SVAR COMPLEMENT ASSAYS A powerful solution for analyzing ...
Transcript of SVAR COMPLEMENT ASSAYS A powerful solution for analyzing ...
SVAR COMPLEMENT ASSAYS
A powerful way of analyzing the inhibitory effect of a drug candidate on the complement systemTHE COMPLEMENT SYSTEM
The complement system is a key component of the body’s innate immune defense. It has an important role as a first-line defense against microorganisms but is also involved in other important functions such as homeostasis, clearance of cellular debris and immune surveillance.
Clearly, such a broad range of functions requires a well-balanced interaction with other biological systems in the body. Indeed, the complement system has been likened to a balancing scale where there needs to be sufficient activity to protect the body but not enough to be harmful. A number of genetic and environmental factors can tip the scale from defense to destruction.
COMPLEMENT THERAPEUTICS
The complement system is an essential part of the innate immune system and plays an important role in defending the body against infection. However, deficiencies or overactivation can instead cause harm to the body, which has made the complement system a promising target for therapeutics. Since the complement system consists of over 30 proteins there are many potential targets to choose from (Fig. 1).
As can be expected from the great diversity of clinical disorders involving abnormal complement activity or regulation, many creative approaches to therapeutically intervene at various stages of the complement cascade have been explored over the years.
A large number of anti-complement drugs are in development, providing tools for blocking complement activity via specific activation pathways, or isolated complement components.
This new focus on complement-targeted therapeutics has raised important questions concerning target selection, point and length of intervention, safety and drug delivery. There can also be side-effects of inhibiting the complement system, e.g., risk of infection, that need to be addressed in the drug development process.
There are also important pathway-dependent issues to consider when developing complement drug candidates. For example, drugs that block the classical pathway may be beneficial in antibody-mediated diseases as the classical pathway is involved in autoimmunity. However, drugs that block downstream targets in the complement cascade, while leaving the early classical pathway intact, may be preferable for other diseases.
Drugs that block specific complement fragments further down the complement cascade may also have fewer side effects than drugs that more completely block the complement system. Indeed, many new anti-complement drugs that target specific activation mechanisms or downstream activation fragments are currently in development.
WHAT IS ECULIZUMAB?
The first antibody-based complement drug to be approved was eculizumab, which is an antibody targeting complement component C5. This drug is used to treat paroxysmal nocturnal hemoglobinuria (PNH), neuromyelitis optica (NMO) and atypical hemolytic uremic syndrome (aHUS) and works by preventing C5 from being cleaved into C5a and C5b.
The success of eculizumab and the importance of the complement system in disease has led to an increased interest in the development of complement inhibitors, especially in those targeting C5.
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Exploration of the Complement Pathway SVAR LIFE SCIENCE
INITIATION
ACTIVATION
AMPLIFICATION
TERMINATION
© Svar Life Science AB, January 2022
Explore our Complement Solutions at svarlifescience.com
Following the Complement PathsComplement System Roadmap
CD59Clusterin
VitronectinCSMD1
C4BPFactor I
CR1/CD35MCP/CD46DAF/CD55
CSMD1
Factor HFactor I
CR1/CD35MCP/CD46DAF/CD55
CSMD1
CPN
CPN
C1-INH
CPN
C4bC2b
Lectin Pathway
Classical PathwayAg-Ab complexes
IgG, IgM, CRP. SAPPentraxins, Polymers
Microbial molecules, IgMFucose, Mannose, Polymers
Acetylated sitesAlternative Pathway
Spontaneously, Microbial moleculesPolysaccarides (LPS)
Host, Foreign & Artificial surfaces
Target cell
C4b
CD59Clusterin
VitronectinCSMD1
C4BPFactor I
CR1/CD35MCP/CD46DAF/CD55
CSMD1
Factor HFactor I
CR1/CD35MCP/CD46DAF/CD55
CSMD1
C3bBbPC3 convertase
Opsonization
Inactivation,Dissociation
Opsonization
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C5b
C3bB
C5b-9
C6C7
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C1q
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CLs
C1rC1s C1-INH
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C3(H2O)BbC3 convertaseC3 convertase
MASP-1 MASP-2
CPN
Anaphylatoxin
Anaphylatoxin
C5aR
C3a
Inactivation
CPN
C4aDesArg
CPN
pro-Factor D
MASP-3
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Non-”complement” proteases
Coagulation & Contact systems
Non-”complement”
proteases
TICKOVER
C3(H2O)B
C3(H2O)
C3
Factor D
Factor B
C3b
Immune cell
Immune cell
TCCTargetcell
MAC
Inflammation
Inflammation
C3aR
Phagocyte
FcγR
C1qR
CR3CR4
Immune cell
CR3CR4
CRIgCR2
CR1
Factor B
C4c
C4d
C2a
Ba
Ba
C4d
iC4b
C3dg
iC3b
C3g
C3d
C3c C3f
C3aDesArg
C5aDesArg
Target cell
Cell Lysis
ClearancePhagocytosisCell Activation
Properdin
C4
C2b
C4a C2
C4biC4b
C3dg iC3b
C4bC2bC3b C3bBbPC3bC5 convertase C5 convertase
C5
C5a
C3b
TCC
Complement TCC ELISA-based biomarker assay2
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Exploration of the Complement Pathway
SVAR’S COMPLEMENT ASSAY HISTORY
Svar Life Science has over 15 years’ experience of complement system assay development. Our complement system assays have been developed by our experienced scientists in close collaboration with key opinion leaders, to yield sensitive, reliable and easy-to-use products for the exploration of most aspects of the complement system.
We offer ELISA-based functional assays1 that lets you analyze the functionality of all three complement pathways.
Figure 1. Schematic representation of the complement system. Complement components analyzed in this paper (C4d, C5a, C5b-9 and TCC) are highlighted.
Go to our website www.svarlifescience.com/get-a-free-poster to order your own copy of the Complement System Roadmap poster. ©Svar Life Science AB, February 2020
In addition, Svar Life Science offers biomarker assays2 that are ideal for detection of complement activation and dysregulation.
Recently, we have added reporter-gene assays3 based on iLite® technology for cell-based complement analysis.
Here, we present examples of complement analysis using our products and how they can be used in efficacy and potency testing in the development of complement therapeutics.
THE COMPLEMENT SYSTEM
C5b-9
TCC
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C4dC4dCCCCC4C4444444ddddd
Complement C4d ELISA-based biomarker assay2
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iLite® C5a cell-based assay3
CP, MBL & AP pathway specific Wieslab® Complement, ELISA-based functional assays1
Complement solutions used for analyzis:
C4d
AP MBL CP
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SVAR LIFE SCIENCE
Figure 2. Eculizumab (ECU) prevents the formation of the terminal complement complex (TCC). TCC concentrations are elevated in the control (PBS and TRZ) assays following addition of the activator, indicating an activation of the complement system. Only low concentrations of TCC are detected upon addition of activator when eculizumab is present in the serum, which is expected as the drug blocks C5, upstream of TCC in the complement cascade.
Figure 3. Eculizumab (ECU) does not affect the generation of C4d. C4d concentrations are elevated in all the samples following addition of the activator, indicating an activation of the complement system.
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MATERIAL & METHODS
The experiments were performed in three steps:
1. A mixture of eculizumab and neat normal human serum (NHS) was incubated for 1 hour at room temperature. In the negative controls, PBS or trastuzumab (a HER2 monoclonal drug) was added instead of eculizumab.
2. In order to induce strong complement activation, an activator consisting of heat-aggregated IgG and zymosan was added to the samples, followed by incubation for 1 hour at 37°C. PBS was used in the negative controls. For the functional complement assay, eculizumab samples were diluted according to the manufacturer’s instructions, followed by activation in the microplate during the first incubation (1 hour at 37°C).
3. Samples were analyzed for complement activation using iLite C5a cell-based assays or Complement C4d or TCC ELISA-based biomarker assays. For the functional assessment, serum was analyzed using the Wieslab® Complement Screen kit.
RESULTS
In the first experiment, we analyzed the formation of the terminal complement complex (TCC) in the presence or absence of eculizumab.
Since C5 is the target of eculizumab and TCC is downstream of C5 in the complement cascade, we would expect that TCC formation is inhibited in the presence of the antibody (Fig. 1).
Indeed, TCC concentration is low in the presence of eculizumab and the complement activator (Fig. 2). In the controls, TCC concentration is highly elevated upon addition of the activator indicating a strong activation of the complement system. As expected, TCC also forms spontaneously in the controls
without addition of activator. Addition of the C5-antibody also inhibits all such spontaneous in vitro activation.
Next, we analyzed the generation of C4d in the presence of the drug. This was performed as a control experiment to verify that complement had been highly activated in all samples, especially those where activator was added.
As shown in figure 3, eculizumab does not prevent C4d generation, which is expected as C4d is upstream of C5 in the complement cascade (Fig. 1). Again, there is a strong induction of the complement system upon addition of the activator.
There are important pathway-dependent issues to consider when developing complement drug candidates
Doc No: S-021-GB01, Feb. 2022
T F E W
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Svar Life Science AB
Mail address: P.O. Box 50117 SE - 202 11 Malmö Sweden
SVAR LIFE SCIENCE
The Wieslab® Complement System Screen kit is an enzyme immunoassay used for functional assessment of the classical, lectin and alternative complement pathways, as well as for determination of complement deficiencies, in human serum.
It combines the specific activation of each pathway with the use of labeled antibodies specific for C5b-9 (Fig. 1).
Figure 4 shows that complement function of all three activation pathways, measured as C5b-9 deposition in the microplate, is inhibited in a concentration-dependent manner upon addition of eculizumab.
Figure 4. Eculizumab inhibits complement function. Complement function of all three activation pathways (classical [CP], lectin [LP] and alternative [AP]), measured as C5b-9 deposition in the microplate, is inhibited by ECU in a concentration-dependent manner.
Figure 5. Eculizumab (ECU) prevents the generation of C5a. C5a concentrations are elevated in the control (PBS and TRZ) assays following addition of the activator, indicating an activation of the complement system. Only low concentrations of C5a are detected upon addition of activator when eculizumab is present in the serum. This assay contained 1% serum.
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SUMMARY
The interest in complement disorders in recent years has led to a surge in the development of complement therapeutics, with dozens of complement therapeutics in various stages of clinical development. Currently, complement drugs target mainly rare diseases, such as PNH and aHUS. In the future, we expect this type of drugs to become available for age-related macular degeneration, periodontitis and other more common disorders. Clearly, there is a need for accurate and sensitive assays for the study of complement drug candidates.
Here, we have used three biomarker assays and a functional assay to study the inhibitory effect of eculizumab on the complement system.
Formation of two complement components, C5a and TCC, remained low despite high complement activation in the presence of the drug, consistent with the fact that these are generated downstream of C5 – the target of eculizumab – in the complement cascade.
Conversely, levels of C4d, which is upstream of C5, were significantly increased in all samples, including those with eculizumab, which is a clear sign of complement activation.
Taken together, these assays offer a powerful way of analyzing the inhibitory effect of a drug candidate on the complement system, which will be needed for the development of future complement therapeutics.
Finally, we used an iLite cell-based assay to analyze the effect of eculizumab on C5a levels. C5a is a cleavage product from C5, hence we expected no increase of C5a levels in the presence of the drug (Fig. 1).
Indeed, C5a levels remained unchanged, in contrast to the controls, following complement activation in the presence of the drug (Fig. 5).