CN115028736A - Targeting molecular probe and application - Google Patents

Targeting molecular probe and application Download PDF

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CN115028736A
CN115028736A CN202210506251.1A CN202210506251A CN115028736A CN 115028736 A CN115028736 A CN 115028736A CN 202210506251 A CN202210506251 A CN 202210506251A CN 115028736 A CN115028736 A CN 115028736A
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protein
fusion protein
elastin
gly val
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CN115028736B (en
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蒋锡群
陈伟芝
胡若凡
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Nanjing University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
    • A61K51/0478—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group complexes from non-cyclic ligands, e.g. EDTA, MAG3
    • A61K51/048—DTPA (diethylenetriamine tetraacetic acid)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
    • A61K51/0482—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group chelates from cyclic ligands, e.g. DOTA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/082—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins the peptide being a RGD-containing peptide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1027—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against receptors, cell-surface antigens or cell-surface determinants
    • A61K51/103—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against receptors, cell-surface antigens or cell-surface determinants against receptors for growth factors or receptors for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses a fusion protein macromolecule, which is formed by sequentially connecting an EGFR antibody, an elastin-like protein and a polypeptide containing an RGD motif, or connecting the EGFR antibody and the elastin-like protein and/or the elastin-like protein and the polypeptide containing the RGD motif through a connecting peptide, wherein the elastin-like protein has a repeating unit consisting of VPGXG, X in each repeating unit is the same or different, and X can be any amino acid except proline. The invention also discloses a molecular imaging probe prepared by coupling the fusion protein with a chelating agent and chelating metal ions. The molecular probe has good targeting capability, can be more concentrated to a tumor part, has longer retention time at the tumor part, has excellent relaxation capability and imaging capability, and effectively improves the signal-to-noise ratio of tumor imaging compared with a commercial small molecular probe.

Description

Targeting molecular probe and application
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a specific and macromolecular imaging probe for tumor imaging.
Background
The gadolinium-containing contrast agent is the most widely used magnetic resonance molecular probe in clinic, the molecular probe is injected into a patient body, the image visibility of tissues and organs can be improved in magnetic resonance imaging,is helpful for improving the accuracy of disease diagnosis. Free Gd 3+ Toxic to human body, and chelating it with carrier molecule chelating agent to obtain stable complex with low toxicity for magnetic resonance imaging. Gadolinium-based molecular probes can be classified into linear and large rings according to the chemical structure of the ligand. The macrocyclic structure is more stable than a linear structure. At present, 7 gadolinium-containing molecular probes are totally approved in China, including linear gadopentetate meglumine injection, gadodiamide injection, gadobenate meglumine injection, gadoxetate disodium injection, and large ring gadotenate meglumine injection, gadoteridol injection, and gadobutrol injection.
In recent years, researchers have found that the use of gadolinium-containing molecular probes has problems with gadolinium deposits in vivo, including renal dysfunction, bone deposition, and neurological diseases, among others. Linear gadolinium-based molecular probes are more likely to cause gadolinium deposition in the brain due to structural instability than large rings. The european medical ministry officially banned the use of partially linear gadolinium molecular probes in 2017. Since 2017, foreign drug administration departments such as the United states and European Union issue messages for many times, and remind that gadolinium may be gradually deposited in the brain after many times of contrast-enhanced MRI scans. In 12 months 2017, the food and drug administration of (former) China also announced that medical staff should use gadolinium-containing molecular probes cautiously, use the lowest approved dose if necessary, and carefully assess the risk of benefit before repeat dosing. Therefore, it is necessary and important to develop a novel gadolinium-based molecular probe.
The main direction for solving the problem of internal deposition of the existing gadolinium-based molecular probe is to improve the relaxation rate of the gadolinium-containing molecular probe so as to reduce the injection concentration of the molecular probe, enhance the targeting property of the molecular probe, prolong the imaging time so as to reduce the imaging times in a short period, and improve the stability of the molecular probe through structural design.
Researches show that materials such as artificially synthesized macromolecules (such as dendritic macromolecules) and nanoparticles can be used as carriers to construct magnetic resonance molecular probes, and the relaxation capacity of the molecular probes is enhanced by a method of increasing the molecular weight of the carriers. However, these macromolecular probes are not usually targeted, and can only be targeted to the lesion site such as tumor by passive targeting (i.e., EPR effect).
In the literature (Oncology Reports,2016,35(6)), it is reported that a nuclear magnetic resonance molecular probe prepared by coupling an EGFR antibody and iRGD fusion protein (anti-EGFR-iRGD) with a chelating agent DTPA and then chelating metal ion gadolinium is used for in vivo imaging of human gastric cancer BGC823 xenograft tumor. Although the relaxation rate of the molecular probe is improved to a certain extent compared with that of a small molecular probe, the relaxation rate is still low, and the in vivo imaging effect is poor. This is probably because the chelator DTPA forms an amide bond via a carboxyl group with an amino group in a lysine in the fusion protein, whereas the fusion protein has a smaller number of lysines available for conjugation, resulting in a chelated Gd on each fusion protein 3+ Is limited, which in turn limits the relaxation rate of the molecular probe. In addition, direct coupling of chelating agent DTPA with EGFR antibody and lysine on short peptide iRGD may affect the targeting binding function of the fusion protein, and further affect the in vivo tumor detection capability.
Chinese patent CN 108187064 a discloses a double-targeting fusion protein drug conjugate of elastin-anti-EGFR nanobody-iRGD, which is prepared by fusing elastin-like protein (ELP) at the N-terminal of double-targeting recombinant protein, and performing drug coupling by using lysine on elastin-like protein, thereby reducing the functional influence on the targeting part as much as possible, but it does not make detailed study on the fusion position and sequence length of ELP.
Disclosure of Invention
The invention aims to provide a dual-targeting fusion protein for improving the relaxation capacity of a molecular image probe, which improves the relaxation rate of the molecular probe on the premise of enhancing the targeting property of the molecular probe by selecting a proper elastin-like protein and adjusting the connection sequence of functional proteins.
The technical scheme of the invention is as follows:
a fusion protein is formed by sequentially connecting an EGFR antibody, an elastin-like protein and a polypeptide containing an RGD motif, or connecting the EGFR antibody and the elastin-like protein and/or the elastin-like protein and the polypeptide containing the RGD motif through a connecting peptide, wherein the elastin-like protein has a repeating unit consisting of VPGXG, X in each repeating unit is the same or different, X can be any amino acid except proline, and at least one X is lysine.
Preferably, X is selected from V, P, G or K.
The connecting peptide is a short peptide consisting of Gly and Ser residues. Among these, the most widely used linker peptide has the sequence (Gly-Gly-Gly-Gly-Ser) n, where n represents a positive integer of 1 to 50, preferably 1 to 5. In a specific embodiment of the invention, the linker peptide is GGGGS.
The number of the VPGXG repeating units in the elastin-like protein is 2-50. More preferably 20.
In one specific example of the present invention, the number of repeating units is 20.
Preferably, the ELP repeat sequence in the elastin-like protein VPGXG has the X amino acid K: the quantity ratio of V is 1: 1.
in a specific example of the invention, the elastin-like amino acid sequence is shown as SEQ ID No. 1.
The EGFR antibody according to the present invention may be any EGFR antibody obtained by the prior art, including but not limited to anti-EGFR monoclonal antibody, anti-EGFR single chain antibody, anti-EGFR nanobody, and the like.
In a specific example of the invention, the amino acid sequence of the EGFR antibody is shown as SEQ ID No. 2.
The short peptide is selected from polypeptides containing RGD motif, including but not limited to RGD, cRGD and iRGD. In a preferred embodiment of the present invention, the polypeptide containing the RGD motif is iRGD (c (CRGDKGPDC)).
In a specific example of the invention, the amino acid sequence of the fusion protein is shown as SEQ ID No. 3.
The invention also aims to provide a targeting molecular probe, which is obtained by coupling the fusion protein with a chelating agent and chelating metal ions.
The invention can utilize one or more of carboxyl, amino or sulfhydryl of the fusion protein to couple the chelating agent with the fusion protein through chemical reaction, such as condensation reaction, addition reaction, substitution reaction and the like, with functional groups on the chelating agent.
The chelation is carried outThe agent is selected from EDTA, DTPA, DOTA, NOTAM, TETA, BAT-TM, EC, HBED, SBAD, NOTA, CB, TE 2 A. One or more of DFO, a specific example of the present invention, is DTPA, and the amino group of lysine residue in the fusion protein reacts with carboxyl group of DTPA to form amide bond.
The metal capable of being chelated by the chelating agent is selected from Gd 3+ 、Ga 3+ 、Y 3+ 、Cu + 、Cu 2+ 、Tc 2+ 、In 3+ 、Zr 4+ 、Zn 2+ 、Fe 2 + 、Fe 3+ 、Eu 2+ Or Eu 3+ One or more of them. In a specific embodiment of the invention, the metal is Gd (III).
According to an example of the invention, a plasmid is constructed by a molecular biology means and a gene recombination technology, a fusion protein anti-EGFR-ELP-iRGD (named aEi and with an amino acid sequence shown as SEQ ID No: 3) obtained by expressing escherichia coli is used, a chelating agent diethyl triaminepentaacetic acid (DTPA) is connected with the fusion protein through a chemical reaction, and Gd is chelated 3+ Forming aEi double-targeting fusion protein tumor molecule imaging probe. The structure is shown in figure 1.
The invention also aims to provide application of the fusion protein or the targeting macromolecular probe in preparing a molecular imaging probe. The molecular imaging probe is used for imaging tumor cells.
The anti-EGFR nano antibody-elastin-like protein-iRGD double-targeting fusion protein tumor molecule imaging probe prepared by the invention has good water solubility and stability, and simultaneously has good imaging capability and biocompatibility.
The invention has the advantages that:
(1) the invention places the elastin-like protein in the middle of the target proteins at two ends, Gd 3+ Mainly located in an elastin-like segment in three segments of proteins, when the fusion protein target is subjected to multi-site combination, the internal motion is limited, so that the relaxation capacity of the polymer probe can be better improved. The injection dosage of the molecular probe can be reduced under the condition of keeping clear imaging, so that the harm of heavy metal ions to human bodies is reduced.
(2) Taking advantage of the elastin-like remodelability, adding multiple lysines provides an amino group for site-directed coupling of chelators; thereby increasing fusion protein sequestration of Gd 3+ The imaging capability of the small molecular probe is improved, and an image with higher sensitivity is obtained.
(3) The elastin-like protein in the fusion protein is positioned in the middle of a macromolecule and chelated metal ions are limited in the middle of the macromolecule, so that the functional influence on the targeting groups at two ends is reduced as much as possible, the relaxation rate is improved, the targeting property is ensured, the molecular probe is more accurately concentrated to a tumor part, the signal-to-noise ratio is improved, the sensitivity of the molecular probe is improved, the injection dosage of imaging is reduced, and the in-vivo deposition of heavy metal ions is further reduced.
Drawings
FIG. 1 is a schematic structural diagram of a gadolinium-based tumor molecular imaging probe with double-targeting recombinant protein aEi.
FIG. 2 is a MALDI-TOF MS graph of the dual-targeted recombinant protein aEi-DTPA.
FIG. 3 shows the longitudinal relaxivity r of each recombinant protein molecular probe aEi-DTPA-Gd, ai-DTPA-Gd, aE' i-DTPA-Gd, Eai-DTPA-Gd 1 Figure (a).
FIG. 4 is an in vitro MR weighted image of the dual targeting recombinant protein molecular probe aEi-DTPA-Gd.
FIG. 5 is the results of in vitro cytocompatibility testing of the dual targeting recombinant protein molecular probe aEi-DTPA-Gd.
FIG. 6 is the results of quantification of dual targeting recombinant protein molecular probe aEi-DTPA-Gd into cells.
FIG. 7 is an in vivo MRI image of a dual targeted recombinant protein molecular probe aEi-DTPA-Gd.
Detailed Description
The invention will be further elucidated with reference to the following examples, which, however, do not limit the scope of the invention.
Example 1: preparation of double-targeted recombinant protein aEi
1. Expression of Dual-Targeted recombinant protein aEi
pET-28a plasmid containing target gene aEi (anti-EGFR-ELP-iRGD) and entrusted with Shanghai biological engineeringLimited company synthesis. The amino acid sequence of the fusion protein is shown as SEQ ID No. 3 (wherein ELP has 12 repetitive units VPGKGVPGVG, X is K: V ═ 1: 1 in VPGXG repetitive sequence, K accounts for 50%), and the gene sequence is shown as SEQ ID No. 4. The plasmid containing the aEi fusion protein gene is transferred into escherichia coli BL21 by a heat shock method, and the screened positive clone is cultured overnight at 37 ℃ and then stored. The preserved strain was inoculated into LB medium, 0.1% antibiotic was added, and shaking culture was carried out overnight at 37 ℃ in a shaker. Inoculating the cultured bacterial liquid into 400ml of sterilized TB culture medium according to the proportion of 0.5% bacterial liquid and 0.1% antibiotic, and performing shake culture on a shaker at 37 ℃ for about 3 h. When OD is measured 600 When the temperature is 0.6-0.8, 0.01% isopropyl-beta-D-thiogalactoside (IPTG) is added for induction, and the shaking culture is continued on a shaking table at 18 ℃ for 20 h.
2. Extraction and purification of double-target recombinant protein aEi
And collecting the bacteria liquid after the induction expression is finished at 4 ℃ by using a high-capacity centrifuge.
After resuspending the pellet using a Binding Buffer, the cells were disrupted using an ultrasonic cell disruptor. After the cells are completely crushed, collecting the crushed solution into a centrifuge tube, and centrifuging to collect the supernatant.
The column packed with HosPur Ni-NTA was equilibrated with Binding Buffer. The collected protein-containing supernatant was filtered and loaded, and the sample was bound through a histidine (His) tag and a nickel column. Wash the column with different concentration gradients of Wash Buffer to Wash away the contaminating proteins. Then, the column was washed with a volume of Eluent Buffer and the Eluent Buffer containing the protein was collected. The molecular weight of the samples was determined by SDS-PAGE and MADLI-TOF MS and was consistent with the theoretical molecular weight.
Example 2 Synthesis of Dual-targeting recombinant protein Macro-Probe aEi-DTPA-Gd
A quantity of the lyophilized fusion protein was weighed and dissolved in Tris-HCl (0.1M pH 9.0) buffer. According to the calculation of the sequence and the molecular weight of the protein, a certain amount of diethylene triamine pentaacetic acid Dianhydride (DTPA) is weighed and dissolved in a small amount of dimethyl sulfoxide, and then the solution is slowly dropped into the protein solution, and the reaction is carried out for 12 hours under the stirring at room temperature. After completion of the reaction, the reaction mixture was collected and filtered, dialyzed against Tris-HCl (0.1M pH 7.4) buffer solution, and the dialyzate was changed every 6 hours to remove unreacted DTPA, thereby obtaining purified fusion protein-DTPA. The MALDI-TOF MS diagram of the fusion protein aEi-DTPA is shown in FIG. 2, and it can be known that the fusion protein is successfully connected with the DTPA, and the molecular weight of the fusion protein is 30135.3Da, that is, 4 DTPAs can be connected to each molecule of the fusion protein.
After the fusion protein-DTPA was dialyzed against Tris-HCl (0.1M pH 7.4) buffer, a certain amount of GdCl was weighed 3 ·6H 2 Dissolving O in a certain amount of ultrapure water, slowly dropping the solution into the buffer solution, and stirring and reacting for 12 hours at room temperature. After the reaction is finished, dialyzing with ultrapure water for 24h, changing the dialyzate every 6h, and removing unreacted Gd 3+ . After dialysis, the solution was lyophilized to a powder in a lyophilizer and stored in a freezer at-80 ℃ for future use.
In order to examine the importance of the fusion sequence and fragment size of elastin-like protein in recombinant protein, a fusion protein ai lacking elastin-like protein (anti-EGFR-iRGD, amino acid sequence SEQ ID No:5), a double-targeted recombinant protein Eai (ELP-anti-EGFR-iRGD, amino acid sequence SEQ ID No:6) of elastin-like protein at the N-terminal of recombinant protein, and a double-targeted recombinant protein aE ' i (anti-EGFR-ELP ' -iRGD, in which the number of repeat units of ELP ' is 10, the sequence is VPGKGVPGVGVPGFGVPGVGVPGKGVPGVGVPGFGVPGVGVPGKGVPGVGVPGFGVPGV, in VPGXG, X is K: V: F: 1: 2: 1, K accounts for 25%, and the amino acid sequence of recombinant protein is SEQ ID No:7) of elastin-like protein in the middle of recombinant protein were prepared according to example 1. And preparing corresponding gadolinium-chelated molecular probes ai-DTPA-Gd, Eai-DTPA-Gd and aE' i-DTPA-Gd by referring to the method.
Example 3: gd in recombinant protein molecular probes 3+ Concentration determination
Gd standard solution purchased from national Standard office and having a concentration of 1000ppm is diluted by a method of using 2 percent of HNO 3 The solutions were diluted to 50, 100, 200, 400, 800, 1000ppb standard solutions and standard curves were measured using an inductively coupled plasma mass spectrometer.
A certain amount of the lyophilized molecular probe of example 2 was weighed and dissolved in water to prepare a solution. Nitrolyzing at high temperature until the solution completely disappearsPost-treatment with 2% HNO 3 And (4) dissolving. The solution was collected and filtered, and the molecular probe was analyzed for gadolinium content by inductively coupled plasma mass spectrometry (ICP-MS).
Example 4: longitudinal relaxation rate r of recombinant protein macromolecular probe 1 Characterization of
Relaxation rate measurements were performed on the molecular probe using a 0.5T MR scanner. According to the gadolinium ion concentration measured by ICP-MS, the molecular probe aqueous solution is diluted by steps with ultrapure water to prepare a series of solutions with different concentration gradients, wherein the gadolinium ion concentration in the solutions is respectively 0.0125mM, 0.025mM, 0.05mM, 0.1mM, 0.15mM and 0.2 mM. Setting parameters of the magnetic resonance scanner, and obtaining T of each sample solution on the 0.5T magnetic resonance scanner through fast spin echo scanning 1 The value is obtained. Relaxation Rate (r) 1 ) Determined as 1/T 1 . Will r is 1 Plotting the gadolinium ion concentration, performing linear fitting on the curves, and finally calculating the longitudinal relaxation rate r of the sample according to the slope of each curve 1 (mM -1 ·s -1 ) As shown in FIG. 3 and Table 1, the longitudinal relaxation rate r of molecular probes aEi-DTPA-Gd 1 About 8.89mM -1 ·s -1 Is a commercial molecular probe Gd-DTPA (r) 1 At 4.41mM -1 ·s -1 ) 2 times of the total weight of the composition. The longitudinal relaxivity r of the molecular probe ai-DTPA-Gd, the molecular probe aE' i-DTPA-Gd and the molecular probe Eai-DTPA-Gd is measured by the same method 1 (mM -1 ·s -1 ) Longitudinal relaxation rate r of aE' i-DTPA-Gd 1 Is 6.53mM -1 ·s -1 Longitudinal relaxation rate r of aEi-DTPA-Gd 1 Is 8.89mM -1 ·s -1 The results show that K: and V is 1: the relaxation rate of the molecular probe aEi-DTPA-Gd is higher at 1 time. Longitudinal relaxation rate r of Eai-DTPA-Gd 1 Is 5.36mM -1 ·s -1 And longitudinal relaxation rate r of aEi-DTPA-Gd 1 8.89mM -1 ·s -1 It shows that when ELP is in the middle of recombinant protein and the molecular weight is about 1 ten thousand, the relaxation rate of the molecular probe is higher.
TABLE 1 longitudinal relaxivity of various molecular probes
Figure BDA0003636277590000061
Example 5: in vitro MR enhancement effect of fusion protein aEi gadolinium-based tumor molecular imaging probe according to gadolinium ion concentration measured by ICP-MS, molecular probe water solution is diluted with ultra pure water to prepare a series of solutions with different concentration gradients, wherein the gadolinium ion concentration in the solution is 0.0125mM, 0.025mM, 0.05mM, 0.1mM, 0.15mM and 0.2mM respectively. In vitro T of molecular probes by 7.0T Micro-MR 1 The result of weighted MRI enhancement effect analysis is shown in fig. 4, and it can be seen that the in vitro MR enhancement effect of the fusion protein aEi gadolinium-based tumor molecular imaging probe has obvious change with concentration, and is better than that of the commercial magnetic resonance molecular probe.
Example 6: cell compatibility experiments with fusion protein aEi gadolinium-based tumor molecular imaging probe human normal mammary epithelial MCF-10A cells were seeded in 96-well plates at a density of 5000 cells per well. After culturing cells and adhering to the wall, discarding the original culture medium of the cells, replacing the original culture medium with a culture medium containing molecular probes with different concentrations to be used as an experimental group, simultaneously setting a negative control group only containing the culture medium and a positive control group only containing the cells, respectively replanting 6 holes, and continuously incubating for 24 hours in an incubator. After completion of incubation, 20. mu.L of MTT staining solution was added to each well, and incubation was continued for 4 hours in the dark, and then the stock solution was discarded, and 150. mu.L of DMSO was added to each well to sufficiently dissolve formazan crystals. Absorbance (Abs) values at 562nm were measured for each well after 10min using a microplate reader. And calculating the cell survival rate according to the measured absorbance value. The results obtained are shown in FIG. 5, and it can be seen that the cell viability was all 80% or more, which indicates that Gd is present 3+ When the concentration of the fusion protein is in the range of 0.313-80 muM, the fusion protein aEi gadolinium-based tumor molecule imaging probe is nontoxic to cells, namely the fusion protein aEi gadolinium-based tumor molecule imaging probe has good biocompatibility.
Example 7: quantification of fusion protein aEi gadolinium-based tumor molecule imaging Probe into cell assay Hela cells were processed according to 1X 10 6 Was seeded in 6cm cell culture dishes. After culturing cells adherent, abandoning original culture medium, replacing with culture medium containing 0.1M molecular probe as experimental group, and setting culture medium containing no molecular probeThe needle medium served as a control group, each group was set in 3-dish replicates and incubated in an incubator for 3 h. After completion of the incubation, the cells were washed 3 times with PBS, then digested with pancreatin, washed 3 times with PBS, cell counted and the cell sample was lysed, after lysis with 2% nitric acid, Gd contained in the sample was measured using ICP-MS 3+ The concentration of (c). And calculating the cell entering amount of each probe according to the concentration result and the cell counting result. The results obtained are shown in FIG. 6, and it can be seen that the same Gd is present 3+ After concentration incubation, probe aEi-DTPA-Gd enters the Gd of the cells 3+ The amount of (a) is 354.5 ng/2X 10 6 cell, and Probe ai-DTPA-Gd into the Gd of the cell 3+ The amount of (A) is 95.3 ng/2X 10 6 cell, commercial probe DTPA-Gd, no detectable concentration (n ═ 3, P ═ P-<0.0001, compared using ANOVA method). This result indicates that probe aEi-DTPA-Gd has a stronger cell-entering ability than probe ai-DTPA-Gd; the ELP is used for separating two targeting groups, and the contrast agent coupling site is provided, so that the important role of improving the receptor binding capacity of the targeting groups in the dual-targeting fusion protein is played.
Example 8: living body magnetic resonance imaging
In vivo magnetic resonance imaging experiments were performed on a 7.0T small animal magnetic resonance scanner. Tumor-bearing mice were randomly selected and injected with 50. mu. mol of Gd]kg -1 Dose of molecular probe. After anesthetizing a tumor-bearing mouse, fixing the tumor-bearing mouse on a mouse bed of a 7.0T magnetic resonance scanner in an advanced prone position of the head, and installing a small animal gas anesthesia device and a respiration electrocardio monitoring device. Tail vein injection aEi-DTPA-Gd molecular probe (50 mu mol [ Gd ]]kg -1 ) In-situ Hela tumor model MR weighted imaging was performed 15min, 1h, 2h, 3h, 4h, 6h, 8h and 12h before and after injection. As a control, tail vein injection of Gd-DTPA commercial molecular Probe (50. mu. mol [ Gd ]]kg -1 ) In-situ Hela tumor model MR weighted imaging is carried out at 15min, 1h, 2h, 3h, 4h, 6h, 8h and 12h before and after injection. The detailed MR imaging parameter settings are as follows: TR is 500.0ms, TE is 10.7 ms. Image signal processing is carried out on the imaging graph by using Image J professional software. The imaging result is shown in fig. 7, and it can be seen that the tumor signal is brightest after 1h of injection of the molecular probe, and a stronger signal still exists at 12 h.
Sequence listing
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ggcaaaggtg taccgggcgt tggcgttcca ggcaaaggtg tgccgggcgt gggtgtgcca 780
ggtaaaggtg ttccaggcgt tggtggtggc ggcggcagcg gcggtggtgg cagctgccgt 840
ggtgataaag gtccggattg ctaa 864
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Claims (10)

1. A fusion protein, characterized in that: the EGFR antibody, the elastin-like protein and the polypeptide containing the RGD motif are sequentially connected, or the EGFR antibody and the elastin-like protein and/or the elastin-like protein and the polypeptide containing the RGD motif are connected through connecting peptides, the elastin-like protein has a repeating unit consisting of VPGXG, X in each repeating unit is the same or different, X is any amino acid except proline, at least one X is lysine, and the number of the repeating unit of VPGXG in the elastin-like protein is 2-50.
2. Fusion protein according to claim 1, characterized in that X is selected from V, P, G or K.
3. The fusion protein of claim 2, wherein X in each repeat unit is selected from K and V, K: the quantity ratio of V is 1: 1.
4. the fusion protein of claim 1, wherein the elastin-like amino acid sequence is set forth in SEQ ID No: 1.
5. The fusion protein of claim 1, wherein the RGD motif-containing polypeptide is selected from the group consisting of RGD, cRGD, and iRGD.
6. The fusion protein of claim 1, wherein the amino acid sequence of the fusion protein is shown in SEQ ID No. 3.
7. A targeting molecular probe, characterized in that the fusion protein according to any one of claims 1 to 6 is coupled to a chelating agent and chelates metal ions.
8. The targeted molecular probe according to claim 7, wherein the chelating agent is selected from the group consisting of EDTA, DTPA, DOTA, NOTAM, TETA, BAT-TM, EC, HBED, SBAD, NOTA, CB, TE 2 A. One or more of DFO, metal ion selected from Gd 3 + 、Ga 3+ 、Y 3+ 、Cu + 、Cu 2+ 、Tc 2+ 、In 3+ 、Zr 4+ 、Zn 2+ 、Fe 2+ 、Fe 3+ 、Eu 2+ Or Eu 3+ One or more of them.
9. Use of the fusion protein of any one of claims 1-6 or the targeted molecular probe of claim 7 or 8 for the preparation of a molecular imaging probe.
10. The use according to claim 9, wherein said molecular imaging probe is a tumor imaging contrast agent.
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CN108187064A (en) * 2017-11-28 2018-06-22 南京大学 The preparation method and purposes of double targent fused protein adriamycin couplets of one type elastin laminin-anti-EGFR nano antibodies-iRGD
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