Recombinant glycosidases, pharmaceutical compositions, and uses in managing autoimmune or inflammatory conditions

Abstract

This disclosure relates to recombinant immunoglobulin endoglycosidases and uses in managing diseases or conditions associated with immune dysregulation or inflammation. In certain embodiments, the recombinant immunoglobulin endoglycosidases comprise sequences disclosed herein. In certain embodiments, this disclosure relates to pharmaceutical compositions and methods of treating or preventing an autoimmune disease or inflammatory condition comprising administering to a subject in need thereof an effective amount of a recombinant immunoglobulin endoglycosidase, or nucleic acids or vector encoding the same, as disclosed herein.

Description

[0001] RECOMBINANT GLYCOSIDASES, PHARMACEUTICAL COMPOSITIONS, AND USES IN MANAGING AUTOIMMUNE OR INFLAMMATORY CONDITIONS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 525,476 filed July 7, 2023 and U.S. Provisional Application No. 63 / 584,527 filed September 22, 2023. The entirety of each of these applications is hereby incorporated by reference for all purposes.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under Al 149297 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED AS AN XML FILE VIA THE OFFICE ELECTRONIC FILING SYSTEM

[0007] The Sequence Listing associated with this application is provided in XML format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing is 23002PCT.xml. The XML file is 48 KB, was created on July 5, 2024, and is being submitted electronically via the USPTO patent electronic filing system.

[0008] BACKGROUND

[0009] Humans produce antibodies containing immunoglobulin G (IgG) proteins which are abundantly present in the blood. As part of the immune system, IgG antibodies play a central role in protecting the body from pathogens by recognizing the presence of biological material that is not a “self-molecule.” When IgG antibodies specifically bind a pathogen, a series of biological events occur that cause the body to attack the pathogen. However, environmental factors, genetic defects, and aging sometimes inadvertently directs these same events to occur abnormally. Thus, the presence of undesirable IgG antibodies are often the cause of autoimmune diseases and inflammatory disorders. Recombinant therapeutic antibody therapies exist for certain autoimmune diseases and inflammatory disorders; however, treatments are not always universally effective and sometimes have side effects. Thus, there is a need to identify improvements. Human TgG proteins contain variable regions and constant regions (Fc). The Fc region contains an asparagine amino acid at position 297, (Asn297 or N297), which is commonly substituted to contain different chains of sugar units, also referred to as a glycan. The state of glycosylation at Asn297 is reported to be involved regulating immunological events. See Flevaris et al. Int J Mol Sci, 2022, 23, 5180.

[0010] Flevaris et al. report immunoglobulin GN-glycan biomarkers for autoimmune diseases. Int J Mol Sci, 2022, 23, 5180

[0011] Shadnezhad et al. report CP40 from Corynebacterium pseudotuberculosis is an endo- -N- acetylglucosaminidase. BMC Microbiology, 2016, 16:261.

[0012] Yamin et al. report human FcyRIIIa activation on splenic macrophages drives dengue pathogenesis in mice. Nat Microbiol, 2023, 8(8): 1468-1479.

[0013] References cited herein are not an admission of prior art.

[0014] SUMMARY

[0015] This disclosure relates to immunoglobulin endoglycosidases and uses in managing diseases or conditions associated with immune dysregulation. In certain embodiments, this disclosure relates to methods of cleaving or altering glycans attached to immunoglobulins comprising contacting a glycosylated immunoglobulin and an immunoglobulin endoglycosidase disclosed herein providing a glycan cleaved immunoglobulin.

[0016] In certain embodiments, this disclosure relates to recombinant immunoglobulin endoglycosidases comprising sequences disclosed herein. In certain embodiments, this disclosure relates to nucleic acids and vectors encoding immunoglobulin endoglycosidases disclosed herein.

[0017] In certain embodiments, this disclosure relates to pharmaceutical compositions comprising recombinant immunoglobulin endoglycosidases disclosed herein and / or nucleic acids or vector encoding the same.

[0018] In certain embodiments, this disclosure relates to methods of treating or preventing an autoimmune disease or condition associated with glycosylated immunoglobulins comprising administering to a subject in need thereof an effective amount of a recombinant immunoglobulin endoglycosidase, or nucleic acids or vectors encoding the same, as disclosed herein.

[0019] In certain embodiments, the recombinant immunoglobulin endoglycosidase comprises the amino acid sequence of (CU43) AALSNAPLAASPGQADKVGAQATCAAKPIFFGYYRTWRDKAIELNDGDKWKDK LHTKLTDIPEQVDMVSLFHVPDNQKSDQRFWETFDKEYHPTLKERGTKVVRTIGAKLLL NKIKEKGL YGQ SREDD SK YREI AHE V YEE Y V AK HNLDGLD VDMELRE VEK YTNLR WQ LRKIMGAFSELMGPKAPGNAGKKPGDDGYKYLIYDTFDNAQLAQVALVADVVDYVLA QTYDKGTEESITRVWNGFRDKINSCQFLAGYAHPEENDTNRFLTAIGDVDTSGAMNVA AWKPEGGEKGGTFAYALDRDGRTYDGDDLTTLKPTDFAFTKRAIELTKGISLTDLG (SEQ ID NO: 19).

[0020] In certain embodiments, the recombinant immunoglobulin endoglycosidase comprises the amino acid sequence of (CP258) ADLSQAPLKASPGHADKVGVQTTCDAKPIFFGYYRTWRDKAIQLKDDDPWKDKLQVK LTDIPEHVNMVSLFHVEDNQKSDDQFWETFRKEYQPKLKERGTRVVRTVGAQLLLNKI KEKGLYGRSVEDDYKYREIARDIYKKYVTDHNLDGLDVDMELRKVEKRIDLQWQLRKI MGAFSELMGPKAPANEGKKPGHEGKKPGHEGYKYLIYDTFDNAQTSQVGLVADLVDY VLAQTYDKGTKESIDQVWNGFRDKINSCQFMAGYAHPEENDTNRFLTAVGEVNKSGA MQVAEWKPDNGVKGGTFAYALDRDGRTYDGDDFTTLKPTDFAFTKRAIELTTGESSTD LG (SEQ ID NO: 20).

[0021] In certain embodiments, the recombinant immunoglobulin endoglycosidase comprises the amino acid sequence of (CM49)

[0022] ASPTSLPLPEHKGFHPDAGERSDCEPIVTAYYRTWRDKEIQQLPDDRVGPNVIAM TDIPHNIDVLSLIHVPDHQQSDQQFWATFASTYLPELHRRGTKVLYTLDISAVLDPSLKPT S SPEEYAAHAQRL VDKYVRPHKLDGLDIDMERDLSFDQRIVLRNTMRALTQLVGPF SNT NTLLTYNTNRNAQKAYIDEVAQYVNYVFVQTYSVTDPNEIQYSYWNTYRFYLSSCQFL PSYANPEEFDRNRFLGAIGPVEETAAVKIAGWQPFQGGVKGGIMTYAIDRDGMTYDQP DISTLRVTTFPVIKRVTAVLKAKKFAAAK (SEQ ID NO: 21).

[0023] In certain embodiments, the recombinant immunoglobulin endoglycosidase comprises the amino acid sequence of (CR39)

[0024] PAIAETSLRGGTGQHETPGSQKCDSEPVFFAYYRTWRDKAIVQNESDKPNRANKI ALTDIPQHVNMVSLFHAGNDHYQSDAEFWKTFDDVYYPELKRRGTRVVRTISATELLK ETD SLRA VGVDTD AAD YREVAEKIKKEYVD AHNLDGLD VDME VLHLERNWRS SWEK RWRIRKTMAALSELLGPKADVNQGKKKDDSGYKFLIYDTFDDVERSQIRAIAELVDYVL PQTYK SGK AEIDQL WNQ SKGIL S SCQF VPGYAHPEEGDT VNRFETAIGD VD S SK AME VA AWQPAGGEKGGAFVYAIDRDGRTYGEDDLKNVKETDFSFTKRGAALARSVTFAKAK (SEQ ID NO: 46).

[0025] In certain embodiments, this disclosure relates to a fusion protein comprising any of the immunoglobulin endoglycosidases as provided herein.

[0026] In certain embodiments, this disclosure relates to a nucleic acid or vector encoding a recombinant immunoglobulin endoglycosidase as provided herein in operable combination with a heterologous promoter. In certain embodiments, this disclosure relates to vectors comprising a nucleic acid or encoding a recombinant immunoglobulin endoglycosidase as provided herein. In certain embodiments, this disclosure relates to attenuated bacterial strains (non-naturally occurring) such as attenuated Corynebacterium pseudotuberculosis strains comprising a nuclei acid encoding a recombinant immunoglobulin endoglycosidase as provided herein. In certain embodiments, the nucleic acid is mRNA, RNA, or DNA.

[0027] In certain embodiments, this disclosure relates to methods of treating or preventing a disease or condition mediated by IgG antibodies or associated with altered Asn297 IgG glycosylation comprising administering to a subject in need thereof an effective amount of a recombinant immunoglobulin endoglycosidase as provided herein, or nucleic acid, vector, or attenuated bacterial strains such as attenuated Corynebacterium pseudotuberculosis strain encoding a recombinant immunoglobulin endoglycosidase as provided herein.

[0028] In certain embodiments, this disclosure relates to methods of treating or preventing a viral infection comprising administering to a subject in need thereof an effective amount of a recombinant immunoglobulin endoglycosidase as provided herein, or nucleic acid, vector, or attenuated bacterial strains such as attenuated Corynebacterium pseudotuberculosis strain encoding a recombinant immunoglobulin endoglycosidase as provided herein.

[0029] In certain embodiments, this disclosure relates to methods of treating or preventing antibody-mediated depletion of B cells comprising administering to a subject in need thereof an effective amount of a recombinant immunoglobulin endoglycosidase as provided herein, or nucleic acid, vector, or attenuated bacterial strains such as attenuated Corynebacterium pseudotuberculosis strain encoding a recombinant immunoglobulin endoglycosidase as provided herein.

[0030] In certain embodiments, this disclosure relates to methods of treating or preventing antibody-mediated cytotoxicity against CD4 T cells comprising administering to a subject in need thereof an effective amount of a recombinant immunoglobulin endoglycosidase as provided herein, or nucleic acid, vector, or attenuated bacterial strains such as attenuated Corynebacterium pseudotuberculosis strain encoding a recombinant immunoglobulin endoglycosidase as provided herein.

[0031] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0032] Figure 1A shows a sequence comparison of CU43 (SEQ ID NO: 19, QI) and CP40 (SEQ ID NO: 47, SI).

[0033] Figure IB shows a sequence comparison of CP258 (SEQ ID NO: 20, QI) and CP40 (SEQ ID NO: 47, SI).

[0034] Figure 1C shows a sequence comparison of CM49 (SEQ ID NO: 21, QI) and CP40 (SEQ ID NO: 51, SI).

[0035] Figure ID shows a sequence comparison of CR39 (SEQ ID NO: 49, QI) and CP40 (SEQ ID NO: 50, SI) (CR39).

[0036] Figure 2A shows data on the percentage of IgGl-Fc using an LC-MS based assay to study the substrate specificity of CP40 and CP40-like IgG-specific proteins.

[0037] Figure 2B shows data on the percentage of HM-IgG.

[0038] Figure 3 shows data on the in vivo half-life of CU-43 and CM-49 Fc fusion constructs in FcyR humanized mice. To determine the in vivo stability and half-life of the EndoS-Fc fusion constructs, FcyR humanized mice (n=3 / group) were administered intravenously with 50 pg of CU- 43 or CM-49 (enzymatically active or inactive). The serum levels of CU-43 or CM-49 at different points following administration were determined by ELISA and CU-43 / CM-49 were detected using anti-human IgG secondary antibodies.

[0039] Figure 4A shows data on the in vivo activity of CU-43 Fc fusion constructs to prevent cytotoxic mAb-mediated depletion of CD4+ T cells in FcyR humanized mice. FcyR humanized mice (n=2-4 / group) were administered intravenously with the indicated dose of the chimeric rat anti-mouse CD4 mAb (clone YTS191) expressed as wild-type (WT) human IgGl (hlgGl), afucosylated hlgGl, and hlgGl N297A. The levels of CD4+ T cells in the blood were determined at various timepoints by flow cytometry. Depletion of CD4+ T cells was observed in YTS191 WT hlgGl and to a greater extent in YTS afucosylated hlgGl -treated mice, whereas the YTS191 hlgGl N297A variant (unable to engage Fey Rs) had minimal impact on CD4+ T cell levels. Figure 4B shows additional data. Since depletion of CD4+ T cells by the YTS191 mAh is dependent on Fc-FcyR interactions, the capacity of CU43 Fc fusion constructs (enzymatically active or inactive) to prevent mAb-mediated depletion was evaluated in vivo in FcyR humanized mice. Mice (n=5-6 / group) were treated with 10 pg afucosylated hlgGl YTS191, followed by the indicated dose of CU43 Fc fusion constructs (enzymatically active or inactive). The abundance of CD4+ T cells in peripheral blood was assessed by flow cytometry.

[0040] Figure 5A shows data wherein titration of CU-43 Fc fusion constructs were used to determine the minimum dose required to prevent cytotoxic mAb-mediated depletion of CD4 T cells in FcyR humanized mice. FcyR humanized mice (n=2-4 / group) were treated with 10 pg afucosylated hlgGl YTS191, followed by the indicated dose of CU43 Fc fusion constructs (1 - 50 pg) and the abundance of CD4+ T cells in peripheral blood at the indicated timepoints was assessed by flow cytometry.

[0041] Figure 5B shows data using 0.01 - 1 pg of CU43 Fc fusion constructs.

[0042] Figure 6 shows data comparing of the activity of CU-43 Fc fusion constructs in FcyR humanized mice with or without the human IgGl transgene. FcyR humanized mice express all classes of human FcyRs, whereas hlgGl / hFcR mice additionally express human IgGl, which has been inserted as transgene into the mouse IgG2c locus (Ighg2c). To determine whether CU43 Fc fusion constructs are capable of preventing cytotoxic mAb-mediated depletion of CD4+ T cells, FcyR humanized or hlgGl / hFcR mice (n=2-6 / group) were treated with 10 pg afucosylated hlgGl YTS191, followed by the indicated dose of CU43 Fc fusion constructs. The abundance of CD4+ T cells in peripheral blood was assessed at the indicated timepoints by flow cytometry and was found to be comparable between the two mouse strains. These results indicate that in the presence of endogenous human IgGl, which could potentially compete with YTS191 for CU-43 -mediated Fc-glycan cleavage, CU-43 still exhibits potent activity and has the capacity to abrogate the YTS191 mAb-mediated cytotoxicity.

[0043] Figure 7 shows data comparing of the ability of CU-43 Fc and CM-49 Fc to prevent cytotoxic mAb-mediated depletion of CD4+ T cells in FcyR humanized mice. The capacity of CU- 43 Fc and CM-49 Fc to prevent mAb-mediated depletion was evaluated in vivo in FcyR humanized mice. Mice (n=3 / 4 / group) were treated with 10 pg afucosylated hlgGl YTS191 (anti -mouse CD4) followed by 0.1 pg CU43-Fc (enzymatically active or inactive) or CM49-Fc (enzymatically active). The abundance of CD4+ T cells in peripheral blood was assessed by flow cytometry one day before injection with YTS191 and Fc-fusion enzymes (baseline) and three days post-injection and expressed as % change from baseline.

[0044] Figure 8A shows data from an evaluation of CU-43 Fc activity in a model of autoimmune hemolytic anemia. The anti-human CD47 monoclonal antibody 5F9 induces the depletion ofRBCs in vivo via an FcR-mediated mechanism. The capacity of CU-43 Fc to modulate 5F9-mediated depletion of red blood cells was evaluated in mice humanized for CD47, SIRPa, and FcyRs. Mice (n=3-4 / group) were injected intravenously with the 5F9 mAb (expressed as human IgGl wild-type or as an IgGl Fc variant with no binding to all FcyR classes (GRLR variant; G236R / L328R) at a dose of 5 mg / kg followed by CU-43 Fc (enzymatically active or inactive (CU43i)) at a dose of 0.1 pg / mouse. RBC counts were measured one day pre-injection (d-1) and one day post-injection (dl), expressed as % change from baseline.

[0045] Figure 8B shows data on mouse survival that was recoded and compared among treatment groups (Mantel Cox log rank test; CU43i vs CU43 active).

[0046] Figure 9A shows data indicating CU-43 Fc has the ability to abrogate antibody-dependent enhancement (ADE) of dengue disease. The ability of CU-43 Fc to modulate ADE of dengue disease was evaluated in a murine model of dengue disease (Yamin et al, 2023 Nat Microbiol). Six hours before infection with DENV2 virus (3.5xl08GE, i.v.), type I interferon-knockout mice humanized for FcyRs (Ifnarl- / - / hFcR mice; n=3 mice / group) were pre-treated with 20 pg (i.v.) of the anti-DENV2 E protein antibody CIO (hlgGl) modified to either enhance (afucosylated) binding to FcyRIIIa or abrogate (GRLR variant (G236R / L328R)) binding to all FcRs followed by i.v. treatment with either 5 pg CU-43 Fc or an equivalent volume of PBS (i.v ). Weights were monitored for six days following infection.

[0047] Figure 9B shows survival data.

[0048] Figure 10 shows data indicating the prevention of antibody -mediated depletion of B cells by CU-43 Fc. FcyR / CD20 humanized mice (n=4 mice / group) were co-treated (i.v.) with the B cell depleting anti -human CD20 antibody 2B8 (expressed as human IgGl GAALIE; G236A / A330L / I332E for enhanced binding to activating human Fey Rs) at a dose of 1 mg / kg followed by 0.5 pg CU-43 Fc (enzymatically active or inactive) or PBS. B cell counts in peripheral blood (B220+ cells) were assessed three days post-injection by flow cytometry and compared.

[0049] Figure 11 shows data comparing the in vivo activity of CU-43-Fc and IdeS-Fc to prevent mAb-mediated cytotoxicity against CD4 T cells. To determine the protective dose of CU43 Fc fusion constructs and IdeS-Fc fusion constructs against mAb-mediated cytotoxicity, FcyR humanized mice (n=3-4 / group) were treated (i.v.) with 10 pg afucosylated human IgGl YTS191, followed by the indicated dose of CU43 or IdeS-Fc fusion constructs. The abundance of CD4+ T cells in peripheral blood was assessed at the indicated timepoints by flow cytometry and normalized to baseline.

[0050] Figure 12A shows time course analysis data of a head-to-head comparison of the in vivo protective activity of CU43-Fc and efgartigimod against mAb-mediated cytotoxicity in FcyR humanized mice. FcyR humanized mice (n=4-6 / group) were treated (i.v.) with 10 pg chimeric rat anti -mouse CD4 mAb (clone YTS191) expressed as afucosylated human IgGl followed by the indicated dose of CU43-Fc fusion construct or efgartigimod (i.v.; dose expressed as mg / kg). The abundance of CD4+ T cells in peripheral blood was assessed at the indicated timepoints by flow cytometry (expressed as CD4+ / CD3+ and normalized to baseline.

[0051] Figure 12B shows a day 1 time point.

[0052] Figure 13 shows data on the impact of CU43-Fc and efgartigimod treatment on the capacity of neutralizing anti-influenza HA mAbs to confer in vivo protection against lethal influenza challenge. CU43-Fc and efgartigimod both prevent mAb-mediated cytotoxicity; CU43-Fc hydrolyses the Fc-associated glycan to prevent Fc-FcyR interactions, whereas efgartigimod targets the FcRn pathway to accelerate IgG catabolism. To determine the impact of these two therapeutic strategies on the in vivo neutralizing activity of anti-HA mAbs during influenza infection, FcyR humanized mice were treated (i.v.) with the indicated dose of CU43-Fc or efgartigimod (EFG) along with the neutralizing anti-HA mAb 7B2 (0.25 mg / kg, i.v.) one day prior to lethal challenge with H1N1 (Neth / 09; i.n. 5mLD5O). Weight was monitored for a period of 14 days post-infection and compared between groups.

[0053] DETAILED DISCUSSION

[0054] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims or as amended during prosecution.

[0055] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0056] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0057] An "embodiment" of this disclosure refers to an example, but not necessarily limited to such example. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0058] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.

[0059] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. In this specification and in the claims that follow reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.

[0060] As used in this disclosure and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") have the meaning ascribed to them in U.S. Patent law in that they are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0061] "Consisting essentially of' or "consists of' or the like, when applied to methods and compositions encompassed by the present disclosure refers to compositions like those disclosed herein that exclude certain prior art elements to provide an inventive feature of a claim, but which may contain additional composition components or method steps, etc., that do not materially affect the basic and novel characteristic(s) of the compositions or methods. A "subject" refers to any animal, preferably a human patient, livestock, or domestic pet.

[0062] As used herein, the terms "treat" and "treating" are not limited to the case where the subject (e.g., patient) is cured and the disease is eradicated. Rather, embodiments, of the present disclosure also contemplate treatment that merely reduces symptoms, and / or delays disease progression.

[0063] As used herein, the terms "prevent" and "preventing" include the prevention of the recurrence, spread or onset. It is not intended that the present disclosure be limited to complete prevention. In some embodiments, the onset is delayed, or the severity of the disease is reduced.

[0064] A “nucleic acid” refers to a DNA- or RNA-molecule and is used synonymous with polynucleotide. Wherever herein reference is made to a nucleic acid or nucleic acid sequence encoding a particular protein and / or peptide, said nucleic acid or nucleic acid sequence, respectively, preferably also comprises regulatory sequences allowing in a suitable host, e.g., a human being, its expression, i.e., transcription and / or translation of the nucleic acid sequence encoding the particular protein or peptide.

[0065] "Amino acid sequence" is defined as a sequence composed of any one of the 20 naturally appearing amino acids, amino acids which have been chemically modified, or composed of synthetic amino acids. The terms "protein" and "peptide" refer to compounds comprising amino acids joined via peptide bonds and are used interchangeably. As used herein, where "amino acid sequence" is recited herein to refer to an amino acid sequence of a protein molecule. An "amino acid sequence" can be deduced from the nucleic acid sequence encoding the protein. Furthermore, unless the context demands otherwise, the term "peptide" and "polypeptide" and "protein" are used interchangeably to refer to amino acids in which the amino acid residues are linked by covalent peptide bonds or alternatively (where post-translational processing has removed an internal segment) by covalent disulfide bonds, etc. The ammo acid chains can be of any length and comprise at least three amino acids, they can include domains of proteins or full-length proteins. Unless otherwise stated the terms peptide, polypeptide, and protein also encompass various modified forms thereof, including but not limited to glycosylated forms, phosphorylated forms, etc.

[0066] The term “comprising” in reference to a peptide having an amino acid sequence refers a peptide that may contain additional N-terminal (amine end) or C-terminal (carboxylic acid end) amino acids, i.e., the term is intended to include the amino acid sequence within a larger peptide. The term “consisting of’ in reference to a peptide having an amino acid sequence refers a peptide having the exact number of amino acids in the sequence and not more or having not more than a range of amino acids expressly specified in the claim. In certain embodiments, the disclosure contemplates that the “N-terminus of a peptide consists of an amino acid sequence,” which refers to the N-terminus of the peptide having the exact number of amino acids in the sequence and not more or having not more than a range of amino acids specified in the claim; however, the C- terminus may be connected to additional amino acids, e.g., as part of a larger peptide. Similarly, the disclosure contemplates that the “C-terminus of a peptide consists of an amino acid sequence,” which refers to the C-terminus of the peptide having the exact number of amino acids in the sequence and not more or having not more than a range of amino acids specified in the claim; however, the N-terminus may be connected to additional amino acids, e.g., as part of a larger peptide. In certain embodiments, this disclosure relates to proteins disclosed herein consisting of sequences disclosed herein having less than an additional 10 or 50 amino acids on the N-terminus. In certain embodiments, this disclosure relates to proteins disclosed herein consisting of sequences disclosed herein having less than an additional 10 or 50 amino acids on the C-terminus.

[0067] The term "recombinant" when made in reference to a nucleic acid molecule refers to a nucleic acid molecule that is comprised of segments of nucleic acid joined together by means of molecular biological techniques. The term "recombinant" when made in reference to a protein or a polypeptide refers to a protein molecule that is expressed using a recombinant nucleic acid molecule.

[0068] A "heterologous" nucleic acid sequence or peptide sequence refers to a nucleic acid sequence or a peptide sequence that does not naturally occur, e.g., because the whole sequence contains a segment from other plants, bacteria, viruses, other organisms, or joinder of two sequences that occur the same organism but are joined together in a manner that does not naturally occur in the same organism or any natural state.

[0069] The terms "vector" or " expression vector " refer to a recombinant nucleic acid containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism or expression system, e.g., cellular or cell-free. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. Protein "expression systems" refer to in vivo (e.g. host cell) and in vitro (cell free) systems. Systems for recombinant protein expression typically utilize somatic cells transfecting with a DNA or mRNA expression vector that contains the template. The cells are cultured under conditions such that they translate the desired protein. Expressed proteins are extracted for subsequent purification. In vivo protein expression systems using prokaryotic and eukaryotic cells are well known. Proteins may be recovered using denaturants and protein-refolding procedures. In vitro (cell-free) protein expression systems typically use translation-compatible extracts of whole cells or compositions that contain components sufficient for transcription, translation, and optionally post-translational modifications such as RNA polymerase, regulatory protein factors, transcription factors, ribosomes, tRNA cofactors, amino acids, and nucleotides. In the presence of an expression vectors, these extracts and components can synthesize proteins of interest. Cell-free systems typically do not contain proteases and enable labeling of the protein with modified amino acids. Some cell free systems incorporated encoded components for translation into the expression vector. See, e.g., Shimizu et al., Cell-free translation reconstituted with purified components, 2001, Nat. Biotechnol., 19, 751-755 and Asahara & Chong, Nucleic Acids Research, 2010, 38(13): el41, both hereby incorporated by reference in their entirety.

[0070] A “selectable marker” is a nucleic acid introduced into a recombinant vector that encodes a polypeptide that confers a trait suitable for artificial selection or identification (report gene), e.g., beta-lactamase confers antibiotic resistance, which allows an organism expressing beta-lactamase to survive in the presence antibiotic in a growth medium. Another example is thymidine kinase, which makes the host sensitive to ganciclovir selection. It may be a screenable marker that allows one to distinguish between wanted and unwanted cells based on the presence or absence of an expected color. For example, the lac-z-gene produces a beta-galactosidase enzyme which confers a blue color in the presence of X-gal (5-bromo-4-chloro-3-indolyl-P-D-galactoside). If recombinant insertion inactivates the lac-z-gene, then the resulting colonies are colorless. There may be one or more selectable markers, e.g., an enzyme that can complement to the inability of an expression organism to synthesize a particular compound required for its growth (auxotrophic) and one able to convert a compound to another that is toxic for growth. URA3, an orotidine-5' phosphate decarboxylase, is necessary for uracil biosynthesis and can complement ura3 mutants that are auxotrophic for uracil. URA3 also converts 5-fluoroorotic acid into the toxic compound 5-fluorouracil. Additional contemplated selectable markers include any genes that impart antibacterial resistance or express a fluorescent protein. Examples include, but are not limited to, the following genes: ampr, camr, tetr, blasticidinr, neor, hygr, abxr, neomycin phosphotransferase type II gene (nptll), p-glucuronidase (gus), green fluorescent protein (gfp), egfp, yfp, mCherry, p- galactosidase (lacZ), lacZa, lacZAM15, chloramphenicol acetyltransferase (cat), alkaline phosphatase (phoA), bacterial luciferase (luxAB), bialaphos resistance gene (bar), phosphomannose isomerase (pmi), xylose isomerase (xylA), arabitol dehydrogenase (atlD), UDP- glucose:galactose-l -phosphate uridyltransferasel (galT), feedback-insensitive a subunit of anthranilate synthase (0ASA1D), 2-deoxy lucose (2-DOGR), benzyladenine-N-3 -glucuronide, E. coli threonine deaminase, glutamate 1 -semialdehyde aminotransferase (GSA-AT), D-amino acidoxidase (DAAO), salt-tolerance gene (rstB), ferredoxin-like protein (pflp), trehalose-6-P synthase gene (AtTPSl), lysine racemase (lyr), dihydrodipicolinate synthase (dapA), tryptophan synthase beta 1 (AtTSBl), dehalogenase (dhlA), mannose-6-phosphate reductase gene (M6PR), hygromycin phosphotransferase (HPT), and D-serine ammonialyase (dsdA).

[0071] A "label" refers to a detectable compound or composition that is conjugated directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymatic linkages, and radioactive isotopes. A label includes the incorporation of a radiolabeled amino acid or the covalent attachment of biotinyl moieties to a polypeptide that can be detected by marked avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Various methods of labeling polypeptides and glycoproteins are known in the art and may be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionucleotides (such as18F,35S or131I) fluorescent labels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic labels (such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (such as a leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), or magnetic agents, such as gadolinium chelates. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0072] In certain embodiments, the disclosure relates to recombinant polypeptides comprising sequences disclosed herein or variants or fusions thereof wherein the amino terminal end or the carbon terminal end of the amino acid sequence are optionally attached to a heterologous amino acid sequence, label, or reporter molecule.

[0073] In certain embodiments, the disclosure relates to the recombinant vectors comprising a nucleic acid encoding a polypeptide disclosed herein or chimeric protein thereof.

[0074] In certain embodiments, the recombinant vector optionally comprises a mammalian, human, insect, viral, bacterial, bacterial plasmid, yeast associated origin of replication or gene such as a gene or retroviral gene or lentiviral LTR, TAR, RRE, PE, SLIP, CRS, and INS nucleotide segment or gene selected from tat, rev, nef, vif, vpr, vpu, and vpx or structural genes selected from gag, pol, and env.

[0075] In certain embodiments, the recombinant vector optionally comprises a gene vector element (nucleic acid) such as a selectable marker region, lac operon, a CMV promoter, a hybrid chicken B-actin / CMV enhancer (CAG) promoter, tac promoter, T7 RNA polymerase promoter, SP6 RNA polymerase promoter, SV40 promoter, internal ribosome entry site (IRES) sequence, cis-acting woodchuck post regulatory element (WPRE), scaffold-attachment region (SAR), inverted terminal repeats (ITR), FLAG tag coding region, c-myc tag coding region, metal affinity tag coding region, streptavidin binding peptide tag coding region, polyHis tag coding region, HA tag coding region, MBP tag coding region, GST tag coding region, polyadenylation coding region, SV40 polyadenylation signal, SV40 origin of replication, Col El origin of replication, fl origin, pBR322 origin, or pUC origin, TEV protease recognition site, loxP site, Cre recombinase coding region, or a multiple cloning site such as having 5, 6, or 7 or more restriction sites within a continuous segment of less than 50 or 60 nucleotides or having 3 or 4 or more restriction sites with a continuous segment of less than 20 or 30 nucleotides.

[0076] The term "fusion" when used in reference to a polypeptide refers to the expression product of two or more coding sequences obtained from different sources such that they do not exist together in a natural environment, that have been cloned together and that, after translation, act as a single polypeptide sequence. Fusion polypeptides are also referred to as "hybrid" polypeptides. The coding sequences include those obtained from the same or from different species of organisms.

[0077] As used herein, an “CP40,” refers to an approximately 40kDa protein produced by Corynebacterium tuberculosis. CP40 is believed to be an endoglycosidase with putative activity on host glycoproteins and all known variants or substantial fragments thereof. One example has the amino acid sequence MHNSPRSVSRLITVGITSALFASTFSAVASAESATLSKEPLKASPGRADTVGVQTT CNAKPIFFGYYRTWRDKAIQLKDDDPWKDKLQVKLTDIPEHVNMVSLFHVEDNQKSDQ QFWETFHREYQPELKKRGTRVVRTVGAQLLLNKIKDKNLYGKHVEDDYKYREIARDV YNEYVVKHNLDGLDVDMELRQVEKQLNLKWQLRKIMGAFSELMGPKAPANEGKKPD HEGYKYLIYDTFDNAQTSQVGLVADLVDYVLAQTYKKDTKESVTQVWNGFRDKINSC QFMAGYAHPEENDTNRFLTAVGEVNKSGAMQVAEWKPEGGEKGGTFAYALDRDGRT YDGDDFTTLKPTDFAFTKRAIELTTGESSTDLGKPTGSR (SEQ ID NO: 18) NCBI Reference Sequence: WP_013242749.1) wherein the N-terminal Methionine (M) is position one. Other examples include those designated by Accession numbers: WP_013242749.1, WP_058831578.1, WP_014522771.1, WP_058156943.1, WP_014733097.1, WP_138130220.1, WP_045421254.1, and WP_072577955.1.

[0078] In certain embodiments, this disclosure relates to a recombinant immunoglobulin endoglycosidase comprising the amino acid sequence of (CU43)

[0079] AALSNAPLAASPGQADKVGAQATCAAKPIFFGYYRTWRDKAIELNDGDKWKDK LHTKLTDIPEQVDMVSLFHVPDNQKSDQRFWETFDKEYHPTLKERGTKVVRTIGAKLLL NK I K EKGLYGQ SREDD SK YREIAHE V YEE Y V AK HNLDGLD VDMELRE VEK YTNLR WQ LRKIMGAFSELMGPKAPGNAGKKPGDDGYKYLIYDTFDNAQLAQVALVADVVDYVLA QTYDKGTEESITRVWNGFRDKINSCQFLAGYAHPEENDTNRFLTAIGDVDTSGAMNVA AWKPEGGEKGGTFAYALDRDGRTYDGDDLTTLKPTDFAFTKRAIELTKGISLTDLG (SEQ ID NO: 19) or variant thereof.

[0080] In certain embodiments, this disclosure relates to a recombinant immunoglobulin endoglycosidase comprising the amino acid sequence of (CP258) ADLSQAPLKASPGHADKVGVQTTCDAKPIFFGYYRTWRDKAIQLKDDDPWKDKLQVK LTDIPEHVNMVSLFHVEDNQKSDDQFWETFRKEYQPKLKERGTRVVRTVGAQLLLNKI KEKGLYGRSVEDDYKYREIARDIYKKYVTDHNLDGLDVDMELRKVEKRIDLQWQLRKI MGAFSELMGPKAPANEGKKPGHEGKKPGHEGYKYLIYDTFDNAQTSQVGLVADLVDY VLAQTYDKGTKESIDQVWNGFRDKINSCQFMAGYAHPEENDTNRFLTAVGEVNKSGA MQVAEWKPDNGVKGGTFAYALDRDGRTYDGDDFTTLKPTDFAFTKRAIELTTGESSTD LG (SEQ ID NO: 20) or variant thereof.

[0081] In certain embodiments, this disclosure relates to a recombinant immunoglobulin endoglycosidase comprising the amino acid sequence of (CM49) ASPTSLPLPEHKGFHPDAGERSDCEPIVTAYYRTWRDKEIQQLPDDRVGPNVIAMTDIPH NIDVLSLIHVPDHQQSDQQFWATFASTYLPELHRRGTKVLYTLDISAVLDPSLKPTSSPEE YAAHAQRLVDKYVRPHKLDGLDIDMERDLSFDQRIVLRNTMRALTQLVGPFSNTNTLL TYNTNRNAQKAYIDEVAQYVNYVFVQTYSVTDPNEIQYSYWNTYRFYLSSCQFLPSYA NPEEFDRNRFLGAIGPVEETAAVKIAGWQPFQGGVKGGIMTYAIDRDGMTYDQPDISTL RVTTFPVIKRVTAVLKAKKFAAAK (SEQ ID NO: 21) or variant thereof.

[0082] In certain embodiments, this disclosure relates to recombinant immunoglobulin endoglycosidases comprising peptide motifs of the following amino acid sequences from an N- terminal to C-terminal order sequentially, YYRTWRDK (SEQ ID NO: 1), TDIP, (SEQ ID NO: 2), LDGLDX1DME (SEQ ID NO: 3), SCQF (SEQ ID NO: 4), NRFL (SEQ ID NO: 5), and DRDG (SEQ ID NO: 6), wherein X1is I or V or any amino acid, or variant thereof, provided the immunoglobulin endoglycosidase does not contain an amino acid sequence KESV (SEQ ID NO: 48, contained in CP40) between LDGLDX1DME (SEQ ID NO: 3) and SCQF (SEQ ID NO: 4).

[0083] In certain embodiments, this disclosure relates to recombinant immunoglobulin endoglycosidases comprise peptide motifs of the following amino acid sequences from an N- terminal to C-terminal order sequentially, AXXXXXPLXXXXGXXXXXG (SEQ ID NO: 7), PIXXXYYRTWRDKXIXXXXXD (SEQ ID NO: 8), TDIPXXXXXXSLXHVXD, (SEQ ID NO: 9), QXSDXXFWXTFXXXYXPXLXXRGTXVXXTXXXXXXL (SEQ ID NO: 10), YXXXAXXXXXXYVXXHXLDGLDXDME (SEQ ID NO: 11)

[0084] LRXXMXAXXXLXGPXXXXN (SEQ ID NO: 12), LXYXTXXNAQXXXXXXV (SEQ ID NO: 13), AXXVXYVXXQTY (SEQ ID NO: 14), WNXXRXXXXSCQFXXXYAXPEEXDX (SEQ ID NO: 15), NRFLXAXGXGXVXXXXAXXXAXWXP (SEQ ID NO: 16), and GXKGGXXXYAXDRDGXTYDXXDXXTLXXTXFXXXKR (SEQ ID NO: 17), wherein X is individually and independently at each position is any amino acid, or variant thereof.

[0085] In certain embodiments, this disclosure relates to a recombinant immunoglobulin endoglycosidase comprising the amino acid sequence of (CR39) PAIAETSLRGGTGQHETPGSQKCDSEPVFFAYYRTWRDKAIVQNESDKPNRANKIALTDI PQHVNMVSLFHAGNDHYQSDAEFWKTFDDVYYPELKRRGTRVVRTISATELLKETDSL RAVGVDTDAADYREVAEKII<I<EYVDAHNLDGLDVDMEVLHLERNWRSSWEI<RWRIR KTMAALSELLGPKADVNQGKKKDDSGYKFLIYDTFDDVERSQIRAIAELVDYVLPQTYK SGKAEIDQLWNQSKGILSSCQFVPGYAHPEEGDTVNRFETAIGDVDSSKAMEVAAWQP AGGEKGGAFVYAIDRDGRTYGEDDLKNVKETDFSFTKRGAALARSVTFAKAK (SEQ ID NO: 46) or variants thereof.

[0086] In certain embodiments, this disclosure relates to recombinant immunoglobulin endoglycosidases comprising peptide motifs of the following amino acid sequences from an N- terminal to C-terminal order sequentially, PVFF (SEQ ID NO: 22), YYRTWRDKAI (SEQ ID NO: 23), LTDIP (SEQ ID NO: 24), MVSLFHA (SEQ ID NO: 25), VRTT (SEQ ID NO: 26), HNLDGLDVDME (SEQ ID NO: 27), GPKA (SEQ ID NO: 28), LIYDTFD (SEQ ID NO: 29), VDYVL (SEQ ID NO: 30), SCQF (SEQ ID NO: 4), GYAHPEE (SEQ ID NO: 31), NRFETAIG (SEQ ID NO: 32), DRDGRTY(SEQ ID NO: 33), TFKR (SEQ ID NO: 34), or variant thereof.

[0087] In certain embodiments, this disclosure relates to recombinant immunoglobulin endoglycosidases comprising peptide motifs of the following amino acid sequences from an N- terminal to C-terminal order sequentially, PXXXXXSLXXXXGXXXXXG (SEQ ID NO: 35), PVFFXYYRTWRDKAIXXXXXD (SEQ ID NO: 36), LTDIPXXVXMVSLFHAXN (SEQ ID NO: 37), YXSDXXFWXTFXXXYXPXLKXRGTXVRTTXXAXXLL (SEQ ID NO: 38), DXXYREXAXXXXXXYVXXHNLDGLDVDME (SEQ ID NO: 39),

[0088] WXIRKXMXAXSELXGPKAXXN (SEQ ID NO: 40), GYKXLIYDTFDXXXXXQ (SEQ ID NO: 41), AXXVDYVLXQTY (SEQ ID NO: 42), WNXXRXXXXSCQFXXGYAHPEEXDT (SEQ ID NO: 43), NRFETAIGXGXVXXSXAMXVAXWXP (SEQ ID NO: 44), and GGXKGGXFXYAXDRDGRTYXXDDXXXXKXTDFXTFKR (SEQ ID NO: 45), wherein X is individually and independently at each position is any amino acid or variant thereof.

[0089] A "variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide and retains essential properties. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall (homologous) and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and / or deletions). A substituted or inserted ammo acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.

[0090] Modifications and changes can be made in the structure of the peptides of this disclosure and still result in a molecule having similar characteristics as the peptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a peptide that defines the biological functional activity of the peptide, certain ammo acid sequence substitutions can be made in a peptide sequence and nevertheless obtain a peptide with like properties. Amino acid substitutions are generally based on the relative similarity of the ammo acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take one or more of the foregoing characteristics into consideration are well known to those of skill in the art and include, but are not limited to (original residue: exemplary substitution): (Ala to Gly or Ser), (Arg to Lys), (Asn to Gin or His), (Asp to Glu, Cys, or Ser), (Gin to Asn), (Glu to Asp), (Gly to Ala), (His to Asn, Gin), (Leu to He or Vai), (Lys to Arg), (Met to Leu, Tyr), (Ser to Thr), (Thr to Ser), (Trp to Tyr), (Tyr to Trp or Phe), and (Vai to He or Leu). Embodiments of this disclosure thus contemplate functional or biological equivalents of a peptide as set forth above. In certain embodiments, peptide variants are those having greater than 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 97%, and 99% sequence identity to the peptide of interest. In general, homologous peptides of the present disclosure are characterized as having one or more amino acid substitutions, deletions, and / or additions.

[0091] "Identity," as known in the art, is a relationship between two or more peptide sequences, as determined by comparing the sequences. In the art, "identity" also refers to the degree of sequence relatedness between peptides as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods. Preferred methods to determine identity are designed to give the largest match between the sequences tested. In certain embodiments, sequence "identity" refers to the number of exactly matching amino acids (expressed as a percentage) in a sequence alignment between two sequences of the alignment calculated using the number of identical positions divided by the greater of the shortest sequence or the number of equivalent positions excluding overhangs wherein internal gaps are counted as an equivalent position.

[0092] The term "sample" is used in its broadest sense, in that it has chemical makeup that is physical for analysis, i.e., analyte. In one sense it can refer to a nasal fluid, saliva, cough droplets, or expelled droplets of saliva into the air, e.g., produced by speaking, or other lung fluid blood. In another sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples include bodily fluids, urine, feces, nasal drip, seminal fluid, hair, skin (dead or epithelial layer of skin), finger or toenail clipping, and blood products such as plasma, serum, and the like. Preferably the sample is from a subject and encompass fluids, solids, tissues, and gases.

[0093] Methods of Use

[0094] In certain embodiments, this disclosure relates to methods of cleaving or altering a glycan attached to immunoglobulin comprising contacting a glycosylated immunoglobulin and an immunoglobulin endoglycosidase disclosed herein providing a glycan cleaved immunoglobulin. In certain embodiments, the immunoglobulin is in a sample from a subject, e g., human patient.

[0095] In certain embodiments, this disclosure relates to methods of treating or preventing a disease or condition mediated by IgG antibodies or associated with altered Asn297 IgG glycosylation comprising administering to a subject in need thereof an effective amount of a recombinant immunoglobulin endoglycosidase as provided herein, or nucleic acid, vector, or live attenuated bacterial strain, such as Corynebacterium pseudotuberculosis strain, encoding a recombinant immunoglobulin endoglycosidase as provided herein.

[0096] In certain embodiments, the disease or conditions is associated with increased or decreased galactosylation, sialylation, fucosylation, and / or the presence of bisecting N-acetylglucosamine (GlcNAc) chains.

[0097] In certain embodiments, the disease or condition is an autoimmune diseases or chronic inflammatory condition such as, including rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, autoimmune liver diseasejuvenile idiopathic arthritis, osteoarthritis, spondyloarthropathies, neonatal lupus, lupus nephritis, Sjogren’s syndrome, antineutrophil cytoplasmic antibody (ANCA)-associated antineutrophil cytoplasmic antibody (ANCA)- associated vasculitis (AAV), Crohn’s disease, ulcerative colitis, Hashimoto’s thyroiditis, multiple sclerosis, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, Myasthenia gravis, Lambert-Eaton myasthenic syndrome, autoimmune hemolytic anemia, and antiphospholipid syndrome.

[0098] Altered Asn297 IgG glycosylation (e.g., increased or decreased galactosylation, sialylation, fucosylation, and / or the presence of bisecting N-acetylglucosamine (GlcNAc) chains) are reported in patients with in autoimmune diseases and conditions such as rheumatoid arthritis, juvenile idiopathic arthritis, osteoarthritis, spondyloarthropathies, systemic lupus erythematosus, neonatal lupus, lupus nephritis, Sjogren’s syndrome, anti neutrophil cytoplasmic antibody (ANCA)-associated antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), Crohn’s disease, ulcerative colitis, Hashimoto’s thyroiditis, multiple sclerosis, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, Myasthenia gravis, Lambert- Eaton myasthenic syndrome, autoimmune hemolytic anemia, and antiphospholipid syndrome. See Flevaris et al. Int J Mol Sci, 2022, 23, 5180.

[0099] In certain embodiments, this disclosure relates to methods of treating or preventing myasthenia gravis or chronic inflammatory demyelinating polyneuropathy (CIDP) or neuromyelitis optica spectrum disorder (NMOSD) comprising administering to a subject in need thereof an effective amount of a recombinant immunoglobulin endoglycosidase as provided herein, or nucleic acid, vector, or attenuated bacterial strains such as attenuated Corynebacterium pseudotuberculosis strain encoding a recombinant immunoglobulin endoglycosidase as provided herein. In certain embodiments, subjects are diagnosed as being anti-acetylcholine receptor (AChR) positive and / or anti-muscle-specific tyrosine kinase (MuSK) antibody positive.

[0100] In certain embodiments, this disclosure relates to methods of treating or preventing primary membranous nephropathy, idiopathic inflammatory myopathy (IIM), anti-neutrophil cytoplasmic antibody-associated vasculitis (ANCA Vasculitis), Graves' ophthalmopathy, antibody-mediated rejection (AMR), immune-mediated necrotizing myopathy, anti -synthetase syndrome, dermatomyositis, polymyositis, immune thrombocytopenia, primary Sjogren's syndrome, rheumatoid arthritis, or pemphigus comprising administering to a subject in need thereof an effective amount of a recombinant immunoglobulin endoglycosidase as provided herein, or nucleic acid, vector, or attenuated bacterial strains such as attenuated Corynebacterium pseudotuberculosis strain encoding a recombinant immunoglobulin endoglycosidase as provided herein. In certain embodiments, subjects are diagnosed as being anti-acetylcholine receptor (AChR) and / or anti- muscle-specific tyrosine kinase (MuSK) antibody positive.

[0101] In certain embodiments, the administering a recombinant immunoglobulin endoglycosidase as provided herein, or nucleic acid, vector, or attenuated bacterial strains such as attenuated Corynebacterium pseudotuberculosis strain encoding a recombinant immunoglobulin endoglycosidase is administered optionally in combination with another pharmaceutical agent such as efgartigimod, efgartigimod alfa, efgartigimod alfa-fcab, hyaluronidase, hyaluronidase- qvfc, ravulizumab, ravulizumab-cwvz, rozanolixizumab, rozanolixizumab-noli, or combinations thereof.

[0102] In certain embodiments, this disclosure relates to methods of treating or preventing a viral infection comprising administering to a subject in need thereof an effective amount of a recombinant immunoglobulin endoglycosidase as provided herein, or nucleic acid, vector, or attenuated bacterial strain, e.g., Corynebacterium pseudotuberculosis strain, encoding a recombinant immunoglobulin endoglycosidase as provided herein. In certain embodiments, the viral infection is from a dengue viral infection. In certain embodiments, the viral infection is from an influenza viral infection.

[0103] In certain embodiments the viral infection may be from an adenovirus, herpesvirus, papilloma virus, polyomavirus, hepadnavirus, parvovirus, astrovirus, calcivirus, picornavirus, coronavirus, flavivirus, togavirus, hepevirus, retrovirus, orthomyxovirus, arenavirus, bunyaviruses, filovirus, paramyxovirus, rhabdovirus, reovirus, poxvirus, or virus that infect humans or other animals and causes an immunodeficiency, e.g., human immunodeficiency virus.

[0104] In certain embodiments, the viral infection is from a dengue viral infection, diphtheria viral infection, ebola viral infection, human coronaviral (HCOV) infection (types 299E, NL63, OC43, HKU1) SARS-CoV-1, SARS-CoV-2 viral infection, the virus that causes COVID-19, respiratory syncytial viral (RSV) infection, human papillomaviral (HPV) infection, varicella zoster viral (shingles), chikungunya viral infection, influenza viral infection, human parainfluenza viral (PIV) infection (types 1, 2, 3, and 4), human metapneumoviral (HMPV) infection, rhinovirus / enteroviral (RV / EV) infection, zika viral infection, respiratory adenoviral infection, rotaviral infection, or noroviral infection.

[0105] In certain embodiments, the recombinant immunoglobulin endoglycosidase as provided herein, or nucleic acid, vector, or attenuated bacterial strain, e.g., Corynebacterium pseudotuberculosis strain, encoding the same may be administered in combination with an antiviral agent(s). Antiviral agents may specifically bind a target to interfere with stages in the viral life cycle such as viral attachment to host cell, uncoating, synthesis of viral mRNA, translation of mRNA, replication of viral RNA and DNA, maturation of new viral proteins, budding, release of newly synthesized virus, and free virus in body fluids. Antiviral agents may be derivatives of naturally occurring nucleotides. Examples of antiviral agents include abacavir, acyclovir, adefovir, amantadine, ampligen, amprenavir, atazanavir, atripla, baloxavir marboxil, bictegravir, boceprevir, bulevirtide, cidofovir, cobicistat, combivir, daclatasvir, darunavir, delavirdine, didanosine, docosanol, dolutegravir, doravirine, edoxudine, efavirenz, elvitegravir, emtricitabine, enfuvirtide, entecavir, etravirine, famciclovir, fomivirsen, fosamprenavir, foscarnet, ganciclovir, ibacitabine, ibalizumab, idoxuridine, imiquimod, imunovir, indinavir, lamivudine, letermovir, lopinavir, loviride, maraviroc, methisazone, moroxydine, nelfinavir, nevirapine, nexavir, nitazoxanide, norvir, oseltamivir, penciclovir, peramivir, penciclovir, pleconaril, podophyllotoxin, raltegravir, remdesivir, ribavirin, rilpivirine, rilpivirine, rimantadine, ritonavir, saquinavir, simeprevir, sofosbuvir, stavudine, taribavirin, telaprevir, telbivudine, tenofovir alafenamide, tenofovir disoproxil, tenofovir disoproxil fumarate tenofovir, tipranavir, trifluridine, trizivir, tromantadine, truvada, umifenovir, valaciclovir, valganciclovir, vicriviroc, vidarabine, zalcitabine, zanamivir, zidovudine, or combinations thereof.

[0106] In certain embodiments, this disclosure relates to methods of treating or preventing of antibody-mediated depletion of B cells comprising administering to a subject in need thereof an effective amount of a recombinant immunoglobulin endoglycosidase as provided herein, or nucleic acid, vector, or attenuated bacterial strain such as a Corynebacterium pseudotuberculosis strain encoding a recombinant immunoglobulin endoglycosidase as provided herein.

[0107] In certain embodiments, this disclosure relates to methods of treating or preventing of antibody-mediated cytotoxicity against CD4 T cells comprising administering to a subject in need thereof an effective amount of a recombinant immunoglobulin endoglycosidase as provided herein, or nucleic acid, vector, or attenuated bacterial strain such as Corynebacterium pseudotuberculosis strain encoding a recombinant immunoglobulin endoglycosidase as provided herein.

[0108] Pharmaceutical Compositions

[0109] In certain embodiments, this disclosure relates to pharmaceutical compositions comprising a recombinant immunoglobulin endoglycosidase, nucleic acid, or vector as reported herein. The pharmaceutical compositions provided herein may generally include one or more pharmaceutically acceptable and / or approved carriers, additives, antibiotics, preservatives, diluents and / or stabilizers. Such auxiliary substances can be water, saline, glycerol, ethanol, wetting or emulsifying agents, pH buffering substances, or the like. Suitable carriers are typically large, slowly metabolized molecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, or the like.

[0110] In certain embodiments, the nucleic acid or recombinant vector encodes a recombinant immunoglobulin endoglycosidase disclosed herein having at least one open reading frame that can be translated by a cell or an organism provided with the nucleic acid, DNA, RNA, or mRNA. If more than one protein is translated, the proteins can be expressed in one vector / nucleic acid or in multiple (a plurality of) separate nucleic acids / vectors wherein the product of translation is a recombinant immunoglobulin endoglycosidase disclosed herein. The product may also be a fusion protein composed of more than one recombinant immunoglobulin endoglycosidase, e.g., a fusion protein that has two or more recombinant immunoglobulin endoglycosidases, wherein recombinant immunoglobulin endoglycosidases are optionally linked by self-cleaving linker sequences.

[0111] In certain embodiments, nucleic acid, DNA, RNA, or mRNA may be designed to have two (bicistronic) or more (multicistronic) open reading frames (ORF). An open reading frame in this context is a sequence including a start codon that can be used as a location to start translation of the encoded nucleic acid into a recombinant immunoglobulin endoglycosidase disclosed herein. Translation of such nucleic acid(s) yields two (bicistronic) or more (multicistronic) identical or distinct translation products / proteins (provided the ORFs are not identical). For expression in eukaryotes such nucleic acids may comprise an internal ribosomal entry site (IRES) sequence which allows for expression of two or more proteins on a single nucleic acid molecule.

[0112] In certain embodiments, the recombinant immunoglobulin endoglycosidase, nucleic acid, or recombinant vector may be administered naked without being associated with any further vehicle, e.g., mRNA or DNA.

[0113] In certain embodiments, the recombinant immunoglobulin endoglycosidase, nucleic acid, or recombinant vector may be administered in a pharmaceutical composition having a pharmaceutically acceptable excipient selected from lactose, sucrose, mannitol, triethyl citrate, dextrose, cellulose, methyl cellulose, ethyl cellulose, hydroxyl propyl cellulose, hydroxypropyl methylcellulose, carboxymethylcellulose, croscarmellose sodium, polyvinyl N-pyrrolidone, crospovidone, ethyl cellulose, povidone, methyl and ethyl acrylate copolymer, polyethylene glycol, fatty acid esters of sorbitol, lauryl sulfate, gelatin, glycerin, glyceryl monooleate, silicon dioxide, titanium dioxide, talc, corn starch, carnauba wax, stearic acid, sorbic acid, magnesium stearate, calcium stearate, castor oil, mineral oil, calcium phosphate, starch, carboxymethyl ether of starch, iron oxide, triacetin, acacia gum, esters, or salts thereof.

[0114] In certain embodiments, the pharmaceutical composition is in the form of a sterilized pH buffered aqueous salt solution or a saline phosphate buffer between a pH of 6 to 8, optionally comprising a saccharide or polysaccharide.

[0115] In certain embodiment, the pharmaceutically acceptable excipient is a cationic or polycationic compound and / or with a polymeric carrier. In certain embodiments, the recombinant immunoglobulin endoglycosidase, nucleic acid, or recombinant vector are in a pharmaceutical composition associated with or complexed with a cationic or polycationic compound or a polymeric carrier. In certain embodiments, the cationic or polycationic compound is protamine, spermine, spermidine, poly-L-lysine (PLL), poly-histidine, or poly-arginine, cationic polysaccharides, such as chitosan, polybrene, cationic polymers, polyethyleneimine (PEI), homo- and co-polymers of lactic acid and glycolic acid, or polymethylmethacrylate.

[0116] Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, albumin, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, citric acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, hydrophilic polymers such as polyvinyl pyrrolidone, cellulose based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, gelatin, polyethylene polyoxypropylene block polymers, polyethylene glycol and antioxidants including ascorbic acid and methionine; preservatives; low molecular weight (less than about 10 residues) polypeptides; proteins; and amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine. In certain embodiments, the excipient may be one or more selected from the list consisting of NaCl, trehalose, sucrose, mannitol, and / or glycine.

[0117] The disclosure also encompasses products obtainable by further processing of a liquid formulation, such as a frozen, lyophilized or spray-dried product. Upon reconstitution, these solid products can become liquid formulations as described herein (but are not limited thereto). In its broadest sense, therefore, the term "formulation" encompasses both liquid and solid formulations. However, solid formulations are understood as derivable from the liquid formulations (e.g. by freezing, freeze-drying or spray-drying), and hence have various characteristics that are defined by the features specified for liquid formulations herein.

[0118] In certain embodiments, the formulations are isotonic in relation to human blood. Isotonic solutions possess the same osmotic pressure as blood plasma, and so can be intravenously infused into a subject without changing the osmotic pressure of the blood plasma.

[0119] In certain embodiments, the pharmaceutical composition is administered either systemically or locally, i.e., parenteral, subcutaneous, intravenous, intramuscular, intranasal, or any other path of administration. The mode of administration, the dose and the number of administrations can be optimized.

[0120] In certain embodiments, this disclosure relates to kits containing materials useful for the treatment, or prevention of diseases or conditions as described above is provided. In certain embodiments, the kit comprises a container, a product label and a package insert. Suitable containers include, for example, bottles, vials, syringes, and boxes containing the same. The containers may be of a variety of materials, e.g., glass, plastic, or cardboard. The container holds the composition which is effective in treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a recombinant immunoglobulin endoglycosidase, nucleic acid, or recombinant vector as disclosed herein. The product label on, or associated with, the container indicates that the composition is used for treating the condition of choice. In certain embodiments, the kit may further comprise a second container comprising a pharmaceutically acceptable buffer, such as a phosphate buffer saline or a citrate buffered saline. It may further include other materials desirable from a user or commercial standpoint, including other buffers, diluents, fdters, needles, and syringes. In certain embodiments, a dosage unit form can be, e.g., in the format of a prefdled syringe, an ampoule, cartridge or a vial.

[0121] In certain embodiments, this disclosure relates to kits or articles of manufacture, comprising a recombinant immunoglobulin endoglycosidase, nucleic acid, or recombinant vector as disclosed herein and instructions for use by, e.g., a healthcare professional. The kits or articles of manufacture may include a container, vial, or a syringe containing the formulation as described herein. Preferably, the container, vial, or syringe is composed of glass, plastic, or a polymeric material. The syringe, ampoule, cartridge, or vial can be manufactured of any suitable material, such as glass or plastic and may include rubber materials, such as rubber stoppers for vials and rubber plungers and rubber seals for syringes and cartridges.

[0122] Recombinant Endo-P-N-acetylglucosaminidases

[0123] Endo-P-N-acetylglucosaminidases, hereafter referred to as endoglycosidases or GH18 enzymes, produced by various organisms catalyze the hydrolysis of N-linked glycans on glycoproteins. Most endoglycosidases recognize their glycoprotein substrates by glycan-specific, but protein-nonspecific, mechanisms. However, a subset of endoglycosidases specifically hydrolyzes the Asn297-linked glycan on IgG antibodies. This glycan is the major molecular determinant of Fc gamma receptor and complement Clq binding by IgG antibodies, interactions that in turn trigger antibody-mediated effector functions that are critical for the signaling properties of these antibodies. IgG-specific endoglycosidases are useful for the treatment or prevention of diseases or conditions mediated by IgG antibodies including, such as, autoimmunity and transplantation rejection. Many IgG-specific endoglycosidases are multi-domain proteins belonging to a family of enzymes exemplified by EndoS and EndoS2, which are secreted by various strains of Streptococcus pyogenes. One single-domain IgG-specific endoglycosidase, CP40, produced by Cory neb acterium tuberculosis has also been identified.

[0124] Reported herein is the identification and validation of additional members of a singledomain IgG specific endoglycosidase family. To identify putative homologs to the single-domain IgG-specific endoglycosidase CP40, sequence similarity networks (SSN) were constructed using all annotated GH18 enzymes from the CAZY database and the Enzyme Similarity Tool (EFI-EST). Cytoscape™ software was used for deep analysis of the resulting SSNs. By selecting an alignment score threshold of 50 (to reach an alignment stringency that allowed separation into distinct clusters in which proteins shared sequence identity lower than 35%), CP40 from C. pseudotuberculosis was located in a cluster containing 120 additional GH18 enzymes. Also located within this cluster were the multi-domain IgG-specific endoglycosidades, EndoS and EndoS2.

[0125] Sequences were filtered these based on sequence length, sequence identity, and alpha-fold model structure prediction. Of the 120 total GH enzymes, 48 were composed of single polypeptide sequences of greater than 600 amino acids, indicating the presence of multiple domains that may be involved in the recognition and / or binding of glycoprotein substrates. ORFs containing less than 300 residues were considered incompletes and discarded for the analysis. The remaining putative single-domain ORFs were subject to comprehensive comparison of their predicted three- dimensional structures using AlphaFold™. From this analysis, four homologs to CP40, were identified and characterized by sequence identities of 40-90% and a highly conserved fold, specifically in certain regions around the active site that appear as short helixes in CP40 and these four CP40 homologs (or CP40-like enzymes).

[0126] To increase sample size, another SSN was performed using Glyco_hydro_18 (PF00704) from the Pfam database, which contains 79,058 sequences of glycoside hydrolases from the GH18 family. By selecting an alignment score threshold of 60 (to reach a 35-40% sequence identity between the sequences in each cluster), CP40 from C. pseudotuberculosis was located within a cluster containing another 160 proteins. Three subclusters were identified, including those: (I) containing multi-domain IgG-specific endoglycosidases including EndoS, EndoS2 and other EndoS / S2-like proteins; (II) containing a single GH18 domain alone or paired with additional glycoside hydrolase domains, such as EndoCoM and EndoE (containing GH18 and GH20 domains); and (III) containing CP40 and its close homologs. Of these CP40 homologs, a strain of C. pseudotuberculosis (strain 258) shares 91% amino acidic sequence identity with CP40. In the interface between sub-clusters II and III, CP40-like proteins were found from a genus of actinobacteria different from Corynebacteria, identified as Winkia. Hidden-Markov analysis of CP40 sequence using HMMER also identified this hit. Winkia neuii BV029A5 or Actinomyces neuii were identified in the top 3 hits (e value 3. le-55), indicating a highly similar sequence pattern with CP40 protein. Also using HMM, it was found that CP40 shares high similarity with C. mustalea (e-value_l 9e-63), although this protein contains a triple helix bundle and a long flexible linker between the GH18 domain and a C-terminal transmembrane domain. Additionally, after BLAST searching CP40-like genes against non-redundant NCBI databases, two additional putative CP40-like genes from C. silvaticum and C. belfantii were found that were not in the Uniprot, Pfam and CAZY databases.

[0127] Multiple sequence alignments of CP40 and its closest homologs from Cory neb acterium species was performed identified in the SSN analysis. DonE, an endoglycosidase from Bacteroides fragilis, was included in the sequence alignment as a negative control, as it has been reported to hydrolyze complex type (CT) N-glycans from transferrin but is unable to deglycosylate IgG or IgA. The GH18 domain sequence of EndoS2, which, by itself, also exhibits IgG-specific ENGase activity without the requirement of additional domains to specifically deglycosylate IgG was also included. At least 23 residues that are 100% conserved in all CP40- like proteins and the EndoS2 GH18 domain that are, additionally, different from those in DonE were identified. All CP40-like proteins contained two highly conserved cysteine residues (Cys57 and Cys284, numeration in CP40) that form a disulfide bond in AlphaFold™ modeled structures. Finally, multiple sequence alignments were performed using CP40 and the four CP404ike enzymes identified by the methods described above (CP258, CU43, CR39 and CM49 following the naming convention established for CP40). These enzymes have a sequence identity with CP40 as high as 91% and as low as 38%.

[0128] To define the enzymatic functionality of CP40 and the CP404ike enzymes, the ORFs of these four CP40 orthologs were cloned into the pET28a vector between BamHI-XhoI sites, containing His6 tag on the N-terminus. Codon optimization based on E. coli codon-usage was performed for all sequences. The signal peptide of each genetic construct was removed based on Signal P 6.0 prediction, the transmembrane domain of one homolog (named as CM49) based on hydrophobicity prediction using the Phobius server was deleted. The long flexible linker of CM49 was also removed based on its AlphaFold™ model prediction, but a C-terminal triple helix bundle was maintained (residues 339-394). After expressing these proteins in E. coli and purifying them to homogeneity by affinity chromatography, their thermal stabilities were determined by differential scanning fluorimetry (DSF) assays using 5-10 pM final concentration of enzyme and SyproOrange™ 25X, in the range of 10-95 °C, at pH 3, 4, 5.5, 6.5, 7.4 and 9. The CU43 and CM49 enzymes exhibited higher thermal stabilities (Tm of 52 and 56 C, respectively) compared to CR39 and CP258 (Tm 34 and 38C, respectively) at physiological conditions of ionic strength and pH (150 mM NaCl, pH 7.4). Higher thermal stability of CR39 and CP258 was registered at pH 5.5 (Tm 43 and 42 C, respectively). CP40-like proteins were thermally unstable at pH about or less than 4 (Tm about 26 C for proteins tested).

[0129] IgG specificity of CP40 and CP40-like proteins

[0130] Enzymes were incubated with a cocktail of glycoprotein substrates, including: (1) IgGl-Fc region (complex type glycan, IgG substrate); (2) gly can-remodeled IgGl (high mannose type glycan, IgG substrate); (3) RNAseB (high mannose type glycan, non-IgG substrate); and (4) Transferrin (complex type glycan, non-IgG substrate). Enzymes were at final concentrations of either 10 nM or IpM and the reactions were run for 12 hours at room temperature before analyzing the progress. Enzymatic reactions were monitored by intact mass spectrometry using an Agilent 1290 Infinity II LC System equipped with a 50 mm PLRP-S column from Agilent with 1000 A pore size, attached to an Agilent 6545XT quadrupole-time of flight (Q-TOF) mass spectrometer. The relative amounts of the substrate and hydrolysis products were quantified after deconvolution of the raw data and peaks were identified. CP40 and the CP40-like enzymes (Figure 1A-D) were all IgG-specific endoglycosidases, with robust activity on IgG substrates bearing complex type glycans and more modest activity on IgG substrates bearing high-mannose type glycans (Figures 2A-2B). No substantial activity for these enzymes on the non-IgG substrates tested were observed.

Claims

CLAIMSWhat is claimed is:

1. A recombinant immunoglobulin endoglycosidase comprising peptide motifs of the following amino acid sequences from an N-terminal to C-terminal order sequentially, YYRTWRDK (SEQ ID NO: 1), TDIP, (SEQ ID NO: 2), LDGLDX’DME (SEQ ID (SEQ ID NO: 4), NRFL (SEQ ID NO: 5), and DRDG (SEQ ID NO: 6), whereinany amino acid and provided the immunoglobulin endoglycosidase does not contain an amino acid sequence KESV (SEQ ID NO: 48, contained in CP40) between LDGLDX’DME (SEQ ID NO: 3) and SCQF (SEQ ID NO: 4).

2. A recombinant immunoglobulin endoglycosidase of claim 1 comprising peptide motifs of the following amino acid sequences from an N-terminal to C-terminal order sequentially, AXXXXXPLXXXXGXXXXXG (SEQ ID NO: 7), PIXXXYYRTWRDKXIXXXXXD (SEQ ID NO: 8),TDIPXXXXXXSLXHVXD, (SEQ ID NO: 9), QXSDXXFWXTFXXXYXPXLXXRGTXVXXTXXXXXXL (SEQ ID NO: 10) YXXXAXXXXXXYVXXHXLDGLDXDME, (SEQ ID NO: 11) LRXXMXAXXXLXGPXXXXN (SEQ ID NO: 12) LXYXTXXNAQXXXXXXV (SEQ ID NO: 13), AXXVXYVXXQTY (SEQ ID NO: 14) WNXXRXXXXSCQFXXXYAXPEEXDX (SEQ ID NO: 15), NRFLXAXGXGXVXXXXAXXXAXWXP (SEQ ID NO: 16), and GXKGGXXXYAXDRDGXTYDXXDXXTLXXTXFXXXKR (SEQ ID NO: 17), wherein X is individually and independently at each position is any amino acid.

3. The recombinant immunoglobulin endoglycosidase of claim 1 comprising the amino acid sequence CU43) AALSNAPLAASPGQADKVGAQATCAAKPIFFGYYRTWRDKAIELNDGDKWKDKLHTK LTDIPEQVDMVSLFHVPDNQKSDQRFWETFDKEYHPTLKERGTKVVRTIGAKLLLNKIK EKGLYGQ SREDD SK YREI AHEVYEEYVAKHNLDGLD VDMELREVEKYTNLRWQLRKIMGAFSELMGPKAPGNAGKKPGDDGYKYLIYDTFDNAQLAQVALVADVVDYVLAQTY DKGTEESITRVWNGFRDKINSCQFLAGYAHPEENDTNRFLTAIGDVDTSGAMNVAAWK PEGGEKGGTFAYALDRDGRTYDGDDLTTLKPTDFAFTKRAIELTKGISLTDLG (SEQ ID NO: 19).

4. A recombinant immunoglobulin endoglycosidase of claim 1 comprising the amino acid sequence of (CP258)ADLSQAPLKASPGHADKVGVQTTCDAKPIFFGYYRTWRDKAIQLKDDDPWKDKLQVK LTDIPEHVNMVSLFHVEDNQKSDDQFWETFRKEYQPKLKERGTRVVRTVGAQLLLNKI KEKGLYGRSVEDDYKYREIARDIYKKYVTDHNLDGLDVDMELRKVEKRIDLQWQLRKI MGAFSELMGPKAPANEGKKPGHEGKKPGHEGYKYLIYDTFDNAQTSQVGLVADLVDY VLAQTYDKGTKESIDQVWNGFRDKINSCQFMAGYAHPEENDTNRFLTAVGEVNKSGA MQVAEWKPDNGVKGGTFAYALDRDGRTYDGDDFTTLKPTDFAFTKRAIELTTGESSTD LG (SEQ ID NO: 20).

5. A recombinant immunoglobulin endoglycosidase of claim 1 comprising the amino acid sequence of (CM49)ASPTSLPLPEHKGFHPDAGERSDCEPIVTAYYRTWRDKEIQQLPDDRVGPNVIAMTDIPH NIDVLSLIHVPDHQQSDQQFWATFASTYLPELHRRGTKVLYTLDISAVLDPSLKPTSSPEE YAAHAQRLVDKYVRPHKLDGLDIDMERDLSFDQRIVLRNTMRALTQLVGPFSNTNTLL TYNTNRNAQKAYIDEVAQYVNYVFVQTYSVTDPNEIQYSYWNTYRFYLSSCQFLPSYA NPEEFDRNRFLGAIGPVEETAAVKIAGWQPFQGGVKGGIMTYAIDRDGMTYDQPDISTL RVTTFPVIKRVTAVLKAKKFAAAK (SEQ ID NO: 21).

6. A recombinant immunoglobulin endoglycosidase comprising peptide motifs of the following amino acid sequences from an N-terminal to C-terminal order sequentially,PVFF (SEQ ID NO: 22), YYRTWRDKAI (SEQ ID NO: 23), LTDIP (SEQ ID NO: 24), MVSLFHA (SEQ ID NO: 25), VRTT (SEQ ID NO: 26), HNLDGLDVDME, (SEQ ID NO: 27), GPKA (SEQ ID NO: 28), LIYDTFD (SEQ ID NO: 29), VDYVL (SEQ ID NO: 30), SCQF (SEQ ID NO: 4), GYAHPEE (SEQ ID NO: 31), NRFETAIG (SEQ ID NO: 32), DRDGRTY(SEQ ID NO: 33), TFKR (SEQ ID NO: 34).

7. A recombinant immunoglobulin endoglycosidase of claim 6 comprising peptide motifs of the following amino acid sequences from an N-terminal to C-terminal order sequentially, PXXXXXSLXXXXGXXXXXG (SEQ ID NO: 35), PVFFXYYRTWRDKAIXXXXXD (SEQ ID NO: 36), LTDIPXXVXMVSLFHAXN, (SEQ ID NO: 37), YXSDXXFWXTFXXXYXPXLKXRGTXVRTTXXAXXLL (SEQ ID NO: 38) DXXYREXAXXXXXXYVXXHNLDGLDVDME, (SEQ ID NO: 39) WXIRKXMXAXSELXGPKAXXN (SEQ ID NO: 40)GYKXLIYDTFDXXXXXQ (SEQ ID NO: 41), AXXVDYVLXQTY (SEQ ID NO: 42) WNXXRXXXXSCQFXXGYAHPEEXDT (SEQ ID NO: 43), NRFETAIGXGXVXXSXAMXVAXWXP (SEQ ID NO: 44), and GGXKGGXFXYAXDRDGRTYXXDDXXXXKXTDFXTFKR (SEQ ID NO: 45), wherein X is individually and independently at each position is any amino acid.

8. A recombinant immunoglobulin endoglycosidase of claim 6 comprising the amino acid sequence of (CR39) PAIAETSLRGGTGQHETPGSQKCDSEPVFFAYYRTWRDKAIVQNESDKPNRANKIALTDI PQHVNMVSLFHAGNDHYQSDAEFWKTFDDVYYPELKRRGTRVVRTISATELLKETDSL RAVGVDTDAADYREVAEKIKKEYVDAHNLDGLDVDMEVLHLERNWRSSWEKRWRIR KTMAALSELLGPKADVNQGKKKDDSGYKFLIYDTFDDVERSQIRAIAELVDYVLPQTYK SGKAEIDQLWNQSKGILSSCQFVPGYAHPEEGDTVNRFETAIGDVDSSKAMEVAAWQP AGGEKGGAFVYAIDRDGRTYGEDDLKNVKETDFSFTKRGAALARSVTFAKAK (SEQ ID NO: 46).

9. A fusion protein comprising any of the immunoglobulin endoglycosidases as provided in claims 1-8.

10. A nucleic acid encoding a recombinant immunoglobulin endoglycosidase as provided in claims 1-8 in operable combination with a heterologous promoter.

11. The nucleic acid of claim 10 which is mRNA, RNA, or DNA.

12. A live attenuated Corynebacterium pseudotuberculosis strain comprising a recombinant immunoglobulin endoglycosidase as provided in any of claims 1-9.

13. A vector comprising a nucleic acid of claim 10 or encoding a recombinant immunoglobulin endoglycosidase as provided in claims 1-9.

15. A method of treating or preventing a disease or condition associates mediated by IgG antibodies or with altered Asn297 IgG glycosylation comprising administering to a subject in need thereof an effective amount of a recombinant immunoglobulin endoglycosidase as provided in claims 1-9, or nucleic acid, vector, or attenuated Corynebacterium pseudotuberculosis strain encoding a recombinant immunoglobulin endoglycosidase as provided in claims 10-12.

16. The method of claim 15 wherein the disease or condition an autoimmune diseases and chronic inflammatory condition such as, including rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, autoimmune liver disease, juvenile idiopathic arthritis, osteoarthritis, spondyloarthropathies, neonatal lupus, lupus nephritis, Sjogren’s syndrome, antineutrophil cytoplasmic antibody (ANCA)-associated antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), Crohn’s disease, ulcerative colitis, Hashimoto’s thyroiditis, multiple sclerosis, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, Myasthenia gravis, Lambert-Eaton myasthenic syndrome, autoimmune hemolytic anemia, and antiphospholipid syndrome.

17. The method of claim 15 wherein the disease or conditions is associated with increased or decreased galactosylation, sialylation, fucosylation, and / or the presence of bisecting N- acetylglucosamine (GlcNAc) chains.

18. A method of treating or preventing a viral infection comprising administering to a subject in need thereof an effective amount of a recombinant immunoglobulin endoglycosidase asprovided in claims 1 -9, or nucleic acid, vector, or attenuated Corynebacterium pseudotuberculosis strain encoding a recombinant immunoglobulin endoglycosidase as provided in claims 10-12.

19. A method of treating or preventing antibody-mediated depletion of B cells comprising administering to a subject in need thereof an effective amount of a recombinant immunoglobulin endoglycosidase as provided in claims 1-9, or nucleic acid, vector, or attenuated Corynebacterium pseudotuberculosis strain encoding a recombinant immunoglobulin endoglycosidase as provided in claims 10-12.

20. A method of treating or preventing antibody-mediated cytotoxicity against CD4 T cells comprising administering to a subject in need thereof an effective amount of a recombinant immunoglobulin endoglycosidase as provided in claims 1-9, or nucleic acid, vector, or attenuated Corynebacterium pseudotuberculosis strain encoding a recombinant immunoglobulin endoglycosidase as provided in claims 10-12.

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