CN106834300B - anti-CD 33 single-chain antibody and photosensitizer compound and preparation method thereof - Google Patents
anti-CD 33 single-chain antibody and photosensitizer compound and preparation method thereof Download PDFInfo
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Classifications
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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
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
- A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
- A61K47/02—Inorganic compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- 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
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
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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
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3061—Blood cells
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- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Molecular Biology (AREA)
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- Veterinary Medicine (AREA)
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- Pharmacology & Pharmacy (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- Inorganic Chemistry (AREA)
- Hematology (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
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- Oil, Petroleum & Natural Gas (AREA)
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Abstract
The invention relates to an anti-CD 33 single-chain antibody and photosensitizer compound and a preparation method thereof, belonging to the technical field of photodynamic therapy. The gene sequence of the anti-CD 33 single-chain antibody provided by the invention is shown in SEQ ID NO. 1. The photosensitizer compound provided by the invention is formed by connecting the single-chain antibody, Ce6 and hydrophilic modified C60. The photosensitizer compound provided by the invention has good targeting property, can kill tumor cells in a targeted manner, and does not damage normal cells; meanwhile, the killing effect of the photosensitizer compound is better than that of the photosensitizer Ce6 alone.
Description
Technical Field
The invention belongs to the technical field of photodynamic therapy, and particularly relates to an anti-CD 33 single-chain antibody and photosensitizer compound and a preparation method thereof.
Background
Acute Myeloid Leukemia (AML) is the most common malignancy of the blood system. CD33 is the primary marker for leukemic stem cells, and is expressed predominantly on myeloid progenitors, but not on the surface of normal hematopoietic stem cells, mature granulocytes, and other tissue cells. It was found that CD33 is expressed in more than 95% of AML patients, and thus CD33 is a good target for AML treatment.
Merotagger (GO) is a humanized anti-CD 33 monoclonal antibody, approved by the FDA in the united states in 2001, for the treatment of senile relapsed AML. Currently, GO is combined with other treatment modes (such as chemotherapy, radiotherapy and photothermal therapy) by multiple global units to comprehensively treat AML patients.
But the chemotherapy and radiotherapy in the traditional treatment have large toxic and side effects; antibodies targeting CD33 are well-targeted, but are milder in therapeutic efficacy.
Disclosure of Invention
The invention aims to provide an anti-CD 33 single-chain antibody and photosensitizer compound and a preparation method thereof. The photosensitizer compound provided by the invention has good targeting property, can kill tumor cells in a targeted manner, has no damage to normal cells and small toxic and side effects, and has wide prospects in the aspects of single medication and combined medication of acute lymphatic leukemia.
The invention provides a gene for encoding anti-CD 33 single-chain antibody, and the nucleotide sequence is shown in SEQ ID NO. 1.
The invention also provides an anti-CD 33 single-chain antibody, and the gene sequence of the coded antibody is shown in SEQ ID NO. 1
The invention also provides an anti-CD 33 single-chain antibody expression vector, wherein the anti-CD 33 single-chain antibody expression vector is a pE-SUMO vector fused with the gene of claim 1.
The invention also provides a photosensitizer compound which is formed by connecting the single-chain antibody of claim 1, Ce6 and PEG-modified C60; c60 modified by PEG is connected with Ce6 through non-covalent bond; ce6 on the ligation product was ligated to the single-chain antibody via an amide bond.
Preferably, the PEG has an Mw molecular weight of 3000.
Preferably, the particle size distribution of the photosensitizer compound is 30-105 nm, and the average particle size of the photosensitizer compound is 48.96 nm.
The invention also provides a preparation method of the photosensitizer compound in the technical scheme, which comprises the following steps:
1) carrying out a Bingle cycloaddition reaction on the C60 and bromomalonic acid ethyl ester in toluene to obtain a C60 malonic acid ethyl ester derivative; hydrolyzing the C60 ethyl malonate derivative to obtain a C60-COOH derivative;
2) respectively carrying out amination modification and methoxy ether modification on hydroxyl groups at two ends of PEG to obtain H2N-PEG-OMe；
3) Reacting the C60-COOH derivative with H2Mixing N-PEG-OMe, stirring overnight, and performing esterification reaction to obtain a coupling product C60-PEG;
4) mixing the coupling product C60-PEG with Ce6, and carrying out ultrasonic oscillation to obtain C60-PEG-Ce 6;
5) activating carboxyl on the surface of Ce6 in the C60-PEG-Ce6 by EDC-HCl and NHS, and performing esterification reaction on the activated product and GOscFv to obtain a photosensitizer compound;
there is no chronological restriction between the step 1) and the step 2).
Preferably, the temperature of hydrolysis in the step 1) is 80 ℃; separately adding NaH into the cycloaddition reaction and the hydrolysis reaction of the step 1).
Preferably, the ultrasonic oscillation in the step 4) is performed in an ethanol aqueous solution; the mass concentration of the ethanol water solution is 50%.
Preferably, the esterification reaction of step 5) is carried out in NaHCO3Is carried out in solution; the molar ratio of the C60-PEG-Ce6 to the GOscFv is 1: 10.
the invention provides an anti-CD 33 single-chain antibody. The photosensitizer compound prepared from the anti-CD 33 single-chain antibody has good targeting property, and the design of the single-chain antibody coding gene can well bring the photosensitizer compound to the surface of acute lymphocytic leukemia cells expressing CD33 molecules, kill tumor cells in a targeted manner, and does not damage normal cells; meanwhile, the killing effect of the photosensitizer compound is better than that of the photosensitizer Ce6 alone.
Drawings
FIG. 1 is a result diagram of colony PCR verification of GOscFv positive recombinant bacteria provided in example 1 of the present invention;
FIG. 2 is a result chart of SDS-PAGE provided in example 1 of the present invention verifying whether recombinant bacteria can express GOscFv-SUMO protein; wherein M: marker; lane 1: uninduced control, Lane 2: adding IPTG to induce group;
FIG. 3 is a WesternBlot validation chart of GOscFv-SUMO provided in example 1 of the present invention; FIG. 3a is a graph showing the results of the verification with an anti-His antibody, and FIG. 3b is a graph showing the results of the verification with an anti-SUMO antibody; wherein Lane 1: uninduced control, Lane 2: adding IPTG to induce group;
FIG. 4 is a SDS-PAGE result chart of GOscFv-SUMO protein purified by Ni column before and after the SUMO enzymolysis provided in example 1 of the present invention; wherein Lane 1: GOscFv-SUMO protein; lane 2: a GOscFv protein;
FIG. 5 is a schematic diagram of the synthetic scheme of the photosensitizer complex C60-PEG-Ce6-GOscFv provided in example 1 of the present invention;
FIG. 6 is a TEM analysis of the photosensitizer complex C60-PEG-Ce6-GOscFv provided in example 1 of the present invention;
FIG. 7 is a graph of particle size analysis of the photosensitizer complex C60-PEG-Ce6-GOscFv provided in example 2 of the present invention;
FIG. 8 is a graph showing the result of detecting the PDT effect of photosensitizer complex C60-PEG-Ce6-GOscFv on AML cells by the MTT method provided in example 2 of the present invention;
FIG. 9 is a graph showing the result of detecting PDT effect of photosensitizer complex C60-PEG-Ce6-GOscFv on normal PBMC cells by MTT method provided in example 2 of the present invention;
FIG. 10 is a graph showing the PDT effect of photosensitizer complex C60-PEG-Ce6-GOscFv in AML model provided in example 3 of the present invention;
FIG. 11 is a graph of the quantitative results of PDT efficacy of photosensitizer complex C60-PEG-Ce6-GOscFv in AML model provided in example 3 of the present invention.
Detailed Description
The invention provides a gene for encoding anti-CD 33 single-chain antibody, and the nucleotide sequence is shown in SEQ ID NO. 1.
In the invention, the anti-CD 33 single-chain antibody gene is designed according to the following principle: number of merotagger (GO) by drug bank: DB00056, searching and obtaining the amino acids of the heavy chain and light chain variable regions, and obtaining the corresponding nucleic acid sequences GO-VL (SEQ ID NO: 2) and GO-VH (SEQ ID NO: 3) by carrying out codon optimization of escherichia coli; by (G)4S)3The two sections of nucleic acid sequences are connected by a Linker to obtain the gene of the anti-CD 33 single-chain antibody (GOscFv)The sequence is as follows: VL-Linker-VH, BamHI and HinderIII cleavage sites were added at the 5 'and 3' ends of VL-Linker-VH, respectively, to give the amino acid sequences shown in SEQ ID NO:1 (GOscFv). In the present invention, the molecular weight of the single chain antibody (GOscFv) is 1/6 of the marketed full-length antibody, and the targeting property to the CD33 antigen is retained while the molecular weight is reduced. The method for optimizing the Escherichia coli codon is not particularly limited, and can be optimized by a codon optimization method known to those skilled in the art, such as DNMAN software.
The invention also provides an anti-CD 33 single-chain antibody, and the gene sequence of the coded antibody is shown in SEQ ID NO. 1.
The invention also provides an anti-CD 33 single-chain antibody expression vector, wherein the anti-CD 33 single-chain antibody expression vector is a pE-SUMO vector fused with the gene of claim 1.
In the present invention, the method for preparing the single-chain antibody comprises the following steps: constructing a pE-SUMO vector containing the gene sequence in the technical scheme, expressing the pE-SUMO vector through an escherichia coli expression system, and purifying an expression product; and carrying out enzymolysis on the purified product by using SUMO protease to obtain the anti-CD 33 single-chain antibody.
The construction method of the vector is not particularly limited, and the expression vector can be constructed by adopting an enzyme digestion ligation construction method well known by the technical personnel in the field. The expression vector is preferably pE-SUMO.
The construction of the E.coli expression system in the present invention is not particularly limited, and may be carried out by using an E.coli expression system known to those skilled in the art. The conditions for expressing the single-chain antibody in the present invention are not particularly limited, and protein expression methods known to those skilled in the art may be used.
In the present invention, the purification method is preferably Ni column purification.
After the purified product is obtained, the SUMO protease is preferably added to carry out enzymolysis on the purified product at 37 ℃, and the single-chain antibody is obtained after the purification by the Ni column is carried out again.
In the present invention, after obtaining the single-chain antibody, ultrafiltration concentration is preferably performed. The method of ultrafiltration concentration in the present invention is not particularly limited, and a protein concentration method known to those skilled in the art may be used, for example, concentration using an ultrafiltration tube, preferably concentration to 5 mg/mL.
The invention also provides a photosensitizer compound, which is formed by connecting the single-chain antibody, Ce6 and PEG-modified C60 in the technical scheme; c60 modified by PEG is connected with Ce6 through non-covalent bond; ce6 on the ligation product was ligated to the single-chain antibody via an amide bond.
In the invention, the Ce6 is chlorin e6, has an absorption wavelength of 663nm, can generate more ROS, and has small side reaction to skin; the C60 is fullerene, the molecular structure is a spherical 32-surface body, the fullerene is formed by connecting 60 carbon atoms by 20 six-membered rings and 12 five-membered rings, the football-shaped hollow molecule is provided with 30 carbon-carbon double bonds, and the diameter of the sphere is about 0.7 nm; c60 is used as a drug carrier in the invention, C60 also has certain photosensitive property, and C60 can efficiently generate ROS after being irradiated by visible light or ultraviolet light to mediate PDT effect; the water solubility and biocompatibility of the C60 with the modified surface are improved, and the cytotoxicity is reduced. The photodynamic therapy effect produced by combining Ce6 and C60 is greatly enhanced. The source of Ce6 and C60 is not particularly limited in the present invention, and conventional commercial products of Ce6 and C60 known to those skilled in the art may be used.
In the present invention, the molecular weight Mw of the PEG is 3000.
In the invention, the particle size distribution of the photosensitizer compound is 30-105 nm, and the average particle size of the photosensitizer compound is 48.96 nm.
The invention also provides a preparation method of the photosensitizer compound in the technical scheme, which comprises the following steps:
1) carrying out a Bingle cycloaddition reaction on the C60 and bromomalonic acid ethyl ester in toluene to obtain a C60 malonic acid ethyl ester derivative; hydrolyzing the C60 ethyl malonate derivative to obtain a C60-COOH derivative;
2) respectively carrying out amination modification on hydroxyl groups at two ends of PEGMethoxy ether modification to obtain H2N-PEG-OMe；
3) Reacting the C60-COOH derivative with H2Mixing N-PEG-OMe, stirring overnight, and performing esterification reaction to obtain a coupling product C60-PEG;
4) mixing the coupling product C60-PEG with Ce6, and carrying out ultrasonic oscillation to obtain C60-PEG-Ce 6;
5) activating carboxyl on the surface of Ce6 in the C60-PEG-Ce6 by EDC-HCl and NHS, and performing esterification reaction on the activated product and GOscFv to obtain a photosensitizer compound;
there is no chronological restriction between the step 1) and the step 2).
The invention carries out a Bingle cycloaddition reaction of C60 and bromomalonic acid ethyl ester in toluene to obtain the C60 malonic acid ethyl ester derivative. The double cycloaddition reaction is not particularly limited, and the conditions of the double cycloaddition reaction known by the technical personnel in the field are adopted, for example, in the embodiment of the invention, C60 is dissolved in toluene, NaH is added, bromomalonic acid ethyl ester is added after stirring, the reaction is carried out for 4-6 h under the protection of nitrogen, NaH is removed through filtration, and the C60 malonic acid ethyl ester derivative is obtained after drying. In the present invention, the ratio of the mass of C60 to the volume of toluene is preferably 1 g: (1-3) L, more preferably 1 g: 1L of the compound. In the present invention, the mass ratio of C60 to NaH is preferably 1: (5-7), more preferably 1: 6. in the invention, the volume ratio of the mass of C60 to the ethyl bromomalonate is (200-300) g: 1L, more preferably 250 g: 1L of the compound. In the invention, the drying preferably adopts a vacuum drying method, and the vacuum drying preferably is carried out at 45-60 ℃ for 20-26 h, and more preferably at 50 ℃ for 24 h.
After obtaining the C60 malonic acid ethyl ester derivative, the invention hydrolyzes the C60 malonic acid ethyl ester derivative to obtain the C60-COOH derivative. In the present invention, the temperature of the hydrolysis is preferably 80 ℃; the hydrolysis time was 10 h. In the present invention, the hydrolysis reaction is carried out in the presence of NaH. The hydrolysis reaction of the present invention is preferably: dissolving a C60 ethyl malonate derivative and NaH in toluene, carrying out ultrasonic treatment for 15-30 min, stirring for 8-12 h at 80 ℃ under the protection of nitrogen, and neutralizing in a water phase to obtain a precipitate C60-COOH derivative. In the present invention, the ratio of the mass of the C60 ethyl malonate derivative to the volume of the toluene for hydrolysis is preferably 1 g: (5-7) L, more preferably 1 g: 6L. In the present invention, the mass ratio of the C60 ethyl malonate derivative to the NaH for hydrolysis is preferably 1: (3-5), more preferably 1: 3.6. in the present invention, the temperature of the ultrasound is preferably 40 ℃, and the frequency is preferably 10 KHz;
the method for neutralizing the water phase is not particularly limited in the invention, and the water phase can be neutralized by using an acid-base regulator well known to those skilled in the art, such as concentrated hydrochloric acid for neutralizing NaH; in the present invention, the precipitate is preferably dissolved in methanol, and insoluble matter is removed by filtration to obtain a C60-COOH derivative.
The invention respectively carries out amination modification and methoxy ether modification on hydroxyl groups at two ends of PEG to obtain H2N-PEG-OMe. The method of the present invention for modifying the amino group and the methoxy ether is not particularly limited, and the methods for modifying the amino group and the methoxy ether known to those skilled in the art may be used.
To obtain H2After N-PEG-OMe, the C60-COOH derivative is mixed with H2Mixing the N-PEG-OMe, stirring overnight, and performing esterification reaction to obtain a coupling product C60-PEG. In the present invention, the stirring reaction is added with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC). According to the invention, after the reaction is carried out overnight under stirring, preferably, ethanol precipitation is carried out on a reaction product, the precipitate is washed for more than 10 times by ethanol, and finally, the ethanol is dried and volatilized to obtain a coupling product. In the invention, the drying is preferably vacuum drying, and the drying conditions are preferably 45-60 ℃ and 20-26 h, more preferably 50 ℃ and 24 h.
After obtaining the coupling product C60-PEG, the coupling product C60-PEG and Ce6 are mixed and ultrasonically oscillated to obtain the product C60-PEG-Ce6 bonded by Van der Waals force. In the invention, the mass ratio of C60-PEG to Ce6 is preferably 1-2.5-3.5, more preferably 1: 3. in the invention, the time of ultrasonic oscillation is preferably 1.5-2.5 h, and more preferably 2 h. In the invention, the ultrasonic oscillation is preferably carried out by dissolving C60-PEG and Ce6 in 50% by mass of ethanol aqueous solution. After ultrasonic agitation, the present invention preferably slowly evaporates the aqueous ethanol solution at 40 ℃. In the invention, the excessive Ce6 is preferably removed in the aqueous solution by a high-speed centrifugation method according to the difference of molecular weight, and the precipitate is taken to obtain C60-PEG-Ce 6.
After C60-PEG-Ce6 is obtained, EDC-HCl and NHS are used for activating carboxyl on the surface of Ce6 in C60-PEG-Ce6, and the activated product and the amino of GOscFv are subjected to esterification reaction to form an ester bond for coupling to obtain a photosensitizer compound: C60-PEG-Ce 6-GOscFv. The concentration of EDC in the EDC-HCl is preferably 0.4mol/L, the concentration of NHS is preferably 0.05mol/L, and the C60-PEG-Ce6 is preferably prepared into an aqueous solution with the mass concentration of 50 mg/L; the volume ratio of EDC, N-hydroxysuccinimide (NHS) and C60-PEG-Ce6 aqueous solution is preferably 1: 1: 2. the EDC and NHS function to activate the carboxyl on the Ce6 surface of C60-PEG-Ce 6. In the present invention, the molar ratio of C60-PEG-Ce6 to GOscFv after carboxyl activation is preferably 1: 10. the esterification reaction is preferably NaHCO at pH73The esterification reaction is carried out in solution, the esterification reaction condition is that the mixture is stirred vigorously at room temperature overnight, and the rotation speed of the vigorous stirring is preferably 400 rpm.
After the esterification reaction, the reaction product is preferably centrifuged, the precipitate is taken to remove free GOscFv, and the obtained precipitate is preferably dissolved in water to obtain the final product C60-PEG-Ce 6-GOscFv. In the invention, the centrifugal condition is preferably 12000-15000 rpm for 20-30 min.
The anti-CD 33 single-chain antibody and photosensitizer complex and the preparation method thereof provided by the present invention are further described in detail with reference to the following embodiments, but the technical solutions of the present invention include, but are not limited to, the following embodiments.
Example 1
Construction of expression vectors
The GOscFv nucleic acid sequence shown as SEQ ID NO. 1 is biosynthesized in Shanghai, and the sequence is inserted into the corresponding site of a vector pE-SUMO through enzyme digestion and enzyme ligation to obtain an expression vector: pE-SUMO-GOscFv.
Construction of expression Strain
The expression vector (pE-SUMO-GOscFv) is transformed into Escherichia coli BL21(DE3), and screened on LB solid medium plate containing 50ug/mL kanamycin to obtain the corresponding expression strain: BL21(DE3)/pE-SUMO-GOscFv, the strain on the plate was picked for colony PCR verification, and the results are shown in FIG. 1. After the positive clone is preserved, the strain is sent to Shanghai biological sequencing, and through comparison with an expected sequence, strains No. 1, 3 and 6 are selected for protein fermentation.
Strain No. 1 was inoculated into 10mLLB (containing 50ug/mL kanamycin) medium, and after 3 hours of culture at 37 ℃ and 220rpm, it was divided into 2 groups: the induction group was induced with 0.1mM IPTG, while the control group was free of IPTG. The 2 groups of bacterial liquid are further cultured at 37 ℃ and 220rpm overnight, and the bacterial liquid is subjected to SDS-PAGE verification, compared with the experimental group, the induced group has protein expression at about 45KDa (the result is shown in figure 2); and performing WB verification, namely verifying the expression product by using an anti-His antibody and an anti-SUMO antibody respectively, wherein verification result graphs are shown in figures 3a and 3b respectively, and the result shows that the expression product at 45KDa is provided with a 6 XHis tag (shown in figure 3 a) and a SUMO tag (shown in figure 3 b).
Expression and purification of Single chain antibodies
1) The positive strain was inoculated into 50mLLB (containing 50ug/mL kanamycin) medium and cultured overnight at 37 ℃ and 220rpm to activate the strain.
2) And (3) mixing the activated bacterium liquid according to the proportion of 1: 100 in proportion, transferred to LB medium without antibiotics, and expanded at 37 ℃ and 220 rpm. After 3h, 0.1mM IPTG was added for induction and further incubation at 160rpm at 20 ℃ for 24 h.
3) The cultured E.coli was collected and centrifuged at 8000rpm for 30min at 4 ℃. The pellet was resuspended in 20mL of a cell lysate containing 0.1mg/mL lysozyme (50mM Tris-HCl,0.5M NaCl, 1% Triton X-100, pH 8.0), sonicated for 10min, and the mixture was magnetically stirred at 4 ℃ for 30 min. Then 14000rpm for 30min, and the supernatant was collected as a soluble cytosol fraction.
4) Passing the supernatant through 0.22um filter membrane, adsorbing by Ni column, gradient eluting with 500mM, 200mM, 100mM, 50mM, 20mM and 10mM sequentially, and determining the elution component containing the target protein by SDS-PAGE verification of the elution product. Combining the components containing the target protein, dialyzing with 500mL PBS for 3 times for 12 hours each time, adding SUMO protease (containing His label) into the obtained dialysate according to 100IU/mL, reacting at 37 ℃ for 2 hours, adsorbing the reaction product by Ni column again, collecting the effluent, ultrafiltering the effluent by using an ultrafiltration tube (WMCO: 50KDa) to obtain a concentrated solution, and verifying the obtained concentrated product by SDS-PAGE, wherein the result is shown in figure 4.
Synthesis of C60-COOH:
1) 50mg of C60(I) was dissolved in 50ml of toluene, and 0.3g of NaH was added thereto, followed by stirring for 1 hour.
2) 0.2ml of bromomalonic acid ethyl ester is measured and dissolved in 3ml of toluene, slowly and dropwise added into the system, and the mixture reacts for 5 hours at room temperature under the protection of nitrogen.
3) The NaH was removed by filtration and dried under vacuum at 50 ℃ for 24h to give the C60 ethyl malonate derivative, as shown in II in FIG. 5.
4) Weighing 50mg of C60 ethyl malonate derivative and 180mg of NaH obtained by the reaction, adding into 30ml of toluene, and carrying out ultrasonic treatment for 20 min;
5) the mixture is stirred for 10 hours at 80 ℃ in a three-way bottle under the protection of nitrogen, and is naturally cooled to room temperature.
6) After the reaction is finished, the toluene removing phase is removed, 20ml of concentrated hydrochloric acid is added into the water phase to neutralize NaH, C60-COOH sediment is obtained,
7) dissolving the C60-COOH precipitate in methanol, filtering to remove insoluble substances, and evaporating methanol to obtain C60-COOH derivative, as shown in III in FIG. 5.
MeO-PEG(3000)-NH2The synthesis of (2):
1) 100.01g of dry, conventional, commercially available MeO-PEG (3000) was weighed into a 500mL three-necked flask, 300mL of thionyl chloride was added, and heated under reflux for 24h under nitrogen.
2) After the reaction is finished, most of the solvent is distilled off under normal pressure, and then the residual thionyl chloride is removed under reduced pressure to obtain a light yellow semisolid compound MeO-PEG (3000) -Cl.
3) 80.023g of MeO-PEG (3000) -Cl were weighed into a 100mL autoclave, 400mL of 25% aqueous ammonia was added, and the autoclave was sealed. The reaction is carried out for 24 hours at the temperature of 140 ℃,
4) evaporating most of ammonia water at 50 deg.C, dissolving the residue in 500mL of water, extracting with 250mL of dichloromethane for 2 times, filtering, and collecting the filtrateThe solvent was distilled off to give the product: MeO-PEG (3000) -NH2。
Synthesis of C60-PEG (3000):
weighing 20mg of C60-COOH derivative, dissolving in 10ml PBS, adding H2N-PEG-OMe 250mg and EDC 5mg reacted at room temperature overnight, washed with water to remove excess PEG and EDC, and dried at 50 deg.C under vacuum for 24h to give C60-PEG derivatives, as shown in IV in FIG. 5.
Synthesis of C60-PEG-Ce 6:
weighing 50mg of C60-PEG-COOH and Ce6150mg into 50mL of mixed solvent of ethanol and water (1: 1), carrying out ultrasonic treatment for 2h, slowly evaporating at 40 ℃, removing ethanol and water, carrying out ultrasonic treatment for 20min, centrifuging the reaction mixture for 25min at 14000rpm, removing free Ce6, adding 200 mu L of water into the precipitate for redissolving to obtain C60-PEG-Ce6 aqueous solution, and storing at 4 ℃, wherein V is shown in figure 5.
Synthesis of C60-PEG-Ce 6-GOscFv:
1) 2.5mL of LEDC (0.4M) and 2.5mL of LNHS (0.05M) were mixed, added to 5mL of an aqueous solution of C60-PEG-Ce6, and incubated for 30min with shaking in a gas bath incubator to activate carboxyl groups on the surface of C60-PEG-Ce 6.
2) C60-PEG-Ce6 and GOscFv after carboxyl activation are mixed according to a molar ratio of 1: 10 portions were mixed in 10mL NaHCO3(pH7) and stirred vigorously at room temperature overnight.
3) The reaction mixture was centrifuged for 25min at 14000rpm to remove free GOscFv, and the resulting pellet was dissolved with 500uL ddH2O as the final product: C60-PEG-Ce6-GOscFv as shown in VI in FIG. 5.
4) The C60-PEG-Ce6-GOscFv was analyzed for particle size distribution to obtain a particle size distribution range of 48.5-110nm and an average particle size of 55nm, as shown in FIG. 6. And the shape thereof was analyzed by transmission scanning electron microscopy (TEM), and the result is shown in fig. 7.
Example 2
In vitro experiments to evaluate the PDT Effect of C60-PEG-Ce6-GOscFv
Blood of leukemia patients is taken for 100mL, and PBMC is separated from Ficoll lymphocyte separating medium.
Adjust the cell concentration to 2X 10 with 16405cells/mL, 2mL of cell suspension per dish 3In a 5mm petri dish. Dividing into 3 groups, adding PBS, Ce6 (final concentration of 5ug/mL), and C60-PEG-Ce6-GOscFv (final concentration of 15ug/mL), incubating in incubator for 4h in dark place, irradiating with 630nm wavelength light for 40min and with light energy of 18J/cm2。
MTT colorimetric method is adopted. 24h after illumination, cells in the culture dish were transferred into a 96-well plate, 2X 104Cells/well, MTT assay. And adding 180 mu L of cell suspension and 20 mu L of MTT solution (5mg/mL) into each group of 3 wells, incubating for 4h, centrifuging, discarding the supernatant, adding 200 mu L of DMSO into each well to dissolve the formazan, and detecting the absorbance at 570nm of an enzyme labeling instrument. Cell inhibition (%) -1-absorbance value of experimental group/absorbance value of control group × 100%.
HL-60 is a human AML cell strain, the surface of which expresses CD33 antigen, and the PDT treatment effect of the HL-60 cell is detected by the method shown above.
The killing effect of the C60-PEG-Ce6-GOscFv on normal blood PBMC and HL-60 cells is shown in figures 8 and 9, and the results show that the PDT curative effect of the C60-PEG-Ce6-GOscFv on HL-60 is stronger than that of Ce6, the C60-PEG-Ce6-GOscFv has almost no inhibiting effect on normal human PBMC, the C60-PEG-Ce6-GOscFv has stronger cytotoxicity on leukemia cells, and the toxic and side effects of normal cells are very slight.
Example 3
In vivo experiments to evaluate PDT Effect of C60-PEG-Ce6-GOscFv
HL-60 cells expressing EGFP were constructed. Transfecting an expression vector (pCDNA3.0-EGFP) containing green fluorescent protein into the HL-60 cell by a liposome method, performing pressurized screening by using G418, and performing 2 rounds of limiting dilution to obtain the HL-60 cell for stably expressing the EGFP: HL-60-EGFP.
Selecting 15C 3H/HeJ mice (7-9 weeks old, female), mixing, and making into 2 × 10 mice6HL-60-EGFP cells are implanted into a C3H/HeJ mouse body through a tail vein for AML modeling. After 5 days, the C3H/HeJ mice implanted with HL-60-EGFP cells were randomly divided into 3 groups, 5/group. 100uL of PBS, Ce6 (concentration of 5mg/mL), and C60-PEG-Ce6-GOscFv (concentration of 20mg/mL) were injected into the tail vein, and the mixture was incubated for 1 day in the dark.
After 1 day, PDT treatment was performed in the dark using a needle-embedding method: immobilizing rat tails at 37 deg.COn a thermostatic plate, a G24 indwelling needle (diameter 0.75mm) was used to puncture into the tail vein of a mouse, and then a 75% ethanol sterilized optical fiber (diameter 0.5mm) was inserted into the indwelling needle, and the needle was held stationary for 3 hours, during which time the tail was held stationary without shaking. The other end of the optical fiber is connected with a Ne-He laser therapeutic instrument for irradiating blood, and the therapeutic power is as follows: 12.5J/cm2。
After PDT treatment, culture was continued for 5 days. Blood was obtained from each treatment group at 1 mL/mouse by cardiac puncture. The obtained 1mL of blood was placed in one well of a 6-well plate, observed under a fluorescent microscope, and the number of EGFP-expressing cells in each group was recorded as shown in fig. 10. The viability of HL-60-EGFP cells in vivo was recorded for each group of AML mice and the results are shown in FIG. 11. Indicating that C60-PEG-Ce6-GOscFv has stronger therapeutic effect in vivo compared with Ce 6.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
SEQUENCE LISTING
<110> Lissvin
<120> anti-CD 33 single-chain antibody and photosensitizer compound and preparation method thereof
<130> 2017
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Claims (10)
1. The gene of the anti-CD 33 single-chain antibody is shown in SEQ ID NO. 1.
2. The anti-CD 33 single-chain antibody has the nucleotide sequence shown in SEQ ID No. 1.
3. An anti-CD 33 single-chain antibody expression vector, wherein the anti-CD 33 single-chain antibody expression vector is a pE-SUMO vector fused with the gene of claim 1.
4. A photosensitizer complex, which is formed by connecting the anti-CD 33 single-chain antibody coded by the gene of claim 1, Ce6 and PEG modified C60; c60 modified by PEG is connected with Ce6 through non-covalent bond; ce6 on the ligation product was ligated to the single-chain antibody via an amide bond.
5. The photosensitizer complex of claim 4, wherein the PEG has an Mw molecular weight of 3000.
6. The photosensitizer complex according to claim 4 or 5, wherein the photosensitizer complex has a particle size distribution of 30 to 105nm, and the average particle size of the photosensitizer complex is 48.96 nm.
7. A method of preparing a photosensitizer complex according to any one of claims 4 to 6, comprising the steps of:
1) carrying out a Bingle cycloaddition reaction on the C60 and bromomalonic acid ethyl ester in toluene to obtain a C60 malonic acid ethyl ester derivative; hydrolyzing the C60 ethyl malonate derivative to obtain a C60-COOH derivative;
2) respectively carrying out amination modification and methoxy ether modification on hydroxyl groups at two ends of PEG to obtain H2N-PEG-OMe；
3) Reacting the C60-COOH derivative with H2Mixing N-PEG-OMe, stirring overnight, and performing esterification reaction to obtain a coupling product C60-PEG;
4) mixing the coupling product C60-PEG with Ce6, and carrying out ultrasonic oscillation to obtain C60-PEG-Ce 6;
5) activating carboxyl on the surface of Ce6 in the C60-PEG-Ce6 by EDC-HCl and NHS, and carrying out esterification reaction on the activated product and an anti-CD 33 single-chain antibody to obtain a photosensitizer compound;
there is no chronological restriction between the step 1) and the step 2).
8. The method for preparing the compound of claim 7, wherein the hydrolysis temperature in the step 1) is 80 ℃; separately adding NaH into the cycloaddition reaction and the hydrolysis reaction of the step 1).
9. The method according to claim 7, wherein the ultrasonic oscillation in the step 4) is performed in an aqueous ethanol solution; the mass concentration of the ethanol water solution is 50%.
10. The method of claim 7, wherein the esterification reaction in step 5) is performed in NaHCO3Is carried out in solution; the molar ratio of the C60-PEG-Ce6 to the anti-CD 33 single-chain antibody is 1: 10.
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| WO2012045752A1 (en) * | 2010-10-04 | 2012-04-12 | Boehringer Ingelheim International Gmbh | Cd33 binding agents |
| CN102952191A (en) * | 2012-09-17 | 2013-03-06 | 浙江大学 | Fully human CD33 single-chain antibody ZJL101 and applications thereof |
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| WO2012045752A1 (en) * | 2010-10-04 | 2012-04-12 | Boehringer Ingelheim International Gmbh | Cd33 binding agents |
| CN104159600A (en) * | 2012-01-26 | 2014-11-19 | Ibc药品公司 | Targeting interferon-lambda with antibodies potently enhances anti-tumor and anti-viral activities |
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