Mutant of immunoglobulin degrading enzyme IdeE

Abstract

The invention provides a coding sequence of an immunoglobulin degrading enzyme and a polypeptide coded by the immunoglobulin degrading enzyme. The function of the polypeptide at least comprises the function of the immunoglobulin degrading enzyme IdeE.

Description

A mutant of immunoglobulin-degrading enzyme IdeE

[0001] Priority Declaration

[0002] This disclosure claims priority to Chinese patent application No. 202311508868.8, filed on November 13, 2023. This disclosure incorporates the entirety of the aforementioned Chinese patent application. Technical Field

[0003] The present disclosure relates to the field of biotechnology, and in particular to a mutant of an immunoglobulin-degrading enzyme and its expression. Background Art

[0004] Immunoglobulin G (IgG) is the primary antibody component of serum, accounting for approximately 75% of serum immunoglobulins. It plays a primary protective role in immunity and effectively prevents infectious diseases. Beyond its protective role, IgG is also associated with disease. In some autoimmune diseases, IgG antibodies react with the body's own molecules. In organ transplants, IgG can cause acute transplant rejection.

[0005] IdeS (Immunoglobulin G-degrading enzyme of S. pyogenes) is an extracellular cysteine ​​protease produced by the human pathogen Streptococcus pyogenes. IdeS catalyzes a single proteolytic cleavage in the lower hinge region of all subclasses of human IgG heavy chains. IdeS effectively cleaves IgG into Fc and F(ab′)2 fragments through a two-stage mechanism. In the first stage, one (first) IgG heavy chain is cleaved to produce a single-cleaved IgG (scIgG) molecule with a non-covalently bound Fc molecule. The scIgG molecule is actually an intermediate product that retains the remaining (second) heavy chain of the original IgG molecule. In the second stage of the mechanism, the second heavy chain is cleaved by IdeS to release the F(ab′)2 fragment and the homodimeric Fc fragment, which helps GAS evade antibody-mediated phagocytosis and cellular action, thereby weakening the host immune system's killing of GAS. IdeE is derived from Streptococcus equi ssp.equi, a pathogenic bacterium of horses (Jonas Bengt Guss. FEMS Microbiol Lett, 2006, 262: 230-235). IdeE and IdeS cleave IgG at exactly the same position, with high reproducibility and specificity, and have very similar substrate ranges.

[0006] In view of the applications of the above-mentioned immunoglobulin cleavage enzymes, there is a great demand for immunoglobulin degrading enzymes and their large-scale production.

[0007] Summary of the Invention

[0008] Some aspects of the present application provide an immunoglobulin degrading enzyme, characterized in that the immunoglobulin degrading enzyme reduces or eliminates bacterial / cell autolysis when induced to express. In some embodiments, the immunoglobulin degrading enzyme of the present disclosure does not cause bacterial / cell autolysis.

[0009] In some embodiments, the OD of the cells / bacteria during the expression of the immunoglobulin degrading enzyme of the present disclosure is 600 No decrease will occur after it is higher than 40, 50, 60, 70 or 80; preferably, during the expression of the immunoglobulin, the OD of the bacteria / cells 600 More preferably, the OD of the bacteria / cells during the expression of the immunoglobulin is 600 There will be no decline for those above 80.

[0010] In some embodiments, during the expression of the immunoglobulin degrading enzyme of the present disclosure, for example, within 4 hours, 8 hours, 12 hours, 16 hours, 24 hours or longer after induced expression, the bacterial OD 600 No decline will occur above 40, 50, 60, 70 or 80.

[0011] Some aspects of the present application provide an immunoglobulin degrading enzyme, characterized in that the immunoglobulin degrading enzyme comprises or consists of the following amino acid sequence:

[0012] (a) the amino acid sequence shown in SEQ ID NO: 2; or

[0013] (b) an amino acid sequence having one or more amino acid substitutions, additions and / or deletions compared to SEQ ID NO: 2.

[0014] In some embodiments, the immunoglobulin degrading enzyme of the present disclosure comprises or consists of the amino acid sequence shown in SEQ ID NO: 2.

[0015] In some embodiments, the immunoglobulin degrading enzyme comprises or consists of the amino acid sequence shown in SEQ ID NO: 2, wherein the coding sequence reduces or eliminates bacterial / cell autolysis upon expression. In some embodiments, the immunoglobulin degrading enzyme does not cause bacterial / cell autolysis upon induced expression.

[0016] In some embodiments, the immunoglobulin degrading enzymes of the present disclosure are derived from Streptococcus equi ssp. equi.

[0017] In some embodiments, the immunoglobulin degrading enzyme disclosed herein is characterized in that the immunoglobulin is expressed intracellularly or extracellularly.

[0018] Some aspects of the present application provide a nucleotide sequence encoding an immunoglobulin degrading enzyme of the present disclosure. In some embodiments, the nucleotide sequence encoding the immunoglobulin degrading enzyme of the present disclosure comprises or consists of the following nucleotide sequence:

[0019] (a) the nucleotide sequence shown in SEQ ID NO: 3; or

[0020] (b) a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% but less than 100% sequence identity to the nucleotide sequence of SEQ ID NO: 3;

[0021] (c) a nucleotide sequence having one or more nucleotide substitutions, additions and / or deletions compared to SEQ ID NO: 3.

[0022] Some aspects of the present application provide an expression vector comprising a nucleotide sequence of the present disclosure.

[0023] Some aspects of the present application provide a host cell comprising a nucleotide sequence of the present disclosure or an expression vector of the present disclosure. In some embodiments, the host cell of the present disclosure is a bacterial cell or a fungal cell. In some embodiments, the host cell of the present disclosure is an Escherichia coli cell or a yeast cell.

[0024] Some aspects of the present application provide a method for intracellular expression of the immunoglobulin degrading enzyme of the present disclosure, comprising the following steps:

[0025] (a) selecting a single clone of the host strain of the present disclosure;

[0026] (b) Seed liquid culture;

[0027] (c) fermentation tank culture;

[0028] (d) inducing expression; and

[0029] (e) optionally collecting the expressed immunoglobulin degrading enzyme.

[0030] Some aspects of the present application provide a composition comprising: an immunoglobulin degrading enzyme of the present disclosure; and optionally a pharmaceutically acceptable carrier or excipient.

[0031] In some embodiments, the composition of the present disclosure further comprises: an antibody or a protein containing an Fc fragment, preferably, wherein the target of the antibody is selected from the group consisting of: cell surface proteins, cytokines, hormones, enzymes, intracellular messengers, intercellular messengers, and immune checkpoints. In some embodiments, the composition of the present disclosure further comprises: a viral vector drug, a drug that can reduce blood IgG levels, preferably, the viral vector drug is selected from the group consisting of: oncolytic viruses, gene therapy viruses, and viral vector vaccines. Preferably, the drug that reduces blood IgG levels is selected from the group consisting of: FcRn antibodies, and Fc fragment variants with high affinity to FcRn.

[0032] Some aspects of the present application provide a kit comprising:

[0033] (1) the immunoglobulin degrading enzyme of the present disclosure; and

[0034] (2) one or more selected from the group consisting of: (a) a pharmaceutically acceptable carrier or excipient; (b) an antibody or an Fc-containing protein; and / or

[0035] (3) viral vector drugs, wherein the viral vector drugs are selected from oncolytic viruses, gene therapy viruses and viral vector vaccines; and / or

[0036] (4) A drug capable of reducing blood IgG levels, wherein the drug capable of reducing blood IgG levels is selected from FcRn antibodies and Fc fragment variants with high affinity to FcRn.

[0037] In some embodiments, the kit of the present disclosure comprises: Kit A and Kit B, wherein Kit A contains the immunoglobulin degrading enzyme described in the present disclosure, and Kit B contains one or more selected from the following groups: (1) a pharmaceutically acceptable carrier or excipient; (2) an antibody or a protein containing Fc; and / or (3) a viral vector drug; and / or (4) a drug that can reduce blood IgG levels. In some embodiments, the viral vector drug is selected from the group consisting of oncolytic viruses, gene therapy viruses, and viral vector vaccines. In some embodiments, the drug that reduces blood IgG levels is selected from the group consisting of FcRn antibodies and Fc fragment variants with high affinity to FcRn. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG1 is the electrophoresis result of the expression product of the shake flask fermentation supernatant of the IdeE-1 expression strain;

[0039] FIG2 is the electrophoresis result of the expression product of IdeE-2 expression fermentation bacteria lysis broth;

[0040] Figure 3a shows the OD values ​​of IdeE-1 expression strains at different induction times in a 5L fermenter. 600Figure 3b shows the OD of IdeE-2 expression strain in 5L fermenter at different induction times 600 Figure 3c shows the OD values ​​of IdeE-1 expression strains at different induction times in a 5L fermenter. 600 and changes in fermentation supernatant turbidity;

[0041] FIG4 shows the electrophoresis results of expression products of IdeE-1 and IdeE-2 expression strains in 5 L fermenters at different induction times;

[0042] Figure 5 shows the changes in the level of IgG degradation in vivo at a dose of 0.25 mg / kg KJ103.

[0043] Detailed Description of the Invention

[0044] I. Functional polypeptides having immunoglobulin degrading enzyme activity

[0045] In a first aspect of the present disclosure, a functional polypeptide is provided, wherein the functional polypeptide has the activity of an immunoglobulin degrading enzyme and comprises an amino acid sequence shown in SEQ ID NO: 2.

[0046] The polypeptides disclosed herein are preferably produced by genetic engineering recombination.

[0047] Preferably, the polypeptide has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the sequence shown in SEQ ID NO: 2. Preferably, the polypeptide has one or more amino acid substitutions, additions and / or deletions compared to SEQ ID NO: 2.

[0048] Preferably, the coding sequence of the polypeptide is as shown in the nucleotide sequence of SEQ ID NO: 3, or a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence similarity to the nucleotide sequence of SEQ ID NO: 3, wherein the coding sequence does not cause bacteria / cell autolysis when expressed. Preferably, the coding sequence of the polypeptide has one or more nucleotide substitutions, additions and / or deletions compared to SEQ ID NO: 3.

[0049] II. Preparation of functional polypeptides with immunoglobulin degrading enzyme activity

[0050] In a second aspect of the present disclosure, a nucleotide encoding the polypeptide or mutant as described above is provided.

[0051] Furthermore, the present disclosure also provides a vector comprising a nucleic acid molecule encoding the polypeptide or mutant. The vector may further comprise an expression control sequence operably linked to the sequence of the nucleic acid molecule to facilitate expression of the protein or mutant.

[0052] A variety of suitable nucleic acid molecules encoding the polypeptides or mutants described above are suitable for use in the present disclosure. The sequences mentioned in the examples below are all suitable for use in the methods of the present disclosure. It should be understood that once the amino acid sequence of a protein or polypeptide is provided, those skilled in the art can readily determine the nucleic acid molecule encoding it.

[0053] A variety of suitable vectors can be used, such as those used for cloning and expression in mammals, bacteria, fungi, and yeast, such as pET, Pouwels et al., Cloning Vectors: A Laboratory Manual (Elsevier latest edition). In a preferred embodiment of the present disclosure, the vector is a vector suitable for use in Escherichia coli cells.

[0054] In some embodiments, the vector may be some viral vectors, such as but not limited to retroviral vectors, phage vectors, adenoviral vectors, herpes simplex virus (HSV) vectors, AAV vectors, or lentiviral vectors.

[0055] Expression vector comprises the albumen that is connected with suitable transcription and translation regulatory sequence or the DNA sequence of mutant, such as mammals, microorganisms, viruses or insect genes.Regulating sequence comprises transcription promoter, operator, enhancer, ribosome bind site or the appropriate sequence that controls transcription and translation initiation and termination.When polypeptide as above or mutant need regulating sequence function, then connect suitable regulating sequence.Like this, promoter sequence is connected to the DNA sequence front end of coded protein or mutant.The ability of replication in host cell is controlled by replication origin usually.The screening gene that is used for transformant identification also can add expression vector.

[0056] Additionally, a leader sequence can be fused to the polypeptide or mutant coding sequence to allow the translated protein or mutant to be secreted extracellularly. A signal peptide can enhance extracellular secretion of the chimeric polypeptide by the host cell. The signal peptide can be cleaved during the process of polypeptide secretion from the cell.

[0057] The present disclosure also provides an expression system (e.g., a host cell) for expressing the polypeptide or mutant as described above, the expression system comprising the expression vector described herein or the polynucleotide described herein integrated into the genome with an exogenous source. Any cell suitable for expression by an expression vector can be used as a host cell, for example, the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell, including but not limited to Escherichia coli; Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, filamentous fungi, plant cells; insect cells of Drosophila S2 or Sf9; CHO, COS, HEK293 cells, or animal cells of Bowes melanoma cells, etc. The method for constructing the expression system should be known to those skilled in the art, for example, it can be a combination of one or more including but not limited to chemical transformation, microinjection, gene gun method, electroporation method, virus-mediated transformation method, electron bombardment method, calcium phosphate precipitation method, etc. As a preferred embodiment of the present disclosure, the expression system is a prokaryotic expression system, such as an Escherichia coli expression system, a Bacillus subtilis expression system, etc. In a more specific embodiment, the Escherichia coli cell is BL21, BL21 (DE3), BL21 (DE3) pLysS, BL21 (DE3) pLysE, BL21 Star (DE3), BL21 Star (DE3) pLysS, Lemo21 (DE3), T7 Express lysY, T7 Express lysY / Iq, SHuffle, Origami, Rosetta, HMS174, etc.

[0058] Methods for producing the polypeptides or mutants described above are also encompassed by the present disclosure. The methods comprise culturing recombinant cells containing nucleic acid encoding the polypeptides or mutant proteins described above. The methods may comprise expressing the encoded polypeptides or mutants described above in the cells, and renaturing the expressed polypeptides or mutant proteins described above. The products of the methods are also protected.

[0059] The steps of intracellular or extracellular expression are: selecting single clones of expression strains, culturing seed liquid, expanding culture in fermentation tanks, inducing expression, and collecting samples after fermentation.

[0060] The further step of the intracellular expression is as follows: inoculating the production strain on an agarose plate, picking a single colony, inoculating it into a test tube / shake flask containing a culture medium, continuing to culture with or without transferring it to a larger volume shake flask / fermenter, inoculating the cultured bacterial liquid into a fermenter of a certain volume for culture, adding an inducer or other induction method for induction, continuing to culture until the end of fermentation, and collecting samples.

[0061] Furthermore, the intracellular expression step described in the embodiment of the present disclosure is as follows: the production strain is inoculated on an LB agarose plate containing 100 μg / ml ampicillin. Culture at 37°C overnight until colonies grow. Pick a single colony, inoculate it in 3 ml of LB medium containing 100 μg / ml ampicillin, and culture it at 37°C and 250 rpm overnight. Take 500 μl of the overnight cultured bacterial liquid and inoculate it in 50 ml of LB medium containing 100 μg / ml ampicillin. After culture at 37°C for 2 to 4 hours, add 0.1 mM IPTG for induction, and continue the induction culture overnight.

[0062] The production strain was inoculated on an LB agarose plate containing 100 μg / ml ampicillin. Cultured at 37°C overnight until colonies grew. Pick a single colony and inoculate it in 3 ml of LB medium containing 100 μg / ml ampicillin, and cultured overnight at 37°C and 250 rpm. Take 500 μl of the overnight cultured bacterial liquid and inoculate it in 50 ml of LB medium containing 100 μg / ml ampicillin. After incubation overnight, take 2.5 ml and inoculate it in 250 ml of medium. After incubation at 37°C overnight, transfer it to a 5 L tank for fermentation. Fermentation was carried out by fed-batch and deep aeration culture. Glycerol or glucose was selected as the limiting carbon source for controlled flow addition before induction, and IPTG / lactose was selected as the inducer and the limiting carbon source for controlled flow addition after induction. The fermentation temperature is 37°C, and the pH is controlled at 6.0-7.2 by adding 25% ammonia water; the fermentation tank ventilation volume is selected to be 0.5-2.0 vvm, and the dissolved oxygen value is kept at around 30% by controlling the feeding rate, stirring rate and ventilation volume. During the fermentation culture, samples are taken regularly to measure the OD 600 , when OD 600 After reaching a certain value, IPTG / lactose was added for induction, and the fermentation was terminated after 8 to 16 hours of induction culture.

[0063] III. Drug Combinations

[0064] The third aspect of the present disclosure provides a composition comprising the polypeptide or mutant as described above, or a protein comprising the polypeptide or mutant thereof as described above, and optionally a pharmaceutically acceptable carrier or excipient.

[0065] 3.1 Antibody Targets

[0066] Preferably, in the composition as described above, the target of the antibody may be cell surface proteins, cytokines, hormones, enzymes, intracellular and intercellular messengers, immune checkpoints, etc.

[0067] 3.2 Targeted drugs

[0068] Preferably, the composition as described above further comprises a targeted drug, a chemotherapy drug, or an immune checkpoint blocker.

[0069] 3.3 Drugs that can lower blood IgG levels

[0070] Preferably, in the composition as described above, the polypeptide drug capable of reducing blood IgG levels can block the binding between blood IgG and FcRn protein.

[0071] 3.4 Viral vector drugs

[0072] Preferably, in the composition as described above, in the viral vector drug, the virus used in the viral vector drug is selected from ssDNA viruses, dsDNA viruses, ssRNA viruses or dsRNA viruses; and / or, the virus used in the viral vector drug is selected from wild-type virus strains or naturally attenuated strains, genetically engineered selectively attenuated strains, gene-loaded virus strains, and gene transcription-targeted virus strains.

[0073] 3.5 Gene therapy drugs

[0074] Preferably, in the composition as described above, the gene therapy virus expresses a foreign gene, and the foreign gene encodes a protein required for a gene defect disease.

[0075] IV. Products

[0076] The present disclosure also provides a product, which contains the mutant or protein as described above and a therapeutic agent; the therapeutic agent is selected from viral vector drugs, antibodies, and polypeptide drugs that can reduce blood IgG levels.

[0077] The present disclosure also provides a kit or a set of kits, comprising: 1) a therapeutically effective amount of a drug comprising the mutant described above; and 2) a therapeutically effective amount of a therapeutic agent; the therapeutic agent is selected from a viral vector drug, an antibody, or a polypeptide drug capable of reducing blood IgG levels; the viral vector drug is preferably an oncolytic virus or a gene therapy virus. The kit may also include 3) a targeted drug, a chemotherapy drug, or an immune checkpoint blocker.

[0078] The test kit or kit comprises kit A and kit B, wherein kit A comprises a therapeutically effective amount of the mutant or protein as described above, and kit B comprises a therapeutically effective amount of a therapeutic agent; the therapeutic agent is selected from a viral vector drug, an antibody, and a polypeptide drug capable of reducing blood IgG levels.

[0079] The kit may include instructions for administering a therapeutically effective amount of a mutant or protein as described above and a therapeutically effective amount of a therapeutic agent (e.g., dosage information, dosing interval information), wherein the therapeutic agent is selected from a viral vector drug, an antibody, or a polypeptide drug capable of reducing blood IgG levels.

[0080] The pharmaceutical carrier can be a liquid, and the pharmaceutical composition can be in the form of a solution. Liquid carriers are used to prepare solutions, suspensions, emulsions, syrups, elixirs, and pressurized compositions. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid carrier, such as water, an organic solvent, a mixture thereof, or a pharmaceutically acceptable oil or fat.

[0081] Pharmaceutical compositions for parenteral administration are sterile, substantially isotonic, pyrogen-free, and prepared in accordance with GMPs of the FDA or similar agencies. Viral vector drugs can be administered as injectable dosage forms of solutions or suspensions of the substance in a physiologically acceptable diluent and pharmaceutical carrier (which can be a sterile liquid, such as water, oil, saline, glycerol, or ethanol). In addition, auxiliary substances such as wetting agents or emulsifiers, surfactants, and pH buffering substances may be present in the composition. Other components of the pharmaceutical composition include components of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, and mineral oil. Generally, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, especially for injectable solutions. Viral vector drugs can be administered as depot injections or implants, which can be formulated to allow for sustained release of the active ingredient. Typically, the composition is prepared as an injectable, i.e., a liquid solution or suspension; it can also be prepared as a solid form suitable for dissolution or suspension in a liquid carrier prior to injection.

[0082] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods, devices, and materials are now described.

[0083] When used in this article, the term "immunoglobulin degrading enzyme" refers to a hydrolytic cleavage from the lower hinge region of the heavy chain of the substrate immunoglobulin, effectively cutting IgG into Fc and F(ab')2 fragments. Any enzyme or functional fragment thereof. The term immunoglobulin degrading enzyme includes variants having one or more amino acid substitutions, deletions or insertions relative to the immunoglobulin degrading enzyme SEQ ID NO:2 sequence and / or fusion proteins or conjugates including sialidase. Immunoglobulin degrading enzymes are also known as IgG degrading enzymes, and unless otherwise indicated, the two terms are used interchangeably herein. When used in this article, the term "having the activity of an immunoglobulin degrading enzyme" retains, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the enzymatic activity of the polypeptide.

[0084] The term "nucleotide" or "polynucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single or double stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioate, phosphoramidate, etc.). Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including but not limited to degenerate codon substitutions) and complementary sequences as well as explicitly specified sequences. In particular, degenerate codon substitutions can be achieved by generating sequences in which position 3 of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Cassol et al., (1992); Rossolini et al., Mol Cell. Probes 8:91-98 (1994)).

[0085] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, a description directed to a polypeptide applies equally to describing a peptide and describing a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally encoded amino acid. As used herein, the terms encompass amino acid chains of any length, including full-length proteins (i.e., antigens), in which the amino acid residues are linked via covalent peptide bonds.

[0086] The term "host cell" means a cell comprising a nucleic acid of the present disclosure, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, mating, or other methods known in the art. The exogenous polynucleotide may be maintained as a non-integrating vector, such as a plasmid, or may be integrated into the host genome. The host cell may be a prokaryotic cell or a eukaryotic cell.

[0087] The term "transformation" refers to a process by which a heterologous DNA sequence is introduced into a host cell or organism.

[0088] The term "expression" means the transcription and / or translation of an endogenous gene or a transgene in a cell.

[0089] The term "scale-up" generally refers to the process of increasing a device from laboratory scale to industrial scale production. As used herein, the term includes scaling up from small shake flask expression to small fermenters or larger fermenters.

[0090] The term "autolysis" refers to the undesirable cell lysis of a strain before the predetermined fermentation endpoint, which often results in an OD 600 Alternatively, the expression level and product separation may be adversely affected by the “no autolysis of bacteria / cells” mentioned herein, which may be manifested as the OD value being 0.001 or 0.001 within 4 hours, 8 hours, 12 hours, 16 hours, 24 hours or longer of the induction expression of bacteria. 600 A decrease of at least 10%, at least 20%, at least 30%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% does not occur within one or more sampling points after 40, 50, 60, 70 or 80°C. Typical OD 600 The reduction is shown in Figure 3a of this document.

[0091] The positive progress of the present disclosure lies in the unexpected discovery that IdeE-1 has low expression levels and an unstable strain, which makes autolysis of the bacteria easy to occur during scale-up culture. Furthermore, the fermentation broth has extremely high viscosity, making effective solid-liquid separation impossible and making downstream purification operations difficult. The present disclosure provides an immunoglobulin degrading enzyme mutant, IdeE-2, and its expression method. Compared with IdeE-1, its expression advantage lies in greatly improving the expression level and providing a stable expression system. During scale-up culture, autolysis of the bacteria and viscosity of the fermentation broth are avoided. After cell lysis, the final liquid volume can be arbitrarily controlled, facilitating subsequent purification operations. DETAILED DESCRIPTION

[0092] The present disclosure is further illustrated by way of examples below, but the present disclosure is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0093] Example 1. Construction of IdeE-1 expression strain

[0094] After codon optimization, a polynucleotide sequence of the IdeE-1 protein sequence (SEQ ID NO: 1) was synthesized, and an N-terminal signal peptide sequence and a C-terminal 6×histidine tag were added. After sequence synthesis, the sequence was inserted into the pET32a expression vector. After correct sequencing, a recombinant plasmid for expressing the mutant IdeE was obtained. The mutant recombinant plasmid was electroporated into Escherichia coli BL21 Star (DE3) and inoculated on LB agarose plates containing 100 μg / ml ampicillin. Culture was carried out at 37°C overnight until colonies grew. A single colony was picked and inoculated into 3 ml of LB medium containing 100 μg / ml ampicillin and incubated at 37°C and 250 rpm overnight. 500 μl of the overnight culture was inoculated into 50 ml of LB medium containing 100 μg / ml ampicillin. After incubation at 37°C for 4 hours, 0.1 mM IPTG was added for induction, and the induction culture was continued overnight. The culture supernatant of the overnight induced culture was collected by centrifugation. The content of mutant protein IdeE-1 in the culture supernatant was detected by SDS-PAGE, as shown in FIG1 .

[0095] Example 2. Construction of IdeE-2 expression strain

[0096] The polynucleotide sequence of the IdeE-2 protein sequence (SEQ ID NO: 2) was synthesized after codon optimization, and the sequence was inserted into the pET32a expression vector after synthesis to obtain a recombinant plasmid for expressing mutant IdeE. The mutant recombinant plasmid was electrotransformed into Escherichia coli BL21 Star (DE3) and inoculated on an LB agarose plate containing 100 μg / ml ampicillin. Cultured overnight at 37°C until colonies grew. Pick a single colony, inoculate it in 3 ml of LB medium containing 100 μg / ml ampicillin, and cultured overnight at 37°C and 250 rpm. Take 500 μl of the overnight cultured bacterial solution and inoculate it in 50 ml of LB medium containing 100 μg / ml ampicillin. After culturing at 37°C for 4 hours, add 0.1 mM IPTG for induction, and continue induction culture overnight. The bacterial cells of the overnight induced culture were collected by centrifugation, and the cells were lysed by ultrasonication. The content of the mutant protein IdeE in the cell precipitate and the supernatant was detected by SDS-PAGE, as shown in FIG2 .

[0097] Example 3. Fermentation culture of IdeE-1 and IdeE-2 expression strains

[0098] The secretion and intracellular expression strains constructed in Examples 1 and 2 were gradually expanded to 5L tank fermentation culture through shake flask culture. Fermentation culture was carried out by feed addition and deep aeration culture. Glycerol or glucose was selected as the limiting carbon source for control addition before induction, and IPTG / lactose was selected as the inducer and the limiting carbon source for control addition after induction. The fermentation temperature was 37°C, and the pH was controlled at 6.0-7.2 by adding 25% ammonia water; the fermentation tank ventilation volume was selected to be 0.5-2.0vvm, and the dissolved oxygen value was achieved at about 30% by controlling the feed rate, stirring rate and ventilation volume. During the fermentation culture, samples were taken regularly to measure the OD 600 , when OD 600 After reaching a certain value, IPTG / lactose was added for induction, and the fermentation was terminated after 8 to 16 hours of induction. 600 The OD value of the bacterial solution gradually decreased, and the turbidity increased significantly. After the fermentation, the bacterial solution was viscous and could not be effectively separated by centrifugation. This phenomenon is a typical autolysis phenomenon of E. coli. Multiple experiments have confirmed that the autolysis phenomenon of the secretory expression strain in the late stage of induction is not accidental (Figure 3a). 600 The protein expression level in the cells after induction also continued to increase with induction time. After placing the culture in the tank, the culture liquid was not sticky and could be harvested by centrifugation. This indicates that the secretory expression of the IdeE-1 mutant protein in E. coli causes autolysis of the cells, making it impossible to purify it from the fermentation supernatant and thus preventing scale-up production.

[0099] As shown in Figure 4, the expression level of IdeE-2 (amino acid sequence SEQ ID NO: 2) was significantly higher than that of IdeE-1 (amino acid sequence SEQ ID NO: 1). In addition, during the production process, the strain encoding the protein IdeE-1 by SEQ ID NO: 1 was more unstable, and the bacteria showed autolysis (Figure 3a). In addition, the viscosity of the fermentation broth expressed by IdeE-1 increased, making it impossible to effectively perform solid-liquid separation. The ultrafiltration liquid exchange volume was larger and more difficult, making purification more difficult.

[0100] Example 4. In vivo efficacy

[0101] The safety and tolerability, pharmacokinetic profile, pharmacodynamic profile, and immunogenicity of IdeE-2 (a novel, low-immunogenic immunoglobulin G degrading enzyme) were evaluated in healthy volunteers from China and New Zealand. A total of 68 healthy volunteers were enrolled in the study, all of whom completed the study. The results of Phase I clinical trials conducted in China and New Zealand were highly consistent, demonstrating that IdeE-2 was safe and well-tolerated in healthy volunteers. The PK profiles of IdeE-2 were reproducible across all dose groups, demonstrating rapid distribution and slow elimination in healthy volunteers. The PK profiles conformed to a two-compartment model, with an optimal dose response achieved at a dose of 0.25 mg / kg. IdeE-2 efficiently, rapidly, and specifically cleaves human IgG. A dose of 0.25 mg / kg cleaved 95% of IgG within 45 minutes to 6 hours after administration, maintaining low IgG levels (average reduction of over 70%) for one week (Figure 5). The pre-existing anti-IdeE-2 antibody ratio and titer are both low, demonstrating significant clinical advantages over the similar foreign product, Imlifidase. The use of IdeE-2 can improve clinical safety and efficacy. It is expected to provide a safer and more effective breakthrough therapy in organ transplantation (whether solid organ transplantation or hematopoietic stem cell transplantation), acute severe autoimmune diseases, and gene therapy.

[0102] Safety is superior to that of Imlifidase: Five escalating dose levels were administered to healthy subjects within a safe and well-tolerated dose range, and safety and tolerability were observed.

[0103] The applicant declares that while the above-described embodiments illustrate the detailed methods of the present disclosure, the present disclosure is not limited to the above-described detailed methods, and does not imply that the present disclosure must rely on the above-described detailed methods in order to be implemented. Persons skilled in the art should understand that any improvements to the present disclosure, equivalent replacements for raw materials in the products of the present disclosure, addition of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present disclosure.

[0104] sequence

Claims

1. An immunoglobulin degrading enzyme, characterized in that The immunoglobulin degrading enzyme does not cause bacterial / cell autolysis when induced to express.

2. The immunoglobulin degrading enzyme according to claim 1, characterized in that The term "not causing autolysis of bacteria / cells" means that during the expression of the immunoglobulin, the OD600 of the bacteria / cells will not decrease after being higher than 40, 50, 60, 70 or 80; preferably, during the expression of the immunoglobulin, the OD600 of the bacteria / cells will not decrease after being higher than 70; more preferably, during the expression of the immunoglobulin, the OD600 of the bacteria / cells will not decrease after being higher than 80.

3. The immunoglobulin degrading enzyme according to claim 1 or 2, characterized in that The immunoglobulin degrading enzyme comprises or consists of the following amino acid sequence: (a) the amino acid sequence shown in SEQ ID NO: 2; or (b) an amino acid sequence having one or more amino acid substitutions, additions and / or deletions compared to SEQ ID NO:

2.

4. The immunoglobulin degrading enzyme according to any one of claims 1 to 3, characterized in that The immunoglobulin degrading enzyme is derived from Streptococcus equi ssp. equi.

5. An immunoglobulin degrading enzyme, characterized in that The immunoglobulin degrading enzyme comprises or consists of the following amino acid sequence: (a) the amino acid sequence shown in SEQ ID NO: 1 or 2; or (b) an amino acid sequence having one or more amino acid substitutions, additions or deletions compared to SEQ ID NO: 1 or 2.

6. The immunoglobulin degrading enzyme according to claim 5, characterized in that The immunoglobulin degrading enzyme is derived from Streptococcus equi ssp.equi.

7. The immunoglobulin degrading enzyme according to claim 5, characterized in that The immunoglobulin degrading enzyme does not cause bacterial / cell autolysis when expressed.

8. The immunoglobulin degrading enzyme according to claim 7, characterized in that The term "not causing autolysis of bacteria / cells" means that during the expression of the immunoglobulin, the OD600 of the bacteria / cells will not decrease after being higher than 40, 50, 60, 70 or 80; preferably, during the expression of the immunoglobulin, the OD600 of the bacteria / cells will not decrease after being higher than 70; more preferably, during the expression of the immunoglobulin, the OD600 of the bacteria / cells will not decrease after being higher than 80.

9. The immunoglobulin degrading enzyme according to claim 1 or 5, characterized in that The immunoglobulin is expressed intracellularly or extracellularly.

10. A nucleotide sequence encoding the immunoglobulin degrading enzyme according to any one of claims 1 to 9; Preferably, the sequence comprises or consists of the following nucleotide sequence: (a) the nucleotide sequence shown in SEQ ID NO: 3; or (b) a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% but less than 100% sequence homology to the nucleotide sequence shown in SEQ ID NO: 3; (c) a nucleotide sequence having one or more nucleotide substitutions, additions and / or deletions compared to SEQ ID NO:

3. An expression vector comprising the nucleotide sequence according to claim 10 .

12. A host cell comprising the nucleotide sequence according to claim 10 or the expression vector according to claim 11, preferably the host cell is a bacterial cell or a fungal cell, more preferably the bacterial cell is an Escherichia coli cell or the fungal cell is a yeast cell.

13. A method for intracellular expression of the immunoglobulin degrading enzyme described in any one of claims 1 to 9, comprising the following steps: (a) selecting a single clone of the host strain of claim 12; (b) seed liquid culture; (c) fermentation tank culture; (d) inducing expression; and optionally (e) collecting the expressed immunoglobulin degrading enzyme.

14. A composition comprising: The immunoglobulin degrading enzyme according to any one of claims 1 to 9; and Optional pharmaceutically acceptable carrier or excipient.

15. The composition according to claim 14, further comprising: an antibody or a protein containing an Fc fragment, preferably, wherein the target of the antibody is selected from the group consisting of: cell surface proteins, cytokines, hormones, enzymes, intracellular messengers, intercellular messengers and immune checkpoints.

16. The composition according to claim 14 or 15, further comprising: Viral vector drugs, drugs that can reduce blood IgG levels, preferably, the viral vector drugs are selected from the group consisting of oncolytic viruses, gene therapy viruses, and viral vector vaccines. Preferably, the drugs that reduce blood IgG levels are selected from the group consisting of FcRn antibodies, and Fc fragment variants with high affinity to FcRn.

17. A kit comprising: (1) The immunoglobulin degrading enzyme according to any one of claims 1 to 9; and (2) one or more selected from the group consisting of: (a) a pharmaceutically acceptable carrier or excipient; (b) an antibody or a protein containing Fc; and / or (3) a viral vector drug selected from an oncolytic virus, a gene therapy virus, and a viral vector vaccine; and / or (4) A drug capable of reducing blood IgG levels, wherein the drug capable of reducing blood IgG levels is selected from FcRn antibodies and Fc fragment variants with high affinity to FcRn.

18. A kit comprising: a kit A and a kit B, characterized in that: The kit A contains the immunoglobulin degrading enzyme according to any one of claims 1 to 9, The medicine box B contains one or more selected from the following groups: (1) a pharmaceutically acceptable carrier or excipient; (2) an antibody or a protein containing Fc; and / or (3) a viral vector drug; and / or (4) a drug capable of reducing blood IgG levels; Wherein, the viral vector drug is selected from oncolytic virus, gene therapy virus and viral vector vaccine; the drug for reducing blood IgG level is selected from FcRn antibody and Fc fragment variant with high affinity to FcRn.

Images