Preparation method for transferrin receptor 1-targeting glycoside hydrolase complex and use thereof

Abstract

A preparation method for a transferrin receptor-targeting glycoside hydrolase complex, and a use thereof. The transferrin receptor-targeting glycoside hydrolase complex is formed by complexing a glycoside hydrolase with a human ferritin heavy chain. The present invention fuses a molecular glue peptide pair (ST-SC) with glycoside hydrolases (FpGalNAc and FpGal) and hFn, respectively, to prepare a glycoside hydrolase and hFn containing molecular glue peptides, and couples the glycoside hydrolase and hFn together to prepare a transferrin receptor 1-targeting glycoside hydrolase complex. The complex significantly improves the activity of the glycoside hydrolase cleaving a type A antigen. The glycoside hydrolase complex can convert a type-A organ into a type-O organ without requiring the use of dextran, overcoming a series of side effects of existing methods which are caused by the use of dextran, can effectively alleviate the problem of supply shortage in clinical organ transplantation, and has wide application prospects.

Description

Preparation method and application of transferrin receptor 1 targeted glycoside hydrolase complex TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology drugs, and particularly relates to a preparation method and application of a transferrin receptor 1 targeted glycoside hydrolase complex. BACKGROUND

[0002] Organ transplantation is the only effective means to save the lives of patients with end-stage organ disease, and the severe shortage of organ donors is a worldwide problem. Blood type compatibility is the most basic requirement for organ transplantation, and under normal circumstances, organ transplantation is required to be carried out between donors and recipients of the same blood type. In emergency situations, O-type blood organs can be considered for transplantation into patients of other blood types. Blood transfusion is also an important means to save lives. Under normal circumstances, blood transfusion also follows the principle of the same blood type. However, in emergency situations, O-type red blood cells can be considered for transfusion into patients of other blood types. However, O-type blood sources and organ donors are particularly scarce. Therefore, if other blood type red blood cells or organs can be engineered to be O-type, the problem of severe shortage of blood sources and organ donors can be expected to be alleviated.

[0003] In the ABO blood group system, human blood types can be divided into A, B, AB and O types according to the expressed antigens. A or B type only expresses A or B antigen, and AB type simultaneously expresses A and B antigens. O type does not express A antigen or B antigen, and its antigen is called H antigen. Studies have found that A and B antigens are formed by adding N-acetylgalactosamine (GalNAc) or galactose (Gal) to the end of the H antigen sugar chain, respectively. Therefore, as long as the GalNAc on the A antigen or the Gal on the B antigen is removed, A and B types can be converted to O type, thereby expanding the donor source.

[0004] It has been found that some glycoside hydrolases can cleave GalNAc and / or Gal, thereby converting A antigen or B antigen into O antigen. For example, Rahfeld et al. found that the combined use of GalNAc deacetylase (Flavonifractor plautii GalNAc Deacetylase, FpGalNAc) and galactosaminidase (Flavonifractor plautii Galactosaminidase, FpGal) can effectively remove A antigen from red blood cells and vascular endothelial cells. When the two enzymes are prepared into an organ perfusion solution and perfused under ex vivo conditions, A-type organs can be converted into O-type organs. However, a large amount (300 mg / ml) of dextran needs to be added to the perfusion solution in this method to improve the activity of FpGalNAc. The use of dextran increases the viscosity and osmotic pressure of the enzyme solution, which can easily damage the organ when used for organ perfusion. Moreover, dextran is a solid substance that is difficult to wash out and remains in the microvessels after perfusing the organ, which is not conducive to the survival of the graft. Therefore, there is an urgent need to use other methods to improve enzyme activity.

[0005] Blood group antigens are mainly expressed on cells such as red blood cells and vascular endothelial cells. If glycoside hydrolases are enriched on target cells through targeted delivery, it is highly likely that their activity will be significantly improved. Studies have found that transferrin receptor 1 (TfR1) is highly expressed on red blood cells and vascular endothelial cells. Human ferritin heavy chain (hFn) can bind to TfR1. Therefore, if glycoside hydrolases are fused or coupled with hFn, it is possible to deliver a large amount of glycoside hydrolases to target cells and make them work more efficiently. However, hFn needs to form a nanocage containing 24 monomer proteins to function. If hFn (21 KD) is directly fused with glycoside hydrolases (70-100 KD) with a larger molecular weight, it will lose the ability to form a nanocage. In order to maintain the nanocage structure of hFn, the ideal way is to express hFn alone first, and then couple it with glycoside hydrolases after it forms a nanocage structure. Although there are many methods to link two proteins together, conventional methods cannot achieve site-specific coupling and face problems such as non-uniform products and poor protein activity.

[0006] Recent studies have found that molecular glue peptides, such as SpyTag / SpyCatcher and SnoopTag / SnoopCatcher, can couple paired Tag and Catcher through spontaneous isopeptide bond formation.

[0007] There is no report on fusing the molecular glue peptide with glycoside hydrolase and hFn respectively, whether the fusion confers the targeting of glycoside hydrolase, retains the functions of pGalNAc and FpGal, and improves the enzyme activity. SUMMARY

[0008] The application aims to provide a preparation method and application of a transferrin receptor 1 targeted glycoside hydrolase complex.

[0009] The application provides a transferrin receptor targeted glycoside hydrolase complex, which is characterized in that it is composed of glycoside hydrolase and human ferritin heavy chain.

[0010] Further, in the complex, the ratio (molar ratio) of glycoside hydrolase to human ferritin heavy chain is 1:24, 2:24, 3:24, 4:24, 5:24, 6:24, 7:24, 8:24, 9:24, 10:24, 11:24, 12:24, 13:24, 14:24, 15:24, 16:24 or 17:24.

[0011] Further, the glycoside hydrolase is GalNAc deacetylase or galactosaminease.

[0012] The amino acid sequence of the GalNAc deacetylase is shown in SEQ ID NO. 2; the amino acid sequence of the galactosaminease is shown in SEQ ID NO. 10; and the amino acid sequence of the human ferritin heavy chain is shown in SEQ ID NO. 14.

[0013] Further, the glycoside hydrolase and the human ferritin heavy chain are connected through a covalent bonding system.

[0014] Further, the covalent bonding system is SpyTag-SpyCatcher system or SnoopTag / SnoopCatcher system.

[0015] Further, the complex is composed of glycoside hydrolase-SpyTag and SpyCatcher-human ferritin heavy chain.

[0016] Further, the glycoside hydrolase-SpyTag is connected by connecting the glycoside hydrolase and SpyTag through a linker, and the human ferritin heavy chain-SpyCatcher is connected by connecting the human ferritin heavy chain and SpyCatcher through a linker; preferably, the linker is (G4S)3, the amino acid sequence of the glycoside hydrolase-SpyTag is shown in SEQ ID NO. 4 or SEQ ID NO. 12; and the amino acid sequence of the SpyCatcher-human ferritin heavy chain is shown in SEQ ID NO. 16.

[0017] Further, the complex is a glycoside hydrolase-SpyTag coupled with a SpyCatcher-human ferritin heavy chain nanocage, wherein the SpyCatcher-human ferritin heavy chain nanocage is self-assembled from 24 SpyCatcher-human ferritin heavy chains, and the ratio (molar ratio) of the glycoside hydrolase-SpyTag to the SpyCatcher-human ferritin heavy chain nanocage is 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1 or 1:1.

[0018] The method for preparing the above-mentioned transferrin receptor-targeted glycoside hydrolase complex comprises the following steps: coupling a glycoside hydrolase with a human ferritin heavy chain; preferably, coupling a glycoside hydrolase-SpyTag with a SpyCatcher-human ferritin heavy chain.

[0019] Further,

[0020] The method for preparing the glycoside hydrolase-SpyTag comprises the following steps: taking a nucleotide sequence for expressing the glycoside hydrolase-SpyTag, preparing a recombinant expression plasmid, and recombinantly expressing in E. coli; the method for preparing the SpyCatcher-human ferritin heavy chain comprises the following steps: taking a nucleotide sequence for expressing the SpyCatcher-human ferritin heavy chain, preparing a recombinant expression plasmid, and recombinantly expressing in E. coli; the nucleotide sequence for expressing the glycoside hydrolase-SpyTag is shown in SEQ ID NO. 3 or SEQ ID NO. 11; and the nucleotide sequence for expressing the SpyCatcher-human ferritin heavy chain is shown in SEQ ID NO. 15.

[0021] The application further provides a gene fragment for recombinant expression, and the nucleotide sequence is shown in SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 11, SEQ ID NO. 15 or SEQ ID NO. 17.

[0022] The application further provides a recombinant vector for recombinant expression, which is a recombinant plasmid containing the gene fragment of claim 10.

[0023] The application further provides the use of the above-mentioned transferrin receptor-targeted glycoside hydrolase complex in cutting the A-type antigens on the surface of red blood cells.

[0024] The application further provides a combination product capable of converting an A-type organ into an O-type organ, and the combination product comprises the above-mentioned transferrin receptor-targeted glycoside hydrolase complex and an organ perfusion solution.

[0025] Further, the organ perfusion solution is a kidney perfusion solution, a University of Wisconsin solution, a histidine-tryptophan-ketoglutarate solution (HTK solution), a Celsior solution, a Polysol solution, an IGL-1 solution, a Vasosol solution or an HCA solution; preferably, the kidney perfusion solution is KPS-1.

[0026] The experimental results show that the application provides a preparation method and application of a transferrin receptor 1 targeted glycoside hydrolase complex. The molecular glue peptide is fused to SpyTag / SpyCatcher and glycoside hydrolase (FpGalNAc and FpGal) and hFn respectively to prepare glycoside hydrolase containing a molecular glue peptide and hFn. Then, the glycoside hydrolase containing the molecular glue peptide is mixed with the hFn containing the paired molecular glue peptide, the glycoside hydrolase is coupled with the hFn to prepare the transferrin receptor 1 targeted glycoside hydrolase complex. The complex improves the activity of the glycoside hydrolase in cutting A type antigens. In particular, SpyTag is fused to the C-terminal end of the FpGalNAc protein through a flexible linker (G4S) 3 to prepare FpGalNAc-ST, SpyCatcher is fused to the N-terminal end of hFn through a (G4S) 3 linker to prepare SC-hFn, and FpGalNAc-ST is coupled with SC-hFn to prepare the glycoside hydrolase complex of the application. The glycoside hydrolase complex can target the connection of transferrin receptor 1, can greatly improve the hydrolysis activity of FpGalNAc on A antigens, and can convert A type organs into O type organs without using dextran, can overcome a series of side effects caused by the use of dextran in the prior art, and has excellent effect for transplantation. The application can effectively alleviate the shortage of organ supply in clinical organ transplantation, and has wide application prospect.

[0027] Obviously, according to the above content of the application, according to the ordinary technical knowledge and conventional means in the art, other various forms of modifications, replacements or changes can be made without departing from the above basic technical idea of the application.

[0028] The above content of the application will be further described in detail through the specific embodiments in the form of examples. However, it should not be understood that the scope of the above subject matter of the application is limited to the following examples. Any technology achieved based on the above content of the application belongs to the scope of the application. BRIEF DESCRIPTION OF DRAWINGS

[0029] Fig. 1 is a recombinant expression of FpGalNAc of molecular glue peptide, A: molecular structure schematic diagram; B: purified product SDS-PAGE;

[0030] Figure 2 is the recombinant expression of FpGalNAc-tri of molecular glue peptide; A: schematic diagram of molecular structure; B: SDS-PAGE of purified product;

[0031] Figure 3 is the recombinant expression of FpGal of molecular glue peptide; A: schematic diagram of molecular structure; B: SDS-PAGE of purified product;

[0032] Figure 4 is the recombinant expression of hFn of molecular glue peptide; A: schematic diagram of molecular structure; B: SDS-PAGE of purified product; C: gel filtration chromatogram of purified product;

[0033] Figure 5 Preparation of FpGalNAc and hFn coupling product; A: schematic diagram of coupling reaction; B: SDS-PAGE of coupling product;

[0034] Figure 6 Preparation of FpGalNAc-tri and hFn coupling product; A: schematic diagram of coupling reaction; B: SDS-PAGE of coupling product;

[0035] Figure 7 Preparation of FpGal and hFn coupling product; A: schematic diagram of coupling reaction; B: SDS-PAGE of coupling product;

[0036] Figure 8 Activity comparison of FpGalNAc-hFn and FpGalNAc;

[0037] Figure 9 Activity comparison of FpGalNAc-tri-hFn and FpGalNAc-tri.

[0038] Figure 10 Binding activity of FpGalNAc-hFn to TfR1 receptor positive cells.

[0039] Figure 11 Activity of FpGalNAc-hFn in different perfusion systems. DETAILED DESCRIPTION

[0040] The raw materials and equipment used in the present application are known products, which are obtained by purchasing commercially available products.

[0041] The glycoside hydrolase used in the embodiment of the present application includes: galactosaminease (FpGal), acetylgalactosamine deacetylase (FpGalNAc), and triacetylgalactosamine deacetylase (FpGalNAc-tri).

[0042] FpGalNAc nucleotide sequence, SEQ ID NO. 1:

[0043] FpGalNAc amino acid sequence, SEQ ID NO. 2:

[0044] FpGalNAc-ST nucleotide sequence, SEQ ID NO. 3:

[0045] FpGalNAc-ST amino acid sequence, SEQ ID NO. 4:

[0046] FpGalNAc-SC nucleotide sequence, SEQ ID NO. 5:

[0047] FpGalNAc-SC amino acid sequence, SEQ ID NO. 6:

[0048] FpGalNAc-tri-ST nucleotide sequence, SEQ ID NO. 7:

[0049] FpGalNAc-tri-ST amino acid sequence, SEQ ID NO. 8:

[0050] FpGal nucleotide sequence, SEQ ID NO. 9:

[0051] FpGal amino acid sequence, SEQ ID NO. 10:

[0052] FpGal-ST nucleotide sequence, SEQ ID NO. 11:

[0053] FpGal-ST amino acid sequence, SEQ ID NO. 12:

[0054] hFn nucleotide sequence, SEQ ID NO. 13:

[0055] hFn amino acid sequence, SEQ ID NO. 14:

[0056] SC-hFn nucleotide sequence, SEQ ID NO. 15:

[0057] SC-hFn amino acid sequence, SEQ ID NO. 16:

[0058] hFn-SC nucleotide sequence, SEQ ID NO. 17:

[0059] hFn-SC amino acid sequence, SEQ ID NO. 18:

[0060] Example 1, fusion expression of molecular glue peptide and glycoside hydrolase FpGalNAc

[0061] I. Experimental methods

[0062] (I) Linking FpGalNAc with SpyTag and SpyCatcher respectively

[0063] The present application designs to fuse SpyTag and SpyCatcher in the SpyTag / SpyCatcher peptide pair through a flexible linker (G4S)3 to the C-terminus of FpGalNAc protein, respectively, to construct glycoside hydrolase FpGalNAc-ST and FpGalNAc-SC containing molecular glue peptides (Figure 1A).

[0064] The nucleotide sequences of FpGalNAc, FpGalNAc-ST, and FpGalNAc-SC are shown in SEQ ID NO. 1, SEQ ID NO. 3, and SEQ ID NO. 5, respectively, and the amino acid sequences of FpGalNAc, FpGalNAc-ST, and FpGalNAc-SC are shown in SEQ ID NO. 2, SEQ ID NO. 4, and SEQ ID NO. 6, respectively.

[0065] FpGalNAc-ST and FpGalNAc-SC genes were synthesized by a commission company and cloned into a commercial pET16b plasmid. Then, according to conventional molecular biology methods, they were transformed into E. coli BL21 (DE3) and induced for expression by Isopropyl β-D-Thiogalactoside (IPTG).

[0066] After induction, the bacterial cells were collected by centrifugation, and the supernatant was collected after high-pressure bacterial disruption. The target protein was purified by Ni-NTA affinity chromatography, and detected by Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis (SDS-PAGE) gel electrophoresis.

[0067] As shown in FIG. IB, the purified FpGalNAc-ST shows a single protein band on the SDS-PAGE gel with a molecular weight of 86KD, which is consistent with the expectation, indicating that the SpyTag is fused with FpGalNAc expressed by the present application, and the pure FpGalNAc-ST is obtained by the Ni-NTA one-step purification method.

[0068] The SDS-PAGE electrophoresis of the purified FpGalNAc-SC shows that the main band is 97KD, which is consistent with the expectation, but there are other impurities in the purified product, indicating that the SpyCatcher is fused with FpGalNAc expressed, and it is not easy to obtain a pure product by simple Ni-NTA purification.

[0069] Example 2, fusion expression of molecular glue peptide and glycoside hydrolase FpGalNAc-tri

[0070] The present application designs to introduce SpyTag at the C-terminal of FpGalNAc-tri to construct FpGalNAc-tri-ST (FIG. 2A), so as to couple FpGalNAc-tri with hFn to obtain FpGalNAc-tri-ST and enhance its activity.

[0071] The nucleotide sequence of FpGalNAc-tri-ST is shown in SEQ ID NO. 7, and the amino acid sequence is shown in SEQ ID NO. 8.

[0072] The full-length coding gene of FpGalNAc-tri-ST is synthesized by entrusting a company. The gene cloning, recombinant expression and protein purification are carried out according to Example 1.

[0073] The SDS-PAGE electrophoresis shows that the purified FpGalNAc-tri-ST shows a single protein band on the gel with a molecular weight of about 93KD (FIG. 2B), which is consistent with the expectation, indicating that the pure FpGalNAc-tri-ST is obtained by the simple Ni-NTA one-step purification method.

[0074] Example 3, fusion expression of molecular glue peptide and glycoside hydrolase FpGal

[0075] In order to couple glycoside hydrolase FpGal with hFn together, the molecular glue peptide pair needs to be fused with FpGal. The present application designs to fuse SpyTag at the C-terminal of FpGal protein through (G4S)3 linker to prepare FpGal-ST (FIG. 3A). The coding genes of FpGal and SpyTag are obtained from the gene library. The (G4S)3 coding gene is referred to Example 1.

[0076] The nucleotide sequences of FpGal and FpGal-ST are shown in SEQ ID NO. 9 and SEQ ID NO. 11, respectively; the amino acid sequences of FpGal and FpGal-ST are shown in SEQ ID NO. 10 and SEQ ID NO. 12, respectively.

[0077] According to the molecular design, the genes encoding each domain were spliced by software, and the full-length gene of FpGal-ST was synthesized by a commissioned company. The gene synthesis, cloning, recombinant expression and protein purification were performed as described in Example 1.

[0078] The results are shown in Figure 3B, and the purified product of FpGal-ST shows multiple protein bands on the SDS-PAGE gel, with the main band having a molecular weight of 118KD, which is consistent with the expectation, indicating that SpyTag can be expressed after fusion with FpGal, but the purity of the product obtained by Ni-NTA one-step purification is slightly low.

[0079] Example 4, Fusion expression of molecular glue peptide and hFn

[0080] In order to enable the glycoside hydrolase containing the molecular glue peptide to be coupled with hFn, the molecular glue peptide also needs to be fused with hFn.

[0081] In the present application, SpyCatcher is fused to the N- or C-terminus of hFn through a (G4S)3 linker to prepare SC-hFn and hFn-SC, respectively (Figure 4A).

[0082] The hFn gene coding sequence was obtained from a gene library. According to the molecular design, the genes of each domain were first spliced together by software, and the full-length genes of SC-hFn and hFn-SC were synthesized by a commissioned company, and then cloned into the commercial pQE30 plasmid, and then transformed into E. coli M15 for induction expression. The recombinant protein was purified according to the method described in Example 1.

[0083] The nucleotide sequences of hFn, SC-hFn and hFn-SC are shown in SEQ ID NO. 13, SEQ ID NO. 15 and SEQ ID NO. 17, respectively, and the amino acid sequences of hFn, SC-hFn and hFn-SC are shown in SEQ ID NO. 14, SEQ ID NO. 16 and SEQ ID NO. 18, respectively.

[0084] The results are shown in Figure 4B. After purification, both SC-hFn and hFn-SC showed a single protein band on SDS-PAGE gel, with a molecular weight of 33KD, which is consistent with the expectation, indicating that the pure SC-hFn and hFn-SC can be obtained by the Ni-NTA one-step purification method. It is worth noting that although SDS-PAGE shows that the molecular weight of SC-hFn and hFn-SC is 33KD, which is consistent with the molecular weight of monomer protein, gel filtration chromatography shows that the molecular weight of SC-hFn and hFn-SC under non-denaturing conditions is more than 600KD, indicating that they exist in the form of polymer in solution, which is consistent with the result reported in the literature that hFn forms a nanocage containing 24 monomer proteins in solution (4C). This indicates that the fusion of the molecular glue peptide SpyCatcher does not affect the formation of hFn nanocage.

[0085] It was found in the experiment that unlike SC-hFn, hFn-SC is easily precipitated in solution and is extremely unstable, which cannot be used for subsequent coupling, suggesting that the molecular glue peptide is preferably fused to the N terminus of hFn.

[0086] Therefore, the present application further fuses SpyTag to the N terminus of hFn to construct ST-hFn (Figure 4A). The splicing, synthesis, cloning, recombinant expression and isolation and purification of the full-length gene are the same as the preparation conditions of SC-hFn.

[0087] The results are shown in Figure 4B. After purification, both SC-hFn and hFn-SC showed a single protein band on SDS-PAGE gel, with a molecular weight of 33KD, which is consistent with the expectation, indicating that the pure SC-hFn and hFn-SC can be obtained by the Ni-NTA one-step purification method. It is worth noting that although SDS-PAGE shows that the molecular weight of SC-hFn and hFn-SC is 33KD, which is consistent with the molecular weight of monomer protein, gel filtration chromatography shows that the molecular weight of SC-hFn and hFn-SC under non-denaturing conditions is more than 600KD, indicating that they exist in the form of polymer in solution, which is consistent with the result reported in the literature that hFn forms a nanocage containing 24 monomer proteins in solution (4C). This indicates that the fusion of the molecular glue peptide SpyCatcher does not affect the formation of hFn nanocage.

[0088] Example 5, coupling of FpGalNAc and hFn mediated by molecular glue peptide (coupling of FpGalNAc-ST and SC-hFn)

[0089] I. Experimental method

[0090] According to the principle of molecular glue pairing reaction (Figure 5A), in order to allow FpGalNAc to be coupled with hFn, FpGalNAc-ST and SC-hFn or hFn-SC should be paired, or FpGalNAc-SC and ST-hFn should be paired. However, due to the instability of hFn-SC and the non-uniformity of ST-hFn product, the present application preferentially pairs and couples FpGalNAc-ST and SC-hFn.

[0091] Since SC-hFn can spontaneously form a nanocage structure composed of 24 monomer proteins in solution, not all SC on SC-hFn is necessarily exposed on the surface of the nanocage for binding with FpGalNAc-ST. Moreover, since the molecular weight of the coupled FpGalNAc-ST is large, once coupled, it can block the coupling of SC with ST next to it, and the number of FpGalNAc coupled to hFn depends on the ratio of the two proteins.

[0092] Therefore, in order to couple more FpGalNAc to hFn, the present application mixes a constant amount of SC-hFn with a gradually increasing number of moles of FpGalNAc-ST and reacts overnight at room temperature, and then analyzes the coupling product simultaneously by SDS-PAGE gel electrophoresis and gel filtration chromatography.

[0093] Specifically, the coupling experiment steps are as follows: SC-hFn protein is diluted with PBS buffer (pH 7.4) to 4 μM (calculated as a nanocage containing 24 monomers), and FpGalNAc-ST (calculated as a protein monomer) is diluted with PBS buffer to a concentration of 8, 16, 24, 32, 40, 48 and 56 μM, respectively), the FpGalNAc-ST buffer is mixed with the SC-hFn nanocage buffer in equal volume, the molar concentration ratio of FpGalNAc-ST to SC-hFn nanocage is 2:1, 4:1, 6:1, 8:1, 10:1, 12:1 and 14:1, after reaction overnight at room temperature, the coupling product is analyzed simultaneously by SDS-PAGE gel electrophoresis and gel filtration chromatography.

[0094] II. Experimental results

[0095] The results of gel filtration chromatography are shown in Figure 5B. After the pairing reaction of SC-hFn and FpGalNAc-ST, SDS-PAGE showed that a band with a molecular weight consistent with the fusion protein of the two appeared in the product, indicating that FpGalNAc-ST and SC-hFn were successfully coupled. Moreover, as the amount of FpGalNAc-ST increased, the coupling product also gradually increased.

[0096] When the molar ratio of FpGalNAc-ST (calculated as a protein monomer) to SC-hFn nanocage (1 nanocage contains 24 SC-hFn monomer proteins) increased to 8:1, the coupling product no longer increased significantly, indicating that the FpGalNAc coupled to hFn at this time was close to saturation. Under this condition, about 70% of FpGalNAc was coupled to hFn, and the coupling product yield was high.

[0097] Example 6, FpGalNAc-tri coupling with hFn mediated by molecular glue peptide (FpGalNAc-tri-ST coupling with SC-hFn)

[0098] I. Experimental Methods

[0099] To determine whether the molecular glue peptide can mediate the coupling of FpGalNAc-tri with hFn (Figure 6A), the present application mixes and reacts FpGalNAc-tri-ST (calculated based on protein monomers) and SC-hFn (calculated based on nanocage 24-mer) at different molar ratios according to the description of Example 5, and then detects the conjugate using SDS-PAGE and gel filtration chromatography after overnight reaction.

[0100] Specifically, the SC-hFn protein is diluted to 4 μM (calculated based on nanocage containing 24 monomers) with PBS buffer, and the corresponding FpGalNAc-tri-ST (calculated based on protein monomers) is diluted to 8, 16, 24, 32, 40 and 48 μM with PBS buffer; then, the FpGalNAc-tri-ST buffer and the SC-Fn buffer are mixed in equal volumes to make the molar concentration ratio of FpGalNAc-tri-ST to SC-hFn nanocage 2:1, 4:1, 6:1, 8:1, 10:1 and 12:1, and the two are reacted overnight at room temperature, and the coupling product is analyzed simultaneously using SDS-PAGE gel electrophoresis and gel filtration chromatography.

[0101] II. Experimental Results

[0102] The results are shown in Figure 6B. After FpGalNAc-tri-ST and SC-hFn are mixed and reacted overnight, a larger molecular weight protein appears in the product, indicating that FpGalNAc-tri-ST and SC-hFn are successfully coupled. Moreover, the amount of conjugate increases with the increase of FpGalNAc-tri-ST.

[0103] However, when the molar ratio of FpGalNAc-tri-ST protein to SC-hFn nanocage exceeds 10:1, the content of conjugate no longer increases significantly, indicating that the coupled FpGalNAc-Tri tends to be saturated. Gel filtration chromatography with PBS as the mobile phase also proves that FpGalNAc-tri-ST can be coupled with hFn after mixing and reacting with SC-hFn, and the optimal molar ratio of FpGalNAc-tri-ST to SC-hFn nanocage in the coupling system is 10:1. Moreover, under this ratio, about 70% of FpGalNAc-tri is coupled to the hFn nanocage, and the yield of conjugate is high (Figure 6C).

[0104] Example 7, FpGal coupling with hFn mediated by molecular glue peptide (FpGal-ST coupling with SC-hFn)

[0105] I. Experimental method

[0106] To determine whether the molecular glue peptide can mediate the coupling of FpGal with hFn, i.e. whether FpGal-ST can couple with SC-hFn (Figure 7A), the present application mixed FpGal-ST (calculated based on protein monomer) and SC-hFn (calculated based on nanocage containing 24 monomer proteins) at different molar ratios according to the description of Example 5, and reacted overnight, and then detected the conjugate using SDS-PAGE and gel filtration chromatography.

[0107] II. Experimental results

[0108] The results are shown in Figure 7B. After FpGal-ST and SC-hFn were mixed and reacted overnight, a protein with a larger molecular weight appeared in the product, indicating that FpGal-ST and SC-hFn were successfully coupled. Moreover, the yield of the conjugate increased with the increase of FpGal-ST. However, when the molar ratio of FpGal-ST to SC-hFn nanocage exceeded 10:1, the content of the conjugate no longer increased significantly, indicating that the FpGal coupled to hFn tended to be saturated. Correspondingly, gel filtration chromatography also proved that FpGal-ST and SC-hFn formed a protein with a larger molecular weight after mixing, indicating that the two were successfully coupled. Moreover, when the molar ratio of FpGal-ST to SC-hFn nanocage exceeded 10:1, the content of the conjugate no longer increased significantly (Figure 7C), indicating that the FpGal coupled to hFn tended to be saturated.

[0109] However, under this ratio condition, only 40% of FpGal was coupled to hFn, and the yield of the conjugate was low. These results indicate that although the molecular glue peptide can successfully couple FpGal with hFn, the coupling efficiency is low.

[0110] The beneficial effects of the present application are demonstrated by the following experimental examples.

[0111] Experimental Example 1, hydrolysis activity of A antigen by FpGalNAc-ST and SC-hFn conjugate (FpGalNAc-hFn)

[0112] I. Experimental method

[0113] The FpGalNAc-ST and the SC-hFn nanocage are mixed in equal volume according to the description of Example 5 at a molar ratio of 8:1 (FpGalNAc-ST is 32 μM and SC-hFn nanocage is 4 μM) to react overnight, the conjugate FpGalNAc-hFn is separated by gel filtration chromatography and collected, and the activity of FpGalNAc-hFn is determined by using the A antigen provided by red blood cells as substrate according to the classical red blood cell agglutination experiment method, and the activity of FpGalNAc-hFn is compared with that of uncoupled FpGalNAc.

[0114] The specific method is as follows. The A type red blood cells are diluted to 1% red blood cell packed volume, washed with PBS for 3 times, and then different concentrations of FpGalNAc-hFn, FpGalNAc or FpGal (control group) are added respectively, the specific concentration of each substance is referred to Figure 8, incubated for 1 hour, washed with PBS for 3 times, resuspended with 100 μl of PBS buffer, collected red blood cells, and then 50 μl of red blood cell suspension is taken, incubated with 40 μl of anti-A antibody at room temperature for 30 minutes, and the agglutination state of red blood cells is observed.

[0115] II. Experimental results

[0116] The results are shown in Figure 8. The A type red blood cells treated with FpGal which cannot directly cut A antigen all appear agglutination within 30 minutes after the addition of anti-A antibody. However, after the treatment of FpGalNAc (FpGalNAc-hFn) coupled on hFn at a concentration of 0.5 μg / ml or more, the red blood cells no longer agglutinate, indicating that the A antigen is removed by FpGalNAc-hFn.

[0117] However, when the red blood cells are treated with 5 μg / ml of FpGalNAc, agglutination still occurs.

[0118] The experimental results show that the activity of FpGalNAc can be increased by at least 10 times after being coupled on hFn.

[0119] The hydrolysis activity of FpGalNAc-tri-hFn on A antigen is determined by the same method, and compared with that of FpGalNAc-tri. The results are shown in Figure 9. The hydrolysis activity of FpGalNAc-tri-hFn on A antigen is similar to that of FpGalNAc-tri, indicating that the coupling of FpGalNAc-tri on hFn does not significantly improve its hydrolysis activity on A antigen. This suggests that after the coupling of FpGalNAc-tri and hFn, there is mutual influence between them, and the activity of FpGalNAc-tri cannot be significantly improved.

[0120] The present application found that the FpGalNAc-hFn after coupling FpGalNAc with hFn can significantly improve the hydrolysis activity of FpGalNAc to A antigen, while the coupling of FpGalNAc-tri with hFn does not enhance the activity of FpGalNAc-tri, which shows that each component in the conjugate may interact with each other, and not any glycoside hydrolase coupled with hFn can improve its activity.

[0121] Experimental Example 2, binding activity of FpGalNAc-ST and SC-hFn conjugate (FpGalNAc-hFn) to TfR1 receptor positive cells

[0122] I. Experimental method

[0123] In order to test the binding activity of FpGalNAc-ST and SC-hFn conjugate (FpGalNAc-hFn) to TfR1 receptor, according to the description of Example 5, FpGalNAc-hFn was prepared, and the molar concentration ratio of FpGalNAc-ST (32 μM) to SC-hFn nanocage (4 μM) used was 8:1.

[0124] Then the binding of the conjugate to TfR1 receptor was detected by a molecular interaction instrument. Specifically, the Biotin-labeled TfR1 receptor was diluted to 2 μM with PBS buffer, and the FpGalNAc-ST and SC-hFn conjugate was diluted according to a certain concentration gradient; then the avidin probe was inserted into the diluted TfR1 receptor for 45 s, and after washing with washing buffer (PBS containing 0.02% TWEEN) for 60 s, the diluted conjugate solution was inserted for 120 s, and then transferred to dissociation buffer for 180 s, the binding and dissociation curve was drawn, and the binding force was calculated. Further, the conjugate was labeled with a fluorescent dye, and the binding of the conjugate to red blood cells and kidney tubular endothelial cells (such as HK-2) was detected by flow cytometry.

[0125] II. Experimental results

[0126] As shown in FIG. 10A, hFn and TfR1 receptor exhibit a typical binding and dissociation curve, and the binding force constant (KD) is 3.5 x 10 -7M, indicating that the hFn can bind to the TfR1 receptor. Further, the flow cytometry test results (Figure 10B) show that the positive rate of TfR1 receptor expression on the renal tubular endothelial cells HK-2 is 99.7%, and the positive rate of the binding of the FpGalNAc-hFn labeled by 5(6)-Carboxyfluorescein (FAM) fluorescent dye to the HK-2 cells is 88.2%, which is similar to the positive rate of the binding of the hFn alone to the HK-2 cells, and these results indicate that the FpGalNAc-hFn in the present application can bind to the renal tubular endothelial cells HK-2 with high expression of TfR1 receptor.

[0127] Experimental Example 3, Preparation and Application of Organ Perfusion Solution Based on FpGalNAc-ST and SC-hFn Conjugate

[0128] I. Experimental Methods

[0129] Some organ perfusion solutions commonly used in the clinic at present include: kidney perfusion solution (KPS-1), University of Wisconsin solution (UW), histidine-tryptophan-ketoglutarate (HTK) solution, Celsior solution, Polysol solution, IGL-1 solution, Vasosol solution, HCA solution, etc.

[0130] In order to test the enzyme activity of the FpGalNAc-ST and SC-hFn conjugate FpGalNAc-hFn in different perfusion solutions, according to the description in Example 5, the FpGalNAc-ST and SC-hFn nanocage were mixed at a molar concentration ratio of 8:1 for reaction, then the PBS was used as the mobile phase, and the conjugate FpGalNAc-hFn was obtained by gel filtration chromatography separation and purification, and then was added into different perfusion solutions, and the enzyme cutting activity of these perfusion solutions on the A antigen of cells, tissues or organs was compared by red blood cell agglutination and flow cytometry experiment.

[0131] II. Experimental Results

[0132] To analyze the activity of FpGalNAc-hFn and FpGalNAc in different perfusion solution systems, according to the method of red blood cell agglutination test in experimental example 1, the enzymatic effect of FpGalNAc-hFn and FpGalNAc on red blood cells under PBS, KPS-1, UW, HTK and Celsior solution systems was detected respectively. The results showed that under the condition of low temperature (4℃) perfusion, the enzyme concentration required for FpGalNAc-hFn to completely remove A antigen in the five systems was 0.5 μg / ml, while FpGalNAc required 1 μg / ml, 1 μg / ml, 50 μg / ml, 50 μg / ml and 250 μg / ml respectively (Figure 11A). Similarly, under the condition of body temperature (37℃) perfusion, the enzyme concentration required for FpGalNAc-hFn to completely remove A antigen in the five systems was 0.5 μg / ml, 0.5 μg / ml, 1 μg / ml, 1 μg / ml and 25 μg / ml respectively, while FpGalNAc required 3 μg / ml, 3 μg / ml, 50 μg / ml, 50 μg / ml and 100 μg / ml respectively (Figure 11B). These results showed that FpGalNAc-hFn in the present application showed significantly better A antigen enzymatic activity than FpGalNAc, whether in low temperature perfusion or body temperature perfusion system.

[0133] In summary, the experimental results show that the application provides a preparation method and application of transferrin receptor 1 targeted glycoside hydrolase complex. The molecular glue peptide is fused to SpyTag / SpyCatcher and glycoside hydrolase (FpGalNAc and FpGal) and hFn, respectively, to prepare glycoside hydrolase containing molecular glue peptide and hFn. Then, the glycoside hydrolase containing molecular glue peptide is mixed with the hFn containing the paired molecular glue peptide, the glycoside hydrolase is coupled with the hFn, and the transferrin receptor 1 targeted glycoside hydrolase complex is prepared. The complex improves the activity of the glycoside hydrolase in cutting A-type antigens. In particular, SpyTag is fused to the C-terminal end of the FpGalNAc protein through a flexible linker (G4S)3 to prepare FpGalNAc-ST, SpyCatcher is fused to the N-terminal end of hFn through a (G4S)3 linker to prepare SC-hFn, and FpGalNAc-ST is coupled with SC-hFn to prepare the glycoside hydrolase complex of the application. The glycoside hydrolase complex can target the connection of transferrin receptor 1, can greatly improve the hydrolysis activity of FpGalNAc on A antigens, and can convert A-type organs into O-type organs without using dextran, can overcome a series of side effects caused by the use of dextran in the prior art, and has excellent effect for transplantation. The application can effectively alleviate the shortage of organ supply in clinical organ transplantation, and has wide application prospect.

Claims

1. A transferrin receptor-targeted glycoside hydrolase complex, characterized in that, The complex is formed by the glycoside hydrolase and the human ferritin heavy chain.

2. The composite of claim 1, wherein, In the complex, the ratio of the glycoside hydrolase to the human ferritin heavy chain is 1:24, 2:24, 3:24, 4:24, 5:24, 6:24, 7:24, 8:24, 9:24, 10:24, 11:24, 12:24, 13:24, 14:24, 15:24, 16:24, or 17:

24.

3. The composite of claim 1, wherein, The glycoside hydrolase is acetyl-galactosamine deacetylase or galactosaminease; the amino acid sequence of the acetyl-galactosamine deacetylase is shown in SEQ ID NO. 2; the amino acid sequence of the galactosaminease is shown in SEQ ID NO. 10; and the amino acid sequence of the human ferritin heavy chain is shown in SEQ ID NO.

14.

4. The composite of claim 1, wherein, The glycoside hydrolase and the human ferritin heavy chain are connected by a covalent coupling system.

5. The composite body according to any one of claims 1 to 4, characterized in that The covalent coupling system is the SpyTag-SpyCatcher system or the SnoopTag / SnoopCatcher system.

6. The composite of claim 5, wherein, The complex is formed by the coupling of the glycoside hydrolase-SpyTag and the SpyCatcher-human ferritin heavy chain.

7. The composite body according to claim 5 or 6, characterized in that The complex is formed by the coupling of the glycoside hydrolase-SpyTag and the SpyCatcher-human ferritin heavy chain nanocage, wherein the SpyCatcher-human ferritin heavy chain nanocage is formed by the self-assembly of 24 SpyCatcher-human ferritin heavy chains, and the ratio of the glycoside hydrolase-SpyTag to the SpyCatcher-human ferritin heavy chain nanocage is 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:

1.

8. A method of preparing the transferrin receptor targeted glycoside hydrolase complex of any one of claims 1-7, characterized by: The complex is formed by the coupling of the glycoside hydrolase-SpyTag and the SpyCatcher-human ferritin heavy chain nanocage, wherein the SpyCatcher-human ferritin heavy chain nanocage is formed by the self-assembly of 24 SpyCatcher-human ferritin heavy chains, and the ratio of the glycoside hydrolase-SpyTag to the SpyCatcher-human ferritin heavy chain nanocage is 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:

1.

9. The method of claim 8, wherein, The method for preparing the glycoside hydrolase-SpyTag comprises the following steps: taking a nucleotide sequence for expressing the glycoside hydrolase-SpyTag, preparing a recombinant expression plasmid, and recombinantly expressing in E. coli; the method for preparing the SpyCatcher-human ferritin heavy chain comprises the following steps: taking a nucleotide sequence for expressing the SpyCatcher-human ferritin heavy chain, preparing a recombinant expression plasmid, and recombinantly expressing in E. coli; the nucleotide sequence for expressing the glycoside hydrolase-SpyTag is shown in SEQ ID NO. 3 or SEQ ID NO. 11; and the nucleotide sequence for expressing the SpyCatcher-human ferritin heavy chain is shown in SEQ ID NO.

15.

10. A gene fragment for recombinant expression, wherein the nucleotide sequence is shown in SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 11, SEQ ID NO. 15, or SEQ ID NO.

17.

11. A recombinant vector for recombinant expression, characterized by: It is a recombinant plasmid containing the gene fragment of claim 10.

12. Use of a transferrin receptor-targeting glycoside hydrolase complex according to any one of claims 1 to 7 for cleaving A-type antigens on the surface of red blood cells.

13. A combined product, characterized in that, The combination product is capable of converting an A-type organ into an O-type organ, and comprises a transferrin receptor-targeting glycoside hydrolase complex according to any one of claims 1 to 5 and an organ perfusion solution.

14. The combination for use according to claim 13, wherein, The organ perfusion solution is kidney perfusion solution KPS-1, University of Wisconsin solution (UW), histidine-tryptophan-ketoglutarate solution, Celsior solution, Polysol solution, IGL-1 solution, Vasosol solution or HCA solution; preferably the organ perfusion solution is kidney perfusion solution KPS-1.

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