Soluble Complement Receptor 1 Therapeutics
Matthew P. Hardy1*, Tony Rowe1, and Sandra Wymann2
1CSL, Bio21 Institute, Victoria, Australia
2CSL, CSL Biological Research Centre, Bern, Switzerland
Abstract
Human Complement Receptor 1 (CR1/CD35) is a potent negative regulator of the complement system. Its mechanism of action is through interaction with the complement activation fragments, C3b and C4b to mediate decay acceleration of the C3 and C5 convertase complexes as well as cleavage of both ligands into inactive fragments via cofactor activity. The result is inhibition of the classical, lectin, and alternative complement pathways. This article will focus on recombinant soluble forms of CR1 that have been generated as potential therapeutics for complement-mediated disorders. Specifically, we will review and contrast the in vitro and in vivo properties of: sCR1 (BRL55730/TP10/CDX-1135), the soluble full-length extracellular domain of human CR1; sCR1-sLex (TP20), a glyco-engineered version of sCR1 additionally targeted to activated endothelium; APT070 (Mirococept), a CR1 fragment conjugated to a myristoylated peptide to enhance tissue targeting; and CSL040, a soluble truncated version of the CR1 extracellular domain which exhibits altered potency and pharmacokinetic properties as compared to the parental molecule. The data obtained from studies on the effects of these CR1-based molecules in animal models of disease and their therapeutic applications will also be discussed.
Introduction
The complement cascade is an arm of the innate immune system that has evolved as a primary defence mechanism against pathogens and other deleterious processes. It consists of multiple proteins found both in plasma and at the surface of many cell types which work to target not only immune complexes and cells for phagocytosis via the process of opsonization, but also to drive inflammation via the formation of the anaphylatoxins C3a and C5a, and to initiate cell lysis directly through the generation of the membrane attack complex. These processes are initiated by the classical, lectin, and alternative complement pathways by a variety of activating factors such as immune complexes, endotoxin, neoepitopes, specific carbohydrate moieties, and by the spontaneous C3 ‘tick-over’ mechanism1-4. To prevent unchecked activation of the complement cascade and damage to host cells or tissues, specific proteins have evolved as regulators of complement. Both transmembrane and soluble forms of these regulators exist, acting at all levels of the complement cascade. Examples include CD465, CD556, CD597, Factor H8, Factor I9, and the complement receptors CR1 – CR410.
Complement Receptor 1
Human CR1 (CD35) is a central regulator of the complement system, acting at the level of complement component C3 to inhibit the classical, lectin and alternative pathways11. It is primarily a cell surface bound membrane protein expressed on erythrocytes (E-CR1) and immune cell types such as monocytes, macrophages, neutrophils, eosinophils, certain B and T cells, and glomerular podocytes11-13. A soluble version of CR1 exists; this is produced at very low levels (approximately 50 ng/mL) by proteolytic cleavage of the extracellular domain from the membrane, rather than produced constitutively14,15. CR1 belongs to a family of complement receptors that include CR2, CR3 and CR410. It also belongs to a wider family of structurally related proteins referred to as the Regulators of Complement Activation. These include CD46, CD55, C4-binding protein, and Factor H, and are characterized by repetitive and highly homologous modules of approximately 60 amino acids known as short consensus repeat (SCR) domains16. The dominant allelic variant of human CR1 (Figure 1) is a 2039 amino acid protein with a 41 amino acid signal peptide, a 1930 amino acid extracellular domain, and a short cytoplasmic tail16,17. The extracellular domain of human CR1 is further comprised of 30 SCR domains which are themselves arranged into four long homologous repeat domains (LHR-A, -B, -C, -D), each composed of seven SCR domains16,18,19. Although SCR domains 29-30 are not technically part of LHR-D, they are often included for experimental purposes20-22. There is a high degree of homology (up to 99%) between SCR domains within CR116,23, such as the SCR8-10 and SCR15-17 domains located within LHR-B and LHR-C, respectively (Figure 1). Several allelic variants of human CR1 exist that contain fewer or additional LHR domains16,17. CR1 has a monomeric and highly flexible structure similar to other family members and adopts a “string of pearls” type conformation as suggested by its domain structure, negative stain microscopy, X-ray scattering and analytical ultracentrifugation24-26. CR1 is also highly glycosylated on multiple glycan sites found within its primary amino acid sequence, with its glycan structure determined to be exclusively N-linked27.
Figure 1. The structure of human CR1: Shown here schematically, human CR1 is a type I membrane protein composed of an N-terminal extracellular domain, a transmembrane region (TM), and a short cytoplasmic tail. CR1 also contains a signal peptide (SP) that is removed upon expression of the mature protein on the cell surface or when soluble CR1 is expressed recombinantly. The extracellular domain is made up of four long homologous repeat (LHR) domains, labelled –A to –D. The numbering above the top schema refers to the number of the last amino acid of each LHR domain and the final amino acid of the cytoplasmic region (based on Met+1), with the exception of “42”, which refers to the number of the first amino acid of LHR-A. The lower schema shows how each LHR domain is comprised of a number of short consensus repeat (SCR) domains to a total of 30 within the extracellular domain of CR1. The green and red bracketed regions with the numerated percentages denote the degree of amino acid homology between sets of SCR domains located within LHR-A and LHR-B. At the bottom of the figure the location (horizontal bars) and details of the ligand binding, decay acceleration, and co-factor activity properties of CR1 are shown.
The biological activity of human CR1 is mediated by binding to the ligands C3b and C4b, which are activated fragments of C3 and C4 zymogens, respectively12,28. Since they share similar binding sites on C3b and C4b, CR1 competitively displaces the Factor Bb and C2a catalytic fragments from the C3 convertases (C4bC2a, C3bBb) and C5 convertases (C4bC2aC3b, C3bBbC3b) which are formed upon complement activation. This mechanism of action is termed decay acceleration activity (DAA) and works to block further complement activation at the cell surface29,30. Human CR1 has a second function which is to act as a co-factor for complement Factor I, a serum protease that can cleave CR1-bound C3b and C4b via this co-factor activity (CFA) into the inactive forms iC3b and iC4b/C4c, to further inhibit complement activation13,29-32. Weak binding of human CR1 to iC3b and cleavage to further degradation products (C3c and C3dg) mediated by Factor I has also been reported24,31,33.
Soluble Complement Receptor 1 Therapeutics
The structure of human CR1 as a type I membrane protein makes it particularly amenable to engineering to create soluble versions via removal of the transmembrane and cytoplasmic regions, plus any part of the extracellular domain deemed undesirable. Soluble CR1 proteins have been used for more than 30 years as negative regulators of the complement system in preclinical and clinical studies of the effects of complement inhibition in various disease and injury settings. Here we review four of them in detail: sCR1 (also known as BRL55730, TP10, and CDX-1135), sCR1-sLex (TP20), APT070 (Mirococept), and finally a recently identified and characterized molecule, CSL040 (Figure 2).
Figure 2. Therapeutic agents based on human CR1: Shown here schematically are the structures of four soluble CR1-based recombinant therapeutics: A) sCR1, also known as BRL55730, TP10, or CDX-1135; B) sCR1-sLex, also called TP20; C) APT070 (Mirococept); D) CSL040. Each LHR domain found within each molecule is indicated with the exception of APT070 which has its SCR domains denoted. The vertical lines and numbering above the schemas shown in A, B and D denote the position and amino acid numbering (based on Met+1) of N-linked glycosylation sites. The red vertical lines capped by diamonds in panel B represent the sialyl Lewis X glycans found on TP20. The red “C” at the C-terminal end of SCR3 in panel C denotes the presence of a C-terminal cysteine on APT070 to which is conjugated the myristoylated peptide (wavy line) needed for tissue targeting.
sCR1 (BRL55730 / TP10 / CDX-1135)
The first description of a soluble recombinant molecule designed as a potential therapeutic inhibitor of complement was published in 199024,34. Using recently developed molecular biology techniques, it was possible to synthesize the cDNA encoding the entire extracellular domain of CR1 containing all four LHR domains (Figure 2A) and to express this protein recombinantly. Studies then showed that sCR1 retained all the biological functions of its parental molecule, such as ligand binding to C3b and C4b, Factor I-mediated CFA, C3 and C5 convertase DAA, and potent inhibition of all three complement pathways in vitro20,22,24,34-36. Broad cross-reactivity across multiple surrogate species also meant that sCR1 was particularly suitable for studies in animals to assess its pharmacokinetic and pharmacodynamic properties, as well as assessing its potency in vivo in animal models of disease or injury where it was hypothesized that complement played a role in the aetiology and/or progression of cellular or tissue damage. However, the in vivo half-life of sCR1 in rats was determined to be approximately 100 minutes37,38. This relatively short half-life limited its use to acute, rather than chronic settings.
This short half-life of sCR1 in vivo has not prevented its widespread use in a host of animal models for proof-of-concept testing, and we were able to identify over one hundred different studies in which it has been tested (Table 1). A breakdown of these studies is shown in Figure 3 where sCR1 has been applied to animal models across multiple therapeutic areas such as inflammation, tissue injury, neurology, auto-immunity and immune-complex mediated diseases, and infection, with a particular emphasis on ischemia-reperfusion injury (IRI) and transplantation (Figure 3A; Table 1). Several different animal species have been employed in these in vivo proof-of-concept studies with sCR1 (Figure 3B; Table 1), with most studies performed in rats as a surrogate species where the strength of the complement system is similar to that of humans39. Other important information that has been gained from these animal studies has revolved around dosing and tolerability, which would have informed subsequent human studies. Soluble CR1 has been found to be safe and well tolerated across species at doses – both single and multiple – of up to 60 mg/kg, and various routes of administration such as intravenous, intraperitoneal, and even intrathecal have been safely employed (Table 1). Most importantly, sCR1 has been found to be protective in the vast majority of studies in which it has been tested (Figure 3C; Table 1), although it has been applied mostly prophylactically, rather than therapeutically, potentially limiting the translatability of data to human disease settings. In vivo studies weren’t the only means to assess the suitability of sCR1 for clinical applications. Two early studies showed that sCR1 is a potent inhibitor of membrane-induced complement activation ex vivo40,41, rendering it of potential clinical utility during haemodialysis.
Figure 3. The use of sCR1 (BRL55730 / TP10 / CDX-1135) in animal models:Graphical representation in pie chart form of some of the key characteristics of the 101 animal models used to test sCR1 in vivo and the proportions of their usage. A) The indications in which sCR1 was tested, separated broadly into therapeutic areas (see key below chart). IRI: ischemia-reperfusion injury; IC: immune complex; CPB: Cardiopulmonary bypass. B) The species in which sCR1 was tested. When Xenografts required the use of two species, both were counted. C) The use of sCR1 prophylactically or therapeutically, and whether that usage was protective (as defined by statistically significant differences in key disease biomarkers compared to controls) or had no significant effect. The degree of protection is not noted here, as this varies and is defined differently from model to model. See Supplementary table I for the complete list and details of each animal model used to assess in vivo efficacy of sCR1.
Table 1: Use of sCR1 (BRL55730 / TP10 / CDX-1135) in vivo and ex vivo – animal models
Species |
Experiment / model |
Route(s) of administration |
Dose(s) |
Prophylactic or Therapeutic |
Effecta |
Reference |
Ischemia-Reperfusion Injury (IRI) |
||||||
Rat |
Myocardial IRI |
intravenous |
1 mg/rat |
Prophylactic |
Protective |
Weisman et al (1990a)34 Weisman et al (1990b)24 |
Rat |
Intestinal IRI |
intravenous |
3, 6 mg/rat x4 |
Prophylactic |
Protective |
Hill et al (1992)42 |
Rat |
Hind Limb IRI |
intravenous |
1, 3, 6 mg/rat |
Prophylactic |
Protective |
Lindsay et al (1992)43 |
Mouse |
Skeletal (Cremaster) Muscle IRI |
intravenous |
100 ug/mouse + 100 ug/hr/mouse infusion |
Prophylactic |
Protective |
Pemberton et al (1993)44 |
Ratb |
Cardiac IRI |
Perfusion |
10 μg/mL |
Prophylactic |
Protective |
Shandelya et al (1993)45 |
Rat |
Myocardial IRI |
intravenous |
5 mg/kg |
Prophylactic |
Protective |
Smith et al (1993)46 |
Rat |
Liver IRI |
intravenous |
25 mg/kg x1, or 50 mg/kg x2 |
Prophylactic |
Protective |
Chavez-Cartaya et al (1995)47 |
Rat |
Intestinal IRI |
intravenous / Perfusion |
20 mg/kg / 0.286 mg/mL |
Prophylactic |
Protective |
Xiao et al (1997)48 |
Mouse |
Intestinal IRI |
intravenous |
10 mg/kg |
Prophylactic |
Protective |
Austen et al (1999)49 |
Rat |
Intestinal IRI |
intravenous |
12 mg/kg x 2 |
Prophylactic |
Protective |
Eror et al (1999)50 |
Rat |
Intestinal IRI |
intravenous |
12 mg/kg |
Therapeutic |
Protective |
Eror et al (1999)50 |
Mouse |
Intestinal IRI |
intravenous |
20 mg/kg |
Prophylactic |
Protective |
Williams et al (1999)51 |
Rat |
Acute Myocardial Infarction |
intravenous |
1, 5, 15 mg/kg |
Prophylactic |
Protective |
Zacharowski et al (1999)52 |
Rat |
Hepatic IRI |
intravenous |
15 mg/kg |
Prophylactic |
Protective |
Lehmann et al (2001)53 |
Rat |
Pancreatic IRI |
intravenous |
15 mg/kg |
Prophylactic |
Protective |
von Dobschuetz et al (2004)54 |
Rat |
Placental Ischemia |
intravenous |
15 mg/kg/day |
Prophylactic |
Protective |
Lillegard et al (2013)55 Regal et al (2019)56 |
Mouse |
Renal IRI |
intravenous |
25 mg/kg |
Prophylactic |
Protective |
Hameed et al (2020)57 |
Transplantation |
||||||
Guinea Pig to Rat |
Cardiac Xenograft |
intravenous |
3, 5.9, 15, 60 mg/kg |
Prophylactic |
Protective |
Pruitt et al (1991)58 |
Guinea Pig to Rat |
Renal Xenograft |
intravenous |
50 mg/kg |
Prophylactic |
Protective |
Chrupcala et al (1994)59 |
Guinea Pig to Rat |
Cardiac Xenograft |
intravenous |
20 mg/kg |
Prophylactic |
Protective |
Zehr et al (1994)60 |
Pig to Cyno |
Cardiac Xenograft |
intravenous |
15 mg/kg |
Prophylactic |
Protective |
Pruitt et al (1994)61 |
Guinea Pig to Rat |
Cardiac Xenograft |
intravenous |
25 mg/kg + 20mg/kg/12hr (repeat), or 25mg/kg + 40 mg/kg/day (infusion) |
Prophylactic |
Protective |
Candinas et al (1996)62 |
Pig to Cyno |
Cardiac Xenograft |
intravenous |
25 mg/kg + 40 mg/kg/day |
Prophylactic |
Protective |
Davis et al (1996)63 |
Rat |
Lung Allograft |
intravenous |
25 mg/kg/day |
Prophylactic |
Protective |
Pratt et al (1996a)64 Pratt et al (1996b)37 Pratt et al (1997)65 |
Guinea Pig to Rat |
Cardiac Xenograft |
intravenous |
20 mg/kg |
Prophylactic |
Protective |
Fujiwara et al (1997)66 |
Rat |
Lung Allograft |
intravenous |
15 mg/kg |
Prophylactic |
Protective |
Naka et al (1997)67 |
Pig to Cyno |
Cardiac Xenograft |
intravenous |
25 mg/kg + 40 mg/kg/day |
Prophylactic |
Protective |
Pruitt et al (1997)68 |
Rat |
Liver Allograft |
intravenous |
15 mg/kg |
Prophylactic |
Protective |
Lehmann et al (1998)69 |
Pig |
Lung Allograft |
intravenous |
15 mg/kg |
Prophylactic |
Protective |
Pierre et al (1998)70 |
Pig |
Lung Allograft |
intravenous |
15 mg/kg |
Prophylactic |
Protective |
Schmid et al (1998)71 |
Pig to Cyno |
Intraportal Xenograft |
intravenous |
40 mg/kg |
Prophylactic |
Protective |
Bennet et al (2000)72 Lundgren et al (2001)73 |
Rat |
Tracheal Allograft |
intraperitoneal |
20 mg/kg/day |
Prophylactic |
Protective |
Kallio et al (2000)74 |
Rat |
Lung allograft |
intracardiac |
10 mg/kg |
Prophylactic |
Protective |
Stammberger et al (2000)75 Schmid et al (2001)76 |
Pig to Cyno |
Renal Xenograft |
intravenous |
40 mg/kg + (17-20 mg/kg Daily) |
Prophylactic |
No effect |
Lam et al (2005)77 |
Rat |
Renal Allograft |
intravenous |
25 mg/kg |
Prophylactic |
Protective |
Damman et al (2011)78 |
Other Injury |
||||||
Rat |
CVF-induced Lung Injury |
intravenous |
5, 10, 15, 20, 25 mg/kg |
Prophylactic |
Protective |
Mulligan et al (1992)79 |
Rat |
Thermal Injury |
intravenous |
5, 10, 15, 20, 25 mg/kg |
Therapeutic |
Protective |
Mulligan et al (1992)79 |
Rat |
LPS-induced Lung Injury |
intravenous |
1, 10 mg/kg |
Prophylactic |
Protective |
Rabinovici et al (1992)80 |
Rabbitb |
NHS-induced Cardiac Injury |
Perfusion |
20 nM |
Prophylactic |
Protective |
Homeister et al (1993)81 |
Pigb |
Human blood-induced Cardiac Injury |
Perfusion |
70, 300 μg/mL |
Prophylactic |
Protective |
Pruitt et al (1994)61 |
Rat |
IL2-induced Lung Injury |
intravenous / intraperitoneal |
10, 30 mg/kg (50% each route) |
Prophylactic |
Protective |
Rabinovici et al (1994)82 |
Rabbitb |
Human Plasma-induced Cardiac injury |
Perfusion |
20 mM |
Prophylactic |
Protective |
Gralinski et al (1996)83 |
Rat |
Acid-induced Lung Injury |
intravenous |
10 mg/kg |
Prophylactic |
Protective |
Nishizawa et al (1996)84 |
Rat |
Acid-induced Lung Injury |
intravenous |
10 mg/kg |
Therapeutic |
Protective |
Nishizawa et al (1996)84 |
Mouse |
Acid-induced Lung Injury |
intravenous |
20 mg/kg |
Prophylactic |
Protective |
Weiser et al (1997)85 |
Guinea Pig |
Porphyrin-induced Phototoxicity |
intraperitoneal |
60 + 20/40 mg/kg |
Prophylactic |
Protective |
Nomura et al (1998)86 |
Rat |
CVF-induced lung injury |
intravenous |
0.3, 1.5, 4.5 mg/rat |
Prophylactic |
Protective |
Mulligan et al (1999)87 |
Rabbitb |
NHS-induced Lung Injury |
Perfusion |
2.0 ug/mL |
Prophylactic |
Protective |
Heller et al (2000)88 |
Rabbitb |
NHS-induced Cardiac Injury |
Perfusion |
20 nM |
Prophylactic |
Protective |
Tanhehco et al (2000)89 |
Mouse |
Acid-induced Lung injury |
intravenous |
5, 10 mg/kg |
Prophylactic |
Protective |
Kyriakides et al (2001)90 |
Mouse |
Acid-induced Lung injury |
intravenous |
10 mg/kg |
Therapeutic |
Protective |
Kyriakides et al (2001)90 |
Pigb |
Human blood-induced Lung Injury |
Perfusion |
100 μg/mL |
Prophylactic |
No effect |
Azimzadeh et al (2003)91 Pfeiffer et al (2005)92 |
Mouseb |
Human blood-induced Lung Injury |
Perfusion |
225 μg/mL |
Prophylactic |
Protective |
Schroder et al (2003)93 |
Ratc |
Hypertension and Renal Injury |
intraperitoneal |
15 mg/kg/day |
Prophylactic |
No effect |
Regal et al (2018)94 |
Ratc |
Hypertension and Renal Injury |
intraperitoneal |
15 mg/kg/day |
Therapeutic |
No effect |
Regal et al (2018)94 |
Immune complex & Inflammation |
||||||
Rat |
Reverse Passive Arthus Reaction |
intradermal |
0.003, 0.03, 0.3, 1, 3, 30 μg/site |
Prophylactic |
Protective |
Yeh et al (1991)95 |
Rat |
Glycogen-induced Peritonitis |
intravenous |
7.5 mg/kg x2 |
Therapeutic |
Protective |
Mulligan et al (1992)79 |
Rat |
IgG immune complex Alveolitis |
intravenous |
3.75 mg/kg x4 |
Prophylactic |
Protective |
Mulligan et al (1992)79 |
Rat |
TNF- and Collagen-induced Arthritis |
intraperitoneal |
20 mg/kg/day |
Prophylactic |
Protective |
Fava et al (1993)96 |
Rat |
Anti-Thy1 Glomerulonephritis |
intraperitoneal |
60 mg/kg/day |
Prophylactic |
Protective |
Couser et al (1995)97 |
Rat |
Anti-Concavalin-A Glomerulonephritis |
intraperitoneal |
60 mg/kg/day |
Prophylactic |
Protective |
Couser et al (1995)97 |
Rat |
Passive Heymann Nephritis |
intraperitoneal |
60 mg/kg/day |
Prophylactic |
Protective |
Couser et al (1995)97 |
Rat |
Experimental Autoimmune Thyroiditis |
intraperitoneal |
10 mg/kg/day |
Prophylactic |
No effect |
Metcalfe et al (1996)98 |
Rat |
Cerulein-Induced Pancreatitis |
intravenous |
15 mg/kg/hour |
Prophylactic |
No effect |
Weiser et al (1996)99 |
Rat |
Cerulein-Induced Pancreatitis |
intravenous |
22.5 mg/kg |
Prophylactic |
Protective |
Acioli et al (1997)100 |
Rat |
Mono-articular Arthritis |
intravenous and/or intra-articular |
20 mg/kg/day and/or 200 μg/joint |
Prophylactic |
Protective |
Goodfellow et al (1997)101 |
Rat |
Mono-articular Arthritis |
intra-articular |
200 μg/joint |
Therapeutic |
No effect |
Goodfellow et al (1997)101 |
Rat |
Antigen-induced Pleural Inflammation |
intravenous |
10 mg/kg or 15 mg/kg x2 |
Prophylactic |
Protective |
Lima et al (1997)102 |
Rat |
Muscle Inflammation |
intraperitoneal |
20 mg/kg + (10 mg/kg x 3) |
Prophylactic |
Protective |
Frenette et al (2000)103 |
Rat |
Collagen-induced Arthritis |
intravenous |
15 mg/kg twice daily |
Prophylactic |
Protective |
Goodfellow et al (2000)104 |
Rat |
Collagen-induced Arthritis |
intravenous |
15 mg/kg twice daily |
Therapeutic |
Protective |
Goodfellow et al (2000)104 |
Rat |
Acute Arthritis/Synovitis |
intra-articular |
0.5 mg/joint |
Prophylactic |
Protective |
Mizuno et al (2000)105 |
Rat |
Acute Arthritis/Synovitis |
intravenous |
20 mg/kg |
Prophylactic |
No effect |
Mizuno et al (2000)105 |
Rat |
Allergic Asthma |
intraperitoneal |
10 mg/kg |
Prophylactic |
Protective |
Abe et al (2001)106 |
Rat |
Thrombotic Glomerulonephritis |
intravenous |
20 mg/kg |
Prophylactic |
Protective |
Kondo et al (2001)107 |
Rat |
Severe Acute Pancreatitis |
intravenous |
12 mg/kg x 2 |
Prophylactic |
Protective |
Hartwig et al (2006)108 |
Moused |
C3 Glomerulonephritis |
intraperitoneal |
25 or 50 mg/kg/day |
Prophylactic |
Protective |
Zhang et al (2013)109 |
Cardiopulmonary Bypass |
||||||
Pig |
Cardiopulmonary Bypass |
intravenous |
6 mg/kg x2 |
Prophylactic |
Protective |
Gillinov et al (1993)110 |
Pig |
Cardiopulmonary Bypass |
intravenous |
10 mg/kg |
Prophylactic |
Protective |
Lazar et al (1999)111 |
Pig |
Cardiopulmonary Bypass |
intravenous |
10 mg/kg |
Prophylactic |
Protective |
Chai et al (2000)112 |
Neurology |
||||||
Rat |
Antibody-mediated demyelinating Experimental Allergic Encephalomyelitis |
intraperitoneal |
20 mg/kg/day |
Prophylactic |
Protective |
Piddlesden et al (1994)113 |
Rat |
Experimental Autoimmune Neuritis |
intraperitoneal |
30 mg/kg/day |
Therapeutic |
Protective |
Jung et al (1995)114 |
Rat |
Traumatic Brain Injury |
intravenous / intraperitoneal |
20 mg/kg + 15 mg/kg |
Prophylactic |
Protective |
Kaczorowski et al (1995)115 |
Rat |
Experimental Autoimmune Myasthenia Gravis |
intraperitoneal |
20 mg/kg/day |
Prophylactic |
Protective |
Piddlesden et al (1996)116 |
Rat |
Experimental Allergic Neuritis |
intraperitoneal |
60 mg/kg x 2 |
Therapeutic |
No effect |
Vriesendorp et al (1997)117 |
Mouse |
Stroke (middle cerebral artery) |
intravenous |
15 mg/kg |
Therapeutic |
Protective |
Huang et al (1999)118 |
Rat |
Sciatic Inflammatory Neuropathy |
intrathecal |
50 μg/rat |
Therapeutic |
Protective |
Twining et al (2005)119 |
Rat |
Chronic Constriction Nerve Injury |
intrathecal |
50 μg/rat |
Therapeutic |
Protective |
Twining et al (2005)119 |
Rat |
gp120-induced Mechanical Allodynia |
intrathecal |
50 μg/rat |
Therapeutic |
Protective |
Twining et al (2005)119 |
Baboon |
Reperfused Stroke |
intravenous |
15 mg/kg |
Prophylactic |
No effect |
Mocco et al (2006)120 |
Rat |
Nerve Crush Injury |
intraperitoneal |
15 mg/kg/day |
Prophylactic |
Protective |
Ramaglia et al (2008)121 Ramaglia et al (2009)122 |
Rat |
Spinal Cord Injury |
intravenous |
6 mg/kg/day |
Therapeutic |
Protective |
Li et al (2010)123 |
Infection & Shock |
||||||
Guinea Pig |
Anaphylaxis (Passive and active) |
intravenous / intraperitoneal |
15, 105 mg/kge |
Prophylactic |
Protective |
Regal et al (1993)124 |
Rat |
Bacterial infection |
intravenous |
10, 25 mg/kg |
Prophylactic |
Protective |
Swift et al (1994)125 |
Rat |
Haemorrhage / Resuscitation |
intravenous |
15 mg/kg |
Prophylactic |
Protective |
Fruchterman et al (1998)126 |
Rat |
Haemorrhage / Resuscitation |
intravenous |
15 mg/kg |
Prophylactic |
Protective |
Spain et al (1999)127 |
Guinea Pig |
Forssman (pulmonary) Shock |
intravenous |
15 mg/kg |
Prophylactic |
Protective |
Wagner et al (1999)128 |
Rat |
Acute Shock |
intravenous |
20 mg/kg |
Prophylactic |
Protective |
Mizuno et al (2002)129 |
Rat |
Acute Shock |
intravenous |
20 mg/kg |
Therapeutic |
Protective |
Mizuno et al (2002)129 |
Mouse |
Red Blood Cell Transfusion |
intravenous |
1.5, 10 mg/kg |
Prophylactic |
Protective |
Yazdanbakhsh et al (2003)130 |
Rat |
Haemorrhagic Shock |
intravenous |
No dose provided |
Prophylactic |
Protective |
Chen et al (2016)131 |
Listings are separated into broad therapeutic areas for ease of reading. Some animal models fit into more than one category; in these cases, the most appropriate was chosen. While every attempt has been made to capture all reported usage of sCR1 in vivo and ex vivo from extant literature, there may be examples that have been missed. aThe degree of protection varies from model to model but must be statistically significant compared to controls to be deemed protective. bExperiment performed ex vivo. cDahl Salt-sensitive (SS) rats. dComplement Factor H knock-out mice (a model of C3 Glomerulonephritis) with or without the transgene for human CR1. e15 mg/kg dose administered intravenously only; 105 mg/kg dose administered cumulatively both intravenously and intraperitoneally over a 24 hr dosing period, rather than as a single dose. IRI: Ischemia-reperfusion injury; CVF: Cobra Venom Factor; LPS: Lipopolysaccharide; TNF: Tumour Necrosis Factor; NHS: Normal Human Serum; Cyno: Cynomolgus monkey. The multipliers added to entries within the Dose(s) column refer to the number of doses used that were administered less frequently than daily.
The strength of the sCR1 in vivo data from animal models led to the initiation of human clinical trials as summarised in Table 2. A Phase I single ascending dose study132 of sCR1 (TP10) in acute lung injury and acute respiratory distress syndrome patients demonstrated its safety and tolerability, with a pharmacokinetic/pharmacodynamic assessment showing an in vivo half-life of approximately 70 hours and complete inhibition of complement ex vivo in hemolytic assays at doses >1 mg/kg. Similar results were observed in a subsequent Phase I/II study in infants undergoing cardiopulmonary bypass (CPB), with evidence suggesting that sCR1 could confer some clinical benefit in this setting133. Given these tentative findings and previous data from pig models of CPB110-112 , a Phase II clinical study with 564 patients undergoing CPB during cardiac surgery was then initiated to determine the efficacy of sCR1 (TP10) in this indication. Unfortunately, the outcome of this clinical trial reported no overall efficacy in this indication for sCR1134 despite showing complete inhibition of complement. When the data from the study was assessed in more detail, there appeared to be a gender-specific effect, with males showing significant improvements in the primary endpoints compared to both females and controls134. A follow-on study of sCR1 (TP10) in female patients undergoing CPB and administered sCR1 (TP10) showed no effect135. Of equal interest is a study in a single patient who inadvertently received an ABO-mismatched lung allograft, where compassionate use of sCR1 (TP10) administration suggested some efficacy136. This led to a Phase II clinical study conducted in 59 patients undergoing lung transplantation, in which sCR1 (TP10) showed significant positive effects on extubation times and time of stay in intensive care, although no significant differences in other parameters such as death and graft rejection rates were found137. More recent attempts to use sCR1 clinically have been made (Table 2), but these failed for various reasons, and it appears that the entire program has been discontinued138.
Table 2: Use of sCR1 (TP10 / CDX-1135) in vivo – human studies
Clinical Trial |
Patient number / indication |
Route of administration |
Dose(s) |
Main Outcomes |
Reference |
Phase I |
24 Acute Lung Injury and ARDS |
intravenous |
0.1, 0.3, 1.0, 3.0, 10 mg/kg |
Single ascending dose safe and well tolerated Half-life of 69 hours Doses >1 mg/kg inhibit Complement activity |
Zimmerman et al (2000)132 |
Case Report |
1 ABO-mismatched Lung Allograft |
intravenous |
15 mg/kg x 5 doses every 3-4 days |
Reduction of anti-A antibody titer No humoral injury or cellular rejection Patient stable for 3 years post-transplant |
Pierson et al (2002)136 |
Phase I/II |
15 (infants <1 year old) Cardiopulmonary Bypass |
intravenous |
10 mg/kg plus 0.1 mg/mL to bypass circuit |
All infants survived and no TP10-related adverse events Half-life of 71 hours TP10 may protect against increase in vascular permeability |
Li et al (2004)133 |
Phase II |
564 Cardiopumonary Bypass |
intravenous |
1, 3, 5, or 10 mg/kg |
Elimination half-life of 55-57 hours; complement activity inhibited No effect on primary end point between TP10 and controls Significant improvement in endpoints in males only |
Lazar et al (2004)134 |
Phase II |
297 (Females) Cardiopulmonary Bypass |
intravenous |
10 mg/kg |
TP10 well tolerated Effective inhibition of complement No effect on primary end points of death or myocardial infarction |
Lazar et al (2007)135 |
Phase III |
59 Lung Allograft |
intravenous |
10 mg/kg |
Significant increase in patients undergoing early extubation Total time on ventilator and in intensive care shorter No difference in operative deaths, infection and rejection rates |
Keshavjee et al (2005)137 |
Phase I |
0 C3 Glomerulopathy |
- |
- |
ClinicalTrials.gov Identifier: NCT02302755 Study withdrawn (2014) – no recruitment |
- |
Phase I |
0 Dense Deposit Disease |
- |
- |
ClinicalTrials.gov Identifier: NCT01791686 Study Terminated (2014) – due to slow enrolment, portfolio prioritization and issues with role of complement in indication |
- |
ARDS: Acute Respiratory Distress Syndrome; ABO: human blood groups.
sCR1-sLex (TP20)
Glyco-engineering recombinant proteins to improve or modify their biological activity has been used for many years139. One such approach was used to generate a glyco-engineered variant of sCR1, denoted sCR1-sLex (TP20). Specific Sialyl-Lewis-X (sLex) tetra-saccharide carbohydrate motifs are found on the glycans of neutrophils and other leucocytes, and are made up of N-acetylglucosamine, galactose, neuraminic acid and fucose. These bind to specific ligands called selectins, particularly P-, L- and E-selectin, expressed on the surface of vascular endothelium140,141. Since leucocyte migration mediated by selectin binding contributes to inflammation and tissue damage, and given that selectins can be upregulated during disease142,143, it was thought that a bi-functional molecule (sCR1-sLex; Figure 2B) able to both inhibit complement and act as a selectin antagonist could be generated by decorating the N-Glycans of sCR1 with sLex motifs. There would be the added benefit of targeting sCR1 to the site of tissue damage144. Earlier studies have also demonstrated the ability of sLex to act as a stand-alone therapeutic, reducing neutrophil infiltration into tissue and protecting against damage145,146.
Engineering soluble CR1 to specifically expresses the sLex motif on its N-glycans was not easily achieved, requiring the use of a Chinese Hamster Ovary (CHO) cell line – LEC11 – to express the α(1,3)-fucosyltransferase needed to add α(1,3)-fucose to the N-glycans present on sCR1144,147,148. The end-result, sCR1-sLex, was shown to have a 10:1 ratio of sLex to sCR1 with an increased sialic acid content as well as a doubling of fucose content144. In vitro assessment of sCR1-sLex demonstrated increased binding to CHO cell-expressed E-selectin relative to its unmodified counterpart, and a dose-dependent blockade of U937 cell adhesion to immobilized P-selectin-IgG. Unmodified sCR1 was ineffective in the latter assay. It should be noted that sCR1-sLex also exhibited a small (<2-fold) but statistically significant decrease in complement inhibitory activity compared to sCR1 alone144.
Over the next few years, sCR1-sLex was tested in a number of animal disease models, several of which also employed sCR1 as a comparator, to determine its potency in vivo (See Table 3). The first model tested was a mouse model of middle cerebral artery occlusion (stroke) in which complement was shown to play a role118. In this model, sCR1 and sCR1-sLex were compared in both prophylactic and therapeutic settings, with equal doses of 15 mg/kg administered. In these experiments, sCR1-sLex was found to be more efficacious than sCR1, showing improvements in animal survival and additional reductions in infarct volume, neural deficit score and intracerebral hemorrhage. In an experimental rat model of cobra venom factor-induced lung injury, sCR1-sLex was almost twice as protective as sCR1, with dose-dependent reductions in both vascular leakage and lung neutrophil accumulation measured87. Of additional note in this study was the binding of sCR1-sLex to the lung vasculature, a phenomenon not observed with sCR1 alone. Addition of an anti-P-selectin antibody blocked this interaction, demonstrating the specificity of the binding of sCR1-sLex to P-selectin.
Table 3: Use of sCR1-sLex (TP20) in vivo – animal models
Species |
Experiment / model |
Route of administration |
Dose(s) |
Prophylactic or Therapeutic |
Effecta |
References |
Mouse |
Stroke (middle cerebral artery) |
intravenous |
15 mg/kg |
Prophylactic |
Protective |
Huang et al (1999)118 |
Mouse |
Stroke (middle cerebral artery) |
intravenous |
15 mg/kg |
Therapeutic |
Protective |
Huang et al (1999)118 |
Rat |
CVF-induced lung injury |
intravenous |
0.3, 1.5, 4.5 mg/rat |
Prophylactic |
Protective |
Mulligan et al (1999)87 |
Rat |
Acute Myocardial Infarction |
intravenous |
1, 5, 15 mg/kg |
Prophylactic |
Protective |
Zacharowski et al (1999)52 |
Rat |
Lung allograft |
intra-cardiac |
10 mg/kg |
Prophylactic |
Protective |
Stammberger et al (2000)75 Schmid et al (2001)76 |
Mouse |
Acid-induced Lung injury |
intravenous |
5, 10 mg/kg |
Prophylactic |
Protective |
Kyriakides et al (2001)90 |
Mouse |
Acid-induced Lung injury |
intravenous |
10 mg/kg |
Therapeutic |
Protective |
Kyriakides et al (2001)90 |
Baboon |
Reperfused stroke |
intravenous |
15 mg/kg |
Prophylactic |
Worse outcomeb |
Ducruet et al (2007)149 |
aThe degree of protection varies from model to model but must be statistically significant compared to controls to be deemed protective. bDoubling of infarct volume measured 3 days post-operatively. Study terminated prematurely following an interim analysis. CVF: Cobra venom factor.
Zacharowski et al52 tested sCR1-sLex in a rat model of myocardial IRI, showing reductions in both infarct size, cardiac troponin T release (a marker of cardiac tissue damage) and polymorphonuclear cell infiltrate at administered doses of 1 and 5 mg/kg, although a 15 mg/kg dose showed no further reduction of infarct size. In another study, a rat allogeneic single lung transplant model75,76 was used to compare sCR1 and sCR1-sLex, both administered prophylactically at 10 mg/kg. Assessment of graft function post-transplant showed improvements in gas exchange for both molecules compared to vehicle controls (383 mmHg, sCR1-sLex; 243 mmHg, sCR1; 56 mmHg, vehicle) with a concomitant reduction in both neutrophil migration and lipid peroxidation. As in previous studies, sCR1-sLex out-performed sCR1 in its ability to protect against tissue damage. The final rodent model described was a mouse model of acid aspiration injury in which sCR1 and sCR1-sLex were compared in both prophylactic and therapeutic settings90. Significant decreases in measured lung permeability index and polymorphonuclear cell infiltrates were observed for both molecules in prophylactic and therapeutic settings, with sCR1-sLex again showing superior efficacy compared to sCR1. The therapeutic efficacy was reduced the longer administration was delayed following induction of lung injury, and at two hours no effect was observed compared to the vehicle control90. Lastly, sCR1-sLex was tested in a NHP (baboon) model of reperfused stroke149. Although a previous study in the same NHP stroke model with unmodified sCR1 (as described above) showed no efficacy120, other studies using sCR1-sLex in a mouse model of stroke118 were successful and the authors hypothesized that adding sLex-mediated functionality to sCR1 would provide increased efficacy. Unfortunately, prophylactic administration of 15 mg/kg sCR1-sLex showed a worse outcome in NHPs than vehicle-treated animals, with a doubling of measured infarct volume at post-operative Day 3 and no change in neurological score, despite complete inhibition of complement activity for 12 hours following dosing149. The experiment was terminated following these interim results, and no further pre-clinical development of sCR1-sLex has occurred since then, given this was the last description of this molecule in the literature.
APT070 (Mirococept)
APT070 was first described150 as an N-terminal fragment of human CR1 containing only the first three SCR domains found within LHR-A, fused to a myristoylated peptide designed to bind to cell surface lipid bilayers for targeted complement inhibition at the disease site (Figure 2C). Generation of unmodified SCR1-3 began several years prior to this, when Dodd et al151 managed to successfully express and purify this protein from E. coli with yields of 6 – 15 mg/L. This and subsequent studies demonstrated that SCR1-3 was able to inhibit classical and alternative complement activity in hemolytic assays, display CFA for both C3b and C4b, and show similar DAA to the entire LHR-A domain36,151,152. However, purified SCR1-3 was still significantly less potent than soluble CR1 for both the classical/lectin and alternative pathways36. To overcome this deficiency in potency, a cysteine residue was added to the C-terminus of SCR1-3 (APT898) which allowed a membrane-localizing peptide (APT542) to be chemically coupled to it via disulphide bond formation, thereby generating APT070153. Comparative assessment of APT070 against its unmodified counterpart APT898 in human- and rat-specific assays showed cross-reactivity and significant improvements in potency (>100-fold) in vitro150,153-155.
In order to assess its efficacy in vivo, APT070 was used in several animal models of disease where complement plays a role in the indication’s pathophysiology (See Table 4 for a summary of all in vivo assessments for APT070). One of the earliest studies was in a rat model of complement-dependent acute intravascular shock, where intravenous prophylactic administration of APT070 up to 5 mg/kg showed a protective effect153,155. This was followed by a study in a model of antigen-induced arthritis154 where APT070 was administered directly to the relevant joint of rats at a 90 μg dose, resulting in a reduction in joint swelling and mean histological score compared to both vehicle control and APT898. A higher (250 μg) subsequent dose resulted in an even greater beneficial effect. A third study involved a rat kidney transplant model in which APT070 was shown to bind both glomerular endothelial and tubular epithelial cells156. Addition of APT070 to the perfusate applied to Donor’s kidneys prior to syngeneic renal transplantation led to improved graft function for up to 20 weeks. At 24 hours post-transplant, reduced acute tubular necrosis, neutrophil activity, complement deposition and blood urea nitrogen levels were observed156. Very little PK data in rats for APT070 is known with the exception of a single report stating a terminal half-life of 1 hour155, also limiting its pre-clinical use to acute settings.
APT070 then gained an additional name, Mirococept, following commercial acquisition, and was then used prophylactically in several additional animal models of disease: a mouse model of Miller-Fisher syndrome (a variant of Guillain-Barre syndrome)157; mild and severe rat intestinal IRI models158; another rat renal transplant model159; a closed-chest pig model of acute myocardial infarction160; and a transplant model whereby human pancreatic islets exposed to allogeneic whole blood were transplanted to the kidney capsule of humanized mice161. In all cases, APT070 was found to attenuate disease. APT070 was also found to be effective when administered in a therapeutic, rather than prophylactic setting. In the mouse model of Miller-Fisher syndrome, therapeutic administration of APT070 also resulted in a neuro-protective effect, although this was not as strong as observed in the prophylactic setting157. Not all animal models where APT070 was tested showed a beneficial effect. APT070 showed no effect on graft survival in a xenotransplantation model where baboons received hearts from transgenic pigs, even when administered both to the perfusate and separately to the donor itself both prior to and after reperfusion162. Similarly, in a rat model of hind limb IRI, APT070 administration had no effect on edema formation and other parameters of tissue damage163. This was in contrast to a comparator, C1-inhibitor, where a protective effect was shown. In vitro, APT070 was shown to inhibit complement activation in a cardiopulmonary bypass circuit model, and a reduction in the neutrophil activation marker CD11b was also observed164.
Table 4: Use of APT070 / Mirococept in vivo
Species |
Experiment / model |
Route(s) of administration |
Dose(s) |
Prophylactic or Therapeutic |
Effecta |
References |
Rat |
Antigen-induced Arthritis |
intra-articular |
90 & 250 μg/joint |
Prophylactic |
Protective |
Linton et al (2000)154 |
Rat |
Intravascular shock |
intravenous |
up to 5 mg/kg |
Prophylactic |
Protective |
Smith and Smith (2001)153 Smith (2002)155 |
Rat |
Kidney Allograft |
Perfusion |
40 μg/mL |
Prophylactic |
Protective |
Smith and Smith (2001)153 Smith (2002)155 Pratt et al (2003)156 |
Mouse |
Miller-Fisher Syndrome |
intravenous |
580 μg/mouse |
Prophylactic |
Protective |
Halstead et al (2005)157 |
Mouse |
Miller-Fisher Syndrome |
intravenous |
580 μg/mouse |
Therapeutic |
Protective |
Halstead et al (2005)157 |
Rat |
Intestinal IRI – mild and severe |
intravenous |
1, 3, 10 mg/kg |
Prophylactic |
Protective |
Souza et al (2005)158 |
Rat |
Kidney Allograft |
Perfusion |
40 μg/mL |
Prophylactic |
Protective |
Patel et al (2006)159 |
Pig |
Acute Myocardial Infarction |
intra-coronary |
0.5 mg/kg |
Prophylactic |
Protective |
Banz et al (2007)160 |
Pig to Baboon |
Heart Xenograft |
Perfusion & Intravenous |
0.1 mg/mL (perfusion) & 3 mg/kg (2 doses i.v.) |
Prophylactic |
No effect |
Wu et al (2007)162 |
Rat |
Hind Limb IRI |
intravenous |
9 mg/kg |
Prophylactic |
No effect |
Duehrkop et al (2013)163 |
Human to Mouseb |
human islet Xenograft |
in vitro |
0.4 μM |
Prophylactic |
Protective |
Xiao et al (2016)161 |
Human |
Phase I healthy volunteers |
intravenous |
2, 5, 10, 20, 40, 70, 100 mg |
N/A |
safe and well tolerated |
Smith (2002)155 Smith et al (2007)165 Kassimatis et al (2017)166 |
Human |
Phase IIa Kidney transplant |
Perfusion |
10 mg |
Prophylactic |
No effect |
Kassimatis et al (2017)166 |
Human |
Phase IIb |
Perfusion |
10 mgc |
Prophylactic |
No effect |
Kassimatis et al (2017)166 Kassimatis et al (2021)167 |
Pig |
Dose finding |
Perfusion |
20, 40, 80, 160 mg |
N/A |
80mg dose suitable |
Kassimatis et al (2021)167 |
aThe degree of protection varies from model to model but must be statistically significant compared to controls to be deemed protective. IRI: Ischemia Reperfusion Injury. bHumanized mice; cEMPIRIKAL study plan was designed with an initial 10 mg dose for Cohort 1 followed by with doses ranging from 5 – 25 mg for subsequent cohorts. However, study was terminated following administration of 10 mg dose due to lack of efficacy. i.v.: intravenous.
In humans, early reports of a Phase I trial of APT070 indicated that intravenous administration to healthy volunteers was safe and well-tolerated155,165. Additional information was provided in a later paper describing the design of the Phase IIb (EMPIRIKAL) trial, where 7 doses of 2, 5, 10, 20, 40, 70 and 100 mg were reported to have been administered166. Adverse events were described for the highest (100 mg) dose cohort, along with data showing the pharmacokinetics of APT070 (a plasma elimination half-life of 3 hours) and a lack of complement inhibition at doses below 10 mg166. The outcome of a pilot Phase IIa study in twelve patients was also described, where 10 mg APT070 perfused into donor kidneys pre-transplant was well tolerated with 80% of drug retained in the grafted kidney, but with no systemic complement inhibition reported and only a “trend to lower creatinine in the Mirococept group” 166. In the Phase IIb EMPIRIKAL trial which was aimed at reducing delayed graft function in transplanted kidneys, a similar (10 mg) dose of APT070 showed no efficacy, resulting in the premature termination of the study before additional doses could be tested167. In an attempt to determine a more suitable and efficacious dose of APT070 for further assessment in humans, the authors have recently conducted a dose-finding study in pigs at doses ranging from 20 to 160 mg, selecting a 80 mg dose (equivalent in humans to 120 mg) indicated as safe and sufficiently potent for further study167.
CSL040
In devising our own therapeutic candidate based on CR1, we were conscious of ensuring that any new molecule had two key benefits over its predecessors: increased potency, and improved pharmacokinetics and pharmacodynamic profiles. We were aware that there was a limit to which soluble CR1 could be truncated to create any new molecule, based on the comparatively weak complement inhibition profile of SCR1-3 compared to sCR136 and existing knowledge on the roles of the LHR domains, as described above. The strategy of adding dual functionality such as tissue/membrane targeting was not pursued, given the negative outcome of sCR1-sLex in the previously described NHP model of stroke149. So, relatively straightforward approach was devised involving the construction of a series of basic N- and C-terminal truncation variants of sCR122, which were expressed using mammalian cells and purified. Comparative assessment in complement inhibition assays quickly identified one variant, truncated at amino acid 1392 and containing the LHR-A, -B and -C domains (designated as CSL040; Figure 2D) which exhibited significantly greater in vitro potency for all three complement pathways than sCR1. Why removal of the LHR-D domain of sCR1 would produce this effect in CSL040 is not fully understood, but it is likely that a combination of increased stability and affinity for ligand, decreased steric hindrance along with the removal of interaction sites located within the LHR-D domain for C1q, MBL and certain Ficolins might all play a role22,168,169.
CSL040 was demonstrated to be a more potent inhibitor of the alternative complement pathway compared to that of the classical and lectin pathways22. This differential pathway activity can be explained mechanistically. In terms of the relative binding affinities of CR1 to C3b and C4b, 10-fold more sCR1 is required to inhibit the binding of C4b to erythrocytes compared to C3b34; a later study showed a 20-fold weaker affinity for the C4b-CR1 interaction compared to C3b-CR1170. This clearly has an effect on the DAA of CR1, with 5-10-fold more sCR1 needed to inhibit classical pathway convertase formation in vitro compared to alternative pathway convertase formation35. Other studies171,172 also showed differences in CFA for C3b compared to C4. C4b cleavage mediated by CR1 with Factor I is slower than that for C3b, with a preference for C3b if both ligands are present with Factor I. Mossakowska et al36 confirmed this finding, showing co-factor IC50 values for sCR1 to C3b of 0.8 nM compared to 15 nM for sCR1 to C4b.
Comparative assessment of CSL040 and sCR1 in a series of pharmacokinetic/ pharmacodynamic studies in both mice and rats was performed next22,173. In these studies, we took steps to ensure that the asialo-N-glycan levels of both molecules tested in these studies were similar, since previous studies showed the importance of protein sialylation for protein clearance174,175. In both species, CSL040 displayed a superior PK profile compared to sCR1. As the only point of difference between the two molecules tested, the LHR-D domain must be responsible for the faster clearance of sCR1 compared to CSL040; it was hypothesized that suboptimal glycosylation of the glycans present within LHR-D might contribute to more rapid clearance via clearance receptors such as the asialoglycoprotein or mannose receptors173. A series of additional experiments determined a relationship between the levels of CSL040 sialylation and in vivo clearance173, making this a critical quality attribute for any future in vivo studies. These experiments also demonstrated that CSL040 was safe and well tolerated at single doses of up to 90 mg/kg in both rats and non-human primates.
An analysis of the pharmacodynamic properties of CSL040 revealed an extended duration of alternative pathway inhibition, relative to the duration of the classical/lectin pathway response. An extended alternative pathway response in vivo was anticipated, given the increased in vitro potency of CSL040 for this pathway, but the extent of the response relative to the other pathways was not, since the studies performed with sCR1, sCR1-sLex, and APT070 discussed above typically only showed classical pathway activity following in vivo administration. Our data suggests some scope to potentially widen indication selection for CSL040 to chronic indications primarily involving the alternative complement pathway, rather than restricting development to acute indications.
The final studies performed to date with CSL040 have been proof-of-concept experiments in animal models of disease to evaluate in vivo efficacy. The vast body of literature for sCR1 in surrogate species (Table 1) informed decisions around indication and model selection for our own in vivo efficacy studies, and two mouse models were selected in which to test CSL040 (Table 5). The first model was a previously described176 model of immune-complex mediated kidney disease, the attenuated passive anti-glomerular basement membrane antibody-induced glomerulonephritis model, in which we found that CSL040 was able to significantly attenuate kidney damage (as measured by urine albumin output) at single 20 and 60 mg/kg doses administered prophylactically22. The second model in which CSL040 was tested was a mouse model of warm kidney IRI177. Here, we also used sCR1 (generated in-house) as a comparator, again ensuring that it retained similar asialo-N-glycan levels to that of CSL040. While two doses of 60 mg/kg CSL040 were able to significantly attenuate kidney damage, equimolar doses of 85.2 mg/kg sCR1 showed no significant effect177. This difference in relative in vivo potency is likely explained by both the 3-fold increased potency of CSL040 in vitro compared to sCR1, as well its significantly improved pharmacokinetic and pharmacodynamic properties22,173. We are now looking to expand our assessment of CSL040 to additional relevant in vivo animal models, and in settings in which CSL040 can be tested therapeutically, rather than prophylactically.
Table 5: Use of CSL040 in vivo in animal models of disease
Species |
Experiment / model |
Route of administration |
Dose(s) |
Prophylactic or Therapeutic |
Effecta |
References |
Mouse |
Glomerulonephritisb |
intraperitoneal |
5, 20, or 60 mg/kg |
Prophylactic |
Protective |
Wymann et al (2021)22 |
Mouse |
Kidney IRI |
intraperitoneal |
15, 30, or 60 mg/kg x 2 doses each |
Prophylactic |
Protective |
Bongoni et al (2021)177 |
aThe degree of protection varies from model to model but must be statistically significant compared to controls to be deemed protective. bThe Glomerulonephritis model used is abbreviation of its full name, the attenuated passive anti-glomerular basement membrane antibody-induced glomerulonephritis model.
Summary
It has now been more than three decades since a soluble regulator of complement and potential therapeutic based on CR1 has been described. As is clear from this review, the various forms of soluble CR1 that have been engineered and developed as therapeutics since that time have shown great promise in multiple disease indications with protective effects shown in a wide variety of animal models of specific indications where complement plays a role in mediating the pathophysiology and/or progression of disease or injury. Unfortunately, it also seems clear that translating efficacy from animal models to humans for the CR1-based molecules we have reviewed has been challenging, with perhaps an over-reliance of prophylactic, rather than therapeutic animal models from which to select indications for non-human primate and human clinical studies. It is hoped that with CSL040, we can learn from past knowledge and with informed indication selection achieve sustained success in the clinic.
Supporting information
None
Conflict of interest statement
M.P.H, T.R. and S.W. are listed as inventors on International Patent Publication number WO2019/218009. All authors are CSL shareholders.
Funding
This work was wholly supported by CSL. No external sources of funding were used.
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