Proteases

Abstract

The invention relates to an IgM specific protease. Specifically, the protease according to the present invention is capable of cleaving IgM, including human IgM, with high specificity. The invention also relates to a method and to the use of said protease, in particular for the analysis and / or modification of IgM.

Description

Technical Field

[0001] This invention relates to IgM-specific proteases. Specifically, the proteases according to the invention are capable of cleaving IgM, including human IgM, with high specificity. The invention also relates to methods and uses of said proteases, particularly for the analysis and / or modification of IgM. Background Technology

[0002] Pathogenic bacteria have evolved many mechanisms to evade the host's immune system, enabling them to successfully colonize in desired host environments.

[0003] One such mechanism is that some bacteria can modulate the functionality of host antibodies to reduce or inactivate them. For example, some known bacteria encode virulence factors with IgG-specific glycolytic or proteolytic activity (Sjögren J. et al., Glycobiology 2020, 30(4):254-267; Brezski RJ, Jordan RE. mAbs 2010, 2(3):212-220), and some have IgA-specific proteolytic activity (Kornfeld SJ, Plaut AG. RevInfect Dis 1981, 3(3):521-534).

[0004] Among the known virulence factors that modulate the host immune system by enzymatically modifying host proteins, most are typically derived from zoonotic or species-limited pathogens. In fact, IgG and IgM specific proteases have been isolated from pathogenic species of the genus Streptococcus (Von Pawel-Rammingen U. et al., EMBO2002, 21(7):1607-1615; Lannergård J & Guss B., IdeE, FEMS Microbiol Lett2006, 262(2):230-5; Spoerry C. et al., J Biol Chem 2016, 291(15):7915-25; Seele J. et al., J Bacteriol 2013, 195(5)).

[0005] The activity and importance of IgG are well-known and widely used in biotechnology and medicine. On the other hand, IgM is often overlooked, but it remains a crucial component of the immune system. For example, in responses to microbial infections, IgM plays a vital role in maintaining the integrity of the primary barrier, neutralizing microorganisms by inhibiting the binding between oral and respiratory microbes and their homologous cell receptors. Furthermore, IgM plays a key role in opsonization of pathogens and complement activation.

[0006] IgM has further applications in biotechnology and medicine, extending beyond responses to microbial infections. This application is becoming increasingly prevalent and attractive for therapeutic development (Samsudin F. et al., Chem Sci 2020, 11(10):2843-2854; Zhang J. et al., mAbs 2022, 14(1):2031483).

[0007] Although IgM is important for the endogenous functionalization of the immune system and for therapeutic and biotechnological applications, there is a lack of existing tools for the effective, widely applicable and target-specific detection, analysis and modification of IgM.

[0008] An IgM-specific protease has been identified in Streptococcus suis, a zoonotic porcine pathogen (Seele J. et al., J Bacteriol 2013, 195(5)). The activity of this protease is limited to substrates including porcine IgM; however, it cannot cleave human IgM.

[0009] Therefore, it is clear that there is a severe lack of biological tools suitable for IgM research and manipulation. In particular, there is an unmet need for biological tools that specifically target human IgM. Summary of the Invention

[0010] This invention relates to the unexpected discovery that peptides isolated from the genus *Lachnoanaerobaculum* (a symbiotic human bacterium) can cleave IgM with high specificity. Therefore, the peptides of this invention allow for the specific and efficient targeting of IgM, including human IgM. The peptides of this invention have practical applications as biological tools for studying and manipulating IgM in numerous settings.

[0011] In the first aspect, a polypeptide with human IgM-specific endonuclease activity is provided.

[0012] In one aspect, a polypeptide having endopeptidase activity is provided, the polypeptide comprising:

[0013] a) The amino acid sequence of SEQ ID NO: 1;

[0014] b) A variant thereof that has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 1; or

[0015] c) fragments of a) or b),

[0016] Preferably, the endopeptide activity is human IgM-specific endopeptide activity.

[0017] In another aspect, a polypeptide with endopeptide activity is provided, the polypeptide comprising:

[0018] a) The amino acid sequence of SEQ ID NO: 8;

[0019] b) A variant thereof that has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 8; or

[0020] c) fragments of a) or b),

[0021] Preferably, the endopeptide activity is human IgM-specific endopeptide activity.

[0022] In one aspect, a composition or combination is provided comprising a first polypeptide and a second polypeptide according to the invention, wherein the first polypeptide is a polypeptide with endopeptide activity according to the invention, and the second polypeptide is a polypeptide with IgG-specific endopeptide activity.

[0023] In one aspect, a polynucleotide is provided that encodes a polypeptide according to the invention.

[0024] In one aspect, a carrier is provided that encodes or contains a polynucleotide according to the invention.

[0025] In one embodiment, an expression vector is provided that encodes or contains a polynucleotide of a encoded polypeptide according to the invention, wherein the expression vector is used to produce the polypeptide according to the invention.

[0026] In one aspect, a kit is provided comprising:

[0027] a) The polypeptide according to the present invention;

[0028] b) The polynucleotide according to the present invention;

[0029] c) The carrier according to the invention; and / or

[0030] d) The composition or combination according to the invention, wherein the first polypeptide and the second polypeptide of the combination are provided separately; and / or

[0031] e) A composition or combination according to the invention, wherein the first polypeptide and the second polypeptide of the combination are provided together.

[0032] In one aspect, a method for hydrolyzing IgM is provided, the method comprising contacting a sample containing IgM with a polypeptide, composition or combination according to the invention.

[0033] In one aspect, a method for detecting the presence of IgM in a sample is provided, the method comprising:

[0034] a) Contacting the sample with the polypeptide according to the invention; and

[0035] b) Identify and / or isolate IgM-specific cleavage products.

[0036] The presence of IgM-specific cleavage products indicates the presence of IgM in the sample.

[0037] In one aspect, the use of peptides, combinations, or kits according to the invention for degrading, removing, or identifying IgM in a sample is provided.

[0038] In one aspect, peptides, compositions, combinations, pharmaceutical compositions, or kits are provided for use in therapeutics. Attached Figure Description

[0039] Figure 1 SDS-PAGE analysis of LU-M1

[0040] SDS-PAGE analysis of a recombinantly expressed modified version of the peptide derived from *L. umeaense* (designated “LU-M1”) showed that the peptide was produced in high yield and high purity. Apparent molecular weight was indicated by a reference molecular weight ladder marker (lanes 1 and 9); the molecular weights of the ladder marker components are shown. The “elution” lanes indicate that a high-purity and homogeneous LU-M1 sample was obtained after a single-round affinity purification using a His tag.

[0041] Figure 2: Peptides derived from Lachnoanaerobaculum represent the first known human IgM-specific endonucleases.

[0042] The activity of different constructs against human and mouse IgG was evaluated using the full-length construct from *L. umeaense* (LU-FL) and the full-length construct from *L. gingivalis* (LG-FL) (A). LG-FL was further evaluated against human IgG subclasses (B). Both LG-FL and LU-FL were mixed with human IgA and IgM (C). The activity of the LU-M1 construct was evaluated by mixing it with all human IgG subclasses as well as polyclonal IgG, IgA, and IgM (D). The general proteolytic activity of LU-M1 was investigated using a fluorescent casein substrate (E), and species specificity was investigated using IgM purified from mice, rabbits, rats, and monkeys (F). SpeB (broad-spectrum protease) and IdeS (IgG-specific protease) were used as controls. Figures 2A to 2D and Figure 2F SDS-PAGE gels were depicted, lanes were marked accordingly, and apparent molecular weight was indicated by a reference molecular weight ladder marker.

[0043] Figure 3: Site-specific cleavage of IgM by peptides derived from Lachnoanaerobaculum

[0044] Analysis of myeloma IgM after PNGase F treatment, reduction, and denaturation by liquid chromatography-mass spectrometry (LC-MS). The untreated sample is shown. Figure 3A ) or samples further processed by LU-M1 ( Figure 3B The total ion current chromatogram (TIC; top) and the unconvolution mass spectrum of the target compound (bottom).

[0045] Figure 4 Size exclusion analysis of IgM-cut products

[0046] Size exclusion chromatography analysis of hydrolyzed IgM samples was performed together with size standards consisting of thyroglobulin (1), deferroferrin (2), and human IgG (3). Intact IgM, as well as hydrolysis products (pentamers CH3-CH4; CH1-CH1-CH2), were indicated on the chromatogram.

[0047] Figure 5: Evaluation of catalytic activity

[0048] The activity of LU-M1 was evaluated under various conditions, including a range of pH values ​​(A), NaCl concentration (B), the presence of divalent cations (C), temperature (D), and the presence of protease inhibitors (E). Figures 5A to 5E SDS-PAGE gels were depicted, lanes were marked accordingly, and apparent molecular weight was indicated by a reference molecular weight ladder marker.

[0049] Figure 6: LU-M1 is a highly efficient enzyme

[0050] The activity of LU-M1 against IgM was evaluated. IgM and LU-M1 were mixed at different enzyme-to-substrate ratios (1:20 to 1:100; w:w) while the total reaction volume remained constant. The reaction mixture was incubated at 37°C for 5 to 60 minutes. Figure 6A SDS-PAGE gels were depicted, lanes were marked accordingly, and apparent molecular weight was indicated by a reference molecular weight ladder marker. Figure 6B and Figure 6C The optical density data obtained from the corresponding SDS-PAGE analysis are depicted.

[0051] Figure 7: LU-M1 is active in serum

[0052] Human serum was incubated with IdeS and LU-M1 at 37°C for 30 minutes, and then analyzed by SDS-PAGE (top) and Western blotting for IgG (A) and IgM (B) (bottom).

[0053] Figure 8: LU-M1 can be freeze-dried and immobilized while retaining its activity.

[0054] The lyophilized LU-M1 was reconstituted with water to the same concentration, and its activity against IgM was evaluated. Figure 8A (A) The numbers above the SDS-PAGE indicate the lyophilization conditions of the sample before reconstitution. (B) Image of the lyophilized particles. Evaluation of the activity of resin-immobilized LU-M1 against IgM ( Figure 8C The numbers above the SDS-PAGE indicate the concentration of immobilized LU-M1. Figure 8A and Figure 8C SDS-PAGE gels were depicted, lanes were marked accordingly, and apparent molecular weight was indicated by a reference molecular weight ladder marker.

[0055] Figure 9 LU-M1 is not easily recognized by IVIG.

[0056] The presence of LU-M1-specific human IgG was assessed using Western blotting. 0.5 μg of each of IdeS, Xork, and LU-M1 was separated by SDS-PAGE, either stained (left panel) or transferred to a nitrocellulose membrane for Western blotting (right panel). The membrane was blocked, incubated with human IVIG, and detected using an alkaline phosphatase-conjugated anti-human IgG Fc antibody. Western blotting data indicated the presence of little or no anti-LU-M1 antibody in the IVIG. Detailed Implementation

[0057] It should be understood that different applications of the disclosed products and methods can be tailored to specific needs in the field. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention and is not intended to be limiting.

[0058] All publications, patents and patent applications cited in this article are incorporated herein by reference in their entirety.

[0059] Unless otherwise stated, the terms will be understood to have the common meaning in the art as assigned to them by those skilled in the art.

[0060] As used herein, the terms “comprising,” “comprises,” and “comprised of” are synonymous with “including,” “includes,” “containing,” and “contains,” and are inclusive or open-ended, not excluding additional, unlisted components, elements, or method steps. The terms “comprising,” “comprises,” and “comprised of” also include the term “composed of.” Thus, the term “comprising” is used to mean “including, but not limited to.” “Comprising” and “including, but not limited to” are used interchangeably. Conversely, the term “composed of” is intended to be exclusive or closed-ended, and does not include additional, unlisted components, elements, or method steps.

[0061] As used in this specification and the appended claims, unless otherwise expressly indicated by the content, the singular forms “a,” “an,” and “the” include plural references. Thus, for example, reference to “a polypeptide” includes “multiple polypeptides,” etc.

[0062] IgM

[0063] IgM is an immunoglobulin (Ig) isotype, typically characterized by its large size and pentamer structure. However, IgM can also exist in other structural forms, such as secreted hexamers or cell surface display monomers that form part of the B cell receptor.

[0064] The pentamer structure of IgM makes it essentially composed of five structures similar to IgG. IgM contains 10 potential antigen-binding sites (or complementary sites), which facilitates high-affinity target binding. This high-affinity binding allows target binding even if monovalent binding affinity is low.

[0065] IgM is produced early in infection following antigen exposure and plays a key role in stimulating other effector functions of the immune system, such as activating complement and Fc receptor-mediated activity.

[0066] IgM has demonstrated favorable therapeutic properties, even when compared to IgG, and has been described as a promising candidate for monoclonal antibody therapy for several conditions (Samsudin F. et al., Chem Sci 2020, 11(10):2843-2854; Zhang J. et al., mAbs 2022, 14(1):2031483).

[0067] IgM-specific endonuclease

[0068] This invention relates to polypeptides with IgM-specific endopeptidase activity.

[0069] It should be understood that, as used herein, the term "peptide with IgM-specific endonuclease activity" can refer to any protein, peptide, or fragment thereof capable of catalyzing IgM cleavage. The terms endonuclease and protease are used interchangeably herein.

[0070] Those skilled in the art can readily determine whether IgM cleavage has occurred, for example using techniques commonly used in the field of protein biochemistry, including SDS-PAGE analysis, Western blotting, chromatography (e.g., size exclusion chromatography), and mass spectrometry. These techniques are illustrated herein (see examples) but are in no way intended to limit the scope of the study.

[0071] Furthermore, it should be understood that IgM-specific endonuclease activity can refer to target (IgM)-specific endonuclease activity. This activity may not be considered as having a broad target protein profile, such as trypsin. However, IgM-specific activity encompasses activity against different IgM molecules, such as IgM molecules derived from different species or modified IgM molecules.

[0072] Modified IgM molecules may include, for example, non-naturally occurring (e.g., synthetic) IgM molecules; fusion proteins containing IgM molecules or portions / sequences thereof; and fragments of IgM molecules.

[0073] Those skilled in the art will understand that the peptide may have residual or low levels of activity against other targets (e.g., IgG).

[0074] In one embodiment, the polypeptide may have IgM-specific and IgG-specific protease activities.

[0075] In some embodiments, the peptide may have low or negligible levels of detectable IgG protease activity.

[0076] In some embodiments, the peptide may have a low level of detectable IgG activity, wherein the IgG activity level is lower than the IgM activity level.

[0077] In some embodiments, the peptide may have a low level of detectable IgG activity, wherein the IgG activity level is less than 70% of the IgM activity level, for example less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of the IgM activity level.

[0078] In some implementations, IgG activity is IgG2-specific activity.

[0079] In a preferred embodiment, the polypeptide has no detectable activity against polypeptides other than IgM.

[0080] In another preferred embodiment, the polypeptide has no detectable activity against any immunoglobulin isotype other than IgM.

[0081] In another preferred embodiment, the peptide has no detectable activity against IgG.

[0082] In a preferred embodiment of the present invention, the IgM-specific endonuclease activity is human IgM-specific endonuclease activity.

[0083] It should be understood that the term "human IgM specificity" does not mean that the polypeptide is limited to human IgM. On the contrary, the endopeptide is specific to IgM, and human IgM is a recognized substrate, but not necessarily the only one. For example, IgM from non-human primates can also be a substrate.

[0084] In one implementation, the peptide activity against IgM is derived from monkeys of the genus *Macaques*, such as macaques and / or cynomolgus monkeys.

[0085] In one implementation, the peptide activity against IgM is derived from monkeys of the genus Baboon.

[0086] Lachnoanaerobaculum

[0087] Lachnoanaerobaculum is a human commensal bacterium found in the human oral cavity and gut. Lachnoanaerobaculum orale and Lachnoanaerobaculum umeaense were first described in 2012, isolated from the oral cavity of healthy individuals and the gut of children with celiac disease, respectively (Hedberg ME, et al., J SystEvol Microbiol 2012, 62(Pt11):2685-2690).

[0088] Although two recent studies have shown that species of Lachnoanaerobaculum, L. orale and L. gingivalis, cause bacteremia in immunocompromised individuals (Ida Y. et al., Anaerobe 2022, 73:102506; Okada N. et al., Anaerobe 2022, 76:102610), species of Lachnoanaerobaculum have not been described as pathogens of any pathology.

[0089] Prior to the work of the inventors, as described herein, no isolates from the genus *Lachnoanaerobaculum* had been shown to have proteolytic activity, nor had any specific immunoglobulin modification activity been identified or studied.

[0090] Unexpectedly, the inventors have identified an IgM-specific endonuclease encoded by bacteria of the genus Lachnoanaerobaculum.

[0091] In one embodiment, the present invention provides a polypeptide having IgM-specific endopeptidase activity, wherein the polypeptide is derived from bacteria of the genus Lachnoanaerobaculum.

[0092] In one embodiment, the present invention provides a polypeptide having IgM-specific endonuclease activity, wherein the polypeptide is encoded by a polynucleotide sequence having at least 80% sequence identity with a polynucleotide sequence encoded by Lachnoanaerobaculum umeaense, for example at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0093] In one embodiment, the present invention provides a polypeptide having IgM-specific endonuclease activity, wherein the polypeptide is encoded by a polynucleotide sequence having at least 80% sequence identity with a polynucleotide sequence encoded by Lachnoanaerobaculumgingivalis, for example at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0094] In one embodiment, the present invention provides a polypeptide having IgM-specific endopeptide activity, wherein the polypeptide has at least 80% sequence identity with the amino acid sequence encoded by Lachnoanaerobaculum umeaense, for example at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0095] In one embodiment, the present invention provides a polypeptide having IgM-specific endopeptide activity, wherein the polypeptide has at least 80% sequence identity with the amino acid sequence encoded by Lachnoanaerobaculum gingivalis, for example at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0096] In one embodiment, the present invention provides a polypeptide having IgM-specific endopeptidase activity, wherein the polypeptide is derived from Lachnoanaerobaculum umeaense.

[0097] In one embodiment, the present invention provides a polypeptide having IgM-specific endopeptidase activity, wherein the polypeptide is derived from Lachnoanaerobaculum gingivalis.

[0098] The polypeptides of the present invention may be fragments or modified versions of naturally occurring polypeptides encoded by bacteria of the genus Lachnoanaerobaculum.

[0099] Engineered IgM-specific endopeptidase

[0100] After identifying candidate protease genes in the Lachnoanaerobaculum species, the inventors engineered many peptides.

[0101] The polypeptide according to the invention may contain a sequence derived from Lachnoanaerobaculum umeaense or consist of a sequence derived from Lachnoanaerobaculum umeaense.

[0102] Exemplary Lachnoanaerobaculum umeaense polypeptide sequences are listed in Table 1 of this paper.

[0103] Table 1: Exemplary Lachnoanaerobaculum umeaense sequences

[0104]

[0105]

[0106] The polypeptide according to the invention may contain a sequence derived from Lachnoanaerobaculum gingivalis or consist of a sequence derived from Lachnoanaerobaculum gingivalis.

[0107] Exemplary polypeptide and polynucleotide sequences of Lachnoanaerobaculum gingivalis are listed in Table 2 of this paper.

[0108] Table 2: Exemplary Lachnoanaerobaculum gingivalis sequences

[0109]

[0110]

[0111] Exemplary polynucleotide sequences encoding polypeptides according to the present invention are listed in Table 3.

[0112] Table 3: Exemplary Lachnoanaerobaculum polynucleotide sequences

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124] It should be understood that, due to the degeneracy of the genetic code, any number of polynucleotide sequences can be used to encode a given polypeptide according to the invention. Therefore, the polynucleotide sequences described herein are merely illustrative.

[0125] polypeptide

[0126] This invention relates to polypeptides having IgM-specific endonuclease activity. In a preferred embodiment, the IgM-specific endonuclease activity is human IgM-specific endonuclease activity.

[0127] In one aspect, a polypeptide having endopeptide activity is provided, comprising:

[0128] d) The amino acid sequence of SEQ ID NO: 1;

[0129] e) A variant thereof that has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 1; or

[0130] f) a) or b) fragments,

[0131] Preferably, the endopeptide activity is human IgM-specific endopeptide activity.

[0132] In another aspect, a polypeptide having endopeptide activity is provided, comprising:

[0133] d) The amino acid sequence of SEQ ID NO: 8;

[0134] e) A variant thereof that has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 8; or

[0135] f) a) or b) fragments,

[0136] Preferably, the endopeptide activity is human IgM-specific endopeptide activity.

[0137] In one embodiment, the polypeptide of the present invention has IgM-specific and IgG-specific protease activities, and the polypeptide comprises or consists of a sequence of any one of SEQ ID NO: 8 to 11 as defined herein, or a variant or fragment thereof.

[0138] In some implementations, IgG specific activity is lower than IgM specific activity.

[0139] In one embodiment of the present invention, a polypeptide having IgM-specific endopeptide activity is provided, wherein the polypeptide comprises a polypeptide of the group consisting of SEQ ID NO: 1 to 7 or a polypeptide selected from the group consisting of SEQ ID NO: 1 to 7.

[0140] In one embodiment of the present invention, a polypeptide having IgM-specific endopeptide activity is provided, wherein the polypeptide comprises or is composed of a polypeptide having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 7.

[0141] In one embodiment of the present invention, a polypeptide having IgM-specific endopeptide activity is provided, wherein the polypeptide comprises or is composed of a polypeptide having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1 to 7, for example, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity.

[0142] In one embodiment of the present invention, a polypeptide having IgM-specific endopeptide activity is provided, wherein the polypeptide comprises or is composed of a polypeptide having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 8 to 11.

[0143] In one embodiment of the present invention, a polypeptide having IgM-specific endopeptide activity is provided, wherein the polypeptide comprises or is composed of the following: a polypeptide having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 8 to 11, for example, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity.

[0144] This invention also covers fragments of other polypeptides of the invention. These fragments should themselves be considered polypeptides.

[0145] In other words, the polypeptides of the present invention may comprise or consist of fragments or shorter segments of longer polypeptides according to the present invention as defined herein.

[0146] A fragment should be understood as any continuous polypeptide sequence shorter than a longer sequence that is identical to it (spanning the amino acids they share). For example, a polypeptide of 200 amino acids in length can be considered a fragment of a longer polypeptide sequence of 250 amino acids in length that has 100% sequence identity with it.

[0147] In one embodiment, the polypeptide comprises or consists of a fragment of a polypeptide sequence selected from the group consisting of SEQ ID NO: 1 to 7.

[0148] In one embodiment, the polypeptide comprises or consists of a fragment of a polypeptide sequence selected from the group consisting of SEQ ID NO: 8 to 11.

[0149] In one embodiment, the polypeptide comprises or consists of a fragment of a polypeptide sequence selected from the group consisting of SEQ ID NO: 1 to 11, wherein the fragment is at least 100 amino acids long, for example, at least 150 amino acids long, at least 200 amino acids long, at least 250 amino acids long, at least 300 amino acids long, at least 350 amino acids long, at least 400 amino acids long, at least 450 amino acids long, at least 500 amino acids long, at least 550 amino acids long, at least 600 amino acids long, or at least 650 amino acids long.

[0150] In one embodiment, the polypeptide comprises or consists of a fragment of a polypeptide sequence selected from the group consisting of SEQ ID NO: 1 to 11, wherein the fragment is at least 300 amino acids in length, for example, at least 310 amino acids, at least 320 amino acids, at least 330 amino acids, at least 340 amino acids, at least 350 amino acids, at least 360 amino acids, at least 370 amino acids, at least 380 amino acids, at least 390 amino acids, or at least 400 amino acids in length.

[0151] The length of a polypeptide is typically at least 75, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, or at least 650 amino acids.

[0152] In a preferred embodiment, the polypeptide length is at least 300 amino acids, for example, at least 310 amino acids, at least 320 amino acids, at least 330 amino acids, at least 340 amino acids, at least 350 amino acids, at least 360 amino acids, at least 370 amino acids, at least 380 amino acids, at least 390 amino acids, or at least 400 amino acids.

[0153] The length of a polypeptide can be no longer than 700 amino acids, for example, no longer than 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, or 100 amino acids.

[0154] It should be understood that any of the lower limits listed above can be combined with any of the upper limits listed above to provide a range of peptide lengths. For example, the length of a peptide can be 75 to 700 amino acids, or 450 to 550 amino acids.

[0155] In one embodiment, the polypeptide length is between 300 and 700 amino acids, for example, between 310 and 690 amino acids, between 320 and 690 amino acids, between 330 and 680 amino acids, between 330 and 670 amino acids, between 320 and 640 amino acids, and between 320 and 630 amino acids.

[0156] In a preferred embodiment, the polypeptide length is between 300 and 700 amino acids, more preferably between 320 and 690 amino acids.

[0157] In one embodiment, a polypeptide having endopeptide activity is provided, the polypeptide comprising or consisting of an amino acid sequence of any one of SEQ ID NO: 1 to 11.

[0158] In one embodiment, a polypeptide having endopeptide activity is provided, the polypeptide comprising or consisting of a polypeptide having at least 80% sequence identity with the amino acid sequence of any one of SEQ ID NO: 1 to 11, such as 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0159] In one embodiment, a fragment of a polypeptide is provided, the fragment having endopeptide activity, wherein the fragment comprises or consists of the following: a fragment of the amino acid sequence of any one of SEQ ID NO: 1 to 11, or a fragment of the amino acid sequence having at least 80% sequence identity with the amino acid sequence of any one of SEQ ID NO: 1 to 11, such as 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0160] In one embodiment, a polypeptide having endopeptide protease activity is provided, the polypeptide comprising:

[0161] a) The amino acid sequence of any one of SEQ ID NO: 1 to 11;

[0162] b) A variant thereof, which has at least 80% sequence identity with the amino acid sequence of any one of SEQ ID NO: 1 to 11; or

[0163] c) fragments of a) or b).

[0164] In one embodiment, a polypeptide having human IgM-specific endonuclease activity is provided, wherein the polypeptide comprises or is composed of an amino acid sequence having at least 80% sequence identity with an amino acid sequence of any one of SEQ ID NO: 1 to 11.

[0165] In one embodiment, a polypeptide having human IgM-specific endonuclease activity is provided, wherein the polypeptide comprises or is composed of an amino acid sequence having at least 85% sequence identity with an amino acid sequence of any one of SEQ ID NO: 1 to 11.

[0166] In one embodiment, a polypeptide having human IgM-specific endonuclease activity is provided, wherein the polypeptide comprises or is composed of an amino acid sequence having at least 90% sequence identity with an amino acid sequence of any one of SEQ ID NO: 1 to 11.

[0167] In one embodiment, a polypeptide having human IgM-specific endonuclease activity is provided, wherein the polypeptide comprises or is composed of an amino acid sequence having at least 95% sequence identity with an amino acid sequence of any one of SEQ ID NO: 1 to 11.

[0168] In one embodiment, a polypeptide having human IgM-specific endonuclease activity is provided, wherein the polypeptide comprises or is composed of an amino acid sequence having at least 98% sequence identity with an amino acid sequence of any one of SEQ ID NO: 1 to 11.

[0169] In one embodiment, a polypeptide having human IgM-specific endonuclease activity is provided, wherein the polypeptide comprises or is composed of an amino acid sequence having at least 99% sequence identity with an amino acid sequence of any one of SEQ ID NO: 1 to 11.

[0170] In one embodiment, a polypeptide having endopeptide activity is provided, the polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 1.

[0171] In one embodiment, a polypeptide having endopeptide activity is provided, the polypeptide comprising or consisting of the following: a polypeptide having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 1, such as 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0172] In one embodiment, a fragment of a polypeptide is provided, the fragment having endopeptide activity, wherein the fragment comprises or consists of the following: a fragment of the amino acid sequence of SEQ ID NO: 1, or a fragment of an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 1, such as 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0173] In one embodiment, a polypeptide having endopeptide protease activity is provided, the polypeptide comprising:

[0174] a) The amino acid sequence of SEQ ID NO: 1;

[0175] b) A variant thereof that has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 1; or

[0176] c) fragments of a) or b).

[0177] In one embodiment, a polypeptide having human IgM-specific endonuclease activity is provided, wherein the polypeptide comprises or is composed of an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 1.

[0178] In one embodiment, a polypeptide having endopeptide activity is provided, the polypeptide comprising the amino acid sequence of SEQ ID NO: 7.

[0179] In one embodiment, a polypeptide having endopeptide activity is provided, the polypeptide consisting of the amino acid sequence of SEQ ID NO: 7.

[0180] In one embodiment, a polypeptide having endopeptide activity is provided, the polypeptide comprising or consisting of the following: a polypeptide having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 7, such as 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0181] In one embodiment, a fragment of a polypeptide is provided, the fragment having endopeptide activity, wherein the fragment comprises or consists of the following: a fragment of the amino acid sequence of SEQ ID NO: 1, or a fragment of an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 7, such as 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0182] In one embodiment, a polypeptide having endopeptide protease activity is provided, the polypeptide comprising:

[0183] d) The amino acid sequence of SEQ ID NO: 7;

[0184] e) A variant thereof that has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 7; or

[0185] f) a) or b) fragments.

[0186] In one embodiment, a polypeptide having human IgM-specific endonuclease activity is provided, wherein the polypeptide comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 7 or consists of an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 7.

[0187] In one embodiment, a polypeptide having endopeptide activity is provided, the polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 8.

[0188] In one embodiment, a polypeptide having endopeptide activity is provided, the polypeptide comprising or consisting of the following: a polypeptide having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 8, such as 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0189] In one embodiment, a fragment of a polypeptide is provided, the fragment having endopeptide activity, wherein the fragment comprises or consists of the following: a fragment of the amino acid sequence of SEQ ID NO: 1, or a fragment of an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 8, such as 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0190] In one embodiment, a polypeptide having endopeptide protease activity is provided, the polypeptide comprising:

[0191] a) The amino acid sequence of SEQ ID NO: 8;

[0192] b) A variant thereof that has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 8; or

[0193] c) fragments of a) or b).

[0194] In one embodiment, a polypeptide having human IgM-specific endonuclease activity is provided, wherein the polypeptide comprises an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 8 or is composed of an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 8.

[0195] The polypeptides according to the invention can be provided in a variety of states. For example, the polypeptides can be provided as isolated polypeptides in solution, as lyophilized solids (e.g., powders), or as conjugates (e.g., conjugated with other biomolecules or polymers).

[0196] In one implementation, the peptide is lyophilized.

[0197] In one implementation, the polypeptide is provided in solution form.

[0198] In one implementation, the polypeptide is coupled to another molecule.

[0199] In one embodiment, the polypeptide is immobilized on a solid (e.g., a solid support resin) or substantially solid support (e.g., beads).

[0200] General characteristics of polypeptides

[0201] The term "polypeptide" is used herein in its broadest sense to refer to a compound containing two or more substituted amino acids, amino acid analogs, or other peptide mimics. Therefore, the term "polypeptide" includes short peptide sequences as well as longer polypeptides and proteins. The terms "protein," "peptide," and "polypeptide" are used interchangeably. As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including D or L optical isomers, as well as amino acid analogs and peptide mimics. Therefore, a polypeptide according to the invention may comprise entirely natural or synthetic amino acids, or a mixture of natural and synthetic amino acids.

[0202] Peptides can be produced by any suitable method, including recombinant or synthetic methods. For example, peptides can be synthesized directly using standard techniques known in the art, such as Fmoc solid-phase chemistry, Boc solid-phase chemistry, or via liquid-phase peptide synthesis. Alternatively, peptides can be produced by transforming cells (e.g., bacterial, insect, or mammalian cells) with a nucleic acid molecule or carrier encoding the peptide.

[0203] This document describes the expression of the polypeptides of the present invention, which can be more easily achieved by those skilled in the art using standard techniques.

[0204] Peptides can be derivatized or modified to aid in production, isolation, or purification. For example, when the peptides of the present invention are produced by recombinant expression in bacterial host cells, the sequence of the peptide may include an additional methionine (M) residue at the N-terminus to improve expression. As another example, the peptides of the present invention can be derivatized or modified by adding a ligand capable of binding directly and specifically to an isolation method. Alternatively, the peptide can be derivatized or modified by adding one member of a binding pair, and the isolation method comprises a reagent derivatized or modified by adding the other member of the binding pair. Any suitable binding pair can be used.

[0205] In some embodiments, the polypeptide of the present invention includes one or more tags, such as polypeptide tags.

[0206] In one implementation, the label is a cuttable label.

[0207] In one implementation, the tag allows for specific modification of the tag or the polypeptide to which it is attached.

[0208] In one implementation, the tag facilitates the identification and / or isolation of the peptide.

[0209] In one implementation, the tag is bound to a binding partner. In some implementations, the binding partner is a polypeptide, such as an antibody or other binding protein (e.g., avidin); a metal (e.g., Ni); and / or a small organic molecule (e.g., biotin).

[0210] The polypeptides according to the invention may include any one or more tags selected from the group consisting of: polyhistidine tags, Fc tags; FLAG tags, Rho1D4 tags, HA tags, strep tags, and Avi tags. However, any suitable tag known to those skilled in the art may be used in combination with any polypeptide of the invention.

[0211] In one implementation, the tag facilitates or improves the expression and / or properties of the peptide (e.g., solubility or polymerization state).

[0212] The polypeptide according to the invention may contain any one or more tags selected from the group consisting of: Fc tag; SUMO tag; GST tag; lysozyme tag; MBP tag; GFP tag; Myc tag; and thioredoxin tag.

[0213] Tags can be located at C-terminals or N-terminals. Furthermore, one or more tags can be separated from each other and / or from other sequence elements via one or more connector sequences. Any number of tags and their combinations can be used.

[0214] In a preferred embodiment, the polypeptide used in the present invention is derivatized or modified by adding a member of a binding pair, the polypeptide preferably being histidine-tagged or biotin-tagged. Typically, the amino acid coding sequence includes a histidine or biotin tag at the gene level, and the polypeptide is recombinantly expressed in *E. coli*. The histidine or biotin tag is typically present at either end of the polypeptide. It can be directly linked to the polypeptide or indirectly linked via any suitable adapter sequence, such as 3, 4, or 5 glycine residues, or a mixture of glycine and serine residues, such as a "GGS" adapter.

[0215] Histidine tags typically consist of six histidine residues; however, the length of the tag can be longer, for example, up to 7, 8, 9, 10, or 20 amino acids. Alternatively, His tags can be shorter, for example, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 amino acid (one or more).

[0216] Peptides can be provided in a substantially isolated or purified form. That is, isolated from most other components present in cell extracts from cells expressing the peptide. “Substantially purified” should be understood as the peptide purified to a homogeneity of at least 50%, 60%, 70%, 80%, or preferably at least 90%. Purity levels can be assessed by any suitable method, but typically involve SDS-PAGE analysis of the sample, followed by Coomassie Brilliant Blue detection. Peptides can be mixed with carriers, diluents, or preservatives that do not interfere with the intended purpose of the peptide and are still considered substantially isolated or purified. When a peptide is provided in a composition or combination with another active component (e.g., another peptide), each said peptide will be individually purified to a high level of homogeneity and then mixed in appropriate proportions for the intended purpose of each peptide. For example, two peptides may each be purified to at least 90% homogeneity before being combined in a 1:1 ratio. Other proportions will be apparent to those skilled in the art.

[0217] Peptides (or mixtures thereof) may be provided in lyophilized form, suitable for reconstitution in aqueous solution prior to use. Lyophilized peptides or compositions exhibit improved stability, facilitating long-term storage of the peptides. This document provides a method for preparing lyophilized peptides (or mixtures thereof) comprising lyophilizing the peptide (or mixture) in a suitable buffer, such as Tris-buffered saline (TBS). The peptide is typically substantially purified prior to lyophilization. The resulting peptide (or mixture) is also provided in lyophilized form. A method for preparing a solution of the peptide (or mixture) is also provided, comprising providing the lyophilized peptide (or mixture) and reconstituted with a suitable carrier or diluent, such as water.

[0218] Peptides can be immobilized using methods known in the art, such as those described in Datta S et al., Enzymeimmobilization: an overview on techniques and support materials, 3 Biotech, 3(1):1-9 (2013). For example, peptides can be immobilized by adsorption, covalent binding, affinity immobilization, or embedding. Materials that can be used as supports include, but are not limited to, natural supports such as agarose, collagen, gelatin, cellulose, pectin, agarose gel, inorganic materials such as ceramics, silica, glass, activated carbon, or charcoal, or synthetic polymers. For example, peptides can be immobilized on agarose gel or agarose, optionally provided as a resin.

[0219] Amino acid identity can be calculated using any suitable algorithm. For example, the BLAST algorithm can be used to calculate identity or aligned sequences, typically using the default settings. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0220] The sequence of the polypeptide of the present invention may comprise a variant of the amino acid sequence of any one of SEQ ID NO: 1 to 11, wherein the sequence of any one of SEQ ID NO: 1 to 11 has been modified, for example, by the addition, deletion or substitution of amino acids.

[0221] Unless otherwise stated, modifications are preferably conservative amino acid substitutions. Conservative substitutions replace amino acids with other amino acids having similar chemical structures, similar chemical properties, or similar side chain volumes. The introduced amino acids may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge as the amino acids they replace. Alternatively, conservative substitutions may introduce another aromatic or aliphatic amino acid to replace a pre-existing aromatic or aliphatic amino acid. Changes in conservative amino acids are well known in the art and can be selected based on the properties of the 20 major amino acids defined in Table 3 below. When amino acids have similar polarity, this can be determined by referring to the hydrophobicity scale of the amino acid side chain (see also Table 4).

[0222] The sequence of the polypeptide of the present invention may include a variant of the amino acid sequence of any one of SEQ ID NO: 1 to 11, wherein up to 10, for example up to 20, up to 30, up to 40, up to 50, up to 60, up to 70, up to 80, up to 90, up to 100, up to 150 or up to 200 conservative substitutions have been made.

[0223] Table 4: Chemical properties of amino acids

[0224]

[0225] Polynucleotides

[0226] The present invention also relates to polynucleotides encoding polypeptides having IgM-specific endonuclease activity.

[0227] In one aspect of the invention, a polynucleotide encoding a polypeptide having IgM-specific endonuclease activity is provided.

[0228] As used herein, the term polynucleotide is intended to encompass polymers of nucleotides or nucleotide analogs. Thus, polynucleotides include, for example, DNA, RNA, or DNA / RNA.

[0229] In one implementation, the polynucleotide is RNA.

[0230] In one implementation, the polynucleotide is DNA.

[0231] In one embodiment, the polynucleotide comprises one or more modified nucleotides or nucleotide analogs.

[0232] The polynucleotides according to the present invention can be naturally occurring, naturally derived, or synthetic.

[0233] In one embodiment, a polynucleotide encoding a polypeptide having endopeptide activity is provided, the polypeptide comprising or consisting of an amino acid sequence of any one of SEQ ID NO: 1 to 11.

[0234] In one embodiment, a polynucleotide encoding a polypeptide having endopeptide activity is provided, the polypeptide comprising or consisting of a polypeptide having at least 80% sequence identity with the amino acid sequence of any one of SEQ ID NO: 1 to 11, such as 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0235] In one embodiment, a polynucleotide fragment encoding a polypeptide fragment having endopeptide activity is provided, wherein the fragment comprises or consists of the following: a fragment of the amino acid sequence of any one of SEQ ID NO: 1 to 11, or a fragment of the amino acid sequence having at least 80% sequence identity with the amino acid sequence of any one of SEQ ID NO: 1 to 11, such as 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0236] In one embodiment, a polynucleotide encoding a polypeptide having endopeptide activity is provided, the polypeptide comprising:

[0237] a) The amino acid sequence of any one of SEQ ID NO: 1 to 11;

[0238] b) A variant thereof, which has at least 80% sequence identity with the amino acid sequence of any one of SEQ ID NO: 1 to 11; or

[0239] c) fragments of a) or b).

[0240] In one embodiment, a polynucleotide is provided encoding a polypeptide having human IgM-specific endonuclease activity, wherein the polypeptide comprises or is composed of an amino acid sequence having at least 80% sequence identity with an amino acid sequence of any one of SEQ ID NO: 1 to 11.

[0241] In one embodiment, a polynucleotide comprising the sequence of any one of SEQ ID NO: 12 to 22 is provided.

[0242] In one embodiment, a polynucleotide consisting of any one of the sequences of SEQ ID NO: 12 to 22 is provided.

[0243] In one embodiment, a polynucleotide is provided that has at least 60% sequence identity, such as 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity, with any one of SEQ ID NO: 12 to 22.

[0244] In one embodiment, a polynucleotide is provided, the polynucleotide comprising:

[0245] a) The polynucleotide sequence of any one of SEQ ID NO: 12 to 22;

[0246] b) A variant thereof that has at least 80% sequence identity with the polynucleotide sequence of any one of SEQ ID NO: 12 to 2; or

[0247] c) fragments of a) or b).

[0248] In one embodiment, a polynucleotide is provided encoding a polypeptide having human IgM-specific endonuclease activity, wherein the polypeptide comprises or is composed of an amino acid sequence having at least 80% sequence identity with an amino acid sequence of any one of SEQ ID NO: 1 to 11, and optionally wherein the polynucleotide comprises or is composed of a sequence according to any one of SEQ ID NO: 12 to 22, or a sequence having at least 80% sequence identity with a sequence of any one of SEQ ID NO: 12 to 22.

[0249] The present invention also provides a carrier comprising any one of the aforementioned polynucleotides.

[0250] The present invention also provides a vector encoding any of the aforementioned polynucleotides.

[0251] In one implementation, the carrier is an expression carrier.

[0252] In one embodiment, the carrier is the pET21a+ carrier.

[0253] General characteristics of polynucleotides

[0254] Therefore, the present invention provides a nucleic acid molecule (i.e., a polynucleotide) encoding the polypeptide of the present invention and a carrier.

[0255] Exemplary polynucleotide molecules encoding the polypeptides disclosed herein are provided in SEQ ID NO: 12 to 22. However, due to the degeneracy of the genetic code, it can be understood that any number of sequences can encode the same polypeptide.

[0256] The polynucleotide of the present invention may include a codon for an N-terminal methionine (ATG) at the 5' end and a codon for a Gly-Ser-Gly linker and a 6×His tag before the stop codon (TAA) at the 3' end, which may optionally be excluded. The optional inclusion of additional methionines and tags will be discussed in more detail below.

[0257] The terms “nucleic acid molecule” and “polynucleotide” are used interchangeably herein and refer to a polymer of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.

[0258] The polynucleotides of this invention encode the polypeptides according to the invention and can be provided in a separated or substantially separated form. "Substantially separated" means that the polypeptide can be substantially separated from any surrounding medium, but not completely separated. The polynucleotides can be mixed with a carrier or diluent that does not interfere with their intended use and are still considered substantially separated.

[0259] A nucleic acid sequence “encoding” a selected polypeptide is a nucleic acid molecule that, when placed under the control of a suitable regulatory sequence (e.g., in an expression vector), is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo or in vitro. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) end and a translation stop codon at the 3' (carboxyl) end. For the purposes of this invention, such nucleic acid sequences may include, but are not limited to, cDNA from viral, prokaryotic, or eukaryotic mRNA, genomic sequences from viral or prokaryotic DNA or RNA, or even synthetic DNA sequences. The transcription termination sequence may be located at the 3' end of the coding sequence.

[0260] Polynucleotides can be synthesized according to methods well known in the art, as illustrated by examples in Sambrook et al. (1989, Molecular Cloning - a laboratory manual; Cold Spring Harbor Laboratory Press). The nucleic acid molecules of the present invention can be provided in the form of expression cassettes comprising control sequences operatively linked to an insert sequence, thereby allowing the polypeptides of the present invention to be expressed in vivo (e.g., in prokaryotic or eukaryotic expression systems). These expression cassettes are typically provided in vectors (e.g., plasmids or recombinant viral vectors). Such expression cassettes can be directly applied to a host object. Alternatively, a vector containing the polynucleotides of the present invention can be applied to a host object. Preferably, the polynucleotides are prepared and / or applied using a genetic vector. A suitable vector can be any vector capable of carrying a sufficient amount of genetic information and allowing expression of the polypeptides of the present invention.

[0261] Therefore, the present invention includes expression vectors containing such polynucleotide sequences. These expression vectors are conventionally constructed in the field of molecular biology and may, for example, contain plasmid DNA and suitable initiators, promoters, enhancers, and other elements, such as necessary polyadenylation signals, and they are oriented in the correct direction to allow expression of the peptides of the present invention. Other suitable vectors will be apparent to those skilled in the art. As another example in this regard, we refer to the literature of Sambrook et al.

[0262] Any suitable algorithm can be used to calculate sequence identity between two or more polynucleotide sequences. For example, the BLAST algorithm can be used to calculate identity or align sequences, typically using the default settings. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0263] cell

[0264] The present invention also provides a cell that expresses or is capable of expressing the polypeptide of the present invention.

[0265] In one embodiment, a cell comprising the polynucleotide according to the invention is provided.

[0266] In one embodiment, a cell comprising a carrier according to the invention is provided.

[0267] In another embodiment, a cell comprising the polypeptide according to the invention is provided. Such cells may contain the polypeptide intracellularly, for example, contained in inclusion bodies, or soluble in cytosol; the polypeptide may exist temporarily within the cell before being secreted into the extracellular environment.

[0268] Cells typically include prokaryotic cells, such as bacterial cells, like Escherichia coli. These cells can be cultured using conventional methods to produce the peptides of this invention.

[0269] In one implementation, the cell is a BL21 (DE3) STAR cell.

[0270] Furthermore, the cells according to the invention can be eukaryotic cells, such as insect cells or mammalian cells.

[0271] Cells according to the invention may have been modified, for example by standard molecular biology techniques, to contain polynucleotides, carriers or polypeptides according to the invention, and / or modified to express polypeptides according to the invention.

[0272] Compositions and combinations

[0273] The polypeptides, polynucleotides, carriers, and cells of the present invention can be provided in combination or in combination with other compounds.

[0274] The present invention provides compositions or combinations comprising the polypeptide of the present invention and other polypeptides.

[0275] Regarding the composition, it should be understood that the individual components of the composition may be mixed; by combining, it should be understood that the individual components of the combination need not be mixed and may be provided separately, for example in separate containers, which may optionally be mixed.

[0276] In one embodiment, a composition or combination comprising a first polypeptide and a second polypeptide according to the invention is provided, wherein the second polypeptide has IgG-specific endopeptide activity.

[0277] In one embodiment, the second polypeptide having IgG-specific endopeptide activity comprises a polypeptide having the sequence shown in any one of SEQ ID NO: 23 to 25 or consists of a polypeptide having the sequence shown in any one of SEQ ID NO: 23 to 25.

[0278] In one embodiment, a composition or combination comprising a first polypeptide and a second polypeptide according to the invention is provided, wherein the second polypeptide has IgG-specific endopeptide activity and comprises a sequence or fragment thereof having at least 85% sequence identity (e.g., at least 90%, at least 95%, at least 98%, or at least 99%) with respect to the sequence shown in SEQ ID NO: 23.

[0279] In particular, it is envisioned that the polypeptide of the present invention can be used in combination with a second polypeptide having IgG-specific endopeptide activity, wherein the second polypeptide comprises or consists of a sequence lacking a C-terminal polyhistidine tag relative to SEQ ID NO: 23 to 25.

[0280] In one embodiment, the second polypeptide having IgG-specific intraprotease activity is IdeS (FabRICATOR), FabALACTICA, and / or Xork.

[0281] Table 5: Exemplary IgG-specific proteases

[0282]

[0283] The present invention also covers compositions comprising polypeptides, polynucleotides, carriers, and / or cells of the present invention mixed with aqueous components (e.g., diluents, salts, and / or buffers) suitable for their storage.

[0284] This document also provides a pharmaceutical composition comprising a mixture of the polypeptides, polynucleotides, carriers and / or cells of the present invention with pharmaceutically acceptable diluents, salts and / or carriers.

[0285] Reagent test kit

[0286] This document provides a kit comprising any polypeptide, polynucleotide, composition, combination, and / or cell according to the present invention. In some embodiments, the kit includes packaging and / or instructions for use.

[0287] In one embodiment, a kit is provided that comprises one or more of the following:

[0288] a) The polypeptide according to the present invention;

[0289] b) The composition according to the invention;

[0290] c) The combination according to the invention;

[0291] d) Polynucleotides according to the present invention;

[0292] e) the carrier according to the invention; and / or

[0293] f) Cells according to the present invention.

[0294] In embodiments where the kit contains combinations, the first and second peptides may be provided separately (i.e., in different containers within the kit or in different kits) or together (i.e., in the same kit and / or in the same container within the kit).

[0295] It should be understood that any component of the kit according to the invention can be used in the methods or uses of the invention as defined herein. Furthermore, the kit according to the invention may include instructions for use related to any of the methods or uses of the invention.

[0296] Methods and uses

[0297] This invention also provides methods and uses related to the polynucleotides, peptides, compositions, combinations, carriers, cells, and kits of this invention.

[0298] Any method or use described herein may be performed in vivo, in vitro, or ex vivo.

[0299] This article provides a method for hydrolyzing IgM, which includes contacting a sample containing IgM with a polypeptide or a composition or combination according to the invention.

[0300] The present invention also provides a method for detecting the presence of IgM in a sample, the method comprising:

[0301] a) Contacting the sample with the polypeptide, composition, or combination of the present invention; and

[0302] b) Identify IgM-specific cleavage products.

[0303] The presence of IgM-specific cleavage products indicates the presence of IgM in the sample.

[0304] The present invention also provides a method for detecting the presence of IgM in a sample, the method comprising:

[0305] a) Contacting the sample with the polypeptide, composition, or combination of the present invention; and

[0306] b) Identification and isolation of IgM-specific cleavage products

[0307] The presence of IgM-specific cleavage products indicates the presence of IgM in the sample.

[0308] In some embodiments, the method of the present invention includes a separation step. The separation step may include the use of gel electrophoresis and / or chromatography.

[0309] In some embodiments, the method of the present invention includes identification and / or analytical steps. The identification and / or analytical steps may include the use of gel electrophoresis, chromatography, immunoblotting, ELISA, and / or mass spectrometry.

[0310] Therefore, in some embodiments, the method of the present invention includes identification, separation, and / or steps, comprising using:

[0311] a) Gel electrophoresis;

[0312] b) Immunoblotting;

[0313] c) Chromatography;

[0314] d) ELISA; and / or

[0315] e) Mass spectrometry.

[0316] The present invention also provides the use of peptides, compositions, combinations, or kits according to the present invention for degrading IgM in samples.

[0317] The present invention also provides the use of the polypeptides, compositions, combinations, or kits according to the invention for removing IgM from a sample.

[0318] The present invention also provides the use of peptides, compositions, combinations, or kits according to the invention for identifying IgM in samples.

[0319] In one embodiment of the aforementioned method or use, IgM is human IgM.

[0320] In one embodiment of the aforementioned method or use, the sample is not an in vivo sample.

[0321] In one embodiment of the aforementioned method or use, the sample is an ex vivo sample.

[0322] In one embodiment of the aforementioned method or use, the sample is a sample derived from a human object.

[0323] The peptides of the present invention exhibit proven activity in both simple and complex media, wherein the substrate may be considered substantially pure (e.g., purified IgM sample) or substantially impure (e.g., crude or complex sample).

[0324] In one implementation, the IgM in the sample is substantially pure.

[0325] In one implementation, the IgM in the sample is substantially impure.

[0326] In one embodiment, the sample comprises or is composed of a composite medium (e.g., a complex biological medium).

[0327] In one embodiment, the sample comprises or consists of whole blood, blood-derived fractions, serum, and / or plasma.

[0328] In a preferred embodiment, the sample is a human sample.

[0329] In some implementations, the sample is an ex vivo sample.

[0330] In some implementations, the sample is obtained during the subject's dialysis.

[0331] In some embodiments, the sample is obtained from a human subject who does not have an identified disease or condition. In other embodiments, the sample is obtained from a human subject who does not have an identified IgM-related disease or condition.

[0332] In some implementations, the sample is obtained from a human subject, wherein the subject suffers from:

[0333] a) Autoimmune diseases or conditions;

[0334] b) Cancer;

[0335] c) Suspected or known microbial infection.

[0336] In some implementations, the microorganism is a bacterium, a virus, or a parasite.

[0337] In some implementations, the microorganism is a pathogenic organism.

[0338] In some embodiments, the aforementioned method includes the steps of identifying and / or separating IgM-specific cleavage products.

[0339] In a further embodiment, the method further includes contacting the sample with a polypeptide having IgG-specific endopeptide activity.

[0340] In some embodiments, the method or use of the present invention may be for the purpose of degrading or removing IgM from a sample.

[0341] In some embodiments, the method or use of the present invention may be for diagnostic purposes.

[0342] In some embodiments, the method or use of the present invention may be for diagnostic purposes, wherein the assay is an anti-drug antibody (ADA) assay.

[0343] Antidrug assays are used to identify the presence of antibodies against or induced by a drug of interest. Typically, these assays are performed using bridging ELISAs, which do not distinguish between antibody isotypes. The IgM-degradable peptides according to the invention can be used to remove or inactivate IgM in a sample, and thus facilitate increased resolution of ADA assays or other similar assays (containing IgM in a mixture of multiple components) by determining the contribution of IgM to any observed activity.

[0344] In some embodiments, the methods or uses of the present invention may be for the purpose of quality control of diagnostic assays, such as the quality control of recombinant or non-recombinant monoclonal and / or polyclonal IgM or IgM-derived molecules.

[0345] To develop IgM, such as IgM for therapeutic purposes, IgM for diagnostic purposes, or IgM-like drugs, extensive quality control is necessary to validate the drug and, for example, the presence of its sequence and / or post-translational modifications. Typically, this type of quality control involves the use of trypsin or similar "bottom-up" methods, which lack specificity and are therefore subject to improvement. Peptide IgM proteases that hydrolyze the hinge region of IgM can facilitate this type of analysis.

[0346] In some embodiments, the methods or uses of the present invention can be used for gene therapy purposes.

[0347] IgM has been shown to significantly (negatively) affect gene transfer in adenovirus type 5 (ADF5), one of the most commonly used adenovirus vectors. IgM reduces vector transduction in the liver. Therefore, the use of peptide degradation, inactivation, or removal of IgM in this invention could facilitate gene therapy applications, particularly those using ADF5 vectors.

[0348] In one embodiment, a method or use according to the invention is provided, wherein the method or use is for removing IgM during gene therapy. "During" can refer to pretreatment, concomitant treatment, or post-treatment of a target or sample containing the polypeptide of the invention.

[0349] In some embodiments, the methods or uses of the present invention can be used to treat or prevent cancer.

[0350] In some embodiments, the methods or uses of the present invention can be used to treat or prevent diffuse large B-cell lymphoma.

[0351] Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL), affecting thousands of people each year, and has a poor prognosis if left untreated. Even with treatment, the overall five-year survival rate is low. Lymphoma B cells typically possess IgM as their B-cell receptor (BCR), and removing or degrading this receptor using the peptides of this invention can aid in cancer treatment, for example, by inducing apoptosis and clearing lymphoma B cells.

[0352] In some embodiments, the methods or uses of the present invention can be used to treat or prevent autoimmune diseases or conditions.

[0353] In some embodiments, the methods or uses of the present invention can be used to treat or prevent systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and / or Sjögren's syndrome.

[0354] Several autoimmune diseases, including SLE, RA, and Sjögren's syndrome, are characterized by high levels of rheumatoid factor (RF), which is commonly used as a prognostic biomarker for these diseases. High RF levels are associated with worse outcomes and more severe pathogenesis. RF is typically an IgM-based antibody against the body's own IgG, which stimulates the formation of immune complexes that contribute to disease, for example, by causing chronic inflammation and joint destruction. Therefore, using the peptides of the present invention to degrade or remove IgM in these diseases could aid in their treatment or prevention.

[0355] In one aspect, the use of the polypeptide, combination, composition or pharmaceutical composition according to the invention in the remanufacturing of a pharmaceutical remedy is provided.

[0356] In one aspect, the polypeptide, combination, composition or pharmaceutical composition according to the invention is provided for use in a therapeutic manner.

[0357] In one aspect, the polypeptide, combination, composition or pharmaceutical composition according to the invention is provided for use in treating or preventing diseases or conditions associated with elevated IgM.

[0358] In one embodiment, a polypeptide, combination, composition, or pharmaceutical composition according to the invention is provided for use in treating or preventing cancer.

[0359] In one embodiment, the use of the polypeptide, combination, composition, or pharmaceutical composition according to the invention for the treatment or prevention of diffuse large B-cell lymphoma is provided.

[0360] In one embodiment, the polypeptide, combination, composition, or pharmaceutical composition according to the invention is provided for use in treating or preventing autoimmune diseases or conditions.

[0361] In one embodiment, the use of the polypeptide, combination, composition, or pharmaceutical composition according to the invention for the treatment or prevention of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and / or Sjögren's syndrome is provided.

[0362] In one embodiment, the use of the polypeptide, combination, composition, or pharmaceutical composition according to the invention for treating or preventing IgM nephropathy is provided.

[0363] Aspects of the present invention

[0364] The present invention will be described in terms of the following numbered aspects.

[0365] Aspect 1. A polypeptide having endopeptide protease activity, the polypeptide comprising:

[0366] a) The amino acid sequence of SEQ ID NO: 1;

[0367] b) A variant thereof that has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 1; or

[0368] c) fragments of a) or b).

[0369] Aspect 2. A polypeptide having endopeptide protease activity, the polypeptide comprising:

[0370] a) The amino acid sequence of SEQ ID NO: 8;

[0371] b) A variant thereof that has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 8; or

[0372] c) fragments of a) or b).

[0373] Aspect 3. The polypeptide according to Aspect 1 or Aspect 2, wherein the variant of said polypeptide has at least 85% sequence identity, at least 90% sequence identity, at least 91% sequence identity, at least 92% sequence identity, at least 93% sequence identity, at least 94% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 8.

[0374] Aspect 4. The polypeptide according to aspect 1 or aspect 3, wherein said polypeptide comprises or is composed of the following:

[0375] a) The amino acid sequence of SEQ ID NO: 2;

[0376] b) A variant thereof that has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 2; or

[0377] c) fragments of a) or b).

[0378] Aspect 5. The polypeptide according to Aspect 2 or Aspect 3, wherein the polypeptide comprises or is composed of the following:

[0379] a) The amino acid sequence of SEQ ID NO: 9;

[0380] b) A variant thereof that has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 9; or

[0381] c) fragments of a) or b).

[0382] Aspect 6. The polypeptide according to any one of Aspects 1 to 5, wherein the fragment length of said polypeptide is at least 100 amino acids, at least 150 amino acids, at least 200 amino acids, at least 250 amino acids, at least 300 amino acids, at least 350 amino acids, at least 400 amino acids, at least 450 amino acids, at least 500 amino acids, at least 550 amino acids, at least 600 amino acids, or at least 650 amino acids.

[0383] Aspect 7. The polypeptide according to any one of Aspects 1 to 6, wherein said polypeptide comprises:

[0384] a) An additional methionine at the N-terminus; and / or

[0385] b) His tag.

[0386] Aspect 8. The polypeptide according to aspect 7, wherein the His tag is located at the C-terminus.

[0387] Aspect 9. The polypeptide according to aspect 7, wherein the His tag is located at the N-terminus.

[0388] Aspect 10. The polypeptide according to any one of Aspects 1 to 8, wherein the polypeptide comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 3, 5, 6, 7 and 10.

[0389] Aspect 11. The polypeptide according to any one of Aspects 1 to 7 and 9, wherein the polypeptide comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 4 and 11.

[0390] Aspect 12. The polypeptide according to any one of Aspects 1 to 8 and 10, wherein the polypeptide comprises or consists of SEQ ID NO: 7.

[0391] Aspect 13. The polypeptide according to any one of Aspects 1 to 12, wherein the endopeptide activity is IgM-specific endopeptide activity.

[0392] Aspect 14. The polypeptide according to any one of Aspects 1 to 13, wherein the polypeptide cleaves human IgM.

[0393] Aspect 15. The polypeptide according to any one of Aspects 1 to 14, wherein the polypeptide is provided in solution, lyophilized or immobilized form.

[0394] Aspect 16. A composition or combination comprising a first polypeptide according to any one of aspects 1 to 15 and a second polypeptide, wherein the second polypeptide has IgG-specific endopeptide activity.

[0395] Aspect 17. A polynucleotide encoding a polypeptide of any one of aspects 1 to 15.

[0396] Aspect 18. A carrier encoding or containing a polynucleotide according to aspect 17.

[0397] Aspect 19. A cell comprising a polypeptide of any one of aspects 1 to 15, a polynucleotide of aspect 17, or a carrier of aspect 18.

[0398] Aspect 20. According to aspect 20, the cell is a bacterial cell.

[0399] Aspect 21. A reagent kit comprising:

[0400] a) A polypeptide according to any one of aspects 1 to 15;

[0401] b) The composition or combination according to aspect 16, wherein the first polypeptide and the second polypeptide are provided individually or together;

[0402] c) Polynucleotides according to aspect 17;

[0403] d) According to aspect 19, the carrier; and / or

[0404] e) According to aspect 20 of the cells.

[0405] Aspect 22. A method for hydrolyzing IgM, the method comprising contacting a sample containing IgM with a polypeptide of any one of aspects 1 to 15 or a composition or combination of aspect 16.

[0406] Aspect 23. The method according to aspect 22, wherein the IgM is human IgM.

[0407] Aspect 24. The method according to aspect 22, wherein the IgM is non-human primate IgM, preferably IgM from monkeys of the genus *Macaques*, such as macaques and / or cynomolgus monkeys, and / or monkeys of the genus *Baboon*.

[0408] Aspect 25. The method according to any one of Aspects 22 to 24, wherein the sample is:

[0409] a) Basically pure; or

[0410] b) It is basically impure.

[0411] Aspect 26. The method according to any one of Aspects 22 to 25, wherein the sample contains complex biological media.

[0412] Aspect 27. The method according to any one of Aspects 22 to 26, wherein the sample comprises blood, serum and / or plasma.

[0413] Aspect 28. The method according to any one of Aspects 22 to 27, wherein the sample comprises human blood, human serum and / or human plasma.

[0414] Aspect 29. The method according to any one of Aspects 22 to 28, wherein the method includes the steps of identifying and / or separating IgM-specific cleavage products.

[0415] Aspect 30. The method according to any one of Aspects 22 to 29, wherein the method further comprises contacting the sample with a polypeptide having IgG-specific endonuclease activity, optionally wherein the polypeptide having IgG-specific endonuclease activity comprises or consists of a polypeptide having the sequence shown in any one of SEQ ID NO: 23 to 25.

[0416] Aspect 31. A method for detecting the presence of IgM in a sample, the method comprising:

[0417] a) Contacting the sample with a polypeptide according to any one of aspects 1 to 15 or a composition or combination according to aspect 16; and

[0418] b) Identify and / or isolate IgM-specific cleavage products.

[0419] The presence of IgM-specific cleavage products indicates the presence of IgM in the sample.

[0420] Aspect 32. The method according to any one of Aspects 22 to 31, wherein the identification and separation steps include the use of:

[0421] a) Gel electrophoresis;

[0422] b) Immunoblotting;

[0423] c) Chromatography;

[0424] d) ELISA; and / or

[0425] e) Mass spectrometry.

[0426] Aspect 33. Use of a polypeptide according to any one of aspects 1 to 15, a composition or combination according to aspect 16, or a kit according to aspect 21 for the degradation, removal, or identification of IgM in a sample.

[0427] Aspect 34. The method according to any one of aspects 22 to 32, or the use according to aspect 33, wherein the sample is an in vitro, in vivo, or ex vivo sample.

[0428] Aspect 35. The method according to any one of aspects 22 to 32, or the application according to aspect 33 or aspect 34, wherein the method or application is:

[0429] a) Used in quality control (QC) processes; and / or

[0430] b) Used for diagnostic testing.

[0431] Aspect 36. The method according to any one of aspects 22 to 32 and 35, or the application according to aspects 33 to 35, wherein the method or application is:

[0432] a) Used to degrade or remove IgM during dialysis;

[0433] b) Quality control of recombinant or non-recombinant monoclonal and / or polyclonal IgM or IgM-derived molecules; and / or

[0434] c) Used for the assay of anti-drug antibodies (ADA);

[0435] Aspect 37. The method or use according to any one of Aspects 22 to 36, wherein the method or use is non-therapeutic.

[0436] Aspect 38. The method or use according to any one of Aspects 22 to 36, wherein the method or use is therapeutic.

[0437] Aspect 39. The method according to any one of Aspects 22 to 32, 35 and 38, or the use according to Aspects 33 to 36 and 38, wherein the method or use is for treatment or prevention:

[0438] a) Kidney disease or dysfunction;

[0439] b) Cancer; and / or

[0440] c) Autoimmune diseases or conditions.

[0441] Aspect 40. The method or use according to aspect 39, wherein the method or use is for treatment or prevention:

[0442] a) Diffuse large B-cell lymphoma;

[0443] b) Systemic lupus erythematosus (SLE);

[0444] c) Rheumatoid arthritis (RA);

[0445] d) Sjögren's syndrome; and / or

[0446] e) IgM nephropathy.

[0447] Aspect 41. The use of a polypeptide according to any one of aspects 1 to 15, a composition or combination according to aspect 16, or a kit according to aspect 21 for therapeutic purposes.

[0448] Aspect 42. A polypeptide according to any one of Aspects 1 to 15, a composition or combination according to Aspect 16, or a kit according to Aspect 21, for treating or preventing a disease or condition, wherein the disease or condition is:

[0449] a) Kidney disease or dysfunction;

[0450] b) Cancer; and / or

[0451] c) Autoimmune diseases or conditions.

[0452] Aspect 43. The polypeptide, composition, combination, or kit according to aspect 42, wherein the disease or condition is:

[0453] a) Diffuse large B-cell lymphoma;

[0454] b) Systemic lupus erythematosus (SLE);

[0455] c) Rheumatoid arthritis (RA);

[0456] d) Sjögren's syndrome; and / or

[0457] e) IgM nephropathy.

[0458] Aspect 44. A polypeptide according to any one of aspects 1 to 15, a composition or combination according to aspect 16, or a kit according to aspect 21, for use in the manufacture of a pharmaceutical product.

[0459] Aspect 45. The polypeptide, composition, combination, or kit according to aspect 44, wherein the medicine is used to treat or prevent a disease or condition, wherein the disease or condition is:

[0460] a) Kidney disease or dysfunction;

[0461] b) Cancer; and / or

[0462] c) Autoimmune diseases or conditions.

[0463] Aspect 46. The polypeptide, composition, combination, or kit according to aspect 44 or aspect 45, wherein the drug is used to treat or prevent a disease or condition, wherein the disease or condition is:

[0464] a) Diffuse large B-cell lymphoma;

[0465] b) Systemic lupus erythematosus (SLE);

[0466] c) Rheumatoid arthritis (RA);

[0467] d) Sjögren's syndrome; and / or

[0468] e) IgM nephropathy.

[0469] The invention will now be further described by way of examples, which are intended to help those skilled in the art to implement the invention, and are not intended to limit the scope of the invention in any way.

[0470] Example

[0471] Example 1: Identification, isolation, and purification of IgM-specific protease from Lachnoanaerobaculum

[0472] Identification of potential proteases in Lachnoanaerobaculum

[0473] The inventors used a combination of bioinformatics and biochemical analysis to identify homologs of the only known IgM-specific endonuclease IdeSSuis (Seele J. et al., J Bacteriol 2013, 195(5)).

[0474] Although none of the known Lachnoanaerobaculum isolates have shown proteolytic or specific immunoglobulin modification activity, the inventors have surprisingly discovered genes containing IdeS-like domains that show low sequence similarity to the known IgM-specific endonuclease IdeSSuis.

[0475] The identification of genes containing IdeS-like domains was unexpected for two reasons: (1) these domains were previously identified primarily in streptococci, and (2) the presence of enzymes containing IdeS-like domains is associated with pathogens rather than with symbiotic organisms that form part of the normal human flora.

[0476] Synthesis of candidate proteins

[0477] Many constructs were designed and synthesized and used to express the desired proteins in recombinant host cells.

[0478] Design and synthesize constructs according to Table 5.

[0479] Table 5: Design and synthesis of peptides

[0480]

[0481] Full-length constructs containing the IdeS / Mac-1 gene from *L. gingivalis* and *L. umeaense* were expressed, with N-terminal or C-terminal His tags (*L. gingivalis* D1-N-10H, *L. umeaense* D1-N-10H, *L. umeaense* D1-C-6His, and *L. umeaense* D1-C-6His, respectively). The yield of the full-length constructs was less than 5 mg / g, and the resulting peptides were relatively low in purity and fragmented.

[0482] Constructs from *L. umeaense* were selected for further engineering to express only certain fragments of the full-length protein. The *L. umeanse Suis* (LU-S) construct expressed well and produced high yields after optimized purification, but the resulting protein was prone to fragmentation under test conditions. Shorter fragments of 38 amino acids (SuisΔ38; LU-d38S) were more stable under test conditions, but yielded lower amounts.

[0483] The construct was further engineered and optimized until the construct (L. umeaense MAC-1; further referred to as LU-M1) was found to be expressed in high yield (> 20 mg / g), high purity and uniformity. Figure 1 LU-M1 expression is scalable, with a yield of approximately 10 mg / g when expressed in a bioreactor where the culture reaches a high density (220 g cells per 600 mL batch).

[0484] LU-M1 shows little sequence identity with many known streptococcal virulence factors (Table 6). However, this construct exhibits a higher level of sequence identity with proteins containing L. gingivalis IdeS / Mac-1.

[0485] Table 6: Sequence identity between LU-M1 and other bacterial proteases

[0486]

[0487] All constructs were codon-optimized for *E. coli* and inserted into the pET21a+ backbone, then transformed into BL21(DE3)STAR cells under ampicillin selection. Clones were cultured in ampicillin-supplemented LB flasks, and protein expression was performed in baffled 2L flasks via OD expression with the addition of 1 mM IPTG. 600Induction was performed at a temperature of 0.5–0.7. After induction at 20–37°C for 4 hours, cells were harvested by centrifugation and maintained at -20°C until further processing. Cells were lysed by sonication, and lysates were removed from cell debris by centrifugation. The material was purified on His GraviTRAP, and purity was assessed by SDS-PAGE.

[0488] To express LU-M1 in a bioreactor system, recombinant E. coli BL21(DE3) STAR was used to produce LU-M1. Fermentation was carried out using a high-cell-density fed-batch strategy and a chemically defined basal medium. The induction, harvesting, and purification steps were similar to those used in small-scale production.

[0489] Example 2: Functional characterization of IgM-specific proteases from Lachnoanaerobaculum

[0490] To investigate the activity of the full-length constructs (LG-FL and LU-FL), the purified proteins were incubated with human and mouse IgG. Figure 2A Only LG-FL exhibited some activity against human serum IgG, further demonstrating its specificity against human IgG2. Figure 2B ).

[0491] Despite their homology with known IgG proteases, and due to their limited activity (LG-FL) or inactivity (LU-FL) against human IgG, purified LU-FL and LG-FL were incubated with human IgA and IgM. Surprisingly, both LG-FL and LU-FL showed activity against IgM; and no IgA-specific activity was observed. Figure 2C ).

[0492] The IgM-specific activity observed in the full-length construct was retained by the shorter engineered fragment LU-M1. Figure 2D ).

[0493] Furthermore, LU-M1 has been shown to have no activity against the broad-spectrum protease substrate casein. Figure 2E It may also lack activity against IgM from rodent species (mice, rabbits, rats). However, LU-M1 showed some activity against IgM purified from monkeys. Figure 2F The purified IgM from monkeys was obtained from Rockland (017-0107) and was a combination of IgM from rhesus monkeys, cynomolgus monkeys and baboons.

[0494] General protease activity was determined using the EnzCheck protease assay kit according to the manufacturer's instructions. Broad-spectrum protease SpeB (FabULOUS) was used as a positive control, and IgG-specific protease IdeS (FabRICATOR) was used as a negative control. All reactions were measured in a fluorescent microplate reader after incubation at 37°C for 1 hour and 2 hours.

[0495] In addition, to assess species specificity, IgM from different species were incubated with LU-M1 at 37°C for 1 hour (1:40, w:w, enzyme:substrate) before SDS-PAGE analysis.

[0496] Myeloma IgM was deglycosylated with PNGase F and incubated with LU-M1 to investigate the hydrolysis sites (Figure 3). Only the heavy chain was hydrolyzed, while the light and J chains remained intact. The total intact mass of the deglycosylated heavy chain fragments (63140.6167 Da) was in excellent agreement with the measured mass of the reduced IgM heavy chain (63140.9971 Da). Upon addition of LU-M1, the heavy chain broke into m / z 37499.6142 and m / z 25659.0200. The C-terminal fragment (25659.0200 Da) could be attributed to the theoretical mass (25657.6646 Da) of amino acids 221-453 in the human IgM constant region (UniProt accession number: P01871).

[0497] IgM myeloma (Sigma-Aldrich) was incubated in PBS buffer at a 1:40 (w:w) enzyme-to-substrate ratio for 1 h with and without purified recombinant LU-M1. The mixture was then denatured and reduced at 90 °C in 50 mM DTT (GBiosciences) and 0.5% (v / v) lauryl sarcosine for 5 min, cooled to room temperature, and then 2 U / μg PNGase F (Genovis) was added. N-deglycosylation was performed at 37 °C for 2 h before TBS dialysis of the samples using a Slide-A-Lyzer MINI device (ThermoFisherScientific). The hydrolysate was denatured at 37 °C for 30 min in 4 M GnHCl (Sigma-Aldrich) and 100 mM DTT. Digestion products were separated on an acetonitrile gradient on a reverse-phase C4 column (Acquity premier BEHC4, 450 Å, 2.7 μm 2.1 x 100 mm, Waters) and analyzed by ESI-QTOF-MS (Bruker Impact II). Deconvolution was performed using Bruker Compass DataAnalysis version 5.2 and the MaxEnt algorithm.

[0498] Digestion occurs below the CH2 region of human IgM (...VPDQDT / AIRVFA...), similar to the porcine IgM protease IdeSSuis. Due to the intermonomer disulfide bond between cysteine ​​residues 413 in CH3, the native reaction produces F(ab')2 (VH-CH1-CH2) and the pentamer Fc (CH3-CH4) (...). Figure 4 ).

[0499] Example 3: Optimization and Analysis of Protease Activity

[0500] To further evaluate the activity of LU-M1, the enzymatic activity of the peptide was assessed in terms of pH, salt (NaCl), divalent cations, temperature, and sensitivity / resistance to protease inhibitors.

[0501] LU-M1 can hydrolyze IgM (5.5 to 9.0) over a wide pH range; Figure 5A The activity was not significantly reduced.

[0502] Enzyme activity appeared to be negatively affected by increasing NaCl concentration, with a 12.5% ​​decrease observed in the presence of 150 mM NaCl, a 40% decrease in 500 mM NaCl, and a 75% decrease in 1000 mM NaCl. Figure 5B ).

[0503] LU-M1 is highly active in the absence of any added divalent cations. Figure 5C Conversely, adding Mg 2+ and Ca 2+ After ionization, the activity appears to decrease slightly. In Zn 2+ Aggregation was observed in the presence of EDTA. Furthermore, the addition of EDTA appeared to cause a concentration-dependent decrease in enzyme activity (30% to 50%, depending on concentration).

[0504] Enzyme activity is enhanced by increasing temperature; however, LU-M1 retains some activity (35% hydrolysis) at 4°C. Although slower than at 37°C, the enzyme can efficiently hydrolyze IgM at room temperature. Figure 1 D).

[0505] Next, the activity of LU-M1 was evaluated in the presence of a set of protease inhibitors. Figure 5E Before adding LU-M1 (1:50 w:w LU-M1:IgM), serum IgM was pre-incubated with a group of protease inhibitors (G-Biosciences) for 15 minutes, and the mixture was incubated at 37°C for 30 minutes before analysis by SDS-PAGE.

[0506] Based on the inhibitory activity of these protease inhibitors, LU-M1 was classified as a cysteine ​​protease; the presence of iodoacetamide completely inhibited its activity, while the presence of AEBSF and chymostatin showed strong inhibitory activity. Other protease inhibitors evaluated had no significant effect on enzyme activity.

[0507] LU-M1 was incubated with IgM (IgM myeloma (20 μg)) at different ratios (1:20, 1:50, and 1:100; LU-M1 to IgM (w:w)) for 5 to 60 minutes to study hydrolysis efficiency. At low enzyme:substrate ratios (i.e., high relative enzyme concentrations), almost all IgM was hydrolyzed within 5 minutes; at a 1:100 ratio, all IgM was hydrolyzed within 60 minutes. Figure 6A , Figure 6B Similarly, higher substrate concentrations lead to faster hydrolysis. Figure 6C ).

[0508] Unless otherwise specified, for all activities related to optimal conditions, use an enzyme:substrate ratio of 1:100 and incubate at 37°C for 30 minutes.

[0509] Next, the enzymes' ability to function in complex media was evaluated. Undiluted serum was incubated with IgG-specific protease IdeS, IgM-specific protease LU-M1 as defined herein, or combinations thereof, and the resulting mixtures were analyzed using SDS-PAGE and Western blotting.

[0510] Human heat-inactivated serum (Sigma, H2667) was incubated with IdeS (1:50, w:w, enzyme:IgG) and LU-M1 (1:40, w:w, enzyme:IgM) at 37°C for 30 min. Materials were analyzed by SDS-PAGE and Western blotting. Anti-human IgM ALP (Mabtech, 3880-9A-1000; 1:1000) and anti-human IgG ALP (Mabtech, 3310-1-1000; 1:2000) were used as primary antibodies, and a Western blotting kit (Invitrogen) was used for blocking, washing, and chromogenic substrate detection.

[0511] Both IdeS and LU-M1 were found to be highly specific, targeting only IgG and IgM in the sample, respectively. Furthermore, it was observed that both enzymes could operate in the same sample without significantly affecting each other's function (Figure 7).

[0512] These data demonstrate the ability of LU-M1 to specifically hydrolyze IgM in essentially impure samples, such as complex biological samples like serum.

[0513] Example 4: Evaluating the ability to operate LU-M1

[0514] The ability to change the state of LU-M1 to facilitate handling of LU-M1 under various conditions was evaluated.

[0515] First, LU-M1 was lyophilized at different concentrations and volumes. All conditions produced highly active material, thus indicating that LU-M1 is stable in lyophilized form and can be reconstituted to produce active enzymes (Figure 8).

[0516] Lyophilization was performed in a Cool-Safe 10-4 Pro (ScanVac). Briefly, the aluminum block in the vial was pre-cooled at -80°C, then the sample (25 μL to 100 μL, 0.5 mg / mL to 2 mg / mL) was resuspended in TBS and added to the block, and incubated overnight at -80°C. The sample was lyophilized for 24 hours, and the particles were stored at -20°C.

[0517] The activity of the lyophilized material was assessed by reconstitution of the material in milliQ water to 40 U / μL (1 U = 0.02 μg). Prior to SDS-PAGE analysis, the recombinase was incubated with 1 U / μg IgM in PBS at 37°C for 1 hour (Figure 8).

[0518] Next, LU-M1 was immobilized at working concentrations of 20 mg / mL to 30 mg / mL according to the manufacturer's instructions (NHS-activated agarose gel, Cytiva). The activity of the immobilized protein was evaluated by mixing 50 μL of resin with 100 μg IgM (100 μL) and incubating at room temperature with end-to-end shaking for 15 to 30 minutes. LU-M1 was readily immobilized at high concentrations (20 mg / mL to 30 mg / mL) with good coupling efficiency (92% to 94%) and retained activity ( Figure 8C ).

[0519] These data indicate that LU-M1 is readily lyophilized / immobilized and retains its functionality after these processes, demonstrating the enzyme's broad applicability in many situations and indicating its suitability for storage and / or transport in forms other than isolated aqueous proteins.

[0520] Example 5: Evaluation of IVIG responsiveness to LU-M1

[0521] The presence of LU-M1-specific human IgG was assessed using SDS-PAGE analysis and Western blotting. Figure 9 ).

[0522] IdeS, Xork, and LU-M1, each at 0.5 μg, were separated by SDS-PAGE and transferred to nitrocellulose membranes for Western blotting. The membranes were blocked in casein solution before adding 5 μg / mL of the polyclonal primary antibody mixture IVIG. The membranes were incubated with the primary antibody at room temperature for 2 hours. After washing, the membranes were incubated with secondary antibody (alkaline phosphatase-conjugated α-human IgG HC, 1:2000) at room temperature for 1 hour. The membranes were thoroughly washed before adding the chromogenic substrate BCIP / NBT.

[0523] Western blot data indicated the presence of few or no anti-LU-M1 antibodies in IVIG. The absence or low levels of anti-enzyme antibodies in human IVIG are advantageous and highly desirable. Specifically, the lack or scarcity of anti-enzyme antibodies facilitates human treatment because it increases the number of patients who are sensitive to treatment and improves treatment efficiency, for example, by preventing enzyme removal or adverse reactions.

Claims

1. A polypeptide having endoprotease activity, comprising: a) the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 8; b) a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 8; or c) a fragment of a) or b).

2. The polypeptide of claim 1, wherein the polypeptide comprises or consists of: a) the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 9; b) a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 9; or c) a fragment of a) or b).

3. The polypeptide of claim 1 or claim 2, wherein the polypeptide comprises: a) an additional methionine at the N-terminus; and / or b) a His tag; wherein the His tag is at the C-terminus or the N-terminus.

4. The polypeptide of any one of claims 1 to 3, wherein the polypeptide comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 3, 4, 5, 6, 7, 10, and 11.

5. The polypeptide of any one of claims 1 to 4, wherein the polypeptide comprises or consists of SEQ ID NO:

7.

6. The polypeptide of any one of claims 1 to 5, wherein the endoprotease activity is an IgM-specific endoprotease activity.

7. The polypeptide of any one of claims 1 to 6, wherein the polypeptide cleaves human IgM.

8. The polypeptide of any one of claims 1 to 7, wherein the polypeptide is provided in solution, lyophilized, or immobilized.

9. A composition or combination comprising a first polypeptide according to any one of claims 1 to 8 and a second polypeptide, wherein the second polypeptide has an IgG-specific endoprotease activity.

10. A polynucleotide encoding a polypeptide according to any one of claims 1 to 8.

11. A vector encoding or comprising a polynucleotide according to claim 10.

12. A cell comprising a polypeptide of any one of claims 1 to 8, a polynucleotide of claim 10, or a vector of claim 11.

13. A kit comprising: a) a polypeptide according to any one of claims 1 to 8; b) a composition or combination according to claim 9, wherein the first polypeptide and the second polypeptide are provided separately or together; c) a polynucleotide according to claim 10; d) a vector according to claim 11; and / or e) a cell according to claim 12.

14. A method of hydrolyzing IgM, the method comprising contacting a sample comprising IgM with a polypeptide according to any one of claims 1 to 8 or a composition or combination according to claim 10.

15. The method according to claim 14, wherein the IgM is human IgM.

16. The method according to any one of claims 13 to 15, wherein the sample comprises a complex biological medium, preferably wherein the sample comprises blood, serum and / or plasma, more preferably wherein the sample comprises human blood, human serum and / or human plasma.

17. The method according to any one of claims 13 to 16, wherein the method comprises the step of identifying and / or isolating an IgM-specific cleavage product.

18. The method according to any one of claims 13 to 17, wherein the method further comprises contacting the sample with a polypeptide having endoprotease activity specific for IgG.

19. A method of detecting the presence of IgM in a sample, the method comprising: a) contacting a sample with a polypeptide according to any one of claims 1 to 8 or a composition or combination according to claim 9; and b) identifying and / or isolating an IgM-specific cleavage product, wherein the presence of the IgM-specific cleavage product is indicative of the presence of IgM in the sample.

20. Use of a polypeptide according to any one of claims 1 to 8, a composition or combination according to claim 9 or a kit according to claim 12 for degrading, removing or identifying IgM in a sample.

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