cells. After more than ten years of research, active sites capable of specifically inhibiting transcription regulation of p19ARF and p21CIP are found from TBX2 and TBX3 genes, the sites are found to be related to growth and migration of tumor cells, and a polypeptide molecule TAP21 capable of specifically identifying and inhibiting the sites is designed, so that the polypeptide can effectively inhibit growth of liver cancer cells in vitro and induce apoptosis of the liver cancer cells, and has no toxicity to normal cells. In animal experiments, TAP21 significantly inhibited tumor growth and migration. The american deficiency is that TAP21 is too long (81 amino acids), thereby resulting in (1) a short half-life requiring daily dosing; (2) a potential immune response; (3) the preparation cost is too high; (4) the technical difficulty is high. Thus, to some extent, the clinical use of TAP21 is greatly limited. Although this research has been granted to Chinese patent (patent application No. 201010110980.2, title: nucleic acids and polypeptides against tumors and their use), there is a considerable distance to develop anticancer drugs.
Disclosure of Invention
Based on the technical problems in the prior art, the invention aims to provide a target-specific anti-tumor polypeptide, which shortens the sequence of the amino terminal of TAP21 on the basis of an anti-cancer polypeptide with independent intellectual property rights, and measures the anti-cancer activity of the polypeptide through chemical synthesis so as to shorten the length of the polypeptide, maintain the target specificity of the polypeptide, reduce the potential immunogenicity of the polypeptide and reduce the technical difficulty of production, thereby obtaining better quality control and reducing the development and production cost. Through a series of chemical modification methods, the conformation of polypeptide amino acid is changed, such as circularity, and unnatural amino acid and the like are introduced, so that the activity, the membrane permeability and the half-life period of the polypeptide amino acid are improved.
The purpose of the invention is realized by the following technical scheme:
in one aspect, the invention provides a polypeptide comprising one or more of a linear polypeptide with a cell-penetrating peptide, a cyclic polypeptide without a cell-penetrating peptide, and a mutant polypeptide;
the amino acid sequence of the linear polypeptide with the cell-penetrating peptide comprises one or more of the following amino acid sequences:
RKKRRQRRRLFPYPTYAAVHRHPFL (shown in SEQ ID NO: 1);
RKKRRQRRRLFPYPTYASVHRHPFL (shown in SEQ ID NO: 2);
RKKRRQRRRLFPYPTYAAAVHRHPFL (shown in SEQ ID NO: 3);
RKKRRQRRRLFPYPTYAASAAVHRHPFL (shown in SEQ ID NO: 4);
RKKRRQRRRLFPYPTYAAVHRHPF (shown in SEQ ID NO: 5);
the amino acid sequence of the cyclic polypeptide without the cell-penetrating peptide comprises one or more of the following amino acid sequences:
LFPYPYTYAAVHRHP (shown in SEQ ID NO: 6);
LFPYPTY (shown as SEQ ID NO: 7);
RGDFLFPYPTYR (shown in SEQ ID NO: 8);
(wherein f represents D-proline, and R are connected to form cyclic peptide)
The amino acid sequence of the mutant polypeptide includes one or more of the following amino acid sequences:
RKKRRQRRRLFPYPYTYAAVHRHPFL (shown in SEQ ID NO: 11);
RKKRRQRRRLFPYPYTYAASHRHPFL (shown in SEQ ID NO: 12);
RKKRRQRRRFPYPYTYAASHRHPFL (shown in SEQ ID NO: 13);
RKKRRQRRRPYPYTYAASHRHPFL (shown in SEQ ID NO: 14);
RKKRRQRRRLFPYPYTAASHRHPFL (shown in SEQ ID NO: 15);
RKKRRQRRRLFPYPYAASHRHPFL (shown in SEQ ID NO: 16);
RKKRRQRRRFPYPYTAASHRHPFL (shown in SEQ ID NO: 17);
RKKRRQRRRLFPYPYTYAASHRHPF (shown in SEQ ID NO: 18);
RKKRRQRRRLFPYPYTYAASHRHP (shown in SEQ ID NO: 19);
RKKRRQRRRFPYPYTYAASHRHPF (shown in SEQ ID NO: 20);
RKKRRQRRRPYPYTYAASHRHP (shown in SEQ ID NO: 21);
PYPYTYAASHRHP (shown in SEQ ID NO: 22).
The polypeptide shortens the peptide segment of TAP21 in the prior application, reduces repeated alanine and serine sequences in the peptide segment, can keep the original activity and has higher activity, and reduces the difficulty of the process and the synthesis cost.
Among the above polypeptides, the polypeptide is preferably synthesized using D-amino acids based on its amino acid sequence, which contributes to the prolongation of the half-life of the polypeptide in cancer cells and in vivo.
In a preferred embodiment, the
In the above polypeptides, preferably, the polypeptide is synthesized by using beta-type amino acids according to the amino acid sequence thereof, which helps to prolong the half-life of the polypeptide in cancer cells and in vivo.
In a preferred embodiment,
On the other hand, the present invention also provides a modified polypeptide, which is modified by N-terminal acetylation and C-terminal amidation of the above-mentioned polypeptide (including chemical synthesis of D-type amino acid and chemical synthesis of beta-type amino acid), and further improves the stability of the polypeptide and has an anti-tumor effect.
In a preferred embodiment,
In still another aspect, the present invention also provides a pharmaceutical composition for treating tumor, comprising the above-mentioned polypeptide, modified polypeptide, and excipient thereof.
The pharmaceutical composition of the present invention enables in vitro drug efficacy observation for a longer period of time.
In still another aspect, the invention also provides the use of the above-mentioned polypeptide, modified polypeptide in the preparation of a medicament, polypeptide medicament or combination medicament for treating tumors or cancers in an individual.
The above use, preferably, the tumor or cancer is selected from lung cancer, nasopharyngeal cancer, laryngeal cancer, gastric cancer, liver cancer, esophageal cancer, intestinal cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, leukemia, lymphoma, hemangioma, bone cancer, cervical cancer, cancer of the organs, ovarian cancer, adipose cancer, breast cancer, brain tumor, squamous cancer, skin cancer, thyroid cancer, lip cancer, melanoma, tongue cancer, thymus cancer, and brain or central nervous system cancer;
more preferably, the tumor or cancer is selected from liver cancer, gastric cancer, colorectal cancer, breast cancer or lung cancer. The cancer may be a primary tumor or a metastatic cancer.
In some preferred embodiments, the polypeptide drug or drug combination can be provided alone with the active polypeptide of the invention in a suitable form suitable for administration to a mammalian subject, e.g., human, monkey, pig, horse, cow, sheep, dog, rat, mouse, guinea pig, house-hold, cat, and the like. If desired, the active polypeptides of the invention can be used in combination with a variety of conventional and existing drugs or means for treating cancer, for example in combination with chemotherapeutic agents and/or radiation therapy.
In the above use, preferably, the administration of an effective amount of said drug, said polypeptide drug or said combination is carried out to an individual.
The present invention may employ conventional pharmaceutical methods to provide suitable pharmaceutical formulations or compositions for administration of the compounds or agents in combination to an individual, preferably a human, suffering from cancer. Any suitable, e.g., parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intraperitoneal, intranasal, aerosol, or oral administration may be employed. The pharmaceutical formulation or formulation may be a liquid solution or suspension; a tablet or capsule; in the form of a powder, nasal drops or aerosol. And methods of making such formulations are well known in the art. For therapeutic or prophylactic compositions, the compound can be administered to a subject in an amount sufficient to halt or delay the proliferation of tumor cells.
The term "effective amount" as used herein includes a therapeutically effective amount or a prophylactically effective amount. A "therapeutically or prophylactically effective amount" is a dose effective amount, and within the requisite practical period, to achieve the desired result of inhibiting a tumor. In general, the term "therapeutically effective amount" is sufficient to effect a desired response or improvement, for example: the amount of symptoms or indications of the tension, size or growth of the metastasis or primary tumor. The therapeutically effective amount for a particular individual may vary depending on factors such as the disease state, the general health of the individual being treated, the method, route and dosage of administration, and the severity of the side effects. When in combination, the ratio of the combination of the relative components is such that the effect does not follow that of the individual components in a therapeutically effective amount.
A therapeutically effective amount of a polypeptide molecule, drug or pharmaceutical composition of the invention is generally capable of ameliorating a symptom of cancer by at least about 15%; typically at least about 25%; preferably at least about 35%; or more preferably at least about 50%. Inhibition of cancer metastasis means that migration of tumor cells or metastasis is affected or inhibited. This will result in, for example, a statistically significant or quantifiable change in the number of affected cells, e.g., a decrease in the number of affected target cells over a period of time or in a target area. The rate, size, spread or growth of progression of the primary tumor can also be monitored.
A preferred therapeutically or prophylactically effective amount of an active ingredient or compound of the invention may range from 0.1nM to 0.1M.
In the invention, according to the previous research basis, the half-life period of the polypeptide and the tumor inhibition effect are effectively improved by shortening, mutating, modifying and the like the sequence of the
On one hand, the length of the TAP21 peptide fragment is shortened, repeated alanine and serine sequences in the middle of the peptide fragment are reduced, and 21 polypeptides are designed in total. On the other hand, the screened active polypeptide is also subjected to structural modification and chemical modification to improve the transmembrane capability and the biological activity. After the structure of the polypeptide is determined, the preparation process is optimized, a synthesis process basically suitable for production is determined, the stability of the physicochemical and biological properties of the medicine is ensured, and the purposes of stably inhibiting and removing tumor cells can be achieved.
The "polypeptide" or "peptide" described in the present invention may be used interchangeably. A "polypeptide" or "peptide" is any chain of two or more amino acids, regardless of post-translational modification, including naturally occurring or non-naturally occurring amino acids or amino acid analogs.
The polypeptides of the invention can also be extended to biologically equivalent polypeptides, or conservative amino acid substitutions to obtain variants of a partial sequence of the polypeptide sequences of the invention; or by non-conservative substitutions of the sequence without affecting the biological function, for example in any one embodiment, a partial sequence variant of a polypeptide sequence to which the invention belongs.
The term "conservative amino acid substitution" as used herein refers to the substitution of one amino acid or more amino acids at a given position in the polypeptide, wherein the substitution of an amino acid can be made without substantial loss of the relevant function.
In certain embodiments, a "conservative substitution" is the substitution of an amino acid for another amino acid having the same property, such that the properties of the secondary structure and hydrophilicity of the polypeptide are not substantially altered. Amino acid substitutions are typically made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or facultative properties of the disability.
In certain embodiments, conservative changes also include chemical derivatization of non-derivatized residues, for example, by functional side chain reaction of amino acids. Polypeptide analogs also include those that are chemically altered, e.g., methylated, by chemically modifying the C-terminal amino acid with an alkylamine, such as ethylamine, ethanolamine, or glycol amine. Also, isosteres in which the amide bond is replaced with a substituted amide or an amide bond are included.
The invention has the following beneficial effects:
(1) the polypeptide and the modified polypeptide thereof shorten the peptide segment of TAP21 in the prior application, reduce repeated alanine and serine sequences in the middle of the peptide segment, can keep the original activity and have higher activity, and reduce the difficulty of the process and the synthesis cost.
(2) The polypeptide and the modified polypeptide thereof have good anti-tumor performance in cells and animal models, the effect is obviously superior to that of the existing polypeptide products, and the application of the polypeptide and the modified polypeptide thereof in inhibiting tumors or blocking tumor cell proliferation provides good prospects for anti-tumor treatment.
Drawings
FIG. 1 shows the amino acid sequences of the polypeptides of example 1 and the IC50 values (NS, IC50) of 40nM determined for the effect on tumor cell proliferation of each polypeptide treated with tumor cells for 48 hours;
FIG. 2 is a graph showing the effect of each of the polypeptides of example 2 on tumor cell proliferation (48 hours);
FIG. 3 is a graph showing the effect of
FIG. 4 shows a graph of the effect of
FIG. 5 shows the IC50 value of the modified polypeptide sequence of example 2 in which the
FIG. 6 is a graph showing the effect of modification of
FIG. 7 is a graph showing the in vivo tumor-inhibiting effect of modified
Detailed Description
The technical solutions of the present invention will be described in detail below in order to clearly understand the technical features, objects, and advantages of the present invention, but the present invention is not limited to the practical scope of the present invention.