WO2025256977A1

WO2025256977A1 - Novel igm and igg cleaving enzymes

Info

Publication number: WO2025256977A1
Application number: PCT/EP2025/065427
Authority: WO
Inventors: Oliver Kensch; Anja BRAKE; Simone BRÜCKNER; Sabine JACH; Philipp KNYPHAUSEN; Mirko MORONI; Florian Richter; Matthias UTHOFF; Lamprinos Frantzeskakis
Original assignee: Bayer AG; Asklepios Biopharmaceutical Inc
Current assignee: Bayer AG; Asklepios Biopharmaceutical Inc
Priority date: 2024-06-13
Filing date: 2025-06-04
Publication date: 2025-12-18
Anticipated expiration: 2026-12-13

Abstract

The disclosure provides the first natural occurring proteases capable of cleaving human and cynomolgus antibodies of type IgM and IgG. They are from the genus of Lachnospiraceae and are only distantly related to all other known specific Ig cleaving proteases. In other aspects, there are provided nucleic acids encoding these proteases, vectors comprising these nucleic acids, and host cells expressing these proteases. Further there are provided in vitro and in vivo uses of said proteases, expression vectors or compositions related to said proteases, including but not limited to organ transplantation, treatment of autoimmune diseases, and treatments with cell and gene therapies.

Description

NOVEL IGM AND IGG CLEAVING ENZYMES

Background of the invention

Antibodies or immunoglobulins (Ig) are the key players in the adaptive immune system and are critical to reduce the long-term severity of pathogens or prevent disease outright. However, under certain circumstances antibodies are undesired i.e., in organ transplantations in cases of HLA incompatibility, in auto-immune diseases in which antibodies target “self’-antigens or in therapies utilizing proteins or vectors such as viruses.

In autoimmune diseases patients develop antibodies with affinities against self-antigens. This can lead to the recruitment of the immune system, inflammation, improper functionality of the affected tissue or cell death. Several auto-immune diseases can have an acute and fast onset or are reoccurring in strong relapses. Example diseases are Guillan-Barre Syndrome, Multiple Sclerosis, Myasthenia Gravis, Pemphigus Vulgaris I Foliaceus, Autoimmune Hemolytic Anemia or the post-COVID19 syndrome. Therapies often include the suppression of antibody production by administration of immunomodulators. However, this leaves the existing antibodies in place which damage tissues or trigger symptoms. Therefore, an immediate removal of the antibodies as a complementary treatment could be beneficial.

Gene therapies (GTs) often utilize adeno associated viruses (AAVs) as delivery vehicles. While mostly harmless, these viruses are common and - depending on the specific AAV serotype - up to 80 % of the population have developed antibodies against them. Such pre-existing antibodies also target the recombinant AAVs used in GTs and are therefore a frequent reason to exclude patients. Since the DNA delivered by AAVs is not integrated into the genome, but present as episomal DNA, (fast) proliferating tissues lose the introduced the gene over time and a redosing of the AAV becomes necessary. However, the first AAV dose usually triggers the build-up of a strong immunity against AAVs by high total and neutralizing antibody (tAB and nAB) titers. Therefore, a method to remove anti-AAV antibodies and to break the immunity, increases the eligible population of patients and allows the redosing of AAV-GT to ensure a prolonged effect.

Conceptually, antibodies are Y-shaped complexes of two heterodimers. Two so-called heavy chains form the “Y” scaffold and the both upper “Y”-arms are complemented with a light chain each. The region where the arms meet is called the “hinge”, the upper joined arms are called the F(ab’)2 fragment and the remaining region below the hinge is the Fc part. These Y-shaped assemblies are often referred to as “monomers” as they can take part in larger arrangements. Igs fall into different classes, each having different tasks Of major importance are IgG (isotypes lgG1 , lgG2, lgG3, lgG4; “monomeric”) and IgM (“penta”- and ’’hexameric”) antibodies. Both trigger the immune system to act on the antigen’s host. IgGs stimulate antibody-dependent cellbased cytotoxicity (ADCC) and to some extent complement dependent cytotoxicity (CDC). IgMs on the other hand stimulate only but strongly CDC. Moreover, antibodies have neutralizing properties if they sterically block the antigen from its task, like blocking interactions or active sites. Typically, IgM antibodies are less matured and have therefore a lower affinity to the antigen than IgG antibodies. However, due to their complex assembly with 5-6 times the number of antigen binding domains, IgM antibodies benefit from avidity. Often IgG antibodies are the cause for auto-immune diseases, but IgM antibodies do as well, for example in Cold Autoimmune Hemolytic Anemia. In the AAV-GT naive population, lgG1 is the dominant class of tABs and nABs against AAVs. Nonetheless, antibodies of class I gG2 , lgG3 and IgM are prevalent as well and while often only one IgG isotype has elevated levels against one AAV type, IgM antibodies appear to be present more abundantly. Hence removal of I gG 1 , 1 gG2 , lgG3 and IgM is preferred (Murphy, Samuel L., Hojun Li, Federico Mingozzi, Denise E. Sabatino, Daniel J. Hui, Shyrie A. Edmonson, and Katherine A. High. 2008. ..Diverse IgG Subclass Responses to Adeno- associated Virus Infection and Vector Administration." J. Med. Virol. 65-74. doi:10.1002/jmv.21360.; Schroeder, Harry W., and Lisa Cavacini. 2010. ..Structure and function of immunglobulins." J. Allergy. Clin. Immunol. S41-S52. doi:10.1016/j.jaci.2009.09.046.).

Reduction of serum antibody levels can be achieved via different methods. The class of plasmapheresis-like methods utilizes special matrices such as Protein A or specific antigens to remove all or specific Igs from the blood ex vivo (Fuchs, Kornelius, Silke Rummler, Wolfgang Ries, Matthias Helmschrott, Jochen Selbach, Friedlinde Ernst, Christian Morath, et al. 2022. ..Performance, clinical effectiveness, and safety of immunoadsorption in a wide range of indications." Ther. Apher. Dial. 229-241. doi:10.1111/1744-9987.13663.). The other two strategies to remove antibodies target the antibody recycling via FcRn. Cells in contact with blood engulf a portion of plasma via pinocytosis and degrade the contents, especially debris and damaged proteins via the endosomes and fusion with lysosomes. FcRn receptors interact with the Fc part of immunoglobulins of class G (IgGs) in the endosomes and prevent their degradation in lysosomes by recycling them back to the cell surface. This yields a long half-life of up to 20 days for IgGs. Recently, FcRn inhibitors such as Efgartigimod (Argenx), Rozanolixizumab (UCB), Nipocalimab (Johnson & Johnson) and Batoclimab (Immunovant) have been approved or are in Phase III clinical studies (Horiuchi, Makoto, Christian J. Hinderer, Hailey N. Shankle, Peter M. Hayashi, Jessica A. Chichester, Casey Kissel, Peter Bell, Cecilia Dyer, and James M. Wilson. 2023. „Neonatal Fc Receptor Inhibition Enables Adeno-Associated Virus Gene Therapy Despite Pre-Existing Humoral Immunity." Human Gene Therapy 1022-1032. doi:10.1089/hum.2022.216.; Bril, Vera, Artur Druzdz, Julian Grosskreutz, Ali A. Habib, Renato Mantegazza, Sabrina Sacconi, Kimiaki Utsugisawa, et al. 2023. ..Safety and efficacy of rozanolixizumab in patients with generalised myasthenia gravis (MycarinG): a randomised, double-blind, placebo-controlled, adaptive phase 3 study." 383-394. doi:10.1016/S1474- 4422(23)00077-7.).

Immunoadsorption and FcRN inhibitors often cannot remove all antibodies and only reduce them down to 20 % of the baseline. Moreover, both approaches require multiple doses or treatments. These disadvantages can be addressed by another approach to remove antibodies: Specific proteases. They cleave both heavy chains in the hinge region of IgGs and produce F(ab’)2 and Fc fragments. F(ab’)2 molecules usually retain a high affinity to the antigen but are cleared from the blood within days as the interaction and recycling via FcRn is abolished. The first Ig protease IdeS/ldefirix has been discovered in 2002 and was granted a conditional approval for desensitization of patients receiving a kidney transplant in the Ell (EP1458861 , W0202016318). IdeS is also evaluated to improve AAV gene therapies (EP3768304). IdeZ, a close relative to IdeS is available for in vitro cleavage and is developed either alone or as an IdeS/ldeZ hybrid (WO2016128558, WO2016128559, WO2021233911). Further alternative proteases are KJ103 (IdeE variant; WO2021244628, EP4162951) and Xork (WO2022223818). These proteases share all the papain-cysteine protease fold and are highly specific for IgG antibodies. Moreover, they all originated from the genus Streptococcus either S. pyogenes (IdeS), S. equus (IdeZ, ldeZ2, IdeE) or S. krosus (Xork).

In 2013 the first IdeS-like IgM-specific protease called Idessuis was discovered (Seele, Jana, Alena Singpiel, Christian Spoerry, Ulrich von Pawel-Rammingen, Peter Valentin-Weigand, and Christoph G. Baums. 2013. ..Identification of a novel host-specific IgM protease in Streptococcus suis." J. Bacteriol. 930-940. doi:10.1128/JB.01875-12.), however it cannot degrade human antibodies as it is porcine specific. Recently, new human specific IgM proteases have been announced: lgMDEi_s_P, lgMDEi_ba IgMDEori; IgMDEMma (Windgassen, Tricia, Nikki Kruse, Brian Ferrer, Faye Du, Kumar Hirdesh, and Adam P. Silverman. 2024. ..Identification of bacterial protease domains that cleave human IgM." Enzyme Microb. Technol. doi: 10.1016/j.enzmictec.2023.110366.) and IgMBrazor (Genovis; in vitro use only) and IceM as well as IceMG (Smith, Timothy, Joshua A. Hull, Robert M. Fusco, Leo Clondel, Zachary Elmore, Aravind Asokan. 2023. “Newly Engineered IgM and IgG Cleaving Enzymes for AAV Gene Therapy" in “Presidential Symposium and Presentation of TOP Abstracts”. Mol. Ther. doi:10.1016/j.ymthe.2023.04.017.). According to the respective authors, except from IceMG, none has cleavage activity on the IgM and IgG antibody type simultaneously. IceMG was only presented orally and its origin, sequence and information about specificity and activity are not fully publicly available.

Igs are generated as outlined briefly: Unmatured B cells produce IgM antibodies and once triggered by an antigen they start the affinity maturation process. This leads to a strong antigen binding Ig. This is usually followed by class switching from IgM to for example the IgG class. B cells present their produced Ig in their B cell receptor (BCR) on the cell surface. Its binding to the antigen triggers the B cell maturation. Matured B cells either produce Igs (plasma cells) or enter a state of long-lived stand-by (memory B cells) so they can be activated in the future (Schroeder, Harry W., and Lisa Cavacini. 2010. ..Structure and function of immunglobulins." J. Allergy. Clin. Immunol. S41-S52. doi:10.1016/j.jaci.2009.09.046.).

IdeS and Idessuis, demonstrated cleavage of their respective substrates (IgGs/IgMs) also if assembled in the BCR on the B cell surface. In both cases the B cells could no longer be activated until about 12-24 h after the BCRs were regenerated. Hence, administration of Ig-proteases simultaneously with an immunogenic compound like another protein or AAV may reduce the immune response to that compound. In case of AAVs this could lower the nABs titers after a first AAV-GT dose and facilitate a redosing (Jarnum, Sofia, Robert Bockermann, Anna Runstrbm, and Lena Winstedt. 2015. „The Bacterial Enzyme IdeS Cleaves the IgG-Type of B Cell Receptor (BCR), Abolishes BCR-Mediated Cell Signaling, and Inhibits Memory B Cell Activation." J. Immunol. 5592-5601. doi:10.4049/jimmunol.1501929.; Breitfelder, Annika Katharina, Wieland Schrddl, Viktoria Rungelrath, Christoph Georg Baums, Gottfried Alber, Nicole Schutze, and Uwe Muller. 2023. ..Immunoglobulin M-degrading enzyme of Streptococcus suis (Idessuis) impairs porcine B cell signaling". Front. immunol. doi: 10.3389/fimmu.2023.1122808.).

In conclusion, cleavage of IgGs leads to their fast clearance from the blood and temporary inhibition of memory B cell activation. Cleavage of IgMs leads to the dissociation of the polymeric assembly and therefore its avidity. This may further reduce the immunity, may reduce a CDC response, and can prevent transiently B cell activation. Therefore, an IgG/IgM-specific protease may reduce prevalent and prevent future antibody titers against AAVs as well as it might reduce the immunogenicity of a compound by administering the protease in advance and simultaneously.

Therefore, a protease with activity against both IgM and IgG is of high interest. The inventors present the first natural occurring proteases capable of cleaving human and cynomolgus antibodies of type IgM and IgG.

Sequences

Table 1 contains all disclosed protein and Table 2 all disclosed DNA sequences. SEQ ID NO: 1- 10 are reference protein sequences from prior art used for example for alignments and in silico comparisons. SEQ ID NO: 15-32 and 38-55 are the core protease and DNA sequences of the disclosure, respectively. An actual gene used for expression consists of the signal peptide (SEQ ID NO: 34) and one putative protease sequence (SEQ ID NO: 38-55) followed by an additional stop codon. Hence, the expressed protein is the sequence of the signal peptide (SEQ ID NO: 11) directly followed by one putative protease sequence (SEQ ID NO: 15-32). Upon secretion, the signal peptide is cleaved off and only the putative protease polypeptide remained (SEQ ID NO: 15-32) and was tested. SEQ ID NO: 12-14+33 and 35-37+56 are the protein and DNA sequences of three selected proteases to be purified and the expressed reference IdeS. The sequences contain all elements like signal peptide, linker and tags. Table 1: Protein sequences.

Table 2: DNA Sequences.

Summary of the invention

Several modern treatment methods as gene therapy, cell therapy, and organ transplantation are interfered by antibodies against said therapeutics. In addition, in auto immune diseases a patient develops antibodies with affinities against body-own antigens. This can lead to the recruitment of the immune system, inflammation, improper functionality of the affected tissue or cell death. Therapies often include antibody production suppression by immunomodulation, but antibody removal could be an improved starting point for the therapy. In these therapies antibodies of the IgG and IgM type play an important role. Therefore, there is a need for methods providing solutions for IgG as well as IgM removal. A protease with activity against both IgM and IgG is of high interest.

The inventors present the first natural occurring proteases capable of cleaving human and cynomolgus antibodies of type IgM and IgG. They are from the genus of Lachnospiraceae and are only distantly related to all other known specific Ig cleaving proteases. The disclosure relates to new enzymes which display IgG and IgM cysteine protease activity. These enzymes form two homology clusters which are related to each other.

Accordingly, in a first aspect there is provided an isolated polypeptide having IgG and IgM cysteine protease activity comprising: a) an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 19, or b) an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 30.

In other aspects, there are provided nucleic acids encoding these polypeptides, vectors comprising nucleic acids encoding these polypeptides, and host cell expressing these polypeptides.

The polypeptides of the disclosure are useful in various in vitro and in vivo methods for cleaving immunoglobulins, IgGs and IgMs. Thus, provided herein are methods comprising administering to a subject in need in which undesired IgG or IgM is present, a polypeptide, a polynucleotide, an expression vector or a composition related to the first aspect. There are provided in vitro and in vivo uses of polypeptides, expression vectors or a composition related to the first aspect including but not limited to organ transplantation, treatment of autoimmune diseases, and treatments with cell and gene therapies.

Detailed description of the invention

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs. The following references, however, can provide one of skill in the art to which this disclosure pertains with a general definition of many of the terms used in this disclosure, and can be referenced and used so long as such definitions are consistent with the meaning commonly understood in the art. Such references include, but are not limited to, Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); Hale & Marham, The Harper Collins Dictionary of Biology (1991); and Lackie et al., The Dictionary of Cell & Molecular Biology (3d ed. 1999); and Cellular and Molecular Immunology, Eds. Abbas, Lichtman and Pober, 2nd Edition, W.B. Saunders Company. Any additional technical resource available to the person of ordinary skill in the art providing definitions of terms used herein having the meaning commonly understood in the art can be consulted. For the purposes of the present disclosure, the following terms are further defined. Additional terms are defined elsewhere in the description. As used herein and in the appended claims, the singular forms "a," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a gene" is a reference to one or more genes and includes equivalents thereof known to those skilled in the art, and so forth.

The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

Amino acids may be referred to herein by their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

An "isolated" polypeptide is one that has been identified and separated from a component of the cell that expressed it. Contaminant components of the cell are materials that would interfere with diagnostic or therapeutic uses of the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.

An "isolated" nucleic acid is one that has been identified and separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extra- chromosomally or at a chromosomal location that is different from its natural chromosomal location.

The term "antibody", as used herein, is intended to refer to immunoglobulin molecules, preferably comprised of polypeptide chains, heavy (H) chains and light (L) chains which are typically interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region can comprise e.g. three domains CH1 , CH2 and CH3 (for IgG) or CH1 , CH2, CH3 and CH4 (for IgM). Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is typically composed of three CDRs and up to four FRs arranged from amino-terminus to carboxy-terminus e.g. in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.

As used herein, the term "Complementarity Determining Regions” (CDRs; e.g., CDR1 , CDR2, and CDR3) refers to the amino acid residues of an antibody variable domain the presence of which are necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDR1 , CDR2 and CDR3. Each complementarity determining region may comprise amino acid residues from a "complementarity determining region" as defined by Kabat (e.g. about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and SI- 35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; (Kabat et al., Sequences of Proteins of Immulological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or those residues from a "hypervariable loop" (e.g. about residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26- 32 (H 1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain (Chothia and Lesk; J Mol Biol 196: 901-917 (1987)). In some instances, a complementarity determining region can include amino acids from both a CDR region defined according to Kabat and a hypervariable loop.

Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different "classes". There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these maybe further divided into "subclasses" (isotypes), e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 , and lgA2.

The heavy-chain constant domains that correspond to the different classes of antibodies are called [alpha], [delta], [epsilon], [gamma], and [mu], respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. As used herein antibodies are conventionally known antibodies and functional fragments thereof.

A “functional fragment” or “antigen-binding antibody fragment” of an antibody/immunoglobulin hereby is defined as a fragment of an antibody/immunoglobulin (e.g., a variable region of an IgG) that retains the antigen-binding region. An “antigen-binding region” of an antibody typically is found in one or more hyper variable region(s) of an antibody, e.g., the CDR1 , -2, and/or -3 regions; however, the variable “framework” regions can also play an important role in antigen binding, such as by providing a scaffold for the CDRs. Preferably, the “antigen-binding region” comprises at least amino acid residues 4 to 103 of the variable light (VL) chain and 5 to 109 of the variable heavy (VH) chain, more preferably amino acid residues 3 to 107 of VL and 4 to 111 of VH, and particularly preferred are the complete VL and VH chains (amino acid positions 1 to 109 of VL and 1 to 113 of VH; numbering according to WO 97/08320).

“Functional fragments”, “antigen-binding antibody fragments”, or “antibody fragments” of the disclosure include but are not limited to Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabodies; single domain antibodies (DAbs), linear antibodies; single-chain antibody molecules (scFv); and multispecific, such as bi- and tri-specific, antibodies formed from antibody fragments (C. A. K Borrebaeck, editor (1995) Antibody Engineering (Breakthroughs in Molecular Biology), Oxford University Press; R. Kontermann & S. Duebel, editors (2001) Antibody Engineering (Springer Laboratory Manual), Springer Verlag). An antibody other than a "multi-specific" or "multifunctional" antibody is understood to have each of its binding sites identical. The F(ab’)2 or Fab may be engineered to minimize or completely remove the intermolecular disulfide interactions that occur between the CH1 and CL domains.

The term "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In case of an IgG antibody the Fc region comprises the CH2 and CH3 domains and in case of an IgM antibody the Fc region comprises the CH3 and CH4 domains. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

"Percent (%) sequence identity" with respect to a reference polynucleotide or polypeptide sequence, respectively, is defined as the percentage of nucleic acid or amino acid residues, respectively, in a candidate sequence that are identical with the nucleic acid or amino acid residues, respectively, in the reference polynucleotide or polypeptide sequence, respectively, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Conservative substitutions are not considered as part of the sequence identity. Preferred are un-gapped alignments. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

"Sequence homology" indicates the percentage of amino acids that either is identical or that represent conservative amino acid substitutions.

Amino acids may be referred to herein by their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The term "pharmaceutical formulation" I “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

The term "vector", as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants", "transformed cells", “transfectants”, “transfected cells”, and “transduced cells”, which include the primary transformed/transfected/transduced cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that has the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

As used herein without limitation, the term “recombinant,” as a modifier of a viral vector, such as a recombinant AAV (rAAV) vector, as well as a modifier of sequences such as recombinant polynucleotides and polypeptides, means that compositions have been manipulated (i.e., engineered) in a fashion that generally does not occur in nature.

Polypeptides having immunoglobulin cysteine protease activity

The inventors present the first natural occurring proteases capable of cleaving human antibodies of type IgM and IgG.

The present disclosure provides a polypeptide having immunoglobulin cysteine protease activity. That is, the polypeptide is able to cleave an immunoglobulin molecule. The immunoglobulin molecule is typically an IgG and/or an IgM molecule. The polypeptide may cleave any immunoglobulin molecule comprising a hinge/CH2 (for IgG) or CH2/CH3 (for IgM) sequence of any one of SEQ ID Nos: 57 to 61 as shown in Table 10. The polypeptide might cleave between the bold/underlined residues as shown for each of these sequences in Table 10.

Cysteine proteases, also known as thiol proteases, are hydrolase enzymes that degrade proteins. These proteases share a common catalytic mechanism that involves a nucleophilic cysteine thiol in a catalytic triad or dyad. Cysteine proteases have a “cysteine protease activity”, as used herein.

The term “polypeptide having IgG and IgM cysteine protease activity”, as used herein, refers to a polypeptide which is a cysteine protease and cleaves human IgG molecules as well as human IgM molecules. IgG and IgM molecules of species other than cynomolgus might be cleaved, too.

The polypeptide may cleave all human IgG subclasses, that is lgG1 , lgG2, lgG3 and lgG4. The polypeptide may exhibit lower activity against human lgG4 as compared to the other human IgG subclasses. The polypeptide may exhibit greater activity against human lgG2 than human I gG 1 . The polypeptide may exhibit activity against non-human IgG molecules.

The polypeptide may efficiently cleave IgG and IgM to Fc and F(ab’)2 fragments via a two-stage mechanism. In the first stage, one (first) heavy chain of IgG or IgM is cleaved to generate a single cleaved molecule with a single non-covalently bound Fc chain. IgG antibodies can be cleaved within the hinge region between the CH1 and CH2 domains and IgM antibodies can be cleaved between the CH2 and CH3 domains.

In the second stage of the mechanism the remaining (second) heavy chain of the original IgG or IgM molecule is quickly cleaved by the polypeptide to release a F(ab’)2 fragment and a Fc fragment which comprises for IgG the CH2-CH3 domains and for IgM the CH3-CH4 domains. In case of an IgM the CH2 domain is part of the F(ab’)2 fragment. These are the products generally observed under physiological conditions. Under reducing conditions, the F(ab’)2 fragment may dissociate to two Fab fragments.

IgG and IgM cysteine protease activity may be assessed by any suitable method, for example by incubating a polypeptide with a sample containing IgG and/or IgM and determining the presence of IgG and/or IgM cleavage products. Assays for assessing immunoglobulin protease activity may also be used to quantify the efficacy of said activity, that is to assess the potency of a polypeptide. Suitable assays to determine activity and/or quantify potency of said activity are well known in the art and any suitable assay may be used. Suitable assays to determine IgG and IgM cysteine protease activity are provided in the Examples.

The polypeptide having IgG and IgM cysteine protease activity cleaves at least 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of total amount of the IgG and/or IgM incubated with said polypeptide into the corresponding F(ab’)2 fragments.

The disclosure relates to new enzymes which display IgG and IgM cysteine protease activity. These enzymes form two homology clusters which are highly related to each other. These polypeptides having IgG and IgM cysteine protease activity are only distantly related to all other known specific IgG proteases. In Example 6 the inventors provide a detailed sequence analysis comparing the identified polypeptides with each other and comparisons with all other known IgG proteases.

The isolated polypeptide having IgG and IgM cysteine protease activity may have a maximum length of 1000, 560, 500, 400, 350, 300, or 250 amino acids.

The isolated polypeptide having IgG and IgM cysteine protease activity may be engineered or modified to assist with production, isolation, or purification. For example, where a polypeptide of the disclosure is produced by recombinant expression in a bacterial host cell, the sequence of the polypeptide may include an additional methionine (M) residue at the N terminus to improve expression.

The polypeptide sequence may include an N terminal methionine followed by a secretion signal sequence. A sequence may include an N terminal methionine followed by a secretion signal sequence. The polypeptide may comprise an amino acid sequence of SEQ ID NO: 11 (MNIKKFAKQATVLTFTTALLAGGATQAFA) at the N terminus. The sequence is often not part of the mature isolated polypeptide.

As another embodiment, the polypeptide of the disclosure may be engineered or modified by addition of protein purification tag and the N or C terminus, preferably at the C terminus. The protein purification tag is preferably a moiety which is not naturally expressed in bacteria. The protein purification tag is preferably a moiety which is not present in a wildtype polypeptide chain as expressed in bacteria. A protein purification tag may be a ligand which is capable of binding directly and specifically to a separation means. Alternatively, the protein purification tag may be one member of a binding pair and the separation means comprises a reagent that includes the other member of the binding pair.

In particular embodiments of the first aspect, the isolated polypeptide having IgG and IgM cysteine protease activity comprises an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 15.

In particular embodiments of the first aspect, the isolated polypeptide having IgG and IgM cysteine protease activity comprises an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 16.

In particular embodiments of the first aspect, the isolated polypeptide having IgG and IgM cysteine protease activity comprises an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 17.

In particular embodiments of the first aspect, the isolated polypeptide having IgG and IgM cysteine protease activity comprises an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 18.

In particular embodiments of the first aspect, the isolated polypeptide having IgG and IgM cysteine protease activity comprises an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 19.

In particular embodiments of the first aspect, the isolated polypeptide having IgG and IgM cysteine protease activity comprises an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 20.

In particular embodiments of the first aspect, the isolated polypeptide having IgG and IgM cysteine protease activity comprises an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 21.

In particular embodiments of the first aspect, the isolated polypeptide having IgG and IgM cysteine protease activity comprises an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 22. In particular embodiments of the first aspect, the isolated polypeptide having IgG and IgM cysteine protease activity comprises an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 23.

In particular embodiments of the first aspect, the isolated polypeptide having IgG and IgM cysteine protease activity comprises an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 24.

In particular embodiments of the first aspect, the isolated polypeptide having IgG and IgM cysteine protease activity comprises an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 25.

In particular embodiments of the first aspect, the isolated polypeptide having IgG and IgM cysteine protease activity comprises an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 26.

In particular embodiments of the first aspect, the isolated polypeptide having IgG and IgM cysteine protease activity comprises an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 27.

In particular embodiments of the first aspect, the isolated polypeptide having IgG and IgM cysteine protease activity comprises an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 28.

In particular embodiments of the first aspect, the isolated polypeptide having IgG and IgM cysteine protease activity comprises an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 29.

In particular embodiments of the first aspect, the isolated polypeptide having IgG and IgM cysteine protease activity comprises an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 30.

In particular embodiments of the first aspect, the isolated polypeptide having IgG and IgM cysteine protease activity comprises an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 31.

In particular embodiments of the first aspect, the isolated polypeptide having IgG and IgM cysteine protease activity comprises an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 32.

In a certain embodiment of the first aspect the isolated polypeptide having IgG and IgM cysteine protease activity is a polypeptide, which comprises: a) an amino acid sequence which is SEQ ID NO: 15, b) an amino acid sequence which is SEQ ID NO: 16, c) an amino acid sequence which is SEQ ID NO: 17, d) an amino acid sequence which is SEQ ID NO: 18, e) an amino acid sequence which is SEQ ID NO: 19, f) an amino acid sequence which is SEQ ID NO: 20, g) an amino acid sequence which is SEQ ID NO: 21 , h) an amino acid sequence which is SEQ ID NO: 22, i) an amino acid sequence which is SEQ ID NO: 23, j) an amino acid sequence which is SEQ ID NO: 24, k) an amino acid sequence which is SEQ ID NO: 25, l) an amino acid sequence which is SEQ ID NO: 26, m) an amino acid sequence which is SEQ ID NO: 27, n) an amino acid sequence which is SEQ ID NO: 28, o) an amino acid sequence which is SEQ ID NO: 29, p) an amino acid sequence which is SEQ ID NO: 30, q) an amino acid sequence which is SEQ ID NO: 31 , or r) an amino acid sequence which is SEQ ID NO: 32.

In a certain embodiment of the first aspect the isolated polypeptide having IgG and IgM cysteine protease activity is a polypeptide, which is or essentially consists of: a) an amino acid sequence which is SEQ ID NO: 15, b) an amino acid sequence which is SEQ ID NO: 16, c) an amino acid sequence which is SEQ ID NO: 17, d) an amino acid sequence which is SEQ ID NO: 18, e) an amino acid sequence which is SEQ ID NO: 19, f) an amino acid sequence which is SEQ ID NO: 20, g) an amino acid sequence which is SEQ ID NO: 21 , h) an amino acid sequence which is SEQ ID NO: 22, i) an amino acid sequence which is SEQ ID NO: 23, j) an amino acid sequence which is SEQ ID NO: 24, k) an amino acid sequence which is SEQ ID NO: 25, l) an amino acid sequence which is SEQ ID NO: 26, m) an amino acid sequence which is SEQ ID NO: 27, n) an amino acid sequence which is SEQ ID NO: 28, o) an amino acid sequence which is SEQ ID NO: 29, p) an amino acid sequence which is SEQ ID NO: 30, q) an amino acid sequence which is SEQ ID NO: 31 , or r) an amino acid sequence which is SEQ ID NO: 32.

Amino Acid Variants

Polypeptide variants of a polypeptide of the first aspect having IgG and IgM cysteine protease activity may be made that conserves the overall molecular structure of a polypeptide described herein. Given the properties of the individual amino acids, some rational substitutions will be recognized by the skilled worker. Amino acid substitutions, i.e., "conservative substitutions," may be made, for instance, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.

For example, (a) nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophane, and methionine; (b) polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positively charged (basic) amino acids include arginine, lysine, and histidine; and (d) negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Substitutions typically may be made within groups (a)-(d). In addition, glycine and proline may be substituted for one another based on their ability to disrupt a-helices. Similarly, certain amino acids, such as alanine, cysteine, leucine, methionine, glutamic acid, glutamine, histidine and lysine are more commonly found in a-helices, while valine, isoleucine, phenylalanine, tyrosine, tryptophan and threonine are more commonly found in p-pleated sheets. Glycine, serine, aspartic acid, asparagine, and proline are commonly found in turns. Some preferred substitutions may be made among the following groups: (i) S and T; (ii) P and G; and (iii) A, V, L and I. Given the known genetic code, and recombinant and synthetic DNA techniques, the skilled scientist readily can construct DNAs encoding the conservative amino acid variants.

Recombinant DNA constructs and recombinant expression

In a second aspect there are provided nucleotide acids encoding a polypeptide of the first aspect having IgG and IgM cysteine protease activity (see for examples, Table 2), DNA constructs comprising said nucleic acids, and host cells comprising said nucleic acids and/or said DNA constructs. The recombinant constructs of the present disclosure may be used in connection with a vector, such as a plasmid, phagemid, phage or viral vector, into which a DNA molecule encoding a polypeptide having IgG and IgM cysteine protease activity is inserted.

A polypeptide having IgG and IgM cysteine protease activity provided herein can be prepared by recombinant expression of nucleic acid sequences encoding said polypeptide in a host cell. To express a polypeptide having IgG and IgM cysteine protease activity recombinantly a host cell can be transfected with one or more recombinant expression vectors carrying DNA fragments encoding said polypeptide. Standard recombinant DNA methodologies are used to prepare and/or obtain nucleic acids encoding said polypeptide, incorporate these nucleic acids into recombinant expression vectors and introduce the vectors into host cells, such as those described in Sambrook, Fritsch and Maniatis (eds.), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F. M. et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and in U.S. Pat. No. 4,816,397 by Boss et al..

To express the polypeptide having IgG and IgM cysteine protease activity standard recombinant DNA expression methods can be used (see, for example, Goeddel; Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). For example, DNA encoding the desired polypeptide can be inserted into an expression vector which is then transfected into a suitable host cell. Suitable host cells are prokaryotic or eukaryotic cells. Examples for prokaryotic host cells are e.g. bacteria, examples for eukaryotic hosts cells are yeasts, insects and insect cells, plants and plant cells, transgenic animals, or mammalian cells. It is understood that the design of the expression vector, including the selection of regulatory sequences is affected by factors such as the choice of the host cell, the level of expression of protein desired and whether expression is constitutive or inducible.

Therefore, further embodiments of the present disclosure are also host cells comprising the vector or a nucleic acid molecule, whereby the host cell can be a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell, and may be a prokaryotic cell, such as a bacterial cell.

Another embodiment of the present disclosure is a method of using the host cell to produce a polypeptide having IgG and IgM cysteine protease activity, comprising culturing the host cell under suitable conditions and recovering said polypeptide having IgG and IgM cysteine protease activity.

Useful expression vectors for bacterial use are constructed by inserting a DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desirable, to provide amplification within the host. Suitable prokaryotic hosts for transformation include but are not limited to E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.

Bacterial vectors may be, for example, bacteriophage-, plasmid- or phagemid-based. These vectors can contain a selectable marker and a bacterial origin of replication derived from commercially available plasmids typically containing elements of the well-known cloning vector pBR322 (ATCC 37017). Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is de-repressed/induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

In bacterial systems, several expression vectors may be advantageously selected depending upon the use intended for the protein being expressed.

Therefore, an embodiment of the present disclosure is an expression vector comprising a nucleic acid sequence encoding for the polypeptide having IgG and IgM cysteine protease activity.

The polypeptide having IgG and IgM cysteine protease activity include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic host, including, for example, E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.

In a specific embodiment, the vector comprising the nucleic acid encoding a polypeptide having IgG and IgM cysteine protease activity is the plasmid pBSX, a derivative of pUB110 (Palva, llkka, Matti Sarvas, Paivi Lehtovaara, Mervi Sibakov, and Leevi Kaariainen. 1982. ..Secretion of Escherichia coli p-lactamase from Bacillus subtilis by the aid of alpha-amylase signal sequence." Proc. Natl. Acad. Sci. USA 5582-5586. doi:10.1073/pnas.79.18.5582.). In a specific embodiment said vector is transformed into Bacillus subtilis WB600 cells.

In vitro methods

In a third aspect there is provided an in vitro method of generating Fc, F(ab’)2 fragments, and/or Fab fragments of IgG or IgM molecules comprising contacting IgG or IgM molecules with an isolated polypeptide according to the first aspect having IgG and IgM cysteine protease activity.

The Fc, F(ab’)2 fragments, and/or Fab fragments may be isolated and further characterized.

In an embodiment of the third aspect, there is provided an in vitro method of generating Fc, F(ab’)2 fragments, and/or Fab fragments of IgG or IgM comprising contacting a solution comprising IgG or IgM with a polypeptide according to the first aspect. In certain embodiments the IgG or IgM molecules are of human or cynomolgus origin.

In certain embodiments the solution comprising IgG or IgM is of human or cynomolgus origin.

In certain embodiments human or cynomolgus samples are incubated with a polypeptide having IgG and IgM cysteine protease activity according to the first aspect.

In certain embodiments the resulting enzymatic products are further analyzed by methods known in the art, e.g., by SDS-PAGE, ELISA, MS, surface plasmon resonance, etc. Examples for such analysis are provided within the Examples.

Therapeutic Methods

In a fourth aspect there are provided therapeutic methods comprising administering to a subject in need of treatment a therapeutically effective amount of a polypeptide according to the first aspect having IgG and IgM cysteine protease activity, or administering to a subject in need of treatment a therapeutically effective amount of a nucleotide acid encoding a polypeptide of the first aspect having IgG and IgM cysteine protease activity, a vector, or other DNA constructs comprising said nucleic acids.

A "therapeutically effective" amount hereby is defined as the amount of a polypeptide according to the first aspect having IgG and IgM cysteine protease activity that is of sufficient quantity to reduce IgG and/or IgM levels of a subject in need for the therapeutic purpose either as a single dose or according to a multiple dose regimen, alone or in combination with other agents. The subject may be a human.

The therapeutic methods may be characterized as: a) A method of treatment or prevention of a disease or condition mediated by IgG and/or IgM antibodies. b) A method of prevention or treatment of a humoral immune response caused by transplants and post-operative treatments. c) A method of prevention or treatment of a humoral immune response caused by administration of a recombinant gene therapy virus vector, wherein the humoral immune response is against the recombinant gene therapy virus vector. d) A method of combination with a recombinant gene therapy virus vector for the treatment of a disease treated by said recombinant gene therapy virus vector in a patient in need thereof. A method of treatment or prevention of a disease or condition mediated by IgG and/or IgM antibodies.

In an embodiment of the fourth aspect there is provided a method of treatment or prevention of a disease or condition mediated by IgG and/or IgM antibodies in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a polypeptide according to the first aspect having IgG and IgM cysteine protease activity or a nucleic acid encoding this polypeptide.

The subject is typically a mammalian subject. In certain embodiments the subject is human.

In certain aspects the IgG and/or IgM antibodies are pathogenic antibodies.

In certain embodiments there is provided a polypeptide having IgG and IgM cysteine protease activity for use as a medicament for the treatment or prevention of a disease or condition mediated by IgG and/or IgM antibodies wherein said polypeptide comprises: a) an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 19, or b) an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 30.

In certain embodiments there is provided a method for the treatment or prevention of a disease or condition mediated by IgG and/or IgM antibodies comprising the administration of a polypeptide having IgG and IgM cysteine protease activity wherein said polypeptide comprises: a) an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 19, or b) an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 30.

In specific embodiments, the polypeptide having IgG and IgM cysteine protease used in the fourth aspect comprises an amino acid sequence which is at least 90%, 95%, 98%, 99%, or 100% identical to: a) an amino acid sequence which is SEQ ID NO: 15, b) an amino acid sequence which is SEQ ID NO: 16, c) an amino acid sequence which is SEQ ID NO: 17, d) an amino acid sequence which is SEQ ID NO: 18, e) an amino acid sequence which is SEQ ID NO: 19, f) an amino acid sequence which is SEQ ID NO: 20, g) an amino acid sequence which is SEQ ID NO: 21 , h) an amino acid sequence which is SEQ ID NO: 22, i) an amino acid sequence which is SEQ ID NO: 23, j) an amino acid sequence which is SEQ ID NO: 24, k) an amino acid sequence which is SEQ ID NO: 25, l) an amino acid sequence which is SEQ ID NO: 26, m) an amino acid sequence which is SEQ ID NO: 27, n) an amino acid sequence which is SEQ ID NO: 28, o) an amino acid sequence which is SEQ ID NO: 29, p) an amino acid sequence which is SEQ ID NO: 30, q) an amino acid sequence which is SEQ ID NO: 31 , or r) an amino acid sequence which is SEQ ID NO: 32.

In a certain embodiment the polypeptide having IgG and IgM cysteine protease activity used in the fourth aspect is a polypeptide, which comprises: a) an amino acid sequence which is SEQ ID NO: 15, b) an amino acid sequence which is SEQ ID NO: 16, c) an amino acid sequence which is SEQ ID NO: 17, d) an amino acid sequence which is SEQ ID NO: 18, e) an amino acid sequence which is SEQ ID NO: 19, f) an amino acid sequence which is SEQ ID NO: 20, g) an amino acid sequence which is SEQ ID NO: 21 , h) an amino acid sequence which is SEQ ID NO: 22, i) an amino acid sequence which is SEQ ID NO: 23, j) an amino acid sequence which is SEQ ID NO: 24, k) an amino acid sequence which is SEQ ID NO: 25, l) an amino acid sequence which is SEQ ID NO: 26, m) an amino acid sequence which is SEQ ID NO: 27, n) an amino acid sequence which is SEQ ID NO: 28, o) an amino acid sequence which is SEQ ID NO: 29, p) an amino acid sequence which is SEQ ID NO: 30, q) an amino acid sequence which is SEQ ID NO: 31 , or r) an amino acid sequence which is SEQ ID NO: 32.

In a certain embodiment the polypeptide having IgG and IgM cysteine protease activity used in the fourth aspect is a polypeptide, which is or essentially consists of: a) an amino acid sequence which is SEQ ID NO: 15, b) an amino acid sequence which is SEQ ID NO: 16, c) an amino acid sequence which is SEQ ID NO: 17, d) an amino acid sequence which is SEQ ID NO: 18, e) an amino acid sequence which is SEQ ID NO: 19, f) an amino acid sequence which is SEQ ID NO: 20, g) an amino acid sequence which is SEQ ID NO: 21 , h) an amino acid sequence which is SEQ ID NO: 22, i) an amino acid sequence which is SEQ ID NO: 23, j) an amino acid sequence which is SEQ ID NO: 24, k) an amino acid sequence which is SEQ ID NO: 25, l) an amino acid sequence which is SEQ ID NO: 26, m) an amino acid sequence which is SEQ ID NO: 27, n) an amino acid sequence which is SEQ ID NO: 28, o) an amino acid sequence which is SEQ ID NO: 29, p) an amino acid sequence which is SEQ ID NO: 30, q) an amino acid sequence which is SEQ ID NO: 31 , or r) an amino acid sequence which is SEQ ID NO: 32.

In certain embodiments the disease or condition mediated by pathogenic IgG and/or IgM antibodies is an autoimmune disease, transplant rejection or acquired hemophilia.

In certain embodiments said transplant rejection is an allograft or a xenograft rejection.

In certain embodiments said autoimmune disease is Addison’s disease, alopecia areata, ankylosing spondilitis, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, antiphospholipid syndrome, aplastic anemia, autoimmune cardiomyopathies, autoimmune gastritis, autoimmune hearing loss, autoimmune hepatitis, autoimmune hypoparathyroidism, autoimmune hypophysitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune polyendocrinopathy, Bechet's disease, bullous pemphigoid, cardiomyopathy, chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, coeliac disease, Crohn's disease, CREST syndrome, Degos disease, epidermolysis bullosa acquisita, essential mixed cryoglobulinemia, giant cells arteritis, glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillan-Barre syndrome, Hashimoto's thyroiditis, idiopathic thrombocytopenic purpura, inflammatory bowel disease, Kawasaki's disease, membranous nephropathy, Meniere's syndrome, mixed connective tissue disease, Mooren's ulcer, multiple sclerosis, myasthenia gravis, pemphigus foliaceous, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polyglandular autoimmune syndrome type 1 (PAS-I), polyglandular autoimmune syndrome type 2 (PAS-2), polyglandular autoimmune syndrome type 3 (PAS-3), polymyositis/dermatomyositis, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's syndrome, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, subacute thyroiditis, sympathetic ophthalmia, systemic lupus erythematosus, Takayasu's arteritis, type 1 diabetes mellitus, vitiligo, Vogt-Koyanagi-Harada disease or Wegener's granulomatosis.

In a particular embodiment said autoimmune disease is rheumatoid arthritis or systemic lupus erythematosus.

Further particular methods of treatment or prevention of a disease or condition mediated by IgG and/or IgM antibodies are the treatment of anti-myelin associated glycoprotein (anti-MAG) neuropathy, autoimmune hemolytic anemia, monoclonal gammopathy, Hyper-IgM syndrome, post-COVID19 syndrome, Waldenstrom macroglobulinemia.

IgG and IgM activate the classical pathway of the complement system. The isolated polypeptide according to the first aspect having IgG and IgM cysteine protease activity or a nucleic acid encoding this polypeptide may therefore be used to treat diseases and conditions where complement activation is detrimental to the patient. For example, isolated polypeptide according to the first aspect having IgG and IgM cysteine protease activity or a nucleic acid encoding this polypeptide may be used to treat transplantation-derived disorders, for example transplant rejection (such as allograft and xenograft rejection) and graft-versus-host disease. The transplantation-derived disorder may occur due to the transplantation of a tissue or an organ in a patient.

The isolated polypeptide according to the first aspect having IgG and IgM cysteine protease activity or a nucleic acid encoding this polypeptide may also of use in post-operative treatment, for example in the treatment of patients who have undergone heart by-pass operations. In certain embodiments of preventing transplant rejection the polypeptide according to the first aspect having IgG and IgM cysteine protease activity or a nucleic acid encoding this polypeptide is administered to the subject before transplantation, or after transplantation treatment. Therefore, the treatment may be therapeutic or prophylactic.

A therapeutically effective amount of the polypeptide according to the first aspect having IgG and IgM cysteine protease activity or a nucleic acid encoding this polypeptide is an amount effective to ameliorate one or more symptoms of a disease or condition.

In certain embodiments there is provided a method for treating a disorder or condition associated with the undesired presence of IgG and/or IgM, comprising administering to a subject in need thereof a therapeutically effective amount of a polypeptide according to the first aspect having IgG and IgM cysteine protease activity or a nucleic acid encoding this polypeptide.

A method of prevention or treatment of a humoral immune response caused by administration of a recombinant gene therapy viral vector & a method of combination with a recombinant gene therapy virus vector for the treatment of a disease treated by said recombinant gene therapy viral vector

In another aspect there is provided a method for utilizing the polypeptide according to the first aspect having IgG and IgM cysteine protease activity or a nucleic acid encoding this polypeptide to reduce antibody IgG and/or IgM levels in human plasma. Methods according to the disclosure may be used, inter alia, to treat patients with pre-existing neutralizing antibodies to gene therapy vectors and to re dose patients previously treated with a gene therapy vector.

In certain embodiments there is provided a method of treating a subject in need of treatment for a disease caused by a gain of function activity or expression, of a protein includes: (a) administering to the subject a recombinant viral vector comprising a heterologous polynucleotide that is transcribed into a nucleic acid that inhibits, decreases or reduces expression of the gain of function, activity or expression of said protein; and (b) administering to the subject the polypeptide according to the first aspect having IgG and IgM cysteine protease activity or a nucleic acid encoding this polypeptide effective to degrade or digest and/or inhibit or reduce effector function of antibodies that bind to said recombinant viral vector.

In certain embodiments there is provided a method of treating a subject in need of treatment for a disease caused by a loss of function or activity of a protein includes: (a) administering to the subject a recombinant viral vector comprising a heterologous polynucleotide that encodes a protein or peptide that provides or supplements a function or activity of the protein; and (b) administering to the subject an amount of the polypeptide according to the first aspect having IgG and IgM cysteine protease activity or a nucleic acid encoding this polypeptide effective to degrade or digest and/or inhibit or reduce effector function of antibodies that bind to said recombinant viral vector and/or the protein or peptide encoded by the heterologous polynucleotide.

In certain embodiments of the method, step (b) is performed within about 14 days before step

(a). In certain embodiments, step (b) is performed within about 7 days before step (a). In certain embodiments, step (b) is performed within about 72 hours before step (a). In certain embodiments, step (b) is performed within about 48 hours before step (a). In certain embodiments, step (b) is performed within about 24 hours before step (a). In certain embodiments, step (b) is performed within about 12 hours before step (a). In certain embodiments, step (b) is performed within about 6 hours before step (a).

In certain embodiments there is provided a polypeptide having IgG and IgM cysteine protease activity for use in the prevention or treatment of an humoral immune response caused by administration of a recombinant gene therapy virus vector, wherein the humoral immune response is against the recombinant gene therapy virus vector, wherein said polypeptide is administered before the recombinant gene therapy vector is administered, and wherein said polypeptide comprises: a) an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 19, or b) an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 30.

In certain embodiments there is provided a polypeptide having IgG and IgM cysteine protease activity for use in combination with a recombinant gene therapy virus vector for the treatment of a disease treated by said recombinant gene therapy virus vector in a patient in need thereof, wherein said polypeptide is administered before the recombinant gene therapy virus vector is administered, wherein said patient has neutralizing anti-virus antibodies that inhibit cell transduction of said recombinant gene therapy virus vector, and wherein said polypeptide comprises: a) an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 19, or b) an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 30.

In certain embodiments, the recombinant gene therapy virus vector comprises a recombinant lentiviral vector, a recombinant parvovirus vector, a recombinant adenoviral vector, or a recombinant adeno-associated virus (AAV) vector.

In certain embodiments, the polypeptide having IgG and IgM cysteine protease activity is administered within about 14 days before the recombinant gene therapy virus vector is administered. In certain embodiments, the polypeptide having IgG and IgM cysteine protease activity is administered within about 7 days before the recombinant gene therapy virus vector is administered. In certain embodiments, the polypeptide having IgG and IgM cysteine protease activity is administered within about 72 hours before the recombinant gene therapy virus vector is administered. In certain embodiments, the polypeptide having IgG and IgM cysteine protease activity is administered within about 48 hours before the recombinant gene therapy virus vector is administered. In certain embodiments, the polypeptide having IgG and IgM cysteine protease activity is administered within about 24 hours before the recombinant gene therapy virus vector is administered. In certain embodiments, the polypeptide having IgG and IgM cysteine protease activity is administered within about 12 hours before the recombinant gene therapy virus vector is administered. In certain embodiments, the polypeptide having IgG and IgM cysteine protease activity is administered within about 6 hours before the recombinant gene therapy virus vector is administered.

In certain embodiments there is provided a method for prevention or treatment of an humoral immune response caused by administration of a recombinant gene therapy virus vector, wherein the humoral immune response is against the recombinant gene therapy virus vector, comprising administration of a polypeptide having IgG and IgM cysteine protease, wherein said polypeptide is administered before the recombinant gene therapy vector is administered, and wherein said polypeptide comprises: a) an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 19, or b) an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 30.

In certain embodiments there is provided a method for prevention or treatment of a disease comprising administration of a polypeptide having IgG and IgM cysteine protease activity in combination with a recombinant gene therapy virus vector to in a patient in need thereof, wherein said polypeptide is administered before the recombinant gene therapy virus vector is administered, wherein said patient has neutralizing anti-virus antibodies that inhibit cell transduction of said recombinant gene therapy virus vector, and wherein said polypeptide comprises: a) an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 19, or b) an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 30. In certain embodiments of these methods, the recombinant gene therapy virus vector comprises a recombinant lentiviral vector, a recombinant parvovirus vector, a recombinant adenoviral vector, or a recombinant adeno-associated virus (AAV) vector.

In certain embodiments of these methods, the polypeptide having IgG and IgM cysteine protease activity is administered within about 14 days before the recombinant gene therapy virus vector is administered. In certain embodiments, the polypeptide having IgG and IgM cysteine protease activity is administered within about 7 days before the recombinant gene therapy virus vector is administered. In certain embodiments, the polypeptide having IgG and IgM cysteine protease activity is administered within about 72 hours before the recombinant gene therapy virus vector is administered. In certain embodiments, the polypeptide having IgG and IgM cysteine protease activity is administered within about 48 hours before the recombinant gene therapy virus vector is administered. In certain embodiments, the polypeptide having IgG and IgM cysteine protease activity is administered within about 24 hours before the recombinant gene therapy virus vector is administered. In certain embodiments, the polypeptide having IgG and IgM cysteine protease activity is administered within about 12 hours before the recombinant gene therapy virus vector is administered. In certain embodiments, the polypeptide having IgG and IgM cysteine protease activity is administered within about 6 hours before the recombinant gene therapy virus vector is administered.

As used herein, the terms “patient” and “subject” interchangeably refer to an animal, typically a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, a subject or patient according to the instant disclosure is a human.

The high prevalence of neutralizing anti-AAV antibodies excludes certain subjects from enrollment in gene transfer trials with AAV vectors and will exclude certain patients from receiving approved AAV gene therapies, leaving certain patients without access to potentially lifesaving therapies. Furthermore, neutralizing anti-AAV antibodies are induced following AAV gene transfer, which prevents the transduction efficiency in case of redosing of the same individual.

In specific embodiments, the recombinant adeno-associated virus (AAV) vector comprises a capsid protein of an AAV virus selected from the Table of AAVs, or a variant of said AAVs.

In certain embodiments, the recombinant adeno-associated virus (AAV) vector comprises a VP1 , VP2 and/or VP3 capsid protein of an AAV virus selected from the group consisting of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV3B, AAV-2i8, or a variant of said AAV viruses.

In certain embodiments, the polypeptide having IgG and IgM cysteine protease comprises an amino acid sequence which is at least 90%, 95%, 98%, 99%, or 100% identical to: a) an amino acid sequence which is SEQ ID NO: 15, b) an amino acid sequence which is SEQ ID NO: 16, c) an amino acid sequence which is SEQ ID NO: 17, d) an amino acid sequence which is SEQ ID NO: 18, e) an amino acid sequence which is SEQ ID NO: 19, f) an amino acid sequence which is SEQ ID NO: 20, g) an amino acid sequence which is SEQ ID NO: 21 , h) an amino acid sequence which is SEQ ID NO: 22, i) an amino acid sequence which is SEQ ID NO: 23, j) an amino acid sequence which is SEQ ID NO: 24, k) an amino acid sequence which is SEQ ID NO: 25, l) an amino acid sequence which is SEQ ID NO: 26, m) an amino acid sequence which is SEQ ID NO: 27, n) an amino acid sequence which is SEQ ID NO: 28, o) an amino acid sequence which is SEQ ID NO: 29, p) an amino acid sequence which is SEQ ID NO: 30, q) an amino acid sequence which is SEQ ID NO: 31 , or r) an amino acid sequence which is SEQ ID NO: 32.

In a certain embodiment the polypeptide having IgG and IgM cysteine protease activity is a polypeptide, which comprises: a) an amino acid sequence which is SEQ ID NO: 15, b) an amino acid sequence which is SEQ ID NO: 16, c) an amino acid sequence which is SEQ ID NO: 17, d) an amino acid sequence which is SEQ ID NO: 18, e) an amino acid sequence which is SEQ ID NO: 19, f) an amino acid sequence which is SEQ ID NO: 20, g) an amino acid sequence which is SEQ ID NO: 21 , h) an amino acid sequence which is SEQ ID NO: 22, i) an amino acid sequence which is SEQ ID NO: 23, j) an amino acid sequence which is SEQ ID NO: 24, k) an amino acid sequence which is SEQ ID NO: 25, l) an amino acid sequence which is SEQ ID NO: 26, m) an amino acid sequence which is SEQ ID NO: 27, n) an amino acid sequence which is SEQ ID NO: 28, o) an amino acid sequence which is SEQ ID NO: 29, p) an amino acid sequence which is SEQ ID NO: 30, q) an amino acid sequence which is SEQ ID NO: 31 , or r) an amino acid sequence which is SEQ ID NO: 32. In a certain embodiment the polypeptide having IgG and IgM cysteine protease activity is a polypeptide, which is or essentially consists of: a) an amino acid sequence which is SEQ ID NO: 15, b) an amino acid sequence which is SEQ ID NO: 16, c) an amino acid sequence which is SEQ ID NO: 17, d) an amino acid sequence which is SEQ ID NO: 18, e) an amino acid sequence which is SEQ ID NO: 19, f) an amino acid sequence which is SEQ ID NO: 20, g) an amino acid sequence which is SEQ ID NO: 21 , h) an amino acid sequence which is SEQ ID NO: 22, i) an amino acid sequence which is SEQ ID NO: 23, j) an amino acid sequence which is SEQ ID NO: 24, k) an amino acid sequence which is SEQ ID NO: 25, l) an amino acid sequence which is SEQ ID NO: 26, m) an amino acid sequence which is SEQ ID NO: 27, n) an amino acid sequence which is SEQ ID NO: 28, o) an amino acid sequence which is SEQ ID NO: 29, p) an amino acid sequence which is SEQ ID NO: 30, q) an amino acid sequence which is SEQ ID NO: 31 , or r) an amino acid sequence which is SEQ ID NO: 32.

In some embodiments, the polypeptide according to the first aspect having IgG and IgM cysteine protease activity described here allows for the administration of a recombinant gene therapy virus vector to a subject who would otherwise not be a good candidate to receive such vector, for example, a subject who has previously received administration of a recombinant gene therapy virus vector and/or who was previously exposed to the recombinant gene therapy virus vector and has subsequently developed an antibody response to the vector. Typically, a subject is considered to be a candidate, i.e. , a good candidate, for administration of a recombinant gene therapy virus vector when they have an antibody titer for binding gene therapy virus vector that is less than 1:5 (e.g., 1 :1 , 1 :2, 1 :3, or 1 :4). In contrast, a subject is considered not to be a suitable candidate for administration of a recombinant gene therapy virus vector when they have an antibody titer for binding gene therapy virus vector that is 1 :5 or greater (e.g., 1 :6, 1 :6, 1 :7, 1 :8, 1 :9, 1 :10, 1 :20, 1 :30, 1 :50, 1 : 100, 1 :1 ,000 or more). One skilled in the art can assess the antibody titer of a subject using standard techniques in the art, e.g., by taking a biological sample from a subject, e.g., the subject’s blood, or, serum, challenging the biological sample with known antigens, and detecting the presence antibodies binding to the known antigens, e.g, viral vectors. An antibody titer is a measure of how much a sample containing the antibodies can be diluted before the antibody can no longer be detected in the sample. A neutralizing antibody to a viral vector is typically measured by an antibody titer that is needed to neutralize 50% of the viral vector antigen, e.g., as described in Guo P et al, Molecular Therapy: Methods & Clinical Development Vol. 13, p 40-46, June 2019. As an exemplary comparison, A titer of 1 :10 of an antibody binding to a viral vector is an indication of a lower-level antibodies than a 1 :100 titer of the same.

Accordingly, in one embodiment, the subject is assessed for the presence of anti-viral vector antibodies to the vector of a gene therapy prior to administration of the gene therapy. In one embodiment, the subject is assessed for the presence of neutralizing anti-viral vector antibodies to the vector of a gene therapy prior to administration of the gene therapy. An exemplary method for methods for detecting neutralizing anti-viral vector antibodies is further described in, e.g., Kasprzyk T., et al. Mol Therapy. Methods & Clinical Dev. Jan 6, 2022; Toon K., et al. Viruses 2021 , 13(2), 217; Vincent T., et al. J. Virol. 2001 75(3): 1516-1521 ; the contents of which are each incorporated herein in their entirety by reference.

Currently, prospective patients with viral neutralizing antibody levels 1 :5 or higher are excluded from treatment with viral vectors, i.e., they are not good candidates. Administration of the polypeptide having IgG and IgM cysteine protease activity described here into a subject having an antibody titer greater than or equal to 1 :5 but less than 1 :10 is expected to decrease the antibody titer present in the subject to less that 1 :5, thereby qualifying the subject as a candidate for administration of the recombinant gene therapy virus vector.

In one embodiments, the polypeptide according to the first aspect having IgG and IgM cysteine protease activity is administered to a subject that was found to have a titer of viral vector binding antibodies present in the biological sample, e.g., a blood sample or serum sample, from the subject that is greater than or equal to 1 :5 and less than about 1 :25 (e.g., 1 :6, 1 :7, 1 :8, 1 :9, 1 :10, 1 :11 , 1 :12, 1 :13, 1 :14, 1 :15, 1 :16, 1 :17, 1 :18, 1 :19, 1 :20, 1 :21 , 1 :22, 1 :23 and1 :24),. For example, administration of the polypeptide according to the first aspect having IgG and IgM cysteine protease activity into a subject having a titer greater than or equal to 1 :5 but less than 1 :15 is expected to decrease the antibody titer present in the subject to less that 1 :5, thereby qualifying the subject as a candidate for administration of the recombinant gene therapy virus vector. In one embodiment, the polypeptide according to the first aspect having IgG and IgM cysteine protease activity is administered to a subject having an antibody titer of gene therapy virus vector binding antibodies present in the biological sample from the subject that is greater than or equal to 1 :5 and less than about 1 :100 (e.g., 1 :6, 1 :7, 1 :8, 1 :9, 1 :10, 1 :11 , 1 :12, 1 :13, 1 :14, 1 :15, 1 :16, 1 :17, 1 :18, 1 :19, 1 :20, 1:21 , 1 :22, 1 :23, 1 :24, 1 :25, 1 :30, 1 :35, 1 :40, 1 :45, 1 :50, 1 :55, 1 :60, 1 :65, 1 :70, 1 :75, 1 :80, 1 :85, 1 :90, 1 :95, and 1 :99), Administration of the polypeptide according to the first aspect having IgG and IgM cysteine protease activity to a subject having an antibody titer greater than or equal to 1 :5 but less than 1 :25 is expected to decrease the antibody titer present in the subject to less that 1 :5, thereby qualifying the subject as a candidate for administration of the recombinant gene therapy virus vector.

In some embodiments of the invention, the polypeptide according to the first aspect having IgG and IgM cysteine protease activity enables repeated dosages, or repeat administration of an AAV vector as disclosed herein. For example, administration of the gene therapy virus vector, e.g., a AAV vector disclosed herein, with the polypeptide having IgG and IgM cysteine protease activity (at substantially the same time, or before or after the administration of the AAV vector) can be administered multiple times (i.e. , greater than one time) over a defined time period. For exemplary purposes, the AAV vector can be administered several times, i.e., more than once, over a several weeks (e.g., 2-weeks) to several months (e.g., 2-months). Without wishing to be limited to theory, administration of the AAV vector with the polypeptide having IgG and IgM cysteine protease activity according to the methods as disclosed herein can be, as non-limiting examples, every month over a period of 6-months, 3-4 times over a period of 6-weeks, every week over a period of 1-month (or about 4 weeks) or 2-months (or about 8-weeks). In some embodiments, where a AAV vector as disclosed herein is administered with a polypeptide having IgG and IgM cysteine protease activity (at substantially the same time, or before, or after the administration of the AAV vector) multiple times (e.g., a repeat dose), the dose of the gene therapy virus vector, e.g., AAV vector is lower than typically used in a single-dose regimen, for example, at a dose lower than a single-dose regimen as described herein. For example, the dose of the AAV vector can be less than or equal to about 10¹²vg, or lower than about 10¹² vg, for example, the dose can be about 10⁷, about 10⁸, about 10⁹, about 10¹⁰, about 10¹¹, or about 10¹², or any dose between 10⁷ vg and 10¹² vg.

In some embodiments, where repeat doses of a gene therapy viral vector as disclosed herein are administered to a subject with the polypeptide having IgG and IgM cysteine protease activity described herein, the polypeptide having IgG and IgM cysteine protease activity can be changed between the doses, i.e., the same or different polypeptide having IgG and IgM cysteine protease activity can be used in repeat doses. In some embodiments, the dosing regimen of the polypeptide having IgG and IgM cysteine protease activity further comprises administration of other immunomodulators. For example, in some embodiments, the IgG and IgM cleaving enzyme compositions of the invention are co-administered with otherAb cleaving enzymes (such as IdeS or IdeZ), and/or, other immunomodulators, e.g. ImmTOR™, or proteosome inhibitors.

One aspect herein provides a method for administering a recombinant gene therapy virus vector to a subject who has previously received a recombinant gene therapy virus vector, for example, the same recombinant gene therapy virus vector or another gene therapy virus vector having a similar serotype, the method comprising, prior to administering the recombinant gene therapy virus vector, administering to the subject at least one polypeptide having IgG and IgM cysteine protease activity. In one embodiment, the previously received recombinant gene therapy virus vector elicits an immune response resulting in anti-viral vector antibodies that target (i.e., recognizes and binds) to the recombinant gene therapy virus vector administered. In one embodiment, the subject has anti-viral vector antibody titer of at least 1 :5-1 :15, at least 1 :5-1 :25, at least 1 :5-1 :50, or at least 1 :5-1 :100, and the method comprises the steps of administering to the subject an polypeptide having IgG and IgM cysteine protease activity described herein, prior to, during, or after administration of the recombinant gene therapy virus vector.

Another aspect herein provides a method for administering a recombinant AAV gene therapy viral vector to a subject who was previously exposed to a the same or similar AAV gene therapy virus vector, wherein the exposure elicits an immune response resulting in anti-AAV antibodies that target the recombinant AAV gene therapy virus vector to be administered, and wherein the subject has anti-AAV antibody titer of at least 1 :5-1 :15, at least 1 :5-1 :25, at least 1 :5-1 :50, or at least 1 :5-1 :100, the method comprising the steps of administering to the subject an polypeptide having IgG and IgM cysteine protease activity described herein, prior to, during, or after administering the recombinant gene therapy virus vector.

In some embodiments, the polypeptide having IgG and IgM cysteine protease activity described herein is co-administered with other immunoglobulin degrading enzymes such as IdeS, IdeZ, IdeS/Z, Endo S, or, their functional variant. Non-limiting examples of such immunoglobulin degrading enzymes and their uses are described in US 7,666,582, US 8,133,483, US 20180037962, US 20180023070, US 20170209550, US 8,889,128, WO 2010057626, US 9,707,279, US 8,323,908, US 20190345533, US 20190262434, US 20210246469 and WO 2020016318, each of which are incorporated in their entirety herein by reference.

In some embodiments, the polypeptide having IgG and IgM cysteine protease activity described herein is co-administered with other immune modulators e.g, a protease or glycosidase or a proteasome inhibitor. In certain aspects, the proteasome inhibitor is Bortezomib. In some aspects of the embodiment, the immune modulator comprises bortezomib and an anti-CD20 antibody, such as Rituximab. In other aspects of the embodiment, the immune modulator comprises bortezomib, Rituximab, methotrexate, and intravenous gamma globulin. Non-limiting examples of proteasome inhibitors and their combinations with Rituximab, methotrexate and intravenous gamma globulin are described in US 10,028,993, US 9,592,247, and US 8,809,282, each of which is incorporated in its entirety herein by reference. In alternative embodiments, the immune modulator is an inhibitor of the NF-kB pathway. In certain aspects of the embodiment, the additional immune modulator is Rapamycin or a functional variant thereof. Non-limiting examples of uses of rapamycin are described in (i.e., ImmTOR™ nanoparticles) Kishimoto, et al., 2016, Nat Nanotechnol, 11(10): 890-899; Maldonado, et al., 2015, PNAS, 112(2): E156-165) and in US20200038463 and US Patent 9,006,254, US 10,071 ,114, US 20160067228, US 20160074531 , US 20160074532, US 20190076458, US 10,046,064, which are each incorporated herein by reference in their entirety. In other aspects of the embodiment, the additional immune modulator is synthetic nanocarriers comprising an immunosuppressant. Non limiting examples of immunosuppressants, immunosuppressants coupled to synthetic nanocarriers, synthetic nanocarriers comprising rapamycin, and/or, tolerogenic synthetic nanocarriers, their doses, administration and use are described in US20150320728, US 20180193482, US 20190142974, US 20150328333, US20160243253, US 10,039,822, US 20190076522, US 20160022650, US 10,441 ,651 , US 10,420,835, US 20150320870, US 2014035636, US 10,434,088, US 10,335,395, US 20200069659, US 10,357,483, US 20140335186, US 10,668,053, US 10,357,482, US 20160128986, US 20160128987, US 20200038462, US 20200038463, each of which is incorporated in its entirety herein by reference.

In some embodiments, the immune modulator is an engineered cell, e.g., an immune cell that has been modified using SQZ technology as described in WO2017192786, which is incorporated herein in its entirety by reference.

In some embodiments, the co-administered immune modulator is a small molecule that inhibits the innate immune response in cells, such as chloroquine (a TLR signaling inhibitor) and/or 2- aminopurine (a PKR inhibitor), which can also be administered in combination with the composition comprising at least one recombinant gene therapy AAV (rAAV) as disclosed herein. Some non-limiting examples of commercially available TLR-signaling inhibitors include BX795, chloroquine, CLI-095, OxPAPC, polymyxin B, and rapamycin (all available for purchase from INVIVOGEN). In addition, inhibitors of pattern recognition receptors (PRR) (which are involved in innate immunity signaling) such as 2-aminopurine, BX795, chloroquine, and H-89, can also be used in the compositions and methods comprising at least one rAAV vector as disclosed herein for in vivo protein expression as disclosed herein.

In some embodiments, the immune modulator is photopheresis, also known as extracorporeal photochemotherapy, or ECP. Photopheresis treatment is performed on a subject’s blood. Using either an IV or a catheter, blood is routed from the subject through a device which separates out a portion of white blood cells (leukocytes). The separated white blood cells are treated with naturally occurring photosensitizing chemicals called 8-methoxypsoralen (8-MOP) and then exposed to specific wavelengths of ultraviolet (UVA) light. Following exposure to the UVA light, the blood is administered back to the subject. Photopheresis can be performed at least once daily. In one embodiment, photopheresis is performed at least 1 , 2, 3, 4, 5, 6, 7 times a week prior to administration of the recombinant gene therapy virus vector. In one embodiment, photopheresis is performed at least 1 , 2, 3, 4, 5, 6, 7 times a week for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12 or more weeks, or for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 months prior to administration of the recombinant gene therapy virus vector. Therefore, administering the polypeptide having IgG and IgM cysteine protease activity to the subject can include performing photopheresis on the subject. It is understood that the photopheresis can be performed in conjunction with administration of a co administered immunomodulator selected from the enzymes, nanoparticles, and chemical compositions described herein and/or as a portion of a multiple dosing regimen.

In some embodiments, a polypeptide having IgG and IgM cysteine protease activity for use in the administration methods as disclosed herein is co-administered with an immunosuppressive drug or agent. As used herein, the term "immunosuppressive drug or agent" refers to pharmaceutical agents that inhibit or interfere with normal immune function. Examples of immunosuppressive drugs or agents suitable for the methods disclosed herein include agents that inhibit T-cell/B- cell co-stimulation pathways, such as agents that interfere with the coupling of T-cells and B-cells via the CTLA4 and B7 pathways, as disclosed in U.S. Patent Pub. No 2002/0182211 , which is incorporated herein by reference in its entirety. In one embodiment, an immunosuppressive agent is cyclosporine A. Other examples of immunosuppressive agents include myophenylate mofetil, rapamycin, and anti-thymocyte globulin. In various embodiments, the immunosuppressive drug is administered in a composition comprising at least one rAAV vector as disclosed herein, or in a separate composition but simultaneously with, or before or after administration of a composition comprising at least one rAAV vector according to the methods of administration as disclosed herein. An immunosuppressive drug is administered in a formulation which is compatible with the route of administration and is administered to a subject at a dosage sufficient to achieve the desired therapeutic effect. In some embodiments, the immunosuppressive drug is administered transiently for a sufficient time to induce tolerance to the rAAV vector as disclosed herein.

In any embodiment of the methods and compositions as disclosed herein, a subject being administered a composition disclosed herein is also administered an immunosuppressive agent. Various methods are known for achieving immunosuppression of an immune response in a patient being administered AAV. Methods known in the art include administering to the patient an immunosuppressive agent, such as a proteasome inhibitor. One such proteasome inhibitor known in the art, for instance as disclosed in U.S. Patent No. 9,169,492 and U.S. Patent Application No. 15/796,137, both of which are incorporated herein by reference in their entireties, is bortezomib. In some embodiments, the immunosuppressive agent is an antibody, including polyclonal, monoclonal, scFv or other antibody-derived molecule that is capable of suppressing the immune response, for instance, through the elimination or suppression of antibody producing cells. In a further embodiment, the immunosuppressive element is a short hairpin RNA (shRNA). In this embodiment, the coding region of the shRNA is included in the rAAV cassette and is generally located downstream, i.e., 3’, of the poly-A tail. The shRNA can be targeted to reduce, reduce, or eliminate expression of immunostimulatory agents, such as cytokines, growth factors (including transforming growth factors pi and (32, TNF and others that are known in the art).

Pharmaceutical Compositions and Administration

To treat any of the foregoing disorders, pharmaceutical compositions for use in accordance with the present disclosure may be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. The polypeptide according to the first aspect having IgG and IgM cysteine protease activity or a nucleic acid encoding this polypeptide can be administered by any suitable means, which can vary, depending on the type of disorder being treated. Possible administration routes include parenteral (e.g., intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous), intrapulmonary and intranasal, and, if desired for local immunosuppressive treatment, intralesional administration. In addition, an IgG and IgM cysteine protease of the disclosure or a variant thereof might be administered by pulse infusion, with, e.g., declining doses of the IgG and IgM cysteine protease. Preferably, the dosing is given by injections, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. The amount to be administered will depend on a variety of factors such as the clinical symptoms, weight of the individual, whether other drugs are administered. The skilled artisan will recognize that the route of administration will vary depending on the disorder or condition to be treated.

An embodiment of the present disclosure are pharmaceutical compositions which comprise the polypeptide according to the first aspect having IgG and IgM cysteine protease activity or a nucleic acid encoding this polypeptide or variants thereof, alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. A further embodiment are pharmaceutical compositions comprising a polypeptide according to the first aspect having IgG and IgM cysteine protease activity or a nucleic acid encoding this polypeptide and a further pharmaceutically active compound that is suitable to treat IgG and/or IgM related diseases. Any of these molecules can be administered to a patient alone, or in combination with other agents, drugs or hormones, in pharmaceutical compositions where it is mixed with excipient(s) or pharmaceutically acceptable carriers. In one embodiment of the present disclosure, the pharmaceutically acceptable carrier is pharmaceutically inert. The present disclosure also relates to the administration of pharmaceutical compositions. Such administration is accomplished orally or parenterally. Methods of parenteral delivery include topical, intra-arterial (directly to the tumor), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration. In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Ed. Maack Publishing Co, Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for ingestion by the patient.

Pharmaceutical preparations for oral use may be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl-cellulose, hydroxypropylmethylcellulose, or sodium carboxymethyl cellulose; and gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

Dragee cores may be provided with suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e. dosage.

Pharmaceutical preparations that may be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. Push- fit capsules can contain active ingredients mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations for parenteral administration include aqueous solutions of active compounds. For injection, the pharmaceutical compositions of the disclosure may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances that increase viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

The pharmaceutical compositions of the present disclosure may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, drageemaking, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can be formed with acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms. In other cases, the preferred preparation may be a lyophilized powder in 1 mM - 50 mM histidine, 0.1%-2% sucrose, 2%-7% mannitol at a pH range of 4.5 to 7.5 that is combined with buffer prior to use.

After pharmaceutical compositions comprising a compound of the disclosure formulated in an acceptable carrier have been prepared, they may be placed in an appropriate container and labeled for treatment of an indicated condition. For administration of the isolated polypeptide according to the first aspect having IgG and IgM cysteine protease activity or a nucleic acid encoding this polypeptide, such labeling would include amount, frequency and method of administration.

Kits

The disclosure further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the disclosure. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacture, use or sale of the product for human administration.

A further embodiment of the disclosure is: ) An isolated polypeptide having IgG and IgM cysteine protease activity comprising: a) an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 19, or b) an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 30. ) The polypeptide according to embodiment 1 , wherein said polypeptide comprises an amino acid sequence which is at least 90%, 95%, 98%, 99%, or 100% identical to: a) an amino acid sequence which is SEQ ID NO: 15, b) an amino acid sequence which is SEQ ID NO: 16, c) an amino acid sequence which is SEQ ID NO: 17, d) an amino acid sequence which is SEQ ID NO: 18, e) an amino acid sequence which is SEQ ID NO: 19, f) an amino acid sequence which is SEQ ID NO: 20, g) an amino acid sequence which is SEQ ID NO: 21 , h) an amino acid sequence which is SEQ ID NO: 22, i) an amino acid sequence which is SEQ ID NO: 23, j) an amino acid sequence which is SEQ ID NO: 24, k) an amino acid sequence which is SEQ ID NO: 25, l) an amino acid sequence which is SEQ ID NO: 26, m) an amino acid sequence which is SEQ ID NO: 27, n) an amino acid sequence which is SEQ ID NO: 28, o) an amino acid sequence which is SEQ ID NO: 29, p) an amino acid sequence which is SEQ ID NO: 30, q) an amino acid sequence which is SEQ ID NO: 31 , or r) an amino acid sequence which is SEQ ID NO: 32. ) The polypeptide according to embodiment 1 or 2, wherein said polypeptide comprises: a) an amino acid sequence which is SEQ ID NO: 15, b) an amino acid sequence which is SEQ ID NO: 16, c) an amino acid sequence which is SEQ ID NO: 17, d) an amino acid sequence which is SEQ ID NO: 18, e) an amino acid sequence which is SEQ ID NO: 19, f) an amino acid sequence which is SEQ ID NO: 20, g) an amino acid sequence which is SEQ ID NO: 21 , h) an amino acid sequence which is SEQ ID NO: 22, i) an amino acid sequence which is SEQ ID NO: 23, j) an amino acid sequence which is SEQ ID NO: 24, k) an amino acid sequence which is SEQ ID NO: 25, l) an amino acid sequence which is SEQ ID NO: 26, m) an amino acid sequence which is SEQ ID NO: 27, n) an amino acid sequence which is SEQ ID NO: 28, o) an amino acid sequence which is SEQ ID NO: 29, p) an amino acid sequence which is SEQ ID NO: 30, q) an amino acid sequence which is SEQ ID NO: 31 , or r) an amino acid sequence which is SEQ ID NO: 32.

4) An in vitro method of generating Fc and/or Fab fragments of IgG or IgM comprising contacting IgG or IgM with the polypeptide according to any one of embodiments 1 to 3.

5) The method according to embodiment 4 wherein the IgG or IgM is a human IgG or IgM.

6) An isolated nucleic acid encoding a polypeptide according to any one of embodiments 1 to 3.

7) A vector comprising a nucleic acid according to embodiment 6.

8) The vector according to embodiment 7, wherein said vector is the plasmid pBSX.

9) An isolated cell expressing a polypeptide according to any one of embodiments 1 to 3 and/or comprising a nucleic acid according to embodiment 6 or a vector according to embodiment 7 or 8.

10) The isolated cell according to embodiment 9, wherein said cell is a prokaryotic or a eukaryotic cell.

11) The cell according to embodiment 9, wherein said cell is Bacillus subtilis, preferentially Bacillus subtilis WB600 cells. ) The polypeptide having IgG and IgM cysteine protease activity according to any one of embodiments 1 to 3, or the nucleic acid according to embodiment 6, or the vector according to embodiment 7 for use as a medicament. ) The polypeptide having IgG and IgM cysteine protease activity according to any one of embodiments 1 to 3, or the nucleic acid according to embodiment 6, or the vector according to embodiment 7 for the treatment or prevention of a disease or condition mediated by IgG and/or IgM. ) The polypeptide, the nucleic acid, or the vector for use according to embodiment 13, wherein the disease or condition is an autoimmune disease, transplant rejection, postoperative treatment or acquired haemophilia. ) The polypeptide, the nucleic acid, or the vector for use according to embodiment 14, wherein said autoimmune disease is Addison’s disease, alopecia areata, ankylosing spondilitis, anti-myelin associated glycoprotein (anti-MAG) neuropathy, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, antiphospholipid syndrome, aplastic anemia, autoimmune cardiomyopathies, autoimmune gastritis, autoimmune haemolytic anemia, autoimmune hearing loss, autoimmune hepatitis, autoimmune hypoparathyroidism, autoimmune hypophysitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune polyendocrinopathy, Bechet's disease, bullous pemphigoid, cardiomyopathy, chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, coeliac disease, Crohn's disease, CREST syndrome, Degos disease, epidermolysis bullosa acquisita, essential mixed cryoglobulinemia, giant cells arteritis, glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillan-Barre syndrome, Hashimoto's thyroiditis, Hyper-IgM syndrome, idiopathic thrombocytopenic purpura, inflammatory bowel disease, Kawasaki's disease, membranous nephropathy, Meniere's syndrome, mixed connective tissue disease, Mooren's ulcer, monoclonal gammopathy, multiple sclerosis, myasthenia gravis, pemphigus foliaceous, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polyglandular autoimmune syndrome type 1 (PAS-I), polyglandular autoimmune syndrome type 2 (PAS-2), polyglandular autoimmune syndrome type 3 (PAS-3), polymyositis/dermatomyositis, post-COVID19 syndrome, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's syndrome, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, subacute thyroiditis, sympathetic ophthalmia, systemic lupus erythematosus, Takayasu's arteritis, type 1 diabetes mellitus, vitiligo, Vogt-Koyanagi-Harada disease, Waldenstrom macroglobulinemia or Wegener's granulomatosis. ) The polypeptide, the nucleic acid, or the vector for use according to embodiment 14, wherein said transplant rejection is an allograft or a xenograft rejection. ) The polypeptide having IgG and IgM cysteine protease activity according to any one of embodiments 1 to 3, or the nucleic acid according to embodiment 6, or the vector according to embodiment 7 for use in the prevention or treatment of an humoral immune response caused by administration of a recombinant gene therapy virus vector, wherein the humoral immune response is against the recombinant gene therapy virus vector, wherein said polypeptide is administered before the recombinant gene therapy vector is administered. ) The polypeptide having IgG and IgM cysteine protease activity according to any one of embodiments 1 to 3, or the nucleic acid according to embodiment 6, or the vector according to embodiment 7 for use in combination with a recombinant gene therapy virus vector for the treatment of a disease treated by said recombinant gene therapy virus vector in a patient in need thereof, wherein said polypeptide is administered before the recombinant gene therapy virus vector is administered, wherein said patient has neutralizing anti-virus antibodies that inhibit cell transduction of said recombinant gene therapy virus vector. ) The polypeptide, the nucleic acid, or the vector for use according to embodiment 17 or 18, wherein said recombinant gene therapy virus vector comprises a recombinant adeno-associated virus (AAV) vector, a recombinant lentiviral vector, a recombinant parvovirus vector, or recombinant adenoviral vector. ) The polypeptide, the nucleic acid, or the vector for use according to embodiment 19, wherein the recombinant adeno-associated virus (AAV) vector comprises a VP1 , VP2 and/or VP3 capsid protein of an AAV virus selected from the group consisting of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV3B, AAV- 2i8, or an AAV virus selected from the Table of AAVs, or a variant of said AAV viruses. ) A method for treating a disorder or condition associated with the undesired presence of IgG and/or IgM, comprising administering to a subject in need thereof an effective amount of the polypeptide having IgG and IgM cysteine protease activity according to any one of embodiments 1 to 3, or the nucleic acid according to embodiment 6, or the vector according to embodiment 7. ) A method according to embodiment 21 wherein said subject is a human. ) A pharmaceutical composition comprising the polypeptide, the nucleic acid, or the vector according to any one of embodiments 1 to 3, 6 and 7 or comprising the polypeptide, the nucleic acid, or the vector for use according to any one of embodiments 12 or 20. ) A kit comprising the polypeptide, the nucleic acid, or the vector according to any one of embodiments 1 to 3, 6 and 7 or comprising the polypeptide, the nucleic acid, or the vector for use according to any one of embodiments 12 or 20, or the pharmaceutical composition according to embodiment 23.

Examples

The present invention is further described by the following examples. The examples are provided solely to illustrate the invention by reference to specific embodiments. These exemplifications, while illustrating certain specific aspects of the invention, do not portray the limitations or circumscribe the scope of the disclosed invention.

All examples were carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail. Routine molecular biology techniques of the following examples can be carried out as described in standard laboratory manuals, such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

Example 1 : Design and production of polypeptides

Vector generation

Polypeptide sequences were derived from metagenomic data and reverse translated into nucleic acid sequences. Further genetic elements were introduced like a sequence encoding an N-terminal signal peptide for secretion which is cleaved upon secretion (SEQ ID NO 11) or a N- or C-terminal Hise tag including short linker regions.

The final nucleotide sequences were synthesized as known in the art and cloned into an expression vector. Reverse-translation, synthesis and cloning are services offered by respective providers such as Eurofins Genomics GmbH (Eurofins Genomics GmbH, Anzinger Str. 7a, 85560 Ebersberg, Germany). The vector herein used is the plasmid pBSX, a derivative of pUB110 (Palva, llkka, Matti Sarvas, Paivi Lehtovaara, Mervi Sibakov, and Leevi Kaariainen. 1982. ..Secretion of Escherichia coli p-lactamase from Bacillus subtilis by the aid of alphaamylase signal sequence." Proc. Natl. Acad. Sci. USA 5582-5586. doi: 10.1073/pnas.79.18.5582.).

Vectors were transformed into electro-competent Bacillus subtilis WB600 cells (Gentaur GmbH). For isolation of single bacterial colonies, appropriate dilutions of B. subtilis cultures were plated onto LB-Agar plates containing suitable concentrations of neomycin and incubated at 37°C until single colonies were obtained. Sequence verification was performed as known in the art.

Glycerol stocks of the B. subtilis cultures transformed with the respective expression plasmids were prepared by adding one volume of 40% glycerol solution to one volume of B. subtilis culture. Expression

Expression for direct usage

For the pre-culture 96-well (Fisher Scientific, 260860) plates were prepared with 100 pl LB medium (10 g/l tryptone (Oxoid, LP 0042), 10 gl NaCI (Merck Millipore, 106400), 5 g/l yeast extract (BD 212720)) supplemented with 10 pg/ml neomycin (Sigma-Aldrich, N1142) per well and inoculated with single colonies from agar-plates. Plates were sealed with breath seal foil and incubated at 37 °C and 160 rpm overnight in a Shaker.

96-well (Fisher Scientific, 260860) plates were prepared with 90 pl per well LB-medium supplemented with 10 pg/ml neomycin and subsequently inoculated with 10 pl of pre-culture. Four expression culture plates were prepared and inoculated from the same pre-culture plate. Plates were sealed with breath seal foil and incubated at 37 °C and 160 rpm for 16 h. Cells were sedimented by centrifugation for 15 min at 2500 g at 4 °C.

Expression for assays with concentrated supernatant

Putative proteases including SEQ ID NO 15-32 were prepared and expressed.

5 ml LB-medium and 20 pg/ml neomycin were inoculated with single colonies in a Tubespin Bioreactor 50 (TPP). The pre-cultures were incubated 2 h at 15 °C and 16 h at 37 °C at 160 rpm in a Multitron incubation shaker (Infors HT).

10 ml LB-medium and 20 pg/ml neomycin were inoculated with 1 ml pre-culture in a Tubespin Bioreactor 50 (TPP). The expression cultures were incubated 6 h at 15 °C without shaking and 16 h at 37 °C at 160 rpm in a Multitron incubation shaker (Infors HT).

Cells in the supernatant were removed by centrifugation and sterile filtration through a 0.22 pm filter. Afterwards, the supernatant was concentrated 40-fold using 10 kDa molecular weight cutoff (MWCO; Sartorius, VS2002) filtration devices by centrifugation. Eventually, the supernatants were washed twice with 1 ml 20 mM NaCI and 50 mM Tris/HCI pH 7.2.

Example 2: Cleavage of substrate antibodies

The substrate was prepared by dilution in DPBS (Sigma-Aldrich, D5562) and usually mixed 1 :1 with protease containing solution (bacterial supernatant or purified proteases) and incubated for 37 °C varying amount of time in 96 well PCR plates (4ti-tude, 4ti-0960). For hlgG1 , hlgG2 and hlgG4 internal isotypes were available. Two with different variable regions were usually mixed in a 1 :1 ratio.

Table 3: Substrate antibodies used.

Example 3: Analysis of Ig digestion via SDS-PAGE

Cleavage was analyzed by SDS-PAGE using the NuPAGE system from Invitrogen according to manufacturer recommendations. Reactions were stopped by addition of 4x LDS sample loading buffer and 10x Reducing agent. Samples were denatured for 10 min at 95°C or 99 °C (Biometra Tadvanced) and loaded in the NuPAGE gels. Following electrophoresis with NuPAGE MES SDS Running Buffer, gels were imaged via the ChemiDoc system (Biorad).

Example 4: Analysis of Ig digestion via LabChip GX microfluidics

Separation and detection of intact substrate and cleavage products were performed with a LabCHip GX microfluidics device (Perkin Elmer) according to manufacturer recommendations. Electropherograms were qualitatively analyzed by visual inspection.

Example 5: New proteases with cleavage activity against human IgG antibodies

Method

All sequences were synthesized in the pBSX vector, transformed and expressed in B. subtilis as outlined in Example 1. The centrifuged bacterial supernatants were assayed for cleavage activity on three human IgG (hlgG) isotypes as outlined in Example 2 and Example 4. Substrates were always a mixture from two different purified antibodies per isotype. Final substrate concentration was 1 .2 mg/ml in the presence of concentrated supernatants. One batch of bacterial supernatants was expressed, concentrated and used as 20-fold concentrates in the presence of the substrate over 24 h (Table 4). A second batch of proteases was expressed, concentrated, and used with equalized concentrations (based on SDS-PAGE analysis, Example 3) and different incubation times per substrate. On average 5-fold supernatants were present in the reaction (Table 5). Results

18 proteases (SEQ ID NO 15-32) with activity on IgG have been found. All proteases appear to have similar substrate profiles. hlgG2 is most efficiently cleaved followed by hlgG 1 and hlgG4. Proteases belonging to the pMC-11902 cluster (see Example 6) appear to be less active on hlgG1 and hlgG4.

Table 4: Scoring of the LabChip GX Microfluidics band patterns of 3 proteases. Substrates tested are IgG 1, lgG2 and lgG4 isotypes. Digest was performed for 24 h with 20-fold concentrates. Scoring: 0: No product bands visible; 1 Product band visible; 2: Product band visible and visible /oss of IgG/heavy chain band compared to negative control (pBSX); 3 Product band visible and (almost) complete loss of IgG/heavy chain band.

Table 5: Scoring of the LabChip GX Microfluidics band patterns of the further 15 proteases with equalized concentrations in IgG cleavage reactions. Substrates tested are lgG1, lgG2 and lgG4 isotypes. Cleavage was aborted by addition of LabChip GX Microfluidics loading buffer after 1 h, 3 h, and 6 h for lgG2, lgG1 and lgG4, respectively. Scoring: 0: No product bands visible; 1 Product band visible; 2: Product band visible and visible /oss of IgG/heavy chain band; 3: Considerable loss of IgG/heavy chain band; 4: Product band visible and (almost) complete loss of IgG/heavy chain band; n/d: not determined.

Example 6: Sequence analysis

The proteases fall into two clusters referred to by their reference sequence (Table 6): pMC-11902 (SEQ ID NO 19) and pMC-11930 (SEQ ID NO 30). The pMC-11902 cluster contains 12 proteases with at least 91 % identity to each other. Proteases from prior art have only sequence identities of lower than 44 %, except from lgMDEi_ba and IgMDEisp. The sequence identity to lgMDEi_ba is 68 % to 70 % and to lgMDEi_sp is 76 % to 79 %.

The pMC-11930 cluster contains 7 proteases with at least 90 % identity to each other. Proteases from prior art have only sequence identities of lower than 44 %, except from lgMDEi_ba and IgMDEisp. The sequence identity to lgMDEi_ba is 67 % to 70 % and to lgMDEi_sp is 81 % to 84 %.

The sequences of IceMG and IgMBRAZOR are not public and could not be compared to.

Table 6: Identity matrix of the newly found proteases and proteases from prior art. Highlighted in boxes are the submatrices of the two clusters (pMC- 11902 and pMC- 11930) of novel IgG/IgM active proteases. Cluster representatives have been highlighted as well. The upper rows represent proteases from prior art and their sequence identity to the novel proteases.

Example 7: New proteases have a high cleavage activity against human IgM antibodies

Method

All 18 proteases (SEQ ID NO 15-32), pBSX (negative control; empty vector) and IdeS (pMC- 11486, SEQ ID NO 33) were expressed again. For the pre-culture 96-well (Eppendorf,

30502337) plates were prepared with 600 l LB medium (10 g/l tryptone (Oxoid, LP 0042), 10 g/l NaCI (Merck Millipore, 106400), 5 g/l yeast extract (BD 212720)) supplemented with 10 pg/ml neomycin per well and inoculated with single colonies from agar-plates. Plates were sealed with breath seal foil and incubated at 37 °C and 250 rpm for 16 h in a shaker. 96-well (Eppendorf, 30502337) plates were prepared with 570 pl per well LB medium supplemented with 10 pg/ml neomycin and subsequently inoculated with 30 pl of pre-culture. Four expression culture plates were prepared and inoculated from the same pre-culture plate. Plates were sealed with breath seal foil and incubated at 37 °C and 250 rpm for 16 h. Cells were sedimented by centrifugation for 30 min at 2500 g at 4 °C.

The supernatants were used as is (“1-fold” concentration) and directly mixed 1 :1 with 0.5 mg/ml IgM and incubated for 1 h at 37 °C. The reaction was stopped by addition of loading buffer and the samples were analyzed via SDS-PAGE (Example 3).

Result

Without concentrating the supernatants and within 1 h, all tested proteases were able to completely cleave the IgM substrate. pMC-11486 (IdeS) and the empty vector negative control pBSX failed to produce any visible product bands.

Example 8: Production of eight selected proteases for further characterization pMC-11486 (IdeS, SEQ ID NO 33), pMC-11886, pMC-11900, pMC-11902, pMC-11914, pMC-11926, pMC-11930, pMC-11933, pMC-11938 and the empty pBSX vector (negative control) were expressed and the bacterial supernatants concentrated for further characterization.

For the pre-culture, TubeSpin Bioreactor 50 were prepared with 10 ml LB (10 g/l Tryptone, 10 g/l sodium chloride, 5 g/l yeast extract) supplemented with 10 pg/ml neomycin and inoculated with one colony each. Incubation was carried out at 37 °C and 160 rpm for 16 hours in a climo shaker ISF1-X (Kuhner AG).

Flasks were prepared with 10 ml (20 g/l Tryptone, 10 g/l sodium chloride, 10 g/l yeast extract) supplemented with 10 pg/ml neomycin and subsequently inoculated with 100 pL of pre-culture. Incubation at 33 °C for 14 hours and 160 rpm in a climo shaker ISF1-X.

Cells were sedimented by centrifugation for 60 min at about 3,428 g and 4 °C. Supernatant was sterile filtered with a syringe having a filter with pore size 0.22 pm. The filtrate was carried over to an ultrafiltration device (MWCO 10 kDa, Sartorius, VS2002) and centrifuged for 30 minutes at 3,428 g. Procedure was repeated until 10 ml of supernatant was concentrated down to 1 ml. These 10-fold concentrated supernatants were stored at 4°C and used for cleavage experiments. Example 9: Activity of eight selected proteases against human lgG3

Method

1/5 diluted in DPBS (Sigma-Aldrich, D5562), 10-fold concentrated bacterial supernatants (Example 8) were mixed 1 : 1 with 0.5 mg/ml lgG3 and incubated for 4 h at 37 °C. Cleavage was evaluated via SDS-PAGE (Example 3).

Result

Heavy chain band intensity decreased in the presence of every protease thus demonstrating all tested proteases cleave lgG3. A product band appears in the presence of all proteases. A second product band is not unambiguously identifiable.

Example 10: Activity of eight selected proteases against IgG in human serum

Method

The concentration of IgGs in human serum was assumed to be between 10 mg/ml and 16 mg/ml.

4 l of 10-fold concentrated proteases (Example 8) were added to 16 pl of substrate. This should yield 8-12.8 mg/ml IgG in serum and 9.92 mg/ml of TPP-5657 during incubation. Samples were first mixed for 1 min at 1 ,200 rpm using the BioSHake iQ and then centrifuged in ‘short’ mode until 1 ,000 g were reached. The samples were subsequently, incubated for 21 h at 37 °C and then heat inactivated for 10 min at 57 °C.

Samples were prepared and analyzed via gel electrophoresis as outlined in Example 3. However, for better visibility the samples were diluted 1/3 with 1x loading buffer prior to electrophoresis.

Result

On SDS-PAGE gel in the lane of the purified hlgG 1 without proteases two bands are visible at 53.4 kDa (heavy chain) and 28.0 kDa (light chain). A band with similar strength is visible at comparable position in the untreated human serum samples (54.8 kDa). This highly likely represents the heavy chain IgG fraction in human serum. The band has a comparable intensity as the reference purified hlgG1 heavy chain band, thus indicating that a similar amount of substrate is available in the human serum samples.

The gel demonstrates cleavage of almost all purified hlgG1 heavy chains (ca. 11 % band intensity remains i.e. 89 % was cleaved) if treated with pMC-11886. A similar result is observed with the IgG heavy chains in human serum with all proteases. There is a faint protein band at higher molecular mass (+0.7-1.1 kDa) visible compared to the actual IgG heavy chain fraction band in the untreated human serum sample. This could be a different protein, though the evaluation in Table 7 assumes it to be IgG fraction heavy chain. Then, the remaining normalized intensity is between 40%-50 % for all tested proteases. This corresponds to 50%-60 % cleavage. Hence, activity in human serum is as high as against purified I gG 1 or at least half as high.

Table 7: Cleavage of IgG fraction in human serum. “Maximum remaining IgG" indicates the ratio of the heavy chain IgG fraction band to the reference heavy chain IgG fraction band (pBSX). The intensity (Adj. Volume) of a clear and distinct band of the serum served as loading control and was used to normalize the IgG fraction heavy chain band’s intensity per lane. “Maximum remaining IgG" indicates the ratio of the normalized heavy chain IgG fraction intensity to the reference normalized heavy chain IgG fraction intensity (pBSX, negative control).

Example 11 : Activity of eight selected proteases against cynomolgus IgGs

Method

The concentration of IgGs in cynomolgus serum was assumed to be comparable to human serum (Example 10) and therefore the experiment was repeated with the same parameters as in example 10 but with cynomolgus instead of human serum.

4 l of 10-fold concentrated proteases (Example 8) were added to 16 pl of substrate. This should yield 8-12.8 mg/ml IgG in serum and 9.92 mg/ml of TPP-5657 during incubation. Samples were first mixed for 1 min at 1 ,200 rpm using the BioSHake iQ and then centrifuged in ‘short’ mode until 1 ,000 g were reached. The samples were eventually, incubated for 21 h at 37 °C and then 10 min at 57 °C heat inactivated.

Samples were prepared and analyzed via gel electrophoresis as outlined in Example 3.

Result

On SDS-PAGE gel in the lane of the purified hlgG 1 without proteases are two bands visible at 52.9 kDa (heavy chain) and 32.7 kDa (light chain). A band with similar strength is visible at comparable position in the untreated cynomolgus serum samples (53.4 kDa). This highly likely represents the heavy chain IgG fraction in cynomolgus serum. The band has a comparable intensity as the reference purified hl gG 1 heavy chain band, thus indicating that a similar amount of substrate is available in the cynomolgus serum samples.

The gel demonstrates cleavage of all purified hlgG1 heavy chain if treated with pMC-11886 or IdeS. The section in the gel surrounding the IgG fraction heavy chain is equivalent to the human serum (Example 10). After cleavage, a less intense band remains with each protease. This clearly indicates that every protease can cleave cynomolgus IgGs under the chosen conditions. Table 8 evaluates the ratio between treated and untreated cynomolgus serum. The remaining intensity is between 27%-56 % for all tested proteases including pMC-11486 (IdeS). This relates to 44%-73 % cleavage, a similar ratio as with human serum (Example 10) tested under the same conditions. Hence, activity in cynomolgus serum is equivalent to the activity in human serum. Table 8: Cleavage of IgG fraction in cynomolgus serum. “Maximum remaining IgG” indicates the ratio of the heavy chain IgG fraction band to the reference heavy chain IgG fraction band (pBSX). The intensity (Adj. Volume) of a clear and distinct band of the serum served as loading control and was used to normalize the IgG fraction heavy chain band’s intensity per lane. “Maximum remaining IgG" indicates the ratio of the normalized heavy chain IgG fraction intensity to the reference normalized heavy chain IgG fraction intensity (pBSX, negative control).

Example 12: Expression and purification for assays with purified protein pMC-11900, pMC-11902 and pMC-11930 (SEQ ID NO: 17, 19, 30) have been modified to include a C-terminal His6-tag for purification. It is fused to the protease sequence via a GSGS- linker. This yields pMC-12237, pMC-12238 and pMC-12241 (SEQ ID NO: 12-14). As with the other protease of the disclosure, they were expressed with the same signal peptide for secretion (SEQ ID NO: 11). Plasmids were generated and transformed as described in Example 1.

100 ml TB-medium containing 10 pg/ml neomycin was inoculated with single colonies or with 100 pl glycerol stocks. The pre-cultures were incubated for 8h-16 h at 37°C and 180 rpm in a Multitron incubation shaker (Infors HT) until an ODeoo of around 3.

For expression cultures, 500-600 ml TB-medium supplemented with 10 pg/ml neomycin was inoculated with pre-culture to achieve an ODeoo of 0.1. The culture was incubated for 16 h at 37 °C, 180 rpm. Afterwards, cells were removed by centrifugation and sterile filtration. The filtrate was used directly for immobilized metal affinity chromatography.

Two buffers were used and mixed with each other: Buffer A 500 mM NaCI, 20 mM HEPES/NaOH pH 7.2; Buffer B 150 mM NaCI, 500 mM Imidazole, 20 mM HEPES, pH 7.2. Two His Trap HP 5 ml (Cytiva, 17524802) were equilibrated with 5 % buffer B. The supernatant was applied with 5 ml/min flow rate and after washing with 10 CV 5 % buffer B, a linear gradient elution was performed over 10 CV up to 50 % buffer B. Fractions containing protein (absorption at 280 nm) were pooled and EDTA was added to a final concentration of 10 mM. The protein was incubated on ice for 15 min and then concentrated and washed with 25 mM NaCI and 20 mM HEPES/NaOH pH 7.2 so that the buffer was diluted at least 1/1000 using a 10 kDa MWCO (Sartorius, VS2002) filtration devices. The protein solution was flash frozen in liquid nitrogen and stored at -80 °C.

Example 13: Cleavage site analysis of IgG isotypes and IgM

Method

For lgG1 cleavage site analysis, 10-fold concentrated proteases (Example 8) were diluted 1/45 in 20 mM NaCI and 50 mM Tris/HCI pH 7.2, mixed 1 :1 with 10 mg/ml TPP-9809 (lgG1) and incubated for 20 h at 37 °C. Reaction was stopped by heat at 57 °C for 15 min. The samples were prepared for protein mass spectrometry with standard protocols known to the expert. The antibodies were deglycosylated and reduced. For IgM cleavage site analysis, pooled lithium heparin plasma from four healthy donors was used. In this protocol substrates are cleaved by trypsin and the mass of the resulting tryptic peptides are evaluated by peptide mass spectrometry. The intensity of the tryptic peptides is used as reference for samples which were in addition previously treated with the protease to be evaluated. The reduction/absence of a reference tryptic peptide indicates a cleavage site of the protease to be analyzed in said peptide. The purified proteases (1 ll/pl IdeS (FabRICATOR; Genovis), 3.5 mg/ml pMC-12238, 1.7 mg/ml pMC-12237 and pMC-12241) were mixed 4:20 with pooled human lithium-heparin plasma, incubated for 19 h at 37 °C and then immediately denatured, reduced, alkylated and further digested with trypsin. Typical protocols are available in the literature (Gaither, Claudia, Robert Popp, and Christoph H. Borchers. 2020. ..Determination of the concentration range for 267 proteins from 21 lots of commercial human plasma using highly multiplexed multiple reaction monitoring mass spectrometry." Analyst 3634- 3644. doi:doi.org/10.1039/C9AN01893J.). The samples were purified via solid phase extraction (SPE), measured via nanoLC-HRMS and analyzed by a data independent acquisition methodology. Typical protocols are available in the literature (Demichev, Vadim, Christoph B. Messner, I. Spyros Vernardis, S. Kathryn Lilley, and Markus Raiser. 2020. „DIA-NN: neural networks and interference correction enable deep proteome coverage in high throughput." Nat. Methods 41-44. doi:doi.org/10.1038/s41592-019-0638-x.).

Result

The mass spectrometry analysis revealed two different sets of cleavage products. Whereas pMC-11486 (IdeS) yields only a 24,537 kDa Fd’ and a 23,760 kDa Fc/2 (dissociated Fc) fragment, all tested protases of the disclosure yield in addition a 24,481 kD Fd’ and a 23,817 kDa Fc/2 fragment (Table 9). The difference is on average 57 g/mol which corresponds to a missing/additional glycyl residue at the neo-termini. The cleavage sites are listed in Table 10. Therefore, lgG1 heavy chains are cleaved effectively like IdeS which is C-terminally of the disulfide bridges linking the F(ab’)2 part.

The only significantly reduced IgM tryptic peptide is found in the IgM sequence between constant region C2 and C3. This yields similarly to IgG F(ab’)2 and Fc/2 fragments (Table 10). However, as IgM is a penta- or hexamer, the Fc/2 fragments of two neighboring IgM “monomers” are potentially still linked together but do not form a true Fc part. The tested proteases correspond to core sequence SEQ ID NO 17, 19, 30.

Table 9: Cleavage products of IgG 1 generated by IdeS and the selected 8 proteases.

Table 10: Cleavage sites of IdeS (pMC-11486, SEQ ID NO 33), Idessms and IgMDEs from all 4 species (SEQ ID NO 1, 7- 10, according to literature) as well as those experimentally determined for pMC-11886, pMC-11900, pMC-11902, pMC-11914, pMC-11926, pMC-11930, pMC-11933 and pMC-11938 (abbreviated as “8 candidates’ as well as pMC-12237, pMC-12238 and pMC-112241 (abbreviated as “3 candidates”, corresponding to pMC-11900, pMC-11902 and pMC-11930). The cleavage site flanking amino acids are highlighted bold and underscored. The tryptic peptide lost due to cleavage by the selected 3 candidates is highlighted in bold font only.

Claims

1) An in vitro method of generating Fc and/or Fab and/or F(ab’)2 fragments of IgG or IgM comprising contacting IgG or IgM with a polypeptide having IgG and IgM cysteine protease activity comprising: a) an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 19, or b) an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 30.

2) The in vitro method according to claim 1, wherein said polypeptide having IgG and IgM cysteine protease activity comprises an amino acid sequence which is at least 90%, 95%, 98%, 99%, or 100% identical to: a) an amino acid sequence which is SEQ ID NO: 15, b) an amino acid sequence which is SEQ ID NO: 16, c) an amino acid sequence which is SEQ ID NO: 17, d) an amino acid sequence which is SEQ ID NO: 18, e) an amino acid sequence which is SEQ ID NO: 19, f) an amino acid sequence which is SEQ ID NO: 20, g) an amino acid sequence which is SEQ ID NO: 21, h) an amino acid sequence which is SEQ ID NO: 22, i) an amino acid sequence which is SEQ ID NO: 23, j) an amino acid sequence which is SEQ ID NO: 24, k) an amino acid sequence which is SEQ ID NO: 25, l) an amino acid sequence which is SEQ ID NO: 26, m) an amino acid sequence which is SEQ ID NO: 27, n) an amino acid sequence which is SEQ ID NO: 28, o) an amino acid sequence which is SEQ ID NO: 29, p) an amino acid sequence which is SEQ ID NO: 30, q) an amino acid sequence which is SEQ ID NO: 31, or r) an amino acid sequence which is SEQ ID NO: 32. 3) The in vitro method according to claim 1, wherein said polypeptide having IgG and IgM cysteine protease activity comprises: a) an amino acid sequence which is SEQ ID NO: 15, b) an amino acid sequence which is SEQ ID NO: 16, c) an amino acid sequence which is SEQ ID NO: 17, d) an amino acid sequence which is SEQ ID NO: 18, e) an amino acid sequence which is SEQ ID NO: 19, f) an amino acid sequence which is SEQ ID NO: 20, g) an amino acid sequence which is SEQ ID NO: 21, h) an amino acid sequence which is SEQ ID NO: 22, i) an amino acid sequence which is SEQ ID NO: 23, j) an amino acid sequence which is SEQ ID NO: 24, k) an amino acid sequence which is SEQ ID NO: 25, l) an amino acid sequence which is SEQ ID NO: 26, m) an amino acid sequence which is SEQ ID NO: 27, n) an amino acid sequence which is SEQ ID NO: 28, o) an amino acid sequence which is SEQ ID NO: 29, p) an amino acid sequence which is SEQ ID NO: 30, q) an amino acid sequence which is SEQ ID NO: 31, or r) an amino acid sequence which is SEQ ID NO: 32.

4) The in vitro method according to any one of claims 1 to 3 wherein the IgG or IgM is a human or cynomolgus IgG or IgM.

5) A polypeptide having IgG and IgM cysteine protease activity comprising: a) an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 19, or b) an amino acid sequence which is at least 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 30, or a nucleic acid encoding said polypeptide, or a vector comprising a nucleic acid encoding said polypeptide for use as a medicament.

6) The polypeptide, the nucleic acid, or the vector for use according to claim 5, wherein said polypeptide having IgG and IgM cysteine protease activity comprises an amino acid sequence which is at least 90%, 95%, 98%, or 99% identical to: a) an amino acid sequence which is SEQ ID NO: 15, b) an amino acid sequence which is SEQ ID NO: 16, c) an amino acid sequence which is SEQ ID NO: 17, d) an amino acid sequence which is SEQ ID NO: 18, e) an amino acid sequence which is SEQ ID NO: 19, f) an amino acid sequence which is SEQ ID NO: 20, g) an amino acid sequence which is SEQ ID NO: 21, h) an amino acid sequence which is SEQ ID NO: 22, i) an amino acid sequence which is SEQ ID NO: 23, j) an amino acid sequence which is SEQ ID NO: 24, k) an amino acid sequence which is SEQ ID NO: 25, l) an amino acid sequence which is SEQ ID NO: 26, m) an amino acid sequence which is SEQ ID NO: 27, n) an amino acid sequence which is SEQ ID NO: 28, o) an amino acid sequence which is SEQ ID NO: 29, p) an amino acid sequence which is SEQ ID NO: 30, q) an amino acid sequence which is SEQ ID NO: 31, or r) an amino acid sequence which is SEQ ID NO: 32.

7) The polypeptide, the nucleic acid, or the vector for use according to claim 5, wherein said polypeptide having IgG and IgM cysteine protease activity comprises: a) an amino acid sequence which is SEQ ID NO: 15, b) an amino acid sequence which is SEQ ID NO: 16, c) an amino acid sequence which is SEQ ID NO: 17, d) an amino acid sequence which is SEQ ID NO: 18, e) an amino acid sequence which is SEQ ID NO: 19, f) an amino acid sequence which is SEQ ID NO: 20, g) an amino acid sequence which is SEQ ID NO: 21, h) an amino acid sequence which is SEQ ID NO: 22, i) an amino acid sequence which is SEQ ID NO: 23, j) an amino acid sequence which is SEQ ID NO: 24, k) an amino acid sequence which is SEQ ID NO: 25, l) an amino acid sequence which is SEQ ID NO: 26, m) an amino acid sequence which is SEQ ID NO: 27, n) an amino acid sequence which is SEQ ID NO: 28, o) an amino acid sequence which is SEQ ID NO: 29, p) an amino acid sequence which is SEQ ID NO: 30, q) an amino acid sequence which is SEQ ID NO: 31, or r) an amino acid sequence which is SEQ ID NO: 32.

8) The polypeptide, the nucleic acid, or the vector for use according to any one of claims 5 to 7 for use in the treatment or prevention of a disease or condition mediated by IgG and/or IgM.

9) The polypeptide, the nucleic acid, or the vector for use according to claim 8, wherein the disease or condition is an autoimmune disease, transplant rejection, post-operative treatment or acquired hemophilia.

10) The polypeptide, the nucleic acid, or the vector for use according to claim 9, wherein said autoimmune disease is Addison’s disease, alopecia areata, ankylosing spondilitis, anti-myelin associated glycoprotein (anti-MAG) neuropathy, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, antiphospholipid syndrome, aplastic anemia, autoimmune cardiomyopathies, autoimmune gastritis, autoimmune haemolytic anemia, autoimmune hearing loss, autoimmune hepatitis, autoimmune hypoparathyroidism, autoimmune hypophysitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune polyendocrinopathy, Bechet's disease, bullous pemphigoid, cardiomyopathy, chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, coeliac disease, Crohn's disease, CREST syndrome, Degos disease, epidermolysis bullosa acquisita, essential mixed cryoglobulinemia, giant cells arteritis, glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillan-Barre syndrome, Hashimoto's thyroiditis, Hyper-IgM syndrome, idiopathic thrombocytopenic purpura, inflammatory bowel disease, Kawasaki's disease, membranous nephropathy, Meniere's syndrome, mixed connective tissue disease, Mooren's ulcer, monoclonal gammopathy, multiple sclerosis, myasthenia gravis, pemphigus foliaceous, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polyglandular autoimmune syndrome type 1 (PAS-I), polyglandular autoimmune syndrome type 2 (PAS-2), polyglandular autoimmune syndrome type 3 (PAS-3), polymyositis/dermatomyositis, post-COVID19 syndrome, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's syndrome, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, subacute thyroiditis, sympathetic ophthalmia, systemic lupus erythematosus, Takayasu's arteritis, type 1 diabetes mellitus, vitiligo, Vogt-Koyanagi-Harada disease, Waldenstrom macroglobulinemia or Wegener's granulomatosis.

11) The polypeptide, the nucleic acid, or the vector for use according to claim 9, wherein said transplant rejection is an allograft or a xenograft rejection.

12) The polypeptide, the nucleic acid, or the vector for use according to any one of claims 5 to 7 for use in the prevention or treatment of an humoral immune response caused by administration of a recombinant gene therapy virus vector, wherein the humoral immune response is against the recombinant gene therapy virus vector, wherein said polypeptide is administered before the recombinant gene therapy vector is administered.

13) The polypeptide, the nucleic acid, or the vector for use according to any one of claims 5 to 7 for use in combination with a recombinant gene therapy virus vector for the treatment of a disease treated by said recombinant gene therapy virus vector in a patient in need thereof, wherein said polypeptide is administered before the recombinant gene therapy virus vector is administered, wherein said patient has neutralizing anti-virus antibodies that inhibit cell transduction of said recombinant gene therapy virus vector. 14) The polypeptide, the nucleic acid, or the vector for use according to claim 12 or 13, wherein said recombinant gene therapy virus vector comprises a recombinant adeno- associated virus (AAV) vector, a recombinant lentiviral vector, a recombinant parvovirus vector, or recombinant adenoviral vector.

15) The polypeptide, the nucleic acid, or the vector for use according to claim 14, wherein the recombinant adeno-associated virus (AAV) vector comprises a VP1 , VP2 and/or VP3 capsid protein of an AAV virus selected from the group consisting of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV3B, AAV-2i8, or an AAV virus selected from the Table of AAVs, or a variant of said AAV viruses.

16) A method for treating a disorder or condition associated with the undesired presence of IgG and/or IgM, comprising administering to a subject in need thereof an effective amount of the polypeptide, the nucleic acid, or the vector for use according to any one of claims 5 to 15.

17) The method according to claim 16 wherein said subject is a human.

18) A pharmaceutical composition comprising the polypeptide, the nucleic acid, or the vector for use according to any one of claims 5 to 7.