cysteine ​​proteases

Abstract

The present invention relates to novel polypeptides that exhibit IgG cysteine ​​protease activity and their uses in vivo and ex vivo. Uses of the polypeptides include methods for preventing or treating IgG-mediated diseases and conditions, and methods for analyzing IgG and generating F(ab')2 fragments in vitro.

Description

[Technical Field]

[0001] The present invention relates to novel polypeptides that exhibit IgG cysteine ​​protease activity and their uses in vivo and ex vivo. Uses of the polypeptides include methods for preventing or treating IgG-mediated diseases and conditions, and methods for analyzing IgG and generating F(ab')2 fragments in vitro. [Background technology]

[0002] IdeS( I mmunoglobulin G- d egrading e nzyme of SIdeS (IgG-degrading enzyme of Streptococcus pyogenes, also known as immunoglobulin G-degrading enzyme or immunofidase) is an extracellular cysteine ​​protease produced by the human pathogen Streptococcus pyogenes. IdeS was originally isolated from serotype M1 of group A streptococcus strains, but the ides gene has now been identified in all group A streptococcus strains tested. IdeS has an exceptionally high degree of substrate specificity, with IgG being its only identified substrate. IdeS catalyzes a single proteolytic cleavage in the lower hinge region of the heavy chains of all subclasses of human IgG. IdeS also catalyzes corresponding cleavage in the heavy chains of several subclasses of IgG in various animal species. IdeS efficiently cleaves IgG into Fc and F(ab')2 fragments via a two-step mechanism. In the first step, one (first) heavy chain of IgG is cleaved to generate a single truncated IgG (scIgG) molecule with a single, noncovalently bound Fc chain. The scIgG molecule is effectively an intermediate product that retains the remaining (second) heavy chain of the original IgG molecule. In the second step of the mechanism, this second heavy chain is cleaved by IdeS to release an F(ab')2 fragment and a homodimeric Fc fragment, which are the products commonly observed under physiological conditions. The homodimeric Fc may dissociate into its constituent monomers. Under reducing conditions, the F(ab')2 fragment may dissociate into two Fab fragments. The ability of IdeS to cleave IgG has been shown to be useful ex vivo in methods for generating Fab, F(ab')2, and Fc fragments, for example, and can be used for IgG analysis and in vitro generation of F(ab')2 fragments. See, e.g., International Publication Nos. 2003051914 and 2009033670. IdeS has also been shown to be useful in vivo as a therapeutic agent, due to its ability to cleave disease-causing or otherwise undesirable IgG molecules in vivo. See, for example, International Publication Nos. 2006131347 and 2013110946. IdeS can be used as a therapy for any disease or condition mediated in whole or in part by IgG. IgG contributes to the pathology of many autoimmune conditions as well as acute rejection of transplanted organs.

[0003] However, IdeS is an immunogenic protein. That is, when IdeS is used as a therapeutic agent, the immune system of a subject administered IdeS often responds to it. The immune system's response to IdeS is usually accompanied by the production of IdeS-specific antibodies. These antibodies are sometimes referred to herein as IdeS-specific anti-drug antibodies (ADAs) or "IdeS-specific ADAs." The immune response to IdeS in general, and the production of IdeS-specific ADAs specifically, can cause two related problems. First, the efficacy of IdeS may be reduced, for example, due to ADA binding, potentially requiring higher doses or repeated administration to achieve the same effect. ADAs with this effect are sometimes referred to as "neutralizing ADAs." Second, undesirable or even harmful complications, such as excessive inflammatory responses triggered by immune complexes between ADA and IdeS, may occur. The higher the amount of IdeS-specific ADAs in a given subject, the greater the likelihood of these problems occurring. The presence and amount of IdeS-specific ADA molecules in a patient can be determined by any suitable method, such as an agent-specific CAP FEIA (ImmunoCAP) test or a titer assay performed on a serum sample from the patient. Above a clinician-defined threshold, the amount of IdeS-specific ADA molecules in a patient may preclude administration of IdeS or indicate the need for a higher dose of IdeS. Such a higher dose may then result in an increase in the amount of IdeS-specific ADA molecules in the patient, thereby precluding further administration of IdeS.

[0004] IdeS is the virulence factor of Streptococcus pyogenes, which causes common infections such as tonsillitis and streptococcal pharyngitis. Therefore, most human subjects will encounter IdeS in this setting and are likely to have anti-IdeS antibodies in their blood. Typically, serum samples from human subjects (likely due to a history of streptococcal infection) are collected, along with IVIg (In Vitro Infectious Diseases), a preparation of IgG extracted from pooled serum of several thousand donors. I ntrav enous I mmuno g Varying levels of IdeS-specific ADAs can be detected in intravenous immunoglobulin (Ig) preparations. Techniques for detecting IdeS-specific ADAs are known in the art. Even if a subject does not possess IdeS-specific ADAs before the first administration of IdeS, it is likely that such molecules will be produced thereafter. Thus, for any given subject, issues related to the immunogenicity of IdeS are likely to pose a barrier to using IdeS as a treatment. These issues may require increasing the dose of IdeS, especially if repeated administration is required, and / or may preclude treatment with IdeS altogether. Existing approaches to these types of problems involve, for example, PEGylation of therapeutic agents to reduce immunogenicity or coadministration of therapeutic agents with immunosuppressants.

[0005] IdeZ is an IgG cysteine ​​protease produced by Streptococcus equi subspecies zooepidemicus, a bacterium found primarily in horses. IdeZ shares approximately 66% identity with IdeS. Because Streptococcus equi subspecies zooepidemicus is not a human pathogen, humans may have fewer or no antibodies (anti-drug antibodies, ADA) against IdeZ, and IdeZ was considered an alternative to IdeS-based therapy. However, the level of IgG cysteine ​​protease activity of IdeZ against human IgG is significantly lower than that of IdeS, especially when cleaving IgG2.

[0006] Thus, there remains a need for IdeZ-derived cysteine ​​proteases that have high activity against human IgG (preferably higher than wild-type IdeZ, and even more preferably higher than IdeS). Specifically, there remains a need for IdeZ-derived cysteine ​​proteases that have high activity against human IgG1 and IgG2 (preferably higher than wild-type IdeZ, and even more preferably higher than IdeS). Summary of the Invention

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

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

[0009] The present inventors have been able to identify specific positions within the sequence of IdeZ that, when modified as described herein, result in novel polypeptides with increased IgG cysteine ​​protease activity against human IgG compared to IdeZ. The IgG cysteine ​​protease activity of the polypeptides of the present invention against human IgG (e.g., in cleaving human IgG) is preferably at least as high as the IgG cysteine ​​protease activity of IdeS against human IgG. The polypeptides of the present invention may be more effective at cleaving human IgG than the IgG cysteine ​​protease of IdeS, particularly when the IgG is of the IgG1 or IgG2 isotype. The polypeptides of the present invention may be more effective at cleaving human IgG than the IgG cysteine ​​protease IdeS, particularly as measured by cleavage of the second chain of IgG1. The polypeptides of the present invention may be more effective at cleaving IgG1 than IgG2. The polypeptides of the present invention are typically less immunogenic than IdeS, and preferably as immunogenic as IdeZ.

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

[0011] Thus, in one aspect, the present invention provides a method for the preparation of an antibody having IgG cysteine ​​protease activity, (i) SEQ ID NO: 1; or (ii) SEQ ID NO: 2; or (iii) a variant of SEQ ID NO: 1 or SEQ ID NO: 2 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modification(s) relative to SEQ ID NO: 1 or SEQ ID NO: 2, respectively; and (c) an asparagine (N) at a position corresponding to position 226 of SEQ ID NO:5, wherein the sequence retains (a) an asparagine (N) at a position corresponding to position 95 of SEQ ID NO:5, (b) an aspartic acid (D) at a position corresponding to position 99 of SEQ ID NO:5, and (c) an asparagine (N) at a position corresponding to position 226 of SEQ ID NO:5, and wherein the polypeptide is at least as effective at cleaving human IgG as a polypeptide consisting of the amino acid sequence of SEQ ID NO:1 or 2, respectively, when measured in the same assay.

[0012] The invention also provides a polynucleotide, expression vector, or host cell encoding or expressing a polypeptide of the invention.

[0013] The present invention also provides a method for treating or preventing a disease or condition mediated by IgG antibodies in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of a polypeptide of the present invention, which may typically include multiple administrations of the polypeptide to the subject.

[0014] The present invention also provides a method for ex vivo treating blood taken from a patient, typically a patient suffering from a disease or condition mediated by IgG antibodies, comprising contacting the blood with a polypeptide of the present invention.

[0015] The present invention also provides a method for improving the benefit of a therapy or therapeutic agent in a subject, comprising: (a) administering to the subject a polypeptide of the invention; and (b) thereafter administering the therapy or therapeutic agent to the subject; - the therapy is an organ transplant or the therapeutic agent is an antibody, a gene therapy such as a viral vector, a replacement for a defective endogenous factor such as an enzyme, a growth or clotting factor, or a cell therapy; - the amount of the polypeptide administered is sufficient to cleave substantially all IgG molecules present in the plasma of the subject; and - steps (a) and (b) are separated by a time interval sufficient to cleave substantially all IgG molecules present in the subject's plasma.

[0016] The present invention also provides a method for producing an Fc, Fab, or F(ab')2 fragment of an IgG, which comprises contacting the IgG with a polypeptide of the present invention, preferably ex vivo.

[0017] Kits for carrying out the methods of the present invention are also provided. [Brief explanation of the drawings]

[0018] [Figure 1] Figure 1 shows SDS-PAGE analysis of expression and purification of pCART239 (SEQ ID NO: 1 with an N-terminal Met and C-terminal His tag). (A) Overexpression of pCART239—Lane 1 and 2 show lysates obtained from cells harvested 1 hour after induction with IPTG; lane 3 shows pooled lysates. (B) Purification of pCART239—Lane 1 shows the flow-through from the NiNTA purification process, demonstrating the removal of impurities found in the lysate, and lanes 2 and 3 represent purified pCART239 (loaded with approximately 0.5 μg and approximately 3.0 μg of protein, respectively). [Figure 2] Figure 2 shows the results of a representative SDS-PAGE gel used to visualize cleavage products generated by incubating IgG1 (Humira) with IdeS and tested IdeZ variants as indicated. The concentrations above the lanes indicate the concentrations of the IdeS / IdeZ variants tested. Panels A and B represent two separate experiments. [Figure 3]Figure 3 shows the results of a representative SDS-PAGE gel used to visualize cleavage products generated by incubating IgG2(XGEVA) with IdeS and tested IdeZ variants as indicated. The concentrations above the lanes indicate the concentrations of the IdeS / IdeZ variants tested. Panels A and B represent two separate experiments. [Figure 4] Figure 4 shows the results of a representative SDS-PAGE gel used to visualize the cleavage products generated by incubating (A) IgG1 (Humira), (B) IgG2 (XGEVA), (C) IgG3, and (D) IgG4 with IdeS and N240, as indicated. The concentrations above the lanes indicate the concentrations of the IdeS / IdeZ variants tested. [Figure 5] Figure 5 shows the fitted titration curve of the mean electrochemiluminescence (ECL) values ​​from triplicate samples in an assay to determine the potency (efficacy of IgG1 cleavage) of N240 compared to IdeS. Error bars represent SD. [Figure 6] Figure 6 shows the digestion of serum IgG by pCART239 with and without a decoy (an inactive version of pCART239). The use of a decoy can reduce the inhibitory effect of ADA present in serum. The concentrations above the lanes indicate the concentrations of the IdeZ variants tested. [Figure 7] Figure 7 shows fitted titration curves of mean electrochemiluminescence (ECL) values ​​from triplicate samples and two separate dilution series in an assay to determine the potency (efficacy of IgG cleavage) of N240 and IdeS in serum. Error bars represent SD. [Figure 8] FIG. 8 shows the mean ECL values ​​corresponding to pre-existing N240 and IdeS ADA levels in sera from 40 healthy individuals and one normal serum pool (n=100). [Figure 9] FIG. 9 shows a schematic diagram illustrating the stepwise cleavage of IgG by the polypeptides of the invention. DETAILED DESCRIPTION OF THE INVENTION

[0019] A brief description of arrays SEQ ID NO: 1 is the nucleotide sequence of the polypeptide of the present invention.

[0020] SEQ ID NO:2 is the sequence of a further polypeptide of the invention which is related to SEQ ID NO:1 and is identical to SEQ ID NO:1 except that the first 20 amino acids at the N-terminus of SEQ ID NO:1, which correspond to the consecutive sequence DDYQRNATEAYAKEVPHQIT, are deleted.

[0021] SEQ ID NO: 3 is the mature sequence of IdeZ, lacking the N-terminal methionine and signal sequence.

[0022] SEQ ID NO: 4 is the mature sequence of IdeS, lacking the N-terminal methionine and signal sequence. Also disclosed as Genbank accession number ADF13949.1.

[0023] SEQ ID NO: 5 is the full sequence of IdeZ, including the N-terminal methionine and signal sequence. Also disclosed as NCBI reference sequence number WP_014622780.1.

[0024] SEQ ID NO: 6 is the complete sequence of IdeS, including the N-terminal methionine and signal sequence, and is also disclosed as NCBI reference sequence number WP_010922160.1.

[0025] SEQ ID NO: 7 is the sequence of pCART207, a variant IdeZ polypeptide.

[0026] SEQ ID NO: 8 is the sequence of pCART229, a variant IdeZ polypeptide.

[0027] SEQ ID NO: 9 is the sequence of pCART239, a variant IdeZ polypeptide of the invention related to SEQ ID NO: 1 by the presence of an additional N-terminal methionine (with a glycine linker) and an additional C-terminal histidine tag.

[0028] SEQ ID NO: 10 is the sequence of N240, a variant IdeZ polypeptide of the invention related to SEQ ID NO: 1 by the presence of an additional N-terminal methionine.

[0029] SEQ ID NO: 11 is the sequence of pCART242, a variant IdeZ polypeptide of the invention related to SEQ ID NO: 2 by the presence of an additional N-terminal methionine (with a glycine linker) and an additional C-terminal histidine tag.

[0030] SEQ ID NO: 12 is the sequence of pCART243, an inactive variant IdeZ polypeptide.

[0031] SEQ ID NO: 13 is the sequence of a control IdeS polypeptide, which comprises the sequence of SEQ ID NO: 4 with an additional N-terminal methionine and histidine tag (with a glycine linker) (internal reference pCART124).

[0032] SEQ ID NO: 14 is the sequence of a control IdeZ polypeptide, which comprises the sequence of SEQ ID NO: 3 with an additional N-terminal methionine and histidine tag (with a glycine linker) (internal reference pCART144).

[0033] SEQ ID NO: 15 is the consecutive sequence DDYQRNATEAYAKEVPHQIT corresponding to positions 35 to 54 of SEQ ID NO: 5.

[0034] SEQ ID NOs: 16-23 are nucleotide sequences encoding specific polypeptides disclosed herein.

[0035] MODE FOR CARRYING OUT THE INVENTION It is to be understood that the various applications of the disclosed products and methods can be tailored to the particular needs of the art, and that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

[0036] Furthermore, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes "polypeptides," and the like.

[0037] "Polypeptide" is used herein in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. Thus, the term "polypeptide" includes short peptide sequences and also includes longer polypeptides and proteins. As used herein, the term "amino acid" refers to natural and / or unnatural, i.e., synthetic, amino acids, including both D- and L-enantiomers, as well as amino acid analogs and peptidomimetics.

[0038] 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.

[0039] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0040] Functional characteristics of polypeptides The present invention relates to novel polypeptides having IgG cysteine ​​protease activity, which are more effective at cleaving human IgG than IdeZ. The IgG cysteine ​​protease activity of the polypeptides of the present invention against human IgG is preferably at least as high as the IgG cysteine ​​protease activity of IdeS against human IgG. Furthermore, the polypeptides of the present invention are typically less immunogenic than IdeS, and preferably at the same level as IdeZ. In the context of a control or comparison to a polypeptide of the present invention, "IdeS" and "IdeZ" refer to polypeptides consisting of the amino acid sequences of SEQ ID NOs: 4 and 3, respectively. Alternatively, or in addition, when used as a control or comparison, "IdeS" and "IdeZ" may refer to polypeptides comprising the amino acid sequences of SEQ ID NOs: 4 and 3, respectively, with an additional N-terminal methionine (M) residue and / or a C-terminal tag to aid expression in and isolation from standard bacterial expression systems. Suitable tags include histidine tags, which may be directly linked to the C-terminus of a polypeptide or indirectly linked via any suitable linker sequence, e.g., three, four, or five glycine residues. Histidine tags typically consist of six histidine residues, but may be longer, typically up to seven, eight, nine, ten, or 20 amino acids, or may be shorter, e.g., five, four, three, two, or one amino acid. The sequence of an exemplary IdeS polypeptide used as a control herein is provided as SEQ ID NO: 13. This polypeptide comprises the sequence of SEQ ID NO: 4 with an additional N-terminal methionine and histidine tag and is sometimes referred to herein as pCART124. The sequence of an exemplary IdeZ polypeptide used as a control herein is provided as SEQ ID NO: 14. This polypeptide comprises the sequence of SEQ ID NO: 3 with an additional N-terminal methionine and histidine tag and is sometimes referred to herein as pCART144.

[0041] IgG cysteine ​​protease activity can be assessed by any suitable method, for example, by incubating a polypeptide with a sample containing IgG and determining the presence of IgG cleavage products. Efficacy can be assessed in the presence or absence of an inhibitor, such as a neutralizing antibody. However, efficacy herein typically refers to efficacy assessed in the absence of such an inhibitor, unless otherwise specified. Suitable methods are described in the Examples. The efficacy of a polypeptide in cleaving IgG is sometimes referred to herein as the "potency" of the polypeptide. The potency of the polypeptides of the present invention is preferably at least 2.0-fold higher than the potency of IdeZ measured in the same assay. The potency of the polypeptides of the present invention may be at least 1.5-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, at least 4.0-fold, at least 4.5-fold, at least 5.0-fold, at least 6.0-fold, at least 7.0-fold, at least 7.5-fold, or at least 8.0-fold higher than the potency of IdeZ measured in the same assay. Alternatively or additionally, the potency of the polypeptides of the present invention is preferably at least equivalent to that of IdeS measured in the same assay. Alternatively or additionally, the potency of the polypeptides of the present invention is preferably higher than that of IdeS measured in the same assay. The potency of the polypeptides of the present invention may be at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2.0-fold, at least 2.5-fold, at least 3.0-fold, or at least 4.0-fold higher than that of IdeS measured in the same assay. The potency of the polypeptides of the present invention is preferably at least 2.0-fold, more preferably at least 3.0-fold, or at least 4.0-fold higher than that of IdeS measured in the same assay.

[0042] The polypeptides of the present invention are typically less immunogenic than IdeS, and therefore increased potency relative to IdeZ and / or equivalent potency to IdeS is the minimum acceptable level of cysteine ​​protease activity against human IgG. However, increased potency relative to IdeS is a desirable improvement. Such increased potency will typically allow for the use of lower doses of the polypeptides of the present invention to achieve the same therapeutic effect as higher doses of IdeS. Lower doses also allow for more frequent repeated administration of the polypeptides of the present invention compared to IdeS. This is because the use of lower doses reduces problems associated with the immunogenicity of therapeutic agents, as the immune system is less likely or less responsive to agents present at lower concentrations.

[0043] Assays for assessing the efficacy of a polypeptide in cleaving IgG, i.e., assays for assessing the efficacy of a polypeptide, are well known in the art, and any suitable assay may be used. Suitable assays include ELISA-based assays, such as those described in the Examples. In such assays, the wells of an assay plate are typically coated with an antibody target, such as bovine serum albumin (BSA). A sample of the polypeptide to be tested is then added to the well, followed by a sample of a target-specific antibody, in this example an antibody specific for BSA (and susceptible to cleavage by IdeS). The polypeptide and antibody are allowed to interact under conditions suitable for IgG cysteine ​​protease activity. After a suitable interval, the assay plate is washed, and a detection antibody that specifically binds to the Fc region of the target-specific antibody is added under conditions suitable for binding to the target-specific antibody. The detection antibody binds to the Fc region of any intact target-specific antibody that binds to the target in each well. After washing, the amount of detection antibody present in the well is proportional to the amount of target-specific antibody bound to that well. The detection antibody can be directly or indirectly conjugated to a label or another reporter system (such as an enzyme), allowing the amount of detection antibody remaining in each well to be determined. The higher the potency of the test polypeptide present in the well, the less intact target-specific antibody will remain, and therefore the less detection antibody will be present. Typically, at least one well of a given assay plate contains IdeS in place of the test polypeptide, allowing the potency of the test polypeptide to be directly compared to that of IdeS. IdeZ or a known variant of IdeZ may also be included for comparison.

[0044] In other assays, the potency of a test polypeptide can be determined by directly visualizing and / or quantifying the IgG fragments generated by cleavage of the IgG by the test polypeptide. This type of assay is also described in the Examples. In such assays, a sample of IgG is typically incubated with different concentrations of the test polypeptide (or one or more of IdeS, IdeZ, and known variants of IdeZ as controls) in a titration series. The products resulting from each concentration are then separated using gel electrophoresis, for example, by SDS-PAGE. Whole IgG and fragments generated by cleavage of the IgG can then be distinguished by size and quantified by the intensity of staining with a suitable dye. The greater the amount of cleavage fragments, the more potent the test polypeptide at a given concentration. Polypeptides of the present invention typically produce detectable amounts of cleavage fragments at lower concentrations (lower points in the titration series) than IdeZ and / or IdeS. This type of assay can also determine the amount of different fragments generated from each cleavage event, thereby enabling the identification of test polypeptides that are more effective at cleaving the first or second heavy chain of IgG molecules. Suitable IgG samples for the potency assays described herein may come from a variety of sources. For example, commercially available antibody preparations may be used. Commercially available antibody preparations are generally pure and isotype- and subclass-specific. Alternatively, samples containing a mixed population of IgG, such as human serum, may be used. IgG samples, including serum, may contain ADAs against IdeS and / or IdeZ. Thus, suitable IgG samples for the potency assays described herein may contain ADAs. Polypeptides of the present invention may be more effective at cleaving the first chain of an IgG molecule (compared to IdeS and / or IdeZ) than the second chain, particularly when the IgG is of the IgG2 isotype (see the schematic diagram in Figure 9). Alternatively, polypeptides of the present invention may be more effective at cleaving the second chain of an IgG molecule (compared to IdeS and / or IdeZ) than the second chain, particularly when the IgG is of the IgG1 isotype (see the schematic diagram in Figure 9).Polypeptides of the invention may be more effective at cleaving IgG1 than IgG2 (when compared to IdeS and / or IdeZ).

[0045] This type of assay can also be adapted to determine the extent to which the presence of IdeS-specific ADAs can reduce the efficacy of a polypeptide of the invention. In an adapted assay, a sample of IgG is incubated with the test polypeptide (or IdeS as a control), and serum or an IVIg preparation containing IdeS-specific ADAs is included in the reaction medium. Preferably, the efficacy of a polypeptide of the invention is not affected by the presence of ADAs or is reduced less by the presence of ADAs than the efficacy of IdeS in the same assay. That is, preferably, the neutralizing effect of IdeS-specific ADAs on a polypeptide of the invention is equal to or less than the neutralizing effect of IdeS-specific ADAs on IdeS measured in the same assay.

[0046] As noted above, the polypeptides of the present invention are typically less immunogenic than IdeS. That is, when present at equivalent doses or concentrations and measured in the same assay, the polypeptides of the present invention may elicit an immune response comparable to or preferably less than that of IdeS. The immunogenicity of the polypeptides of the present invention is typically 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, or 25% or less of the immunogenicity of IdeS measured in the same assay. Preferably, the immunogenicity of the polypeptides of the present invention is 25% or less of the immunogenicity of IdeS measured in the same assay.

[0047] 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 ADA specific for IdeS also binds to a polypeptide of the invention. This type of assay is described in the Examples. The presence and amount of IdeS-specific ADA molecules in a patient can be determined by any suitable method, such as an agent-specific CAP FEIA (ImmunoCAP) test or a titer assay performed on a serum sample from the patient.

[0048] One such assay involves testing the competition between IdeS and a test polypeptide for binding to an IdeS-specific ADA. Typically, IdeS is coated onto the wells of an assay plate, followed by the administration of a preincubated mixture of a solution containing an IdeS-specific ADA, such as an IVIg preparation, and the test polypeptide (or IdeS as a control). The preincubation is performed in the presence of an inhibitor of IgG cysteine ​​protease activity, such as iodoacetic acid (IHAc), and at a high salt concentration to allow only high-affinity binding between the protein and the ADA. The preincubated mixture is allowed to interact with the IdeS-coated wells. Any IdeS-specific ADA not bound to the test polypeptide binds to IdeS in the wells. After a suitable interval, the assay plate is washed, and a detection antibody that specifically binds to IgG is added under conditions suitable for binding. The detection antibody binds to any ADA bound to IdeS in each well. After washing, the amount of detection antibody present in the well is inversely proportional to the amount of ADA bound to the test polypeptide. The detection antibody can be directly or indirectly conjugated 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 of a given assay plate is tested with a pre-incubated mixture of IVIg and IdeS in place of the test polypeptide, so that binding of ADA to the test polypeptide can be directly compared with binding to IdeS. IdeZ may also be included as an additional control.

[0049] Another suitable assay involves testing the degree to which different concentrations of IdeS-specific ADA, e.g., a titration series of IVIg preparations, bind to a test polypeptide, compared with IdeS and / or IdeZ as controls. Preferably, the polypeptides of the present invention require a higher concentration of ADA for detectable binding compared to the concentration of ADA at which binding to IdeS becomes detectable. Such an assay is described in the Examples. Such an assay typically involves coating the wells of an assay plate with a test polypeptide or control, followed by incubation of each well with different concentrations of IdeS-specific ADA from a titration series. Incubation is performed in the presence of an inhibitor of IgG cysteine ​​protease activity, e.g., iodoacetic acid (IHAc), and at high salt concentrations to allow only high-affinity binding between the protein and ADA. After a suitable interval, the assay plate is washed, and a detection antibody that specifically binds to IgG F(ab')2 is added under conditions suitable for binding. The detection antibody binds to any ADA bound to the test polypeptide or IdeS coated in each well. After washing, the amount of detection antibody present in the well is directly proportional to the amount of ADA bound to the test polypeptide or control. The detection antibody can be directly or indirectly conjugated 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. To establish a threshold level for detection of binding in the test wells, at least one well on a given assay plate is incubated with buffer without ADA as a blank.

[0050] Structural features of polypeptides This section describes the structural features of the polypeptides of the invention, which apply in addition to the functional features outlined in the section above.

[0051] Polypeptides of the present invention are typically at least 100 amino acids long, at least 150 amino acids long, at least 200 amino acids long, at least 250 amino acids long, at least 260 amino acids long, at least 270 amino acids long, at least 280 amino acids long, at least 290 amino acids long, at least 300 amino acids long, or at least 310 amino acids long. Polypeptides of the present invention are typically no longer than 400 amino acids long, no longer than 350 amino acids long, no longer than 340 amino acids long, no longer than 330 amino acids long, no longer than 320 amino acids long, or no longer than 315 amino acids long. It will be understood that any of the above lower limits can be combined with any of the above upper limits to provide a range for the length of a polypeptide of the present invention. For example, a polypeptide may be 100 to 400 amino acids long or 250 to 350 amino acids long. A polypeptide is preferably 290 to 320 amino acids long, most preferably 300 to 315 amino acids long.

[0052] The primary structure (amino acid sequence) of the polypeptide of the present invention is SEQ ID NO: 1, which is a specific variant based on the wild-type mature IdeZ sequence (SEQ ID NO: 3). In other words, SEQ ID NO: 1 is related to SEQ ID NO: 3 through a specific set of point mutations in the primary polypeptide sequence that are responsible for increased potency against human IgG (when compared to SEQ ID NO: 3).

[0053] Another polypeptide of the invention is SEQ ID NO:2, which is related to SEQ ID NO:1. SEQ ID NO:2 is identical to SEQ ID NO:1 except for the deletion of the first 20 amino acids at the N-terminus of SEQ ID NO:1, which correspond to the consecutive sequence DDYQRNATEAYAKEVPHQIT. In other words, SEQ ID NO:2 is identical to SEQ ID NO:1 with respect to point mutations in the primary polypeptide sequence relative to the wild-type IdeZ sequence (SEQ ID NO:3).

[0054] The present invention also relates to variants of SEQ ID NO:1 or SEQ ID NO:2 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications relative to SEQ ID NO:1 or SEQ ID NO:2, respectively, with the proviso that the sequence retains (a) an asparagine (N) at a position corresponding to position 95 of SEQ ID NO:5, (b) an aspartic acid (D) at a position corresponding to position 99 of SEQ ID NO:5, and (c) an asparagine (N) at a position corresponding to position 226 of SEQ ID NO:5, and wherein the polypeptide is at least as effective at cleaving human IgG as a polypeptide consisting of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2, respectively, when measured in the same assay. Optionally, one or more of the amino acid modifications in the variant of SEQ ID NO:1 or SEQ ID NO:2 do not result in the same amino acid as is present at the corresponding position in the polypeptide sequence of SEQ ID NO:3, and preferably, none of the modifications result in the same amino acid as is present at the corresponding position in the polypeptide sequence of SEQ ID NO:3.

[0055] When the sequence of a polypeptide of the present invention comprises a variant of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 in which 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications, e.g., amino acid additions, deletions, or substitutions, have been made compared to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, the sequence must retain (a) an asparagine (N) at the position corresponding to position 95 of SEQ ID NO: 5, (b) an aspartic acid (D) at the position corresponding to position 99 of SEQ ID NO: 5, and (c) an asparagine (N) at the position corresponding to position 226 of SEQ ID NO: 5. Otherwise, the modifications are preferably conservative amino acid substitutions. A variant of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 may contain one or more modifications (made to SEQ ID NO: 1 or SEQ ID NO: 2, respectively) that do not result in the same amino acid present at the corresponding position in the polypeptide sequence of SEQ ID NO: 3. Preferably, in a variant of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, none of the modifications (made to SEQ ID NO: 1 or SEQ ID NO: 2, respectively) result in the same amino acid being present at the corresponding position in the polypeptide sequence of SEQ ID NO: 3.

[0056] Conservative substitutions replace an amino acid with another amino acid of similar chemical structure, similar chemical properties, and / or similar side chain volume. The introduced amino acid may have a similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge to the amino acid being replaced. Alternatively, conservative substitutions may introduce another aromatic or aliphatic amino acid in place of an existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and may be selected according to the properties of the 20 major amino acids defined in Table A1 below. When amino acids have similar polarity, this can be determined by referring to the hydropathy scale of amino acid side chains in Table A2.

[0057] [Table 1]

[0058] [Table 2]

[0059] Specific residues in the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 (other than positions 95, 99, and 226 corresponding to SEQ ID NO:5) are preferably retained in variant sequences containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid alterations. For example, the variant sequences typically retain specific residues known to be necessary for IgG cysteine ​​protease activity. Thus, the cysteine ​​corresponding to position 102 of SEQ ID NO:5 must be retained in the amino acid sequence of a polypeptide of the invention. Optionally, the lysine (K) corresponding to position 92 of SEQ ID NO:5, the histidine (H) corresponding to position 272 of SEQ ID NO:5, and the aspartic acid (D) corresponding to each of positions 294 and 296 of SEQ ID NO:5 are also retained. Thus, polypeptide variants of SEQ ID NO: 1 or SEQ ID NO: 2 of the present invention typically have a cysteine ​​(C) at a position corresponding to position 102 of SEQ ID NO: 5; and optionally have a lysine (K), a histidine (H), an aspartic acid (D), and an aspartic acid (D) at positions corresponding to positions 92, 272, 294, and 296 of SEQ ID NO: 5, respectively.

[0060] The present inventors have also found that certain other modifications to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 may increase the potency of the polypeptide of the invention and / or reduce its recognition by IdeS-specific ADA. Thus, a polypeptide variant of SEQ ID NO: 1 or SEQ ID NO: 2 according to the invention may comprise amino acid substitutions at one, two, three, four, five, six, seven, eight, nine, or ten of the positions corresponding to positions 84, 93, 97, 137, 139, 140, 147, 150, 162, 165, 166, 171, 174, 205, 226, 237, 239, 243, 250, 251, 254, 255, 282, 288, 312, 315, 347, and 349 of SEQ ID NO: 5.

[0061] Substitutions typically replace an existing amino acid with another amino acid with different properties. For example, an uncharged amino acid may be replaced with a charged amino acid, or vice versa. Preferred substitutions at these positions are shown in Table B below using one-letter codes.

[0062] [Table 3]

[0063] Each substitution may be referred to herein using the term obtained by combining the entries in columns 1, 2, and 3 of each row from left to right. For example, the substitution in row 1 of Table B may be referred to herein as "H84N," the substitution in row 2 may be referred to herein as "A93T," and so on. 7

[0064] Table C below summarizes the changes made to the wild-type IdeZ sequence (SEQ ID NO: 3) to generate the amino acid sequences of certain polypeptides described herein.

[0065] [Table 4]

[0066] The amino acid sequences of the specific polypeptides referred to herein are reproduced in full below.

[0067] SEQ ID NO: 1 DDYQRNATEAYAKEVPHQITSVWTKGVTPPEQFTQGEDVIHAPYLAHQGWYDITKAFNGKDDLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFRNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYY LNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGNQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLS

[0068] SEQ ID NO: 2 SVWTKGVTPPEQFTQGEDVIHAPYLAHQGWYDITKAFNGKDDLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFRNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVFKTQSTD VNRPYQDKDKRGGIFDAVFTRGNQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLS

[0069] SEQ ID NO: 3 (IdeZ mature sequence) DDYQRNATEAYAKEVPHQITSVWTKGVTPLTPEQFRYNNEDVIHAPYLAHQGWYDITKAFDGKDNLL CGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFNNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRG DQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLS

[0070] SEQ ID NO: 4 (IdeS mature sequence) DSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATA GNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHL GVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLI KKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAIS AKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

[0071] SEQ ID NO: 5 (IdeZ complete sequence) MKTIAYPNKPHSLSAGLLTAIAIFSLASSNITYADDYQRNATEAYAKEVPHQITSVWTKGVTPLTPEQFRYNNEDVIHAPYLAHQGWYDITKAFDGKDNLL CGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFNNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRG DQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLS

[0072] SEQ ID NO: 6 (IdeS complete sequence) MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFNGEQMFDVKEAIDTKNHQLDS KLFEYFKEKAFPYLSTKHLGVFPDHVIDMFINGYRLSLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSNGNLKAIYVTDSDSNASIGMKKYFVGV NSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

[0073] SEQ ID NO: 7 (pCART207) MDDYQRNATEAYAKEVPHQITSVWTKGVTPPEQFTQGEDVIHAPYLAHQGWYDITKAFDGKDNLL CGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFRNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRG DQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLS

[0074] SEQ ID NO: 8 (pCART229) MDDYQRNATEAYAKEVPHQITSVWTKGVTPPEQFTQGEDVIHAPYLAHQGWYDITKAFDGKDNLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFRNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGY YLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGNQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLS

[0075] SEQ ID NO: 9 (pCART239) MDDYQRNATEAYAKEVPHQITSVWTKGVTPPEQFTQGEDVIHAPYLAHQGYDITKAFNGKDDLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFRNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVF KTQSTDVNRPYQDKDKRGGIFDAVFTRGNQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLSGGGHHHHHHH

[0076] SEQ ID NO: 10 (N240) MDDYQRNATEAYAKEVPHQITSVWTKGVTPPEQFTQGEDVIHAPYLAHQGWYDITKAFNGKDDLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFRNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGY YLNVFKTQSTDVNRPYQDKDKRGGIFDAVFTRGNQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLS

[0077] SEQ ID NO: 11 (pCART242) MSVWTKGVTPPEQFTQGEDVIHAPYLAHQGWYDITKAFNGKDDLLCGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFRNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVFKTQSTDVNRP YQDKDKRGGIFDAVFTRGNQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLEAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLSGGGHHHHHH

[0078] SEQ ID NO: 12 (pCART243-inactive IdeZ variant) MDDYQRNATEAYAKEVPHQITSVWTKGVTPPEQFTQGEDVIHAPYLAHQGYDITKAFNGKDDLLGGAATAGNMLHWWFDQNKTEIEAYLSKHPEKQKIIFRNQELFDLKAAIDTKDSQTNSQLFNYFRDKAFPNLSARQLGVMPDLVLDMFINGYYLNVF KTQSTDVNRPYQDKDKRGGIFDAVFTRGNQTTLLTARHDLKNKGLNDISTIIKQELTEGRALALSHTYANVSISHVINLWGADFNAEGNLAIYVTDSDANASIGMKKYFVGINAHGHVAISAKKIEGENIGAQVLGLFTLSSGKDIWQKLSGGGHHHHHHH

[0079] The polypeptides of the present invention may comprise, consist essentially of, or consist of the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. SEQ ID NO: 1 or SEQ ID NO: 2, respectively, may optionally comprise 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 linked to the C-terminus by a linker of 3 glycine residues or 5 glycine residues.

[0080] If relevant, any suitable algorithm can be used to calculate amino acid identity. For example, the PILEUP and BLAST algorithms can be used to calculate identity and align sequences (typically with their default settings, such as to identify equivalent or corresponding sequences), 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 / ). This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet some positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act 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 is stopped when the cumulative alignment score falls by an amount X from its maximum achieved value; when the cumulative score falls below zero due to the accumulation of one or more negative-scoring residue alignments; or when 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 as defaults 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, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

[0081] 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 in a comparison of a first sequence 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 package provides the BESTFIT program (e.g., used with its default settings) that can be used to calculate identity (Devereux et al. (1984) Nucleic Acids Research 12, 387-395).

[0082] Polypeptide production The polypeptides disclosed herein can be produced by any suitable means. For example, polypeptides can be directly synthesized using standard methods known in the art, such as Fmoc solid-phase chemistry, Boc solid-phase chemistry, or solution-phase peptide synthesis. Alternatively, polypeptides can be produced by transforming cells, typically bacterial cells, with a nucleic acid molecule or vector encoding the polypeptide. Production of polypeptides by expression in bacterial host cells is described below and illustrated in the Examples. The present invention provides nucleic acid molecules and vectors encoding the polypeptides of the invention. The present invention also provides host cells containing such nucleic acids or vectors. Exemplary polynucleotide molecules encoding the polypeptides of the invention and others disclosed herein are provided as SEQ ID NOS: 16-23. These sequences each include an N-terminal methionine (ATG) codon at the 5' end and codons for a 3xGly linker and a 6xHis histidine tag before the stop codon (TAA) at the 3' end, although these may optionally be omitted.

[0083] 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 invention can be provided in isolated or substantially isolated form. By substantially isolated, it is meant that the polypeptide can be largely, but not completely, isolated from any surrounding medium. A polynucleotide can be mixed with a carrier or diluent that will not interfere with its intended use and still be considered substantially isolated. A nucleic acid sequence "encoding" a selected polypeptide is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences, for example, in an expression vector. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. For purposes of the present invention, such nucleic acid sequences may include, but are not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic sequences derived from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence.

[0084] Polynucleotides can be synthesized according to methods well known in the art, as described, for 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 comprising regulatory sequences operably linked to the inserted sequence, thus allowing for in vivo expression of the polypeptides of the present invention. These expression cassettes are then typically provided within vectors (e.g., plasmids or recombinant viral vectors). Such expression cassettes can be administered directly to the host subject. Alternatively, vectors comprising the polynucleotides of the present invention can be administered to the host subject. Preferably, the polynucleotides are prepared and / or administered using genetic vectors. Suitable vectors can be any vectors that carry a sufficient amount of genetic information and are capable of expressing the polypeptides of the present invention.

[0085] Thus, the present invention includes expression vectors containing such polynucleotide sequences. Such expression vectors are routinely constructed in the field of molecular biology and may involve, for example, the use of plasmid DNA and appropriate initiators, promoters, enhancers, and other elements, such as polyadenylation signals, where necessary and positioned in the correct orientation, to express the peptides of the present invention. Other suitable vectors will be apparent to those skilled in the art. For further examples in this regard, see Sambrook et al.

[0086] The present invention also includes cells modified to express a polypeptide of the present invention. Such cells typically include prokaryotic cells, e.g., bacterial cells such as E. coli. Such cells may be cultured using conventional methods to produce the polypeptide of the present invention.

[0087] A polypeptide may be derivatized or modified to aid in the production, isolation, or purification of the polypeptide. For example, when the polypeptide of the present invention is produced by recombinant expression in a bacterial host cell, the polypeptide sequence may include an additional methionine (M) residue at the N-terminus to improve expression. As another example, a polypeptide of the present invention may be derivatized or modified by adding a ligand capable of directly and specifically binding to a separation means. Alternatively, the polypeptide may be derivatized or modified by adding one member of a binding pair, and the separation means comprises a reagent that has been derivatized or modified by adding the other member of the binding pair. Any suitable binding pair may be used. In a preferred embodiment in which a polypeptide for use in the present invention is derivatized or modified by adding one member of a binding pair, the polypeptide is preferably tagged with histidine or biotin. Typically, the amino acid coding sequence for the histidine or biotin tag is included at the genetic level, and the polypeptide is recombinantly expressed in E. coli. The histidine or biotin tag is typically present at either end of the polypeptide, preferably the C-terminus. The tag may be linked directly to the polypeptide or indirectly by any suitable linker sequence, for example, 3, 4, or 5 glycine residues. Histidine tags typically consist of 6 histidine residues, but may be longer, typically up to 7, 8, 9, 10, or 20 amino acids, or may be shorter, for example, 5, 4, 3, 2, or 1 amino acid.

[0088] The amino acid sequence of a polypeptide may be modified to include non-naturally occurring amino acids, for example, to enhance stability. If the polypeptide is produced by synthetic means, such amino acids may be introduced during production. Alternatively, the polypeptide may be modified after either synthetic or recombinant production. Also, D-amino acids may be used to produce the polypeptide. In such cases, the amino acids will be linked in reverse order in a C→N orientation. This is conventional in the art for producing such polypeptides.

[0089] Many side chain modifications are known in the art and can be made to the side chains of polypeptides, provided that the polypeptide retains any additional desired activity or characteristic, as may be specified herein. It will also be understood that polypeptides can be chemically modified, e.g., post-translationally modified, e.g., glycosylated, phosphorylated, or contain modified amino acid residues.

[0090] The polypeptide may be PEGylated. The polypeptide of the present invention may be in a substantially isolated form. The polynucleotide may be mixed with a carrier or diluent that does not interfere with its intended use (as discussed below) and still be considered substantially isolated. It may also be in a substantially purified form, which generally comprises at least 90%, e.g., at least 95%, at least 98%, or at least 99% of the protein in the preparation.

[0091] Compositions and formulations comprising polypeptides In another aspect, the present invention provides compositions comprising the 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. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not harmful to the subject to which it is administered. Typically, the carrier and final composition are sterile and pyrogen-free.

[0092] Suitable compositions can be formulated using standard pharmaceutical formulation chemistry and methodology, all of which are readily available to those skilled in the art. For example, the agent may be combined with one or more pharmaceutically acceptable excipients or vehicles. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, reducing agents, etc., may be present in the excipient or vehicle. Suitable reducing agents include cysteine, thioglycerol, thioredoxin, glutathione, etc. Excipients, vehicles, and auxiliary substances are generally pharmaceutical agents that do not induce an immune response in the individual receiving the composition and 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 therein, such as mineral acid salts, such as hydrochlorides, hydrobromides, phosphates, and sulfates; and salts of organic acids, such as acetates, propionates, malonates, and benzoates. A thorough discussion of pharmaceutically acceptable excipients, vehicles, and auxiliary substances is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0093] Such compositions may be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable compositions may be prepared, packaged, or sold in unit dosage form, such as in ampoules or multi-dose containers containing a preservative. Compositions include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such compositions may further comprise one or more additional ingredients, including, but not limited to, suspending agents, stabilizers, or dispersing agents. In one embodiment of a composition for parenteral administration, the active ingredient is provided in a dry form (e.g., for powders or granules) for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water), after which the reconstituted composition is administered parenterally. The composition may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. The suspension or solution may be formulated according to art-known methods and may contain, in addition to the active ingredient, additional ingredients such as dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations can be prepared using a non-toxic parenterally acceptable diluent or solvent, such as water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils, such as synthetic mono- or diglycerides.

[0094] Other useful parenterally administrable compositions include those containing the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for controlled release or implantation may contain pharmaceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts. The compositions may be suitable for administration by any suitable route, including, for example, intradermal, subcutaneous, transdermal, intramuscular, intraarterial, intraperitoneal, intraarticular, intraosseous, intrathecal, or other suitable administration route. Preferred compositions are suitable for administration by intravenous infusion.

[0095] Methods of using polypeptides The present invention provides uses of the polypeptides of the present invention in various methods. For example, the polypeptides can provide useful tools in biotechnology. The polypeptides can be used for the specific ex vivo cleavage of IgG, particularly human IgG. In such methods, the polypeptides can be incubated with a sample containing IgG under conditions that allow specific cysteine ​​protease activity to occur. Specific cleavage can be confirmed and the cleavage products isolated using any suitable method, such as those described in WO 2003051914 and WO 2009033670. Thus, this method can be used, in particular, to produce Fc and F(ab')2 fragments. Fab fragments can then be generated by performing a reduction step (e.g., in 2-mercaptoethanolamine or cysteamine) on the F(ab')2 fragments generated by cleaving IgG with the polypeptides of the present invention.

[0096] The methods can also be used to detect or analyze IgG in a sample or to remove IgG from a sample. Methods for detecting IgG in a sample typically involve incubating a polypeptide with the sample under conditions that allow IgG-specific binding and cleavage. The presence of IgG can be confirmed by detection of specific IgG cleavage products, which can then be analyzed.

[0097] The polypeptides of the present invention can also be used for treatment or prevention. In therapeutic applications, a polypeptide or composition is administered to a subject already suffering from a disorder or condition in an amount sufficient to cure, alleviate, or partially halt the condition or one or more of its symptoms. Such therapeutic treatment may result in a decrease in the severity of disease symptoms or an increase in the frequency or duration of symptom-free periods. An amount adequate to accomplish this is defined as a "therapeutically effective amount." In prophylactic applications, a polypeptide or composition is administered to a subject not yet exhibiting symptoms of a disorder or condition in an amount sufficient to prevent or delay the onset of symptoms. Such an amount is defined as a "prophylactically effective amount." The subject may have been identified by any suitable means as being at risk for developing a disease or condition. Accordingly, the present invention also provides polypeptides of the present invention for use in treating the human or animal body. Also provided herein are methods for preventing or treating a disease or condition in a subject, comprising administering to the subject a prophylactically or therapeutically effective amount of a polypeptide of the present invention. 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, intrathecal, or other suitable administration route. The amount of polypeptide administered may be 0.01 mg / kg BW to 2 mg / kg BW, 0.05 to 1.5 mg / kg BW, 0.1 mg / kg BW to 1 mg / kg BW, preferably 0.15 mg / kg to 0.7 mg / kg BW, most preferably 0.2 mg / kg to 0.3 mg / kg BW, and particularly 0.25 mg / kg BW. The polypeptide may be administered multiple times to the same subject, provided that the amount of ADA in the subject's serum capable of binding to the polypeptide does not exceed a threshold determined by the clinician. The amount of ADA in the subject's serum capable of binding to the polypeptide can be determined by any suitable method, such as an agent-specific CAP FEIA (ImmunoCAP) test or a titer assay.

[0098] The polypeptides of the present invention may be particularly useful in the treatment or prevention of diseases or conditions mediated by pathogenic IgG antibodies. Accordingly, the present invention provides polypeptides of the present invention for use in the treatment or prevention of diseases or conditions mediated by pathogenic IgG antibodies. The present invention also provides a method for treating or preventing diseases or conditions mediated by pathogenic IgG antibodies, comprising administering a polypeptide of the present invention to an individual. The method may comprise repeated administration of the polypeptide. The present invention also provides polypeptides of the present invention for use in the manufacture of a medicament for treating or preventing diseases or conditions mediated by pathogenic IgG antibodies, particularly autoimmune diseases mediated in whole or in part by pathogenic IgG antibodies.

[0099] Pathogenic antibodies may be specific for antigens that are typically targeted in autoimmune diseases or other conditions mediated in whole or in part by antibodies. Table D provides 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 in treating or preventing autoimmune diseases mediated in whole or in part by pathogenic IgG antibodies.

[0100] [Table 5-1]

[0101] [Table 5-2]

[0102] [Table 5-3]

[0103] In another embodiment, the polypeptides of the invention may be used in a method for improving the benefit of a therapy or therapeutic agent in a subject, the method comprising two steps, referred to herein as steps (a) and (b).

[0104] 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) then comprises administering the therapy or therapeutic agent to the subject. Steps (a) and (b) are preferably separated by a time interval sufficient to cleave substantially all IgG molecules present in the subject's plasma. The interval may typically be at least 30 minutes and up to 21 days.

[0105] The therapeutic agent whose benefit is improved is typically an antibody administered for the treatment of cancer or another disease. The therapeutic agent may be IVIg. In the context of this embodiment, the present invention alternatively relates to a method for treating cancer or another disease in a subject, comprising: (a) administering to the subject a polypeptide of the present invention; and (b) subsequently administering to the subject a therapeutically effective amount of an antibody that treats the cancer or another disease, - the amount of the polypeptide administered is sufficient to cleave substantially all IgG molecules present in the plasma of the subject; and The method may also be described as providing steps (a) and (b) separated by a time interval of at least 30 minutes and up to 21 days.

[0106] In other words, the present invention also provides polypeptides for use in such methods for treating cancer or another disease. The present invention also provides use of agents in the manufacture of a medicament for treating cancer or another disease by such methods. Cancers include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendiceal cancer, astrocytoma, childhood cerebellar or cerebral, basal cell carcinoma, bile duct cancer, extrahepatic, bladder cancer, bone cancer, osteosarcoma / malignant fibrous histiocytoma, brain stem glioma, brain cancer, brain tumor, cerebellar astrocytoma, brain tumor, cerebral astrocytoma / malignant glioma, brain tumor, ependymoma, brain tumor, medulloblastoma, brain tumor, supratentorial primitive neuroectodermal tumor, brain tumor, visual pathway and hypothalamic glioma, breast cancer, and bronchial adenoma. / Carcinoid, Burkitt lymphoma, carcinoid tumor, carcinoid tumor, digestive system, carcinoma of unknown primary site, central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, cervical cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing sarcoma in Ewing family tumors, extracranial germ cell tumor, children, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, intraocular melanoma , eye cancer, retinoblastoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumors: extracranial, extragonadal, or ovarian, gestational trophoblastic tumor, brain stem glioma, glioma, childhood cerebral astrocytoma, glioma, childhood visual pathway and hypothalamus, gastric carcinoid, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, pancreatic islet cell carcinoma (endocrine pancreas), Kaposi's sarcoma, kidney cancer (renal cells ), laryngeal cancer, leukemia, leukemia, acute lymphoblastic (also called acute lymphocytic leukemia), leukemia, acute myeloid (also called acute myeloid leukemia), leukemia, chronic lymphocytic (also called chronic lymphocytic leukemia), leukemia, chronic myeloid (also called chronic myeloid leukemia), leukemia, hairy cell, lip and oral cancer, liposarcoma, liver cancer (primary), lung cancer, non-small cell lung cancer, small cell, lymphoma, lymphoma, AIDS-related, lymphoma, Burkitt's, lymphoma, cutaneous T-cell, lymphoma, Hodgkin's, lymphoma,Non-Hodgkin's (old classification of all lymphomas except Hodgkin's), lymphoma, primary central nervous system, macroglobulinemia, Waldenstrom, malignant fibrous histiocytoma / osteosarcoma of bone, medulloblastoma, melanoma, melanoma, intraocular (eye), Merkel cell carcinoma, mesothelioma, adult malignancy, mesothelioma, occult primary metastatic squamous cell neck cancer, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorder, myeloid leukemia, chronic myeloid leukemia, adult Acute, myeloid leukemia, childhood acute, myeloma, multiple (cancer of the bone marrow), myeloproliferative disorders, nasal and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer (surface epithelial-stromal tumor), ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, pancreatic islet cell, paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pineoblastoma and tetanus Epithelial primitive neuroectodermal tumor, pituitary adenoma, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), renal pelvis and ureter, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Ewing family tumor, Kaposi's sarcoma, sarcoma, soft tissue, sarcoma, uterus, Sezary syndrome, skin cancer (non-melanoma), skin cancer (melanoma), skin carcinoma, Merkel cell, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, occult The cancer may be primary squamous cell carcinoma of the neck, metastatic, gastric cancer, supratentorial primitive neuroectodermal tumor, T-cell lymphoma, cutaneous - see mycosis fungoides and Sézary syndrome, testicular cancer, pharyngeal cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, thyroid carcinoma, transitional cell carcinoma of the renal pelvis and ureter, choriocarcinoma, ureter and renal pelvis, transitional cell carcinoma of the urethra, uterine cancer, endometrium, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, Waldenström's macroglobulinemia and Wilms' tumor (kidney cancer).

[0107] 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.

[0108] The antibody administered in step (b) is preferably specific to the tumor antigen associated with one or more of the above-mentioned cancer types.The target of interest of the antibody used in the method includes CD2, CD3, CD19, CD20, CD22, CD25, CD30, CD32, CD33, CD40, CD52, CD54, CD56, CD64, CD70, CD74, CD79, CD80, CD86, CD105, CD138, CD174, CD205, CD227, CD326, CD340, MUC16, GPNMB, PSMA, Cripto, ED-B, TMEFF2, EphA2, EphB2, FAP, αv integrin, mesothelin, EGFR, TAG-72, GD2, CA1X, 5T4, α4β7 integrin, and Her2. Other targets are cytokines such as interleukins IL-1 to IL-13, tumor necrosis factors α and β, interferons α, β, and γ, transforming growth factor β (TGF-β), colony-stimulating factors (CSFs), and granulocyte-monocyte colony-stimulating factor (GMCSF). See Human Cytokines: Handbook for Basic & Clinical Research (Aggrawal et al. eds., Blackwell Scientific, Boston, MA 1991). Other targets are hormones, enzymes, and intracellular and intercellular messengers, such as adenylyl cyclase, guanyl cyclase, and phospholipase C. Other targets of interest are leukocyte antigens, such as CD20 and CD33. Drugs may also be targets of interest. Target molecules may be human, mammalian, or bacterial. Other targets are antigens, such as proteins, glycoproteins, and carbohydrates of microbial pathogens, both viral and bacterial, and tumors. Still other targets are described in US Pat. No. 4,366,241.

[0109] The antibody may be directly or indirectly conjugated to a cytotoxic moiety or a detectable label. The antibody may be administered via one or more routes of administration using one or more of a variety of methods known in the art. The route and / or mode of administration will vary depending on the desired results. Preferred routes of administration of the antibody include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, such as injection or infusion. The term "parenteral administration," as used herein, refers to modes of administration other than enteral and topical administration, typically by injection. Alternatively, the antibody may be administered via a parenteral route, such as a topical, epidermal, or mucosal route of administration. Topical administration is also preferred, including peritumoral, juxtatumoral, intratumoral, intralesional, perilesional, intracavitary infusion, intravesical administration, and inhalation.

[0110] Appropriate dosages of antibodies of the present invention can be determined by skilled physicians. Actual dosage levels of antibodies may be varied to obtain an amount of active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular antibody used, 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 used, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and similar factors well known in the medical field.

[0111] A suitable dose of the antibody can range, for example, from about 0.1 μg / kg (body weight of the patient to be treated) to about 100 mg / kg (body weight). For example, a suitable dosage may be from about 1 μg / kg (body weight) to about 10 mg / kg per day, or from about 10 μg / kg (body weight) to about 5 mg / kg per day.

[0112] The dosage regimen may be adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus may be administered, or step (b) of the method may comprise several divided doses administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation, provided that the necessary interval between steps (a) and (b) is not exceeded. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions in unit dosage form. As used herein, unit dosage form refers to physically discrete units suitable as unitary dosages for the treated subject, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0113] The antibody of step (b) may be administered in combination with chemotherapy or radiation therapy. The method may further include administering an additional anti-cancer antibody or other therapeutic agent, which may be administered with the antibody of step (b) in a single composition or in a separate composition as part of a combination therapy. For example, the antibody of step (b) may be administered before, after, or simultaneously with the other agent.

[0114] Antibodies include abagovomab, abciximab, actoxumab, adalimumab, adecatumumab, afelimomab, afutuzumab, alacizumab pegol, ALD518, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, anatumomab mafenatox, anrukinzumab, apolizumab, arcitumomab, acelizumab, atinumab, atulizumab (= tocilizumab), atrolimumab, bapineuzumab, basiliximab, bavituximab, bectumomab, belimumab, benralizumab, bertilimumab, besilesomab, and bevacizumab. Zumab, bezlotoxumab, biciromab, bimagrumab, bivatuzumab mertansine, blinatumomab, brosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab mertansine, cantuzumab vedotin, caplacizumab, capromab pendetide, carlumab, catumaxomab, CC49, cedelizumab, certolizumab pegol, cetuximab, Ch.14.18, sitatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, conatumumab, con Cizumab, crenezumab, CR6261, dacetuzumab, daclizumab, dalotuzumab, daratumumab, demcizumab, denosumab, detumomab, dorlimomab alitox, drozitumab, durigotumab, dupilumab, dusigitumab, ecromeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab, elotuzumab, ersilimomab, enavatuzumab, enlimomab pegol, enokizumab, enoticumab, ensituximab, epitumomab cituxetan, epratuzumab, erlizumab, ertumaxomab, Thalasizumab, etrolizumab, evolocumab, exibirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, FBTA05, felvizumab, fezakinumab, ficlatuzumab, figitumumab, framvotumab, fontolizumab, foralumab, foravirumab, fresolimumab, furanumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab, girentuximab, glembatumumab vedotin, golimumab, gomiliximab, GS6624,Ibalizumab, ibritumomab tiuxetan, icrucumab, igovomab, imciromab, imgatuzumab, inlacumab, indatuximab ravtansine, infliximab, intetumumab, inolimomab, inotuzumab ozogamicin, ipilimumab, iratumumab, itolizumab, ixekizumab, keliximab, labetuzumab, lampalizumab, lebrikizumab, remaresomab, lerdelimumab, lexatumumab, ribivirumab, ligelizumab, lintuzumab, lirilumab, roderucizumab, lorvotuzumab mertansine, lucatumumab, Miriximab, mapatumumab, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimumab, milatuzumab, minletumomab, mitumomab, mogamulizumab, morolimumab, motavizumab, moxetumomab pasudotox, muromonab CD-3, nacolomab butafenatox, namilumab, naptumomab estafenatox, narunatumab, natalizumab, nebacumab, necitumumab, nerelimomab, nesbacumab, nimotuzumab, nivolumab, nofetumomab merpentan, obinutuzumab, ocralizumab, ocrelizumab, odulimomab , ofatumumab, olaratumab, olokizumab, omalizumab, onartuzumab, oportuzumab monatox, oregovomab, olticumab, otelixizumab, oxelumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, panobacumab, palsatuzumab, pascolizumab, pateclizumab, patritumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pinatuzumab vedotin, pintumomab, placurab, polatuzumab vedotin, ponezumab, priliximab, pritoxaximab Mab, Pritumumab, PRO140, Quilizumab, Racotumomab, Ladletumab, Rafivirumab, Ramucirumab, Ranibizumab, Raxibacumab, Regavirumab, Reslizumab, Rilotumumab, Rituximab, Lobatumumab, Loredumab, Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab, Samalizumab, Sarilumab, Satumomabpendetide, Secukinumab, Seribantumab, Cetoxaximab, Sevirumab, Sibrotuzumab, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab, Sirukumab, Solanezumab, Solitomab,Sonepcizumab, sontuzumab, stamulumab, sulesomab, subizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tefibazumab, terimomab alitox, tenatumomab, teneliximab, teplizumab, teprotumumab, TGN1412, ticilimumab (= tremelimumab), tildrakizumab, tigatuzumab, TNX-650, tocilizumab (= atulizumab), toralizumab, tositumomab, tocilizumab It may be larokinumab, trastuzumab, TRBS07, tregalizumab, tremelimumab, tucotuzumab celmoleukin, tuvilumab, ublituximab, urelumab, urtoxazumab, ustekinumab, bapaliximab, batelizumab, vedolizumab, veltuzumab, bepalimomab, besencumab, visilizumab, volociximab, borsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, diralimumab, or zolimomab alitox.

[0115] Preferred antibodies include natalizumab, vedolizumab, belimumab, atacicept, alefacept, otelixizumab, teplizumab, rituximab, ofatumumab, ocrelizumab, epratuzumab, alemtuzumab, abatacept, eculizumab, omalizumab, canakinumab, meplizumab, reslizumab, tocilizumab, and ustekinumab. Mab, briakinumab, etanercept, infliximab, adalimumab, certolizumab pegol, golimumab, trastuzumab, gemtuzumab, ozogamicin, ibritumomab, tiuxetan, tostitumomab, cetuximab, bevacizumab, panitumumab, denosumab, ipilimumab, brentuximab, and vedotin.

[0116] The therapy of improved benefit is typically organ transplantation. The organ may be selected from kidney, liver, heart, pancreas, lung, or small intestine. The treated subject may preferably be sensitized or highly sensitized. "Sensitized" means that the subject has developed antibodies against human major histocompatibility (MHC) antigens (also called human leukocyte antigens (HLA)). Anti-HLA antibodies originate from allosensitized B cells and are usually present in patients who are already sensitized by blood transfusion, previous transplantation, or pregnancy (Jordan et al., 2003).

[0117] Whether a prospective transplant recipient is sensitized can be determined by any suitable method. For example, a panel reactive antibody (PRA) test can be used to determine whether the recipient is sensitized. A PRA score of >30% is typically interpreted as meaning that the patient is at "high immunological risk" or "sensitized." Alternatively, a cross-match test can be performed in which a blood sample from the prospective transplant donor is mixed with a blood sample from the intended recipient. A positive cross-match means that the recipient has antibodies that react with the donor's sample, indicating that the recipient is sensitized and should not undergo transplantation. A cross-match test is typically performed as a final check immediately before transplantation.

[0118] The presence of high-titer antibodies (i.e., donor-specific antibodies [DSA]) against the MHC antigens of a potential donor is a direct contraindication to transplantation due to the risk of acute antibody-mediated rejection. Sensitization to donor MHC antigens therefore hampers the identification of suitable donors. A positive crossmatch is a clear obstacle to transplantation. Approximately one-third of patients waiting for kidney transplants are sensitized, and as many as 15% are highly sensitized, leading to a backlog of patients waiting for transplants. In the United States, the median waiting list time for kidney transplants in 2001–2002 was 1,329 days for those with a panel reactive antibody (PRA) score of 0–9%, 1,920 days for those with a PRA of 10–79%, and 3,649 days for those with a PRA of 80% or greater (OPTN-Database, 2011).

[0119] One accepted strategy to overcome the DSA barrier is to apply plasma exchange or immunoadsorption, often in combination with intravenous gamma globulin (IVIg) or rituximab, to reduce DSA levels 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 involve repeated treatments over a long period of time, making them inefficient and requiring careful planning. When organs from deceased donors become available, they must be transplanted within hours because prolonged cold ischemic time is one of the most significant risk factors for delayed graft function and allograft loss in kidney transplantation (Ojo et al., (1997) Transplantation 63:968-74).

[0120] In contrast, the method of the present invention can rapidly, temporarily, and safely eliminate DSA in prospective transplant recipients. Administration of the polypeptide of the present invention immediately before transplantation has the potential to effectively desensitize highly sensitized patients, thereby enabling transplantation and avoiding acute antibody-mediated rejection. A single administration of the polypeptide before transplantation will enable transplantation in thousands of patients with donor-specific IgG antibodies.

[0121] In the context of this embodiment, the method is alternatively a method of treating organ failure in a subject, comprising: (a) administering to the subject a polypeptide of the invention; and (b) thereafter transplanting a transplant organ into the subject; - the amount of the polypeptide administered is sufficient to cleave substantially all IgG molecules present in the plasma of the subject; and The method may also be described as one in which steps (a) and (b) are separated by a time interval of at least 30 minutes and at most 21 days.

[0122] That is, this embodiment can be described as a method for preventing rejection of a transplanted organ, particularly acute antibody-mediated transplant rejection, in a subject, comprising administering a polypeptide of the present invention to the subject at least 30 minutes and up to 21 days prior to transplantation of the organ, 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 use of a 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 use of a polypeptide of the present invention in the manufacture of a medicament for treating organ failure or preventing transplant rejection by such a method. In this embodiment, the method of the present invention may further comprise a step performed at or immediately before transplantation, which step comprises the induction of T cells and / or B cells in the patient. The induction of T cells and / or B cells may typically comprise the administration of an effective amount of an agent that kills or inhibits T cells and / or an effective amount of an agent that kills or inhibits B cells. Agents that kill or inhibit T cells include muromonab, basiliximab, daclizumab, antithymocyte globulin (ATG) antibodies, and lymphocyte immunoglobulin, antithymocyte globulin preparation (ATGAM). Rituximab is known to kill or inhibit B cells.

[0123] Polypeptides having IgG cysteine ​​protease activity, such as the polypeptides of the present invention, may also be useful in methods of inducing hematopoietic chimerism in a subject, for example, in the context of transplantation of hematopoietic stem / progenitor cells (HSPCs) into the subject. Thus, the polypeptides of the present invention can be used in methods of inducing hematopoietic chimerism in a subject, comprising administering a conditioning regimen of the present invention to the subject, followed by administration of HSPCs to the subject in an amount sufficient and under suitable conditions to induce hematopoietic chimerism in the subject. Alternatively, the method can be described as a method of stably transplanting HSPCs. HSPCs may be autologous (the subject's / patient's own cells are used), syngeneic (the cells are derived from a genetically identical twin), or allogeneic (the cells are derived from a separate, non-identical donor). Immune complications that reduce the likelihood of successful engraftment of HSPCs in the recipient are most pronounced in allogeneic cells; therefore, methods of inducing hematopoietic chimerism are of greatest benefit to such cells. However, even autologous cells can lead to immune complications if they express products to which the recipient has not previously been exposed. If autologous cells are genetically modified to express a gene therapy, the cells may be altered sufficiently to elicit an immune response. For example, there may be an immune response to the expressed gene therapy product. The same is true if HSPCs are genetically modified to express a different HLA type that is incompatible with the recipient's HLA.

[0124] Polypeptides having IgG cysteine ​​protease activity, such as the polypeptides of the present invention, can also be used in combination with adoptive cell transfer immunotherapy. The limited survival and sustained activity of transplanted cells, such as CAR-T cells, can reduce the efficacy of adoptive cell transfer immunotherapy. Proteins with IgG cysteine ​​protease activity can protect the transferred cells. Specifically, pre-existing antibodies and antibodies generated after administration of the transferred cells can shorten the potency of the transferred cells, and the therapeutic effect of the transferred cells can benefit from removing antibody effector function through conditioning of the recipient. Thus, administering a protein with IgG cysteine ​​protease activity can increase the viability and activity of the transferred cells, thereby improving the patient benefit of adoptive cell transfer immunotherapy and improving therapy and prognosis in situations such as cancer treatment. In this context, a method for treating cancer may include administering a polypeptide of the present invention having IgG cysteine ​​protease activity before and / or after administering one or more doses of adoptive cell transfer immunotherapy. Polypeptides having IgG cysteine ​​protease activity, such as the polypeptides of the present invention, can also be used in conjunction with adoptive cell transfer immunotherapy in the context of methods for treating autoimmune conditions, infectious diseases, and conditions mediated by harmful antibodies, making it possible to inactivate both pre-existing anti-drug antibodies (ADAs) and antibodies elicited by adoptive cell transfer immunotherapy. [Example]

[0125] Unless otherwise noted, the methods used are standard biochemical and molecular biological techniques. Examples of suitable methodological 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.

[0126] Example 1 - Expression and purification of pCART239 The mature IdeZ molecule and sequence were analyzed to identify regions suitable for mutation. In some cases, in silico evaluation was used to assess the likely consequences of mutations. pCART239 was sequenced, and cDNAs encoding each polypeptide were generated at GeneCust (Luxembourg) by site-directed mutagenesis of the starting sequence or synthesis, depending on the number of mutations to be introduced. The cDNAs were sequenced and transferred into the pET9a expression vector (Novagen) in frame with a C-terminal 6xHis tag linked to the C-terminus by a short glycine linker (3xGly). For bacterial expression, a methionine was added to the N-terminus. Thus, the pCART239 polypeptide is equivalent to SEQ ID NO: 1 with an additional (1) N-terminal methionine and (2) a C-terminal 6xHis tag linked to the C-terminus by a short glycine linker (3xGly).

[0127] The pCART239 expression plasmid was transformed into Escherichia coli (E. coli) T7E2 (SOURCE) and plated onto LB agarose plates containing 50 μg / mL kanamycin. Single colonies were picked and overnight cultures (10 mL LB medium) were started at 37°C and 250 rpm. The following day, 5 mL of the overnight culture was inoculated into two flasks containing 125 mL of LB medium + 50 μg / mL kanamycin + 1:100,000 diluted antifoam. The culture flasks were already at 37°C at the time of inoculation, and the cultures were grown (37°C, 300 rpm) until the OD reached 0.6-0.7. At this point, IPTG (final concentration 1 mM) was added to induce expression, and the cultures were further incubated for at least 2 hours. After incubation, the bacterial suspension was collected by centrifugation (10 min, 4000 × g, 4°C), and the supernatant was discarded. The pellet was washed once with PBS (30 mL), centrifuged again, and the supernatant discarded. The final pellet was frozen at -20°C and stored in the freezer overnight. Bacterial lysis was performed using a Panda Plus 2000 homogenizer (GEA) according to the manufacturer's instructions, or using a freeze-thaw protocol (three freeze / thaw cycles in 10 mL PBS with sterile glass beads). After the freeze-thaw cycle, the tubes were centrifuged (20 min, 25,000 × g, 4°C) to isolate the bacterial lysate (supernatant) and then stored on ice. The final bacterial lysates were pooled and sterile filtered through a 0.2 μm nylon filter (HPF Millex®-Nylon), and the protein was purified using Ni-NTA prepacked spin columns (ThermoFisher). After purification, DTT (final concentration 5 mM) and EDTA (final concentration 5 mM) were added to the eluate before buffer exchange (Amicon Ultra-4 10K). Protein concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific). Purified protein purity and stability during the expression purification process were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using stainless steel 12% Mini-PROTEAN® TGX™ precast gels (Biorad) (Figure 1). Figure 1A shows successful IPTG induction and subsequent overexpression of pCART239.FIG. 1B shows that the purification process was also successful, resulting in a pCART239 sample of high purity and stability.

[0128] Example 2 - Efficacy of pCART207, pCART229, N240, and pCART242 against Humira (IgG1) and XGEVA (IgG2) In this example, human IgG1 is represented by Humira (Abbvie) and IgG2 by XGEVA (Amgen). These are used to compare the activity of IdeZ variants and the IdeS positive control in cleaving monoclonal human IgG. The IdeZ variant enzymes (and IdeS) were titrated in 0.05% BSA in PBS in 1:3 dilution steps from a starting concentration of 3.3 μg / mL, and cleavage products were analyzed by SDS-PAGE (4% to 20% gradient gel). The pCART242 polypeptide tested is equivalent to SEQ ID NO: 2, with an additional (1) N-terminal methionine and (2) a C-terminal 6xHis-tag linked to the C-terminus by a short glycine linker (3xGly).

[0129] method: 1. 25 μL of the enzyme and control (buffer) dilutions were transferred to a multititer plate. 2. The reaction was initiated by adding 25 μL of a 2 mg / mL solution of Humira or XGEVA to each well, resulting in a 1 mg / mL concentration of each antibody in the reaction. Taking into account serial dilutions, the highest concentration of IdeZ (or IdeS) tested was 3.3 μg / mL, decreasing to a minimum of 0.057 ng / mL. 3. Incubate the plate at 37°C for 2 hours with gentle rotation. 4. After incubation, 10 μL of each sample was mixed with 30 μL of 2×SDS loading buffer in the microtiter plate. After overnight storage (4-8°C), the mixture was transferred to a 1.5 mL tube and incubated at 92°C for 5 minutes. 10 μL of the sample was loaded onto a 15-well 4→20% Mini-PROTEAN® TGX™ precast gel, and the samples were analyzed by SDS-PAGE under non-reducing conditions.

[0130] Gels showing the digestion of IgG1 (Humira) and IgG2 (XGEVA) by IdeZ variants are shown in Figures 2 and 3, respectively. Different panels represent different sets of experiments (although the protocol was identical in both cases).

[0131] The approximate concentrations for cleaving the first and second heavy chains of IgG1 / IgG2 were estimated from the cleavage patterns shown on the gel as follows:

[0132] [Table 6]

[0133] [Table 7]

[0134] [Table 8]

[0135] [Table 9]

[0136] overview: The efficacies of IdeS, pCART207, and pCART229 for cleaving IgG1 (Humira) are very similar. pCART239 and N240 show higher efficacies than both IdeS and the very similar variant pCART229. N240 and pCART242 appear to be more potent than IdeS in cleaving the second IgG1 heavy chain. N240 and pCART242 are not only more potent than pCART229 and pCART207 primarily for the second cleavage of IgG2 (Xgeva), but are also more potent in cleaving the first heavy chain of IgG2.

[0137] Example 3 - Efficacy of N240 against human IgG subclasses This report describes the characterization of N240 activity in vitro by digestion of various IgG subclasses, in which two different in-house produced N240 batches were compared, and the reference material used was IdeS.

[0138] method: 1. 25 μL of N240 or IdeS enzyme and control (buffer) dilutions were transferred to a multititer plate. Between each successive well, the enzyme was serially diluted 1:3 in 0.05% BSA in PBS. 2. The reaction was initiated by adding 25 μL of a 2 mg / mL solution of human IgG to each well. This resulted in 1 mg / mL of each antibody in the reaction. The IgG1 used was Humira (Abbvie), the IgG2 used was XGEVA (Amgen), the IgG3 used was from Sigma (I5654, lot number SLBW0899), and the IgG4 used was from Abcam (ab90286, lot number GR3180469). 3. Incubate the plate at 37°C for 2 hours with gentle rotation. 4. After incubation, 10 μL of each sample was mixed with 30 μL of 2x SDS loading buffer in the microtiter plate. After overnight storage (4-8°C), the mixture was transferred to a 1.5 mL tube and incubated at 92°C for 5 minutes. 10 μL of the sample was then loaded onto a 15-well 4→20% Mini-PROTEAN® TGX™ precast gel.

[0139] A gel showing the digestion of IgG subclasses by N240 and IdeS is shown in FIG.

[0140] Estimated EC obtained by visual inspection of the gel 50 The values ​​are listed in Table 3 below.

[0141] [Table 10]

[0142] overview: 1. All four human subclasses IgG1, IgG2, IgG3, and IgG4 are cleaved by N240. 2. N240 clearly shows higher activity (against IdeS), especially for second chain cleavage. EC for IgG1 50 F(ab')2 was approximately 5-fold higher for IdeS compared to N240. 3. For IgG2, IgG3, and IgG4, N240 had similar or slightly lower potency than IdeS.

[0143] Example 4 - Comparison of MSD potency assays between N240 and IdeS method: A more detailed overview of the principles behind the MSD potency assay is provided below in Example 5.

[0144] Briefly, goat anti-human IgG, F(ab')2 fragment specific, was coated onto a 96-well MSD plate. After blocking, the plate was washed, and serially diluted N240 reference material and IdeS test samples were added to the plate. A fixed concentration of human IgG was added, and the plate was incubated at 37°C. After incubation and washing, a detection mix containing biotinylated mouse anti-human IgG (Fc specific) and SULFO-TAG-labeled streptavidin was added to the plate. After the final incubation and wash, Read Buffer was added. The MSD instrument measured the luminescence intensity from the SULFO-TAG to provide a quantitative measure of the uncleaved and single-cleaved IgG in the sample. The N240 reference material and IdeS test samples were analyzed in duplicate wells and triplicate plates. The EC values ​​for each reference and test sample were determined by fitting the results to a five-parameter curve. 50 EC 50 Reference / EC 50 The relative efficacy (%) of each test sample was determined by dividing the test and multiplying by 100%. Reported values ​​are the average from three different plates.

[0145] The resulting dilution curves representing IgG cleavage for IdeS and N240 are shown in FIG.

[0146] The mean relative efficacy of N240 was calculated to be approximately 300% compared to IdeS.

[0147] Example 5 - Efficacy of pCART239 against IgG in human serum The IdeZ variant pCART239 was further characterized by measuring its activity in serum.

[0148] As an IgG substrate, a human serum pool from 100 individuals was used.

[0149] SDS-PAGE The dilution series used for pCART239 was as follows: 30, 15, 7.5, 3.75, 1.9, 0.9, 0.2, 0.2, 0.1, 0.06, and 0.03 μg / mL, following the activity assay and SDS-PAGE analysis protocols outlined in Examples 2 and 3.

[0150] A gel showing the digestion of serum IgG by pCART239 is shown in Figure 6. This figure shows that pCART239 has IgG cleavage activity in the serum pool, and the gel estimates that approximately 0.9 μg / mL of enzyme cleaves IgG into scIgG (Figure 6, upper panel). The scIgG is further digested into F(ab')2 and Fc fragments at approximately 1.9 μg / mL of enzyme.

[0151] The activity values ​​obtained in serum were lower than those obtained in buffer, due to the presence of inhibitory anti-drug antibodies (ADAs) in serum, a phenomenon known from previous studies. To confirm the absence of specific inhibitors other than inhibitory ADAs in serum, we also performed experiments in which an inactive variant of pCART239 (pCART243) was added at 0.1 mg / mL. This addition was in high molar excess and should bind to the inactivated ADAs sufficiently to restore pCART239 activity in serum. This was shown to be the case (Figure 6, lower panel), with the presence of pCART243 resulting in IgG cleavage at much lower pCART239 concentrations. Intact IgG began to be digested already at 0.03 μg / mL, and all IgG was converted to scIgG at 0.5 μg / mL.

[0152] Electrochemiluminescence Mesoscale Discovery (MSD) Platform To further investigate N240 activity in serum, an MSD potency assay was performed in serum matrix.

[0153] The principle of this assay was to coat the wells of a multititer plate with F(ab)2 fragments of a human IgG antibody with specificity for the Fab region. Then, titrated concentrations of IgG cysteine ​​protease polypeptides (test or control) were incubated with human serum in the wells. The amount of intact or single-cleaved human IgG bound to the wells was measured using a detection antibody against human IgG with specificity for the Fc portion of the antibody. The higher the concentration of a given IgG cysteine ​​protease polypeptide in the well, the less intact human IgG antibody bound to the well, and therefore the lower the signal. Similarly, a more potent IgG cysteine ​​protease polypeptide will give a lower signal than a less potent IgG cysteine ​​protease polypeptide when present at the same concentration. A titration dose-response curve was performed for the IdeS control (pCART124) and N240. The EC values ​​of the tested cysteine ​​proteases were then calculated. 50 The efficacy was estimated by calculating the EC 50Lower values ​​represent more potent IgG cysteine ​​proteases. In this assay, cleavage of the first IgG heavy chain from IgG to scIgG is not observed, as the Fc portion of the IgG is still present and can be detected by the Fc-specific detection antibody.

[0154] A brief summary of the laboratory protocol: Wells of a multititer plate were coated overnight (+2–8°C) with goat anti-human Fab-specific F(ab)2-fragment (0.5 μg / mL) (Jackson #109-006-097), washed with PBS + 0.05% Tween 20 (PBS-T), and blocked for 45–120 min at room temperature in 0.45% fish gelatin in PBS-T (blocking buffer). Control IdeS (pCART124) and test IgG cysteine ​​protease polypeptides were prepared as a titration series of 1:4 dilutions in blocking buffer starting at 80 μg / mL. Equal volumes (25 μL) of human serum and titrated amounts of IgG cysteine ​​protease polypeptides were added to the wells and incubated with shaking in a controlled temperature environment at 37°C for 2 h, followed by washing with PBS-T. Biotinylated mouse anti-human IgG Fc-specific (ma-hIgG Bio II, lot: C0013-ZC43C, Southern Biotech) antibody (600 ng / mL) was mixed with strep-sulfo (200 ng / mL) and added to a multititer plate. The plate was sealed with aluminum tape and incubated at +25°C for 1 hour with shaking. The plate was then washed with PBS-T, and 150 μL of 2x diluted Read Buffer T (MSD Read Buffer T, catalog number R92TC-2) was added to each well. The plate was immediately read on a plate reader, MSD (Meso Scale Discovery) QuickPlex SQ 120 Model 1300.

[0155] The resulting standard curves representing IgG cleavage for IdeS and N240 are shown in Figure 7. The table below shows the EC values ​​determined from the dose-response curves. 50 The relative potency estimates based on the values ​​are shown.

[0156] [Table 11]

[0157] overview N240 is active in serum. While the potency of N240 in buffer solution was shown in the previous example to be approximately four-fold higher than that of ImmuRifidase, this difference is smaller when the two enzymes are compared in serum. In the MSD potency assay, N240 also showed a substantial increase in activity against human IgG in serum (approximately 142%) when compared to IdeS.

[0158] Example 6 - ADA levels of N240 and IdeS in healthy individuals The general MSD protocol described in Example 5 was followed with the following exceptions.

[0159] Multi-array MSD plates were coated with 20 μg / mL N240 or IdeS. Plates were blocked with fish gelatin before incubation with sera (1:100 dilution) from healthy individuals (n = 40) and one human serum pool (n = 100). The detection reagent used was anti-human F(ab')2-specific F(ab')2-bio antibody (JAX, 109-066-097) with streptavidin sulfo (MSD #R32AD-1). After adding Read Buffer (MSD #R92TC-2), plates were read using an MSD QuickPlex SQ 120.

[0160] The results are presented in Figure 8. The results show that the level of ADA to N240 is lower than that to IdeS.

Claims

1. has IgG cysteine ​​protease activity, (i) SEQ ID NO: 1; or (ii) SEQ ID NO: 2; or (iii) a variant of SEQ ID NO: 1 or SEQ ID NO: 2 having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid modifications relative to SEQ ID NO: 1 or SEQ ID NO: 2, respectively; 5, (b) an aspartic acid (D) at a position corresponding to position 99 of SEQ ID NO:5, and (c) an asparagine (N) at a position corresponding to position 226 of SEQ ID NO:5, wherein the polypeptide is at least as effective at cleaving human IgG as a polypeptide consisting of the amino acid sequence of SEQ ID NO:1 or 2, respectively, when measured in the same assay.

2. 2. The polypeptide of claim 1, wherein at least one of the modifications in (iii) does not result in the same amino acid present at the corresponding position in the polypeptide sequence of SEQ ID NO:

3.

3. A polypeptide described in claim 1 or 2, wherein none of the modifications in (iii) result in the same amino acid being present at the corresponding position in the polypeptide sequence of SEQ ID NO:

3.

4. 4. The polypeptide of claim 1, further comprising an additional methionine at the N-terminus and / or an additional histidine tag at the C-terminus.

5. A polypeptide described in any one of claims 1 to 4, which is more effective at cleaving human IgG than IdeZ polypeptide and / or is at least as effective at cleaving human IgG as IdeS polypeptide when measured in the same assay.

6. 6. A polypeptide according to any one of claims 1 to 5, which is more effective at cleaving human IgG than an IdeS polypeptide when measured in the same assay, and optionally the effectiveness is measured in vitro in a sample of blood or serum taken from a human subject.

7. 7. The polypeptide of claim 6, which is at least 1.2 times more effective at cleaving human IgG than the IdeS polypeptide when measured in the same assay.

8. A polypeptide according to claim 6 or 7, which is at least 1.3 times more effective at cleaving human IgG than the IdeS polypeptide when measured in the same assay.

9. A polypeptide according to any one of claims 6 to 8, which is at least 1.4 times more effective at cleaving human IgG than the IdeS polypeptide when measured in the same assay.

10. 10. The polypeptide of any one of claims 1 to 9, which is less immunogenic than an IdeS polypeptide and / or does not exceed the immunogenicity of an IdeZ polypeptide when measured in the same assay.

11. 11. The polypeptide of any one of claims 1 to 10, which is less immunogenic than the IdeS polypeptide when measured in the same assay.

12. A polypeptide described in any one of claims 1 to 11, wherein the immunogenicity of the polypeptide is 85% or less of the immunogenicity of the IdeS polypeptide when measured in the same assay.

13. A polynucleotide encoding a polypeptide described in any one of claims 1 to 12.

14. An expression vector comprising the polynucleotide of claim 13.

15. A host cell comprising the polynucleotide of claim 13 or the expression vector of claim 14.

16. The host cell described in claim 15, wherein the host cell is a bacterial cell.

17. The cell described in claim 14 or 15, wherein the host cell is an Escherichia coli (E. coli) cell.

18. A composition comprising a polypeptide according to any one of claims 1 to 12 and at least one pharmaceutically acceptable carrier or diluent.

19. A polypeptide according to any one of claims 1 to 12 for use in the treatment of the human or animal body.

20. 20. The composition of claim 18, for use in preventing or treating a disease or condition in a subject.

21. 21. The composition of claim 20, wherein the disease or condition is a disease or condition mediated in whole or in part by pathogenic IgG antibodies.

22. The disease or condition is ABO-incompatible transplantation, Addison's disease, anti-GBM glomerulonephritis (Goodpasture-associated), antineutrophil cytoplasmic antibody-associated vasculitis (ANCA-associated vasculitis) (Wegener's granulomatosis, Churg-Strauss syndrome, microscopic polyangiitis), anti-NMDAR encephalitis, antiphospholipid syndrome (APS) and refractory APS, autoimmune bullous skin disease (pemphigus), pemphigus foliaceus (PF), Brazilian pemphigus (FS) (endemic type), pemphigus vulgaris (PV), autoimmune lytic anemia Hematopoietic ulcers (AIHA), autoimmune hepatitis (AIH), autoimmune neutropenia (AIN), bullous pemphigoid (BP), celiac disease, chronic urticaria, complete congenital heart block (CCHB), type 1A diabetes mellitus (T1DM), epidermolysis bullosa acquisita (EPC), essential mixed cryoglobulinemia, Goodpasture's syndrome (also known as Goodpasture's disease and anti-glomerular basement membrane disease), Graves' disease (goiter and hyperthyroidism, infiltrative exophthalmos, and infiltrative pemphigoid). skin diseases), Guillain-Barré syndrome (GBS), acute inflammatory demyelinating polyneuropathy (AIDP), acute motor axonal neuropathy (AMAN), hemophilia-acquired FVIII deficiency, IgA nephritis, idiopathic thrombocytopenic purpura (ITP), Lambert-Eaton myasthenic syndrome (LEMS), mixed connective tissue disease (MCTD), multiple myeloma, myasthenia gravis, myasthenic crisis, myocarditis, dilated cardiomyopathy (DCM) (congestive cardiomyopathy), neuromyelitis optica (NMO), primary biliary cirrhosis 22. The composition of claim 20 or 21, wherein the disease is selected from the group consisting of primary progressive multiple sclerosis (PBC), primary progressive multiple sclerosis (PPMS), rheumatic heart disease (RHD), (rheumatic fever), rheumatoid arthritis (RA), serum sickness, immune complex hypersensitivity (type III), Sjogren's syndrome (SS), SLE including lupus nephritis, stiff person syndrome (SPS), systemic sclerosis (scleroderma), transplant rejection, thrombotic thrombocytopenic purpura (TTP), Wegener's granulomatosis (granulomatosis with polyangiitis), and anti-drug antibodies (ADA).

23. 13. An ex vivo method for cleaving IgG, comprising contacting a sample containing IgG with a polypeptide according to any one of claims 1 to 12 under conditions that result in IgG cysteine ​​protease activity.

24. Fc, Fab, and / or F(ab') ２ 24. The method of claim 23, wherein the method is carried out to generate a fragment of

25. 25. The method of claim 23 or 24, wherein the sample is a blood sample taken from a subject suffering from a disease or condition as defined in claim 21 or 22.

26. The composition of claim 18 for use in therapy, comprising: (a) administering the composition to the subject; and (b) thereafter administering the therapy to the subject. the amount of the polypeptide administered in the composition is sufficient to cleave substantially all IgG molecules present in the plasma of the subject; The composition, wherein steps (a) and (b) are separated by a time interval sufficient to cleave substantially all IgG molecules present in the subject's plasma.

27. ​​The composition described in claim 26, wherein the therapy is organ transplantation.

28. The composition of claim 18 for use in improving the benefit of a therapeutic agent in a subject, comprising: (a) administering the composition to the subject; and (b) thereafter administering the therapeutic agent to the subject; the amount of the polypeptide administered in the composition is sufficient to cleave substantially all IgG molecules present in the plasma of the subject; The composition, wherein steps (a) and (b) are separated by a time interval sufficient to cleave substantially all IgG molecules present in the subject's plasma.

29. The therapeutic agent an antibody; Gene therapy such as viral vectors; Substitutes for defective endogenous factors such as enzymes; a growth or clotting factor; or 29. The composition of claim 28, which is a cell therapy.

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