Cysteine ​​proteases

Abstract

The present invention relates to novel polypeptides showing 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 disorders mediated by IgG, and methods for analyzing IgG.

Description

[0001] This application is a divisional application of an invention patent application which entered the Chinese national phase on October 12, 2017, with application number 201680021601.8, application date February 12, 2016, and invention name “Cysteine ​​Protease”. Technical Field

[0002] The present invention relates to novel polypeptides showing 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 disorders mediated by IgG, and methods for analyzing IgG.

[0003] IdeS (Immunoglobulin G degrading enzyme of Streptococcus pyogenes, I mmunoglobulin G- d egrading e nzyme of S .pyogenes) 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, and its only recognized substrate is IgG. IdeS catalyzes a single proteolytic cleavage in the lower hinge region of all subclasses of heavy chains of human IgG. IdeS also catalyzes equivalent cleavage of the heavy chains of some subclasses of IgG in various animals. IdeS effectively cleaves IgG into Fc and F(ab′)2 fragments through a two-stage mechanism. In the first stage, one (first) IgG heavy chain is cleaved to produce a single cleaved IgG (scIgG) molecule with a non-covalently bound Fc molecule. 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, the second heavy chain is cleaved by IdeS to release the F(ab′)2 fragment and the homodimeric Fc fragment. These are the products normally observed under physiological conditions. Under reducing conditions, the F(ab')2 fragment can be dissociated into two Fab fragments, and the homodimeric Fc can be dissociated into its component monomers. Summary of the invention

[0004] The IgG cleavage ability of IdeS has been shown to have in vitro utility, for example in methods for producing Fab and Fc fragments, which can be used to analyze IgG. See, for example, WO2003051914 and WO2009033670. IdeS has also been shown to have in vivo utility as a therapeutic agent, as it is able to cleave disease-mediated or otherwise unwanted IgG molecules in vivo. See, for example, WO2003051914, WO2006131347 and WO2013110946. IdeS can be used as a treatment for any disease or condition that is mediated in whole or in part by IgG. Many autoimmune diseases are mediated in whole or in part by IgG, as is acute rejection of donated organs.

[0005] 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, the immune response to IdeS (particularly the production of IdeS-specific ADA) may cause two related types of problems. First, the effectiveness of IdeS may be reduced, for example, due to ADA binding, higher or repeated doses may be required to achieve the same effect. ADA with this effect may be referred to as "neutralizing ADA". Secondly, there may be unwanted or even harmful complications, such as a high inflammatory response triggered by an immune complex of ADA and IdeS. The higher the number of ADA specific to IdeS in a given subject, the greater the possibility of these problems. The presence and quantity of IdeS-specific ADA molecules in the patient can be determined by any suitable method (such as a reagent-specific CAP FEIA (ImmunoCAP) test or a titer determination performed on a serum sample from the 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.

[0006] 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. In serum samples from random human subjects (likely due to previous streptococcal infection) and in IVIg (intravenous immunoglobulin, Intra v enous I IdeS-specific ADA are routinely detected in IVIg products, which are IgG products extracted from the pooled sera of thousands of donors. Even if a subject does not have IdeS-specific ADA before 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 excluding treatment with IdeS entirely, especially if repeated administration is required. Existing solutions to such problems involve, for example, pegylation of therapeutic agents to reduce the immunogenicity of the therapeutic agents, or co-administration of therapeutic agents with immunosuppressants.

[0007] The present inventors have taken a completely different approach. The present inventors have identified specific positions within the IdeS sequence that, when modified as described herein, produce novel polypeptides that have reduced problems associated with immunogenicity compared to IdeS. Relative to IdeS, some modifications may increase the potency of the polypeptides of the invention to cleave IgG, thereby indirectly reducing immunogenicity by allowing the use of lower doses or concentrations to achieve the same effect. Alternatively or in addition, relative to IdeS, other modifications may directly reduce immunogenicity by reducing the ability of IdeS-specific antibodies to recognize the polypeptides of the invention.

[0008] The full sequence of IdeS is publicly available as NCBI reference sequence number WP_010922160.1 and is provided herein as SEQ ID NO: 1. The sequence includes an N-terminal methionine followed by a 28 amino acid secretion signal sequence. The N-terminal methionine and signal sequence (a total of 29 amino acids 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: 2.

[0009] 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 position in SEQ ID NO: 1 starting from the N-terminus. Thus, since SEQ ID NO: 2 lacks the N-terminal methionine and 28 amino acid signal sequence of SEQ ID NO: 1, the aspartic acid (D) residue at the N-terminus of SEQ ID NO: 2 is referred to as position 30, as is the corresponding position in this SEQ ID NO: 1. Applying this numbering scheme, the most critical residue for the IgG cysteine ​​protease activity of IdeS is the cysteine ​​(C) at position 94 (the 65th residue from the N-terminus of SEQ ID NO: 2). Other residues that may be important for IgG cysteine ​​protease activity are lysine (K) at position 84, histidine (H) at position 262, and aspartic acid (D) at each of positions 284 and 286. These are the 55th, 233rd, 255th, and 257th residues from the N-terminus of SEQ ID NO: 2, respectively.

[0010] According to the present invention, there is therefore provided a polypeptide having IgG cysteine ​​protease activity and comprising a variant of the sequence SEQ ID NO: 2, said variant:

[0011] (a) at least 50% identical to SEQ ID NO: 2;

[0012] (b) having cysteine ​​(C) at the position corresponding to position 94 of SEQ ID NO: 1 in the variant sequence; and optionally

[0013] (c) having lysine (K), histidine (H), aspartic acid (D) and aspartic acid (D) at the positions corresponding to positions 84, 262, 284 and 286 of SEQ ID NO: 1, respectively, in the variant sequence;

[0014] Wherein the polypeptide is more effective than IdeS in cleaving IgG and / or the polypeptide has lower immunogenicity than IdeS. The polypeptide of the present invention may be more effective in cleaving IgG1 than IgG2.

[0015] Preferably, the variant of SEQ ID NO: 2:

[0016] (1) having a positively charged amino acid at the position corresponding to position 130 of SEQ ID NO: 1 in the variant, optionally wherein the positively charged amino acid is arginine (R) or lysine (K); and / or

[0017] (2) having a positively charged amino acid at the position corresponding to position 131 of SEQ ID NO: 1 in the variant, optionally wherein the positively charged amino acid is arginine (R) or lysine (K); and / or

[0018] (3) does not include a continuous sequence of NQTNs; and / or

[0019] (4) Does not include the continuous sequence DSFSANQEIR YSEVTPYHVT.

[0020] The present invention also provides a polynucleotide, an expression vector or a host cell encoding or expressing the polypeptide of the present invention.

[0021] 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 preventively effective amount of the polypeptide of the present invention. The method may generally comprise administering the polypeptide to the subject multiple times.

[0022] 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.

[0023] The present invention also provides a method for improving the benefit of a treatment or therapeutic agent to 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:

[0024] - 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;

[0025] - the amount of the polypeptide administered is sufficient to cleave substantially all IgG molecules present in the plasma of the subject; and

[0026] - 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.

[0027] The present invention also provides a method for producing an Fc fragment, a Fab fragment or a F(ab')2 fragment of IgG, which comprises contacting IgG with a polypeptide of the present invention (preferably in vitro).

[0028] Also provided are kits for carrying out the methods according to the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1Shown are the results of a representative assay to determine the potency (efficacy for IgG cleavage) of polypeptides of the invention compared to a control.

[0030] Figure 2 Shown are the results of a representative SDS-PAGE gel used to visualize the cleavage products resulting from incubation of IgG1 with a polypeptide of the invention or a control.

[0031] Figure 3 Shown are the results of a representative SDS-PAGE gel used to visualize the cleavage products resulting from incubation of IgG1 with more polypeptides of the invention or controls.

[0032] Figure 4 Shown are the results of a representative SDS-PAGE gel used to visualize the cleavage products generated by incubation of IgG2 with a polypeptide of the invention or a control.

[0033] Figure 5 and Figure 6 Shown are the results of a representative competition assay to determine the level of recognition of a polypeptide of the invention by an IdeS-specific antibody compared to a control.

[0034] Figure 7 Representative titration curves for cleavage of IgG1 by different IgG cysteine ​​protease polypeptides are shown.

[0035] Figure 8 Representative titration curves for cleavage of IgG2 by different IgG cysteine ​​protease polypeptides are shown.

[0036] Fig. 9 Shown are the results of a representative SDS-PAGE used to visualize the cleavage products generated by incubation of IgG with the polypeptides of the invention or controls.

[0037] Fig.10 Shown are the results of a representative SDS-PAGE used to visualize the cleavage products generated by incubation of IgG with the polypeptides of the invention or controls.

[0038] Fig.11 Shown are the results of a representative SDS-PAGE used to visualize the cleavage products generated by incubation of IVIg with a polypeptide of the invention or a control.

[0039] Fig.12 Shown are the results of a representative SDS-PAGE used to visualize the cleavage products generated by incubation of IVIg with a polypeptide of the invention or a control.

[0040] Fig.13 Schematic diagram of cleavage of immunoglobulins by polypeptides of the present invention.

[0041] Fig.14 Representative results of % competition of ADA binding sites with polypeptides of the invention or controls are shown.

[0042] Fig.15 Further representative results of % competition of ADA binding sites with polypeptides of the invention or controls are shown.

[0043] Fig.16 Shown are the results of a representative potency ELISA for determining the potency of the polypeptides of the invention to cleave human IgG in vivo.

[0044] Fig.17 Shown are the results of representative SDS-PAGE used to visualize IgG cleavage products produced by the polypeptides of the present invention in vivo.

[0045] A brief description of the sequence

[0046] SEQ ID NO: 1 is the complete sequence of IdeS including the N-terminal methionine and signal sequence. Also published as NCBI reference sequence number WP_010922160.1

[0047] SEQ ID NO: 2 is the mature sequence of IdeS lacking the N-terminal methionine and signal sequence. Also published as Genbank Accession No. ADF13949.1

[0048] SEQ ID NOs: 3 to 16 are sequences of exemplary polypeptides of the present invention

[0049] SEQ ID NO: 17 is the sequence of the IdeS polypeptide used as a control herein, including the sequence SEQ ID NO: 2 with an additional N-terminal methionine and histidine tag (internal reference pCART124).

[0050] SEQ ID NO:18 is the contiguous sequence NQTN, which corresponds to positions 336-339 of SEQ ID NO:1.

[0051] SEQ ID NO:19 is the contiguous sequence DSFSANQEIR YSEVTPYHVT, which corresponds to positions 30-49 of SEQ ID NO:1.

[0052] SEQ ID NOs: 20 to 34 are nucleotide sequences encoding the polypeptides disclosed herein.

[0053] SEQ ID NO:35 is the sequence SFSANQEIRY SEVTPYHVT corresponding to positions 31-49 of SEQ ID NO:1.

[0054] SEQ ID NO: 36 is the sequence DYQRNATEAY AKEVPHQIT corresponding to positions 36-54 of the IdeZ polypeptide NCBI Reference Sequence No. WP_014622780.1.

[0055] SEQ ID NO: 37 is the sequence DDYQRNATEAYAKEVPHQIT which may be present at the N-terminus of the polypeptide of the present invention. DETAILED DESCRIPTION

[0056] 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 terms used herein are only used for the purpose of describing specific embodiments of the present invention and are not limiting.

[0057] 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.

[0058] "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.

[0059] The terms "patient" and "subject" are used interchangeably and typically refer to a human. References to IgG typically refer to human IgG unless otherwise specified.

[0060] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0061] Functional characteristics of peptides

[0062] The present invention relates to novel polypeptides having IgG cysteine ​​protease activity, wherein the polypeptide is more effective than IdeS in cleaving IgG and / or has lower immunogenicity than IdeS. In the context of a control or comparison relative to the polypeptide of the present invention, "IdeS" refers to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2. Alternatively or in addition, when used as a control or comparison, "IdeS" may refer to a polypeptide comprising the sequence of the amino acid sequence of SEQ ID NO: 2, which has 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 can be directly attached to the C-terminus of the polypeptide or indirectly attached via any suitable linker sequence (such as 3, 4 or 5 glycine residues). The histidine tag is typically composed of six histidine residues, although it can be longer (typically up to 7, 8, 9, 10 or 20 amino acids) or shorter (e.g., 5, 4, 3, 2 or 1 amino acids) than this. The sequence of an exemplary IdeS polypeptide used herein is a control provided as SEQ ID NO: 14. The polypeptide comprises the sequence SEQ ID NO: 2 with an additional N-terminal methionine and histidine tag, and may be referred to herein as pCART124.

[0063] IgG cysteine ​​protease activity can be evaluated 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 evaluated in the presence or absence of an inhibitor (such as a neutralizing antibody). However, potency herein generally refers to potency evaluated in the absence of such an inhibitor, unless otherwise specified. Suitable methods are described in the Examples. The potency of a polypeptide on cleavage IgG may be referred to herein as the "potency" of a polypeptide. The potency of the polypeptide of the present invention is generally 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 or 7.5 times greater than the potency of IdeS measured in the same assay. The potency of the polypeptide of the present invention is preferably at least 4.5 times, more preferably at least 6.0 times, and most preferably at least 7.5 times greater than the potency of IdeS measured in the same assay. Regardless of the problems associated with the immunogenicity of IdeS, an increase in potency relative to IdeS is an ideal improvement. However, this increase in titer will also 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 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 because the immune system is less likely to respond or will respond less to an agent present in a lower concentration. Therefore, when present at the same dose, the polypeptide of the invention may have the same immunogenicity as IdeS, or even a higher immunogenicity than IdeS, but the problems associated with immunogenicity are reduced or avoided because a lower dose is required to achieve the same therapeutic effect. In an alternative embodiment, if the polypeptide of the invention has a lower immunogenicity than IdeS when present at the same dose, the polypeptide of the invention may have an equivalent titer to IdeS.

[0064] Assays for assessing the effectiveness of polypeptides on 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 a target of the antibody, 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 specific to BSA in this example. 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 target-specific antibody will be added under conditions suitable for binding to the target-specific antibody. The detection antibody will bind to any intact target-specific antibody that has been bound to the target in each well. After washing, the amount of the detection antibody present in the well will be proportional to the amount of the target-specific antibody bound to the well. The detection antibody can be directly or indirectly conjugated to a marker or another reporting system, such as an enzyme, so that the amount of the detection antibody remaining in each well can be determined. The higher the titer of the tested polypeptide present in a 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 rather than the polypeptide to be tested, so that the titer of the tested polypeptide can be directly compared to the titer of IdeS. The polypeptides of the invention may be more effective at cleaving IgG1 than IgG2.

[0065] 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 an assay typically incubates the IgG sample with the test polypeptide (or with IdeS as a control) 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 polypeptide of the present invention typically produces a detectable amount of cleavage fragments at a lower concentration than IdeS (at a lower point in the titration series). This type of assay can also be able to identify a more effective test polypeptide on the first or second heavy chain of the cleavage IgG molecule, because the number of different fragments produced by each cleavage event can also be determined. 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 present invention. In this adaptive assay, serum or IVIg preparations containing IdeS-specific ADA are included together with the reaction medium when the IgG sample is incubated with the test polypeptide (or with IdeS as a control). Preferably, the titer of the polypeptide of the present invention is not affected by the presence of ADA, or the titer of the polypeptide of the present 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 present 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, since problems associated with this immunogenicity are reduced or avoided by requiring a lower dose of the polypeptide of the present invention to achieve the same therapeutic effect, the polypeptide of the present invention may have the same immunogenicity as IdeS, or even a higher immunogenicity than IdeS when present at the same dose. However, the polypeptide of the present invention generally does not have a higher immunogenicity than IdeS, and preferably, it has a lower immunogenicity than IdeS. That is, when present at an equal dose or concentration and measured in the same assay, the polypeptide of the present invention may cause the same immune response as IdeS or preferably a lower immune response. The immunogenicity of the polypeptide of the present invention is generally no more than 90%, no more than 85%, no more than 80%, no more than 70%, no more than 60% or no more than 50% of the immunogenicity of IdeS measured in the same assay. Preferably, the immunogenicity of the polypeptide of the present invention does not exceed 85% of the immunogenicity of IdeS measured in the same assay. More preferably, the immunogenicity of the polypeptide of the present invention does not exceed 70% of the immunogenicity of IdeS measured in the same assay.

[0067] Assays for assessing the immunogenicity of a polypeptide 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 an ADA specific for IdeS also binds to a polypeptide of the invention. This type of assay is described in the Examples.

[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 been 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 been 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 the binding of the ADA to the polypeptide being tested can be directly compared to the binding to IdeS.

[0069] Another suitable assay includes testing a titration series of different concentrations of IdeS-specific ADA (e.g., IVIg preparations) to determine the extent to which IdeS binds to a test polypeptide. 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 assays are described in the Examples. Such assays typically include coating the wells of an assay plate with a test polypeptide or IdeS, followed by incubation of each well with different concentrations of IdeS-specific ADA from the titration series. The 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 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 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 IdeS. 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 a 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 or 300 amino acids. The polypeptide of the present invention generally has a length of no more than 400, 350, 340, 330, 320 or 310 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 310 amino acids.

[0073] The primary structure (amino acid sequence) of the polypeptide of the present invention is based on the primary structure of IdeS, specifically the amino acid sequence of SEQ ID NO: 2. The sequence of the polypeptide of the present invention comprises a variant of the amino acid sequence of SEQ ID NO: 2, which is at least 50% identical to the amino acid sequence of SEQ ID NO: 2. The variant sequence may be at least 60%, at least 70%, at least 80%, at least 85%, preferably at least 90%, at least 95%, at least 98% or at least 99% identical to the sequence SEQ ID NO: 2. The variant may be identical to the sequence SEQ ID NO: 2 except that it includes one or more specific modifications identified herein. The identity relative to the sequence SEQ ID NO: 2 may be measured over a region of at least 50, at least 100, at least 200, at least 300 or more consecutive amino acids of the sequence shown in SEQ ID NO: 2, or more preferably over the entire length of SEQ ID NO: 2.

[0074] Any suitable algorithm can be used to calculate amino acid identity. For example, the PILEUP and BLAST algorithms can be used to calculate identity or align sequences (such as identifying identical or corresponding sequences (usually on their default settings), for example, as described in Altschul SF (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 words of length W in the query sequence that match or satisfy some positive-valued threshold score T when aligned with a word of the same length in the database sequence. T is called the neighborhood word score threshold (Altschul et al., supra). These initial hits are considered to be neighbors. The word hits are used as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction ceases when: the cumulative alignment score falls by an amount X from its maximum achieved value; the cumulative score goes to 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, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919), alignments (B) of 50, an expectation (E) of 10, M=5, N=4, and a comparison of both chains as defaults.

[0075] 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 that 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).

[0076] The sequence of the polypeptide of the present invention comprises a variant of the amino acid sequence of SEQ ID NO: 2, wherein modifications such as amino acid addition, deletion or substitution are made relative to the sequence SEQ ID NO: 2. Unless otherwise indicated, the modification is preferably a conservative amino acid substitution. Conservative substitution replaces an amino acid with another amino acid of similar chemical structure, similar chemical properties or similar side chain volume. 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 main amino acids as defined in Table A1 below. When the amino acids have similar polarities, this can be determined by referring to the hydropathy scale of the amino acid side chains in Table A2.

[0077] Table A1 - Chemical properties of amino acids

[0078]

[0079]

[0080] Table A2 - Hydrophilicity Scale

[0081]

[0082] The amino acid sequence of the polypeptide of the present invention comprises a variant of the amino acid sequence of SEQ ID NO: 2. However, certain residues in the amino acid sequence of SEQ ID NO: 2 are preferably retained in the variant sequence. For example, the variant sequence generally retains certain residues known to be required for IgG cysteine ​​protease activity. Therefore, the cysteine ​​at position 94 of SEQ ID NO: 1 (the 65th residue of SEQ ID NO: 2) must be retained in the amino acid sequence of the polypeptide of the present invention. Optionally, the lysine at position 84, the histidine at position 262, and the aspartic acid at each of positions 284 and 286 of SEQ ID NO: 1 are also retained. These are the 55th, 233rd, 255th, and 257th residues of SEQ ID NO: 2, respectively. Therefore, the polypeptide of the present invention generally comprises a variant of the amino acid sequence of SEQ ID NO: 2, which has cysteine ​​(C) at the position corresponding to position 94 of SEQ ID NO: 1 in the variant sequence; and optionally has lysine (K), histidine (H), aspartic acid (D) and aspartic acid (D) at the positions corresponding to position 84, position 262, position 284 and position 286 of SEQ ID NO: 1 in the variant sequence, respectively.

[0083] Starting from the above structural constraints, the inventors identified specific locations for modifications to adjust the functional properties of IdeS by evaluating the three-dimensional model of IdeS. The inventors have determined the following:

[0084] (1) Substituting the asparagine (N) at position 130 of SEQ ID NO: 1 with a positively charged amino acid enhances the potency of the polypeptide comprising the change. Therefore, the polypeptide of the present invention may comprise a variant of the amino acid sequence of SEQ ID NO: 2, which has a positively charged amino acid at the position corresponding to position 130 of SEQ ID NO: 1 in the variant. Common positively charged amino acids are identified in Table A1 above. The positively charged amino acid is preferably arginine (R) or lysine (K). Therefore, this specific modification may be identified herein by the term "N130R / K".

[0085] (2) Substituting a positively charged amino acid for glycine (G) at position 131 of SEQ ID NO: 1 enhances the potency of the polypeptide comprising the change. Therefore, the polypeptide of the present invention may comprise a variant of the amino acid sequence of SEQ ID NO: 2, wherein the variant has a positively charged amino acid at the position corresponding to position 131 of SEQ ID NO: 1. Common positively charged amino acids are identified in Table A1 above. The positively charged amino acid is preferably arginine (R) or lysine (K). Therefore, this particular modification may be identified herein by the term "G131R / K".

[0086] ](3) Deleting the last four residues at the C-terminus of SEQ ID NO:2 enhances the potency of the polypeptide comprising the change. The last four residues at the C-terminus of SEQ ID NO:2 consist of the continuous sequence NQTN. Therefore, the polypeptide of the present invention may comprise a variant of the amino acid sequence of SEQ ID NO:2 that does not include the continuous sequence NQTN. That is, the last four residues at the C-terminus of SEQ ID NO:2 may not be present in the variant of SEQ ID NO:2. The last four residues of SEQ ID NO:2 correspond to positions 336-339 of SEQ ID NO:1. Therefore, this particular modification may be identified herein by the term "N336N339del".

[0087] ](4) Deleting or changing the sequence of the first 20 residues at the N-terminus of SEQ ID NO: 2 can enhance the potency of the polypeptide containing the change and / or can reduce the immunogenicity without adversely affecting the potency.

[0088] The first 20 residues at the N-terminus of SEQ ID NO: 2 consist of the continuous sequence DSFSANQEIRYSEVTPYHVT (SEQ ID NO: 19). Therefore, the polypeptide of the present invention may include a variant of the amino acid sequence of SEQ ID NO: 2 that does not include the continuous sequence DSFSANQEIRYSEVTPYHVT.

[0089] As an example, the contiguous sequence of SEQ ID NO: 19 may be deleted in its entirety. That is, the first 20 residues at the N-terminus of SEQ ID NO: 2 may not be present in the variant of SEQ ID NO: 2. The first 20 residues of SEQ ID NO: 2 correspond to positions 30-49 of SEQ ID NO: 1. Thus, the deletion of this contiguous sequence may be identified herein by the term "D30_T49del".

[0090] ] Alternatively, the polypeptide of the present invention may comprise a variant of the amino acid sequence of SEQ ID NO: 2, wherein the sequence of the first twenty residues at the N-terminus of SEQ ID NO: 2 is altered by replacing one or more amino acids in the first twenty residues. The altered sequence preferably has reduced immunogenicity compared to the first 20 amino acids of SEQ ID NO: 2. As an example, the amino acids at positions 2 to 20 of SEQ ID NO: 2 (which correspond to positions 31-49 of SEQ ID NO: 1) may optionally be replaced by the consecutive sequence DYQRNATEAY AKEVPHQIT (SEQ ID NO: 36). In other words, the first 20 amino acids of the polypeptide of the present invention may consist of the sequence DDYQRNATEA YAKEVPHQIT (SEQ ID NO: 37) instead of the sequence of SEQ ID NO: 19. The inserted sequence SEQ ID NO: 36 is taken from the N-terminal region of IdeZ and corresponds to positions 36-54 of the IdeZ NCBI reference sequence number WP_014622780.1. Human subjects do not typically express antibodies to IdeZ, and thus polypeptides comprising this sequence are less prone to ADA.Substitution of SEQ ID NO: 35 with SEQ ID NO: 37 may be identified herein by the term "S31_T49replZ".

[0091] Therefore, in summary, the polypeptide of the present invention comprises a variant of the sequence SEQ ID NO: 2, which variant:

[0092] (a) at least 50% identical to SEQ ID NO: 2;

[0093] (b) having cysteine ​​(C) at the position corresponding to position 94 of SEQ ID NO: 1 in the variant sequence; and optionally

[0094] (c) having lysine (K), histidine (H), aspartic acid (D) and aspartic acid (D) at the positions corresponding to positions 84, 262, 284 and 286 of SEQ ID NO: 1, respectively, in the variant sequence;

[0095] Preferably, the variant of SEQ ID NO: 2:

[0096] (1) having a positively charged amino acid at the position corresponding to position 130 of SEQ ID NO: 1 in the variant, optionally wherein the positively charged amino acid is arginine (R) or lysine (K); and / or

[0097] (2) having a positively charged amino acid at the position corresponding to position 131 of SEQ ID NO: 1 in the variant, optionally wherein the positively charged amino acid is arginine (R) or lysine (K); and / or

[0098] (3) does not include a continuous sequence of NQTNs; and / or

[0099] (4) Does not include the continuous sequence DSFSANQEIR YSEVTPYHVT.

[0100] The variant of SEQ ID NO: 2 may include at least one, two, three or all four of the modifications (1) to (4) listed above. The variant may include any combination of two or three of the modifications (1) to (4) listed above. A preferred variant includes modification (3) and at least one of modifications (1) and (2). Another preferred variant includes modifications (3) and (4) and at least one of modifications (1) and (2).

[0101] The inventors have also determined that certain other modifications to the sequence SEQ ID NO: 2, which may be applied alternatively or in addition to any combination of the above modifications (1) to (4), may increase the potency of the polypeptide of the invention and / or may reduce the recognition of the polypeptide of the invention by an IdeS-specific ADA. Thus, the polypeptide of the invention may comprise a variant of the sequence SEQ ID NO: 2, wherein substitutions are made at one or more positions corresponding to positions 115, 119, 139, 142, 198, 216, 226, 241, 245, 302, 316 and 333 of SEQ ID NO: 1. The variant may include substitutions in all of these positions, but typically includes substitutions in two, three, four, five, six or seven of these positions. Substitutions in these positions typically replace the existing amino acid with another amino acid having different properties. For example, an uncharged amino acid may 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:

[0102] Table B

[0103] Amino acids present in SEQ ID NO: 2 Position in SEQ ID No: 1 Preferred alternative

[0104] K 115 E E 119 R E 139 K D 142 R E 198 K D 216 N D 226 N K 241 E E 245 K S 302 K D 316 K D 333 K

[0105] Each substitution in Table B 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 may be referred to herein as "K115E", the substitution in the second row may be referred to as "E119R", and so on. The specific modification "D226N" is intended to disrupt a known cell adhesion motif in the sequence of IdeS, which is a continuous RGD sequence at positions 224-226 of SEQ ID NO: 1.

[0106] Table C below summarizes modifications made to the amino acid sequences that resulted in certain exemplary polypeptides of the invention.

[0107] Table C

[0108]

[0109]

[0110] The full length amino acid sequences of SEQ ID NOs: 1 and 2 are reproduced below, followed by the amino acid sequence of each exemplary polypeptide of the invention described in Table C.

[0111] SEQ ID NO: 1

[0112]

[0113] SEQ ID NO: 2

[0114]

[0115] SEQ ID NO: 3 (pCART152)

[0116]

[0117] SEQ ID NO: 4 (pCART183)

[0118]

[0119] SEQ ID NO: 5 (pCART184)

[0120]

[0121] SEQ ID NO:6(pCART185)

[0122]

[0123] SEQ ID NO:7(pCART186)

[0124]

[0125] SEQ ID NO:8(pCART187)

[0126]

[0127] SEQ ID NO:9(pCART188)

[0128]

[0129] SEQ ID NO:10(pCART189)

[0130]

[0131]

[0132] SEQ ID NO:11(pCART190)

[0133]

[0134] SEQ ID NO:12(pCART209)

[0135]

[0136] SEQ ID NO:13(pCART125)

[0137]

[0138] SEQ ID NO:14(pCART213)

[0139]

[0140] SEQ ID NO: 15 (pCART214)

[0141]

[0142] SEQ ID NQ:16(pCART228)

[0143]

[0144] The polypeptide of the present invention may comprise, consist essentially of or consist of the sequence of any one of SEQ ID NOs: 3 to 16. Each of SEQ ID NOs: 3 to 16 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. For SEQ ID NO: 13, the histidine tag is preferably attached to the C-terminus via a linker of 5×glycine residues.

[0145] Peptide production

[0146] 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 disclosed herein are provided as SEQ ID NOs: 20 to 34. Each of these sequences includes a codon (ATG) for the N-terminal methionine at the 3′ end, and before the stop codon (TAA) at the 5′ end, 3× sugar linkers and 6× codons for histidine tags, which can be optionally excluded.

[0147] 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 analogs thereof. 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 may 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 substantial but not complete. The polynucleotide may be mixed with carriers or diluents that do not interfere with its intended use and still be considered substantially isolated. A nucleic acid sequence that "encodes" 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 appropriate regulatory sequences (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 the 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] The polypeptide can be derivatized or modified to help 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 the 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 the binding pair, the polypeptide is preferably histidine-tagged or biotin-tagged. Typically, the amino acid coding sequence of the histidine or biotin tag is included at the gene level, and the polypeptide is 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 than this, typically up to 7, 8, 9, 10 or 20 amino acids or shorter, for example 5, 4, 3, 2 or 1 amino acid.

[0152] 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 synthetic means, such 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 field of producing such polypeptides.

[0153] 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.

[0154] 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 does 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.

[0155] Compositions and preparations containing polypeptides

[0156] 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 whom the composition is administered. Typically, the carrier and the final composition are sterile and pyrogen-free.

[0157] 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., NJ 1991).

[0158] 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 multidose containers containing preservatives. Compositions include, but are not limited to suspensions, solutions, emulsions, pastes and implantable sustained release or biodegradable preparations in oily or aqueous vehicles. Such compositions can 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 recovery with a suitable vehicle (e.g., sterile pyrogen-free water), followed by parenteral administration of the recovered composition. Compositions can be prepared, packaged or sold in the form of sterile injectable aqueous or oily suspensions or solutions. Such suspensions or solutions can be prepared according to known techniques, and in addition to the active ingredient, can 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 this 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 monoglycerides or diglycerides).

[0159] 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 or other suitable routes of administration. Preferred compositions are suitable for use by intravenous infusion.

[0160] How to use peptides

[0161] 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, the method can be particularly used to produce Fc and F (ab ') 2 fragments. Then, the Fab fragment can 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.

[0162] The method can also be used to detect or analyze IgG in a sample or to remove IgG from a sample. The method for detecting IgG in a sample generally includes incubating the polypeptide with the sample under conditions that allow IgG specific binding and cleavage. The presence of IgG can be verified by detecting specific IgG cleavage products, which can be analyzed subsequently.

[0163] 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 a subject who 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 a subject who 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 subject 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 in a subject, the method comprising administering a polypeptide of the present invention to the subject 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 or other suitable administration routes. The amount of the polypeptide administered can have a lower limit of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25 mg / kg BW (body weight). The amount of the polypeptide administered can have an upper limit of about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 mg / kg BW. It should be understood that any of the above upper limits can be combined with any of the above lower limits to provide the scope of the administered dosage. For example, the amount of the polypeptide administered can be between about 0.01mg / kg BW to 2mg / kg BW, between 0.10 to 1mg / kg BW, and preferably between 0.25mg / kg BW to 0.5mg / kg BW. Polypeptide can be repeatedly applied to the same subject, provided that the number of ADA that can be combined to the polypeptide in the subject's serum does not exceed the threshold value determined by the clinician. The number of ADA that can be combined to the polypeptide in the subject's serum can be determined by any suitable method, such as reagent-specific CAP FE IA (ImmunoCAP) test or titer determination.

[0164] 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.

[0165] Pathogenic antibodies may often be specific for an antigen, targeting the antigen in autoimmune diseases or other conditions that are mediated in whole or in part by the antibodies. Table D lists a list of such 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.

[0166] Table D

[0168]

[0169]

[0170] 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).

[0171] 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 administering the treatment or therapeutic agent to the subject subsequently. 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.

[0172] 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:

[0173] - the amount of the polypeptide administered is sufficient to cleave substantially all IgG molecules present in the plasma of the subject; and

[0174] - Step (a) and step (b) are separated by a time interval of at least 2 hours and at most 21 days.

[0175] 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 manufacture of a medicament for treating cancer or other diseases by this method. The cancer may be acute lymphoblastic 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 (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 (Burkitt lymphoma), carcinoid tumors, gastrointestinal carcinoid tumors, cancer of unknown primary, central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, cervical cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, 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, 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, Glioma, Childhood cerebral astrocytoma, 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 Brain 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 cancer, liposarcoma, liver cancer ( primary), 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 central nervous system lymphoma, macroglobulinemia, Waldenstrom's macroglobulinemia, 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 Cancer, Multiple Endocrine Neoplasms Syndrome, Multiple Myeloma / Plasma Cell Neoplasms, Mycosis Fungoides, Myelodysplastic Syndrome, Myelodysplastic / Myeloproliferative Disorders, Chronic Myeloid Leukemia, Acute Myeloid Leukemia in Adults, Acute Myeloid Leukemia in Children, Myeloma, Multiple (Bone Marrow Cancer) Myeloproliferative Disorders, Nasal and Paranasal Sinus Cancer, Nasopharyngeal Carcinoma, Neuroblastoma, Non-Hodgkin Lymphoma, Non-Small Cell Lung Cancer, Oral Cancer Cancer), Oropharyngeal cancer, Osteosarcoma / malignant fibrous histiocytoma of the 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 cells), Paranasal sinus and nasal cavity cancer, Parathyroid cancer, Penile cancer, Pharyngeal cancer, Pheochromocytoma, Pineal astrocytoma, Pineal germ cell tumor, Pineoblastoma and supratentorial primitive neuroectodermal tumor, Pituitary adenoma, Plasma cell neoplasia / multiple myeloma, Pleuropulmonary blastoma, Primary central nervous system lymphoma, Prostate cancer, Rectal cancer, Renal cell carcinoma (kidney cancer), Transitional cell carcinoma of the renal pelvis and ureter, Retinoblastoma, Rhabdomyosarcoma, Salivary gland cancer, Sarcoma, Ewing family tumor, Kaposi's sarcoma Tumors, Soft Tissue Sarcomas, Uterine Sarcomas, Sezary Syndrome, Skin Cancer (Non-Melanoma), Skin Cancer (Melanoma), Skin Cancer (Merkel Cell), Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcomas, Squamous Cell Carcinoma, Metastatic Squamous Cell Neck Cancer with Occult Primary, Gastric Cancer, Supratentorial Primitive Neuroectodermal Tumors, T-Cell Lymphoma, Cancer 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, Urethra Cancer, Uterine Cancer (Endometrial), Uterine Sarcomas, Vaginal Cancer, Visual Pathway and Hypothalamic Gliomas, Vulvar Cancer, Waldenstrom's Macroglobulinemia, and Wilms' Tumor (Kidney Cancer).,

[0176] 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.

[0177] The antibody administered in step (b) is 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, av 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 factors alpha and beta, interferons alpha, beta and gamma, tumor growth factor beta (TGF-β), colony stimulating factor (CSF) and granulocyte monocyte colony stimulating factor (GMCSF). See Human Cytokines: Handbook for Basic & Clinical Research (Aggrawal et al., ed., 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 US4,366,241.

[0178] The antibody can be directly or indirectly attached to the cytotoxic moiety or to a detectable label. The antibody 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 the antibody 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, the antibody 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 inhalation.

[0179] 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.

[0180] 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.

[0181] 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 dosage can be proportionally reduced or increased as indicated by the urgency of the treatment situation, as long as the interval required between step (a) and step (b) is not exceeded. It is particularly advantageous to prepare parenteral compositions in a dosage unit form for the administration and uniformity of the dosage. The dosage unit form used herein refers to a physically discrete unit suitable as a unit dose for a subject to be treated; each unit contains a predetermined amount of active compound calculated to produce a desired therapeutic effect associated with a desired pharmaceutical carrier.

[0182] 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.

[0183] The antibody can be Abagovomab, Abciximab, Actoxumab, Adalimumab, Adecatumumab, Afelimomab, Afutuzumab, Alacizumab pegol, ALD518, Alemtuzumab, Alirocumab, Altumomab pentetate, Amatuximab, Anatumomab mafenatox、Anrukinzumab、Apolizumab、Arcitumomab、Aselizumab、Atinumab、Atlizumab(=tocilizumab)、Atorolimumab、Bapineuzumab、Basiliximab、Batiximab 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 (chimeric human / murine anti-ganglioside G(D2) monoclonal antibody), Citatuzumab bogatox, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumab Tetraxetan, 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, Enlimomab pegol, Enokizumab, Enoticumab, Ensituximab, Epitumomabcituxetan), Epratuzumab, Erlizumab, Ertumaxomab, Etaracizumab, Etrolizumab, Evolocumab, Exbivirumab, Fanolesomab, Faralimomab, Farletuzumab, Fasinumab, FBTA05, Felvizumab, Fezakinumab, Fencla Ficlatuzumab, Figitumumab, Flanvotumab, Fontolizumab, Foralumab, Foravirumab, Fresolimumab, Fulranumab, Futuximab, Galiximab, Ganitumab, Gantenerumab, Gavilimomab, Gemtuzumab Ozomicin ozogamicin), Gevokizumab, Girentuximab, Glembatumumabvedotin, Golimumab, Gomiliximab, GS6624, Ibalizumab, Ibritumomab tiuxetan, 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, Rovotuzumab Mertan Lorvotuzumabmertansine, Lucatumumab, Lumiliximab, Mapatumumab, Maslimomab, Mavrilimumab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mitumomab, Mogamulizumab, Morolimumab, Motavizumab, Moxetumomab pasudotox), Muromonab-CD3, Nacolomab tafenatox, Namilumab, Naptumomabestafenatox, Namatumab, 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, Polatuzimab 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.

[0184] Preferred antibodies include natalizumab, vedolizumab, belimumab, atacicept, alefacept, otelixizumab, teplizumab, rituximab, oratumumab, ocrelizumab, epratuzumab, alemtuzumab, abatacept, Abatacept, Eculizamab, 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.

[0185] The treatment for which the benefit is improved is typically 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 to human major histocompatibility (MHC) antigens, also known as human leukocyte antigens (HLA). Anti-HLA antibodies are derived from heterologous sensitized B cells and are typically present in patients who have been previously sensitized by transfusion, previous transplant or pregnancy (Jordan et al., 2003).

[0186] 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 typically performed as a final check immediately prior to a transplant.

[0187] Due to the risk of acute antibody-mediated rejection, the presence of high titer antibodies (i.e., donor-specific antibodies (DSA)) to the MHC antigens of potential donors is a direct contraindication to transplantation. In short, sensitization to the donor MHC antigens hinders the identification of suitable donors. Positive cross-matching tests are a clear obstacle to transplantation. Since approximately one-third of patients waiting for kidney transplants are sensitized, and up to 15% of patients are highly sensitized, this leads to the accumulation of patients waiting for transplantation. In the United States, the median time on the kidney transplant waiting list during 2001-2002 was 1329 days for those with a group 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 more (OPTN database, 2011).

[0188] 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 (Oio et al., 1997).

[0189] 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.

[0190] 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:

[0191] - the amount of the polypeptide administered is sufficient to cleave substantially all IgG molecules present in the plasma of the subject; and

[0192] - Steps (a) and (b) are separated by a time interval of at least 2 hours and at most 21 days.

[0193] In other words, this embodiment 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 2 hours 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 subject's plasma. 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.

[0194] Example

[0195] 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.

[0196] Example 1 - Design, production and purification of polypeptides

[0197] Mature IdeS molecules were analyzed and regions suitable for mutation were identified. In some cases, in silico assessment was used to estimate the likely outcome of the mutation. It was determined that on the sequence of each polypeptide, the cDNA encoding each polypeptide was generated by site-directed mutagenesis of the starting sequence or by synthesis at GeneCust, Luxembourg, according to the number of mutations introduced. The cDNA was sequenced and transferred in-frame to a pET9a expression vector (Novagene) with a C-terminal 6× histidine tag connected to the C-terminus via a short glycine linker (3× glycine). An N-terminal methionine was added to improve bacterial expression. The plasmids were transformed (heat shocked) into E. coli BL21 (DE3) (Stratagene) and inoculated on LB agarose plates containing 30 μg / ml kanamycin. Single colonies were picked and overnight cultures (3 ml LB-medium) were started at 37°C, 250rpm. The next day, a glycerol stock was prepared and 10 ml TB-medium supplemented with 30 μg / ml kanamycin and antifoam was inoculated with an overnight culture and grown to OD 0.6-0.8 (37°C, 300 rpm). At this point, IPTG (1 mM) was added and culture was continued for 1 hour before the bacteria were harvested by centrifugation. The precipitate was washed in PBS and frozen at -20°C. A freeze-thaw protocol for bacterial lysis (three freeze / thaw cycles in 1 ml PBS each time) was used and the protein was purified using a Ni-NTA pre-packed spin column (Pierce). After purification, the eluted protein was activated with 10 mM DTT and then the buffer was changed (3 volumes of PBS in a MWCO 9K Millipore cfg design). 12% of the uncontaminated SDS-PAGE was used. TGX TM Precast gels (Biorad) SDS-PAGE were used to evaluate the purity and stability of each protein.

[0198] The following table summarizes the changes of each tested polypeptide relative to mature IdeS, excluding the additional N-terminal methionine and histidine tag. Thus, the sequence of each polypeptide used in the experiments described herein generally comprises the sequence of the SEQ ID NO indicated in the table, plus an additional N-terminal methionine and a histidine tag connected to the C-terminus via a short glycine linker.

[0199]

[0200]

[0201] As a control, a variant of IdeS was produced using the same method as described above. This variant of IdeS comprises the sequence SEQ ID NO: 2 plus an additional N-terminal methionine and a histidine tag connected to the C-terminus via a short glycine linker. This variant of IdeS may be referred to herein as pCART124. The sequence of pCART124 is provided below:

[0202]

[0203] IdeS lacking the tag was also produced independently using automated multi-step chromatography purification to GMP standards for use as an additional control. This polypeptide is referred to herein as BX1001865.

[0204] Provided below is the cDNA sequence for producing each polypeptide tested and pCART124.Each cDNA sequence includes a codon (ATG) for N-terminal methionine at the 3' end and a codon for a glycine linker and a histidine tag before the stop codon (TAA) at the 5' end.

[0205] pCART124(IdeS; SEQ ID NO: 20)

[0206]

[0207] pCART152 (SEQ ID NO: 21)

[0208]

[0209] pCART183 (SEQ ID NO: 22)

[0210]

[0211]

[0212] pCART|84 (SEQ ID NO: 23)

[0213]

[0214] pCART185 (SEQ ID NO: 24)

[0215]

[0216] pCART186(SEQ ID NO:25)

[0217]

[0218]

[0219] pCART187(SEQ ID NO:26)

[0220]

[0221] pCART188(SEQ ID NO:27)

[0222]

[0223] pCART189(SEQ ID NO:28)

[0224]

[0225]

[0226] pCART190(SEQ ID NO:29)

[0227]

[0228] pCART209(SEQ ID NO:30)

[0229]

[0230] pCART125(SEQ ID NO:31)

[0231]

[0232]

[0233] pCART213(SEQ ID NO:32)

[0234]

[0235] pCART214 (SEQ ID NO: 33)

[0236]

[0237] pCART228 (SEQ ID NO: 34)

[0238]

[0239]

[0240] Example 2 - Evaluation of potency (IgG cleavage potency)

[0241] ELISA

[0242] Enzyme activity is measured using an ELISA-based titer assay. The principle of ELISA is to coat the wells of a multi titer plate with an antibody target (BSA), then incubate different concentrations of IgG cysteine ​​protease polypeptides (test or control) with anti-BSA antibodies in the wells, and then use a detection antibody to detect the number of anti-BSA antibodies bound to the wells. The higher the concentration of a given IgG cysteine ​​protease polypeptide in the well, the fewer intact anti-BSA polypeptides will be bound to the wells, giving a lower signal. Similarly, when present at the same concentration, a more titer IgG cysteine ​​protease polypeptide will produce a lower signal than a less titer IgG cysteine ​​protease polypeptide.

[0243] The reference IdeS BX1001865 was prepared as a titration series from 320 nM down to 0.16 nM in 1:2 dilution steps to allow the drawing of a standard calibration curve for the assay. The results achieved in the assay for various known concentrations of each test polypeptide were compared against the linear portion of the calibration curve to determine the concentration of the reference IdeS that achieved the same potency. The known concentration of each polypeptide was divided by the determined equivalent concentration of the reference IdeS from the curve, resulting in a score as the fold change in potency relative to the reference IdeS BX1001865. For example, if 5 nM of the test polypeptide achieves a result equivalent to 10 nM of the reference IdeS on the calibration curve, the test polypeptide has a potency 2 times greater than the reference IdeS BX1001865. The average score for the fold change in potency relative to the reference IdeS BX1001865 was calculated from all scores obtained with each test polypeptide at different concentrations, as long as they fell within the linear portion of the calibration curve. The average score is then compared to the average score obtained for the pCART124 reference IdeS, which is included on each plate to allow comparison between plates. The average score of pCART124 is divided by the average score of the test polypeptide to produce a "pCART124 ratio", which effectively becomes the fold change in the potency of each polypeptide relative to IdeS. The pCART124 ratio can then be visualized on a bar graph.

[0244] Briefly summarize the laboratory protocol: The wells of the multi titer plate were coated with BSA (10 μg / ml) overnight, then washed with PBS-T and blocked with 2% fish skin gelatin in PBS for 1 hour. The IdeS BX1001865 polypeptide was prepared in PBS with 0.1% gelatin in a 1:2 dilution step as a titration series from 320nM to 0.16nM. Then, in PBS with 0.1% gelatin, each of the test polypeptides and pCART124 controls at 15, 7.5, 3.75 and 1.9nM was prepared. 50μl of polypeptide sample and 50μl of rabbit anti-BSA (ACRIS, #R1048P, 10nM) as a substrate were added to each well. The plate was incubated at room temperature for 1 hour and then washed with PBS-T. Biotinylated goat anti-rabbit Fc specific antibody (30000 times diluted) was added as a detection antibody and incubated for 30 minutes. The plate was washed, 40000 times diluted SA-horseradish peroxidase (HRP; Pierce) was added and incubated for 30 minutes. The plate was washed and developed for 7 minutes using TMB One Component as a chromogenic substrate for HRP, and terminated with 0.5M H2SO4. The absorbance (OD) was measured at λ=450nm. The average score of the multiple change relative to the potency of BX1001865 was determined for each test polypeptide and for pCART124. The "pCART124 ratio" for each test polypeptide was then calculated as described above.

[0245] The "pCART124 ratio" results for pCART152, 183, 184, 185, 186, 187, 189, and 190 are shown together with the results for pCART124. Figure 1 All exemplary polypeptides of the invention shown here achieve at least a 1.5-fold increase in titer relative to the IdeS control (pCART124). pCART152, 1S3, 184, 188, 189, and 190 all achieve much higher titers, even exceeding the pCART189 control by up to 8.0 times. Each of the six tested polypeptides includes N130R / K modification. The pCART125 polypeptide was tested alone and obtained a titer comparable to that of IdeS (data not shown).

[0246] Visualization of IgG cleavage patterns

[0247] By showing the cleavage products produced by the titration series of each polypeptide in different substrates on SDS-PAGE, the effectiveness of different pCART polypeptides was further evaluated. In order to test the effectiveness in pure IgG substrates, adalimumab (Humira) is used for IgG1 and denosumab (XGEVA) is used for IgG2. In order to test the effectiveness in a more complex physiological environment, some polypeptides are also titrated in IVIg (Octagam). This allows the evaluation of the effect of neutralizing anti-IdeS antibodies on polypeptide activity. The cleavage pattern of each polypeptide is compared with the cleavage pattern of IdeS (BX1001865 and pCART124) in the same substrate. The scheme is as follows:

[0248] For pure IgG testing, each test polypeptide or control was diluted in a 1:3 step titration series (from 6.7 μg / ml down to 0.04 ng / ml) in PBS with 0.05% BSA as a supporting protein. 25 μl of each concentration was transferred to a multi-titer plate and the cleavage reaction was started by adding 25 μl of Humira or XGEVA (2 mg / ml). Therefore, each starting concentration of the polypeptide was diluted 1:2 in the well to obtain a titration series from 3.3 μg / ml down to 0.02 ng / ml.

[0249] For IVIg testing, each test polypeptide or control was diluted in a 1:2 step titration series (from 30 μg / ml down to 0.015 ng / ml) in PBS with 0.05% BSA as a supporting protein. 25 μl of each concentration was transferred to a multi-titer plate and the cleavage reaction was started by adding 25 μl of 10 mg / ml of IVIg. Therefore, each starting concentration of the polypeptide was diluted 1:2 in the well to obtain a titration series from 15 μg / ml down to 0.0075 ng / ml.

[0250] The plate was incubated at 37°C for 1.5 hours. The samples were mixed 1:4 in 2× SDS loading buffer and heated at 92°C for 5 minutes. 10 μl was loaded on a polyacrylamide gel (15-well 4-20% Mini- TGX TM Precast gels (Biorad) were loaded and read according to standard protocols.

[0251] Figure 2 The cleavage pattern produced by using IgG1 (adalimumab) substrate is shown for pCART183, 184, 185, 186, 187, 189 and 190 compared to IdeS (pCART|24). The polypeptide concentration was reduced from 3.33 μg / ml (lane 1) to 0.02 ng / ml (lane 12) in a 1:3 step dilution series. The complete adalimumab (no enzyme) is shown to the left (lane C).

[0252] The arrows on the right represent the different cleavage products from IgG. Arrow 1: intact IgG; Arrow 2: scIgG (single cleavage IgG - produced by cleavage of the first IgG heavy chain); Arrow 3: F(ab')2 fragment (produced by cleavage of the second IgG heavy chain). Vertical lines were added to facilitate comparison at the first IgG heavy chain cleavage and at the second IgG heavy chain cleavage, where intact IgG becomes scIgG at the first IgG heavy chain cleavage (between lanes 6 and 7) and where scIgG becomes F(ab')2 fragment at the second IgG heavy chain cleavage (between lanes 2 and 3).

[0253] Compared to pCART124 at the same concentration, pCART183, 184, 188, 189, and 190 in particular all showed increased cleavage efficiency of the second heavy chain, resulting in a more intense F(ab')2 (arrow 3) band and a less intense scIgG band (arrow 2). The difference can be seen from the 0.12 μg / ml concentration (lane 4), but is more obvious at 0.37 μg / ml (lane 3). Therefore, overall Figure 2 It was shown that changing to a positive amino acid at position 130 (N130R / K) increased the cleavage efficiency of the second IgG heavy chain (pCART183, 184, 188, 189 and 190).

[0254] Figure 3 The cleavage patterns produced with IgG1 substrates are shown for pCART152 and pCART209 compared to two IdeS controls (pCART124 and BX1001865). The enzyme concentration was reduced from 3.33 μg / ml (lane 1) to 0.02 ng / ml (lane 12) in a 1:3 step dilution series. The arrows on the right represent different cleavage products from IgG. Arrow 1: complete IgG; Arrow 2: scIgG (single-cleaved IgG-produced by cleavage of the first IgG heavy chain); Arrow 3: F(ab')2 fragment (produced by cleavage of the second IgG heavy chain); Arrow 4: Fc fragment; (*) represents carrier protein (BSA) in titration buffer. Vertical lines are added to facilitate comparison at the first IgG heavy chain cleavage and at the second IgG heavy chain cleavage, where the complete IgG becomes scIgG at the first IgG heavy chain cleavage (between lanes 6 and 7) and where scIgG becomes F(ab')2 fragment at the second IgG heavy chain cleavage (between lanes 2 and 3).

[0255] Compared with pCART124 and BX1001865 (arrows 3 and 2) at the same concentration, pCART152 and pCART209 both show an improved cleavage efficiency of the second heavy chain, resulting in a stronger F (ab ') 2 band (arrow 3) and less scIgG residue (arrow 2). The difference can be seen from the enzyme concentration of 41ng / ml (lane 5), but it is even more obvious at 0.12μg / ml and 0.37μg / ml (lanes 4 and 3). Both pCART152 and pCART209 show that the cleavage efficiency of the second heavy chain is about 3 times that of the original IdeS (BX1001865 and pCART124), that is, 0.37μg / ml of pCART152 and pCART209 (lane 3) produce similar results to 1.11μg / ml of BX100186 and pCART124 (lane 2).

[0256] Compared with the original IdeS (BX1001865 and pCART124) at the same concentration, pCART152 and pCART209 also show improved efficacy in the cleavage of the first IgG heavy chain, resulting in a more intense scIgG band (arrow 2) and a less intense IgG band (arrow 1). For pCART152, this can be observed at 1.5ng / ml of enzyme (lane 8), but even more obvious at 4.6ng / ml (lane 7). pCART209 also shows that the improved cleavage efficacy for the first heavy chain cleavage is about 3 times that of the original IdeS (BX1001865 and pCART124), i.e., 4.6ng / ml (lane 7) of pCART209 produces similar results to 14ng / ml (lane 6) of BX1001865 and pCART124.

[0257] therefore, Figure 3 It was shown that a single amino acid substitution at position 130 (N130K in pCART152) increased the cleavage efficacy of the major second IgG1 heavy chain, but also increased the cleavage efficacy of the first IgG1 heavy chain to some extent, and the deletion of the NQTN sequence (pCART209) increased the cleavage efficacy of the first and second IgG1 heavy chains by 3-fold.

[0258] Figure 4The cleavage patterns produced with IgG2 substrates are shown for pCART152 and pCART209 compared to two IdeS controls (pCART124 and BX1001865). The enzyme concentration was reduced from 3.33 μg / ml (lane 1) to 0.02 ng / ml (lane 12) in a 1:3 step dilution series. The arrows on the right represent different cleavage products from IgG. Arrow 1: complete IgG; Arrow 2: scIgG (single-cleaved IgG-produced by cleavage of the first IgG heavy chain); Arrow 3: F(ab')2 fragment (produced by cleavage of the second IgG heavy chain); Arrow 4: Fc fragment; (*) represents carrier protein (BSA) in titration buffer. Vertical lines are added to facilitate comparison at the first IgG heavy chain cleavage and at the second IgG heavy chain cleavage, where the complete IgG becomes scIgG at the first IgG heavy chain cleavage (between lanes 6 and 7) and where scIgG becomes F(ab')2 fragment at the second IgG heavy chain cleavage (between lanes 2 and 3).

[0259] Compared with pCART124 and BX1001865 (arrows 3 and 2) at the same concentration, pCART152 and pCART209 both showed improved cleavage efficiency to the second heavy chain, resulting in a more intense F(ab')2 band (arrow 3) and a less intense scIgG (arrow 2). The difference can be seen from the enzyme concentration of 0.37 μg / ml (lane 3), but it is even more obvious at 1.11 μg / ml (lane 2).

[0260] Compared with BX1001865 and pCART124, the results of pCART209 in lane 6 (enzyme concentration 14ng / ml) show that pCART209 is also more effective in cutting the first IgG heavy chain. Since most of the protein has been converted to scIgG, it leads to a less intense complete IgG band (arrow 1) and a more intense scIgG band (arrow 2).

[0261] therefore, Figure 4 A single amino acid substitution at position 130 (N130K in pCART152) was shown to increase the cleavage efficacy of the predominant second IgG2 heavy chain, and the deletion of the NQTN sequence (pCART209) was shown to increase the cleavage efficacy of both the first and second IgG2 heavy chains.

[0262] In general, it can be seen that the tested polypeptides are generally more effective than IdeS in cleaving IgG1 and IgG2. Consistent with the ELISA results, pCART152, 183, 184, 188, 189 and 190 appear to be particularly effective. The increased efficacy of these polypeptides appears to be primarily related to the cleavage of the second Ig6 heavy chain.

[0263] Changing from G to a positively charged amino acid at position 131 is expected to produce similar results as the N130R / K substitution. Positions 130 and 131 are located in the loop of the beta hairpin structure spanning positions 126 to 136 of SEQ ID NO: 1. Based on the results obtained herein, changing to a positively charged amino acid in either or both positions 130 and 131 is expected to increase IgG cysteine ​​protease activity.

[0264] Example 3 - Evaluation of Immunogenicity

[0265] Competitive ADA assay

[0266] The assay is based on competition between the test polypeptide and IdeS for binding to anti-IdeS antibodies. Pre-incubation of the test enzyme and IVIg will enable the anti-IdeS antibody to bind to the pCART enzyme tested. Thereafter, the IVIg-enzyme mixture is added to the IdeS-coated plate, and any anti-IdeS antibody that does not bind to the test polypeptide will bind to the IdeS on the plate instead. All binding incubations are carried out in the presence of 2mM iodoacetic acid (IHAc) that inhibits IgG cleavage and in high salts that allow only high-affinity binding to occur. After washing, biotinylated goat anti-human F(ab')2-specific F(ab')2 fragments are used as detectors. Poor recognition of the test polypeptide by the anti-IdeS antibody in IVIg will result in a high degree of binding of the anti-IdeS antibody in IVIg to the plate, producing a high signal. Good recognition of the test polypeptide by the anti-IdeS antibody in IVIg will produce the opposite result. The detailed scheme is as follows:

[0267] The reference IdeS (BX1001865) was coated overnight on a multi-titer plate (5 μg / ml), then washed with PBS-T and blocked for 1 hour with 2% BSA in PBS supplemented with 2 mM IHAc and 1 M NaCl. Mixed plates were prepared with stepwise dilutions of the test polypeptide and 20 μg / ml IVIg in PBS supplemented with 0.1% BSA, 2 mM IHAc and 1 M NaCl. The mixed plates were incubated on a shaker at room temperature for 1 hour. After incubation, the blocking solution was discarded from the IdeS-coated plate, and 50 μl of each mixture from the mixed plate was transferred to the wells of the coated plate. After incubation on a shaker at room temperature for 1 hour, the plate was washed with PBS-T and the detection substance, i.e., biotinylated goat anti-human F(ab′)2-specific F(ab′)2 fragment (20,000-fold diluted), was added. After incubation for 30 minutes, the plate was washed, and 40,000 times diluted SA-HRP (Pierce) was added and incubated for 30 minutes. The plate was washed and developed for 7 minutes using TMB One Component as a chromogenic substrate for HRP, and terminated with 0.5M H2SO4. The absorbance (OD) was measured at λ = 450nm. The results were reciprocated (1 / OD value) and expressed as a ratio (1 / (test polypeptide / pCART124)) compared to pCART124 for visualization in a bar graph.

[0268] The results for pCART183, 184, 185, 186, 187, 189, and 190 are shown in Figure 5 The results of pCART125 are shown in Figure 6 Compared to IdeS, all tested polypeptides showed some reduction in recognition of anti-IdeS antibodies, usually at least 10%. pCART185, 186, and 187 in particular showed a greater reduction in recognition, about 40% reduction relative to IdeS. pCART125 showed about 60% reduction.

[0269] summary

[0270] All tested polypeptides showed increased potency and / or decreased immunogenicity compared to IdeS.

[0271] Example 4 - Evaluation of potency

[0272] Titer ELISA

[0273] In order to address the cleavage ability of human IgG1 and IgG2, two ELISA-based titer assays were established. One assay measures IgG1 cleavage and the other measures IgG2 cleavage. EC50 (half maximum effective concentration) values ​​were calculated for the different IgG cysteine ​​protease polypeptides tested. The principle of these assays is to coat the wells of multiple titer plates with F(ab)2 fragments, which are directed to human IgG antibodies with specificity for the Fab region. Then the titrated concentration of IgG cysteine ​​protease polypeptides (test or control) is incubated together with human IgG1 antibodies (Xiu Mei Le) or human IgG2 antibodies (XGEVA) in the wells. The amount of complete or single-cut human IgG (Xiu Mei Le or XGEVA) bound to the wells is measured using a detection antibody for human IgG with specificity for the Fc portion of the antibody. The higher the concentration of a given IgG cysteine ​​protease polypeptide in the wells, the less complete human IgG antibodies will bind to the wells, thereby obtaining a lower signal. Similarly, when there is the same concentration, the IgG cysteine ​​protease polypeptide with higher titer will give a lower signal than the IgG cysteine ​​protease polypeptide with lower titer. In IgG1 (Ximei Le) and IgG2 (XGEVA) assays, titration dose-response curves were prepared for IdeS controls (pCART124) and all tested IgG cysteine ​​protease polypeptides. The EC50 value of each test variant was also calculated, representing the concentration at which 50% of the maximum effect of the polypeptide on the second cutting of the IgG molecule, i.e., half of the IgG was completely cut. Lower EC50 values ​​represent more effective IgG cysteine ​​proteases. Since the Fc portion of IgG is still present and can be detected by Fc-specific detection antibodies, the cutting of the first IgG heavy chain (i.e., IgG to scIgG) is invisible in this assay ( Fig.13 ).

[0274] Brief summary of laboratory protocol: The wells of the multi-titer plate 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 phosphate buffered saline-Tween (PBS-T) (blocking buffer) at room temperature for 45-120 minutes. The control IdeS (pCART124) and the IgG cysteine ​​protease polypeptide to be tested were prepared as a titration series with a starting concentration of 80 μg / ml in blocking buffer in a 1:4 dilution step. An equal volume (25 μl) of human IgG1 (Humira) with a concentration of 0.5 μg / ml and a titration amount of IgG cysteine ​​protease polypeptide were added to the wells and incubated in a controlled temperature environment at 37°C for 2 hours with shaking, then washed with PBS-T. Biotinylated mouse anti-human IgG Fc specific (ma-hIgGBioII, Lot: C0013-ZC43C, Southern Biotech) (600ng / ml) antibody was mixed with Strep-sulfo (200ng / 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 150μl of 2-fold 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 (plate reader, microplate reader, Plate reader) i.e. MSD (Meso Scale Discovery) QuickPlex SQ 120Model 1300.

[0275] Potency assay visualized on gel: Assays were performed as described in Example 2 for cleavage of IgG1 (Humira), IgG2 (XGEVA) and cleavage of human IgG pool IVIg (Octagam).

[0276] result

[0277] Titer ELISA

[0278] The dose-response curves obtained in the potency assay for the tested IgG cysteine ​​proteases are shown in Figure 7 (IgG1 cleavage) and Figure 8 Compared to the IdeS control pCART124, pCART125, 213, and 214 in the exemplary polypeptides of the invention tested here were significantly more efficient in cleaving IgG1 ( Figure 7 ) and IgG2( Figure 8) had increased titers on the heavy chain of both pCART125 and pCART214, with reduced EC50 values ​​(Table 1), while having fold increases in titers on cleaved IgG1, 1.5-fold for pCART125, 2.2-fold for pCART214, and up to 3.0-fold for pCART213. Figure 8 ), the fold increase compared to pCART124(IdeS) was 1.6-fold for pCART125, 2.1-fold for pCART214, and 3.5-fold for pCART213.

[0279] Potency assay visualized on gel

[0280] IgG1 ( Fig. 9 A) and IgG2( Fig. 9 B) cleavage clearly shows the first and second heavy chain cleavage (the vertical lines in the figure mark the first and second IgG heavy chain cleavages cut by BX1001865 and pCART124). The * in the figure represents the approximate EC50 value, i.e., the concentration at which 50% of the IgG is single-cut (scIgG) and 50% is completely cut (F(ab')2). The data from the gel are summarized in Table 2 (IgG1 cleavage) and Table 3 (IgG2 cleavage). The concentration of IgG cysteine ​​protease required for cutting the first heavy chain of IgG1 (Humira) is about the same as for all tested polypeptides, 1.5ng / ml for BX1001865 and pCART124 (IdeS control), pCART125, 213 and 214 ( Fig. 9 A). For cleavage of the second heavy chain of IgG1IdeS, approximately 120 ng / ml of pCART125 and 214 were required to obtain a significant F(ab′)2 band on the gel, and approximately one titration step less, 40 ng / ml, for pCART213 ( Fig. 9 A). Fig. 9 The visible IgG2 (XGEVA) cleavage in B shows that all tested polypeptides BX1001865, pCART124, 125, 213 and 214 show roughly the same potency, where 14 ng / ml is required to cut the first IgG heavy chain, and about 370 ng / ml is required to produce scIg62 to produce F(ab')2 fragments (Table 3). In another set of efficacy experiments, BX1001865 and pCART124 were used as IdeS controls, and pCART228 ( Fig.10 A and Fig.10 B, Table 4). In these experiments, Fig. 9Compared to Table 3, the controls (BX1001865 and pCART124) require concentrations approximately one titration step higher, and small differences between experiments can be observed due to laboratory precision in sample handling and sample titration dilutions. In order to obtain a reliable comparison, the cleavage of pCART228 must be compared to the cleavage of the IdeS control in the same experiment. Very similar IgG cleavage patterns are seen for pCART228 and IdeS controls on IgG1 and IgG2 cleavage ( Fig.10 A and Fig.10 B), but pCART228 has about 3 times more efficient cleavage of the second Ig6 heavy chain of IgG1 (one titration step) ( Fig.10 A), where pCART228 required 120 ng / ml compared to 370 ng / ml required by IdeS (Table 4).

[0281] The IgG cysteine ​​protease peptides pCART125, 213 and 214 were also titrated in the human IgG library IVIg (Octagam) with IdeS (BX1001865 and pCART124) as a control ( Fig.11 All tested polypeptides required 1.5 μg / ml for the first IgG heavy chain cleavage and about 6 μg / ml for the second IgG heavy chain cleavage ( Fig.11 and Table 5). Fig.11 A broader titration profile (with a 1:2 dilution starting at 30 μg / ml) was used to analyze the cleavage of IVIg by pCART228 compared to the variants tested in Fig.12 ). The same potency was seen for IdeS (BX1001865 and pCART124) and pCART228 ( Fig.12 ), with a concentration of 1.9 μg / ml for scIgG and 7.5 μg / ml for F(ab′)2 fragments (Table 6), which represents the same titration steps for pCART125, 213 and 214 ( Fig.11 and Table 5).

[0282] Summary of the Figures of Example 4

[0283] Figure 7 . Titration curves of cleavage of IgG1 (Humira) by different IgG cysteine ​​protease peptides.

[0284] Figure 8 Titration curves of Ig62 (X6EVA) cleavage by different Ig6 cysteine ​​protease peptides.

[0285] Fig. 9.IgG cleavage analyzed by SDS-PAGE using titrated (1:3 dilution starting from 3300nng / ml) amounts of pCART125, 213 and 214 with BX1001865 and pCART124 (original IdeS) as controls in the same cleavage experiment. A: Cleavage of Humira (IgG1) and B: Cleavage of XGEVA (denosumab) (IgG2). The vertical lines mark the concentrations of IdeS (BX1001865 and pCART124) required to cause cleavage of the first and second IgG heavy chains (where the amount of cleavage products exceeds that of uncut products). The * in the figure represents the approximate EC50 value in this experiment.

[0286] Fig.10 IgG cleavage analyzed by SDS-PAGE using titrated (1:3 dilution starting from 3300 ng / ml) amounts of pCART228 with BX1001865 and pCART124 (original IdeS) as controls in the same cleavage experiment. A: cleavage of Humira (IgG1) and B: cleavage of XGEVA (denosumab) (IgG2). The vertical lines mark the concentration of IdeS (BX1001865 and pCART124) required to cause cleavage of the first and second IgG heavy chains (where the amount of cleaved product exceeds that of uncleaved product to dominate).

[0287] Fig.11 IVIg cleavage analyzed by SDS-PAGE using titrated (1:2 dilution starting from 6 μg / ml) amounts of the tested IgG cysteine ​​protease polypeptides and IdeS (BX1001865 and pCART124) as controls in the same cleavage experiment.

[0288] Fig.12 IVIg cleavage analyzed by SDS-PAGE using titrated amounts (1:2 dilution starting from 30 μg / ml) of pCART228 and IdeS (BX|001865 and pCART124) as controls in the same cleavage experiment.

[0289] Fig.13 Schematic diagram of immunoglobulin cleavage by polypeptides of the present invention. Enzymatic cleavage of IgG is performed in two steps. First, one heavy chain of the complete IgG is cleaved and a single-cleaved IgG (scIgG) is produced. Secondly, the next IgG heavy chain is cleaved and the Fc-part is released. The Fc part is still attached to the Fab part in the scIgG molecule, and since the detection antibody in the titer ELISA recognizes the Fc part of the IgG molecule, the assay will not distinguish between complete IgG and scIgG.

[0290] Discussion and Conclusion

[0291] The lower EC50 values ​​of pCART125, 213 and 214 in the titer ELISA indicate that these polypeptides have an increased titer in the second heavy chain cleavage (from scIgG to F(ab')2) of both IgG1 and IgG2 compared to pCART124 (original IdeS). The IgG cleavage visualized on the gel shows the cleavage of the first heavy chain (from IgG to scIgG) that cannot be measured in the titer ELISA using the Fc-specific detection antibody. Most of the Fc-mediated effects of IgG are lost in single-cut molecules (data not shown), which is important in clinical situations where the main focus is to disable pathogenic IgG molecules. pCART228 is as effective as the original IdeS in both IgG1 and IgG2 cleavage. This is important when an IgG cysteine ​​protease of the same molecular size as IdeS is required to have an ideal lower recognition of IdeS-specific antibodies. IVIg is a human IgG library containing about 65-70% IgG1, 35-30% IgG2 and about 1% IgG3 / IgG4. Human IVIg also naturally contains anti-IdeS antibodies from IgG donors' earlier exposure to S. pyogenes. Anti-IdeS antibodies in human IVIg are neutralized whenever binding to IdeS may attenuate or completely destroy the IgG protease activity of IdeS. The results of IVIg cleavage showed overall cleavage of all different IgG subclasses with a predominance of IgG1 and IgG2 and the presence of neutralizing anti-IdeS antibodies. In general, all IgG cysteine ​​protease polypeptides tested had lower potency on IgG2 cleavage compared to IgG1.

[0292] Table 1 EC50 (ng / ml) measured by potency ELISA and fold difference in potency compared to original IdeS (pCART124).

[0293]

[0294]

[0295] Table 2 Data of IgG1 (Humira) cleavage shown on gel ( Fig. 9 A). The concentration of polypeptides required to achieve cleavage of the first and second IgGs (ng / ml), wherein the cleaved product predominates in amount over the uncleaved product. Approximate EC50 values ​​(in Fig. 9 A).

[0296]

[0297] Table 3 Data of IgG2 (X6EVA) cleavage shown on gel ( Fig. 9B). The concentration of polypeptides required to achieve cleavage of the first and second IgGs (ng / ml), wherein the cleaved product predominates in amount over the uncleaved product. Approximate EC50 values ​​(in Fig. 9 (* in B).

[0298]

[0299] Table 4. Data of pCART228 cleavage of IgG1 (Humira) and IgG2 (XGEVA) shown on the gel ( Fig.10 ). The concentration of polypeptides (ng / ml) required to achieve cleavage of the first and second IgGs (wherein the cleaved product predominates in amount over the uncleaved product).

[0300]

[0301] Table 5. Data of IVIg cleavage by pCART125, 213 and 214 shown on gel ( Fig.11 ). The concentration of polypeptides (ng / ml) required to achieve cleavage of the first and second IgGs, wherein the cleaved product predominates over the uncleaved product.

[0302]

[0303] Table 6. Data of IVIg cleavage by pCART228 shown on gel ( Fig.12 ). The concentration of polypeptide (ng / ml) required to achieve cleavage of the first and second IgG heavy chains, wherein the cleaved product predominates over the uncleaved product.

[0304]

[0305] Example 5 - ADA ELISA, ie, competition ELISA for ADA-IdeS binding site.

[0306] Using ELISA, Meso Scale Discovery (MSD) based assay, the anti-drug antibody (ADA) binding site for IdeS was measured for the "ADA" modified polypeptides (pCART125, 213 and 214) of the present invention. The principle of ELISA is to coat the wells of the multi-titer plate with the original histidine-tagged IdeS (pCART124). Due to early group A streptococcal infection, most people have antibodies against IdeS in their serum. Two different clinical human serum libraries are used here as standards for ADA detection. The first library is normal human serum from 100 individuals, referred to as human serum library 1191807, and the second library is a serum library from patients in Phase II study 13-HMedIdeS-02, referred to as Phase II library-2. These patients have been administered IdeS once with a dosage range of 0.24-0.5 mg / kg body weight, and thus have an induced level (about 50 times) of anti-IdeAADA in their serum.

[0307] The overview of the competitive ADA ELISA is that IdeS (pCART124) is coated at the bottom of a microtiter plate. The human serum pool is pre-incubated with the polypeptide of the present invention to be tested for ADA recognition or with the positive control IdeS (pCART124) at a molar ratio of 1: 100 (with a 100-fold excess of the polypeptide of the present invention). The concentrations of the two different serum pools used for pre-incubation are estimated from the standard curve to provide about 80% binding to the original IdeS. If the ADA binding site has been eliminated in the tested polypeptide, these variants cannot compete with the binding of ADA to the original IdeS at the bottom of the well, i.e., low signals show strong ADA similarity to the original IdeS (pCART124) and high signals show weak ADA similarity to the original IdeS.

[0308] The concentration of the two standards that achieve about 80% binding in the linear part of the standard curve is about 200ng ADA (IdeS) / ml. In the competitive pre-incubation, the concentrations of the two standards are used separately, and the concentration of the polypeptide of the present invention is used at a concentration of 100 times the concentration of ADA, including the molar weight difference between the antibody of 150kDa and the IdeS of about 35kDa, i.e. 4.2 times, providing 100 times 200ng / ml divided by 4.2, i.e. providing about 5μg / ml of test polypeptide. Standard serum containing 200ng / ml ADA was pre-incubated with IdeS (pCART124) or the polypeptide of the present invention at room temperature (RT) for 1 hour. As a control for maximum ADA binding, the same concentration of standard was pre-incubated in the absence of IdeS (pCART124) or the tested polypeptide and used as 80% binding-maximum. The lowest level of the standard curve is used as the lower limit of the competition calculation range. The average score of the standard pre-incubated with IdeS (pCART124) or the tested polypeptide minus 80% standard binding value divided by 80% standard binding value minus lower limit value provides % competition value. The tested polypeptide with the lowest % competition means that most ADA binding epitopes have been eliminated compared to the original IdeS (pCART124).

[0309] Brief summary of the laboratory protocol: wells of multititer plates were coated with pCART124 (1 μg / ml) overnight, washed three times with PBS-T, and blocked for 1 hour with 0.45% fish skin gelatin and 2 mM of the cysteine ​​protease inhibitor iodinated acetic acid (IHAc) in PBS.

[0310] Both standards were prepared in 0.45% fish skin gelatin and 2mM IHAc in PBS with 1:3 dilution steps as a titration series (from 5000ng ADA (IdeS) / mi to 2.5ng ADA (IdeS) / ml) to allow the standard calibration curve for the assay to be drawn and measured at the linear part of the standard curve as well as the maximum and minimum parts. While the plate was blocked, the standards were pre-incubated with IdeS (pCART124) or the tested polypeptide at room temperature (RT) for 1 hour, i.e., samples in the competition step using 200ng / ml of ADA (standard) and 5μg / ml of IdeS (pCART124) or the polypeptide to be tested.

[0311] The pCART124-coated plates were washed 3 times, and 50 μl of pre-incubated samples or 50 μl of standards were added to each well of the multititer plate.

[0312] The plate was incubated at room temperature (RT) for 2 hours and then washed with PBS-T. Goat anti-human F(ab) specific F(ab)2 fragment-bio (Jackson #109-066-097, 0.65 mg / ml) (1000-fold dilution) was added as the detection antibody and Streptavidin-Sulfo (MSD catalog number: R32AD-1 or R32AD-5) (2000-fold dilution) in blocking buffer was added and incubated in the dark for 1 hour at room temperature (RT). The plate was washed 3 times, 4-fold diluted Read buffer T (MSD Read buffer T (4x)) was added, and the plate was directly analyzed on the Plate Reader MSD (Meso Scale Discovery) QuickPlex SQ 120 Model 1300.

[0313] Results and Conclusions

[0314] Compared with the original IdeS (pCART124), all tested IgG cysteine ​​protease polypeptides pCART125, 213 and 214 are rarely recognized by IdeS-specific ADA in human serum. Some ADA recognition epitopes are found in the N-terminal part of IdeS. Compared with IdeS, all these polypeptides of the present invention are N-terminal deletions, and the only sequence difference between pCART125 and the original IdeS is the N-terminal deletion.

[0315] The percentage (%) of IdeS-ADA binding sites blocked for pCART125, 213, and 214 is shown in Fig.14 and Fig.15 , and the original IdeS pCART124 was used as a 100% similar positive control.

[0316] Example 6 - Evaluation of in vivo efficacy of Octagam (human IVIg) in a mouse model

[0317] In this study, BALB / c mice were injected intraperitoneally (ip) with human IVIg (Octagam). The concentration of human IVIg was administered at a dosage of 900mg / kg, related to human IgG plasma concentration (10mg / ml). Human IVIg was injected intraperitoneally (ip) on day 0. 24 hours (day 1) after human IVIg was injected, PBS, IdeS controls (BX1001865 and pCART124) or IgG cysteine ​​proteases to be tested pCART125, pCART213 and pCART214 were administered intravenously (iV) at a dosage of 1mg / kg. Two hours later, serum samples were collected and mice were killed.

[0318] Potency ELISA

[0319] The principle of the assay is to coat the wells of a multi-titer plate with F (ab') 2 fragments for specific human IgG antibodies to the Fab region. Then add the serum from mice treated with IVIg and IdeS controls (BX1001865 and pCART124) or tested IgG cysteine ​​protease polypeptides. The amount of complete or single-cut human IgG (IVIg) bound to the wells is measured using a specific detection antibody for the Fc portion of the antibody for human IgG (IVIg). The lower the concentration of the detected complete human IgG antibody (IVIg), the more effective the IgG cysteine ​​protease polypeptide is expected to be.

[0320] Brief summary of the laboratory protocol: Wells of multititer plates were coated overnight (+2-8°C) with goat anti-human Fab specific F(ab)2 fragments (0.5 μg / ml) (Jackson #109-006-097), then washed with PBS+0.05% Tween 20 (PBS-T) and blocked in 2% BSA in PBS-T (blocking buffer) for 45-120 minutes at RT (room temperature). Human serum protein calibrator (DAKO #X0908) was used as a standard and added in a range of 0.5-300 ng / ml. Serum samples taken from mice treated with IVIg and different IgG cysteine ​​protease polypeptides were thawed and diluted 100,000 times in blocking buffer and then added to the assay multititer plates. The plates were incubated with shaking for 2 hours at room temperature (RT) and then washed with PBS-T. Biotinylated mouse anti-human IgG Fc specific (600nng / ml) (Jackson #109-066-098) antibody was mixed with streptavidin-sulfo (200ng / ml) (MSD #R32AD-1) and added to a multi-titer plate. The plate was sealed with aluminum tape and incubated with shaking for 1 hour at room temperature (RT). The plate was then washed in PBS-T and 150 μl of a 2-fold dilution of reading buffer T (MSD #R92TC-2) was added to each well. The plate was directly analyzed on a plate reader MSD (Meso Scale Discovery) QuickPlex SQ120 Model 1300.

[0321] Efficacy visualized on gel

[0322] 10 μl of mouse serum was diluted 1:10 in 90 μl of PBS. Then 10 μl of diluted serum was mixed with 30 μl of 4x SDS-PAGE loading buffer. 5 μl of IgG in-house marker was used to visualize different IgG fragments (IgG, scIgG, and F(ab′)2). The samples were heated at 92°C for 3 min (ThermoMixer compact, eppendorf) and incubated at 4-20% TGX, Stain-freeTM gel (Cat. No. 456-8096, Biorad) was centrifuged briefly before loading 10 μl of the sample. The gel was run at 200 V for 40 minutes.

[0323] Results and Conclusions

[0324] The in vivo cleavage of human IVIg (Octagam) by IdeS control (BX1001865 and pCART124) and pCART125, 213 and 214 was compared by studying the levels of human IgG in serum by potency ELISA and by analyzing the degradation of IgG by SDS-PAGE. Treatment in mice with IdeS control (BX1001865 and pCART124) and different IgG cysteine ​​proteases pCART125, pCART213 and pCART214 showed a significant effect on the cleavage of human IgG in vivo (Table 7 and Figure 6 ).

[0325] For the IdeS controls (BX1001865 and pCART124), pCART125 and pCART213 showed complete cleavage with no visible scIgG band on the gel but a significant F(ab′)2 band ( Fig.17 pCART214 showed lower efficacy in this mouse model, where scIgG molecules were present in the serum after two hours (in Fig.17 * in C). However, intact IVIg could not be detected on the gel, which means Fig.16 The higher band of pCART214 in the scIgG represents rather than the complete IgG. This indicates that the polypeptide of the present invention cleaves IgG in the in vivo model.

[0326] Table 7. Analysis of in vivo cleavage of human IgG in sera from mice treated with IdeS (BX1001865 and pCART 124) / IdeS variants by potency ELISA (mean ± Stdev (estimated standard deviation of samples)).

[0327] Average value (mg / mL) Stdev (estimates the standard deviation of a sample) Control (PBS) 6.58 0.80 BX1001865 0.30 0.05 pCART124 0.39 0.12 pCART125 0.38 0.39 pCART213 0.26 0.06 pCART214 1.29 0.05 . SEQUENCE LISTING <110> Hansa Biopharmaceuticals AG <120> Cysteine ​​proteases <130> 191266CN2 <160> 37 <170> PatentIn version 3.5 <210> 1 <211> 339 <212> PRT <213> Streptococcus pyogenes <400> 1 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> 2 <211> 310 <212> PRT <213> Streptococcus pyogenes <400> 2 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> 3 <211> 310 <212> PRT <213> Streptococcus pyogenes <400> 3 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 Arg 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> 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 Lys 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 Arg Gly Ser Lys Asp Pro Arg Gly 165 170 175 Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn 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> 310 <212> PRT <213> Streptococcus pyogenes <400> 5 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 Arg 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 Lys Gly Ser Lys Asp Pro Arg Gly 165 170 175 Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn 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 Lys 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> 6 <211> 310 <212> PRT <213> Streptococcus pyogenes <400> 6 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 Arg 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 Asn 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 Asp 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> 7 <211> 310 <212> PRT <213> Streptococcus pyogenes <400> 7 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 Lys 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 Arg 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 Asn 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 Glu 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 Lys 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> 8 <211> 310 <212> PRT <213> Streptococcus pyogenes <400> 8 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 Glu 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 Asn 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 Glu Glu Leu Thr Lys 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 Lys Asn 275 280 285 Ile Gly Ala Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Lys 290 295 300 Ser Trp Asn Gln Thr Asn 305 310 <210> 9 <211> 310 <212> PRT <213> Streptococcus pyogenes <400> 9 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 Lys Glu His Pro Glu Lys Gln 85 90 95 Lys Ile Asn Phe Arg Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile 100 105 110 Arg 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 Asn 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 Ser Glu Leu Glu Asn 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 Lys 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> 10 <211> 310 <212> PRT <213> Streptococcus pyogenes <400> 10 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 Lys Glu His Pro Glu Lys Gln 85 90 95 Lys Ile Asn Phe Arg Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile 100 105 110 Arg 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 Lys Gly Ser Lys Asp Pro Arg Gly 165 170 175 Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn 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 Glu 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> 11 <211> 310 <212> PRT <213> Streptococcus pyogenes <400> 11 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 Glu Arg Tyr Leu Glu Glu His Pro Glu Lys Gln 85 90 95 Lys Ile Asn Phe Arg 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 Lys Gly Ser Lys Asp Pro Arg Gly 165 170 175 Gly Ile Phe Asp Ala Val Phe Thr Arg Gly Asn 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 Glu Glu Leu Thr Lys 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 Lys 290 295 300 Ser Trp Asn Gln Thr Asn 305 310 <210> 12 <211> 306 <212> PRT <213> Streptococcus pyogenes <400> 12 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 305 <210> 13 <211> 290 <212> PRT <213> Streptococcus pyogenes <400> 13 Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln Gly 1 5 10 15 Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr Asp 20 25 30 Ile Thr Lys Thr 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 Asp Gln 50 55 60 Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn Phe 65 70 75 80 Asn Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp Thr Lys Asn 85 90 95 His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys Ala Phe 100 105 110 Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro Asp His Val Ile 115 120 125 Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr Asn His Gly Pro 130 135 140 Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe Asp 145 150 155 160 Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr Ser Arg His 165 170 175 Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys Lys 180 185 190 Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala Asn 195 200 205 Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp Ser 210 215 220 Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn Ala 225 230 235 240 Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly Lys 245 250 255 Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala Gln 260 265 270 Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn Gln 275 280 285 Thr Asn 290 <210> 14 <211> 290 <212> PRT <213> Streptococcus pyogenes <400> 14 Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln Gly 1 5 10 15 Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr Asp 20 25 30 Ile Thr Lys Thr 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 Asp Gln 50 55 60 Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn Phe 65 70 75 80 Lys Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp Thr Lys Asn 85 90 95 His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys Ala Phe 100 105 110 Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro Asp His Val Ile 115 120 125 Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr Asn His Gly Pro 130 135 140 Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe Asp 145 150 155 160 Ala Val Phe Thr Arg Gly Asn Gln Ser Lys Leu Leu Thr Ser Arg His 165 170 175 Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys Lys 180 185 190 Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala Asn 195 200 205 Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp Ser 210 215 220 Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn Ala 225 230 235 240 Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly Lys 245 250 255 Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala Gln 260 265 270 Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn Gln 275 280 285 Thr Asn 290 <210> 15 <211> 290 <212> PRT <213> Streptococcus pyogenes <400> 15 Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln Gly 1 5 10 15 Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr Asp 20 25 30 Ile Thr Lys Thr 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 Asp Gln 50 55 60 Ile Glu Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn Phe 65 70 75 80 Lys Gly Glu Gln Met Phe Asp Val Lys Lys Ala Ile Asp Thr Lys Asn 85 90 95 His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys Ala Phe 100 105 110 Pro Tyr Leu Ser Thr Lys His Leu Gly Val Phe Pro Asp His Val Ile 115 120 125 Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr Asn His Gly Pro 130 135 140 Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe Asp 145 150 155 160 Ala Val Phe Thr Arg Gly Asn Gln Ser Lys Leu Leu Thr Ser Arg His 165 170 175 Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys Glu 180 185 190 Glu Leu Thr Lys Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala Asn 195 200 205 Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp Ser 210 215 220 Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn Ala 225 230 235 240 Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly Lys 245 250 255 Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala Gln 260 265 270 Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Lys Ser Trp Asn Gln 275 280 285 Thr Asn 290 <210> 16 <211> 319 <212> PRT <213> Streptococcus pyogenes <400> 16 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 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 Glu Arg Tyr Leu Glu Glu His Pro Glu Lys Gln 85 90 95 Lys Ile Asn Phe Lys Gly Glu Gln Met Phe Asp Val Lys Lys 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 Asn 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 Glu Glu Leu Thr Lys 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 Lys 290 295 300 Ser Trp Asn Gln Thr Asn Gly Gly Gly His His His His His His 305 310 315 <210> 17 <211> 320 <212> PRT <213> Streptococcus pyogenes <400> 17 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> 18 <211> 4 <212> PRT <213> Streptococcus pyogenes <400> 18 Asn Gln Thr Asn 1 <210> 19 <211> 20 <212> PRT <213> Streptococcus pyogenes <400> 19 Asp Ser Phe Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro 1 5 10 15 Tyr His Val Thr 20 <210> 20 <211> 963 <212> DNA <213> Streptococcus pyogenes <400> 20 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> 21 <211> 963 <212> DNA <213> Streptococcus pyogenes <400> 21 atggatagtt tttctgctaa tcaagagatt agatattcgg aagtaacacc tttcacgtt 60 acttccgttt ggaccaaagg agttactcct ccagcaaact tcactcaagg tgaagatgtt 120 tttcacgctc cttatgttgc taaccaagga tggtatgata ttaccaaaac attcaatgga 180 aaagacgatc ttctttgcgg ggctgccaca gcagggaata tgcttcactg gtggttcgat 240 caaaacaaag accaaattaa acgttatttg gaagagcatc cagaaaagca aaaaataaac 300 ttccgtggcg 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 atgttataaa cctgtgggga 720 gctgactttg attctaacgg gaaccttaaa gctatttatg taacagactc tgatagtaat 780 gcatctattg gtatgaagaa atactttgtt ggtgttaatt ccgctgggaaa agtagctatt 840 tctgctaaag aaataaaaga agataatata ggtgctcaag tactagggtt atttacactt 900 tcaacagggc aagatagttg gaatcagacc aatggcggtg gccatcatca ccatcaccac 960 taa 963 <210> 22 <211> 963 <212> DNA <213> Streptococcus pyogenes <400> 22 atggatagtt tttctgctaa tcaagagatt agatattcgg aagtaacacc ttatcacgtt 60 acttccgttt ggaccaaagg agttactcct ccagcaaact tcactcaagg tgaagatgtt 120 tttcacgctc cttatgttgc taaccaagga tggtatgata ttaccaaaac attcaatgga 180 aaagacgatc ttctttgcgg ggctgccaca gcagggaata tgcttcactg gtggttcgat 240 caaaacaaag accaaattaa acgttatttg gaagagcatc cagaaaagca aaaaataaac 300 ttcaaaggcg aacagatgtt tgacgtaaaa gaagctatcg acactaaaaaa ccaccagcta 360 gatagtaaat tatttgaata ttttaaagaa aaagctttcc cttatctatc tactaaacac 420 ctaggagttt tccctgatca tgtaattgat atgttcatta acggctaccg ccttagtcta 480 actaaccacg gtccaacgcc agtaaaacgt ggtagtaaag atccccgagg tggtattttt 540 gacgccgtat ttacaagagg taaccaaagt 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> 963 <212> DNA <213> Streptococcus pyogenes <400> 23 atggatagtt tttctgctaa tcaagagatt agatattcgg aagtaacacc tttcacgtt 60 acttccgttt ggaccaaagg agttactcct ccagcaaact tcactcaagg tgaagatgtt 120 tttcacgctc cttatgttgc taaccaagga tggtatgata ttaccaaaac attcaatgga 180 aaagacgatc ttctttgcgg ggctgccaca gcagggaata tgcttcactg gtggttcgat 240 caaaacaaag accaaattaa acgttatttg gaagagcatc cagaaaagca aaaaataaac 300 ttccgtggcg aacagatgtt tgacgtaaaa gaagctatcg acactaaaaa ccaccagcta 360 gatagtaaat tatttgaata ttttaaagaa aaagctttcc cttatctatc tactaaacac 420 ctaggagttt tccctgatca tgtaattgat atgttcatta acggctaccg ccttagtcta 480 actaaccacg gtccaacgcc agtaaaaaaa ggtagtaaag atccccgagg tggtattttt 540 gacgccgtat ttacaagagg taaccaaagt aagctattga caagtcgtca tgattttaaa 600 gaaaaaaatc tcaaagaaat cagtgatctc attaagaaag agttaaccga aggcaaggct 660 ctaggcctat cacacaccta cgctaacgta cgcatcaacc atgttataaa cctgtgggga 720 gctgactttg attctaacgg gaaccttaaa gctatttatg taacagactc tgatagtaat 780 gcatctattg gtatgaagaa atactttgtt ggtgttaata aagctggaaa agtagctatt 840 tctgctaaag aaataaaaga agataatata ggtgctcaag tactagggtt atttacactt 900 tcaacagggc aagatagttg gaatcagacc aatggcggtg gccatcatca ccatcaccac 960 city ​​963 <210> 24 <211> 963 <212> DNA <213> Streptococcus pyogenes <400> 24 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 cgtgagcatc 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 taaccaaagt aagctattga caagtcgtca tgattttaaa 600 gaaaaaaatc tcaaagaaat cagtgatctc attaagaaag agttagatga 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> 25 <211> 963 <212> DNA <213> Streptococcus pyogenes <400> 25 atggatagtt tttctgctaa tcaagagatt agatattcgg aagtaacacc tttcacgtt 60 acttccgttt ggaccaaagg agttactcct ccagcaaact tcactcaagg tgaagatgtt 120 tttcacgctc cttatgttgc taaccaagga tggtatgata ttaccaaaac attcaatgga 180 aaagacgatc ttctttgcgg ggctgccaca gcagggaata tgcttcactg gtggttcgat 240 caaaacaaag accaaattaa acgttatttg aaagagcatc cagaaaagca aaaaataaac 300 ttcaatggcg aacagatgtt tgacgtaaaa gaagctatcc gtactaaaaa 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 taaccaaagt aagctattga caagtcgtca tgattttaaa 600 gaaaaaaatc tcaaagaaat cagtgatctc attaagaaag agttagaaga aggcaaggct 660 ctaggcctat cacacaccta cgctaacgta cgcatcaacc atgttataaa cctgtgggga 720 gctgactttg attctaacgg gaaccttaaa gctatttatg taacagactc tgatagtaat 780 gcatctattg gtatgaagaa atactttgtt ggtgttaata aagctgggaaa agtagctatt 840 tctgctaaag aaataaaaga agataatata ggtgctcaag tactagggtt atttacactt 900 tcaacagggc aagatagttg gaatcagacc aatggcggtg gccatcatca ccatcaccac 960 taa 963 <210> 26 <211> 963 <212> DNA <213> Streptococcus pyogenes <400> 26 atggatagtt tttctgctaa tcaagagatt agatattcgg aagtaacacc ttatcacgtt 60 acttccgttt ggaccaaagg agttactcct ccagcaaact tcactcaagg tgaagatgtt 120 tttcacgctc cttatgttgc taaccaagga tggtatgata ttaccaaaac attcaatgga 180 aaagacgatc ttctttgcgg ggctgccaca gcagggaata tgcttcactg gtggttcgat 240 caaaacaaag accaaattga acgttatttg gaagagcatc cagaaaagca aaaaataaac 300 ttcaatggcg aacagatgtt tgacgtaaaa gaagctatcg acactaaaaaa 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 taaccaaagt aagctattga caagtcgtca tgattttaaa 600 gaaaaaaatc tcaaagaaat cagtgatctc attaaggaag agttaaccaa 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 aaaaatata ggtgctcaag tactagggtt atttacactt 900 tcaacagggc aaaaaagttg gaatcagacc aatggcggtg gccatcatca ccatcaccac 960 city ​​963 <210> 27 <211> 963 <212> DNA <213> Streptococcus pyogenes <400> 27 atggatagtt tttctgctaa tcaagagatt agatattcgg aagtaacacc tttcacgtt 60 acttccgttt ggaccaaagg agttactcct ccagcaaact tcactcaagg tgaagatgtt 120 tttcacgctc cttatgttgc taaccaagga tggtatgata ttaccaaaac attcaatgga 180 aaagacgatc ttctttgcgg ggctgccaca gcagggaata tgcttcactg gtggttcgat 240 caaaacaaag accaaattaa acgttatttg aaagagcatc cagaaaagca aaaaataaac 300 ttccgtggcg aacagatgtt tgacgtaaaa gaagctatcc gtactaaaaa 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 taaccaaagt aagctattga caagtcgtca tgattttaaa 600 gaaaaaaatc tcaaagaaat cagtgatctc attaagagtg agttagaaaa cggcaaggct 660 ctaggcctat cacacaccta cgctaacgta cgcatcaacc atgttataaa cctgtgggga 720 gctgactttg attctaacgg gaaccttaaa gctatttatg taacagactc tgatagtaat 780 gcatctattg gtatgaagaa atactttgtt ggtgttaata aagctggaaa agtagctatt 840 tctgctaaag aaataaaaga agataatata ggtgctcaag tactagggtt atttacactt 900 tcaacagggc aagatagttg gaatcagacc aatggcggtg gccatcatca ccatcaccac 960 city ​​963 <210> 28 <211> 963 <212> DNA <213> Streptococcus pyogenes <400> 28 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 aaagagcatc cagaaaagca aaaaataaac 300 ttccgtggcg aacagatgtt tgacgtaaaa gaagctatcc gtactaaaaa ccaccagcta 360 gatagtaaat tatttgaata ttttaaagaa aaagctttcc cttatctatc tactaaacac 420 ctaggagttt tccctgatca tgtaattgat atgttcatta acggctaccg ccttagtcta 480 actaaccacg gtccaacgcc agtaaaaaaa ggtagtaaag atccccgagg tggtattttt 540 gacgccgtat ttacaagagg taaccaaagt aagctattga caagtcgtca tgattttaaa 600 gaaaaaaatc tcaaagaaat cagtgatctc attaagaaag agttagaaga 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> 29 <211> 963 <212> DNA <213> Streptococcus pyogenes <400> 29 atggatagtt tttctgctaa tcaagagatt agatattcgg aagtaacacc tttcacgtt 60 acttccgttt ggaccaaagg agttactcct ccagcaaact tcactcaagg tgaagatgtt 120 tttcacgctc cttatgttgc taaccaagga tggtatgata ttaccaaaac attcaatgga 180 aaagacgatc ttctttgcgg ggctgccaca gcagggaata tgcttcactg gtggttcgat 240 caaaacaaag accaaattga acgttatttg gaagagcatc cagaaaagca aaaaataaac 300 ttccgtggcg aacagatgtt tgacgtaaaa gaagctatcg acactaaaaa ccaccagcta 360 gatagtaaat tatttgaata ttttaaagaa aaagctttcc cttatctatc tactaaacac 420 ctaggagttt tccctgatca tgtaattgat atgttcatta acggctaccg ccttagtcta 480 actaaccacg gtccaacgcc agtaaaaaaa ggtagtaaag atccccgagg tggtattttt 540 gacgccgtat ttacaagagg taaccaaagt aagctattga caagtcgtca tgattttaaa 600 gaaaaaaatc tcaaagaaat cagtgatctc attaaggaag agttaaccaa aggcaaggct 660 ctaggcctat cacacaccta cgctaacgta cgcatcaacc atgttataaa cctgtgggga 720 gctgactttg attctaacgg gaaccttaaa gctatttatg taacagactc tgatagtaat 780 gcatctattg gtatgaagaa atactttgtt ggtgttaatt ccgctggaaa agtagctatt 840 tctgctaaag aaataaaaga agataatata ggtgctcaag tactagggtt atttacactt 900 tcaacagggc aaaaaagttg gaatcagacc aatggcggtg gccatcatca ccatcaccac 960 city ​​963 <210> 30 <211> 957 <212> DNA <213> Streptococcus pyogenes <400> 30 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 atgttataaa 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 gggtggcggc ggtggccatc atcaccatca ccactaa 957 <210> 31 <211> 903 <212> DNA <213> Streptococcus pyogenes <400> 31 atgtccgttt ggaccaaagg agttactcct ccagcaaact tcactcaagg tgaagatgtt 60 tttcacgctc cttatgttgc taaccaagga tggtatgata ttaccaaaac attcaatgga 120 aaagacgatc ttctttgcgg ggctgccaca gcagggaata tgcttcactg gtggttcgat 180 caaaacaaag accaaattaa acgttatttg gaagagcatc cagaaaagca aaaaataaac 240 ttcaatggcg aacagatgtt tgacgtaaaa gaagctatcg acactaaaaa ccaccagcta 300 gatagtaaat tatttgaata ttttaaagaa aaagctttcc cttatctatc tactaaacac 360 ctaggagttt tccctgatca tgtaattgat atgttcatta acggctaccg ccttagtcta 420 actaaccacg gtccaacgcc agtaaaagaa ggtagtaaag atccccgagg tggtattttt 480 gacgccgtat ttacaagagg tgatcaaagt aagctattga caagtcgtca tgattttaaa 540 gaaaaaaatc tcaaagaaat cagtgatctc attaagaaag agttaaccga aggcaaggct 600 ctaggcctat cacacaccta cgctaacgta cgcatcaacc atgttataaa cctgtgggga 660 gctgactttg attctaacgg gaaccttaaa gctatttatg taacagactc tgatagtaat 720 gcatctattg gtatgaagaa atactttgtt ggtgttaatt ccgctggaaa agtagctatt 780 tctgctaaag aaataaaaga agataatata ggtgctcaag tactagggtt atttacactt 840 tcaacagggc aagatagttg gaatcagacc aatggcggtg gccatcatca ccatcaccac 900 city ​​903 <210> 32 <211> 903 <212> DNA <213> Streptococcus pyogenes <400> 32 atgtccgttt ggaccaaagg agttactcct ccagcaaact tcactcaagg tgaagatgtt 60 tttcacgctc cttatgttgc taaccaagga tggtatgata ttaccaaaac attcaatgga 120 aaagacgatc ttctttgcgg ggctgccaca gcagggaata tgcttcactg gtggttcgat 180 caaaacaaag accaaattaa acgttatttg gaagagcatc cagaaaagca aaaaataaac 240 ttcaaaggcg aacagatgtt tgacgtaaaa gaagctatcg acactaaaaa ccaccagcta 300 gatagtaaat tatttgaata ttttaaagaa aaagctttcc cttatctatc tactaaacac 360 ctaggagttt tccctgatca tgtaattgat atgttcatta acggctaccg ccttagtcta 420 actaaccacg gtccaacgcc agtaaaagaa ggtagtaaag atccccgagg tggtattttt 480 gacgccgtat ttacaagagg taaccaaagt aagctattga caagtcgtca tgattttaaa 540 gaaaaaaatc tcaaagaaat cagtgatctc attaagaaag agttaaccga aggcaaggct 600 ctaggcctat cacacaccta cgctaacgta cgcatcaacc atgtataaa cctgtgggga 660 gctgactttg attctaacgg gaaccttaaa gctatttatg taacagactc tgatagtaat 720 gcatctattg gtatgaagaa atactttgtt ggtgttaatt ccgctggaaa agtagctatt 780 tctgctaaag aaataaaaga agataatata ggtgctcaag tactagggtt atttacactt 840 tcaacagggc aagatagttg gaatcagacc aatggcggtg gccatcatca ccatcaccac 900 city ​​903 <210> 33 <211> 903 <212> DNA <213> Streptococcus pyogenes <400> 33 atgtccgttt ggaccaaagg agttactcct ccagcaaact tcactcaagg tgaagatgtt 60 tttcacgctc cttatgttgc taaccaagga tggtatgata ttaccaaaac attcaatgga 120 aaagacgatc ttctttgcgg ggctgccaca gcagggaata tgcttcactg gtggttcgat 180 caaaacaaag accaaattga acgttatttg gaagagcatc cagaaaagca aaaaaataaac 240 ttcaaaggcg aacagatgtt tgacgtaaaa aaagctatcg acactaaaaaa ccaccagcta 300 gatagtaaat tatttgaata ttttaaagaa aaagctttcc cttatctatc tactaaacac 360 ctaggagttt tccctgatca tgtaattgat atgttcatta acggctaccg ccttagtcta 420 actaaccacg gtccaacgcc agtaaaagaa ggtagtaaag atccccgagg tggtattttt 480 gacgccgtat ttacaagagg taaccaaagt aagctattga caagtcgtca tgattttaaa 540 gaaaaaatc tcaaagaaat cagtgatctc attaaggaag agttaaccaa aggcaaggct 600 ctaggcctat cacacaccta cgctaacgta cgcatcaacc atgttataaa cctgtgggga 660 gctgactttg attctaacgg gaaccttaaa gctatttatg taacagactc tgatagtaat 720 gcatctattg gtatgaagaa atactttgtt ggtgttaatt ccgctgggaaa agtagctatt 780 tctgctaaag aaataaaaga agataatata ggtgctcaag tactagggtt atttacactt 840 tcaacagggc aaaaaagttg gaatcagacc aatggcggtg gccatcatca ccatcaccac 900 lamp 903 <210> 34 <211> 963 <212> DNA <213> Streptococcus pyogenes <400> 34 atggacgatt accaaaggaa tgctacggaa gcttatgcca aagaagtacc acatcagatc 60 acttccgttt ggaccaaagg agttactcct ccagcaaact tcactcaagg tgaagatgtt 120 tttcacgctc cttatgttgc taaccaagga tggtatgata ttaccaaaac attcaatgga 180 aaagacgatc ttctttgcgg ggctgccaca gcagggaata tgcttcactg gtggttcgat 240 caaaacaaag accaaattga acgttatttg gaagagcatc cagaaaagca aaaaataaac 300 ttcaaaggcg aacagatgtt tgacgtaaaa aaagctatcg 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 taaccaaagt aagctattga caagtcgtca tgattttaaa 600 gaaaaaaatc tcaaagaaat cagtgatctc attack agttacca aggcaaggct ctaggcctat cacacaccta cgctaacgta cgcatcaacc atgttataaa cctgtgggga gctgactttg attctaacgg gaaccttaaa gctatttatg taacagactc tgatagtaat gcatctattg gtatgaagaa atactttgtt ggtgttaatt ccgctggaaa agtagctatt tctgctaaag aaataaaaga agataatata ggtgctcaag tactagggtt atttacactt tcaacagggc aaaaaagttg gaatcagacc aatggcggtg gccatcatca ccatcaccac taa 963 <210> 35 <211> 19 <212> PRT <213> Antibiotic(Streptococcus pyogenes) <400> 35 Ser Phe Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr 1 5 10 15 His Val Thr <210> 36 <211> 19 <212> PRT <213> Antibiotic(Streptococcus pyogenes) <400> 36 Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val Pro His 1 5 10 15 Gln Ile Thr <210> 37 <211> 20 <212> PRT <213> Streptococcus pyogenes <400> 37 Asp Asp Tyr Gln Arg Asn Ala Thr Glu Ala Tyr Ala Lys Glu Val Pro 1 5 10 15 His Gln Ile Thr 20

Claims

1. A polypeptide having IgG cysteine ​​protease activity and consisting of the sequence of any one of SEQ ID NOs: 3-5, 9-11 and 14-16.

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

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

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

8. A method for cleaving IgG in vitro, the method comprising contacting a sample comprising IgG with a polypeptide according to claim 1 or 2 under conditions that allow IgG cysteine ​​protease activity to occur.

9. The method according to claim 8, which is performed to produce an Fc fragment and a Fab fragment.

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