Humanized recombinant single-chain antibody, nano-particle, nano-drug composition and application thereof

Abstract

The invention relates to the technical field of antibody drugs, in particular to a human recombinant single-chain antibody, nano-particles, a nano-drug composition and application of the human recombinant single-chain antibody, the nano-particles and the nano-drug composition. The invention provides a human recombinant single-chain antibody of specific anti-PHD2. The amino acid sequence of the human recombinant single-chain antibody is as shown in SEQ ID NO: 2. The obtained human recombinant single-chain antibody is more stable than INP and has good organ and tissue targeting. The human recombinant single-chain antibody is combined with meso-porous silicon nanoparticles and an erythrocyte membrane to prepare a nano-drug composition, and the nano-drug composition has a better effect of treating diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody drugs, and in particular to a humanized recombinant single-chain antibody, nanoparticles and nanomedicine composition and applications thereof. Background Art

[0002] Hypoxia-inducible factor-1 (HIF-1) is an oxygen-sensitive transcriptional activator that can specifically bind to the transcription factor of the hypoxia response element. HIF-1 is a heterodimer composed of HIF-1α and HIF-1β, of which HIF-1β is constitutively expressed and distributed in the cell nucleus, while HIF-1α is mainly distributed in the cytoplasm. Under hypoxic conditions, HIF-1α can be transported to the cell nucleus to form a HIF-1 heterodimer with HIF-1β, and this step is a prerequisite for the function of HIF-1.

[0003] HIF-1 can activate more than 100 genes under hypoxic conditions. These genes are involved in various hypoxic adaptation processes of cells, including angiogenesis, erythropoiesis, energy metabolism, cell proliferation and cell cycle control. HIF-1 reduces cell death caused by hypoxia by regulating the expression of these genes. For example, HIF-1 regulation of glycolysis or angiogenesis genes constitutes an early adaptive response to oxidative stress. Secondly, hypoxia and HIF-1 can induce the expression of growth factors such as insulin-like growth factor-2 (IGF2) and transforming growth factor-α (TGF-α), which bind to their corresponding receptors and activate intracellular signaling pathways related to cell proliferation / survival. In addition, a large number of genes involved in angiogenesis have been shown to increase in expression under hypoxic conditions. Therefore, the presence of HIF-1 plays a key role in the recovery process of diseases such as injuries.

[0004] However, under normoxic conditions, HIF-1α is constantly degraded and is extremely unstable. This oxygen-dependent degradation is mediated by (prolyl hydroxylase) PHD. Hydroxylation of HIF-1α at prolyl residues 402 or 564 promotes binding to von Hippel-Lindau protein (VHL), which recruits an E3 ubiquitin-protein ligase complex to promote the ubiquitination and degradation of HIF-1α. Prolyl hydroxylases include PHD1, PHD2, and PHD3. The biochemical characteristics of PHDs are similar to collagen proline-4-hydroxylases. They are both 2-OG-dependent dioxygenases that use molecular oxygen (O2) and 2-oxoglutarate as co-substrates to hydroxylate the two proline residues of HIF-1α, thereby mediating the degradation and inactivation of HIF-α. Among them, PHD2 is considered to be the rate-limiting enzyme that regulates the prolyl hydroxylation and ubiquitination degradation of HIF-1α.

[0005] Studies have shown that HIF-1 is associated with the development of many diseases. Currently, PHD inhibitors have been widely used, but PHD inhibitors often lack subtype specificity and have significant toxic side effects. Therefore, there is an urgent need to develop new, efficient and highly specific PHD inhibitors.

[0006] Antibodies are an important part of the human immune system. Due to their superior targeting specificity, pharmacokinetic and pharmacodynamic properties, safety and toxicity characteristics, and adaptability of multifunctional engineering, they have become the fastest-growing drugs for treating a variety of human diseases, including cancer. Advances in genetic engineering technology, humanization technology, transgenic mice, and antibody screening technology have promoted the development of antibody technology, and also provided opportunities for the construction of various small molecule antibodies such as antibody fragments, such as single-chain variable fragments (scFv, also known as single-chain antibodies). In medical diagnosis and treatment applications, antibody fragments are increasingly used as substitutes for monoclonal whole antibodies. Among them, single-chain antibodies (scFv) are a recombinant molecule composed of the variable regions of the light chain (VL) and heavy chain (VH) of IgG and a flexible linker sequence connecting the two. Due to its advantages of small molecular weight, strong tissue penetration and high targeting, it has been widely used in disease diagnosis and treatment.

[0007] Although single-chain antibodies are currently widely used, unmodified single-chain antibodies have disadvantages such as poor organ or tissue targeting and easy clearance, resulting in poor efficacy and potential toxicity. In addition, single-chain antibodies cannot effectively penetrate the cell membrane to act on intracellular target molecules. These problems limit their further clinical application. Therefore, how to prolong the half-life in vivo, achieve tissue or organ targeted enrichment and improve the efficiency of single-chain antibodies in cellular entry has become the main research direction in the field of single-chain antibodies that act on intracellular target molecules.

[0008] Based on this, the present invention is proposed. Summary of the invention

[0009] The purpose of the present invention is to develop a human recombinant single-chain antibody, nanoparticles and nanomedicine composition and their application.

[0010] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0011] The present invention provides a human recombinant single-chain antibody, the amino acid sequence of the human recombinant single-chain antibody is shown in SEQ ID NO:2.

[0012] The present invention also provides nucleotides encoding the amino acid sequence of the human recombinant single-chain antibody, and the nucleotides are shown in SEQ ID NO:1.

[0013] The present invention also provides a primer set for amplifying the nucleotide, wherein the primer set comprises a primer pair 1 and a primer pair 2;

[0014] The primer pair 1 is shown in SEQ ID NO: 3-4;

[0015] The primer pair 2 is shown in SEQ ID NOs: 5-6.

[0016] The present invention also provides the use of the humanized recombinant single-chain antibody in the preparation of a prolyl hydroxylase inhibitor, an agent for activating hypoxia-inducible factor-1, or a drug for protecting and treating liver damage.

[0017] The present invention also provides an expression vector comprising the nucleotide.

[0018] The present invention also provides an expression strain, which comprises the nucleotide.

[0019] The present invention also provides the use of the expression vector or the expression strain in preparing a prolyl hydroxylase inhibitor, an agent for activating hypoxia-inducible factor-1, or a drug for protecting and treating liver damage.

[0020] The present invention also provides a nanoparticle, wherein the nanoparticle comprises the human recombinant single-chain antibody.

[0021] The present invention also provides a nano drug composition, comprising the nano particles and a drug carrier.

[0022] The present invention also provides the use of the nanoparticles or the nanomedicine composition in the preparation of prolyl hydroxylase inhibitors, reagents for activating hypoxia-inducible factor-1, or drugs for protecting and treating liver damage.

[0023] The present invention provides a human recombinant single-chain antibody, nanoparticles and nano drug composition and its application. The human single-chain antibody INP described in the present invention is a human single-chain antibody, which has the characteristics of low immunogenicity, high tissue penetration efficiency and strong specificity, and is suitable as a therapeutic drug. However, as a macromolecular drug, it is difficult to effectively enter cells to play a role. The present invention successfully constructs a recombinant single-chain antibody IRB and verifies the feasibility of the method by coupling the single-chain antibody INP with a cell-penetrating peptide to improve the cell entry efficiency of INP. In addition, unmodified single-chain antibodies have poor organ or tissue targeting and are easily cleared by proteases in the body. In order to use the recombinant single-chain antibody to treat diseases, the present invention also prepares the recombinant single-chain antibody into a nano drug composition, and the nanoparticles in the nano drug composition have a size effect during in vivo transportation. Nanoparticles of different sizes have different retention capacities for different organs or tissues. Controlling the particle size of nanoparticles can effectively control the organ or tissue targeting of nanoparticles, thereby effectively treating diseases. The particle size of the nano drug composition constructed by the present invention is about 70nm, which effectively improves the targeting of human recombinant single-chain antibodies to the liver (nanoparticles of 6 to 100nm are easy to be retained in the liver). The nano drug composition has good biosafety and a long retention time at the lesion point, and can effectively treat diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Preparation of human recombinant single-chain antibody IRB (Figure A represents the amplification of IRB and identification of the expression plasmid; Figure B represents the expression and purification of IRB, wherein 1 represents protein marker, 2 represents bacterial lysate without IPTG induction, 3 represents bacterial lysate after 1 mM IPTG induction at 30°C for 20 h, 4 represents column protein sample solution, 5 represents nickel column loading effluent, 6 represents Binding Buffer effluent through the nickel column, 7 represents Washing Buffer effluent from the nickel column, 8-13 represent Elution Buffer effluent from protein elution);

[0025] Figure 2Characterization of human recombinant single-chain antibody IRB (A is the ELISA identification of INP and IRB (ns represents no significant difference, ** represents P < 0.01, and the ordinate represents the absorbance value at 492 nm); B is the Western blot identification of IRB, where 1 represents the bacterial lysate before induction, 2 represents the bacterial lysate after induction, 3 represents the protein sample solution, and 4 represents the purified target protein; C is the ELISA analysis of the binding activity of INP and IRB with recombinant PHD2 (*** represents P < 0.001), and the ordinate represents the absorbance value; D is the affinity curve of INP and IRB with PHD2, and the abscissa represents concentration, the ordinate represents the absorbance value; E is Co-IP detection of human recombinant single-chain antibody IRB that can bind to intracellular PHD2; F is MTT verification of INP and IRB without cytotoxicity (# represents that the control group (0 μM group) has no significant difference compared with other experimental groups, and the ordinate represents the relative cell survival rate); G is Western blot verification of the intracellular efficiency of INP and IRB; H is immunofluorescence detection of the localization of IRB in cells (red fluorescence is PHD2, green fluorescence is IRB, blue fluorescence is cell nucleus, and the scale is 20 μm); I is Western blot detection of the in vitro biological activity of IRB);

[0026] Figure 3 Construction and characterization of the drug delivery system coated with erythrocyte membrane (A is the particle size and potential of mesoporous silicon nanoparticles (MSN), erythrocyte membrane (RM) and mesoporous silicon nanoparticles coated with erythrocyte membrane, i.e., nanoparticles (MSN@RM); B is the transmission electron microscopy image of MSN@RM; C is the SDS-PAGE image of erythrocyte membrane proteins in the nanoparticles; D is the transmission electron microscopy image of MSN; E is the EDS analysis of MSN and IRB@MSN and the mass percentages of the four elements C, N, O and S in the two nanoparticles; F is the encapsulation efficiency of MSN loaded with IRB (IRB@MSN) and MSN loaded with IRB wrapped with erythrocyte (IRB@MSN@RM) (the ordinate is the encapsulation efficiency %); G is the drug loading of IRB@MSN and IRB@MSN@RM (the ordinate is the drug loading %));

[0027] Figure 4In vitro safety and immune escape of the drug delivery system coated with red blood cell membrane (A is MTT detection of cytotoxicity of MSN and MSN@RM (# represents that there is no significant difference between the control group (0μM group) and other experimental groups (the horizontal axis is the concentration of nanoparticles, and the vertical axis is the relative cell survival rate %)); B is live-dead cell staining detection of cytotoxicity of MSN and MSN@RM (red fluorescence represents dead cells, green cells represent live cells, and the scale is 200μm); C is hemolytic assay analysis of MSN and MSN@RM (*** represents the positive control group (Water group) compared with other experimental groups, P<0.001) (the horizontal axis represents the concentration of nanoparticles, and the vertical axis represents the absorbance at 540nm); D is fluorescence detection of macrophage uptake of MSN@RM (red fluorescence represents Rhodamine-labeled nanoparticles, blue fluorescence represents DAPI-labeled cell nuclei, and the scale is 50μm));

[0028] Figure 5 The in vivo circulation and in vivo biosafety of the drug delivery system wrapped by red blood cell membrane (A is the blood half-life of MSN and MSN@RM in mice (the horizontal axis represents time, and the vertical axis represents the relative fluorescence intensity of fluorescent nanoparticles in serum); B is the distribution of MSN and MSN@RM in various tissues and organs of mice (heart, liver, spleen, lungs and kidney); CF is the levels of ALT (C), AST (D), UA (E) and CRE (F) in mice after drug injection (# represents that there is no significant difference between the control group (Ctrl group) and other experimental groups); G is the H&E staining image of paraffin sections of various organ tissues of mice after drug injection (scale bar is 50μm));

[0029] Figure 6Figure 3 is the therapeutic effect of the drug delivery system wrapped in red blood cell membrane on APAP-induced acute liver injury (A is the level of ALT in mice 8h, 24h, 48h and 72h after the drug was injected into AILI mice (* represents the comparison between IRB@MSN@RM group and other experimental groups (except Crtl group) at the same time point, P<0.05); B is the level of AST in mice 8h, 24h, 48h and 72h after the drug was injected into AILI mice (* represents the comparison between IRB@MSN@RM group and other experimental groups (except Crtl group) at the same time point, P<0.05, ** represents the comparison between IRB@MSN@ The RM group was compared with other experimental groups (except the Crtl group) at the same time point, P<0.01); C is the H&E staining image of the liver paraffin sections of mice 8h, 24h, 48h and 72h after the drug was injected into AILI mice (scale bar, 50μm); DG are the levels of MDA (D), GSH (E), CAT (F) and SOD (G) in the mice 24h after the drug was injected into AILI mice (ns means there is no significant difference between IRB@MSN@RM and Ctrl groups, * means P<0.05 between IRB@MSN@RM and other experimental groups (except the Ctrl group)). DETAILED DESCRIPTION

[0030] In the present invention, the amino acid sequence of the human recombinant single-chain antibody is shown in SEQ ID NO: 2, SEQ ID NO: 2: EIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQTPTFGQGTKLTVLGSGGSTITSSDVYHTKLSSSGTQVQLVESGGGVVQPGRSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAVTSYDGSDKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDMAVYYCAKSGGYNGYGVDYWGQGTLVTVSSEAAAKEAAAKEAAAKKLPVM.

[0031] In the present invention, the nucleotide encoding SEQ ID NO:2 is as shown in SEQ ID NO:1, SEQ ID NO:1: GAAATTGTGATGACGCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCTTGCAGGTCTAGTCAGTCCCTCCTGCATTCTAATGGATATAACTATTTGGACTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATTTATTTGGGTAGCAATCGGGCCTCTGGAGTCCCAGACAGGTTTAGTGGCAGTGGGTCAGGTACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGCATGCAAGGTCTACAAACTCCGACGTTCGGCCAAGGGACCAAGCTCACCGTCCTAGGTTCCGGAGGGTCGACCATAACTTCGTCTGATGTATACCATACGAAGTTATCCTCGAGCGGTACCCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGCTTCACCTTCAGTGACTACGGAATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTACATCATATGATGGATCTGATAAGTACTACGGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTACCTGCAAATGAACAGCCTGAGAGCCGAGGACATGGCTGTGTACTATTGTGCGAAGTCAGGCGGATATAATGGTTACGGAGTTGACTACTGGGGCCAGGGAACCCTGGTCACTGTCTCCTCAGAGGCTGCAGCTAAGGAGGCTGCTGCTAAGGAGGCTGCTGCTAAGAAACTGCCGGTTATG。

[0032] The primer set for amplifying the nucleotide described in SEQ ID NO:1 includes primer pair 1 and primer pair 2;

[0033] The primer pair 1 is shown in SEQ ID NOs: 3 to 4; the primer pair 2 is shown in SEQ ID NOs: 5 to 6. Forward primer SEQ ID NO: 3: TTGGCGCGCATGCCGAAATTGTGATGACGCAG; reverse primer SEQ ID NO: 4: GCCTCCTTAGCAGCAGCCTCCTTAGCTGCAGCCTCTGAGGAGACAGTG ACCAG; forward primer SEQ ID NO: 5: TTGGCGCGCATGCCGAAATTGTGATGACGCAG; reverse primer SEQ ID NO: 6: CTAGCTAGCCATAACCGGCAGTTTCTTAGCAGCAGCCTCCTTAGCAGCA GCC.

[0034] In the present invention, the construction of human recombinant single-chain antibody is based on human single-chain antibody INP, and the human single-chain antibody INP is prepared according to the method disclosed in application number: 201711449190.5 and patent name "A human single-chain antibody and its application", and the amino acid sequence is shown in SEQ ID NO: 1 in 201711449190.5.

[0035] In the present invention, the human recombinant single-chain antibody (IRB) specific to PHD2 is prepared by adding a rigid linker (EAAAK)×3 and a cell-penetrating peptide BIP gene to the 3' end of the human single-chain antibody INP gene to obtain the NP-RL-BIP (IRB) gene, and prokaryotic expression and purification are performed in Escherichia coli HB2151 to prepare a human recombinant single-chain antibody specific to PHD2. The gene sequence of the cell-penetrating peptide BIP is shown in SEQ ID NO:7, and the amino acid sequence of the cell-penetrating peptide BIP is shown in SEQ ID NO:8.

[0036] SEQ ID NO:7: AAACTGCCGGTTATG;

[0037] SEQ ID NO:8: KLPVM.

[0038] In the present invention, when preparing an expression vector, it is preferred to construct it by adding a nucleotide sequence to a pINP vector.

[0039] In the present invention, when preparing the expression strain, it is preferred to add nucleotides to Escherichia coli HB2151 for construction.

[0040] In the present invention, the nanoparticles include not only human recombinant single-chain antibodies, but also nanoparticles, which include metal nanoparticles, oxide nanoparticles, carbon-based nanomaterials, high molecular polymer nanomaterials, etc., preferably oxide nanoparticles, and more preferably mesoporous silica nanoparticles.

[0041] In the present invention, the drug carrier in the nano drug composition includes liposomes, nano micelles, cell membranes, etc., preferably red blood cell membranes.

[0042] The solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be understood as limiting the protection scope of the present invention.

[0043] The human single-chain antibody INP described in the embodiment is prepared according to the method disclosed in the application number: 201711449190.5 and the patent name "A human single-chain antibody and its application".

[0044] Example 1

[0045] Preparation of human recombinant single-chain antibody specific for PHD2

[0046] 1. Experimental Materials

[0047] 1.1 Escherichia coli HB2151 and XL1-Blue were purchased from Beijing Dingguo Biotechnology Co., Ltd.; INP / XL1-Blue and INP / HB2151 strains were prepared and preserved in our laboratory; BSSH II and Nhe I restriction endonucleases were purchased from Promega; T4 DNA ligase and its corresponding buffer were purchased from Takara Biotechnology (Dalian) Co., Ltd.

[0048] 1.2 PCR primers used in this step:

[0049] The primers for PCR amplification of human recombinant single-chain antibody were synthesized by Shanghai Sangon Biotechnology Technology Service Co., Ltd., and the primers included primer pair 1 and primer pair 2;

[0050] The primer pair 1 is shown in SEQ ID NOs: 3-4; the primer pair 2 is shown in SEQ ID NOs: 5-6.

[0051] 2. Specific implementation steps

[0052] 1. Construction and identification of prokaryotic expression vector of human recombinant single-chain antibody (IRB)

[0053] In order to prepare a human recombinant single-chain antibody IRB coupled with a cell-penetrating peptide BIP and a rigid linker (EAAAK) × 3, it is necessary to construct a recombinant expression vector of IRB. First, the recombinant plasmid (pINP) of INP was extracted by alkaline lysis. Using the pINP plasmid as a template, two rounds of overlapping extension PCR were used to connect BIP with a rigid linker (RL) at the N-terminus, i.e., RL-BIP, to the C-terminus of INP. The PCR reaction procedure is: (1) 95°C pre-denaturation for 2min; (2) 95°C for 20s; (3) 60°C for 20s; (4) 72°C for 10s; (5) 72°C extension for 5min; (2)-(4) 35 cycles. The specific construction method of the recombinant plasmid (pINP) is detailed in the application number: 201711449190.5, and the patent name is the method disclosed in "A human single-chain antibody and its application".

[0054] After the pINP plasmid vector and IRB fragment were digested with BSSH II and Nhe I, the target fragment was inserted into the pINP plasmid vector between the BSSH II and Nhe I restriction sites using T4 DNA ligase to construct a secretory recombinant IRB prokaryotic expression vector. The ligation product was transformed into E. coli XL1-Blue to construct clones. The positive clones were selected and cultured to extract the plasmids, and double restriction digestion and 1% agarose gel electrophoresis were performed for identification. Figure 1 As shown in A, the secretory prokaryotic expression vector pIRB of IRB was successfully constructed.

[0055] 2. Purification of IRB

[0056] The correctly sequenced secretory prokaryotic expression vector pIRB was transformed into Escherichia coli HB2151 to obtain the prokaryotic expression strain IRB / HB2151. The prokaryotic expression strain was induced by IPTG to achieve soluble expression of the human recombinant IRB single-chain antibody, and the culture supernatant rich in the target protein was collected. Ammonium sulfate was added to it and the precipitate was collected. After dissolving the precipitate and dialyzing, a protein solution was obtained. Since there is a His tag at the C-terminus of IRB, the obtained solution was filtered through HisTrap HPNi 2+ The target protein IRB (SEQ ID NO: 2, the nucleotide encoding the amino acid is shown in SEQ ID NO: 1) can be obtained by affinity chromatography purification using a column. The purified IRB is identified by SDS-PAGE gel electrophoresis. Figure 1 As shown in B, the target protein was successfully induced to express, and there was an obvious protein band at 31 kDa, which was consistent with the expected size of IRB, indicating that the specific anti-PHD2 human recombinant single-chain antibody IRB was successfully purified.

[0057] Example 2

[0058] Identification and activity characterization of human recombinant single-chain antibody IRB specific for PHD2

[0059] 1. Experimental Materials

[0060] Human single-chain antibody INP, human recombinant single-chain antibody IRB prepared in Example 1.

[0061] 2. Specific implementation steps

[0062] 2.1 ELISA Identification of IRB

[0063] Since IRB carries a V5 tag at the C-terminus, we used this as an example to identify IRB. The purified single-chain antibody was added to an ELISA plate and coated overnight at 4°C. After blocking, mouse anti-V5 monoclonal antibody and rabbit anti-mouse HRP-conjugated IgG were added in turn for incubation. ELISA was used to identify whether the IRB protein was successfully purified. The results are shown in Figure 2. Figure 2 As shown in A, Figure 2 A results showed that human recombinant single-chain antibody IRB was successfully purified.

[0064] 2.2 Western blot identification of IRB

[0065] The prokaryotic expression strain IRB / HB2151 before induction, the prokaryotic expression strain IRB / HB2151 after IPTG induction, the protein sample solution and the purified IRB were mixed with the loading buffer and boiled, and the protein samples were separated by SDS-PAGE gel electrophoresis. Western blot was used to identify whether the IRB protein was successfully purified. The results are shown in 2B, which further show that the human recombinant single-chain antibody IRB with a size of 31 kDa was successfully obtained.

[0066] 2.3 ELISA detection of PHD2 binding ability of IRB

[0067] In order to ensure that the human recombinant single-chain antibody can effectively function, the recombinant human PHD2 protein was expressed and purified (purified according to the purification method disclosed in Example 1 of Patent Application No.: 201711449190.5), and the purified recombinant human PHD2 protein rhPHD2 was obtained. ELISA was used to detect whether IRB can effectively bind to PHD2. 1-10μg / mL recombinant human PHD2 was added to the enzyme-linked well plate, coated overnight at 4°C and blocked, and then 100μL of purified single-chain antibody (10μg / mL), 100μL of mouse anti-V5 monoclonal antibody (0.02μg / mL) and 100μL of rabbit anti-mouse HRP-conjugated IgG (0.01μg / mL) were added in sequence. ELISA was used to detect whether IRB can bind to PHD2. The results are as follows. Figure 2 As shown in C, it shows that IRB can effectively bind to PHD2.

[0068] 2.4 Antigen affinity determination of IRB

[0069] The affinity of IRB binding to rhPHD2 was tested. 100 μL of gradient diluted rhPHD2 was added to the enzyme-linked plate and coated overnight at 4°C. Next, the plate was blocked with 3% skim milk powder-PBST. Subsequently, 100 μL of gradient diluted purified human recombinant single-chain antibody IRB was added. 100 μL of mouse anti-V5 monoclonal antibody (0.02 μg / mL) and 100 μL of rabbit anti-mouse HRP-conjugated IgG (0.01 μg / mL) were added in sequence. After adding OPD colorimetric solution for color development and terminating with H2SO4, the reading of the microplate reader was read at 492 nm. The affinity constant (K) of the single-chain antibody aff ) is calculated as K aff =(n-1) / (n[Ab′]-[Ab]), n=[Ag] / [Ag′], where [Ab], [Ab′] refer to the antibody concentrations used when the wells coated with [Ag] and [Ag′], respectively, reach half the maximum absorbance at a wavelength of 492 nm calculated by the nonlinear regression method. The results are shown in Figure 2 As shown in D, IRB can effectively bind to PHD2 and has a good affinity for PHD2, with an affinity of 1.69×10 7 M -1 .

[0070] 2.5 Co-IP detection of IRB binding to intracellular PHD2

[0071] To further verify whether IRB can effectively bind to endogenously expressed PHD2 in cells, 100 μg of IRB was added to the extracted HepG2 cell proteins, and then 20 μL of ProteinA / G agarose beads (SANTACRUZ BIOTECHNOLOGY) pre-incubated with anti-V5 monoclonal antibody (5 μg) was added to the mixture. After incubation overnight, the agarose beads of each group were collected for Western blot analysis. The results are shown in Figure 2. Figure 2 As shown in E, PHD2 and V5 were detected in the IRB group of IP, indicating that IRB can effectively bind to intracellular PHD2.

[0072] Example 3

[0073] In vitro biosafety and membrane penetration efficiency testing of human recombinant single-chain antibody IRB specific for PHD2

[0074] 1. Experimental Materials

[0075] Human single-chain antibody INP, human recombinant single-chain antibody IRB prepared in Example 1.

[0076] 2. Specific implementation steps

[0077] 3.1 MTT assay of IRB cytotoxicity

[0078] 10 μM, 5 μM, 2 μM, 1 μM and 0.1 μM of recombinant single-chain antibody INP or IRB were added to RAW264.7 or LO-2 cells, and the cytotoxicity of the single-chain antibody was detected by MTT. The cytotoxicity results of the human recombinant single-chain antibody are shown in Figure 2 As shown in F, INP and IRB still had no detectable cytotoxicity to LO-2 cells and Raw 264.7 cells at a concentration of 10 μM. These results indicate that IRB and INP are not cytotoxic to RAW264.7 and LO2 cells.

[0079] 3.2 Detection of IRB transmembrane efficiency

[0080] In order to detect whether the addition of membrane-penetrating peptides effectively improved the cell entry efficiency of single-chain antibody INP, INP and IRB were co-incubated with HepG2 cells for 9 h, and then the whole cell protein was extracted and analyzed by Western blot. The results are as follows Figure 2 As shown in G, the level of IRB in HepG2 cells was significantly higher than that in INP, proving that the addition of the membrane-penetrating peptide BIP effectively improved the membrane-penetrating efficiency of the single-chain antibody INP. To further prove this result, IRB was co-incubated with HepG2 cells for 12 hours, and the intracellular localization of IRB was detected by immunofluorescence experiment. The results are shown in Figure 2 As shown in H, IRB efficiently entered HepG2 cells and colocalized well with PHD2.

[0081] 3.3 Analysis of intracellular biological activity of IRB

[0082] In order to prove that IRB can exert biological activity in cells to inhibit PHD2 function and increase the level of HIF-1α in cells, HepG2 cells were co-incubated with IRB for 12 hours, and then the whole protein of HepG2 cells was extracted and analyzed by Western blot. The results are as follows: Figure 2 As shown in I, the PHD2 level in HepG2 cells remained unchanged, but the HIF-1α level in HepG2 cells added with IRB increased significantly, proving that IRB enters the cells and effectively inhibits the enzyme activity of PHD2, and has good biological activity.

[0083] Example 4

[0084] Construction and characterization of membrane-camouflaged nanosystems (nanodrug compositions)

[0085] 1. Experimental Materials

[0086] Human single-chain antibody INP, human recombinant single-chain antibody IRB prepared in Example 1.

[0087] 2. Specific implementation steps

[0088] 4.1 Synthesis of single-chain antibody-loaded mesoporous silica nanoparticles

[0089] First, mesoporous silicon nanoparticles (MSN) were synthesized by template method: deionized water was added to a round-bottom flask, and hexadecyltrimethylammonium chloride (CTAC) and triethanolamine (TEA) (the ratio was deionized water: 60 mL, CTAC: 6 g, TEA: 0.18 g) were added thereto, and stirred slowly at 30°C for 1 h. Subsequently, 20 mL of cyclohexane-ethyl orthosilicate (TEOS) solution (the volume ratio of cyclohexane to ethyl orthosilicate was 2:5) was added dropwise to the above liquid at 30°C, and stirred slowly at 30°C for 12 h. After stirring, ethanol equal to the volume of the previous mixed solution was added to precipitate mesoporous silicon, centrifuged at 12000 rpm for 10 min, and the precipitate was collected to collect the mesoporous silicon. The collected product was condensed and refluxed with 100 mL of 1% hydrochloric acid ethanol solution at 80°C for 6 h to remove the template, and this process was repeated three times. The final product was washed three times with ethanol and water and freeze-dried to obtain mesoporous silicon nanoparticles (MSN) and stored.

[0090] After synthesizing MSN, MSN and IRB were added to PBS so that the final concentration of both was 1 mg / mL, and they were incubated at 4°C for 12 h with rotation, centrifuged at 12000 rpm for 10 min, and the precipitate was taken to obtain mesoporous silica nanoparticles loaded with IRB, i.e., nanoparticles (IRB@MSN). The precipitate was washed three times with PBS and set aside. The preparation method of mesoporous silica nanoparticles loaded with INP (INP@MSN) was the same as that of IRB@MSN.

[0091] 4.2 Extraction of red blood cell membrane

[0092] After the mice were anesthetized, blood was collected from the heart. After centrifugation, the upper serum was discarded and washed. The precipitate was added to 40 times the volume of 0.25× PBS to achieve the swelling effect. The precipitate collected after centrifugation is the red blood cell membrane, which can be used after washing.

[0093] 4.3 Construction of membrane-camouflaged nanosystems (nano-drug compositions)

[0094] After the MSN and red blood cell membrane were evenly mixed at a mass ratio of 3:1, the mixture was ultrasonicated for 3 min to prepare MSN@RM;

[0095] The prepared IRB@MSN and red blood cell membrane were evenly mixed at a mass ratio of 3:1, and then ultrasonicated for 3 min to prepare IRB@MSN@RM;

[0096] 4.4 Characterization of membrane-camouflaged nanosystems

[0097] After the red blood cell membrane was coated onto the surface of MSN by ultrasound, the particle size was characterized by dynamic light scattering analysis. Figure 3 As shown in Figure A, it was found that after the red blood cell membrane was coated, the particle size of the nanoparticles increased from 63.57nm to 77.77nm. In addition, the zeta potential analysis of the surface potential of the nanoparticles was as follows Figure 3 As shown in A, it is found that the potential of MSN@RM is similar to that of RM. TEM observation is also performed, such as Figure 3 As shown in B, the surface of MSN in the MSN@RM group was obviously coated with membrane, which indicated that the MSN was successfully coated with the red blood cell membrane. SDS-PAGE was further used to analyze the membrane proteins of the red blood cell membrane before and after coating, as shown in Figure 3 As shown in C, it was found that the red blood cell membrane proteins were well retained after coating.

[0098] The structure of mesoporous silicon nanoparticles was observed using a transmission electron microscope (TEM). Figure 3 As shown in D, it can be observed that it has an obvious mesoporous structure. In order to verify that MSN can load IRB, EDS spectrum analysis was performed on MSN and IRB@MSN. The results are shown in Figure 3 As shown in Figure E, after incubation with IRB, the ratio of C, N, and S in the nanoparticles increased significantly, while the ratio of O decreased because it is also an important component of MSN and its ratio in MSN is higher than that in protein. These results indicate that MSN has been successfully loaded with protein sample IRB. The encapsulation rate and drug loading of the two nanoparticles for single-chain antibody were measured, as shown in Figure 1. Figure 3 F and 3G, where the encapsulation efficiency of IRB@MSN is 37.06% and the drug loading is 27.02%, and the encapsulation efficiency of IRB@MSN@RM is 46.70% and the drug loading is 25.86%.

[0099] Example 5

[0100] Biosafety and immune escape of membrane-camouflaged nanosystems (nano-drug compositions) in vitro 1. Experimental materials

[0101] MSN, MSN@RM, IRB@MSN@RM, INP@MSN@RM.

[0102] 2. Specific implementation steps

[0103] 5.1 Cytotoxicity detection of membrane-camouflaged nanosystems

[0104] 1, 10, 50, and 100 μg / mL of nanoparticles were added to LO-2, HepG2, or RAW264.7 cells. The cytotoxicity of the camouflage system was detected using MTT. Figure 4 As shown in A, the camouflage system showed no obvious cytotoxicity. This result was further verified by live-dead cell staining experiments. Figure 4 As shown in B, green fluorescence represents living cells, and no red fluorescence representing dead cells is seen in the field of view.

[0105] 5.2 Blood compatibility testing of membrane-camouflaged nanosystems

[0106] After blood was drawn, 1, 10, 50, and 100 μg / mL of nanoparticles were added to the collected red blood cells. The hemolysis test was used to test the blood compatibility of the camouflage system. The results are shown in Figure 2. Figure 4 As shown in C, the camouflage system has good blood compatibility.

[0107] 5.3 Detection of immune escape effect of membrane-camouflaged nanosystems

[0108] CD47 on the red blood cell membrane can avoid recognition by macrophages, thereby achieving immune escape and long-term circulation in the body. To this end, MSNs were first labeled with rhodamine B (the synthesis method of rhodamine-labeled MSNs was based on the MSN synthesis method of Example 4, and 100 μL of 3-aminopropyltriethoxysilane APTES modified with rhodamine B was added after adding TEOS), and then the red blood cell membrane was coated with rhodamine B-labeled MSNs to treat RAW264.7 cells. The uptake of nanoparticles by macrophages RAW264.7 was observed under a fluorescence microscope. The results are as follows: Figure 4 As shown in D, it was found that the uptake of MSN by macrophages was significantly reduced after coating with the red blood cell membrane, indicating that the camouflage system coated with the red blood cell membrane is conducive to immune escape and effectively reduces the clearance of nanoparticles by the immune system.

[0109] Example 6

[0110] In vivo targeting and in vivo safety of membrane-camouflaged nanosystems (nanodrug compositions)

[0111] 1. Experimental Materials

[0112] Single-chain antibody INP, recombinant single-chain antibody IRB, MSN, MSN@RM, INP@MSN, IRB@MSN, INP@MSN@RM, IRB@MSN@RM.

[0113] 2. Specific implementation steps

[0114] 6.1 In vivo circulation time of membrane-camouflaged nanosystems

[0115] MSN and MSN@RM labeled with rhodamine B were injected into mice via the tail vein at a dose of 1 mg / kg, and blood was collected from the eye sockets at 0.5, 1, 2, 3, 4, 7, and 24 hours, and the relative concentration of nanoparticles in the blood was detected by a fluorescence spectrophotometer. The results are shown in 5A, and the coating of the red blood cell membrane prolongs the blood half-life of the nanoparticles.

[0116] 6.2 In vivo targeting of membrane-camouflaged nanosystems

[0117] MSN and MSN@RM labeled with rhodamine B were injected into mice via the tail vein at a dose of 2.5 mg / kg. The hearts, livers, spleens, lungs, and kidneys of the mice were removed at 1.5, 6, 12, 24, and 48 hours, and the relative number of nanoparticles in each organ was observed using a small animal in vitro imaging system. Figure 5 As shown in B, the coating of the red blood cell membrane effectively increased the enrichment and retention time of the camouflage system in the liver.

[0118] 6.3 In vivo safety of membrane-camouflaged nanosystems

[0119] IRB and nanoparticles of each group were injected into mice via tail vein at a dose of 0.8 mg / kg of single-chain antibody (the dose of MSN@RM group was based on the amount of MSN). Simple injection of PBS was used as a negative control. Blood was collected from the eye sockets at 24 hours to detect the levels of AST (aspartate aminotransferase), ALT (alanine aminotransferase), UA (uric acid) and CRE (creatinine) in the mice. The results are shown in Table 1. Figure 5 As shown in C-5F, the AST, ALT, UA and CRE levels of mice in the IRB and nanoparticles administration groups were not significantly different from those of the control mice. The hearts, livers, spleens, lungs and kidneys of the mice were removed at 24 h for H&E staining. The results are shown in Figure 5 As shown in G, no structural changes were observed in the organs of the mice in each group of drug administration, further proving that the nanoparticles are non-toxic and have good in vivo safety.

[0120] Example 7

[0121] Therapeutic effect of IRB membrane-camouflaged nanosystem (nanodrug composition) on acetaminophen (APAP)-induced acute liver injury

[0122] 1. Experimental Materials

[0123] Single-chain antibody INP, recombinant single-chain antibody IRB, MSN, MSN@RM, INP@MSN, IRB@MSN, INP@MSN@RM, IRB@MSN@RM.

[0124] 2. Specific implementation steps

[0125] Eight-week-old male Balb / c mice were selected. They were fasted but not watered from the night before the injection to deplete the glutathione in the mouse liver. The next day, 250 mg / kg of APAP solution was intraperitoneally injected. After the injection, the mice resumed their normal diet to construct an APAP-induced acute liver injury mouse model. IRB and nanoparticles in each group were injected into the mice through the tail vein at a dose of 0.8 mg / kg of single-chain antibody (the dose of the MSN@RM group was based on the amount of MSN). Simple injection of PBS was used as a negative control. Blood was collected from the eye sockets at 8, 24, 48, and 72 hours to measure the ALT and AST levels in the mice. The results are shown in Figure 2. Figure 6 As shown in A and 6B, the ALT and AST levels in the IRB@MSN@RM group were significantly reduced, showing the best therapeutic effect. The livers of mice treated for 24, 48, and 72 hours were subjected to H&E staining and pathological analysis. Figure 6 As shown in C, the IRB@MSN@RM group significantly inhibited liver damage, and the liver damage was repaired at 48h, with the best therapeutic effect. In addition, the test results of GSH (glutathione), MDA (malondialdehyde), CAT (catalase) and SOD (superoxide dismutase) levels in the liver of each group at 24h were shown in Figure 6 As shown in D-6G, it further shows that the IRB@MSN@RM group significantly improved the oxidative stress state of the liver. These results all indicate that IRB@MSN@MSN has an excellent therapeutic effect on APAP-induced acute liver injury.

[0126] As can be seen from the above embodiments, the present invention provides a human recombinant single-chain antibody, nanoparticles and nanopharmaceutical composition and their applications. The present invention utilizes cell-penetrating peptides and cell membrane-coated drug delivery systems to improve the organ targeting and cell entry efficiency of anti-PHD2 human single-chain antibodies INP, thereby effectively inhibiting the hydroxylation of HIF-1α by PHD2 and increasing the level of HIF-1 in cells, thereby achieving the treatment of diseases targeting PHD2 such as liver damage. The present invention constructs a nanopharmaceutical composition with a particle size of about 70nm, which effectively improves the targeting of single-chain antibodies to the liver (nanoparticles of 6 to 100nm are easily retained in the liver). The pharmaceutical composition has good biosafety, a long liver retention time, and effectively promotes the recovery of liver damage.

[0127] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A human recombinant single-chain antibody, characterized in that: The amino acid sequence of the human recombinant single-chain antibody is shown in SEQ ID NO:

2.

2. A nucleotide encoding the amino acid sequence of the human recombinant single-chain antibody according to claim 1, characterized in that: The nucleotide is shown in SEQ ID NO:

1.

3. A primer set for amplifying the nucleotide according to claim 2, characterized in that: The primer set includes primer pair 1 and primer pair 2; The primer pair 1 is shown in SEQ ID NO: 3-4; The primer pair 2 is shown in SEQ ID NOs: 5-6.

4. Use of the humanized recombinant single-chain antibody according to claim 1 in the preparation of a prolyl hydroxylase inhibitor, an agent for activating hypoxia-inducible factor-1, or a drug for protecting or treating liver damage.

5. An expression vector, characterized in that: Comprising the nucleotide according to claim 2.

6. An expression strain, characterized in that The expression strain comprises the nucleotide sequence of claim 2.

7. Use of the expression vector according to claim 5 or the expression strain according to claim 6 in the preparation of a prolyl hydroxylase inhibitor, an agent for activating hypoxia-inducible factor-1, or a drug for protecting or treating liver damage.

8. A nanoparticle, characterized in that: The nanoparticles include the human recombinant single-chain antibody according to claim 1.

9. A nano drug composition, characterized in that: The invention comprises the nanoparticles according to claim 8 and a drug carrier.

10. Use of the nanoparticles according to claim 8 or the nanopharmaceutical composition according to claim 9 in the preparation of a prolyl hydroxylase inhibitor, an agent for activating hypoxia-inducible factor-1, or a drug for protecting or treating liver damage.

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