CN111909275A - Targeted drug-loading system for prolonging circulating half-life period of polypeptide drug and construction method thereof - Google Patents

Targeted drug-loading system for prolonging circulating half-life period of polypeptide drug and construction method thereof Download PDF

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CN111909275A
CN111909275A CN201910380691.5A CN201910380691A CN111909275A CN 111909275 A CN111909275 A CN 111909275A CN 201910380691 A CN201910380691 A CN 201910380691A CN 111909275 A CN111909275 A CN 111909275A
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polypeptide
peptide
kla
tumor
drug
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朱毅敏
田庆美
李海洋
孟祥州
王姣姣
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Suzhou Institute of Nano Tech and Nano Bionics of CAS
University of Shanghai for Science and Technology
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University of Shanghai for Science and Technology
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
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    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

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Abstract

The invention discloses a targeted drug loading system for prolonging the circulating half-life period of a polypeptide drug and a construction method thereof. The construction method comprises the following steps: adding a polypeptide targeting tumor cells at the N end of the KLA toxic peptide, and connecting the C end of the KLA toxic peptide with albumin binding protein through a response peptide to construct a targeting drug loading system; the response peptide is capable of being cleaved by highly expressed proteases in the tumor microenvironment. The construction method is realized based on biotechnology, has simple process and low cost, and is beneficial to popularization and application. The targeted drug-loading system has high biocompatibility, low cost and long in-vivo circulation half-life, and can be specifically combined with tumor cells when circulating along with blood, and the over-expressed MMP2 enzyme in a tumor microenvironment cracks response peptide, so that KLA enters the tumor cells, and further apoptosis of the tumor cells is induced.

Description

Targeted drug-loading system for prolonging circulating half-life period of polypeptide drug and construction method thereof
Technical Field
The invention relates to a recombinant protein, in particular to a recombinant protein of a targeted drug loading system capable of being applied to prolonging the circulating half-life of a polypeptide drug and a construction method thereof, belonging to the technical field of biology and medicine.
Background
With the increasing maturity of biotechnology and polypeptide synthesis technology, more and more polypeptide drugs are developed and applied clinically. Because of wide adaptation, high safety and obvious curative effect, the polypeptide medicament is widely applied to the prevention, diagnosis and treatment of diseases such as tumor, hepatitis, diabetes, AIDS and the like at present, and has wide development prospect. The polypeptide drug has a small molecular structure, is easy to modify and synthesize, does not need a large-flow device in production, can reach production conditions in a common large laboratory, discharges less waste in the production process, and belongs to green pharmacy, so the polypeptide drug is one of the most promising drugs in the 21 st century.
Polypeptide drugs have many advantages but have disadvantages, and are rapidly cleared by the body after entering the body. Therefore, it is a worth discussing to find a drug carrier system suitable for polypeptide drugs.
Disclosure of Invention
The invention mainly aims to provide a targeted drug delivery system for prolonging the circulating half-life period of a polypeptide drug and a construction method thereof, so as to overcome the defects of the prior art,
in order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:
the embodiment of the invention provides a recombinant protein which comprises:
a KLA toxic peptide;
a tumor cell targeting polypeptide linked to the N-terminus of the KLA toxic peptide;
and an albumin binding protein linked to the C-terminus of the KLA toxic peptide by a response peptide that is cleavable by a highly expressed protease in the tumor microenvironment.
The embodiment of the invention also provides application of the recombinant protein in preparing functional products, wherein the functional products comprise tumor targeted drugs or targeted drug-loaded systems for prolonging the circulating half-life of polypeptide drugs.
The embodiment of the invention also provides a gene for coding the recombinant protein.
The embodiment of the invention also provides a host cell containing the recombinant vector.
The embodiment of the invention also provides a preparation method of the recombinant protein, which comprises the following steps: and inserting the recombinant vector into a host cell to enable the host cell to express the target protein, and then purifying and carrying out enzyme digestion.
The embodiment of the invention also provides a construction method of the targeted drug loading system for prolonging the circulating half life of the polypeptide drug, which comprises the following steps: adding a polypeptide targeting tumor cells at the N end of the KLA toxic peptide, and connecting the C end of the KLA toxic peptide with albumin binding protein through a response peptide to construct a targeting drug loading system; the response peptide is capable of being cleaved by highly expressed proteases in the tumor microenvironment.
Compared with the prior art, the invention has the beneficial effects that:
(1) the albumin conjugated protein is used as a drug loading system, the polypeptide circulation half-life period is obviously prolonged, and compared with PEG polymer, the polypeptide drug loading system has the advantages of higher biocompatibility, lower cost and the like.
(2) The combination of the targeting polypeptide to the tumor cells has diversity, is not limited to the HCBP1 polypeptide of the targeting H460 stem cells described in the invention, can be added with other polypeptide molecules sensitively responding to the tumor microenvironment by utilizing the characteristics of the tumor microenvironment, and can further design a drug loading system with more intelligence by utilizing other targeting polypeptides and other response peptides, thereby having wider prospect.
(3) The targeting drug-loaded system for prolonging the circulating half-life of the polypeptide is obtained by a biotechnology, avoids the complex steps of synthesis and modification of other drug-loaded systems, has simple and convenient obtaining mode, is beneficial to popularization and application, and reduces the production cost.
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Fig. 1 shows that when a targeted drug delivery system for prolonging the circulating half-life of polypeptide drugs is combined with tumor cells based on HCBP1 along with blood circulation, the over-expressed MMP2 enzyme in the tumor microenvironment cleaves PLGLAG response peptide, and KLA enters the tumor cells to induce apoptosis of the tumor cells in an exemplary embodiment of the invention.
Fig. 2 is a gel image of a targeted drug delivery system obtained by prokaryotic expression in an exemplary embodiment of the present invention.
Fig. 3 is a schematic diagram of targeted drug delivery system and human serum albumin binding verified by Elisa experiments in an exemplary embodiment of the invention.
FIG. 4A shows the flow assay results of H460 stem cells incubated with FITC-labeled HSA in an exemplary embodiment of the invention.
Fig. 4B shows flow assay results of H460 after incubation with targeted drug delivery system and then with FITC-labeled HSA in an exemplary embodiment of the invention.
Fig. 5 is a schematic diagram of verification of killing of H460 stem cells by a targeted drug delivery system by MTT assay in an exemplary embodiment of the invention.
Fig. 6 shows the results of testing the targeted drug delivery system to prolong the circulating half-life of the polypeptide in vivo by pharmacokinetic experiments in an exemplary embodiment of the invention.
Figure 7 shows that the targeted drug-loaded system can be enriched in tumor tissue in mice observed by a small animal imaging system in an exemplary embodiment of the invention.
Fig. 8A-8C show that the drug-loaded system has certain anti-tumor activity in mice according to an exemplary embodiment of the present invention, which is verified by in vivo experiments in mice.
Detailed Description
One aspect of an embodiment of the present invention provides a recombinant protein, including:
a KLA toxic peptide;
a tumor cell targeting polypeptide linked to the N-terminus of the KLA toxic peptide;
and albumin binding protein (ABD) linked to the C-terminus of the KLA toxic peptide by a response peptide that is cleavable by a highly expressed protease in the tumor microenvironment.
Further, the tumor cell targeting polypeptide may be linked to the N-terminus of the KLA toxic peptide through one or more flexible amino acids, including G and/or G.
Further, the tumor cell targeting polypeptide comprises polypeptide HCBP1 specifically binding to H460 tumor stem cells. In some embodiments, a polypeptide that specifically binds to tumor cells (e.g., the H460 tumor stem cell-specific binding polypeptide HCBP1) can be linked N-terminal to the KLA toxic peptide by GG two flexible amino acids.
Further, the response peptide includes an MMP2 response peptide.
The sequence of the MMP2 response peptide may be PLGLAG.
In another aspect of the embodiments of the present invention, there is provided a use of the recombinant protein in the preparation of a functional product, wherein the functional product comprises a tumor targeted drug or a targeted drug delivery system for extending the circulating half-life of a polypeptide drug.
When the recombinant protein provided by the previous embodiment of the invention is applied in vivo, ABD is combined with albumin in vivo, the circulating half-life of the polypeptide in vivo is prolonged, and based on the targeting function of HCBP1, when the fusion protein reaches a tumor microenvironment, the over-expressed MMP2 cleaves PLGLAG, KLA enters tumor stem cells, and then apoptosis of the tumor cells is caused.
In another aspect of the embodiments of the present invention, a gene encoding the recombinant protein is provided.
In another aspect of the embodiments of the present invention, there is provided a recombinant vector comprising the gene.
Preferably, the recombinant vector comprises the pET32a plasmid.
Another aspect of the embodiments provides a host cell comprising the recombinant vector.
Further, the host cell is Escherichia coli, and may be, for example, Escherichia coli BL21(DE 3).
Another aspect of the embodiments of the present invention provides a method of preparing a recombinant protein, including: and inserting the recombinant vector into a host cell to enable the host cell to express the target protein, and then purifying and carrying out enzyme digestion.
Preferably, the purification comprises purification using Ni + magnetic beads.
Preferably, the enzyme digestion comprises enzyme digestion with TEV enzyme.
In some embodiments, the gene sequence encoding the recombinant protein can be cloned into a vector such as a pET32a plasmid, successfully expressed by a host cell such as Escherichia coli BL21(DE3), and purified to obtain the target protein.
Among them, thioredoxin, a chaperone protein in the pET32a plasmid, is also beneficial to improving the stability of target protein.
Wherein, the cost can be greatly reduced by using a prokaryotic expression system to obtain the target protein.
Another aspect of the embodiments of the present invention provides a method for constructing a targeted drug delivery system for prolonging the circulating half-life of a polypeptide drug, which is characterized by comprising: adding a polypeptide targeting tumor cells at the N end of the KLA toxic peptide, and connecting the C end of the KLA toxic peptide with albumin binding protein through a response peptide to construct a targeting drug loading system; the response peptide is capable of being cleaved by highly expressed proteases in the tumor microenvironment.
Further, the tumor cell targeting polypeptide may be linked to the N-terminus of KLA toxic peptide through one or more flexible amino acids, including G and/or G.
Further, the tumor cell targeting polypeptide comprises polypeptide HCBP1 specifically binding to H460 tumor stem cells.
Further, the response peptide includes an MMP2 response peptide.
In the construction method provided by the embodiment of the invention, the KLA can be increased to enter the cells by utilizing the targeting property of the HCBP1, so that the killing capacity of the KLA on tumor cells can be increased.
In the construction method provided by the embodiment of the invention, PLGLAG can be identified and cleaved by MMP2 overexpressed in a tumor microenvironment, so that the drug carrier system is ensured to release KLA at a target site in vivo, and the safety of the polypeptide drug carrier system in vivo is improved.
In the aforementioned construction method provided in the embodiments of the present invention, the ABD at the C-terminal of the drug carrier (i.e., recombinant protein, also referred to as fusion protein) can be combined with albumin in human body, so as to prolong the circulation half-life of the active polypeptide in vivo by virtue of albumin.
The whole drug-carrying system constructed by the construction method provided by the embodiment of the invention is protein, and has high biocompatibility. Some of the terms referred to in the foregoing embodiments of the invention are defined as follows:
the polypeptide targeting tumor cells, also called targeting polypeptide, can be selected as one of targeting molecules of a drug delivery system, such as HCBP-1 polypeptide used in the embodiment of the invention, and is a polypeptide which is obtained by screening through a bacterial surface display technology and specifically binds to H460 stem cells without depending on a known Marker. MMP2 and MMP9 in the MMP family can decompose type IV collagen of the main component of basement membrane, and the over-expression of the MMP2 and the MMP9 is related to the infiltration and metastasis of malignant tumors. MMP2, whose expression is upregulated, is considered a biomarker in the diagnosis and prognosis of many cancers.
Albumin, the most abundant protein in plasma, is a protein. Due to its size above the kidney filtration threshold, human serum albumin has a half-life of up to 19 days and high ABD affinity for albumin mediated circulation by neonatal Fc receptors.
In some more specific embodiments of the present invention, a method for constructing a targeted drug delivery system (which can also be considered as an intelligent drug delivery system) for extending the circulating half-life of a polypeptide drug can comprise the following steps:
(1) cloning a target gene (a gene encoding a recombinant protein) into a pet32a plasmid through NcoI and XhoI enzyme cutting sites;
(2) electrically transferring the recombinant plasmid into escherichia coli BL21(DE3), selecting a single clone for sequencing, and displaying that the recombinant plasmid is successfully electrically transferred into BL21(DE3) through a sequencing result;
(3) and (3) inducing the expression of the target protein by using IPTG.
(4) And purifying the target protein by using Ni + magnetic beads.
(5) And (3) adopting TEV to carry out enzyme digestion on the fusion protein, and purifying the enzyme-digested protein by using Ni + magnetic beads again.
In the step (1), a TEV cleavage site may be added to the N-terminus of the target protein, and a stop codon TAA may be added to the C-terminus of the target protein. After the chaperonin thioredoxin is cut by TEV, the targeting polypeptide HCBP1 at the N end of the target protein can be exposed. Ensuring that the targeting polypeptide can exert the property of binding with H460 cells.
In the aforementioned step (3), preferred process conditions include: the OD value of the induced phage is 0.8, the concentration of the inducer is 0.1mM, the induction temperature is 37 ℃, and the protein can be expressed in the supernatant. 15-20 mg of fusion protein can be obtained from 1L of bacterial liquid.
In the aforementioned step (4), preferred process conditions include: the imidazole concentration for eluting the hetero protein is 80mM, and the imidazole concentration for eluting the target protein is 500 mM. The incubation conditions of the protein and the magnetic beads are room temperature, and the incubation is performed for 30min in a rotating way. Under the purification condition, the fusion protein with higher purity can be obtained. And the magnetic bead purification is simple and easy to implement, and the purification cost is low.
In the step (5), when designing the target gene, adding TEV restriction enzyme sites to the N segments, where the restriction enzyme sites may be: ENLYFQ. The digestion buffer was 25mM Tris pH 8.0. The enzyme digestion conditions are explored, the temperature is selected to be 4 ℃, and the enzyme digestion is carried out overnight. And (3) purifying the enzyme-digested protein again by using Ni + magnetic beads, and concentrating the obtained target protein to a certain concentration by using a 3K ultrafiltration tube after purification, and storing at-80 ℃.
The invention is described in more detail below with reference to examples and figures, but the scope of the invention is not limited thereto.
Referring to fig. 1, in an exemplary embodiment of the invention, when the intelligent drug delivery system (shown as ABD fusion protein) circulates with the blood, based on the specific binding of HCBP1 to tumor cells, the overexpressed MMP2 enzyme in the tumor microenvironment cleaves PLGLAG-responsive peptides and KLA enters the tumor cells to induce apoptosis of the tumor cells.
In an exemplary embodiment of the present invention, a method for constructing a targeted drug delivery system (hereinafter referred to as "drug delivery system" or "fusion protein") for extending the circulating half-life of a polypeptide comprises the following steps:
step S1: and constructing a recombinant plasmid.
In one embodiment, the primer design and the choice of cleavage sites are used.
HCBP1-KLA-ABD Froward：
Figure BDA0002053269480000061
HCBP1-KLA-ABD Reverse：
Figure BDA0002053269480000062
CR-amplified target gene:
HCBP1-KLA-ABD PCR reaction system
Figure BDA0002053269480000063
PCR procedure: 30S Pre-denaturation at 98 ℃, degree of cycling: 10S at 98 ℃, 20S at 55 ℃ and 20S at 72 ℃ for 30 cycles. 72 finish for 2 minutes.
And (3) recovering the reaction product by using a PCR purification kit, measuring the concentration, carrying out double digestion on the PCR product and pET32a plasmid, and inserting the digested target gene into pET32 a.
Enzyme digestion system
Figure BDA0002053269480000064
Figure BDA0002053269480000071
Connection system
Figure BDA0002053269480000072
Electric conversion: 0.7ul of plasmid is added into DH5 alpha competent bacteria liquid, after being blown evenly by a gun, the liquid is transferred into an electric shock cup, and is electrified and transferred on an electric transfer instrument, and 1ml of LB culture medium is added after the electric transfer is finished. After being uniformly blown, the mixture is put into a shaking table at 37 ℃ and 200rpm for activation for 1 h. After activation, 100ul of the solution was applied to LB ampicillin solid plates. The mixture was placed in a 37 ℃ incubator overnight. The next day, single clones were picked for sequencing and the sequencing results were analyzed using the Sequencher software. Clones with correct sequencing were picked, expanded, plasmids extracted, and electrotransferred into BL21(DE 3). Step S2: and (3) expressing and purifying a targeted drug loading system.
The expression mentioned in the step adopts low-cost prokaryotic expression, and the purification adopts Ni + magnetic beads for purification.
In one embodiment, the specific steps of E.coli expression include: the optimal growth temperature of the thalli is selected to be 37 ℃, and the influence of the inducer on the protein expression condition is seen. BL21(DE3) electroporated with the recombinant plasmid was cultured at 37 ℃ until OD600 became 0.5, and then 0.1mM of IPTG as an inducer was added. The protein expression level was observed after 3 to 4 hours of induction at 37 ℃ and the fusion protein was found to be expressed in the supernatant.
In one embodiment, the purification with Ni + magnetic beads comprises the following steps: inoculating the overnight activated bacterial liquid into 500mL LB liquid culture medium containing corresponding antibiotics according to the proportion of 1-2% (v/v), and carrying out shake culture at 37 ℃ for 2-3h at the rotating speed of 200 rpm; when OD600 reached 0.5, adding 0.01mM IPTG, inducing overnight at 37 deg.C for 3-4h, at 200 rpm; 4 ℃ for 15min, rotating speed 12000g, centrifugally collecting thalli sediment and abandoning supernatant. 30ml of binding buffer (40mM imidazole, 1Xpbs) is used for resuspending thalli precipitation, ultrasonic crushing is carried out on an ice water bath, the ultrasonic power is 300 and 400W, the ultrasonic is carried out for 2s, the interval is 3s, and the total time is 20 min; taking 20ul of the whole bacteria as a sample, adding 1% Tritonx-100 into the whole bacteria, and incubating for 10min on ice; after crushing, the sample is centrifuged for 15min at 4 ℃ and 12000 g. The supernatant and pellet were each 20 ul.
Taking 5ml of magnetic bead suspension, removing the preservation solution, incubating and rinsing by using ddH2O with 10 times of column volume, placing on a magnetic frame, removing supernatant after the magnetic beads are completely adsorbed on the tube wall, and repeating the step twice; using binding buffer with 10 times of column volume to balance and pre-treat the magnetic beads, and removing supernatant; mixing the centrifuged supernatant with magnetic beads balanced by binding buffer, and incubating for 2h at 4 ℃ on a rotary mixer; performing magnetic treatment, and taking out 20ul of reserved sample; 30ml of binding buffer (40mM imidazole, 1XPbs, 1% Tritonx-100) was incubated for 5min at room temperature and rinsed three times. Wash buffer1(75mM imidazole, 1XPbs) incubation rinse for 3min at room temperature, repeated three times. At room temperature, elute buffer (300 imidazole, 1Xpbs) was incubated for 5 min. The 300mM imidazole eluent is dialyzed, ultrafiltered, concentrated and cut.
In one embodiment, TEV is used to cleave the fusion protein, the cleavage sequence is: ENLYFQ. Enzyme cutting conditions are as follows: 1mg/ml protein 1ml, 60ug TEV enzyme, 4 ℃ overnight.
Step S3: after the enzyme digestion in step S2, the enzyme digestion is performed again using Ni + magnetic beads, where the fusion protein and thioredoxin that are not enzyme digested have His tag, and the target protein obtained by the enzyme digestion does not have His tag. The supernatant after incubation of the magnetic beads contains a large amount of target protein, but the purity is not high, and the supernatant also contains fusion protein which is not subjected to enzyme digestion.
Referring to FIG. 2, a gel image of the drug loading system obtained by prokaryotic expression is shown. Wherein: m: maker, 1: expressed fusion protein, 2: protein band after Tev cleavage, 3: and (3) the purified target drug-carrying system protein and the fusion protein which is not removed.
Step S4: the resulting drug-loaded system of interest was diluted to different concentrations as shown in figure 3. 100ul of each well was 200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.125nM in 96-well high adsorption plates at 4 ℃ overnight. After overnight PBST was washed three times for 3min each. 100ul of 2% BSA was added to each well and blocked for 45min, and PBST was washed three times for 3min each. 200ng of biotin-labeled HSA was added to each well and incubated for 1 h. PBST was washed three times for 3min each. 0.1ul of 1mg/ml SA-PE was added to each well and incubated for 45min PBST washes three times for 3min each. 100ul PBS was added to each well and the fluorescence intensity was measured using a microplate reader at the position where the excitation light 488 emitted light 578. The results show that the drug carrier can be well combined with albumin.
Step S5: taking H460 stem cells growing for 7 days, centrifuging at 300rpm for 3min, adding pancreatin, immediately blowing for about 10 times, putting into an incubator at 37 ℃ for digestion for 2min, adding a culture medium after digestion to stop digestion, centrifuging for 5min at 300g, counting to enable the cell density to reach 1000000/ml, adding a drug-loaded system, incubating for half an hour at 4 ℃, incubating to end PBS, centrifuging and washing twice at 300g, adding HSA-FITC for incubating for half an hour at 4 ℃, incubating to end PBS, centrifuging and washing twice at 300g
As shown in fig. 4A-4B, using H460 cells not bound by HCBP1 as a control, and separately incubating the experimental groups with the vehicle, it was found that the vehicle could bind to H460-sphere cells, but not to H460 cells.
Step S6: the drug-carrying system and H460 stem cells are co-cultured to see the killing effect of the drug-carrying system on target cells. The H460 stem cells cultured normally were digested and plated in 96-well plates at a density of 50000/ml per well, with 100ul per well. After overnight incubation, the addition of different concentration gradients of the drug loaded system was started. And (3) putting the 96-well plate back to the cell culture box, culturing for 24h, adding 10% of CCK8 into each well, continuously culturing for 1h, and detecting the light absorption value at the wavelength of 450 on an enzyme-labeling instrument.
As shown in fig. 5, with the increase of the drug-carrying system, the survival rate of the H460 stem cells is reduced, and the targeting polypeptide in the drug-carrying system can help KLA to enter the cells, so as to exert the function of killing the H460 stem cells.
Step S7: injecting the drug-loaded system HCBP1-KLA-ABD into a mouse body through tail vein, taking mouse serum at different time points, and detecting the concentration of the drug-loaded system in the mouse serum by using an ELISA experiment.
As shown in FIG. 6, HCBP1-KLA-ABD significantly prolonged the circulating half-life in mice compared to the control group. The data were fitted using pharmacokinetic software. The control group was in accordance with the single chamber model with a circulation half-life of only 0.145 hours, the HCBP1-KLA-ABD was in accordance with the dual chamber model with a circulation half-life of 13.592 hours.
Step S8: the drug carrier HCBP1-KLA-ABD is marked with near-infrared fluorescent molecules, tail veins are injected into a mouse body, and the distribution condition of HCBP1-KLA-ABD in the mouse body is observed by using a small animal imaging system.
As shown in FIG. 7, HCBP1-KLA-ABD was cleared mainly by the kidney in mice and had some accumulation in tumor tissues. And further demonstrates that HCBP1-KLA-ABD extends the circulating half-life of the polypeptide in vivo as compared to the control. Step S9: the drug carrier HCBP1-KLA-ABD tail vein is injected into the mouse body, and refraction is performed for 6 times in every other day. The mice were observed for tumor volume, body weight, and tumor tissue weight after the experiment was completed.
As shown in FIG. 8A, the tumor volume of mice in the experimental group injected with HCBP1-KLA-ABD was significantly smaller than that in the control group. The tumor tissue weighing results after the experiment showed that the weight of the tumor tissue in the experimental mice was significantly less than that of the control mice (shown in fig. 8B). Observation of the body weight of the mice showed no significant difference in body weight between the mice in the experimental group and the control group (shown in fig. 8C). Indicating that HCBP1-KLA-ABD has biosafety in mice.
It should be noted that the tumor cells and the polypeptides specifically cleavable by the tumor microenvironment in the above embodiments correspond to each other, for example, the responsive polypeptide recognizable by the tumor microenvironment in this embodiment is MMP2 responsive peptide PLGLAG, while in other embodiments, the tumor cells, the targeting polypeptide, and the responsive polypeptide may be replaced by any combination specifically binding to each other.
In conclusion, the invention is based on biotechnology, realizes the specificity recognition and killing of tumor cells by skillfully combining different functional polypeptides, has low cost and higher biocompatibility, is convenient for obtaining and using a drug-carrying system compared with a method for achieving long circulation of the polypeptides by PEG modification, and has wide application prospect in the field of prolonging the half-life period of the polypeptide circulation.
It should be noted that the above-mentioned embodiments of the present invention do not limit the scope of the present invention. Any other corresponding changes and modifications made according to the technical idea of the present invention should be included in the protection scope of the claims of the present invention.

Claims (10)

1. A recombinant protein, comprising:
a KLA toxic peptide;
a tumor cell targeting polypeptide linked to the N-terminus of the KLA toxic peptide;
and an albumin binding protein linked to the C-terminus of the KLA toxic peptide by a response peptide that is cleavable by a highly expressed protease in the tumor microenvironment.
2. The recombinant protein according to claim 1, wherein: the polypeptide targeting tumor cells is connected with the N end of the KLA toxic peptide through more than one flexible amino acid, wherein the flexible amino acid comprises G and/or G; and/or, the tumor cell targeting polypeptide comprises polypeptide HCBP1 that specifically binds to H460 tumor stem cells; and/or, the response peptide comprises an MMP2 response peptide.
3. Use of the recombinant protein of any one of claims 1-2 for the preparation of a functional product comprising a tumor targeted drug or a targeted drug delivery system for extending the circulating half-life of a polypeptide drug.
4. A gene encoding the recombinant protein of any one of claims 1-2.
5. A recombinant vector comprising the gene of claim 4; preferably, the recombinant vector comprises the pET32a plasmid.
6. A host cell comprising the recombinant vector of claim 5.
7. The host cell of claim 6, wherein: the host cell is Escherichia coli.
8. A method for producing a recombinant protein, comprising: inserting the recombinant vector of claim 5 into a host cell to allow the host cell to express a target protein, and then purifying and carrying out enzyme digestion; preferably, the purification comprises purification using Ni + magnetic beads; preferably, the enzyme digestion comprises enzyme digestion with TEV enzyme.
9. A construction method of a targeting drug-loading system for prolonging the circulating half-life of a polypeptide drug is characterized by comprising the following steps: adding a polypeptide targeting tumor cells at the N end of the KLA toxic peptide, and connecting the C end of the KLA toxic peptide with albumin binding protein through a response peptide to construct a targeting drug loading system; the response peptide is capable of being cleaved by highly expressed proteases in the tumor microenvironment.
10. The building method according to claim 9, characterized by comprising: connecting the polypeptide targeting tumor cells with the N end of the KLA toxic peptide through more than one flexible amino acid, wherein the flexible amino acid comprises G and/or G; and/or, the tumor cell targeting polypeptide comprises polypeptide HCBP1 that specifically binds to H460 tumor stem cells; and/or, the response peptide comprises an MMP2 response peptide.
CN201910380691.5A 2019-05-08 2019-05-08 Targeted drug-loading system for prolonging circulating half-life period of polypeptide drug and construction method thereof Pending CN111909275A (en)

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Application publication date: 20201110