Immunoglobulin proteases and their uses

Abstract

The present invention relates to IgG specific proteases, and also IgG and IgM specific proteases. Specifically, proteases according to the present invention are able to cleave IgG, including human IgG, with high specificity, including IgG subclass specificity. The invention further relates to methods and uses of said proteases, particularly for gene therapy applications.

Description

[0001] IMMUNOGLOBULIN PROTEASES AND THEIR USES

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to novel polypeptides which display protease activity against immunoglobulins, particularly human IgG and also IgG and IgM, and in vivo and ex vivo uses thereof. Uses of the polypeptide include methods for the analysis of immunoglobulins, particularly IgG and also IgG and IgM, and the generation of antibody fragments, as well as methods for the enhancement of gene therapy applications, and for the prevention or treatment of diseases and conditions mediated by immunoglobulins, particularly, IgG and also IgG and IgM.

[0004] BACKGROUND OF THE INVENTION

[0005] Immunoglobulin (Ig) proteases are enzymes that specifically cleave immunoglobulins, which are commonly referred to as antibodies.

[0006] For example, the IgG protease IdeS efficiently cleaves IgG to Fc and F(ab')2 fragments via a two-stage mechanism. In the first stage, one (first) heavy chain of IgG is cleaved to generate a single cleaved IgG (scIgG) molecule with a single noncovalently bound Fc chain. The scIgG molecule is effectively an intermediate product which retains the remaining (second) heavy chain of the original IgG molecule. In the second stage of the mechanism this second heavy chain is cleaved by IdeS to release a F(ab')2 fragment and a homodimeric Fc fragment. These are the products generally 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.

[0007] The Ig cleaving ability of Ig protease has been shown to have utility ex vivo, for example in methods for production of Fab, F(ab')2 and Fc fragments, which may be used for the analysis of IgG and in vitro generation of F(ab')2 fragments, including the validation and quality control of therapeutic antibodies. See, for example, WG2003051914 and WG2009033670.

[0008] Additionally, Ig proteases have garnered significant interest for their potential therapeutic applications due to their ability to modulate immune responses, with several IgG cleaving proteins having been shown to have in vivo utility as therapeutic agents, see for example, W02003051914 and W02009033670. Additionally, modified IgG cleaving proteins have also been shown to have in vivo utility as therapeutic agents, see for example, WO2006131347, WO2013110946, WO2016012285, WO2016128558, and WO2016128559.

[0009] Specific Ig proteases exhibit specificity towards different classes of immunoglobulins, which facilitates targeted Ig protease applications in research and therapeutic settings, enabling precise modulation of immune responses without broadly suppressing the immune system. For example, IdeS has an extraordinarily high degree of substrate specificity, with its only identified substrate being IgG. IdeS catalyses a single proteolytic cleavage in the lower hinge region of the heavy chains of all subclasses of human IgG. Such proteins are useful in therapeutic contexts because they are capable of the specific in vivo cleavage of Ig molecules which mediate disease or which are otherwise undesirable. The removal of specific classes of Ig, e.g. IgG and IgM, can be beneficial because IgG and IgM contribute to the pathology of many autoimmune conditions as well as to acute rejection of transplanted organs, and may also contribute to anti-drug responses. Furthermore, antibodies specific for a given therapeutic agent may reduce its efficacy.

[0010] This is particularly the case for antibody-based therapeutics, gene therapy vectors, and cell therapies including adoptive cell transfer (ACT) immunotherapies e.g. using CAR-T cells. In other words, IgG and IgM cleaving proteins and modified IgG and IgM cleaving proteins may be used as a therapy for any disease or condition wholly or partly mediated by IgG and / or IgM, which may include anti-drug responses.

[0011] However, many Ig proteases act as virulence factors of their species of origin, which is responsible for common infections such as tonsillitis and strep throat. Accordingly, most human subjects have encountered many Ig proteases in this context and are likely to have raised antibodies against these proteases in the bloodstream, which may provoke an immune response and thus reduce their utility as a therapeutic agent. Likewise, patients having previously been treated with an Ig protease, e.g. a ‘first- line Ig protease treatment’ will also have raised antibodies against these proteases, similarly reducing their utility as a therapeutic agent. Therefore, there is a clear need to identify further Ig proteases with low prevalence of existing anti-enzyme antibodies, which may be used as ‘second-’ or ‘third-line’ treatment.

[0012] Additionally, whilst the therapeutic potential of immunoglobulin proteases is significant, there is a clear need to develop a repertoire of proteases having finely tuned specificities for specific disease or immune response Ig proteins in order to maximize therapeutic efficacy and avoid off-target effects and unintentional degradation of non-pathogenic immunoglobulins.

[0013] SUMMARY OF THE INVENTION

[0014] The present inventors have identified, purified and characterised novel polypeptides having protease activity with a surprisingly high degree of substrate specificity towards IgG and IgG subfamilies. Furthermore, the present inventors have identified and characterised novel polypeptides naturally comprising two linked IgG proteases which may be suitable for the generation of chimeric Ig proteases.

[0015] The present invention relates to one or more engineered polypeptides of the identified polypeptides of the invention, having desirable / beneficial properties.

[0016] The polypeptides of the invention allow for the specific and effective targeting of IgG and IgG subfamilies, including human IgG and human IgG subfamilies. The polypeptides of the invention have utility in numerous applications as biological tools for the study and manipulation of IgG and IgG subfamilies. In aspects of the invention, a polypeptide, or engineered polypeptide, having immunoglobulin protease activity is provided, wherein said polypeptide comprises, consists essentially of, or consists of:

[0017] (a) the amino acid sequence of any one of SEQ ID NOs: 1 , 4, 7, 10, 13, 41 , or 42; or

[0018] (b) a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 1 , 4, 7, 10, 13, 41 , or 42; or

[0019] (c) an amino acid sequence which is a fragment of the sequence of either (a) or (b).

[0020] In embodiments of the invention, the fragment of the sequence of SEQ ID NO: 1 , 4, 7, 10, 13, 41 , or 42 is: at least about 100 amino acids in length, at least about 200 amino acids in length, or at least about 300 amino acids in length; or wherein the fragment of the sequence of SEQ ID NO: 1 , 4, 10, 13, 41 , or 42 is: at least about 400 amino acids in length, at least about 500 amino acids in length, or at least about 600 amino acids in length; or wherein the fragment of the sequence of SEQ ID NO: 1 , 4, 13, 41 , or 42 is: at least about 700 amino acids in length; or wherein the fragment of the sequence of SEQ ID NO: 1 , 4 or 13 is: at least about 800 amino acids in length, at least about 900 amino acids in length, at least about 1000 amino acids in length, at least about 1100 amino acids in length, at least about 1200 amino acids in length, at least about 1300 amino acids in length, at least about 1400 amino acids in length, at least about 1500 amino acids in length; or wherein the fragment of the sequence of SEQ ID NO: 1 or 4 is: at least about 1600 amino acids in length, or at least about 1700 amino acids in length; or wherein the fragment of the sequence of SEQ ID NO: 4 is: at least about 1800 amino acids in length.

[0021] In aspects of the invention, a polypeptide, or engineered polypeptide, having immunoglobulin protease activity is provided, wherein said polypeptide comprises, consists essentially of, or consists of:

[0022] (a) the amino acid sequence of SEQ ID NO: 2, 5, 8, 11 , or 14; or

[0023] (b) a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 2, 5, 8, 11 , or 14; or

[0024] (c) an amino acid sequence which is a fragment of the sequence of

[0025] (a) or (b).

[0026] In embodiments of the invention, the fragment of the sequence of SEQ ID NO: 2, 5, 8, 11 , or 14 is: at least about 100 amino acids in length, at least about 150 amino acids in length, at least about 200 amino acids in length, at least about 250 amino acids in length, at least about 300 amino acids in length, or wherein the fragment of the sequence of SEQ ID NO: 5 is: at least about 350 amino acids in length at least about 400 amino acids in length, at least about 450 amino acids in length at least about 500 amino acids in length at least about 550 amino acids in length, at least about 600 amino acids in length, at least about 650 amino acids in length, at least about 700 amino acids in length, at least about 750 amino acids in length, at least about 800 amino acids in length.

[0027] In embodiments of the invention, the polypeptide is engineered to include an additional methionine at the N terminus and / or a protein purification tag at the C terminus, optionally wherein the tag may be joined to the C terminus by a linker.

[0028] In embodiments of the invention, the polypeptide has protease activity against an immunoglobulin molecule comprising a CH2 / hinge sequence, optionally wherein the CH2 / hinge sequence may be selected from any one of SEQ ID NOs: 28 to 37.

[0029] In embodiments of the invention, the polypeptide cleaves the CH2 / hinge sequence between amino acid positions 6 and 7 of any one or more of SEQ ID NOs: 28, or 30 to 34, and / or between positions 5 and 6 and / or 6 and 7 of SEQ ID NO: 29, and / or between positions 10 and 11 of one or more of SEQ ID NOs: 35 or 36, and / or between amino acid positions 5 and 6 of SEQ ID NO: 37.

[0030] In embodiments of the invention, the polypeptide comprises, consists essentially of, or consists of the amino acid sequence of any of SEQ ID NOs: 3, 6, 9, 12, 15, 16, 18 or 40.

[0031] In embodiments of the invention, the polypeptide has protease activity against IgG, optionally wherein the IgG is from a mammal; optionally wherein the mammal is selected from human, primate, horse, cow, sheep, goat, rabbit, or rodent (such as mouse, guinea pig, rat), optionally wherein the IgG is human IgG.

[0032] In embodiments of the invention, the immunoglobulin protease activity is IgG specific protease activity, optionally wherein the polypeptide has lower protease activity against one or more of IgA, IgD, IgE and IgM as compared to IgG, optionally wherein the level of activity against IgA, IgD, IgE or IgM is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the level of activity against IgG.

[0033] In embodiments of the invention, the polypeptide exhibits lower protease activity against human lgG2 as compared to any one other human IgG subclass, optionally wherein the polypeptide has lower protease activity against lgG2 as compared to one or more of IgG 1 , lgG3, and lgG4, optionally wherein the level of activity against lgG2 is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the level of activity against lgG1 , lgG3, and / or lgG4.

[0034] In embodiments of the invention, the polypeptide exhibits lower protease activity against mouse lgG1 , lgG2a, or lgG3 as compared to lgG2b, optionally wherein the level of activity against lgG1 , lgG2a, and / or lgG3 is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the level of activity against lgG2b.

[0035] In embodiments of the invention, the polypeptide exhibits lower protease activity against a human lgG1 and / or lgG3 as compared to a human lgG2 and / or lgG4 subclass.

[0036] In embodiments of the invention, the polypeptide exhibits lower protease activity against lgG1 and / or lgG3 as compared to lgG2 and / or lgG4, optionally wherein the level of activity against lgG1 and / or lgG3 is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the level of activity against lgG2 and / or lgG4.

[0037] In embodiments of the invention, the polypeptide exhibits lower protease activity against lgG1 , lgG3 and / or lgG4 as compared to lgG2, optionally wherein the level of activity against lgG1 , lgG3 and / or lgG4 is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the level of activity against lgG2.

[0038] In embodiments of the invention, the polypeptide exhibits lower protease activity against lgG3 and / or lgG4 as compared to lgG1 and / or lgG2, optionally wherein the level of activity against lgG3 and / or lgG4 is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the level of activity against lgG1 and / or lgG2.

[0039] In embodiments of the invention, the polypeptide has protease activity against an lgG1 variant, optionally against a modified IgG 1 variant, optionally against a hinge-modified IgG 1 variant.

[0040] In embodiments of the invention, the polypeptide has protease activity against a human lgG1 variant, optionally a modified human lgG1 variant, optionally a hinge-modified human lgG1 variant, optionally hlgG1 -LALA, hlgG1-LAGA, or hlgG1-LFLE.

[0041] In embodiments of the invention, the lgG1 variant, optionally human lgG1 variant, comprises the amino acid substitution L234A and / or L235A based on the sequence of SEQ ID NO: 43.

[0042] In embodiments of the invention, the lgG1 variant, optionally human lgG1 variant, comprises the amino acid substitution L234F, and / or L235E, and / or P331 S based on the sequence of SEQ ID NO: 43.

[0043] In embodiments of the invention, the lgG1 variant, optionally human lgG1 variant, comprises the amino acid substitution L235A, and / or G237A based on the wild-type sequence of SEQ ID NO: 43. In embodiments of the invention, the polypeptide has protease activity against an lgG4 variant, optionally against a modified lgG4 variant, optionally against a hinge-modified lgG4 variant.

[0044] In embodiments of the invention, the polypeptide has protease activity against a human lgG4 variant, optionally a modified human lgG4 variant, optionally a hinge-modified human lgG4 variant, optionally lgG4-FALA.

[0045] In embodiments of the invention, the lgG4 variant, optionally human lgG4 variant, comprises the amino acid substitution F234A, and / or L235A based on the wild-type sequence of SEQ ID NO: 38.

[0046] In embodiments of the invention, the polypeptide has protease activity against IgG and IgM, optionally wherein the IgG and / or IgM is from a mammal; optionally wherein the mammal is selected from human, primate, horse, cow, sheep, goat, rabbit, or rodent (such as mouse, guinea pig, rat), optionally wherein the IgG and / or IgM is human IgG and / or IgM.

[0047] In embodiments, the IgM may comprise the heavy chain sequence of SEQ ID NO: 44, or a sequence having at least 90%, at least 95%, at least 98% or at least 99% identity thereto.

[0048] In embodiments, the IgG 1 may comprise the heavy chain sequence of SEQ ID NO: 43, or a sequence having at least 90%, at least 95%, at least 98% or at least 99% identity thereto.

[0049] In embodiments, the lgG4 may comprise the heavy chain sequence of SEQ ID NO: 38, or a sequence having at least 90%, at least 95%, at least 98% or at least 99% identity thereto.

[0050] In embodiments of the invention, the immunoglobulin protease activity is IgG and IgM specific protease activity, optionally wherein the polypeptide has lower protease activity against one or more of IgA, IgD and IgE as compared to IgG and / or IgM, optionally wherein the level of activity against IgA, IgD and / or IgE is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the level of activity against IgG and / or IgM.

[0051] In aspects of the invention, a polynucleotide encoding a polypeptide of the invention having protease activity is provided, the polynucleotide comprising:

[0052] (a) the polynucleotide sequence of any one of SEQ ID NOs: 20 to 27;

[0053] (b) a variant thereof having at least 80% sequence identity to the polynucleotide sequence of any one of SEQ ID NOs: 20 to 27; or

[0054] (c) a polynucleotide fragment of either (a) or (b).

[0055] In embodiments of the invention, the polynucleotide fragment is at least about 500 nucleotides in length, at least about 600 nucleotides in length, at least about 700 nucleotides in length, at least about 800 nucleotides in length, at least about 900 nucleotides in length, or at least about 1000 nucleotides in length, or optionally at least about 1100 nucleotides in length, at least about 1200 nucleotides in length, at least about 1300 nucleotides in length, at least about 1400 nucleotides in length, at least about 1500 nucleotides in length, at least about 1600 nucleotides in length, at least about 1700 nucleotides in length, at least about 1800 nucleotides in length, at least about 1900 nucleotides in length, at least about 2000 nucleotides in length, at least about 2100 nucleotides in length, at least about 2200 nucleotides in length, at least about 2300 nucleotides in length, at least about 2400 nucleotides in length, or at least about 2500 nucleotides in length.

[0056] In aspects of the invention, an Ig protease fusion protein is provided, comprising (i) an Ig protease or Ig protease domain according to this disclosure, and (ii) an Fc domain. In embodiments, the Fc domain may be fused with the N-terminal or with the C-terminal end of the Ig protease or Ig protease domain. In embodiments, the Ig protease or Ig protease domain may comprise a polypeptide sequence according to any of SEQ ID NOs: 1 to 19, 40 to 42, or fragments thereof. In embodiments, the Fc-fusion protein may have beneficial properties over an Ig protease alone, for example, the fusion protein may have similar or increased protease activity relative to an Ig protease or Ig protease domain. In embodiments, the beneficial property may be an increased circulating half-life relative to the non-fused Ig protease or Ig protease domain. Another aspect of the invention relates to polynucleotides encoding the Ig protease fusion proteins of the disclosure.

[0057] In aspects of the invention, a vector, optionally an expression vector, is provided, comprising a polynucleotide of the invention.

[0058] In aspects of the invention, a cell comprising a polypeptide of the invention, a polynucleotide of the invention, or a vector of the invention, is provided.

[0059] BRIEF DESCRIPTION OF THE FIGURES

[0060] Figure 1 Sequence homology of novel polypeptides compared to IdeS and Xork

[0061] All proteases exhibit a low degree of primary sequence identity to existing IgG proteases (e.g. IdeS and Xork), with 27.6 to 32.0% identity to IdeS and 29.9 to 41.3% homology to Xork. O_mod and R_mod share some similarity to each other (60.2%).

[0062] Figure 2 SDS-PAGE analysis of the expressed and purified polypeptides

[0063] The polypeptides were produced according to the methods described herein. All seven recombinantly engineered proteins could be successfully produced in E. coli, solubilized in the supernatant, and affinity- purified to high purity and yield. (A) The full-length protein St1_mod is expressed as a 38.6 kDa protein. The full-length protein St2_mod is expressed as a 94.65 kDa protein. The full-length protein C_mod is expressed as a 40.52 kDa protein. The full-length protein O_mod is expressed as a 37.66 kDa protein. The full-length protein R_mod is expressed as a 38.78 kDa protein. (B) The full-length protein Xork-St2L-lgMBRAZOR is expressed as a 92.73 kDa protein. The full-length protein St1-St2L- IgMBRAZOR is expressed as a 94.65 kDa protein. With respect to each lane, and where applicable, P indicates the insoluble cell fraction after cell lysis, S indicates the soluble cell fraction after lysis, FT indicates the flow-through, i.e. the cell fraction which did not bind to a column, W indicates any protein obtained from any solution used to wash a purification means, E indicates the eluted protein obtained in the final fraction from a purification means (e.g. a column, such as an IMAC column, for example an Ni2+ column configured to bind His-tagged polypeptides), and contains the polypeptides of the invention.

[0064] Figure 3 SDS-PAGE analysis of General protease activity

[0065] The positive control SpeB is a general protease which gives a high fluorescence signal when exposed to the general protease substrate Casein (conjugated to FiTC) for 1 hour. The negative controls IdeS and Xork are IgG-specific proteases which yield almost no signal. The novel polypeptides of the invention, St1_mod, St2_mod, O_mod, R_mod, and C_mod, yielded a similar signal to the negative controls used in the assay.

[0066] Figure 4 SDS-PAGE analysis of the specificity of the novel proteases towards Ig classes and IgG subclasses

[0067] The novel polypeptides of the invention and the pan-subclass IgG proteases IdeS and Xork were mixed with different Ig antibody subclasses and incubated overnight at 37°C before analysis on SDS-PAGE. The activity of the analysed proteases is reported as having no activity (-), low activity (+), moderate activity (++), or high activity (+++), which relates to the extent of the decrease in intensity of band corresponding to the heavy chain of each respective Ig tested, indicating degradation of the target Ig molecule. (A) Summary of protease activity vs human IgG subclasses (IgG 1 to 4), IgA, and IgM. All proteases had activity, though varying, against human lgG1 to 4, and no activity against IgA or IgM. (B) Activity of ST1_mod and ST2_mod vs human IgG subclasses (IgG 1 to 4). St1_mod and St2_mod exhibit exceptional IgG subclass specificity, hydrolyzing human lgG1 , lgG3, and lgG4 (indicated by the reduction of the intensity of the approx. 55 kDa bands and presence of lower molecular weight bands), but demonstrating weak activity towards lgG2, wherein the approx. 55 kDa band does not decrease in intensity. (C) Activity of O_mod, R_mod, and C_mod vs human IgG subclasses. The protease O_mod exhibits comparatively lower activity / preference vs lgG3 and lgG4, and comparatively higher activity / preference vs lgG1 and lgG2. The protease R_mod exhibits comparatively lower activity / preference vs lgG1 , lgG3, and lgG4, and comparatively higher activity / preference vs lgG2. R_mod has a very high preference for human lgG2. The protease C_mod exhibits comparatively high activity vs all IgG subclasses tested, particularly lgG4. (D) Activity of St1_mod, O_mod, R_mod, and C_mod vs human IgA and IgM. The proteases do not exhibit activity / preference towards IgA, wherein the approx. 60 kDa bands corresponding to the IgA heavy chain and the approx. 80 kDa bands corresponding to IgM heavy chain do not decrease in intensity. (E) Activity of St1_mod and St2_mod vs human IgG and IgM. The proteases exhibit activity towards IgG, but not towards IgM.

[0068] Figure 5 SDS-PAGE analysis of the specificity of the novel proteases towards Ig classes and IgG subclasses from non-human species

[0069] The novel IgG proteases exhibit IgG subclass specificity and cross species reactivity. (A) Summary of protease activity vs mouse, rabbit, and monkey IgG. A single asterisk (*) indicates activity towards mouse lgG2a and no activity towards lgG1 , lgG2b, and lgG3. A double asterisk (**) indicates activity towards mouse lgG2a and no activity towards lgG1 , lgG2b. (B) Activity of ST1_mod and ST2_mod, vs mouse IgG subclasses. The proteases exhibit comparatively lower activity vs mlgG1 (approx. 55 kDa band), and comparatively higher activity vs mlgG2a, mlgG2b and mlgG3 (approx. 55 kDa bands). (C) Activity of O_mod, R_mod, and C_mod vs rabbit and mouse IgG subclasses. The proteases do not exhibit activity / preference vs rabbit or mouse IgG or IgG subclasses.

[0070] Figure 6 SDS-PAGE analysis of the specificity of novel proteases O_mod, R_mod, and C_mod towards modified lgG1 variants

[0071] The C_mod protease exhibits high activity against hinge-modified IgG variant lgG1-LALA (L234A, L235A) and also a degree of activity towards hinge-modified IgG variant lgG1-LFLE (L234F, L235E, P331 S). The O_mod protease also exhibits activity against hinge-modified IgG variant lgG1 -LALA (L234A, L235A). The R_mod protease does not exhibit activity towards any of the hinge-modified IgG variants tested.

[0072] Figure 7: Specificity of novel engineered chimeric proteases St1 -St2L-lgMBRAZOR; Xork-St2L- IgMBRAZOR towards IgM and IgG

[0073] The engineered chimeric proteins, comprising an IgM protease and an IgG protease for increasing the variety of target substrates, St1-St2L-lgMBRAZOR and Xork-St2L-lgMBRAZOR exhibit activity against human IgM and IgG.

[0074] Figure 8: Activity of St1_mod and St2_mod in serum

[0075] SDS-page analysis of St1_mod and St2_mod proteases in serum.

[0076] The thickest band at around 60 kDa indicates albumin, with the heavy chain of IgG situated directly below as highlighted by the arrow at around 55kDa. Upon the addition of either IdeS, Xork, St1_mod, or St2_mod, the IgG heavy chain band disappears or at least gets much weaker, wherein the two hydrolysis products from the hinge-cleaved heavy chain shows up as two bands around 25-30 kDa as shown by the bracket, with one of the hydrolysis product bands being mostly hidden behind the light chain at approximately 25 kDa, which is also visible in the untreated plasma.

[0077] Figure 9: Activity of O_mod, C_mod, and R_mod against IVIG

[0078] The O_mod, C_mod, and R_mod constructs are active against Intravenous Immunoglobulin (IVIG), wherein the intensity of the approximately 55kDa band corresponding to IVIG decreases in the protease treated conditions, yielding an approximately 30 to 33 kDa band. The 55 kDa band corresponding to IVIG is present at a weaker intensity for the IVIG samples treated with the proteases O_mod and C_mod, suggesting that O_mod and C_mod hydrolyse IVIG more efficiently than R_mod.

[0079] Figure 10: SDS-PAGE analysis of the prevalence of anti-IgG protease antibodies

[0080] SDS-PAGE analysis of anti-enzyme antibodies. Bands corresponding to the molecular weight of each protease are visible in the ‘stain’ panel, and bands corresponding to each protease which is recognized by an anti-IVIG antibody are visible in the ‘anti-enzyme blot’ panel. (A) Of all the polypeptides tested, only IdeS had significant antibodies directed to the enzyme, as indicated by the approximately 38 kDa band. (B) Of all the polypeptides tested, only IdeS had significant antibodies directed to the enzyme, as indicated by the approximately 38 kDa band. Figure 11 : Protease target digestion sites

[0081] (A) Summary of hydrolysis site by all the different enzymes. (B to G) Electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS) data for lgG1 (B), lgG2 (C), lgG3 (D), lgG4 (E), lgG1 - LALA (F), and St1_mod activity on lgG1 (G). All enzymes cleave at, or in the vicinity of the IdeS cleavage site. St1_mod and St2_mod hydrolyze both lgG1 and lgG4 at PAPELL / GGPSVF and PAPEFL / GGPSVF. O_mod, C_mod, and R_mod all cleave at the same site as IdeS. For lgG1 , both O_mod and R_mod hydrolyze APELL / G / GPSVFL, e.g. at two sites.

[0082] Figure 12: SDS-PAGE analysis of the expressed and purified polypeptide C_mod_v2

[0083] SDS-PAGE analysis of polypeptide C_mod_v2. A single band with very little to no fragmentation of the protease is visible, indicating very little to no fragmentation of the protease.

[0084] Figure 13: St1_mod target digestion sites of mouse immunoglobulins

[0085] Electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS) data for mouse lgG2b and lgG3 treated with St1_mod. (A) Upon treatment of mouse lgG2b with St1_mod, the heavy chain (HC) peak of mouse lgG2b at a mass / charge (m / z) of approximately 49600 in the control samples gives rise to m / z peaks of approximately 25400 and 24200 which correspond to the Fd and Fc fragments of mouse lgG2b respectively. (B) Upon treatment of mouse lgG3 with St1_mod, the heavy chain (HC) peak of mouse lgG3 at a m / z of approximately 50500 in the control samples gives rise to m / z peaks of approximately 25400 and 24200 which correspond to the Fd and Fc fragments of mouse lgG3 respectively.

[0086] Figure 14: Mass spectrometry analysis of the activity of novel protease C_mod towards modified lgG4 variants

[0087] Mass chromatogram depicting electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QTOF-MS) data for (A) monospecific lgG4-FALA (Galcanezumab) and (B) bispecific lgG4-FALA (Talquestamab). (A) Upon treatment of Galcanezumab with FabRICATOR® (Genovis AB, IdeS derived IgG protease) the heavy chain (HC) of Galcanezumab (having an intensity peak with a retention time of approximately 7.5 minutes), shown in the IgG control panel at the bottom of the figure, is hydrolysed by FabRICATOR® as indicated in the middle panel by the appearance of the Fd’ and scFc fragments, having an intensity peak corresponding to a retention time of approximately 8 minutes and 5.2 minutes respectively. As can be seen in the top panel depicting the results of C_mod treated Galcanezumab, Galcanezumab is more efficiently hydrolysed by C_mod, wherein the intensity of the heavy chain (HC) peak is significantly decreased compared to the IgG control and FabRICATOR® treated IgG, such that Galcanezumab is effectively fully hydrolysed. (B) Upon treatment of Talquestamab with FabRICATOR® (Genovis AB, IdeS derived IgG protease) the first heavy chain (HC1) of Talquestamab (having an intensity peak with a retention time of approximately 7.8 minutes) and the second heavy chain (HC2) of Talquestamab (having an intensity peak with a retention time of approximately 7 minutes), shown in the IgG control panel at the bottom of the figure, is hydrolysed by FabRICATOR® as indicated in the middle panel by the appearance of the Fd’1 and Fd’2 having an intensity peak corresponding to a retention time of approximately 8.3 minutes and 7.3 minutes respectively; and the appearance of the scFd , and scFc2 fragments, having an intensity peak corresponding to a retention time of approximately 5.1 minutes. As can be seen in the top panel depicting the results of C_mod treated Talquestamab, Talquestamab is more efficiently hydrolysed by C_mod, wherein the intensity of HC1 and HC2 peaks are significantly decreased compared to the IgG control and FabRICATOR® treated IgG, such that Talquestamab is effectively fully hydrolysed.

[0088] DETAILED DESCRIPTION OF THE INVENTION

[0089] It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. 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.

[0090] All references cited herein are incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein will be understood as having the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs, such as molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization. Standard techniques are used for nucleic acid synthesis.

[0091] The techniques and procedures are generally performed according to conventional methods in the art (see generally, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. 30 (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated herein by reference).

[0092] Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995; and other similar references. In case of conflict, the terms in this specification will control.

[0093] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps and features referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only.

[0094] As used herein, the term "comprising" means any of the recited elements are necessarily included and other elements may optionally be included as well. "Consisting essentially of’ means any recited elements are necessarily included, other elements may optionally be included, and that elements that would materially affect the basic and novel characteristics of the listed elements are excluded. “Consisting of’ means that all elements other than those listed are excluded. Embodiments defined by each of these terms are within the scope of this invention. As used herein, the term “contacting” means placement in direct physical association, for example both in solid form and / or in liquid form. It should also be understood that the term "and / or" as used herein refers to and encompasses any or all possible combinations of one or more associated listed items.

[0095] Polypeptides

[0096] The term "polypeptide" as used herein relates 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. Thus, a polypeptide according to the invention may comprise entirely natural or synthetic amino acids, or a mixture of natural and synthetic amino acids.

[0097] A polypeptide may be produced by any 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. Production of polypeptides by expression in bacterial host cells is described below and is exemplified in the Examples.

[0098] Expression of polypeptides of the invention is described herein and would further be readily achievable by the person skilled in the art using standard techniques.

[0099] The sequence of a polypeptide of the invention may comprise a variant of the amino acid sequence of any of SEQ ID NOs: 1 to 19 and 40 to 42, in which modifications, such as amino acid additions, deletions or substitutions are made relative to the sequence of any of SEQ ID NOs: 1 to 19 and 40 to 42.

[0100] Unless otherwise specified, the modifications are preferably 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 the 20 main amino acids as defined in Table 1 below. Where amino acids have similar polarity, this can be determined by reference to the hydropathy scale for amino acid side chains (see Table 1).

[0101] A sequence of a polypeptide of the invention may comprise a variant of the amino acid sequence of any of SEQ ID NOs: 1 to 19 and 40 to 42 in which up to about 10, such as up to about 20, up to about 30, up to about 40, up to about 50, up to about 60, up to about 70, up to about 80, up to about 90, up to about 100, up to about 150, up to about 200, up to about 300, up to about 400, up to about 500, up to about 600, up to about 700, up to about 800, up to about 900, up to about 1000 conservative substitutions are made. Taking into account that minor modifications to the primary sequence of the peptides / proteins of the invention can be made without substantially altering the scope of the claimed invention, the invention should be considered to encompass, in addition, any polypeptide sequences that are substantially the same as the specific amino acid sequences disclosed herein. For example, the claimed invention encompasses polypeptide sequences that have at least 80% identity to the SEQ ID NOs: 1 to 19 and 40 to 42 of the polypeptide sequences disclosed herein; at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% identity or approximately, 100% identity to the polypeptide sequences of the SEQ ID NOs explicitly disclosed herein.

[0102] Table 1 : Chemical properties of amino acids

[0103] The term ‘protein activity’, as used herein refers to the specific function performed by a protein, which may include, but is not limited to, enzymatic activity and binding affinity to ligands or substrates. The activity of a protein is typically characterized by measurable parameters, which may include, but are not limited to, reaction rates (kinetics), binding constants, catalytic efficiency, and substrate specificity. Protein activity may be quantified using various assays and techniques, including but not limited to, enzyme assays, binding assays, functional cell-based assays, and structural analysis methods. The activity of a given protein may be influenced by the protein's conformation, post-translational modifications, presence / interaction with other molecules, such as co-factors, or reaction conditions, including but not limited to, pH, temperature, or presence of additional reactants, such as salts.

[0104] Oligonucleotides

[0105] The invention provides oligonucleotides, nucleic acid molecules, and vectors which encode a polypeptide of the invention. The term ‘oligonucleotide’ as used herein, is a polymeric nucleotide macromolecule of any length comprising at least two or more covalently-linked nucleotides in which the 3' and 5' ends on each nucleotide are joined by phosphodiester bonds, such as a nucleic acid. The oligonucleotide may be natural or synthetic. For the purposes of the present invention such oligonucleotides may constitute nucleic acids. Typical nucleic acids include DNA and RNA. The oligonucleotide may be made up of deoxyribonucleotide bases or ribonucleotide bases. An oligonucleotide may be linear or circular and may be single-stranded or double-stranded or comprise one or more double stranded regions and one or more single-stranded regions. The terms ‘oligonucleotide’, ‘polynucleotide’, and ‘nucleic acid molecule’ are used interchangeably.

[0106] The terms and ‘polynucleotide’ are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Nonlimiting examples of polynucleotides include a gene, a gene fragment, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.

[0107] Non-limiting examples of oligonucleotides include a gene, a gene fragment, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.

[0108] Oligonucleotides are comprised of nucleotides. The term ‘nucleotide’ as used herein, typically contains a nucleobase, a sugar and at least one phosphate group. The nucleobase is typically heterocyclic. Nucleobases include but are not limited to purines and pyrimidines and more specifically include but are not limited to, adenine, guanine, thymine, uracil and cytosine. The sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The nucleotide is typically a ribonucleotide or deoxyribonucleotide. The nucleotide typically contains a monophosphate, diphosphate or triphosphate. Phosphates may be attached on the 5' or 3' side of a nucleotide. For the purposes of this invention, the terms ‘nucleotide’ and ‘base’ are used interchangeably.

[0109] The nucleotides can be naturally occurring or artificial. One or more nucleotides in any chromatin fragment or oligonucleotide of the invention may be modified, comprising one or more chemical modifications of a nucleotide. Modified nucleotides may occur singularly or in a plurality with other nucleotide modifications that are the same modification or a different modification, which may be contiguous or non-contiguous with other nucleotide modifications that are the same modification or a different modification.

[0110] One or more nucleotides in any oligonucleotide of the invention may be non-natural or synthetic nucleotides. Non-natural nucleotides may include analogues of natural nucleotides, as well as nucleotides that are modified in the base, sugar and / or phosphate moieties (e.g. phosphorothioate backbones). The oligonucleotides of the invention may also comprise any synthetic nucleic acid known in the art, including but not limited to peptide nucleic acids (PNA), glycerol nucleic acids (GNA), threose nucleic acids (TNA), locked nucleic acids (LNA) or other synthetic polymers with nucleotide side chains. One or more nucleotides in the oligonucleotides may be modified with a label or a tag. The oligonucleotides may comprise one or more spacers.

[0111] Polynucleotides of the invention may include at the 5’ end a codon forthe N terminal methionine (ATG) and, prior to the stop codon (TAA) at the 3’ end, codons for a linker, e.g. a Gly-Ser-Gly linker, and a tag, e.g. a 6x His tag, which may optionally be excluded. The optional inclusion of an additional methionine and a tag are discussed in more detail below.

[0112] A polynucleotide of the invention encodes a polypeptide of the invention 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.

[0113] 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).

[0114] Sequence identity between two or more polynucleotide sequences may be calculated using any suitable algorithm. For example, the BLAST algorithm can be used to calculate identity or align sequences, typically utilising default settings. Software for performing BLAST analyses is publicly available through the National Centre for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0115] In embodiments, an oligonucleotide of the invention encodes a polypeptide of the invention.

[0116] An oligonucleotide sequence which ‘encodes’ a selected polypeptide is an oligonucleotide which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo or in vitro 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.

[0117] For the purposes of the invention, oligonucleotides of the invention can include, but are not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic sequences from viral or prokaryotic DNA or RNA, and synthetic DNA sequences.

[0118] The nucleic acid molecules of the present invention 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 the polypeptide of the invention in vitro (e.g. in prokaryotic or eukaryotic expression systems) and / or in vivo (e.g. a host organism, subject, or patient).

[0119] These expression cassettes, in turn, are typically provided within vectors (e.g., plasmids or recombinant viral vectors). Such an expression cassette may be administered directly to a host subject. Alternatively, a vector comprising a polynucleotide of the invention may be administered to a host subject. Typically, the polynucleotide is prepared and / or administered using a genetic vector. A suitable vector may be any vector which is capable of carrying a sufficient amount of genetic information, and allowing expression of a polypeptide of the invention.

[0120] The present invention thus includes 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 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 peptide of the invention. 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.

[0121] Exemplary polynucleotide molecules encoding polypeptides disclosed herein are provided as SEQ ID NOs: 20 to 27. However, due to the degeneracy of the genetic code it will be understood that any number of sequences may encode identical polypeptides.

[0122] Cells

[0123] The invention also provides for cells expressing or capable of expressing polypeptides of the invention. 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 of the invention. The invention also includes cells that have been modified to express a polypeptide of the invention.

[0124] In embodiments, there is provided a cell comprising a polynucleotide according to the invention.

[0125] In embodiments, there is provided a cell comprising a vector according to the invention.

[0126] In another embodiment, there is provided a cell comprising a polypeptide according to the invention. Such a cell may comprise the polypeptide intracellularly, such as within inclusion bodies, or in solution within the cytosol; the polypeptide may be transiently present within the cell prior to secretion to the extracellular environment.

[0127] 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 of the invention.

[0128] In embodiments the cell is a BL21 (DE3) STAR cell.

[0129] Further, cells according to the invention may be eukaryotic cells, such as insect cells or mammalian cells.

[0130] A cell according to the invention may have been modified, for example by standard molecular biology techniques, to comprise a polynucleotide, vector, or polypeptide according to the invention, and / or modified to express a polypeptide according to the invention. Polypeptide engineering and modifications

[0131] A polypeptide may be engineered or modified to assist with production, isolation or purification. For example, where a polypeptide of the invention 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, the polypeptide of the invention may be engineered or modified by addition of protein purification tag at the N or C terminus, preferably at the C terminus.

[0132] The protein purification tag is preferably a moiety which is not naturally expressed in a streptococcus bacterium. The protein purification tag is preferably a moiety which is not present in a wildtype polypeptide chain as expressed in streptococcus bacteria.

[0133] 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.

[0134] Where the polypeptide is engineered or modified by addition of one member of a binding pair, the polypeptide may be histidine-tagged or biotin-tagged. 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. The histidine or biotin tag is typically present at either end of the polypeptide, preferably at the C-terminus.

[0135] 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 Microbial. 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.

[0136] Typically, the amino acid coding sequence of the protein purification tag is included at the gene level and the polypeptide is expressed recombinantly, for example, in E. coli.

[0137] The protein purification tag 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.

[0138] 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.

[0139] 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 of the invention may comprise at least one such unnatural or synthetic amino acid, or at least one non-L configuration amino acid.

[0140] 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.

[0141] 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.

[0142] 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. For example, two polypeptides may be each be purified to at least 90% homogeneity prior to combining in a 1 : 1 ratio.

[0143] Exemplary polypeptides that may be included in a mixture with a polypeptide of the invention may include any one or more of: an exoglycosidase; an endoglycosidase ( e.g. EndoS, EndoS2, EndoE, including any polypeptide with endoglycosidase activity as disclosed in WO2013 / 037824); a protease (preferably an immunoglobulin protease with different specificity to the polypeptide of the invention, e.g. a protease as disclosed in WO2017 / 134274 - such as a polypeptide comprising SEQ ID NO: 3 of that document or a variant thereof as disclosed in that document); or a G4S-linker specific protease such as the GlySERIAS enzyme, which digests flexible glycine-rich fusion protein linkers such as Gly4Ser and GlyxSery (GS), and polyglycine (G) linkers, or an amidase (e.g. PngaseF).

[0144] 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), Trisbuffered 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.

[0145] A polypeptide may be immobilised using methods known in the art, for example as described in Datta Set 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. igG

[0146] IgG is a class of immunoglobulin (Ig) that is typically characterised by its small size and monomeric structure.

[0147] IgG polypeptides are typically composed of four polypeptide chains: two identical heavy (y) chains, comprising approximately 450 amino acids, and two identical light chains, comprising about 214 amino acids. A light chain and heavy chain are connected to one another by disulfide bonds. Similarly, the two heavy chains are connected in the hinge region by disulfide bonds to form a Y-shaped structure.

[0148] IgG polypeptides consist of four distinct subclasses, designated as lgG1 , lgG2, lgG3, and lgG4, each having unique features and functions. lgG1 is characterized by a heavy chain gamma 1 (y1) with a typical structure comprising three constant domains (CH1 , CH2, CH3) and one variable domain (VH). The hinge region of lgG1 is relatively short and flexible, comprising 15 amino acids and 2 disulphide bonds. lgG1 is the most abundant subclass, constituting approximately 66% of the total IgG in serum. It is highly effective in opsonization, complement activation, and antibody-dependent cell-mediated cytotoxicity (ADCC). lgG1 is particularly efficient in binding to protein antigens and is commonly used in therapeutic applications such as monoclonal antibody therapies for cancer and autoimmune diseases, due to their robust effector functions. lgG2 comprises a heavy chain gamma 2 (y2) with a similar domain organization to lgG1 but a shorter and more rigid hinge region, comprising 12 amino acids and 4 disulfide bonds. lgG2 represents about 23% of the total IgG in serum. lgG2 is less effective in complement activation and ADCC compared to lgG1 , however lgG2 is specialized for responses against carbohydrate antigens and is critical in the defence against encapsulated bacteria. Consequently, lgG2 is often utilized in vaccines and diagnostic assays targeting polysaccharide antigens, leveraging its specificity for carbohydrate epitopes. lgG3 comprises a heavy chain gamma 3 (y3) with an extended hinge region of 62 amino acids and 11 disulphide bonds, the longest among the IgG subfamilies. This hinge region confers increased flexibility but also greater susceptibility to proteolysis. lgG3 constitutes about 7% of total IgG in serum. lgG3 is the most potent subclass for complement activation due to its long hinge region and high affinity for C1q. lgG3 is effective in mediating opsonization and ADCC. Consequently, lgG3 is particularly useful in therapeutic and diagnostic applications where strong complement activation is desired. Its enhanced effector functions make it suitable for targeting pathogens and cancer cells. lgG4 comprises a heavy chain gamma 4 (y4) with a hinge region of 12 amino acids and 2 disulphide bonds, similar in length to lgG2 but with distinct structural properties that influence its function. lgG4 molecules can undergo half-molecule exchange, resulting in functionally monovalent antibodies. lgG4 is the least abundant subclass, making up about 4% of total IgG in serum. It is unique in its inability to activate the complement system and its reduced capacity for ADCC. lgG4 is associated with noninflammatory responses and plays a role in immune tolerance. Consequently, lgG4 antibodies are employed in therapeutic contexts where minimal immune activation is desired, such as in the treatment of chronic inflammatory diseases and in allergen-specific immunotherapy. igM

[0149] IgM is a class of immunoglobulin (Ig) that is typically characterised by its large size and pentameric structure. However, IgM may also exist in other structural forms such as a secreted hexamer or cellsurface displayed monomer that forms part of the B-cell receptor.

[0150] The pentameric structure of IgM is such that it is made up essentially of five structures analogous to IgG. IgM comprises 10 potential antigen-binding sites (or paratopes), which facilitate high avidity target binding. This high avidity binding can allow target binding even when monovalent binding affinities are low.

[0151] IgM is produced early during infection following antigen exposure and plays a key role in stimulating other effector functions of the immune system, such as activating complement and Fc receptor mediated activities.

[0152] IgM has been demonstrated to have favorable therapeutic characteristics, even when compared to IgG, and has been described as a promising candidate for monoclonal antibody therapies for several conditions (Samsudin F., et al., Chem Sci 2020, 11 (10):2843-2854; Zhang J., et al., mAbs 2022, 14(1):2031483).

[0153] In comparison with IgG, IgM represents the early-stage response to a disease or infection, produced immediately after the exposure to a particular antigen, whereas IgG represents the late-stage response to a disease or infection.

[0154] Throughout this disclosure, the IgM may comprise a heavy chain sequence according to SEQ ID NO: 44, or a sequence having at least 90%, at least 95%, at least 98% or at least 99% identity thereto.

[0155] Polypeptides having immunoglobulin protease activity, including IgG specific and IgG and IgM specific proteases

[0156] The term ‘protease activity’, as used herein, which for the purposes of this application may be used interchangeably with simply ‘activity’, refers to the biochemical function of protease enzymes, which catalyse the hydrolysis of peptide bonds within proteins and peptides, leading to the breakdown of these molecules into smaller polypeptides or amino acids. This activity may be specific, wherein the protease recognises and hydrolyses / cleaves specific peptide bonds at particular amino acid sequences or structures. The activity of a protease is typically characterized by measurable parameters, which may include, but are not limited to, reaction rates (kinetics), binding constants, catalytic efficiency, and substrate specificity. Thus, the skilled person would appreciate that protease activity may encompass one or more of these parameters. The activity of a given protease may be influenced by the protease’s conformation, post-translational modifications, presence / interaction with other molecules, such as co-factors, or reaction conditions, including but not limited to pH, temperature, or presence of additional reactants, such as salts.

[0157] Protease activity can be measured, and optionally quantified, using various assays and techniques that determine the rate and extent of peptide bond cleavage. Any suitable method known in the art may be used to analyse the activity of the protease, including spectrophotometric assays, Fluorescence Resonance Energy Transfer (FRET) assays, Kinetic Assays, Gel Electrophoresis, and HPLC or Mass Spectrometry. Gel Electrophoresis analysis of protease activity may comprise analysing the degradation pattern of protein substrates on SDS-PAGE, where the decrease in size or disappearance of specific protein bands indicates protease activity. HPLC or Mass Spectrometry analysis of protease activity may comprise quantifying the cleavage products of specific peptide or protein substrates to determine the extent and specificity of proteolysis, e.g. using liquid chromatography to separate and quantify peptide fragments followed by mass spectrometric identification, which may enable identification of cleavage sites present in the substrate.

[0158] The present invention provides polypeptides having immunoglobulin protease activity. That is, the polypeptides are able to cleave an immunoglobulin molecule.

[0159] In embodiments, the immunoglobulin molecule may be a monospecific immunoglobulin, a bispecific immunoglobulin, or a multispecific immunoglobulin, e.g. a trispecific or tetraspecific immunoglobulin.

[0160] In embodiments, the immunoglobulin molecule is typically an IgG molecule, and preferably the polypeptide does not cleave, or exhibits lower cleavage activity against other classes of immunoglobulin.

[0161] In embodiments, the polypeptide has IgG specific protease activity.

[0162] In embodiments, the IgG specific protease activity is endoprotease activity.

[0163] It will be understood that, as used herein, a polypeptide having ‘IgG specific protease activity’ may refer to any protein, polypeptide, or fragment thereof, that is able to catalyze the cleavage of IgG.

[0164] Further, it will be understood that IgG specific protease activity may refer to protease activity that is target (IgG) specific, and may not be considered to have a broad spectrum of target proteins, for example as trypsin does, and that the polypeptide may have residual or lower level activity for other targets, such as IgM, IgA, IgD, and IgE unless otherwise indicated. Furthermore, IgG specific activity may encompass activity against different IgG molecules, for example, IgG derived from different species or modified IgG molecules. It will be understood that the term ‘human IgG specificity’ does not imply that the polypeptide is restricted to human IgG, rather, the protease is specific for IgG and human IgG is a recognised substrate, but not necessarily the only substrate. For example, IgG from other species, such as non-human primates, may also be a substrate.

[0165] Determination of whether cleavage of IgG has occurred may be readily performed by the person skilled in the art, for example using common techniques in the art of protein biochemistry, such techniques including SDS-PAGE analysis, western blotting, chromatography (e.g., size-exclusion chromatography), and mass spectrometry. Such techniques are exemplified herein (see Examples) but are in no way intended to be limiting.

[0166] Modified IgG molecules may include, for example, non-naturally occurring (e.g., synthetic) IgG molecules; fusion proteins comprising IgG molecules or parts / sequences thereof; and fragments of IgG molecules.

[0167] In embodiments, the polypeptide may have low or negligible levels of detectable IgM protease activity.

[0168] In embodiments, the polypeptide may have low levels of detectable IgM protease activity, wherein the level of IgM protease activity is less than the level of IgG protease activity; for example, less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the level of IgG protease activity.

[0169] In embodiments, the polypeptide may have low or negligible levels of detectable IgA protease activity.

[0170] In embodiments, the polypeptide may have low levels of detectable IgA protease activity, wherein the level of IgA protease activity is less than the level of IgG protease activity; for example, less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the level of IgG protease activity.

[0171] In embodiments, the polypeptide may have low or negligible levels of detectable IgD protease activity.

[0172] In embodiments, the polypeptide may have low levels of detectable IgD protease activity, wherein the level of IgD protease activity is less than the level of IgG protease activity; for example, less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the level of IgG protease activity.

[0173] In embodiments, the polypeptide may have low or negligible levels of detectable IgE protease activity. In embodiments, the polypeptide may have low levels of detectable IgE protease activity, wherein the level of IgE protease activity is less than the level of IgG protease activity; for example, less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the level of IgG protease activity.

[0174] In a preferred embodiment of the invention, the IgG specific protease activity is human IgG specific protease activity.

[0175] In another particularly suitable embodiment, the polypeptide has no detectable activity against IgM.

[0176] In embodiments, the polypeptide has no detectable activity against polypeptides other than IgG.

[0177] The invention also concerns polypeptides having IgG and IgM specific protease activity.

[0178] In embodiments, the IgG and IgM specific protease activity is endoprotease activity.

[0179] In embodiments, the immunoglobulin molecule is typically an IgG or IgM molecule, and preferably the polypeptide does not cleave, or exhibits lower cleavage activity against other classes of immunoglobulin.

[0180] In embodiments, the immunoglobulin molecule is typically an IgG molecule and suitably the polypeptide, in addition, cleaves only an IgM molecule.

[0181] In embodiments, the immunoglobulin molecule is typically an IgM molecule, and suitably the polypeptide, in addition, cleaves only an IgG molecule, or a specific IgG subclass molecule.

[0182] In embodiments, the polypeptides of the invention may comprise a first active site specific for IgG and a second active site specific for IgM.

[0183] It will be understood that, as used herein, a polypeptide having ‘IgG and IgM specific protease activity’ may refer to any protein, polypeptide, or fragment thereof, that is able to catalyze the cleavage of IgG and IgM.

[0184] Further, it will be understood that IgG and IgM specific protease activity may refer to protease activity that is target (IgG and IgM) specific, and may not be considered to have a broad spectrum of target proteins, for example as trypsin does; and that the polypeptide may have residual or lower level activity for other targets, such as IgA, IgD, and IgE unless otherwise indicated. Furthermore, IgG and IgM specific activity may encompass activity against different IgG and IgM molecules, for example, IgG and IgM derived from different species or modified IgG and IgM molecules.

[0185] It will be understood that the term ‘human IgG and IgM specificity’ does not imply that the polypeptide is restricted to human IgG and IgM, rather, the protease is specific for IgG and IgM and that human IgG and IgM are recognised substrates, but not necessarily the only substrates. For example, IgG and IgM from other species, such as non-human primates, may also be a substrate.

[0186] Modified IgM molecules may include, for example, non-naturally occurring (e.g., synthetic) IgM molecules; fusion proteins comprising IgM molecules or parts / sequences thereof; and fragments of IgM molecules. For example, polypeptides comprising the sequence of SEQ ID NO: 44, or a sequence having at least 90%, at least 95%, at least 98% or at least 99% identity thereto; or a fragment thereof.

[0187] The skilled person will appreciate that the polypeptide may have residual or lower level activity for other targets, including Ig targets, such as IgA, IgD, and IgE.

[0188] In embodiments, the polypeptide may have low or negligible levels of detectable IgA protease activity.

[0189] In embodiments, the polypeptide may have low levels of detectable IgA protease activity, wherein the level of IgA protease activity is less than the level of IgG protease activity; for example, less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the level of IgG protease activity.

[0190] In embodiments, the polypeptide may have low or negligible levels of detectable IgD protease activity.

[0191] In embodiments, the polypeptide may have low levels of detectable IgD protease activity, wherein the level of IgD protease activity is less than the level of IgG protease activity; for example, less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the level of IgG protease activity.

[0192] In embodiments, the polypeptide may have low or negligible levels of detectable IgE protease activity.

[0193] In embodiments, the polypeptide may have low levels of detectable IgE protease activity, wherein the level of IgE protease activity is less than the level of IgG protease activity; for example, less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the level of IgG protease activity.

[0194] In a preferred embodiment of the invention, the IgG and IgM specific protease activity is human IgG and IgM specific protease activity.

[0195] In another particularly suitable embodiment, the polypeptide has no detectable activity against IgA, IgD, or IgE.

[0196] In embodiments, the polypeptide has no detectable activity against polypeptides other than IgG and IgM. In embodiments, the immunoglobulin molecule is typically an IgG molecule of one or more specific IgG subclasses, and preferably the polypeptide does not cleave, or exhibits lower cleavage activity against one or more specific subclass or subclasses of IgG.

[0197] The polypeptide may exhibit lower activity against lgG2 as compared to the other IgG subclasses.

[0198] The polypeptide may exhibit greater activity against IgG 1 than lgG2.

[0199] The polypeptide may exhibit greater activity against lgG3 than lgG2.

[0200] The polypeptide may exhibit greater activity against lgG4 than lgG2.

[0201] The polypeptide may exhibit lower activity against human lgG2 as compared to the other human IgG subclasses.

[0202] The polypeptide may exhibit greater activity against human lgG1 than human lgG2.

[0203] The polypeptide may exhibit greater activity against human lgG3 than human lgG2.

[0204] The polypeptide may exhibit greater activity against human lgG4 than human lgG2.

[0205] In embodiments, the polypeptide may have low levels of detectable lgG2 protease activity, wherein the level of lgG2 protease activity is less than about 99% of the level of lgG1 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG1 protease activity.

[0206] In embodiments, the polypeptide may have low levels of detectable lgG2 protease activity, wherein the level of lgG2 protease activity is less than about 99% of the level of lgG3 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG3 protease activity.

[0207] In embodiments, the polypeptide may have low levels of detectable lgG2 protease activity, wherein the level of lgG4 protease activity is less than about 99% of the level of lgG4 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG4 protease activity.

[0208] In embodiments, the polypeptide may have low levels of detectable lgG1 protease activity, wherein the level of IgG 1 protease activity is less than about 99% of the level of lgG2 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG2 protease activity.

[0209] In embodiments, the polypeptide may have low levels of detectable lgG1 protease activity, wherein the level of IgG 1 protease activity is less than about 99% of the level of lgG3 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG3 protease activity.

[0210] In embodiments, the polypeptide may have low levels of detectable lgG1 protease activity, wherein the level of IgG 1 protease activity is less than about 99% of the level of lgG4 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG4 protease activity.

[0211] In embodiments, the polypeptide may have low levels of detectable lgG3 protease activity, wherein the level of lgG3 protease activity is less than about 99% of the level of lgG1 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG1 protease activity.

[0212] In embodiments, the polypeptide may have low levels of detectable lgG3 activity, wherein the level of lgG3 activity is less than about 99% of the level of lgG2 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG2 protease activity.

[0213] In embodiments, the polypeptide may have low levels of detectable lgG3 protease activity, wherein the level of lgG3 protease activity is less than about 99% of the level of lgG4 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG4 protease activity.

[0214] In embodiments, the polypeptide may have low levels of detectable lgG4 protease activity, wherein the level of lgG4 protease activity is less than about 99% of the level of IgG 1 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG1 protease activity. In embodiments, the polypeptide may have low levels of detectable lgG4 protease activity, wherein the level of lgG4 protease activity is less than about 99% of the level of lgG2 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG2 protease activity.

[0215] In embodiments, the polypeptide may have low levels of detectable lgG4 protease activity, wherein the level of lgG4 protease activity is less than about 99% of the level of lgG3 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG3 protease activity.

[0216] In embodiments, the polypeptide exhibits lower protease activity against human lgG1 and / or lgG3 as compared to the human lgG2 and / or lgG4 subclasses.

[0217] In embodiments, the polypeptide may exhibit greater activity against human lgG2 than human IgG 1 .

[0218] In embodiments, the polypeptide may have low levels of detectable lgG1 protease activity, wherein the level of IgG 1 protease activity is less than about 99% of the level of lgG2 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG2 protease activity.

[0219] In embodiments, the polypeptide may exhibit greater activity against human lgG4 than human IgG 1 .

[0220] In embodiments, the polypeptide may have low levels of detectable lgG1 protease activity, wherein the level of IgG 1 protease activity is less than about 99% of the level of lgG4 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG4 protease activity.

[0221] In embodiments, the polypeptide may exhibit greater activity against human lgG2 than human lgG3.

[0222] In embodiments, the polypeptide may have low levels of detectable lgG3 protease activity, wherein the level of lgG3 protease activity is less than about 99% of the level of lgG2 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 1 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG2 protease activity.

[0223] In embodiments, the polypeptide may exhibit greater activity against human lgG4 than human lgG3.

[0224] In embodiments, the polypeptide may have low levels of detectable lgG3 protease activity, wherein the level of lgG3 protease activity is less than about 99% of the level of lgG4 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG4 protease activity.

[0225] In embodiments, the polypeptide has protease activity against lgG1 variants, optionally modified lgG1 variants, optionally hinge-modified lgG1 variants.

[0226] In embodiments, the polypeptide has protease activity against human IgG 1 variants, optionally modified human lgG1 variants, optionally hinge-modified human lgG1 variants.

[0227] In embodiments, the lgG1 variant, optionally human lgG1 variant, comprises the amino acid substitution L234A and / or L235A.

[0228] In embodiments, the lgG1 variant, optionally human lgG1 variant, comprises the amino acid substitution L234F, and / or L235E, and / or P331 S.

[0229] In embodiments, the polypeptide may exhibit greater activity against an lgG1 variant, optionally a human lgG1 variant, than human lgG3.

[0230] In embodiments, the polypeptide may have low levels of detectable lgG3 protease activity, wherein the level of lgG3 protease activity is less than about 99% of the level of lgG1 variant activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG1 variant activity.

[0231] In embodiments, the polypeptide may exhibit greater activity against an lgG1 variant, optionally a human lgG1 variant, than human lgG4.

[0232] In embodiments, the polypeptide may have low levels of detectable lgG4 protease activity, wherein the level of lgG4 protease activity is less than about 99% of the level of lgG1 variant activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG1 variant activity.

[0233] In embodiments, the polypeptide exhibits lower protease activity against human lgG1 and / or lgG3 and / or lgG4 Ig subclasses as compared to the human lgG2 Ig subclass. In embodiments, the polypeptide may exhibit greater activity against human lgG2 than human IgG 1 .

[0234] In embodiments, the polypeptide may have low levels of detectable lgG1 protease activity, wherein the level of IgG 1 protease activity is less than about 99% of the level of lgG2 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG2 protease activity.

[0235] In embodiments, the polypeptide may exhibit greater activity against human lgG2 than human lgG3.

[0236] In embodiments, the polypeptide may have low levels of detectable lgG3 protease activity, wherein the level of lgG3 protease activity is less than about 99% of the level of lgG2 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG2 protease activity.

[0237] In embodiments, the polypeptide may exhibit greater activity against human lgG2 than human lgG4.

[0238] In embodiments, the polypeptide may have low levels of detectable lgG4 protease activity, wherein the level of lgG4 protease activity is less than about 99% of the level of lgG2 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG2 protease activity.

[0239] In embodiments, the polypeptide exhibits lower protease activity against human lgG3 and / or lgG4 as compared to human IgG 1 and / or lgG2.

[0240] In embodiments, the polypeptide may exhibit greater activity against human lgG1 than human lgG3.

[0241] In embodiments, the polypeptide may have low levels of detectable lgG3 protease activity, wherein the level of lgG3 protease activity is less than about 99% of the level of lgG1 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG1 protease activity.

[0242] In embodiments, the polypeptide may exhibit greater activity against human lgG2 than human lgG3. In embodiments, the polypeptide may have low levels of detectable lgG3 protease activity, wherein the level of lgG3 protease activity is less than about 99% of the level of lgG2 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG2 protease activity.

[0243] In embodiments, the polypeptide may exhibit greater activity against human lgG1 than human lgG4.

[0244] In embodiments, the polypeptide may have low levels of detectable lgG4 protease activity, wherein the level of lgG4 protease activity is less than about 99% of the level of lgG1 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG1 protease activity.

[0245] In embodiments, the polypeptide may exhibit greater activity against human lgG2 than human lgG4.

[0246] In embodiments, the polypeptide may have low levels of detectable lgG4 protease activity, wherein the level of lgG4 protease activity is less than about 99% of the level of lgG2 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG2 protease activity.

[0247] In embodiments, the polypeptide exhibits lower protease activity against human lgG3 and / or lgG4 as compared to human IgG 1 variants.

[0248] In embodiments, the polypeptide may exhibit greater activity against human lgG1 variants than human igG3.

[0249] In embodiments, the polypeptide may have low levels of detectable lgG3 protease activity, wherein the level of lgG3 protease activity is less than about 99% of the level of lgG1 variant activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG1 variant activity.

[0250] In embodiments, the polypeptide may exhibit greater activity against human lgG1 variants than human !gG4.

[0251] In embodiments, the polypeptide may have low levels of detectable lgG4 protease activity, wherein the level of lgG4 protease activity is less than about 99% of the level of lgG1 variant activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG1 variant activity.

[0252] In embodiments, the polypeptide exhibits lower protease activity against human lgG2 as compared to any one of the other human IgG subclasses.

[0253] In embodiments, the polypeptide may exhibit greater activity against human lgG1 than human lgG2.

[0254] In embodiments, the polypeptide may have low levels of detectable lgG2 protease activity, wherein the level of lgG2 protease activity is less than about 99% of the level of lgG1 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG1 protease activity.

[0255] In embodiments, the polypeptide may exhibit greater activity against human lgG3 than human lgG2.

[0256] In embodiments, the polypeptide may have low levels of detectable lgG2 protease activity, wherein the level of lgG2 protease activity is less than about 99% of the level of lgG3 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG3 protease activity.

[0257] In embodiments, the polypeptide may exhibit greater activity against human lgG4 than human lgG2.

[0258] In embodiments, the polypeptide may have low levels of detectable lgG2 protease activity, wherein the level of lgG2 protease activity is less than about 99% of the level of lgG4 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG4 protease activity.

[0259] In embodiments, the polypeptide exhibits lower protease activity against human lgG2 as compared to any one of the other human IgG subclasses.

[0260] In embodiments, the polypeptide may exhibit greater activity against human lgG1 than human lgG2.

[0261] In embodiments, the polypeptide may have low levels of detectable lgG2 protease activity, wherein the level of lgG2 protease activity is less than about 99% of the level of lgG1 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG1 protease activity.

[0262] In embodiments, the polypeptide may exhibit greater activity against human lgG3 than human lgG2.

[0263] In embodiments, the polypeptide may have low levels of detectable lgG2 protease activity, wherein the level of lgG2 protease activity is less than about 99% of the level of lgG3 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG3 protease activity.

[0264] In embodiments, the polypeptide may exhibit greater activity against human lgG4 than human lgG2.

[0265] In embodiments, the polypeptide may have low levels of detectable lgG2 protease activity, wherein the level of lgG2 protease activity is less than about 99% of the level of lgG4 protease activity, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the level of lgG4 protease activity.

[0266] In embodiments, the immunoglobulin molecule is typically an IgG molecule of a specific IgG subclass, and suitably the polypeptide, in addition, cleaves only an IgM molecule.

[0267] In embodiments, the immunoglobulin molecule is typically an IgG molecule or one or more specific IgG subclass molecule, and suitably the polypeptide, in addition, cleaves only an IgM molecule.

[0268] The polypeptide may exhibit activity against non-human IgG molecules, including mouse lgG2a and lgG3. The polypeptide may exhibit activity against IgG from guinea pig or horse.

[0269] The polypeptides of the invention allow for the specific and effective targeting of IgG and IgG subfamilies, including human IgG and human IgG subfamilies. The polypeptides of the invention have utility in numerous applications as biological tools for the study and manipulation of IgG and IgG subfamilies.

[0270] The polypeptide may cleave any immunoglobulin molecule comprising a hinge / CH2 sequence, such as any one or combination of SEQ ID Nos: 28 to 37 as shown in Table 2.

[0271] Table 2: Exemplary IgG hinge / CH2 cleavage sequences

[0272]

[0273] The polypeptide preferably cleaves the sequence at the position indicated by the slash character as shown for each of these sequences in Table 2. The cleavage site may alternatively be described as being between amino acid positions 6 and 7 of any of SEQ ID NOs: 28, or 30 to 34, between amino acid positions 5 and 6 and / or amino acid positions 6 and 7 of SEQ ID NO: 29, or between amino acid positions 10 and 11 of SEQ ID NO 35 or 36, or between amino acid positions 5 and 6 of SEQ ID NO: 37.

[0274] In embodiments, the polypeptide may have protease activity against any immunoglobulin molecule comprising a CH2 / hinge sequence, for example, wherein the CH2 / hinge sequence comprises any one of SEQ ID NOs: 28, 31 , 33, 35 or 36.

[0275] In embodiments, the polypeptide cleaves the CH2 / hinge sequence between amino acid positions 5 and 6 of SEQ ID NO: 28, 31 , 33, 35 or 36.

[0276] In embodiments, the polypeptide may have protease activity against any immunoglobulin molecule comprising a CH2 / hinge sequence, for example, wherein the CH2 / hinge sequence comprises any one of SEQ ID NOs: 30, 31 , 32, or 34.

[0277] In embodiments, the polypeptide cleaves the CH2 / hinge sequence between amino acid positions 5 and 6 of SEQ ID NO: 30, 31 , 32, or 34.

[0278] In embodiments, the polypeptide may have protease activity against any immunoglobulin molecule comprising a CH2 / hinge sequence, for example, wherein the CH2 / hinge sequence comprises any one of SEQ ID NOs: 29 or 31 .

[0279] In embodiments, the polypeptide cleaves the CH2 / hinge sequence between amino acid positions 5 and 6 and / or 6 and 7 of SEQ ID NO: 29, or between amino acid positions 6 and 7 of SEQ ID NO: 31 .

[0280] In embodiments, the polypeptide may have protease activity against any immunoglobulin molecule comprising a CH2 / hinge sequence, for example, wherein the CH2 / hinge sequence comprises any one of SEQ ID NOs: 29, 31 , 32, or 34. In embodiments, the polypeptide cleaves the CH2 / hinge sequence between amino acid positions 5 and 6 and / or 6 and 7 of SEQ ID NO: 29, or between amino acid positions 6 and 7 of any one of SEQ ID NOs: 31 , 32, or 34.

[0281] In embodiments, the polypeptide may have protease activity against any immunoglobulin molecule comprising a CH2 / hinge sequence, for example, wherein the CH2 / hinge sequence comprises any one of SEQ ID NOs: 28, 31 , 33, 35, 36, or 37.

[0282] In embodiments, the polypeptide cleaves the CH2 / hinge sequence between amino acid positions 6 and 7 of any one of SEQ ID NOs: 28, 31 , 33, 35, 36 or between amino acid positions 5 and 6 of SEQ ID NO: 37.

[0283] In embodiments, the polypeptide may have protease activity against any immunoglobulin molecule comprising a CH2 / hinge sequence, for example, wherein the CH2 / hinge sequence comprises any one of SEQ ID NOs: 28, 30, 31 , 33, 35, 36, or 37.

[0284] In embodiments, the polypeptide cleaves the CH2 / hinge sequence between amino acid positions 6 and 7 of any one of SEQ ID NOs: 28, 30, 31 , 33, 35, 36, or between amino acid positions 5 and 6 of SEQ ID NO: 37.

[0285] The polypeptides of the invention were identified in several rare, and in some cases recently discovered, streptococcal species.

[0286] The full-length polypeptides identified and provided according to the present disclosure include:

[0287] A first polypeptide derived from Streptococcus thoraltensis, consisting of 1752 amino acids and having a molecular weight of approximately 187.06 kDa, referred to herein as polypeptide St1_FL, the full-length sequence of which is shown in SEQ ID NO: 1 ;

[0288] A second polypeptide derived from Streptococcus thoraltensis, consisting of 1829 amino acids and having a molecular weight of approximately 198.89 kDa, referred to herein as polypeptide St2_FL, the full-length sequence of which is shown in SEQ ID NO: 4;

[0289] A third polypeptide derived from Streptococcus castoreus, consisting of 377 amino acids and having a molecular weight of approximately 42.91 kDa, referred to herein as polypeptide C_FL, the full-length sequence of which is shown in SEQ ID NO: 7;

[0290] A fourth polypeptide derived from Streptococcus ruminantium, consisting of 604 amino acids and having a molecular weight of approximately 68.44 kDa, referred to herein as polypeptide R_FL, the full-length sequence of which is shown in SEQ ID NO: 10; A fifth polypeptide derived from Streptococcus ovuberis, consisting of 1556 amino acids and having a molecular weight of approximately 177.15 kDa, referred to herein as polypeptide O_FL, the full-length sequence of which is shown in SEQ ID NO: 13.

[0291] The sequences of these polypeptides are considerably different to that of other known IgG specific proteases, exhibiting a low degree of primary sequence identity (see Figure 1).

[0292] The present inventors are the first to identify the surprising finding that the polypeptides derived from Streptococcus thoraltensis, St1 and St2, and the modified versions of these polypeptides, St1_mod and St2_mod, hydrolyze a novel site in the IgG hinge region. Consequently, St1 and St2, and St1_mod and St2_mod, exhibit exceptional IgG subclass specificity, hydrolyzing human lgG1 , lgG3, and lgG4, but demonstrating weak activity towards lgG2.

[0293] In mouse lgG2 possesses three different isoforms termed lgG2A, lgG2A / B, and lgG2B with different hinge disulfide bonds. St1 and St1_mod are the first enzymes discovered that are capable of specifically hydrolyzing mouse lgG2b.

[0294] The inventors are also the first to identify that the St2 polypeptide naturally contains two linked IgG proteases which can serve as a platform / backbone to construct chimeric proteases. These chimeric proteases may be configured to have protease activity towards multiple, e.g. two or more, target Ig classes or subclasses, e.g. both IgG and IgM, by selecting suitable protease domains, facilitating combining multiple different protease functions with low-immunogenic characteristics. Furthermore, the larger size of the chimeric proteases compared to typical non-chimeric proteases may increase half-life in circulation, facilitating therapeutic usage.

[0295] As used herein, the term “Ig variant” or “variant Ig” relates to an immunoglobulin comprising any amino acid substitution relative to a consensus Ig amino acid sequence, wherein the amino acid substitution may be naturally occurring or artificial. The term “Ig variant” encompasses disease-associated variants, and also Ig variants present both within and across one or more populations and superpopulations.

[0296] In embodiments, the proteases of the invention may be active against an Ig variant; for example, wherein one more amino acid relative to the corresponding wild-type sequence is substituted, deleted or inserted (i.e. an Ig variant comprises a mutated amino acid sequence).

[0297] In embodiments, the proteases of the invention may be active against an Ig variant, wherein the amino acid mutation(s) is / are not within the hinge region. In embodiments, the proteases of the invention may be active against an Ig variant, wherein the amino acid mutation(s) is / are within the hinge region.

[0298] As used herein, the term “modified Ig variant” relates to an Ig variant wherein the amino acid substitution is artificial. The term “modified Ig variant” encompasses engineered Ig variants, for example, having desirable properties e.g. reduced binding of the Fc receptor to one or more Fc receptor binding sites. Significantly, the inventors are the first to identify that the polypeptide C_mod exhibits high activity against modified IgG variants, specifically hinge-modified IgG variants, more specifically hinge modified human IgG variants, e.g. comprising the ‘LALA’ modification, e.g. lgG1 -LALA; or comprising the ‘LFLE’ modification, e.g. lgG1-LFLE; or comprising the LAGA modification e.g. lgG1 -LAGA; or comprising the FALA modification, e.g. lgG4-FALA, constituting the first identification of a naturally occurring IgG protease exhibiting high cleavage activity towards IgG proteins comprising the LALA or LFLE modification, e.g. lgG1-LALA, lgG1-LFLE, lgG1-LAGA, or lgG4-FALA.

[0299] The ‘LALA’ modification relates to artificially generated sequences of IgG proteins comprising the amino acid substitutions of the combination of two adjacent leucine residues to alanine, typically Leu234Ala and Leu235Ala in the hinge region of the heavy chain of an IgG protein, e.g. lgG1 (see Table 3).

[0300] The ‘LFLE’ modification relates to artificially generated sequences of IgG proteins comprising the amino acid substitutions of the combination of two adjacent leucine residues to phenylalanine and glutamic acid respectively, and a proline residue to serine, typically Leu234Phe, Leu235Glu, and Pro331 Ser, in the hinge region of the heavy chain of an IgG protein, e.g. lgG1 (see Table 3).

[0301] The ‘LAGA’ modification relates to artificially generated sequences of IgG proteins comprising the amino acid substitutions of the combination of a leucine residue and a glycine residue both to alanine, and optionally a proline residue to serine, typically Leu234Ala, and Gly331Ala, in the hinge region of the heavy chain of an IgG protein, e.g. lgG1 (see Table 3).

[0302] The ‘FALA’ modification relates to artificially generated sequences of IgG proteins comprising the amino acid substitutions ofthe combination of a phenylalanine and an adjacent leucine residue both to alanine, and optionally a proline residue to serine, typically F234A and L235A, and Pro331 Ser, in the hinge region of the heavy chain of an IgG protein, e.g. lgG4 (see Table 3).

[0303] Without wishing to be bound by theory, any one or suitable combination of the LALA, LFLE, LAGA, or FALA modifications .alters the conformation of the Fc region of the antibody, which comprises one or more Fc receptor binding sites, negatively impacting the interaction between one or more Fc receptor binding sites of modified antibodies comprising the aforementioned modifications, and an Fc receptor, e.g. FcyRI, FcyRII, and FcyRIII, as well as to complement component C1 q. The reduced binding of the Fc receptor and the Fc receptor binding sites reduces the immune effector functions of IgG proteins, in turn decreasing the resulting cell or tissue damage e.g. introduced by FcyR-mediated immune effector functions. As a result of these properties, the LALA, LFLE, LAGA, or FALA modifications are typically used in therapeutic antibodies and / or fusion proteins to mitigate or eliminate inflammatory responses upon administration to a subject. Furthermore, the LALA, LFLE, LAGA, or FALA modifications typically significantly reduce or eliminate the cleavage of the hinge region by IgG proteases, as, without wishing to be bound by theory, IgG proteases typically interact with IgG at their Fc region, which as described above is conformationally altered by the LALA, LFLE, LAGA, or FALA modifications.

[0304] In embodiments, the polypeptides of the invention may hydrolyze a polypeptide comprising the sequence of SEQ ID NO: 43, further comprising the substitutions L234A, L235A. In embodiments, the polypeptides of the invention may hydrolyze a polypeptide comprising the sequence of SEQ ID NO: 43, further comprising the substitutions L234A, L235A, and P331S.

[0305] In embodiments, the polypeptides of the invention may hydrolyze a polypeptide comprising the sequence of SEQ ID NO: 43, further comprising the substitutions L234F and L235E.

[0306] In embodiments, the polypeptides of the invention may hydrolyze a polypeptide comprising the sequence of SEQ ID NO: 43, further comprising the substitutions L234F, L235E, and P331S.

[0307] In embodiments, the polypeptides of the invention may hydrolyze a polypeptide comprising the sequence of SEQ ID NO: 43, further comprising the substitutions L235A, G237A.

[0308] In embodiments, the polypeptides of the invention may hydrolyze a polypeptide comprising the sequence of SEQ ID NO: 38, further comprising the substitutions F234A and L235A.

[0309] In embodiments, the polypeptides of the invention may hydrolyze a polypeptide comprising the sequence of SEQ ID NO: 38, further comprising the substitutions F234A and L235A and P331S.

[0310] In embodiments, the polypeptides of the invention may hydrolyze a polypeptide comprising the sequence of SEQ ID NO: 39, further comprising the substitutions L232A, L233A.

[0311] In embodiments, the polypeptides of the invention may hydrolyze a polypeptide comprising the sequence of SEQ ID NO: 39, further comprising the substitutions L232A, L233A, and P329S.

[0312] In embodiments, the polypeptides of the invention may hydrolyze a polypeptide comprising the sequence of SEQ ID NO: 39, further comprising the substitutions L233A, G235A.

[0313] Table 3: Examples of Human IgG heavy chain sequences

[0314] Exemplary positions of amino acids which may substituted are highlighted in bold in Table 3.

[0315] The skilled person would appreciate that aspects of the heavy chain sequence are variable, and thus the sequences presented in Table 3 are not intended to be limiting, and show an exemplary sequence context for the purposes of indicating where exemplary amino acid substitutions may be made.

[0316] The skilled person would also appreciate that the proteases of the invention may exhibit activity towards modified IgG variants, suitably modified lgG1 or lgG4 variants, more suitably hinge-modified lgG1 or lgG4 variants, comprising one or more amino acid substitutions of an L (Leucine) residue and / or one or more amino acid substitutions of a P (Proline) residue and / or one or more amino acid substitutions of an F (Phenylalanine) residue, one or more amino acid substitutions of a G (Glycine) residue, suitably wherein the amino acid substitutions may be adjacent to one another, suitably wherein the amino acid substitutions may be spaced by one or more amino acids from one another.

[0317] The skilled person would further appreciate that the proteases of the invention may exhibit activity towards a broad spectrum of modified IgG variants, suitably hinge modified IgG variants, suitably hinge modified lgG1 or lgG4 variants, wherein the type of substitution and the position of the amino acid substitution may be varied. For example, modified IgG 1 polypeptides may comprise the sequence of SEQ ID NO: 43, or a sequence having at least 90%, at least 95%, at least 98% or at least 99% identity thereto. For example, modified lgG4 polypeptides may comprise the sequence of SEQ ID NO: 38, or a sequence having at least 90%, at least 95%, at least 98% or at least 99% identity thereto.

[0318] In embodiments, the polypeptides of the invention may hydrolyze a polypeptide comprising the sequence of SEQ ID NO: 43, comprising amino acid substitutions at any one or more of positions 234, 235, or 331. In embodiments, the polypeptides of the invention may hydrolyze a polypeptide comprising the sequence of SEQ ID NO: 39, comprising amino acid substitutions at any one or more of positions 234, 235, or 331.

[0319] The skilled person would also appreciate that the exact position of the one or more amino acid substitutions in a modified IgG variant as described herein is dependent upon the position of the hinge region or hinge regions of a modified IgG variant.

[0320] In light of the above, the high activity of hydrolysis / cleavage towards IgG proteins comprising the LALA, LFLE, LAGA, or FALA modifications is a surprising feature of the polypeptide C_mod, given that IgG proteins comprising the LALA, LFLE, LAGA, or FALA modifications are not a naturally occurring IgG protease substrate.

[0321] In embodiments, the polypeptides of the invention, e.g. C_FL, may cleave a hinge-modified immunoglobulin, comprising the sequence of any immunoglobulin selected from any one of the following:

[0322] Trastuzumab,

[0323] Vedolizumab,

[0324] Galcanezumab,

[0325] Risankizumab,

[0326] Durvalumab, or

[0327] Talquetamab.

[0328] In embodiments, the polypeptides St1_FL and / or St2_FL may cleave an immunoglobulin, suitably lgG1 , more suitably human IgG 1 , comprising the sequence of SEQ ID NO: 28, suitably wherein the cleavage site is between amino acid positions 6 and 7 of SEQ ID NO: 28.

[0329] In embodiments, the polypeptides O_FL and / or R_FL may cleave an immunoglobulin, suitably lgG1 , more suitably human IgG 1 , comprising the sequence of SEQ ID NO: 29, suitably wherein the cleavage site is between amino acid positions 5 and 6 and / or amino acid positions 6 and 7 of SEQ ID NO: 29.

[0330] In embodiments, the polypeptides C_FL and / or a polypeptide comprising a fragment of IdeS or Xork, (e.g. SEQ ID NO: 19) may cleave an immunoglobulin, suitably lgG1 , more suitably human lgG1 , comprising the sequence of SEQ ID NO: 30, suitably wherein the cleavage site is between amino acid positions 6 and 7 of SEQ ID NO: 30.

[0331] In embodiments, the polypeptides of the invention may cleave an immunoglobulin, suitably lgG2, more suitably human lgG2, comprising the sequence of SEQ ID NO: 31 , suitably wherein the cleavage site is between amino acid positions 6 and 7 of SEQ ID NO: 31.

[0332] In embodiments, the polypeptides C_FL and / or O_FL may cleave an immunoglobulin, suitably lgG3, more suitably human lgG3, comprising the sequence of SEQ ID NO: 32, suitably wherein the cleavage site is between amino acid positions 6 and 7 of SEQ ID NO: 32. In embodiments, the polypeptides St1_FL and / or St2_FL may cleave an immunoglobulin, suitably lgG4, more suitably human lgG4, comprising the sequence of SEQ ID NO: 33, suitably wherein the cleavage site is between amino acid positions 6 and 7 of SEQ ID NO: 33.

[0333] In embodiments, the polypeptides C_FL and / or O_FL and / or a polypeptide comprising a fragment of IdeS may cleave an immunoglobulin, suitably lgG4, more suitably human lgG4, comprising the sequence of SEQ ID NO: 34, suitably wherein the cleavage site is between amino acid positions 6 and 7 of SEQ ID NO: 34.

[0334] In embodiments, the polypeptides St1_FL and / or St2_FL may cleave an immunoglobulin, suitably lgG2b, more suitably mouse lgG2b, comprising the sequence of SEQ ID NO: 35, suitably wherein the cleavage site is between amino acid positions 10 and 11 of SEQ ID NO: 35.

[0335] In embodiments, the polypeptides St1_FL and / or St2_FL may cleave an immunoglobulin, suitably lgG3, more suitably mouse lgG3, comprising the sequence of SEQ ID NO: 36, suitably wherein the cleavage site is between amino acid positions 10 and 11 of SEQ ID NO: 36.

[0336] In aspects of the invention, a polypeptide according to the present invention may comprise, consist essentially of, or consist of a sequence derived from Streptococcus thoraltensis, Streptococcus castoreus, Streptococcus ruminantium, Streptococcus ovuberis, and Lachnoanaerobaculum umeaense.

[0337] Exemplary polypeptide sequences are set out in Table 4 herein.

[0338] Table 4: Exemplary Streptococcus thoraltensis, Streptococcus castoreus, Streptococcus ruminantium, Streptococcus ovuberis, Lachnoanaerobaculum umeaense sequences

[0339] Modified proteases having Ig class and subclass specificity

[0340] A polypeptide may be derivatised or modified to assist with production, isolation or purification.

[0341] The present invention relates to one or more engineered polypeptides of the polypeptides of the invention.

[0342] Following identification of candidate protease genes, the present inventors engineered a number of polypeptides.

[0343] An engineered polypeptide according to the present invention may comprise, consist essentially of, or consist of a sequence derived from Streptococcus thoraltensis, Streptococcus castoreus, Streptococcus ruminantium, Streptococcus ovuberis, Lachnoanaerobaculum umeaense sequences.

[0344] Typically, the full-length protein sequence of an Ig protease comprises further domains in addition to the domains required for the proteolytic cleavage of Ig substrates. These additional domains may be involved in binding or anchoring the protein to a cell structure such as a cell wall or membrane. Alternatively, these additional domains may have unknown functions.

[0345] Often, these additional domains are not necessary, and in some cases are unfavourable, for the Ig protease activity of a polypeptide, e.g. may hinder the Ig protease from working efficiently. Consequently, it is often desirable to remove these domains.

[0346] Furthermore, and without wishing to be bound by theory, smaller proteins are typically expressed at higher levels compared to larger proteins, as for example, smaller proteins are less prone to fragmentation, which results in a higher yield of protein which is beneficial with respect to the production of a protein. The inventors have also noted that a polypeptide of the invention, e.g. SEQ ID NO: 7 exhibited degradation products, potentially indicative of sub-optimal stability. In order to improve the stability of the specific polypeptide, a modified protease was generated having the sequence of SEQ ID NO: 40. As demonstrated in Figure 12, the modified protease comprising the sequence of SEQ ID NO: 40 exhibited a single band with very little to no fragmentation of the protease visible, indicating improved stability vs SEQ ID NO: 7 which exhibits fragmented bands in Figure 2A. The modified protease maintained activity against its target Ig polypeptides.

[0347] In embodiments, all other domains except for the domain responsible for proteolytic cleavage of a target Ig are removed from a full-length sequence of the polypeptides of the invention.

[0348] In embodiments, the polypeptides of the invention comprise a domain of the identified polypeptides having proteolytic cleavage of a target Ig.

[0349] In embodiments, an additional methionine (M) residue is added at the N terminus to improve expression. In embodiments, the polypeptides of the invention include a N terminal methionine (M).

[0350] In embodiments, the polypeptide of the invention may be modified by addition of a ligand which is capable of binding directly and specifically to a separation means.

[0351] In embodiments, the polypeptide may be modified by addition of one member of a binding pair and the separation means comprises a reagent that is derivatised or modified by addition of the other member of a binding pair. Any suitable binding pair can be used.

[0352] In some embodiments, the polypeptide of the invention comprises one or more tags, such as polypeptide tags.

[0353] In embodiments, the tag is a cleavable tag.

[0354] In embodiments, the tag permits the specific modification of said tag or the polypeptide to which it is attached.

[0355] In embodiments, the tag facilitates the identification and / or isolation of the polypeptide.

[0356] In one embodiment, the tag is bound by a binding partner. In some embodiments, the binding partner is a polypeptide such as an antibody or other binding protein (e.g., avidin); a metal (e.g., Ni); and / or an organic small molecule (e.g., biotin).

[0357] The polypeptide according to the invention may include any one or more tag selected from the group consisting of: a histidine-tag, a poly-histidine tag, an Fc-tag, a FLAG tag, a Rho1 D4 tag, a HA tag, a strep tag, a SUMO tag, a GST tag, a lysozyme tag, an MBP tag, a GFP tag, a Myc tag, a thioredoxin tag, or an Avi tag. However, any suitable tag known to the skilled person may be utilised in combination with any polypeptide of the invention. The tag may be included at either the C or N terminus. Further the one or more tags may be separated from one another and / or other sequence elements by one or more linker sequence. Any number and combination of tags may be used.

[0358] In embodiments, the tag facilitates or improves the expression and / or properties (e.g., solubility or polymeric state) of the polypeptide.

[0359] In preferred embodiments where the polypeptide for use in the invention is derivatised or modified by addition of one member of a binding pair, the polypeptide may be a 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. 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, e.g., a ‘GGS’ linker.

[0360] In embodiments, the histidine tag typically consists of six histidine residues, however the tag can be longer than, such as up to 7, up to 8, up to 9, up to 10 or up to 20 amino acids in length. Alternatively, the His tag can be shorter, for example 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer or 1 amino acid(s).

[0361] In embodiments, the invention provides a polypeptide having IgG specific protease activity, e.g. endoprotease activity, wherein the polypeptide is derived from Streptococcus thoraltensis.

[0362] In embodiments, the invention provides a polypeptide having IgG specific protease activity, e.g. endoprotease activity, wherein the polypeptide is derived from Streptococcus castoreus.

[0363] In embodiments, the invention provides a polypeptide having IgG specific protease activity, e.g. endoprotease activity, wherein the polypeptide is derived from Streptococcus ruminantium.

[0364] In embodiments, the invention provides a polypeptide having IgG specific protease activity, e.g. endoprotease activity, wherein the polypeptide is derived from Streptococcus ovuberis.

[0365] In embodiments, the invention provides a polypeptide having IgM specific protease activity, e.g. endoprotease activity, wherein the polypeptide is derived from Lachnoanaerobaculum umeaense.

[0366] In embodiments, the invention provides a polypeptide having IgM specific protease activity, e.g. endoprotease activity, wherein the polypeptide is derived from Streptococcus krosus.

[0367] In embodiments, the invention provides a polypeptide having IgM specific protease activity, e.g. endoprotease activity, wherein the polypeptide is derived from Streptococcus thoraltensis.

[0368] Polypeptides of the invention may comprise, consist essentially of, or consist of any combination of naturally occurring polypeptides encoded by any bacterial species of Streptococcus thoraltensis, Streptococcus castoreus, Streptococcus ruminantium, Streptococcus ovuberis, Lachnoanaerobaculum umeaense, or Streptococcus krosus.

[0369] Polypeptides of the invention may comprise, consist essentially of, or consist of fragments or modified versions of naturally occurring polypeptides encoded by any bacterial species of Streptococcus thoraltensis, Streptococcus castoreus, Streptococcus ruminantium, Streptococcus ovuberis, Lachnoanaerobaculum umeaense, or Streptococcus krosus.

[0370] In embodiments, the invention provides a polypeptide having IgG specific protease activity, e.g. endoprotease activity, wherein the polypeptide is encoded by a polynucleotide sequence that has at least about 80% sequence identity, such as at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity, to a polynucleotide sequence, suitably an Ig protease polynucleotide sequence, more suitably an IgG protease polynucleotide sequence, encoded by Streptococcus thoraltensis.

[0371] In embodiments, the invention provides a polypeptide having IgG specific protease activity, e.g. endoprotease activity, wherein the polypeptide is encoded by a polynucleotide sequence that has at least about 80% sequence identity, such as at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity, to a polynucleotide sequence, suitably an Ig protease polynucleotide sequence, more suitably an IgG protease polynucleotide sequence, encoded by Streptococcus castoreus.

[0372] In embodiments, the invention provides a polypeptide having IgG specific protease activity, e.g. endoprotease activity, wherein the polypeptide is encoded by a polynucleotide sequence that has at least about 80% sequence identity, such as at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity, to a polynucleotide sequence, suitably an Ig protease polynucleotide sequence, more suitably an IgG protease polynucleotide sequence, encoded by Streptococcus ruminantium.

[0373] In embodiments, the invention provides a polypeptide having IgG specific protease activity, e.g. endoprotease activity, wherein the polypeptide is encoded by a polynucleotide sequence that has at least about 80% sequence identity, such as at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity, to a polynucleotide sequence, suitably an Ig protease polynucleotide sequence, more suitably an IgG protease polynucleotide sequence, encoded by Streptococcus ovuberis.

[0374] In embodiments, the invention provides a polypeptide having IgM specific protease activity, e.g. endoprotease activity, wherein the polypeptide is encoded by a polynucleotide sequence that has at least about 80% sequence identity, such as at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity, to a polynucleotide sequence, suitably an Ig protease polynucleotide sequence, more suitably an IgM protease polynucleotide sequence, encoded by Lachnoanaerobaculum umeaense. In embodiments, the invention provides a polypeptide having IgM specific protease activity, e.g. endoprotease activity, wherein the polypeptide is encoded by a polynucleotide sequence that has at least about 80% sequence identity, such as at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity, to a polynucleotide sequence, suitably an Ig protease polynucleotide sequence, more suitably an IgM protease polynucleotide sequence, encoded by Streptococcus krosus.

[0375] In alternative embodiments, e.g. in the case of a chimeric multi-domain polypeptide, the invention provides a polypeptide having IgG and IgM specific protease activity, e.g. endoprotease activity, wherein the polypeptide comprises at least one domain or region which is encoded by a polynucleotide sequence that has at least about 80% sequence identity, such as at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity, to a polynucleotide sequence, suitably an Ig protease polynucleotide sequence, encoded by Lachnoanaerobaculum umeaense.

[0376] In alternative embodiments, e.g. in the case of a chimeric multi-domain polypeptide, the invention provides a polypeptide having IgG and IgM specific protease activity, e.g. endoprotease activity, wherein the polypeptide comprises at least one domain or region which is encoded by a polynucleotide sequence that has at least about 80% sequence identity, such as at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity, to a polynucleotide sequence, suitably an Ig protease polynucleotide sequence, encoded by Streptococcus krosus.

[0377] In alternative embodiments, e.g. in the case of a chimeric multi-domain polypeptide, the invention provides a polypeptide having IgG and IgM specific protease activity, e.g. endoprotease activity, wherein the polypeptide comprises a first domain or region which is encoded by a polynucleotide sequence that has at least about 80% sequence identity, such as at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity, to a polynucleotide sequence, suitably an Ig protease polynucleotide sequence, encoded by Streptococcus thoraltensis; and a second domain or region which is encoded by a polynucleotide sequence that has at least about 80% sequence identity, such as at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity, to a polynucleotide sequence, suitably an Ig protease polynucleotide sequence, encoded by Lachnoanaerobaculum umeaense.

[0378] In alternative embodiments, e.g. in the case of a chimeric multi-domain polypeptide, the invention provides a polypeptide having IgG and IgM specific protease activity, e.g. endoprotease activity, wherein the polypeptide comprises a first domain or region which is encoded by a polynucleotide sequence that has at least about 80% sequence identity, such as at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity, to a polynucleotide sequence, suitably an Ig protease polynucleotide sequence, encoded by Streptococcus thoraltensis; and a second domain or region which is encoded by a polynucleotide sequence that has at least about 80% sequence identity, such as at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity, to a polynucleotide sequence, suitably an Ig protease polynucleotide sequence, encoded by Streptococcus krosus.

[0379] Polypeptides of the invention may comprise, consist essentially of, or consist of any combination of fragments or modified versions of naturally occurring polypeptides encoded by any bacterial species of Streptococcus thoraltensis, Streptococcus castoreus, Streptococcus ruminantium, Streptococcus ovuberis, Lachnoanaerobaculum umeaense, or Streptococcus krosus.

[0380] In embodiments, the invention provides a polypeptide having IgG specific protease activity, e.g. endoprotease activity, comprising, consisting essentially of, or consisting of a polypeptide sequence, wherein the polypeptide sequence has at least about 80% sequence identity, such as at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity, to a polypeptide sequence, suitably an Ig protease polypeptide sequence, encoded by Streptococcus thoraltensis.

[0381] In one embodiment, the invention provides a polypeptide having IgG specific protease activity, e.g. endoprotease activity, comprising, consisting essentially of, or consisting of a polypeptide sequence, wherein the polypeptide sequence has at least about 80% sequence identity, such as at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity, to a polypeptide sequence, suitably an Ig protease polypeptide sequence, encoded by Streptococcus castoreus.

[0382] In one embodiment, the invention provides a polypeptide having IgG specific protease activity, e.g. endoprotease activity, comprising, consisting essentially of, or consisting of a polypeptide sequence, wherein the polypeptide sequence has at least about 80% sequence identity, such as at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity, to a polypeptide sequence, suitably an Ig protease polypeptide sequence, encoded by Streptococcus ruminantium.

[0383] In one embodiment, the invention provides a polypeptide having IgG specific protease activity, e.g. endoprotease activity, comprising, consisting essentially of, or consisting of a polypeptide sequence, wherein the polypeptide sequence has at least about 80% sequence identity, such as at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity, to a polypeptide sequence, suitably an Ig protease polypeptide sequence, encoded by Streptococcus ovuberis.

[0384] In one embodiment, the invention provides a polypeptide having IgM specific protease activity, e.g. endoprotease activity, comprising, consisting essentially of, or consisting of a polypeptide sequence, wherein the polypeptide sequence has at least about 80% sequence identity, such as at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity, to a polypeptide sequence, suitably an Ig protease polypeptide sequence, encoded by Lachnoanaerobaculum umeaense. In one embodiment, the invention provides a polypeptide having IgM specific protease activity, e.g. endoprotease activity, comprising, consisting essentially of, or consisting of a polypeptide sequence, wherein the polypeptide sequence has at least about 80% sequence identity, such as at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity, to a polypeptide sequence, suitably an Ig protease polypeptide sequence, encoded by Streptococcus krosus.

[0385] In one embodiment, the invention provides a polypeptide having IgM and IgG specific protease activity, e.g. endoprotease activity, comprising, consisting essentially of, or consisting of a polypeptide sequence, wherein the polypeptide sequence has at least about 80% sequence identity, such as at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity, to a polypeptide sequence, suitably an Ig protease polypeptide sequence, encoded by Lachnoanaerobaculum umeaense.

[0386] In one embodiment, the invention provides a polypeptide having IgM and IgG specific protease activity, e.g. endoprotease activity, comprising a polypeptide sequence, wherein the polypeptide sequence has at least about 80% sequence identity, such as at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity, to a polypeptide sequence, suitably an Ig protease polypeptide sequence, encoded by Streptococcus krosus.

[0387] In embodiments, the invention provides a polypeptide having activity against Intravenous immunoglobulin (IVIG) (see Figure 9). Typically, IVIG is pooled from hundreds or thousands of individuals, and therefore polypeptides of the invention exhibiting activity towards IVIG may be considered to possess broad IgG activity, e.g. which is not dependent on specific IgG glycosylation patterns.

[0388] In embodiments, the polypeptide of the invention has IgG specific protease activity and said polypeptide comprises, consists essentially of, or consists of a sequence of any one of SEQ ID NOs: 1 , 4, 7, 10, or 13, or a variant or fragment thereof, as defined herein.

[0389] In embodiments, the polypeptide comprises, consists essentially of, or consists of a fragment of a polypeptide sequence selected from the group consisting of SEQ ID NOs: 1 , 4, 7, 10, or 13, wherein the fragment is at least about 100 amino acids in length, such as at least about 120 amino acids in length, at least about 140 amino acids in length, at least about 160 amino acids in length, at least about 180 amino acids in length, at least about 200 amino acids in length, at least about 220 amino acids in length, at least about 240 amino acids in length, at least about 260 amino acids in length, at least about 280 amino acids in length, at least about 300 amino acids in length, at least about 320 amino acids in length, at least about 340 amino acids in length, at least about 360 amino acids in length, at least about 380 amino acids in length, at least about 400 amino acids in length, at least about 420 amino acids in length, at least about 440 amino acids in length, at least about 460 amino acids in length, at least about 480 amino acids in length, at least about 500 amino acids in length, at least about 520 amino acids in length, at least about 540 amino acids in length, at least about 560 amino acids in length, at least about 580 amino acids in length, at least about 600 amino acids in length, at least about 620 amino acids in length, at least about 640 amino acids in length, at least about 660 amino acids in length, at least about 680 amino acids in length, at least about 700 amino acids in length, at least about 720 amino acids in length, at least about 740 amino acids in length, at least about 760 amino acids in length, at least about 780 amino acids in length, at least about 800 amino acids in length, at least about 820 amino acids in length, at least about 840 amino acids in length, at least about 860 amino acids in length, at least about 880 amino acids in length, or at least about 900 amino acids in length.

[0390] In preferred embodiments, the polypeptide comprises, consists essentially of, or consists of a fragment of a polypeptide sequence selected from the group consisting of SEQ ID NOs: 1 , 4, 7, 10, or 13, wherein the fragment is at least about 300 amino acids in length, such as at least about 320 amino acids in length, at least about 340 amino acids in length, at least about 360 amino acids in length, at least about 380 amino acids in length, at least about 400 amino acids in length, at least about 420 amino acids in length, at least about 440 amino acids in length, at least about 460 amino acids in length, at least about 480 amino acids in length, at least about 500 amino acids in length, at least about 520 amino acids in length, at least about 540 amino acids in length, at least about 560 amino acids in length, at least about 580 amino acids in length, at least about 600 amino acids in length, at least about 620 amino acids in length, at least about 640 amino acids in length, at least about 660 amino acids in length, at least about 680 amino acids in length, at least about 700 amino acids in length, at least about 720 amino acids in length, at least about 740 amino acids in length, at least about 760 amino acids in length, at least about 780 amino acids in length, at least about 800 amino acids in length, at least about 820 amino acids in length, at least about 840 amino acids in length, at least about 860 amino acids in length, at least about 880 amino acids in length, or at least about 900 amino acids in length.

[0391] Exemplary fragments of polypeptides as disclosed herein are provided as SEQ ID NOs: 2, 5, 8, 1 1 , 14, 17 and 19 (see Table 5).

[0392] Table 5: Exemplary polypeptide fragment sequences

[0393] Exemplary modified polypeptide disclosed herein are provided as SEQ ID NOs: 3, 6, 9, 12, 15, 16, 18 and 40 (see Table 6).

[0394] Table 6: Exemplary polypeptide sequences

[0395] In embodiments, the polypeptide of the invention has IgG specific protease activity and said polypeptide comprises, consists essentially of, or consists of a sequence of any one of SEQ ID NOs: 3, 6, 9, 12, or 15, or a variant or fragment thereof, as defined herein.

[0396] In embodiments, the polypeptide of the invention has IgM specific protease activity and said polypeptide comprises, consists essentially of, or consists of a sequence of any one of SEQ ID NOs: 17 or 19, or a variant or fragment thereof, as defined herein.

[0397] In embodiments of the invention, there is provided a polypeptide having IgG and IgM specific protease activity, wherein the polypeptide comprises, consists essentially of, or consists of a polypeptide selected from the group consisting of SEQ ID NOs: 16 or 18.

[0398] In embodiments of the invention, there is provided a polypeptide having IgG specific protease activity, wherein the polypeptide comprises, consists essentially of, or consists of a polypeptide having at least about 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 6, 9, 12, or 15.

[0399] In embodiments of the invention, there is provided a polypeptide having IgG specific protease activity, wherein the polypeptide comprises, consists essentially of, or consists of a polypeptide having at least about 80% sequence identity, such as at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity, to a sequence selected from the group consisting of SEQ ID NOs: 3, 6, 9, 12, or 15.

[0400] In embodiments of the invention, there is provided a polypeptide having IgM specific protease activity, wherein the polypeptide comprises, consists essentially of, or consists of a polypeptide having at least about 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 or 19. In embodiments of the invention, there is provided a polypeptide having IgM specific protease activity, wherein the polypeptide comprises, consists essentially of, or consists of a polypeptide having at least about 80% sequence identity, such as at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity, to a sequence selected from the group consisting of SEQ ID NOs: 17 or 19.

[0401] In embodiments of the invention, there is provided a polypeptide having IgG and IgM specific protease activity, wherein the polypeptide comprises, consists essentially of, or consists of a polypeptide having at least about 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 16 or 18 and optionally any one of SEQ ID NOs: 1 to 15.

[0402] In embodiments of the invention, there is provided a polypeptide having IgG and IgM specific protease activity, wherein the polypeptide comprises, consists essentially of, or consists of a polypeptide having at least about 80% sequence identity, such as at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity, to a sequence selected from the group consisting of SEQ ID NOs: 16 or 18 and optionally any one of SEQ ID NOs: 1 to 15.

[0403] In embodiments, the polypeptide is at least about 100 amino acids in length, such as at least about 150 amino acids in length, at least about 200 amino acids in length, at least about 250 amino acids in length, at least about 300 amino acids in length, at least about 350 amino acids in length, at least about 400 amino acids in length, at least about 450 amino acids in length, at least about 500 amino acids in length, at least about 550 amino acids in length, at least about 600 amino acids in length, at least about 650 amino acids in length, at least about 700 amino acids in length, at least about 750 amino acids in length, at least about 800 amino acids in length, at least about 850 amino acids in length, at least about 900 amino acids in length, at least about 950 amino acids in length, at least about 1000 amino acids in length, at least about 1050 amino acids in length, at least about 1 100 amino acids in length, at least about 1 150 amino acids in length, at least about 1200 amino acids in length, at least about 1250 amino acids in length, at least about 1300 amino acids in length, at least about 1350 amino acids in length, at least about 1400 amino acids in length, at least about 1450 amino acids in length, at least about 1500 amino acids in length, at least about 1550 amino acids in length, at least about 1600 amino acids in length, at least about 1650 amino acids in length, at least about 1700 amino acids in length, at least about 1750 amino acids in length, at least about 1800 amino acids in length, at least about 1850 amino acids in length, at least about 1900 amino acids in length, at least about 1950 amino acids in length, or at least about 2000 amino acids in length.

[0404] The polypeptide may be no longerthan about amino acids in length, such as no longerthan about 2000, no longerthan about 1900, no longerthan about 1800, no longerthan about 1700, no longerthan about 1600, no longerthan about 1500, no longer than about 1400 no longer than about 1300, no longerthan about 1200, no longer than about 1100, or no longer than about 1000 amino acids in length. It will be appreciated that any of the above listed lower limits may be combined with any of the above listed upper limits to provide a range for the length the polypeptide. For example, the polypeptide may be between about 300 to about 2000 amino acids in length, between about 400 to 1900 amino acids in length, between about 500 to 1800 amino acids in length, between about 600 to 1700 amino acids in length, between about 700 to 1600 amino acids in length, or between about 800 to 1500 amino acids in length.

[0405] The present invention also encompasses fragments of other polypeptides of the invention. Said fragments are to be considered polypeptides in their own right.

[0406] In other words, polypeptides of the invention may comprise, consist essentially of, or consist of fragments or shorter sections of longer polypeptides according to the invention, as defined herein.

[0407] A fragment will be understood to be any continuous I contiguous polypeptide (or polynucleotide) sequence of the defined I specified sequence, that is shorter than the defined longer sequence with which it is identical (across the amino acids that they have in common). For example, a polypeptide that is 200 amino acids in length may be considered a fragment of a longer polypeptide sequence of 250 amino acids in length with which it shares 100% sequence identity.

[0408] In embodiments, the polypeptide comprises, consists essentially of, or consists of a fragment of a polypeptide sequence selected from the group consisting of SEQ ID NOs: 3, 6, 9, 12, 15, 16, 18 or 40.

[0409] In embodiments of the invention, the fragment of the sequence of SEQ ID NO: 1 , 4, 7, 10, 13, 41 , or 42 is: at least about 100 amino acids in length, at least about 200 amino acids in length, or at least about 300 amino acids in length; or wherein the fragment of the sequence of SEQ ID NO: 1 , 4, 10, 13, 41 , or 42 is: at least about 400 amino acids in length, at least about 500 amino acids in length, or at least about 600 amino acids in length; or wherein the fragment of the sequence of SEQ ID NO: 1 , 4, 13, 41 , or 42 is: at least about 700 amino acids in length; or wherein the fragment of the sequence of SEQ ID NO: 1 , 4 or 13 is: at least about 800 amino acids in length, at least about 900 amino acids in length, at least about 1000 amino acids in length, at least about 1100 amino acids in length, at least about 1200 amino acids in length, at least about 1300 amino acids in length, at least about 1400 amino acids in length, at least about 1500 amino acids in length; or wherein the fragment of the sequence of SEQ ID NO: 1 or 4 is: at least about 1600 amino acids in length, or at least about 1700 amino acids in length; or wherein the fragment of the sequence of SEQ ID NO: 4 is: at least about 1800 amino acids in length.

[0410] In aspects of the invention, a polypeptide, or engineered polypeptide, having immunoglobulin protease activity is provided, wherein said polypeptide comprises, consists essentially of, or consists of: (a) the amino acid sequence of SEQ ID NO: 2, 5, 8, 11 , or 14; or

[0411] (b) a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 2, 5, 8, 11 , or 14; or

[0412] (c) an amino acid sequence which is a fragment of the sequence of

[0413] (a) or (b).

[0414] In embodiments of the invention, the fragment of the sequence of SEQ ID NO: 2, 5, 8, 11 , or 14 is: at least about 100 amino acids in length, at least about 150 amino acids in length, at least about 200 amino acids in length, at least about 250 amino acids in length, at least about 300 amino acids in length, or wherein the fragment of the sequence of SEQ ID NO: 5 is: at least about 350 amino acids in length at least about 400 amino acids in length, at least about 450 amino acids in length at least about 500 amino acids in length at least about 550 amino acids in length, at least about 600 amino acids in length, at least about 650 amino acids in length, at least about 700 amino acids in length, at least about 750 amino acids in length, at least about 800 amino acids in length.

[0415] In one embodiment, there is provided a polypeptide having endoprotease activity comprising, consisting essentially of, or consisting of the amino acid sequence of any one of SEQ ID NOs: 3, 6, 9, 12, 15, 16, 18 or 40.

[0416] In embodiments, the polypeptides St1_mod and / or St2 mod may cleave an immunoglobulin, suitably lgG1 , more suitably human lgG1 , comprising the sequence of SEQ ID NO: 28, suitably wherein the cleavage site is between amino acid positions 6 and 7 of SEQ ID NO: 28.

[0417] In embodiments, the polypeptides O_mod and / or R mod cleave an immunoglobulin, suitably IgG 1 , more suitably human lgG1 , comprising the sequence of SEQ ID NO: 29, suitably wherein the cleavage site is between amino acid positions 5 and 6 and / or amino acid positions 6 and 7 of SEQ ID NO: 29.

[0418] In embodiments, the polypeptides C_mod and / or a polypeptide comprising a fragment of IdeS or Xork, (e.g. SEQ ID NO: 19) may cleave an immunoglobulin, suitably lgG1 , more suitably human lgG1 , comprising the sequence of SEQ ID NO: 30, suitably wherein the cleavage site is between amino acid positions 6 and 7 of SEQ ID NO: 30.

[0419] In embodiments, the polypeptides of the invention may cleave an immunoglobulin, suitably lgG2, more suitably human lgG2, comprising the sequence of SEQ ID NO: 31 , suitably wherein the cleavage site is between amino acid positions 6 and 7 of SEQ ID NO: 31.

[0420] In embodiments, the polypeptides C_mod and / or O mod may cleave an immunoglobulin, suitably lgG3, more suitably human lgG3, comprising the sequence of SEQ ID NO: 32, suitably wherein the cleavage site is between amino acid positions 6 and 7 of SEQ ID NO: 32. In embodiments, the polypeptides St1_mod and / or St2 mod may cleave an immunoglobulin, suitably lgG4, more suitably human lgG4, comprising the sequence of SEQ ID NO: 33, suitably wherein the cleavage site is between amino acid positions 6 and 7 of SEQ ID NO: 33.

[0421] In embodiments, the polypeptides C_mod and / or O mod and / or a polypeptide comprising a fragment of IdeS may cleave an immunoglobulin, suitably lgG4, more suitably human lgG4, comprising the sequence of SEQ ID NO: 34, suitably wherein the cleavage site is between amino acid positions 6 and 7 of SEQ ID NO: 34.

[0422] In embodiments, the polypeptides St1_mod and / or St2 mod may cleave an immunoglobulin, suitably lgG2b, more suitably mouse lgG2b, comprising the sequence of SEQ ID NO: 35, suitably wherein the cleavage site is between amino acid positions 10 and 11 of SEQ ID NO: 35.

[0423] In embodiments, the polypeptides St1_mod and / or St2 mod may cleave an immunoglobulin, suitably lgG3, more suitably mouse lgG3, comprising the sequence of SEQ ID NO: 36, suitably wherein the cleavage site is between amino acid positions 10 and 11 of SEQ ID NO: 36.

[0424] Exemplary Polynucleotides

[0425] The present invention also relates to polynucleotides that encode polypeptides having IgG specific or IgG and IgM specific protease activity, e.g. endoprotease activity.

[0426] In aspects of the invention, polynucleotides that encode polypeptides having IgG specific protease activity, e.g. endoprotease activity are also provided.

[0427] In embodiments, the polynucleotide is an RNA. In embodiments the polynucleotide is a DNA.

[0428] In embodiments, the polynucleotide comprises one or more modified nucleotides or nucleotide analogues.

[0429] Polynucleotides according to the invention may be naturally occurring, naturally derived, or synthetic.

[0430] In embodiments, the polynucleotides of the invention comprise a codon for the N terminal methionine (ATG).

[0431] In embodiments, the polynucleotides of the invention contain one or more codons encoding a protein purification tag. In embodiments, the codons encoding a protein purification tag are situated prior to the stop codon (TAA) at the 3' end of the polynucleotides of the invention, e.g. codons encoding a 6x His tag.

[0432] In embodiments, the polynucleotides of the invention comprise an additional N terminal methionine (ATG) codon added to improve expression. In embodiments, the polynucleotides of the invention may encode a polypeptide comprising a ligand which is capable of binding directly and specifically to a separation means, such as one member of a binding pair, wherein the separation means comprises a reagent that is derivatised or modified by addition of the other member of a binding pair. Any suitable binding pair can be used.

[0433] In embodiments, the polynucleotides of the invention may encode a polypeptide comprising one or more tags, such as polypeptide tags. In embodiments, the tag is a cleavable tag. In embodiments, the tag permits the specific modification of said tag or the polypeptide to which it is attached. In embodiments, the tag facilitates the identification and / or isolation of the polypeptide encoded by the polynucleotides of the invention. In embodiments, the tag is bound by a binding partner. In some embodiments, the binding partner is a polypeptide such as an antibody or other binding protein (e.g., avidin); a metal (e.g., Ni); and / or an organic small molecule (e.g., biotin).

[0434] The polynucleotides of the invention may encode a polypeptide comprising any one or more tag selected from the group consisting of: a histidine-tag, a poly-histidine tag, an Fc-tag, a FLAG tag, a Rho1 D4 tag, a HA tag, a strep tag, a SUMO tag, a GST tag, a lysozyme tag, an MBP tag, a GFP tag, a Myc tag, a thioredoxin tag, or an Avi tag. However, any suitable polynucleotide sequence encoding a tag known to the skilled person may be utilised in combination with any polynucleotide of the invention.

[0435] The polynucleotide sequence tag may be included at either the 5’ or 3’ terminus of the polynucleotide, which may correspond to the N or C terminus of the polypeptide. Furthermore, the one or more polynucleotide sequence(s) encoding the polypeptide tag(s) may be separated from one another and / or other polynucleotide sequence elements by one or more polynucleotide sequences encoding a linker sequence. Any number and combination of polynucleotide sequences encoding polypeptide tags may be used.

[0436] In embodiments, the invention provides a polynucleotide encoding a polypeptide having IgG specific protease activity, e.g. endoprotease activity, comprising a polynucleotide sequence, wherein the polynucleotide sequence has at least about 70% sequence identity, such as at least about 75% sequence identity, at least about 80% sequence identity at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity, to a polynucleotide sequence, suitably an Ig protease polynucleotide sequence, encoded by Streptococcus thoraltensis.

[0437] In embodiments, the invention provides a polynucleotide encoding a polypeptide having IgG specific protease activity, e.g. endoprotease activity, comprising, consisting essentially of, or consisting of a polynucleotide sequence, wherein the polynucleotide sequence has at least about 70% sequence identity, such as at least about 75% sequence identity, at least about 80% sequence identity, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity, to a polynucleotide sequence, suitably an Ig protease polynucleotide sequence, encoded by Streptococcus castoreus.

[0438] In embodiments, the invention provides a polynucleotide encoding a polypeptide having IgG specific protease activity, e.g. endoprotease activity, comprising, consisting essentially of, or consisting of a polynucleotide sequence, wherein the polynucleotide sequence has at least about 70% sequence identity, such as at least about 75% sequence identity, at least about 80% sequence identity, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity, to a polynucleotide sequence, suitably an Ig protease polynucleotide sequence, encoded by Streptococcus ruminantium.

[0439] In embodiments, the invention provides a polynucleotide encoding a polypeptide having IgG specific protease activity, e.g. endoprotease activity, comprising, consisting essentially of, or consisting of a polynucleotide sequence, wherein the polynucleotide sequence has at least about 70% sequence identity, such as at least about 75% sequence identity, at least about 80% sequence identity, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity, to a polynucleotide sequence, suitably an Ig protease polynucleotide sequence, encoded by Streptococcus ovuberis.

[0440] In embodiments, the invention provides a polynucleotide encoding a polypeptide having IgM specific protease activity, e.g. endoprotease activity, comprising, consisting essentially of, or consisting of a polynucleotide sequence, wherein the polynucleotide sequence has at least about 70% sequence identity, such as at least about 75% sequence identity, at least about 80% sequence identity, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity, to a polynucleotide sequence, suitably an Ig protease polynucleotide sequence, encoded by Lachnoanaerobaculum umeaense.

[0441] In embodiments, the invention provides a polynucleotide encoding a polypeptide having IgM specific protease activity, e.g. endoprotease activity, comprising, consisting essentially of, or consisting of a polynucleotide sequence, wherein the polynucleotide sequence has at least about 70% sequence identity, such as at least about 75% sequence identity, at least about 80% sequence identity, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity, to a polynucleotide sequence, suitably an Ig protease polynucleotide sequence, encoded by Streptococcus krosus.

[0442] In embodiments, the invention provides a polynucleotide encoding a polypeptide having IgG and IgM specific protease activity, e.g. endoprotease activity, comprising, consisting essentially of, or consisting of a polynucleotide sequence, wherein the polynucleotide sequence has at least about 70% sequence identity, such as at least about 75% sequence identity, at least about 80% sequence identity, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity, to a polynucleotide sequence, suitably an Ig protease polynucleotide sequence, encoded by Lachnoanaerobaculum umeaense.

[0443] In embodiments, the invention provides a polynucleotide encoding a polypeptide having IgG and IgM specific protease activity, e.g. endoprotease activity, comprising, consisting essentially of, or consisting of a polynucleotide sequence, wherein the polynucleotide sequence has at least about 70% sequence identity, such as at least about 75% sequence identity, at least about 80% sequence identity, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% sequence identity, to a polynucleotide sequence, suitably an Ig protease polynucleotide sequence, encoded by Streptococcus krosus.

[0444] Exemplary polynucleotide sequences that encode polypeptides according to the invention are set out in Table 7.

[0445] Table 7: Exemplary polynucleotide sequences

[0446] In view of codon redundancy, it will be appreciated that many slightly different nucleic acid sequences may accurately code for each of the polypeptides of the invention, and each of these variants is encompassed within the scope of the present invention. As such, it will be understood that any number of polynucleotide sequences may be utilized to encode a given polypeptide according to the invention due to the degeneracy of the genetic code, and that the polynucleotide sequences described herein are illustrative only. The skilled person can readily determine suitable nucleic acid sequences for encoding each of the polypeptides of the invention, and may select appropriate codon codes according to the system in which the polypeptide is to be expressed (e.g. bacteria or mammals such as human and mouse).

[0447] In embodiments, there is provided a polynucleotide encoding a polypeptide having protease activity, suitably Ig protease activity, comprising, consisting essentially of, or consisting of the amino acid sequence of any of SEQ ID NOs: 1 to 19 or 40 to 42.

[0448] In embodiments, there is provided a polynucleotide encoding a polypeptide having endoprotease activity comprising, consisting essentially of, or consisting of a polypeptide having at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to the amino acid sequence of any of SEQ ID NOs: 1 to 19 or 40 to 42.

[0449] In embodiments, there is provided a polynucleotide encoding a fragment of polypeptide, the fragment having protease activity, wherein the fragment comprises, consists essentially of, or consists of a fragment of the amino acid sequence of any one of SEQ ID NOs: 1 to 19 or 40 to 42, or a fragment of an amino acid sequence having at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to the amino acid sequence of any of SEQ ID NOs: 1 to 19 or 40 to 42.

[0450] In embodiments, there is provided a polynucleotide encoding a polypeptide having protease activity, the polypeptide comprising: a) the amino acid sequence of any of SEQ ID NOs: 1 to 19 and 40 to 42; b) a variant thereof having at least 80% sequence identity to the amino acid sequence of any of SEQ ID NOs: 1 to 19 and 40 to 42; or c) a fragment of either a) or b).

[0451] In embodiments, there is provided a polynucleotide encoding a polypeptide having human IgG specific protease activity, wherein the polypeptide comprises, consists essentially of, or consists of an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 1 to 15.

[0452] In embodiments, there is provided a polynucleotide encoding a polypeptide having human IgM and IgG specific protease activity, wherein the polypeptide comprises, consists essentially of, or consists of an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 16 or 18.

[0453] In embodiments, there is provided a polynucleotide comprising the sequence of any one of SEQ ID NOs: 20 to 27. In embodiments, there is provided a polynucleotide having at least about 60% sequence identity, such as at least about 70% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to sequence of any of SEQ ID NOs: 20 to 27.

[0454] In embodiments, there is provided a polynucleotide comprising: a) the polynucleotide sequence of any one of SEQ ID NOs: 20 to 27; or b) a variant thereof having at least 80% sequence identity to the polynucleotide sequence of any of SEQ ID NOs: 20 to 27; or c) a polynucleotide fragment of either a) or b).

[0455] In embodiments, the polynucleotide fragment is at least about 500 nucleotides in length, at least about 600 nucleotides in length, at least about 700 nucleotides in length, at least about 800 nucleotides in length, at least about 900 nucleotides in length, at least about 1000 nucleotides in length, at least about 1100 nucleotides in length, at least about 1200 nucleotides in length, at least about 1300 nucleotides in length, at least about 1400 nucleotides in length, at least about 1500 nucleotides in length, at least about 1600 nucleotides in length.

[0456] In embodiments, there is provided a polynucleotide encoding a polypeptide having IgG specific protease activity, suitably human IgG specific protease activity, wherein the polypeptide comprises, consists essentially of, or consists of an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 20 to 25, and optionally wherein the polynucleotide comprises, consists essentially of, or consists of a sequence according to any one of SEQ ID NOs: 20 to 25, or a sequence having at least about 80% sequence identity to a polynucleotide sequence according to any one of SEQ ID NOs: 20 to 25.

[0457] In embodiments, there is provided a polynucleotide encoding a polypeptide having IgG and IgM specific protease activity, suitably human IgG and IgM specific protease activity, wherein the polypeptide comprises, consists essentially of, or consists of an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 26 or 27, and optionally wherein the polynucleotide comprises, consists essentially of, or consists of a sequence according to any one of SEQ ID NOs: 26 or 27, or a sequence having at least about 80% sequence identity to a polynucleotide sequence according to any one of SEQ ID NOs: 26 to 27.

[0458] The invention also provides for vectors comprising any of the foregoing polynucleotides.

[0459] The invention also provides for vectors encoding any of the foregoing polypeptides.

[0460] In embodiments, the vector is an expression vector.

[0461] In embodiments, the vector is a pET21 a+ vector. Compositions and formulations comprising polypeptides

[0462] The polypeptides, polynucleotides, vectors and cells of the invention may be provided in a composition or combination with other compounds.

[0463] By composition, it will be understood that the components of said composition may be mixed; by combination it will be understood that the components of said combination are not necessarily mixed and may be provided separately, e.g., in separate containers, which may optionally be mixed.

[0464] In embodiments, the present invention provides compositions comprising a polypeptide of the invention. For example, the invention provides a composition comprising one or more polypeptides of the invention and at least one excipient, preferably a preservative or stabiliser, and optionally also one or more additional carriers, diluents or vehicles.

[0465] The invention also encompasses compositions comprising polypeptides, polynucleotides, vectors and / or cells of the invention in admixture with aqueous components suitable for the storage thereof, such as a diluent, salt, and / or buffer.

[0466] Also provided herein are pharmaceutical compositions comprising polypeptides, polynucleotides, vectors and / or cells of the invention in admixture with a pharmaceutically acceptable diluent, salt, and / or carrier.

[0467] Typically, the final composition is sterile and pyrogen free.

[0468] In embodiments, the pharmaceutical composition is in admixture with TBS.

[0469] In embodiments, the pharmaceutical composition is in admixture with PBS.

[0470] In particular embodiments, all of the present 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.

[0471] 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.

[0472] 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).

[0473] Such 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.

[0474] 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 embodiments 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.

[0475] 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.

[0476] Other parentally-administrable compositions which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt. The compositions may be suitable for administration by any suitable route including, for example, intradermal, subcutaneous, percutaneous, intramuscular, intraarterial, intraperitoneal, intraarticular, intraosseous or other appropriate administration routes.

[0477] In embodiments, the compositions are suitable for administration by intravenous infusion.

[0478] Kits

[0479] Provided herein is a kit comprising any polypeptide, polynucleotide, composition, combination, and / or cell according to the invention. In certain embodiments, the kit comprises packaging and / or instructions for use of said kit. In embodiments, the polypeptides of the invention may be purified after production, wherein purification comprises contacting, optionally attaching, the polypeptides to a separation means, which may comprise a suitable substrate or object as a means of separating and isolating the polypeptide, e.g. from a medium or lysed cells. In embodiments, the polypeptide may be purified from Fc and / or Fab fragments. In embodiments, the Fc and / or Fab fragments do not bind, or bind with low affinity to the separation means or substrate. Alternatively, the separation means or substrate may be one member of a binding pair, wherein a protein purification or affinity tag / moiety includes the other member of the binding pair. Any suitable binding pair can be used. In embodiments, the separation means or substrate may be a coated object, such as a bead, a well, a tube, a membrane, a filter, or a surface thereof, wherein the coating may comprise, for example, a resin or a gel. In embodiments, the separation means or substrate may be a resin.

[0480] Any suitable resin can be used. Non limiting examples include immobilized metal affinity chromatography (IMAC) Resins, immobilized biotin and iminobiotin resins, calmodulin resins, immobilized p-aminophenyl phosphoryl choline resins, immobilized o-phosphorylethanolamine resins, agarose resins, preactivated resins (for the coupling to sulfhydryls, amines, carboxyls, carbohydrates, and active hydrogens), and avidin and streptavidin purification resins.

[0481] As a non-limiting example, the separation means or substrate may comprise the CaptureSelect Technology from ThermoFisher Scientific.

[0482] As a non-limiting example, the separation means or substrate may comprise the Profinity IMAC Resins from BIO-RAD.

[0483] In embodiments, there is provided a kit comprising any combination of one or more of: a) a polypeptide according to the invention; b) a composition according to the invention; c) a polynucleotide according to the invention; d) a vector according to the invention; e) a cell according to the invention; and / or f) a separation means.

[0484] In embodiments wherein the kit comprises a combination, for example of two or more proteases, a first polypeptide and a second polypeptide, and any further polypeptides, may be provided separately (i.e., in different containers within the kit or in different kits) or together (i.e., within the same kit and / or within the same container within the kit).

[0485] It will be understood that any substituent parts of a kit according to the invention may be used in the methods or uses of the invention as defined herein. Further, a kit according to the invention may comprise directions for use that relate to any one of the methods or uses of the invention. Methods and uses

[0486] The invention also provides for methods and uses relating to the polynucleotides, polypeptides, compositions, combinations, vectors, cells, and kits of the invention.

[0487] Any of the methods or uses herein may be performed in vivo, in vitro or ex vivo.

[0488] The polypeptides, polynucleotides, expression vectors and / or compositions of the invention are useful in various methods. Thus, provided herein are methods comprising administering a polypeptide, polynucleotide, expression vector and / or composition of the invention, for example to a sample or a subject. The method may be a method treating a disease, the method comprising administering a polypeptide of the invention to a subject. Also provided are methods comprising contacting IgG with a polypeptide, polynucleotide, expression vector or composition of the invention.

[0489] The method may be a method of cleaving an immunoglobulin, and may optionally further comprises the detection or analysis of the cleavage products. For example, the methods may comprise a step of identifying and / or isolating the IgG and / or IgM specific cleavage products.

[0490] In embodiments, the methods or uses may additionally comprise contacting the sample with a polypeptide having IgG specific protease activity.

[0491] In embodiments, the methods or uses may additionally comprise contacting the sample with a polypeptide having IgG and IgM specific protease activity.

[0492] In some embodiments, the methods or uses of the invention may be for the purpose of degradation or removal of IgG and / or IgM from a sample.

[0493] In some embodiments, the methods or uses of the invention may be for the purpose of a diagnostic assay.

[0494] In some embodiments, the methods or uses of the invention may be for the purpose of a diagnostic assay, wherein the assay is an anti-drug antibody (ADA) assay.

[0495] Anti-drug assays (ADAs) are utilised to identify the presence of antibodies directed to or elicited against a drug of interest. Often, these assays are conducted using bridging ELISAs, which do not distinguish between antibody classes or subclasses. A polypeptide according to the present invention, which can degrade IgG, may be used to remove or inactivate IgG in a sample and therefore facilitate increased resolution in ADA assays, or other similar assays that comprise IgG within a mix of components, by determining the contribution of IgG to any observed activity.

[0496] In some embodiments, the methods or uses of the invention may be for the purpose of a diagnostic assay quality control, such as quality control of recombinant or non-recombinant monoclonal and / or polyclonal IgG or IgG-derived molecules, and / or monoclonal and / or polyclonal IgM or IgM-derived molecules. For development of IgG and IgM for therapeutic purposes, diagnostic purposes, or IgG or IgM-like drugs, it is imperative to perform extensive quality control in order to validate the drug and verify, for example, its sequence and / or the presence of post-translational modifications. Often, such quality control involves the use of trypsin or similar ‘bottom-up‘ approaches which lack specificity and therefore can be improved. The polypeptides of the invention, e.g. the IgG specific and / or IgG and IgM specific proteases which hydrolyse IgG and / or IgM, are suitable for facilitating such analysis.

[0497] The present polypeptides may provide useful tools for biotechnology.

[0498] The polypeptides may be used in a method for the ex vivo cleavage of IgG. The IgG may be human, mouse, horse or guinea pig IgG. The IgG may be a recombinant, monoclonal or polyclonal, suitably monoclonal, antibody derived from any of these species. The polypeptide may be administered to a sample containing IgG and incubated under conditions which permit immunoglobulin protease activity to occur.

[0499] Suitable conditions include incubation for at least 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 120 minutes, 3 hours, 5 hours, 10 hours, or overnight, typically with mixing e.g. end-over-end mixing. Particularly suitable incubation times are between about 20 minutes and 3 hours, or between about 30 minutes and 2 hours (120 minutes).

[0500] Suitable incubation conditions may take place at room temperature, preferably at approximately 20°C, 25°C, 30°C, 35°C, 40°C or 45°C, and more preferably at approximately 37°C.

[0501] 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 8.0, most preferably at around pH 7.5.

[0502] 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, cofactors or reducing agents, although the presence of salts (e.g. NaCI) at up to 200mM is preferably tolerated by the polypeptide of the invention.

[0503] The approximate ratio of the polypeptide of the invention 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).

[0504] A preferred ratio of the polypeptide of the invention to the protein content of the sample is between 1 :20 and 1 :40 (wt:wt).

[0505] Specific cleavage of Ig according to the methods can be verified, and the cleavage products isolated using any suitable method, such as those described elsewhere in this document, including in the Examples, or in WG2003051914 and WG2009033670. The method can be used in particular 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 IgG with a polypeptide of the invention.

[0506] The method may also be used to detect or analyse IgG and / or IgM in a sample, or to remove IgG and / or IgM from a sample. A method for the detection of IgG and / or IgM in a sample typically involves incubating the polypeptide with the sample under conditions which permit binding and cleavage. The presence of IgG and / or IgM can be verified by detection of the specific IgG and / or IgM cleavage products, which may subsequently be analysed.

[0507] The invention provides a method of detecting the presence of IgG in a sample, the method comprising: a) contacting a sample with a polypeptide, composition, or combination of the invention; and b) identifying the IgG specific cleavage products, wherein the presence of IgG specific cleavage products is indicative of the presence of IgG in the sample.

[0508] The invention provides a method of detecting the presence of one or more IgG subclass or subclasses, e.g. lgG1 , lgG2, lgG3, lgG4, in a sample, the method comprising: a) contacting a sample with a polypeptide, composition of the invention; and b) identifying the specific cleavage products of the one or more IgG subclass, wherein the presence of the specific cleavage products of the one or more IgG subclass is indicative of the presence of the one or more specific IgG subclass in the sample.

[0509] The invention also provides a method of detecting the presence of IgM in a sample, the method comprising: a) contacting a sample with a polypeptide, composition, or combination of the invention; and b) identifying and isolating the IgM specific cleavage products, wherein the presence of IgM specific cleavage products is indicative of the presence of IgM in the sample.

[0510] The invention also provides a method of detecting the presence of IgG and IgM in a sample, the method comprising: a) contacting a sample with a polypeptide, composition, or combination of the invention; and b) identifying and isolating the IgG and IgM specific cleavage products, wherein the presence of IgG and IgM specific cleavage products is indicative of the presence of IgG and IgM in the sample.

[0511] In some embodiments a method of the invention comprises an isolation step. The isolation step may comprise the use of gel electrophoresis and / or chromatography. In some embodiments a method of the invention comprises an identification and / or analysis step. The identification and / or analysis step may comprise the use of gel electrophoresis, chromatography, immunoblotting, ELISA and / or mass spectrometry.

[0512] In some embodiments the methods of the invention comprise identifying, isolating, and / or analysis step(s), comprising the use of any of or combination of the following: a) gel electrophoresis, b) immunoblotting, c) chromatography, d) ELISA, e) mass spectrometry.

[0513] Therapeutic methods and uses

[0514] The polypeptides, polynucleotides, expression vectors and / or compositions 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.

[0515] 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. Thus, the invention also provides a polypeptide of the invention for use in the treatment of the human or animal body.

[0516] Also provided herein is a method of prevention or treatment of disease or condition in a subject, which method comprises administering a polypeptide of the invention to the subject in a prophylactically or therapeutically effective amount. The polypeptide 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, intraosseous or other appropriate administration routes.

[0517] Polypeptides of the invention may be particularly useful in a method for the treatment or prevention of a disease or condition mediated by IgG and / or IgM antibodies, which in this context may be described as pathogenic IgG or IgM antibodies. Accordingly, the invention provides a polypeptide of the invention for use in the treatment or prevention of a disease or condition, particularly a disease or condition mediated by pathogenic IgG or IgM antibodies. The invention also provides a method of treating or preventing a disease or condition, particularly a disease or condition mediated by pathogenic IgG or IgM antibodies, comprising administering to an individual a polypeptide of the invention. The method may comprise repeat administration of the said polypeptide. The invention also provides a polypeptide of the invention for use in the manufacture of a medicament for the treatment or prevention of a disease or condition mediated by pathogenic IgG or IgM antibodies, particularly a disease or condition mediated by pathogenic IgG or IgM antibodies.

[0518] Numerous autoimmune diseases are mediated in whole or in part by pathogenic IgG or IgM 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 or IgM 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 of the invention may be used in a method to treat any of these diseases or conditions.

[0519] Pathogenic antibodies may recognise a tissue or organ transplant received by the subject. Thus, the disease treated or prevented by the polypeptide 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 the polypeptide may be any antibody response of the subject which reduces the efficacy or benefit to the subject of said therapeutic agent.

[0520] Polypeptides of the invention may also 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 IgG and / or IgM proteases. For example, the subject may comprise antibodies against IdeS and / or IdeZ and / or a protease derived from IdeS and / or IdeZ, such as Xork, due to a previous streptococcal infection, such as a S. pyogenes infection, or prior treatment with an IgG and / or IgM protease. Thus, the polypeptides of the invention may constitute a ‘second (or more)-line’ treatment.

[0521] Methods of the invention may involve cleavage of a sample containing a known antibody.

[0522] The antibody is preferably a recombinant monoclonal antibody. The antibody may be Abagovomab, Abciximab.Actoxumab, Adalimumab, Adecatumumab, Afelimomab, Afutuzumab, Alacizumab pegol, ALD5 I 8, Alemtuzumab, Alirocumab, Altumomabpentetate, 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, Epitumomabcituxetan, 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, Narnatumab, Natalizumab, Nebacumab, Necitumumab, Nerelimomab, Nesvacumab, Nimotuzumab, Nivolumab, Nofetumomabmerpentan, 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, U stekinumab, V apaliximab, V atelizumab, V edolizumab, Veltuzumab, Vepalimomab Vesencumab, Visilizumab, Volociximab, Vorsetuzumab mafodotin, Votumumab, Zalutumumab, Zanolimumab, Zatuximab, Ziralimumab or Zolimomab aritox.

[0523] Polypeptides of the invention may be useful in a method for improving the benefit to a subject of a therapeutic antibody or therapeutic agent, such as whose effect is mediated through Fc receptor binding. For example, the polypeptides of the invention may rapidly, temporarily and safely eliminate Fc receptor binding by all or substantially all endogenous IgG in a subject. Thus, an administered, such as a subsequently administered, therapeutic antibody (or therapeutic agent, such as that binds Fc receptor) would have enhanced efficacy because it does not need to compete with endogenous IgG for binding to Fc receptor. There is provided herein, a method of hydrolysing IgG, the method comprising contacting a sample comprising IgG with the polypeptide according to the invention, or the composition or combination according to the invention.

[0524] There is also provided herein, a method of hydrolysing IgG and IgM, the method comprising contacting a sample comprising IgG and IgM with the polypeptide according to the invention, or the composition or combination according to the invention.

[0525] The invention also provides for the use of the polypeptide according to the invention, the composition according to the invention, the combination according to the invention, or the kit according to the invention, for the degradation IgG in a sample.

[0526] The invention also provides for the use of the polypeptide according to the invention, the composition according to the invention, the combination according to the invention, or the kit according to the invention, for the degradation of IgG and IgM in a sample.

[0527] The invention also provides for the use of the polypeptide according to the invention, the composition according to the invention, the combination according to the invention, or the kit according to the invention, for the removal of IgG from a sample.

[0528] The invention also provides for the use of the polypeptide according to the invention, the composition according to the invention, the combination according to the invention, or the kit according to the invention, for the removal of IgG and IgM from a sample.

[0529] The invention also provides for the use of the polypeptide according to the invention, the composition according to the invention, the combination according to the invention, or the kit according to the invention, for the identification of IgG in a sample.

[0530] The invention also provides for the use of the polypeptide according to the invention, the composition according to the invention, the combination according to the invention, or the kit according to the invention, for the identification of IgG and IgM in a sample.

[0531] In embodiments of the methods or uses the IgG is human IgG.

[0532] In embodiments of the methods or uses the IgM is human IgM.

[0533] In embodiments of the methods or uses the sample is not an in vivo sample.

[0534] In embodiments of the methods or uses the sample is an ex vivo sample.

[0535] In embodiments of the methods or uses the sample is a sample derived from a human subject. The polypeptides of the invention have demonstrable activity in both simple and complex media, wherein the substrate may be considered substantially pure (e.g., a purified IgM sample) or substantially impure (e.g., a crude or complex sample).

[0536] In embodiments, the IgG in the sample is substantially pure. In embodiments, the IgG in the sample is substantially impure. In embodiments, the IgM in the sample is substantially pure. In embodiments, the IgM in the sample is substantially impure.

[0537] In embodiments, the sample comprises or consists of a complex medium, such as a complex biological medium. In embodiments, the sample comprises or consists of whole blood, a blood derived fraction, serum, and / or plasma.

[0538] In embodiments, the sample is a human sample.

[0539] In embodiments, the sample is an ex vivo sample.

[0540] In embodiments, the sample is a sample obtained during dialysis of a subject.

[0541] In some embodiments the sample is a sample obtained from a human subject, wherein the subject has no recognised disease or disorder. In some embodiments the sample is a sample obtained from a human subject, wherein the subject has no recognised disease or disorder associated with IgG. In some embodiments the sample is a sample obtained from a human subject, wherein the subject has no recognised disease or disorder associated with IgM.

[0542] In some embodiments the sample is a sample obtained from a human subject, wherein the subject has a recognised disease or disorder. In some embodiments the sample is a sample obtained from a human subject, wherein the subject has a recognised disease or disorder associated with IgG. In some embodiments the sample is a sample obtained from a human subject, wherein the subject has a recognised disease or disorder associated with IgM.

[0543] In some embodiments the sample is a sample obtained from a human subject, wherein the subject has: a) an autoimmune disease or disorder; b) cancer; c) suspected or known infection with a microorganism.

[0544] In some embodiments, the methods or uses of the invention may be for the treatment or prevention of an autoimmune disease or disorder.

[0545] In some embodiments, the methods or uses of the invention may be for the treatment or prevention of cancer.

[0546] Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL) and affects several thousand individuals on a yearly basis, with poor prognosis if left untreated. Even with treatment, the overall five-year survivability is low. The lymphoma B-cells most often have IgM as their B-cell receptor (BCR), and the removal or degradation of this receptor using a polypeptide of the invention may aid in the treatment of the cancer, e.g., due to the induction of apoptosis and the clearance of the lymphoma B-cells.

[0547] In some embodiments, the methods or uses of the invention may be for the treatment or prevention of diffuse large B-cell lymphoma.

[0548] In some embodiments, the microorganism is a bacterium, a virus, or a parasite.

[0549] In some embodiments, the microorganism is a pathogenic organism.

[0550] Gene Therapy

[0551] In some embodiments, the methods or uses of the invention may be used for gene therapy applications.

[0552] One of the primary challenges in gene therapy is the host immune response against the therapeutic vectors, often viral in nature (e.g. adenovirus vectors), used to deliver genes, including transgenes and edited genes. The immune system can recognize and target these vectors for inactivation, removal, and degradation, reducing their effectiveness and rendering repeated treatments less effective.

[0553] IgG and IgM have been demonstrated to significantly negatively affect gene transfer by adenovirus type 5, which is one of the most commonly used adenovirus vectors. For example, IgG and IgM reduces the transduction of the vector in the liver.

[0554] IgG and IgM proteases can enhance gene therapy treatments by degrading IgG or IgM antibodies that specifically target these therapeutic vectors, which may beneficially prolong the presence of the vectors in the body, allowing for more effective gene delivery and expression.

[0555] Furthermore, patients undergoing gene therapy often require multiple doses to achieve the desired therapeutic effect. However, the immune system can develop memory responses against the vectors after the first administration, leading to rapid neutralization in subsequent doses. By using IgG and / or IgM proteases to degrade pre-existing and newly formed IgG and / or IgM antibodies, the IgG and / or IgM mediated neutralizing effect may be reduced, improving the effectiveness and efficiency of repeated administration.

[0556] In addition, IgG and IgM antibodies can bind to viral vectors and both sequester the vectors to nontarget regions of the body, and also facilitate their clearance from the bloodstream. This sequestration and rapid clearance limits the distribution and time therapeutic vectors have to reach their target cells. Consequently, IgG and / or IgM proteases which are used to degrade these antibodies can promote a more widespread distribution of the vectors and increase the circulation time of therapeutic vectors in the bloodstream, thus improving their chances of reaching and transducing target cells. Typically, IgG and IgM antibodies trigger inflammatory responses in response to the delivery of therapeutic vectors to a subject through the activation of immune cells such as macrophages and complement pathways. This inflammation can damage tissues and cause side-effects, which may reduce the overall effectiveness of gene therapy. IgG and / or IgM proteases can mitigate these inflammatory responses by reducing the levels of IgG and / or IgM antibodies that activate these immune pathways, resulting in a safer therapeutic profile. Additionally, IgG proteases can be used in combination with other strategies to modulate the immune system, such as using immunosuppressive drugs or designing less immunogenic vectors. This multifaceted approach can further enhance the effectiveness and safety of gene therapies.

[0557] As such, degradation, inactivation, or removal of IgG and / or IgM using the polypeptides of the invention may be particularly useful in improving the efficiency, efficacy, and safety profile of gene therapy applications, in particular those utilising adenovirus type 5 vectors.

[0558] In embodiments, there is provided a method or use according to the invention, wherein the method or use is for the removal of IgG and / or IgM during gene therapy. ‘During’ may refer to the pre-treatment, concomitant treatment, or post-treatment of a subject or sample with a polypeptide of the invention.

[0559] IgG and IgM mediated diseases

[0560] In some embodiments, the methods or uses of the invention may be for the treatment or prevention of Systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and / or Sjogren’s syndrome.

[0561] Several autoimmune diseases, including SLE, RA, and Sjogren’s syndrome, are characterised by high levels of Rheumatoid factor (RF), which is often used as a prognostic biomarker for the diseases. High levels of RF are associated with worse outcomes and more severe pathogenesis. RF is typically an IgM-based antibody directed towards self IgG, which stimulates the formation of immune complexes that contribute to the disease, e.g., by causing chronic inflammation and joint destruction. The degradation or removal of IgM and / or IgG in such diseases using a polypeptide of the invention may therefore help in the treatment or prevention of these diseases.

[0562] In aspects of the invention, there is provided a polypeptide, combination, composition, or pharmaceutical composition according to the invention, for use in the manufacture of a medicament.

[0563] In aspects of the invention, there is provided a polypeptide, combination, composition, or pharmaceutical composition according to the invention, for use in therapy.

[0564] In aspects of the invention, there is provided a polypeptide, combination, composition, or pharmaceutical composition according to the invention, for use in the treatment or prevention of a disease or disorder associated with elevated IgM.

[0565] In one embodiment, there is provided a polypeptide, combination, composition, or pharmaceutical composition according to the invention, for use in the treatment or prevention of cancer. In one embodiment, there is provided a polypeptide, combination, composition, or pharmaceutical composition according to the invention, for use in the treatment or prevention of diffuse large B-cell lymphoma.

[0566] In one embodiment, there is provided a polypeptide, combination, composition, or pharmaceutical composition according to the invention, for use in the treatment or prevention of an autoimmune disease or disorder.

[0567] In one embodiment, there is provided a polypeptide, combination, composition, or pharmaceutical composition according to the invention, for use in the treatment or prevention of Systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and / or Sjogren’s syndrome.

[0568] In one embodiment, there is provided a polypeptide, combination, composition, or pharmaceutical composition according to the invention, for use in the treatment or prevention of IgG nephropathy.

[0569] In one embodiment, there is provided a polypeptide, combination, composition, or pharmaceutical composition according to the invention, for use in the treatment or prevention of IgM nephropathy.

[0570] EXAMPLES

[0571] Materials and Methods

[0572] Expression system

[0573] All genes were codon optimized for expression in E. coli and put into a pET21 a(+) vector with ampicillin selection, and a C-terminal His6 tag. The vector was added into competent BL21 (DE3) STAR E. coli cells.

[0574] Cultivation and expression

[0575] Cultivation of E. coli expressing recombinant IgG proteases was conducted in 400 mL cultures of LB broth (Vegitone), 0.1 mg / mL ampicillin, in 2 L baffled flasks. The shake-flasks were inoculated with 1 .3% pre-culture (grown at 37°C and 200 rpm o / n) at 120 rpm, 37°C, and induced with 1 mM IPTG at GD600 ca 0.8. The temperature was decreased to 20°C after induction, and protein expression was allowed to proceed for 4 h. Cells were harvested at 10,000 g for 10 minutes at 4°C, and stored at -80°C until further usage.

[0576] Small-scale IMAC purification

[0577] Harvested cells were resuspended in IMAC loading buffer (3 x PBS, 20 mM imidazole) at 5 mL / g of cells. The cell suspension was sonicated for 5 cycles at 5 min each (70%) and the cell debris removed via centrifugation at 16,000 g for 10 min at 4°C. The supernatant was applied to pre-equilibrated His GraviTrap columns washed with loading buffer, and the bound protein finally eluted in 3 x PBS, 500 mM imidazole buffer. The protein solution was buffer exchanged to TBS using PD-10 desalting columns, and all material analyzed on SDS-PAGE and NanoDrop 2000 for quality assessment. Activity

[0578] The activity of each of the enzymes was evaluated by incubation with different substrates at 37°C. Varying enzyme to substrate ratios and varying times were used as indicated in the specific experiments. Analysis was performed by reducing SDS-PAGE analysis.

[0579] Determination of digestion site on IgG (human and mouse)

[0580] Human IgG of subclasses 1 , 2 and 4 was incubated with and without addition of purified recombinant IgG proteases at 37°C for 1 hour using an enzyme to substrate ratio of 1 :20 (pg : pg) in TBS buffer. The commercially available human monoclonal antibodies, Trastuzumab-Herceptin hlgG1 , Panitumumab- Vectibix hlgG2 and Nivolumab - Orencia hlgG4 were used as substrates. In addition, two human lgG1 monoclonal antibodies with hinge-mutations were tested, Durvalumab - Imfinzi (LFLE mutation) and Rizankizumab - Skyrizi (LALA mutation). The mouse IgG samples were deglycosylated with GlyCINATOR after protease digestion to facilitate data evaluation. The hydrolysed IgG and IgG control samples were denatured in 4 M GnHCI (Sigma-Aldrich) and 100 mM DTT at 37°C for 30 min. The digestion products were separated on a reversed phase C4 column (Acquity premier BEHC4, 450A, 2.7pm 2.1 x 100 mm, Waters) using an acetonitrile gradient and analyzed by ESI-QTOF-MS (Bruker Impact II). Deconvolution was done using Bruker Compass DataAnalysis version 5.2 and the MaxEnt algorithm.

[0581] Presence of anti-enzyme antibodies in IVIG

[0582] IgG proteases were separated using SDS-PAGE. One of the gels was stained with coomassie blue, and the proteins on the corresponding gel were transferred to a membrane using the Trans-Blot Turbo system (Bio-Rad). After blocking of the membrane in a casein solution, the primary antibody (IVIG derived from human plasma- Privigen, 5 pg / mL) was added and incubated for 2 h in RT. The membrane was washed, and a secondary antibody (alkaline phosphatase conjugated a-human IgG HC, 1 :2000) was added and incubated for 1 h at RT. The membrane was thoroughly washed before the addition of the chromogenic substrate BCIP / NBT.

[0583] Digestion in human serum

[0584] The ability to digest human IgG in serum was evaluated by the addition of IgG proteases at E:S ratio 1 :20-1 :40 (pg:pg) using the estimation that the serum holds approximately 10 mg IgG / ml. The digestion was performed for 2 h at 37°C and the samples were evaluated on SDS-PAGE.

[0585] Example 1

[0586] Sequence homology

[0587] All IgG proteases in this application share limited sequence homology to existing IgG proteases (e.g. IdeS and Xork), with 27.6-32.0% identity to IdeS and 29.9-41 .3% homology to Xork (Figure 1). Protease O_mod and R_mod share some similarity to each other (60.2%), and which is not demonstrated in this graph, St1 and St2, since St2 consists essentially of two St1 domains with a linker structure between the two domains, and are derived from the same species.

[0588] Example 2

[0589] Production and purification of proteases All recombinantly engineered proteins could be produced in E. coli to high purity and homogeneity. (Figure 2A and B) and could be produced in E. coli, solubilized (supernatant), and affinity-purified to high purity and yield. The full-length protein St1_mod is expressed as a 38.6 kDa protein. The full-length protein St2_mod is expressed as a 94.65 kDa protein. The full-length protein C_mod is expressed as a 40.52 kDa protein, exhibiting a smaller 30 kDa fragment. The full-length protein O_mod is expressed as a 37.66 kDa protein. The full-length protein R_mod is expressed as a 38.78 kDa protein. (B) The full-length protein Xork-St2L-lgMBRAZOR is expressed as a 92.73 kDa protein. The full-length protein St1-St2L-lgMBRAZOR is expressed as a 94.65 kDa protein. The full-length protein C_mod_V2 is expressed as a 40.52 kDa protein as can be seen in Figure 12, and does not exhibit the smaller 30 kDa fragment present in the purification of C_mod as seen in Figure 2A, indicating improved stability compared to C_mod.

[0590] Example 3

[0591] Specificity of proteases

[0592] The proteases of the invention and the protease SpeB, a broad-acting protease utilized as a positive control, were incubated with the general protease substrate Casein (conjugated to FiTC) for 1 hour at 37°C using 3 pg of each enzyme and 0.5 pg of substrate at an enzyme to substrate ratio of 6:1 (pg:pg). None of the proteases of the invention exhibited activity against the general protease substrate casein conjugated to FITC (Figure 3), yielding similar background levels of fluorescence as the IgG-specific protease IdeS, while the positive control SpeB exhibited high activity.

[0593] Example 4

[0594] Specificity of the novel proteases towards Ig classes and IgG subclasses

[0595] Specific proteases of the invention were incubated with specific target IgG molecules. Figure 4B: proteases St1_mod and St2_mod were incubated with IgG 1 , lgG2, lgG3, and lgG4 for 1 hours at 37°C at an enzyme to substrate ratio of 1 :10 (pg:pg). Figure 4C: proteases O_mod, R_mod, and C_mod were incubated with lgG1 , lgG2, lgG3, and lgG4 for 2 hours at 37°C at an enzyme to substrate ratio of 1 :20 (pg:pg). Figure 4D: proteases St1_mod, O_mod, R_mod, and C_mod were incubated with IgA and IgM for 2 hours at 37°C at an enzyme to substrate ratio of 1 :10 (pg:pg). Figure 4E: proteases St1_mod and St2_mod were incubated with IgA and IgM overnight at 37°C at an enzyme to substrate ratio of 1 :1 (pg:pg). As summarized in Figure 4A, and with data in Figures 4B to 4E, all constructs had activity, though varying, against human lgG1 to 4, and no activity against IgM except forthe engineered chimeric proteins (St1-St2L-lgMBRAZOR; Xork-St2L-lgMBRAZOR) where an IgM protease has been fused to an IgG protease for increased functionality. Additionally, none of the proteases had protease activity vs IgA, as evidenced by Figure 4D, or in the instances of the engineered chimeric proteins (St1-St2L- IgMBRAZOR; Xork-St2L-lgMBRAZOR), by virtue of the absence of a domain having IgA protease activity. R_mod, while not demonstrating high activity against any of the modified hinge-region sequences (Figure 6), has a very high preference for hlgG2 (Figure 4C).

[0596] Example 5

[0597] Specificity of the novel proteases towards Ig classes and IgG subclasses of non-human species Several relevant model animals have IgG being targeted by the identified IgG proteases. The ability to digest non-human IgG in serum was evaluated by the addition of the IgG proteases at Enzyme:Substrate (E:S) ratio 1 :10 (pg:pg), using 1 mg / mL of substrate (i.e. Ig) and the digestion was performed for 3 hours at 37°C and the samples were evaluated on SDS-PAGE. The novel IgG proteases exhibit IgG subclass specificity and cross species reactivity. Summary of protease activity vs mouse, rabbit, and monkey IgG (Figure 5A). A single asterisk (*) indicates activity towards mouse lgG2a and no activity towards IgG 1 , lgG2b, and lgG3. A double asterisk (**) indicates activity towards mouse lgG2a and no activity towards lgG1 , lgG2b. Activity of ST1_mod and ST2_mod, vs mouse IgG subclasses is shown in Figure 5B, wherein the proteases of the invention were incubated with the specific target IgG molecules for 3 hours at 37°C. The proteases exhibit comparatively lower activity vs mlgG1 (approx. 55 kDa band), and comparatively higher activity vs mlgG2a, mlgG2b and mlgG3 (approx. 55 kDa bands).

[0598] Activity of O_mod, R_mod, and C_mod vs rabbit and mouse IgG subclasses is depicted in Figure 5C. The proteases do not exhibit activity preference vs rabbit or mouse IgG or IgG subclasses.

[0599] Example 6

[0600] Analysis of the specificity of novel proteases O_mod, R_mod, and C_mod towards modified IgG variants

[0601] The O_mod, C_mod, and R_mod proteases of the invention were incubated with specific target hinge- modified IgG molecules for 2 hours at 37°C at an enzyme to substrate ratio of 1 :20 (pg:pg). The C_mod protease exhibits high activity against hinge-modified IgG variant lgG1 -LALA (L234A, L235A) and also a degree of activity towards hinge-modified IgG variant lgG1-LFLE (L234F, L235E, P331 S) (see Figure 6). The O_mod protease also exhibits activity against hinge-modified IgG variant lgG1-LALA (L234A, L235A). The R_mod protease does not exhibit activity towards any of the hinge-modified IgG variants tested (see Figure 6).

[0602] The protease C_mod and a reference IdeS derived IgG protease, the protease FabRICATOR® (Genovis AB) were also incubated with the hlgG4-FALA antibodies Galcanezumab and Talquetamab at a concentration of 1 U of enzyme per pg of antibody for 30 minutes at 37°C. The C_mod protease exhibits high activity against both hinge-modified lgG4-FALA variants (see Figure 14), and significantly improved hydrolysis efficiency compared to the IdeS derived IgG protease FabRICATOR® (Genovis AB).

[0603] Example 7

[0604] Specificity of novel engineered chimeric proteases St1-St2L-lgMBRAZOR; Xork-St2L- IgMBRAZOR towards IgM and IgG.

[0605] The chimeric proteases of the invention were incubated with specific target IgG and IgM molecules for 3 hours at 37°C at an enzyme to substrate ratio of 1 :10 (pg:pg). The engineered chimeric proteins, comprising an IgM protease and an IgG protease for increasing the variety of target substrates, St1- St2L-lgMBRAZOR and Xork-St2L-lgMBRAZOR, were incubated with IgM or IgG to evaluate their activity towards each Ig class. As evidenced by Figure 7, both chimeric proteases exhibit activity against human IgM and IgG. Example 8

[0606] Protease Active in serum and vs IVIG

[0607] The ability of the proteases to digest human IgG in serum was evaluated by the addition of IgG proteases IdeS, Xork, St1_mod, or St2_mod to serum at Enzyme:Substrate (E:S) ratio 1 :20-1 :40 (pg:pg) using the estimation that the serum holds approximately 10 mg / mL of IgG. The digestion was performed for 2 hours at 37°C and the samples were evaluated on SDS-PAGE. As evidenced in Figure 8, St1_mod and St2_mod proteases are active in serum, wherein the heavy chain of IgG as highlighted by the arrow at around 55kDa (situated directly below the thickest band at around 60 kDa indicating albumin), disappears or at least the visual intensity of the band gets much weaker upon the addition of either IdeS, Xork, St1_mod, or St2_mod. Additionally, the two hydrolysis products from the hinge- cleaved heavy chain shows up as two bands around 25-30 kDa as shown by the bracket, with one of the hydrolysis product bands being mostly hidden behind the light chain at approximately 25 kDa, which is also visible in the untreated plasma.

[0608] The ability of the proteases to digest human IgG in serum was evaluated by the addition of IgG proteases O_mod, C_mod, and R_mod to IVIG at an Enzyme:Substrate (E:S) ratio of 1 :10 (pg:pg). The digestion was performed for 2 hours at 37°C in PBS and the samples were evaluated on SDS-PAGE. As evidenced in Figure 9, the O_mod, C_mod, and R_mod constructs are active against IVIG, wherein the intensity of the approximately 55 kDa band corresponding to IVIG decreases in the protease treated conditions, yielding an approximately 30 to 33 kDa band. The 55 kDa band corresponding to IVIG is present at a weaker intensity for the IVIG samples treated with the proteases O_mod and C_mod, suggesting that O_mod and C_mod hydrolyse IVIG more efficiently than R_mod.

[0609] Example 9

[0610] Presence of anti-enzyme antibodies (IgG)

[0611] All enzymes were separated using SDS-PAGE gel and blotted towards IVIG to investigate presence and prevalence of anti-enzyme antibodies. One of the gels was stained with coomassie blue, and the proteins on the corresponding gel were transferred to a membrane using the Trans-Blot Turbo system (Bio-Rad). After blocking of the membrane in a casein solution, the primary antibody (IVIG derived from human plasma- Privigen, 5 pg / mL) was added and incubated for 2 hours at RT. The membrane was washed, and a secondary antibody (alkaline phosphatase conjugated a-human IgG HC, 1 :2000) was added and incubated for 1 hour at RT. The membrane was thoroughly washed before the addition of the chromogenic substrate BCIP / NBT.

[0612] As evidenced in Figure 10A, of the group of polypeptides: Ides, Xork, St1_mod, and St2_mod, only IdeS had significant antibodies directed to the enzyme, as indicated by the approximately 38 kDa band. Similarly, as evidenced in Figure 10B, of the group of polypeptides: Ides, Xork, St1_mod, and O_mod, R_mod, and C_mod, only IdeS had significant antibodies directed to the enzyme, as indicated by the approximately 38 kDa band on the ‘anti-enzyme blot’ panel.

[0613] Example 10

[0614] Analysis of site-specific hydrolysis of IgG by ESI-QTOF-MS The hydrolysis sites targeted by the different proteases of the invention as detected by ESI-QTOF-MS data is summarized in Figure 11A and also in Table 2, and presented in detail for lgG1 (Figure 1 1 B), lgG2 (Figure 11 C), lgG3 (Figure 1 1 D), lgG4 (Figure 1 1 E), lgG1 -LALA (Figure 11 F), and St1_mod activity on lgG1 (Figure 11 G). Figure 1 1 B: the Fd’ fragments of approximately 25300 Da could be assigned to the theoretical mass value of amino acids 1 -239, and the scFc fragments of approximately 23750 Da could be assigned to the theoretical mass value of amino acids 240-449. Figure 11 C: the Fd’ fragments of approximately 24900 Da could be assigned to the theoretical mass value of amino acids 1-234, and the scFc fragments of approximately 25000 Da could be assigned to the theoretical mass value of amino acids 235-444. Figure 11 D: the Fd’ fragments of approximately 30500 Da could be assigned to the theoretical mass value of amino acids 1 -234, and the scFc fragments of approximately 25000 Da could be assigned to the theoretical mass value of amino acids 235-444. Figure 11 E: the Fd’ fragments of approximately 24500 Da could be assigned to the theoretical mass value of amino acids 1 -229, and the scFc fragments of approximately 23700 Da could be assigned to the theoretical mass value of amino acids 230-449. Figure 11 F: the Fd’ fragments of approximately 25500 Da could be assigned to the theoretical mass value of amino acids 1-239 or amino acids 1-238, and the scFc fragments of approximately 25000 to 25500 Da could be assigned to the theoretical mass value of amino acids 239-449 or 240-449. Figure 11 G: the Fd’ fragments of approximately 25300 Da could be assigned to the theoretical mass value of amino acids 1 -238, and the scFc fragments of approximately 25050 Da could be assigned to the theoretical mass value of amino acids 239-449.

[0615] All enzymes cleave at, or in the vicinity of the IdeS cleavage site. St1_mod and St2_mod hydrolyze both lgG1 and lgG4 at PAPELL / GGPSVF and PAPEFL / GGPSVF respectively. O_mod, C_mod, and R_mod all cleave at the same site as IdeS, but for lgG1 , both O_mod and R_mod hydrolyze APELL / G / GPSVFL, e.g., at two sites. St1_mod hydrolyzes both mouse lgG2b at CHKCPAPNLE / GGPSVFIFPPNI and lgG3 at GSSCPPGNIL I GGPSVFIFPPKP (Figure 13A and B). Figure 13A: the Fc fragments of approximately 24200 Da could be assigned to the theoretical mass value of amino acids 124-335 based on the sequence of sequence of Uniprot constant HC secreted P01867-2. Figure 13B: The Fc fragments of approximately 24000 Da could be assigned to the theoretical mass value of amino acids 118-328 based on the sequence of sequence of Uniprot constant HC secreted P02987-2.

[0616] CLAUSES

[0617] Clause Group A:

[0618] A1. A polypeptide, or engineered polypeptide, having immunoglobulin protease activity, wherein said polypeptide comprises, consists essentially of, or consists of:

[0619] (a) the amino acid sequence of SEQ ID NO: 1 ; or

[0620] (b) a variant of SEQ ID NO: 1 having at least 80% sequence identity thereto; or

[0621] (c) an amino acid sequence which is a fragment of the sequence of either (a) or (b).

[0622] A2. A polypeptide, or engineered polypeptide, having immunoglobulin protease activity, wherein said polypeptide comprises, consists essentially of, or consists of: (a) the amino acid sequence of SEQ ID NO: 2; or

[0623] (b) a variant of SEQ ID NO: 2 having at least 80% sequence identity thereto; or

[0624] (c) an amino acid sequence which is a fragment of the sequence of either (a) or (b).

[0625] A3. The polypeptide according to clause A1 , wherein the variant has at least about 85% sequence identity, at least about 90% sequence identity, at least about 91 % sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 .

[0626] A4. The polypeptide according to clause A2, wherein the variant has at least about 85% sequence identity, at least about 90% sequence identity, at least about 91 % sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0627] A5. The polypeptide according to clause A1 , wherein the fragment of the sequence of SEQ ID NO: 1 is at least about 100 amino acids in length, at least about 200 amino acids in length, at least about 300 amino acids in length, at least about 400 amino acids in length, at least about 500 amino acids in length, at least about 600 amino acids in length, at least about 700 amino acids in length, at least about 800 amino acids in length, at least about 900 amino acids in length, at least about 1000 amino acids in length, at least about 1 100 amino acids in length, at least about 1200 amino acids in length, at least about 1300 amino acids in length, at least about 1400 amino acids in length, at least about 1500 amino acids in length, at least about 1600 amino acids in length, or at least about 1700 amino acids in length.

[0628] A6. The polypeptide according to clause A1 , wherein the fragment of the sequence of SEQ ID NO:

[0629] 1 is at least about 100 amino acids in length, at least about 150 amino acids in length, at least about 200 amino acids in length, at least about 250 amino acids in length, at least about 300 amino acids in length, at least about 350 amino acids in length, at least about 400 amino acids in length, at least about 450 amino acids in length, at least about 500 amino acids in length, at least about 550 amino acids in length, or at least about 600 amino acids in length.

[0630] A7. The polypeptide according to clause A2, wherein the fragment of the sequence of SEQ ID NO:

[0631] 2 is at least about 100 amino acids in length, at least about 150 amino acids in length, at least about 200 amino acids in length, at least about 250 amino acids in length, at least about 300 amino acids in length, at least about 350 amino acids in length, at least about 400 amino acids in length, at least about 450 amino acids in length, at least about 500 amino acids in length, at least about 550 amino acids in length, or at least about 600 amino acids in length.

[0632] A8. The polypeptide according to any one of clauses A1 to A7, wherein the fragment comprises, consists essentially of, or consists of an N terminal fragment. A9. The polypeptide according to any one of clauses A1 to A7, wherein the fragment comprises, consists essentially of, or consists of a C terminal fragment.

[0633] A10. The polypeptide according to any one of clauses A1 to A9, wherein the polypeptide comprises a methionine at the N terminus.

[0634] A11 . The polypeptide according to any one of clauses A1 to A10, wherein the polypeptide comprises a protein purification tag at the C terminus.

[0635] A12. The polypeptide according to any one of clauses A1 to A11 , wherein the polypeptide comprises a protein purification tag at the N terminus.

[0636] A13. The polypeptide according any one of clauses A11 to A12, wherein the tag is joined to the N and / or C terminus by a linker.

[0637] A14. The polypeptide according to clause A13, wherein the linker comprises one, or more than one, of any or a combination of glycine, glycine-serine, serine-glycine, or serine residues.

[0638] A15. The polypeptide according to clause A13 or A14, wherein the linker is GSS.

[0639] A16. The polypeptide according to any one of clauses A11 to A15, wherein the tag is a His tag.

[0640] A17. The polypeptide according to any one of clauses A11 to A15, wherein the tag is a polyhistidine- tag.

[0641] A18. The polypeptide according to any one of clauses A11 to A15, wherein the tag is a 6xHis tag.

[0642] A19. The polypeptide according to any one of clause A1 , A3, A5, A6, or clause A8 to A18 when dependent on clause A1 , wherein the fragment of the sequence of SEQ ID NO: 1 comprises the sequence from about sequence position 100 to 500, about 110 to 490, about 120 to 480, about 130 to 470, about 140 to 460, about 150 to 450, or about 160 to 440, e.g. from sequence position 138 to 466.

[0643] A20. The polypeptide according to any one of the preceding clauses, comprising, consisting essentially of, or consisting of SEQ ID NO: 3.

[0644] A21 . The polypeptide according to any one of the preceding clauses, wherein the polypeptide is provided in solution, lyophilised, or immobilised.

[0645] A22. The polypeptide according to any one of the preceding clauses which has protease activity against an immunoglobulin molecule comprising a CH2 / hinge sequence, optionally wherein the CH2 / hinge sequence comprises any one of SEQ ID NOs: 28, 31 , 33, 35, or 36. A23. The polypeptide according to clause A22, wherein the immunoglobulin molecule is lgG1 and the CH2 / hinge sequence comprises SEQ ID NO: 28.

[0646] A24. The polypeptide according to any one of clauses A22 to A23, wherein the immunoglobulin molecule is lgG2 and the CH2 / hinge sequence comprises SEQ ID NO: 31 .

[0647] A25. The polypeptide according to any one of clauses A22 to A24, wherein the immunoglobulin molecule is lgG4 and the CH2 / hinge sequence comprises SEQ ID NO: 33.

[0648] A26. The polypeptide according to any one of clauses A22 to A25, wherein the immunoglobulin molecule is mlgG2b and the CH2 / hinge sequence comprises SEQ ID NO: 35.

[0649] A27. The polypeptide according to any one of clauses A22 to A26, wherein the immunoglobulin molecule is mlgG3 and the CH2 / hinge sequence comprises SEQ ID NO: 36.

[0650] A28. The polypeptide according to any one of clauses A22 to A27, wherein the polypeptide cleaves the CH2 / hinge sequence between amino acid positions 6 and 7 of at least one of SEQ ID NOs: 28, 31 , or 33, or between amino acid positions 10 and 11 of at least one of SEQ ID NOs: 35 or 36.

[0651] A29. The polypeptide according to any one of clauses A22 to A28, wherein the polypeptide cleaves the CH2 / hinge sequence between amino acid positions 6 and 7 of all of SEQ ID NOs: 28, 31 , or 33, or between amino acid positions 10 and 11 of all of SEQ ID NOs: 35 or 36.

[0652] A30. The polypeptide according to any one of the preceding clauses, wherein the polypeptide cleaves IgG.

[0653] A31. The polypeptide according to any one of the preceding clauses, wherein the polypeptide cleaves human IgG.

[0654] A32. The polypeptide according to any one of the preceding clauses, wherein the immunoglobulin protease activity is IgG specific protease activity.

[0655] A33. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits lower protease activity against IgM as compared to IgG.

[0656] A34. The polypeptide according to clause A33, wherein the protease activity against IgM is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the protease activity against IgG.

[0657] A35. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits lower protease activity against IgA as compared to IgG. A36. The polypeptide according to clause A35, wherein the protease activity against IgA is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the protease activity against igG.

[0658] A37. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits lower protease activity against IgD as compared to IgG.

[0659] A38. The polypeptide according to clause A37, wherein the protease activity against IgD is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the protease activity against IgG.

[0660] A39. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits lower protease activity against IgE as compared to IgG.

[0661] A40. The polypeptide according to clause A39, wherein the protease activity against IgE is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the protease activity against IgG.

[0662] A41. The polypeptide according to any one of the preceding clauses, wherein the polypeptide has protease activity against an IgG comprising a CH2 / hinge sequence, optionally wherein the CH2 / hinge sequence comprises any one of SEQ ID NOs: 28, 31 , or 33.

[0663] A42. The polypeptide according to any one of the preceding clauses, wherein the polypeptide has protease activity against any of IgG 1 , lgG2, lgG3, or lgG4, or mouse lgG2b or lgG3.

[0664] A43. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits lower protease activity against human lgG2 as compared to any one other human IgG subclass.

[0665] A44. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits greater protease activity against human lgG1 than against human lgG2.

[0666] A45. The polypeptide according to clause A44, wherein the protease activity against lgG2 is less than about 99% of the protease activity against lgG1 , such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the protease activity against lgG1 . A46. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits greater protease activity against human lgG3 than against human lgG2.

[0667] A47. The polypeptide according to clause A46, wherein the activity against lgG2 is less than about 99% of the protease activity against lgG3, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the protease activity against lgG3.

[0668] A48. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits greater protease activity against human lgG4 than against human lgG2.

[0669] A49. The polypeptide according to clause A48, wherein the protease activity against lgG2 is less than about 99% of the protease activity against lgG4, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the protease activity against lgG4.

[0670] A50. The polypeptide according to any one of the preceding clauses, wherein the immunoglobulin is from a mammal; optionally wherein the mammal is selected from human, primate, horse, cow, sheep, goat, rabbit, or rodent (such as mouse, guinea pig, rat).

[0671] A51 . The polypeptide according to any one of the preceding clauses, wherein the polypeptide is provided in solution, lyophilised, or immobilised, optionally together with an effective amount of at least one excipient, for example, wherein the excipient is a preservative.

[0672] A52. A polynucleotide encoding a polypeptide having protease activity according to any one of clauses A1 to A51 , the polynucleotide comprising:

[0673] (a) the polynucleotide sequence of SEQ ID NO: 20;

[0674] (b) a variant of SEQ ID NO: 20 having at least 70% sequence identity thereto; or

[0675] (c) a polynucleotide fragment of either (a) or (b).

[0676] A53. The polynucleotide according to clause A52, wherein the variant has at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, or at least about 99% sequence identity to the polynucleotide sequence of SEQ ID NO: 20.

[0677] A54. The polynucleotide according to clause A52, wherein the polynucleotide fragment is at least about 300 nucleotides in length, at least about 400 nucleotides in length, at least about 500 nucleotides in length, at least about 600 nucleotides in length, at least about 700 nucleotides in length, at least about 800 nucleotides in length, at least about 900 nucleotides in length, or at least about 1000 nucleotides in length. A55. A polynucleotide encoding a polypeptide according to any one of clauses A1 to A51 .

[0678] A56. A vector encoding or comprising a polynucleotide according to any one of clauses A52 to A55.

[0679] A57. An expression vector which comprises the polynucleotide sequence according to any one of clauses A52 to A55.

[0680] A58. A host cell comprising the polynucleotide, vector, or expression vector of any one of clauses A52 to A57.

[0681] A59. The host cell of clause A58, wherein the host cell is a bacterial cell.

[0682] A60. The host cell of clause A58 or A59, wherein the host cell is not streptococcal species.

[0683] A61 . The host cell of clause A58 or A59X, wherein the host cell is E. coll.

[0684] A62. The polynucleotide, vector, or expression vector according to any one of clauses A52 to A57, wherein the polynucleotide, vector, or expression vector is provided in solution, lyophilised, or immobilised, optionally together with an effective amount of at least one excipient, for example, wherein the excipient is a preservative.

[0685] A63. A cell comprising the polypeptide of any one of clauses A1 to A51 , the polynucleotide of any one of clause A52 to A55, the vector of clause A56, or expression vector of A57.

[0686] Clause Group B:

[0687] B1. A polypeptide, or engineered polypeptide, having immunoglobulin protease activity, wherein said polypeptide comprises, consists essentially of, or consists of:

[0688] (a) the amino acid sequence of SEQ ID NO: 4; or

[0689] (b) a variant of SEQ ID NO: 4 having at least 80% sequence identity thereto; or

[0690] (c) an amino acid sequence which is a fragment of either (a) or (b).

[0691] B2. A polypeptide, or engineered polypeptide, having immunoglobulin protease activity, wherein said polypeptide comprises, consists essentially of, or consists of:

[0692] (a) the amino acid sequence of SEQ ID NO: 5; or

[0693] (b) a variant of SEQ ID NO: 5 having at least 80% sequence identity thereto; or

[0694] (c) an amino acid sequence which is a fragment of the sequence of either (a) or (b).

[0695] B3. The polypeptide according to clause B1 , wherein the variant has at least about 85% sequence identity, at least about 90% sequence identity, at least about 91 % sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 4.

[0696] B4. The polypeptide according to clause B2, wherein the variant has at least about 85% sequence identity, at least about 90% sequence identity, at least about 91 % sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 5.

[0697] B5. The polypeptide according to clause B1 , wherein the fragment of the sequence of SEQ ID NO: 4 is at least about 100 amino acids in length, at least about 200 amino acids in length, at least about 300 amino acids in length, at least about 400 amino acids in length, at least about 500 amino acids in length, at least about 600 amino acids in length, at least about 700 amino acids in length, at least about 800 amino acids in length, at least about 900 amino acids in length, at least about 1000 amino acids in length, at least about 1 100 amino acids in length, at least about 1200 amino acids in length, at least about 1300 amino acids in length, at least about 1400 amino acids in length, at least about 1500 amino acids in length, at least about 1600 amino acids in length, at least about 1700 amino acids in length, or at least about 1800 amino acids in length.

[0698] B6. The polypeptide according to clause B1 , wherein the fragment of the sequence of SEQ ID NO:

[0699] 4 is at least about 100 amino acids in length, at least about 150 amino acids in length, at least about 200 amino acids in length, at least about 250 amino acids in length, at least about 300 amino acids in length, at least about 350 amino acids in length, at least about 400 amino acids in length, at least about 450 amino acids in length, at least about 500 amino acids in length, at least about 550 amino acids in length, or at least about 600 amino acids in length.

[0700] B7. The polypeptide according to clause B2, wherein the fragment of the sequence of SEQ ID NO:

[0701] 5 is at least about 100 amino acids in length, at least about 150 amino acids in length, at least about 200 amino acids in length, at least about 250 amino acids in length, at least about 300 amino acids in length, at least about 350 amino acids in length, at least about 400 amino acids in length, at least about 450 amino acids in length, at least about 500 amino acids in length, at least about 550 amino acids in length, or at least about 600 amino acids in length.

[0702] B8. The polypeptide according to any one of clauses B1 to B7, wherein the fragment comprises, consists essentially of, or consists of an N terminal fragment.

[0703] B9. The polypeptide according to any one of clauses B1 to B7, wherein the fragment comprises, consists essentially of, or consists of a C terminal fragment.

[0704] B10. The polypeptide according to any one of clauses B1 to B9, wherein the polypeptide comprises a methionine at the N terminus. B11 . The polypeptide according to any one of clauses B1 to B10, wherein the polypeptide comprises a protein purification tag at the C terminus.

[0705] B12. The polypeptide according to any one of clauses B1 to B11 , wherein the polypeptide comprises a protein purification tag at the N terminus.

[0706] B13. The polypeptide according to any one of clauses B1 to B12, wherein the tag is joined to the N and / or C terminus by a linker.

[0707] B14. The polypeptide according to clause B13, wherein the linker comprises one, or more than one, of any or a combination of glycine, glycine-serine, serine-glycine, or serine residues.

[0708] B15. The polypeptide according to clause B13 or B14, wherein the linker is GSS.

[0709] B16. The polypeptide according to any one of clauses B11 to B15, wherein the tag is a His tag.

[0710] B17. The polypeptide according to any one of clauses B1 to B15, wherein the tag is a polyhistidine- tag.

[0711] B18. The polypeptide according to any one of clauses B1 to B15, wherein the tag is a 6xHis tag.

[0712] B19. The polypeptide according to any one of clause B1 , B3, B5, B6, or clause B8 to B18 when dependent on clause B1 , wherein the fragment of the sequence of SEQ ID NO: 4 comprises the sequence from about sequence position 50 to 1400, about 100 to 1300, about 110 to 1250, about 115 to 1200, about 120 to 1150, about 125 to 1100, about 130 to 1050, about 135 to 1000, about 140 to 975, about 145 to 950, about 150 to 925, e.g. from sequence position 138 to 964.

[0713] B20. The polypeptide according to any one of the preceding clauses, comprising SEQ ID NO: 6.

[0714] B21. The polypeptide according to any one of the preceding clauses, wherein the polypeptide is provided in solution, lyophilised, or immobilised.

[0715] B22. The polypeptide according to any one of the preceding clauses which has protease activity against an immunoglobulin molecule comprising a CH2 / hinge sequence, optionally wherein the CH2 / hinge sequence comprises any one of SEQ ID NOs: 28, 31 , 33, 35, or 36.

[0716] B23. The polypeptide according to clause B22, wherein the immunoglobulin molecule is lgG1 and the CH2 / hinge sequence comprises SEQ ID NO: 28.

[0717] B24. The polypeptide according to any one of clauses B22 or B23, wherein the immunoglobulin molecule is lgG2 and the CH2 / hinge sequence comprises SEQ ID NO: 31 . B25. The polypeptide according to any one of clauses B22 to B24, wherein the immunoglobulin molecule is lgG4 and the CH2 / hinge sequence comprises SEQ ID NO: 33.

[0718] B26. The polypeptide according to any one of clauses B22 to B25 wherein the immunoglobulin molecule is mlgG2b and the CH2 / hinge sequence comprises SEQ ID NO: 35.

[0719] B27. The polypeptide according to any one of clauses B22 to B26, wherein the immunoglobulin molecule is mlgG3 and the CH2 / hinge sequence comprises SEQ ID NO: 36.

[0720] B28. The polypeptide according to any one of clauses B22 to B27, wherein the polypeptide cleaves the CH2 / hinge sequence between amino acid positions 6 and 7 of at least one of SEQ ID NOs: 28, 31 , or 33, or between amino acid positions 10 and 11 of at least one of SEQ ID NOs: 35 or 36.

[0721] B29. The polypeptide according to any one of clauses B22 to B28, wherein the polypeptide cleaves the CH2 / hinge sequence between amino acid positions 6 and 7 of all of SEQ ID NOs: 28, 31 , or 33, or between amino acid positions 10 and 11 of all of SEQ ID NOs: 35 or 36.

[0722] B30. The polypeptide according to any one of the preceding clauses, wherein the polypeptide cleaves IgG.

[0723] B31. The polypeptide according to any one of the preceding clauses, wherein the polypeptide cleaves human IgG.

[0724] B32. The polypeptide according to any one of the preceding clauses, wherein the immunoglobulin protease activity is IgG specific protease activity.

[0725] B33. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits lower protease activity against IgM as compared to IgG.

[0726] B34. The polypeptide according to clause B33, wherein the protease activity against IgM is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the level of protease activity against IgG.

[0727] B35. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits lower protease activity against IgA as compared to IgG.

[0728] B36. The polypeptide according to clause B35, wherein the protease activity against IgA is less than less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the protease activity against IgG. B37. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits lower protease activity against IgD as compared to IgG.

[0729] B38. The polypeptide according to clause B37, wherein the protease activity against IgD is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the protease activity against IgG.

[0730] B39. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits lower protease activity against IgE as compared to IgG.

[0731] B40. The polypeptide according to clause B39, wherein the protease activity against IgE is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the protease activity against IgG.

[0732] B41. The polypeptide according to any one of the preceding clauses, wherein the polypeptide has protease activity against an IgG comprising a CH2 / hinge sequence, optionally wherein the CH2 / hinge sequence comprises any one of SEQ ID NOs: 28, 31 , or 33.

[0733] B42. The polypeptide according to any one of the preceding clauses, wherein the polypeptide has protease activity against any of IgG 1 , lgG2, lgG3, or lgG4, or mouse lgG2b or lgG3.

[0734] B43. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits lower protease activity against human lgG2 as compared to any one other human IgG subclasses.

[0735] B44. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits greater protease activity against human lgG1 than against human lgG2.

[0736] B45. The polypeptide according to clause B44, wherein the protease activity against lgG2 is less than about 99% of the level of activity against lgG1 , such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the protease activity against IgG 1 .

[0737] B46. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits greater protease activity against human lgG3 than human lgG2. B47. The polypeptide according to clause B46, wherein the protease activity against lgG2 is less than about 99% of the level of activity against lgG3, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the protease activity against lgG3.

[0738] B48. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits greater protease activity against human lgG4 than human lgG2.

[0739] B49. The polypeptide according to clause B48, wherein the protease activity against lgG2 is less than about 99% of the level of activity against lgG4, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the protease activity against lgG4.

[0740] B50. The polypeptide according to any one of the preceding clauses, wherein the immunoglobulin is from a mammal; optionally wherein the mammal is selected from human, primate, horse, cow, sheep, goat, rabbit, or rodent (such as mouse, guinea pig, rat).

[0741] B51. The polypeptide according to any one of the preceding clauses, wherein the polypeptide is provided in solution, lyophilised, or immobilised, optionally together with an effective amount of at least one excipient, for example, wherein the excipient is a preservative.

[0742] B52. A polynucleotide encoding a polypeptide having protease activity of any one of clauses B1 to B51 , the polynucleotide comprising:

[0743] (a) the polynucleotide sequence of SEQ ID NO: 21 ;

[0744] (b) a variant of SEQ ID NO: 21 having at least 70% sequence identity thereto; or

[0745] (c) a polynucleotide fragment of either (a) or (b).

[0746] B53. The polynucleotide according to clause B52, wherein the variant has at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, or at least about 99% sequence identity to the polynucleotide sequence of SEQ ID NO: 21 .

[0747] B54. The polynucleotide according to clause B52, wherein the polynucleotide fragment is at least about 300 nucleotides in length, at least about 400 nucleotides in length, at least about 500 nucleotides in length, at least about 750 nucleotides in length, at least about 1000 nucleotides in length, at least about 1250 nucleotides in length, at least about 1500 nucleotides in length, at least about 1750 nucleotides in length, at least about 2000 nucleotides in length, at least about 2250 nucleotides in length, or at least about 2500 nucleotides in length.

[0748] B55. A polynucleotide encoding a polypeptide according to any one of clauses B1 to B51 . B56. A vector encoding or comprising a polynucleotide according to any one of clauses B52 to B55.

[0749] B57. An expression vector which comprises the polynucleotide sequence according to any one of clauses B52 to B55.

[0750] B58. A host cell comprising the polynucleotide, vector, or expression vector of clause B52 or B57.

[0751] B59. The host cell of clause B58, wherein the host cell is a bacterial cell.

[0752] B60. The host cell of clause B58 or B59, wherein the host cell is not streptococcal species.

[0753] B61 . The host cell of clause B58 or B59, wherein the host cell is E. coll.

[0754] B62. The polynucleotide, vector, or expression vector according to any one of clauses B52 to B57, wherein the polynucleotide, vector, or expression vector is provided in solution, lyophilised, or immobilised, optionally together with an effective amount of at least one excipient, for example, wherein the excipient is a preservative.

[0755] B63. A cell comprising the polypeptide of any one of clauses B1 to B51 , the polynucleotide of any one of clauses B52 to B55, the vector of clause B56, or expression vector of clause B57.

[0756] Clause Group C:

[0757] C1. A polypeptide, or engineered polypeptide, having immunoglobulin protease activity, wherein said polypeptide comprises, consists essentially of, or consists of:

[0758] (a) the amino acid sequence of SEQ ID NO: 7; or

[0759] (b) a variant of SEQ ID NO: 7 having at least 80% sequence identity thereto; or

[0760] (c) an amino acid sequence which is a fragment of the sequence of either (a) or (b).

[0761] C2. A polypeptide, or engineered polypeptide, having immunoglobulin protease activity, wherein said polypeptide comprises, consists essentially of, or consists of:

[0762] (a) the amino acid sequence of SEQ ID NO: 8; or

[0763] (b) a variant of SEQ ID NO: 8 having at least 80% sequence identity thereto; or

[0764] (c) an amino acid sequence which is a fragment of the sequence of either (a) or (b).

[0765] C3. The polypeptide according to clause C1 , wherein the variant has at least about 85% sequence identity, at least about 90% sequence identity, at least about 91 % sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 7. C4. The polypeptide according to clause C2, wherein the variant has at least about 85% sequence identity, at least about 90% sequence identity, at least about 91 % sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 8.

[0766] C5. The polypeptide according to clause C1 , wherein the fragment of the sequence of SEQ ID NO:

[0767] 7 is at least about 100 amino acids in length, at least about 150 amino acids in length, at least about 200 amino acids in length, at least about 250 amino acids in length, at least about 300 amino acids in length, or at least about 350 amino acids in length.

[0768] C6. The polypeptide according to clause C2, wherein the fragment of the sequence of SEQ ID NO:

[0769] 8 is at least about 100 amino acids in length, at least about 150 amino acids in length, at least about 200 amino acids in length, at least about 250 amino acids in length, or at least about 300 amino acids in length.

[0770] C7. The polypeptide according to any one of clauses C1 to C6, wherein the fragment comprises, consists essentially of, or consists of an N terminal fragment.

[0771] C8. The polypeptide according to any one of clauses C1 to C6, wherein the fragment comprises, consists essentially of, or consists of a C terminal fragment.

[0772] C9. The polypeptide according to any one of clauses C1 to C8, wherein the polypeptide comprises a methionine at the N terminus.

[0773] C10. The polypeptide according to any one of clauses C1 to C9, wherein the polypeptide comprises a protein purification tag at the C terminus.

[0774] C11 . The polypeptide according to any one of clauses C1 to C10, wherein the polypeptide comprises a protein purification tag at the N terminus.

[0775] C12. The polypeptide according to clause C10 or C11 , wherein the tag is joined to the N and / or C terminus by a linker.

[0776] C13. The polypeptide according to clause C12, wherein the linker comprises one, or more than one, of any or a combination of glycine, glycine-serine, serine-glycine, or serine residues.

[0777] C14. The polypeptide according to clause C12 or C13, wherein the linker is GSS.

[0778] C15. The polypeptide according to any one of clauses C10 to C14, wherein the tag is a His tag. C16. The polypeptide according to any one of clauses C10 to C14, wherein the tag is a polyhistidine- tag.

[0779] C17. The polypeptide according to any one of clauses C10 to C14, wherein the tag is a 6xHis tag.

[0780] C18. The polypeptide according to clause C1 , C3, C5, or C7 to C17 when dependent on clause C1 , wherein the fragment of the sequence of SEQ ID NO: 7 comprises the sequence from about sequence position 0 to 377, about 10 to 377, about 20 to 377, about 30 to 377, about 40 to 360, about 50 to 350, or about 60 to 340, e.g. sequence position 30 to 377.

[0781] C19. The polypeptide according to any one of the preceding clauses, comprising, consisting essentially of, or consisting of SEQ ID NO: 9 or 40.

[0782] C20. The polypeptide according to any one of the preceding clauses, wherein the polypeptide is provided in solution, lyophilised, or immobilised.

[0783] C21 . The polypeptide according to any one of the preceding clauses which has protease activity against an immunoglobulin molecule comprising a CH2 / hinge sequence, optionally wherein the CH2 / hinge sequence comprises any one of SEQ ID NOs: 30, 31 , 32, or 34.

[0784] C22. The polypeptide according to clause C21 , wherein the immunoglobulin molecule is lgG1 and the CH2 / hinge sequence comprises SEQ ID NO: 30.

[0785] C23. The polypeptide according to any one of clauses C21 or C22, wherein the immunoglobulin molecule is lgG2 and the CH2 / hinge sequence comprises SEQ ID NO: 31 .

[0786] C24. The polypeptide according to any one of clauses C21 to C23, wherein the immunoglobulin molecule is lgG3 and the CH2 / hinge sequence comprises SEQ ID NO: 32.

[0787] C25. The polypeptide according to any one of clauses C21 to C24, wherein the immunoglobulin molecule is lgG4 and the CH2 / hinge sequence comprises SEQ ID NO: 34.

[0788] C26. The polypeptide according to any one of clauses C21 to C25, wherein the polypeptide cleaves the CH2 / hinge sequence between amino acid positions 6 and 7 of at least one of SEQ ID NOs: 30, 31 , 32, or 34.

[0789] C27. The polypeptide according to any one of the preceding clauses, wherein the polypeptide cleaves IgG.

[0790] C28. The polypeptide according to any one of the preceding clauses, wherein the polypeptide cleaves human IgG. C29. The polypeptide according to any one of the preceding clauses, wherein the immunoglobulin protease activity is IgG specific protease activity.

[0791] C30. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits lower protease activity against IgM as compared to IgG.

[0792] C31 . The polypeptide according to clause C30, wherein the protease activity against IgM is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the protease activity against IgG.

[0793] C32. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits lower protease activity against IgA as compared to IgG.

[0794] C33. The polypeptide according to clause C32, wherein the protease activity against IgA is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the protease activity against IgG.

[0795] C34. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits lower protease activity against IgD as compared to IgG.

[0796] C35. The polypeptide according to clause C34, wherein the protease activity against IgD is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the protease activity against IgG.

[0797] C36. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits lower protease activity against IgE as compared to IgG.

[0798] C37. The polypeptide according to clause C36, wherein the protease activity against IgE is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the protease activity against IgG.

[0799] C38. The polypeptide according to any one of the preceding clauses, wherein the polypeptide has protease activity against an IgG comprising a CH2 / hinge sequence, optionally wherein the CH2 / hinge sequence comprises any one of SEQ ID NOs: 30, 31 , 32, or 34. C39. The polypeptide according to any one of the preceding clauses, wherein the polypeptide has protease activity against any of IgG 1 , lgG2, lgG3, or lgG4, or mouse lgG2b or lgG3.

[0800] C40. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits lower protease activity against human lgG1 and / or lgG3 as compared to a human lgG2 and / or lgG4 subclass.

[0801] C41. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits greater protease activity against human lgG2 than against human IgG 1 .

[0802] C42. The polypeptide according to clause C41 , wherein the protease activity against lgG1 is less than about 99% of the protease activity against lgG2, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1% of the protease activity against lgG2.

[0803] C43. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits greater protease activity against human lgG4 than against human IgG 1 .

[0804] C44. The polypeptide according to clause C43, wherein the protease activity against lgG1 is less than about 99% of the protease activity against lgG4, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1% of the protease activity against lgG4.

[0805] C45. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits greater protease activity against human lgG2 than human lgG3.

[0806] C46. The polypeptide according to clause C45, wherein the protease activity against lgG3 is less than about 99% of the protease activity against lgG2, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1% of the protease activity against lgG2.

[0807] C47. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits greater protease activity against human lgG4 than human lgG3.

[0808] C48. The polypeptide according to clause C47, wherein the protease activity against lgG3 is less than about 99% of the protease activity against lgG4, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1% of the protease activity against lgG4. C49. The polypeptide according to any one of the preceding clauses, wherein the immunoglobulin is from a mammal; optionally wherein the mammal is selected from human, primate, horse, cow, sheep, goat, rabbit, or rodent (such as mouse, guinea pig, rat).

[0809] C50. The polypeptide according to any one of the preceding clauses, wherein the polypeptide has protease activity against an lgG1 variant, optionally wherein the lgG1 variant is a modified lgG1 variant, optionally wherein the lgG1 variant is a hinge-modified lgG1 variant.

[0810] C51. The polypeptide according to any one of the preceding clauses, wherein the polypeptide has protease activity against a human IgG 1 variant, optionally wherein the human lgG1 variant is a modified human IgG 1 variant, optionally wherein the human IgG 1 variant is a hinge-modified human IgG 1 variant.

[0811] C52. The polypeptide according to clause C51 , wherein the human lgG1 variant comprises an amino acid substitution in at least one amino acid position in the hinge region.

[0812] C53. The polypeptide according to clause C52, wherein the at least one amino acid position in the hinge region is selected from any one of amino acid position 234, 235, 237, or 331 of SEQ ID NO: 43.

[0813] C54. The polypeptide according to clause C51 to C53, wherein the human lgG1 variant comprises the amino acid substitution L234A and / or L235A based on the numbering of SEQ ID NO: 43.

[0814] C55. The polypeptide according to clause C51 to C54, wherein the human lgG1 variant comprises the amino acid substitution L235A and / or G237A based on the numbering of SEQ ID NO: 43.

[0815] C56. The polypeptide according to clause C51 to C55, wherein the human lgG1 variant, comprises the amino acid substitution L234F, and / or L235E, and / or P331 S based on the numbering of SEQ ID NO: 43.

[0816] C57. The polypeptide according to any one of the preceding clauses, wherein the polypeptide has protease activity against an lgG4 variant, optionally wherein the lgG4 variant is a modified lgG4 variant, optionally wherein the IgG variant is a hinge-modified lgG4 variant.

[0817] C58. The polypeptide according to any one of the preceding clauses, wherein the polypeptide has protease activity against a human lgG4 variant, optionally wherein the human lgG4 variant is a modified human lgG4 variant, optionally wherein the human lgG4 variant is a hinge-modified human lgG4 variant.

[0818] C59. The polypeptide according to clause C58, wherein the human lgG4 variant comprises an amino acid substitution in at least one amino acid position in the hinge region.

[0819] C60. The polypeptide according to clause C58, wherein the at least one amino acid position in the hinge region is selected from any one of amino acid position 234 or 235 of SEQ ID NO: 38. C61. The polypeptide according to clause C58 to C60, wherein the human lgG4 variant comprises the amino acid substitution F234A and / or L235A based on the numbering of SEQ ID NO: 38.

[0820] C62. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits greater protease activity against an lgG1 variant or an lgG4 variant, optionally a human lgG1 variant or lgG4 variant, than against a human lgG3.

[0821] C63. The polypeptide according to clause C62, wherein the protease activity against the lgG3 is less than about 99% of the protease activity against the lgG1 variant or lgG4 variant, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the protease activity against the lgG1 variant or lgG4 variant.

[0822] C64. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits greater protease activity against an lgG1 variant or lgG4 variant, optionally a human lgG1 variant or lgG4 variant, than against a human lgG4.

[0823] C65. The polypeptide according to clause C64, wherein the protease activity against the lgG4 is less than about 99% of the protease activity against the lgG1 variant or lgG4 variant, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the protease activity against the lgG1 variant or lgG4 variant.

[0824] C66. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits greater protease activity against an lgG1 variant or lgG4 variant, optionally a human lgG1 variant or lgG4 variant, than against a non-variant human IgG 1 or lgG4, optionally an unmodified human lgG1 or lgG4.

[0825] C67. The polypeptide according to clause C66, wherein the protease activity against non-variant human IgG 1 , optionally unmodified human lgG1 , is less than about 99% of the protease activity against the lgG1 variant, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the protease activity against the lgG1 variant.

[0826] C68. The polypeptide according to any one of the preceding clauses, wherein the polypeptide is provided in solution, lyophilised, or immobilised, optionally together with an effective amount of at least one excipient, for example, wherein the excipient is a preservative.

[0827] C69. A polynucleotide encoding a polypeptide having protease activity of any one of clauses C1 to C68, the polynucleotide comprising: (a) the polynucleotide sequence of SEQ ID NO: 22;

[0828] (b) a variant of SEQ ID NO: 22 having at least 70% sequence identity thereto; or

[0829] (c) a polynucleotide fragment of either (a) or (b).

[0830] C70. The polynucleotide according to clause C69, wherein the variant has at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, or at least about 99% sequence identity to the polynucleotide sequence of SEQ ID NO: 22.

[0831] C71 . The polynucleotide according to clause C69, wherein the polynucleotide fragment is at least about 300 nucleotides in length, at least about 400 nucleotides in length, at least about 500 nucleotides in length, at least about 600 nucleotides in length, at least about 700 nucleotides in length, at least about 800 nucleotides in length, at least about 900 nucleotides in length, or at least about 1000 nucleotides in length.

[0832] C72. A polynucleotide encoding a polypeptide according to any one of clauses C1 to C67.

[0833] C73. A vector encoding or comprising a polynucleotide according to any one of clause C69 to C72.

[0834] C74. An expression vector which comprises the polynucleotide sequence according to any one of clause C69 to C72.

[0835] C75. A host cell comprising the polynucleotide, vector, or expression vector of any one of clauses C69 to C74.

[0836] C76. The host cell of clause C75, wherein the host cell is a bacterial cell.

[0837] C77. The host cell of clause C75 or 76, wherein the host cell is not streptococcal species.

[0838] C78. The host cell of clause C75 or 76, wherein the host cell is E. coll.

[0839] C79. The polynucleotide, vector, or expression vector according to any one of clauses C69 to C74, wherein the polynucleotide, vector, or expression vector is provided in solution, lyophilised, or immobilised, optionally together with an effective amount of at least one excipient, for example, wherein the excipient is a preservative.

[0840] C80. A cell comprising the polypeptide of any one of clauses C1 to C68, the polynucleotide of any one of clause C69 to C72, the vector of clause C73, or the expression vector of clause 74.

[0841] Clause Group D:

[0842] D1 . A polypeptide, or engineered polypeptide, having immunoglobulin protease activity, wherein said polypeptide comprises, consists essentially of, or consists of: (a) the amino acid sequence of SEQ ID NO: 10; or

[0843] (b) a variant of SEQ ID NO: 10 having at least 80% sequence identity thereto; or

[0844] (c) an amino acid sequence which is a fragment of the sequence of either (a) or (b).

[0845] D2. A polypeptide, or engineered polypeptide, having immunoglobulin protease activity, wherein said polypeptide comprises, consists essentially of, or consists of:

[0846] (a) the amino acid sequence of SEQ ID NO: 11 ; or

[0847] (b) a variant of SEQ ID NO: 11 having at least 80% sequence identity thereto; or

[0848] (c) an amino acid sequence which is a fragment of the sequence of either (a) or (b).

[0849] D3. The polypeptide according to clause D1 , wherein the variant has at least about 85% sequence identity, at least about 90% sequence identity, at least about 91 % sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 10.

[0850] D4. The polypeptide according to clause D2, wherein the variant has at least about 85% sequence identity, at least about 90% sequence identity, at least about 91 % sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 11 .

[0851] D5. The polypeptide according to clause D1 , wherein the fragment of the sequence of SEQ ID NO:

[0852] 10 is at least about 100 amino acids in length, at least about 150 amino acids in length, at least about 200 amino acids in length, at least about 250 amino acids in length, at least about 300 amino acids in length, at least about 350 amino acids in length, at least about 400 amino acids in length, at least about 450 amino acids in length, at least about 500 amino acids in length, at least about 550 amino acids in length, or at least about 600 amino acids in length.

[0853] D6. The polypeptide according to clause D2, wherein the fragment of the sequence of SEQ ID NO:

[0854] 11 is at least about 50 amino acids in length, at least about 100 amino acids in length, at least about 150 amino acids in length, at least about 200 amino acids in length, at least about 250 amino acids in length, or at least about 300 amino acids in length.

[0855] D7. The polypeptide according to any one of clauses D1 to D6, wherein the fragment comprises, consists essentially of, or consists of an N terminal fragment.

[0856] D8. The polypeptide according to any one of clauses D1 to D6, wherein the fragment comprises, consists essentially of, or consists of a C terminal fragment. D9. The polypeptide according to any one of clauses D1 to D8, wherein the polypeptide comprises a methionine at the N terminus.

[0857] D10. The polypeptide according to any one of clauses D1 to D9, wherein the polypeptide comprises a protein purification tag at the C terminus.

[0858] D11 . The polypeptide according to any one of clauses D1 to D10, wherein the polypeptide comprises a protein purification tag at the N terminus.

[0859] D12. The polypeptide according to clause D10 or clause D11 , wherein the tag is joined to the N and / or C terminus by a linker.

[0860] D13. The polypeptide according to clause D12, wherein the linker comprises one, or more than one, of any or a combination of glycine, glycine-serine, serine-glycine, or serine residues.

[0861] D14. The polypeptide according to clause D12 or D13, wherein the linker is GSS.

[0862] D15. The polypeptide according to any one of clauses D10 to D14, wherein the tag is a His tag.

[0863] D16. The polypeptide according to any one of clauses D10 to D15, wherein the tag is a polyhistidine- tag.

[0864] D17. The polypeptide according to any one of clauses D10 to D16, wherein the tag is a 6xHis tag.

[0865] D18. The polypeptide according to any one of clauses D1 , D2, D4, D5, or clause D7 to D17 when dependent on clause D1 , wherein the fragment of the sequence of SEQ ID NO: 10 comprises the sequence from about sequence position 0 to 600, about 50 to 600, about 100 to 575, about 150 to 550, about 200 to 550, or about 250 to 500, e.g. from sequence position 200 to 528.

[0866] D19. The polypeptide according to any one of the preceding clauses, comprising, consisting essentially of, or consisting of SEQ ID NO: 12.

[0867] D20. The polypeptide according to any one of the preceding clauses, wherein the polypeptide is provided in solution, lyophilised, or immobilised.

[0868] D21 . The polypeptide according to any one of the preceding clauses, which has protease activity against an immunoglobulin molecule comprising a CH2 / hinge sequence, optionally wherein the CH2 / hinge sequence comprises any one of SEQ ID NOs: 29 or 31 .

[0869] D22. The polypeptide according to clause D21 , wherein the immunoglobulin molecule is lgG1 and the CH2 / hinge sequence comprises SEQ ID NO: 29. D23. The polypeptide according to any one of clauses D21 or D22, wherein the immunoglobulin molecule is lgG2 and the CH2 / hinge sequence comprises SEQ ID NO: 31 .

[0870] D24. The polypeptide according to any one of clauses D21 to D23, wherein the polypeptide cleaves the CH2 / hinge sequence between amino acid positions 5 and 6 and / or 6 and 7 of SEQ ID NO: 29, and / or between amino acid positions 6 and 7 of SEQ ID NO: 31 .

[0871] D25. The polypeptide according to any one of the preceding clauses, wherein the polypeptide cleaves IgG.

[0872] D26. The polypeptide according to any one of the preceding clauses, wherein the polypeptide cleaves human IgG.

[0873] D27. The polypeptide according to any one of the preceding clauses, wherein the immunoglobulin protease activity is IgG specific protease activity.

[0874] D28. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits lower protease activity against IgM as compared to IgG.

[0875] D29. The polypeptide according to clause D28, wherein the protease activity against IgM is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the protease activity against IgG.

[0876] D30. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits lower protease activity against IgA as compared to IgG.

[0877] D31 . The polypeptide according to clause D30, wherein the protease activity against IgA is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the protease activity against IgG.

[0878] D32. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits lower protease activity against IgD as compared to IgG.

[0879] D33. The polypeptide according to clause D32, wherein the protease activity against IgD activity is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the protease activity against IgG. D34. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits lower protease activity against IgE as compared to IgG.

[0880] D35. The polypeptide according to clause D34, wherein the protease activity against IgE is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the protease activity against IgG.

[0881] D36. The polypeptide according to any one of the preceding clauses, wherein the polypeptide has protease activity against IgG comprising a CH2 / hinge sequence, optionally wherein the CH2 / hinge sequence comprises any one of SEQ ID NOs: 29 or 31 .

[0882] D37. The polypeptide according to any one of the preceding clauses, wherein the polypeptide has protease activity against any of lgG1 , lgG2, lgG3, or lgG4.

[0883] D38. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits lower protease activity against a human IgG 1 and / or lgG3 and / or lgG4 Ig subclass as compared to a human lgG2 Ig subclass.

[0884] D39. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits greater protease activity against human lgG2 than against human IgG 1 .

[0885] D40. The polypeptide according to clause D39, wherein the protease activity against lgG1 is less than about 99% of the protease activity against lgG2, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the protease activity against lgG2.

[0886] D41. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits greater protease activity against human lgG2 than against human lgG3.

[0887] D42. The polypeptide according to clause D41 , wherein the protease activity against lgG3 is less than about 99% of the protease activity against lgG2, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the protease activity against lgG2.

[0888] D43. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits greater protease activity against human lgG2 than against human lgG4.

[0889] D44. The polypeptide according to clause D43, wherein the protease activity against lgG4 is less than about 99% of the protease activity against lgG2, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the protease activity against lgG2.

[0890] D45. The polypeptide according to any one of the preceding clauses, wherein the immunoglobulin is from a mammal; optionally wherein the mammal is selected from human, primate, horse, cow, sheep, goat, rabbit, or rodent (such as mouse, guinea pig, rat).

[0891] D46. The polypeptide according to any one of the preceding clauses, wherein the polypeptide is provided in solution, lyophilised, or immobilised, optionally together with an effective amount of at least one excipient, for example, wherein the excipient is a preservative.

[0892] D47. A polynucleotide encoding a polypeptide having protease activity of any one of clauses D1 to D46, the polynucleotide comprising:

[0893] (a) the polynucleotide sequence of SEQ ID NO: 24;

[0894] (b) a variant of SEQ ID NO: 24 having at least 80% sequence identity thereto; or

[0895] (c) a polynucleotide fragment of either (a) or (b).

[0896] D48. The polynucleotide according to clause D47, wherein the variant has at least about 75% sequence identity, at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, or at least about 99% sequence identity to the polynucleotide sequence of SEQ ID NO: 24.

[0897] D49. The polynucleotide according to clause D48, wherein the polynucleotide fragment is at least about 300 nucleotides in length, at least about 400 nucleotides in length, at least about 500 nucleotides in length, at least about 600 nucleotides in length, at least about 700 nucleotides in length, at least about 800 nucleotides in length, at least about 900 nucleotides in length, or at least about 1000 nucleotides in length.

[0898] D50. A polynucleotide encoding a polypeptide according to any one of clauses D1 to D45.

[0899] D51 . A vector encoding or comprising a polynucleotide according to any one of clauses D47 to D50.

[0900] D52. An expression vector which comprises the polynucleotide sequence according to any one of clauses D47 to D50.

[0901] D53. A host cell comprising the polynucleotide, vector, or expression vector of any one of clauses D50 to D52.

[0902] D54. The host cell of clause D53, wherein the host cell is a bacterial cell.

[0903] D55. The host cell of clause D54, wherein the host cell is not streptococcal species. D56. The host cell of clause D54 or D55, wherein the host cell is E. coli.

[0904] D57. The polynucleotide, vector, or expression vector according to any one of clauses D47 to D52, wherein the polynucleotide, vector, or expression vector is provided in solution, lyophilised, or immobilised, optionally togetherwith an effective amount of at least one excipient, for example, wherein the excipient is a preservative.

[0905] D58. A cell comprising the polypeptide of any one of clauses D1 to D46, the polynucleotide of any one of clauses D47 to D50, the vector of clause D51 , or the expression vector of clause D52.

[0906] Clause Group E:

[0907] E1. A polypeptide, or engineered polypeptide, having immunoglobulin protease activity, wherein said polypeptide comprises, consists essentially of, or consists of:

[0908] (a) the amino acid sequence of SEQ ID NO: 13; or

[0909] (b) a variant of SEQ ID NO: 13 having at least 80% sequence identity thereto; or

[0910] (c) an amino acid sequence which is a fragment of the sequence of either (a) or (b).

[0911] E2. A polypeptide, or engineered polypeptide, having immunoglobulin protease activity, wherein said polypeptide comprises, consists essentially of, or consists of:

[0912] (a) the amino acid sequence of SEQ ID NO: 14; or

[0913] (b) a variant of SEQ ID NO: 14 having at least 80% sequence identity thereto; or

[0914] (c) an amino acid sequence which is a fragment of the sequence of either (a) or (b).

[0915] E3. The polypeptide according to clause E1 , wherein the variant has at least about 85% sequence identity, at least about 90% sequence identity, at least about 91 % sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 13.

[0916] E4. The polypeptide according to clause E2, wherein the variant has at least about 85% sequence identity, at least about 90% sequence identity, at least about 91 % sequence identity, at least about 92% sequence identity, at least about 93% sequence identity, at least about 94% sequence identity, at least about 95% sequence identity, at least about 96% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 14.

[0917] E5. The polypeptide according to clause E1 , wherein the fragment of the sequence of SEQ ID NO: 13 is at least about 100 amino acids in length, at least about 200 amino acids in length, at least about 300 amino acids in length, at least about 400 amino acids in length, at least about 500 amino acids in length, at least about 600 amino acids in length, at least about 700 amino acids in length, at least about 800 amino acids in length, at least about 900 amino acids in length, at least about 1000 amino acids in length, at least about 1 100 amino acids in length, at least about 1200 amino acids in length, at least about 1300 amino acids in length, at least about 1400 amino acids in length, or at least about 1500 amino acids in length.

[0918] E6. The polypeptide according to clause E1 , wherein the fragment of the sequence of SEQ ID NO:

[0919] 13 is at least about 100 amino acids in length, at least about 150 amino acids in length, at least about 200 amino acids in length, at least about 250 amino acids in length, at least about 300 amino acids in length, at least about 350 amino acids in length, at least about 400 amino acids in length, at least about 450 amino acids in length, at least about 500 amino acids in length, at least about 550 amino acids in length, or at least about 600 amino acids in length.

[0920] E7. The polypeptide according to clause E2, wherein the fragment of the sequence of SEQ ID NO:

[0921] 14 is at least about 100 amino acids in length, at least about 150 amino acids in length, at least about 200 amino acids in length, at least about 250 amino acids in length, or at least about 300 amino acids in length.

[0922] E8. The polypeptide according to any one of clauses E1 to E7, wherein the fragment comprises, consists essentially of, or consists of an N terminal fragment.

[0923] E9. The polypeptide according to any one of clauses E1 to E7, wherein the fragment comprises, consists essentially of, or consists of a C terminal fragment.

[0924] E10. The polypeptide according to any one of clauses E1 to E9, wherein the polypeptide comprises a methionine at the N terminus.

[0925] E11 . The polypeptide according to any one of clauses E1 to E10, wherein the polypeptide comprises a protein purification tag at the C terminus.

[0926] E12. The polypeptide according to any one of clauses E1 to E1 1 , wherein the polypeptide comprises a protein purification tag at the N terminus.

[0927] E13. The polypeptide according to clause E11 or clause E12, wherein the tag is joined to the N and / or C terminus by a linker.

[0928] E14. The polypeptide according to clause E13, wherein the linker comprises one, or more than one, of any or a combination of glycine, glycine-serine, serine-glycine, or serine residues.

[0929] E15. The polypeptide according to clause E13 or E14, wherein the linker is GSS.

[0930] E16. The polypeptide according to any one of clauses E13 to E15, wherein the tag is a His tag.

[0931] E17. The polypeptide according to any one of clauses E13 to E16, wherein the tag is a polyhistidine- tag.

[0932] Ill E18. The polypeptide according to any one of clauses E13 to E17, wherein the tag is a 6xHis tag.

[0933] E19. The polypeptide according to any one of clauses E1 , E3, E5, E6, or clause E8 to E18 when dependent on clause E1 , wherein the fragment of the sequence of SEQ ID NO: 13 comprises the sequence from about sequence position 500 to 1550, about 550 to 1450, about 600 to 1400, about 650 to 1350, about 700 to 1300, about 725 to 1300, about 750 to 1250, about 775 to 1200, about 800 to 1150, about 850 to 1100, or about 900 to 1050, e.g. from sequence position 809 to 1132.

[0934] E20. The polypeptide according to any one of the preceding clauses, comprising, consisting essentially of, or consisting of SEQ ID NO: 15.

[0935] E21. The polypeptide according to any one of the preceding clauses, wherein the polypeptide is provided in solution, lyophilised, or immobilised.

[0936] E22. The polypeptide according to any one of the preceding clauses, which has protease activity against an immunoglobulin molecule comprising a CH2 / hinge sequence, optionally wherein the CH2 / hinge sequence comprises any one of SEQ ID NOs: 29, 31 , 32, or 34.

[0937] E23. The polypeptide according to clause E22, wherein the immunoglobulin molecule is lgG1 and the CH2 / hinge sequence comprises SEQ ID NO: 29.

[0938] E24. The polypeptide according to any one of clauses E22 to E23, wherein the immunoglobulin molecule is lgG2 and the CH2 / hinge sequence comprises SEQ ID NO: 31 .

[0939] E25. The polypeptide according to any one of clauses E22 to E24, wherein the immunoglobulin molecule is lgG3 and the CH2 / hinge sequence comprises SEQ ID NO: 32.

[0940] E26. The polypeptide according to any one of clauses E22 to E25, wherein the immunoglobulin molecule is lgG4 and the CH2 / hinge sequence comprises SEQ ID NO: 34.

[0941] E27. The polypeptide according to any one of clauses E22 to E26, wherein the polypeptide cleaves the CH2 / hinge sequence between amino acid positions 5 and 6 and / or 6 and 7 of SEQ ID NO: 29, and / or between amino acid positions 6 and 7 of any one of SEQ ID NOs: 31 , 32, or 34.

[0942] E28. The polypeptide according to any one of the preceding clauses, wherein the polypeptide cleaves IgG.

[0943] E29. The polypeptide according to any one of the preceding clauses, wherein the polypeptide cleaves human IgG.

[0944] E30. The polypeptide according to any one of the preceding clauses, wherein the immunoglobulin protease activity is IgG specific protease activity. E31. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits lower protease activity against IgM as compared to IgG.

[0945] E32. The polypeptide according to clause E31 , wherein the protease activity against IgM is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the protease activity against IgG.

[0946] E33. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits lower protease activity against IgA as compared to IgG.

[0947] E34. The polypeptide according to clause E33, wherein the protease activity against IgA is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the protease activity against IgG.

[0948] E35. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits lower protease activity against IgD as compared to IgG.

[0949] E36. The polypeptide according to clause E35, wherein the protease activity against IgD is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the protease activity against IgG.

[0950] E37. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits lower protease activity against IgE as compared to IgG.

[0951] E38. The polypeptide according to clause E37, wherein the protease activity against IgE is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the protease activity against IgG.

[0952] E39. The polypeptide according to any one of the preceding clauses, wherein the polypeptide has protease activity against IgG comprising a CH2 / hinge sequence, optionally wherein the CH2 / hinge sequence comprises any one of SEQ ID NOs: 29, 31 , 32, or 34.

[0953] E40. The polypeptide according to any one of the preceding clauses, wherein the polypeptide has protease activity against any of IgG 1 , lgG2, lgG3, or lgG4. E41. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits lower protease activity against a human lgG3 and / or lgG4 as compared to a human lgG1 and / or igG2.

[0954] E42. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits greater protease activity against human lgG1 than against human lgG3.

[0955] E43. The polypeptide according to clause E42, wherein the protease activity against lgG3 is less than about 99% of the protease activity against lgG1 , such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the protease activity against lgG1 .

[0956] E44. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits greater protease activity against human lgG2 than human lgG3.

[0957] E45. The polypeptide according to clause E44, wherein the protease activity against lgG3 is less than about 99% of the protease activity against lgG2, such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2%, or less than about 1 % of the protease activity against lgG2.

[0958] E46. The polypeptide according to any one of the preceding clauses, wherein the polypeptide exhibits greater protease activity against human IgG 1 than human lgG4.

[0959] E47. The polypeptide according to clause E46, wherein the protease activity against lgG4 is less than about 99% of the protease activity against lgG1 , such as less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, l...

Claims

CLAIMS1 . A polypeptide, or engineered polypeptide, having immunoglobulin protease activity, wherein said polypeptide comprises, consists essentially of, or consists of:(a) the amino acid sequence of any one of SEQ ID NOs: 1 , 4, 7, 10, or 13; or(b) a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ IDNO: 1 , 4, 7, 10, or 13; or(c) an amino acid sequence which is a fragment of the sequence of either (a) or (b).

2. The polypeptide according to claim 1 , wherein the fragment of the sequence of SEQ ID NO: 1 ,4, 7, 10, or 13 is: at least about 100 amino acids in length, at least about 200 amino acids in length, or at least about 300 amino acids in length; or wherein the fragment of the sequence of SEQ ID NO: 1 , 4, 10, or 13 is: at least about 400 amino acids in length, at least about 500 amino acids in length, or at least about 600 amino acids in length; or wherein the fragment of the sequence of SEQ ID NO: 1 , 4, or 13 is: at least about 700 amino acids in length; or wherein the fragment of the sequence of SEQ ID NO: 1 , 4 or 13 is: at least about 800 amino acids in length, at least about 900 amino acids in length, at least about 1000 amino acids in length, at least about 1100 amino acids in length, at least about 1200 amino acids in length, at least about 1300 amino acids in length, at least about 1400 amino acids in length, at least about 1500 amino acids in length; or wherein the fragment of the sequence of SEQ ID NO: 1 or 4 is: at least about 1600 amino acids in length, or at least about 1700 amino acids in length; or wherein the fragment of the sequence of SEQ ID NO: 4 is: at least about 1800 amino acids in length.

3. A polypeptide, or engineered polypeptide, having immunoglobulin protease activity, wherein said polypeptide comprises, consists essentially of, or consists of:(d) the amino acid sequence of SEQ ID NO: 2, 5, 8, 11 , or 14; or(e) a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 2, 5, 8, 11 , or 14; or(f) an amino acid sequence which is a fragment of the sequence of(a) or (b).

4. The polypeptide according to claim 3, wherein the fragment of the sequence of SEQ ID NO: 2,5. 8, 1 1 , 14 is: at least about 100 amino acids in length, at least about 150 amino acids in length, at least about 200 amino acids in length, at least about 250 amino acids in length, at least about 300 amino acids in length, or wherein the fragment of the sequence of SEQ ID NO: 5 is:at least about 350 amino acids in length at least about 400 amino acids in length, at least about 450 amino acids in length at least about 500 amino acids in length at least about 550 amino acids in length, at least about 600 amino acids in length, at least about 650 amino acids in length, at least about 700 amino acids in length, at least about 750 amino acids in length, at least about 800 amino acids in length.

5. A polypeptide, or engineered polypeptide, having immunoglobulin protease activity, wherein said polypeptide comprises, consists essentially of, or consists of:(a) the amino acid sequence of SEQ ID NO: 41 or 42; or(b) a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 41 or 42; or(c) an amino acid sequence which is a fragment of the sequence of either (a) or (b), wherein the fragment of the sequence of SEQ ID NO: 41 or 42 is at least about 700 amino acids in length, at least about 750 amino acids in length, or at least about 800 amino acids in length.

6. The polypeptide of any one of the preceding claims, wherein the polypeptide is engineered to include an additional methionine at the N terminus and / or a protein purification tag at the C terminus, optionally wherein the tag may be joined to the C terminus by a linker.

7. The polypeptide according to any one of the preceding claims, wherein the polypeptide has immunoglobulin protease activity against an immunoglobulin molecule comprising a CH2 / hinge sequence, optionally wherein the CH2 / hinge sequence may be selected from any one of SEQ ID NOs: 28 to 37.

8. The polypeptide according to claim 7, wherein the polypeptide cleaves the CH2 / hinge sequence between amino acid positions 6 and 7 of any one or more of SEQ ID NOs: 28, or 30 to 34, and / or between positions 5 and 6 and / or 6 and 7 of SEQ ID NO: 29, and / or between positions 10 and 1 1 of one or more of SEQ ID NOs: 35 or 36, and / or between amino acid positions 5 and 6 of SEQ ID NO: 37.

9. The polypeptide according to any one of the preceding claims, which comprises, consists essentially of, or consists of the amino acid sequence of any of SEQ ID NOs: 3, 6, 9, 12, 15, 16, 18 or 40.

10. The polypeptide according to any one of the preceding claims, wherein the polypeptide has immunoglobulin protease activity against IgG, optionally wherein the IgG is from a mammal; optionally wherein the mammal is selected from human, primate, horse, cow, sheep, goat, rabbit, or rodent (such as mouse, guinea pig, rat), optionally wherein the IgG is human IgG.

11. The polypeptide according to any one of the preceding claims, wherein the immunoglobulin protease activity is IgG specific protease activity, optionally wherein the polypeptide has lower immunoglobulin protease activity against one or more of IgA, IgD, IgE and IgM as compared to IgG, optionally wherein the level of immunoglobulin protease activity against IgA, IgD, IgE or IgM is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, lessthan about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the level of immunoglobulin protease activity against IgG.

12. The polypeptide according to any one of the preceding claims, wherein the polypeptide exhibits lower immunoglobulin protease activity against human lgG2 as compared to any one other human IgG subclass, optionally wherein the polypeptide has lower immunoglobulin protease activity against lgG2 as compared to one or more of lgG1 , lgG3, and lgG4, optionally wherein the level of immunoglobulin protease activity against lgG2 is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the level of immunoglobulin protease activity against lgG1 , lgG3, and / or lgG4.

13. The polypeptide according to any one of the preceding claims, wherein the polypeptide exhibits lower immunoglobulin protease activity against mouse lgG1 , lgG2a, or lgG3 as compared to lgG2b, optionally wherein the level of immunoglobulin protease activity against lgG1 , lgG2a, and / or lgG3 is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the level of immunoglobulin protease activity against lgG2b.

14. The polypeptide according to claim 1 1 , wherein the polypeptide exhibits lower immunoglobulin protease activity against a human lgG1 and / or lgG3 as compared to a human lgG2 and / or lgG4 subclass.

15. The polypeptide according to claim 1 1 or claim 14, wherein the polypeptide exhibits lower immunoglobulin protease activity against lgG1 and / or lgG3 as compared to lgG2 and / or lgG4, optionally wherein the immunoglobulin protease activity against lgG1 and / or lgG3 is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the immunoglobulin protease activity against lgG2 and / or lgG4.

16. The polypeptide according to claim 1 1 , wherein the polypeptide exhibits lower immunoglobulin protease activity against lgG1 , lgG3 and / or lgG4 as compared to lgG2, optionally wherein the immunoglobulin protease activity against lgG1 , lgG3 and / or lgG4 is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1 % of the immunoglobulin protease activity against igG2.

17. The polypeptide according to claim 1 1 , wherein the polypeptide exhibits lower immunoglobulin protease activity against lgG3 and / or lgG4 as compared to lgG1 and / or lgG2, optionally wherein theimmunoglobulin protease activity against lgG3 and / or lgG4 is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1% of the immunoglobulin protease activity against lgG1 and / or lgG2.

18. The polypeptide according to claim 11 , wherein the polypeptide has immunoglobulin protease activity against an lgG1 variant, optionally against a modified IgG 1 or lgG4 variant, optionally against a hinge-modified lgG1 or lgG4 variant.

19. The polypeptide according to claim 11 or claim 18, wherein the polypeptide has immunoglobulin protease activity against a human lgG1 or lgG4 variant, optionally a modified human lgG1 or lgG4 variant, optionally a hinge-modified human lgG1 or lgG4 variant, optionally lgG1-LALA, IgG-LAGA, lgG1-LFLE„ or lgG4-FALA.

20. The polypeptide according to claim 18 or claim 19, wherein the IgG 1 variant, optionally human lgG1 variant, comprises the amino acid substitution L234A and / or L235A based on the sequence of SEQ ID NO: 43.

21. The polypeptide according to any one of claims 18 to 20, wherein the lgG1 variant, optionally human lgG1 variant, comprises the amino acid substitution L234F, and / or L235E, and / or P331S; optionally based on the residue numbering of SEQ ID NO: 43.

22. The polypeptide according to any one of claims 18 to 21 , wherein the lgG1 variant, optionally human lgG1 variant, comprises the amino acid substitution L235A, and / or G237A; optionally based on the residue numbering of SEQ ID NO: 43.

23. The polypeptide according to any one of claims 18 to 22, wherein the lgG4 variant, optionally human lgG4 variant, comprises the amino acid substitution F234A, and / or L235A; optionally based on the residue numbering of SEQ ID NO: 38.

24. The polypeptide according to any one of claims 1 to 13, wherein the polypeptide has immunoglobulin protease activity against IgG and IgM, optionally wherein the IgG and / or IgM is from a mammal; optionally wherein the mammal is selected from human, primate, horse, cow, sheep, goat, rabbit, or rodent (such as mouse, guinea pig, rat), optionally wherein the IgG and / or IgM is human IgG and / or IgM.

25. The polypeptide according to any one of claims 1 to 13 or 24, wherein the immunoglobulin protease activity is IgG and IgM specific protease activity, optionally wherein the polypeptide has lower protease activity against one or more of IgA, IgD and IgE as compared to IgG and / or IgM, optionally wherein the level of activity against IgA, IgD and / or IgE is less than about 99%, less than about 95%, less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about50%, less than about 40%, less than about 30%, less than about 20%, less than 10%, less than 5%, less than about 2%, or less than about 1% of the level of activity against IgG and / or IgM.

26. A polynucleotide encoding a polypeptide having immunoglobulin protease activity of any one of claims 1 to 4 or 6 to 25 when dependent on any one of claims 1 to 4, the polynucleotide comprising:(a) the polynucleotide sequence of any one of SEQ ID NOs: 20 to 25;(b) a variant thereof having at least 80% sequence identity to the polynucleotide sequence of any one of SEQ ID NOs: 20 to 25; or(c) a polynucleotide fragment of either (a) or (b).

27. The polynucleotide according to claim 26, wherein the polynucleotide fragment is at least about 500 nucleotides in length, at least about 600 nucleotides in length, at least about 700 nucleotides in length, at least about 800 nucleotides in length, at least about 900 nucleotides in length, or at least about 1000 nucleotides in length, or optionally at least about 1100 nucleotides in length, at least about 1200 nucleotides in length, at least about 1300 nucleotides in length, at least about 1400 nucleotides in length, at least about 1500 nucleotides in length, at least about 1600 nucleotides in length, at least about 1700 nucleotides in length, at least about 1800 nucleotides in length, at least about 1900 nucleotides in length, at least about 2000 nucleotides in length, at least about 2100 nucleotides in length, at least about 2200 nucleotides in length, at least about 2300 nucleotides in length, at least about 2400 nucleotides in length, or at least about 2500 nucleotides in length.

28. A polynucleotide encoding a polypeptide having immunoglobulin protease activity of claim 5 or any of claims 6 to 25 when dependent on claim 5, the polynucleotide comprising:(a) the polynucleotide sequence of SEQ ID NO: 41 or 42;(b) a variant thereof having at least 80% sequence identity to the polynucleotide sequence of SEQ ID NO: 41 or 42; or(c) a polynucleotide fragment of either (a) or (b), wherein the polynucleotide fragment is at least about 2100 nucleotides in length, at least about 2200 nucleotides in length, at least about 2300 nucleotides in length, or at least about 2400 nucleotides in length.

29. A vector, optionally an expression vector, comprising a polynucleotide according to any one of claims 25 to 28.

30. A cell comprising the polypeptide of any one of claims 1 to 25, the polynucleotide of any one of claims 25 to 28, or the vector of claim 29.

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