WO2019223740A1 - Chimeric peptide or chimeric protein targeting tumor cells and application thereof - Google Patents

Abstract

Disclosed are a chimeric peptide or chimeric protein that targets tumor cells and an application thereof, relating to the field of biotechnology. The disclosed chimeric peptide or chimeric protein that targets tumor cells comprises: an iRGD peptide used for targeting a tumor of interest and a specific peptide or protein used for killing the tumor of interest, the iRGD peptide being linked to a carboxyl terminus of the specific peptide or protein. The present chimeric peptide or chimeric protein may target the tumor of interest and specifically kill the tumor cells, is safe for normal diploid cells, may achieve the purpose of preventing or treating tumors, and provides a brand-new treatment idea for treating tumors.

Description

Chimeric peptide or chimeric protein targeting tumor cells and application thereof

Cross-reference to related applications

This application claims the priority of the Chinese patent application filed with the Chinese Patent Office on May 24, 2018 under the application number CN201810511274.5 and entitled "A Chimeric Peptide or Chimeric Protein Targeting Tumor Cells and Applications" , The entire contents of which are incorporated herein by reference.

Technical field

The present application relates to the field of biotechnology, and in particular, to a chimeric peptide or chimeric protein targeted to tumor cells and application thereof.

Background technique

Tumors are a big issue for human health, so they are also a hot issue in modern biological and medical research. Although the annual human and material investment is huge, the results received are far from satisfactory. The cause of the tumor is unclear and the pathogenesis is unclear. However, in recent years, tumor biology research and medical workers have agreed that immunology problems have arisen. Therefore, it is believed that tumor immunology research can bring hope to the diagnosis, treatment and prevention of tumors. In theory, immunologically active cells, lymphocytes can recognize tumors, attack and expel and destroy tumors. At the same time, lymphocytes will also remember tumor markers, quickly initiate anti-tumor immunity, and do not allow tumor recurrence and metastasis. However, research has also found that tumor tissues have established an immunosuppressed internal environment, inhibited and reduced and eliminated the ability of lymphocytes to recognize tumors, attack tumors, and destroy tumors. Suppressor lymphocytes and highly expressed immunosuppressive factors such as TGF-beta, IL6 and IL10 in tumor tissues negate the immune clearance of tumors and promote rapid growth and metastasis. How to deal with the immune status of tumors, different laboratories take their own choices. Excited immune attack tumors use vaccine to activate immune response, use Th1 cytokines IL2, IL12, IL15 and TNFa, IFN-r, GM-CSF to stimulate lymphocytes, establish cell immunotherapy such as LIK and DC-CIK, clinical use results Unsatisfactory. Deregulation of immunosuppressive factors and suppressor cells Treg and MDSCs rely on chemical anticancer drugs, so there is currently no specific method to combat suppressor T cells. PD-1 and PD-1L are important mechanisms that have been found to prevent lymphocytes from attacking tumor cells in recent years. The FDA has approved the use of PD-1 and PD-1L dual antibodies to treat solid tumors. The results of clinical use show that its effective rate is only about 15% of the patients accepted, and the recurrence rate is very high after discontinuation. Patients receive antibody doses of 150mg per day and the cost reaches tens of thousands of yuan. It takes more than 1.5 million yuan to complete a course of medication. The patient and family cannot afford it. Moreover, it has recently been discovered that PD1 and PD1L are not tumor-specific cellular biological phenomena. Highly inhibiting this biological process can lead to autoimmune diseases.

The biggest recent development in tumor cell immunotherapy is the CAR-T (chimeric antigen receptor) T cell therapy technology established by researchers. The key to the technology is to find the antigen specific to the cancer cell itself and the antibody with high affinity for the antigen, use the variable regions on the heavy and light chains of the antibody, and then use genetic engineering technology to transform the T cells into CAR-T cells and become A cell with a precise navigation system that can launch suicide attacks against cancer cells. Scientists have modified this antibody so that it can recognize cancer cell antigens and chimeric to T cells to play a role. Linked to T cells, after completing the design of the above antibody, the next step is to chimeric this antibody to T cells in vitro to culture this CAR-T cell in large numbers. The cells to be cultured will be in the order of billions to tens of billions. These CAR-T cells, which can initiate suicide attack on cancer cells, are introduced into patients for treatment. CAT-T cells directed against B-cell undifferentiated CD19 antigen have proven effective in experimental applications of B lymphoma and lymphocytic leukemia. A large number of CAR-T cells are cultured in vitro, and the number of cells to be cultured will be in the order of billions to tens of billions. These CAR-T cells, which can initiate suicide attack on cancer cells, are introduced into patients for treatment. The General Hospital of the Chinese People's Liberation Army (Hospital 301) has placed the Chinese research and development team among the forefront of global CAR-T cell technology translation medical research. Published Phase I clinical data on CAR-T CD19 for acute lymphoblastic leukemia, CAR-T CD20 for diffuse large B-cell lymphoma, and CAR-T CD30 for Hodgkin's lymphoma, which initially show CAR 301 hospital CAR -T cell targeted tumor immunotherapy is relatively safe, feasible and effective.

There are three most common side effects of CAR T therapy: cytokine storm, encephalopathy, and B cell hypoplasia (meaning that the B cells in the patient cannot grow into normal and functional lymphatic B cells), so how many of these side effects occur What about high? The probability of severe cytokine storms is 27-53%, the incidence of encephalopathy is 25-47%, and the incidence of B cell hypoplasia is 86-100% in patients who have received lymphadenectomy pretreatment and CAR T treatment. What are the symptoms of these side effects? Patients developed fever, tachycardia, hypotension, capillary permeability syndrome, and respiratory symptoms during cytokine storms. The manifestation of encephalopathy is blurred consciousness, unable to speak, and convulsions. Patients with B-cell dysplasia will have repeated infections because poor immunity in the body cannot attack bacterial and viral infections, placing a burden on patients for lifelong treatment. However, there are many problems with CAR-T.

For example: 1. High cost: Because this immunotherapy requires "private order" according to individual differences, the cost is very high.

2. Narrow scope of application: This therapy is currently mainly applied to leukemia, lymphoma, melanoma, especially in the improvement of leukemia patients, and it has a good effect even on advanced leukemia and lymphoma that are not curable. But our most common cancer is lung cancer, liver cancer, a solid tumor that lacks a specific tumor antigen. CAR-T therapy is possible.

3. There are huge risks: Most of the immunotherapy-related ones are still in the experimental stage. Compared with the previous immunotherapy, the CAR-T idea is really good, but it is quite troublesome to operate. Just to find the patient It is very troublesome for specific cancer antigens without damaging normal cells, not to mention the following series of chimeric antibodies, in vitro appreciation of immune cells, immune escape of cancer cells, etc., which require very strong technical strength. In addition, the CAR-T cells imported into the patient need to be the patient's own cells, otherwise the rejection reaction will occur like organ transplantation. Therefore, although the idea is good, whether CAR-T therapy can be applied on a large scale and become a conventional treatment method still has a long way to go. Therefore, CAR-T research is still in the academic stage and cannot meet the requirements for clinicians and patients to treat tumors.

In view of this, this application is hereby filed.

Summary of the Invention

The purpose of this application is to provide a chimeric peptide or chimeric protein targeted to tumor cells. The chimeric peptide or chimeric protein can target the tumor of interest, specifically kill tumor cells, achieve the purpose of preventing or treating tumors, and provides a new treatment idea for treating tumors.

Another object of the present application is to provide a nucleic acid fragment. The nucleic acid fragment can encode the aforementioned chimeric peptide or chimeric protein targeting tumor cells.

Another object of the present application is to provide a chimeric expression cassette. It contains the aforementioned nucleic acid fragment and can express the aforementioned chimeric peptide or chimeric protein targeted to tumor cells.

Another object of the present application is to provide a carrier. This vector is used to transfect host cells and express the aforementioned chimeric peptides or chimeric proteins targeted to tumor cells.

Another object of the present application is to provide a virus. The virus can be used for direct gene therapy in vivo and in vivo.

Another object of the present application is to provide an application of the aforementioned nucleic acid fragment, chimeric expression cassette, vector, and virus.

Another object of the present application is to provide a modern malignant solid tumor treatment method with specific non-toxic and side effects.

This application is implemented as follows:

A chimeric peptide or chimeric protein targeted to tumor cells, comprising: an iRGD peptide for targeting a tumor of interest and a specific peptide or protein for killing the tumor of interest, the iRGD peptide is connected to the tumor The carboxy terminus of a specific peptide or protein.

The iRGD peptide is a circular membrane penetrating peptide. Its linear amino acid sequence is: CRGDKRGPDEC (SEQ ID NO. 8). The specific peptide or protein linked to the iRGD peptide can increase its targeting effect, so that the specific peptide or protein can Specific kill the tumor or tumor cell or tumor tissue of interest.

When the chimeric peptide or chimeric protein reaches the tumor cell, the metalloproteinase secreted by the tumor cell cuts the circular membrane penetrating peptide at the K-R or D-E peptide bond, so that the tumor targeting ability and membrane permeability of iRGD can be achieved. Moreover, normal diploid cells lack this metalloproteinase, and the recombinant protein or peptide with cyclic peptide lacks membrane penetration effect on normal cells and increases the half-life in vivo.

The type of the specific peptide or protein of the tumor to be killed can be selected according to the type of the tumor, as long as it has a killing effect on the tumor cells.

Among them, the term "killing" means: the sum of the two effects of specific peptides or proteins on tumor cells to promote their apoptosis, inhibit proliferation, and damage the tumor cell membrane by pore-breaking or directly lysing the mitochondria of the cells. .

Further, in some embodiments of the present application, the specific peptide or protein is selected from any one of the peptides or proteins shown in SEQ ID Nos. 1-7.

SEQ ID NO. 1 shows the P53 amino-terminal 15 peptide (hereinafter may be referred to as the N-15 peptide).

SEQ ID NO. 2 shows P53 carboxy-terminal 22 peptide (hereinafter may be abbreviated as C-22 peptide).

SEQ ID No. 3 shows Apoptin protein.

SEQ ID ID NO.4 shows the ORF4 protein.

SEQ ID NO. 5 shows a Sphervin peptide.

SEQ ID NO. 6 shows par-4 SAC peptide.

SEQ ID NO. 7 shows a P73 activating peptide.

As a cell's transcript, P53 plays an important role in cell biology in the occurrence of tissues, apoptosis and monitoring cell mutations. More than 50% of tumors have mutations in the P53 gene, which in turn causes them to lose their antitumor effect. P53 related peptides such as its amino terminal 15 peptide (SEQ ID NO.1), carboxy terminal 22 peptide (SEQ ID NO.2), and coding region P73 activating peptide (SEQ ID ID NO.7) and other peptides (P53 and P63 P73 belongs to the same transcription factor family and has the same cancer suppressing function. P53 is the most active factor among them, but it is also the most frequently mutated, deleted and abnormal cell localization and metabolic factor. However, P73 and P53 are different because P73 is affected by iASPP Inhibition does not show transcriptional function under normal conditions and malignant transformation of cells. There are two peptide sequences in the P53 amino acid sequence, with a length of 37aa, which can combine with iASPP to release the transcriptional activity of P73, so it can be activated by expressing 37aa in P53 deficient tumor cells Peptide to make tumor cells apoptotic) has the effect of killing tumors and inducing apoptosis.

Since normal diploid cells do not have P53 mutations, P53 amino-terminal 15 peptide and P53 carboxy-terminal 22 peptide cannot kill normal tissues, which are non-toxic and harmless to normal tissues. This is the theoretical basis of specific tumor-killing cells using P53 amino terminal 15 peptide and P53 carboxy terminal 22 peptide in tumor treatment.

However, some patients have no abnormalities in histochemical pathology and P53 gene detection, and the tumorigenesis and development of these patients may be independent of P53. In this case, tumor suppressor proteins or peptides other than the P53 mechanism can be used, such as chick poplar anemia virus V3P protein Apoptin (shown in SEQ ID NO. 3), adenovirus E4 early-start ORF4 protein (SEQ ID ID NO. 4 ), Sphervin peptide (SEQ ID NO. 5) that inhibits survivin, Pro-4 anti-cancer gene Par-4 SAC peptide (SEQ ID NO. 6), and the like.

Apoptin protein (SEQ ID NO. 3) has the ability to specifically induce apoptosis and necrosis of transformed cells and tumor cells, and does not damage normal human diploid cells. Apoptin's therapeutic effect is related to its carboxyl-terminus, which has a strong nuclear localization signal for tumor cells and a DNA strand that binds to chromatin of the cell. The examples of the present application construct a scAAV virus that secretes and expresses the Apoptin-iRGD chimeric peptide. Cytological experiments and animal experiments show the potential value of the virus that secretes and expresses the Apoptin-iRGD chimeric peptide to treat tumors.

Further, in some embodiments of the present application, the chimeric peptide or chimeric protein is a chimeric peptide or chimeric protein composed of an Apoptin protein (SEQ ID NO. 3) and an iRGD peptide.

ORF4 protein (SEQ ID NO.4) refers to the fourth coding framework protein controlled by the early promoter of adenovirus. The combination of ORF4 protein and PP2A and Src caused apoptosis of tumor cells. An scAAV virus that secretes and expresses an ORF4-iRGD chimeric peptide is constructed in the examples of this application. Cytological experiments and animal experiments show the potential value of the ORF4-iRGD chimeric peptide in treating tumors.

Further, in some embodiments of the present application, the chimeric peptide or chimeric protein is a chimeric peptide or chimeric protein composed of an ORF4 protein (SEQ ID NO. 4) and an iRGD peptide.

Sphervin peptide (SEQ ID NO. 5) is an inhibitor of survivin. It inhibits tumor cell apoptosis by overexpressing survivin. Survivin is an intracellular anti-apoptotic protein with a short half-life. It enters the proteasome degradation process through the action of ubiquitinase. Tumor cells highly express heat shock protein (HSP-90). The combination of HSP-90 and survivin blocks its ubiquitination and degradation. Therefore, Spherin peptide can block the binding of survivin and HSP-90, accelerate the degradation of survivin, and promote tumor apoptosis. In the examples of the present application, the scAAV virus secreting and expressing the Sphervin-iRGD chimeric peptide was constructed, and ideal results were observed in experiments with cells and tumor-bearing animals.

Further, in some embodiments of the present application, the chimeric peptide or chimeric protein is a chimeric peptide or chimeric protein composed of a Sphervin peptide (SEQ ID NO. 5) and an iRGD peptide.

The par-4 SAC peptide (SEQ ID NO. 6) is a peptide encoding a core region domain of a prostate-responsive apoptotic gene. Humans and animals that are sensitive to hormones can undergo spontaneous apoptosis of prostate cancer after castration. This process is the result of prostate reactive apoptotic gene expression. Studies on the relationship between the structure and function of Par-4 protein suggest that the core region (SAC) of the protein has similar functions and is not restricted by organ specificity. It also shows that the prostate reactive apoptotic protein SAC can apoptotic tumors in different organs without harming normal cells. With the prospect of clinical treatment of tumors, the scAAV encoding the Par-4 SAC-iRGD chimeric peptide was cloned in the examples of this application. Satisfactory results have been seen in cells, tumor-bearing animals, and a few human patients with advanced cancer.

Further, in some embodiments of the present application, the chimeric peptide or chimeric protein is a chimeric peptide or chimeric protein composed of a par-4 SAC peptide (SEQ ID NO. 6) and an iRGD peptide.

Further, in some embodiments of the present application, the chimeric peptide or chimeric protein further has a linker peptide between the specific peptide or protein and the iRGD peptide.

Further, in some embodiments of the present application, the amino acid sequence of the linker peptide is GG or GS.

Using GG or GS as the linking peptide can make the chimeric peptide or chimeric protein more stable, and ensure that each part of the peptide or protein can exert normal physiological activity.

A nucleic acid fragment encoding the chimeric peptide or chimeric protein targeted to tumor cells according to any one of the above.

A chimeric expression cassette containing a nucleic acid fragment as described above.

Further, in some embodiments of the present application, the chimeric expression cassette is a plurality of nucleic acid fragments as described above. It should be noted that, by inserting multiple nucleic acid fragments into the expression cassette, the chimeric expression cassette can express multiple types of chimeric peptides or chimeric proteins.

Further, in some embodiments of the present application, the chimeric expression cassette comprises: a signal peptide nucleic acid sequence encoding a signal peptide; and the signal peptide nucleic acid sequence is located upstream of the nucleic acid fragment.

The signal peptide is also necessary for the secretion and expression of the target protein. If the amino acid length of the primary protein of the expression product exceeds 60-70aa, a signal peptide with a length of about 20aa, such as a signal peptide that secretes IgG, is usually used. If the length of the translated pre-peptide sequence is less than 60aa, a signal peptide longer than 40aa must be used. The initial amino acid sequence is shorter than 60aa. The ribosomal protease system will digest and decompose the amino acid according to the length of the amino acid.

The generalized signal peptide is the intracellular localization signal including cellular structural proteins, such as nuclear localization signal, mitochondrial localization signal, membrane anchor localization signal, etc. Therefore, some authors refer to it as the zip code of synthetic proteins. Functional proteins synthesized by cells must be secreted into the extracellular space, and biological effects can be achieved through cell autocrine, endocrine and endocrine and membrane receptors of target cells. For the therapeutic recombinant virus to infect cells, secretion of effector peptides and proteins, secretion of expressed signal peptides is very important.

The introduction of the signal peptide nucleic acid sequence can ensure that the chimeric peptide or chimeric protein is not broken down and secreted outside the cell smoothly.

Of course, the specific signal peptide used can be determined according to the actual situation. For example, 80aa NT4 signal peptide, short peptide secretion expression. IgG signal peptides can be used for proteins whose secreted and expressed peptides are longer than 100aa, and the length is usually between 20-30aa.

Further, in some embodiments of the present application, the amino acid sequence of the NT4 signal peptide is shown in SEQ ID NO.9.

Further, in some embodiments of the present application, the signal peptide nucleic acid sequence encoding the NT4 signal peptide is shown in SEQ ID NO. 25.

Further, in some embodiments of the present application, when the number of nucleic acid fragments is plural, a splicer sequence for controlling the expression efficiency or ratio of the specific peptide or protein is inserted into the chimeric expression cassette. A splicer sequence is inserted between two adjacent nucleic acid fragments.

To enhance the stability of the transcript, it is necessary to insert the splicer sequence into the expression cassette. The insertion of the splicer is conducive to the expression of stable mRNA. For the mRNA generated during the initial transcription, the splicer sequence will be selectively cut and then spliced to produce mature mRNA.

The splicing sequence is inserted between the two nucleic acid sequences that express the target peptide (the two target peptides may be the same or different). It has intron function and contains the sequence of the acceptor site and the donor site, which can be controlled at the same time. Upstream and downstream expression ratio of transcription and translation expression.

The sequence selection of the splicer needs to be determined according to the ratio of the expressed peptides or proteins described upstream and downstream.

Further, in some embodiments of the present application, the sequence of the splicing member is selected from one of SEQ ID Nos. 10-20.

The ratio of downstream protein or peptide to upstream protein or peptide controlled by different splicer sequences is given in Table 1 below.

Table 1 Splicer sequences and the expression ratios of the upstream and downstream proteins or peptides they control

Downstream Protein: Upstream Protein Ratio	Nucleotide sequence (5’-3 ’)	Sequence identifier
10: 0	CCTTTCTCTCCACAGGT	SEQ ID NO.10
8.8: 1.2	CCTTTCTCTC G ACAGGT	SEQ ID NO.11
7.6: 2.4	CCTT C CTCTC A ACAGGT	SEQ ID NO.12
6.4: 3.6	CCTT C CTCTCCACAGGT	SEQ ID NO.13
5.2: 4.8	CCTT A CTCTCCACAGGT	SEQ ID NO.14
4.0: 6.0	CCTT G CTCTC A A T AGGT	SEQ ID NO.15

2.8: 7.2	CCTT A CTCTC A A A AGGT	SEQ ID NO.16
1.2: 8.8	CCTT C CTCTCCA G AGGT	SEQ ID NO.17
0:10	CCTT G CTCTC G A G AGGT	SEQ ID NO.18
4.0: 6.0	CCTT G CTCTC A A T AGGT	SEQ ID NO.19
5.2: 4.8	CCTT A CTCTCCACAGGT	SEQ ID NO.20

Further, in some embodiments of the present application, there are three kinds of multiple nucleic acid fragments, which are a first nucleic acid fragment, a second nucleic acid fragment, and a third nucleic acid fragment, respectively, which respectively express an N-15 peptide and an iRGD peptide. The first chimeric peptide composed of the fusion, the second chimeric peptide composed of the fusion of the P73 activating peptide and the iRGD peptide, and the third chimeric peptide composed of the fusion of the C-22 peptide and the iRGD peptide.

Further, in some embodiments of the present application, in the chimeric expression cassette, a first splicer sequence, a second nucleic acid fragment, and a third nucleic acid are inserted between the downstream of the first nucleic acid fragment and the upstream of the second nucleic acid fragment. A second splicing subsequence is inserted between the fragments.

Further, in some embodiments of the present application, the first splicing sequence is SEQ ID NO. 19, and the second splicing sequence is SEQ ID NO. 20.

Further, in some embodiments of the present application, an artificial intron sequence is inserted downstream of the signal peptide nucleic acid sequence and upstream of the nucleic acid fragment.

Further, in some embodiments of the present application, the artificial intron sequence is as shown in SEQ ID NO.26.

The use of artificial introns in expression cassettes is different from natural introns because the natural intron nucleic acid sequence is too long, and the structure and function of artificial introns are the same as natural introns, but the sequence length is short and easy Shearing to produce mature mRNA.

Further, in some embodiments of the present application, the expression cassette includes sequentially connecting the following expression elements:

Nucleic acid sequence encoding NT4 signal peptide, artificial intron, nucleic acid sequence encoding first chimeric peptide, first splicer, nucleic acid sequence encoding second chimeric peptide, second splicer, and Nucleic acid sequence.

The base sequence of the expression cassette is shown in SEQ ID NO.27.

Further, in some embodiments of the present application, a TRX partial nucleic acid sequence is inserted upstream and / or downstream of the nucleic acid fragment in the chimeric expression cassette, and the TRX partial nucleic acid sequence encodes a thiooxidized protein partial sequence.

Further, in some embodiments of the present application, the thiooxidized protein partial sequence is a TRX amino terminal sequence or a TRX carboxy terminal sequence.

The TRX amino terminal sequence is shown in SEQ ID NO.21, and the TRX carboxy terminal sequence is shown in SEQ ID NO.22.

Further, in some embodiments of the present application, the TRX partial nucleic acid sequence encoding the TRX amino terminal sequence is shown in SEQ ID NO.23.

Further, in some embodiments of the present application, the TRX partial nucleic acid sequence encoding the TRX carboxy-terminal sequence is shown in SEQ ID NO.24.

Adding a TRX sequence to the amino and / or carboxyl terminus of a chimeric peptide or chimeric protein can enhance the formation of disulfide bonds in the reduced environment of the secreted protein into the oxidized environment outside the cell, ensuring the advanced structure and biology of the secreted peptide and protein Features.

TRX stands for thiooxidized protein.

A vector comprising the chimeric expression cassette according to any one of the above.

A virus containing the above vector.

Further, in some embodiments of the present application, the virus is a double-stranded adeno-associated virus (scAAV).

Application of a chimeric peptide or chimeric protein, a nucleic acid fragment, a chimeric expression cassette, a vector, or a virus targeted to tumor cells as described above in the manufacture of a medicament for preventing or treating tumors.

A specific non-toxic and side effect modern malignant solid tumor treatment method includes: transfecting a virus containing the above vector into a subject in need of tumor treatment.

Further, in some embodiments of the present application, the subject is a human or non-human animal.

This application has the following beneficial effects:

Chimeric peptides or chimeric proteins targeted to tumor cells provided by the present application include: iRGD peptides for targeting tumors of interest and specific peptides or proteins for killing tumors of interest, iRGD peptides are linked to specific peptides or The carboxy terminus of the protein. The chimeric peptide or chimeric protein can target a tumor of interest, specifically kill tumor cells, be safe for normal diploid cells, achieve the purpose of preventing or treating tumors, and provide a new treatment idea for treating tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to explain the technical solutions of the embodiments of the present application more clearly, the drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present application, so It should be regarded as a limitation on the scope. For those of ordinary skill in the art, other related drawings can be obtained according to these drawings without paying creative work.

1 is a schematic structural diagram of a chimeric expression cassette of a P53-related peptide scAAV in Example 1 of the present application;

2 is a schematic structural diagram of a chimeric expression cassette of a non-P53-related peptide scAAV in Example 2 of the present application;

3 is a schematic structural diagram of a pscAAV vector used in an embodiment of the present application;

4 is a cell morphology of an inverted microscope under Hela cells infected with a P53-related peptide scAAV in Experimental Example 1 of the present application at 24, 48 and 72 hours;

5A-D are the results of flow cytometry detection at 0, 24, 48 and 72 hours after Hela cells infected with P53-related peptide scAAV in Experimental Example 2 of the present application;

6 is a result of immunohistochemical staining of Hela cells infected with P53-related peptide scAAV in Experimental Example 3 of the present application;

FIG. 7 is the observation results of the ICR mice inoculated with S180 tumor cells intraperitoneally and non-inoculated ascites ascites in Experimental Example 4 of the present application;

8 is an anatomical view of the abdominal mucosa of a mouse with ascites tumor formed in Experimental Example 4 of the present application;

9 is a body shape observation result of ICR mice in Experimental Example 5 of the present application inoculated with S180 ascites tumor cells to form ascites tumor, and then injected intraperitoneally with P53-related peptide scAAV for one week;

FIG. 10 shows the local tumor growth of ICR mice in Experimental Example 6 of this application after 6 weeks of subcutaneous inoculation with H22 liver cancer cells;

FIG. 11 is an observation result of an ICR mouse subcutaneously inoculated with H22 hepatoma cells to form tumors and locally injected with P53-related peptide scAAV in Experimental Example 6 of the present application; FIG.

FIG. 12 is an observation result of an ICR mouse subcutaneously inoculated with H22 hepatocellular carcinoma cells into a tumor and injected locally with a non-P53-related peptide scAAV in Experimental Example 6 of the application;

FIG. 13 shows the intraperitoneal injection of ICR mice inoculated with S180 ascites tumor in Experimental Example 7 of the present application, secreted P53-related peptide scAAV, peptide and non-P53-related peptide scAAV, and untreated Number of surviving mice and survival time within 12 weeks;

FIG. 14 shows that after inoculation of H22 mouse liver cancer cells by subcutaneous inoculation with ICR mice in Experimental Example 8 of the present application, local injection of P53-related peptide scAAV and non-P53-related peptide scAAV, and untreated tumor-bearing mice at 20 weeks Comparison of the number of surviving mice and survival time within the range;

FIG. 15 shows the results of immunological detection of the subcutaneous tumor injected with the P53-related peptide scAAV in the experimental example 9 of the present application by the ABC method.

Detailed ways

To make the objectives, technical solutions, and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. If the specific conditions are not indicated in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. If the reagents or instruments used are not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

The features and performance of the present application are further described in detail in combination with the following embodiments.

Example 1

1 Construction of a chimeric expression cassette

The structure of the chimeric expression cassette provided in this embodiment is shown in Figure 1 (arrows in the figure indicate the direction of transcription), and the upstream to downstream include the following expression elements in order:

Nucleic acid sequence encoding NT4 signal peptide, artificial intron, nucleic acid sequence encoding first chimeric peptide, first splicer, nucleic acid sequence encoding second chimeric peptide, second splicer, and Nucleic acid sequence.

Among them, the first chimeric peptide is a fusion peptide in which N-15 peptide and iRGD peptide are fused, the second chimeric peptide is a fusion peptide in which P73 activation peptide and iRGD peptide are fused, and the third chimeric peptide is C-22 peptide and iRGD peptide Fusion composed of a third chimeric peptide.

The amino acid sequence of the NT4 signal peptide is shown in SEQ ID NO.9; the nucleic acid sequence encoding the NT4 signal peptide is shown in SEQ ID NO.25.

The base sequence of the artificial intron is shown in SEQ ID NO.26.

The amino acid sequence of the first chimeric peptide is shown in SEQ ID NO. 28, and the N-15 peptide is fused to the iRGD peptide through the GS linker peptide.

The base sequence of the first splicer is shown in SEQ ID NO.19.

The amino acid sequence of the second chimeric peptide is shown in SEQ ID NO. 29. The P73 activation peptide is fused to the iRGD peptide through the GS linker peptide.

The base sequence of the second splicer is shown in SEQ ID NO.20.

The amino acid sequence of the third chimeric peptide is shown in SEQ ID NO. 30. The C-22 peptide is fused to the iRGD peptide through the GS linker peptide.

The base sequence of the expression cassette provided in this example is shown in SEQ ID NO.27. Among them, the two bases of "CG" added between the NT4 signal peptide and the artificial intron are for the recognition and alignment of the endopeptidase.

2Construction vector

The above-mentioned chimeric expression cassette was synthesized by chemical methods. EcoR and BamH I restriction sites were introduced at both ends, and the chimeric expression cassette was inserted into the pscAAV vector (its structure is shown in Figure 3. The deletion of the D region of ITR occurred. △ ITR mutated), an expression vector was obtained, and was named P53-related peptide scAAV.

When pscAAV was transfected into cells, three chimeric peptides were expressed, the first chimeric peptide consisting of the fusion of N-15 peptide and iRGD peptide, and the second chimeric peptide consisting of the fusion of P73 activating peptide and iRGD peptide. Peptide, a third chimeric peptide consisting of a fusion of a C-22 peptide and an iRGD peptide. The three chimeric peptides of the expression cassette structure constructed in this embodiment are expressed and can be simultaneously targeted to tumor cells, and play a role of "killing" tumor cells or inhibiting their proliferation, for the purpose of treating tumors.

The use of a single expression cassette and a promoter Promoter to express more than two proteins or peptides is an important progress in recombinant expression in recent years. In the early days, more than two Promoters were used in a recombinant to drive their respective expressions, increasing the recombinant nucleic acid capacity, and different Promote comes from different organisms, such as the CMV early promoter and the SV40 promoter. The promoter capacity is different. The amount and ratio of expressed proteins and peptides are difficult to control. Later, the ribosome entry site IRES is used to drive upstream and downstream protein or peptide expression. There is a problem that the ratio of upstream and downstream products is uncontrollable. At the same time, IRES is composed of more than 500 bp nucleic acids. For recombinant AAV parvovirus, it often exceeds the virus packaging capacity. The use of classical swine fever virus FA2 lysates to express two peptides or proteins under a promoter has long been reported, but the FA2 recognition sequence needs to be added between the upstream and downstream expression products in order to generate a missing shell (amino acid chain) in upstream and downstream translation expression. There are discontinuous Nike) proteins and peptides, and both the upstream and downstream products add undesired amino acid sequences, affecting the biological activity of the expressed peptide. Not suitable for short biopeptide secretion applications. Therefore, this patent uses recombinant scAAV secretion and expression technology. It is necessary to pay attention to the indispensable ATR ITR on both sides of the AAV, hairpin sequence ΔITR, CMV promoter, MCS (multicloning site), SV40 PolyA, in order to make short peptide secretion expression The long signal peptide and guide peptide sequences are installed so that the vector can be inserted into the vector to successfully package the infectious ability. The viral nucleic acid capacity is about 1000 bp. Therefore, in order to use scAAV for gene therapy, it must have a strict recombination design.

In the embodiment of the present application, artificial introns and mRNA selective cutting and splicing technology are used to generate mature mRNA expression, and an expression cassette of three biological short peptides is used to treat tumors. In our earlier work, we used three tag peptides, 6XHis, C-Myc, and Flage tags as markers. The feasibility of expressing three short peptides in one expression cassette was tested with commercially available antibodies. It is proved to be completely feasible, but over-expression of short peptides is prone to under-expression and splicing errors. Therefore, the embodiment of the present application uses a scAAV containing an expression cassette to express three kinds of antitumor peptides, which is called a one-shot three-star technology in our research group.

Example 2

The structure of the chimeric expression cassette provided in this embodiment is shown in FIG. 2, and the upstream to downstream thereof include the following expression elements in order:

Nucleic acid sequence encoding NT4 signal peptide, artificial intron, nucleic acid sequence encoding fourth chimeric protein, first splicer, nucleic acid sequence encoding fifth chimeric peptide, second splicer, encoding of sixth chimeric peptide Nucleic acid sequence.

The fourth chimeric protein is a fusion protein of Apoptin protein (shown in SEQ ID No. 3) fused to an iRGD peptide through a GS linker peptide; the corresponding coding sequence is shown in SEQ ID NO. 31.

The fifth chimeric peptide is a par-4 SAC peptide (shown in SEQ ID No. 6), a fusion peptide fused to the iRGD peptide through a GS linking peptide, and the corresponding coding sequence is shown in SEQ ID NO. 32.

The sixth chimeric peptide is a fusion peptide in which the amino terminus of TRX (SEQ ID NO. 21), the Sphervin peptide (SEQ ID NO. 5), and the carboxyl terminus of TRX (SEQ ID NO. 22) are fused to the iRGD peptide through a GS linking peptide, corresponding The coding sequence is shown in SEQ ID NO.33.

The remaining expression element sequences are basically the same as in Example 1.

The base sequence of the chimeric expression cassette provided in this example is shown in SEQ ID NO.34.

2Construction vector

The nucleic acid sequence of the chimeric expression cassette of this example was synthesized by a chemical method, and EcoR Im restriction sites were added at the 5 ~ end and BamH restriction sites were added at the 3 ~ end to facilitate the insertion of the expression cassette into the pscAAV vector, and the expression vector was named. It is a non-P53 related peptide scAAV.

Experimental example 1

Figure 4 shows the morphology of the cells under inverted microscope at 24, 48, and 72 hours after Hela cells were infected with the p53-related peptide scAAV. It can be seen that the cells began to appear spherical detachment after 24 hours, and the cells showed circular contraction and apoptosis at 48 hours. Necrosis and lysis of cells after 72 hours.

Experimental example 2

Figures 5A-D show the results of flow cytometry at 24, 48, and 72 hours after Hela cells were infected with the p53-related peptide scAAV. It can be seen that with the extension of the recombinant virus infection time, the number of apoptotic cells continues to increase, and S phase after 72 hours Cells diminish and disappear, and G1 phase cell growth arrests.

Experimental example 3

Figure 6 shows the results of immunohistochemical staining of Hela cells infected with the P53-related peptide scAAV. Figures 6-A and 6-B are cells not infected with the P53-related peptide scAAV. Both the C22 and N15 antibodies using P53 were negative. Figures 6-C and 6-D show that cells infected with the p53-related peptide scAAV are all positive. Figure 6-C shows the expression of the N15 peptide of P53, which uniformly colored the cytoplasm. Fig. 6-D shows the expression of P53 in the nucleus.

Experimental Example 4

Figure 7 shows the observation results of ascites ascites type in the 20 days after ICR mice were inoculated with S180 tumor cells (3 × 10 ⁴ cells / head). The mice injected with S180 tumor cells weighed 41 grams and had a large amount of ascites in the abdominal cavity (Figure 7 -A); abdominal type of mice without tumor cells in the normal abdominal cavity, weighing only 21 grams (Figure 7-B); mice with ascites tumors can see a large number of tumors on the abdominal mucosa after drainage of ascites, as shown in Figure 8 -A shows the pathological results of abdominal tumors shown in Figure 8-B;

Experimental Example 5

Figure 9 shows the results of ICR mice inoculated with S180 ascites tumor cells to form ascites tumors. One week after intraperitoneal injection of P53-related peptide scAAV (4 mice represent 4 replicates), the injection amount is 3 × 10 ⁵ pfu It can be seen that the ascites disappeared after one week, and no tumor recurrence was observed after continued feeding for 60 days. The effective therapeutic effect of scAAV of the recombinant virus P53-related peptide is shown.

Experimental Example 6

Figure 10 shows the local tumor growth of ICR mice subcutaneously inoculated with H22 hepatocellular carcinoma cells after 6 weeks without treatment. Although the mouse strain, the age of the mice, and the same number of tumor cells were the same, the tumor size and tumor growth behavior of each mouse big difference.

Figure 11 shows ICR mice were inoculated subcutaneously with tumor cells H22 hepatoma local injection P53 observations (two mice receiving injections shown in the figure) after a scAAV-related peptide, injection volume 3 × 10 ⁵ pfu / only, 3 times Second, the local tumor nodules of the mice disappeared 6 weeks after injection,

Figure 12 shows the ICR mice H22 hepatoma cells were inoculated subcutaneously into non-tumor topical injection of observation (two mice receiving injections shown in the figure) after a scAAV related peptide P53, injection volume 3 × 10 ⁵ pfu / only, the number of Three times, local tumor nodules disappeared 6 weeks after injection.

Experimental Example 7

Figure 13 shows the survival of ICR mice inoculated with S180 ascites tumors by intraperitoneal injection of P53-related peptide scAAV, peptides and non-P53-related peptide scAAV, and untreated ascites tumor mice (control group) within 12 weeks. Count and survival time. Twenty mice were injected with P53-related peptide scAAV, and 19 mice survived. 20 mice were injected with non-P53-related peptide scAAV, and 18 mice survived. Treatment group mice (P53-related peptide scAAV and non-P53-related peptide). (scAAV) No ascites tumor recurred when the survival time exceeded 12 weeks, and the mice ate and behaved as usual. The mice in the control group died from the third week, and all the mice died at the seventh week.

Experimental Example 8

Figure 14 shows the number of surviving mice within 20 weeks after the inoculation of ICR mice with H22 mouse liver cancer cells by subcutaneous injection of P53-related peptide scAAV and non-P53-related peptide scAAV, and untreated tumor-bearing mice. Comparison of survival time. Local injection of P53-related peptide scAAV resulted in 2 deaths and 18 survived at 20 weeks. Locally non-P53-related peptide scAAV, 4 died and 16 survived at 20 weeks. Sixteen mice that were injected with the reported virus scAAV and left untreated died locally, but four mice survived with tumors.

Experimental Example 9

Figure 15 shows the results of immunoassay using anti-P53 antibody as primary antibody and ABC method for subcutaneous tumors injected with P53-related peptide scAAV 96 hours later. It can be seen that the expression of P53-related peptide is not only seen under the X100 microscope, but also seen at high magnification X200 and X400 saw apoptotic and necrotic morphology of virus-infected tumor cells.

In summary, the purpose of using the recombinant virus (P53-related peptide scAAV or non-P53-related peptide scAAV) provided in the present application to treat solid tumors is based on the transduction of the patient's own lymphocytes for the recombinant virus. MHC is homologous, there is no rejection between the graft and the host, and lymphocytes have the ability to accumulate chemotaxis in the tumor. More importantly, the recombinant virus WHO for gene therapy must be non-replicating to ensure gene therapy safety. Sex. Although conditional replication viruses are currently being reported for tumor treatment, their safety remains questionable. However, the current packaging and production cannot meet the number of viruses required to treat tumors. MOI (Each tumor cell is killed by 1000pfu, a tumor patient has a tumor load of 10 ^15-18 tumor cells, and theoretically requires 10 ^18-22 Pfu recombination. Virus production technology is difficult and possible, and it is also the main reason for the low efficacy of current gene therapy tumors. This patented technology uses the principle and method of eukaryotic cell secretion and expression, and the therapeutic proteins and peptides produced can be enlarged by 10 ^5-7 orders of magnitude. There are also many technical difficulties in inputting 10 ^15-17 pfu recombinant virus to patients at one time. The easiest way to solve the problem of dosage of recombinant virus is to use replication virus. Some reports use conditional replication virus, but its biological safety is still questionable. WHO The use of a recombinant virus as a gene vector file requires the use of non-replicating viruses, which makes it difficult to overcome obstacles in the use of recombinant viruses to treat tumors in vivo, but it ensures the safety of recombinant virus technology. The technology provided by this application provides Recombinant viral gene technology that is safe, effective and can be industrially produced

The scAAV of the present application secretes tumor specific killer peptides and protein chimeric iRGD cyclic peptides. On the one hand, it reduces the degradation of expression products by carboxypeptidase, and at the same time enables the target peptide and tumor and tumor vascular endothelial cell integrin receptors through RGD peptide sequence Combining to achieve iRGD chimeric peptides is targeted for tumor treatment, and tumor cells secrete metalloproteinases to digest the chimeric peptide leak CRGDK / R and the tumor cell membrane's Neuropilin receptor bind and perforate into the cells, achieving tumor-targeting peptide killing Tumor effect.

The above description is only a preferred embodiment of the present application, and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and changes. Any modification, equivalent replacement, or improvement made within the spirit and principle of this application shall be included in the protection scope of this application.

Claims (10)

1. A chimeric peptide or chimeric protein targeting tumor cells, comprising: an iRGD peptide for targeting a tumor of interest and a specific peptide or protein for killing the tumor of interest, the iRGD peptide Linked to the carboxy terminus of the specific peptide or protein.
2. The chimeric peptide or chimeric protein targeting tumor cells according to claim 1, wherein the specific peptide or protein is selected from any one of the peptides or proteins shown in SEQ ID Nos. 1-7. Species

   Preferably, the amino acid sequence of the iRGD peptide is shown in SEQ ID NO.8.
3. A nucleic acid fragment, characterized in that it encodes a chimeric peptide or chimeric protein targeted to tumor cells according to claim 1 or 2.
4. A chimeric expression cassette, comprising the nucleic acid fragment according to claim 3.
5. The chimeric expression cassette according to claim 4, wherein the chimeric expression cassette comprises: a signal peptide nucleic acid sequence encoding a signal peptide; and the signal peptide nucleic acid sequence is located upstream of the nucleic acid fragment.
6. The chimeric expression cassette according to claim 4, wherein an intron sequence is inserted into the chimeric expression cassette upstream and / or downstream of the nucleic acid fragment.
7. The chimeric expression cassette according to claim 4, wherein a TRX sequence is inserted upstream and / or downstream of the nucleic acid fragment in the chimeric expression cassette.
8. A vector, comprising the chimeric expression cassette according to any one of claims 4-7.
9. A virus comprising the vector according to claim 8.
10. The chimeric peptide or chimeric protein targeting tumor cells according to claim 1, the nucleic acid fragment according to claim 2 or 3, the chimeric expression cassette according to any one of claims 4 to 7, and claim 8 The use of the vector or the virus according to claim 9 in the preparation of a medicament for preventing or treating tumors.

Images