Cysteine ​​proteases

Abstract

The present invention relates to novel polypeptides that display IgG cysteine protease activity and their in vivo and in vitro uses. The uses of the polypeptides include methods for preventing or treating IgG-mediated diseases and disorders, as well as methods for analyzing IgG and in vitro F(ab’)2 fragment generation.

Description

Technical Field

[0001] The present invention relates to novel polypeptides exhibiting IgG cysteine ​​protease activity and their in vivo and in vitro uses. The uses of the polypeptides include methods for preventing or treating diseases and conditions mediated by IgG, and methods for analyzing IgG and in vitro F(ab')2 fragment production. Background Art

[0002] IdeS (Immunoglobulin G degrading enzyme of Streptococcus pyogenes, I mmunoglo bulin G- d egradingenzyme of S .pyogenes , also known as imlifidase) is an extracellular cysteine ​​protease produced by the human pathogen Streptococcus pyogenes. IdeS was originally isolated from a group A Streptococcus strain of serotype M1, but the ides gene has now been identified in all tested group A Streptococcus strains. IdeS has a very high degree of substrate specificity, with the only substrate it recognizes being IgG. IdeS catalyzes a single proteolytic cleavage in the lower hinge region of the heavy chains of all subclasses of human IgG. IdeS also catalyzes equivalent cleavages of the heavy chains of some subclasses of IgG in various animals. IdeS efficiently cleaves IgG into Fc and F(ab')2 fragments via a two-stage mechanism. In the first stage, one (first) IgG heavy chain is cleaved to produce a singly cleaved IgG (scIgG) molecule with a single non-covalently bound Fc chain. The scIgG molecule is actually an intermediate product that 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 the F(ab')2 fragment and the homodimeric Fc fragment. These are products commonly observed under physiological conditions. Homodimeric Fc can be dissociated into its constituent monomers. Under reducing conditions, the F(ab')2 fragment can be dissociated into two Fab fragments. It has been shown that the IgG cleavage ability of IdeS has in vitro utility, such as in methods for producing Fab, F(ab')2 and Fc fragments, which can be used, for example, to analyze IgG and generate F(ab')2 fragments in vitro. See, for example, WO2003051914 and WO2009033670. It has also been shown that IdeS has in vivo utility as a therapeutic agent because it can cut disease-mediated or otherwise unwanted IgG molecules in vivo. See, for example, WO2006131347 and WO2013110946. IdeS can be used as a treatment for any disease or condition mediated entirely or in part by IgG. IgG is the pathological cause of many autoimmune diseases and acute rejection of transplanted organs.

[0003] However, IdeS is an immunogenic protein. That is, when IdeS is used as a therapeutic agent, the immune system of the subject receiving IdeS will often respond to it. The immune system's response to IdeS will generally involve the production of antibodies specific to IdeS. These antibodies may be referred to herein as anti-drug antibodies (ADA) specific to IdeS or "IdeS-specific ADA". In general, immune responses to IdeS (particularly the production of IdeS-specific ADA) may cause two related types of problems. First, the efficacy of IdeS may be reduced, for example, due to ADA binding, and higher or repeated doses may be required to achieve the same effect. ADA with this effect may be referred to as "neutralizing ADA". Second, there may be unwanted or even harmful complications, such as a high inflammatory response triggered by an immune complex of ADA with IdeS. The higher the number of ADA specific to IdeS in a given subject, the greater the likelihood of these problems. The presence and number of IdeS-specific ADA molecules in a patient can be determined by any suitable method, such as an agent-specific CAP FEIA (ImmunoCAP) test or a titer assay performed on a serum sample from a patient. Above a threshold determined by a clinician, the number of IdeS-specific ADA molecules in a patient may preclude the administration of IdeS, or indicate the need for a higher dose of IdeS. Such a higher dose may in turn lead to an increase in the number of IdeS-specific ADA molecules in the patient, thereby precluding further administration of IdeS.

[0004] IdeS is a virulence factor of Streptococcus pyogenes, which is responsible for common infections such as tonsillitis and strep throat. Therefore, most human subjects have already encountered IdeS in this setting and are likely to have anti-IdeS antibodies in the bloodstream. IdeS antibodies have been found in serum samples from human subjects (perhaps due to previous streptococcal infection) and in IVIg (intravenous immunoglobulin, I ntravenous IDifferent levels of IdeS-specific ADA are routinely detected in IVIg preparations, which are preparations of IgG extracted from the pooled sera of thousands of donors. Techniques for detecting IdeS-specific ADA are known in the art. Even if a subject does not have IdeS-specific ADA prior to the initial administration of IdeS, such molecules may be produced subsequently. Therefore, for any given subject, problems related to the immunogenicity of IdeS may pose an obstacle to the use of IdeS as a treatment. These problems may require increasing the dose of IdeS and / or precluding treatment with IdeS entirely, particularly if repeated administration is required. Existing solutions to such problems involve, for example, pegylation of the therapeutic agent to reduce the immunogenicity of the therapeutic agent, or co-administration of the therapeutic agent with an immunosuppressant.

[0005] IdeZ is a bacterial pathogen that is caused by Streptococcus equi subsp. zooepidemicus ( Streptococcus equi ssp .zooepidemicus equi), a bacterium found primarily in horses. IdeZ is approximately 66% identical to IdeS. Since S. equi subsp. zooepidemicus is not a human pathogen, IdeZ has been considered an alternative to IdeS-based therapeutics because humans may have fewer or no antibodies (anti-drug antibodies, ADA) to IdeZ. However, IdeZ has levels of IgG cysteine ​​protease activity against human IgG that are much lower than those of IdeS, particularly when cleaving IgG2.

[0006] Therefore, there is still a need for cysteine ​​proteases derived from IdeZ that have high activity against human IgG (preferably higher than wild-type IdeZ, even more preferably higher than IdeS). In particular, there is still a need for cysteine ​​proteases derived from IdeZ that have high activity against human IgG1 and IgG2 (preferably higher than wild-type IdeZ, even more preferably higher than IdeS). Summary of the invention

[0007] The full sequence of IdeS is publicly available as NCBI reference sequence number WP_010922160.1 and is provided herein as SEQ ID NO: 6. The sequence includes an N-terminal methionine followed by a 28 amino acid secretion signal sequence. The N-terminal methionine and signal sequence (29 amino acids total at the N-terminus) are typically removed to form a mature IdeS protein, the sequence of which is publicly available as Genbank accession number ADF13949.1 and is provided herein as SEQ ID NO: 4.

[0008] The full sequence of IdeZ is publicly available as NCBI reference sequence number WP_014622780.1 and is provided herein as SEQ ID NO: 5. The sequence includes an N-terminal methionine followed by a 33 amino acid secretion signal sequence. The N-terminal methionine and signal sequence (34 amino acids total at the N-terminus) are typically removed to form a mature IdeZ protein, the sequence of which is provided herein as SEQ ID NO: 3.

[0009] The inventors have been able to identify specific positions in the sequence of IdeZ that, when modified as described herein, result in novel polypeptides having increased IgG cysteine ​​protease activity against human IgG relative to IdeZ. The IgG cysteine ​​protease activity of the polypeptides of the present invention against human IgG (e.g., when cleaving human IgG) is preferably at least as high as the IgG cysteine ​​protease activity of IdeS against human IgG. The polypeptides of the present invention may be more effective than the IgG cysteine ​​protease of IdeS at cleaving human IgG, particularly when the IgG is of the IgG1 or IgG2 isotype. The polypeptides of the present invention may be more effective than the IgG cysteine ​​protease of IdeS at cleaving human IgG, particularly when measured by cleavage of the second chain of IgG1. The polypeptides of the present invention may be more effective than IgG2 at cleaving IgG1. The polypeptides of the present invention are generally less immunogenic than IdeS and may preferably be no more immunogenic than IdeZ.

[0010] Unless otherwise indicated, all references to the numbering of amino acid positions in the polypeptides disclosed herein are based on the numbering of the corresponding positions in SEQ ID NO: 3 starting from the N-terminus. Thus, since SEQ ID NO: 1 lacks the N-terminal methionine and 33 amino acid signal sequence of SEQ ID NO: 5, the aspartic acid (D) residue at the N-terminus of SEQ ID NO: 1 is referred to as position 35, as this is the corresponding position in SEQ ID NO: 5. Applying this numbering scheme, the most critical residue for the IgG cysteine ​​protease activity of IdeS is the cysteine ​​(C) at position 102 corresponding to SEQ ID NO: 5. Other residues that may be important for IgG cysteine ​​protease activity are the lysine (K) at position 92, the histidine (H) at position 272, and the two aspartic acids (D) at positions 294 and 296 of SEQ ID NO: 5. It has also been found that deleting the first 20 residues at the N-terminus of SEQ ID NO: 1 can increase the potency of a polypeptide containing this change, and / or can reduce immunogenicity without negatively affecting potency. The first 20 residues at the N-terminus of SEQ ID NO: 1 consist of the continuous sequence DDYQRNATEAYAKEVPHQIT. Therefore, the polypeptide according to the present invention may comprise the amino acid sequence of SEQ ID NO: 2 (excluding the continuous sequence DDYQRNATEAYAKEVPHQIT). The first 20 residues of SEQ ID NO: 1 correspond to positions 35-54 of SEQ ID NO: 5. This particular modification can be identified herein by the term "D35_T54del". Therefore, since SEQ ID NO: 2 lacks the N-terminal methionine, the 33 amino acid signal sequence of SEQ ID NO: 5, and the sequence DDYQRNATEAYAKEVPHQIT corresponding to positions 35-54 of SEQ ID NO: 5 is further deleted, the serine (S) residue at the N-terminus of SEQ ID NO: 2 is referred to as position 55, as if this is the corresponding position in SEQ ID NO: 5.

[0011] Therefore, in one aspect, the present invention provides a polypeptide having IgG cysteine ​​protease activity and comprising or consisting of the following amino acid sequence:

[0012] (i) SEQ ID NO: 1; or

[0013] (ii) SEQ ID NO: 2; or

[0014] (iii) a variant of SEQ ID NO: 1 or SEQ ID NO: 2 having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications relative to SEQ ID NO: 1 or SEQ ID NO: 2, respectively, provided that the sequence retains:

[0015] (a) asparagine (N) at the position corresponding to position 95 of SEQ ID NO: 5,

[0016] (b) aspartic acid (D) at the position corresponding to position 99 of SEQ ID NO: 5, and

[0017] (c) asparagine (N) at the position corresponding to position 226 of SEQ ID NO: 5,

[0018] And provided that the polypeptide is at least as effective at cleaving human IgG as a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2, respectively, when measured in the same assay.

[0019] The present invention also provides a polynucleotide, an expression vector or a host cell encoding or expressing the polypeptide of the present invention.

[0020] The present invention also provides a method for treating or preventing a disease or condition mediated by IgG antibodies in a subject, the method comprising administering to the subject a therapeutically or prophylactically effective amount of the polypeptide of the present invention. The method may generally comprise administering the polypeptide to the subject multiple times.

[0021] The invention also provides a method for the ex vivo treatment of blood taken from a patient, typically a patient suffering from a disease or condition mediated by IgG antibodies, the method comprising contacting the blood with a polypeptide of the invention.

[0022] The present invention also provides a method for improving the benefit of a treatment or therapeutic agent on a subject, the method comprising (a) administering a polypeptide of the present invention to a subject; and (b) subsequently administering the treatment or therapeutic agent to the subject; wherein:

[0023] - the treatment is an organ transplant or the therapeutic agent is an antibody, gene therapy (such as a viral vector), replacement of a defective endogenous factor (such as an enzyme, growth or coagulation factor), or cell therapy;

[0024] - the amount of the polypeptide administered is sufficient to cleave substantially all IgG molecules present in the plasma of the subject; and

[0025] - Step (a) and step (b) are separated by a time interval sufficient to cleave substantially all IgG molecules present in the plasma of the subject.

[0026] The present invention also provides a method for producing an Fc fragment, a Fab fragment or a F(ab')2 fragment of IgG, the method comprising contacting IgG with a polypeptide of the present invention (preferably in vitro).

[0027] Also provided are kits for carrying out the methods according to the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 SDS-PAGE analysis of expression and purification of pCART239 (SEQ ID NO: 1, Met at the N-terminus and His tag at the C-terminus) is shown. (A) Overexpression of pCART239 - Lanes 1 and 2 show lysates obtained from cells harvested 1 hour after induction with IPTG; Lane 3 shows pooled lysates. (B) Purification of pCART239 - Lane 1 shows the flow through during NiNTA purification, showing the removal of impurities in the lysate, and Lanes 2 and 3 represent purified pCART239 (~0.5 µg and ~3.0 µg protein loaded, respectively).

[0029] Figure 2 Results of representative SDS-PAGE gels used to visualize cleavage products generated by incubation of IgG1 (Humira) with IdeS and tested IdeZ variants are shown, as indicated. The concentrations above the lanes represent the concentrations of the IdeS / IdeZ variants tested. Panels A and B are representative of two independent experiments.

[0030] Figure 3 Results of representative SDS-PAGE gels used to visualize cleavage products generated by incubation of IgG2 (XGEVA) with IdeS and tested IdeZ variants are shown, as indicated. The concentrations above the lanes represent the concentrations of the IdeS / IdeZ variants tested. Panels A and B are representative of two independent experiments.

[0031] Figure 4 Results are shown for representative SDS-PAGE gels used to visualize cleavage products resulting from incubation of (A) IgG1 (Humira), (B) IgG2 (XGEVA), (C) IgG3, (D) IgG4 with IdeS and N240, as indicated. The concentrations above the lanes represent the concentrations of the IdeS / IdeZ variants tested.

[0032] Figure 5 Shown are fitted titration curves of average electrochemiluminescence (ECL) values ​​of triplicate samples in the assay to determine the potency (cleavage efficiency of IgG1) of N240 compared to IdeS. Error bars represent SD.

[0033] Figure 6Digestion of serum IgG by pCART239 with and without decoy (an inactive version of pCART239) is shown. The use of decoys can reduce the inhibitory effect of ADA present in serum. The concentration above the lanes indicates the concentration of the IdeZ variant tested.

[0034] Figure 7 Fitted titration curves of mean electrochemiluminescence (ECL) values ​​of triplicate samples and two separate dilution series in the assay to determine the potency (cleavage efficiency of IgG) of N240 and IdeS in serum are shown. Error bars represent SD.

[0035] Figure 8 Shown are mean ECL values ​​corresponding to the levels of pre-existing N240 and IdeS ADA in sera from 40 healthy individuals and one normal serum pool (n=100).

[0036] Fig. 9 A schematic diagram showing the step-by-step cleavage of IgG by the polypeptide of the present invention is shown.

[0037] A brief description of the sequence

[0038] SEQ ID NO: 1 is the sequence of a polypeptide of the present invention.

[0039] SEQ ID NO: 2 is the sequence of another polypeptide of the present invention, which is related to SEQ ID NO: 1 and is identical to SEQ ID NO: 1 except that the first 20 amino acids at the N-terminus of SEQ ID NO: 1 are missing (corresponding to the continuous sequence DDYQRNATEAYAKEVPHQIT).

[0040] SEQ ID NO: 3 It is the mature sequence of IdeZ, lacking the N-terminal methionine and signal sequence.

[0041] SEQ ID NO: 4 It is the mature sequence of IdeS, lacking the N-terminal methionine and signal sequence. It is also published as Genbank accession number ADF13949.1.

[0042] SEQ ID NO: 5 The full sequence of IdeZ including the N-terminal methionine and signal sequence is also published as NCBI reference sequence WP_014622780.1.

[0043] SEQ ID NO: 6 The full sequence of IdeS including the N-terminal methionine and signal sequence is also published as NCBI reference sequence WP_010922160.1.

[0044] SEQ ID NO: 7is the sequence of pCART207, a variant IdeZ polypeptide.

[0045] SEQ ID NO: 8 is the sequence of pCART229, a variant IdeZ polypeptide.

[0046] SEQ ID NO: 9 is the sequence of pCART239, a variant IdeZ polypeptide of the invention, which is related to SEQ ID NO: 1 by the presence of an additional N-terminal methionine and an additional C-terminal histidine tag (with a glycine linker).

[0047] SEQ ID NO: 10 is the sequence of N240, a variant IdeZ polypeptide of the invention, which is related to SEQ ID NO: 1 by the presence of an additional N-terminal methionine.

[0048] SEQ ID NO: 11 is the sequence of pCART242, a variant IdeZ polypeptide of the invention, which is related to SEQ ID NO: 2 by the presence of an additional N-terminal methionine and an additional C-terminal histidine tag (with a glycine linker).

[0049] SEQ ID NO: 12 is the sequence of pCART243, an inactive variant IdeZ polypeptide.

[0050] SEQ ID NO: 13 is the sequence of the control IdeS polypeptide, including the sequence SEQ ID NO: 4 with an additional N-terminal methionine and histidine tag (with a glycine linker) (internal reference pCART124).

[0051] SEQ ID NO: 14 is the sequence of the control IdeZ polypeptide, including the sequence SEQ ID NO: 3 with an additional N-terminal methionine and histidine tag (with a glycine linker) (internal reference pCART144).

[0052] SEQ ID NO: 15 is the consecutive sequence DDYQRNATEAYAKEVPHQIT, which corresponds to positions 35-54 of SEQ ID NO:5.

[0053] SEQ ID NO: 16 23 are nucleotide sequences encoding certain polypeptides disclosed herein. DETAILED DESCRIPTION

[0054] It should be understood that different applications of the disclosed products and methods can be adjusted according to the specific needs of the art. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments of the present invention and is not limiting.

[0055] In addition, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes "a plurality of polypeptides" and the like.

[0056] "Polypeptide" is used herein in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or other peptide mimetics. Thus, the term "polypeptide" includes short peptide sequences as well as longer polypeptides and proteins. As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids (including both D or L optical isomers), as well as amino acid analogs and peptide mimetics.

[0057] The terms "patient" and "subject" are used interchangeably and generally refer to a human. References to IgG generally refer to human IgG unless otherwise specified.

[0058] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0059] Functional characteristics of peptides

[0060] The present invention relates to novel polypeptides having IgG cysteine ​​protease activity, wherein the polypeptides are more effective than IdeZ in cleaving human IgG. The IgG cysteine ​​protease activity of the polypeptides of the present invention against human IgG is preferably at least as high as the IgG cysteine ​​protease activity of IdeS against human IgG. In addition, the polypeptides of the present invention are generally less immunogenic than IdeS, and may preferably not exceed the immunogenicity of IdeZ. In the context of a control or comparison relative to the polypeptides of the present invention, "IdeS" and "IdeZ" refer to polypeptides consisting of the amino acid sequences SEQ ID NOs: 4 and 3, respectively. Alternatively or in addition, when used as a control or comparison, "IdeS" and "IdeZ" may refer to polypeptides comprising the amino acid sequences SEQ ID NOs: 4 and 3, respectively, having a methionine (M) residue attached to the N-terminus and / or a tag at the C-terminus to facilitate expression in and separation from standard bacterial expression systems. Suitable tags include histidine tags, which may be directly linked to the C-terminus of the polypeptide or indirectly linked via any suitable linker sequence (such as 3, 4 or 5 glycine residues). The histidine tag typically consists of six histidine residues, although it can be longer than this (typically up to 7, 8, 9, 10 or 20 amino acids) or shorter (e.g., 5, 4, 3, 2 or 1 amino acids). The sequence of an exemplary IdeS polypeptide used herein is a control provided as SEQ ID NO: 13. The polypeptide comprises the sequence SEQ ID NO: 4 with an additional N-terminal methionine and a histidine tag, and may be referred to herein as pCART124. The sequence of an exemplary IdeZ polypeptide used herein is a control provided as SEQ ID NO: 14. The polypeptide comprises the sequence SEQ ID NO: 3 with an additional N-terminal methionine and a histidine tag, and may be referred to herein as pCART144.

[0061] IgG cysteine ​​protease activity can be assessed by any suitable method, for example, by incubating the polypeptide with a sample containing IgG and determining the presence of IgG cleavage products. Potency can be assessed in the presence or absence of an inhibitor (such as a neutralizing antibody). However, potency herein generally refers to potency assessed in the absence of such an inhibitor, unless otherwise specified. Suitable methods are described in the Examples. The potency of a polypeptide in cleaving IgG may be referred to herein as the "potency" of the polypeptide. The potency of the polypeptide of the present invention is preferably at least 2.0 times greater than the potency of IdeZ measured in the same assay. The potency of the polypeptide of the present invention may be at least 1.5 times, 2.0 times, 2.5 times, 3.0 times, 4.0 times, 4.5 times, 5.0 times, 6.0 times, 7.0 times, 7.5 times or 8.0 times greater than the potency of IdeZ measured in the same assay. Alternatively or additionally, the potency of the polypeptide of the present invention is preferably at least equal to the potency of IdeS measured in the same assay. Alternatively or additionally, the titer of the polypeptide of the invention is preferably at least greater than the titer of IdeS measured in the same assay. The titer of the polypeptide of the invention may be at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.5 times, 3.0 times, 4.0 times greater than the titer of IdeS measured in the same assay. The titer of the polypeptide of the invention is preferably at least 2.0 times, more preferably at least 3.0 or at least 4.0 times greater than the titer of IdeS measured in the same assay.

[0062] The polypeptides of the invention are generally less immunogenic than IdeS, and thus an increased titer relative to that of IdeZ and / or a titer comparable to that of IdeS is an acceptable minimum standard for cysteine ​​protease activity against human IgG. However, an increased titer relative to IdeS is a desirable improvement. Such an increase in titer will generally allow the use of a lower dose of the polypeptide of the invention to have the same therapeutic effect as a higher dose of IdeS. A lower dose may also allow for a greater number of repeated administrations of the polypeptide of the invention relative to IdeS. This is because the use of a lower dose reduces problems associated with the immunogenicity of the therapeutic agent, as the immune system is less likely to respond or will respond less to an agent present at a lower concentration.

[0063] Assays for assessing the effectiveness of polypeptides in cleaving IgG, i.e., assays for assessing the potency of polypeptides, are well known in the art, and any suitable assay can be used. Suitable assays include ELISA-based assays, such as those described in the examples. In such assays, the wells of the assay plate will typically be coated with an antibody target, such as bovine serum albumin (BSA). A sample of the polypeptide to be tested is then added to the well, followed by a sample of a target-specific antibody, which is an antibody specific for BSA in this example (and which is sensitive to cleavage by IdeS). The polypeptide and antibody are allowed to interact under conditions suitable for IgG cysteine ​​protease activity. After an appropriate interval, the assay plate will be washed, and a detection antibody that specifically binds to the Fc region of the target-specific antibody will be added under conditions suitable for binding to the target-specific antibody. The detection antibody will bind to the Fc region of any intact target-specific antibody that has been bound to the target in each well. After washing, the amount of detection antibody present in the well will be proportional to the amount of target-specific antibody bound to the well. The detection antibody may be conjugated directly or indirectly to a label or another reporter system (such as an enzyme) so that the amount of detection antibody remaining in each well can be determined. The higher the titer of the tested polypeptide present in the well, the less intact target-specific antibody will remain, and therefore the less detection antibody will be present. Typically, at least one well on a given assay plate will include IdeS in place of the polypeptide to be tested so that the titer of the tested polypeptide can be directly compared to the titer of IdeS. IdeZ or known variants of IdeZ may also be included for comparison.

[0064] Other assays can determine the titer of the tested polypeptide by directly visualizing and / or quantifying the IgG fragments produced by the tested polypeptide cleaving the IgG. This type of assay is also described in the Examples. Such assays typically incubate IgG samples with test polypeptides (or with one or more of the known variants of IdeS, IdeZ, and IdeZ as controls) at different concentrations in a titration series. The products produced by incubation at each concentration are then separated using gel electrophoresis (e.g., by SDS-PAGE). The intact IgG and the fragments produced by the cleavage of the IgG can then be identified by size and quantified by the intensity of staining with a suitable dye. The greater the number of cleavage fragments, the higher the titer of the tested polypeptide at a given concentration. The polypeptides of the present invention typically produce detectable amounts of cleavage fragments at lower concentrations (at lower points in the titration series) than IdeZ and / or IdeS. This type of assay can also be able to identify test polypeptides that are more effective at cleaving the first or second heavy chain of the IgG molecule, because the number of different fragments produced by each cleavage event can also be determined. IgG samples suitable for titer determinations described herein can come from different sources. For example, commercial antibody preparations can be used. Commercial antibody preparations are typically pure, isotype- and subclass-specific. Alternatively, samples containing a mixed IgG population, such as human serum, may be used. Samples containing serum containing IgG may contain ADA against IdeS and / or IdeZ. Thus, IgG samples suitable for use in the titer assays described herein may contain ADA. The polypeptides of the present invention may be more efficient at cleaving the first chain of an IgG molecule than the second chain (compared to IdeS and / or IdeZ) (see Fig. 9 ), particularly when the IgG is of the IgG2 isotype. Alternatively, the polypeptides of the invention may be more efficient at cleaving the second chain of an IgG molecule than the second chain (compared to IdeS and / or IdeZ) (see Fig. 9 ), particularly when the IgG is of the IgG1 isotype. The polypeptides of the invention may be more efficient at cleaving IgG1 than IgG2 (when compared to IdeS and / or IdeZ).

[0065] This type of assay can also be adapted to determine the extent to which the presence of IdeS-specific ADA can reduce the titer of the polypeptide of the invention. In this adaptive assay, serum or IVIg preparations containing IdeS-specific ADA are included together with a reaction medium when an IgG sample is incubated with a test polypeptide (or with IdeS as a control). Preferably, the titer of the polypeptide of the invention is not affected by the presence of ADA, or the titer of the polypeptide of the invention is reduced less by the presence of ADA than the titer of IdeS in the same assay. In other words, preferably, the neutralizing effect of IdeS-specific ADA on the polypeptide of the invention is the same as or lower than the neutralizing effect of IdeS-specific ADA on IdeS measured in the same assay.

[0066] As described above, the polypeptides of the invention are generally less immunogenic than IdeS. That is, when present at equal doses or concentrations and measured in the same assay, the polypeptides of the invention may cause the same immune response as IdeS or preferably a lower immune response. The immunogenicity of the polypeptides of the invention is generally no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, or no more than 25% of the immunogenicity of IdeS measured in the same assay. Preferably, the immunogenicity of the polypeptides of the invention is no more than 25% of the immunogenicity of IdeS measured in the same assay.

[0067] Assays for assessing the immunogenicity of polypeptides are also well known in the art, and any suitable assay may be used. A preferred assay for assessing the immunogenicity of a polypeptide relative to the immunogenicity of IdeS involves assessing the extent to which ADA specific for IdeS also binds to the polypeptides of the invention. This type of assay is described in the Examples. The presence and quantity of IdeS-specific ADA molecules in a patient may be determined by any suitable method, such as a formulation-specific CAP FEIA (ImmunoCAP) test or a titer assay performed on a patient serum sample.

[0068] One such assay involves testing for competition between IdeS and a test polypeptide for binding to an IdeS-specific ADA. Typically, the wells of an assay plate are coated with IdeS, followed by application of a pre-incubated mixture of a solution containing an IdeS-specific ADA (e.g., an IVIg preparation) and a test polypeptide (or IdeS as a control). The pre-incubation is performed in the presence of an inhibitor of IgG cysteine ​​protease activity (e.g., iodoacetic acid (IHAc)) and at a high salt concentration, so that only high affinity binding between the protein and the ADA is allowed. The pre-incubated mixture is allowed to interact with the wells coated with IdeS. Any IdeS-specific ADA that does not bind to the test polypeptide will bind to the IdeS on the wells. After a suitable interval, the assay plate will be washed and a detection antibody that specifically binds to IgG will be added under conditions suitable for binding. The detection antibody will bind to any ADA that has bound to IdeS in each well. After washing, the amount of detection antibody present in the well will be inversely proportional to the amount of ADA that has bound to the test polypeptide. The detection antibody can be conjugated directly or indirectly to a label or another reporter system (such as an enzyme) so that the amount of detection antibody remaining in each well can be determined. Typically, at least one well on a given assay plate will be tested with a pre-incubated mixture of IVIg and IdeS instead of the polypeptide to be tested so that binding of the ADA to the polypeptide being tested can be directly compared to binding to IdeS. IdeZ can also be included as a further control.

[0069] Another suitable assay includes testing a titration series of different concentrations of IdeS-specific ADA (e.g., IVIg preparations) for the extent of binding to the test polypeptide compared to IdeS and / or IdeZ as a control. Preferably, the polypeptide of the present invention will require a higher ADA binding concentration to become detectable relative to the concentration at which ADA binding to IdeS can be detected. Such an assay is described in the Examples. Such an assay typically includes coating the wells of an assay plate with a test polypeptide or a control, followed by incubation of each well with different concentrations of IdeS-specific ADA from the titration series. The incubation is carried out in the presence of an inhibitor of IgG cysteine ​​protease activity (e.g., iodoacetic acid (IHAc)) and at a high salt concentration, so that only high affinity binding between the protein and ADA is allowed. After a suitable interval, the assay plate will be washed and a detection antibody that specifically binds to IgG F(ab′)2 will be added under conditions suitable for binding. The detection antibody will bind to any ADA that has bound to the test polypeptide or IdeS coated in each well. After washing, the amount of detection antibody present in the well will be proportional to the amount of ADA that has bound to the test polypeptide or control. The detection antibody may be conjugated directly or indirectly to a label or another reporter system (such as an enzyme) so that the amount of detection antibody remaining in each well can be determined. At least one well on a given assay plate will be incubated with buffer lacking ADA as a blank to establish a threshold level for detecting binding in the test wells.

[0070] Structural characteristics of peptides

[0071] This section lists the structural features of the polypeptides of the invention, which additionally apply to the functional features outlined in the previous sections.

[0072] The polypeptide of the present invention generally has a length of at least 100, 150, 200, 250, 260, 270, 280, 290, 300 or 310 amino acids. The polypeptide of the present invention generally has a length of no more than 400, 350, 340, 330, 320 or 315 amino acids. It should be understood that any one of the lower limits listed above can be combined with any one of the upper limits listed above to provide a range of lengths of the polypeptide of the present invention. For example, the polypeptide can have a length of 100 to 400 amino acids, or a length of 250 to 350 amino acids. The polypeptide preferably has a length of 290 to 320 amino acids, and most preferably has a length of 300 to 315 amino acids.

[0073] The primary structure (amino acid sequence) of the polypeptide of the present invention is SEQ ID NO: 1, which is a specific variant based on the wild-type mature IdeZ sequence (SEQ ID NO: 3). In other words, SEQ ID NO: 1 is related to SEQ ID NO: 3 by a set of specific point mutations in the primary polypeptide sequence (compared to SEQ ID NO: 3), which are responsible for increasing its potency against human IgG.

[0074] Another polypeptide of the present invention is SEQ ID NO: 2, which is related to SEQ ID NO: 1. SEQ ID NO: 2 is identical to SEQ ID NO: 1 except that the first 20 amino acids (corresponding to the continuous sequence DDYQRNATEAYAKEVPHQIT) are missing at the N-terminus of SEQ ID NO: 1. In other words, SEQ ID NO: 2 is also identical to SEQ ID NO: 1 with respect to the point mutations in the primary polypeptide sequence compared to the wild-type IdeZ sequence (SEQ ID NO: 3).

[0075] The present invention also relates to variants of SEQ ID NO: 1 or SEQ ID NO: 2, which variants have 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications relative to SEQ ID NO: 1 or SEQ ID NO: 2, respectively, provided that the sequence retains: (a) asparagine (N) at the position corresponding to position 95 of SEQ ID NO: 5, (b) aspartic acid (D) at the position corresponding to position 99 of SEQ ID NO: 5, and (c) asparagine (N) at the position corresponding to position 226 of SEQ ID NO: 5, and provided that when measured in the same assay, the polypeptide is at least as effective at cleaving human IgG as a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2, respectively. Optionally, one or more amino acid modifications in the variant of SEQ ID NO: 1 or SEQ ID NO: 2 do not result in the same amino acid as that present in the corresponding position in the polypeptide sequence of SEQ ID NO: 3, preferably, wherein all modifications do not result in the same amino acid as that present in the corresponding position in the polypeptide sequence of SEQ ID NO: 3.

[0076] The polypeptide sequence of the present invention comprises a variant of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications are made relative to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, such as amino acid additions, deletions or substitutions, and the sequence must retain: (a) asparagine (N) at the position corresponding to position 95 of SEQ ID NO: 5, (b) aspartic acid (D) at the position corresponding to position 99 of SEQ ID NO: 5, and (c) asparagine (N) at the position corresponding to position 226 of SEQ ID NO: 5. In addition, these modifications are preferably conservative amino acid substitutions. The variant of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 may comprise one or more modifications (made relative to SEQ ID NO: 1 or SEQ ID NO: 2, respectively) that do not result in the same amino acid as that present in the corresponding position in the polypeptide sequence of SEQ ID NO: 3. Preferably, in a variant of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, all modifications (made relative to SEQ ID NO: 1 or SEQ ID NO: 2, respectively) do not result in an amino acid identical to that present in the corresponding position in the polypeptide sequence of SEQ ID NO: 3.

[0077] Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties and / or similar side chain volumes. The introduced amino acid may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acid it replaces. Alternatively, conservative substitutions may introduce another aromatic or aliphatic amino acid to replace a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and can be selected based on the properties of the 20 major amino acids as defined in Table A1 below. When amino acids have similar polarity, this can be determined by referring to the hydropathy scale of the amino acid side chains in Table A2.

[0078] Table A1 - Chemical properties of amino acids

[0079] Alanine (A) Aliphatic, hydrophobic, neutral Methionine (M) Hydrophobic, neutral Cysteine ​​(C) Polar, hydrophobic, neutral Asparagine (N) Polar, hydrophilic, neutral Aspartic acid (D) Polar, hydrophilic, charged (-) Proline (P) Hydrophobic, neutral Glutamate (E) Polar, hydrophilic, charged (-) Glutamine (Q) Polar, hydrophilic, neutral Phenylalanine (F) Aromatic, hydrophobic, neutral Arginine(R) Polar, hydrophilic, charged (+) Glycine (G) Aliphatic, neutral Serine (S) Polar, hydrophilic, neutral Histidine (H) Aromatic, polar, hydrophilic, charged (+) Threonine (T) Polar, hydrophilic, neutral Isoleucine (I) Aliphatic, hydrophobic, neutral Valine (V) Aliphatic, hydrophobic, neutral Lysine (K) Polar, hydrophilic, charged (+) Tryptophan (W) Aromatic, hydrophobic, neutral Leucine (L) Aliphatic, hydrophobic, neutral Tyrosine (Y) Aromatic, polar, hydrophobic

[0080] Table A2 – Hydrophilicity Scale

[0081] <![CDATA[ Sidechain ]]> <![CDATA[ Hydrophilic <!-- 9 -->]]> Isoleucine (Ile) 4.5 Valine (Val) 4.2 Leucine (Leu) 3.8 Phenylalanine (Phe) 2.8 Cysteine ​​(Cys) 2.5 Methionine (Met) 1.9 Alanine (Ala) 1.8 Glycine (Gly) -0.4 Threonine (Thr) -0.7 Serine (Ser) -0.8 Tryptophan (Trp) -0.9 Tyrosine (Tyr) -1.3 Proline (Pro) -1.6 Histidine (His) -3.2 Glutamate (Glu) -3.5 Glutamine (Gln) -3.5 Aspartic acid (Asp) -3.5 Asparagine (Asn) -3.5 Lysine (Lys) -3.9 Arginine (Arg) -4.5

[0082] Certain residues in the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2 (except positions 95, 99 and 226 corresponding to SEQ ID NO: 5) are preferably retained in a variant sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications. For example, the variant sequence generally retains certain residues known to be required for IgG cysteine ​​protease activity. Thus, the cysteine ​​corresponding to position 102 of SEQ ID NO: 5 must be retained in the amino acid sequence of the polypeptide of the present invention. Optionally, lysine (K) corresponding to position 92 of SEQ ID NO: 5, histidine (H) corresponding to position 272 of SEQ ID NO: 5, and aspartic acid (D) corresponding to each of positions 294 and 296 of SEQ ID NO: 5 are also retained. Therefore, the polypeptide variant of SEQ ID NO: or SEQ ID NO: 2 according to the present invention generally has cysteine ​​(C) at the position corresponding to position 102 of SEQ ID NO: 5; and optionally has lysine (K), histidine (H), aspartic acid (D) and aspartic acid (D) at the positions corresponding to position 92, position 272, position 294 and position 296 of SEQ ID NO: 5, respectively.

[0083] The inventors have also determined that certain other modifications to SEQ ID NO: 1 or SEQ ID NO: 2 sequences can increase the titer of the polypeptide of the invention and / or can reduce the recognition of the polypeptide of the invention by IdeS-specific ADA. Therefore, according to the present invention, SEQ ID NO: 1 or SEQ ID NO: 2 polypeptide variants can be included in SEQ ID NO: 5 positions 84, 93, 97, 137, 139, 140, 147, 150, 162, 165, 166, 171, 174, 205, 226, 237, 239, 243, 250, 251, 254, 255, 282, 288, 312, 315, 347, 349 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 positions of amino acid substitutions.

[0084] Substitutions typically replace an existing amino acid with another amino acid having different properties. For example, an uncharged amino acid can be replaced by a charged amino acid, and vice versa. Preferred substitutions at these positions are listed in Table B below using single letter codes:

[0085] Table B

[0086] Amino acids present in SEQ ID NO: 1 or 2 The position corresponding to SEQ ID NO: 5 Preferred alternative H 84 N A 93 T K 97 A F 137 I N 139 R / K Q 140 E A 147 E D 150 R N 162 E R 165 K D 166 E N 171 Y A 174 T N 205 K L 237 F N 239 E N 243 K K 250 S Q 251 E T 254 E E 255 K N 282 D E 288 K A 312 K H 315 K K 347 Q S 349 N

[0087] Each substitution may be referred to herein using a term obtained by combining the entries in the first, second, and third columns of each row from left to right. For example, the substitution in the first row of Table B may be referred to herein as "H84N", the substitution in the second row may be referred to as "A93T", and so on.

[0088] Table C below summarizes the changes made relative to the wild-type IdeZ sequence (SEQ ID NO: 3) to produce the amino acid sequences of certain polypeptides described herein.

[0089] Table C

[0090]

[0091] The full length amino acid sequences of certain polypeptides mentioned herein are reproduced below.

[0092] SEQ ID NO: 1

[0093] DDYQRNATEAYAKEVPHQITSVWTKGVTPPEQFTQGEDVIHAPYLAHQGWYDITKAFNGKDDLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFRNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYY LNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGNQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFFTLSSGKDIWQKLS

[0094] SEQ ID NO: 2

[0095] SVWTKGVTPPEQFTQGEDVIHAPYLAHQGWYDITKAFNGKDDLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFRNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGNQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLS

[0096] SEQ ID NO: 3 (Mature IdeZ sequence)

[0097] DDYQRNATEAYAKEVPHQITSVWTKGVTPLTPEQFRYNNEDVIHAPYLAHQGWYDITKAFDGKDNLL C GAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFNNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGDQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLS

[0098] SEQ ID NO: 4 (Mature IdeS sequence)

[0099] DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

[0100] SEQ ID NO: 5 (Full sequence of IdeZ)

[0101] MKTIAYPNKPHSLSAGLLTAIAIFSLASSNITYADDYQRNATEAYAKEVPHQITSVWTKGVTPLTPEQFRYNNEDVIHAPYLAHQGWYDITKAFDGKDNLL C GAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFNNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGDQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLS

[0102] SEQ ID NO: 6 (Full sequence of IdeS)

[0103] MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

[0104] SEQ ID NO: 7 (pCART207)

[0105] MDDYQRNATEAYAKEVPHQITSVWTKGVTPPEQFTQGEDVIHAPYLAHQGWYDITKAFDGKDNLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFRNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGDQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLS

[0106] SEQ ID NO: 8 (pCART229)

[0107] MDDYQRNATEAYAKEVPHQITSVWTKGVTPPEQFTQGEDVIHAPYLAHQGWYDITKAFDGKDNLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFRNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGNQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLS

[0108] SEQ ID NO: 9 (pCART239)

[0109] MDDYQRNATEAYAKEVPHQITSVWTKGVTPPEQFTQGEDVIHAPYLAHQGWYDITKAFNGKDDLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFRNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGNQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLSGGGHHHHHH

[0110] SEQ ID NO: 10 (N240)

[0111] MDDYQRNATEAYAKEVPHQITSVWTKGVTPPEQFTQGEDVIHAPYLAHQGWYDITKAFNGKDDLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFRNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGNQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLS

[0112] SEQ ID NO: 11 (pCART242)

[0113] MSVWTKGVTPPEQFTQGEDVIHAPYLAHQGWYDITKAFNGKDDLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFRNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGNQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLSGGGHHHHHH

[0114] SEQ ID NO: 12 (pCART243 – Inactive IdeZ Variant)

[0115] MDDYQRNATEAYAKEVPHQITSVWTKGVTPPEQFTQGEDVIHAPYLAHQGWYDITKAFNGKDDLLGGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFRNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVF KTQSTDVNRPYQDKDKRGGIFDAVFTRGNQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFFTLSSGKDIWQKLSGGGHHHHHHH

[0116] The polypeptide of the present invention may comprise, consist essentially of or consist of the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. Each of SEQ ID NO: 1 or SEQ ID NO: 2 may optionally include an additional methionine at the N-terminus and / or a histidine tag at the C-terminus. The histidine tag preferably consists of six histidine residues. The histidine tag is preferably attached to the C-terminus via a linker of 3×glycine or 5×glycine residues.

[0117] Where relevant, any suitable algorithm may be used to calculate amino acid identity. For example, the PILEUP and BLAST algorithms may be used to calculate identity or align sequences (such as identifying identical or corresponding sequences (usually on their default settings), e.g., as described in Altschul S.F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al. (1990) J Mol Biol 215:403-10. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short characters of length W in the query sequence that match or satisfy some positive threshold score T when aligned with a character of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood characters (initialneighbourhood word Hits are used as seeds for initiating searches to find HSPs containing them. Hits are extended in both directions along each sequence as far as the cumulative alignment score can be increased. Extension of hits in each direction ceases when: the cumulative alignment score falls by an amount X from its maximum achieved value; the cumulative score reaches zero or below due to the accumulation of one or more negative scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses a word length (W) of 11 and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc .Natl .Acad .Sci. USA 89: 10915-10919) with alignment (B) of 50, expectation (E) of 10, M = 5, N = 4, and comparison of both strands as default values.

[0118] The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see, e.g., Karlin and Altschul (1993). Proc .Natl. Acad. Sci.USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two polynucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability of a first sequence compared to a second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. Alternatively, the UWGCG software package provides the BESTFIT program (e.g., used on its default settings) that can be used to calculate identity (Devereux et al. (1984) Nucleic Acids Research 12, 387-395).

[0119] Peptide production

[0120] The polypeptides disclosed herein can be produced by any suitable means. For example, the polypeptides can be directly synthesized using standard techniques known in the art (such as Fmoc solid phase chemistry, Boc solid phase chemistry or by solution phase peptide synthesis). Alternatively, the polypeptides can be produced by transforming cells (usually bacterial cells) with nucleic acid molecules or vectors encoding the polypeptides. The production of polypeptides by expression in bacterial host cells is described below and exemplified in the examples. The present invention provides nucleic acid molecules and vectors encoding the polypeptides of the present invention. The present invention also provides host cells comprising such nucleic acids or vectors. Exemplary polynucleotide molecules encoding the polypeptides of the present invention and other disclosed herein are provided as SEQ ID NOs: 16 to 23. Each of these sequences includes a codon (ATG) for an N-terminal methionine at the 5' end, and before a stop codon (TAA) at the 3' end, codons for a 3× Gly linker and a 6× His histidine tag, which can be optionally excluded.

[0121] The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. Non-limiting examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. The polynucleotides of the present invention can be provided in an isolated or substantially isolated form. By substantially isolated, it is meant that the separation of the polypeptide from any surrounding medium may be basic but not complete. The polynucleotide can be mixed with a carrier or diluent that does not interfere with its intended use and is still considered to be substantially isolated. A nucleic acid sequence "encoding" a selected polypeptide is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide when placed under the control of an appropriate regulatory sequence (e.g., 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' (carboxyl) terminus. For purposes of the present invention, such nucleic acid sequences may include, but are not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic sequences from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. The transcription termination sequence may be located 3' to the coding sequence.

[0122] Polynucleotides can be synthesized according to methods well known in the art, as described by way of example in Sambrook et al. (1989, Molecular Cloning-a laboratory manual; Cold Spring Harbor Press). The nucleic acid molecules of the present invention can be provided in the form of expression cassettes, which include control sequences operably linked to the inserted sequences, thereby allowing the in vivo expression of the polypeptides of the present invention. These expression cassettes are then typically provided in vectors (e.g., plasmids or recombinant viral vectors). Such expression cassettes can be directly administered to a host subject. Alternatively, a vector comprising a polynucleotide of the present invention can be administered to a host subject. Preferably, a genetic vector is used to prepare and / or administer the polynucleotides. A suitable vector can be any vector capable of carrying a sufficient amount of genetic information and allowing the expression of the polypeptide of the present invention.

[0123] Therefore, the present invention includes expression vectors comprising such polynucleotide sequences. Such expression vectors are routinely constructed in the field of molecular biology and may, for example, involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements that may be necessary and located in the correct orientation, such as, for example, polyadenylation signals, to allow expression of the peptides of the present invention. Other suitable vectors will be apparent to those skilled in the art. For another example in this regard, we refer to Sambrook et al.

[0124] The present invention also includes cells that have been modified to express the polypeptide of the present invention. Such cells generally include prokaryotic cells (such as bacterial cells), for example Escherichia coli. Such cells can be cultured using conventional methods to produce the polypeptide of the present invention.

[0125] The polypeptide can be derivatized or modified to aid in its production, separation or purification. For example, when the polypeptide of the present invention is produced by recombinant expression in a bacterial host cell, the sequence of the polypeptide can include an additional methionine (M) residue at the N-terminus to improve expression. As another example, the polypeptide of the present invention can be derivatized or modified by adding a ligand that can directly and specifically bind to the separation means. Alternatively, the polypeptide can be derivatized or modified by adding a member of a binding pair, and the separation means includes an agent that is derivatized or modified by adding another member of the binding pair. Any suitable binding pair can be used. In a preferred embodiment in which the polypeptide of the present invention is derivatized or modified by adding a member of a binding pair, the polypeptide is preferably histidine-tagged or biotin-tagged. Typically, the amino acid coding sequence of the histidine or biotin tag is included at the genetic level, and the polypeptide is recombinantly expressed in Escherichia coli. The histidine or biotin tag is usually present at either end of the polypeptide, preferably at the C-terminus. It can be directly connected to the polypeptide, or indirectly connected by any suitable linker sequence (such as 3, 4 or 5 glycine residues). A histidine tag typically consists of six histidine residues, although it may be longer (typically up to 7, 8, 9, 10 or 20 amino acids) or shorter (e.g. 5, 4, 3, 2 or 1 amino acid).

[0126] The amino acid sequence of a polypeptide can be modified to include non-naturally occurring amino acids, for example to increase stability. When a polypeptide is produced by a synthetic method, these amino acids can be introduced during the production process. The polypeptide can also be modified after synthesis or recombinant production. D-amino acids can also be used to produce polypeptides. In these cases, the amino acids will be connected in the reverse order of the C to N direction. This is conventional in the technology for producing such polypeptides.

[0127] Many side chain modifications are known in the art and can be made to the side chains of the polypeptides so that the polypeptides retain any further desired activity or property that may be specified herein. It will also be understood that polypeptides can be chemically modified, such as post-translationally modified. For example, they can be glycosylated, phosphorylated or contain modified amino acid residues.

[0128] The polypeptide may be pegylated. The polypeptide of the invention may be in substantially isolated form. It may be mixed with a carrier or diluent (as described below) that will not interfere with the intended use and still be considered substantially isolated. It may also be in substantially purified form, in which case it will typically contain at least 90%, such as at least 95%, 98% or 99% of the protein in the preparation.

[0129] Compositions and preparations containing polypeptides

[0130] On the other hand, the present invention provides compositions comprising polypeptides of the present invention. For example, the present invention provides compositions comprising one or more polypeptides of the present invention and at least one pharmaceutically acceptable carrier or diluent. One or more carriers must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not harmful to the subject to which the composition is administered. Typically, the carrier and the final composition are sterile and pyrogen-free.

[0131] The preparation of suitable compositions can be carried out using standard pharmaceutical preparation chemistry and methodology, all of which are readily available to fairly skilled technicians. For example, the reagent can be combined with one or more pharmaceutically acceptable excipients or vehicles. Auxiliary substances (such as wetting agents or emulsifiers), pH buffer substances, reducing agents, etc. can be present in the excipients or vehicles. Suitable reducing agents include cysteine, thioglycerol, thiolactones, glutathione, etc. Excipients, vehicles and auxiliary substances are generally pharmaceutical agents that do not induce immune responses in individuals receiving the composition, and they can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids (such as water), saline, polyethylene glycol, hyaluronic acid, glycerol, thioglycerol and ethanol. Pharmaceutically acceptable salts may also be included, for example, inorganic acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, etc.; and organic acid salts such as acetates, propionates, malonates, benzoates, etc. A detailed discussion of pharmaceutically acceptable excipients, vehicles, and auxiliary substances is found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N. J. 1991).

[0132] Such compositions can be prepared, packaged or sold in a form suitable for bolus administration or continuous administration. Injectable compositions can be prepared, packaged or sold in unit dosage form, such as in ampoules or multi-dose containers containing preservatives. Compositions include, but are not limited to suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable preparations. Such compositions may also include one or more additional ingredients, including but not limited to suspending agents, stabilizers or dispersants. In one embodiment of a composition for parenteral administration, the active ingredient is provided in a dry (e.g., powder or granule) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water), followed by parenteral administration of the reconstituted composition. Compositions may be prepared, packaged or sold in the form of sterile injectable aqueous or oily suspensions or solutions. Such suspensions or solutions may be prepared according to known techniques, and in addition to the active ingredient, may also include additional ingredients, such as dispersants, wetting agents or suspending agents as described herein. For example, a nontoxic parenterally acceptable diluent or solvent (such as water or 1,3-butylene glycol) can be used to prepare such a sterile injectable preparation. 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).

[0133] Other useful parenteral compositions include those comprising active ingredients in microcrystalline form, in liposome preparations or as components of biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion exchange resins, slightly soluble polymers or slightly soluble salts. Compositions may be suitable for use by any suitable route, including, for example, intradermal, subcutaneous, percutaneous, intramuscular, intraarterial, intraperitoneal, intraarticular, intraosseous, intracapsular or other suitable routes of administration. Preferred compositions are suitable for use by intravenous infusion.

[0134] How to use peptides

[0135] The present invention provides the use of the polypeptide of the present invention in various methods. For example, the polypeptide of the present invention can provide a useful tool for biotechnology. The polypeptide can be used for specific in vitro cleavage of IgG (particularly human IgG). In this method, the polypeptide can be incubated with a sample containing IgG under conditions that allow specific cysteine ​​protease activity to occur. The specific cleavage can be verified and the cleavage products can be separated using any suitable method (such as those described in WO2003051914 and WO2009033670). Therefore, this method can be particularly used to produce Fc and F(ab')2 fragments. Fab fragments can then be prepared by performing a reduction step (e.g., in 2-mercaptoethanolamine or cysteamine) on the F(ab')2 fragment produced by cleaving IgG with the polypeptide of the present invention.

[0136] The method can also be used to detect or analyze the IgG in the sample or remove IgG from the sample. The method for detecting IgG in the sample is generally included in the conditions that allow IgG specific binding and cutting, and the polypeptide is hatched together with the sample. The presence of IgG can be verified by detecting the specific IgG cleavage product, which can be analyzed subsequently.

[0137] The polypeptide according to the present invention can also be used for treatment or prevention. In therapeutic applications, the polypeptide or composition is applied to an object that has already suffered from a disease or condition in an amount sufficient to cure, alleviate or partially prevent the disease or one or more symptoms thereof. This therapeutic therapy may result in a disease with a reduced severity of disease symptoms, or an increased frequency or duration of asymptomatic periods. An amount sufficient to achieve this purpose is defined as a "therapeutically effective amount". In preventive applications, the polypeptide or composition is applied to an object that has not yet shown symptoms of a disease or condition in an amount sufficient to prevent or delay the development of symptoms. Such an amount is defined as a "preventive effective amount". The object may be determined to be at risk of developing a disease or condition by any suitable means. Therefore, the present invention also provides a polypeptide of the present invention for use in the treatment of a human or animal body. Also provided herein is a method for preventing or treating a disease or condition of an object, the method comprising administering a polypeptide of the present invention to the object in a preventive or therapeutically effective amount. The polypeptide may be co-administered with an immunosuppressant. The polypeptide is preferably administered by intravenous infusion, but may be administered by any suitable route, including, for example, intradermal, subcutaneous, transdermal, intramuscular, intraarterial, intraperitoneal, intraarticular, intraosseous, intracapsular or other suitable administration routes. The amount of the polypeptide administered may be between 0.01 mg / kg BW and 2 mg / kg BW, between 0.05 and 1.5 mg / kg BW, between 0.1 mg / kg BW and 1 mg / kg BW, preferably between 0.15 mg / kg and 0.7 mg / kg BW, and most preferably between 0.2 mg / kg and 0.3 mg / kg BW, in particular 0.25 mg / kg BW. The polypeptide may be administered to the same subject multiple times, provided that the number of ADA in the subject's serum that is capable of binding to the polypeptide does not exceed a threshold value determined by a clinician. The number of ADA in the subject's serum that is capable of binding to the polypeptide may be determined by any suitable method, such as an agent-specific CAP FEIA (ImmunoCAP) test or a titer assay.

[0138] The polypeptides of the present invention are particularly useful for treating or preventing diseases or conditions mediated by pathogenic IgG antibodies. Therefore, the present invention provides polypeptides of the present invention for treating or preventing diseases or conditions mediated by pathogenic IgG antibodies. The present invention also provides methods for treating or preventing diseases or conditions mediated by pathogenic IgG antibodies, comprising administering the polypeptides of the present invention to an individual. The method may comprise repeated administration of the polypeptides. The present invention also provides polypeptides of the present invention for use in the manufacture of a medicament for treating or preventing diseases or conditions mediated by pathogenic IgG antibodies (particularly autoimmune diseases mediated in whole or in part by pathogenic IgG antibodies).

[0139] Pathogenic antibodies can often be specific for an antigen, targeting the antigen in autoimmune diseases that are mediated in whole or in part by the antibodies. Table D lists a list of these diseases and associated antigens. The polypeptides of the present invention can be used to treat any of these diseases or conditions. The polypeptides are particularly effective for treating or preventing autoimmune diseases that are mediated in whole or in part by pathogenic IgG antibodies.

[0140] Table D

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149] In another embodiment, the polypeptides of the invention can be used in a method for improving the benefit of a treatment or therapeutic agent to a subject. The method comprises two steps, which are referred to herein as step (a) and step (b).

[0150] Step (a) comprises administering a polypeptide of the present invention to a subject. The amount of the polypeptide administered is preferably sufficient to cleave substantially all IgG molecules present in the subject's plasma. Step (b) comprises subsequently administering the treatment or therapeutic agent to the subject. Steps (a) and (b) are separated by a time interval that is preferably sufficient to cleave substantially all IgG molecules present in the subject's plasma. The interval can generally be at least 30 minutes and up to 21 days.

[0151] The therapeutic agent whose benefit is improved is typically an antibody administered for the treatment of cancer or other diseases. The therapeutic agent may be IVIg. In the context of this embodiment, the invention may alternatively be described as providing a method for treating cancer or other diseases in a subject, the method comprising: (a) administering a polypeptide of the invention to the subject; and (b) subsequently administering to the subject a therapeutically effective amount of an antibody as a treatment for said cancer or said other disease; wherein:

[0152] - the amount of the polypeptide administered is sufficient to cleave substantially all IgG molecules present in the plasma of the subject; and

[0153] - Step (a) and step (b) are separated by a time interval of at least 30 minutes and at most 21 days.

[0154] In other words, the present invention also provides polypeptides for use in this method for treating cancer or other diseases. The present invention also provides the use of the agent in the preparation of a medicament for treating cancer or other diseases by this method. The cancer may be acute lymphocytic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendix cancer, childhood cerebellar or cerebral astrocytoma, basal cell carcinoma, extrahepatic bile duct cancer, bladder cancer, bone cancer, osteosarcoma / malignant fibrous histiocytoma, brain stem glioma, brain cancer, brain tumor (cerebellar astrocytoma), brain tumor (cerebral astrocytoma / malignant glioma), brain tumor (ependymoma), brain tumor (medulloblastoma), brain tumor (supratentorial primitive neuroectodermal tumor), brain tumor (visual pathway and hypothalamic glioma), breast cancer, bronchial adenoma / carcinoid, Burkitt's lymphoma (Burkittlymphoma), carcinoid tumors, gastrointestinal carcinoid tumors, unknown primary cancer, central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, cervical cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disease, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma in the Ewing family of tumors, extracranial germ cell tumors in children, extragonadal germ cell tumors, extrahepatic bile duct cancer, eye Cancer (intraocular melanoma), Eye cancer (retinoblastoma), Gallbladder cancer, Gastric (stomach) cancer, Gastrointestinal carcinoma-like tumors, Gastrointestinal stromal tumors (GIST), Germ cell tumors (extracranial, extragonadal, or ovarian), Gestational trophoblastic tumors, Brain stem gliomas, Gliomas, Childhood cerebral astrocytomas, Childhood visual pathway and hypothalamic gliomas, Gastric carcinoid, Hairy cell leukemia, Head and neck cancer, Heart cancer, Hepatocellular (liver) cancer, Hodgkin lymphoma, Hypopharyngeal cancer, Hypothalamic and visual pathway gliomas, Intraocular melanoma , Islet cell carcinoma (endocrine pancreas), Kaposi sarcoma, Kidney cancer (renal cell carcinoma), Laryngeal cancer, Leukemia, Acute lymphoblastic leukemia (also known as acute lymphocytic leukemia), Acute myeloid leukemia (also known as acute myeloid leukemia), Chronic lymphocytic leukemia (also known as chronic lymphocytic leukemia), Chronic myeloid leukemia (also known as chronic myeloid leukemia), Hairy cell leukemia, Lip and oral cavity cancer, Liposarcoma, Liver cancer (primary), Lung cancer, Non-small cell lung cancer, Small cell lung cancer, Lymphoma , AIDS-related lymphoma, Burkitt's lymphoma, Cutaneous T-cell lymphoma, Hodgkin's lymphoma, Non-Hodgkin's lymphoma (old classification of all lymphomas except Hodgkin's), Primary CNS lymphoma, Macroglobulinemia, Waldenström's, Malignant fibrous histiocytoma / osteosarcoma of bone, Medulloblastoma, Melanoma, Intraocular (eye) melanoma, Merkel cell carcinoma, Malignant mesothelioma in adults, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Mouth cancerCancer), Multiple Endocrine Neoplasms Syndrome, Multiple Myeloma / Plasma Cell Neoplasms, Mycosis Fungoides, Myelodysplastic Syndrome, Myelodysplastic / Myeloproliferative Diseases, Chronic Myeloid Leukemia, Adult Acute Myeloid Leukemia, Childhood Acute Myeloid Leukemia, Myeloma, Multiple (Bone Marrow Cancer) Myeloproliferative Diseases, Nasal and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin's Lymphoma, Non-Small Cell Lung Cancer, Oral Cancer, Oropharyngeal Cancer, Bone Sarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer (surface epithelial-stromal tumor), ovarian germ cell tumor, ovarian low-grade malignant potential tumor, pancreatic cancer, pancreatic cancer (islet cell), paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germ cell tumor, pinealoblastoma and supratentorial primitive neuroectodermal tumor, pituitary adenoma, plasma cell neoplasia / multiple myeloma, pleuropulmonary blastoma, primary central nervous system Lymphoma, prostate cancer, colorectal cancer, renal cell carcinoma (kidney cancer), transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Ewing family tumors, Kaposi sarcoma, soft tissue sarcoma, uterine sarcoma, Sézary syndrome, skin cancer (non-melanoma), skin cancer (melanoma), skin cancer (Merkel cell), small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, metastatic squamous cell neck cancer with occult primary, gastric cancer , supratentorial primitive neuroectodermal tumors, T-cell lymphomas, cancers of the skin - see mycosis fungoides and Sezary syndrome, testicular cancer, laryngeal cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumors, transitional cell carcinoma of the ureter and renal pelvis, urethral cancer, uterine cancer (endometrium), uterine sarcoma, vaginal cancer, optic pathway and hypothalamic gliomas, vulvar cancer, Waldenström's macroglobulinemia, and Wilms tumor (kidney cancer).

[0155] The cancer is preferably prostate cancer, breast cancer, bladder cancer, colon cancer, rectal cancer, pancreatic cancer, ovarian cancer, lung cancer, cervical cancer, endometrial cancer, kidney (renal cell) cancer, esophageal cancer, thyroid cancer, skin cancer, lymphoma, melanoma or leukemia.

[0156] The antibodies administered in step (b) are preferably specific for tumor antigens associated with one or more of the above cancer types. Targets of interest for antibodies used in this method include CD2, CD3, CD19, CD20, CD22, CD25, CD30, CD32, CD33, CD40, CD52, CD54, CD56, CD64, CD70, CD74, CD79, CD80, CD86, CD105, CD138, CD174, CD205, CD227, CD326, CD340, MUC16, GPNMB, PSMA, Cripto, ED-B, TMEFF2, EphA2, EphB2, FAP, αv integrin, mesothelin, EGFR, TAG-72, GD2, CA1X, 5T4, α4β7 integrin, Her2. Other targets are cytokines (such as interleukins IL-1 to IL-13), tumor necrosis factor α & β, interferon α, β and γ, tumor growth factor β (TGF-β), colony stimulating factor (CSF) and granulocyte monocyte colony stimulating factor (GMCSF). See Human Cytokines: Handbook for Basic & Clinical Research (Aggrawal et al. eds., Blackwell Scientific, Boston, MA 1991). Other targets are hormones, enzymes, and intracellular and intercellular messengers (such as adenylate cyclase, guanylate cyclase and phospholipase C). Other targets of interest are leukocyte antigens such as CD20 and CD33. Drugs may also be targets of interest. The target molecule may be human, mammalian or bacterial. Other targets are antigens from microbial pathogens (both viral and bacterial) and tumors, such as proteins, glycoproteins and carbohydrates. Other targets are also described in US 4,366,241.

[0157] The antibody can be directly or indirectly attached to the cytotoxic moiety or to a detectable label. Antibodies can be administered by one or more routes of administration using one or more of the various methods known in the art. The route and / or mode of administration will vary according to the desired result. Preferred routes of administration for antibodies include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as by injection or infusion. The phrase "parenteral administration" used herein refers to a mode of administration generally by injection rather than enteral administration and topical administration. Alternatively, antibodies can be administered by non-parenteral routes, such as topical, epidermal or mucosal routes of administration. Local administration is also preferred, including peri-tumoral, juxtatumoral, intra-tumoral, intralesional, perilesional, intracavity infusion, intravesicle administration and suction.

[0158] The appropriate dosage of the antibodies of the present invention can be determined by a skilled physician. The actual dosage level of the antibodies can be varied to obtain an amount of active ingredient that is effective and non-toxic to the patient to achieve the desired therapeutic response for a particular patient, composition, and mode of administration. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular antibody employed, the route of administration, the time of administration, the rate of antibody excretion, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and other factors well known in the medical field.

[0159] Suitable dosages of the antibody may be, for example, in the range of about 0.1 μg / kg to about 100 mg / kg body weight of the patient to be treated. For example, a suitable dosage may be about 1 μg / kg to about 10 mg / kg body weight per day or about 10 μg / kg to about 5 mg / kg body weight per day.

[0160] The dosage regimen can be adjusted to provide the best desired response (e.g., therapeutic response). For example, a single bolus can be administered, or step (b) of the method can include several separate doses administered over time, or the dose can be proportionally reduced or increased as indicated by the urgency of the therapeutic situation, as long as the required interval between step (a) and step (b) is not exceeded. It is particularly advantageous to formulate parenteral compositions in a dosage unit form that facilitates administration and uniformity of dosage. The dosage unit form used herein refers to physically discrete units suitable as unit doses for subjects to be treated; each unit contains a predetermined amount of active compound calculated to produce a desired therapeutic effect in association with a desired pharmaceutical carrier.

[0161] The antibody of step (b) can be administered in combination with chemotherapy or radiation therapy. The method can further include administering additional anti-cancer antibodies or other therapeutic agents, which can be administered together with the antibody of step (b) in a single composition or in separate compositions as part of a combined therapy. For example, the antibody of step (b) can be administered before, after, or simultaneously with the other agents.

[0162] The antibody can be Abagovomab, Abciximab, Actoxumab, Adalimumab, Adecatumumab, Afelimomab, Afutuzumab, Alacizumab pegol, ALD518, Alemtuzumab, Alirocumab, Altumomab pentetate, Amatuximab, Anatumomab mafenaox), Anrukinzumab, Apolizumab, Arcitumomab, Aselizumab, Atinumab, Atlizumab (= tocilizumab), Atorolimuumab, Bapineuzumab, Basiliximab, Batuximab 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, Certolizumabpegol), Cetuximab, Ch .14.18, Citatuzumab bogatox, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumabtetraxetan, Conatumumab, Concizumab, Crenezumab, CR6261, Dacetuzumab, Daclizumab, Dalotuzumab, Daratumumab, Demcizumab, Denosumab, Detumomab, Dorlimomab aritox), Drozitumab, Duligotumab, Dupilumab, Dusigitumab, Ecormeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Elotuzumab, Elsilimomab, Enavatuzumab, Enlimomabpegol), Enokizumab, Enoticumab, Ensituximab, Epitumomabcituxetan, Epratuzumab, Erlizumab, Ertumaxomab, Etaracizumab, Etrolizumab, Evolocumab, Exbivirumab, Fanolesomab, Faralimomab, Farletuzumab, Fasinumab, FBTA0 5. 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, Ibritumomabtiuxetan, Icrucumab, Igovomab, Imciromab, Imgatuzumab, Inclacumab, Indatuximab ravtansine), Infliximab, Intetumumab, Inolimomab, Inotuzumab ozogamicinozogamicin), Ipilimumab, Iratumumab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lampalizumab, Lebrikizumab, Lemalesomab, Lerdelimumab, Lexatumumab, Libivirumab, Ligelizumab, Lintuzumab, Lirilumab, Lodelcizumab, Lorvotuzumab bmertansine), Lucatumumab, Lumiliximab, Mapatumumab, Maslimomab, Mavrilimumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mitumomab, Mogamulizumab, Morolimumab, Motavizumab, Moxetumomabpasudotox, 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, PRO140. 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、Telimomabaritox), Tenatumomab, Teneliximab, Teplizumab, Teprotumumab, TGN1412, Ticilimumab (= tremelimumab), Tildrakizumab, Tigatuzumab, TNX-650, Tocilizumab (= atlizumab), Toralizumab, Tositumomab, Tralokinumab, Trastuzumab, TRBS07, Tregalizumab, Tremelimumab, Tucotuzumab celmoleukin), Tuvirumab, Ublituximab, Urelumab, Urtoxazumab, Ustekinumab, Vapaliximab, Vatelizumab, Vedolizumab, Veltuzumab, Vepalimomab, Vesencumab, Visilizumab, Volociximab, Vorsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, ziralimumab, or zolimomab aritox.

[0163] Preferred antibodies include Natalizumab, Vedolizumab, Belimumab, Atacicept, Alefacept, Otelixizumab, Teplizumab, Rituximab, Ofatumumab, Ocrelizumab, Epratuzumab, Alemtuzumab, Abatacept, Abatacept, Eculizumab, Omalizumab, Canakinumab, Meplizumab, Reslizumab, Tocilizumab, Ustekinumab, Briakinumab, Etanercept, Inlfliximab, Adalimumab, Certolizumab pegol), Golimumab, Trastuzumab, Gemtuzumab, Ozogamicin, Ibritumomab, Tiuxetan, Tostitumomab, Cetuximab, Bevacizumab, Panitumumab, Denosumab, Ipilimumab, Brentuximab, and Vedotin.

[0164] The treatment for which the benefit is improved is usually an organ transplant. The organ may be selected from the kidney, liver, heart, pancreas, lung or small intestine. The subject to be treated may preferably be sensitized or highly sensitized. "Sensitized" means that the subject has developed antibodies against human major histocompatibility (MHC) antigens, also known as human leukocyte antigens (HLA). Anti-HLA antibodies are derived from heterologous sensitized B cells and are usually present in patients who have been previously sensitized by transfusion, previous transplant or pregnancy (Jordan et al., 2003).

[0165] Whether a potential transplant recipient is sensitized can be determined by any suitable method. For example, a population reactive antibody (PRA) test can be used to determine whether the recipient is sensitized. A PRA score >30% is generally considered to mean that the patient is "high immunological risk" or "sensitized." Alternatively, a crossmatch test can be performed in which a blood sample from a potential transplant donor is mixed with a blood sample from the intended recipient. A positive crossmatch means that the recipient has antibodies that react with the donor sample, indicating that the recipient is sensitized and that the transplant should not occur. A crossmatch test is often performed as a final check immediately prior to transplantation.

[0166] The presence of high titer antibodies against the MHC antigens of a potential donor, i.e., donor-specific antibodies (DSA), is a direct contraindication to transplantation due to the risk of acute antibody-mediated rejection. In short, sensitization to the donor MHC antigens hampers the identification of a suitable donor. A positive cross-match test is a clear barrier to transplantation. Since approximately one-third of patients awaiting a kidney transplant are sensitized, and up to 15% are highly sensitized, this leads to an accumulation of patients awaiting a transplant. In the United States, the median time on the kidney transplant waiting list during 2001-2002 was 1329 days for those with a population reactive antibody (PRA) score of 0-9%, 1920 days for those with a PRA of 10-79%, and 3649 days for those with a PRA of 80% or above (OPTN Database, 2011).

[0167] An accepted strategy to overcome the DSA barrier is to use plasma exchange or immunoadsorption, which is usually combined with, for example, intravenous gamma-globulin (IVIg) or rituximab, to reduce the level of DSA to a level at which transplantation can be considered (Jordan et al., 2004; Montgomery et al., 2000; Vo et al., 2008a; Vo et al., 2008b). However, plasma exchange, immunoadsorption, and IVIg treatments have the disadvantages of being inefficient and requiring strict planning because they involve repeated treatments over a long period of time. When organs from deceased donors become available, they must be transplanted within a few hours because prolonged cold ischemia time is one of the most important risk factors for delayed graft function and allograft loss in renal transplantation (Ojo et al., (1997) Transplantation 63:968-74).

[0168] In contrast, the method of the present invention allows for rapid, short-term and safe removal of DSA in potential transplant recipients. Administration of the polypeptide of the present invention immediately prior to transplantation has the ability to effectively desensitize highly sensitized patients, thereby allowing transplantation and avoiding acute antibody-mediated rejection. A single dose of the polypeptide prior to transplantation will make transplantation possible for tens of thousands of patients with donor-specific IgG antibodies.

[0169] In the context of this embodiment, the method may alternatively be described as a method for treating organ failure in a subject, the method comprising: (a) administering to the subject a polypeptide of the invention, and (b) subsequently transplanting a replacement organ into the subject; wherein:

[0170] - the amount of the polypeptide administered is sufficient to cleave substantially all IgG molecules present in the plasma of the subject; and

[0171] - Steps (a) and (b) are separated by a time interval of at least 30 minutes and at most 21 days.

[0172] In other words, the implementation can be described as a method for preventing rejection of a transplanted organ in a subject (particularly acute antibody-mediated transplant rejection), the method comprising administering a polypeptide of the present invention to a subject at least 30 minutes and up to 21 days before organ transplantation, wherein the amount of the polypeptide administered is sufficient to cleave substantially all IgG molecules present in the plasma of the subject. The present invention also provides the use of the polypeptide of the present invention in such a method for treating organ failure or preventing transplant rejection (particularly acute antibody-mediated transplant rejection). The present invention also provides the use of the polypeptide of the present invention in the manufacture of a medicament for treating organ failure or preventing transplant rejection by this method. In this embodiment, the method of the present invention may further include a step performed at the time of transplantation or immediately before transplantation, which step includes induction inhibition of T cells and / or B cells in the patient. The induction inhibition may generally include administering an effective amount of an agent that kills or inhibits T cells, and / or administering an effective amount of an agent that kills or inhibits B cells. Agents that kill or inhibit T cells include Muromonab, Basiliximab, Daclizumab, anti-thymocyte globulin (ATG) antibodies and lymphocyte immunoglobulins, anti-thymocyte globulin preparations (ATGAM). Rituximab is known to kill or suppress B cells.

[0173] Polypeptides with IgG cysteine ​​protease activity (such as the polypeptides of the present invention) can also be used in methods for inducing hematopoietic chimerism in a subject, for example, in the case of transplanting hematopoietic stem / progenitor cells (HSPC) to a subject. Therefore, the polypeptides of the present invention can be used in methods for inducing hematopoietic chimerism in a subject, the method comprising carrying out a conditioning regimen of the present invention, followed by administering HSPC to the subject in sufficient amounts and under conditions suitable for inducing hematopoietic chimerism in the subject. The method can alternatively be described as a method for stable transplantation of HSPC. HSPC can be autologous (using the subject / patient's own cells) or homologous (cells from genetically identical twins), or can be allogeneic (cells from separate, non-identical donors). Immune complications reduce the likelihood of successful transplantation of HSPC in a recipient, which is most important for allogeneic cells, so methods for inducing hematopoietic chimerism are most favorable to such cells. However, even for autologous cells, immune complications can occur if there is expression of a product that the recipient has not been exposed to before. If autologous cells are genetically modified to express gene therapy, the cells can be sufficiently altered to cause an immune response. For example, an immune response may arise to the expressed gene therapy product. A similar situation could occur if HSPCs were genetically modified to express a different HLA type that did not match the recipient's HLA.

[0174] Polypeptides with IgG cysteine ​​protease activity (such as the polypeptides of the present invention) can also be used in combination with adoptive cell transfer immunotherapy. The efficacy of adoptive cell transfer immunotherapy may be reduced by the limited survival and limited sustained activity of transferred cells (such as CAR-T cells). Proteins with IgG cysteine ​​protease can protect transferred cells. In particular, pre-existing antibodies and antibodies produced after administration with transferred cells may shorten the potential of transferred cells, and the therapeutic effect of transferred cells will benefit from the removal of antibody effector functions through receptor conditioning. Therefore, the administration of proteins with IgG cysteine ​​protease activity can increase the survival and activity of transferred cells, thereby increasing the benefits of patients with adoptive cell transfer immunotherapy and providing better treatment and prognosis in, for example, cancer treatment. In this case, the method for treating cancer may include administering a polypeptide of the present invention with IgG cysteine ​​protease activity before and / or after administering one or more doses of adoptive cell transfer immunotherapy. In the case of methods for treating autoimmune diseases, infections, and diseases mediated by harmful antibodies, polypeptides with IgG cysteine ​​protease activity (such as the polypeptides of the present invention) can also be used in combination with adoptive cell transfer immunotherapy. This approach will inactivate both pre-existing anti-drug antibodies (ADA) and antibodies induced by adoptive cell transfer immunotherapy. Example

[0175] Unless otherwise indicated, the methods used are standard biochemistry and molecular biology techniques. Examples of suitable method textbooks include Sambrook et al., Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al., Current Protocols in Molecular Biology (1995), John Wiley and Sons, Inc.

[0176] Example 1 - Expression and purification of pCART239

[0177] The mature IdeZ molecules and sequences were analyzed and regions suitable for mutation were identified. In some cases, computer evaluation was used ( in silicoThe possible consequences of mutations were estimated by PCR assessment. Having determined the sequence of pCART239, cDNAs encoding each polypeptide were generated by site-directed mutagenesis of the starting sequence or by synthesis at GeneCust, Luxembourg, depending on the number of mutations introduced. The cDNAs were sequenced and transferred in-frame to the pET9a expression vector (Novagene) with a C-terminal 6× histidine tag linked to the C-terminus via a short glycine linker (3× glycine). An N-terminal methionine was added to improve bacterial expression. Thus, the pCART239 polypeptide corresponds to SEQ ID NO: 1 with the addition of (1) an N-terminal methionine and (2) a C-terminal 6× histidine tag linked to the C-terminus via a short glycine linker (3× glycine).

[0178] The pCART239 expression plasmid was transformed into E. coli T7E2 (SOURCE) and plated on LB agarose plates containing 50 μg / ml kanamycin. A single colony was picked and an overnight culture (10 ml LB-medium) was started at 37°C, 250 rpm. The next day, 2 flasks containing 125 ml LB-medium + 50 µg / ml kanamycin + 1:100,000 dilution of antifoam were inoculated with 5 ml of the overnight culture. The flasks were already at 37°C at the time of inoculation, and the cultures were grown to an OD value of 0.6-0.7 (37°C, 300 rpm). At this point, IPTG (final concentration 1 mM) was added to induce expression, and the cultures were further incubated for at least 2 hours. After incubation, the bacterial suspension was harvested by centrifugation (10 minutes, 4000 xg, 4°C), and the supernatant was discarded. The pellet was washed once in PBS (30 ml), the supernatant was discarded by re-centrifugation, and the final pellet was frozen at -20 °C and stored in the freezer overnight. To lyse the bacteria, a Panda Plus 2000 homogenizer (GEA) was used according to the manufacturer's instructions, or a freeze-thaw protocol was used (three freeze-thaw cycles in 10 ml PBS with the aid of sterile glass beads). After the freeze-thaw cycle, the tubes were centrifuged (20 min, 25 000 x g, 4 °C) to separate the bacterial lysate (supernatant) and then stored on ice. The final bacterial lysate was pooled, sterile filtered through a 0.2 µm nylon filter (HPF Millex®-Nylon), and the protein was purified using Ni-NTA pre-packed spin columns (ThermoFisher). After purification, DTT (final concentration 5 mM) and EDTA (final concentration 5 mM) were added to the eluate, after which the buffer was changed (Amicon Ultra-4 10K). Protein concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific). Contamination-free 12% Mini-PROTEAN® TGX TM Precast gels (Biorad) were used to evaluate the purity and stability of the purified protein during expression and purification ( Figure 1 ). Figure 1 A shows that IPTG induction and subsequent overexpression of pCART239 was successful. Figure 1 B shows that the purification process was also successful and resulted in a pCART239 sample of high purity and stability.

[0179] Example 2 - pCART207, pCART229, N240, pCART242 vs Humira (IgG1) and XGEVA (IgG2) Effectiveness

[0180] In this example, human IgG1 is represented by Humira (Abbvie) and IgG2 is represented by XGEVA (Amgen). These were used to compare the activity of the IdeZ variants and the IdeS positive control in cleaving monoclonal human IgG. The IdeZ variant enzymes (and IdeS) were titrated in PBS with 0.05% BSA in 1:3 dilution steps from a starting concentration of 3.3 µg / ml and the cleavage products were analyzed by SDS-PAGE (4-20% gradient gel). The pCART242 polypeptide tested corresponded to SEQ ID NO: 2 with the addition of (1) an N-terminal methionine and (2) a C-terminal 6× histidine tag attached to the C-terminus via a short glycine linker (3× glycine).

[0181] method:

[0182] 1. Transfer 25 µl of enzyme and control (buffer) dilutions into a multiwell plate.

[0183] 2. Start the reaction by adding 25 µl of 2 mg / ml Humira or XGEVA solution to each well. This results in 1 mg / ml of each antibody in the reaction. Taking into account the serial dilutions, the maximum IdeZ (or IdeS) concentration tested was 3.3 µg / ml and the lowest was 0.057 ng / ml.

[0184] 3. Incubate the plate at 37°C with gentle rotation for 2 hours.

[0185] 4. After incubation, mix 10 µl of each sample with 30 µl 2x SDS loading buffer in a microtiter plate. After overnight storage (4-8°C), transfer to a 1.5 ml tube and incubate at 92°C for 5 minutes. Load 10 µl of the sample onto a 15-well 4-20% Mini-PROTEAN® TGX™ precast gel and analyze the samples by SDS-PAGE under non-reducing conditions.

[0186] Figure 2 and Figure 3 Gels showing digestion of IgG1 (Humira) and IgG2 (XGEVA) by IdeZ variants are shown separately. The different panels represent different experimental sets (but the protocol is the same in each example).

[0187] Based on the cleavage pattern shown in the gel, the approximate concentrations of cleavage of the first and second heavy chains of IgG1 / IgG2 were estimated as follows:

[0188] Table 1A: Approximate concentrations of cleavage of the first and second heavy chains of IgG1 ( Figure 2 A).

[0189]

[0190] Table 1B: Approximate concentrations of cleavage of the first and second heavy chains of IgG1 ( Figure 2 B).

[0191]

[0192] Table 2A: Approximate concentrations of cleavage of the first and second heavy chains of IgG2 ( Figure 3 A).

[0193]

[0194] Table 2B: Approximate concentrations of cleavage of the first and second heavy chains of IgG2 ( Figure 3 B).

[0195]

[0196] Overview:

[0197] IdeS, pCART207, pCART229 have very similar potency at cleavage of IgG1 (Humira). pCART239 and N240 are more potent than IdeS and the very similar variant pCART229. N240 and pCART242 appear more potent than IdeS at cleaving the second IgG1 heavy chain. N240 and pCART242 have higher potency than pCART229 and pCART207 at the primary second cleavage of IgG2 (Xgeva), and are also more potent at cleaving the first heavy chain of IgG2.

[0198] Example 3 - Potency of N240 against human IgG subclasses

[0199] This report describes the characterization of the in vitro activity of N240 by digestion of various IgG subclasses. In the case of IgG subclasses, two different in-house produced N240 batches were compared and the reference material used was IdeS.

[0200] method:

[0201] 1. Transfer 25 µl of N240 or IdeS enzyme and control (buffer) dilutions to a multiwell plate. Enzyme is serially diluted 1:3 in 0.05% BSA in PBS between each successive well.

[0202] 2. At the start of the reaction, 25 µl of a 2 mg / ml human IgG solution was added to each well. This resulted in 1 mg / ml of each antibody in the reaction. The IgG1 used was Humira (Abbvie), the IgG2 used was XGEVA (Amgen), the IgG3 used was from Sigma (I5654 Lot#SLBW0899), and the IgG4 used was from Abcam (ab90286 Lot#GR3180469).

[0203] 3. Incubate the plate at 37°C with gentle rotation for 2 hours.

[0204] 4. After incubation, mix 10 µl of each sample with 30 µl 2x SDS loading buffer in a microtiter plate. After overnight storage (4-8°C), transfer to a 1.5 ml tube and incubate at 92°C for 5 minutes. Load 10 µl of the sample onto a 15-well 4-20% Mini-PROTEAN® TGX™ precast gel.

[0205] A gel showing digestion of IgG subclasses by N240 and IdeS is shown in Figure 4 middle.

[0206] EC estimated from visual inspection of the gel 50 The values ​​are shown in Table 3 below.

[0207] Table 3: EC of IgG1, IgG2, IgG3 and IgG4 digestion 50 result

[0208]

[0209]

[0210] Overview:

[0211] 1. All four human subclasses, IgG1, IgG2, IgG3, and IgG4, are cleaved by N240.

[0212] 2. It is clear that N240 has higher activity (relative to IdeS), especially for second-chain cleavage. Compared with N240, IdeS has a lower EC value for IgG1. 50 F(ab')2 is approximately 5 times higher.

[0213] 3. In the case of IgG2, IgG3, and IgG4, the potency of N240 was similar or slightly lower than that of IdeS.

[0214] Example 4 - MSD potency assay comparison between N240 and IdeS

[0215] method:

[0216] A more detailed summary of the principles behind the MSD potency assay is discussed below in Example 5.

[0217] Briefly, goat anti-human IgG, F(ab')2 fragment specific, was coated on a 96-well MSD plate. After blocking, the plates were washed and N240 reference material and IdeS test samples diluted in a series of concentrations were added to the plates. A fixed concentration of human IgG was added and the plates were incubated at 37°C. After incubation and washing, a detection mixture containing biotinylated mouse anti-human IgG (Fc specific) and SULFO-TAG labeled streptavidin was added to the plates. After the last incubation and wash, Read Buffer was added. Using the MSD instrument, the luminescence intensity of SULFO-TAG was measured to provide a quantitative measurement of non-cleaved and single-cleaved IgG in the sample. N240 reference material and IdeS test samples were analyzed in duplicate wells and triplicate plates. The EC values ​​for each reference and test sample were 90.89 for each reference and test sample. 50 The relative potency (%) of each test sample was calculated by fitting the results to a 5-parameter curve. 50 Ref / EC 50 Determined by dividing Test by 100%. The reported values ​​are the average of three different plates.

[0218] Figure 5 The resulting dilution curves representing IgG cleavage of IdeS and N240 are shown.

[0219] The average relative potency of N240 was calculated to be approximately 300% compared to IdeS.

[0220] Example 5 - Efficacy of pCART239 on IgG in human serum

[0221] The IdeZ variant pCART239 was further characterized by measuring its activity in serum.

[0222] A human serum pool from 100 individuals was used as IgG substrate.

[0223] SDS-PAGE

[0224] The dilution series used for pCART239 was as follows: 30, 15, 7.5, 3.75, 1.9, 0.9, 0.2, 0.2, 0.1, 0.06 and 0.03 µg / mL, and the activity assay and SDS-PAGE analysis protocols described in Examples 2 and 3 were followed.

[0225] A gel showing the digestion of serum IgG by pCART239 is shown in Figure 6The figure shows that pCART239 has IgG cleavage activity in the serum pool, and the gel estimates that the cleavage of IgG to scIgG is about 0.9 µg / ml of enzyme ( Figure 6 , upper panel). scIgG was further digested into F(ab')2 and Fc fragments with approximately 1.9 µg / ml of enzyme.

[0226] The activity values ​​obtained in serum were lower than those obtained in buffer, a phenomenon known from previous studies due to the presence of inhibitory anti-drug antibodies (ADA) in serum. To verify that there were no other specific inhibitory factors in the serum besides the inhibitory ADA, an experiment was performed in which an inactive variant of pCART239 (pCART243) was added at 0.1 mg / ml. This addition was in high molar excess and should bind to the inactive ADA to the extent that the activity of pCART239 in serum was restored. This turned out to be the case ( Figure 6 , bottom), the presence of pCART243 caused IgG cleavage to occur at lower pCART239 concentrations. Intact IgG began to be digested at 0.03 µg / ml, and all IgG was converted to scIgG at 0.5 µg / ml.

[0227] Electrochemiluminescence Meso Scale Discovery (MSD) Platform

[0228] To further investigate the activity of N240 in serum, an MSD potency assay was performed in serum matrix.

[0229] The principle of these assays is to coat the wells of a multititer plate with F(ab)2 fragments directed against human IgG antibodies specific for the Fab region. Titrated concentrations of IgG cysteine ​​protease polypeptides (test or control) are then incubated with human serum in the wells. The amount of intact or singly cleaved human IgG bound to the wells is measured using a detection antibody directed against human IgG specific for the Fc portion of the antibody. The higher the concentration of a given IgG cysteine ​​protease polypeptide in the well, the less intact human IgG antibody will bind to the well, resulting in a lower signal. Similarly, a more potent IgG cysteine ​​protease polypeptide will produce a lower signal than a less potent IgG cysteine ​​protease polypeptide when present at the same concentration. Titration dose-response curves were prepared for the IdeS control (pCART124) and N240. The EC values ​​of the cysteine ​​proteases tested were calculated by 50 The lower EC 50 Values ​​represent more efficient IgG cysteine ​​proteases. Since the Fc part of IgG is still present and can be detected by Fc-specific detection antibodies, cleavage of the first IgG heavy chain (IgG to scIgG) is not visible in this assay.

[0230] Brief summary of the laboratory protocol: Wells of multititer plates were coated with goat anti-human Fab specific F(ab)2 fragments (0.5 μg / ml) (Jackson #109-006-097) overnight (+2-8°C), then washed with PBS + 0.05% Tween 20 (PBS-T) and blocked in 0.45% fish gelatin in PBS-T (blocking buffer) for 45-120 minutes at room temperature. Control IdeS (pCART124) and the IgG cysteine ​​protease polypeptides to be tested were prepared as a titration series with a starting concentration of 80 μg / ml in blocking buffer in 1:4 dilution steps. Equal volumes (25 μl) of human serum and titrated amounts of IgG cysteine ​​protease polypeptides were added to the wells and incubated for 2 hours at a controlled temperature of 37°C with shaking, then washed with PBS-T. Biotinylated mouse anti-human IgG Fc specific (ma-hIgG Bio II, Lot: C0013-ZC43C, SouthernBiotech) (600 ng / ml) antibody was mixed with Strep-sulfo (200 ng / ml) and added to the multi-titer plate. The plate was sealed with aluminum tape and incubated at +25°C for 1 hour with shaking. The plate was then washed in PBS-T and 150 μl of 2x diluted Read buffer T (MSD Reading Buffer T, Cat. No. R92TC-2) was added to each well. The plate was immediately read on a plate reader (MSD (Meso Scale Discovery) QuickPlex SQ 120 Model 1300).

[0231] The resulting standard curve representing IgG cleavage by IdeS and N240 is shown in Figure 7 The following table shows the EC values ​​based on the dose-response curves. 50 Values ​​are estimates of relative potency.

[0232] Table 4: According to EC 50 Values ​​to estimate the relative titer of IgG cleavage in serum

[0233]

[0234] Overview:

[0235] N240 is active in serum. In the previous example, the titer of N240 in buffer solution was approximately 4-fold higher than that of Ilifidase, but this difference was smaller when the two enzymes were compared in serum. In the MSD titer analysis, N240 still showed a substantial increase in activity against human IgG in serum (~142%) compared to IdeS.

[0236] Example 6 - N240 and IdeS ADA levels in healthy individuals

[0237] The general MSD protocol described in Example 5 was followed with the following exceptions.

[0238] Multiarray MSD plates were coated with 20 µg / ml N240 or IdeS. Plates were blocked with fish gelatin before incubation with sera (1:100 dilution) from healthy individuals (n = 40) and a human serum pool (n = 100). Detection reagents were anti-human F(ab')2-specific F(ab')2-bioantibody (JAX, 109-066-097) and Streptavidin-Sulfo (MSD #R32AD-1). Plates were read using an MSD QuickPlex SQ 120 after addition of reading buffer (MSD #R92TC-2).

[0239] Results Figure 8 This result indicated that the level of ADA against N240 was lower than that against IdeS. Sequence Listing <110> Hansa Biopharmaceuticals AG <120> Cysteine ​​proteases <130> 195420-22F-CNP <150> GB 2007431.6 <151> 2020-05-19 <160> twenty three <170> PatentIn version 3.5 <210> 1 <211> 312 <212> PRT <213> Streptococcus equi ssp. Zooepidemicus <400> 1 Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val Pro 1 5 10 15 His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Glu Gln 20 25 30 Phe Thr Gln Gly Glu Asp Val Ile His Ala Pro Tyr Leu Ala His Gln 35 40 45 Gly Trp Tyr Asp Ile Thr Lys Ala Phe Asn Gly Lys Asp Asp Leu Leu 50 55 60 Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln 65 70 75 80 Asn Lys Thr Glu Ile Glu Ala Tyr Leu Ser Lys His Pro Glu Lys Gln 85 90 95 Lys Ile Ile Phe Arg Asn Gln Glu Leu Phe Asp Leu Lys Ala Ala Ile 100 105 110 Asp Thr Lys Asp Ser Gln Thr Asn Ser Gln Leu Phe Asn Tyr Phe Arg 115 120 125 Asp Lys Ala Phe Pro Asn Leu Ser Ala Arg Gln Leu Gly Val Met Pro 130 135 140 Asp Leu Val Leu Asp Met Phe Ile Asn Gly Tyr Tyr Leu Asn Val Phe 145 150 155 160 Lys Thr Gln Ser Thr Asp Val Asn Arg Pro Tyr Gln Asp Lys Asp Lys 165 170 175 Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn Gln Thr Thr 180 185 190 Leu Leu Thr Ala Arg His Asp Leu Lys Asn Lys Gly Leu Asn Asp Ile 195 200 205 Ser Thr Ile Ile Lys Gln Glu Leu Thr Glu Gly Arg Ala Leu Ala Leu 210 215 220 Ser His Thr Tyr Ala Asn Val Ser Ile Ser His Val Ile Asn Leu Trp 225 230 235 240 Gly Ala Asp Phe Asn Ala Glu Gly Asn Leu Glu Ala Ile Tyr Val Thr 245 250 255 Asp Ser Asp Ala Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly 260 265 270 Ile Asn Ala His Gly His Val Ala Ile Ser Ala Lys Lys Ile Glu Gly 275 280 285 Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Ser Gly 290 295 300 Lys Asp Ile Trp Gln Lys Leu Ser 305 310 <210> 2 <211> 292 <212> PRT <213> Streptococcus equi ssp. Zooepidemicus <400> 2 Ser Val Trp Thr Lys Gly Val Thr Pro Pro Glu Gln Phe Thr Gln Gly 1 5 10 15 Glu Asp Val Ile His Ala Pro Tyr Leu Ala His Gln Gly Trp Tyr Asp 20 25 30 Ile Thr Lys Ala Phe Asn Gly Lys Asp Asp Leu Leu Cys Gly Ala Ala 35 40 45 Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Thr Glu 50 55 60 Ile Glu Ala Tyr Leu Ser Lys His Pro Glu Lys Gln Lys Ile Ile Phe 65 70 75 80 Arg Asn Gln Glu Leu Phe Asp Leu Lys Ala Ala Ile Asp Thr Lys Asp 85 90 95 Ser Gln Thr Asn Ser Gln Leu Phe Asn Tyr Phe Arg Asp Lys Ala Phe 100 105 110 Pro Asn Leu Ser Ala Arg Gln Leu Gly Val Met Pro Asp Leu Val Leu 115 120 125 Asp Met Phe Ile Asn Gly Tyr Tyr Leu Asn Val Phe Lys Thr Gln Ser 130 135 140 Thr Asp Val Asn Arg Pro Tyr Gln Asp Lys Asp Lys Arg Gly Gly Ile 145 150 155 160 Phe Asp Ala Val Phe Thr Arg Gly Asn Gln Thr Thr Leu Leu Thr Ala 165 170 175 Arg His Asp Leu Lys Asn Lys Gly Leu Asn Asp Ile Ser Thr Ile Ile 180 185 190 Lys Gln Glu Leu Thr Glu Gly Arg Ala Leu Ala Leu Ser His Thr Tyr 195 200 205 Ala Asn Val Ser Ile Ser His Val Ile Asn Leu Trp Gly Ala Asp Phe 210 215 220 Asn Ala Glu Gly Asn Leu Glu Ala Ile Tyr Val Thr Asp Ser Asp Ala 225 230 235 240 Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Ile Asn Ala His 245 250 255 Gly His Val Ala Ile Ser Ala Lys Lys Ile Glu Gly Glu Asn Ile Gly 260 265 270 Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Ser Gly Lys Asp Ile Trp 275 280 285 Gln Lys Leu Ser 290 <210> 3 <211> 315 <212> PRT <213> Streptococcus equi ssp. Zooepidemicus <400> 3 Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val Pro 1 5 10 15 His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Leu Thr Pro 20 25 30 Glu Gln Phe Arg Tyr Asn Asn Glu Asp Val Ile His Ala Pro Tyr Leu 35 40 45 Ala His Gln Gly Trp Tyr Asp Ile Thr Lys Ala Phe Asp Gly Lys Asp 50 55 60 Asn Leu Leu Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp 65 70 75 80 Phe Asp Gln Asn Lys Thr Glu Ile Glu Ala Tyr Leu Ser Lys His Pro 85 90 95 Glu Lys Gln Lys Ile Ile Phe Asn Asn Gln Glu Leu Phe Asp Leu Lys 100 105 110 Ala Ala Ile Asp Thr Lys Asp Ser Gln Thr Asn Ser Gln Leu Phe Asn 115 120 125 Tyr Phe Arg Asp Lys Ala Phe Pro Asn Leu Ser Ala Arg Gln Leu Gly 130 135 140 Val Met Pro Asp Leu Val Leu Asp Met Phe Ile Asn Gly Tyr Tyr Leu 145 150 155 160 Asn Val Phe Lys Thr Gln Ser Thr Asp Val Asn Arg Pro Tyr Gln Asp 165 170 175 Lys Asp Lys Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asp 180 185 190 Gln Thr Thr Leu Leu Thr Ala Arg His Asp Leu Lys Asn Lys Gly Leu 195 200 205 Asn Asp Ile Ser Thr Ile Ile Lys Gln Glu Leu Thr Glu Gly Arg Ala 210 215 220 Leu Ala Leu Ser His Thr Tyr Ala Asn Val Ser Ile Ser His Val Ile 225 230 235 240 Asn Leu Trp Gly Ala Asp Phe Asn Ala Glu Gly Asn Leu Glu Ala Ile 245 250 255 Tyr Val Thr Asp Ser Asp Ala Asn Ala Ser Ile Gly Met Lys Lys Tyr 260 265 270 Phe Val Gly Ile Asn Ala His Gly His Val Ala Ile Ser Ala Lys Lys 275 280 285 Ile Glu Gly Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu 290 295 300 Ser Ser Gly Lys Asp Ile Trp Gln Lys Leu Ser 305 310 315 <210> 4 <211> 310 <212> PRT <213> Streptococcus pyogenes <400> 4 Asp Ser Phe Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro 1 5 10 15 Tyr His Val Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn 20 25 30 Phe Thr Gln Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln 35 40 45 Gly Trp Tyr Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu 50 55 60 Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln 65 70 75 80 Asn Lys Asp Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln 85 90 95 Lys Ile Asn Phe Asn Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile 100 105 110 Asp Thr Lys Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys 115 120 125 Glu Lys Ala Phe Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro 130 135 140 Asp His Val Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr 145 150 155 160 Asn His Gly Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly 165 170 175 Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu 180 185 190 Thr Ser Arg His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp 195 200 205 Leu Ile Lys Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His 210 215 220 Thr Tyr Ala Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala 225 230 235 240 Asp Phe Asp Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser 245 250 255 Asp Ser Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn 260 265 270 Ser Ala Gly Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn 275 280 285 Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp 290 295 300 Ser Trp Asn Gln Thr Asn 305 310 <210> 5 <211> 349 <212> PRT <213> Streptococcus equi ssp. Zooepidemicus <400> 5 Met Lys Thr Ile Ala Tyr Pro Asn Lys Pro His Ser Leu Ser Ala Gly 1 5 10 15 Leu Leu Thr Ala Ile Ala Ile Phe Ser Leu Ala Ser Ser Asn Ile Thr 20 25 30 Tyr Ala Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu 35 40 45 Val Pro His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Leu 50 55 60 Thr Pro Glu Gln Phe Arg Tyr Asn Asn Glu Asp Val Ile His Ala Pro 65 70 75 80 Tyr Leu Ala His Gln Gly Trp Tyr Asp Ile Thr Lys Ala Phe Asp Gly 85 90 95 Lys Asp Asn Leu Leu Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His 100 105 110 Trp Trp Phe Asp Gln Asn Lys Thr Glu Ile Glu Ala Tyr Leu Ser Lys 115 120 125 His Pro Glu Lys Gln Lys Ile Ile Phe Asn Asn Gln Glu Leu Phe Asp 130 135 140 Leu Lys Ala Ala Ile Asp Thr Lys Asp Ser Gln Thr Asn Ser Gln Leu 145 150 155 160 Phe Asn Tyr Phe Arg Asp Lys Ala Phe Pro Asn Leu Ser Ala Arg Gln 165 170 175 Leu Gly Val Met Pro Asp Leu Val Leu Asp Met Phe Ile Asn Gly Tyr 180 185 190 Tyr Leu Asn Val Phe Lys Thr Gln Ser Thr Asp Val Asn Arg Pro Tyr 195 200 205 Gln Asp Lys Asp Lys Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg 210 215 220 Gly Asp Gln Thr Thr Leu Leu Thr Ala Arg His Asp Leu Lys Asn Lys 225 230 235 240 Gly Leu Asn Asp Ile Ser Thr Ile Ile Lys Gln Glu Leu Thr Glu Gly 245 250 255 Arg Ala Leu Ala Leu Ser His Thr Tyr Ala Asn Val Ser Ile Ser His 260 265 270 Val Ile Asn Leu Trp Gly Ala Asp Phe Asn Ala Glu Gly Asn Leu Glu 275 280 285 Ala Ile Tyr Val Thr Asp Ser Asp Ala Asn Ala Ser Ile Gly Met Lys 290 295 300 Lys Tyr Phe Val Gly Ile Asn Ala His Gly His Val Ala Ile Ser Ala 305 310 315 320 Lys Lys Ile Glu Gly Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe 325 330 335 Thr Leu Ser Ser Gly Lys Asp Ile Trp Gln Lys Leu Ser 340 345 <210> 6 <211> 339 <212> PRT <213> Streptococcus pyogenes <400> 6 Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala Val Leu Ala Ala Val 1 5 10 15 Thr Leu Phe Val Leu Ser Val Asp Arg Gly Val Ile Ala Asp Ser Phe 20 25 30 Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val 35 40 45 Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln 50 55 60 Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr 65 70 75 80 Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu Cys Gly Ala 85 90 95 Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Asp 100 105 110 Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn 115 120 125 Phe Asn Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp Thr Lys 130 135 140 Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys Ala 145 150 155 160 Phe Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro Asp His Val 165 170 175 Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr Asn His Gly 180 185 190 Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe 195 200 205 Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr Ser Arg 210 215 220 His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys 225 230 235 240 Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala 245 250 255 Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp 260 265 270 Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn 275 280 285 Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly 290 295 300 Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala 305 310 315 320 Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn 325 330 335 Gln Thr Asn <210> 7 <211> 313 <212> PRT <213> Streptococcus equi ssp. Zooepidemicus <400> 7 Met Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val 1 5 10 15 Pro His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Glu 20 25 30 Gln Phe Thr Gln Gly Glu Asp Val Ile His Ala Pro Tyr Leu Ala His 35 40 45 Gln Gly Trp Tyr Asp Ile Thr Lys Ala Phe Asp Gly Lys Asp Asn Leu 50 55 60 Leu Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp 65 70 75 80 Gln Asn Lys Thr Glu Ile Glu Ala Tyr Leu Ser Lys His Pro Glu Lys 85 90 95 Gln Lys Ile Ile Phe Arg Asn Gln Glu Leu Phe Asp Leu Lys Ala Ala 100 105 110 Ile Asp Thr Lys Asp Ser Gln Thr Asn Ser Gln Leu Phe Asn Tyr Phe 115 120 125 Arg Asp Lys Ala Phe Pro Asn Leu Ser Ala Arg Gln Leu Gly Val Met 130 135 140 Pro Asp Leu Val Leu Asp Met Phe Ile Asn Gly Tyr Tyr Leu Asn Val 145 150 155 160 Phe Lys Thr Gln Ser Thr Asp Val Asn Arg Pro Tyr Gln Asp Lys Asp 165 170 175 Lys Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asp Gln Thr 180 185 190 Thr Leu Leu Thr Ala Arg His Asp Leu Lys Asn Lys Gly Leu Asn Asp 195 200 205 Ile Ser Thr Ile Ile Lys Gln Glu Leu Thr Glu Gly Arg Ala Leu Ala 210 215 220 Leu Ser His Thr Tyr Ala Asn Val Ser Ile Ser His Val Ile Asn Leu 225 230 235 240 Trp Gly Ala Asp Phe Asn Ala Glu Gly Asn Leu Glu Ala Ile Tyr Val 245 250 255 Thr Asp Ser Asp Ala Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val 260 265 270 Gly Ile Asn Ala His Gly His Val Ala Ile Ser Ala Lys Lys Ile Glu 275 280 285 Gly Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Ser 290 295 300 Gly Lys Asp Ile Trp Gln Lys Leu Ser 305 310 <210> 8 <211> 313 <212> PRT <213> Streptococcus equi ssp. Zooepidemicus <400> 8 Met Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val 1 5 10 15 Pro His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Glu 20 25 30 Gln Phe Thr Gln Gly Glu Asp Val Ile His Ala Pro Tyr Leu Ala His 35 40 45 Gln Gly Trp Tyr Asp Ile Thr Lys Ala Phe Asp Gly Lys Asp Asn Leu 50 55 60 Leu Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp 65 70 75 80 Gln Asn Lys Thr Glu Ile Glu Ala Tyr Leu Ser Lys His Pro Glu Lys 85 90 95 Gln Lys Ile Ile Phe Arg Asn Gln Glu Leu Phe Asp Leu Lys Ala Ala 100 105 110 Ile Asp Thr Lys Asp Ser Gln Thr Asn Ser Gln Leu Phe Asn Tyr Phe 115 120 125 Arg Asp Lys Ala Phe Pro Asn Leu Ser Ala Arg Gln Leu Gly Val Met 130 135 140 Pro Asp Leu Val Leu Asp Met Phe Ile Asn Gly Tyr Tyr Leu Asn Val 145 150 155 160 Phe Lys Thr Gln Ser Thr Asp Val Asn Arg Pro Tyr Gln Asp Lys Asp 165 170 175 Lys Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn Gln Thr 180 185 190 Thr Leu Leu Thr Ala Arg His Asp Leu Lys Asn Lys Gly Leu Asn Asp 195 200 205 Ile Ser Thr Ile Ile Lys Gln Glu Leu Thr Glu Gly Arg Ala Leu Ala 210 215 220 Leu Ser His Thr Tyr Ala Asn Val Ser Ile Ser His Val Ile Asn Leu 225 230 235 240 Trp Gly Ala Asp Phe Asn Ala Glu Gly Asn Leu Glu Ala Ile Tyr Val 245 250 255 Thr Asp Ser Asp Ala Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val 260 265 270 Gly Ile Asn Ala His Gly His Val Ala Ile Ser Ala Lys Lys Ile Glu 275 280 285 Gly Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Ser 290 295 300 Gly Lys Asp Ile Trp Gln Lys Leu Ser 305 310 <210> 9 <211> 322 <212> PRT <213> Streptococcus equi ssp. Zooepidemicus <400> 9 Met Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val 1 5 10 15 Pro His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Glu 20 25 30 Gln Phe Thr Gln Gly Glu Asp Val Ile His Ala Pro Tyr Leu Ala His 35 40 45 Gln Gly Trp Tyr Asp Ile Thr Lys Ala Phe Asn Gly Lys Asp Asp Leu 50 55 60 Leu Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp 65 70 75 80 Gln Asn Lys Thr Glu Ile Glu Ala Tyr Leu Ser Lys His Pro Glu Lys 85 90 95 Gln Lys Ile Ile Phe Arg Asn Gln Glu Leu Phe Asp Leu Lys Ala Ala 100 105 110 Ile Asp Thr Lys Asp Ser Gln Thr Asn Ser Gln Leu Phe Asn Tyr Phe 115 120 125 Arg Asp Lys Ala Phe Pro Asn Leu Ser Ala Arg Gln Leu Gly Val Met 130 135 140 Pro Asp Leu Val Leu Asp Met Phe Ile Asn Gly Tyr Tyr Leu Asn Val 145 150 155 160 Phe Lys Thr Gln Ser Thr Asp Val Asn Arg Pro Tyr Gln Asp Lys Asp 165 170 175 Lys Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn Gln Thr 180 185 190 Thr Leu Leu Thr Ala Arg His Asp Leu Lys Asn Lys Gly Leu Asn Asp 195 200 205 Ile Ser Thr Ile Ile Lys Gln Glu Leu Thr Glu Gly Arg Ala Leu Ala 210 215 220 Leu Ser His Thr Tyr Ala Asn Val Ser Ile Ser His Val Ile Asn Leu 225 230 235 240 Trp Gly Ala Asp Phe Asn Ala Glu Gly Asn Leu Glu Ala Ile Tyr Val 245 250 255 Thr Asp Ser Asp Ala Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val 260 265 270 Gly Ile Asn Ala His Gly His Val Ala Ile Ser Ala Lys Lys Ile Glu 275 280 285 Gly Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Ser 290 295 300 Gly Lys Asp Ile Trp Gln Lys Leu Ser Gly Gly Gly His His His His 305 310 315 320 His His <210> 10 <211> 313 <212> PRT <213> Streptococcus equi ssp. Zooepidemicus <400> 10 Met Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val 1 5 10 15 Pro His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Glu 20 25 30 Gln Phe Thr Gln Gly Glu Asp Val Ile His Ala Pro Tyr Leu Ala His 35 40 45 Gln Gly Trp Tyr Asp Ile Thr Lys Ala Phe Asn Gly Lys Asp Asp Leu 50 55 60 Leu Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp 65 70 75 80 Gln Asn Lys Thr Glu Ile Glu Ala Tyr Leu Ser Lys His Pro Glu Lys 85 90 95 Gln Lys Ile Ile Phe Arg Asn Gln Glu Leu Phe Asp Leu Lys Ala Ala 100 105 110 Ile Asp Thr Lys Asp Ser Gln Thr Asn Ser Gln Leu Phe Asn Tyr Phe 115 120 125 Arg Asp Lys Ala Phe Pro Asn Leu Ser Ala Arg Gln Leu Gly Val Met 130 135 140 Pro Asp Leu Val Leu Asp Met Phe Ile Asn Gly Tyr Tyr Leu Asn Val 145 150 155 160 Phe Lys Thr Gln Ser Thr Asp Val Asn Arg Pro Tyr Gln Asp Lys Asp 165 170 175 Lys Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn Gln Thr 180 185 190 Thr Leu Leu Thr Ala Arg His Asp Leu Lys Asn Lys Gly Leu Asn Asp 195 200 205 Ile Ser Thr Ile Ile Lys Gln Glu Leu Thr Glu Gly Arg Ala Leu Ala 210 215 220 Leu Ser His Thr Tyr Ala Asn Val Ser Ile Ser His Val Ile Asn Leu 225 230 235 240 Trp Gly Ala Asp Phe Asn Ala Glu Gly Asn Leu Glu Ala Ile Tyr Val 245 250 255 Thr Asp Ser Asp Ala Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val 260 265 270 Gly Ile Asn Ala His Gly His Val Ala Ile Ser Ala Lys Lys Ile Glu 275 280 285 Gly Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Ser 290 295 300 Gly Lys Asp Ile Trp Gln Lys Leu Ser 305 310 <210> 11 <211> 302 <212> PRT <213> Streptococcus equi ssp. Zooepidemicus <400> 11 Met Ser Val Trp Thr Lys Gly Val Thr Pro Pro Glu Gln Phe Thr Gln 1 5 10 15 Gly Glu Asp Val Ile His Ala Pro Tyr Leu Ala His Gln Gly Trp Tyr 20 25 30 Asp Ile Thr Lys Ala Phe Asn Gly Lys Asp Asp Leu Leu Cys Gly Ala 35 40 45 Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Thr 50 55 60 Glu Ile Glu Ala Tyr Leu Ser Lys His Pro Glu Lys Gln Lys Ile Ile 65 70 75 80 Phe Arg Asn Gln Glu Leu Phe Asp Leu Lys Ala Ala Ile Asp Thr Lys 85 90 95 Asp Ser Gln Thr Asn Ser Gln Leu Phe Asn Tyr Phe Arg Asp Lys Ala 100 105 110 Phe Pro Asn Leu Ser Ala Arg Gln Leu Gly Val Met Pro Asp Leu Val 115 120 125 Leu Asp Met Phe Ile Asn Gly Tyr Tyr Leu Asn Val Phe Lys Thr Gln 130 135 140 Ser Thr Asp Val Asn Arg Pro Tyr Gln Asp Lys Asp Lys Arg Gly Gly 145 150 155 160 Ile Phe Asp Ala Val Phe Thr Arg Gly Asn Gln Thr Thr Leu Leu Thr 165 170 175 Ala Arg His Asp Leu Lys Asn Lys Gly Leu Asn Asp Ile Ser Thr Ile 180 185 190 Ile Lys Gln Glu Leu Thr Glu Gly Arg Ala Leu Ala Leu Ser His Thr 195 200 205 Tyr Ala Asn Val Ser Ile Ser His Val Ile Asn Leu Trp Gly Ala Asp 210 215 220 Phe Asn Ala Glu Gly Asn Leu Glu Ala Ile Tyr Val Thr Asp Ser Asp 225 230 235 240 Ala Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Ile Asn Ala 245 250 255 His Gly His Val Ala Ile Ser Ala Lys Lys Ile Glu Gly Glu Asn Ile 260 265 270 Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Ser Gly Lys Asp Ile 275 280 285 Trp Gln Lys Leu Ser Gly Gly Gly His His His His His His 290 295 300 <210> 12 <211> 322 <212> PRT <213> Streptococcus equi ssp. Zooepidemicus <400> 12 Met Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val 1 5 10 15 Pro His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Glu 20 25 30 Gln Phe Thr Gln Gly Glu Asp Val Ile His Ala Pro Tyr Leu Ala His 35 40 45 Gln Gly Trp Tyr Asp Ile Thr Lys Ala Phe Asn Gly Lys Asp Asp Leu 50 55 60 Leu Gly Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp 65 70 75 80 Gln Asn Lys Thr Glu Ile Glu Ala Tyr Leu Ser Lys His Pro Glu Lys 85 90 95 Gln Lys Ile Ile Phe Arg Asn Gln Glu Leu Phe Asp Leu Lys Ala Ala 100 105 110 Ile Asp Thr Lys Asp Ser Gln Thr Asn Ser Gln Leu Phe Asn Tyr Phe 115 120 125 Arg Asp Lys Ala Phe Pro Asn Leu Ser Ala Arg Gln Leu Gly Val Met 130 135 140 Pro Asp Leu Val Leu Asp Met Phe Ile Asn Gly Tyr Tyr Leu Asn Val 145 150 155 160 Phe Lys Thr Gln Ser Thr Asp Val Asn Arg Pro Tyr Gln Asp Lys Asp 165 170 175 Lys Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn Gln Thr 180 185 190 Thr Leu Leu Thr Ala Arg His Asp Leu Lys Asn Lys Gly Leu Asn Asp 195 200 205 Ile Ser Thr Ile Ile Lys Gln Glu Leu Thr Glu Gly Arg Ala Leu Ala 210 215 220 Leu Ser His Thr Tyr Ala Asn Val Ser Ile Ser His Val Ile Asn Leu 225 230 235 240 Trp Gly Ala Asp Phe Asn Ala Glu Gly Asn Leu Glu Ala Ile Tyr Val 245 250 255 Thr Asp Ser Asp Ala Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val 260 265 270 Gly Ile Asn Ala His Gly His Val Ala Ile Ser Ala Lys Lys Ile Glu 275 280 285 Gly Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Ser 290 295 300 Gly Lys Asp Ile Trp Gln Lys Leu Ser Gly Gly Gly His His His His 305 310 315 320 His His <210> 13 <211> 320 <212> PRT <213> Streptococcus pyogenes <400> 13 Met Asp Ser Phe Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr 1 5 10 15 Pro Tyr His Val Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala 20 25 30 Asn Phe Thr Gln Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn 35 40 45 Gln Gly Trp Tyr Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu 50 55 60 Leu Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp 65 70 75 80 Gln Asn Lys Asp Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys 85 90 95 Gln Lys Ile Asn Phe Asn Gly Glu Gln Met Phe Asp Val Lys Glu Ala 100 105 110 Ile Asp Thr Lys Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe 115 120 125 Lys Glu Lys Ala Phe Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe 130 135 140 Pro Asp His Val Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu 145 150 155 160 Thr Asn His Gly Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg 165 170 175 Gly Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu 180 185 190 Leu Thr Ser Arg His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser 195 200 205 Asp Leu Ile Lys Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser 210 215 220 His Thr Tyr Ala Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly 225 230 235 240 Ala Asp Phe Asp Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp 245 250 255 Ser Asp Ser Asn Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val 260 265 270 Asn Ser Ala Gly Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp 275 280 285 Asn Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln 290 295 300 Asp Ser Trp Asn Gln Thr Asn Gly Gly Gly His His His His His His 305 310 315 320 <210> 14 <211> 325 <212> PRT <213> Streptococcus equi ssp. Zooepidemicus <400> 14 Met Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val 1 5 10 15 Pro His Gln Ile Thr Ser Val Trp Thr Lys Gly Val Thr Pro Leu Thr 20 25 30 Pro Glu Gln Phe Arg Tyr Asn Asn Glu Asp Val Ile His Ala Pro Tyr 35 40 45 Leu Ala His Gln Gly Trp Tyr Asp Ile Thr Lys Ala Phe Asp Gly Lys 50 55 60 Asp Asn Leu Leu Cys Gly Ala Ala Thr Ala Gly Asn Met Leu His Trp 65 70 75 80 Trp Phe Asp Gln Asn Lys Thr Glu Ile Glu Ala Tyr Leu Ser Lys His 85 90 95 Pro Glu Lys Gln Lys Ile Ile Phe Asn Asn Gln Glu Leu Phe Asp Leu 100 105 110 Lys Ala Ala Ile Asp Thr Lys Asp Ser Gln Thr Asn Ser Gln Leu Phe 115 120 125 Asn Tyr Phe Arg Asp Lys Ala Phe Pro Asn Leu Ser Ala Arg Gln Leu 130 135 140 Gly Val Met Pro Asp Leu Val Leu Asp Met Phe Ile Asn Gly Tyr Tyr 145 150 155 160 Leu Asn Val Phe Lys Thr Gln Ser Thr Asp Val Asn Arg Pro Tyr Gln 165 170 175 Asp Lys Asp Lys Arg Gly Gly Ile Phe Asp Ala Val Phe Thr Arg Gly 180 185 190 Asp Gln Thr Thr Leu Leu Thr Ala Arg His Asp Leu Lys Asn Lys Gly 195 200 205 Leu Asn Asp Ile Ser Thr Ile Ile Lys Gln Glu Leu Thr Glu Gly Arg 210 215 220 Ala Leu Ala Leu Ser His Thr Tyr Ala Asn Val Ser Ile Ser His Val 225 230 235 240 Ile Asn Leu Trp Gly Ala Asp Phe Asn Ala Glu Gly Asn Leu Glu Ala 245 250 255 Ile Tyr Val Thr Asp Ser Asp Ala Asn Ala Ser Ile Gly Met Lys Lys 260 265 270 Tyr Phe Val Gly Ile Asn Ala His Gly His Val Ala Ile Ser Ala Lys 275 280 285 Lys Ile Glu Gly Glu Asn Ile Gly Ala Gln Val Leu Gly Leu Phe Thr 290 295 300 Leu Ser Ser Gly Lys Asp Ile Trp Gln Lys Leu Ser Gly Gly Gly His 305 310 315 320 His His His His His 325 <210> 15 <211> 20 <212> PRT <213> Streptococcus equi ssp. Zooepidemicus <400> 15 Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val Pro 1 5 10 15 His Gln and Thr 20 <210> 16 <211> 969 <212> DNA <213> Streptococcus equi ssp. Zooepidemicus) <400> 16 atggacgatt accaaaggaa tgctacggaa gcttatgcca aagaagtacc acatcagatc 60 acttctgtat ggaccaaagg tgttacacca cccgagcagt ttactcaagg tgaagatgtg 120 atccatgcgc catatcttgc tcatcaaggc tggtacgata tcaccaagc cttcgatggg 180 aaggataatc tcttgtgtgg cgcagcaacg gcaggtaata tgctgcattg gtggtttgat 240 caaaataaaa cagagattga agcctattta agtaaacacc ctgaaaagca aaaaatcatt 300 tttcgtaacc aagagctatt tgatttgaaa gctgctatcg ataccaagga cagtcaaacc 360 aatagtcagc ttttaatta ttttagagat aaagcctttc caaatctatc agcacgtcaa 420 ctcggggtta tgcctgatct tgttctagat atgtttatca atggttacta cttaaatgtg 480 tttaaaacac agtctactga tgtcaatcga ccttatcagg acaaggacaa acgaggtggt 540 attttcgatg ctgttttcac cagaggagat cagacaacgc tcttgacagc tcgtcatgat 600 ttaaaaaata aaggactaaa tgacatcagc accattatca agcaagaact gactgaagga 660 agagcccttg ctttatcaca tacctacgcc aatgttagca ttagccatgt gattaacttg 720 tggggagctg attttaatgc tgaaggaaac cttgaggcca tctatgtcac agactcagat 780 gctaatgcgt ctattggtat gaaaaaatat tttgtcggca ttaatgctca tggacatgtc 840 gccatttctg ccaagaaaat agaaggagaa aacattggcg ctcaagtatt aggcttattt 900 acgctttcca gtggcaagga catttggcag aaactgagcg gcggtggcca tcatcaccat 960 caccactaa 969 <210> 17 <211> 969 <212> DNA <213> Streptococcus equi ssp. Zooepidemicus <400> 17 atggacgatt accaaaggaa tgctacggaa gcttatgcca aagaagtacc acatcagatc 60 acttctgtat ggaccaaagg tgttacacca cccgagcagt ttactcaagg tgaagatgtg 120 atccatgcgc catatcttgc tcatcaaggc tggtacgata tcaccaaggc cttcgatggg 180 aaggataatc tcttgtgtgg cgcagcaacg gcaggtaata tgctgcattg gtggtttgat 240 caaaataaaa cagagattga agcctattta agtaaacacc ctgaaaagca aaaaatcatt 300 tttcgtaacc aagagctatt tgatttgaaa gctgctatcg ataccaagga cagtcaaacc 360 aatagtcagc tttttaatta ttttagagat aaagcctttc caaatctatc agcacgtcaa 420 ctcggggtta tgcctgatct tgttctagat atgtttatca atggttacta cttaaatgtg 480 tttaaaacac agtctactga tgtcaatcga ccttatcagg acaaggacaa acgaggtggt 540 attttcgatg ctgttttcac cagaggaaac cagacaacgc tcttgacagc tcgtcatgat 600 ttaaaaaata aaggactaaa tgacatcagc accattatca agcaagaact gactgaagga 660 agagcccttg ctttatcaca tacctacgcc aatgttagca ttagccatgt gattaacttg 720 tggggagctg attttaatgc tgaaggaaac cttgaggcca tctatgtcac agactcagat 780 gctaatgcgt ctattggtat gaaaaaatat tttgtcggca ttaatgctca tggacatgtc 840 gccatttctg ccaagaaaat agaaggagaa aacattggcg ctcaagtatt aggcttattt 900 acgctttcca gtggcaagga catttggcag aaactgagcg gcggtggcca tcatcaccat 960 caccactaa 969 <210> 18 <211> 969 <212> DNA <213> Streptococcus equi ssp. Zooepidemicus <400> 18 atggacgatt accaaaggaa tgctacggaa gcttatgcca aagaagtacc acatcagatc 60 acttctgtat ggaccaaagg tgttacacca cccgagcagt ttactcaagg tgaagatgtg 120 atccatgcgc catatcttgc tcatcaaggc tggtacgata tcaccaaggc cttcaatggg 180 aaggatgatc tcttgtgtgg cgcagcaacg gcaggtaata tgctgcattg gtggtttgat 240 caaaataaaa cagagattga agcctattta agtaaacacc ctgaaaagca aaaaatcatt 300 tttcgtaacc aagagctatt tgatttgaaa gctgctatcg ataccaagga cagtcaaacc 360 aatagtcagc tttttaatta ttttagagat aaagcctttc caaatctatc agcacgtcaa 420 ctcggggtta tgcctgatct tgttctagat atgtttatca atggttacta cttaaatgtg 480 tttaaaacac agtctactga tgtcaatcga ccttatcagg acaaggacaa acgaggtggt 540 attttcgatg ctgttttcac cagaggaaac cagacaacgc tcttgacagc tcgtcatgat 600 ttaaaaaata aaggactaaa tgacatcagc accattatca agcaagaact gactgaagga 660 agagcccttg ctttatcaca tacctacgcc aatgttagca ttagccatgt gattaacttg 720 tggggagctg attttaatgc tgaaggaaac cttgaggcca tctatgtcac agactcagat 780 gctaatgcgt ctattggtat gaaaaaatat tttgtcggca ttaatgctca tggacatgtc 840 gccatttctg ccaagaaaat agaaggagaa aacattggcg ctcaagtatt aggcttattt 900 acgctttcca gtggcaagga catttggcag aaactgagcg gcggtggcca tcatcaccat 960 caccactaa 969 <210> 19 <211> 942 <212> DNA <213> Streptococcus equi ssp. Zooepidemicus <400> 19 atggacgatt accaaaggaa tgctacggaa gcttatgcca aagaagtacc acatcagatc 60 acttctgtat ggaccaaagg tgttacacca cccgagcagt ttactcaagg tgaagatgtg 120 atccatgcgc catatcttgc tcatcaaggc tggtacgata tcaccaaggc cttcaatggg 180 aaggatgatc tcttgtgtgg cgcagcaacg gcaggtaata tgctgcattg gtggtttgat 240 caaaataaaa cagagattga agcctattta agtaaacacc ctgaaaagca aaaaatcatt 300 tttcgtaacc aagagctatt tgatttgaaa gctgctatcg ataccaagga cagtcaaacc 360 aatagtcagc tttttaatta ttttagagat aaagcctttc caaatctatc agcacgtcaa 420 ctcggggtta tgcctgatct tgttctagat atgtttatca atggttacta cttaaatgtg 480 tttaaaacac agtctactga tgtcaatcga ccttatcagg acaaggacaa acgaggtggt 540 attttcgatg ctgttttcac cagaggaaac cagacaacgc tcttgacagc tcgtcatgat 600 ttaaaaaata aaggactaaa tgacatcagc accattatca agcaagaact gactgaagga 660 agagcccttg ctttatcaca tacctacgcc aatgttagca ttagccatgt gattaacttg 720 tggggagctg attttaatgc tgaaggaaac cttgaggcca tctatgtcac agactcagat 780 gctaatgcgt ctattggtat gaaaaaatat tttgtcggca ttaatgctca tggacatgtc 840 gccatttctg ccaagaaaat agaaggagaa aacattggcg ctcaagtatt aggcttattt 900 acgctttcca gtggcaagga catttggcag aaactgagct aa 942 <210> 20 <211> 909 <212> DNA <213> Streptococcus equi ssp. Zooepidemicus <400> 20 atgtctgtat ggaccaaagg tgttacacca cccgagcagt ttactcaagg tgaagatgtg 60 atccatgcgc catatcttgc tcatcaaggc tggtacgata tcaccaaggc cttcaatggg 120 aaggatgatc tcttgtgtgg cgcagcaacg gcaggtaata tgctgcattg gtggtttgat 180 caaaataaaa cagagattga agcctattta agtaaacacc ctgaaaagca aaaaatcatt 240 tttcgtaacc aagagctatt tgatttgaaa gctgctatcg ataccaagga cagtcaaacc 300 aatagtcagc tttttaatta ttttagagat aaagcctttc caaatctatc agcacgtcaa 360 ctcggggtta tgcctgatct tgttctagat atgtttatca atggttacta cttaaatgtg 420 tttaaaacac agtctactga tgtcaatcga ccttatcagg acaaggacaa acgaggtggt 480 attttcgatg ctgttttcac cagaggaaac cagacaacgc tcttgacagc tcgtcatgat 540 ttaaaaaata aaggactaaa tgacatcagc accattatca agcaagaact gactgaagga 600 agagcccttg ctttatcaca tacctacgcc aatgttagca ttagccatgt gattaacttg 660 tggggagctg attttaatgc tgaaggaaac cttgaggcca tctatgtcac agactcagat 720 gctaatgcgt ctattggtat gaaaaaatat tttgtcggca ttaatgctca tggacatgtc 780 gccatttctg ccaagaaaat agaaggagaa aacattggcg ctcaagtatt aggcttattt 840 acgctttcca gtggcaagga catttggcag aaactgagcg gcggtggcca tcatcaccat 900 caccactaa 909 <210> 21 <211> 969 <212> DNA <213> Streptococcus equi ssp. Zooepidemicus <400> 21 atggacgatt accaaaggaa tgctacggaa gcttatgcca aagaagtacc acatcagatc 60 acttctgtat ggaccaaagg tgttacacca cccgagcagt ttactcaagg tgaagatgtg 120 atccatgcgc catatcttgc tcatcaaggc tggtacgata tcaccaaggc cttcaatggg 180 aaggatgatc tcttgggtgg cgcagcaacg gcaggtaata tgctgcattg gtggtttgat 240 caaaataaaa cagagattga agcctattta agtaaacacc ctgaaaagca aaaaatcatt 300 tttcgtaacc aagagctatt tgatttgaaa gctgctatcg ataccaagga cagtcaaacc 360 aatagtcagc tttttaatta ttttagagat aaagcctttc caaatctatc agcacgtcaa 420 ctcggggtta tgcctgatct tgttctagat atgtttatca atggttacta cttaaatgtg 480 tttaaaacac agtctactga tgtcaatcga ccttatcagg acaaggacaa acgaggtggt 540 attttcgatg ctgttttcac cagaggaaac cagacaacgc tcttgacagc tcgtcatgat 600 ttaaaaaata aaggactaaa tgacatcagc accattatca agcaagaact gactgaagga 660 agagcccttg ctttatcaca tacctacgcc aatgttagca ttagccatgt gattaacttg 720 tggggagctg attttaatgc tgaaggaaac cttgaggcca tctatgtcac agactcagat 780 gctaatgcgt ctattggtat gaaaaaatat tttgtcggca ttaatgctca tggacatgtc 840 gccatttctg ccaagaaaat agaaggagaa aacattggcg ctcaagtatt aggcttattt 900 acgctttcca gtggcaagga catttggcag aaactgagcg gcggtggcca tcatcaccat 960 caccactaa 969 <210> 22 <211> 963 <212> DNA <213> Streptococcus pyogenes <400> 22 atggatagtt tttctgctaa tcaagagatt agatattcgg aagtaacacc tttcacgtt 60 acttccgttt ggaccaaagg agttactcct ccagcaaact tcactcaagg tgaagatgtt 120 tttcacgctc cttatgttgc taaccaagga tggtatgata ttaccaaaac attcaatgga 180 aaagacgatc ttctttgcgg ggctgccaca gcaggggaata tgcttcactg gtggttcgat 240 caaaacaaag accaaattaa acgttatttg gaagagcatc cagaaaagca aaaaataaac 300 ttcaatggcg aacagatgtt tgacgtaaaa gaagctatcg acactaaaaa ccaccagcta 360 gatagtaaat tatttgaata ttttaaagaa aaagctttcc cttatctatc tactaaacac 420 ctaggagttt tccctgatca tgtaattgat atgttcatta acggctaccg ccttagtcta 480 actaaccacg gtccaacgcc agtaaaagaa ggtagtaaag atccccgagg tggtattttt 540 gacgccgtat ttacaagagg tgatcaaagt aagctattga caagtcgtca tgattttaaa 600 gaaaaaaatc tcaaagaaat cagtgatctc attaagaaag agttaaccga aggcaaggct 660 ctaggcctat cacacaccta cgctaacgta cgcatcaacc atgtataaa cctgtgggga 720 gctgactttg attctaacgg gaaccttaaa gctatttatg taacagactc tgatagtaat 780 gcatctattg gtatgaagaa atactttgtt ggtgttaatt ccgctggaaa agtagctatt 840 tctgctaaag aaataaaaga agataatata ggtgctcaag tactagggtt atttacactt 900 tcaacagggc aagatagttg gaatcagacc aatggcggtg gccatcatca ccatcaccac 960 city ​​963 <210> 23 <211> 978 <212> DNA <213> Streptococcus equi ssp. Zooepidemicus) <400> 23 atggacgatt accaaaggaa tgctacggaa gcttatgcca aagaagtacc acatcagatc 60 acttctgtat ggaccaaagg tgttacacca ctaacacccg agcagtttcg atataataac 120 gaagatgtga tccatgcgcc atactctgct catcaaggct ggtacgatat caccaaggcc 180 ttcgatggga aggataatct cttgtgtggc gcagcaacgg caggtaatat gctgcattgg 240 tggtttgatc aaaataaac agagattgaa gcctatttaa gtaaaacaccc tgaaaagcaa 300 aaaatcattt ttacaacca agagctattt gatttgaaag ctgctatcga taccaaggac 360 agtcaaacca atagtcagct ttattat tttagagata aagcctttcc aaatctatca 420 gcacgtcaac tcggttt gcctgatctt gttctagata tgtttatcaa tggttactac 480 ttaaatgtgt ttaaaacaca gtctactgat gtcaatcgac cttatcagga caggacaaa 540 cgaggtgta ttttcgatgc tgtttcacc agaggagatc agacaacgct cttgacagct 600 cgtcatgatt taaaaaataa aggactaat gatacagca ccattatca gcaagactg 660 actgaaggaa gagcccttgc tttatcacat acctacgcca atgttagcat tagccatgtg 720 attaacttgt ggggagctga ttttaatgct gaaggaaacc ttgaggccat ctatgtcaca 780 gactcagatg ctaatgcgtc tattggtatg aaaaatatt tgtcggcat taatgctcat 840 ggacatgtcg ccattctgc caaaaata gaaggaaa acatggcgc tcaagtatta 900 ggcttattta cgctttccag tggcaaggac atttggcaga aactgagcgg cggtggccat 960 catcaccatc accactaa 978

Claims

1. A polypeptide having IgG1 and IgG2 cysteine ​​protease activity and consisting of an amino acid sequence, wherein the amino acid sequence is: (i) SEQ ID NO: 1; or (ii) SEQ ID NO:

2.

2. The polypeptide according to claim 1, wherein the polypeptide further comprises an additional methionine at the N-terminus and / or an additional histidine tag at the C-terminus.

3. A polynucleotide encoding the polypeptide according to claim 1 or 2. An expression vector comprising the polynucleotide according to claim 3 . A host cell comprising the polynucleotide according to claim 3 or the expression vector according to claim 4. The host cell according to claim 5 , wherein the host cell is a bacterial cell. The host cell according to claim 5 , wherein the host cell is an Escherichia coli cell.

8. A composition comprising the polypeptide according to claim 1 or 2 and at least one pharmaceutically acceptable carrier or diluent.

9. An in vitro method for cleaving IgG1 and IgG2, the method comprising contacting a sample comprising IgG1 and IgG2 with a polypeptide according to claim 1 or 2 under conditions that allow IgG1 and IgG2 cysteine ​​protease activity to occur.

10. The method of claim 9, which is performed to produce Fc, Fab and / or F(ab')2 fragments.

Images