CN115197328A - Recombinant fusion protease capable of clearing in-vivo immunoglobulin A, preparation method thereof and application thereof in treating IgA nephropathy - Google Patents

Recombinant fusion protease capable of clearing in-vivo immunoglobulin A, preparation method thereof and application thereof in treating IgA nephropathy Download PDF

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CN115197328A
CN115197328A CN202110385443.7A CN202110385443A CN115197328A CN 115197328 A CN115197328 A CN 115197328A CN 202110385443 A CN202110385443 A CN 202110385443A CN 115197328 A CN115197328 A CN 115197328A
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吕继成
金京
解新芳
张宏
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NORTHWEST UNIVERSITY
Peking University First Hospital
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Abstract

The invention provides an AK183 protease-based recombinant fusion protein and application thereof in treating IgA nephropathy and other IgA complex deposition-mediated related diseases, wherein the recombinant fusion protein structurally comprises a human commensal bacterium protease AK183 active region and a human immunoglobulin fragment; the human immunoglobulin fragment is positioned at the N terminal of the active region of the protease AK 183. The invention also provides a method for preparing the recombinant fusion protein. The recombinant fusion protein has the comprehensive advantages of prolonging the half-life period of plasma, effectively clearing circulation and kidney IgA compounds, greatly reducing the side effect of protease on immunogenicity of human bodies and the like by recombining and fusing the natural AK183 protease into human immunoglobulin fragments, and is suitable for specifically treating IgA nephropathy and other IgA compound deposition-mediated related diseases.

Description

Recombinant fusion protease capable of clearing in vivo immunoglobulin A, preparation method thereof and application thereof in treating IgA nephropathy
Technical Field
The invention relates to the field of fusion protein recombination, in particular to an artificially synthesized polypeptide with protease activity, a synthesis method thereof and a method for treating IgA nephropathy and other diseases caused by a poly-IgA complex by using the pharmaceutical and enzymatic activity of the polypeptide.
Background
IgA nephropathy is one of the most common primary glomerular diseases in the world at present, and particularly accounts for 40% -50% of primary glomerular nephritis diagnosed by renal biopsy in east Asia region, and more than 30% -40% of patients progress to end-stage nephropathy-uremia after 20-30 years of attack 1,2 It imposes a heavy burden on the patient and the society. There is currently a lack of specific treatments for IgA nephropathy. Clinically, RAS blockers based supportive treatment is used to slow down the worsening of renal function. Patients who failed to support treatment were treated with a combination hormone immunosuppressant. However, the use of hormonal immunosuppressants has poor long-term treatment and causes serious side effects to patients 3,4 。
In recent years, the focus of pharmaceutical research has been on the development of effective and low-side-effect therapeutic drugs. The main direction includes the development of drugs that reduce and specifically clear renal IgA deposits. These include treatments that systematically reduce immune responses and IgA production, and that reduce IgA-induced complement activation. IgA nephropathy is caused by the deposition of circulating IgA1 complex in glomerulus, and both human body and its pathogenic or symbiotic microorganisms possess the biological mechanism of eliminating IgA. IgA1 consists of two heavy chainsA chain and two light chains. Between the Fab and Fc regions there is a Hinge Region (HR) 6 This is the site of action of the microorganism for cleaving IgA1. A variety of pathogenic and commensal microorganisms possess such proteases for immunization against humans. Wherein the AK183 is IgA protease derived from human intestinal symbiotic bacterium Clostridium (Clostridium). The invention aims to develop a medicine based on AK183 protease by utilizing the excellent tolerance of a human body to the symbiotic bacteria, and further optimize the half-life period of the medicine by a recombinant fusion protein technology so as to effectively clear circulating IgA and renal deposition thereof. The fusion protein medicine is expected to have the characteristics of low immunogenicity, high specificity and long-acting property, and is used for rapidly controlling progressive IgA nephropathy to stabilize renal function.
Disclosure of Invention
In view of the above background, the present invention contemplates the modification of commensal IgA enzymes by means of recombinant techniques to achieve superior pharmacological activity and pharmacokinetic properties, and methods for the specific treatment of IgA nephropathy (including primary and secondary IgA nephropathy) and a wide range of diseases caused by deposition of pathogenic polyiga complexes.
An aspect of the invention aims to: provides a fusion protease capable of clearing pathogenic IgA1 complex to realize the super long blood medicine half life of the protease, so as to prolong the medicine effect of clearing pathogenic IgA1 complex in vivo.
It is another object of the invention to provide methods of using recombinant fusion proteins for the treatment of IgA nephropathy (both primary and secondary IgA nephropathy) and a wide range of diseases caused by pathogenic poly IgA complexes.
It is still another object of the present invention to provide a method for preparing the fusion protease that can scavenge pathogenic IgA1 complexes.
The above purpose of the invention is realized by the following technical scheme:
first, the present invention provides a recombinant fusion protein, comprising a human commensal protease AK183 active region and a human immunoglobulin fragment in its structure; the human immunoglobulin fragment is positioned at the N-terminal of the active region of the protease AK183, and the amino acid sequence of the human immunoglobulin fragment is at least 90% identical to the sequence shown in SEQ ID No.1, preferably at least 95% identical to the sequence, and most preferably 100% identical to the sequence; the active region of the protease AK183 is derived from an amino acid sequence which is at least 90% identical, preferably at least 95% identical, and most preferably 100% identical to the sequence shown in SEQ ID No. 3. The recombinant fusion protein provided by the invention is named as Fc-AK 183. In the recombinant fusion protein, the AK183 active region part can provide a drug active center to degrade IgA, and the human immunoglobulin fragment can enhance the compatibility of the drug and a human body and prolong the half-life period of the drug.
In the proposed process of the present invention, to determine the region of the enzymatically active fragment of wild AK183 (the region of protease AK183 activity), we first constructed a series of truncated AK183 fragments. By in vitro enzyme activity validation of the series of truncated AK183 fragments, we found that the inactive regulatory region of the C-segment inhibits enzyme activity, and that the wild-type AK183 precursor protease needs to complete maturation after C-terminal autostomy. Thus, we grafted the human immunoglobulin fragment to the amino-terminus of the protease AK183 active region, so that the target recombinant fusion protein Fc-AK83 can always retain the human immunoglobulin fusion fragment during its passage from precursor to maturation.
The most preferred recombinant fusion protein of the invention has an amino acid sequence shown as SEQ ID No. 5.
Since IgA nephropathy is caused by chronic deposition of circulating polyIgA complex in glomerular mesangial region, polyIgA complex can also cause diseases with different clinical manifestations, causing different organs to be affected, and in a word, all the related diseases mediated by such IgA complex deposition can be taken as indications of the medicine of the present invention regardless of whether the kidney is affected. Accordingly, a second aspect of the present invention provides a method for treating a related disease mediated by IgA complex deposition using a recombinant fusion protein based on AK183 protease; namely, the recombinant fusion protein based on AK183 protease is used for the production of a medicament for treating an IgA complex deposition-mediated related disease; the recombinant fusion protein structure based on the AK183 protease comprises a human symbiotic protease AK183 active region and a human immunoglobulin fragment; the human immunoglobulin fragment is positioned at the N end of the active region of the protease AK183, and the amino acid sequence of the human immunoglobulin fragment is at least 90 percent identical with the sequence shown in SEQ ID No.1, preferably at least 95 percent identical, and most preferably 100 percent identical; the active region of the protease AK183 is derived from an amino acid sequence which is at least 90% identical, preferably at least 95% identical, and most preferably 100% identical to the sequence shown in SEQ ID No. 3; the most preferred amino acid sequence of the recombinant fusion protein is shown as SEQ ID No. 5.
Furthermore, the research and development team verifies the efficacy of the recombinant fusion protein serving as a novel test drug in a disease model by using in vitro experiments and in vivo animal models. In our in vitro drug validation Fc-AK183 could cleave patient-derived IgA in a variety of diseases, such as samples from IgA nephropathy, purpuric nephritis or kawasaki disease patients of different clinical pathological manifestations. Thus in a preferred use of the invention, the associated disease mediated by deposition of IgA complexes may comprise: igA nephropathy, igA vasculitis, renal damage (or Henoch-Schonlein purpura nephritis) or other secondary IgA nephropathy, or other IgA complex-induced diseases such as Kawasaki disease, igA rheumatoid factor-positive rheumatoid arthritis, igA type anti-GBM disease or IgA type ANCA-associated vasculitis.
In the application of the invention, based on the fact that the treatment is crucial to clearing the poly-IgA complex in the tissues (kidney) or circulation, the immunoglobulin-derived fusion fragment is used for realizing long-acting blood circulation half-life so as to achieve the effect of systematically clearing pathological deposits, therefore, in a preferred scheme, the recombinant fusion protein based on AK183 protease is prepared into a liquid preparation for administration to mammals suffering from related diseases mediated by the deposition of the IgA complex; further preferably, it is prepared into an injection and administered by intravenous injection or infusion route.
In the application of the present invention, the mammal may be a human or other mammal (e.g., a transgenic mammal model useful for drug testing).
Various humanized mouse models were used by the research and development team to verify the potency of Fc-AK183 and to obtain pharmacokinetic parameters. Our experimental results suggest that when the mammal is an animal model for drug testing, the animal model may be a passive human IgA injected murine model, a human IgA1 transgenic murine model, or a primate model, and the intravenous administration regimen comprises: injecting the injection once every 5-10 days at the dose of 5-10 mg/kg; preferably, the injection is administered at a dose of 5mg/kg once every 5 days.
Since the immunoglobulin fragments we used are of human origin and have the best match to the human-related receptor (FcRn), previous data suggest that such fusion drugs have significantly longer half-lives in humans than mice. We therefore speculate that when the mammal is a human, the dosing interval may be longer than in mice. The intravenous administration schedule for humans is expected to be approximately: 5-10mg/kg is injected once every 10-15 days; preferably, the dosage of 10-15mg/kg is used as the initial injection dosage, and then the maintenance dosage of 5-10mg/kg is used for once injection every 10 days. According to our experimental pharmacokinetic data, this dosing regimen can achieve complete clearance of blood circulating IgA over several weeks; during the period, the kidney is fully cleared of the deposition of IgA, so that glomerular injury can be partially repaired, and the kidney is shifted from a progressive injury period to a stable repair period.
In a third aspect, the invention provides a recombinant fusion protein gene expression vector, which is a plasmid formed by recombinant fusion of a carrier and a target gene, wherein the target gene is a recombinant fusion gene and comprises a gene sequence coding a human immunoglobulin fragment (human IgG hinge region CH2 and CH 3) and a gene sequence coding an active region of protease AK 183; the nucleotide sequence of the gene sequence for coding the human immunoglobulin fragment is at least 90 percent identical with the sequence shown in SEQ ID No.2, preferably at least 95 percent identical, and most preferably 100 percent identical; the gene sequence of the coding protease AK183 activity region is at least 90% identical, more preferably at least 95% identical, and most preferably 100% identical with the sequence shown in SEQ ID No. 4.
In the preferred recombinant fusion protein gene expression vector of the present invention, the vector is a vector suitable for prokaryotic expression system, further preferably a vector compatible with E.coli expression system, more preferably a vector with purification tag, and most preferably pET30a plasmid.
In the most preferred embodiment of the invention, the recombinant fusion protein gene expression vector is a plasmid formed by recombinant fusion of a carrier and a target gene, wherein the carrier is an escherichia coli expression vector pET30a plasmid; the target gene is a recombinant fusion gene, and sequentially comprises a gene sequence for coding a human immunoglobulin fragment from the 5' end, wherein the nucleotide sequence is shown as SEQ ID No.2, and a gene sequence for coding an active region containing protease AK183, and the nucleotide sequence is shown as SEQ ID No. 4.
In a fourth aspect, the present invention provides a method for constructing a recombinant fusion protein gene expression vector, comprising: selecting an expression system, respectively synthesizing two cDNA sequences suitable for expression of the expression system according to an amino acid sequence containing a protease AK183 active region and an amino acid sequence of a human immunoglobulin fragment, respectively carrying out DNA recombination on the two cDNA sequences serving as target genes and the expression system to obtain two recombinant plasmids, and finally recombining and fusing the target genes in the two recombinant plasmids in the same recombinant plasmid, wherein in the recombination and fusion, the cDNA sequence of the human immunoglobulin fragment is inserted into the 5' end of the cDNA sequence of the protease AK183 active region to obtain a recombinant fusion protein gene expression vector; the amino acid sequence of the human immunoglobulin fragment is at least 90% identical, preferably at least 95% identical, and most preferably 100% identical to the sequence shown in SEQ ID No. 1.
In a preferred embodiment of the present invention, the method for constructing the recombinant fusion protein gene expression vector comprises the following steps:
(I) Synthesizing a cDNA sequence I 'suitable for escherichia coli expression based on an amino acid sequence of a protease AK183 active region from natural symbiotic clostridium in human intestinal tracts through codon optimization, and synthesizing a cDNA sequence II' suitable for escherichia coli expression based on an amino acid sequence of a human immunoglobulin fragment which is at least 90% identical to a sequence shown in SEQ ID No.1 through codon optimization;
(II) taking the cDNA sequence I 'obtained in the step (I) as a target gene, and carrying out DNA recombination on the target gene and escherichia coli to obtain a subcloned plasmid I'; taking the cDNA sequence II 'obtained in the step (I) as a target gene, and carrying out DNA recombination on the target gene and escherichia coli to obtain a subcloned plasmid II';
(III) cutting the subcloned plasmid II 'obtained in the step (II) to obtain a cDNA sequence II' fragment with a cohesive end; cleaving the subcloned plasmid I 'obtained in (II) such that a nick with a sticky end is formed upstream of the cDNA sequence I';
(IV) carrying out DNA recombination on the cDNA sequence II ' fragment obtained in the step (III) and the cut subcloned plasmid I ', wherein in the DNA recombination, the cDNA sequence II ' is inserted into the 5' end of the cDNA sequence I ' to obtain the recombinant fusion protein gene expression vector.
In a further preferred embodiment of the invention, the cDNA sequence II' described in step (I) is preceded by a base to prevent frameshifting and followed by a fragment of GGGGGGGS linker peptide based on the sequence shown in SEQ ID No.1, in order to protect the protease activity from the fusion IgG Fc fragment.
In a further preferred embodiment of the present invention, in the step (IV), in the DNA recombination of the cDNA sequence II 'fragment obtained in (III) and the cleaved subcloning plasmid I', the N-terminus of the cDNA sequence II 'fragment is fused with a His-tag attached to the vector of the subcloning plasmid I'.
In a fifth aspect, the present invention further provides a method for expressing and purifying the recombinant fusion protein, comprising: transfecting the recombinant fusion protein gene expression vector of the third aspect of the invention into escherichia coli (BL 21-DE 3) competent cells, and carrying out protein expression under the induction of an inducer to obtain the recombinant fusion protein; the inducer is preferably isopropyl-beta-D-thiogalactoside (IPGT) with the concentration of 0.1-0.5 mM; the protein expression temperature is preferably 15-18 ℃; the protein expression time is preferably 20-30 hours; and after the expression is finished, treating the escherichia coli cell bodies by a conventional method, performing ultrasonic fragmentation, performing high-speed centrifugation, retaining supernatant, and then purifying by adopting affinity chromatography and a molecular sieve to obtain the recombinant fusion protein.
In a further preferred expression purification method of the invention, the inducer is IPGT at a concentration of 0.3 mM; the protein expression temperature is 16-18 ℃; the protein expression time is 24 hours.
In a further preferred expression purification method of the invention, the affinity chromatography and molecular sieve purification are performed at 4 ℃ and protease activity is maximally protected using a buffer containing 0.8mM EDTA. Finally, the obtained purified protein is frozen and stored in a neutral phosphate buffer solution.
In a sixth aspect, the present invention also provides a pharmaceutical composition comprising a recombinant fusion protein according to the first aspect of the invention.
In conclusion, the construction strategy of the recombinant fusion protein of the present invention aims to obtain the maximum exertion of the pharmacological advantages: the bacterial source strong IgA hydrolase (AK 183) is used as the core part of the medicine effect and combines the long-acting biological and pharmacological advantages of human immunoglobulin fragment to reach optimal treating effect. Compared with the natural AK183 protease in the prior art, the recombinant fusion protein of the invention has improved drug efficacy in the following aspects:
1. plasma half-life extension
The recombinant fusion protein of the present invention, as an AK 183-based protease fused to the human immunoglobulin fragment, has a sufficiently long plasma half-life in mice compared to the prior natural protease AK183 (see experimental example 1 for details).
2. Effective ablation of circulating and renal IgA complexes
The half-life of the medicine in vivo circulation is obviously prolonged, so the medicine effect of clearing the circulating IgA compound and the kidney IgA deposition is obviously enhanced. In vitro experiments prove that the fusion protease can effectively cleave the blood-derived poly-IgA 1 complex of IgA nephropathy patients, purpura nephritis patients and Kawasaki patients (the effects are shown in figures 15, 16 and 17 in detail in the detailed description of the examples). By experimental treatment against two different humanized IgA mouse models, the recombinant fusion protease of the present invention can clear not only human IgA in the mouse circulation but also IgA1 complex which has been deposited on the glomerulus (experimental example 2).
3. Non-needle generation of neutralizing antibodies to AK183 protease
In order to greatly reduce the immune resistance reaction of a human body to a heterologous protein sequence after multiple administrations, the IgA protease from human intestinal symbiotic bacteria is specially selected. Most of the bacteria reported in the prior art to have IgA protease activity are pathogenic bacteria, including diplococcus meningitidis, haemophilus influenzae, streptococcus pneumoniae, neisseria gonorrhoeae, and the like. Such pathogenic microorganisms use the function of cleaving the human IgA hinge region to protect against host immunity. We note that a few human gut commensals have also acquired an IgA-mediated immune response against the host in their evolution, thus gaining symbiotic capacity. At the same time, human beings are highly tolerant to their symbiotic bacteria and their secreted proteins. Thus, we specifically selected clostridial enterobacter-derived IgA protease AK183 as the drug template.
Animal experiments of mice preliminarily verify that after the recombinant fusion protease is repeatedly injected, a neutralizing antibody which obviously influences the activity of the AK183 protease is not generated in the bodies of the mice (experimental example 3), so that the recombinant fusion protease still has the activity of the AK183 protease after being repeatedly injected into the bodies, and still has the effect of quickly removing the IgA1 compound.
In conclusion, the recombinant fusion protein is suitable for preparing a medicament for specifically treating IgA nephropathy, and indications of the recombinant fusion protein can comprise induction remission treatment of chronic progressive IgA nephropathy and rapid control treatment of clinical rapid progressive patients such as neonatal IgA nephropathy. The medicine prepared by the recombinant fusion protein can effectively reduce the circulating IgA compound in time, thereby improving disease prognosis. In addition, the recombinant fusion protein can be used for treating ANCA-related vasculitis, anti-glomerular basement membrane disease and IgA1 heavy chain myeloma which take anaphylactoid purpura, kawasaki disease and IgA as main pathogenic antibodies, is expected to replace plasma replacement to quickly and efficiently remove the pathogenic IgA antibodies, and improves the survival and disease prognosis of patients.
Drawings
FIG. 1A shows the structure of example 1 in which a base is added in front of a human immunoglobulin fragment to prevent frame shift, and GGGGS is added as a linker.
FIGS. 1B and 1C show the positions of the restriction enzyme cleavage in example 1.
FIG. 2 is a schematic diagram showing the construction process of the recombinant fusion protein gene expression vector described in example 1.
FIG. 3 is a structural diagram of functional regions of a natural protease AK 183.
FIG. 4 is a schematic diagram showing the structure of the functional region of the recombinant fusion protein expressed by E.coli cells described in example 2.
FIG. 5 is an elution profile of Superdex S200 molecular sieves as described in example 2 after purification.
FIG. 6 is an immunoblot of protein peaks F1, F2 and F3 after purification of Superdex S200 molecular sieves described in example 2.
FIG. 7 shows protein peaks F1, F2 and F3 purified as described in example 2, and their in vitro activity confirmation.
FIG. 8 shows the cleavage activity of protein peak F1 obtained by purification described in example 2, after it was added to a sample of purified IgA nephropathy patients with circulating origin polyIgA 1 at different ratios in vitro.
FIG. 9 shows the IgA1 cleavage activity in serum environment of the purified protein peak F1 described in example 2.
FIG. 10 is a schematic diagram of the structure of the functional region of the fusion protein constructed by the method described in example 3.
FIG. 11 is a structural diagram showing the functional regions of the fusion protein constructed by the method described in comparative example 1.
FIG. 12 shows that the fusion protein constructed by the method described in comparative example 1 is self-cut in the human immunoglobulin fragment after expression.
FIG. 13 is a graph showing the change of the concentration of the fusion protease in the serum of mice with time after the injection of the fusion protease into the mice at a dose of 5mg/kg in Experimental example 1.
FIG. 14 is a graph of the concentration of native IgA protease from Lechner et al (J Am Soc Nephrol.2016Sep;27 (9): 2622-9) in mouse plasma as a function of time.
FIG. 15 shows the in vitro cleavage activity of the fusion protease prepared in example 2 on poly-IgA 1 purified from plasma of 9 cases of IgA nephropathy patients.
FIG. 16 shows the in vitro cleavage activity of the fusion protease prepared in example 2 on poly-IgA 1 purified from plasma of 4 patients with Henoch Schonlein purpura nephritis.
FIG. 17 shows the in vitro cleavage activity of the fusion protease prepared in example 2 on plasma-purified IgA1 from 1 example Kawasaki patient.
FIG. 18 enzymatic cleavage activity of the fusion protease described in example 3 on IgA nephropathy patients and healthy controls IgA1 in vitro in a serum environment.
FIG. 19 shows the passive human IgA1 mouse model described in Experimental example 1 and the effect of rapidly and effectively eliminating IgA1 after administration by different routes. .
FIG. 20 shows the therapeutic effect of the immunoblotting method described in Experimental example 1 for detecting fusion protease in passive IgA1 mouse model.
FIG. 21 is a graph showing the in vivo metabolism of fusion protease and the reduction of the concentration at different time points of the ability to cleave passively injected human IgA1 after intravenous injection of the fusion protease into wild-type BALB/C mice as described in Experimental example 2, indicating the ability of the protease to cleave IgA1 for a long period of time in vivo.
Fig. 22 shows that the Fc-tag-free AK183 protease described in experimental example 2 had no cleavage activity after 24 hours in mice.
FIG. 23 is a graph showing the change in body weight after the injection of the fusion protease in the passive IgA1 mouse model described in Experimental example 1.
FIG. 24 shows humanized IgA 1-. Alpha.1 KI described in Experimental example 1 +/WT Circulating IgA1 levels rapidly declined to undetectable within 1 hour and persisted for 5-7 days after injection of fusion protease in the mouse model.
FIG. 25 shows humanized IgA 1-. Alpha.1 KI described in Experimental example 1 +/WT Graph of body weight change after intravenous injection of fusion protease in mouse model.
FIG. 26 is the modeling of passive human IgA deposition on mouse kidney.
FIG. 27 shows that in Experimental example 3, compared with the mice without the fusion protease, the fusion protease in the administered group can scavenge IgA1 deposited by glomeruli in the mouse model of passive human IgA1 renal deposition, and the Fc fragment of hIgA1 after cleavage is reabsorbed by renal tubules by glomerular filtration.
FIG. 28 shows humanized IgA 1-. Alpha.1 KI raised in ordinary environment after injection of recombinant fusion protease in Experimental example 3 +/WT IgA1 deposited chronically in the kidneys of mice can be cleared by proteases.
FIG. 29 shows that injection of recombinant fusion protease did not affect humanized IgA1- α 1KI +/WT The immune barrier function of the mouse intestinal mucosa secretory IgA.
FIG. 30 shows that no antibodies against AK183 were significantly produced in vivo at 30 days after the fusion protease injection described in Experimental example 3 in mice.
FIG. 31 is a graph showing that there was no significant difference in the antibody titer recognizing AK183 in the serum at 30 days after the injection of the fusion protease described in Experimental example 3, compared with that in the non-injected control mouse.
FIG. 32 shows that serum incubated with the fusion protease in vitro at 30 days after the injection of the fusion protease described in Experimental example 3 had no significant effect on the cleavage activity of the fusion protease.
FIG. 33 is a graph showing that mice injected with the fusion protease described in Experimental example 3 were able to more rapidly eliminate human IgA1 passively injected into the mice after re-intravenous administration of the same dose of the fusion protease after day 23.
FIG. 34 shows humanized IgA 1-. Alpha.1 KI described in Experimental example 3 +/WT The circulating IgA1 level of the equivalent dose of the fusion protease rapidly decreased 30 days after intravenous injection of the fusion protease in the mouse model and was still below the baseline level 7 days after intravenous injection of the fusion protease.
Detailed Description
The technical solutions of the present invention will be further described in detail by way of examples, but the scope of the present invention is not limited to the examples.
Example 1 construction of fusion protein expression plasmid
As shown in FIG. 2, the steps for constructing the fusion protein expression plasmid are as follows:
(1) subclone plasmid construction
AK183 protease derived from naturally symbiotic Clostridium in intestinal tract was selected as a natural enzyme to be fused, and its gene structure is shown in FIG. 3, and it comprises a signal peptide protease active region and a transmembrane anchor region. According to the previous studies, it was confirmed (fig. 11, fig. 12) that the autocleavage site of AK183 was located downstream of the C-terminal transmembrane anchor region of its amino acid sequence.
The natural AK183 proteinase amino acid sequence (shown as SEQ ID No. 3) and the amino acid sequence of human immunoglobulin are obtained through NCBI database search, the corresponding cDNA sequence suitable for colibacillus expression is obtained through codon optimization, different restriction endonucleases are added at two ends, the base is added in front of the codon of the human immunoglobulin fragment to prevent frame shift, and GGGGS is added as a linker (shown as figure 1A), so that the biological activity of the fusion protein is protected to the maximum. The above cDNA sequences (shown as SEQ ID No.2 and SEQ ID No.4, wherein, as shown in FIG. 1B and FIG. 1C, restriction enzyme cutting sites are shown by underlines) were synthesized, and cDNA fragments were ligated into the PET30a plasmid vector using DNA ligase, respectively, to obtain subcloned PET30a-AK183 and a plasmid carrying SEQ ID No. 2.
(2) Construction of fusion protein plasmids
Cutting a plasmid carrying SEQ ID No.2 by using restriction enzyme, recovering a fragment I of the SEQ ID No.2 after being cut according to the position shown in figure 1B, cutting a PET30a-AK183 plasmid (the cutting position is shown in figure 1C) by using the same restriction enzyme to form a splicing viscous tail end, putting a DNA ligase linking fragment I into an upstream (N end) of an AK183 sequence far away from a self-cutting site and an active center thereof to form a recombinant fusion vector, and simultaneously adding a His label attached to the N end fusion vector to facilitate later-stage protein purification. The His label is before the PET30a plasmid Enterokinase, and the purified His label of the protease can be cut off by the Enterokinase at the later stage, so that the influence of the His label on the activity of the protease is avoided.
Example 2 expression purification of recombinant fusion proteins
1) Coli expression recombinant fusion protease conditions
The recombinant fusion vector obtained in example 1 was transfected into BL21-DE3 E.coli competent cells, and the expression was carried out at 16 ℃ for 24 hours under the induction of 0.3mM IPGT to obtain cells expressing the recombinant fusion protein, and the gene structure of the recombinant fusion protein expressed in the cells is shown in FIG. 4.
2) Separation and purification of recombinant fusion protein
Collecting 1) the obtained thallus, adding high salt solution (500 mM sodium chloride, 20mM sodium dihydrogen phosphate, 30mM imidazole, 0.8mM EDTA) lysozyme, carrying out ultrasonic DNA lysis, carrying out Ni-column affinity chromatography and Superdex S200 molecular sieve purification on the recombinant fusion protease with His tag to obtain four protein peaks F1, F2, F3 and F4 (figure 5), and identifying the complete expression of the F1 protein peak (figure 6). Adding three protein peaks F1, F2 and F3 into a serum sample of the same IgA nephropathy patient at 37 ℃ overnight, and carrying out Western blot verification after Jacalin purifies IgA1 to find that the protein peaks F1 and F2 have enzyme digestion IgA1 activity in the serum sample of the IgA nephropathy patient (figure 7), and the protein peak F1 has higher protease activity in vitro, and can still well cleave IgA1 when the mass ratio of the protein peak F1 to a substrate IgA1 is 1. From the above detection results, the peak of the F1 protein obtained in this example completely expresses the recombinant fusion protein, the single-chain molecular weight thereof is about 150kDa, the dimer thereof is-300 KD, and the expressed recombinant fusion protein has high-efficiency in vitro enzyme activity.
Example 3
Selecting a eukaryotic expression system HEK293 cell, constructing a fusion protein expression vector suitable for the HEK293 cell, selecting a signal peptide of human IL-2, constructing a fusion protein by using a human immunoglobulin fragment and AK183 which are the same as those in example 1, fusing the human immunoglobulin fragment at the N end of the AK183 protein to form a plasmid, transfecting the HEK293 cell, expressing, separating and purifying, and obtaining a completely expressed recombinant fusion protein from a cell culture solution and a cell body (the gene structure is shown in figure 10).
Comparative example 1.
With reference to the construction method described in example 3, HEK293 was selected as an expression system to construct an expression plasmid (the gene structure is shown in fig. 11), which is different from the fusion protein structure in example 3 in that: the DNA ligase ligases the human immunoglobulin fragment into the C-terminus of the natural enzyme sequence of AK183 (downstream of the transmembrane anchor region) to form a recombinant fusion vector.
After purification, it was found that C-terminal self-cleavage occurred due to maturation of AK183, resulting in self-cleavage of the human immunoglobulin fragment located at the C-terminal end of the fusion protein (see fig. 12).
EXAMPLE 1 in vivo pharmacokinetics of recombinant fusion proteins
The pharmacokinetics in vivo of the recombinant fusion protease prepared in example 2 was evaluated in wild-type BALB/c mice.
3 wild BALB/c mice were injected with 5mg/kg body weight of the recombinant fusion protease with His-tag in tail vein, and tail vein plasma was collected 5min, 1.5h, 4h, 1d, 2d, 3d, 4d, 6d, 8d, and 11d after intravenous injection. An ELISA method is used for detecting the concentration of the recombinant fusion protease in plasma at different time points by taking an anti-His tag monoclonal antibody as a coating antibody and HRP-labeled anti-human IgG Fc as a detection antibody, and the related parameters of the pharmacokinetics are calculated by using a 2Distribution phase. As shown in FIG. 13, the recombinant fusion protease of the present invention was detected in plasma after 11 days of intravenous injection of 5mg/kg body weight of the recombinant fusion protease into mice, AUC (area under the curve) 93063, half-life 62.5 hours. In contrast, in Lechner et al (J Am Soc Nephrol.2016Sep;27 (9): 2622-9), no AK183 protease was completely detected in the plasma of mice 24 hours after the mice were injected with native AK183 protease at a dose of 10mg/kg body weight (see FIG. 14). It can be seen that the recombinant fusion protease prepared in example 2 of the present invention has a significantly extended half-life and can maintain stable activity.
Experimental example 2 in vivo and in vitro functional verification of recombinant fusion protease
The activity of the recombinant fusion protease prepared in example 2 in vitro was verified according to the following method:
this experiment was performed using plasma or plasma purified IgA complexes from patients with clinical renal biopsy confirmed to be primary IgA nephropathy. The IgA nephropathy diagnostic standard is defined as KIDIGO guideline that the kidney puncture pathology mainly takes IgA deposition in glomerular mesangial area and/or capillary loop with or without complement C3 deposition, and clinical manifestations are hematuria and/or proteinuria of different degrees and renal function injury.
FIG. 15 shows the in vitro recombinant fusion protease activity assay using plasma purified poly-IgA complexes from 9 patients diagnosed with IgA nephropathy (PT.1, PT.3, PT.6, PT.7, PT.9, PT.12, PT.19, PT.22 and PT.26, wherein PT.1, PT.3, PT.6 and PT.9 are crescent IgA nephropathy patients), and the number of poly-IgA and clinical and pathological data of the patients after purification are shown in Table 1.
TABLE 1 clinical case data on renal transrenal status of IgA nephropathy patients
Figure BDA0003014600240000121
The recombinant fusion protease prepared in example 2 was added to 9 purified IgA complex samples, using the IgA complex samples with the numbers corresponding to table 1 as the experimental group; taking a poly-IgA complex sample which is not added with recombinant fusion protease and has the number of PT.1 as a control group; all experimental groups and control groups sample at 37 ℃ overnight, and the sizes of IgA1 heavy chain fragments are detected by immunoblotting detection of anti-human IgAalpha chain antibodies to verify the activity of the recombinant fusion protease added in each experimental group. As a result, as shown in FIG. 15, the recombinant fusion protease of example 2 was able to cleave 100% of the purified poly-IgA 1 complex from the circulation of IgA nephropathy patients overnight at 37 ℃ and the cleaved IgA1 heavy chain was cleaved into an Fc fragment having a smaller molecular weight recognized by an anti-human IgA alpha antibody as compared with the complex in the control group.
FIG. 16 shows that the in vitro recombinant fusion protease activity assay was performed using purified polyIgA complexes from 4 patients (HSPN 1, HSPN2, HSPN3 and HSPN 4) with purpuric nephritis diagnosis, and the purified polyIgA complexes and clinical and pathological data of the patients are shown in Table 2.
TABLE 2 clinical case data of patients with purpura nephritis
Figure BDA0003014600240000122
Figure BDA0003014600240000131
FIG. 16 shows a case where the samples of the poly-IgA complex having the numbers corresponding to Table 2 were used as experimental groups, and 4 samples of the purified poly-IgA complex were added with the recombinant fusion protease prepared in example 2; taking a poly IgA compound sample which is not added with recombinant fusion protease and is numbered HSPN.1 as a control group; all experimental groups and control groups are subjected to sample 37 ℃ overnight, and the sizes of IgA1 heavy chain fragments are detected by anti-human IgAalfa chain antibodies through immunoblotting to verify the activity of the recombinant fusion protease added in each experimental group. As a result, as shown in FIG. 16, the recombinant fusion protease of example 2 was able to cleave 100% of the poly-IgA 1 complex purified from the circulation of Henoch Schonlein purpura nephritis patients under the overnight condition at 37 ℃, and the cleaved IgA1 heavy chain was cleaved into an Fc fragment having a smaller molecular weight recognized by the anti-human IgA alpha antibody, as compared with the complex in the control group.
FIG. 17 shows the results of using 1 example of plasma-purified IgA1 confirmed to be diagnosed with Kawasaki disease as a substrate for the experimental group and the recombinant fusion protease prepared in example 2 as a control group, using the same sample of poly-IgA complex without recombinant fusion protease; immunoblotting detection is carried out on samples of an experimental group and a control group after overnight at 37 ℃, and the result shows that the fusion protease can obviously remove IgA1 in circulation of Kawasaki patients.
To further verify the enzyme digestion activity of the recombinant fusion protease of the present invention in a serum environment, the recombinant fusion protease prepared in example 2 and human IgA1 were added to the sera of different IgA nephropathy patients and healthy persons (IgA nephropathy patients numbered pt.3 and pt.6 and healthy persons numbered hc.1 and hc.2 described above) in vitro at a mass ratio of 1; all experimental groups and control groups are subjected to samples at 37 ℃ overnight, and the sizes of IgA1 heavy chain fragments are detected by immunoblotting to verify the activity of the recombinant fusion protease added in each experimental group. As a result, as shown in FIG. 18, the recombinant fusion protease of example 2 was able to cleave the poly-IgA 1 complex in the circulation of IgA nephropathy patients and healthy persons 100% in a serum environment under an overnight condition at 37 ℃ in vitro, and the cleaved IgA1 heavy chain was cleaved into an IgA-Fc fragment having a smaller molecular weight recognized by an anti-human IgAalfa antibody, as compared with the complex in the control group.
The activity of the recombinant fusion protease prepared in example 2 in the humanized mouse model was verified according to the following method:
the in vivo protease activity of the recombinant fusion protease obtained in example 2 was verified in a wild-type BALB/C mouse model injected with human IgA and a humanized IgA1alpha chain knock-in (α 1 KI-Tg) C57BL/6 mouse model, respectively.
All wild type passive models BALB/c mice were randomly divided into dosing group a (i.v.), dosing group B (i.p.) and control group, 3 per group. The verification scheme A of the passive model is as follows: the tail vein of BALB/c mice which are administrated to the group A and the control group is injected with 25mg/kg of human IgA1 to ensure that the level of human IgA1 in blood is rapidly increased, and a wild type BALB/c mouse model injected with passive human IgA is obtained. After 5 minutes, the recombinant fusion protease prepared in example 2 was administered to mice of group A via tail vein injection at a dose of 5mg/kg body weight, and PBS was injected to tail vein of control group. Respectively collecting blood plasma after 3min, 15min, 45min, 75min, 105min and 135min after injection, collecting EP tube, adding EDTA in advance for anticoagulation, and avoiding in vitro enzyme digestion reaction.
In order to prove that the intraperitoneal drug delivery can still be well absorbed into blood, a verification scheme B of the passive model is as follows: the recombinant fusion protease prepared in example 2 was intraperitoneally injected into BALB/c mice administered to group B, and the blood concentration of the recombinant fusion protein injected intraperitoneally after about 2 hours from the past experience reached a peak, so that after 1.5 hours, 25mg/kg IgA1 was injected into the tail vein of human, and after 3min, 30min, 90min and 150min after injection, plasma was collected by cutting the tail, and an EP tube was collected and EDTA was added in advance for anticoagulation, thereby avoiding the in vitro enzyme digestion reaction from continuing.
The ELISA method is used for coating an anti-human IgA Fc antibody, an HRP-labeled anti-human Ig Fab antibody is used as a detection antibody, and the concentration of complete IgA in plasma samples collected at different time points in the verification schemes is detected (5 mM EDTA is added into plasma diluent to prevent recombinant fusion protease in blood from continuously enzyme-cutting hIgA1 in the experiment process), the result is shown in figure 19, the initial blood IgA1 concentration of all mouse models passively injected with human IgA1 is similar to the human blood IgA1 concentration and is slowly metabolized in mice, and the human IgA1 concentration in circulation is reduced by 35% compared with the initial 3 minutes in a PBS control group at 2.25h (which reflects the natural clearance of non-protease-mediated heterologous IgA in mice), and the passive model can be used for detecting the activity of the in-vivo recombinant fusion protease. Compared with the PBS treatment group, the tail vein administration (figure 19A) and the intraperitoneal administration of the recombinant fusion protease (figure 19B) can quickly eliminate passively injected human IgA1 in a mouse body, the blood IgA level of the intravenous recombinant fusion protease administration group is obviously lower than that of the PBS control group from 3 minutes, only 5 percent of the whole human IgA1 is remained in the blood of the administration group within 1.75 hours, and the intravenous administration mode can quickly lead the blood concentration of the recombinant fusion protease to reach the peak value so as to quickly eliminate the human IgA1 in the blood.
Plasma collected at various time points in the above administration group A was purified of human IgA1 using Jacalin beads (plasma 6. Mu.l was added with 100. Mu.l of PBS containing 5mM EDTA), and the collected human IgA1 heavy chain fragments were detected with anti-human IgA alpha antibody by immunoblotting. As shown in FIG. 20A, consistent with the above ELISA results, the recombinant fusion protease of example 2 significantly reduced the IgA1Fc (molecular weight 65 KD) fragment in plasma at 3 minutes in group A administered intravenously with recombinant fusion protease, and a portion of the IgA1Fc fragment was cleaved into Fd fragments of smaller molecular weight (theoretical molecular weight 27KD, and a reduced state was found in a protein band of 37 KD), and no IgA1Fc band was found at 1.75 hours after administration with group A. FIG. 20B is the results of quantification of the full band intensity of human IgA1Fc in FIG. 20A. The result proves that the recombinant Fc-AK183 has obvious effect of cutting IgA1 in mice.
Furthermore, in order to examine the process of the attenuation of the pharmaceutical activity of the recombinant fusion protease of the present invention in vivo, 12 wild BALB/c mice were administered tail vein injection of the recombinant fusion protease prepared in example 2 at a dose of 5mg/kg at 0 point, and then the experiment was divided into 4 groups of 3 mice each, 25mg/kg of human IgA1 was administered tail vein injection on days 1, 3, 7, and 10 after the injection of the recombinant fusion protease, respectively, and plasma was collected at 0.05h, 0.5h, 1.5h, 3h, 5h, and 7h after the injection in each group, while 3 mice in the control group were administered intravenous injection of PBS and human IgA1 (fig. 21A). The results of the ELISA method for detecting intact human IgA1 in plasma (the specific method is as described in Experimental example 1) compared with the PBS control treatment group, the blood IgA level of each mouse at 3 minutes is taken as a baseline 100%, the curve of the rate of decrease of the human IgA1 level in the blood of the mouse with time is taken as the enzyme activity evaluation, and the decrease rate of the IgA1 concentration of the mice of the experimental group which receive the intravenous injection of the recombinant fusion protease in example 2 is faster than that of the PBS group within 7 hours after the injection of the human IgA1 on the 1 st, 3 th, 7 th and 10 th days, and the decrease rate of the IgA1 concentration is faster than that of the PBS group on the 1 st day than that of the 3 rd, 7 th and 10 th days, which indicates that the enzyme digestion activity of the fusion protease is slowly decreased with the time in vivo (see FIG. 21B).
In order to more clearly verify that the recombinant fusion protein can play a role for a long time, the Fc-tag-free recombinant AK183 protease is expressed through escherichia coli recombination by a conventional method to serve as a control protease, BALB/c mice are injected with 5mg/kg of the control protease intravenously, 25mg/kg of human IgA1 is injected into the tail vein after 1 day, then blood plasma of 0.05h, 0.5h, 1.5h and 3h is collected, and the concentration of the human IgA1 is detected by an ELISA method. The time-dependent concentration profile of human IgA1 compared to the recombinant fusion protease of example 2 showed no significant acceleration of IgA1 degradation by the control protease group and coincided with the IgA1 metabolism profile of the PBS group (fig. 22). The control protease was suggested to be metabolically cleared of non-protease activity after 1 day in mice, consistent with the Lechner et al study.
The body weights of the 6 passive IgA1 mouse models injected with the recombinant fusion protease of example 2 in the group a and the group B were measured every day within 10 days after the injection, and no body weight reduction tendency was observed (as shown in fig. 23), and the behavior and response of the mice were normal, indicating that the injection of the recombinant fusion protease of the present invention did not exert adverse effects such as weight reduction on the mice.
In order to solve the problem of no stable level of IgA1 in the passive IgA1 mouse model circulation, a humanized IgA1 transgenic animal model mouse (IgA 1-alpha 1 KI) is further adopted +/WT Mouse model) (fig. 24A). The heterozygotes expressed stable levels of human IgA1 and mouse IgA (see Baseline Baseline time points in immunoblot FIG. 24C). In this experiment mouse IgA was not cleaved by Fc-AK183 due to the hinge-free sequence and was therefore drug-specificControl for cleavage of human IgA1. 3 IgA 1-. Alpha.1 KI was administered 3 recombinant fusion proteases of example 2 by intravenous injection at 5mg/kg +/WT In mice, tail vein EDTA anticoagulated plasma was collected at 0.05H, 2H, 4H, 24H, 48H, 72H, 96H, 120H, 168H, 240H and 336H before and after injection (see FIG. 24B), and the intact IgA-H (IgA heavy chain) level in plasma was detected at each time point by an anti-human IgA1alpha antibody immunoblotting method and the mouse plasma IgA level at the above time points was detected by an anti-mouse IgA antibody immunoblotting method. The results show that circulating IgA1 levels rapidly decreased to no distinct band within 1 hour and persisted for 5-7 days after injection of the recombinant fusion protease of example 2, slowly increased thereafter, and rose back to baseline after 14 days, during which there was no significant change in mouse IgA levels (fig. 24C). FIG. 24D is a semi-quantitative analysis of human IgA immunoblot bands for three mice in 24A at different time points. At the same time, the three mice observed no significant weight loss for 14 days (fig. 25). It was thus revealed that when the recombinant fusion protease of the present invention was administered to a passive IgA1 mouse model, a dosage regimen of 5mg/kg by injection once every 5 to 7 days was effective.
In addition, a passive IgA1 deposition mouse model and IgA 1-alpha 1KI fed in a common environment are respectively established +/WT Mice led to a renal IgA1 deposition model to investigate the drug-clearing capacity of the recombinant fusion proteases of the invention for renal deposited IgA1. A study protocol for the passive IgA1 deposition model is shown in fig. 26. Earlier studies found that immunofluorescence staining at about 2.5 hours after a single injection of human IgA1 at a dose of-25 mg/kg revealed significant IgA1 deposition on the glomeruli, mainly on capillary loops and mesangial regions, in a manner similar to that of renal IgA1 deposition in human IgA nephropathy (FIG. 26). In the experiment, 25mg/kg of human IgA1 is injected into the tail vein of a BABL/c mouse every 1 hour, the injection is performed twice (the scheme is shown in figure 27A), after the last IgA1 injection of a PBS control treatment group is performed for 2.5 hours, an OCT-embedded frozen kidney tissue 4-micron section is subjected to immunofluorescence staining by using an anti-human IgA1Fc FITC labeled antibody, obvious IgA1 is found to be only deposited on a glomerular mesangial region and a part of capillary loops, and the model establishment is proved to be successful (see the lower left part and the lower right part of figure 27B). In contrast to the PBS control group, the recombinant fusion protease of example 2 was injected into the tail vein 1 hour after the last IgA1 injection at a dose of 5mg/kgAfter 1.5 hours of in vivo reaction, no IgA1 deposition is found in glomeruli (see upper left of FIG. 27B), and an IgA1 positive signal is seen in renal tubular epithelial cells (see upper right of FIG. 27B), which indicates that IgA1 deposited in the kidney can be rapidly digested by the recombinant fusion protease of example 2 within 1.5 hours, and the IgA1-Fc fragment after digestion can be filtered and cleared and then metabolized by the renal tubular epithelial cells due to the fact that the molecular weight is lower than the mechanical filtration barrier of the kidney.
Furthermore, igA 1-alpha 1KI is fed in the common environment +/WT Mice, present with the deposition of transgenic human IgA1 in the kidney. To more specifically verify the efficacy of Fc-AK183 on renal clearance of deposited IgA1, two kidneys of the same mouse were harvested before and after administration. The contralateral kidney was removed 3.5 hours after removal of 5mg/kg of the recombinant fusion protease of example 2 of the invention intravenously after one side of the kidney was removed (FIG. 28A). Renal IgA1 deposition was then detected by immunofluorescent staining with FITC-labeled anti-human IgA antibodies. The results found that pre-dosed harvested kidneys had significant human IgA deposition (2 + -3 +) in the glomerular mesangial region. Whereas the contralateral kidney IgA1 harvested 2 hours after administration was significantly attenuated (+ -) (see FIG. 28B), which in turn demonstrated the therapeutic effect of Fc-AK183 on the clearance of chronic cumulative IgA1 deposition. Meanwhile, the intestinal secretory IgA1 was found to be still strongly positive in the staining of the intestinal tissue (see fig. 29), suggesting that the recombinant protease for injection of the present invention had no effect of clearing the extravascular mucosal IgA1. It can be concluded that this drug does not affect the gut mucosal defense barrier mediated by secretory IgA1.
EXAMPLE 3 detection of neutralizing antibodies to recombinant fusion proteins and Effect on their Activity
To test the antigenic response of the recombinant fusion protease Fc-AK183 after multiple injections, we performed an intermittent injection protocol of the drug to verify: 1. whether mice had developed anti-AK 183 antibody, 2. Whether the antibody inhibited protease activity.
Sera of BALB/c mice were collected at day 30 after intravenous injection of the recombinant fusion protease of example 2 at a dose of 5mg/kg, and the lower wells of an equal-concentration BSA plate were set using 2.5 μ g/ml of AK183 and the recombinant fusion protease as plate-coating antigens by ELISA method, and after 1% BSA blocking, the mice were incubated with an HRP-labeled anti-mouse IgG as a detection antibody, and antibodies against AK183 and the recombinant fusion protease were detected by an HRP-labeled anti-mouse IgG. As shown in fig. 30, high titer antibodies recognizing the intact recombinant fusion protease Fc-AK83, but no significant antibodies recognizing AK183, were present in the sera of mice at day 30 after the treatment with the recombinant fusion protease Fc-AK183 of example 2, suggesting that the Fc fragment is more antigenic than AK 183. The results of 4 wild BALB/c not injected with AK183 as a negative control group and mice treated with AK183 were tested again by ELISA for antibodies against AK183 and human IgG1Fc as shown in fig. 31, and compared to the non-dried group, the recombinant fusion protease Fc-AK83 treatment group of example 2 was verified to have very low titer of the antibody against AK183 and high titer of the antibody against human Fc from different angles.
Further, we tested in vitro whether antibodies against recombinant proteases would affect the activity of the protease. Mu.g of the recombinant fusion protease of example 2 was first added to different volumes of the above day 30 antiserum (20ul, 10ul, 2ul) in vitro, reacted at 37 ℃ for 1 hour, and then 10. Mu.g of human IgA was added thereto, and digested at 37 ℃ overnight. And detecting whether the activity of the enzyme-digested human IgA1 still exists in vitro after the recombinant fusion protease reacts with the serum antibody capable of recognizing Fc by using an anti-human IgA1Fc antibody by adopting an immunoblotting method. As a result, as shown in FIG. 32, human IgA1 was cleaved well as the IgA-Fc fragment, suggesting that the neutralizing antibody against fusion protease IgG1Fc did not affect the enzymatic activity of the recombinant fusion protease under in vitro whole serum conditions.
Experimental design of whether neutralizing antibody affects in vivo activity of recombinant fusion protease 3 mice receiving 5mg/kg of recombinant fusion protease of example 2 were intravenously injected, 5mg/kg of recombinant fusion protease of example 2 was intravenously injected again on the 23 th day after administration, 20mg/kg of human IgA1 was posteri-venously injected after 1 hour, plasma was collected at 0.05h, 0.25h, 0.75h, 1.25h, 1.75h and 2.25h after injection of IgA1, human IgA1 concentration in plasma was measured by ELISA method in experiment 1, and the recombinant fusion protease group and PBS group of example 2 injected for the first time in experiment 1 were used as control groups. As a result, as shown in FIG. 33, the recombinant fusion protease of example 2 was injected into the mouse again after 23 days, and the mouse was allowed to react with the neutralizing antibody in vivo and then still had a strong IgA1 cleavage activity. Suggesting that the enzyme activity of the recombinant fusion protease was not affected in vivo by neutralizing antibodies.
3 of the experimental examples 2 were simultaneously injected intravenously with humanized IgA 1-. Alpha.1 KI receiving the recombinant fusion protease Fc-AK83 of example 2 +/WT The mice were treated with 5mg/kg of the recombinant fusion protease Fc-AK183 of example 2 again intravenously at the same dose on the 30 th day after injection, and plasma was collected before administration, 3min, 4 hours, 24 hours, 72 hours, and 120 hours after administration, and plasma human IgA and mouse IgA levels at different time points after administration were measured by the immunoblotting method in example 2. The results are shown in FIG. 34, igA 1-. Alpha.1 after re-administration KI+/WT Mouse levels decreased rapidly within 4 hours and maintained at the 24 hour immunoblot assay threshold level, with elevated blood-to-human IgA at day 3, but still significantly below the pre-dose baseline level (approximately baseline 50% level), and was maintained steady to day 7, while mouse IgA remained stable before and after dosing. Indication of IgA 1-alpha 1 KI+/WT The mice had little effect on the in vivo activity of the re-administration of antibodies generated after the initial injection of the recombinant protease.
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8.Oruc Z,Oblet C,Boumediene A,et al.IgA Structure Variations Associate with Immune Stimulations and IgA Mesangial Deposition.J Am Soc Nephrol,27:2748-2761,2016.
9.Lamm ME,Emancipator SN,Robinson JK,et al.Microbial IgA protease removes IgA immune complexes from mouse glomeruli in vivo:potential therapy for IgA nephropathy.Am J Pathol,172:31-36,2008.
10.Wang L,Li X,Shen H,et al.Bacterial IgA protease-mediated degradation of agIgA1 and agIgA1 immune complexes as a potential therapy for IgA Nephropathy.Sci Rep,6:30964,2016.
Sequence listing
<110> first Hospital of Beijing university
Northwest University (Northwestern University)
<120> a recombinant fusion protease capable of clearing in vivo immunoglobulin A, a preparation method thereof and application thereof in treating IgA nephropathy
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 230
<212> PRT
<213> human (human)
<400> 1
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro
225 230
<210> 3
<211> 720
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atccatggga gcccaaatct tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg 60
aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac accctcatga 120
tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa gaccctgagg 180
tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca aagccgcggg 240
aggagcagta caacagcacg taccgggtgg tcagcgtcct caccgtcctg caccaggact 300
ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca gcccccatcg 360
agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc 420
catcccggga tgagctgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct 480
atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga 540
ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg 600
acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat gaggctctgc 660
acaaccacta cacgcagaag agcctctccc tgtctccggg taaaggtggc ggtggcggcg 720
<210> 2
<211> 1174
<212> PRT
<213> Clostridium clostridia (Clostridium)
<400> 2
Gly Ser Ser Lys Pro Asp Ile Lys Val Gly Asp Tyr Val Lys Met Gly
1 5 10 15
Val Tyr Asn Asn Ala Ser Ile Leu Trp Arg Cys Val Ser Ile Asp Asn
20 25 30
Asn Gly Pro Leu Met Leu Ala Asp Lys Ile Val Asp Thr Leu Ala Tyr
35 40 45
Asp Ala Lys Thr Asn Asp Asn Ser Asn Ser Lys Ser His Ser Arg Ser
50 55 60
Tyr Lys Arg Asp Asp Tyr Gly Ser Asn Tyr Trp Lys Asp Ser Asn Met
65 70 75 80
Arg Ser Trp Leu Asn Ser Thr Ala Ala Glu Gly Lys Val Asp Trp Leu
85 90 95
Cys Gly Asn Pro Pro Lys Asp Gly Tyr Val Ser Gly Val Gly Ala Tyr
100 105 110
Asn Glu Lys Ala Gly Phe Leu Asn Ala Phe Ser Lys Ser Glu Ile Ala
115 120 125
Ala Met Lys Thr Val Thr Gln Arg Ser Leu Val Ser His Pro Glu Tyr
130 135 140
Asn Lys Gly Ile Val Asp Gly Asp Ala Asn Ser Asp Leu Leu Tyr Tyr
145 150 155 160
Thr Asp Ile Ser Glu Ala Val Ala Asn Tyr Asp Ser Ser Tyr Phe Glu
165 170 175
Thr Thr Thr Glu Lys Val Phe Leu Leu Asp Val Lys Gln Ala Asn Ala
180 185 190
Val Trp Lys Asn Leu Lys Gly Tyr Tyr Val Ala Tyr Asn Asn Asp Gly
195 200 205
Met Ala Trp Pro Tyr Trp Leu Arg Thr Pro Val Thr Asp Cys Asn His
210 215 220
Asp Met Arg Tyr Ile Ser Ser Ser Gly Gln Val Gly Arg Tyr Ala Pro
225 230 235 240
Trp Tyr Ser Asp Leu Gly Val Arg Pro Ala Phe Tyr Leu Asp Ser Glu
245 250 255
Tyr Phe Val Thr Thr Ser Gly Ser Gly Ser Gln Ser Ser Pro Tyr Ile
260 265 270
Gly Ser Ala Pro Asn Lys Gln Glu Asp Asp Tyr Thr Ile Ser Glu Pro
275 280 285
Ala Glu Asp Ala Asn Pro Asp Trp Asn Val Ser Thr Glu Gln Ser Ile
290 295 300
Gln Leu Thr Leu Gly Pro Trp Tyr Ser Asn Asp Gly Lys Tyr Ser Asn
305 310 315 320
Pro Thr Ile Pro Val Tyr Thr Ile Gln Lys Thr Arg Ser Asp Thr Glu
325 330 335
Asn Met Val Val Val Val Cys Gly Glu Gly Tyr Thr Lys Ser Gln Gln
340 345 350
Gly Lys Phe Ile Asn Asp Val Lys Arg Leu Trp Gln Asp Ala Met Lys
355 360 365
Tyr Glu Pro Tyr Arg Ser Tyr Ala Asp Arg Phe Asn Val Tyr Ala Leu
370 375 380
Cys Thr Ala Ser Glu Ser Thr Phe Asp Asn Gly Gly Ser Thr Phe Phe
385 390 395 400
Asp Val Ile Val Asp Lys Tyr Asn Ser Pro Val Ile Ser Asn Asn Leu
405 410 415
His Gly Ser Gln Trp Lys Asn His Ile Phe Glu Arg Cys Ile Gly Pro
420 425 430
Glu Phe Ile Glu Lys Ile His Asp Ala His Ile Lys Lys Lys Cys Asp
435 440 445
Pro Asn Thr Ile Pro Ser Gly Ser Glu Tyr Glu Pro Tyr Tyr Tyr Val
450 455 460
His Asp Tyr Ile Ala Gln Phe Ala Met Val Val Asn Thr Lys Ser Asp
465 470 475 480
Phe Gly Gly Ala Tyr Asn Asn Arg Glu Tyr Gly Phe His Tyr Phe Ile
485 490 495
Ser Pro Ser Asp Ser Tyr Arg Ala Ser Lys Thr Phe Ala His Glu Phe
500 505 510
Gly His Gly Leu Leu Gly Leu Gly Asp Glu Tyr Ser Asn Gly Tyr Leu
515 520 525
Leu Asp Asp Lys Glu Leu Lys Ser Leu Asn Leu Ser Ser Val Glu Asp
530 535 540
Pro Glu Lys Ile Lys Trp Arg Gln Leu Leu Gly Phe Arg Asn Thr Tyr
545 550 555 560
Thr Cys Arg Asn Ala Tyr Gly Ser Lys Met Leu Val Ser Ser Tyr Glu
565 570 575
Cys Ile Met Arg Asp Thr Asn Tyr Gln Phe Cys Glu Val Cys Arg Leu
580 585 590
Gln Gly Phe Lys Arg Met Ser Gln Leu Val Lys Asp Val Asp Leu Tyr
595 600 605
Val Ala Thr Pro Glu Val Lys Glu Tyr Thr Gly Ala Tyr Ser Lys Pro
610 615 620
Ser Asp Phe Thr Asp Leu Glu Thr Ser Ser Tyr Tyr Asn Tyr Thr Tyr
625 630 635 640
Asn Arg Asn Asp Arg Leu Leu Ser Gly Asn Ser Lys Ser Arg Phe Asn
645 650 655
Thr Asn Met Asn Gly Lys Lys Ile Glu Leu Arg Thr Val Ile Gln Asn
660 665 670
Ile Ser Asp Lys Asn Ala Arg Gln Leu Lys Phe Lys Met Trp Ile Lys
675 680 685
His Ser Asp Gly Ser Val Ala Thr Asp Ser Ser Gly Asn Pro Leu Gln
690 695 700
Thr Val Gln Thr Phe Asp Ile Pro Val Trp Asn Asp Lys Ala Asn Phe
705 710 715 720
Trp Pro Leu Gly Ala Leu Asp His Ile Lys Ser Asp Phe Asn Ser Gly
725 730 735
Leu Lys Ser Cys Ser Leu Ile Tyr Gln Ile Pro Ser Asp Ala Gln Leu
740 745 750
Lys Ser Gly Asp Thr Val Ala Phe Gln Val Leu Asp Glu Asn Gly Asn
755 760 765
Val Leu Ala Asp Asp Asn Thr Glu Thr Gln Arg Tyr Thr Thr Val Ser
770 775 780
Ile Gln Tyr Lys Phe Glu Asp Gly Ser Glu Ile Pro Asn Thr Ala Gly
785 790 795 800
Gly Thr Phe Thr Val Pro Tyr Gly Thr Lys Leu Asp Leu Thr Pro Ala
805 810 815
Lys Thr Leu Tyr Asp Tyr Glu Phe Ile Lys Val Asp Gly Leu Asn Lys
820 825 830
Pro Ile Val Ser Asp Gly Thr Val Val Thr Tyr Tyr Tyr Lys Asn Lys
835 840 845
Asn Glu Glu His Thr His Asn Leu Thr Leu Val Ala Ala Lys Ala Ala
850 855 860
Thr Cys Thr Thr Ala Gly Asn Ser Ala Tyr Tyr Thr Cys Asp Gly Cys
865 870 875 880
Asp Lys Trp Phe Ala Asp Ala Thr Gly Ser Val Glu Ile Thr Asp Lys
885 890 895
Thr Ser Val Lys Ile Pro Ala Pro Gly His Thr Ala Gly Thr Glu Trp
900 905 910
Lys Ser Asp Asp Thr Asn His Trp His Glu Cys Thr Val Ala Gly Cys
915 920 925
Gly Val Ile Ile Glu Ser Thr Lys Ser Ala His Thr Ala Gly Glu Trp
930 935 940
Ile Val Asp Thr Pro Ala Thr Ala Thr Thr Ala Gly Thr Lys His Lys
945 950 955 960
Glu Cys Thr Val Cys His Arg Val Leu Glu Thr Gln Pro Ile Pro Ser
965 970 975
Thr Gly Thr Glu Leu Lys Ile Ile Ala Gly Asp Asn Gln Ile Tyr Asn
980 985 990
Lys Ala Ser Gly Ser Asp Val Thr Ile Thr Cys Asn Gly Asp Phe Ala
995 1000 1005
Lys Phe Thr Gly Ile Lys Val Asp Gly Ser Val Val Asp Ser Ser Asn
1010 1015 1020
Tyr Thr Ala Val Ser Gly Ser Thr Val Leu Thr Leu Lys Ala Ser Tyr
1025 1030 1035 1040
Leu Gly Thr Leu Thr Asp Gly Ser His Thr Ile Thr Phe Val Tyr Thr
1045 1050 1055
Asp Gly Glu Ala Asn Ala Asn Leu Thr Val Arg Thr Ala Gly Ser Gly
1060 1065 1070
His Ile His Asp Tyr Gly Thr Glu Trp Lys Ser Asn Ala Asp Asn His
1075 1080 1085
Trp His Glu Cys Asn Cys Gly Asp Lys Lys Asp Glu Ala Ala His Ser
1090 1095 1100
Phe Lys Trp Val Val Asp Lys Glu Ala Thr Ala Thr Lys Lys Gly Ser
1105 1110 1115 1120
Lys His Glu Glu Cys Lys Ile Cys Gly Tyr Lys Arg Ser Ala Val Glu
1125 1130 1135
Ile Pro Ala Thr Gly Thr Ser Thr Ala Pro Thr Asp Thr Thr Lys Pro
1140 1145 1150
Asn Asp Thr Thr Lys Pro Gly Asn Thr Asn Gly Ser Glu Lys Ser Pro
1155 1160 1165
Gln Thr Gly Asp Asn Ser
1170
<210> 4
<211> 3534
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ggatccagca aaccggacat caaagtgggc gactacgtga aaatgggtgt gtataataac 60
gcaagcatcc tgtggcgctg tgtgagcatc gacaacaatg gcccgctgat gctggccgat 120
aaaattgttg acacgctggc gtatgatgct aaaaccaacg acaattcgaa cagcaaatct 180
catagtcgtt cctacaaacg cgatgactac ggcagcaact attggaaaga tagtaatatg 240
cgctcctggc tgaactcaac cgcggccgag ggtaaagtgg attggctgtg cggcaatccg 300
ccgaaagacg gttacgtcag cggcgtgggt gcatataatg aaaaagctgg ttttctgaac 360
gcgttctcaa aatcggaaat tgcagctatg aaaacggtga cccagcgtag cctggtttct 420
catccggaat ataataaagg cattgttgat ggtgacgcga actcggatct gctgtattac 480
accgacatca gcgaagcagt ggctaactac gatagctctt attttgaaac cacgaccgaa 540
aaagttttcc tgctggatgt caaacaggcg aacgccgtct ggaaaaatct gaaaggctat 600
tacgtggctt acaacaatga tggtatggca tggccgtatt ggctgcgtac cccggtgacg 660
gattgtaatc atgacatgcg ctatattagt tcctcaggcc aggttggtcg ttacgctccg 720
tggtattctg atctgggcgt ccgtccggcg ttttacctgg acagtgaata tttcgtgacg 780
accagcggct ctggtagtca gtcgagcccg tacattggtt ccgcgccgaa caaacaagaa 840
gatgactata ccatctcaga accggcggaa gatgccaacc cggactggaa tgtttcgacg 900
gaacagagca ttcaactgac cctgggcccg tggtactcga atgatggtaa atatagcaac 960
ccgaccattc cggtgtatac catccagaaa acgcgctcgg ataccgaaaa catggtggtt 1020
gtcgtgtgcg gcgaaggtta taccaaatca cagcaaggca aatttatcaa tgatgttaaa 1080
cgtctgtggc aggacgctat gaaatatgaa ccgtaccgta gctatgcgga tcgctttaat 1140
gtgtatgcac tgtgtacggc ttccgaatca accttcgata acggcggttc tacctttttc 1200
gatgtgatcg ttgacaaata caactctccg gttatcagta acaatctgca tggcagtcag 1260
tggaaaaatc acatttttga acgctgcatc ggtccggaat tcattgaaaa aatccatgat 1320
gcccacatta agaaaaaatg tgacccgaac accatcccgt cgggtagcga atacgaaccg 1380
tattactatg tgcatgatta tattgcacag tttgctatgg ttgtcaatac caaatccgac 1440
ttcggcggtg catataacaa tcgcgaatac ggctttcact atttcatctc tccgagtgat 1500
tcctaccgtg cctctaaaac ctttgcacat gaattcggcc acggtctgct gggcctgggt 1560
gatgaatact cgaatggtta tctgctggat gacaaagaac tgaaaagcct gaacctgtct 1620
agtgtggaag atccggaaaa aattaaatgg cgtcagctgc tgggctttcg caatacgtac 1680
acctgccgta acgcgtatgg ttctaaaatg ctggtttcct catacgaatg tatcatgcgc 1740
gataccaact atcaattttg cgaagtctgt cgcctgcagg gcttcaaacg tatgagccaa 1800
ctggttaaag atgtcgacct gtatgtggcc acgccggaag ttaaagaata caccggtgca 1860
tatagtaaac cgtccgattt tacggacctg gaaacctcga gctactacaa ctacacctac 1920
aaccgtaacg atcgcctgct gagtggcaac tcaaaatcgc gtttcaatac gaacatgaat 1980
ggcaagaaaa ttgaactgcg caccgttatt cagaacatca gcgataaaaa cgcccgtcaa 2040
ctgaaattca aaatgtggat caaacattca gatggctcgg tggcaaccga ctctagtggt 2100
aacccgctgc agaccgtcca aacgtttgat attccggtgt ggaacgacaa agccaatttc 2160
tggccgctgg gcgcactgga tcacatcaaa tccgacttta attcaggtct gaaaagctgc 2220
tctctgattt atcagatccc gtctgatgct caactgaaaa gtggcgacac cgtggcgttc 2280
caggttctgg atgaaaacgg taatgtgctg gcggatgaca acacggaaac ccagcgctac 2340
acgaccgttt ctatccaata caaattcgaa gatggcagtg aaatcccgaa tacggcgggc 2400
ggtaccttca ccgttccgta tggtaccaaa ctggatctga cgccggccaa aaccctgtac 2460
gattacgaat tcatcaaagt tgacggcctg aataaaccga tcgtcagcga tggtaccgtg 2520
gttacgtact actacaaaaa caaaaacgaa gaacatacgc acaacctgac cctggtggcg 2580
gccaaagcag ctacctgtac gaccgcgggc aatagcgcct attacacctg cgatggttgt 2640
gacaaatggt ttgcagatgc taccggctcc gtggaaatta ccgacaaaac gtcagttaaa 2700
atcccggctc cgggtcatac cgccggtacg gaatggaaaa gcgatgacac gaaccattgg 2760
cacgaatgca ccgtcgcagg ctgtggtgtg attatcgaaa gcacgaaatc tgcgcacacc 2820
gccggcgaat ggattgtgga taccccggca acggcaacga ccgccggtac gaaacataaa 2880
gaatgcaccg tctgtcaccg tgtgctggaa acccagccga tcccgagcac gggtaccgaa 2940
ctgaaaatta tcgccggtga taaccaaatc tacaacaaag caagtggctc cgatgtcacg 3000
atcacctgca acggtgactt tgccaaattc accggcatta aagtggatgg tagcgtcgtg 3060
gactcctcaa attacaccgc cgtttcaggc tcgaccgtcc tgacgctgaa agcatcctat 3120
ctgggcacgc tgaccgatgg ttcacatacg attaccttcg tttacaccga cggtgaagca 3180
aacgctaatc tgaccgtccg cacggctggc tctggtcata tccacgatta tggcaccgaa 3240
tggaaaagta acgcggacaa tcattggcac gaatgcaatt gtggtgataa aaaagacgaa 3300
gcggcccatt cctttaaatg ggttgtcgat aaagaagcga cggccaccaa aaaaggctca 3360
aaacacgaag aatgcaaaat ctgtggttac aaacgttcgg ccgtggaaat cccggcaacg 3420
ggtaccagca cggcaccgac cgatacgacc aaaccgaacg acacgacgaa accgggtaat 3480
acgaatggct ccgaaaaatc tccgcaaacg ggcgacaata gttaatgaaa gctt 3534
<210> 5
<211> 1418
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
65 70 75 80
Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
130 135 140
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
145 150 155 160
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Gly Gly Gly Ser
225 230 235 240
Ser Lys Pro Asp Ile Lys Val Gly Asp Tyr Val Lys Met Gly Val Tyr
245 250 255
Asn Asn Ala Ser Ile Leu Trp Arg Cys Val Ser Ile Asp Asn Asn Gly
260 265 270
Pro Leu Met Leu Ala Asp Lys Ile Val Asp Thr Leu Ala Tyr Asp Ala
275 280 285
Lys Thr Asn Asp Asn Ser Asn Ser Lys Ser His Ser Arg Ser Tyr Lys
290 295 300
Arg Asp Asp Tyr Gly Ser Asn Tyr Trp Lys Asp Ser Asn Met Arg Ser
305 310 315 320
Trp Leu Asn Ser Thr Ala Ala Glu Gly Lys Val Asp Trp Leu Cys Gly
325 330 335
Asn Pro Pro Lys Asp Gly Tyr Val Ser Gly Val Gly Ala Tyr Asn Glu
340 345 350
Lys Ala Gly Phe Leu Asn Ala Phe Ser Lys Ser Glu Ile Ala Ala Met
355 360 365
Lys Thr Val Thr Gln Arg Ser Leu Val Ser His Pro Glu Tyr Asn Lys
370 375 380
Gly Ile Val Asp Gly Asp Ala Asn Ser Asp Leu Leu Tyr Tyr Thr Asp
385 390 395 400
Ile Ser Glu Ala Val Ala Asn Tyr Asp Ser Ser Tyr Phe Glu Thr Thr
405 410 415
Thr Glu Lys Val Phe Leu Leu Asp Val Lys Gln Ala Asn Ala Val Trp
420 425 430
Lys Asn Leu Lys Gly Tyr Tyr Val Ala Tyr Asn Asn Asp Gly Met Ala
435 440 445
Trp Pro Tyr Trp Leu Arg Thr Pro Val Thr Asp Cys Asn His Asp Met
450 455 460
Arg Tyr Ile Ser Ser Ser Gly Gln Val Gly Arg Tyr Ala Pro Trp Tyr
465 470 475 480
Ser Asp Leu Gly Val Arg Pro Ala Phe Tyr Leu Asp Ser Glu Tyr Phe
485 490 495
Val Thr Thr Ser Gly Ser Gly Ser Gln Ser Ser Pro Tyr Ile Gly Ser
500 505 510
Ala Pro Asn Lys Gln Glu Asp Asp Tyr Thr Ile Ser Glu Pro Ala Glu
515 520 525
Asp Ala Asn Pro Asp Trp Asn Val Ser Thr Glu Gln Ser Ile Gln Leu
530 535 540
Thr Leu Gly Pro Trp Tyr Ser Asn Asp Gly Lys Tyr Ser Asn Pro Thr
545 550 555 560
Ile Pro Val Tyr Thr Ile Gln Lys Thr Arg Ser Asp Thr Glu Asn Met
565 570 575
Val Val Val Val Cys Gly Glu Gly Tyr Thr Lys Ser Gln Gln Gly Lys
580 585 590
Phe Ile Asn Asp Val Lys Arg Leu Trp Gln Asp Ala Met Lys Tyr Glu
595 600 605
Pro Tyr Arg Ser Tyr Ala Asp Arg Phe Asn Val Tyr Ala Leu Cys Thr
610 615 620
Ala Ser Glu Ser Thr Phe Asp Asn Gly Gly Ser Thr Phe Phe Asp Val
625 630 635 640
Ile Val Asp Lys Tyr Asn Ser Pro Val Ile Ser Asn Asn Leu His Gly
645 650 655
Ser Gln Trp Lys Asn His Ile Phe Glu Arg Cys Ile Gly Pro Glu Phe
660 665 670
Ile Glu Lys Ile His Asp Ala His Ile Lys Lys Lys Cys Asp Pro Asn
675 680 685
Thr Ile Pro Ser Gly Ser Glu Tyr Glu Pro Tyr Tyr Tyr Val His Asp
690 695 700
Tyr Ile Ala Gln Phe Ala Met Val Val Asn Thr Lys Ser Asp Phe Gly
705 710 715 720
Gly Ala Tyr Asn Asn Arg Glu Tyr Gly Phe His Tyr Phe Ile Ser Pro
725 730 735
Ser Asp Ser Tyr Arg Ala Ser Lys Thr Phe Ala His Glu Phe Gly His
740 745 750
Gly Leu Leu Gly Leu Gly Asp Glu Tyr Ser Asn Gly Tyr Leu Leu Asp
755 760 765
Asp Lys Glu Leu Lys Ser Leu Asn Leu Ser Ser Val Glu Asp Pro Glu
770 775 780
Lys Ile Lys Trp Arg Gln Leu Leu Gly Phe Arg Asn Thr Tyr Thr Cys
785 790 795 800
Arg Asn Ala Tyr Gly Ser Lys Met Leu Val Ser Ser Tyr Glu Cys Ile
805 810 815
Met Arg Asp Thr Asn Tyr Gln Phe Cys Glu Val Cys Arg Leu Gln Gly
820 825 830
Phe Lys Arg Met Ser Gln Leu Val Lys Asp Val Asp Leu Tyr Val Ala
835 840 845
Thr Pro Glu Val Lys Glu Tyr Thr Gly Ala Tyr Ser Lys Pro Ser Asp
850 855 860
Phe Thr Asp Leu Glu Thr Ser Ser Tyr Tyr Asn Tyr Thr Tyr Asn Arg
865 870 875 880
Asn Asp Arg Leu Leu Ser Gly Asn Ser Lys Ser Arg Phe Asn Thr Asn
885 890 895
Met Asn Gly Lys Lys Ile Glu Leu Arg Thr Val Ile Gln Asn Ile Ser
900 905 910
Asp Lys Asn Ala Arg Gln Leu Lys Phe Lys Met Trp Ile Lys His Ser
915 920 925
Asp Gly Ser Val Ala Thr Asp Ser Ser Gly Asn Pro Leu Gln Thr Val
930 935 940
Gln Thr Phe Asp Ile Pro Val Trp Asn Asp Lys Ala Asn Phe Trp Pro
945 950 955 960
Leu Gly Ala Leu Asp His Ile Lys Ser Asp Phe Asn Ser Gly Leu Lys
965 970 975
Ser Cys Ser Leu Ile Tyr Gln Ile Pro Ser Asp Ala Gln Leu Lys Ser
980 985 990
Gly Asp Thr Val Ala Phe Gln Val Leu Asp Glu Asn Gly Asn Val Leu
995 1000 1005
Ala Asp Asp Asn Thr Glu Thr Gln Arg Tyr Thr Thr Xaa Xaa Xaa Xaa
1010 1015 1020
Xaa Xaa Val Ser Ile Gln Tyr Lys Phe Glu Asp Gly Ser Glu Ile Pro
1025 1030 1035 1040
Asn Thr Ala Gly Gly Thr Phe Thr Val Pro Tyr Gly Thr Lys Leu Asp
1045 1050 1055
Leu Thr Pro Ala Lys Thr Leu Tyr Asp Tyr Glu Phe Ile Lys Val Asp
1060 1065 1070
Gly Leu Asn Lys Pro Ile Val Ser Asp Gly Thr Val Val Thr Tyr Tyr
1075 1080 1085
Tyr Lys Asn Lys Asn Glu Glu His Thr His Asn Leu Thr Leu Val Ala
1090 1095 1100
Ala Lys Ala Ala Thr Cys Thr Thr Ala Gly Asn Ser Ala Tyr Tyr Thr
1105 1110 1115 1120
Cys Asp Gly Cys Asp Lys Trp Phe Ala Asp Ala Thr Gly Ser Val Glu
1125 1130 1135
Ile Thr Asp Lys Thr Ser Val Lys Ile Pro Ala Pro Gly His Thr Ala
1140 1145 1150
Gly Thr Glu Trp Lys Ser Asp Asp Thr Asn His Trp His Glu Cys Thr
1155 1160 1165
Val Ala Gly Cys Gly Val Ile Ile Glu Ser Thr Lys Ser Ala His Thr
1170 1175 1180
Ala Gly Glu Trp Ile Val Asp Thr Pro Ala Thr Ala Thr Thr Ala Gly
1185 1190 1195 1200
Thr Lys His Lys Glu Cys Thr Val Cys His Arg Val Leu Glu Thr Gln
1205 1210 1215
Pro Ile Pro Ser Thr Gly Thr Glu Leu Lys Ile Ile Ala Gly Asp Asn
1220 1225 1230
Gln Ile Tyr Asn Lys Ala Ser Gly Ser Asp Val Thr Ile Thr Cys Asn
1235 1240 1245
Gly Asp Phe Ala Lys Phe Thr Gly Ile Lys Val Asp Gly Ser Val Val
1250 1255 1260
Asp Ser Ser Asn Tyr Thr Ala Val Ser Gly Ser Thr Val Leu Thr Leu
1265 1270 1275 1280
Lys Ala Ser Tyr Leu Gly Thr Leu Thr Asp Gly Ser His Thr Ile Thr
1285 1290 1295
Phe Val Tyr Thr Asp Gly Glu Ala Asn Ala Asn Leu Thr Val Arg Thr
1300 1305 1310
Ala Gly Ser Gly His Ile His Asp Tyr Gly Thr Glu Trp Lys Ser Asn
1315 1320 1325
Ala Asp Asn His Trp His Glu Cys Asn Cys Gly Asp Lys Lys Asp Glu
1330 1335 1340
Ala Ala His Ser Phe Lys Trp Val Val Asp Lys Glu Ala Thr Ala Thr
1345 1350 1355 1360
Lys Lys Gly Ser Lys His Glu Glu Cys Lys Ile Cys Gly Tyr Lys Arg
1365 1370 1375
Ser Ala Val Glu Ile Pro Ala Thr Gly Thr Ser Thr Ala Pro Thr Asp
1380 1385 1390
Thr Thr Lys Pro Asn Asp Thr Thr Lys Pro Gly Asn Thr Asn Gly Ser
1395 1400 1405
Glu Lys Ser Pro Gln Thr Gly Asp Asn Ser
1410 1415

Claims (18)

1. Use of a recombinant fusion protein based on an AK183 protease for the preparation of a medicament for the treatment of a disease related to IgA complex deposition mediated, characterized in that: the recombinant fusion protein structure comprises a human symbiotic protease AK183 active region and a human immunoglobulin fragment; the human immunoglobulin fragment is positioned at the N-terminal of the active region of the protease AK183, and the amino acid sequence of the human immunoglobulin fragment is at least 90% identical to the sequence shown in SEQ ID No.1, preferably at least 95% identical to the sequence, and most preferably 100% identical to the sequence; the active region of the protease AK183 is derived from an amino acid sequence which is at least 90% identical, preferably at least 95% identical, and most preferably 100% identical to the sequence shown in SEQ ID No. 3; most preferably, the amino acid sequence of the recombinant fusion protein is shown as SEQ ID No. 5.
2. The use of claim 1, wherein: the IgA complex deposition mediated related diseases comprise IgA nephropathy, igA vasculitis kidney damage (or Henoch-Schonlein purpura nephritis), other secondary IgA nephropathy, kawasaki disease, igA rheumatoid factor positive rheumatoid arthritis, igA type anti-GBM disease or IgA type ANCA related vasculitis.
3. The use of claim 1, wherein: said recombinant fusion protein based on AK183 protease is prepared into a liquid formulation for administration to a mammal suffering from said diseases associated with IgA complex deposition-mediated; further preferably, it is prepared into an injection and administered by intravenous injection or infusion route.
4. The use of claim 3, wherein: the mammal is a human and the intravenous administration regimen comprises: injecting the injection once every 10-15 days at the dose of 5-10 mg/kg; preferably, a dose of 10-15mg/kg is used as the initial injection dose, followed by a maintenance dose of 5-10mg/kg once every 10 days.
5. The use of claim 3, wherein: the mammal is a passive human IgA injection mouse model, a human IgA1 transgenic mouse model or a primate model, and the intravenous injection administration scheme comprises: injecting the injection once every 5-10 days at the dosage of 5-10 mg/kg; preferably, the injection is administered at a dose of 5mg/kg once every 5 days.
6. A recombinant fusion protein, the structure of which comprises a human symbiotic protease AK183 active region and a human immunoglobulin fragment; the human immunoglobulin fragment is positioned at the N end of the active region of the protease AK183, and the amino acid sequence of the human immunoglobulin fragment is at least 90 percent identical with the sequence shown in SEQ ID No.1, preferably at least 95 percent identical, and most preferably 100 percent identical; the active region of the protease AK183 is derived from an amino acid sequence which is at least 90% identical, preferably at least 95% identical, and most preferably 100% identical to the sequence shown in SEQ ID No. 3.
7. The recombinant fusion protein of claim 6, wherein: the amino acid sequence is shown in SEQ ID No. 5.
8. A recombinant fusion protein gene expression vector is a plasmid formed by recombinant fusion of a carrier and a target gene, wherein the target gene is a recombinant fusion gene and comprises a gene sequence for coding a human immunoglobulin fragment and a gene sequence for coding a protease AK183 active region; the nucleotide sequence of the gene sequence for coding the human immunoglobulin fragment is at least 90 percent identical with the sequence shown in SEQ ID No.2, preferably at least 95 percent identical with the sequence, and most preferably 100 percent identical with the sequence; the gene sequence of the coding protease AK183 activity region is at least 90% identical, more preferably at least 95% identical, and most preferably 100% identical with the sequence shown in SEQ ID No. 4.
9. The recombinant fusion protein gene expression vector of claim 8, wherein: the vector is a vector suitable for a prokaryotic expression system, further preferably a vector compatible with an E.coli expression system, more preferably a vector with a purification tag, and most preferably a pET30a plasmid.
10. The recombinant fusion protein gene expression vector of claim 8, wherein: the expression vector is a plasmid formed by recombination and fusion of a carrier and a target gene, wherein the carrier is an escherichia coli expression vector pET30a plasmid; the target gene is a recombinant fusion gene, and sequentially comprises a gene sequence for coding a human immunoglobulin fragment from the 5' end, wherein the nucleotide sequence is shown as SEQ ID No.2, and a gene sequence for coding an active region containing protease AK183, and the nucleotide sequence is shown as SEQ ID No. 4.
11. A method for constructing a recombinant fusion protein gene expression vector, comprising: selecting an expression system, respectively synthesizing two cDNA sequences suitable for the expression of the expression system according to an amino acid sequence containing a protease AK183 active region and an amino acid sequence of a human immunoglobulin fragment, respectively carrying out DNA recombination on the two cDNA sequences serving as target genes and the expression system to obtain two recombinant plasmids, and finally recombining and fusing the target genes in the two recombinant plasmids in the same recombinant plasmid, wherein in the recombination and fusion, the cDNA sequence of the human immunoglobulin fragment is inserted into the 5' end of the cDNA sequence of the protease AK183 active region to obtain a recombinant fusion protein gene expression vector; the amino acid sequence of the human immunoglobulin fragment is at least 90% identical, preferably at least 95% identical, and most preferably 100% identical to the sequence shown in SEQ ID No. 1.
12. The method for constructing the recombinant fusion protein gene expression vector comprises the following steps:
(I) Based on an amino acid sequence which has at least 90 percent of identity with the sequence shown in SEQ ID No.3 and contains a protease AK183 active region, synthesizing a cDNA sequence I 'suitable for expressing escherichia coli through codon optimization, and based on an amino acid sequence of a human immunoglobulin fragment which has at least 90 percent of identity with the sequence shown in SEQ ID No.1, synthesizing a cDNA sequence II' suitable for expressing the escherichia coli through codon optimization;
(II) taking the cDNA sequence I 'obtained in the step (I) as a target gene, and carrying out DNA recombination on the target gene and escherichia coli to obtain a subcloned plasmid I'; taking the cDNA sequence II 'obtained in the step (I) as a target gene, and carrying out DNA recombination on the target gene and escherichia coli to obtain a subcloned plasmid II';
(III) cutting the subcloned plasmid II 'obtained in the step (II) to obtain a cDNA sequence II' fragment with a cohesive end; cutting the subcloned plasmid I 'obtained in (II) to form a nick with a sticky end upstream of the cDNA sequence I';
(IV) carrying out DNA recombination on the cDNA sequence II ' fragment obtained in the step (III) and the cut subcloned plasmid I ', wherein in the DNA recombination, the cDNA sequence II ' is inserted into the 5' end of the cDNA sequence I ' to obtain the recombinant fusion protein gene expression vector.
13. The method of claim 12, wherein: the cDNA sequence II' in step (I) is a fragment in which a base is added before a codon based on the sequence shown in SEQ ID No.1 to prevent frame shift and a GGGGGS linker peptide is added after the fragment.
14. The method of any one of claims 12 or 13, wherein: in the step (IV), in the DNA recombination of the cDNA sequence II 'fragment obtained in the step (III) and the cut subcloning plasmid I', the N end of the cDNA sequence II 'fragment is fused with the His tag attached to the vector of the subcloning plasmid I'.
15. The AK183 protease based recombinant fusion protein expression and purification method includes: transfecting the recombinant fusion protein gene expression vector of claim 8 into an escherichia coli competent cell, and carrying out protein expression under the induction of an inducer to obtain the recombinant fusion protein; the inducer is preferably isopropyl-beta-D-thiogalactoside (IPGT) with the concentration of 0.1-0.5 mM; the protein expression temperature is preferably 15-18 ℃; the protein expression time is preferably 20-30 hours; and (3) after the expression is finished, treating the escherichia coli cell bodies by a conventional method, performing ultrasonic fragmentation, performing high-speed centrifugation, retaining a supernatant, and purifying by adopting affinity chromatography and a molecular sieve to obtain the recombinant fusion protein.
16. The method of claim 15, wherein: the inducer is IPGT with the concentration of 0.3 mM; the protein expression temperature is 16-18 ℃; the protein expression time is 24 hours.
17. The method of claim 15, wherein: the affinity chromatography and molecular sieve purification are carried out at 4 ℃, the protease activity is protected to the maximum extent by using a buffer solution containing 0.8mM EDTA, and finally the obtained purified protein is frozen in a neutral phosphate buffer solution.
18. A pharmaceutical composition comprising the recombinant fusion protein of claim 6.
CN202110385443.7A 2021-04-09 2021-04-10 Recombinant fusion protease capable of clearing in-vivo immunoglobulin A, preparation method thereof and application thereof in treating IgA nephropathy Pending CN115197328A (en)

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