Patent Application
20220276261
The present invention concerns a method for classifying a disorder associated with the complement system comprising the detection of the presence of C3/C5 convertase complexes. Moreover, the invention refers to a method for determining whether a patient will be responsive to a modulator of the complements system, a method for determining the disorder course of a patient with a disorder associated with the complement system and a method for determining whether a candidate compound is suitable for the treatment of a disorder of the complement system and the respective kits.
There is growing evidence that deregulated complement activation contributes to or drives the pathogenesis of various diseases. Thus, targeting complement is a therapeutic option (Thurman et al., 2015; Zipfel et al., 2015). C5 convertase inhibition is an established therapy in atypical hemolytic uremic syndrome and several novel drugs target other components of the complement cascade (Reddy et al., 2017, Ricklin et al. 2018). Currently, the diagnosis of complement activation in kidney diseases is primarily based on immunohistochemical detection of deposited complement activation products in tissue, the detection of consumption of complement components in plasma or body fluids and the generation of activation fragments or cleavage products.
The activation of the complement system occurs by three different pathways; the classical/lectin pathway and the alternate pathway. The diseases of the complement system differ in their prognosis and therapy. Classifying, i.e. discriminating the various diseases and subgroups of diseases would be advantageous in order to get more insights into the pathogenesis, provide better diagnosis and more specific and efficient treatment strategies, and thus to achieve better prognosis for the specific diseases. Moreover, assays for testing potential drug candidates for the diseases associated with the deregulated complement system are needed.
Complement activation comprises three different pathways, the classical, the lectin and the alternative pathway.
In the classical pathway and the lectin pathway, the C3 convertase consisting of C4b and C2b cleaves C3 to C3b and C3a. In this classical pathway, downstream thereof, the C5 convertase consisting of C4b, C2b and C3b cleaves C5 to C5b and C5a.
In the alternative pathway, the cleavage of C3 to C3b and C3a is catalyzed by the C3 convertase consisting of C3b and Bb and subsequently the cleavage of C5 to C5b and C5a is promoted by the C5 convertase consisting of two C3b subunits and Bb.
Up to now, determining whether a disease is caused or aggravated by aberrations in the classical/lectin and/or alternative pathway has been challenging and often impossible.
Moreover, a method to identify, localize and differentiate complement convertases directly in tissues and other biological specimen has been lacking.
It is an objective of the invention to establish a method to detect molecular complexes of the complement activation pathway or to discriminate between aberration of the classical/lectin and alternative pathways. More specifically, a method to discriminate assembled C3/C5 convertases of the classical/lectin and alternative pathways is provided. Close proximity of C2b and C4b is used to identify assembled C3/C5 convertase of the classical/lectin pathway and close proximity of C3b and Bb for measuring the C3/C5 convertase of the alternative pathway. This allows the diagnosis, assessment of disease course/activity and/or treatment efficacy of disorders associated the complement system.
The present invention further provides a method that allows discriminating between the classical pathway, the lectin pathway and the alternative pathway.
In general, the invention relates to a method for classifying a disorder associated with the complement system, comprising the following steps:
(a) Providing a sample of a subject
(b) Detecting complexes of the complement activation pathway in the sample of the subject;
(c) Classifying the disorder associated with the complement system based on the result of step (b).
More particular, the method for classifying a disorder associated with the complement system, comprising the following steps:
(a) providing a sample of a subject
(b) detecting the presence of C3/C5 convertase complex in the sample of the subject;
(c) classifying the disorder associated with the complement system based on the result of step (b).
In particular step b) comprises the following steps:
In one embodiment, step b) comprises the following steps:
In one embodiment, step b) comprises the following steps:
Since the C3 convertase and the C5 convertase of the classical and the lectin pathways comprise the complex of C4b and C2b, the determination of the interaction of C4b and C2b allows the specific detection of the C3/C5 convertase of the classical/lectin pathway. By the accurate measurement of the C3/C5 convertase specific for the classical/lectin pathway, the invention thus provide for the first time an assay that allows to discriminate whether a specific disorder of the complement system is due to increased activity of the classical/lectin pathway, in particular due to the increased number of C3/C5 convertase complex of the classical/lectin pathway or upstream events that lead to an increased number of C3/C5 convertase complexes of the classical/lectin pathway.
The invention thus provide for the first time an assay that allows to identify the complexes in the biological specimen and furthermore to determine whether a specific disorder of the complement system correlates with or is due to increased activity of the classical/lectin pathway, in particular due to the increased number of C3/C5 convertase complex of the classical/lectin pathway or upstream events that lead to an increased number of C3/C5 convertase complexes of the classical/lectin pathway.
Correspondingly, the C3 convertase and the C5 convertase of the alternative pathway comprise the complex of C3b and Bb, the determination of the interaction of C3b and Bb allows the specific detection of the C3/C5 convertase of the alternative pathway.
By the accurate measurement of the C3/C5 convertase specific for the alternative pathway, the invention thus provide for the first time an assay that allows to directly determine whether a specific disease like a disorder of the complement system is due to aberrations of the alternative pathway, in particular due to the increased activity of the C3/C5 convertase complex of the alternative pathway or upstream events that lead to an increased number of C3/C5 convertase complexes of the alternative pathway.
Further, measuring Ig/C1q complex allows determining whether the classical pathway is activated. Measuring MASP1/3/MBL complex and/or MASP2/MBL allows determining whether the lectin pathway is activated. Hence, the provided method also distinguishes between the lectin pathway and the classical pathway.
The invention thus provides for the first time an assay that allows to directly determine whether a specific disease like a disorder of the complement system is due to aberrations of the classical pathway or the lectin pathway.
The invention also provides for the first time an assay that allows to directly determine whether a specific disease like a disorder of the complement system is due to aberrations of the classical pathway, the lectin pathway or the alternative pathway.
Thus, the method of the invention is able to identify the convertases complexes in tissue and biological specimen and be used to discriminate between a disorder associated with an aberration of the classical/lectin pathway and a disorder associated with an aberration of the alternative pathway.
The combined determination of the interaction of (i) C4b with C2b and (ii) C3b with Bb allows a particularly precise determination whether a disease associated with the complement system is due to aberrations of the classical/lectin pathway or the alternative pathway.
Typically, an increased amount of C4b/C2b complexes compared to a control is indicative for a disorder associated with the classical/lectin pathway. In some embodiments, the increased amount of C4b/C2b complexes compared to a predetermined threshold may be indicative for a disorder associated with the classical/lectin pathway.
Correspondingly, the increased amount of C3b/Bb complexes compared to a control or is indicative for a disorder associated with the alternative pathway. In some embodiments, the increased amount of C3b/Bb complexes above a predetermined threshold may be indicative for a disorder associated with the alternative pathway.
In specific embodiments, a value of the amount of C4b/C2b complexes divided by the amount of C3b/Bb complexes larger than 1 may be indicative for a disorder associated with an aberration of the classical/lectin pathway. Correspondingly, the value of the amount of C3b/Bb complexes divided by the amount of C4b/C2b complexes is larger than 1 may be indicative for a disorder associated with an aberration of the alternative pathway.
Alternatively, an increased value of the amount of C4b/C2b complexes divided by the amount of C3b/Bb complexes compared to a control may be indicative for a disorder associated with an aberration of the classical/lectin pathway. Correspondingly, an increased value of the amount of C3b/Bb complexes divided by the amount of C4b/C2b complexes compared to a control may be indicative for a disorder associated with an aberration of the alternative pathway.
The combined determination of (i) the interaction of Ig with C1q and of (ii) the interaction of MASP1/3 with MBL and/or MASP2 with MBL allows a particularly precise determination whether a disease associated with the complement system is due to aberrations of the classical pathway or the lectin pathway.
The combined combination of (i) the interaction of C4b with C2b, of (ii) the interaction of C3b with Bb, of (iii) the interaction of Ig with C1q and of (iv) the interaction of MASP1/3 with MBL and/or MASP2 with MBL allows a particularly precise determination whether a disease associated with the complement system is due to aberrations of the classical pathway, the lectin pathway or the alternative pathway.
Exemplarily, in step (b) the detection of the presence of the respective complement complex may be determined by in situ proximity ligation in the sample. Alternatively, in step (b) the detection of the presence of the respective complement complex is carried out by fluorescence resonance energy transfer (FRET) or a related method that allows to show proximity of two proteins or by Dexter energy transfer.
Accordingly, typically in step (b) the detection of the presence of the respective complement complex is carried out by microscopy including light microscopy, such as bright field microscopy or fluorescence microscopy.
Exemplarily, in step (b) the detection of the presence of the C3/C5 convertase complex may be determined by in situ proximity ligation in the sample. Alternatively, in step (b) the detection of the presence of the C3/C5 convertase complex s carried out by fluorescence resonance energy transfer (FRET) or a related method that allows to show proximity of two proteins or by Dexter energy transfer.
Accordingly, typically in step (b) the detection of the presence of the C3/C5 convertase complex is carried out by light microscopy, such as bright field microscopy or fluorescence microscopy.
In some embodiments measurement may be carried out in the biopsy sample. Thereby an assessment step for identifying regions that are useful for measurement can be avoided.
Alternatively, measurement is carried out in intact glomeruli which may lead to highly accurate results in some cases, especially in glomerulonephritis.
Typically, the disorder associated with the complement system is selected from the group consisting of kidney disease, cancer, autoimmunity, infection, degeneration and neuropathy. Examples of kidney diseases are systemic lupus erythematodes (SLE), thrombotic microangiopathy caused by atypical hemolytic uremic syndrome (aHUS), Shiga-toxin producing E. coli hemolytic uremic syndrome (STEC-HUS), secondary hemolytic uremic syndrome (secondary HUS), C3 glomerulopathy, C3 glomerulonephritis, dense deposit disease, post-/parainfectious glomerulonephritis, IgA nephropathy, cryoglobulinemia, and membranoproliferative glomerulonephritis (MPGN).
In some embodiments, the disorder associated with an aberration of the classical/lectin pathway is selected from the group consisting of cryoglobulinemia, membranous nephropathy and SLE, preferably SLE.
In some embodiments, the disorder associated with an aberration of the alternative pathway is aHUS, post-/parainfectious glomerulonephritis and C3 glomerulopathy.
The sample may be selected from the group consisting of body fluid or tissue sample.
Examples of body fluids are urine, liquor, blood, plasma, serum, effusion and synovial fluid.
Preferably, the tissue sample is a kidney tissue sample, more preferably a kidney biopsy.
Preferably, the body fluid is blood, plasma or serum, more preferably serum.
Another aspect of the invention refers to a method for determining whether a patient with a disorder associated with the complement system will be responsive to a modulator of the classical/lectin pathway or the alternative pathway, comprising the following steps:
(a) providing a sample of the patient;
(b) detecting the presence of C3/C5 convertase complex comprising the steps:
(c) predicting a whether the patient will be responsive to the inhibitor of the classical/lectin pathway or alternative pathway based on the result of step (b).
Preferably, the modulator is an inhibitor.
In some embodiments, the increased amount of C4b/C2b complexes indicates that the patient will be responsive to an inhibitor of the classical/lectin pathway and/or the increased amount of C3b/Bb complexes indicates that the patient will be responsive to an inhibitor of the alternative pathway or alternatively any complement targeting inhibitor, like also the terminal complement pathway.
In some embodiments, the method may further comprise the additional detection of Ig/C1q complex in the sample; and/or detection of the amount of MASP1/3/MBL complex and/or MASP2/MBL complex in the sample in order to determine whether the patient with a disorder associated with the complement system will be responsive to a modulator of the classical pathway or the lectin pathway.
Another aspect of the invention refers to a method for determining the disorder course of a patient with a disorder associated with the complement system, comprising the following steps:
(a) Providing samples of a subject of at least two time points of the disease course;
(b) Detecting the presence of C3/C5 convertase complex in the samples comprising the steps:
(c) Determining the disorder course based on the result of step (b).
In some embodiments, the decreased amount of C4b/C2b complexes in the sample of the later time point compared to the sample of the earlier time point indicates an amelioration of the disorder of the classical/lectin pathway and wherein the increased amount of C4b/C2b complexes in the sample of the later time point compared to the sample of the earlier time point indicates worsening of the disorder of the classical/lectin pathway.
In some embodiments, the decreased amount of C3b/Bb complexes in the sample of the later time point compared to the sample of the earlier time point indicates an amelioration of the disorder of the alternative pathway and/or the increased interaction of amount of C3b/Bb complexes in the sample of the later time point compared to the sample of the earlier time point indicates worsening of the disorder of the alternative pathway.
Another aspect of the invention refers to a method for determining the disorder course of a patient with a disorder associated with the complement system, comprising the following steps:
(a) Providing samples of a subject of at least two time points of the disease course;
(b) Determining the amount of Ig/C1q complex in the sample; and/or determining the amount of MASP1/3/MBL complex and/or MASP2/MBL complex in the sample
(c) Determining the disorder course based on the result of step (b).
In some embodiments, a decreased amount of Ig/C1q complexes in the sample of the later time point compared to the sample of the earlier time point indicates an amelioration of the disorder of the classical pathway and wherein an increased amount of Ig/C1q complexes in the sample of the later time point compared to the sample of the earlier time point indicates worsening of the disorder of the classical pathway.
In some embodiments, a decreased amount of MASP1/3/MBL complexes and/or MASP2/MBL complexes in the sample of the later time point compared to the sample of the earlier time point indicates an amelioration of the disorder of the lectin pathway and/or an increased MASP1/3/MBL complexes and/or MASP2/MBL complexes in the sample of the later time point compared to the sample of the earlier time point indicates worsening of the disorder of the lectin pathway.
Another aspect of the invention relates to a method for determining whether a candidate compound is suitable for the treatment of a disease like a disorder of the complement system wherein the prediction of a suitable candidate compound is based on step (c), comprising the following steps:
(a) Incubating a sample of a subject with a candidate compound
(b) Detecting the presence of C3/C5 convertase complex comprising the steps:
(c) Predicting whether a candidate compound is suitable for the treatment of a disorder of the complement system based on the result of step (b).
In some embodiments, the decreased amount of C4b/C2b complexes compared to a control may indicate that the candidate compound is suitable for the treatment of the disorder of the complement system. Accordingly, the decreased amount of C3b/Bb complexes compared to a control compound may indicate that the candidate compound is suitable for the treatment of the disorder of the complement system.
In some embodiments, the method may further comprise the additional detection of Ig/C1q complex in the sample; and/or detection of the amount of MASP1/3/MBL complex and/or MASP2/MBL complex in the sample.
Another aspect of the invention refers to a kit comprising:
a) probes for detecting the complexes of C4b and C2b comprising:
b) probes for detecting complexes of C3b and Bb comprising:
In one embodiment, the kit further comprises
c) probes for detecting the complexes of Ig and C1q comprising:
In a further embodiment, the kit further comprises
d) probes for detecting the complexes of MASP1/3 and/or MASP2 and MBL comprising:
In some embodiments, the analyte binding moiety (also termed “moiety specifically binding”) and the signal generating moiety are part of the same molecule. In alternative embodiments, the analyte binding moiety and the signal generating moiety are different molecules.
A moiety specifically binding a complement factor, such as C4b, C2b, C3b or Bb, may for example be an antibody and binding fragment thereof, a nanobody, a non-antibody protein scaffold protein (Vazquez-Lombardi R. et al.), aptamere, nucleotide based molecule and a small molecule.
In specific embodiments, the first and the second signal generating moieties build a FRET or Dexter energy transfer pair or by a related method. In other embodiments, the first and the second signal generating moieties build an in situ proximity ligation pair.
Before the invention is described in detail with respect to some of its preferred embodiments, the following general definitions are provided.
The present invention as illustratively described in the following may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.
The present invention will be described with respect to particular embodiments and with reference to certain figures but the invention is not limited thereto but only by the claims.
Where the term “comprising” is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term “consisting of” is considered to be a preferred embodiment of the term “comprising of”. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group which preferably consists only of these embodiments.
For the purposes of the present invention, the term “obtained” is considered to be a preferred embodiment of the term “obtainable”. If hereinafter e.g. an antibody is defined to be obtainable from a specific source, this is also to be understood to disclose an antibody which is obtained from this source.
Where an indefinite or definite article is used when referring to a singular noun, e.g. “a”, “an” or “the”, this includes a plural of that noun unless something else is specifically stated. The terms “about” or “approximately” in the context of the present invention denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value of ±10%, and preferably of ±5%.
Technical terms are used by their common sense. If a specific meaning is conveyed to certain terms, definitions of terms will be given in the following in the context of which the terms are used.
C3 convertase is a protein complex comprising several protein subunits that acts as serine protease converting the protein C3 to C3a and C3b fragments. In particular embodiments, the term C3 convertase denotes two different protein complexes that can be distinguished by the present invention: (i) the C3 convertase of the classical/lectin pathway that contains the two proteins C4b and C2b; (ii) the C3 convertase of the alternative pathway that contains Bb and one C3b subunit.
C5 convertase is a protein complex comprising several proteins that acts as serine protease converting the protein C5 to C5a and C5b fragments. In particular embodiments, the term C5 convertase denotes two different protein complexes that can be distinguished by the present invention: (i) the C5 convertase of the classical/lectin pathway that contains the two proteins C4b and C2b and C3b; (ii) the C5 convertase of the alternative pathway that contains the protein Bb and two C3b proteins.
C3/C5 convertase complex denotes protein complexes that contain at least the components of the C3 convertase, i.e. (i) the C3 convertase of the classical/lectin pathway that contains the two proteins C4b and C2b; (ii) the C3 convertase of the alternative pathway that contains Bb and one C3b subunit, and may further contain (i) C3b resulting in the C5 convertase of the classical/lectin pathway or (ii) a second C3b subunit resulting in the C5 convertase of the alternative pathway.
C3 denotes human complement protein 3.
C4 denotes human complement protein 4.
C5 denotes human complement protein 5.
C2b also (formerly) termed C2a as used herein refers to the dissociated protein which is capable of binding to C4b.
C4b denotes the cleavage fragment of the complement component C4.
C3b denotes the cleavage fragment of the complement component C3.
Bb denotes the cleaved fragment/cleavage product of complement factor B.
C4b, C2b, C3b and Bb may for example be detected by commercially available antibodies such as anti-C3b antibody (mouse, Abcam, Cambridge, UK), anti-Factor B antibody (ANTI-CFB antibody produced in rabbit, HPA001832-100UL, Sigma Aldrich, Merck Darmstadt, Germany) anti-C4b antiserum (1:500, rabbit, Abcam, Cambridge, UK) and anti-C2 antibody (E-7) (1:10, mouse, Santa Cruz, sc-373809, Dallas, USA)
Ig/C1q complexes include immunoglobulin proteins (Ig) of all classes and subclasses, i.e. IgG with subfragments IgG1, IgG2, IgG3, IgG4, as well as IgA, IgA1, IgA2, IgE, IgD and IgM. The Igs associate with C1 the complement component of the classical pathway to initiate complement.
MASP1/3/MBL complex and MASP2/MBL complex: MASP1, MAPS2, MASP3 are pattern recognition proteins that interact with Mannan binding lectin (MBL) and initiate the lectin pathway of complement.
The terms “convertase” and “convertase complex” are used interchangeable herein.
A first aspect of the invention refers to a method for classifying a disorder associated with the complement system, comprising the following steps:
(a) providing a sample of a subject
(b) detecting the presence of C3/C5 convertase complex comprising the steps:
(c) classifying the disorder associated with the complement system based on the result of step (b).
In one embodiment, step b) further comprises the following steps:
The subject may be a mammal, such as a rodent (e.g. a mouse or rat), cat, dog, leporid (e.g. rabbit) or a primate, such as a monkeys, human. Preferably, the subject is a mouse or a human. More preferably, the subject is a human. The subject may be a healthy or suffering from a disease associated with the complement system.
Determining the amount of C4b/C2b complexes in the sample and/or determining the amount of C3b/Bb complexes in the sample means detecting the complexes of C4b and C2b and detecting complexes of C3b with Bb.
An increased amount of C4b/C2B complexes or C3b/Bb complexes refers in particular to a higher number of C4b/C2b complexes or C3b/Bb complexes, in particular to a higher number of signal spots each representing in general one C4b/C2b complex or C3b/Bb complex; in some embodiments it may also refer increased signal spots or spots with increased signal intensity, that may represent a higher number of complexes compared to smaller signal spots or spots with less signal intensity which may represent a smaller number of complexes. Vice versa, an decreased amount of C4b/C2B complexes or C3b/Bb complexes refers in particular to a lower number of C4b/C2b complexes or C3b/Bb complexes, in particular to a lower number of signal spots each representing in general one C4b/C2b complex or C3b/Bb complex; in some embodiments it may also refer to decreased signal spots or spots with decreased signal intensity, that may represent a lower number of complexes compared to larger signal spots or spots with higher signal intensity which may represent a higher number of complexes.
The skilled person understands that the with higher resolutions each spot may represent a single complex while in a lower resolution a spot, in particular an enlarged spot or a spot with higher intensity may represent several complexes. The skilled person can differentiate between these two scenarios; There may be settings in which both scenarios occur in one sampled.
“Determining the amount of complexes” means measuring a signal representative for the amount of the respective complement complex.
“Determining the amount of C4b/C2b complexes” or “determining the amount of C3b/Bb complexes” means measuring a signal representative for the amount of C4b/C2b complexes or C3b/Bb complexes.
Aberration in the context of the present invention refers to an altered activity. Thus “aberration of the classical/lectin pathway” means “altered activity of the classical/lectin pathway”. Accordingly “aberration of the alternative pathway” means “altered activity of the alternative pathway”. “Aberration of the classical pathway” means “altered activity of the classical pathway”. “Aberration of the lectin pathway” means “altered activity of the lectin pathway”.
Classifying the disorder of the complement system allows a more detailed specification of the disorder, in particular the classification, also termed discrimination, whether the disorder is associated with an aberration of the classical/lectin pathway and/or the diagnosis whether the disorder is associated with an aberration of the alternative pathway. In preferred embodiments, the classification discriminates between a disorder associated with an aberration of the classical/lectin pathway and a disorder associated with an aberration of the alternative pathway.
The skilled person understands that the methods described herein are in vitro, in particular in situ, methods. Therefore, diagnostic methods practiced on the human or animal body are not encompassed by the present description.
In some embodiments, the increased amount of C4b/C2b complexes compared to a control is indicative for a disorder associated with the classical/lectin pathway. Accordingly, the increased amount of C4b/C2b complexes compared to a predetermined threshold is indicative for a disorder associated with the classical/lectin pathway.
The skilled person is aware of methods for determining a threshold for the interaction of C4b and C2b that indicates whether a disorder is associated with the classical/lectin pathway. The threshold may be determined in a study with an appropriate number of patients.
Exemplarily, the threshold may be determined by the mean value of 30 healthy persons plus two standard deviations.
In some embodiments, the increased amount of C3b/Bb complexes compared to a control is indicative for a disorder associated with the alternative pathway. In other embodiments, the increased amount of C3b/Bb complexes above a predetermined threshold is indicative for a disorder associated with the alternative pathway.
In specific embodiments, a value of the amount of C3b/Bb complexes divided by the amount of C4b/C2b complexes larger than 1 is indicative for a disorder associated with an aberration of the alternative pathway.
Preferably the value of the amount of C3b/Bb complexes divided by the amount of C4b/C2b complexes is a ratio larger than 2, more preferably larger than 3, even more preferably larger than 4, most preferably larger than 5, such as larger than 10.
In some embodiments, an increased value of the amount of C3b/Bb complexes divided by the amount of C4b/C2b complexes compared to a control is indicative for a disorder associated with an aberration of the alternative pathway.
The term “control” as used herein refers to a sample, such as a kidney biopsy of an individual which has no aberrations of the complement system, in particular not aberrations of the classical/lectin pathway and the alternative pathway. The skilled person understands that also an average of several such samples could is understood as control. The control may be for example a “zero-hour biopsy” of the same tissue as the sample to be tested. The term “zero-hour biopsy” as used herein refers to a biopsy that is taken from a (presumably healthy) transplanted kidney at the time of transplantation (time-point “zero-hour”).
In some embodiments, a value of the amount of C4b/C2b complexes divided by the amount of C3b/Bb complexes larger than 1 is indicative for a disorder associated with an aberration of the classical/lectin pathway.
In step (b) the detection of the presence of the complement complex, e.g., the C3/C5 convertase complex, may be determined by any method in the art useful the detection of in situ protein interaction, for example by in situ proximity ligation, by fluorescence resonance energy transfer (FRET) or by Dexter energy transfer. Thus, in some embodiments, in step (b) the detection of the presence of the complement complex, e.g., the C3/C5 convertase complex, may be carried out by light microscopy, such as light microscopy is bright field microscopy or fluorescence microscopy.
FRET is based on the transfer of energy between two fluorophores, i.e. a donor and an acceptor, when these fluorophores are in close proximity. When the two fluorophores come close enough to each other, excitation of the donor by an energy source triggers an energy transfer towards the acceptor, which in turn emits specific fluorescence at a given wavelength. The skilled person is aware of FRET imaging techniques and how to adapt them (Jares-Erijman, Nature Biotechnology 21, 1387-1395 (2003)). In the present case, for the detection of the C3/C5 convertase of the classical/lectin pathway, a probe containing the analyte binding moiety and the signal generating moiety, such as an antibody targeting C4b linked to a FRET donor or FRET acceptor, may be used in combination with a further probe, such as an antibody targeting C3b and containing the respective counterpart of the FRET pair. Alternatively, probes, in which the analyte binding moiety and the signal generating moiety are separate molecules may be use. For example, the molecules containing the analyte binding moieties may be primary antibodies targeting C4b and C2b which may be used in combination with signal generating moieties, such as secondary antibodies containing an FRET donor, the other an FRET acceptor. Correspondingly, for the detection of the C3/C5 convertase of the alternative pathway, a probe, such as an antibody linked to an FRET donor or FRET acceptor would be used to detect C3b and a further probe, such as an antibody targeting Bb fused to the respective counterpart of the FRET pair. Alternatively, probes having separate molecules for detection and signal generation, such as primary antibodies targeting C3b and Bb in combination with secondary antibodies containing a FRET donor and a FRET acceptor, respectively, may be used.
The proximity ligation assay is commercially available, e.g. the Duolink® assay provided by SIGMA-Aldrich 8USA). The assay is described inter alia in Söderberg et al.. As described in the method section, for the detection of the C3/C5 convertase of the classical pathway, probe molecules containing the analyte binding moiety, such as primary antibodies targeting C4b and C2b may be used in combination with probe molecules containing the signal generating moiety, i.e. proximity ligation assay secondary antibodies. Correspondingly, for the detection of the C3/C5 convertase of the alternative pathway secondary proximity ligation antibodies targeting the primary antibodies targeting C3b and Bb may be used. In principle, alternatively, also probes containing both the analyte binding moiety that target the complex subunits (i.e. C4b, C2b, C3b and Bb) and the signal generating moiety containing the proximity ligation assay domains can be used.
In some embodiments, the measurement may be carried out in the whole biopsy sample or in intact glomeruli.
The term whole biopsy sample refers to the entire sample without any discrimination of substructures such as glomeruli.
The term “measurement is carried out in intact glomeruli” means that only glomeruli that are intact are measured. The term intact glomeruli” refers to glomeruli of normal appearance, i.e. not scarred, not sclerosed, not necrotic, and not collapsed.
The term “disorder associated with the complement system” refers to diseases due to aberrations of the complement system. In particular, to diseases that lead to an aberrant number of C3/C5 convertases of the classical pathway or to an aberrant number of C3/C5 convertases of the alternative pathway.
In some embodiments, the disorder associated with the complement system is selected from the group consisting of kidney disease, cancer, autoimmunity, infection, degeneration and neuropathy. The kidney disease may be selected from the group consisting of systemic lupus erythematodes (SLE), atypical hemolytic uremic syndrome (aHUS), Shiga-toxin producing E. coli hemolytic uremic syndrome (STEC-HUS), secondary hemolytic uremic syndrome (secondary HUS), thrombotic microangiopathy caused by atypical hemolytic uremic syndrome (aHUS), C3 glomerulopathy, cryoglobulinemia, glomerulonephritis and dense deposit disease.
The glomerulonephritis may be selected from the group IgA nephritis, ANCA-associated nephritis, C3 glomerulonephritis, dense deposit disease, membranoproliferative glomerulonephritis (MPGN), membranous nephropathy, post-/parainfectious glomerulonephritis.
In some embodiments, the disorder associated with an aberration of the classical/lectin pathway is selected from the group consisting of cryoglobulinemia, membranous nephropathy and SLE, preferably SLE.
In other embodiments, the disorder associated with an aberration of the alternative pathway is aHUS post-/parainfectious glomerulonephritis and C3 glomerulopathy.
Typically, the sample is selected from the group consisting of body fluid or tissue sample. In some embodiments, the body fluid is selected from the group consisting of urine, liquor, blood, plasma, serum, effusion and synovial fluid. Preferably, the tissue sample is a kidney tissue sample.
Another aspect of the invention refers to a method for determining whether a patient with a disorder associated with the complement system will be responsive to a modulator of the classical/lectin pathway or alternative pathway, comprising the following steps:
(a) providing a sample of the patient;
(b) detecting the presence of C3/C5 convertase complex comprising the steps:
(i) determining the amount of C4b/C2b complexes in the sample and/or
(ii) determining the amount of C3b/Bb complexes in the sample;
(c) predicting a whether the patient will be responsive to the modulator of the classical/lectin pathway or alternative pathway based on the result of step (b).
In preferred embodiments, the modulator is an inhibitor.
The term “modulator”, in particular “inhibitor”, includes small molecules, antibodies and binding fragments thereof, non-antibody protein scaffold proteins, aptameres and nucleotide based molecules.
In some embodiments, the inhibitor is selected from the group consisting of Eculizumab and Ravulizumab (C5 inhibitors, both Alexion); C1 inhibitors Berinert and Cinryze; soluble C5a peptide binding mAb (IFX-1; InflaRx); C5a receptor antagonist (Avacopan, Chemocentryx); OMS 721 also termed, narsoplimap, a MASP2 inhibitor (Omeros); Amy 101 (Amyndas); APL2 a C3 targeting peptide (Apellis); Danicopan (ACH-4471), a Factor D binding antagonist (Achillion); LNP023 a Factor B blocking compound (Novartis).
In some embodiments, the increased amount of C4b/C2b complexes indicates that the patient will be responsive to an inhibitor of the classical/lectin pathway and/or the increased amount of C3b/Bb complexes indicates that the patient will be responsive to an inhibitor of the alternative pathway.
Another aspect of the invention refers to a method for determining the disorder course of a patient with a disorder associated with the complement system, comprising the following steps:
(a) Providing samples of a subject of at least two time points of the disease course;
(b) Detecting the presence of C3/C5 convertase complex in the samples comprising the steps:
(c) Determining the disorder course based on the result of step (b).
In some embodiments, the decreased amount of C4b/C2b complexes in the sample of the later time point compared to the sample of the earlier time point indicates an amelioration of the disorder of the classical/lectin pathway and wherein the increased amount of C4b/C2b complexes in the sample of the later time point compared to the sample of the earlier time point indicates worsening of the disorder of the classical/lectin pathway.
In some embodiments, the decreased amount of C3b/Bb complexes in the sample of the later time point compared to the sample of the earlier time point indicates an amelioration of the disorder of the alternative pathway
and/or the increased amount of C3b/Bb complexes in the sample of the later time point compared to the sample of the earlier time point indicates worsening of the disorder of the alternative pathway.
The method may further comprise the additional detection of Ig/C1q complex in the sample; and/or detection of the amount of MASP1/3/MBL complex and/or MASP2/MBL complex in the sample.
Another aspect of the invention relates to a method for determining whether a candidate compound is suitable for the treatment of a disorder of the complement system wherein the prediction of a suitable candidate compound is based on step (c), comprising the following steps:
(a) Incubating a sample of a subject with a candidate compound
(b) Detecting the presence of C3/C5 convertase complex comprising the steps:
(c) Predicting whether a candidate compound is suitable for the treatment of a disorder of the complement system based on the result of step (b).
In some embodiments, the decreased amount of C4b/C2b complexes compared to a control may indicate that the candidate compound is suitable for the treatment of the disorder of the complement system. Accordingly, the decreased amount of C3b/Bb complexes compared to a control compound may indicate that the candidate compound is suitable for the treatment of the disorder of the complement system.
The methods described herein may be carried out with a sample that has been obtained from a subject or a patient.
Another aspect of the invention refers to a kit comprising:
a) probes for detecting the complexes of C4b and C2b comprising:
b) probes for detecting complexes of C3b and Bb comprising:
In one embodiment, the kit further comprises
c) probes for detecting the complexes of Ig and C1q comprising:
In a further embodiment, the kit further comprises
d) probes for detecting the complexes of MASP1/3 and/or MASP2 and MBL comprising:
In some embodiments, the analyte binding moiety and the signal generating moiety are part of the same molecule.
In alternative embodiments, the analyte binding moiety and the signal generating moiety are different molecules.
In specific embodiments, the first and the second signal generating moieties build a FRET/Dexter energy transfer pair. In other embodiments, the first and the second signal generating moieties build an in situ proximity ligation pair.
Ten kidney biopsies with lupus nephritis (SLE, class IV), nine cases with thrombotic microangiopathy (TMA) due to atypical hemolytic uremic syndrome, and five zero hour transplant biopsies (control) were analyzed. Preserved glomeruli were defined as regions of interest while scarred or collapsed glomeruli were excluded (Fig. S1 in the Supplementary Appendix). Proximity ligation signal densities (number of signals per glomerular area [mm2]) were determined using image analysis software (hs analysis), and compared between the groups (Fig. S2 in the Supplementary Appendix).
As expected, SLE cases revealed higher densities of classical/lectin convertases within the glomerular mesangium and around capillary walls (median 7,685 signals/mm2), as compared to TMA biopsies (median 393 signals/mm2) and to control biopsies (median 207 signals/mm2). In contrast, TMA cases showed a predominance of alternative convertases with most signals being located within the glomerular capillary lumens (median 3,032 signals/mm2), as compared to SLE cases and control biopsies (median 1,329 signals/mm2 and 1,418 signals/mm2, respectively) (Fig. S3 and S4 in the Supplementary Appendix). Thus, we introduce the first methodological workflow for the visualization, differentiation and quantification of classical/lectin and alternative C3/C5 convertases directly within the tissue specimen. This new approach represents a tool to discriminate complement pathways in tissue and to show the dynamics of activation, enhancing diagnosis and potentially allowing future efficacy monitoring of individualized therapy.
Patients and Biopsies
From the archive of the pathology institute of the University Hospital Hamburg-Eppendorf (UKE), 10 cases of systemic lupus erythematodes nephritis class IV and 9 cases of atypical hemolytic uremic syndrome were selected. Five transplant zero hour biopsies were chosen as 1 control. The study was approved by the local ethics committee for Hamburg (Ethics Commission Hamburg, PV 5541). All work was carried out in accordance with the Declaration of Helsinki.
Methods
Immunohistochemistry and Proximity Ligation Assay
Slides (1 μm) from paraffin embedded kidney biopsies were deparaffinized, pretreated and blocked using normal horse serum (Vector Industries S-2000). As pretreatment for C3b and Factor B detection Dako Target Retrieval Solution (S1699, Sigma-Aldrich, Darmstadt, Germany) was applied for 15 min at 120° C. Primary antibodies against C3b (1:1000, mouse) (Abcam, Cambridge, UK) and Factor B (1:100, rabbit) (Sigma Aldrich, Darmstadt, Germany) were applied at 4° C. for 16 hours. For detection of the classical C3/C5 convertase (C4b and C2) the tissue sections were pretreated with protease for 30 min at 40° C. (P8038, Sigma-Aldrich, Darmstadt, Germany). Afterwards C4b antiserum (1:500, rabbit, Abcam, Cambridge, UK) and C2 antiserum (1:10, mouse, Santa Cruz, Dallas, USA) were applied at 4° C. for 16 hours. Next, proximity ligation assay secondary antibodies against mouse (Duolink® In Situ PLA Probe Anti-Mouse Minus, Sigma Aldrich, Darmstadt, Germany) and rabbit (Duolink® In Situ PLA Probe Anti-Rabbit Plus, Sigma Aldrich, Darmstadt, Germany) antibodies were added. According to protocol the brightfield detection system (Duolink® In Situ Detection Reagents Brightfield, Sigma Aldrich, Darmstadt, Germany) was applied to the slides. The same primary antibodies were used on subsequent slides to visualize the deposition of C4b, C2, C3b and Factor B individually. For staining of C3 (1:3000, rabbit, polyclonal), C1q (1:1500, rabbit, polyclonal) (both DAKO, Eching, Germany) and IgG (1:7500, mouse, monoclonal) (Dianova, Hamburg, Germany), slides were pretreated with protease (15 min, 40° C.) (P8038, Sigma-Aldrich, Darmstadt, Germany) and a standard APAAP-Protocol (monoclonal or polyclonal) was used. Detection was performed with the ZytoChem Plus AP Polymer System (POLAP-100, Zytomed, Berlin, Germany) using new fuchsin red chromogen and haematoxylin nuclear counterstain.
Quantification of Proximity Ligation Assay Signals
Densities of signals in glomeruli and in whole biopsies were analyzed using image analysis software (HSA KIT, HS Analysis GmbH, Karlsruhe, Germany). Scarred or collapsed glomeruli were excluded from glomerular analyses. With the software HSA KIT the PLA signals were detected, counted and measured using the Maximally Stable Extremal Regions (MSER) algorithm, which is described below. The following parameters were defined for this purpose: color ranges separating the foreground from the background of the signals within the biopsy as well as intervals for the intensity and for the size of the signals. Two categories of false positive signals occurred; intramembranous pseudo signals and intranuclear pseudo signals in cases with strong nuclear counterstaining. False positive intramembranous pseudo signals occurred only very rarely, so that they were negligible for the evaluation. In order to minimize the number of false positive intranuclear signals, a further parameter set was defined to determine the intensity and color values for negative signals. These negative signals are also detected via the MSER algorithm using an additional parameter set consisting of intensity and color values.
Negative signals and especially regions where positive and negative signals overlapped were excluded from the evaluation. A live overlay showing both the positive and negative signals allowed for easy parameter tuning until satisfactory values were found.
The MSER algorithm detects a number of features in an image. These MSERs are stable connected components that are chosen from a binary threshold representation of the image. The algorithm works as follows. A set of n equidistant threshold levels for the image signals T={T1 . . . Tn} is chosen. For every threshold level a pixel set Si={x: I(x)≤Ti} is generated from image I(x) where
is a two-dimensional index. For every set Si connected regions Ri⊂Si are detected and arranged as a tree, Ri is a child region of Ri+1 if Ri⊂Ri+1. The goal is now to select regions of maximal stability, in the sense that they do not change over different threshold levels. For that the algorithm assumes that regions that have a similar size as their child regions also do not change much in shape. Every region is assigned an instability score
The regions with minimal instability score are selected. Optionally the score can be modified to exclude regions that are too small (|Ri|<min) or too big (c>max) in size:
Statistical Analyses
Data were presented as mean±standard deviation and median. A two-tailed Mann Whitney test was used to evaluate differences between the values. All statistical analyses were performed using R Software version 3.4.3 packages rcorr and ggplot2 (R Foundation for Statistical Computing, Vienna, Austria). Visualization of the statistical results was performed using the GraphPad Prism software (GraphPad Software, La Jolla Calif. USA). P<0.05 was considered statistically significant.
The aim is to extend the method of in-situ visualization of C3/C5 convertases in order to dissect initiation of the lectin pathway and the classical pathway upstream as well as visualization and quantification of the membrane attack complex (C5b-9) downstream (see
Methods: Proximity ligation assays were performed for both convertases as described above. In addition, the same technique was applied for IgG with C1q for classical pathway initiation and for MASP1/3 with MBL as well as MASP2 with MBL for lectin pathway initiation. Brightfield proximity ligation assays were used and quantification of the signals was done as described. The method can also be applied to the detection of assembled C5b-9, e.g. by the detection of close proximity of C8 and C9. For the other reactions, the following antibodies were used:
Complement mapping was performed as a proof of concept study in 19 biopsy cases with membranous nephropathy (10 PLA2R associated and 9 THSD7A associated forms) and 9 zero hour transplant biopsies as normal controls.
Results: Specific signals were detected in membranous nephropathy cases at the expected location of the subepithial spaces at the outer aspects of the glomerular basement membranes (
Conclusion: In membranous nephropathy complement is activated likely via the classical pathway (IgG/C1q and C2/C4b) in both THSD7A- and PLA2R-associated cases (s.
In order to estimate the capacity of the serum of individual patients at a given time to activate complement, the assay as described above can also be applied to serum. For this purpose cell lines (i.e. HUVEC cells) could be grown as a monolayer in cell culture on a transparent microscope glass slide (see
This technique provides a tool to monitor complement activation capacity of patient serum at the time of diagnosis and during therapy. Furthermore, it allows to test the efficacy of different complement targeting drugs in a given patient.
The invention also relates to the following embodiments:
1. Method for classification a disorder associated with the complement system, comprising the following steps:
2. Method according to embodiment 1 wherein step b) comprises the steps:
3. Method according to embodiment 1, wherein the classification discriminates between an increased activity of the classical/lectin pathway and an increased activity of the alternative pathway.
4. Method according to any one of the preceding embodiments, wherein an increase amount of C4b/C2b complexes compared to a control is indicative for a disorder associated with the classical/lectin pathway.
5. Method according to any one of the preceding embodiments, wherein the increased amount of C4b/C2b complexes compared to a predetermined threshold is indicative for a disorder associated with the classical/lectin pathway.
6. Method according to any one of the preceding embodiments, wherein the increased amount of C3b/Bb complexes compared to a control or is indicative for a disorder associated with the alternative pathway.
7. Method according to any one of the preceding embodiments, wherein the increased amount of C3b/Bb complexes above a predetermined threshold is indicative for a disorder associated with the alternative pathway.
8. Method according to any one of the preceding embodiments, wherein a value of the amount of C3b/Bb complexes divided by the amount of C4b/C2b complexes larger than 1 is indicative for a disorder associated with an aberration of the alternative pathway.
9. Method according to any one of the preceding embodiments, wherein an increased value of the amount of C3b/Bb divided by the amount of C4b/C2b compared to a control is indicative for a disorder associated with an aberration of the alternative pathway.
10. Method according to any one of the preceding embodiments, wherein a value of the amount of C4b/C2b divided by the amount of C3b/Bb larger than 1 is indicative for a disorder associated with an aberration of the classical/lectin pathway.
11. Method according to any one of the preceding embodiments, wherein in step (b) the detection of the amount of the C3/C5 convertase complex is determined by in situ proximity ligation in the sample.
12. Method according to any one of the preceding embodiments, wherein in step (b) the detection of the presence of the C3/C5 convertase complex is carried out by fluorescence resonance energy transfer (FRET) or dexter energy transfer.
13. Method according to any of the preceding embodiments, wherein in step (b) the detection of the presence of the C3/C5 convertase complex is carried out by light microscopy.
14. Method according to embodiment 13, wherein the light microscopy is bright field microscopy or fluorescence microscopy.
15. Method according to any one of the preceding embodiments, wherein the measurement is carried out in the whole biopsy sample.
16. Method according to any one of the preceding embodiments, wherein the measurement is carried out in intact glomeruli.
17 Method according to any one of the preceding embodiments, wherein the disorder associated with the complement system is selected from the group consisting of kidney disease, cancer, autoimmunity, infection, degeneration and neuropathy.
18. Method according to embodiment 17, wherein the kidney disease is selected from the group consisting of systemic lupus erythematodes (SLE), thrombotic microangiopathy caused by atypical hemolytic uremic syndrome (aHUS), Shiga-toxin producing E. coli hemolytic uremic syndrome (STEC-HUS), secondary hemolytic uremic syndrome (secondary HUS), C3 glomerulopathy, cryoglobulinemia, C3 glomerulonephritis, dense deposit disease, post-/parainfectious glomerulonephritis, IgA nephropathy, cryoglobulinemia, and membranoproliferative glomerulonephritis (MPGN).
19. Method according to any one of the preceding embodiments, wherein the disorder associated with an aberration of the classical/lectin pathway is selected from the group consisting of cryoglobulinemia, membranous nephropathy and SLE, preferably SLE.
20. Method according to any one of the preceding embodiments, wherein the disorder associated with an aberration of the alternative pathway is aHUS, post-/parainfectious glomerulonephritis and C3 glomerulopathy.
21. Method according to any one of the proceeding embodiments, wherein the sample is selected from the group consisting of body fluid or tissue sample.
22. Method according to embodiment 21, wherein the body fluid is selected from the group consisting of urine, liquor, blood, plasma, serum, effusion and synovial fluid.
23. Method according to any one of the proceeding embodiments, wherein the tissue sample is a kidney tissue sample.
24. Method for determining whether a patient with a disorder associated with the complement system will be responsive to a modulator of the classical/lectin pathway or alternative pathway, comprising the following steps:
(a) Providing a sample of the patient;
(b) Detecting the presence of C3/C5 convertase in the sample of the patient:
amount of C4b/C2b complexes (c) Predicting a whether the patient will be responsive to the inhibitor of the classical/lectin pathway or alternative pathway based on the result of step (b).
25. Method according to embodiment 24, wherein step b) comprises the steps:
26. Method according to embodiment 24 or 25, wherein the modulator is an inhibitor.
27. Method according to embodiment 25, wherein an increased amount of C4b/C2b complexes indicates that the patient will be responsive to an inhibitor of the classical/lectin pathway and/or the increased amount of C3b/Bb complexes indicates that the patient will be responsive to an inhibitor of the alternative pathway.
28. Method for determining the disorder course of a patient with a disorder associated with the complement system, comprising the following steps:
(a) Providing samples of a subject of at least two time points of the disease course;
(b) Detecting the presence of C3/C5 convertase complex in the sample of the subject;
(c) Determining the disorder course based on the result of step (b).
29. Method according to embodiment 28, wherein step b) comprises the steps:
30. Method according to embodiment 29, wherein the decreased amount of C4b/C2b complexes in the sample of the later time point compared to the sample of the earlier time point indicates an amelioration of the disorder of the classical/lectin pathway and wherein the increased amount of C4b/C2b complexes in the sample of the later time point compared to the sample of the earlier time point indicates worsening of the disorder of the classical/lectin pathway.
31. Method according to embodiment 29 or 30, wherein the decreased amount of C3b/Bb complexes in the sample of the later time point compared to the sample of the earlier time point indicates an amelioration of the disorder of the alternative pathway and/or the increased amount of C3b/Bb complexes in the sample of the later time point compared to the sample of the earlier time point indicates worsening of the disorder of the alternative pathway.
32. Method for determining whether a candidate compound is suitable for the treatment of a disorder of the complement system wherein the prediction of a suitable candidate compound is based on step (c), comprising the following steps:
(a) Incubating a sample of a subject with a candidate compound
(b) Detecting the presence of C3/C5 convertase complex in the sample of step (a);
(c) Predicting whether a candidate compound is suitable for the treatment of a disorder of the complement system based on the result of step (b).
33. Method of embodiment 32, wherein step (b) comprises the following steps:
34. Method according to embodiment 32 or 33, wherein the decreased amount of C4b/C2b complexes compared to a control indicates that the candidate compound is suitable for the treatment of the disorder of the complement system.
35. Method according to embodiment 32 to 34, wherein the decreased amount of C3b/Bb compared to a control compound indicates that the candidate compound is suitable for the treatment of the disorder of the complement system.
36. Kit comprising:
a) probes for detecting the complexes of C4b and C2b comprising:
b) probes for detecting complexes of C3b and Bb comprising:
37. Kit according to embodiment 36, wherein the analyte binding moiety and the signal generating moiety are part of the same molecule.
38. Kit according to embodiment 36, wherein the analyte binding moiety and the signal generating moiety are different molecules.
39. Kit according to embodiments 36 to 38, wherein the first and the second signal generating moieties build a FRET pair.
40. Kit according to embodiments 36 to 38, wherein the first and the second signal generating moieties build an in situ proximity ligation pair.
| Number | Date | Country | Kind |
|---|---|---|---|
| 19189415.3 | Jul 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/071688 | 7/31/2020 | WO |
