WO2024057095A1 - Mutant protease - Google Patents

Abstract

Provided herein are truncated forms of an Ig protease, which truncated forms exhibit protease activity against immunoglobulins, particularly human IgG, and related compositions and uses thereof.

Description

MUTANT PROTEASE

RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application Serial No. 63/406,491, filed on September 14, 2022, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates, at least in part, to a novel polypeptide that has protease activity against immunoglobulins, such as human IgG, and uses thereof.

BACKGROUND OF THE INVENTION

There are a number of possible applications for immunoglobulin-cleaving enzymes, such as IgG-cleaving proteases. However, levels of activity and fit for such applications can vary. The availability of additional molecules is beneficial.

SUMMARY OF THE INVENTION

A novel protease was previously identified in a previously unknown streptococcal species isolated as part of a study in multiple rodents. One particular isolated species, referred to as Streptococcus krosus, was sequenced and found to express several glycosidases and proteases. A polypeptide of approximately 56.4 kDa, consisting of 557 amino acids was identified and referred to as IdeSORK (also referred to herein as Xork in the Examples and Figures). The full-length sequence of which is shown in SEQ ID NO: 1. N-terminal fragments had been found previously to have preserved cleaving activity and enhanced expression. Surprisingly, N-terminally truncated forms of this enzyme has been found to possess similar, and even better in some embodiments, activity as compared to N-terminal fragments (C-terminally truncated forms) of the full-length sequence. Provided herein are these N-terminally truncated forms, as well as related compositions and methods.

Provided herein in one aspect is a polypeptide, which has immunoglobulin protease (Ig protease) activity, which polypeptide comprises an N-terminally truncated form of a polypeptide with IdeSORK activity, which polypeptide with IdeSORK activity may be any one of the polypeptides as provided in PCT Publication WO2022223818A1, the polypeptides of which (and sequences that encode them) are incorporated herein by reference in their entirety. In one embodiment, a polypeptide with IdeSORK activity comprises a sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 3.

In one aspect, any one of the polypeptides as provided herein does not comprise SKRKLLKKIEKKDTSSVLTQKKQTK (SEQ ID NO: 4) in its entirety. Therefore, in one embodiment, a polypeptide as provided herein comprises the sequence of SEQ ID NO: 1, or an N-terminal fragment thereof, but does not comprise SEQ ID NO: 4 in its entirety. In one embodiment, a polypeptide as provided herein comprises the sequence of SEQ ID NO: 3 but without SEQ ID NO: 4 in its entirety. In one embodiment, a polypeptide as provided herein comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 3 but without at least one of amino acids 1-25 at the N-terminus thereof. In one embodiment, a polypeptide as provided herein comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 3 but without amino acids 1 and 2 of the N-terminus thereof. In one embodiment, a polypeptide as provided herein comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 3 but without amino acids 1-3, 1-4, 1-5, 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 or 1- 25 of the N-terminus thereof. In one embodiment, a polypeptide with IdeSORK activity comprises a sequence as shown in SEQ ID NO: 2.

In one aspect, a polypeptide, comprising an N-terminally truncated form of an IdeSORK, which truncated form has an activity of the IdeSORK, the IdeSORK having the sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 3 is provided.

In one embodiment, the polypeptide comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 3 but without the sequence of SEQ ID NO: 4 in its entirety.

In one embodiment, the polypeptide comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 3 but without amino acids 1, 1-2, 1-3, 1-4, 1-5, 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 or 1-25 of the N-terminus thereof.

In one embodiment, the polypeptide comprises the sequence of SEQ ID NO:2.

In one aspect are compositions comprising any one of the polypeptides provided herein. In one embodiment, the composition further comprises at least one carrier or excipient, which composition is pharmaceutically acceptable.

In one aspect, a polynucleotide that comprises a sequence that encodes any one of the polypeptides provided herein is provided. In one aspect are compositions comprising any one of the polynucleotides provided herein. In one embodiment, the composition further comprises and at least one carrier or excipient, which composition is pharmaceutically acceptable. In one aspect, an expression vector that comprises any one of the polynucleotides provided herein is provided. In one aspect are compositions comprising any one of the expression vectors provided herein. In one embodiment, the composition further comprises and at least one carrier or excipient, which composition is pharmaceutically acceptable.

In one aspect, a host cell comprising any one of the polynucleotides or any one of the expression vectors provided herein is provided. In one aspect are compositions comprising any one of the host cells provided herein. In one embodiment, the composition further comprises and at least one carrier or excipient, which composition is pharmaceutically acceptable.

Any one of the polypeptides provided herein can further comprise an N-terminal methionine and/or a His tag or other tag at the C-terminus. In one embodiment, the polypeptide comprises the sequence of SEQ ID NO: 2, optionally further comprising an N-terminal methionine and/or a His tag or other tag at the C-terminus. In one embodiment, the His tag or other tag may be coupled to the rest of the polypeptide by a linker. The polypeptides provided herein also include those that comprise an amino acid sequence that share at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of the foregoing polypeptides. Preferably, any one of the polypeptides provided herein also exhibits a protease activity, such as Ig-cleaving activity, that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% or more as that of a polypeptide comprising the sequence of SEQ ID NO: 1 or SEQ ID NO: 3, which activity can be assessed in vitro such as provided herein or otherwise known in the art.

In another aspect, any one of the polypeptides provided herein may be part of a fusion polypeptide or conjugated to another molecule.

In another aspect, compositions comprising any one of the polypeptides, including fusion polypeptides thereof, provided herein are provided.

In another aspect, a method of using any one of the polypeptides, including fusion polypeptides thereof, provided here is provided.

In another aspect are nucleic acids that encode any one of the polypeptides, including fusion polypeptides thereof, provided herein.

The compositions and methods may be for in vitro or in vivo purposes, such as cleaving of Ig, such as IgG, treatment of a subject, etc. Thus, in one aspect, methods of administering any one of the polypeptides or other compositions provided herein to a subject in need thereof, such as for the treatment of a disease where Ig (e.g., IgG) cleavage, reduction, elimination, etc. may be a benefit, are provided.

In one aspect, a method of cleaving an immunoglobulin (Ig) comprising contacting the immunoglobulin with any one of the polypeptides or any one of the compositions provided herein is provided. In one embodiment, the contacting is in vitro. In one embodiment, the contacting is in vivo. In one embodiment, the contacting is by administering the polypeptide or composition to a subject. In one embodiment, the subject is any one of the subjects provided herein.

In one aspect, a method of treating a subject, wherein any one of the polypeptides provided herein or any one of the compositions provided herein is administered to the subject is provided. In one embodiment, the subject is in need thereof.

In one aspect, a method for the ex vivo cleavage of Ig in a sample comprising administering any one of the polypeptides provided herein or any one of the compositions provided herein to the sample and incubating under conditions suitable for Ig protease activity is provided. In one embodiment, the method further comprises the separation, detection and/or analysis of the resulting cleavage products, and/or wherein the method generates Fc and Fab fragments.

In one aspect, a method comprising administering any one of the polynucleotides or any one of the expression vectors to a sample or a subject in which Ig is present is provided.

In one aspect, a method comprising administering any one of the compositions provided herein to a sample or a subject in which Ig is present is provided.

In one embodiment of any one of the methods provided herein, the method is for the prevention or treatment of a disease or condition in a subject, and which method comprises administering said polypeptide, polynucleotide, expression vector, or composition to the subject in a prophylactically or therapeutically effective amount.

In one embodiment of any one of the methods provided herein, the disease or condition is a disease or condition mediated at least in part by pathogenic Ig antibodies.

In one embodiment of any one of the methods provided herein, the disease or condition is (i) an autoimmune disease mediated at least in part by IgG antibodies; (ii) IgG-mediated rejection of an organ or tissue transplant received by the subject; or (iii) IgG-mediated anti-drug responses to a therapeutic agent administered to the subject.

In one aspect, any one of the polypeptides provided herein, any one of the polynucleotides provided herein, any one of the expression vectors provided herein, or any one of the compositions provided herein, for use in the treatment or prevention of a disease or condition in a subject is provided.

In one aspect, any one of the polypeptides provided herein, any one of the polynucleotides provided herein, any one of the expression vectors provided herein, or any one of the compositions provided herein, in the preparation of a medicament for the treatment or prevention of a disease or condition in a subject is provided.

In one aspect, any one of the polypeptides provided herein, any one of the polynucleotides provided herein, any one of the expression vectors provided herein, or any one of the compositions provided herein is for any one of the uses provided herein, wherein the disease or condition is a disease or condition mediated at least in part by pathogenic Ig antibodies.

In one aspect, any one of the polypeptides provided herein, any one of the polynucleotides provided herein, any one of the expression vectors provided herein, or any one of the compositions provided herein is for any one of the uses provided herein, wherein the disease or condition is (i) an autoimmune disease mediated in whole or in part by IgG antibodies; (ii) IgG mediated rejection of an organ or tissue transplant received by the subject; or (iii) IgG mediated anti-drug responses to a therapeutic agent administered to the subject.

In one aspect, a method for improving the benefit to a subject of a therapeutic agent, such as a therapeutic antibody, comprising administering any one of the polypeptides provided herein, any one of the polynucleotides provided herein, any one of the expression vectors provided herein, or any one of the compositions provided herein is provided, which method comprises i) administering said polypeptide, polynucleotide, expression vector, or composition to the subject in a prophylactically or therapeutically effective amount and ii) administering, such as subsequently administering, a therapeutic agent, such as a therapeutic antibody to the subject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides cleaving data of a polypeptide of SEQ ID NO: 3 (wild-type (Xork 1.0)).

FIG. 2 provides cleaving data of a polypeptide of SEQ ID NO: 2 (Xork 1.2). The data demonstrates the ability of a polypeptide of SEQ ID NO: 2 to cleave Ig. The N-terminally truncated form seems to cleave more completely into F(ab)2 and Fc in this experiment.

FIG. 3 provides cleavage data over time comparing a polypeptide of SEQ ID NO: 3 (wild-type (Xork 1.0)) with a polypeptide of SEQ ID NO: 2 (Xork 1.2). Both cleave greater than 95% IgG in human serum after 1 hr. The polypeptide of SEQ ID NO: 2 had slightly more activity. IgG is stable in general without such treatment. The ELISA signal of human serum without IgG (IgG depleted serum) is as low as blank (approximately 0.005 a.u.)

FIG. 4 provides similar data to FIG. 3 with the ELISA signal normalized to time 0.

FIG. 5 shows that a polypeptide of SEQ ID NO: 3 wild-type (Xork 1.0) and N-terminally truncated mutant (Xork 1.2) (a polypeptide of SEQ ID NO: 2) have comparable activities of cleaving both endogenous IgG. The reactions were sampled at indicated time points and analyzed with SDS-PAGE and Coomassie staining. FIG. 5A is in human sera and shows 30% human sera from three individuals (#595, left panel; #596, middle panel; #598, right panel) were cleaved by 10 uM Xork 1.0 or Xork 1.2 at 37°C. The human IgG abundance in the samples at indicated time points were measured by ELISA and plotted as the absorbance at 450 nm and 570 nm. FIG. 5B is with purified IgG Is and shows 22 uM IgGl substrates (Humira, upper panel or Infliximab, lower panel) were cleaved by 160 nM Xork 1.0 or Xork 1.2 at 37°C.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to be understood that this invention is not limited to particularly exemplified materials or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting of the use of alternative terminology to describe the present invention.

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety for all purposes.

As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to "a polypeptide" includes a mixture of two or more such polypeptides or a plurality of such polypeptides, and the like.

As used herein, the term “comprise” or variations thereof such as “comprises” or “comprising” are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein, the term “comprising” is inclusive and does not exclude additional, unrecited integers or method/process steps. In embodiments of any one of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of’ or “consisting of’. The phrase “consisting essentially of’ is used herein to require the specified integer(s) or steps as well as those which do not materially affect the character or function of the claimed invention. As used herein, the term “consisting” is used to indicate the presence of the recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) alone.

Polypeptide Features

Provided herein are polypeptides that can have immunoglobulin protease activity (i.e., can cleave an Ig molecule, such as an IgG molecule (e.g., IgGl, IgG2, IgG3 and/or IgG4)). As used herein, “immunoglobulin (Ig) protease” refers to an enzyme that cleaves/hydrolyzes one or more peptide bonds in an immunoglobulin molecule. In one embodiment, the polypeptide is specific for cleaving an IgG molecule (e.g., IgGl, IgG2, IgG3 and/or IgG4) and does not cleave other classes of Ig at all or substantially. In one embodiment, the polypeptide cleaves all human IgG subclasses, that is IgGl, IgG2, IgG3 and IgG4, and does not cleave other human immunoglobulin classes. The polypeptide may exhibit lower activity against human IgG2 as compared to the other human IgG subclasses. The polypeptide may exhibit greater activity against human IgGl than human IgG2. The polypeptide may exhibit activity against non-human IgG molecules, including mouse IgG2a and IgG3. The polypeptide may exhibit activity against IgG from guinea pig or horse.

In one embodiment, the polypeptides can efficiently cleave IgG to Fc and F(ab’)2 fragments via a two-stage mechanism. In the first stage, one (first) heavy chain of IgG can be cleaved to generate a single cleaved IgG (scIgG) molecule with a single non-covalently bound Fc chain. The scIgG molecule is effectively an intermediate product which retains the remaining (second) heavy chain of the original IgG molecule. The complex of a polypeptide provided herein and a single cleaved IgG molecule is also provided herein. In this context, the IgG cleaved by the polypeptide is preferably a recombinant, monoclonal antibody. Thus, a complex of a polypeptide provided herein and a single cleaved recombinant, monoclonal IgG molecule is provided. A complex of a polypeptide provided herein and a single cleaved version of an antibody is also provided. In the second stage of the mechanism the remaining (second) heavy chain of the original IgG molecule can be cleaved by the polypeptide to release a F(ab’)2 fragment and a homodimeric Fc fragment. These are the products that generally can be observed under physiological conditions. The homodimeric Fc may dissociate into its component monomers. Under reducing conditions the F(ab’)2 fragment may dissociate to two Fab fragments.

Immunoglobulin protease activity may be assessed by any suitable method, for example by incubating a polypeptide with a sample containing Ig, such as IgG, and determining the presence of Ig 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. Efficacy may be assessed in the presence or absence of an inhibitor. However, efficacy herein will typically mean efficacy as assessed in the absence of such an inhibitor unless otherwise stated. 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 include an ELISA-based assay. In such an assay, the wells of an assay plate will typically be coated with an antibody target, such as bovine serum albumin (BSA). Samples of the polypeptide to be tested are then added to the wells, followed by samples of target- specific antibody, that is antibody specific for BSA in this example. The polypeptide and antibody are allowed to interact under conditions suitable for Ig protease activity. After a suitable interval, the assay plate will be washed and a detector antibody which specifically binds to the target- specific antibody will be added under conditions suitable for binding to the target- specific antibody. The detector antibody will bind to any intact target- specific antibody that has bound to the target in each well. After washing, the amount of detector antibody present in a well will be proportional to the amount of target- specific antibody bound to that well. The detector antibody may be conjugated directly or indirectly to a label or another reporter system (such as an enzyme), such that the amount of detector antibody remaining in each well can be determined. The higher the potency of the tested polypeptide that was in a well, the less intact target- specific antibody will remain and thus there will be less detector antibody. In an embodiment, at least one well on a given assay plate can include wildtype IdeSORK or a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 3 instead of a polypeptide to be tested, so that the potency of the tested polypeptides may be directly compared to the potency of wild-type IdeSORK or polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 3. Other assays may determine the potency of a tested polypeptide by directly visualizing and/or quantifying the fragments of Ig, such as IgG, which result from cleavage of Ig by a tested polypeptide. Such an assay will typically incubate a sample of Ig, such as IgG, with a test polypeptide (or wild-type IdeSORK or polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 3 as a control) at differing concentrations in a titration series. The products which result from incubation at each concentration are then separated using gel electrophoresis, for example by SDS-PAGE. Whole Ig and the fragments which result from cleavage of Ig can then be identified by size and quantified by the intensity of staining with a suitable dye. The greater the quantity of cleavage fragments, the greater the potency of a tested polypeptide at a given concentration. This type of assay may also enable the identification of test polypeptides that are more effective at cleaving the first or the second heavy chain of an Ig molecule, as the quantities of the different fragments resulting from each cleavage event may also be determined. A polypeptide provided herein may be more effective at cleaving the first chain of an IgG molecule than the second chain. A polypeptide provided herein can, in some embodiments, be at least as effective at cleaving human IgGl as wild-type IdeSORK or polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 3.

An immunoglobulin protease activity of a polypeptide provided herein is preferably at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the activity against human Ig (e.g., IgG) of wild-type IdeSORK or a polypeptide as set forth in SEQ ID NO: 1 or 3, when measured in the same assay. In some embodiments, an immunoglobulin protease activity of a polypeptide provided herein is superior.

Suitable methods to assess immunoglobulin protease activity and efficacy are also described in the Examples or are otherwise known in the art.

The sequences referred to herein are provided below.

SEQ ID NO : 1

SKRKLLKKIEKKDTS SVLTQKKQTKTLWADGVQVDDKDFKP STENFGTNYLAAEYGI GKGYYD INKKFDGTDDDLCSGWAANQLHWWLDRNKDYIEKYRQQSKDNGVTIGNT D IFELNKLHDEDQSNFFDF IKKSFGNKFLQPERLLNMYINGYGYLTSQDKAKTSQP SP SKLNFFQKVFKNNLLTDKTP IND IDEFS SQTKNALQNHKVLAVSFAS IKNRGLGHWTV WGADFDENGKWALYVTDSDDRSKNIGNAKLGMKKLRIEVSAQDS ST IKLTGFEDKN SGGSLRHLYSLSTGEQIWKKYFEETEKERIRLEEEADKAKLEQDRIQKEAEEKLALEKA EKERIRLEEEADKAKLEQDRIQKEAEEKLALEKAEKERIRLEEEADKAKLEQDRIQKEAEE KLALEKAEKERIRLEEEADKAKLEQDRIQKEAEEKLALEKAEKERIRLEEEAAKAKLEQEK QIATAPQPDKKQENTTSEQEKPAPTELPPLVNKADETETPRETAPDQTP SATNTFRKIL PKMNAVSQFFSQLMGT IQIVFAF ILKIFK

SEQ ID NO : 2 TLWADGVQVDDKDFKP STENFGTNYLAAEYGIGKGYYD INKKFDGTDDDLC SGWAANQLHWWLDRNKDYIEKYRQQS KDNGVT IGNTD IFELNKLHDEDQSNFFDF IKKSFGNKFLQPERLLNMYINGYGYLTSQDKAKTSQP SP SKLNFFQKVF KNNLLTDKTP IND IDEFS SQTKNALQNHKVLAVSFAS IKNRGLGHWTVWGADFDENGKWALYVTDSDDRSKNIGNA KLGMKKLRIEVSAQDS ST IKLTGFEDKNSGGSLRHLYSLSTGEQIWKKYFEET

SEQ ID NO : 3 . N terminal fragment SKRKLLKKIEKKDTS SVLTQKKQTKTLWADGVQVDDKDFKP STENFGTNYLAAEYGI GKGYYD INKKFDGTDDDLCSGWAANQLHWWLDRNKDYIEKYRQQSKDNGVTIGNT D IFELNKLHDEDQSNFFDF IKKSFGNKFLQPERLLNMYINGYGYLTSQDKAKTSQP SP SKLNFFQKVFKNNLLTDKTP IND IDEFS SQTKNALQNHKVLAVSFAS IKNRGLGHWTV WGADFDENGKWALYVTDSDDRSKNIGNAKLGMKKLRIEVSAQDS ST IKLTGFEDKN SGGSLRHLYSLSTGEQIWKKYFEET

The polypeptide provided herein is typically less than the length of SEQ ID NO: 1 and does not comprise SEQ ID NO: 4 in its entirety. For example, a polypeptide provided herein may comprise the sequence of SEQ ID NO: 1 or SEQ ID NO: 3 but without amino acids 1, 1-2, 1-3, 1- 4, 1-5, 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 or 1-25 of the N-terminus thereof. A polypeptide provided herein may comprise the sequence of SEQ ID NO:2.

The polypeptides provided herein include those that share at least 80%, at least, 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to any one of the sequences provided herein. Identity relative to the sequence can be measured over a region of at least 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318 319, 320, 330, 340, 350, 360, 370, 380, 390, 400 or more contiguous amino acids of the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 or an N-terminally truncated form as provided herein. In one embodiment, the identity is at a foregoing level as compared to the sequence shown in SEQ ID NO: 2. In one embodiment, identity is measured over the full length of any one of the foregoing sequences.

In one embodiment, the polypeptide provided comprises or consists of a sequence with at least 80%, at least, 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 1.

In one embodiment, the polypeptide provided comprises or consists of a sequence with at least 80%, at least, 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 3.

In an embodiment, the polypeptide provided comprises a sequence with at least 80%, at least, 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 2. In a further embodiment, the polypeptides provided consists of a sequence with at least 80%, at least, 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 2.

In one embodiment, the polypeptide provided comprises a sequence with at least 80% identity to SEQ ID NO: 2.

In one embodiment, the polypeptide provided comprises a sequence with at least 85% identity to SEQ ID NO: 2.

In one embodiment, the polypeptide provided comprises a sequence with at least 90% identity to SEQ ID NO: 2.

In one embodiment, the polypeptide provided comprises a sequence with at least 95% identity to SEQ ID NO: 2.

In one embodiment, the polypeptide provided comprises a sequence with at least 96% identity to SEQ ID NO: 2.

In one embodiment, the polypeptide provided comprises a sequence with at least 97% identity to SEQ ID NO: 2.

In one embodiment, the polypeptide provided comprises a sequence with at least 98% identity to SEQ ID NO: 2.

In one embodiment, the polypeptide provided comprises a sequence with at least 99% identity to SEQ ID NO: 2.

In a preferred embodiment, the polypeptide provided comprises the sequence of SEQ ID NO: 2.

In each of the foregoing embodiments wherein the polypeptide comprises or consists of a sequence as defined by reference to SEQ ID NO: 1, 2 or 3, it will be understood that said polypeptide:

(a) is N-terminally truncated relative to SEQ ID NO: 1 and/or SEQ ID NO: 3;

(b) does not comprise a sequence as set forth in SEQ ID NO: 4;

(c) does not comprise amino acids 1, 1-2, 1-3, 1-4, 1-5, 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 or 1-25 of the N- terminus of SEQ ID NO: 1; and/or

(d) does not comprise amino acids 1, 1-2, 1-3, 1-4, 1-5, 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 or 1-25 of the N- terminus of SEQ ID NO: 3. In another preferred embodiment, the polypeptide provided consists of the sequence of SEQ ID NO: 2.

Amino acid identity may be calculated using any suitable algorithm. For example, the PILEUP and BLAST algorithms can be used to calculate identity or line up sequences (such as identifying equivalent or corresponding sequences (typically on their default settings), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two polynucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. Alternatively, the UWGCG Package provides the BESTFIT program which can be used to calculate identity (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, 387-395).

The sequence of a polypeptide provided may be a variant, in which modifications, such as amino acid additions, deletions or substitutions can be made relative to any one of the sequences of any one of the polypeptides provided herein. The modifications can be conservative amino acid substitutions. Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties or similar side-chain volume. The amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acids they replace. Alternatively, the conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well-known in the art and may be selected in accordance with the properties of amino acids as desired. Where amino acids have similar polarity, this can be determined by reference to the hydropathy scale for amino acid side chains.

Any one of the polypeptides provided herein may be included as part of a fusion polypeptide or conjugated to another molecule. In one embodiment, any one of the polypeptides provided herein may be conjugated to a Fc domain. Such a domain, is one that can interact with a Fc receptor or a portion thereof the interacts with a Fc receptor. In some embodiments of the disclosure, the Fc domain is of IgGl, IgG2, IgG3, or IgG4. In some embodiments, the Fc domain is of a mouse Ig. In some embodiments, the Fc domain is of a human Ig. As used herein, the term refers to a full Fc molecule or a portion thereof that interacts with Fc receptors and/or provides the necessary activity consistent with the disclosure and desired outcomes as provided herein.

Any one of the polypeptides provided herein may be engineered to include one or more additional amino acids that are typically to assist with production, isolation or purification. Any one of the polypeptides provided herein may be engineered to include an additional methionine at the N-terminus and/or protein purification or other tag at the C-terminus. The tag can be a sequence which is not naturally expressed in streptococcal bacteria as a contiguous domain. A preferred protein purification tag is a histidine tag. A histidine tag preferably consists of six histidine residues. The histidine tag may be linked to the C-terminus by a linker, which can be a short sequence of amino acids, such as 3 - 5 amino acids. Such a linker can comprise glycine and serine residues, such as the sequence GSG or GSGLE.

Polypeptide Production

A “polypeptide” is used herein in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. The term “polypeptide” thus includes short peptide sequences and also longer polypeptides and proteins. The terms “protein”, “peptide” and “polypeptide” may be used interchangeably. The term “amino acid” may refer to either natural and/or unnatural or synthetic amino acids, including both D or L optical isomers, and amino acid analogs and peptidomimetics.

A polypeptide may be produced by suitable method, including recombinant or synthetic methods. For example, the polypeptide may be synthesised directly using standard techniques known in the art, such as Fmoc solid phase chemistry, Boc solid phase chemistry or by solution phase peptide synthesis. Alternatively, a polypeptide may be produced by transforming a cell, typically a bacterial cell, with a nucleic acid molecule or vector which encodes said polypeptide. Provided herein are nucleic acid molecules and vectors which encode a polypeptide provided herein in one aspect. Also provided, in an aspect, is a host cell comprising such a nucleic acid or vector. A polypeptide may be engineered or modified to assist with production, isolation or purification. For example, where a polypeptide provide herein 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. As another example, a polypeptide provided herein 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 streptococcus bacteria. The protein purification tag is preferably a moiety which is not present in a wildtype polypeptide chain as expressed in streptococcus 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. Any suitable binding pair can be used.

Where the polypeptide is engineered or modified by addition of one member of a binding pair, the polypeptide is preferably histidine-tagged or biotin-tagged. Typically, the amino acid coding sequence of the histidine or biotin tag is included at the gene level and the polypeptide is expressed recombinantly in E. coli. The histidine or biotin tag is typically present at either end of the polypeptide, preferably at the C-terminus. It may be joined directly to the polypeptide or joined indirectly by any suitable linker sequence, such as 3, 4 or 5 glycine residues, or a mixture of glycine and serine residues. The histidine tag typically consists of six histidine residues, although it can be longer than this, typically up to 7, 8, 9, 10 or 20 amino acids or shorter, for example 5, 4, 3, 2 or 1 amino acids.

Alternative tags useful for protein purification include Maltose-binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX), ubiquitin (Ub), small ubiquitin related modifier (SUMO), solubility-enhancer peptide sequences (SET), and N-utilization substance (NusA). Suitable tags are also discussed in Costa et al Front Microbiol. 2014; 5: 63, which is incorporated by reference, including in particular each of the tags that are disclosed in Table 1 of Costa et al.

The amino acid sequence of a polypeptide may be modified or engineered to include at least one non-naturally occurring amino acid, for example to increase stability. When the polypeptides are produced by synthetic means, such amino acids may be introduced during production. The polypeptides may also be modified following either synthetic or recombinant production. Polypeptides may also be produced using D-amino acids. In such cases the amino acids will be linked in reverse sequence in the C to N orientation. This is conventional in the art for producing such polypeptides. Preferred polypeptides provided herein may be engineered to include at least one such unnatural or synthetic amino acid, or at least one non-L configuration amino acid.

A number of side chain modifications are known in the art and may be made to the side chains of the polypeptides, subject to the polypeptides retaining any further required activity or characteristic as may be specified herein. It will also be understood that polypeptides may be chemically modified, e.g., post-translationally modified. For example, they may be glycosylated, phosphorylated or comprise modified amino acid residues. The polypeptide may be PEGylated.

A polypeptide may be provided in a substantially isolated or purified form. That is, isolated from the majority of the other components present in a cellular extract from a cell in which the polypeptide was expressed. The polypeptide may be mixed with carriers or diluents (as discussed below) which will not interfere with the intended use and still be regarded as substantially isolated. It may also be in a substantially purified form, in which case it will generally comprise at least 90%, e.g., at least 95%, 98% or 99%, of the protein in the preparation. Where a polypeptide is provided in a composition with an additional active component, such as another polypeptide, each said polypeptide will individually be purified to a high level of homogeneity prior to mixing in an appropriate ratio for the intended purpose of each.

A polypeptide may be provided in lyophilised form, suitable for reconstitution in aqueous solution prior to use. The lyophilised composition has improved stability enabling longer storage of the polypeptide. A polypeptide is typically substantially purified prior to freeze-drying. A method of preparing a polypeptide in lyophilised form, comprising freeze-drying said polypeptide in a suitable buffer, such as Phosphate-buffered saline (PBS), Tris-buffered saline (TBS), or another Tris-buffer is provided herein. The resulting polypeptide in lyophilised form is also provided. A method of preparing a solution of a polypeptide, comprising providing the polypeptide in lyophilised form and reconstituting with a suitable carrier or diluent, such as water, is also provided.

A polypeptide may be immobilised using methods known in the art, for example as described in Datta S el al., Enzyme immobilization: an overview on techniques and support materials, 3 Biotech, 3(1): 1-9 (2013). For example, the polypeptide may be immobilised by adsorption, covalent binding, affinity immobilization or entrapment. Materials that can be used as supports include but are not limited to for example, natural supports such as agarose, sepharose, collagen, gelatin, cellulose, pectin, sepharose, inorganic materials such as ceramics, silica, glass, activated carbon or charcoal, or synthetic polymers, such as Poly(styrene- divinylbenzene), or latex. Any of these may be provided as a resin or in any other suitable format. The polypeptide may be immobilised on magnetic beads.

Exemplary polynucleotide molecules encoding a polypeptide disclosed herein can include at the 5’ end a codon for the N terminal methionine (ATG) and, prior to the stop codon (TAA) at the 3’ end, codons for a protein purification tag, such as a 6x His tag, which may optionally be excluded. A polynucleotide provided herein can encode a polypeptide provided herein and may be provided in isolated or substantially isolated form. By substantially isolated, it is meant that there may be substantial, but not total, isolation of the polypeptide from any surrounding medium. The polynucleotides may be mixed with carriers or diluents which will not interfere with their intended use and still be regarded as substantially isolated. A nucleic acid sequence which “encodes” a selected polypeptide can be a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide when placed under the control of appropriate regulatory sequences, for example in an expression vector. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A transcription termination sequence may be located 3' to the coding sequence.

Polynucleotides can be synthesised according to methods well known in the art, as described by way of example in Sambrook et al (1989, Molecular Cloning - a laboratory manual; Cold Spring Harbor Press). The nucleic acid molecules provided herein may be provided in the form of an expression cassette which includes control sequences operably linked to the inserted sequence, thus allowing for expression of a polypeptide provided herein (e.g. in prokaryotic or eukaryotic expression systems). These expression cassettes, in turn, are typically provided within vectors (e.g., plasmids or recombinant viral vectors). A suitable vector may be any vector which is capable of carrying a sufficient amount of genetic information, and allowing expression of a polypeptide provided herein.

Provided herein in one aspect are expression vectors that comprise such polynucleotide sequences. Such expression vectors are routinely constructed in the art of molecular biology and may for example involve the use of plasmid DNA and appropriate expression control sequences or initiators, promoters, enhancers and other elements, such as for example polyadenylation signals which may be necessary, and which are positioned in the correct orientation, in order to allow for expression of a polypeptide provided herein. Other suitable vectors would be apparent to persons skilled in the art. By way of further example in this regard we refer to Sambrook et al. “Expression control sequences” are any sequences that can affect expression. In one embodiment, the expression control sequence is a promoter, such as a constitutive promoter. Any one of the nucleic acids or vectors provided herein can comprise an expression control sequence.

Also provided are cells that have been modified to express a polypeptide provided herein. Such cells typically include prokaryotic cells such as bacterial cells, for example E. coli. Such cells may be cultured using routine methods to produce a polypeptide provided herein.

Compositions

In another aspect, compositions comprising a polypeptide provided herein are provided. For example, a composition comprising at least one polypeptide provided herein and at least one excipient, preferably a preservative or stabiliser, and optionally also one or more additional carriers, diluents or vehicles, is provided. In a particular embodiment, the excipients are preferably pharmaceutically acceptable, in the sense of being compatible with the other ingredients of the composition and not deleterious to a subject to which the composition is administered. In this case, the composition may be referred to as a pharmaceutical composition.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier” means a pharmacologically inactive material used together with a pharmacologically active material to formulate the compositions. Pharmaceutically acceptable excipients comprise a variety of materials known in the art, including but not limited to saccharides (such as glucose, lactose, and the like), preservatives such as antimicrobial agents, reconstitution aids, colorants, saline (such as phosphate buffered saline), and buffers.

Compositions provided herein may comprise inorganic or organic buffers (e.g., sodium or potassium salts of phosphate, carbonate, acetate, or citrate) and pH adjustment agents (e.g., hydrochloric acid, sodium or potassium hydroxide, salts of citrate or acetate, amino acids and their salts) antioxidants (e.g., ascorbic acid, alpha- tocopherol), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxyethylene9-10 nonyl phenol, sodium desoxy cholate), solution and/or cryo/lyo stabilizers (e.g., sucrose, lactose, mannitol, trehalose), osmotic adjustment agents (e.g., salts or sugars), antibacterial agents (e.g., benzoic acid, phenol, gentamicin), antifoaming agents (e.g., polydimethylsilozone), preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymeric stabilizers and viscosity-adjustment agents (e.g., polyvinylpyrrolidone, poloxamer 488, carboxymethylcellulose) and co-solvents (e.g., glycerol, polyethylene glycol, ethanol).

It is to be understood that the compositions may be made in any suitable manner, and the invention is in no way limited to compositions that can be produced using the methods described herein. Selection of an appropriate method of manufacture may require attention to the properties of the molecules of a composition. The compositions may be made using conventional pharmaceutical manufacturing and compounding techniques to arrive at useful dosage forms. Techniques suitable for use in practicing the present invention may be found in Handbook of Industrial Mixing: Science and Practice, Edited by Edward L. Paul, Victor A. Atiemo-Obeng, and Suzanne M. Kresta, 2004 John Wiley & Sons, Inc.; and Pharmaceutics: The Science of Dosage Form Design, 2nd Ed. Edited by M. E. Auten, 2001, Churchill Livingstone.

Formulation of a suitable composition can be carried out using standard pharmaceutical formulation chemistries and methodologies all of which are readily available to the reasonably skilled artisan. For example, the agent can be combined with a preservative, and one or more carriers, excipients or vehicles. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, reducing agents and the like, may be present in the excipient or vehicle. Suitable reducing agents include cysteine, thioglycerol, thioreducin, glutathione and the like. Excipients, vehicles and auxiliary substances are generally pharmaceutical agents that do not induce an immune response in the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as polyethyleneglycol, hyaluronic acid, glycerol, thioglycerol and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients, vehicles and auxiliary substances is available in Remington’s Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

Compositions may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable compositions may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Compositions include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such compositions may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a composition for parenteral administration, the active ingredient is provided in dry (for e.g., a powder or granules) form for reconstitution with a suitable vehicle (e. g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. The compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono-or di-glycerides.

In some embodiments, compositions are manufactured under sterile conditions or are terminally sterilized. This can ensure that resulting compositions are sterile and non-infectious, thus improving safety when compared to non-sterile compositions. This provides a valuable safety measure, especially when subjects receiving the compositions have immune defects, are suffering from infection, and/or are susceptible to infection. In some embodiments, the compositions may be lyophilized and stored in suspension or as lyophilized powder depending on the formulation strategy for extended periods without losing activity.

Administration according to the present invention may be by a variety of routes, including but not limited to subcutaneous, intravenous, or intraperitoneal routes. The compositions referred to herein may be manufactured and prepared for administration using conventional methods.

The compositions provided herein can be administered in effective amounts, such as the effective amounts described elsewhere herein. The amount of respective molecules present in a composition can be varied according to the nature of the molecules, the therapeutic benefit to be accomplished, and other such parameters. In embodiments, dose ranging studies can be conducted to establish optimal therapeutic amounts.

In some embodiments, there is provided a combination comprising any one or more of a polypeptide, polynucleotide, expression vector, host cell and/or composition as provided herein, wherein the components of said combination may be provided separately or mixed in any appropriate combination.

Another aspect of the disclosure relates to kits. In some embodiments, the kit comprises a polypeptide, polynucleotide, expression vector, host cell and/or other composition as provided herein. Each of the components of a kit can be contained within separate containers or within the same container in the kit. In some embodiments, the container is a vial or an ampoule. In some embodiments, each of the components can be contained within a solution separate from the container, such that the component may be added to a container at a subsequent time. In some embodiments, the components are in lyophilized form each in a separate container or in the same container, such that they may be reconstituted at a subsequent time. In some embodiments, the kit further comprises instructions for reconstitution, mixing, administration, etc. In some embodiments, the instructions include a description of the methods described herein. Instructions can be in any suitable form, e.g., as a printed insert or a label. In some embodiments, the kit further comprises one or more syringes.

Methods of Use

The polypeptides, polynucleotides, expression vectors, combinations, compositions and/or kits provided herein are useful in various methods. Thus, provided herein are methods comprising administering a polypeptide, polynucleotide, expression vector and/or composition provided, for example, to a sample or a subject. The method may be a method of cleaving an immunoglobulin, and may optionally further comprise the detection or analysis of the cleavage products. The method may be a method treating a disease, the method comprising administering a polypeptide or other composition provided to a subject. The method may be therapeutic or non- therapeutic in nature.

The present polypeptides or other composition may be used in a method for the in vivo, in vitro and/or ex vivo cleavage of Ig, such as IgG. Suitably, the method may be an in vitro method. In some embodiments the method is not an in vivo method. The Ig may be human, mouse, horse or guinea pig Ig, such as an Ig isolated or purified from said species. The Ig may be a recombinant, monoclonal antibody derived from any of these species. The polypeptide or other composition may be administered to a sample containing Ig, such as IgG, and incubated under conditions which permit immunoglobulin protease activity to occur.

Suitable conditions include incubation for at least 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes or 120 minutes, 3 hours, 5 hours, 10 hours, or overnight, typically with mixing e.g., end-over-end mixing. Incubation preferably takes place at room temperature, more preferably at approximately 20°C, 25°C, 30°C, 35°C, 40°C or 45°C, and most preferably at approximately 37°C. The methods described may be carried out under any suitable pH. Suitable pH values include, for example, around pH 6.5 to around pH 8.5, preferably around pH 7.0 to around pH 8.5, most preferably at around pH 7.5. The method may be conducted in any suitable buffer, such as tris buffered saline (TBS) or phosphate buffered saline (PBS). There is preferably no requirement for salts, co-factors or reducing agents, although the presence of salts (e.g., NaCl) at up to 200mM is preferably tolerated by a polypeptide provided herein. The approximate ratio of a polypeptide provided herein to the protein content of the sample may be 1: 1, 2: 1, 4: 1, 6: 1, 10: 1, 15: 1, 20: 1, 1:2, 1:4, or 1:6, 1: 10, 1: 15, 1:20, 1:40, 1: 100, 1: 200, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:1000, 1: 1500, 1:2000, or up to 1:2500 (wt:wt). A preferred ratio is between 1:20 and 1:40 (wt:wt).

Specific cleavage can be verified, and the cleavage products isolated using any suitable method, such as those described elsewhere in this document or in W02003051914 and W02009033670. The method can be used to generate Fc and F(ab’)2 fragments. Fab fragments may then be produced by carrying out a reduction step (for example in 2-mercaptoethanolamine or Cysteamine) on the F(ab’)2 fragments that result from cleavage of Ig with a polypeptide provided herein.

The method may also be used to detect or analyse Ig in a sample, such as IgG, or to remove Ig from a sample. A method for the detection of Ig in a sample typically involves incubating the polypeptide with the sample under conditions which permit binding and cleavage. The presence of Ig can be verified by detection of the specific Ig cleavage products, which may subsequently be analysed.

The polypeptides, polynucleotides, expression vectors, compositions, combinations and/or kits may also be used in therapy or prophylaxis. In therapeutic applications, polypeptides or compositions are administered to a subject already suffering from a disorder or condition, in an amount sufficient to cure, alleviate or partially arrest the condition or one or more of its symptoms. Such therapeutic treatment may result in a decrease in severity of disease symptoms, or an increase in frequency or duration of symptom-free periods. An amount adequate to accomplish this is defined as "therapeutically effective amount". In prophylactic applications, polypeptides or compositions are administered to a subject not yet exhibiting symptoms of a disorder or condition, in an amount sufficient to prevent or delay the development of symptoms. Such an amount is defined as a “prophylactically effective amount”. The subject may have been identified as being at risk of developing the disease or condition by any suitable means. Also provided herein is a method of prevention or treatment of disease or condition in a subject, which method comprises administering a polypeptide, polynucleotide, expression vector, combination or composition provided herein to the subject in a prophylactically or therapeutically effective amount. The polypeptide or composition is preferably administered by intravenous infusion, but may be administered by any suitable route including, for example, intradermal, subcutaneous, percutaneous, intramuscular, intra-arterial, intraperitoneal, intraarticular, or other appropriate administration route. "Administering" or "administration" or “administer” means providing a material to a subject in a manner that is pharmacologically useful. The term is intended to include “causing to be administered” in some embodiments. “Causing to be administered” means causing, urging, encouraging, aiding, inducing or directing, directly or indirectly, another party to administer the material.

Polypeptides, polynucleotides, expression vectors, combinations, compositions and/or kits provided herein may be particularly useful in a method for the treatment or prevention of a disease or condition mediated by Ig antibodies, such as IgG antibodies, which in this context may be described as pathogenic Ig antibodies. Accordingly, there is provided a polypeptide, polynucleotide, expression vector, combination, composition or kit provided herein for use in the treatment or prevention of a disease or condition. In one embodiment there is provided a polypeptide, polynucleotide, expression vector, combination, composition, or kit for use in the treatment or prevention of a disease or condition, wherein the disease or condition is mediated by pathogenic Ig antibodies, such as IgG antibodies. In one embodiment, there is provided a polypeptide, polynucleotide, expression vector, combination, composition or kit for use in treating or preventing a disease or condition, particularly a disease or condition mediated by pathogenic Ig antibodies, such as IgG antibodies, comprising administering to an individual a polypeptide, polynucleotide, expression vector, combination or composition provided herein. The method may comprise repeat administration of the polypeptide, polynucleotide, expression vector, combination, or composition. Also provided herein is a polypeptide, polynucleotide, expression vector, combination, composition and/or kit provided herein for use in the manufacture of a medicament. In one embodiment, there is provided a polypeptide, polynucleotide, expression vector, combination, composition, and/or kit for use in the manufacture of a medicament for the treatment or prevention of a disease or condition mediated by pathogenic Ig antibodies, such as IgG antibodies, particularly a disease or condition mediated by pathogenic Ig antibodies, such as IgG antibodies. Numerous autoimmune diseases are mediated in whole or in part by pathogenic IgG antibodies. Thus, pathogenic antibodies may typically be specific for an antigen which is targeted in an autoimmune disease or other condition mediated wholly or in part by IgG antibodies. Exemplary diseases and conditions mediated by antibodies and the associated antigens are listed in detail in Table D of WO 2016/128559. A polypeptide, polynucleotide, expression vector, combination, composition, and/or kit provided herein may be used in a method to treat any of these diseases or conditions.

Pathogenic antibodies may recognise a tissue or organ transplant received by the subject. Thus, the disease treated or prevented by a polypeptide or composition provided herein may be antibody-mediated transplant rejection. The pathogenic antibodies may recognise another therapeutic agent administered to the subject, such as an antibody, a gene therapy (e.g., a viral or other vector), a replacement for a defective endogenous factor such as an enzyme, a growth or a clotting factor, or a cell therapy. Thus, the disease or condition treated or prevented by a polypeptide or composition provided herein may be any antibody response of the subject which reduces the efficacy or benefit to the subject of said therapeutic agent. In one embodiment, there is provided a polypeptide, polynucleotide, expression vector, combination, composition, and/or kit for use as a concomitant medicament in therapy, optionally wherein said concomitant medicament reduces or eliminates antibody-mediated suppression of a second therapeutic agent (i.e., not the polypeptide, polynucleotide, expression vector, combination, composition, and/or kit provided herein).

Polypeptides, polynucleotides, expression vectors, combinations, compositions, and/or kits provided herein may be particularly useful for the treatment or prevention of a disease or condition in a subject, wherein the subject comprises pre-existing immunity to other Ig proteases, such as IgG proteases. For example, the subject may comprise antibodies against IdeS or IdeZ, due to a previous S. pyogenes infection or prior treatment with Ig protease, such as IgG protease. In one embodiment, there is provided a polypeptide, polynucleotide, expression vector, combination, composition, and/or kit for use in the treatment or prevention of a disease or condition in a subject, wherein the subject has pre-existing immunity one or more Ig protease that is immunologically distinct from the protease of the invention. In one embodiment, there is provided a polypeptide, polynucleotide, expression vector, combination, composition, and/or kit for use according to the foregoing, wherein the subject has antibodies against IdeS or IdeZ. Methods provided herein may involve cleavage of a sample containing a known antibody. The antibody is preferably a recombinant monoclonal antibody. The antibody may be Abagovomab, Abciximab,Actoxumab, Adalimumab, Adecatumumab, Afelimomab, Afutuzumab, Alacizumab pegol, ALD518, Alemtuzumab, Alirocumab, Altumomab pentetate, Amatuximab, Anatumomab mafenatox, Anrukinzumab, Apolizumab, Arcitumomab, Aselizumab, Atinumab, Atlizumab (= tocilizumab), Atorolimumab, Bapineuzumab, Basiliximab, Bavituximab, Bectumomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bivatuzumab mertansine, Blinatumomab, Blosozumab, Brentuximab vedotin, Briakinumab, Brodalumab, Canakinumab, Cantuzumab mertansine, Cantuzumab ravtansine, Caplacizumab, Capromab pendetide, Carlumab, Catumaxomab, CC49, Cedelizumab, Certolizumab pegol, Cetuximab, Ch.14.18, Citatuzumab bogatox, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumab tetraxetan, Conatumumab, Concizumab, Crenezumab, CR6261, Dacetuzumab, Daclizumab, Dalotuzumab, Daratumumab, Demcizumab, Denosumab, Detumomab, Dorlimomab aritox, Drozitumab, Duligotumab, Dupilumab, Dusigitumab, Ecromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Elotuzumab Elsilimomab, Enavatuzumab, Enlimomab pegol, Enokizumab, Enoticumab, Ensituximab, Epitumomab cituxetan, Epratuzumab, Erlizumab, Ertumaxomab, Etaracizumab, Etrolizumab, Evolocumab, Exbivirumab, Fanolesomab, Faralimomab Farletuzumab, Fasinumab, FBTA05, Felvizumab, Fezakinumab, Ficlatuzumab, Figitumumab, Flanvotumab, Fontolizumab, Foralumab, Foravirumab, Fresolimumab, Fulranumab, Futuximab, Galiximab,Ganitumab, Gantenerumab, Gavilimomab, Gemtuzumab ozogamicin, Gevokizumab, Girentuximab,Glembatumumab vedotin, Golimumab, Gomiliximab,GS6624, Ibalizumab, Ibritumomab tiuxetan, Icrucumab, Igovomab, Imciromab, Imgatuzumab, Inclacumab, Indatuximab ravtansine, Infliximab, Intetumumab, Inolimomab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lampalizumab, Lebrikizumab, Lemalesomab, Lerdelimumab, Lexatumumab, Libivirumab, Ligelizumab, Lintuzumab, Lirilumab, Lodelcizumab, Lorvotuzumab mertansine, Lucatumumab, Lumiliximab, Mapatumumab, Maslimomab, Mavrilimumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mitumomab, Mogamulizumab, Morolimumab, Motavizumab, Moxetumomab pasudotox, Muromonab-CD3, Nacolomab tafenatox, Namilumab, Naptumomab estafenatox, Namatumab, Natalizumab, Nebacumab, Necitumumab, Nerelimomab, Nesvacumab, Nimotuzumab, Nivolumab, Nofetumomab merpentan, Obinutuzumab, Ocaratuzumab, Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Olokizumab, Omalizumab, Onartuzumab, Oportuzumab monatox, Oregovomab, Orticumab, Otelixizumab, Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Panitumumab, Panobacumab, Parsatuzumab, Pascolizumab, Pateclizumab, Patritumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab vedotin, Pintumomab, Placulumab, Polatuzumab vedotin, Ponezumab, Priliximab, Pritoxaximab, Pritumumab, PRO 140, Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ramucirumab, Ranibizumab,Raxibacumab, Regavirumab, Reslizumab, Rilotumumab, Rituximab, Robatumumab, Roledumab, Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab, Samalizumab, Sarilumab, Satumomab pendetide, Secukinumab, Seribantumab, Setoxaximab, Sevirumab, Sibrotuzumab, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab, Sirukumab, Solanezumab, Solitomab, Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab, Suvizumab, Tabalumab, Tacatuzumab tetraxetan, Tadocizumab, Talizumab, Tanezumab, Taplitumomab paptox, Tefibazumab, Telimomab aritox, Tenatumomab, Teneliximab, Teplizumab, Teprotumumab, TGN1412, Ticilimumab (= tremelimumab), Tildrakizumab, Tigatuzumab, TNX-650, Tocilizumab (= atlizumab), Toralizumab, Tositumomab, Tralokinumab, Trastuzumab, TRBS07, Tregalizumab, Tremelimumab Tucotuzumab celmoleukin, Tuvirumab, Ublituximab, Urelumab, Urtoxazumab, Ustekinumab, Vapaliximab, Vatelizumab, Vedolizumab, Veltuzumab,Vepalimomab Vesencumab, Visilizumab, Volociximab, Vorsetuzumab mafodotin, Votumumab, Zalutumumab, Zanolimumab, Zatuximab, Ziralimumab or Zolimomab aritox.

Polypeptides, polynucleotides, expression vectors, combinations, compositions, and/or kits provided herein may be useful in a method for improving the benefit to a subject of a therapeutic antibody or therapeutic agent whose effect is mediated through Fc receptor binding. For example, the polypeptides, polynucleotides, expression vectors, combinations, compositions, and/or kits provided herein maybe used to rapidly, temporarily and safely eliminate Fc receptor binding by all or substantially all endogenous Ig, such as IgG, in a subject. Thus, a subsequently administered therapeutic antibody (or therapeutic agent that binds Fc receptor) would have enhanced efficacy because it does not need to compete with endogenous Ig for binding to Fc receptor.

The following Examples are illustrative.

EXAMPLES Example 1

SDS-PAGE-based IgGP cleavage assay

3.3 pM IgGl substrate (Humira) was incubated with 330 nM IgGP muteins at 37°C. The reactions were sampled at indicated time points (0.5, 1, 5, 10, 30, 60, 120, 180 and 240 min) and stopped by immediately mixing with LDS buffer (NuPAGE™ LDS Sample Buffer, Life Technologies) and heat treatment at 70°C for 10 minutes. The samples were analyzed with SDS- PAGE (NuPAGE™ 4 to 12% Bis-Tris gel, Life Technologies). The proteins bands were visualized with Coomassie staining.

EEISA-based IgGP cleavage assay with human sera

Human sera (HMN403595, HMN403596, HMN403598, BIOIVT, ~10 mg/ml endogenous human IgG) were mixed with IgGP muteins at 37°C to start the reactions containing 20 pM IgG and 10 pM IgGPs. Reactions were sampled at indicated time points and stopped with immediately diluting samples into casein/PBS on the ice. The diluted samples were incubated in the ELISA plates precoated with 2 pg/ml goat anti-human IgG F(ab)’ (Life Technologies #31122, 2.4 mg/ml) for 1 hour at room temperature. After washing with PBST, the ELISA plates were incubated with goat anti-monkey IgG_Fc HRP antibody (Novus Biologicals, 1: 10,000 dilution) for 1 hour at room temperature. The ELISA signal was generated by TMB substrates (TMB Substrate Reagent Set, BD Biosciences) and measured as the absorbance at 450 nm and 570 nm on plate reader.

Results

Wild-type (Xork 1.0) and N-terminal truncated mutant (Xork 1.2) of Xork have comparable activities of cleaving both purified IgGl (Humira) (FIG. 1 and FIG. 2) and endogenous IgG in human sera (FIG. 3 and FIG. 4). 3.3 pM IgGl substrates (Humira) were cleaved by 330 nM Xork 1.0 (FIG. 1) or Xork 1.2 (FIG. 2) at 37°C. The reactions were sampled at indicated time points and analyzed with SDS-PAGE and Coomassie staining. The full-length Humira decreases over the time course, corresponding to the increase of F(ab’)2 and Fc fragments. The intermediate product, single chain cleaved Humira, accumulates at early stage and decreases at latter stages. 30% human sera from three individuals (#595, upper left panel; #596, upper right panel; #598, lower panel) were cleaved by 10 pM Xork 1.0 or Xork 1.2 at 37°C. The human IgG abundance in the samples at indicated time points were measured by ELISA and plotted as the absorbance at 450 nm and 570 nm (FIG. 3) or normalized to the absorbance value at the beginning of the reaction (t=0) (FIG. 4).

Example 2

EEISA-based. IgGP cleavage assay with human sera

Human sera (HMN403595, HMN403596, HMN403598, BIOIVT, ~10 mg/ml endogenous human IgG) were mixed with IgGP muteins at 37°C to start the reactions containing 20 uM IgG and 10 uM IgGPs. Reactions were sampled at indicated time points and stopped with immediately diluting samples into casein/PBS on the ice. The diluted samples were incubated in the ELISA plates precoated with 2 ug/ml goat anti-human IgG F(ab)’ (Life Technologies #31122, 2.4 mg/ml) for 1 hour at room temperature. After washing with PBST, the ELISA plates were incubated with goat anti-monkey IgG_Fc HRP antibody (Novus Biologicals, 1: 10,000 dilution) for 1 hour at room temperature. The ELISA signal was generated by TMB substrates (TMB Substrate Reagent Set, BD Biosciences) and measured as the absorbance at 450 nm and 570 nm on plate reader.

SDS-PAGE-based IgGP cleavage assay

22 uM substrate (Humira or Infliximab) was incubated with 160 nM IgGP muteins at 37°C. The reactions were sampled at indicated time points (0, 1, 2, 4,6,10,15 and 30 min) and stopped by immediately mixing with LDS buffer (NuPAGE™ LDS Sample Buffer, Life Technologies, containing 2.5% 2-mercaptoethanol) and heat treatment at 95°C for 10 minutes. The samples were analyzed with SDS-PAGE (NuPAGE™ 4 to 12% Bis-Tris gel, Life Technologies). The proteins bands were visualized with Coomassie staining. Results

FIG. 5 also shows comparisons of wild-type (Xork 1.0) and N-terminal truncated mutant (Xork 1.2) cleaving activity against Humira and Infliximab as well as against human sera. The activities were again comparable.

Claims

What is claimed is: CLAIMS

1. A polypeptide, comprising an N-terminally truncated form of an IdeSORK, which truncated form has an activity of the IdeSORK, the IdeSORK having the sequence as set forth in SEQ ID NO: 1 or SEQ ID NO: 3.

2. The polypeptide of claim 1, wherein the polypeptide comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 3 but without the sequence of SEQ ID NO: 4 in its entirety.

3. The polypeptide of claim 1 or 2, wherein the polypeptide comprises the sequence of SEQ ID NO: 1 or SEQ ID NO: 3 but without amino acids 1, 1-2, 1-3, 1-4, 1-5, 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 or 1-25 of the N- terminus thereof.

4. The polypeptide of any one of claims 1-3, wherein the polypeptide comprises the sequence of SEQ ID NO:2.

5. A composition that comprises the polypeptide of any one of claims 1-4 and at least one carrier or excipient, which composition is pharmaceutically acceptable.

6. A polynucleotide that comprises a sequence that encodes the polypeptide of any one of claims 1-4.

7. A composition comprising the polynucleotide of claim 6 and at least one carrier or excipient, which composition is pharmaceutically acceptable.

8. An expression vector that comprises the polynucleotide of claim 6.

9. A composition comprising the expression vector of claim 8 and at least one carrier or excipient, which composition is pharmaceutically acceptable.

10. A host cell comprising the polynucleotide of claim 6 or expression vector of claim 8.

11. A composition comprising the host cell of claim 10 and at least one carrier or excipient, which composition is pharmaceutically acceptable.

12. A method of cleaving an immunoglobulin (Ig) comprising contacting the immunoglobulin with the polypeptide of any one of claims 1-4 or the composition of claim 5.

13. The method of claim 12, wherein the contacting is in vitro.

14. The method of claim 12, wherein the contacting is in vivo.

15. The method of claim 12 or 14, wherein the contacting is by administering the polypeptide or composition to a subject.

16. The method of claim 15, wherein the subject is any one of the subjects provided herein.

17. A method of treating a subject, wherein the polypeptide of any one of claims 1-4 or the composition of claim 5 is administered to the subject.

18. The method of claim 17, wherein the subject is in need thereof.

19. A method for the ex vivo cleavage of Ig in a sample, the method comprising administering the polypeptide of any one of claims 1-4 or the composition of claim 5 to the sample and incubating under conditions suitable for Ig protease activity.

20. The method of claim 19, wherein the method further comprises the separation, detection and/or analysis of the resulting cleavage products, and/or wherein the method generates Fc and Fab fragments.

21. A method comprising administering the polynucleotide of claim 6 or the expression vector of claim 8 to a sample or a subject in which Ig is present.

22. A method comprising administering the composition of claim 7 or 9 to a sample or a subject in which Ig is present.

23. The method of any one of claims 12, 17, 21 and 22, wherein the method is for the prevention or treatment of a disease or condition in a subject, and which method comprises administering said polypeptide, polynucleotide, expression vector, or composition to the subject in a prophylactically or therapeutically effective amount.

24. The method of claim 23, wherein the disease or condition is a disease or condition mediated at least in part by pathogenic Ig antibodies.

25. The method of claim 23 or 24, wherein the disease or condition is (i) an autoimmune disease mediated at least in part by IgG antibodies; (ii) IgG-mediated rejection of an organ or tissue transplant received by the subject; or (iii) IgG-mediated anti-drug responses to a therapeutic agent administered to the subject.

26. A polypeptide according to any one of claims 1 to 4, a polynucleotide of claim 6 or expression vector according to claim 8, or a composition according to any one of claims 5, 7 and 9, for use in the treatment or prevention of a disease or condition in a subject.

27. Use of a polypeptide according to any one of claims 1 to 4, a polynucleotide of claim 6 or expression vector according to claim 8, or a composition according to any one of claims 5, 7 and 9, in the preparation of a medicament for the treatment or prevention of a disease or condition in a subject.

28. The polypeptide, polynucleotide, expression vector or composition for use according to claim 26, or the use according to claim 27, wherein the disease or condition is a disease or condition mediated at least in part by pathogenic Ig antibodies.

29. The polypeptide, polynucleotide, expression vector or composition for use according to claim 26, or the use according to claim 27, wherein the disease or condition is (i) an autoimmune disease mediated in whole or in part by IgG antibodies; (ii) IgG mediated rejection of an organ or tissue transplant received by the subject; or (iii) IgG mediated anti-drug responses to a therapeutic agent administered to the subject.

30. A method for improving the benefit to a subject of a therapeutic agent, such as a therapeutic antibody, comprising administering the polypeptide according to any one of claims 1- 4, the polynucleotide of claim 6 or expression vector according to claim 8, or a composition according to any one of claims 5, 7 and 9, which method comprises i) administering said polypeptide, polynucleotide, expression vector, or composition to the subject in a prophylactically or therapeutically effective amount and ii) administering, such as subsequently administering, a therapeutic agent, such as a therapeutic antibody to the subject.