CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent application Ser. No. 15/328,879, filed Jan. 24, 2017, which is a national stage entry under 35 U.S.C. § 371 of International Application No. PCT/EP2015/065895, filed Jul. 10, 2015, which claims the benefit of United Kingdom Application No. 1413240.1, filed Jul. 25, 2014, the entire contents of which are incorporated by reference in their entireties for all purposes herein.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety for all purposes. Said ASCII copy, created Jan. 28, 2016, is named pctep2015065895-seql.txt and is 13,515 bytes in size.

FIELD OF THE INVENTION

The invention relates to a method for improving the benefit of a therapy or a therapeutic agent to a subject. The method comprises (a) administering to the subject an agent which reduces Fc receptor binding of serum IgG molecules in the subject; and (b) subsequently administering said therapy or said therapeutic agent to the subject. The invention also relates to a method for reducing the effect of pathogenic autoantibodies in a subject, the method comprising (a) administering to the subject an agent which reduces Fc receptor binding of serum IgG molecules in the subject and optionally (b) subsequently subjecting the subject to a treatment which removes endogenous autoantibodies. The invention also relates to a kit for carrying out a method of the invention.

BACKGROUND TO THE INVENTION

Antibodies are components of the immune system, which recruit other immune system elements to particular targets within the body. Antibodies are specific to target antigens through the specificity of the Fab domains. Antibodies recruit other elements of the immune system through the interaction of the antibody fragment crystallisable (Fc) domain with Fc receptors (FcRs) expressed on the surface of immune cells. The predominant antibodies in mammalian serum are usually of the immunoglobulin G (IgG) class: IgG1, IgG2, IgG3 and IgG4. These antibodies bind the human FcRs: FcγRI, RγIIa, RγIIb, RγIIIa and FcγRn, and the complement Fc receptor C1q. The efficacy of the recruitment of the cellular immune system by IgG molecules is influenced by the affinity of the Fc to the FcR(s). The interaction between the Fc domain of an antibody and an FcR is important both for the action of antibodies which are administered as therapeutic agents and also of antibodies which play a pathogenic role in various autoimmune conditions including antibody-mediated transplant rejection.

SUMMARY OF THE INVENTION

The inventors have surprisingly shown that it is possible to use an agent to completely, rapidly, temporarily and safely eliminate Fc receptor binding by all or substantially all IgG molecules in the serum of a patient. This creates a window of a defined length in which, if a therapeutic antibody is administered, it will have enhanced efficacy because it does not need to compete for binding to Fc receptors with endogenous IgG. Thus, in one embodiment, the method may be used to treat a disease which is treated by a therapeutic antibody.

The window of defined length may also be used to administer a therapy, such as an organ transplant, which would otherwise be ineffective due to the action of anti-donor IgG antibodies present in the serum of the patient. Thus, in one embodiment, the method may be used to desensitize a patient prior to organ transplantation.

Thus, the present invention provides a method for improving the benefit to a subject of a therapy or a therapeutic agent, the method comprising (a) administering to the subject an agent which reduces Fc receptor binding of serum IgG molecules in the subject; and (b) subsequently administering said therapy or said therapeutic agent to the subject; wherein:

- the amount of said agent administered is sufficient to eliminate Fc receptor binding by all or substantially all IgG molecules present in the serum of the subject; and
- steps (a) and (b) are separated by a time interval which is sufficient for Fc receptor binding by substantially all IgG molecules present in the serum of the subject to be eliminated.
  
  The said interval may typically be of at least 30 minutes and at most 21 days.

The invention may also be used to remove or to reduce the effect of antibodies in a subject. This may be particularly helpful in a patient suffering from an autoimmune disease which is wholly or partly mediated by pathogenic autoantibodies, such as Guillain-Barre syndrome or Goodpastures syndrome. Thus the invention also relates to a method for removing or reducing the effect of antibodies in a subject, the method comprising (a) administering to the subject an agent which reduces Fc receptor binding of serum IgG molecules in the subject, and optionally (b) subsequently subjecting the subject to a treatment which removes endogenous autoantibodies; wherein

- steps (a) and (b) are separated by a time interval of at least 2 weeks; and
- said treatment which removes endogenous autoantibodies is plasmapharesis or immunoadsoprtion, or is administration of an agent (such as an anti-FcRn antibody) which prevents recycling of antibodies in serum by the FcRn receptor, thereby reducing antibody half-life.
  
  The invention also provides a method for assessing the quantity of intact IgG in a sample taken from an individual, the method comprising:
- (i) incubating the sample with a first agent which specifically binds to the F(ab′)₂portion of IgG;
- (ii) incubating the sample with a second agent which specifically binds to the Fc portion of IgG;
- (iii) determining the concentration of intact IgG in the sample by determining the presence of both agents
  
  The invention also provides a kit for carrying out a method of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Schematic representation of IgG cleavage by IdeS. Intact human IgG, regardless of isotype, is cleaved by IdeS in two steps. The first step generates a single-cleaved IgG (scIgG) with one intact heavy chain. The second step generates the fully cleaved products consisting of one F(ab′)₂fragment and one homo-dimeric Fc-fragment hold together by non-covalent interactions.

FIGS. 2A and 2B. Proteinuria was monitored as a safety assessment throughout the human study of Example 1. Multistix (Siemens) were routinely used at the hospital and transient proteinuria was detected in several subjects which correlated to IgG cleavage. FIG. 2A is a graph showing the monitoring of proteinuria in subjects given placebo (n=9). FIG. 2B is a graph showing the monitoring of proteinuria in subjects given a single dose of 0.24 mg/kg BW IdeS (n=4).

FIGS. 3A and 3B. Pharmacokinetics of IdeS in serum. IdeS concentrations in serum were detected by a LC-MS/MS method based on four peptides derived from IdeS. FIG. 3A is a graph comparing serum IdeS concentration one minute before end of infusion versus dose levels of IdeS (0.01, 0.04, 0.12, and 0.24 mg/kg BW) (logarithmic scale: circles individual concentrations). Analyte: peptide LFEYFK (n=20). FIG. 3B is a graph comparing serum concentration of mean values of four peptides (AFPYLSTK, AIYVTDSDSNASIGMK, GGIFDAVFTR and LFEYFK) versus time profiles up to 24 hours after infusion of 0.12 or 0.24 mg/kg BW IdeS (n=8).

FIGS. 4A-4C. Qualitative pharmacodynamics analysis by SDS-PAGE showed rapid degradation of IgG. FIG. 4A shows the results of SDS-PAGE analysis of serum from subjects dosed with 0.12 mg/kg BW IdeS. FIG. 4B shows the results of SDS-PAGE analysis of serum from subjects dosed with 0.24 mg/kg BW IdeS. FIGS. 4A and 4B show protein banding patterns at pre-dosing, 14 min, 20 min, 1, 2, 6 and 24 hours after dosing. FIG. 4C shows the results of analyzing IgG recovery in serum from one subject in the 0.24 mg/kg BW group at pre-dosing, 2 hours, 24 hours, 7 days, 14, 21, 28 and 35 days after dosing. Arrows to the right in each figure show the different bands in the IgG-marker containing a mix of human IgG, scIgG, F(ab′)₂and Fc. Lines to the left in each figure show the molecular mass of the kD standard. The gels show a representative subject in the 0.12 and 0.24 mg/kg BW IdeS dose groups.

FIG. 5. Quantitative pharmacodynamics analysis by ELISA showed rapid degradation of IgG. Serum IgG levels from individual subjects dosed with 0.24 mg/kg BW IdeS determined using a validated ELISA method (detecting both intact IgG and scIgG). To be able to follow both early, rapid degradation as well as recovery of IgG, the x-axis has been split in two. The first part shows time in hours (0-24 hours) and the second shows time in days (7-64 days).

FIG. 6. In vitro titration of IdeS on human serum. Human serum samples from healthy subjects were used as substrates for IdeS and titrated by ELISA (n=20; error bars, mean±SEM). The highest dose group, 0.24 mg/kg BW IdeS, corresponds to approximately 6 mg/L IdeS in vitro, 0.12 mg/kg BW to 3 mg/L, 0.04 mg/kg BW to 1 mg/L and 0.01 mg/kg BW to 0.2 mg/L IdeS in vitro. The results are given as percent remaining IgG on the y-axis compared to the start value for each subject. IdeS dose in mg/L is on the x-axis.

FIG. 7. Antigen-specific pharmacodynamics. Human serum samples from the 0.24 mg/kg BW group (n=4) were addressed for presence of IgG against a mixture of antigens (diphteria, pertussis, tetanus, polio and Haemophilus influenza type b). The results are given as percent remaining IgG on the y-axis compared to the start value for each subject. To be able to follow both early, rapid degradation as well as recovery of IgG, the x-axis has been split in two. The first part shows time in hours (0-24 hours) and the second shows time in days (7-64 days).

FIGS. 8A and 8B. Serum from subjects dosed with IdeS showed impaired phagocytosis capacity. The opsonizing capacity of IgG in human serum was measured as percent of effector cells with at least one engulfed fluorescent bead. FIG. 8A is a graph showing the phagocytosis level before and 24 hours after dosing of 0.24 mg/kg BW IdeS vs. placebo treated subjects. Pre-dose phagocytosis level for each individual was set to 100% and background is spontaneous uptake of beads in the absence of serum, n=4 in the IdeS group and n=2 in the placebo group.

FIG. 8B is a graph showing kinetics of the phagocytic potential in serum is shown for one representative subject in the 0.24 mg/kg BW group at different time-points (pre-dose, 2, 6, 24, 48 hours, 4, 7 and 14 days). The spontaneous uptake of beads in the absence of IgG is shown as an open box. P-value was calculated using Mann-Whitney, ***=P<0.01.

FIGS. 9A-9D. Anti-IdeS antibodies were followed before and throughout the study. Human serum samples were analysed using an IdeS specific CAP-FEIA (ImmunoCAP) assay (Thermo Fisher Scientific) on a Phadia® 250 instrument. The cut-off (LLOQ) for IgG was 2 mg/L. FIG. 9A shows the analysis of samples from 130 human donors (reference) when compared to the 78 healthy human male subjects screened in this study (screening). The highlighted lines show median for the reference group (6.1 mg/L) and the screening group (10.6 mg/L). FIG. 9B is a graph showing kinetics of the anti-IdeS IgG levels shown as a mean for the 0.12 and 0.24 mg/kg groups (n=8; error bars, mean±SEM). No increase in anti-IdeS IgG is seen in any of the subjects prior to day 14. FIG. 9C shows Anti-IdeS IgG levels shown for the separate groups at day 14. FIG. 9D shows Anti-IdeS IgG levels shown for the separate groups at day 182. The lines show median level for each group. P-values were calculated using Kruskal-Wallis, One-Way ANOVA and Dunn's Multiple Comparison: *=P<0.05 and **=P<0.02.

FIG. 10. Efficacy of IdeS in serum from twenty tested donors (healthy volunteers and stage 5 CID patients). Remaining IgG after treatment of human sera with different concentrations of IdeS was determined using ELISA. Figure shows the sigmoid dose-response curves of the individual human sera where remaining IgG in mg/ml is plotted against IdeS dose (g/L). Calculated MABEL (0.0031 g/L) and MED (0.025 g/L) are indicated in the graphs (dark blue line). The selected patient sera P02, P04, P07, P08 and P09 are highlighted in different colours.

FIG. 11. Efficacy of IdeS on anti-HLA IgG in serum from sensitized patient No. P02. Graph shows the MFI (Raw) against individual antigens for (upper graph) MEW class-I (A, B and C) and (lower graph) MEW class-II (DP, DQ and DR) after mock (blue) and IdeS (red) treatment. MFI: Mean fluorescent intensity.

FIGS. 12, 13, 14, 15. Equivalent to FIG. 11 for sensistised patients P04, P07, P08 and P09, respectively.

FIG. 16. Balb/c spleenocytes (gate P1) stained with serum (10 μl non-DTT treated) from subjects 503 and 504 collected pre-dosing (black), 24 h after dosing (red), 48 h after dosing (green) and 96 h (blue) after dosing with IdeS or placebo. Binding was detected using a secondary reagent against human Fcγ. For the IdeS graph, the pre-dosing plot is to the right of all three after dosing plots. Thus, there is a reduction in the ability of serum to bind to mouse splenocytes at 24 hrs that is maintained at 96 hrs.

FIG. 17. Xenogenic cross-match between serum from healthy subject (504) dosed with 0.24 mg/kg BW of IdeS and spleen cells from Balb/c mouse. The serum samples were collected pre-dose and at the indicated time-points post-dose. Sera were treated with DTT to inactivate IgM. Overlay photographs of Terasaki-wells showing living cells (green/bright) and dead cells (red/dull). Spleen cells treated with PBS only (no serum) were used as control for spontantaneous cell death.

FIG. 18 shows a schematic representation of the cleavage of the N-linked glycan at Asn-297 (Kabat numbering) of IgG by EndoS.

FIG. 19 shows a comparison of two methods for measuring IgG levels (turbidimetry and PD-ELISA) in serum from a subject (#101) treated with IdeS. Serum was collected at different time point post dosing with IdeS and measured using the standard p-IgG turbidimetry test at the hospital and using the PD-ELISA assay developed by the inventors to discriminate between intact IgG and F(ab′)₂-fragments generated upon IdeS cleaving IgG. ULN=upper limit of normality and LLN=lower limit of normality for IgG in human healthy subjects.

FIGS. 20A and 20B show that IdeS cleaves IgG-type but not IgM-type of BCR on B-cells. FIG. 20A shows flow cytometry analysis of anti-Fab signal on IgG-type (Nu-DUL-1) and IgM-type (Daudi) of BCR expressing cells after treatment with indicated amounts of IdeS. The y-axis shows mean fluorescent intensity in FL4. FIG. 20B shows flow cytometry analysis of anti-Fab and anti-Fc on the surface of Nu-DUL-1 cells after treatment with different amounts of IdeS.

FIGS. 21A and 21B show that IdeS cleaves the IgG-type of BCR with similar efficacy as soluble IgG. In FIG. 21A, Heparinized peripheral blood was treated with PBS or different amounts of IdeS. After incubation period plasma was isolated and separated on an SDS-PAGE gel as shown. Intact IgG, scIgG and F(ab′)₂fragments are indicated to the right. FIG. 21B is a graph showing the analysis of PBMC's purified from the same PBS or IdeS treated blood when double stained for CD19⁺ and anti-Fc or anti-Fab.

FIGS. 22A and 22B show that IdeS cleaves surface IgG on memory B-cells. FIG. 22A is a graph showing that negative selection of B-cells using RosetteSep resulted in >90% CD19⁺ cells. FIG. 22B is a graph showing that the F(ab′)₂part of surface IgG from CD19⁺/CD27⁺ cells is efficiently cleaved by IdeS. The amount of cell-membrane anchored Fc epitopes does not change after IdeS treatment.

FIGS. 23A-23C show the recovery of cells after IdeS treatment. FIG. 23A is a graph showing flow cytometry analysis of anti-Fab on the surface of Nu-DUL-1 cells after treatment with different amounts of IdeS. IdeS was removed and cells were cultured and analyzed after 1 hour and after 24 hours. FIG. 23B is a graph showing the proliferation of cells by measuring BrdU incorporation, where the Nu-DUL-1 cells were treated with different amounts of IdeS or anti-proliferative control substances (cytochalasin D and puromycin) and cultured for 24 hours prior to BrdU 6 hours pulse time. FIG. 23C is a graph showing viability of Nu-DUL-1 cells were treated with PBS or 30 μg/ml IdeS for 24 hours before an intracellular hydrogenase-activity based viability assay (CCK-8) was used as read-out.

FIGS. 24A and 24B show the recovery of IgG-type BCR expression on ex vivo IdeS treated PBMC's. FIG. 24A shows flow cytometry analysis of anti-Fab signal on CD19⁺ cells immediately after PBS or IdeS treatment (30 μg/ml) and after 16 hours of IdeS-free culturing. Double positive cells are found in R2. FIG. 24B shows flow cytometry analysis of anti-Fc signal on CD19⁺ cells immediately after PBS or IdeS treatment (30 μg/ml) and after 16 hours of culturing. Double positive cells are found in R2 and expressed as percentage of cells in gate P3.

FIGS. 25A and 25B show recovery of IgG-type of BCR on enriched B-cells (RosetteSep, >90% CD19⁺ cells). FIG. 25A is a graph showing flow cytometry analysis of anti-Fab signal on enriched B-cells immediately after PBS or IdeS treatment (30 μg/ml) and at indicated time points after treatment. FIG. 25B is a graph showing flow cytometry analysis of anti-Fc signal on enriched B-cells immediately after PBS or IdeS treatment (30 μg/ml) and at different time points after treatment.

FIG. 26 shows IdeS does not affect viability of B-cells. RosetteSep enriched B-cells, containing >90% CD19⁺ cells were kept in culture for several days after PBS or IdeS (30 μg/ml) treatment and viability was measured using the colorimetric CCK-8 assay.

FIGS. 27A and 27B show IdeS treatment inhibits BCR signalling. Nu-DUL-1 cells were treated with PBS or IdeS (30 μg/ml) prior to cross-linking using a F(ab′)₂specific antibody. FIG. 27A is a graph following ERK1/2 phosphorylation at different time points after stimulation using a phospho-specific antibody in flow cytometry. FIG. 27B is a graph following PLC-γ2 phosphorylation at different time points after stimulation using a phospho-specific antibody in flow cytometry.

FIGS. 28A-28D show IdeS specifically blocks B-cell maturation of IgG-producing cells. PBMC's were treated with IdeS and stimulated with recombinant IL2 and R848 in order to activate memory B-cells and differentiate them into Ig-producing cells. ELISPOT filter plates were evaluated for number of IgG-producing cells. FIG. 28A is an image of a filter plate, where the filter plate was seeded with 50 000 or 100 000 cells and treated with/without IdeS and with/without rIL2/R848 on day 0. In one set-up, IdeS was added at day 3 of stimulation with R848 and IL2. FIG. 28B is a graph showing the number of IgA, IgM and IgG producing cells after stimulation with rIL2/R848 in the presence or absence of 30 μg/ml IdeS for 96 hours. FIG. 28C is a graph showing the number of IgG producing cells after stimulation with rIL2/R848 in the presence or absence of 0.3-30 μg/ml IdeS for 72 hours. FIG. 28D is a graph showing the number of IgG producing cells after pre-treating cells for one hour with 0.3-30 μg/ml IdeS prior to removing IdeS and subjected cells to 72 hours of stimulation with rIL2/R848.

FIG. 29 shows Flow cytometry analysis of CD19⁺/IgG⁺ cells at different time points after IdeS treatment in a human healthy subject after a single i.v. dose of 0.24 mg/kg BW of IdeS. Purified PBMCs were gated using forward-side scatter (P1) and the B-cells (CD19⁺) were monitored as M1 in P1. The upper panel shows double positive cells for CD19 (FL2) and the Fc-part of IgG (FL4) pre-dose and up to 96 hours post dosing. The lower panel shows double positive cells for CD19 (FL2) and the Fab-part of IgG (FL4) pre-dose and up to 96 hours post dosing.

FIG. 30 shows IdeS cleaves the IgG-type of BCR in vivo in humans. Healthy human subjects were dosed with 0.24 mg/kg BW IdeS and PBMCs were collected at different time point after dosing. The percentage of double positive cells for CD19 and F(ab′)₂was analyzed using flow cytometry. Hours post-dosing is shown on the x-axis and MFI x cell frequency on the y-axis.

FIG. 31 shows B cell viability after antibody cross-linking of IdeS-treated cells (top) and PBS-treated cells (bottom) during a 48 hour assay period.

DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 shows the amino acid sequence of mature Immunoglobulin G-degrading enzyme of S. pyogenes (IdeS). This protein is sometimes referred to as MAC1. The full sequence of MAC1 including secretion signal is available as Genbank Accession no. WP_010922160.1.

SEQ ID NO: 2 shows the amino acid sequence of mature Endoglycosidase S (EndoS). Full sequence including secretion signal is available at Genbank Accession no. AAK00850.1.

SEQ ID NO: 3 shows the amino acid sequence of mature MAC2, a variant of IdeS. The full sequence of MAC 2 including secretion signal is available as Genbank Accession no. AFC67907.1.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that different applications of the disclosed methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

In addition as used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a lung” includes “lungs”, reference to “an antigen” includes two or more such antigens, reference to “a subject” includes two or more such subjects, and the like.

The terms “patient” and “subject” are used interchangeably and typically refer to a human.

As used herein, “an agent which reduces Fc receptor binding to serum IgG molecules” means an agent which achieves this effect by any suitable mechanism. Various agents are known to reduce the Fc receptor interaction of IgG molecules. These agents are often proteins of bacterial origin and may act in a variety of different ways.

For example, such a protein may be an IgG cysteine protease which cleaves IgG such that the antigen binding domains and Fc interacting domains are separated from each other. In such cases, Fc receptor interaction of serum IgG molecules is reduced because the quantity of intact IgG molecules in the serum is reduced.

As another example, such a protein may be an IgG endoglycosidase which cleaves a glycan structure on the Fc interacting domain of IgG, particularly the N-linked bi-antennary glycan at position Asn-297 (Kabat numbering). This glycan structure has a critical role in Fc receptor binding. Thus, when it is wholly or partially removed by a protein, this will lead to reduced Fc receptor binding by an otherwise intact IgG molecule. In such cases, the reduction in binding preferably results in an increase in the equilibrium binding constant for the IgG:FcγR interaction by a factor of at least two. Preferably, the agent increases the equilibrium binding constant for the IgG:FcγR interaction by a factor of at least two, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7 or at least 8. More preferably, the agent increases the equilibrium binding constant for the IgG:FcγR interaction by a factor of at least eight. An increase in the equilibrium binding constant represents a decrease in the binding between IgG and an FcγR (e.g. between IgG and FcγRIIA).

As used herein, the term “serum IgG molecule” refers to any gamma immunoglobulin (IgG1, IgG2, IgG3 and IgG4) molecule which is present in human tissue prior to a method of the invention being carried out. Such IgG molecules may have been produced endogenously from an individual's B-cells or may be exogenous gamma immunoglobulins which have been administered to a subject prior to the method of the invention being carried out.

As used herein, the term “Fc receptor” refers to Fc gamma immunoglobulin receptors (FcγRs) which are present on cells. In humans, FcγR refers to one, some, or all of the family of receptors comprising FcγRI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a) and FcγRIIIB (CD16b). As used herein, the term FcγR includes naturally occurring polymorphisms of FcγRI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a) and FcγRIIIB (CD16b).

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

Methods for Improving Benefit of a Therapy or a Therapeutic Agent

The present invention provides a method for improving the benefit to a subject of a therapy or a therapeutic agent. The method comprises two steps, which are referred to herein as steps (a) and (b).

Step (a) comprises administering to the subject an agent which reduces Fc receptor binding of serum IgG molecules in the subject. The amount of the agent administered is preferably sufficient to eliminate Fc receptor binding by all or substantially all IgG molecules present in the serum of the subject.

Step (b) comprises subsequently administering to the subject the said therapy or therapeutic agent.

Steps (a) and (b) are separated by a time interval which is preferably sufficient for Fc receptor binding by all or substantially all IgG molecules present in the serum of the subject to be eliminated. The said interval may typically be of at least 30 minutes and at most 21 days.

The invention also provides an agent which reduces Fc receptor binding of serum IgG molecules in a subject for use in a method for improving the benefit to said subject of a therapy or a therapeutic agent, wherein the method comprises: (a) administering to the subject an amount of the agent sufficient to eliminate Fc receptor binding by all or substantially all IgG molecules present in the serum of the subject; and (b) subsequently administering said therapy or said therapeutic agent to the subject, wherein steps (a) and (b) are separated by a time interval sufficient for Fc receptor binding by substantially all IgG molecules present in the serum of the subject to be eliminated. The said interval may typically be of at least 30 minutes and at most 21 days.

The invention also provides the use of an agent which reduces Fc receptor binding of serum IgG molecules in a subject in the manufacture of a medicament for improving the benefit to said subject of a therapy or a therapeutic agent, wherein said improving comprises: (a) administering to the subject an amount of the agent sufficient to eliminate Fc receptor binding by all or substantially all IgG molecules present in the serum of the subject; and (b) subsequently administering said therapy or said therapeutic agent to the subject, wherein steps (a) and (b) are separated by a time interval sufficient for Fc receptor binding by substantially all IgG molecules present in the serum of the subject to be eliminated. The said interval may typically be of at least 30 minutes and at most 21 days.

Timing and Order of Steps (a) and (b)

Step (a) is conducted before step (b), and steps (a) and (b) are separated by a time interval sufficient for Fc receptor binding by all or substantially all IgG molecules present in the serum of the subject to be eliminated. By “substantially all” it is typically meant that Fc receptor binding by serum IgG is reduced to less than 5% of the level that was present prior to step (a). For example, if the agent administered is (a) is a protease (such as IdeS), the interval will be the time required for the agent to cleave at least 95% of serum IgG in the subject, as measured by any suitable assay. The said interval may typically be of at least 30 minutes and at most 21 days.

The lower limit of the time interval between steps (a) and (b) is determined by the time that it takes for the agent administered in step (a) to eliminate Fc receptor binding by substantially all IgG molecules present in the serum of the subject. This may optionally be determined by testing a serum sample taken from the individual and applying any suitable assay. Some exemplary suitable assays are described in the Examples.

Such an assay may directly test for the presence of IgG molecules in a serum sample that are able to bind to one or more Fc receptors, for example in an ELISA. Alternatively, such an assay may be indirect, in that it may test for the presence of one or more reaction products that are expected to result from the treatment of IgG with the agent administered in step (a). For example, where the agent is an enzyme which cleaves the IgG protein, a serum sample may be assayed for the presence of intact IgG molecules or the fragments which result from cleavage. This may be achieved by any suitable method, such as by separating the molecules and fragments based on molecular weight, e.g. by mass spectrometry or SDS-PAGE, or by specific detection of the molecules or fragments, e.g. by ELISA. Alternatively IgG may be detected by mixing serum from a subject with cells expressing FcgR's and monitoring IgG binding by flow cytometry using fluorochrome conjugated anti-human IgG.

Conventional methods for assessing the quantity of IgG in a sample, such as a serum sample, in a clinical setting rely on nephelometry and turbidimetry because of their speed, ease of use and precision. In both nephelometry and turbidimetry, a light source is projected through a liquid sample within a transparent container. Turbidimetry measures the decrease in the intensity of light and nephelometry measures scatter of light as it passes through the sample, which is proportional to the concentration of the immunoglobulin in the solution. Both principles are based on added anti IgG antibodies that react with antigen in the sample to form an antigen/antibody complex (agglutination). Addition of PEG allows the reaction to progress rapidly to the end point, increases sensitivity, and reduces the risk of samples containing excess antigen producing false negative results. In the case of IgG analysis, the F(ab′)₂-part of IgG is cross-linked by the anti-IgG antibody and cause the agglutination reaction. However, such methods may not be appropriate when some or all of the IgG present may not be intact. For example, if an IgG cysteine protease (such as IdeS) has been administered to the subject from whom the sample is taken, e.g. in a method of the invention, or if such a protease has been administered to the sample, cleavage fragments such as F(ab′)₂- and Fc-fragments will be present. This does not affect the agglutination reaction of conventional nephelometry and turbidimetry methods as long as the F(ab′)₂fragments are still present in the sample. Due to the shorter half-life of F(ab′)₂fragments compared to intact IgG, the agglutination will decrease over time though it is not proportional to the amount of intact IgG present in the sample. Thus, samples affected by the presence of an IgG cysteine protease (such as IdeS) cannot be assessed by conventional methods. The inventors developed a new assay for IgG concentration which is compatible with samples affected by the presence of an IgG cysteine protease (such as IdeS) and may be used in any clinical setting, including (but not limited to) uses in combination with other methods of the invention.

Said method is able to discriminate between intact IgG and IdeS-generated F(ab′)₂-fragments. This was accomplished by making use of antibodies that detect the different fragments i.e. an anti-Fab antibody and an anti-Fc antibody. The antibodies used in the assay must not be a substrate for the IgG cysteine protease affecting the sample (typically IdeS). This avoids the assay reagents being affected by any active protease which may be present in a sample. This can be accomplished by testing IgG from different species or by using antibody fragments (i.e. Fab fragments or F(ab′)₂fragments) in place of whole antibodies. Typically, an anti-F(ab′)₂agent is incubated with the sample as a capture reagent. The capture reagent is typically immobilized, for example in the wells of an assay plate. Bound IgG is then detected by incubation with an anti-Fc agent as the detector reagent. Thus, only IgG which possess both Fab and Fc parts will be detected, contrary to the nephelometry and turbidimetry methods. The detector reagent may typically be conjugated directly or indirectly to a moiety to facilitate detection, such as a fluorescent dye or an enzyme which reacts with a chromogenic substrate. The capture and detector reagents can be any other molecule that specifically recognizes the Fab- or Fc-part of IgG and can be used in the reverse order i.e. capture using anti-Fc and detect using anti-Fab. The assay may be conducted in any suitable format, such as a conventional ELISA or Meso Scale Discovery format.

In some cases, such as when the IgG cysteine protease is IdeS, the sample may include intermediate fragments such as scIgG in which only one heavy chain is cleaved, and the F(ab′)₂remains attached to the other, intact heavy chain. In such cases, the scIgG fragment may be incorrectly identified by the assay as an intact IgG. Thus, the method may include a complimentary step of assessing the sizes of the fragments present in the sample. Since there are no disulphide bridges between the heavy chains below the hinge region, the Fc-part of the heavy chain in an scIgG fragment will separate from the intact heavy-chain under denaturating conditions as an approximately 20-25 kDa protein. The different fragment sizes can be detected and quantified using any suitable method, such as SDS-PAGE. A specific embodiment of the method, including the optional complimentary step is described in Example 1 (see Efficacy assessment). The method is particularly useful for assessing the efficacy of IdeS in a clinical setting.

Where the agent of step (a) is an enzyme which cleaves a glycan moiety on IgG, a serum sample may be assayed for the presence of IgG molecules which possess either normal or truncated glycans, or for the glycan fragments that result from cleavage. This may be achieved by any suitable method, such as by separating the molecules and/or fragments based on molecular weight, e.g. by mass spectrometry or SDS-PAGE, or by specific detection of the molecules or fragments, e.g. by ELISA.

The lower limit of the time interval between steps (a) and (b) may be selected from: at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, or at least 6 hours. The lower limit may be shorter than any of the above should it be determined that Fc receptor binding by substantially all IgG molecules present in the serum of the subject has been eliminated at an earlier time point.

The upper limit of the time interval between steps (a) and (b) may be selected independently of the lower limit, and may be determined by the time that it takes for endogenous production of IgG to begin to replace or to completely replace the IgG molecules that were present in the serum of the subject prior to carrying out the method. This may be determined by testing a serum sample taken from the individual and applying any suitable assay, such as those described above with respect to the lower limit. Newly-synthesised IgG typically starts to reappear in serum within 3-4 days, with total replacement complete by around 3 weeks (21 days).

The upper limit of the time interval between steps (a) and (b) may be selected independently from the lower limit, and may be selected from: at most 21 days, at most 18 days, at most 14 days, at most 13 days, at most 12 days, at most 11 days, at most 10 days, at most 9 days, at most 8 days, at most 7 days, at most 6 days, at most 5 days, at most 4 days, at most 3 days, at most 2 days, at most 24 hours, at most 18 hours, at most 12 hours, at most 10 hours, at most 8 hours, at most 7 hours, at most 6 hours, at most 5 hours, at most 4 hours, at most 3 hours, at most 2 hours, or at most 1 hour.

Preferably the time interval between steps (a) and (b) is at most 24 hours, more preferably at most 12 hours, most preferably at most 6 hours, so that steps (a) and (b) may be carried out on the same day or during the same visit to a treatment centre. This is highly advantageous, particularly where access to treatments centres may be limited. As such the time interval between steps (a) and (b) should be long enough for the agent administered in step (a) to eliminate Fc receptor binding by substantially all IgG molecules present in the serum of the subject, but is at most around 6 hours. Thus, the interval between steps (a) and (b) is preferably 30 minutes to 1 hour, 30 minutes to 2 hours, 30 minutes to 3 hours, 30 minutes to 4 hours, 30 minutes to 5 hours, 30 minutes to 6 hours, 1 to 2 hours, 1 to 3 hours, 1 to 4 hours, 1 to 5 hours, 1 to 6 hours, 2 to 3 hours, 2 to 4 hours, 2 to 5 hours, 2 to 6 hours, 3 to 4 hours, 3 to 5 hours, 3 to 6 hours, 4 to 5 hours, 4 to 6 hours, or 5 to 6 hours.

Step (a)

In step (a), an effective amount of an agent which reduces Fc receptor binding of serum IgG molecules in a subject is administered to the subject. By “effective amount” it is meant that the amount of the agent is sufficient to eliminate Fc receptor binding by substantially all IgG molecules present in the serum of the subject.

The Agent

The agent is typically a protein, typically of bacterial origin. The agent may be a protein which has IgG cysteine protease activity, preferably cleaving in the hinge region of the immunoglobulin molecule. An example of such a protein is IdeS (Immunoglobulin G-degrading enzyme of S. pyogenes). IdeS is a streptococcal protease with a unique degree of specificity; it cleaves Immunoglobulin G (IgG) antibodies but no other substrate (including IgA, IgD, IgE and IgM). IdeS cleaves human IgG into F(ab′)₂and Fc fragments at a defined site COOH-terminally of the hinge region (see FIG. 1). The mature sequence of IdeS is provided as SEQ ID NO: 1. The agent may be a protein comprising or consisting of the amino acid sequence of SEQ ID NO: 1, or may be a homologue thereof from an alternative bacterium.

Alternatively the agent may be a variant of the IdeS protein which comprises or consists of any amino acid sequence which has at least 80%, 85%, 90% or 95% identity with SEQ ID NO: 1 and has IgG cysteine protease activity. A preferred variant is the protein MAC2, the full sequence of which is available as Genbank Accession no. AFC67907.1. The sequence of MAC2 without signal sequence is provided as SEQ ID NO: 3. The agent may be a protein comprising or consisting of the amino acid sequence of SEQ ID NO: 3, or may be a homologue thereof from an alternative bacterium.

A variant of the IdeS protein may comprise or consist of an amino acid sequence in which up to 1, 2, 3, 4, 5, 10, 20, 30 or more, amino acid substitutions, insertions or deletions have been made relative to the amino acid sequence of SEQ ID NO: 1, provided the variant has IgG cysteine protease activity. Said amino acid substitutions are preferably conservative. Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties or similar side-chain volume. The amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acids they replace. Alternatively, the conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well-known in the art and may be selected in accordance with the properties of the 20 main amino acids as defined in Table 1 below. Where amino acids have similar polarity, this can be determined by reference to the hydropathy scale for amino acid side chains in Table 2.

TABLE 1

Chemical properties of amino acids

Ala
aliphatic, hydrophobic, neutral
Met
hydrophobic, neutral

Cys
polar, hydrophobic, neutral
Asn
polar, hydrophilic,

neutral

Asp
polar, hydrophilic, charged (−)
Pro
hydrophobic, neutral

Glu
polar, hydrophilic, charged (−)
Gln
polar, hydrophilic,

neutral

Phe
aromatic, hydrophobic, neutral
Arg
polar, hydrophilic,

charged (+)

Gly
aliphatic, neutral
Ser
polar, hydrophilic,

neutral

His
aromatic, polar, hydrophilic,
Thr
polar, hydrophilic,

charged (+)

neutral

Ile
aliphatic, hydrophobic, neutral
Val
aliphatic, hydrophobic,

neutral

Lys
polar, hydrophilic, charged(+)
Trp
aromatic, hydrophobic,

neutral

Leu
aliphatic, hydrophobic, neutral
Tyr
aromatic, polar,

hydrophobic

TABLE 2

Hydropathy scale

Side Chain
Hydropathy

Ile
4.5

Val
4.2

Leu
3.8

Phe
2.8

Cys
2.5

Met
1.9

Ala
1.8

Gly
−0.4

Thr
−0.7

Ser
−0.8

Trp
−0.9

Tyr
−1.3

Pro
−1.6

His
−3.2

Glu
−3.5

Gln
−3.5

Asp
−3.5

Asn
−3.5

Lys
−3.9

Arg
−4.5

Alternatively the agent may be a protein, which comprises or consists of a fragment of SEQ ID NO: 1 or SEQ ID NO: 3, and has IgG cysteine protease activity, preferably wherein said fragment is 100 to 300, 150 to 300 or 200 to 300 amino acids in length. The fragment may be created by the deletion of one or more amino acid residues of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3. Up to 1, 2, 3, 4, 5, 10, 20, 30, 40 or 50 residues may be deleted, or more. The deleted residues may be contiguous with each other.

The agent may be a protein which has IgG endoglycosidase activity, preferably cleaving the glycan moiety at Asn-297 (Kabat numbering) in the Fc region of IgG. An example of such a protein is EndoS (Endoglycosidase of S. pyogenes). EndoS hydrolyzes the β-1,4-di-N-acetylchitobiose core of the asparagine-linked glycan of normally-glycosylated IgG (see FIG. 18). The mature sequence of EndoS is provided as SEQ ID NO: 2. The agent may be a protein comprising or consisting of the amino acid sequence of SEQ ID NO: 2, or may be a homologue thereof from an alternative bacterium, such as Streptococcus equi or Streptococcus zooepidemicus, or Corynebacterium pseudotuberculosis, Enterococcus faecalis, or Elizabethkingia meningoseptica. The agent may be CP40, EndoE, or EndoF2.

Alternatively the agent may be a variant of the EndoS protein which comprises or consists of any amino acid sequence which has at least 80%, 85%, 90% or 95% identity with SEQ ID NO: 2 and has IgG endoglycosidase activity. A variant of the EndoS protein may comprise or consist of an amino acid sequence in which up to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or more, amino acid substitutions, insertions or deletions have been made relative to the amino acid sequence of SEQ ID NO: 2, provided the variant has IgG endoglycosidase activity. Said amino acid substitutions are preferably conservative. Conservative substitutions are as defined above in respect of SEQ ID NO: 1.

Alternatively the agent may be a protein which comprises or consists of a fragment of SEQ ID NO: 2 and has IgG enodglycosidase activity, preferably wherein said fragment is 400 to 950, 500 to 950, 600 to 950, 700 to 950 or 800 to 950 amino acids in length. A preferred fragment consists of amino acids 1 to 409 of SEQ ID NO: 2, which corresponds to the enzymatically active α-domain of EndoS generated by cleavage by the streptococcal cysteine proteinase SpeB. The fragment may be created by the deletion of one or more amino acid residues of the amino acid sequence of SEQ ID NO: 1. Up to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500 or 550 residues may be deleted, or more. The deleted residues may be contiguous with other.

Any fragment or variant of SEQ ID NO: 2 preferably includes residues 191 to 199 of SEQ ID NO: 2, i.e. Leu-191, Asp-192, Gly-193, Leu-194, Asp-195, Val-196, Asp-197, Val-198 and Glu-199 of SEQ ID NO: 1. These amino acids constitute a perfect chitinase family 18 active site, ending with glutamic acid. The glutamic acid in the active site of chitinases is essential for enzymatic activity. Most preferably, therefore, a variant of SEQ ID NO: 2 contains Glu-199 of SEQ ID NO: 2. The variant of SEQ ID NO: 2 may contain residues 191 to 199 of SEQ ID NO: 2 having one or more conservative substitutions, provided that the variant contains Glu-199 of SEQ ID NO: 2.

Administration and Dose

In step (a), the agent is preferably administered by intravenous infusion, but may be administered by any suitable route including, for example, intradermal, subcutaneous, percutaneous, intramuscular, intra-arterial, intraperitoneal, intraarticular, intraosseous or other appropriate administration routes. The amount of said agent that is administered may be between 0.01 mg/kg BW and 2 mg/kg BW, between 0.04 and 2 mg/kg BW, between 0.12 mg/kg BW and 2 mg/kg BW, preferably between 0.24 mg/kg and 2 mg/kg BW and most preferably between 1 mg/kg and 2 mg/kg BW. The agent may be present in a substantially isolated form. It may be mixed with carriers or diluents (as discussed below) which will not interfere with the intended use and still be regarded as substantially isolated. It may also be in a substantially purified form, in which case it will generally comprise at least 90%, e.g. at least 95%, 98% or 99%, of the protein in the preparation.

Formulations and Compositions

The agent is preferably administered together with one or more pharmaceutically acceptable carriers or diluents and optionally one or more other therapeutic ingredients. The carrier (s) must be ‘acceptable’ in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Typically, carriers for injection, and the final formulation, are sterile and pyrogen free.

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

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

Other parentally-administrable compositions which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

Step (b)

In step (b), a therapy or therapeutic agent is administered to the subject. The therapy or therapeutic agent will typically be administed or practised in precisely the same fashion as would have been used had step (a) not been conducted first.

Therapeutic Agent

In one embodiment, the therapeutic agent is an antibody which is administered for the treatment of cancer or another disease. The therapeutic agent may be intravenous immunoglobulin (IVIG). In the context of this embodiment, the method may be alternatively described as a method for the treatment of cancer or another disease in a subject, the method comprising (a) administering to the subject an agent which reduces Fc receptor binding of serum IgG molecules in the subject; and (b) subsequently administering to the subject a therapeutically effective amount of an antibody which is a treatment for said cancer or said other disease; wherein:

- the amount of said agent administered is sufficient to eliminate Fc receptor binding by substantially all IgG molecules present in the serum of the subject; and
- steps (a) and (b) are separated by a time interval of at least 2 hours and at most 21 days.

The invention also provides the agent for use in such a method for the treatment of cancer or another disease. The invention also provides use of the agent in the manufacture of a medicament for the treatment of cancer or another disease by such a method.

The cancer may be Acute lymphoblastic leukemia, Acute myeloid leukemia, Adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, Anal cancer, Appendix cancer, Astrocytoma, childhood cerebellar or cerebral, Basal cell carcinoma, Bile duct cancer, extrahepatic, Bladder cancer, Bone cancer, Osteosarcoma/Malignant fibrous histiocytoma, Brainstem glioma, Brain cancer, Brain tumor, cerebellar astrocytoma, Brain tumor, cerebral astrocytoma/malignant glioma, Brain tumor, ependymoma, Brain tumor, medulloblastoma, Brain tumor, supratentorial primitive neuroectodermal tumors, Brain tumor, visual pathway and hypothalamic glioma, Breast cancer, Bronchial adenomas/carcinoids, Burkitt lymphoma, Carcinoid tumor, Carcinoid tumor, gastrointestinal, Carcinoma of unknown primary, Central nervous system lymphoma, Cerebellar astrocytoma, Cerebral astrocytoma/Malignant glioma, Cervical cancer, Chronic lymphocytic leukemia, Chronic myelogenous leukemia Chronic myeloproliferative disorders, Colon Cancer, Cutaneous T-cell lymphoma, Desmoplastic small round cell tumor, Endometrial cancer, Ependymoma, Esophageal cancer, Ewing's sarcoma in the Ewing family of tumors, Extracranial germ cell tumor, Childhood, 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 tumor: extracranial, extragonadal, or ovarian, Gestational trophoblastic tumor, Glioma of the brain stem, Glioma, Childhood Cerebral Astrocytoma, Glioma, Childhood Visual Pathway and Hypothalamic, Gastric carcinoid, Hairy cell leukemia, Head and neck cancer, Heart cancer, Hepatocellular (liver) cancer, Hodgkin lymphoma, Hypopharyngeal cancer, Hypothalamic and visual pathway glioma, Intraocular Melanoma, Islet Cell Carcinoma (Endocrine Pancreas), Kaposi sarcoma, Kidney cancer (renal cell cancer), Laryngeal Cancer, Leukemias, Leukemia, acute lymphoblastic (also called acute lymphocytic leukemia), Leukemia, acute myeloid (also called acute myelogenous leukemia), Leukemia, chronic lymphocytic (also called chronic lymphocytic leukemia), Leukemia, chronic myelogenous (also called chronic myeloid leukemia), Leukemia, hairy cell, Lip and Oral Cavity Cancer, Liposarcoma, Liver Cancer (Primary), Lung Cancer, Non-Small Cell, Lung Cancer, Small Cell, Lymphomas, Lymphoma, AIDS-related, Lymphoma, Burkitt, Lymphoma, cutaneous T-Cell, Lymphoma, Hodgkin, Lymphomas, Non-Hodgkin (an old classification of all lymphomas except Hodgkin's), Lymphoma, Primary Central Nervous System, Macroglobulinemia, Waldenström, Malignant Fibrous Histiocytoma of Bone/Osteosarcoma, Medulloblastoma, Melanoma, Melanoma, Intraocular (Eye), Merkel Cell Carcinoma, Mesothelioma, Adult Malignant, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Mouth Cancer, Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma/Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes, Myelodysplastic/Myeloproliferative Diseases, Myelogenous Leukemia, Chronic, Myeloid Leukemia, Adult Acute, Myeloid Leukemia, Childhood Acute, Myeloma, Multiple (Cancer of the Bone-Marrow), Myeloproliferative Disorders, Nasal cavity and paranasal sinus cancer, Nasopharyngeal carcinoma, Neuroblastoma, Non-Hodgkin lymphoma, Non-small cell lung cancer, Oral 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 cancer, islet cell, Paranasal sinus and nasal cavity cancer, Parathyroid cancer, Penile cancer, Pharyngeal cancer, Pheochromocytoma, Pineal astrocytoma, Pineal germinoma, Pineoblastoma and supratentorial primitive neuroectodermal tumors, Pituitary adenoma, Plasma cell neoplasia/Multiple myeloma, Pleuropulmonary blastoma, Primary central nervous system lymphoma, Prostate cancer, Rectal cancer, Renal cell carcinoma (kidney cancer), Renal pelvis and ureter, transitional cell cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary gland cancer, Sarcoma, Ewing family of tumors, Kaposi Sarcoma, Sarcoma, soft tissue, Sarcoma, uterine, Sézary syndrome, Skin cancer (nonmelanoma), Skin cancer (melanoma), Skin carcinoma, Merkel cell, Small cell lung cancer, Small intestine cancer, Soft tissue sarcoma, Squamous cell carcinoma, Squamous neck cancer with occult primary, metastatic, Stomach cancer, Supratentorial primitive neuroectodermal tumor, T-Cell lymphoma, cutaneous—see Mycosis Fungoides and Sézary syndrome, Testicular cancer, Throat cancer, Thymoma, Thymoma and Thymic carcinoma, Thyroid cancer, Thyroid cancer, Transitional cell cancer of the renal pelvis and ureter, Trophoblastic tumor, Ureter and renal pelvis, transitional cell cancer Urethral cancer, Uterine cancer, endometrial, Uterine sarcoma, Vaginal cancer, Visual pathway and hypothalamic glioma, Vulvar cancer, Waldenström macroglobulinemia and Wilms tumor (kidney cancer).

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, oesophageal cancer, thyroid cancer, skin cancer, lymphoma, melanoma or leukemia.

The antibody administered in step (b) is preferably specific for a tumour antigen associated with one or more of the above cancer types. Targets of interest for an antibody for use in the method include CD2, CD3, CD19, CD20, CD22, CD25, CD30, CD32, CD33, CD40, CD52, CD54, CD56, CD64, CD70, CD74, CD79, CD80, CD86, CD105, CD138, CD174, CD205, CD227, CD326, CD340, MUC16, GPNMB, PSMA, Cripto, ED-B, TMEFF2, EphA2, EphB2, FAP, av integrin, Mesothelin, EGFR, TAG-72, GD2, CA1X, 5T4, α4β7 integrin, Her2. Other targets are cytokines, such as interleukins IL-I through IL-13, tumour necrosis factors α & β, interferons α, β and γ, tumour growth factor Beta (TGF-β), colony stimulating factor (CSF) and granulocyte monocyte colony stimulating factor (GMCSF). See Human Cytokines: Handbook for Basic & Clinical Research (Aggrawal et al. eds., Blackwell Scientific, Boston, Mass. 1991). Other targets are hormones, enzymes, and intracellular and intercellular messengers, such as, adenyl 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 can be human, mammalian or bacterial. Other targets are antigens, such as proteins, glycoproteins and carbohydrates from microbial pathogens, both viral and bacterial, and tumors. Still other targets are described in U.S. Pat. No. 4,366,241.

By “another disease” it is meant any other disease which is treatable by administration of an antibody. The other disease may be malignant ascites, in which case the antibody which is a treatment for the disease is typically catumaxomab or an antibody which binds to the same target as catumaxomab.

Whether it is a treatment for cancer or another disease, the antibody may be attached directly or indirectly to a cytotoxic moiety or to 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 upon the desired results. Preferred routes of administration for antibodies include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection. Alternatively, an antibody can be administered via a non-parenteral route, such as a topical, epidermal or mucosal route of administration. Local administration is also preferred, including peritumoral, juxtatumoral, intratumoral, intralesional, perilesional, intra cavity infusion, intravesicle administration, and inhalation.

A suitable dosage of an antibody may be determined by a skilled medical practitioner. Actual dosage levels of an antibody may be varied so as to obtain an amount of the active ingredient which is 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 upon a variety of pharmacokinetic factors including the activity of the particular antibody employed, the route of administration, the time of administration, the rate of excretion of the antibody, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

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

Dosage regimens may be adjusted to provide the optimum desired response (e.g., a 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 the required interval between steps (a) and (b) is not exceeded. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

The antibody of step (b) may be administered in combination with chemotherapy or radiation therapy. The method may further comprises the administration of an additional anti-cancer antibody or other therapeutic agent, which may be administered together with the antibody of step (b) in a single composition or in separate compositions as part of a combined therapy. For example, the antibody of step (b) may be administered before, after or concurrently with the other agent.

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

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

Therapy

In another embodiment, the therapy is an organ transplant. The organ may be selected from kidney, liver, heart, pancreas, lung, or small intestine.

The subject to be treated may preferably be sensitized or highly sensitised. By “sensitized” it is meant that the subject has developed antibodies to human major histocompatibility (MHC) antigens (also referred to as human leukocyte antigens (HLA)). The anti-HLA antibodies originate from allogenically sensitized B-cells and are usually present in patients that have previously been sensitized by blood transfusion, previous transplantation or pregnancy (Jordan et al., 2003).

Whether or not a potential transplant recipient is sensitized may be determined by any suitable method. For example, a Panel Reactive Antibody (PRA) test may be used to determine if a recipient is sensitized. A PRA score >30% is typically taken to mean that the patient is “high immulogic risk” or “sensitized”. Alternatively, a cross match test may be conducted, in which a sample of the potential transplant donor's blood is mixed with that of the intended recipient. A positive cross-match means that the recipient has antibodies which react to the donor sample, indicating that the recipient is sensitized and transplantation should not occur. Cross-match tests are typically conducted as a final check immediately prior to transplantation.

The presence of high titer antibodies against MHC antigens of the potential donor (i.e. donor specific antibodies (DSA)) is a direct contraindication to transplantation because of the risk of acute antibody-mediated rejection. In short, sensitization to donor MEW antigens hampers the identification of a suitable donor. A positive cross-match test is an unambiguous barrier to transplantation. Since approximately one third of patients waiting for kidney transplantation are sensitized, with as many as 15% being highly sensitized, this leads to an accumulation of patients waiting for transplant. In the US, the median time on the waiting list for renal transplantation in 2001-2002 was 1329 days for those with Panel Reactive Antibody (PRA) score 0-9%, 1920 days for those with PRA 10-79%, and 3649 days for those with PRA 80% or greater (OPTN-database, 2011).

One accepted strategy to overcome the DSA barrier is to apply plasma exchange or immune adsorption, often in combination with e.g. intravenous gamma globulin (IVIG) or Rituximab, to lower the levels of DSA to a level where transplantation can be considered (Jordan et al., 2004; Montgomery et al., 2000; Vo et al., 2008a; Vo et al., 2008b). However, plasma exchange, immune adsorption and IVIG treatments have the disadvantage of being inefficient and requiring rigorous planning since they involve repeated treatments over an extended period of time. When an organ from a deceased donor becomes available it has to be transplanted within hours since prolonged cold ischemia time is one of the most important risk factors for delayed graft function and allograft loss in renal transplantation (Ojo et al., 1997).

By contrast, the method of the present invention allows the rapid, temporary and safe removal of DSAs in a potential transplant recipient. Administering the agent just prior to transplantation has the capacity to effectively desensitize a highly sensitized patient, thereby allowing transplantation and avoiding acute antibody-mediated rejection. A single dose of agent prior to transplantation will enable transplantation of thousands of patients with donor specific IgG antibodies.

In the context of this embodiment, the method may be alternatively described as a method for the treatment of organ failure in a subject, the method comprising (a) administering to the subject an agent which reduces Fc receptor binding of serum IgG molecules in the subject; and (b) subsequently transplanting a replacement organ into the subject; wherein:

- the amount of said agent administered is sufficient to eliminate Fc receptor binding by substantially all IgG molecules present in the serum of the subject; and
- steps (a) and (b) are separated by a time interval of at least 2 hours and at most 21 days.

This embodiment may be described as a method for preventing rejection of a transplanted organ in a subject, particularly acute antibody-mediated transplant rejection, the method comprising, at least 2 hours and at most 21 days prior to transplantation of the organ, administering to the subject an agent which reduces Fc receptor binding of serum IgG molecules in the subject, wherein the amount of said agent administered is sufficient to eliminate Fc receptor binding by substantially all IgG molecules present in the serum of the subject. It will be appreciated that administration of the agent and subsequent transplantation are separated by a time interval which is equivalent to the time interval between steps (a) and (b) in the alternative phrasings of the method presented above. Thus, the various upper and lower limits for the time interval between steps (a) and (b) described above apply equally to this time interval. In this embodiment it is particularly preferred that the time interval is short enough to allow the method to be conducted during a single hospital visit. Thus, preferred intervals are 1 to 6 hours or 1 to 12 hours.

The invention also provides use of the agent in such a method of treating organ failure or preventing transplant rejection, particularly acute antibody-mediated transplant rejection. The invention also provides use of the agent in the manufacture of a medicament for the treatment of organ failure or for the prevention of transplant rejection by such a method.

In this embodiment, the method of the invention may additionally comprise a step conducted at or immediately prior to transplantation, which step comprises induction suppression of T cells and/or B cells in the patient. Said induction suppression may typically comprise administering an effective amount of an agent which kills or inhibits T cells, and/or administering an effective amount of an agent which kills or inhibits B cells. Agents which kill or inhibit T cells include

Muromonab, Basiliximab, Daclizumab, an antithymocyte globulin (ATG) antibody and a lymphocyte immune globulin, anti-thymocyte globulin preparation (ATGAM). Rituximab is known to kill or inhibit B cells.

Method for Removing Antibodies or Reducing the Effect of Antibodies in a Subject

The invention also provides a method for removing antibodies or reducing the effects of antibodies in a subject. The antibodies to be affected by the method are typically pathogenic autoantibodies. The method comprises a first step, referred to as step (a) and an optional second step, referred to as step (b).

Step (a) comprises administering to the subject an agent which reduces Fc receptor binding of serum IgG molecules in the subject. The amount of the agent administered is preferably sufficient to eliminate Fc receptor binding by substantially all IgG molecules present in the serum of the subject.

If conducted, step (b) comprises, subsequent to step (a), subjecting the subject to a treatment which removes endogenous antibodies; wherein said treatment which removes endogenous antibodies is plasmapharesis or immunoadsoprtion, or is administration of an agent (such as an anti-FcRn antibody) which prevents recycling of antibodies in serum by the FcRn receptor, thereby reducing half-life, and wherein steps (a) and (b) are separated by a time interval of at least 2 weeks.

The method may further comprise repeating step (a). Step (a) is preferably only repeated if the patient has a low level of anti-agent antibody responses. The quantity of anti-agent IgG molecules in the serum of a patient may be determined by any suitable method, such as an agent specific CAP FEIA (ImmunoCAP) test. A repetition of step (a) would only be conducted if the result of the CAP FEIA is below a threshold to be determined by the clinician. Typically, to avoid the development of an excessive anti-agent response, step (a) should be repeated no more frequently than once every 6 months.

The affected by the method may typically pathogenic autoantibodies specific for an auto-antigen which is targeted in an autoimmune disease mediated wholly or in part by autoantibodies. Table 3 sets out a list of such diseases and the associated autoantigens.

TABLE 3

DISEASE
AUTOANTIGENS

Addison's disease
Steroid 21-hydroxylase, 17 alpha-Hydroxylase (17OH) and

side-chain-cleavage enzyme (P450scc), Thyroperoxidase,

thyroglobulin and H+/K(+)-

Anti-GBM glomerulonephritis
Anti-glomerular basement membrane (anti-GBM):

(related to Goodpasteur)
noncollagenous (NC1) domains of the alpha3alpha4alpha5(IV)

collagen

Anti-neutrophil cytoplasmic
Myeloperoxidase, proteinase 3

antibody-associated vasculitides

(ANCA associated

vasculitis)(Wegener granulomatosis,

Churg-Strauss syndrome, microscopic

polyangiitis)

Anti-phospholipid antibody syndrome
Negatively-charged phospholipids complexed with

(APS)
phospholipid binding plasma proteins (e.g. beta2GPI),

cardiolipin, beta2-glycoprotein I, and (beta2GPI)

Autoimmune bullous skin diseases
IgG against keratinocytes. Specific target is desmoglein (Dsg)

(Pemphigus). Pemphigus foliaceus
1 (desmosomal

(PF), fogo selvagem (FS)(endemic
Cadherins)

form), pemphigus vulgaris (PV)

Autoimmune hemolytic anemia
Self-antigens on red-blood-cells

(AIHA)

Autoimmune hepatitis (AIH)
Actin, antinuclear antibody (ANA), smooth muscle antibody

(SMA), liver/kidney microsomal antibody (LKM-1), anti

soluble liver antigen (SLA/LP) and anti-mitochondrial antibody

(AMA), CYP2D6, CYP2C9-tienilic acid, UGT1A, CYP1A2,

CYP2A6, CYP3A, CYP2E1, CYP11A1, CYP17 and CYP21

Autoimmune neutropenia (AIN)
FcgRIIIb

Bullous pemphigoid (BP)
Hemidesmosomal proteins BP230 and BP180 (type XVII

collagen), laminin 5, the alpha6 subunit of the integrin

alpha6beta4 and p200

Celiac disease
transglutaminase 2 (TG2), transglutaminase 3, actin,

ganglioside, collagen, calreticulin and zonulin, thyroid,

endocrine pancreas, anti-gastric and liver, anti-nuclear

constituents, anti-reticulin, actin, smooth muscle, calreticulin,

desmin, collagens, bone, anti-brain, ganglioside, neuronal, blood

vessel

Chronic utricaria
Alpha-subunit of the high-affinity IgE receptor, IgE

Complete congenital heart block
Ro (Sjögens syndrome antigen A (SSA)), La (Sjögens syndrome

(CCHB)
antigen B(SSB))

Diabetes type 1A (T1DM)
Islet cell autoantibodies (ICA), antibodies to insulin (IAA),

glutamic acid decarboxylase (GAA or GAD), protein tyrosine

phosphatase (IA2 or ICA512), Insulinoma Associated Peptide-

2. The number of antibodies, rather than the individual antibody,

is thought to be most predictive of progression to overt diabetes.

Essential mixed cryoglobulinemia
Essential mixed cryoglobulinemia antigens

Goodpasture's syndrome (also known
alpha3(IV) collagen (=Goodpasture antigen)

as Goodpasture's disease and

anti-glomerular basement membrane

disease

Graves'disease (Basedow's disease),
Thyrotropin receptor (TSHR) Thyroid peroxidase (TPO)

includes Goitre and hyperthyroidism,

infiltrative exopthalmos and

infiltarative dermopathy.

Guillain-Barré syndrome (GBS).
Gangliosides GM1, GM1b, GD1a, and GalNAc-GD1a,

Acute inflammatory demyelinating
glycosphingolipid, myelin proteins PMP22 and P0

polyneuropathy (AIDP), acute motor

axonal neuropathy (AMAN)

Hemophilia - Acquired FVIII
Factor VIII

deficiency

Idiopathic thrombocytopenic purpura
Platelet glycoprotein (GP) IIb-IIIa and/or GPIb-IX

(ITP)

Lambert-Eaton myasthenic syndrome
voltage gated calcium channels

(LEMS)

Mixed Connective Tissue Disease
IgG directed against the spliceosome, U1-snRNP

(MCTD)

Multiple Myeloma
Multiple Myeloma antigens

Myasthenia gravis
Acetylcholine receptors (AchR), muscle-specific kinase

(MuSK)

Myocarditis, dilated cardiomyopathy
heart-reactive autoantibodies against multiple antigens e.g.

(DCM)(congestive cardiomyopathy)
cardiac myosin

Primary biliary cirrhosis (PBC)
pyruvate dehydrogenase complex (PDC)-E2 and other members

of the oxaloacid dehydrogenase family, Glycoprotein-210, p62,

sp100

Primary Progressive Multiple
Myelin oligodendrocyte glycoprotein (MOG), Myelin

Sclerosis (PPMS)
proteolipid protein (PLP),

transketolase (TK), cyclic nucleotide phosphodiesterase type I

(CNPase I),

collapsin response mediator protein 2, tubulin beta4,

neurofascin

Rheumatic heart disease
Cardiac myosin

(RHD), (Rheumatic fever)

Rheumatoid Arthritis (RA)
Type II collagen, citrullin (-ated proteins (e.g. (fibrinogen,

vimentin, filaggrin, type II collagen, enolase)), G6PI, RFs (anti-

Fc/IgG), Vimentin, and cytokeratin

Sjögren Syndrome (SS)
Ro (Sjögens syndrome antigen A (SS-A)), La (Sjögens

syndrome antigen B(SS-B)), p80 coilin, antinuclear antibodies,

anti-thyroid, anti-centromere antibodies (Raynaud's

phenomenon), anti-carbonic anhydrase II (distal renal

tubular acidosis), anti-mitochondrial antibodies (liver

pathology), cryoglobulins (evolution to non-Hodgkin's

lymphoma). alpha- and beta-fodrin, islet cell autoantigen,

poly(ADP)ribose polymerase (PARP), NuMA, Golgins, NOR-

90, M3-muscarinic receptor

SLE including Lupus nephritis
Autoantibodies to nuclear constituents (e.g. dsDNA and

nucleosomes), dsDNA, PARP, Sm, PCDA, rRNA Ribosome P

proteins, C1q

Stiff-person syndrome (SPS)
glutamic acid decarboxylase (GAD), amphiphysin.

Systemic sclerosis (scleroderma)
DNA-topoisomerase I (Scl-70), U3 snRNP, U2 snRNP, 7-2

RNP, NOR-90, centromere-associated proteins, and nucleolar

antigens, Anti-Th/To, Anti-RNA

polymerase I/III, Anti-PDGF receptor, Anti-fibrillin-1, M3-

muscarinic receptor,

Transplant rejection
Transplant rejection antigens

In this embodiment, the method may be alternatively described as a method for the treatment of an autoimmune disease in a subject, the method comprising (a) administering to the subject an agent which reduces Fc receptor binding of serum IgG molecules in the subject; and optionally (b) subsequently administering to the subject a therapeutically effective amount of an antibody which is a treatment for said cancer or autoimmune disease; wherein the amount of said agent administered is sufficient to eliminate Fc receptor binding by substantially all IgG molecules present in the serum of the subject; and steps (a) and (b) are separated by a time interval of at least 2 hours and at most 21 days. The invention also provides the agent for use in such a method for the treatment of autoimmune disease. The invention also provides use of the agent in the manufacture of a medicament for the treatment of autoimmune disease by such a method.

The autoimmune disease is preferably a chronic autoimmune disease which is mediated wholly or in part by autoantibodies. The autoimmune disease may be one of the diseases listed in Table 3.

Optional Additional Method Step

In the methods of the invention, the agent administered in step (a) typically does not act only on serum IgG molecules. The inventors have also made the surprising discovery that the agent may also act upon membrane bound IgG molecules which are present as part of a B cell receptor complex (BCR).

The BCR contains one ligand binding and one signalling part. The ligand-binding part consists of an antibody with a transmembrane domain and the signalling part consists of a heterodimer called Ig-α/Ig-β (CD79a/CD79b). The CD79 proteins span the plasma membrane and have a cytoplasmic tail bearing an immunoreceptor tyrosine-based activation motif (ITAM). Upon receptor ligation ITAM is phosphorylated by the SRC family kinase LYN and recruits the spleen tyrosine kinase (SYK) to the receptor. Activation of SYK leads to formation of a plasma membrane-associated signalling complex, named signalosome, which assembles signalling molecules, such as phospholipase-Cγ2 (PLC γ2), (phosphoinositide 3-kinase (PI3K), Bruton's tyrosine kinase (BTK), VAV1 and adaptor molecules. Two fundamental and intensively studied intermediates in the BCR signalling cascades, PLC γ2 and PI3K, generate key second messengers, which in turn, activate IκB kinase (IKK) and extracellular-signal regulated kinases (ERK1/2; AKA MAPK3 and 1). B-cell fate decisions i.e. proliferation, survival, differentiation and cell death are closely regulated by the balance between these signalling events. During B-cell development, naïve mature B-cells leave the bone marrow, go through somatic hyper mutation in germinal centres and class switching before becoming high affinity long-lived plasma cells and memory B-cells ready to respond heavily when activated by antigenic stimulation. Memory B-cells respond to antigen through binding to the BCR and a substantial portion of memory B-cells in circulation have an IgG-type of BCR.

Thus, the agent administered in step (a) of a method of the invention may also act upon the IgG part of the BCR of memory B-cells and may inhibit the normal activation of these cells by ligand binding. As a result, there will be an interval in which activation of memory B cells in the individual is reduced. This interval typically ends at around 12 hours after completion of step (a), but may be longer. At the end of the interval, levels of intact membrane bound IgG (and thus normal BCR) have recovered, typically as a result of membrane turnover in the affected cell. Subsequently, there is then a further interval at the end of which newly-synthesised IgG starts to re-appear in serum. This interval typically ends around 3-4 days after completion of step (a).

The action on memory B cells of the agent administered in step (a) of the methods of the invention therefore provides the opportunity to include an optional additional step in any method of the invention. This step, referred to as step (a1), is conducted after step (a) and, if step (b) is present, before step (b) in a method of the invention. Step (b) will then typically be conducted as soon as is possible or practical after step (a1). Step (a1) may be conducted (i) in the interval after step (a) but before the recovery of levels of intact membrane bound IgG on cell surfaces in the subject, or (ii) in the interval after (i) but before newly-synthesised IgG starts to re-appear in serum of the subject. The recovery of the level of intact membrane-bound IgG or the re-appearance of serum IgG may be determined by any suitable method. Exemplary methods are described in the Examples.

The interval of (i) typically ends at around 12 hours, 16 hours or 24 hours after step (a). Therefore if step (a1) is conducted in interval (i), it may be conducted at up to 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 16 hours or 24 hours after step (a), preferably at up to 1 or 2 hours after step (a).

The interval of (ii) starts at the end of the interval of (i) and typically ends 3 or 4 days after step (a). Thus, the interval of (ii) is thus typically at most from 12 hours to 4 days (96 hours) after step (a). Therefore if step (a1) is conducted in interval (ii) it is conducted between 12 hours and 96 hours after step (a1), and may be conducted between 12 hours and 24 hours, between 12 hours and 48 hours, or between 12 hours and 72 hours after step (a). For the convenience of the subject, it is generally preferable to conduct step (a1) as soon as possible within interval (ii). Thus, conducting step (a1) between 12 hours and 24 hours after step (a) is preferred.

If step (a1) is conducted in interval (i), it typically comprises administration of an agent which specifically targets an epitope present on the IgG or IgG fragment which results from the action on a B cell of the agent of step (a). For example, step (a1) may comprise administration of an agent which specifically binds to a membrane bound Fc fragment (such as that produced by the action of IdeS) or which specifically binds to a membrane bound IgG with altered glycosylation (such as that produced by the action of EndoS). The epitope may be newly created by the action of the agent of step (a), or may be an epitope which is already present in intact IgG, provided that it is retained by the IgG or IgG fragment which results from the action of the agent of step (a).

In other words, the invention may also provide a method in which an additional step (a1) is conducted after step (a) and, if step (b) is present, before step (b), wherein step (a1) comprises administering to the subject an agent which specifically binds to an epitope produced by the action of the agent administered in step (a) on membrane-bound IgG in the BCR complex, wherein said administering is conducted in an interval after step (a) but before the level of intact membrane-bound IgG in BCR complexes has recovered to the same level as was present before step (a). That is interval (i) as described above. The epitope may be, for example, a membrane-bound Fc fragment (such as that produced by the action of IdeS). The agent administered in step (a1) of said method may be any agent which specifically binds to the epitope, such as an antibody. Binding of the agent will typically result in reduced activation and/or death of a cell upon which the target is present. Said cell is typically a memory B cell. The agent may optionally be conjugated to a cytotoxin (suitable examples include those listed in Table 4), radioisotope or other moiety to promote said reduced activation or death of said cell. Thus, in this embodiment, administration of an agent in step (a1) typically results in death of memory B cells which display an IgG molecule which has been altered by the action of the agent of step (a). Thus the inclusion of step (a1) may increase the beneficial effects of a method of the invention, for example by prolonging or maintaining the absence of serum IgG molecules.

TABLE 4

Name
Target
Mode of action

Doxorubicinderivatives
Topoisomerase II
Inhibit DNA religation, leading to

DNA complexes
DNA double-strand breaks

Maytansinoids
α-Tubulin
Prevent tubulin polymerization

Auristatins
α-Tubulin
Prevent tubulin polymerization

Calicheamicins
Sequence-specific
Cause double-strand DNA breaks

minor groove of DNA

CC-1065
Sequence-specific
Induces adenine alkylation

minor groove of DNA

Duocarmycins
Bind to specific minor
Break down adenine-specific

groove of DNA
molecules in the DNA structure

Anthracyclines
DNA, RNA
Inhibit DNA and RNA synthesis by

complexes
intercalating between base pairs,

preventing replications

If step (a1) is conducted in interval (ii), it typically comprises administration of an agent which specifically targets intact membrane bound IgG. Said agent will only affect memory B cells for which levels of membrane bound IgG in the BCR complex have recovered. Within this interval, all other forms of intact IgG (e.g. circulating IgG or IgG bound to effector cells by Fc receptors in the cell membrane) will have been removed by the action of the agent administered in step (a) and the resulting fragments will have been cleared. Thus, the agent administered in step (a1) may be used to target all memory B cells which have recovered an intact BCR. Alternatively the agent may be used to target the specific Fab region of the BCR of memory B cells which are specific for a particular antigen, that is the agent administered in step (a1) may be anti-idiotypic. For example, the agent administered in step (a1) may be used to target the Fab region of donor specific antibodies in a transplant recipient, or the Fab region of antibodies specific for autoimmune antigens in an autoimmune patient, such as a patient suffering from a disorder as listed in Table 3.

In other words, the invention may also provide a method in which an additional step (a1) is conducted after step (a) and before step (b) if step (b) is present, wherein step (a1) comprises administering to the subject an agent which specifically binds to an epitope of intact, membrane-bound IgG, wherein said administering is conducted in the interval after step (a) in which the level of intact membrane-bound IgG in BCR complexes has returned to a level similar to, substantially the same as, or the same as the level that as was present before step (a), but newly-synthesised IgG has not yet re-appeared in serum. That is interval (ii) as described above. The agent administered in step (a1) of said method may be any agent which specifically binds to the epitope, such as an antibody. Binding of the agent will typically result in reduced activation and/or death of a cell upon which the target is present. The agent may optionally be conjugated to a cytotoxin (suitable examples include those listed in Table 4), radioisotope or other moiety to promote said reduced activation or death of said cell. In this embodiment, administration of an agent in step (a1) may result in the death of all memory B cells which display an intact membrane bound IgG molecule. Alternatively it may result in the death only of memory B cells which display a particular specificity of membrane bound IgG molecule. In either case, the inclusion of step (a1) may increase the beneficial effects of a method of the invention, for example by prolonging or maintaining the absence of all serum IgG molecules, or prolonging or maintaining the absence of a specific sub-set of serum IgG molecules specific for a particular target. The latter may be particularly advantageous in that it will allow for the selective removal of unwanted subsets of IgG molecules from the newly-synthesised population of IgG in the serum of the subject to which the method of the invention is applied.

Kit

The invention also provides a kit suitable for use in a method of the invention, the kit containing an amount of an agent which reduces Fc receptor binding of serum IgG molecules in a subject, which amount is sufficient to eliminate Fc receptor binding by all or substantially all IgG molecules present in the serum of a subject.

The kits of the invention may additionally comprise one or more other reagents or instruments which enable any of the embodiments mentioned above to be carried out. Such reagents or instruments include one or more of the following: a therapeutically effective amount of a therapeutic agent, which is an antibody, suitable buffer(s) (aqueous solutions), means to administer the agent to a subject as an intravenous infusion (such as a vessel or an instrument comprising a needle). Reagents may be present in the kit in a dry state such that a fluid sample resuspends the reagents. The kit may also, optionally, comprise instructions to enable the kit to be used in the method of the invention or details regarding which patients the method may be used for.

The following Examples illustrate the invention.

Example 1
Pre-Clinical Study

GLP-compliant pre-clinical investigations designed to investigate toxicology and pharmacology in New Zealand White rabbit of a GMP-produced IdeS demonstrated that IdeS cleaved the complete plasma pool of IgG within 5 minutes upon IdeS administration with <1% remaining IgG one day after treatment. The IgG level reached its lowest level 24-48 hours after IdeS-treatment and then gradually increased during the following days. Normal IgG levels were restored approximately 3 weeks after a single IdeS dose. The end-products, i.e. the F(ab′)₂- and Fc-fragments, had significantly shorter half-lives compared to intact IgG and only low levels were detectable the day after a single dose of IdeS. IdeS had a rapid distribution, showed dose proportional pharmacokinetics and a multi-phase elimination with a plasma half-life of approximately 1 hour in rabbits. Based on repeat dose toxicology studies the No Observed Adverse Effect Level (NOAEL) for IdeS was set to 2 mg/kg body weight (BW). Data not shown.

The effect of IdeS in the above in vitro and in vivo experiments was dramatic and provided the basis for the further investigation in healthy human volunteers.

Human Study
Materials and Methods
Study Design

This was a double-blind, randomized, single-center trial in healthy, male subjects (EudraCT number: 2012-000969-21) conducted in the Phase One unit at the University Hospital in Lund, Sweden. The protocol was approved by the local ethics committee prior to recruitment and all subjects provided signed informed consent before undergoing any study-specific procedures. The primary objective was to assess the safety and tolerability of IdeS following intravenous administration of single ascending doses. Secondary objectives were to evaluate IdeS efficacy (i.e. reduction in serum IgG), pharmacokinetics, and immunogenicity in healthy human subjects.

The diluted infusion solution of the GMP-produced IdeS (Hansa Medical AB, Sweden) was prepared in a phosphate buffered isotonic salt solution by the hospital pharmacy in an infusion syringe with an infusion set including a 0.2 μm filter (B. Braun, Germany). The selected starting dose of 0.010 mg/kg BW was 10-times below the pre-clinically determined Minimal Anticipated Biological Effect Level (MABEL) and 200-times below the No Observed Adverse Effect Level (NOAEL) determined during animal toxicology. The study design allowed gradual escalation of the dose with intensive safety monitoring.

To meet the inclusion criteria the subjects had to be healthy according to the screening medical examination, aged 18-45 years, have suitable veins for cannulation, a body mass index (BMI) between 19 and 30 kg/m²and weigh 50-100 kg. Subjects excluded from the study were those who had (or had a history of) any clinically significant immunodeficiency including but not limited to immunoglobulin A deficiency, had elevated levels of anti-IdeS IgG (>15 mg/L), tested positive for serum hepatitis B surface antigen, hepatitis C antibody, human immunodeficiency virus (HIV), ongoing tuberculosis, ongoing syphilis, active herpes simplex or herpes zoster infection during screening.

Each subject had a three days admission period at the Clinical Trials Unit and was randomized to IdeS or placebo (phosphate buffered saline) and dosed the morning after admission. Two subjects in each dose group were dosed on the first day (one IdeS and one placebo) and the next subjects in the group were dosed after one week. After each cohort the Data Monitoring Committee assessed the safety data and decided the next dose level. The time from the last dose at one dose level to the initiation of next dose level was at least 14 days. The infusions were given during 30 minutes for the first two subjects in each group and during 15 minutes for subsequent subjects in that group. During the admission period intensive safety monitoring and serial blood samplings for safety, pharmacokinetics, efficacy and anti-drug antibodies were performed. The subjects were discharged on day 4 and conducted at least eight intermediate follow-up visits with medical examination and blood sampling until the end of study at day 64.

All subjects participating in the study were treated with antibiotics (Spektramox if not hypersensitive to beta-lactams) as prophylaxis against bacterial infections. Prophylaxis treatment started on the dosing day and continued until plasma IgG levels were >4.5 g/L. No other concomitant medication or therapy was allowed except paracetamol during the first 28 days following dosing unless prescribed by the investigator and considered necessary for the subject's safety and well-being.

Safety Assessments

Adverse events (AE) were collected from the time of admission and throughout the study period including the follow-up period. All information about an AE was recorded including description, start/stop time, common Toxicity Criteria grade (according to CTCAE), severity, causality (unlikely, possible or probable), action taken, discontinuation and outcome. Vital signs, body temperature, heart rate and supine blood pressure were recorded regularly during the admission period and at all subsequent visits. In addition, the subjects were monitored with a 5-lead telemetric ECG during the infusion and the following 24 hours. Safety samples for clinical chemistry, hematology, coagulation, safety biomarkers (IL-6, IL-8 and TNFα) and plasma IgG were analyzed using routine methods at Labmedicin Skåne, Sweden. Urinalysis (U-glucose, U-hemoglobin and U-protein) was assessed using Multistix (Siemens, Germany).

Serum Samples for Efficacy, Pharmacokinetics and Anti-IdeS Antibody Evaluation

Blood samples intended for efficacy studies were collected in modified CAT serum BD vacutainers (BD Diagnostics, NJ, USA) containing 2 mM iodoacetic acid in order to prevent further proteolytic cleavage by IdeS. Blood sampling was performed at the following time-points: pre-dose, 1 minute before end of infusion (14 or 29 min), 5 minutes after end of infusion (20 or 35 min) and 45 minutes after end of infusion (1 h or 1 h and 15 min). In addition samples were collected 2, 6, 24, 48 and 72 hours after start of infusion as well as on day 7, 14, 21, 28 and 64 after infusion. Blood samples intended for pharmacokinetic studies were collected in regular serum BD vacutainers at the following time-points: pre-dose, 1 minute before end of infusion, 5 minutes after end of infusion, 45 min after end of infusion and 2, 6, 24, 48, 72 and 144 hours after dosing. Blood samples intended for anti-IdeS antibody analysis were collected in regular serum BD vacutainers at day 1 (pre-dose), 2 (24 h), 3 (48 h), 4 (72 h), 7 (1 week), 14, (2 weeks), 21 (3 weeks), 28 (4 weeks) and 64 (2 months). Outside the study protocol the subjects were asked for optional serum samples on day 182 (6 months) and 365 (1 year). All samples were stored below −60° C. until analyzed.

Efficacy Assessment

IdeS cleavage and processing of IgG was investigated with different methods; Enzyme-linked immune-sorbent assays (ELISAs) were used to determine IgG and IgG fragments in serum and to investigate the dynamics of the Fab- and Fc-containing fragments. The quantitative assays could not completely differentiate between the IdeS cleavage products; i.e. F(ab′)₂, Fc and single cleaved IgG (scIgG) (where one of the heavy chains is cleaved). The ELISA developed and performed by Covance Ltd, UK, measured intact IgG and scIgG. The Fab-ELISA measured all Fab-containing IgG fragments; i.e. intact IgG, scIgG and F(ab′)₂and the Fc-ELISA measured all Fc containing fragments; i.e. intact IgG, scIgG and free Fc.

The assay performed by Covance Ltd, UK, was formally validated. Briefly, serum samples were analyzed by an ELISA where IgG was allowed to bind to the catcher antibody, goat anti-human IgG F(ab′)₂(#109-006-097, Jackson ImmunoResearch Labs Inc., PA, USA). Quantified human serum protein calibrator (IgG) (X0908, Dako, Denmark) was used for preparation of standards and quality samples. Bound IgG was detected by the subsequent addition of peroxidase-conjugated F(ab′)₂fragment goat anti-human IgG, Fcγ fragment specific (#109-036-098, Jackson ImmunoResearch Labs) and a chromogenic substrate (TMB). The lower limit of quantification was 5 ng/mL (in 100% serum). The serum analyses were performed at Covance Laboratories Limited (Harrogate, UK).

The Fc-ELISA used a goat anti-human IgG (Fcγ fragment specific) F(ab′)₂fragment (#109-006-098 Jackson ImmunoResearch Labs Inc., PA, USA) as catcher antibody and a biotin conjugated goat anti-human IgG (Fcγ fragment specific) F(ab′)₂fragment (#109 066 098 Jackson ImmunoResearch) as detector. In the Fab-ELISA an affinity purified mouse anti-human IgG, F(ab′)₂fragment specific antibody (#209-005-097 Jackson ImmunoResearch Labs Inc., PA, USA) was used as catcher antibody and biotinylated CaptureSelect IgG-CH1 (#710.3120.100 BAC B.V., Naarden, Netherlands) as detector. A streptavidin-horseradish peroxidase conjugate (SA-HRP) (#21126 Pierce, Thermo Fisher Scientific, Rockford, Ill.) was used for secondary detection. Calibrator and QC-samples (ULOQ, LLOQ, H-OC, M-QC and L-QC) were prepared from human intravenous gamma globulin IVIg (Octagam®). All dilutions were performed in PBS+0.1% BSA and the Nunc MaxiSorp® flat-bottom 96-well microtiter plates (Nunc A/S, Roskilde, Denmark) were washed with PBS containing 0.05% Tween 20. The serum samples and QC-samples were analyzed in triplicates. TMB One component HRP Microwell substrate (TMBW-1000-01, BioFx Laboratories Inc., MD, USA) was used as a chromogenic substrate and the enzyme reaction was stopped by the addition of 0.5 M H₂SO₄. The absorbance was measured in an ELISA plate reader (Multiscan EX, Thermo Electron Corp.) (Software: Ascent Software v. 2.6) at λ=450 nm.

A comparison of the Fab-ELISA and a conventional turbidimetric analysis of serum IgG is shown in FIG. 19. As is shown, the turdidimetric assay detects only a small change in the level of intact IgG over time following IdeS treatment, because it cannot discriminate between F(ab′)₂and intact IgG. By contrast, the Fab-ELISA shows almost complete removal and recovery of IgG levels in the same time period.

In order to further evaluate the quantitative data from the ELISA, the serum samples were also analyzed using qualitative SDS-PAGE analyses. The SDS-PAGE analyses were performed according to the manufacturer's instructions (Bio-Rad Laboratories, CA, USA). Briefly, 0.25 μl of serum was separated on 4-20% Mini-PROTEAN® TGX™ precast gels (BioRad) at 200 V for 40 minutes under non-reduced conditions. SeeBlue MW standard (Life Technologies) and an in house prepared mix of human IgG, scIgG, F(ab′)₂and Fc were used as markers. The gels were stained with GelCode Blue stain reagent (Pierce, Thermo Fisher Scientific, MA, USA) according to the manufacturer's instructions and the gels were scanned.

Pharmacokinetics Assessment

Four IdeS derived peptides, i.e. AFPYLSTK, AIYVTDSDSNASIGMK, GGIFDAVFTR and LFEYFK, were assayed in serum samples by a qualified LC-MS/MS assay (Karlsson et al. 2012). Samples were prepared for MS analysis as previously described (Karlsson et al. 2012). The selected reaction monitoring (SRM) measurements were performed on a TSQ Vantage triple quadrupole mass spectrometer (Thermo Fisher Scientific, MA, USA) equipped with a PicoChip column packed with Reprosil-PUR C18 (New Objective, MA, USA) and a Easy-nLC II system (Thermo Fisher Scientific). The raw data was processed and analyzed with SRM analysis software Skyline (MacLean et al. 2010) with manual validation and inspection of the results. The injection volume was 1 μl corresponding to 12.5 nl serum (i.e. 1 μg total protein). Un-normalized peptide Total Peak Areas from IdeS-spiked serum was used for fitting a linear regression curve (label-free protein quantification). The concentrations of the individual peptides in the unknown human samples were interpolated from the linear regression. A commercial equimolar mixture of tryptic peptides from 6 bovine proteins (6 Bovine Tryptic Digest Equal Molar Mix, Michrom Bioresources) was used as QC-sample and run every 4-6 analytical sample (Teleman et al. 2012).

Serum concentration versus time data was analysed by non-compartmental analysis (NCA) in Phoenix™ WinNonlin® version 6.3, build 6.2.0.495 (Pharsight®, St. Louis, Mo., USA). As no major deviations (>20%) between nominal and actual sampling times and doses were observed, nominal sampling times and doses were used for the NCA calculations. The LC-MS/MS assay has not been validated and no formal lowest limit of detection (LLOQ) has been defined. A cut off for the PK calculations was set to 24 hours post dose for all four peptides and individuals.

Anti-IdeS IgG Assessment

A CAP-FEIA (ImmunoCAP) test for quantification of anti-IdeS specific IgG was developed by Thermo Fisher Scientific (Phadia®) in Uppsala, Sweden. Initial testing indicated that a 3-logaritmic measuring range was possible using the test and the limit of detection (LOD) for the IgG IdeS-specific CAP-FEIA assay was shown to be seven times below the suggested low assay cut-off (i.e. 2.0 mg/L). Analyses of the clinical samples were performed on a Phadia® 250 instrument using the test with one replicate according to the Phadia® 250 user manual. The test was intended for research use only.

Antigen-Specific Efficacy

A research grade ELISA assay was developed at Hansa Medical AB in order to address antigen-specific efficacy at the end of the study. The subjects IgG-response against a vaccine included in the Swedish childhood vaccination schedule was utilized as a surrogate for lack of auto-antigens in the healthy subjects included in the phase 1 study. Briefly, DTaP˜IPV//PRP˜T vaccine (Pentavac®/Pentaxim®-Sanofi Pasteur) was diluted 100-times in PBS prior to coating MaxiSorp plates (Nunc) at 4° C. Normal human IgG (IVIg, Octagam®) was utilized as calibrator and a goat anti-human Fc-specific biotin-SP-conjugated F(ab′)₂(Jackson #109-066-098) as detector. Furthermore, SA-HRP (Pierce #21126) was used and the signals were developed with TMB One component HRP Microwell Substrate (BioFX Laboratories #TMBW-1000-01), stopped with 0.5M H₂SO₄and read at 450 nm.

Functional In Vitro Assay

A phagocytosis assay was developed with modifications from (Ackerman et al., 2011). Fluorescent neutravidin beads (#F8776, Molecular Probes) were coated over night with biotinylated anti-IgG CH1 (CaptureSelect, #710.3120.100 BAC B.V., Naarden, Netherlands) at 0.1 mg/ml. The CaptureSelect reagent is specific for human heavy chain IgG on the CH1 domain i.e. intact IgG, scIgG and F(ab′)₂fragments but not IgM will be captured by this protein. Coated beads were washed and mixed with 1:100 diluted serum from study subjects and incubated at 37° C. for 2 hours to allow IgG in serum to bind to the coated beads. A control was prepared by mixing coated beads with dilution buffer (PBS with 0.1% BSA). All samples were prepared in duplicate. After incubation, IgG-loaded beads were washed and mixed with 75 000 THP-1 cells/sample and incubated in a CO₂-incubator at 37° C. for 1.5-3 hours. After incubation samples were fixed in ice-cold 2% phosphate buffered formalin and the fluorescent uptake in THP-1 cells was monitored using an Accuri C6 flow cytometer.

Statistical Analysis

Means, medians, standard deviations, and basic statistical analysis were performed using the GraphPad Prism 6 software (GraphPad Software, CA, USA).

Results
Study Description

A phase I, first in man, randomized double-blind study with single ascending doses of IdeS was conducted after approval from Swedish regulatory and ethical authorities. The objectives were to assess the safety, efficacy, pharmacokinetics, and immunogenicity of IdeS in healthy human subjects following intravenous administration.

A total of 29 healthy subjects were included and divided into four dose groups. The subjects in each dose group were randomized and received either IdeS or placebo. Infusions were given intravenously over 30 minutes for the first two subjects in each group and over 15 minutes for the subsequent subjects. The starting dose was 0.01 mg/kg BW (M_IdeS: 8 and N_Placebo: 4) and after evaluation by the Data Monitoring Committee the dose was stepwise increased to 0.04 mg/kg BW (M_IdeS: 4 and N_Placebo: 2), 0.12 mg/kg BW (M_IdeS: 4 and N_Placebo: 1) and finally 0.24 mg/kg BW (M_IdeS: 4 and N_Placebo: 2). The subjects were monitored until day 64 after dosing with more intensive monitoring during the first week. All subjects were male Caucasians with a median age of 23 (range: 20-41) years, weight 76 (range: 59-100) kg with a BMI of 23 (range: 20-30) kg/m²and there were no statistical significant differences in demographics between the groups.

Assessment of Safety

A total of 77 adverse events (AE) were observed in 24 of the 29 subjects with 39 AEs (in 14 subjects) classified as related (i.e. possible or probable) (Table 1.1). Among these 39 AEs, 35 had a common Toxicity Criteria grade of 1. Four AEs were grade 2 and these were all from one subject (506) who experienced a probable infusion reaction. The symptoms resolved within 15 minutes after treatment with antihistamine (2 mg Tavegyl i.v.) and corticosteroids (8 mg Betapred i.v.) and the IdeS infusion was not interrupted. None of the AEs were reported as serious, met any dose limiting toxicity criteria, or lead to withdrawal of study drug.

Among the 77 AEs the most commonly reported were nasopharyngitis, headache and fatigue. Nasopharyngitis was reported for ten out of twenty subjects on IdeS and for six out of nine subjects on placebo. Seven subjects reported headache at nine occasions (all on IdeS) whereas six subjects (five on IdeS and one on placebo) reported seven incidences of fatigue.

No clinically significant changes in hematology, clinical chemistry or coagulation were identified. However, a transient proteinuria was observed after 24-48 hours in subjects administered an IdeS dose that resulted in significant cleavage of IgG (FIG. 2). This peak probably reflected the clearance of IgG cleavage products from the circulation. Since IdeS degrades IgG antibodies there was an initial concern that study subjects would have an increased risk of infection and the subjects were screened for inherited immunoglobulin disorders, e.g. IgA deficit, before inclusion in the study. Furthermore, concerns were raised that study subjects could be subclinical carriers of bacterial agents (for example pneumococci) with an increased risk of infection due to reduction of plasma IgG. Thus, subjects received antibiotic prophylaxis until serum IgG levels had returned to >4.5 g/L. All study subjects compiled to the antibiotic treatment and there were no signs of an increasing rate of infections.

TABLE 1.1

Summary of adverse events reported for each subject.

Onset of AE

Dose
Subject
0-24 hours
2-7 days
8-21 days
22-64 days
Related
Action
Outcome

0.01
103
Nasopharyngitis (1)

Fatigue (1)
Possible
None
Resolved

Daydreaming (1)

Tinnitus (1)

106
Nasopharyngitis (1)
Epistaxis (1)

Possible
None
Resolved

Chest discomfort (1)

Nightmare (1)

206

Headache (1)
Possible
None
Resolved

Fatigue (1)

Nausea (1)

Dizziness (1)

0.04
303
Nausea (1)

Possible
None
Resolved

Flushing (1)

304

Oropharyngeal pain (1)

Possible
None
Resolved

Nasopharyngitis (1)

305

Blister (1)

Possible
None
Resolved

Headache (1)

0.12
404

Diarrhoea (1)

Possible
None
Resolved

Abdominal distension (1)

0.24
501
Headache (1)
Nasopharyngitis (1)

Possible
None
Resolved

Abdominal discomfort (1)

504

Asthenia (1)
Possible
None
Resolved

506
Flushing (2)
Throat irritation (1)
Oropharyngeal pain (1)
Probable
Tavegyl
Resolved

Sinus tachycardia (1)

Myalgia (2)
Possible
Betapred

Chest discomfort (2)

None

Pharyngeal oedema (2)

Nasal congestion (1)

Placebo
101
Peripheral coldness (1)
Fatigue (1)

Possible
None
Resolved

Infusion related reaction (1)

104
Dysgeusia (1)
Nasal congestion (1)

Possible
None
Resolved

201

Herpes simplex (1)

Possible
None
Resolved

302

Chills (1)

Possible
None
Resolved

Pharmacokinetics of IdeS

IdeS concentrations in serum were measured by a LC-MS/MS method based on four peptides derived from IdeS, and serum concentration versus time data were analysed by non-compartmental analysis. The pharmacokinetic parameters were calculated up to 24 hours post dosing, as the remaining concentrations were around or below the estimated quantitative range of the method.

Out of 29 subjects, nine received placebo and 20 received IdeS in the dose groups 0.01, 0.04, 0.12 and 0.24 mg/kg BW. None of the analyzed peptides could be detected in the pre-dose samples or in samples from the nine placebo subjects. However, IdeS could be detected in samples from the 20 subjects given IdeS, thereby confirming dosing. The concentration of IdeS increased with dose and the increase in the serum concentration one minute before end of infusion, was dose proportional (FIG. 3 A).

In subjects dosed with 0.12 and 0.24 mg/kg BW, a total of 10 blood samples/subject were collected up to 1 week post dose. The serum concentration of IdeS could be described as a multi-phase elimination curve where the main fraction of the exposure was eliminated during the first 24 hours after dosing. During the first 6 hours after dosing, the mean t½ was 4.1 (±2.6) hours at 0.12 mg/kg BW and 4.9 (±2.8) hours at 0.24 mg/kg BW. The C_maxwas 5.0 (±2.5) mg/L at 0.12 mg/kg BW and 8.3 (±3.7) mg/L at 0.24 mg/kg BW (FIG. 3 B).

Efficacy and Pharmacodynamics of IdeS

The efficacy and pharmacodynamics of IgG cleavage by IdeS was evaluated by SDS-PAGE analysis and ELISAs of serum samples from the subjects. IdeS cleaves IgG in two steps (Ryan et al., 2008; Vindebro et al., 2013). The first reaction is a very rapid and efficient cleavage of one of the two heavy chains generating a single cleaved IgG (scIgG), still having one of the two heavy chains intact. The scIgG is a less sensitive but still a functional substrate for IdeS, and this second cleavage generates F(ab′)₂and Fc fragments.

SDS-PAGE analysis revealed that IdeS had full or close to full effect within 6 hours in all 8 subjects dosed with 0.12 or 0.24 mg/kg BW, i.e. the IgG pool was converted into F(ab′)₂and Fc fragments (FIGS. 4 A and B). The effect was remarkably rapid and the IgG pool was converted into scIgG already during dosing (14 min after starting administration, i.e. 1 min prior to full dose) and maximal effect was accomplished 2-6 hours after dosing in all subjects. Newly synthesized intact IgG was detectable in serum two weeks after dosing and after three weeks the level of intact IgG had further increased and constituted the main IgG fraction in serum. (FIG. 4 C).

The dynamics of the Fab- and Fc-containing fragments in serum was analyzed using one Fab- and one Fc-specific ELISA method. The ELISAs did not completely distinguish between the different IgG specimens; i.e. F(ab′)₂, Fc, scIgG and intact IgG. The Fab-ELISA measured all Fab-containing IgG fragments; i.e. intact IgG, scIgG, and F(ab′)₂, and the Fc-ELISA measured all Fc containing fragments; i.e. intact IgG, scIgG and free Fc.

The F(ab′)₂- as well as the Fc-fragments reached bottom levels between one and seven days after dosing after which the levels increased in all subjects due to synthesis of intact IgG (data not shown). The elimination of Fc-fragments was somewhat faster than elimination of F(ab′)₂-fragments and plateau levels were reached already one day after dosing. The rapid cleavage of human IgG into F(ab′)₂and Fc detected by SDS-PAGE analysis was confirmed by ELISA (FIG. 5), showing that 2-6 hours after dosing low plateau levels were reached at less than 5% remaining signal. It was concluded that this signal mainly originated from scIgG. Degradation of IgG in the human subjects correlated well to the previously titrated IdeS concentrations needed to cleave IgG in serum samples from 20 human healthy subjects (FIG. 6).

IdeS and Antigen-Specific IgG Antibodies

A majority of the Swedish population has IgG antibodies against the antigen components of the DTaP-IPV//PRP-T (Pentavac®) vaccine (diphtheria, tetanus, pertussis, polio and Haemophilus type b). The vaccine is part of the Swedish childhood vaccination schedule and most individuals have received repeated injections of this or of a similar vaccine. This was utilized in an exploratory study where pre-existing IgG against these antigens were measured. The results showed that the effect of IdeS on antigen-specific IgG showed the same pattern as on the total IgG in each individual. In addition, the reappearance of these antigen-specific IgG antibodies was similar to that of total IgG (FIG. 7).

IdeS Treatment and Phagocytic Activity

To evaluate the functional activity of the remaining IgG after dosing with IdeS, serum from the subjects were tested in a phagocytosis assay. IgG from serum samples collected pre-dose and at different time-points after IdeS dosing were captured on fluorescent beads, washed and mixed with effector cells and phagocytosis was measured as percent of the effector cells with at least one phagocytized bead. As background in the assay, beads without serum were used to monitor the spontaneous uptake of empty beads by effector cells. The phagocytosis assay showed that all subjects dosed with 0.24 mg/kg BW IdeS reached background phagocytic levels 24 hours after dosing (FIG. 8 A). It further showed that remaining IgG or IgG-fragments in serum from subjects treated with 0.24 mg/kg BW IdeS had a significantly decreased phagocytic capacity already at the two hour sampling point and that the phagocytic capacity remained reduced for seven days (FIG. 8 B).

IdeS and Immunogenicity

Previous work has shown that a significant proportion of the population has pre-formed IgG antibodies against IdeS (Åkesson et al., 2004). It could be presumed that individuals with preformed IdeS antibodies have an increased risk of hypersensitivity/infusion-like reactions against IdeS. Therefore, a specific in vitro test for the quantitative measurement of IdeS-specific antibodies was developed. The test is a CAP Fluoro enzyme immunoassay (CAP-FEIA/ImmunoCap) assay and it was used to screen study subjects before inclusion. Subjects with elevated IgG antibody titers (>15 mg/L) were excluded from this study. A reference group of 130 human subjects were screened with the test prior to study start. Results shown in FIG. 9 A (first column). Ten out of 130 had IdeS specific IgG below the cut-off (<2 mg/L). The median level of anti-IdeS IgG was 6.1 mg/L (range <2-78 mg/L; n=130) with the 80% percentile at 15 mg/L. The 78 healthy human male subjects screened in this study all had detectable IgG against IdeS before treatment. Results shown in FIG. 9 A second column. Median level of anti-IdeS IgG was 10.6 mg/L (range 2.1-90.8 mg/L). 28% of the tested individuals had anti-IdeS IgG titers over 15 mg/L and were excluded from the study.

The majority of the study subjects responded with an increase of anti-IdeS IgG. The response was non-detectable one week after dosing but had reached close to peak levels two weeks after dosing and then slowly decreased (FIG. 9 B). The median pre-dose level (all subjects) of anti-IdeS IgG was 5.3 mg/L (range: 2.0-10.6 mg/L), and on day 14 the median level of all subjects dosed with IdeS was 104 mg/L (range: 3.1-1744 mg/L). Two months after dosing the levels of anti-IdeS IgG had started to decrease in the majority of individuals and the median anti-IdeS IgG level of all subjects dosed with IdeS was 87.8 mg/L (range: 10.5-764 mg/L). Although the individual variation in the magnitude of the anti-IdeS IgG response was large, there was clearly a stronger response among the subjects receiving 0.12 or 0.24 mg/kg BW IdeS compared to subjects receiving 0.01 or 0.04 mg/kg (FIG. 9 C). At day 182, the anti-IdeS IgG levels for 19 out of 20 individuals dosed with IdeS were within the normal range of the previously analysed subjects (range <2-90.8 mg/L; N=208) (FIG. 9 D). Only one subject still had anti-IdeS IgG levels slightly above the normal range on day 182 (101 mg/L). This subject was in the 0.01 mg/kg dose group and at day 365, the anti-IdeS IgG levels were within the normal range for this subject (40.5 mg/L). It can be concluded that the anti-IdeS IgG response is very similar in kinetics and magnitude to the response reported for other protein drugs of bacterial origin, such as streptokinase and staphylokinase.

Discussion

This first in class clinical study shows that IdeS converts plasma IgG into single cleaved IgG (scIgG) only minutes after administration. ScIgG has been demonstrated to have compromised effector functions with reduced binding to Fcγ-receptors and reduced Fc-mediated cytotoxicity (Brerski et al., 2009). Despite the lack of pathogenic autoantibodies in the healthy subjects included in the study, normal IgG could be monitored as a biomarker and IdeS showed impressive efficacy in IgG cleaving within the tested dose-range. Full or close to full effect on total IgG, i.e. conversion into F(ab′)₂and Fc fragments, was seen in all subjects dosed with 0.12 and 0.24 mg/kg BW IdeS and the study drug showed a favorable safety profile. Only six hours after administration only low concentrations of IgG (<5%) could be detected in blood and the IgG persistently remained low for more than a week until newly synthesized IgG had repopulated the plasma. These results could be compared to the results generally obtained using e.g. plasma exchange where a single plasma volume exchange results in a reduction in IgG to approximately 35% of the original level and 24 h after the plasma exchange the IgG levels have raised to 60% mainly due to lymphatic drainage into the vascular space (Ismail et al., 2001).

As a consequence of IdeS being a bacterial protein and most humans have had previous contact with S. pyogenes, all subjects had pre-formed anti-IdeS IgG antibodies and reacted as expected with an IgG response which peaked 2-3 weeks after the IdeS infusion. The amplitude of the anti-drug response varied substantially between individuals, although a dose-response pattern was noted. Six-twelve months after dosing all subjects were back to anti-IdeS antibody levels within the normal range (i.e. <2-91 mg/L) and considering potentially neutralizing antibodies and the safety aspect it is anticipated that IdeS treatment could be repeated after 6-12 months. The IdeS specific CAP FEIA test developed in parallel with this clinical trial could be a valuable tool to guide clinicians when considering repeated dosing.

In addition to total plasma IgG the study investigated IdeS effect on a specific biomarker utilized as a vaccine against diphtheria, tetanus, pertussis, polio and haemophilus type b within the Swedish childhood vaccination schedule. The results showed that there was no major difference in antigen-specificity with regard to the IdeS efficacy on total IgG, and all subjects had fully recovered their antigen-specific IgG at the time when the entire IgG pool was back.

The study also evaluated functional relevance of cleaving IgG with IdeS in a phagocytosis assay, where interaction with the Fcγ-receptor is expected to play a major role. This assay showed that already a few hours post administration of IdeS, the phagocytic effect of remaining IgG/IgG-fragments was significantly reduced in all tested subjects, an effect that remained seven days later. The results show that IdeS has the capacity to inactivate Fc-mediated effector function in vivo in humans.

Taken together the data presented here demonstrate that a single dose of IdeS safely, rapidly and efficiently inactivates IgG in humans and that the effect remains for several weeks. IdeS alone and/or in combination with other B-cell attenuating drugs (e.g. Rituximab or Bortezumib) is a very attractive therapeutic approach for many conditions where IgG autoantibodies contribute to the pathology. The immunogenic nature of IdeS most likely prevents chronic treatment although repeated treatment once or twice per year most likely will be possible. However, by applying a judicious therapeutic approach utilizing the high efficacy of IdeS in combination with other drugs or technologies such as immune adsorption or plasma exchange, it should be possible to maintain low plasma levels of pathogenic antibodies for an extended timeframe.

The removal of IgG by IdeS was temporary, suggesting that its best use may be for conditions with a monophasic course, such as antibody mediated graft rejection. This is currently being investigated in a phase II study with IdeS.

REFERENCES

Ackerman, M. E., B. Moldt, R. T. Wyatt, A. S. Dugast, E. McAndrew, S. Tsoukas, S. Jost, C. T. Berger, G. Sciaranghella, Q. Liu, D. J. Irvine, D. R. Burton, and G. Alter. 2011. A robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples. J Immunol Methods 366:8-19.

Agniswamy, J., B. Lei, J. M. Musser, and P. D. Sun. 2004. Insight of host immune evasion mediated by two variants of group a Streptococcus Mac protein. J Biol Chem 279:52789-52796.

Brerski, R. J., O. Vafa, D. Petrone, S. H. Tam, G. Powers, M. H. Ryan, J. L. Luongo, A. Oberholtzer, D. M. Knight, and R. E. Jordan. 2009. Tumor-associated and microbial proteases compromise host IgG effector functions by a single cleavage proximal to the hinge. Proc Natl Acad Sci USA 106:17864-17869.

Carapetis, J. R., A. C. Steer, E. K. Mulholland, and M. Weber. 2005. The global burden of group A streptococcal diseases. Lancet Infect Dis 5:685-694.

Collen, D., F. De Cock, E. Demarsin, S. Jenne, I. Lasters, Y. Laroche, P. Warmerdam, and L. Jespers. 1997. Recombinant staphylokinase variants with altered immunoreactivity. III: Species variability of antibody binding patterns. Circulation 95:455-462.

Declerck, P. J., S. Vanderschueren, J. Billiet, H. Moreau, and D. Collen. 1994. Prevalence and induction of circulating antibodies against recombinant staphylokinase. Thromb Haemost 71:129-133.

Ismail, N., R. Neyra, and R. Hakim. 2001. Plasmapheresis. In Handbook of dialysis, 3rd edn. J. T. Daugirdas, P. G. Blake, and T. S. Ing, editors. Lippincott Williams Wilkins, Philadelphia. 231-262.

Iyer, S. P., L. E. Nikkel, K. K. Nishiyama, E. Dworakowski, S. Cremers, C. Zhang, D. J. McMahon, S. Boutroy, X. S. Liu, L. E. Ratner, D. J. Cohen, X. E. Guo, E. Shane, and T. L. Nickolas. 2014. Kidney Transplantation with Early Corticosteroid Withdrawal: Paradoxical Effects at the Central and Peripheral Skeleton. J Am Soc Nephrol

Johansson, B. P., O. Shannon, and L. Björck. 2008. IdeS: a bacterial proteolytic enzyme with therapeutic potential. PLoS One 3: e1692.

Jordan, S. C., D. Tyan, D. Stablein, M. McIntosh, S. Rose, A. Vo, M. Toyoda, C. Davis, R. Shapiro, D. Adey, D. Milliner, R. Graff, R. Steiner, G. Ciancio, S. Sahney, and J. Light. 2004. Evaluation of intravenous immunoglobulin as an agent to lower allosensitization and improve transplantation in highly sensitized adult patients with end-stage renal disease: report of the NIH IG02 trial. J Am Soc Nephrol 15:3256-3262.

Karlsson, C., L. Malmstrom, R. Aebersold, and J. Malmstrom. 2012. Proteome-wide selected reaction monitoring assays for the human pathogen Streptococcus pyogenes. Nat Commun 3:1301.

MacLean, B., D. M. Tomazela, N. Shulman, M. Chambers, G. L. Finney, B. Frewen, R. Kern, D. L. Tabb, D. C. Liebler, and M. J. MacCoss. 2010. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 26:966-968.

Mainet, D., M. del Rosario, A. Toruncha, P. Prats, C. Valenzuela, and P. Lopez Saura. 1998. Similar, more than 6-months persisted, antibody and neutralizing activity responses in patients with acute myocardial infarction treated with recombinant or natural streptokinase. Fibrinolysis Proteol 12:301-309.

Montgomery, R. A., A. A. Zachary, L. C. Racusen, M. S. Leffell, K. E. King, J. Burdick, W. R. Maley, and L. E. Ratner. 2000. Plasmapheresis and intravenous immune globulin provides effective rescue therapy for refractory humoral rejection and allows kidneys to be successfully transplanted into cross-match-positive recipients. Transplantation 70:887-895.

Nandakumar, K. S., B. P. Johansson, L. Björck, and R. Holmdahl. 2007. Blocking of experimental arthritis by cleavage of IgG antibodies in vivo. Arthritis Rheum 56:3253-3260.

Ojo, A. O., R. A. Wolfe, P. J. Held, F. K. Port, and R. L. Schmouder. 1997. Delayed graft function: risk factors and implications for renal allograft survival. Transplantation 63:968-974.

Ryan, M. H., D. Petrone, J. F. Nemeth, E. Barnathan, L. Björck, and R. E. Jordan. 2008. Proteolysis of purified IgGs by human and bacterial enzymes in vitro and the detection of specific proteolytic fragments of endogenous IgG in rheumatoid synovial fluid. Mol Immunol 45:1837-1846.

Teleman, J., S. Waldemarson, J. Malmstrom, and F. Levander. 2013. Automated quality control system for LC-SRM setups. J Proteomics 95:77-83.

Tradtrantip, L., N. Asavapanumas, and A. S. Verkman. 2013. Therapeutic cleavage of anti-aquaporin-4 autoantibody in neuromyelitis optica by an IgG-selective proteinase. Mol Pharmacol 83:1268-1275.

Wenig, K., L. Chatwell, U. von Pawel-Rammingen, L. Björck, R. Huber, and P. Sondermann. 2004. Structure of the streptococcal endopeptidase IdeS, a cysteine proteinase with strict specificity for IgG. Proc Natl Acad Sci USA 101:17371-17376.

Vincents, B., U. von Pawel-Rammingen, L. Björck, and M. Abrahamson. 2004. Enzymatic characterization of the streptococcal endopeptidase, IdeS, reveals that it is a cysteine protease with strict specificity for IgG cleavage due to exosite binding. Biochemistry 43:15540-15549.

Vindebro, R., C. Spoerry, and U. von Pawel-Rammingen. 2013. Rapid IgG heavy chain cleavage by the streptococcal IgG endopeptidase IdeS is mediated by IdeS monomers and is not due to enzyme dimerization. FEBS Lett 587:1818-1822.

Vo, A. A., M. Lukovsky, M. Toyoda, J. Wang, N. L. Reinsmoen, C. H. Lai, A. Peng, R. Villicana, and S. C. Jordan. 2008a. Rituximab and intravenous immune globulin for desensitization during renal transplantation. N Engl J Med 359:242-251.

Vo, A. A., E. A. Wechsler, J. Wang, A. Peng, M. Toyoda, M. Lukovsky, N. Reinsmoen, and S. C. Jordan. 2008b. Analysis of subcutaneous (SQ) alemtuzumab induction therapy in highly sensitized patients desensitized with IVIG and rituximab. Am J Transplant 8:144-149.

von Pawel-Rammingen, U., B. P. Johansson, and L. Björck. 2002a. IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G. EMBO J 21:1607-1615.

von Pawel-Rammingen, U., B. P. Johansson, H. Tapper, and L. Björck. 2002b. Streptococcus pyogenes and phagocytic killing. Nat Med 8:1044-1045; author reply 1045-1046.

Yang, R., M. A. Otten, T. Hellmark, M. Collin, L. Björck, M. H. Zhao, M. R. Daha, and M. Segelmark. 2010. Successful treatment of experimental glomerulonephritis with IdeS and EndoS, IgG-degrading streptococcal enzymes. Nephrol Dial Transplant 25:2479-2486.

Åkesson, P., M. Rasmussen, E. Mascini, U. von Pawel-Rammingen, R. Janulczyk, M. Collin, A. Olsen, E. Mattsson, M. L. Olsson, L. Björck, and B. Christensson. 2004. Low antibody levels against cell wall-attached proteins of Streptococcus pyogenes predispose for severe invasive disease. J Infect Dis 189:797-804.

Example 2
Introduction

Transplantation in the presence of donor specific antibodies (DSA) risks resulting in a hyper-acute antibody-mediated rejection with acute allograft loss. The study in Example 1 demonstrates that IdeS is safe and well tolerated up to 0.24 mg/kg BW. At this dose IdeS completely cleaved the pool of plasma-IgG within 14 minutes after initiation of infusion. The level of intact IgG was reduced to less than 5% of its original level. The data clearly indicated that a single dose of IdeS is superior to both plasmapheresis and immunoadsorption with respect to efficiency and rate of plasma IgG reduction.

Therefore IdeS treatment of sensitized kidney patients just prior to transplantation should rapidly and efficiently cleave IgG into F(ab′)₂- and Fc-fragments thereby reducing the serum levels of cytotoxic DSA to a level where living and deceased donor (LD and DD) transplantation is possible. The donor specific F(ab′)₂-fragments still in circulation at the time-point of transplantation may also prevent binding of e.g. low affinity IgM or residual IgG to the transplant thereby further protecting the organ from rejection. The objective of this study was to investigate if treatment with a clinically relevant dose of IdeS can turn a positive cross-match test into a negative using serum from sensitized patients and to investigate the correlation between the reduction in levels of total IgG and IgG specific to HLA class I and II.

Material and Methods
Serum Samples

The investigated patients were diagnosed with stage 5 CKD and were on the waiting list for kidney transplantation. The patients were all sensitized and positive for anti-HLA. The patients were recruited by Prof. H. Ekberg at the Transplant Unit, Dept. of Nephrology and Transplantation, Skåne University Hospital in Malmö, Sweden and Prof. G. Tufveson at the Dept. of Transplant Surgery, Uppsala University Hospital, Uppsala, Sweden. The patients received written patient information and signed the informed consent before any study related procedures were started. Serum was isolated from 10 ml venous blood according to the hospitals procedure. To ensure confidentiality the principal investigator made the identity of the patients unavailable to the investigating scientists by assigning an identification number (PXX) to the serum samples. The samples were sent to the Clinical Immunology Division at the University Hospital in Uppsala for banking and a fraction of each serum was then sent to Hansa Medical AB in Lund for IdeS related analyses.

IdeS Cleavage in Serum from Patients

Sera (100 μl) from five patients (P02, P04, P07, P08 and P09) were treated with IdeS (batch BX1001865; 9.9 g/l). An IdeS stock at 6 g/l was prepared in PBS/0.1% BSA. 100 μL sera were added to 12 μl M HCl in order to adjust serum pH to a physiological level (pH 7.4) and then 2.4 μl of the IdeS stock (6 g/l) were added to reach a final concentration of 125 μg/ml IdeS in 115 μl. All preparations were made on ice. Cleavage was performed at 37° C. (Thermomixer; Eppendorf) for 2 hours and stopped by putting the samples in the freezer (−20° C.) until further analyses. Control samples from each patient were identically mock treated with dilution buffer (PBS/0.1% BSA) replacing IdeS.

Quantification of Human IgG

The serum samples were sent to the Dept. of Clinical Chemistry at Skåne University Hospital in Lund, Sweden for determination of total IgG concentrations. Human serum samples treated with IdeS were analyzed for intact IgG using an ELISA assay developed by Hansa Medical AB. MaxiSorp 96-well ELISA plates were coated in carbonate buffer (pH 9.6) o/n at +4-8° C. with 100 ng/well of AffiniPure F(ab′)₂fragment goat anti-human, F(ab′)₂fragment specific (Jackson #109-006-097). The plates were washed with PBS+Tween20 (0.05%) and blocked with PBS+2% BSA for one hour at RT. Calibrators and samples were diluted in PBS+0.1% BSA (dilution buffer). After washing the diluted calibrators (M−1, serum from healthy volunteer; conc. 11.2 g/l) and serum samples were added on the plate and left to incubate for one hour at RT. Plates were washed again and 500 biotin-SP-AffiniPure F(ab′)₂fragment goat anti-human IgG, Fcγ fragment specific (Jackson #109-066-098) diluted 1:20 000, in dilution buffer, was added and incubated for 30 minutes. After another washing, 50 μl of SA-HRP (Pierce #21126) diluted 1:40 000 in dilution buffer was added and after 30 minutes incubation the plate was washed and the reaction was developed with TMB (BioFX Laboratories #TMBW-1000-01), stopped with 0.5M H₂SO₄and read at λ=450 nm. The calibrators formed a curve with a four parameter logistic fit (y=b+(a−b)/(1+xc){circumflex over ( )}d) within the analyzed range and sample dilutions producing OD-values as close as possible to the IC50 value of the standard curve were preferably used for quantifications.

IdeS Efficacy and Immunogenicity Assessment

The ELISA assay for IdeS efficacy was conducted as in in Example 1, as was the CAP FEIA (ImmunoCap) assay for IdeS specific antibody responses. The results were used only for comparative purposes against the results reported in Example 1 and are not shown.

Complement-Dependent Cytotoxicity (CDC) and Single Antigen Bead (SAB) Analyses (Luminex)

The IdeS and placebo treated sera were analysed for anti-HLA IgG antibodies using SAB analyses against a panel of MHC class-I and -II antigens (LAB Screen Single Antigen, One Lambda). The sera were also tested and scored for reactivity in a complement-dependent cytotoxicity (CDC) screen test on T and B cells from 23 donors. T-cells and B-cells were enriched using CD8 and MHC class-II magnetic beads (Dynal), respectively. The SAB and CDC analyses were conducted using validated methods in a clinical setting by Dr. Mats Bengtsson at the Clinical Immunology Division, Department of Oncology, Radiology and Clinical Immunology, Rudbeck Laboratory, University Hospital, Uppsala, Sweden. The CDC reactions were scored according to the International Workshop procedure (Fuller et al., 1982).

Complement-Dependent Lymphocytotoxic Crossmatch (CDC-CXM) Tests

Splenocytes were prepared from Balb/c mice by Ficoll separation. Cells were washed in PBS (×2) and re-suspended to 2×10⁶cells/ml in DMEM:F12 (Difco) with 0.1% heat inactivated BSA. The serum samples were treated with DTT to inactivate IgM by mixing 45 μl serum with 5 μl 50 mM DTT and incubate for 30-45 minutes at 37° C. The CXM test was performed by adding 1 μl cell suspension (i.e. 2000 cells) and 1 μl of sample (i.e. serum or controls) to a 60-well Terasaki-plate (Nunc). After 30 minutes of incubation at room temperature, Baby Rabbit Complement (5 μl) (Cedarlane) was added and the mix was further incubated for 60 minutes at RT. FluoroQuench AO/EB Stain Quench (50) (One Lambda inc.) was added to each well and the mix was incubated for 15 minutes at RT. The cytotoxicity was scored (score 1-8) and documented with fluorescence microscopy.

Data Processing

The graphs were constructed using GraphPad Prism version 5.0d for Mac OS X, GraphPad Software, San Diego Calif. USA, www.graphpad.com.

Results and Discussion
IdeS-Treatment of Patient Sera

Serum was collected from twelve sensitized patents with stage 5 CKD awaiting kidney transplantation. IdeS was dose titrated in each serum to determine the efficacy of IdeS treatment and it could be concluded that IdeS effectively cleaved IgG in all patient sera within the tested concentration-range although there were individual differences in the minimal concentration of IdeS required to reach maximum effect (FIG. 10). Total IgG levels in the patient sera were analyzed and found to be within the normal range (median: 9.3 g/L; range 6.5-16.2 g/L). Furthermore, the anti-IdeS levels were determined using IdeS-ImmunoCAP analysis and all 12 serum contained low levels (median: 4.5 mg/L; range <2-14.8 mg/L) of anti-IdeS IgG (HMed Doc. No: 2012-041). There was no clear correlation between individual anti-IdeS levels or the levels of total IgG and IdeS efficacy. Five representative sera; i.e. P02, P04, P07, P08 and P09, were selected for further analyses of anti-HLA antibodies.

The sera were treated with IdeS or placebo (PBS) for two hours at 37° C. and analyzed for remaining IgG using the described ELISA. As a comparison we performed exhaustive immune-adsorption with Protein-A sepharose and analyzed remaining IgG in parallel to the IdeS treated samples. The results demonstrated that IdeS treatment reduced the level of IgG from 7.5-15.9 g/L to 0.17-0.4 g/L (table 2.1). The immunoadsorption was generally less effective with 0.18-1.5 g/L remaining IgG.

TABLE 2.1

Sera from five patients (P02, P04, P07, P08 and P09) treated with

IdeS, PBS or subjected to immunoadsorption (IA). IgG in g/L.

P02
P04
P07
P08
P09

PBS
11.0
7.5
11.6
15.9
12.0

IdeS
0.22
0.21
0.17
0.40
0.34

IA
0.50
0.31
0.18
1.5
0.80

These results should be compared to the results generally obtained using e.g. plasmapheresis where a single plasma volume exchange results in a reduction in IgG to approximately 35% of the original level and 24 h after the plasma exchange the IgG levels have raised to 60% mainly due to lymphatic drainage into the vascular space (Ismail et al., 2001). Even repeated, up to five, cycles of plasmapheresis results in oscillating IgG levels between 10% at the end of the procedure to 20-25% before the next procedure. The pre-clinical studies described in Example 1 demonstrated that a single intravenous injection of IdeS in rabbits results in a rapid (within 1 h) reduction of intact IgG down to 2-3% of the original level and that the IgG level remains low for several days after treatment. Similar results were obtained in the clinical phase I trial described in Example 1 after administering IdeS to healthy human subjects. The pool of plasma IgG was completely converted to scIgG already during administration of 0.24 mg/kg BW of IdeS (14 minutes after initiation of infusion) and two hours after dosing to pool IgG had been further converted to F(ab′)₂and Fc. The data showed that levels corresponding to <5% of the original levels, most of which consisted of scIgG, could be reached already between 2-6 hours after dosing and that it took several days before the level started to gradually increase.

CDC-Analyses

IdeS and placebo treated serum from patients P02, P04, P07, P08 and P09 were subjected to a sera-sceen CDC test against a panel of T-cells (i.e. cells enriched for CD8+) and B cells (i.e. cells enriched for MEW class-II+) from selected and well-characterized donors. The results, presented in table 2.2 and 2.3 (also see Summary of individual patient results, below), clearly demonstrated that IdeS treatment had the capacity to completely abrogate complement-dependent cytotoxicity mediated by serum containing donor-specific IgG. In the T-cell test, that mainly addressed anti-MHC class I antibodies, IdeS treatment could completely desensitize patients P02, P04, P07 and P09 and significantly improve the grade of sensitization for patient P08 (table 2.2). In the B cell test, that addressed anti-MHC class I and class II antibodies, IdeS treatment improved the grade of sensitization for all patients (table 2.3). It could be concluded that IdeS-treatment clearly reduced the CDC reactivity against potential donors thereby increasing the chance of finding a suitable donor for all tested patients. It was also clear from the data presented here that IdeS-treatment had the capacity to turn a positive pre-transplantation cross-match into a negative thereby making a sensitized patient transplantable.

Anti-HLA Analyses

To verify that the reduction in total IgG after IdeS treatment was reflected in a reduction in the levels of anti-HLA antibodies in the sera from sensitized patients the samples treated with IdeS or placebo were subjected to SAB analyses. The array included 188 allelic variants of MEW including 97 MEW class I (HLA-A, -B and -C) and 91 MEW class II (HLA-DP, -DQ and -DR) antigens. The results confirmed that IdeS treatment could reduce the levels of anti-HLA IgG in serum from sensitized patients and it could be concluded that the reactivity of serum from all tested patients to individual MHC molecules of class I and class II was significantly reduced after IdeS treatment (FIGS. 11-15 and Appendix I and II). A threshold at an MFI (raw)>1000 (sometimes >2000) is quite often used as a cut-off for a significant reactivity against a specific HLA antigen when considering transplantation of a sensitized patient. IdeS treatment could clearly reduce the number of HLA-antigens above these thresholds in all tested patients both at MHC class I and class II (table 2.4 and 2.5; Appendix I-II).

IdeS treatment has the capacity to turn a positive CXM negative Naturally occurring antibodies against [Gal α-1,3-Gal] structures (anti-Gal antibodies) are the primary effectors of human hyperacute rejection (HAR) of nonhuman tissue. Unlike most mammals, humans lack a functional α-1,3-galactosyltransferase (GalT) gene and produce abundant anti-Gal antibodies, putatively in response to GalT+ enteric bacteria (Ding et al., 2008 and Pierson 2009). The objective was to investigate if the primate v. s. non-primate anti-Gal reactivity can be exploited as a pseudo marker to analyse the effect of IdeS using clinical serum samples from the phase I study of Example 1.

The level of IgG was measured in consecutive serum samples collected before and after dosing of 0.24 mg/kg IdeS to human healthy subjects using a validated PD-ELISA (table 2.6). The data demonstrated that there was approximately a 10-fold decrease in IgG two hours after dosing and a 20-fold decrease 24 hours after dosing of IdeS. The PD-ELISA does not discriminate between intact fully functional IgG and scIgG with an attenuated Fc-effector function. SDS-PAGE analyses indicated that scIgG constituted the dominating fraction of the remaining IgG in these sera suggesting that the level of fully functional IgG is low already minutes after IdeS treatment (see Example 1).

TABLE 2.2

Sera-screen test of sera from sensitized patients (P02, P04, P07, P08 and

P09) treated with IdeS or placebo (PBS) against T cells from a panel of

donors (N = 23). Reactivity was scored by assigning a number 1-8 where

1 corresponds to 0% cytotoxicity and 8 corresponds to >80% cytotoxicity.

P02
P04
P07
P08
P09

+PBS
+IdeS
+PBS
+IdeS
+PBS
+IdeS
+PBS
+IdeS
+PBS
+IdeS

PC:8
1
1
1
1
8
1
8
1
4
1

PC:10
1
1
1
1
8
1
8
1
1
1

PC:19
6
1
8
1
1
1
8
8
8
1

PC:20
1
1
1
1
8
1
8
1
1
1

PC:9B
8
1
1
1
8
1
8
8
1
1

PC:11
1
1
1
1
8
1
8
8
1
1

PC:24B
6
1
1
1
8
1
8
1
8
1

PC:25
1
1
1
1
4
6
8
1
8
1

PC:27
1
1
1
1
1
1
8
8
1
1

PC:29
1
1
1
1
1
1
8
2
1
1

PC:15
1
1
6
1
8
1
8
1
8
1

PC:4
1
1
1
1
8
1
8
1
8
1

PC:14
2
1
6
1
8
1
8
1
1
1

PC:30
1
1
8
1
8
1
8
6
1
1

PC:28
1
1
1
1
1
1
8
1
8
1

PC:26
1
1
1
1
8
1
8
8
1
1

PC:21
1
1
1
1
8
1
8
1
1
1

PC:22
1
1
1
1
8
1
8
1
1
1

PC:12
1
1
1
1
1
1
8
1
1
1

PC:17
1
1
8
1
8
1
8
8
1
1

PC:18
1
1
1
1
8
1
8
6
8
1

PC:13
1
1
1
1
1
1
8
1
1
1

PC:16
1
1
1
1
1
1
8
1
1
1

Median
1
1
1
1
8
1
8
1
1
1

T-PRA (%)
17
0
22
0
70
4
100
39
35
0

TABLE 2.3

Sera-screen test of sera from sensitized patients (P02, P04, P07, P08 and

P09) treated with IdeS or placebo (PBS) against B cells from a panel of

donors (N = 23). Reactivity was scored by assigning a number 1-8 where

1 corresponds to 0% cytotoxicity and 8 corresponds to >80% cytotoxicity.

P02
P04
P07
P08
P09

+PBS
+IdeS
+PBS
+IdeS
+PBS
+IdeS
+PBS
+IdeS
+PBS
+IdeS

PC:8
8
1
1
1
8
1
8
1
8
1

PC:10
8
1
1
1
8
1
8
6
8
4

PC:19
1
1
8
1
8
1
8
8
8
1

PC:20
1
1
1
1
8
8
8
8
8
1

PC:9B
8
6
2
4
8
6
8
8
8
4

PC:11
8
1
8
4
8
6
8
8
8
1

PC:24B
8
1
1
1
8
1
8
8
8
1

PC:25
1
1
1
1
6
4
8
1
8
1

PC:27
6
1
1
1
1
1
8
8
8
1

PC:29
1
1
2
2
1
1
8
6
8
1

PC:15
6
1
4
1
8
2
8
6
8
4

PC:4
2
1
1
1
8
6
8
6
8
1

PC:14
8
1
8
4
8
6
8
8
8
2

PC:30
8
1
4
2
8
2
8
8
8
1

PC:28
8
1
4
2
4
6
8
1
8
1

PC:26
6
4
8
1
8
6
8
8
8
4

PC:21
1
1
1
1
8
8
8
4
1
1

PC:22
8
1
8
4
8
4
8
4
8
4

PC:12
1
2
1
1
4
1
8
1
8
1

PC:17
8
1
8
1
8
1
8
8
8
1

PC:18
8
1
8
1
8
2
8
8
8
1

PC:13
8
1
8
1
1
2
8
1
6
1

PC:16
1
1
1
1
1
1
8
8
8
1

Median
8
1
2
1
8
2
8
8
8
1

B-PRA (%)
70
13
57
30
83
61
100
78
96
26

TABLE 2.4

SAB analyses against 31 HLA-A, 50 HLA-B and 16HLA-C antigens.

The table gives the number of antigens having an MFI (Raw)

above 1000 or above 2000 in each patient before and after

IdeS treatment. Patients P02, P04, P07, P08 and P09.

HLA-A
HLA-B
HLA-C

+PBS
+IdeS
+PBS
+IdeS
+PBS
+IdeS

P02
>1000
12
0
38
9
4
0

>2000
12
0
33
0
4
0

P04
>1000
9
0
44
18
12
2

>2000
5
0
42
3
8
0

P07
>1000
0
0
34
0
0
0

>2000
0
0
34
0
0
0

P08
>1000
29
3
48
0
16
4

>2000
24
1
44
0
16
0

P09
>1000
20
0
7
0
1
0

>2000
17
0
0
0
1
0

TABLE 2.5

SAB analyses against 26 HLA-DP, 29 HLA-DQ and 36

HLA-DR antigens. The table gives the number of

antigens having an MFI (Raw) above 1000 or above

2000 in each patient before and after IdeS treatment.

Patients P02, P04, P07, P08 and P09.

HLA-DP
HLA-DQ
HLA-DR

+PBS
+IdeS
+PBS
+IdeS
+PBS
+IdeS

P02
>1000
8
0
19
0
23
2

>2000
7
0
14
0
23
0

P04
>1000
0
0
7
0
16
2

>2000
0
0
7
0
11
0

P07
>1000
0
0
0
0
13
0

>2000
0
0
0
0
12
0

P08
>1000
7
0
27
23
33
23

>2000
1
0
27
16
33
2

P09
>1000
0
0
22
7
23
0

>2000
0
0
16
3
20
0

TABLE 2.6

Level of IgG measured in serum samples from healthy subjects

dose with placebo (503) or IdeS (504-506) using a validated

PD-ELISA that measures intact IgG (plus scIgG).

[IgG] (g/l)

Pre-dose
2 h
24 h

503
10.6
14.1
12.6

504
12.8
1.6
0.53

505
8.9
0.91
0.62

506
9.5
0.81
0.65

In order to investigate if the human serum samples contained IgG that bind to murine antigens (e.g. anti-gal), mouse spleen cells were stained for FACS analyses with undiluted consecutive serum samples collected before and at different time-point after dosing of 0.24 mg/kg IdeS in human healthy subjects. The binding of IgG to the cells was detected using a hFcγ-specific reagent. The data demonstrated a clear shift 24 hours after IdeS treatment that was sustained up to 96 hours after treatment (representative graph in FIG. 16) consistent with the demonstrated reduction in total IgG. The cleavage products, i.e. F(ab′)₂- and Fcγ-fragments, have a rapid elimination from circulation and reaches low plateau levels 1-2 days after IdeS treatment (see Example 1). Consequently, competition between potentially remaining intact IgG and F(ab′)₂-fragments for binding to target antigens was expected to be insignificant in this assay. It was concluded that the pre-dose samples collected in the phase I trial contain IgG that bind mouse cells and that IdeS treatment reduced this reactivity.

It has been demonstrated that human serum contains reactivity against Gal-antigens that results from complement fixating IgG and IgM (Pierson, 2009). This was confirmed by demonstrating that human serum reacted strongly in a complement dependent cross-match test (CDC-CXM)(Terasaki test) against spleen cells from mouse (Balb/c) (data not shown). Since IdeS is very specific for IgG all samples were DTT-treated in order to inactivate IgM present in the tested sera. Consecutive serum samples collected before dosing, two hours after dosing and 24 hours after dosing from three healthy subjects (504, 505 and 506) dosed with 0.24 mg/kg BW were tested in a CDC-CXM against spleen cells from Balb/c mouse. All pre-dose samples reacted strongly (score 8) whereas the samples collected at 2 and 24 hours after IdeS treatment were completely negative (score 1) (table 2.7 and FIG. 17).

TABLE 2.7

Xenogenic cross-match test with sera from healthy subjects (504-

506) dosed with 0.24 mg/kg IdeS and spleen cells from Balb/c mouse.

Reactivity was scored by assigning a number 1-8, where 1 corresponds

to no cytotoxicity and 8 corresponds maximum cytotoxicity.

Pre-dose
2 h
24 h

504
8
1
1

505
8
1
1

506
8
1
1

Taken together it was concluded that IdeS-treatment can reduce the serum level of specific IgG with the ability to bind murine cell-surface targets and that this effect is sustained for several days after IdeS-treatment. The fact that the IgG did not recover already within the first day(s) following IdeS treatment clearly indicated that IdeS not only cleaved plasma IgG but also IgG located outside the vascular system, i.e. in the interstitial fluid. Furthermore, serum collected two and 24 hours after IdeS-treatment from subjects treated with 0.24 mg/kg IdeS could not mediate complement-dependent cytotoxicity (CDC) against mouse target cells, clearly demonstrating that IdeS can turn a positive CMX result into a negative result.

Summary of Individual Patient Results
Patient P02

Serum from patient P02 demonstrated CDC-reactivity against T-cells from 4 donors and IdeS treatment could completely neutralize this reactivity (score: 1)(table 2.2). In addition, the pre-treatment serum reacted against 16 out of the 23 B-cell donors and after treatment (reduced) reactivity remained against only two donors (table 2.3) whereas the remaining were negative (score: 1). The SAB analyses demonstrated that before IdeS treatment the patient serum had reactivity (i.e. MFI>1000) against HLA-A, —B and -C antigens as well as HLA-DP, -DQ and -DR antigens (tables 2.4 and 2.5; FIG. 11). IdeS treatment reduced the reactivity against all antigens and very few (i.e. two HLA-DR antigens) had reactivity above MFI: 1000 (non were above MFI: 2000)(table 2.4 and 2.5; FIG. 11). The overall conclusion is that IdeS can close to completely desensitize serum from patient P02.

Patient P04

Serum from patient P04 reacted against five donors in the T cell CDC-test and IdeS treatment could completely neutralize this reactivity (i.e. score: 1)(table 2.2). The MHC class I SAB analyses demonstrated strong reactivity mainly against HLA-B antigens but also against some HLA-A and HLA-C antigens (table 2.4; FIG. 12). After IdeS treatment a reduced but significant reactivity remained against some HLA-B antigens. Noteworthy, donor PC:17 has the genotype HLA-B*35:01 and the P04 serum reacted strongly in the CDC assay against this donor (table 2.3). Also, in the SAB assay the serum reacted strongly against the HLA-B*35:01 antigen (MFI: 21463)(Appendix-I). However, IdeS treatment completely neutralized the reactivity against the PC:17 donor (from score 8 to 1) even though the reactivity against the HLA-B*35:01 antigen in the SAB assay was still one of the highest (MFI: 2517).

In the B cell CDC test the serum reacted strongly (i.e. score: 8) against 8 out of the tested 23 donors and IdeS treatment reduced the reactivity against all of these donors (table 3). The serum from patient P04 was positive in the SAB assay against both HLA-DQ and HLA-DR antigens (table 2.5; FIG. 12). However, the IdeS treatment was very effective and after treatment only two of the tested HLA-DR antigens had a significant reactivity. The overall conclusion is that IdeS treatment is highly effective in reducing anti-HLA reactivity in serum from patient P04.

Patient P07

Serum from patient P07 had a broad reactivity in the CDC-test and demonstrated strong reactivity (i.e. CDC-score: 8) against 15 of the 23 tested donors in the T-cell CDC-test. IdeS treatment completely neutralized (i.e. score: 1) the reactivity against all of these 15 donors (table 2.2). The MHC class-I SAB analyses demonstrated strong reactivity against 34 of the tested HLA-B antigens with no reactivity against HLA-A or -C antigens (table 2.5; FIG. 13 and appendix-I). After IdeS treatment no MHC class-I antigens had a significant signal, i.e. the measured MFIs were all below 1000.

In the B cell CDC test the serum reacted strongly (i.e. CDC-score: 8) against 16 out of the tested 23 donors and, with one exception (donor PC:19), they were the same donors that were strongly positive in the T cell CDC (table 2.2 and 2.3). This indicates that the majority of the reactivity could be attributed to MHC class I reactivity since the B cells are both class-I and -II positive. Interestingly, although IdeS reduced the score against the majority of the tested donors IdeS was not as effective as in the corresponding T cell CDC. There were two donors (PC:20 and PC:21) where IdeS completely neutralized the score in the T cell CDC but had no effect in the B cell CDC. Potential explanation for this could be e.g. very high titres against class II antigens or that this patient has significant titres of IgM antibodies to class II antigens. However, the SAB analysis clearly demonstrates that the patient does not have antibodies to HLA-DP or -DQ (table 2.5; FIG. 13 and appendix-II). The patient has low to intermediate (i.e. MFI 1200-6500) reactivity against 13 of the tested HLA-DR antigens and this reactivity is completely neutralized by IdeS treatment (MFI<160). Consequently, it is difficult to explain the remaining reactivity in the B cell test by high titres to class-II antigens. The two donors (PC:20 and PC:21) where IdeS treatment had full effect in the T cell CDC but no effect in the B-cell CDC carries the following HLA-DR alleles; PC:20-DRB1*11:01, DBR3*02:02 and PC:21-DRB1*01:01, DBR1*16:01:01, DRB5*0202. All of these antigens are present on the SAB array. The serum from patient P07 reacts with intermediate reactivity against DRB1*11:01 (MFI: 4552) and weakly against DBR3*02:02 (MFI: 1203) but after IdeS treatment the signal is below 100 for both antigens. The serum has no reactivity against DRB1*01:01 DRB1*16:01:01 or DRB5*0202 neither before nor after IdeS treatment. The conclusion is that IgG against the MHC class II antigens cannot explain the lack of effect in the B-cell CDC using these donors and it is tempting to speculate that IgM could be involved. The overall conclusion is that IdeS is highly effective in reducing the levels of anti-HLA antibodies in serum from patient P07.

Patient P08

The serum from patient P08 is highly reactive against all tested donors in the T and B cell CDC tests (table 2.2 and 2.3). In the T-cell test there are 14 donors where IdeS can completely neutralize the reactivity and 6 donors where IdeS has no measurable effect. In the B cell test there are 5 donors where IdeS can completely neutralize the reactivity and since IdeS also have full activity in the T-cell test using the same donors it is tempting to attribute this reactivity to being merely MHC class I reactivity. There are a number of donors where IdeS has no effect neither in the T nor the B cell tests or where IdeS has only partial effect in these tests. However, there are also 3 donors (PC:14, PC:16 and PC:20) where IdeS has full effect in the T cell test and no measurable effect in the B cell test.

The SAB analyses clearly demonstrate that patient P08 has antibodies to HLA-A, -B and -C as well as HLA-DP, -DQ and -DR. Before IdeS treatment the serum has the broadest reactivity among the tested sera (tables 2.4 and 2.5; FIG. 14). In addition, the SAB analyses indicate that the patient has the highest titres of anti-HLA antibodies (FIG. 15 and Appendixes I and II) among the tested patients. IdeS can clearly reduce the levels of MHC class I and class II antibodies although the reactivity is still significant (i.e. MFI>1000) to a rather high proportion of HLA-DQ and -DR antigens after treatment. It is noteworthy that IdeS was least effective in the P08 serum when measuring remaining total IgG (0.4 g/L) and this is naturally reflected in the level of remaining anti-HLA antibodies.

Patient P09

The serum from patient P09 had strong CDC-reactivity (i.e. CDC-score: 8) against T cells from 7 donors and IdeS treatment could completely neutralize this reactivity (table 2.2). In addition, the serum reacted against 22 out of the 23 B-cell donors and after treatment (reduced) reactivity remained against 6 donors (table 2.3). The SAB analyses demonstrated that before IdeS treatment the patient serum had reactivity (i.e. MFI>1000) mainly against HLA-A-antigens as well as HLA-DQ and -DR antigens (tables 2.4 and 2.5; FIG. 15). IdeS reduced the reactivity against all antigens and only a few HLA-DQ antigens had reactivity above MFI: 1000 after treatment. The overall conclusion is that IdeS can close to completely desensitize serum from patient P09.

Conclusions

Treatment of sera from sensitized patients suffering from stage 5 CKD using a clinically relevant dose of IdeS could rapidly and substantially reduce the level of total-IgG. Furthermore, this activity was directly reflected in a reduction in the levels of specific and/or broad-reactive anti-HLA IgG in serum from these patients. SAB analyses clearly demonstrated that IdeS treatment reduced the level of IgG-antibodies to all MHC-antigens tested positive in serum from all analyzed patients. In the majority of cases the reactivity to individual MHC-antigens after IdeS treatment was below the critical MFI, i.e. below 1000. In CDC-CXM against T and B cells from hypothetical donors IdeS could reduce the reactivity in all tested patient serum samples and had the capacity to turn a positive cross-match into a negative. Furthermore, serum collected from healthy subjects before treatment with 0.24 mg/kg IdeS reacted strongly in CDC-CXM against mouse target cells, whereas serum collected two and 24 hours after IdeS-treatment were negative, which further proves that IdeS-treatment has the capacity turn a positive CXM negative. Taken together the data presented here clearly show that IdeS treatment just prior to transplantation has the potential to desensitize a highly sensitized patient, thereby allowing transplantation and avoiding an acute antibody mediated rejection.

Example 2—Appendix I—MFI Raw Data—MHC Class-I Antigens

TABLE A

MFI (raw) against individual HLA-A antigens measured using SAB assay.

P02
P04
P07
P08
P09

Allele specificity
+PBS
+IdeS
+PBS
+IdeS
+PBS
+IdeS
+PBS
+IdeS
+PBS
+IdeS

A*01:01, —, —,
64
12
258
19
368
16
23322
2047
3670
131

—, —, —

A*11:02, —, —,
76
12
1170
38
155
28
1012
46
647
30

—, —, —

A*11:01, —, —,
95
14
772
34
117
17
1127
40
332
30

—, —, —

A*02:01, —, —,
14010
754
471
28
123
16
377
25
4416
176

—, —, —

A*02:06, —, —,
13081
724
419
27
119
16
682
29
4549
184

—, —, —

A*02:03, —, —,
14679
848
336
22
93
12
3313
54
6714
266

—, —, —

A*23:01, —, —,
6931
184
140
21
170
18
20719
892
3438
118

—, —, —

A*24:02, —, —,
5720
163
146
22
167
19
18660
747
6522
261

—, —, —

A*24:03, —, —,
7963
218
147
23
134
17
3296
118
4950
171

—, —, —

A*25:01, —, —,
16017
708
5330
154
116
15
3941
110
214
29

—, —, —

A*26:01, —, —,
70
15
1792
60
156
19
11061
359
230
33

—, —, —

A*29:01, —, —,
237
19
498
46
179
20
16918
574
1055
67

—, —, —

A*29:02, —, —,
347
23
405
39
174
22
16535
551
763
53

—, —, —

A*03:01, —, —,
481
19
180
22
104
15
2809
114
98
24

—, —, —

A*30:01, —, —,
99
14
155
21
131
16
19053
697
181
26

—, —, —

A*30:02, —, —,
96
11
167
17
219
13
18951
642
156
25

—, —, —

A*31:01, —, —,
108
13
191
21
126
15
20556
975
1473
48

—, —, —

A*32:01, —, —,
15853
659
490
36
154
22
16963
377
378
41

—, —, —

A*33:03, —, —,
130
17
1432
47
114
22
5081
140
7330
243

—, —, —

A*33:01, —, —,
2084
40
297
25
117
18
4557
125
3851
101

—, —, —

A*34:02, —, —,
115
13
2330
63
147
15
12354
241
4128
130

—, —, —

A*34:01, —, —,
122
17
989
49
152
20
8166
164
3602
99

—, —, —

A*36:01, —, —,
97
13
258
26
190
19
21627
1463
1139
54

—, —, —

A*43:01, —, —,
169
19
1343
55
158
22
12911
445
304
33

—, —, —

A*66:02, —, —,
130
15
256
29
155
22
1733
51
4878
163

—, —, —

A*66:01, —, —,
107
16
6368
171
130
17
1510
47
2815
87

—, —, —

A*68:02, —, —,
3975
131
2415
80
166
18
2659
93
12805
656

—, —, —

A*68:01, —, —,
3940
124
2818
75
167
18
3653
110
13413
665

—, —, —

A*69:01, —, —,
10448
484
280
26
132
20
1798
55
12645
611

—, —, —

A*74:01, —, —,
107
13
271
26
115
16
14934
338
722
38

—, —, —

A*80:01, —, —,
82
14
279
27
182
19
21455
1273
3102
76

—, —, —

Sum
117431
5325
32402
1349
4728
564
311736
12941
110522
4671

Reactivity (%)
100%
5%
100%
4%
100%
12%
100%
4%
100%
4%

TABLE B

MFI (raw) against individual HLA-B antigens measured using SAB assay.

P02
P04
P07
P08
P09

Allele specificity
+PBS
+IdeS
+PBS
+IdeS
+PBS
+IdeS
+PBS
+IdeS
+PBS
+IdeS

—, —, B*13:01,
6568
239
2156
96
11940
415
5049
134
612
52

—, —, —

—, —, B*13:02,
10690
467
2597
102
14834
625
6532
140
602
43

—, —, —

—, —, B*18:01,
13289
640
17859
955
9574
282
4412
116
1011
64

—, —, —

—, —, B*27:05,
82
12
156
23
471
17
10312
295
609
36

—, —, —

—, —, B*27:08,
74
13
152
21
283
18
6053
120
288
29

—, —, —

—, —, B*35:01,
14040
735
21463
2517
14472
566
6441
146
175
62

—, —, —

—, —, B*37:01,
6773
187
12918
476
8835
228
17098
655
206
60

—, —, —

—, —, B*38:01,
14695
712
7209
189
11384
337
5136
90
307
55

—, —, —

—, —, B*39:01,
625
21
10465
304
12569
424
5273
77
177
33

—, —, —

—, —, B*41:01,
1423
40
2978
89
16496
839
3424
78
128
33

—, —, —

—, —, B*42:01,
1565
51
422
29
123
16
7160
174
128
33

—, —, —

—, —, B*44:03,
7408
286
4611
151
14513
614
17506
446
1576
62

—, —, —

—, —, B*44:02,
12112
541
3225
108
11467
396
14585
306
318
52

—, —, —

—, —, B*45:01,
905
25
7774
291
17110
907
20330
605
462
48

—, —, —

—, —, B*46:01,
4880
123
16562
1035
169
30
17200
673
222
79

—, —, —

—, —, B*47:01,
1700
41
1045
56
13404
535
2187
62
1193
58

—, —, —

—, —, B*48:01,
2621
53
16060
597
14815
569
16613
811
1493
78

—, —, —

—, —, B*49:01,
19412
1338
18626
1265
16059
743
10511
178
169
53

—, —, —

—, —, B*50:01,
8854
343
20304
1509
17500
932
7763
155
121
48

—, —, —

—, —, B*51:01,
19766
1510
20906
1895
9227
240
3099
72
159
73

—, —, —

—, —, B*51:02,
20506
1611
20995
2547
13718
515
3801
86
183
87

—, —, —

—, —, B*52:01,
19436
1585
20028
1726
10231
275
3682
71
160
61

—, —, —

—, —, B*53:01,
18707
1261
21303
2272
12708
410
7582
160
267
86

—, —, —

—, —, B*54:01,
770
28
12971
405
632
28
14088
316
835
71

—, —, —

—, —, B*55:01,
2044
64
7628
186
691
22
16497
397
162
39

—, —, —

—, —, B*56:01,
6751
224
17712
1039
385
22
3469
110
247
43

—, —, —

—, —, B*57:03,
18455
1192
17761
1170
150
24
4147
107
274
59

—, —, —

—, —, B*57:01,
19337
1338
18733
1350
136
21
4007
110
342
58

—, —, —

—, —, B*58:01,
16725
911
15316
856
170
23
8670
196
294
55

—, —, —

—, —, B*59:01,
15856
819
6399
177
8852
274
14144
330
653
80

—, —, —

—, —, B*40:01,
372
19
8620
267
15827
738
14576
630
735
42

—, —, —

—, —, B*40:02,
318
17
10941
289
16694
856
890
34
1354
55

—, —, —

—, —, B*40:06,
304
26
5967
119
14189
594
2623
69
1283
60

—, —, —

—, —, B*15:01,
9919
349
19678
1672
14553
640
529
33
337
63

—, —, —

—, —, B*15:16,
17630
1094
16364
993
190
27
9578
210
199
63

—, —, —

—, —, B*14:01,
7479
218
3726
105
5556
136
7704
162
292
62

—, —, —

—, —, B*14:02,
8348
280
2889
81
3724
102
8668
175
256
68

—, —, —

—, —, B*67:01,
1276
50
8260
329
219
30
13904
365
393
65

—, —, —

—, —, B*07:02,
79
12
221
23
129
14
4083
89
93
29

—, —, —

—, —, B*15:10,
10301
389
18614
1329
12626
454
1122
46
262
77

—, —, —

—, —, B*15:03,
8867
311
18677
1297
13782
579
2173
38
126
48

—, —, —

—, —, B*73:01,
95
16
1842
65
825
32
14648
466
789
51

—, —, —

—, —, B*15:11,
10379
411
18185
1319
10037
356
3259
78
286
79

—, —, —

—, —, B*15:02,
11482
478
19823
1615
12558
423
1327
39
287
59

—, —, —

—, —, B*15:12,
2830
54
9906
508
12454
501
19323
713
843
52

—, —, —

—, —, B*15:13,
18141
1108
18294
1317
10436
287
1812
55
241
69

—, —, —

—, —, B*78:01,
15049
865
20478
1730
7461
217
1111
38
116
61

—, —, —

—, —, B*08:01,
1581
36
168
23
7192
163
12749
426
181
35

—, —, —

—, —, B*81:01,
81
13
10132
236
134
17
15231
590
1194
54

—, —, —

—, —, B*82:01,
166
21
407
28
439
24
14058
267
227
47

—, —, —

Sum
410769
22178
559523
36782
421945
16539
416138
11738
22865
2830

Reactivity (%)
100%
5%
100%
7%
100%
4%
100%
3%
100%
12%

TABLE C

MFI (raw) against individual HLA-C antigens measured using SAB assay.

P02
P04
P07
P08
P09

Allele specificity
+PBS
+IdeS
+PBS
+IdeS
+PBS
+IdeS
+PBS
+IdeS
+PBS
+IdeS

—, —, —,
144
27
10024
280
275
27
23683
1730
2036
89

—, C*01:02, —

—, —, —,
4722
140
15280
894
193
32
2261
65
131
41

—, C*03:02, —

—, —, —,
2668
75
16963
1176
222
31
2719
85
153
46

—, C*03:04, —

—, —, —,
148
25
4439
133
166
24
16059
463
479
42

—, C*12:03, —

—, —, —,
122
32
6994
177
166
29
21253
1035
780
51

—, C*14:02, —

—, —, —,
5524
181
1606
70
277
34
21755
859
953
56

—, C*15:02, —

—, —, —,
141
29
2493
97
217
37
14945
414
347
64

—, C*16:01, —

—, —, —,
181
36
228
34
189
37
19817
1256
231
45

—, C*17:01, —

—, —, —,
159
31
1037
50
188
32
19501
786
651
61

—, C*18:02, —

—, —, —,
186
28
239
32
198
31
12090
269
558
50

—, C*02:02, —

—, —, —,
186
38
337
37
209
37
11939
315
293
49

—, C*04:01, —

—, —, —,
164
26
1046
42
177
28
15526
424
435
46

—, C*05:01, —

—, —, —,
170
32
1701
62
169
31
17296
491
598
50

—, C*06:02, —

—, —, —,
173
33
396
37
204
31
23743
1643
599
54

—, C*07:02, —

—, —, —,
145
21
8555
244
155
25
18497
558
650
49

—, C*08:01, —

—, —, —,
2107
59
19947
1599
270
35
2816
84
148
64

—, C*03:03, —

Sum
16941
812
91284
4964
3274
500
243901
10477
9039
856

Reactivity (%)
100%
5%
100%
5%
100%
15%
100%
4%
100%
9%

Example 2—Appendix II—MFI Raw Data—MHC Class-II Antigens

TABLE D

MFI (raw) against individual HLA-DP antigens measured using SAB assay.

P02
P04
P07
P08
P09

Allele specificity
+PBS
+IdeS
+PBS
+IdeS
+PBS
+IdeS
+PBS
+IdeS
+PBS
+IdeS

—, —, —, —,
256
28
426
45
273
32
499
31
177
42

—, —, —, —,

DPA1*02:01, —, DPB1*01:01, —

—, —, —, —,
184
35
215
39
175
36
649
35
156
43

—, —, —, —,

DPA1*01:03, —, DPB1*01:01, —

—, —, —, —,
591
26
253
32
122
23
439
27
97
34

—, —, —, —,

DPA1*02:01, —, DPB1*10:01, —

—, —, —, —,
707
39
367
41
144
33
773
47
509
49

—, —, —, —,

DPA1*01:05, —, DPB1*11:01, —

—, —, —, —,
283
42
434
56
184
41
1252
59
234
66

—, —, —, —,

DPA1*01:03, —, DPB1*11:01, —

—, —, —, —,
303
39
323
46
148
43
1576
57
252
52

—, —, —, —,

DPA1*01:05, —, DPB1*13:01, —

—, —, —, —,
123
22
152
26
115
24
380
26
99
29

—, —, —, —,

DPA1*02:01, —, DPB1*13:01, —

—, —, —, —,
5844
156
305
40
148
30
558
31
115
33

—, —, —, —,

DPA1*02:01, —, DPB1*14:01, —

—, —, —, —,
198
37
571
103
236
53
583
66
172
87

—, —, —, —,

DPA1*02:01, —, DPB1*15:01, —

—, —, —, —,
6441
177
291
34
128
25
498
29
295
40

—, —, —, —,

DPA1*02:01, —, DPB1*17:01, —

—, —, —, —,
155
21
188
33
94
24
2631
21
94
29

—, —, —, —,

DPA1*02:01, —, DPB1*18:01, —

—, —, —, —,
189
26
260
42
114
28
506
55
219
39

—, —, —, —,

DPA1*01:04, —, DPB1*18:01, —

—, —, —, —,
213
28
529
69
184
34
575
40
151
48

—, —, —, —,

DPA1*01:05, —, DPB1*18:01, —

—, —, —, —,
1084
64
921
147
216
51
920
55
183
64

—, —, —, —,

DPA1*01:03, —, DPB1*19:01, —

—, —, —, —,
308
32
324
118
158
35
531
37
156
43

—, —, —, —,

DPA1*01:03, —, DPB1*02:01, —

—, —, —, —,
914
80
457
72
235
73
791
70
210
89

—, —, —, —,

DPA1*02:01, —, DPB1*23:01, —

—, —, —, —,
306
58
414
83
240
57
669
67
182
71

—, —, —, —,

DPA1*02:01, —, DPB1*28:01, —

—, —, —, —,
283
38
373
60
166
40
1303
38
137
49

—, —, —, —,

DPA1*01:05, —, DPB1*28:01, —

—, —, —, —,
5508
155
315
44
145
34
563
38
204
47

—, —, —, —,

DPA1*01:05, —, DPB1*03:01, —

—, —, —, —,
6101
168
284
39
124
28
502
29
209
38

—, —, —, —,

DPA1*01:03, —, DPB1*03:01, —

—, —, —, —,
5481
148
352
46
173
34
1259
43
128
42

—, —, —, —,

DPA1*02:01, —, DPB1*03:01, —

—, —, —, —,
250
27
613
72
232
24
317
41
127
54

—, —, —, —,

DPA1*01:03, —, DPB1*04:02, —

—, —, —, —,
156
18
359
43
152
37
474
28
90
36

—, —, —, —,

DPA1*01:03, —, DPB1*04:01, —

—, —, —, —,
170
28
265
37
162
31
1319
41
173
39

—, —, —, —,

DPA1*02:01, —, DPB1*05:01, —

—, —, —, —,
5980
162
373
42
149
35
1342
42
359
55

—, —, —, —,

DPA1*02:01, —, DPB1*06:01, —

—, —, —, —,
7300
220
186
24
89
19
452
19
77
23

—, —, —, —,

DPA1*02:01, —, DPB1*09:01, —

Sum
49327
1874
9549
1435
4306
924
21360
1073
4806
1242

Reactivity (%)
100%
4%
100%
15%
100%
21%
100%
5%
100%
26%

TABLE E

MFI (raw) against individual HLA-DQ antigens measured using SAB assay.

P02
P04
P07
P08
P09

Allele specificity
+PBS
+IdeS
+PBS
+IdeS
+PBS
+IdeS
+PBS
+IdeS
+PBS
+IdeS

—, —, —, —,
133
15
147
20
221
20
21824
3652
20118
3389

DQA1*05:01, —, DQB1*02:01,

—, —, —, —, —

—, —, —, —,
127
18
5588
206
99
23
20701
2023
16486
1044

DQA1*02:01, —, DQB1*02:02,

—, —, —, —, —

—, —, —, —,
112
14
4347
161
92
17
22333
1826
15483
844

DQA1*02:01, —, DQB1*02:01,

—, —, —, —, —

—, —, —, —,
124
23
162
26
120
26
22318
2785
18755
1598

DQA1*04:01, —, DQB1*02:01,

—, —, —, —, —

—, —, —, —,
114
18
112
18
103
20
22232
3023
19425
1575

DQA1*03:01, —, DQB1*02:01,

—, —, —, —, —

—, —, —, —,
1331
49
5717
212
145
28
21762
1109
374
60

DQA1*02:01, —, DQB1*04:02,

—, —, —, —, —

—, —, —, —,
1473
49
5509
175
157
31
23347
1009
453
59

DQA1*02:01, —, DQB1*04:01,

—, —, —, —, —

—, —, —, —,
1235
43
220
26
102
20
20977
1724
14869
702

DQA1*04:01, —, DQB1*04:02,

—, —, —, —, —

—, —, —, —,
2215
69
243
30
97
24
22189
2769
636
139

DQA1*03:03, —, DQB1*04:01,

—, —, —, —, —

—, —, —, —,
1250
52
191
25
122
27
331
29
2641
69

DQA1*01:02, —, DQB1*05:02,

—, —, —, —, —

—, —, —, —,
2344
78
266
24
102
21
660
19
6670
146

DQA1*01:01, —, DQB1*05:01,

—, —, —, —, —

—, —, —, —,
4501
77
281
34
182
25
18442
380
234
47

DQA1*01:01, —, DQB1*06:02,

—, —, —, —, —

—, —, —, —,
7767
181
286
32
136
38
17789
983
9668
281

DQA1*01:03, —, DQB1*06:03,

—, —, —, —, —

—, —, —, —,
4760
127
280
59
192
55
21132
724
357
115

DQA1*01:02, —, DQB1*06:04,

—, —, —, —, —

—, —, —, —,
5321
100
164
25
158
24
20512
1107
543
103

DQA1*01:02, —, DQB1*06:09,

—, —, —, —, —

—, —, —, —,
4236
68
234
37
169
31
21040
358
189
46

DQA1*01:02, —, DQB1*06:02,

—, —, —, —, —

—, —, —, —,
8661
192
304
25
167
35
20774
1892
8624
290

DQA1*01:03, —, DQB1*06:01,

—, —, —, —, —

—, —, —, —,
328
27
7971
330
164
31
22290
2390
1494
234

DQA1*02:01, —, DQB1*03:01,

—, —, —, —, —

—, —, —, —,
642
21
203
18
89
17
22598
3231
17969
1575

DQA1*06:01, —, DQB1*03:01,

—, —, —, —, —

—, —, —, —,
225
37
325
145
151
30
22928
2696
1477
274

DQA1*03:01, —, DQB1*03:01,

—, —, —, —, —

—, —, —, —,
216
22
594
37
169
27
22888
3371
20671
2742

DQA1*05:03, —, DQB1*03:01,

—, —, —, —, —

—, —, —, —,
222
26
529
35
161
27
22683
3319
20511
2678

DQA1*05:05, —, DQB1*03:01,

—, —, —, —, —

—, —, —, —,
2123
46
5785
212
141
30
21968
1912
1342
197

DQA1*02:01, —, DQB1*03:02,

—, —, —, —, —

—, —, —, —,
1509
41
195
32
143
30
21810
2125
1192
204

DQA1*01:01, —, DQB1*03:02,

—, —, —, —, —

—, —, —, —,
2522
39
158
23
109
22
22088
2364
1367
222

DQA1*03:01, —, DQB1*03:02,

—, —, —, —, —

—, —, —, —,
5925
85
145
24
102
28
22349
3391
3207
486

DQA1*03:02, —, DQB1*03:02,

—, —, —, —, —

—, —, —, —,
6583
91
424
21
84
17
22488
3351
2651
441

DQA1*03:02, —, DQB1*03:03,

—, —, —, —, —

—, —, —, —,
6720
109
10667
469
94
23
22211
2934
2710
464

DQA1*02:01, —, DQB1*03:03,

—, —, —, —, —

—, —, —, —,
2768
82
197
26
117
23
22331
2422
1364
243

DQA1*03:01, —, DQB1*03:03,

—, —, —, —, —

Sum
75489
1800
51244
2505
3891
767
586994
58920
211481
20264

Reactivity (%)
100%
2%
100%
5%
100%
20%
100%
10%
100%
10%

TABLE F

MFI (raw) against individual HLA-DR antigens measured using SAB assay.

P02
P04
P07
P08
P09

Allele specificity
+PBS
+IdeS
+PBS
+IdeS
+PBS
+IdeS
+PBS
+IdeS
+PBS
+IdeS

DRB1*01:02, —, —, —,
17110
487
675
42
115
20
431
25
255
32

—, —, —, —, —,

—, —, —

DRB1*01:01, —, —, —,
18574
696
402
31
106
17
340
23
107
24

—, —, —, —, —,

—, —, —

DRB1*10:01, —, —, —,
21681
1351
327
28
691
26
23510
849
188
54

—, —, —, —, —,

—, —, —

DRB1*01:03, —, —, —,
16759
508
2850
51
82
18
487
21
116
22

—, —, —, —, —,

—, —, —

DRB1*11:04, —, —, —,
216
37
1575
66
5395
140
22704
1252
3740
513

—, —, —, —, —,

—, —, —

DRB1*11:01, —, —, —,
159
20
705
33
4552
95
21697
956
3207
380

—, —, —, —, —,

—, —, —

DRB1*12:02, —, —, —,
8815
293
7903
139
3381
63
23939
1668
2763
366

—, —, —, —, —,

—, —, —

DRB1*12:01, —, —, —,
9541
322
13327
341
3622
67
23623
1761
2803
344

—, —, —, —, —,

—, —, —

DRB1*13:03, —, —, —,
207
16
4923
104
6523
156
23331
1541
4764
550

—, —, —, —, —,

—, —, —

DRB1*13:01, —, —, —,
409
22
269
22
5257
119
22827
1247
4399
430

—, —, —, —, —,

—, —, —

DRB1*14:54, —, —, —,
5152
115
319
55
4412
114
22371
1320
3796
471

—, —, —, —, —,

—, —, —

DRB1*14:01, —, —, —,
6374
115
203
23
5847
135
23061
1611
4316
577

—, —, —, —, —,

—, —, —

DRB1*14:02, —, —, —,
2374
70
253
45
3609
96
21144
978
3310
336

—, —, —, —, —,

—, —, —

DRB1*15:02, —, —, —,
289
16
2435
68
94
16
22630
715
600
79

—, —, —, —, —,

—, —, —

DRB1*15:01, —, —, —,
363
18
3604
87
87
15
22151
929
845
102

—, —, —, —, —,

—, —, —

DRB1*15:03, —, —, —,
988
37
1943
72
150
35
22653
766
620
90

—, —, —, —, —,

—, —, —

DRB1*16:01, —, —, —,
322
29
1946
70
151
30
22645
782
868
130

—, —, —, —, —,

—, —, —

DRB1*16:02, —, —, —,
374
31
2495
80
142
28
22465
686
829
112

—, —, —, —, —,

—, —, —

DRB1*03:01, —, —, —,
264
20
182
22
6129
134
23891
2299
7907
778

—, —, —, —, —,

—, —, —

DRB1*03:02, —, —, —,
592
40
268
38
4394
104
22857
1681
5032
524

—, —, —, —, —,

—, —, —

DRB1*04:02, —, —, —,
13287
377
970
45
920
33
21202
1269
2770
330

—, —, —, —, —,

—, —, —

DRB1*04:03, —, —, —,
11768
304
927
79
762
41
22904
1174
2415
321

—, —, —, —, —,

—, —, —

DRB1*04:05, —, —, —,
13364
356
306
36
786
36
21299
1151
2439
318

—, —, —, —, —,

—, —, —

DRB1*04:04, —, —, —,
14417
431
1125
67
933
35
21760
1426
2682
369

—, —, —, —, —,

—, —, —

DRB1*04:01, —, —, —,
13713
413
807
60
897
36
21712
1323
2723
381

—, —, —, —, —,

—, —, —

—, —, DRB5*01:01, —,
13374
364
679
48
194
27
12086
140
147
37

—, —, —, —, —,

—, —, —

—, —, DRB5*02:02, —,
15426
490
1677
47
99
16
8798
241
692
37

—, —, —, —, —,

—, —, —

—, —, DRB3*03:01, —,
8925
415
927
95
667
103
22575
1013
1211
249

—, —, —, —, —,

—, —, —

—, —, DRB3*01:01, —,
8394
284
270
36
572
40
20467
1862
4190
479

—, —, —, —, —,

—, —, —

—, —, DRB3*02:02, —,
295
25
441
41
1203
45
22652
1251
1561
226

—, —, —, —, —,

—, —, —

—, —, DRB4*01:03, —,
21616
1327
247
32
104
32
23348
2341
439
213

—, —, —, —, —,

—, —, —

—, —, DRB4*01:01, —,
19130
944
686
64
304
22
23279
1917
870
196

—, —, —, —, —,

—, —, —

DRB1*07:01, —, —, —,
16524
691
25134
1957
107
16
24193
1793
2057
301

—, —, —, —, —,

—, —, —

DRB1*08:01, —, —, —,
171
29
2716
59
4588
100
22391
1101
4075
543

—, —, —, —, —,

—, —, —

DRB1*09:01, —, —, —,
17862
712
15740
648
199
42
22643
1466
1650
257

—, —, —, —, —,

—, —, —

DRB1*09:02, —, —, —,
20587
820
19870
1099
133
28
23199
1416
3767
351

—, —, —, —, —,

—, —, —

Sum
319416
12223
119123
5831
67208
2080
723269
41992
84154
10522

Reactivity (%)
100%
4%
100%
5%
100%
3%
100%
6%
100%
13%

REFERENCES

Ding J W, Zhou T, Zeng H, Ma L, Verbeek J S, Yin D, m.fl. Hyperacute rejection by anti-Gal IgG1, IgG2a, and IgG2b is dependent on complement and Fc-gamma receptors. J Immunol. 1 Jan. 2008; 180(1):261-8.

Jordan S C, Vo A, Bunnapradist S, Toyoda M, Peng A, Puliyanda D, Kamil E, Tyan D. Intravenous immune globulin treatment inhibits crossmatch positivity and allows for successful transplantation of incompatible organs in living-donor and cadaver recipients. Transplantation 2003 August; 76(4):631-636.

Moll S and Pascual M. Humoral rejection of organ allografts. Am. J. Transplant 2005 November; 5(11):2611-2618.

Montgomery R A, Hardy M A, Jordan S C, Racusen L C, Ratner L E, Tyan D B, Zachary A A. Consensus opinion from the antibody working group on the diagnosis, reporting, and risk assessment for antibody-mediated rejection and desensitization protocols. Transplantation 2004 July; 78(2):181-185.

Organ Procurement and Transplantation Network (OPTN) Database. US Department of Health and Human Services, Health Resources and Services Administration; May 11, 2011.

Thomas B. Martins. Development of Internal Controls for the Luminex Instrument as Part of a Multiplex Seven-Analyte Viral Respiratory Antibody Profile

Patel R, Terasaki P I. Significance of the positive crossmatch test in kidney transplantation. N. Engl. J. Med. 1969 April; 280(14):735-739.

Pierson R N 3rd. Antibody-mediated xenograft injury: mechanisms and protective strategies. Transpl Immunol. juni 2009; 21(2):65-9.

Terasaki P I, Ozawa M. Predicting kidney graft failure by HLA antibodies: a prospective trial. Am. J. Transplant 2004 March; 4(3):438-443.

Vo A A, Petrozzino J, Yeung K, Sinha A, Kahwaji J, Peng A, m.fl. Efficacy, outcomes, and cost-effectiveness of desensitization using IVIG and rituximab. Transplantation. 27 Mars 2013; 95(6):852-8.

von Pawel-Rammingen U, Johansson B P, Björck L. IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G. EMBO J 2002 April; 21(7):1607-1615.

Åkesson P, Moritz L, Truedsson M, Christensson B, von Pawel-Rammingen U. IdeS, a highly specific immunoglobulin G (IgG)-cleaving enzyme from Streptococcus pyogenes, is inhibited by specific IgG antibodies generated during infection. Infect. Immun 2006 January; 74(1):497-503.

Example 3
Introduction

As is demonstrated in Examples 1 and 2, IdeS rapidly cleaves all plasma IgG after intraqvenous administration to human subjects. The following in vitro and ex vivo data show that IdeS not only cleaves soluble IgG as previously shown, but also cuts off the F(ab′)₂part of the B-cell receptor complex from surface IgG-positive B-cells. The truncation of the BCR through IdeS has strong inhibiting effects on the induction of secreted IgG from R848 and IL-2 activated CD27 positive memory B-cells, while the IgM secretion of surface IgM-positive BCR cells are not reduced by the treatment with IdeS. This suggests that the treatment with IdeS of patients with donor specific antibodies not only removes said antibodies, but also renders donor-specific memory B cells (at least initially) incapable of responding to donor antigens. Thus, any initial activation of the memory B cells and generation of plasma cells which could produce more donor specific antibodies is also affected.

Material and Methods
Screening Cell Lines for Surface Immunoglobulin

Different human B-cell lymphoma cell lines i.e. U-2940 (ACC634), NU-DUL-1 (ACC579), Raji (CCL-86) and Daudi (CCL-213) were screened for the presence of membrane bound IgG or IgM. Briefly, cells were cultured at 37° C. in 5% CO₂in RPMI1640 supplemented with 10% FCS and PEST. Cells were treated for 30 min at 37° C. with PBS or different concentrations of IdeS prior to acid wash (0.1 M glycine pH 2.7, 0.5 M NaCl) for 5 min on ice. Acid wash removes antibodies present in FcγR's or bound to antigen while leaving transmembrane molecules intact (Gilden et al., 1978, Jennings et al., 2011). Cells were stained with biotinylated antibodies specific for the F(ab′)₂part (#109-066-097, Jackson, cross-reacts with light chain present in all immunoglobulin subclasses) and the Fc-part of IgG (#109-066-098, Jackson, specific for IgG heavy chain) or IgM (GM-80A, ICL). Streptavidin-APC (#016-130-084, Jackson) was used to monitor cells in FL4 using an Accuri C6 flow cytometer.

Cell Proliferation and Viability Assays

Proliferation was measured by BrdU incorporation. Cells were treated with PBS or different concentrations of IdeS and 5×10⁴cells/well were seeded in 96-well plates and cultured for 24 hours. BrdU was added to cells and incubated for 6 hours prior to measuring proliferation according to the manufacturers recommendation (Cell Proliferation ELISA, BrdU colorimetric, Roche #11 647 229 001). Cytochalasin D (C2618, Sigma) and Puromycin (Invitrogen) were used at different concentrations as anti-proliferative controls.

A sensitive colorimetric assay (CCK-8) was used to measure cell viability. Cells were treated with PBS or 30 μg/ml IdeS and 2×10⁴cells/well were seeded in 96-well plates and cultured for 24 hours. CCK-8 (CCK-8 cell counting kit 8, Dojindo Laboratories, Japan) was added and the absorbance at 450 nm was followed at different time points. In experiments with Nu-DUL-1 cells they were treated with PBS or 30 μg/ml IdeS and different amount of cells were seeded in 96-well plates and cultured for 24 hours prior to addition of CCK-8. In experiments with enriched B-cells peripheral blood was collected in heparin tubes supplemented with IdeS at 30 μg/ml or PBS and incubated at 37° C., 5% CO₂for 30 minutes. 250 μl RosetteSep® Human B cell Enrichment cocktail (#07905, StemCell Technologies) was added to 5 ml blood, mixed well and incubate for 20 minutes at room temperature. Samples were diluted with an equal volume of PBS supplemented with 2% FCS prior to density gradient separation (Ficoll-PaquePLUS). Harvested B-cells were counted and adjusted to 20×10⁴cells/ml in RPMI1640 supplemented with 10% FCS and PEST. 2×10⁴cells/well were seeded in triplicates in 96-well plates and cultured for 24 hours prior to addition of CCK-8.

Addressing IdeS Efficacy in Plasma by SDS-PAGE

Plasma collected during density gradient separation of heparin blood treated with PBS or different amounts of IdeS was used to verify IdeS efficacy on soluble IgG. The SDS-PAGE analyses were performed according to the manufacturer's instructions (Bio-Rad Laboratories, CA, USA). Briefly, 1 μl of plasma was separated on 4-20% Mini-PROTEAN®TGX™ precast gels (Bio-Rad) at 200 V for 40 minutes under non-reduced conditions. The gels were stained with GelCode Blue stain reagent (Pierce, Thermo Fisher Scientific, MA, USA) according to the manufacturer's instructions and the gels were scanned.

Recovery of Cleaved BCR

Nu-DUL-1 cells were treated with PBS or different amounts of IdeS for one hour at 37° C. prior to extensive washing in order to remove any remaining IdeS. The cells were seeded in 96-well plates in RPMI1640 supplemented with 10% FCS and PEST. One plate was immediately used for flow cytometry analysis of intact IgG and the other was cultured (37° C., 5% CO₂) for 24 hours prior to analysis. Cells were stained with a biotinylated antibody specific for the F(ab′)₂part (#109-066-097, Jackson) followed by Streptavidin-APC (#016-130-084, Jackson) and cells were monitored in FL4 using an Accuri C6 flow cytometer.

Peripheral blood was collected in heparin tubes (BD Vacutainer, #367876) from healthy volunteers and treated with either 30 μg/ml IdeS or PBS for one hour at 37° C. prior to isolating PBMC using density gradient separation (Ficoll-PaquePLUS). The PBMC interface was collected, washed in PBS and re-suspended in culture medium (RPMI1640 supplemented with 10% FCS and PEST). PBMCs were counted, adjusted to 2×10⁶cells/ml and a sample was removed, fixed in PFA, washed in PBS supplemented with 0.1% BSA and stored at 4° C. until flow cytometry analysis. The remaining cells were cultured and samples were removed and PFA fixed at indicated time points. For detection of the F(ab′)₂part of IgG, biotinylated anti-CH1-IgG (#710.3202.100, BAC) was used, for detection of the Fc-part of IgG goat anti-human Fc-specific F(ab′)₂fragment (#109-066-098, Jackson) was used. Cells were further double stained with PE-conjugated anti-CD19 (#IP-305-T100, ExBio) and Streptavidin-APC (#016-130-084, Jackson). The lymphocyte population was gated using forward-side scatter and double positive cells were monitored in FL2 and FL4 using an Accuri C6 flow cytometer.

Intracellular Phospho-Specific Flow Cytometry (BCR Signalling)

Nu-DUL-1 cells were cultured overnight in serum free medium in order to minimize background phosphorylation prior to start of signalling experiments. The next day PBS or 30 μg/ml IdeS was added and the cells were cultured (37° C., 5% CO₂) for 30 min. 1×10⁶cells were removed and fixed for 5 min in PFA followed by 10 min permeabilization in 70% ethanol on ice. Cells were washed in PBS supplemented with 0.1% BSA and stored at 4° C. until analysis (zero sample). After collection of the zero sample, the BCR of the remaining cells was cross-linked by addition of 10 μg/ml goat anti-human F(ab′)₂specific F(ab′)₂(Jackson #109-006-097) and cell-samples were collected at different time points, fixed and permeabilized. The fixed cells were stained for flow cytometry analysis using APC-conjugated phospho-specific ERK1/2 (#17-9109-42, eBioscience) and PE-conjugated phospho-specific PLC-γ2 (#558490, BD). Cells were monitored in FL2 and FL4 using an Accuri cytometer C6.

Memory B-Cell Differentiation

Peripheral blood was collected in heparin tubes (BD Vacutainer, #367876) from healthy volunteers and PBMC were isolated using density gradient separation (Ficoll-PaquePLUS). The PBMC interface was collected, washed in PBS and re-suspended in culture medium (RPMI1640 supplemented with 10% FCS and PEST). PBMCs were adjusted to 2×10⁶cells/ml and seeded either with IdeS (final concentration 0.3, 3 and 30 μg/ml) or PBS. Cells were stimulated with a mixture of R848 and rIL-2 according to the manufacturer's recommendation (MabTech) and cultured for 72-96 hours. Cells intended for the short time treatments were left in tubes supplemented with PBS or IdeS and incubated for one hour at 37° C. prior to washing 3×12 ml with PBS and 1×12 ml in culture medium. These cells were seeded and treated with R848/rIL-2 as above.

ELISpot filter plates were pre-wetted with 70% ethanol, washed with sterile water and incubated at 4° C. overnight with capture antibody (ELISpotPLUS Mabtech kit #3850-2HW-Plus for monitoring IgG producing cells, ELISpotPLUS Mabtech kit #3845-2HW-Plus for monitoring IgM producing cells and ELISpotBASIC Mabtech kit #3860-2H for monitoring IgA producing cells). The ELISpot filter plates were wash and blocked for at least 30 min with culture medium prior to seeding cells.

The cells were transferred to 15 ml tubes, extensively washed, counted and adjusted to 0.5×10⁶cells/ml and 2-fold dilutions were prepared before cells were seed in the prepared ELISpot filter plates and cultured for 24 hours. ELISpot-plates were washed and biotinylated detection antibodies for total IgG, IgM and IgA analysis (included in the named kits) were incubated for two hours at room temperature. Plates were wash and incubated for one hour at room temperature with Streptavidin-HRP before they were washed and incubated with TMB ready-to-use solution and developed until distinct spots emerged. The plates were washed in tap water and allowed to dry in the dark. The filters were photo documented and spots were manually counted.

B-Cell Enrichment and Flow Cytometry

For B-cell enrichment peripheral blood was collected in heparin tubes supplemented with IdeS at 30 μg/ml or PBS and incubated at 37° C., 5% CO₂for 30 minutes. 250 μl RosetteSep® Human B cell Enrichment cocktail (#07905, StemCell Technologies) was added to 5 ml blood, mixed well and incubate for 20 minutes at room temperature. Samples were diluted with an equal volume of PBS supplemented with 2% FCS prior to density gradient separation (Ficoll-PaquePLUS). Harvested B-cells were counted and adjusted to 15×10⁴cells/ml in PBS supplemented 2% FCS prior to seeding in V-shaped 96-well plates for flow cytometry staining. Plates were cfg at 1500 rpm for 3 minutes and the supernatant was flicked off. For detection of the F(ab′)₂part of IgG, 10 μg/mlbiotinylated anti-CH1-IgG (#710.3202.100, BAC) was used, for detection of the Fc-part of IgG 0.5 μg/ml goat anti-human Fc-specific F(ab′)₂fragment (#109-066-098, Jackson) was used. Cells were further double stained with either PE-conjugated anti-CD19 (#IP-305-T100, ExBio) or PE-conjugated anti-CD27 (#555441, Pharmingen) followed by Streptavidin-APC ((#016-130-084, Jackson). Cells were monitored in FL2 and FL4 using an Accuri cytometer C6.

IdeS Cleaves the IgG-Type of BCR in a First in Man Clinical Study

A phase I, double blind and randomized study with single ascending doses of IdeS was conducted at the Phase 1 Unit, Lund, after approval from Swedish regulatory and ethical authorities (ClinicalTrials.gov Identifier: NCT01802697). All subjects signed written informed consent before any study related procedures were initiated. As an exploratory part of the study, the integrity of the IgG-type of BCR on CD19⁺ cells was monitored at different time-points after intravenous treatment with 0.12 or 0.24 mg/kg BW IdeS. Peripheral blood was collected in heparin tubes and PBMCs were isolated within 2 hours from collection using density gradient separation (Ficoll-PaquePLUS). The PBMC interface was collected, washed in PBS and fixed in PFA for 30 min on ice. Cells were washed and stored in PBS supplemented with 0.5% BSA until all time points were collected. Cells were stained with 10 μg/ml biotinylated anti-CH1-IgG (#710.3202.100, BAC) for detection of the F(ab′)₂part of IgG. For detection of the Fc-part of IgG 0.5 μg/ml goat anti-human Fc-specific F(ab′)₂fragment (#109-066-098, Jackson) was used. Cells were further double stained with PE-conjugated anti-CD19 (#21270194, Immunotools) and Streptavidin-APC (#016-130-084, Jackson). The lymphocyte population was gated in the pre-dose sample for each individual and this gate was then used for all time points for a subject. CD19⁺ cells were monitored in FL2 and the F(ab′)₂/Fc-signal was monitored in FL4. CD19⁺ cells were monitored in M1 (FL2) and these cells were further monitored for presence of a signal upon anti-Fc and anti-Fab staining (FL4). In each sample the cell counts in upper right (UR) as well as mean fluorescent intensity (MFI) were collected.

Furthermore, the frequency of double positive cells was calculated using the following formula:

$\frac{MFI in UR \times cell counts in UR}{cell counts in M 1}$

This formula was used to be able to appreciate the difference in MFI when only low cell counts were present in UR.

Results

IdeS Cleaves the IgG-Type of BCR with Similar Efficacy as Soluble IgG

Four different B-lymphoma cell lines were screened for the presence of transmembrane IgG or IgM using a flow cytometry approach including an acid wash step to remove IgG or IgM not attached to the membrane via a transmembrane domain (Gilden et al., 1978, Jennings et al., 2011). After verifying the presence of IgG- or IgM-type of BCR on the cell lines, Nu-DUL-1 (IgG-type of BCR) and Daudi (IgM-type of BCR) were selected as models for further analysis. A Fab-fragment specific F(ab′)₂antibody was used to detect the presence of the Fab-part of BCR since the antibody cross-reacts with the light-chain present in both IgG and IgM. To detect the presence of the Fc-part, antibodies directed at the Fc-part of IgG was used. Intact membrane-bound IgG could not be detected on the cell surface at an IdeS concentrations above 4 μg/ml. Daudi cells having an IgM-type of BCR were not affected even at high concentrations of IdeS (FIG. 20A). Nu-DUL-1 cells were treated with different concentrations of IdeS and incubated at 37° C. for 30 min prior to FACS staining. IdeS was shown to efficiently remove the F(ab′)₂part of IgG present in the BCR leaving the cleaved Fc-part attached to the membrane (FIG. 20B).

We next moved to addressing BCR cleavage on PBMCs purified from healthy volunteers. Due to the cross-reactivity of the Fab-specific antibody with light-chains on IgM, the anti-IgG-CH1-domain specific CaptureSelect fragment was used for staining the heterogenic PBMC population. Blood collected in heparin-tubes was treated for 30 minutes (37° C.) with different concentrations of IdeS. The PBMCs were density-gradient separated after IdeS treatment and both plasma and PBMCs were collected. The plasma was analysed on SDS-PAGE to confirm IdeS efficacy on soluble IgG (FIG. 21A). ScIgG was generated already at 0.9 μg/ml IdeS and full cleavage was achieved at 9 μg/ml.

Since CD19 is a hallmark for B-cell linages the PBMCs were double stained with anti-CD19 and anti-Fab or anti-Fc in order to monitor the presence of intact IgG-BCR on B-cells. Flow cytometry showed that IdeS could remove the F(ab′)₂part of IgG from CD19⁺ cells while leaving the Fc-part in the membrane (FIG. 21B). As single-cleaved membrane bound IgG present in the BCR is still attached to the membrane, the effect is not fully visible by flow cytometry as long as the scIgG product is present and attached to the membrane. Full effect was reached on membrane bound IgG at 9 μg/ml IdeS. Thus these results show for the first time that there is a direct correlation between IdeS efficacy on free IgG and membrane bound IgG correspondingly present in B-cell receptors.

In order to further define the effect of IdeS on the memory subset of B-cells the effect on CD19⁺/CD27⁺ memory B-cells was investigated. CD19⁺ B-cells only constitute a few percent of the total PBMC population. Hence, CD19⁺ B-cells were enriched using negative selection (RosetteSep), which generated >90% CD19⁺ cells (FIG. 22A). Approximately 10% of this population stained double positive for surface IgG and CD27 prior to IdeS treatment (FIG. 22B). After IdeS treatment less than 1% of the CD19⁺/CD27⁺ cells stained positive for cell surface IgG (FIG. 22B). Thus, these data show for the first time that the BCR on class-switched memory B cells i.e. CD19⁺/CD27⁺/surface IgG⁺ cells is efficiently cleaved by IdeS.

Cleaved BCR is Rapidly Regenerated on Both Cell Lines and PBMCs and has No Effect on Cell Viability

In order to investigate the membrane turnover of the IgG-type of BCR the Nu-DUL-1 cells were treated with different concentrations of IdeS, washed to remove IdeS and cultured. Fractions of cells were removed one and 24 hours after treatment and analysed for membrane bound IgG by flow cytometry. One hour after treatment there was no detectable IgG at IdeS concentrations >4 μg/ml but 24 hours after treatment, the Fab specific signal was back at the original levels demonstrating that the membrane bound IgG had recovered (FIG. 23A). Nu-DUL-1 cells were also analysed for proliferative capacity using BrdU incorporation and there was no difference in proliferation after cleaving the IgG-type of BCR even when IdeS treatment was continued over 24 hours (FIG. 23B). Substances with known anti-proliferative capacity (puromycin and cytochalasin D) had a strong anti-proliferative effect on the cells. The viability of Nu-DUL-1 cells was also investigated by treating cells with a high dose of IdeS (30 μg/ml) for 24 hours and viability was analysed using the CCK-8 assay and there was no effect on cell viability after IdeS treatment (FIG. 23C).

The findings that the IgG-type of BCR was regenerated within 24 hours on a highly proliferating lymphoma cell line could be expected since membrane turn-over on proliferating cells is usually very high. PBMCs from healthy volunteers were subjected to a similar treatment in order to further investigate the turn-over of the IgG-type of BCR on primary cells. Blood was collected in heparin-tubes and treated with a high dose (30 μg/ml) of IdeS. After treatment of the blood the PBMCs were separated on Ficoll. The PBMCs were washed with large volumes of buffer in order to remove all IdeS prior to culturing. A fraction of cells were removed at different time points, fixed and stained with anti-CD19 for B-cell linage and further stained with anti-Fab or anti-Fc to monitor IgG-BCR. The IgG-type of BCR was rapidly regenerated also on normal human CD19⁺ cells and already within 16 hours after cleavage the number of anti-Fab positive cells was back to pre-treatment levels though still not reaching the full MFI. This indicates that 16 hours post IdeS treatment of PBMCs the cells again have intact IgG-BCR on the surface even though all IgG-BCR are not yet replaced (FIG. 24A). The anti-Fc signal was not affected by the treatment demonstrating that IdeS treatment shed the F(ab′)₂from the IgG-type of BCR (FIG. 24B). Because B-cells only account for a few percent of the total PBMC population we also used a B-cell enrichment kit (RosetteSep), which generated >90% CD19⁺ cells. Approximately 20% of the CD19 enriched cell population stained positive for IgG using both the F(ab′)₂and the Fc specific reagents (FIG. 25). The cell surface recovery experiment was repeated using these purified cells and IdeS treatment efficiently removed the F(ab′)₂part of the membrane bound IgG leaving the Fc-part intact (FIGS. 25A and 25B). Again, the turn-over was rapid and already 16 hours after treatment the cell surface IgG had recovered (FIG. 25A). Cell viability was followed for several days using the CCK-8 assay in order to evaluate survival of primary human B-cells with or without an intact BCR. There was no significant effect on cell viability when temporally removing the IgG-type of BCR from the CD19 enriched cells by IdeS treatment (FIG. 26).

IdeS Treatment Inhibits BCR Signalling

BCR signalling is important in the activation, survival, and differentiation of B lymphocytes. The initial event after BCR engagement is the activation of Lyn and Syk, which is then further propagated into activation of PLC-γ2 and ERK1/2. The described experiments clearly showed that IdeS could cleave the IgG-type of BCR, which should have implications on the BCR signalling. To verify this PLC-γ2 and ERK1/2 phosphorylation were monitored as downstream indicators for the BCR signalling cascade. In a series of experiments where the BCR on Nu-DUL-1 cells was cross-linked using a F(ab′)₂specific antibody it was shown that the cells were unable to signal through the BCR after IdeS treatment (FIGS. 27A and 27B). Neither PLC-γ2 nor ERK1/2, were phosphorylated in response to attempted BCR ligation using a F(ab′)₂specific antibody after IdeS treatment. The mock treated cells responded normally. These data demonstrate that IdeS treated cells with a cleaved IgG-type of BCR cannot respond to antigenic stimulation.

IdeS Blocks B-Cell Maturation

Due to the finding that IdeS does not affect the viability of cell lines or primary B-cells but renders them unable to respond to antigen, we decided to further explore the functionality of primary memory B-cells. Thus, PBMCs were collected, treated with IdeS and stimulated with recombinant IL2 and R848 in order to activate memory B-cells and differentiate them into Ig-producing cells (Jahnmatz et al., 2013). After 72-96 hours the cells were extensively washed in order to remove IdeS and analysed for frequency of Ig-producing cells. IdeS was also added on day three of IL2/R848 culture as additional control. At this time point it is not possible to stop the secretion of IgG of IL2/R848 differentiated B-cells. This control also shows that the loss of signal is not due to a carryover effect of IdeS interfering with the antibodies of the ELISPOT assay (FIG. 28A). In most experiments, IdeS was present throughout the stimulation period (96 hours) and the results showed that IdeS treatment inhibited memory B-cell differentiation and the number of IgG-producing cells while it had no effect on the maturation of IgM- or IgA-producing cells (FIGS. 28A and 28B). There was a significant effect on B-cell differentiation at all tested IdeS concentrations from 0.3 to 30 μg/ml (FIG. 28C). A second set of experiments where IdeS was washed away prior to stimulation with IL2 and R848 also resulted in a significant reduction in the number of IgG-producing cells (FIG. 28D) showing that the initial removal of the antigen-binding part of IgG-BCR is an important step in inhibiting memory B-cell activation into Ig-producing cells.

IdeS Cleaves the IgG-Type of BCR In Vivo in Humans

IdeS has recently been tested in a first in man study where healthy human subjects were given single ascending i.v. doses (ClinicalTrials.gov Identifier: NCT01802697) (submitted manuscript). The highest tested dose given to four subjects was 0.24 mg/kg BW. An exploratory part of the trial was to analyse the integrity of the IgG-type of BCR on circulating CD19⁺ lymphocytes at different time-points after IdeS administration. Peripheral blood was collected and PBMCs were purified at pre-dose, 2 h, 24 h, 48 h and 96 h post administration. Cells were immediately fixed to prevent further cell metabolism and stored until all time-points from a subject could be analysed. The PBMCs were double-stained for CD19 and F(ab′)₂respectively Fc-fragments and analysed using flow cytometry. The method can measure the frequency and mean fluorescence intensity of cells having F(ab′)₂(i.e. intact IgG-type of BCR) and Fc on their cell-surface. However, the method does not discriminate between intact and single-cleaved BCR. The results demonstrated that the number of CD19⁺ cells that stained positive for F(ab′)₂was reduced already 2 h after treatment with IdeS (0.24 mg/kg BW IdeS) while the number of cells that stained positive for Fc was not reduced (FIG. 29). This clearly demonstrates that IdeS in vivo cleaved the surface IgG on CD19⁺ cells. Data from the four subjects, plus two placebos demonstrate that IdeS efficiently cleaved the IgG-type of BCR in humans and that the frequency of CD19⁺ cells that were positive for surface IgG gradually recovered over the days following treatment (FIG. 30).

Discussion

The data presented here clearly show that IdeS cleaves the IgG-type of BCR and completely inhibits BCR signalling in response to receptor ligation. Thus, B-cells with IdeS cleaved IgG-type of BCR are rendered incapable of antigen binding resulting in loss of the major cellular events downstream of receptor ligation i.e. internalization, processing and presentation on MHC class II molecules. B-cells are very potent antigen-presenting cells (Lanzavecchia 1990, Avalos & Plough 2015) and can with high efficiency present an antigen on HLA after specific BCR-mediated endocytosis, therefore the loss of the antigen-binding fragment of the BCR upon IdeS cleavage is likely to have an impact on antigen presentation to CD4⁺ T-cells.

Cleaving the IgG-type of BCR has no impact on cell viability neither on cell lines nor on B-cells purified from healthy human subjects (FIGS. 23, 24 and 26). However, cells seem to recover from the treatment slightly slower in vivo than in vitro (FIG. 30). Due to very limited amounts of cells available for analysis from the human phase 1 study, other B-cell markers could not be followed in this exploratory study. Hence, it is not possible to draw conclusions regarding the fate of any specific subpopulation of B-cells. The evidence is consistent with recovery of IgG-type of BCR on the cleaved cells by means of membrane turnover as we have seen in vitro, but it cannot be ruled out that the slight delay in recovery seen in vivo is due to maturation of new B-cells rather than mere membrane turnover.

The results presented here show that IdeS blocks development of IgG but not IgA or IgM antibody secreting cells if IdeS is used prior to activation with polyclonal stimulation (IL2 and R848). However, if IdeS is used later on it has no blocking effect. Upon in vitro stimulation of PBMCs in culture antigens bound to the BCR are internalized and loaded on MHCII molecules and presented to T-cells which induce B-cell proliferation and differentiation (Tangye & Tralington 2009). In the absence of antigen stimulation i.e. as in the case when the IgG-BCR is cleaved by IdeS, the B-cell will not get a second signal and proliferate.

Additionally, this indicates an important role for the IgG-BCR complex in responding to stimulation even in the absence of antigen, i.e. in maintaining tonic signal. It has been published that a single amino acid mutation in the extracellular region of CD79b of the BCR results in agammaglobulia (Dobbs et al., 2007). This finding indicates that the interaction between the proteins in the BCR on the extracellular part is important for cell activation and we speculate that the interaction between CD79a/b and the IgG is important for the generation of a proper signalosome in response to external stimuli. Further support for this theory comes from a publication targeting CD79b extracellular domain using a non-lytic antibody (Hardy et al., 2014). They found that binding to CD79b resulted in B-cell anergy and loss of IgG producing cells both in vitro and in vivo. We propose that disassembling the BCR by cleaving of the F(ab′)₂part of IgG results in unresponsiveness to antigen-dependent activation due to loss of proper intracellular protein assembling and generation of a functional signalosome.

IdeS is currently developed for desensitisation of highly sensitized patients on the waiting list for kidney transplantation. These patients have developed antibodies against most donors and there is little chance of finding a matching donor. By removing donor specific antibodies (DSA) using IdeS prior to transplantation patients can be made eligible for transplantation despite a positive cross-match before treatment. An additional effect of IdeS treatment is the instant generation of free F(ab′)₂fragments from DSA with retained binding capacity. These F(ab′)₂fragments may bind and block epitopes in the graft and since the F(ab′)₂fragments have lost their Fc-mediated functions such as complement fixation (CDC), antibody dependant cellular cytotoxicity (ADCC) and antibody dependant cellular phagocytosis (ADCP) the F(ab′)₂fragments may have the capacity to block out IgM and newly formed IgG DSA and thereby provide an additional protection of the graft. The results presented here show that IdeS also cleaves the IgG-type of BCR on CD27⁺ positive memory B-cells and renders them incapable of answering to antigenic stimulation. Thus, not only are DSA removed by IdeS, but furthermore, the DSA-specific memory B-cells are initially not capable of responding to donor antigens. This may potentially have long term effects on the outcome of graft survival as the initial activation of memory B-cells and generation of long-lived plasma cells is likely to be affected by IdeS treatment.

REFERENCES

Avalos A. M. & Ploegh H. (2014) Early BCR Events and Antigen Capture, Processing and Loading on MHC Class II on B cells. Front. Immunol. March 10:5:92

Dal Porto J M, Gauld S B, Merrell K T, Mills D, Pugh-Bernard A E, Cambier J. B cell antigen receptor signaling 101. Mol. Immunol. 41(6-7), 599-613 (2004).

Dobbs A K, Yang T, Farmer D, Kager L, Parolini O, Conley M E. (2007) Cutting edge: a hypomorphic mutation in Igbeta (CD79b) in a patient with immunodeficiency and a leaky defect in B cell development. J Immunol. 15; 179(4):2055-9

Hardy I, Anceriz N, Rousseau F, Seefeldt M, Irla M et al. (2014) Anti-CD79 Antibody Induces B Cell Anergy That Protects against Autoimmunity. J Immunol. 192: 1641-1650

Jahnmatz M, Kesa G, Netterlid E, Buisman A M, Thorstensson R, et al. (2013) Optimization of a human IgG B-cell ELISpot assay for the analysis of vaccine-induced B-cell responses. J Immunol Methods 391: 50-59

Johansson B P, Shannon O, Björck L (2008) IdeS: a bacterial proteolytic enzyme with therapeutic potential. PLoS One 3: e1692

Johnson G L and Lapadat R. (2002). Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science, 298, 1911-1912

Kurosaki T. Regulation of B-cell signal transduction by adaptor proteins. Nat. Rev. Immunol. 2(5), 354-363 (2002)

Lanzavecchia A. Receptor-mediated antigen uptake and its effect on antigen presentation to class II-restricted T lymphocytes. Annu Rev Immunol (1990) 8:773-93

Manz R, Hauser A, Hiepe F, Radbruch A. (2005) Maintenance Of Serum Antibody Levels. Annu. Rev. Immunol. 2005. 23:367-86

Nandakumar K S, Johansson B P, Björck L, Holmdahl R (2007) Blocking of experimental arthritis by cleavage of IgG antibodies in vivo. Arthritis Rheum 56: 3253-3260

Rajewsky K. Clonal selection and learning in the antibody system. Nature 1996:381(6585):751-8

Reth M. Antigen receptor tail clue. Nature 338(6214), 383-384 (1989)

Reth M, Wienands J. Initiation and processing of signals from the B cell antigen receptor. Annu. Rev. Immunol. 15, 453-479 (1997)

Scharenberg A M, Humphries L A, Rawlings D J. Calcium signalling and cell-fate choice in B cells. Nat. Rev. Immunol. 7(10), 778-789 (2007)

Stuart G. Tangye and Kim L. Good Human IgM+CD27+B Cells: Memory B Cells or “Memory” B Cells? The Journal of Immunology, 2007, 179: 13-19

Su Y F, Chuang W J, Wang S M, Chen W Y, Chiang-Ni C, Lin Y S, Wu J J, Liu C C. (2011) The deficient cleavage of M protein-bound IgG by IdeS: insight into the escape of Streptococcus pyogenes from antibody-mediated immunity. Mot Immunol. 49(1-2):134-42

Tangye & Tralington. (2009) Memory B cells: Effectors of long-lived immune responses. Eur. J. Immunol. 39: 2065-2075

Tradtrantip L, Asavapanumas N, Verkman A S (2013) Therapeutic cleavage of anti-aquaporin-4 autoantibody in neuromyelitis optica by an IgG-selective proteinase. Mol Pharmacol 83: 1268-1275

Vincents B, von Pawel-Rammingen U, Björck L and Abrahamson M, (2004). Enzymatic characterization of the streptococcal endopeptidase, IdeS, reveals that it is a cysteine protease with strict specificity for IgG cleavage due to exosite binding. Biochemistry 43: 15540-9

von Pawel-Rammingen U, Johansson B P and Björck L, (2002). IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G. EMBO J 21: 1607-15

Wenig K, Chatwell L, von Pawel-Rammingen U, Björck L, Huber R and Sondermann P, (2004). Structure of the streptococcal endopeptidase IdeS, a cysteine proteinase with strict specificity for IgG. Proc Natl Acad Sci USA 101: 17371-6

Yang R, Otten M A, Hellmark T, Collin M, Björck L, et al. (2010) Successful treatment of experimental glomerulonephritis with IdeS and EndoS, IgG-degrading streptococcal enzymes. Nephrol Dial Transplant 25: 2479-2486

Example 4

The following study was conducted to determine whether it is possible to target the BCR of a B cell which has been treated with IdeS.

Materials and Methods

Briefly, two-step dilutions of cells (Nu-DUL-1, B-cell lymphoma with IgG-type of BCR; ACC 579 from DSMZ) from 80 000/well to 1250/well in R10 medium (RPMI1640, PEST and 10% FCS) were seeded in duplicates in a 96-well flat bottom polystyrene plate and used as a calibrator for viable cells. 20 000 cells/well were seeded in the plate and incubated with or without 30 μg/ml IdeS at 37° C. in the CO₂-incubator for 1 hour. Anti-Fab, Anti-Fc, or control was added to the test wells to a final conc. of 10 μg/ml and the plate was incubated at 37° C. in the CO₂-incubator. The first plate was removed after 3 hours, the second after 24 hours and the third after 48 hours. After removing the plate from the incubator, CCK-8 reagent (from CCK-8 cell counting kit; Dojindo Laboratories, Japan) was added and continued incubation for 1 hour prior to reading the plate at 450 nm in an ELISA-plate reader (spectrophotometer). The CCK-8 assay allows sensitive colorimetric assays for the determination of cell viability in cell proliferation and cytotoxicity assays. The anti-Fab agent used was F(ab′)₂specific goat F(ab′)₂fragment (Jackson #109-006-097, 1.3 mg/ml). The anti-Fc agent used was Fc specific goat F(ab′)₂fragment (Jackson #109-006-098, 1.3 mg/ml). The control was mouse gamma globulin (Jackson #015-000-002, 11.4 mg/ml). The control was selected to be from the same manufacturer as the tested anti-Fab and anti-Fc and because mouse IgG is not cleaved by IdeS.

Results and Conclusions

Anti-Fab
Anti-FC
Ctrl IgG

3 h
24 h
48 h
3 h
24 h
48 h
3 h
24 h
48 h

PBS
25665
18514
7969
21569
8568
3112
21569
23598
24386
Mean no of

IdeS
25026
29347
25211
23005
9104
979
21569
21583
22469
viable cells

PBS
1013
288
212
1015
332
446
884
504
1114
StDEV

IdeS
1718
664
355
1940
1780
427
1665
489
374

The results show that cross-linking the BCR on a target cell expressing the IgG-type of BCR (in this case a B-cell lymphoma cell line) induces cell death using antibodies directed against either the F(ab′)₂or the Fc-part. Making direct use of the BCR as a target is however not possible in a human prior to IdeS treatment due to the presence of normal IgG-levels (˜10 mg/mL) in circulation. However, pre-treatment with IdeS can decrease the antibody levels left in circulation while leaving an Fc-fragment on target cells still available for therapeutic intervention. Targeting the Fc-fragment after IdeS cleavage is at least as efficient on IdeS treated cells as on mock treated cells. Therapeutic intervention can be accomplished by means of an antibody targeting an epitope which is created in the BCR as a consequence of IdeS cleavage or even by targeting a common epitope on the Fc (as shown here). The therapeutic antibody is preferably one that is not cleaved by IdeS and has high degree of Fc-effector functions i.e. CDC, ADCC and ADCP. The antibody could also be coupled to a cytotoxic agent i.e. radioisotope or toxin.

Another possibility is provided by the considerably quicker recovery of intact IgG on membrane bound BCR compared to recovery of IgG in circulation. This makes it possible to use the F(ab′)₂part as target and not only the Fc-part. Recovery of the IgG-BCR on memory B-cells opens up the possibility to use antigens (linked to toxins or radioisotopes) to specifically target memory B-cells with particular non-desired specificities (i.e. anti-HLA or anti-insulin).