Multiple experimental sepsis studies have emphasized the detrimental effects of excessive complement activation for the host (**Figure 1**) (49, 50). This can be considered as a "complement paradox" since complement per se is central to the innate immune defense against invading microorganisms. However, in the context of MODS development caused by sepsis, complement activation seems to enhance rather than protecting against several organ dysfunctions, especially in the heart, lungs and kidneys, representing three central organs in MODS (51–53). The multiorgan gene expression profiles in experimental sepsis seems to be either organ-specific, or common to more than one organ, or distinctly opposite in some organs (54). Furthermore, a balanced pro- and anti-inflammatory genetic response was observed and a differential gene expression for mediators responsible for preventing tissue damage, e.g., protease inhibitors, oxidant neutralizing enzymes, decoy receptors, and proteins which can protect tissue barriers (54). Concerning complement, pre-procomplement C3 was highly expressed in all organs except in the brain during the whole course of sepsis (54). However, genetic deficiency of C3 resulted in significantly enhanced lethality in comparison to C3-sufficient mice most likely due to a loss of C3b-dependent opsonization of invaded pathogens (55). In contrast, a blockade of C5a by various strategies in sepsis models, e.g., by anti-C5a antibodies, C5aR antibodies, small peptide C5aR1 antagonists, C5a-neutralizing mirror-image (l-)aptamer C5a aptamers, was coherently protective against biochemical and histological evidence of MODS and in general improved survival of sepsis (53, 55–61). All these experimental results demonstrate that C5a-C5aR interaction is clearly involved in the pathogenesis of MODS during sepsis and represents an important therapeutic sepsis target when the novel definitions of sepsis are applied (**Figure 1**) (47).

In translation to the clinical setting, several non-human primate experiments and studies in humans are in line with the findings in the rodent sepsis model. Evidence of systemic complement activation with reduction of complement hemolytic activity, C3 depletion and enhanced levels of C3a and C5a and corresponding loss of C5aR on neutrophils have been described in several human studies (62–64). Of note, the reduction of C5aR1 and C5aR2 on neutrophils has been correlated with the occurrence of infectious complications in ICU patients (30) and sepsis-induced MODS (62, 65, 66). Since loss of C5aR1 and C5aR2 has been concurrently correlated to the sequential organ failure assessment (SOFA) score (65), a flow-based rapid testing, might have a bedside monitoring potential to predict infectious problems and MODS development.

### MODS—PATHOMECHANSISMS CAUSED BY MULTIPLE COMPLEMENT DYSFUNCTIONS

Several pathomechanisms contribute to the development of MODS, such as enhanced levels of DAMPs and PAMPs, reduced cytochrome P450 metabolism, macrophage activation syndrome, and cytokine-driven cellular dysfunction (67). Overall, it is clear that complement dysregulation contributes to MODS after trauma (**Figure 2**). Some of these aspects have been already mentioned and will be further discussed in this section.

#### Immune Paralysis

Though severely injured patients receive modern ICU management, many of them show signs of immunosuppression known as persistent inflammation-immunosuppressive catabolism syndrome (PICS) (**Figure 1**) (68). Clinically, PICS patients suffer from persistent inflammation, immune suppression and protein catabolism, which can lead to recurrent nosocomial infections with sepsis, MODS and death (68). Severe immune suppression of the fluid-phase and cellular immune response has been proposed as "immune paralysis" of the host response to sterile and infectious insults. In a clinical case report, an inadequate response to infection with signs of systemic depletion of complement (dropping C3 and C4 levels) has been associated with the development of acute kidney injury and multiple organ failure in a 17-day old newborn (69). C5b-9 and C5a have been described as contributors to cell death, immune paralysis, cardiac dysfunction, and multiple organ failure (**Figure 2**) (49, 70). In support, a baboon model of Escherichia coli sepsis showed that blockade of C5 protected organs from "immune paralysis" and improved the sepsis survival rate (**Table 1**) (75).

### Dysregulated Complement Regulators

Additional connection between MODS and signs of complementopathy are supported by the fact that soluble and membrane-bound regulators of complement activity show alterations after trauma, sepsis and hemorrhagic shock. It is well-established that severe tissue injury causes an excessive systemic intravascular activation of the complement system resulting in a loss over the control mechanisms (8). In this context, the soluble form of the complement receptor 2 (sCR2) was shown to be present after nerve injury in rodents (80). After polytrauma in humans, leukocyte expression profiles of the complement regulators (CRegs) CD55 (decay accelerating factor), CD59 (membrane attack complex inhibitor), CD46

(membrane cofactor protein), and CD35 (complement receptor 1) were cell-type- and time-dependently altered, reflecting manifestation of posttraumatic complementopathy (4, 29). Furthermore, an observational study including critically ill patients with multiple injuries and sepsis revealed that trauma patients with MODS have significantly lower C1-inhibitor activities (**Figure 2**) (7). A rare mutation in the complement regulatory gene factor H has been also implicated with a severe form of complement-mediated hemolytic uremic syndrome with multiple organ involvement, showing the general importance of complement regulation for organ (dys)functions (81).

#### Alteration of Signaling Pathway Activity

Experimental studies showed an interplay of complement with specific signaling pathways including the activation of PKC, MAPKs, and ERK (82). In a non-traumatic renal damage model, NF-κB contributed to enhanced complement activation (83), while in a similar mouse model the C5a/C5aR axis activated MAPK signaling (84). During sepsis, the activation of MAPKs and Akt signaling was complement-induced (51). These mechanistic insights require further elaboration to identify other participating mediators downstream to complement activation in trauma and hemorrhagic shock.

# Changes in Protease Activity

Excessive systemic activation of proteases feed in the pathomechanisms of MODS and is known to create a vicious cycle of complement activation. Such proteases include immunoreactive trypsins and neutrophil elastase, which may directly interact with complement and have been extensively described in the case of polytrauma (9). A specific subtype of MODS has been introduced as thrombocytopenia-associated MODS, with low ADAMTS13 activity and defects in inhibitory complement regulators which may result in hyperactivation of coagulation and complement with resultant thrombosis (85). This form of MODS has been successfully treated by C5 blockade (eculizumab) and ADMTS13 reduction by plasma exchange (67). Furthermore, an unspecific and unregulated protease hyperactivity may directly cleave various complement components in a non-canonical manner, which represents a promising future research field for drug development. In this case, the intestine plays an important role in trauma and during shock, which can proceed to a proposed autodigestion phenomenon, since digestive enzymes from the pancreas are activated by enterokinases (86). Under physiological conditions, the auto-digestion is prevented by epithelial and mucosal barriers. However, during shock digestive peptides can pass the mucus and reach the epithelial cell membranes, where they disrupt junctional complexes, further activate complement and contribute barrier and organ dysfunction (87).

# Microcirculatory Alteration

Patients who succumbed to traumatic-hemorrhagic shock showed an impaired microcirculation for at least 72 h, which also was a reliable predictor for a high Sequential Organ Failure Assessment (SOFA) score (88). In an ischemia rat model, application of soluble complement receptor type 1 significantly improved microvascular perfusion in the liver assessed by in vivo microscopy, suggesting an essential role of complement in microcirculatory disorders (89). Activated complement with microvascular alterations can also cause a disruption of cellular barriers leading to edema formation in lung, brain, and liver (84, 90, 91). However, hypothetical complement-caused damage of specific tight junction molecules in specific organs needs still scientific verification. Such mechanisms of MODS development may also be supported by simultaneous activation of complement and coagulation e.g., by complement-dependent generation of thrombin, which is known to efficiently break down various endothelial barriers. Furthermore, complement hyperactivation has been associated with thrombotic microangiopathies causing

TABLE 1 | Most representative preclinical studies addressing trauma, HS and sepsis, respectively.

HS, hemorrhagic shock; MAC, membrane attack complex; NHP, non-human primate; TBI, traumatic brain injury; TNF, tumor necrosis factor.

endothelial cell activation and thrombus formation leading to hemolytic anemia, thrombocytopenia, and organ failure (91).

#### Metabolic Changes

Besides microcirculatory problems, massive blood loss due to hemorrhage can cause acute circulatory failure resulting in lactic acidosis. Acute liver or renal dysfunctions are most often associated with decreased lactate clearance and a pronounced increase in blood lactate levels compared with shock patients without signs of liver or renal dysfunction (92). It is also known that shock states initiate a pronounced compensatory vasoconstriction, consequently leading to hypoxia and accumulation of metabolites associated with lactate acidosis and a low pH milieu (92, 93). In this context, complement dysfunction has a deleterious influence and correlates with a worse outcome after shock. Especially the anaphylatoxin C5a and its following signaling via the C5aR1 have been described to further enhance acidosis after septic shock. More precisely, in vitro stimulation of neutrophils from healthy donors with C5a causes C5aR1 signaling-mediated immunometabolic changes in neutrophils with an enhanced glucose uptake and enhanced glycolytic flux (**Figure 2**) (94). In turn, it can initiate an increased proton secretion and further lower the extracellular pH. In response to C5a exposure, intracellular pH in neutrophils significantly increases via activation of the Na+/H+ exchanger type 1 (NHE-1) leading to an impairment in neutrophil function in vitro. Supporting this evidence, neutrophils isolated from septic patients has been shown to exhibit an increased intracellular pH compared to healthy donors (94). These recent findings indicate that inflammatory processes with produced C5a is solely capable to significantly change the micro-milieu with lactate acidotic features even in the absence of an oxygen deficit. It only can be speculated on, that in the case of an additional oxygen deficit, caused by shock conditions and vascular dysfunction, the anaphylatoxins may even function as a metabolic switch toward lactate acidosis and MODS.

#### Microbiome Alterations

The intestinal complement is suggested to cooperate in a close relationship with the gut microbiome (95). Therefore, another contributor to MODS seems to be the alteration of the gut microbiome after trauma/hemorrhagic shock and/or sepsis. The microbiome, which is described by the phylogenetic composition and taxon relative abundance of the bacteria, is significantly altered in the first 72 h after injury. This rapid change in intestinal microbiota represents a critical phenomenon that may influence outcomes after severe trauma (15). The composition of the microbiome may influence the activation/dysregulation of complement pathways or dysregulated complement may change the microbiome composition. In the skin, complement activation modulates the inflammatory milieu by changing the cutaneous microbiota (96). Considering MODS, a mechanistic explanation for complement-microbiome interaction still remains elusive and needs further research.

#### Alterations in Microvesicle (MV) Shedding

Communication is essential for cellular homeostasis and vesicle shedding has been described to play a crucial role for maintaining proper immune cell function. Extracellular vesicle shedding is altered after inflammation and is considered as a crucial contributor to MODS after multiple injury and sepsis (97). Especially shedding of microvesicles (MV) have been implicated in several inflammatory conditions including sepsis and trauma. Increased amounts of CD41+ and CD31+/CD41–/AnnexinV– MV after sepsis, released by activated platelets and leukocytes have been shown to correlate with unfavorable outcomes (98).

Furthermore, MVs from patients with multiple organ failure support the coagulation system in triggering inflammation. In respect to complement, phosphatidylserine containing MVs also serve as platform for complement activation (99). Besides activation of complement on their surfaces, MVs represent transport vehicles sending complement as cargo to neighboring as well as cells in distance (99). Hence, MV from different cellular origins may contain complement receptors including C5aR but also CRegs such as CD46 and CD59, suggesting a putative role for complement activity (99). In accordance with this, loss of C5aR1, C5aR2, and C3aR on neutrophils after multiple injury was clinically present and was correlated to infectious complications and multiple organ dysfunction (**Figure 2**) (18).

#### Electrophysiological Changes

Another concurrent theory of MODS addresses electrophysiological changes of the cellular membrane which have recently been found in neutrophils from septic pigs (14). The anaphylatoxin C5a was able to alter the membrane potential of neutrophils but not in the case of neutrophils during septic MODS where the electrophysiological response to C5a was somehow frozen (**Figure 2**) (14). Whether complement inhibitory strategies will stabilize the cellular membrane electrophysiology is currently under investigation.

#### Cellular Hibernation

Trauma causes an alarming stress situation for the whole body with an extensive inflammatory response (9). Some studies indicate that trauma especially followed by additional sepsis causes hibernation in the cellular as well as fluid phase of innate immunity including the complement system. Reflecting the evolved habit of conserving physiological resources in the event of environmental stress, with inflammation ensues a similar mechanism where energy-consuming processes are shut down in the organism. Supporting this evidence, hibernation has been observed in the septic heart with ongoing metabolic changes including the upregulation of specific glucose transporters in cardiomyocytes (100). Besides its effects on metabolism, hibernation is known to affect various immune function including leukocyte migration, as well as adaptive immune responses and interestingly complement function, by lowering complement levels and reduced expression of C3 mRNA in the liver, which depicts a suggestive link post-shock. (101). However, another study demonstrated that hibernators are protected from shock-induced injury, inflammation, and organ function (102). Strikingly, arctic ground squirrels challenged with cardiac arrest or hemorrhagic shock showed no markers of organ damage, systemic inflammation, or loss of acid/base balance as indicated by a negative base excess. Neither reduced body temperature nor hibernation season are components of this protection, indicating still unknown mechanisms involved (102).

Unfortunately, no supporting data is available indicating that future research on the complement function during hibernation, especially following trauma and shock is needed. Further, the ability to induce a fully reversible state of immune suppression in humans by artificial hibernation might aid the treatment of several inflammatory and immune-mediated diseases.

# TARGETING COMPLEMENT PATHWAYS IN MODS

Despite improvements in trauma care, the morbidity and mortality of MODS remains very high.

Therefore, new therapeutic strategies are urgently needed. Since complement is critically involved in initiation and progression of MODS, targeting complement as well as molecules contributing to complement activation represent promising future clinical approaches. Other complementassociated inflammatory conditions already addressed such a targeting strategy. Above 20 complement-interfering drugs have been evaluated in clinical settings so far. A few of them received FDA approval for inflammatory indications including eculizumab targeting the terminal complement pathway starting from C5, which is currently used for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) (103). As existing complement therapeutics cannot target every complement-driven disease status, it is a requisite to evaluate the different complement stages, their relevance, and target them in a disease-specific manner (10).

In the context of trauma, hemorrhagic shock and sepsis a few preclinical experimental studies focus on complement-targeting studies (**Table 1**). Besides complement activation products, other molecules including CRP, HMGB1, or mitochondrial DNA play crucial roles in contributing to complement dysregulation after sepsis and trauma, thus profoundly influencing secondary outcomes. Therefore, a rational approach seems to address molecules, which may further boost complement activity. Likewise, HMGB1 is a relevant danger molecule and complement has been described to regulate HMGB1 release from human neutrophils (104) such that, complement inhibition has proved to be protective in blast-induced acute lung injury in rats by ameliorating HMGB1-mediated inflammation (105).

#### Targeting Complement After (poly)Trauma

In polytrauma patients, the only study of complement intervention early after trauma with RCT quality used C1 esterase inhibitor. However, this study has been terminated based on the heterogeneity of the patients (106). Experimentally, the majority of the complement targeting studies were performed in mouse traumatic brain injury (TBI) models. In this case, various interventions to inhibit complement activity, such as inhibitors for MAC formation and C3 deposition inhibitors have been applied, analyzed and reviewed elsewhere (9, 107, 108). To further clarify the impact of distinct complement activation pathways on neuroinflammation, a recent study compared the effect of complement inhibitors targeting upstream as well as downstream complement activity, respectively. This study brought into light that instead of downstream complement activation complexes, upstream complement activation products of the alternative pathway predominantly modulate and propagate chronic inflammation (74) (**Table 1**).

# Targeting Complement in Hemorrhagic Shock

Based on the deduced potential of complement inhibitors to improve micro-perfusion disturbances, ischemia/reperfusion injury and the inflammatory response during hemorrhagic shock, various shock models have been tested for possible benefits of complement interventions. In a rat model of pressure controlled hemorrhagic shock, complement depletion by CVF improved the recovery of the mean arterial pressure (MAP) post shock (109). In a pig model of pressure controlled hemorrhagic shock, application of a C1-inhibitor improved the functional performance and reduced C3 deposition on a multi-organ level including liver, small intestine and lungs (**Table 1**) (79). Limitation of organ injury and sustained survival by C1 inhibitor therapy has also been reported for a porcine injury model mimicking battlefield injury (110). Further downstream, C3 deficient mice with bilateral femur fracture and hemorrhagic shock resulted in protective effects with reduced circulating DAMPs (e.g., dsDNA), decreased systemic inflammatory response and improved organ performance (e.g., liver enzymes) (111). In a rat model of hemorrhagic shock, C3 depletion by CVF, and the soluble form of CR1 to inhibit C3 action restored vascular reactivity to norepinephrine in the superior mesenteric artery (112). In the clinical setting, C3 inhibition has not been tested in hemorrhagic shock so far except in a recent study from our group, where non-human primates were modeled with severe hemorrhagic shock of 30 mmHg MAP for 1 h, and a delayed application of CP40 was tested (78). C3 blockade by the compstatin compound CP40 seems most promising since there was evidence of improved renal function, attenuated intestinal edema and reduced signs of systemic inflammation and coagulopathy (78). However, the transfer into clinical reality is pending although C3 inhibition seems rather safe in the case of hemorrhagic shock. Most likely, an early inhibition to avoid excessive C3 activation might be enough to improve the outcome (113).

On a C5 level, intestinal injury caused by hypovolemic shock in mice was ameliorated by application of a small peptide C5aR1 antagonist and C5 deficient littermates (114). Similarly, in a rodent model of ruptured aortic abdominal aneurysma with shock, C5 blockade by an anti-C5 antibody revealed some protective effects on remote lung injury with improvement of the bronchoalveolar permeability and myeloperoxidase (MPO) concentrations reflecting reduced neutrophil recruitment and activation (115). Combined inhibition of the complement component C5 and the Toll-like receptor co-factor CD14 in a porcine sepsis model, showed beneficial effects in regard to survival, hemodynamic parameters and systemic inflammation including complement activation (**Figure 1**, **Table 1**). First preclinical studies in non-human primates indicate appearance of sC5b-9 in the serum of traumatic-hemorrhagic shock and ex vivo effectiveness of C5 inhibitors in prevention of hemolysis, e.g., the small, inhibitory peptide RA101348, commerciallyavailable C5 inhibitory antibodies and Quidel's A217 antibody (106). However, a RCT-clinical study with C5 inhibitory strategies in hemorrhagic shock has not been performed so far most likely based on the rareness and uncertain evidence of a great impact on MODS development in the so far performed preclinical non-human primate studies. Therefore, research providing information about the histological and biochemical data on the multi-organ level in non-human primate studies is necessary for final assessment.

# Targeting Complement in Sepsis

In the case of septic shock, targeting the C5a/C5aR axis seems to be of practical importance since it correlated with the disease severity and mortality and showed promising improvements in different sepsis models (**Table 1**). Concerning complementtargeted therapies in sepsis, application of RA101295, a 2-kDa macrocyclic peptide inhibitor of C5 cleavage, improved organ performance and survival in an E. coli-sepsis model of baboons (75) with lessened evidence of coagulopathy and preserved endothelial and barrier functions. Furthermore, the inhibition of C5 cleavage lead to improved histomorphology of lungs, liver, kidneys, spleen, and adrenal glands suggesting improvement of sepsis-induced MODS (75).

In the same sepsis model, systemic blockade of C3 by compstatin also revealed organ protection on multiple levels. Compstatin showed evidence of sepsis-induced coagulopathy and preserved anti-coagulatory features of the endothelium. Furthermore, C3 blockade improved hemodynamics and heart function and biochemical damage markers of the kidney and liver, indicating protective effects in sepsis-induced MODS (5). For blocking the central complement component C3 during development of sepsis-caused MODS, clinical trials might be safe (113) but these trials are pending and require a special focus on the benefits or maladies for mental alterations during sepsis.

In terms of applicability in the clinical setting, it has been recently shown that blocking solely C5 activation by canonical C5-convertase (e.g., by eculizumab) might not be specific enough

cC5aR, circulating form of C5aR; CRegs, complement-regulating proteins; CRP, C—reactive protein.

since other serine proteases such as trypsin or thrombin can still cleave and activate C5. Therefore, a C5a blocking approach e.g., by IFX-1 anti-C5a antibody has been proposed as a targeted approach in local or systemic infection (116, 117). Overall, the specific complement targets for sepsis-induced MODS might be different than for polytrauma- or hemorrhagic shock-caused MODS especially when given early (**Figure 1**). The complement target might also change along the course of the disease. It is for example feasible, that early after polytrauma or during hemorrhagic shock a specific C3 inhibition is required, whereas later during development of septic complications, C5a inhibition might help against MODS development (**Figure 1**). And if complete depletion of a specific complement factor occurs, it might be even wise to replace it, which bolsters the importance of monitoring complement for any clinical trial.

In conclusion, MODS is a culmination of highly heterogeneous events which can involve different and complex stages of complement activation, making it nearly impossible to generate a drug addressing MODS generally. More precisely, the most prominent complement pathways and their down-stream signaling pathways involving multiple trauma with or without hemorrhagic shock and septic shock may be addressed in further preclinical studies.

#### Clinical Trials

Considering clinical trials of MODS caused by polytrauma, hemorrhagic shock and sepsis, three observational studies addressed complement activation after polytrauma and severe abdominal sepsis (**Table 2**). One study focused on the complement activation and expression on leukocytes in polytrauma patients revealing increased complement activity early after trauma and an increased shedding of complement receptors from neutrophil surfaces (NTC00710411; **Table 2**) (2, 62). Another observational study focuses on the molecular danger response after polytrauma. It is still recruiting patients, and the study aims to include up to 1,000 patients in a collective national data bank in collaboration with the Trauma Research Network (NTF) of the German Society for Orthopedics and Trauma (DGOU) and thus to obtain novel insight into the molecular pattern of complement activity following severe multiple injury (NTF-PT; NCT02682550; **Table 2**).

A further observational study has set its focus on the central complement C3 by investigation of C3 alterations in patients with severe abdominal sepsis. The aim was to evaluate the impact of C3 on patients' prognosis (NCT01568853; **Table 2**). This study revealed that C3 depletion is associated with a poor prognosis due to dysregulated coagulation and increased susceptibility for infections (118).

In regard to human studies, three interventional trials have been initiated so far (**Table 3**). One study addressed the inhibition of upstream complement component (C1) by targeting C1 esterase. This mono-centered double-blind randomized placebocontrolled trial (CAESAR; NCT01275976, **Table 3**) has been designed using a C1-esterase inhibitor in severely injured patients with femur fracture. Although the rationale of a complement blockade on this level is very well founded because it may also reveal beneficial effects by synchronically inhibition of excessive activation of the coagulation pathway (119), the study has been terminated based on the heterogeneity of patients and challenges in recruitment. A further study has addressed the effect of the C1-esterase inhibitor on human endotoxemia by evaluating its effect on inflammation and marker of organ dysfunction (VECTOR; NCT00785018, **Table 3**). Detailed results of the study are still pending.

Another clinical trial has been designed and performed to study complement inhibition in early, newly developing septic organ dysfunction (SCIENS; NCT02246595, **Table 3**) applying a monoclonal antibody against C5a, though the detailed results of this trial have not been published so far. Regarding one cardinal clinical sign of sepsis, the occult or evident alterations of the mental status, inhibitory strategies against C5a might reveal "Janus faced" effects (120). On one hand, C5a inhibition could improve sepsis-impaired blood-brain-barrier, on the other hand, neuroprotective effects by C5a might be compromised (120, 121). Therefore, alterations of the mental status need to be carefully addressed and monitored in any clinical trial using C5a inhibitory strategies.

It is important to note, that it is in general rather difficult to perform interventional studies on polytrauma patients since an informed written consent cannot be provided and the legal representatives are usually difficult to determine within the first hours after severe injury. Therefore, innovative studies early after polytrauma addressing the complement cascade are rather rare and have not been performed on the C3 or C5 level yet.

#### CLINICAL PERSPECTIVES

Although various clinically relevant models of trauma, hemorrhagic shock and sepsis have been tested already in nonhuman primates for the benefit of complement interventions (75, 78, 106), clinical trials in these multidimensional pathophysiologic conditions remain rare (119, 122). When complement intervention strategies are designed for the clinics,

#### REFERENCES

the targeted complement factor or activation product needs to be measured before the therapy can be applied. Especially when complex intensive care is necessary which can alter complement levels within a short time period, e.g., by infusion of blood products which contain highly variable concentrations of complement (activation) factors (123) or by extra-corporal circulation devices with large artificial surfaces which may deplete key complement components (124, 125), the exact status of complement activation needs to be determined. This would allow precise and timely intervention either by inhibiting or supporting the complement response after trauma or during sepsis in order to rebalance the immune response. Whereas, various highly effective and specific complement intervention strategies have been developed within the last two decades and are available now (10), in the context of the complex immune response after trauma, hemorrhagic shock and sepsis (9, 126), specific organ damage and function assessment including the immune function at bedside seems far beyond. Therefore, functional monitoring of the organ and immune systems can be considered as a prerequisite before complement interventions move into clinical routine in diseases with a complex pathophysiology. In conclusion, further scientific knowledge and translational efforts are demanded for targeting complement pathways in the setting of trauma, hemorrhagic shock and sepsis with the aim to offer causal therapy and improved outcome.

#### AUTHOR CONTRIBUTIONS

EK and MH-L wrote the manuscript. All authors edited and commented on the manuscript. All authors read and approved the final manuscript.

#### FUNDING

This review was supported by grants from the German Research Foundation (DFG) (EI 866/5-1) and by grants from the DFG within the Collaborative Research Center CRC1149/2 Danger Response, Disturbance Factors and Regenerative Potential after Acute Trauma to MH-L.

organ protection in a baboon model of Escherichia coli sepsis. Blood. (2010) 116:1002–10. doi: 10.1182/blood-2010-02-269746

of subarachnoid hemorrhage. Acta Neurochir Suppl. (2013) 118:157–61. doi: 10.1007/978-3-7091-1434-6\_29

paroxysmal nocturnal hemoglobinuria. Oncologist. (2008) 13:993–1000. doi: 10.1634/theoncologist.2008-0086

126. Hotchkiss RS, Moldawer LL, Opal SM, Reinhart K, Turnbull IR, Vincent JL. Sepsis and septic shock. Nat Rev Dis Primers. (2016) 2:16045. doi: 10.1038/nrdp.2016.45

**Conflict of Interest Statement:** JL is the founder of Amyndas Pharmaceuticals, which is developing complement inhibitors (including third-generation compstatin analogs, such as AMY-101) and is the inventor of patents or patent applications that describe the use of complement inhibitors for therapeutic purposes, some of which are developed by Amyndas Pharmaceuticals. JL is also the inventor of the compstatin technology licensed to Apellis Pharmaceuticals [i.e., 4(1MeW)7W/POT-4/APL-1 and PEGylated derivatives].

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Karasu, Nilsson, Köhl, Lambris and Huber-Lang. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Corrigendum: Targeting Complement Pathways in Polytrauma- and Sepsis-Induced Multiple-Organ Dysfunction

#### Ebru Karasu<sup>1</sup> , Bo Nilsson<sup>2</sup> , Jörg Köhl 3,4, John D. Lambris <sup>5</sup> and Markus Huber-Lang<sup>1</sup> \*

1 Institute for Clinical and Experimental Trauma-Immunology, University Hospital of Ulm, Ulm, Germany, <sup>2</sup> Department of Immunology, Genetics and Pathology (IGP), Laboratory C5:3, Uppsala University, Uppsala, Sweden, <sup>3</sup> Institute for Systemic Inflammation Research (ISEF), University of Lübeck, Lübeck, Germany, <sup>4</sup> Division of Immunobiology, Cincinnati Children's Hospital, Cincinnati, OH, United States, <sup>5</sup> Department of Pathology & Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, United States

Keywords: trauma, sepsis, hemorrhagic shock, MODS, complement activation, complement dysregulation, complement therapeutics, clinical trial

#### **A Corrigendum on**

#### Approved by:

Frontiers in Immunology Editorial Office, Frontiers Media SA, Switzerland

#### \*Correspondence:

Markus Huber-Lang<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology

Received: 16 April 2019 Accepted: 17 April 2019 Published: 03 May 2019

#### Citation:

Karasu E, Nilsson B, Köhl J, Lambris JD and Huber-Lang M (2019) Corrigendum: Targeting Complement Pathways in Polytrauma- and Sepsis-Induced Multiple-Organ Dysfunction. Front. Immunol. 10:994. doi: 10.3389/fimmu.2019.00994

#### **Targeting Complement Pathways in Polytrauma- and Sepsis-Induced Multiple-Organ Dysfunction**

by Karasu, E., Nilsson, B., Köhl, J., Lambris, J. D., and Huber-Lang, M. (2019). Front. Immunol. 10:543. doi: 10.3389/fimmu.2019.00543

In the original article, we neglected to include a conflict of interest statement of Prof. John D. Lambris.

JL is the founder of Amyndas Pharmaceuticals, which is developing complement inhibitors (including third-generation compstatin analogs, such as AMY-101) and is the inventor of patents or patent applications that describe the use of complement inhibitors for therapeutic purposes, some of which are developed by Amyndas Pharmaceuticals. JL is also the inventor of the compstatin technology licensed to Apellis Pharmaceuticals [i.e., 4(1MeW)7W/POT-4/APL-1 and PEGylated derivatives].

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.

Copyright © 2019 Karasu, Nilsson, Köhl, Lambris and Huber-Lang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Complement Therapeutics in Autoimmune Disease

#### Joshua M. Thurman\* and Roshini Yapa

*Department of Medicine, University of Colorado School of Medicine, Aurora, CO, United States*

Many autoimmune diseases are characterized by generation of autoantibodies that bind to host proteins or deposit within tissues as a component of immune complexes. The autoantibodies can activate the complement system, which can mediate tissue damage and trigger systemic inflammation. Complement inhibitory drugs may, therefore, be beneficial across a large number of different autoimmune diseases. Many new anti-complement drugs that target specific activation mechanisms or downstream activation fragments are in development. Based on the shared pathophysiology of autoimmune diseases, some of these complement inhibitory drugs may provide benefit across multiple different diseases. In some antibody-mediated autoimmune diseases, however, unique features of the autoantibodies, the target antigens, or the affected tissues may make it advantageous to block individual components or pathways of the complement system. This paper reviews the evidence that complement is involved in various autoimmune diseases, as well as the studies that have examined whether or not complement inhibitors are effective for treating these diseases.

#### Edited by:

*John D. Lambris, University of Pennsylvania, United States*

#### Reviewed by:

*Leendert Trouw, Leiden University Medical Center, Netherlands Lubka T. Roumenina, INSERM U1138 Centre de Recherche des Cordeliers, France*

#### \*Correspondence:

*Joshua M. Thurman<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology*

Received: *28 January 2019* Accepted: *12 March 2019* Published: *03 April 2019*

#### Citation:

*Thurman JM and Yapa R (2019) Complement Therapeutics in Autoimmune Disease. Front. Immunol. 10:672. doi: 10.3389/fimmu.2019.00672* Keywords: complement, autoimmunity, antibody, immune complex, therapeutic

# INTRODUCTION

Autoimmune diseases are conditions in which immunologic tolerance for specific self-proteins is lost, and the adaptive immune system orchestrates injury of organs expressing those proteins. The skin, joints, and kidneys are commonly affected, but any organ in the body can be the target of autoimmunity. Most autoimmune disorders are associated with autoantibodies that are reactive against self-proteins. Systemic lupus erythematosus (SLE), for example, is associated with autoantibodies to nuclear antigens as well as many other intracellular and extracellular antigens (1, 2). Myasthenia gravis is associated with autoantibodies specific for the acetylcholine receptor (AChR). Although not all of the identified autoantibodies have been established as causative of disease, in some cases experiments have convincingly shown that the autoantibodies replicate the disease when transferred into animals that express the target antigen. In membranous nephropathy, for example, there are reported cases in which passage of autoantibodies from mother to fetus have caused antenatal disease (3).

Autoimmunity can be regarded as having two components: (1) the loss of tolerance to self-antigens, and (2) downstream immune-mediated effector mechanisms of injury. Autoantibodies may contribute to disease pathogenesis via several effector mechanisms. They can crosslink target antigens or activate receptors directly on cell surfaces. In Grave's disease, for example, autoantibodies to the thyrotropin receptor trigger release of thyroid hormone, and in myasthenia gravis antibodies to the AChR can cross-link the receptor (4, 5). The inflammatory effects of autoantibodies can also be mediated through ligation of Fc receptors on leukocytes. In

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some models of autoimmunity this accounts for nearly all of the downstream tissue injury (6). Autoantibodies can also directly damage target tissues, and complement activation is a downstream mediator of this injury in some diseases.

Antibodies bound to cell surface antigens activate complement on the target cells, usually through activation of the classical pathway. Immune-complexes (ICs) can also deposit in small capillaries where they activate complement on bystander cells. Once activated, the complement cascade generates multiple different biologically active fragments. C3a and C5a contribute to chemoattraction and activation of leukocytes (7). C3 fragments (C3b, iC3b, C3dg) fixed to host tissues can activate leukocytes through ligation of complement receptors (CRs) 1–4 (8, 9). C5b-9 causes direct cell activation and cytotoxicity. Although most C5b-9 forms directly on cell membranes, it can also insert into nearby cells causing "bystander" injury (10). Thus, once the complement cascade is activated within a tissue it can trigger multiple local and systemic effects.

Given the prevalence of autoantibodies in the various autoimmune and rheumatic diseases, complement inhibition holds promise as an effective strategy for blocking multiple pathways of injury common to these diseases. In many of the diseases there is also pre-clinical or biomarker evidence to support the use of anti-complement therapeutics. Therapeutic complement inhibitors may be effective at blocking direct tissue injury by the complement cascade. Consequently, they may have a role in rapidly reducing tissue inflammation while other immunosuppressive drugs to block the adaptive immune response. Complement inhibitors may also reduce the adaptive immune response by decreasing stimulation of dendritic cells, T cells, and B cells via the complement receptors (11). This class of drugs could therefore be effective in patients with acute disease flares, but also for chronic treatment of these diseases.

A large number of anti-complement drugs are in development, providing tools for blocking all complement activity, specific activation pathways, or isolated complement fragments (12, 13). Intuitively, drugs that block the classical pathway should be beneficial in antibody-mediated diseases. The classical pathway also helps to prevent autoimmunity and to solubilize ICs, however, so drugs that block downstream targets within the complement cascade while leaving the early classical pathway intact may be preferable in some diseases (discussed below). Drugs that block specific complement fragments, such as C5a receptor (C5aR) antagonists, may also have fewer side effects than drugs that more completely block the complement system. It is also noteworthy that complement activation is not always an important component of disease pathogenesis, even in models in which ICs and complement are deposited within tissues. In some models this is due to the effects of other pathways, such as signaling through Fc receptors (6). In other instances this may be due to intrinsic differences in susceptibility or resistance of target tissues to injury (14). Given these considerations, a benefit to complement inhibition cannot simply be inferred from detection of autoantibodies or complement activation. The efficacy of complement inhibition in autoimmune diseases needs to be studied on a case-by-case basis.

# SYSTEMIC AUTOIMMUNE DISEASES

# Systemic Lupus Erythematosus

SLE is the paradigmatic autoimmune disease. It is a complex disorder characterized by formation of autoantibodies to multiple different nuclear antigens, including DNA, histones, and ribonucleoproteins. There is also evidence that autoantibodies react with antigens expressed in specific tissues, including alphaenolase, annexin A1, and annexin A2 (15, 16). SLE frequently affects the joints, skin, and kidneys, but essentially any organ can be affected. Organ involvement is usually associated with the deposition of ICs and complement activation. Other downstream mediators of immunologic injury have been identified, however, including cytokines and chemokines, Fc receptor ligation, cellular infiltrates, and toll-like receptor activation (6, 17).

#### The Role of Complement in SLE

The complement system plays a paradoxical role in the pathogenesis of SLE—it seems to lower the risk of developing SLE while also mediating end organ injury in the disease (**Figure 1**). Congenital deficiency of classical pathway proteins are strong risk factors for developing SLE, presumably because they help protect against autoimmunity. Individuals with deficiencies of C1q, C1s, C1r, C2, and C4, for example, are all at increased risk of the disease (18).

Classical pathway proteins may be protective against SLE though several mechanisms. C1q and C4 opsonize apoptotic cell debris and ICs, and facilitate their clearance [**Figure 2**, and (19)]. C1q binds directly to molecules displayed on the surface of apoptotic bodies, including DNA and phosphatidylserine (20, 21). Mice with targeted deletion of the C1qa gene have a greater abundance of apoptotic bodies in the kidney than control mice, and they develop auto-antibodies to nuclear antigens (22). C1q deficient mice on a pure C57BL/6 background do not develop disease, however, demonstrating that other background factors are also important (23). A recent study also demonstrated that C1q can directly modulate CD8 T cell metabolism via receptors on the cell surface, reducing the response of the cells to self-antigens (24). In this study, C1q deficiency increased renal pathology in a CD8-dependent fashion. Work in animals demonstrates that C4 helps to maintain immune tolerance to selfantigens (25). An intact classical pathway, therefore, may reduce the abundance of nuclear proteins in the extracellular space and also suppress the immune response to these proteins.

Although the classical pathway helps to prevent development of SLE, the complement system is also an important mediator of tissue injury. Complement proteins are seen in kidney biopsies from patients with lupus nephritis and co-localize with deposited ICs (26). Glomerular complement deposits are associated with a worse prognosis (27), suggesting that complement activation contributes to tissue injury. Work in mouse models of lupuslike disease demonstrate that deficiency of classical pathway proteins is not protective [consistent with a protective role for these proteins (28)], whereas deficiency of alternative pathway proteins is protective (29, 30). Several therapies have been shown effective in preclinical studies using models of lupus-like disease, including agents that block the alternative pathway (31, 32),

FIGURE 1 | The complement system has both pathogenic and protective roles in systemic lupus erythematosus. Complement proteins have multiple functions that affect lupus, both positively and negatively. The classical pathway may help facilitate the removal of apoptotic and damaged cells as well as immune complexes, reducing the risk of developing autoimmunity to nuclear components. C1q may also directly reduce CD8 T cell activation, thereby attenuating downstream immunity and autoantibody generation. Once activated, however, the complement system can promote tissue injury and disease severity. The complement system can directly cause cytotoxic injury in tissues, and also increases inflammation by attracting leukocytes and inducing interferon production by dendritic cells. Complement fragments may also increase T cell and B cell responsiveness, increasing the response to autoantigens.

activation at the level of C3 (33), C3a activity (34), and C5a activity (35). Furthermore, treatment with an inhibitory antibody to mouse C5 for 6 months improved renal function and survival in a mouse model of disease (36).

Based on the complex role of complement in SLE, a clinical trial of a complement inhibitory drugs in SLE patients must be designed with equipoise. The treatment should probably not block the classical pathway upstream of C4, even though this is an IC-mediated disease characterized by classical pathway activation.

#### Therapeutic Complement Inhibitors in SLE

A clinical trial of eculizumab in patients with lupus nephritis was planned, but was canceled after enrolling only one patient (37). Eculizumab has been used off-label in patients with proliferative lupus nephritis, and it may be effective in patients with severe disease (38, 39). Several papers have also reported use of eculizumab in SLE patients with thrombotic microangiopathy (40, 41). Apellis is a company that is developing an agent (APL-2) that blocks complement activation at the level of C3. The company is currently conducting a phase 2 trial of the drug that will include patients with lupus nephritis.

# Anti-neutrophil Cytoplasmic Antibody Vasculitis

Anti-neutrophil cytoplasmic antibody (ANCA) vasculitis comprises a group of severe diseases that can affect many organs, including the lungs and upper airways, kidneys, skin, and joints. ANCA associated vasculitis (AAV) is often referred to as "pauciimmune" because immunoglobulin and complement proteins are not prominently seen in tissue biopsies. These diseases are antibody-mediated, however, and the ANCA can cause disease when injected into rodents (42). Antibodies and complement fragments probably function on the surface of neutrophils, and thus are not abundantly deposited within affected tissues. That said, complement fragments are frequently present in tissues biopsies, even if they are less abundant than in IC deposition diseases such as SLE (43).

#### The Role of Complement in ANCA Vasculitis

The important role of the complement system in AAV was revealed by an elegant series of experiments using a murine model of the disease. Using mice with various complement deficiencies, investigators found that factor B deficiency was protective but that C4 deficiency was not (44). Therefore, even though this an antibody-mediated disease, the alternative pathway appears to be more important than the classical pathway for disease pathogenesis. C5 deficiency is also protective in this model but C6 deficiency was not, suggesting that C5a is more important than C5b-9 formation in disease pathogenesis. Although referred to as "pauci-immune," there is clinical evidence of complement activation in human patients with AAV. Glomerular C3 deposits can be seen in more than 40% of patients (45). Complement activation fragments are also elevated in plasma and urine of patients with AAV, including the fragment Bb [an alternative pathway activation fragment (46)].

#### Therapeutic Complement Inhibitors in ANCA Vasculitis

In the murine model of AAV, a monoclonal antibody to C5 (BB5.1) and an orally delivered small molecule C5a receptor antagonist (CCX168) were both nearly completely protective (47, 48). These results demonstrated that that C5a blockade is sufficient for blocking the complement effects in AAV. A subsequent phase 2 study of CCX168 (conducted by the company ChemoCentryx) tested whether the drug could be used in lieu of corticosteroids (49). This study included a group of patients that received cyclophosphamide and CCX168 without corticosteroids. That treatment approach was noninferior when compared to standard therapy (corticosteroids + cyclophosphamide). A phase 3 study is currently underway evaluating the efficacy of the drug in combination with rituximab or cyclophosphamide. The study will compare CCX168 to prednisone in combination with the same other immunosuppressive agents.

# Antiphospholipid Antibody Syndrome

Anti-phospholipid antibodies (APLA) are autoantibodies that bind to endothelial cells, triggering thrombosis. The antiphospholipid syndrome (APS) is diagnosed by detection of these antibodies in patients who have had a thromboembolic event (50). The antibodies associated with APS can be reactive to cardiolipin, β2-glycoprotein 1, or they can be detected as "lupus anticoagulants." The lupus anticoagulant assay detects the antibodies indirectly, through the interference of clotting on phospholipid surfaces in vitro.

#### The Role of Complement in APS

Clinically, APS is associated with thrombosis or fetal loss. In a passive transfer model in rats, anti-β2 glycoprotein I antibodies caused thrombosis in rats only if they had previously been treated with lipopolysaccharide (51). Complement deficiency or inhibition prevented thrombosis in this model. A murine model of fetal loss caused by passive transfer of antiphospholipid antibodies also demonstrated a role for complement activation in the disease (52). The disease requires an intact alternative pathway, and treatment of mice with an inhibitory antibody to factor B protected them from fetal loss (53). Use of a C5a receptor antagonist was also protective in this model (54). There is evidence of complement consumption in patients with APS. Plasma C3 and C4 levels are lower than in healthy and disease-control subjects, and complement activation fragments are elevated (55). Detailed examination of a patient with APS has also demonstrated elevated levels of circulating complement fragments and also complement fragments deposited within an area of arterial thrombosis (56).

#### Therapeutic Complement Inhibitors in APS

The standard therapy for APS is anticoagulation. Catastrophic APS (CAPS) is a syndrome in which patients have thrombosis of multiple organs, and eculizumab has been used in patients with disease refractory to anticoagulation or conventional immunosuppression (57). There are also published case series of CAPS patients who were treated with eculizumab to prevent disease recurrence in renal transplants (58). An open-label phase 3 trial of eculizumab for preventing recurrence of CAPS in renal transplant patients was started, although it is apparently not recruiting patients currently.

# ORGAN SPECIFIC AUTOIMMUNE DISEASES

#### Membranous Nephropathy

Membranous nephropathy (MN) is a form of kidney disease in which ICs deposit in the glomerular capillary wall between the podocyte and the basement membrane ("subepithelial ICs"). The podocyte is a specialized cell that sits on the urine side of the glomerular capillary, and it provides a barrier to leakage of cells and protein into the urine. The subepithelial ICs cause podocyte injury, thereby disrupting the filtration barrier. This leads to the leakage of protein into the urine, and over time it can cause irreversible glomerular damage and a loss of kidney function.

In most cases of MN, autoantibodies bind to proteins expressed on the podocyte surface (referred to as primary MN). In some settings, however, antigens from elsewhere in the body become trapped under the podocyte. Antibodies can then bind the planted antigens in situ (referred to as secondary MN). Primary MN is an autoimmune disease in which the antibodies bind to self-proteins expressed by the podocyte, and several different target podocyte antigens have been identified. The first podocyte antigen identified in MN was neutral endopeptidase (NEP) (3). Mothers who are genetically deficient in NEP can be exposed to the protein during pregnancy, eliciting an immune response. During subsequent pregnancies, the maternal anti-NEP antibodies are transferred to the infant, leading to transient MN in the baby.

The most common target antigen in primary MN is the M-type phospholipase A2 receptor (PLA2R), a transmembrane protein expressed by the podocyte (59). Antibodies to PLA2R are seen in 70–80% of cases of MN. Approximately 5% of patients with primary MN have autoantibodies against thrombospondin type-1 domain-containing 7A (THSD7A) (60). It is not known why immune tolerance to these podocyte proteins is lost in patients with MN, but the disease is associated with certain HLA alleles (61), and the autoantibodies bind to specific epitopes on the target proteins (62, 63).

Autoantibodies to several intracellular proteins have also been found, including antibodies reactive with alpha enolase, aldose reductase, and manganese superoxide dismutase (SOD2) (64, 65). Initial injury to the podocyte may be required to cause release of these proteins into the extracellular space. Secondary MN is associated with numerous different systemic diseases, including SLE, several types of cancer, and various infections. Cancerassociated antigens and infection-associated antigens have been eluted from the glomerular deposits in cases of secondary MN, suggesting that these proteins reach the kidney via the circulation and become trapped under the podocytes. Antibodies then bind to the target antigens, and presumably injure the podocyte through the same inflammatory mechanisms as in primary disease.

#### The Role of Complement in MN

The subepithelial ICs in MN activate complement either directly on the surface of podocytes or immediately adjacent to the cells, and C3 is detected in most biopsies. The C3a and C5a generated by complement activation at this location likely pass directly into the urine, explaining why neutrophils are not usually seen in kidney biopsies (**Figure 3**). Similarly, histologic hallmarks of inflammation are not usually seen in biopsies. C5b-9 that is formed on the podocyte, on the other hand, can directly injure the cell. Early studies using animal models of MN indicated that it is a complement dependent disease (66). Furthermore, animals deficient in C6 are protected from injury, further implicating C5b-9 in podocyte injury and the pathogenesis of the disease (67, 68).

Interestingly, in primary MN the autoantibodies are predominantly of the IgG4 subclass, which does not activate the classical pathway of complement (69). Furthermore, C1q is

not detected in most biopsies, whereas C4 is usually detected. These observations suggest that the autoantibodies may activate complement through the mannose binding lectin (MBL) pathway. Investigators have proposed that the carbohydrate side chains of IgG4 may bind to MBL and activate the lectin pathway (70). There are, however, reports of patients with congenital MBL deficiency who have developed MN (71). These patients had C3 deposits in the absence of C1q or C4 deposits, indicating that activation can occur solely through the alternative pathway and that the MBL pathway is not always required. Further complicating things, some IgG1 and IgG3 autoantibodies are detected in the circulation and in the glomerular deposits in primary MN (59, 72). IgG1, IgG2, and IgG3 are also usually seen in biopsies from patients with secondary MN (73, 74). Therefore, although complement is activated in both primary and secondary MN, the mechanisms may vary from patient to patient.

why leukocytes do not typically infiltrate the glomeruli in this disease.

#### Therapeutic Complement Inhibitors in MN

Based on the pre-clinical evidence that C5b-9 formation is important to the pathogenesis of MN, a clinical trial was conducted in which 122 MN patients were treated with eculizumab or placebo for 16 weeks (75). Treatment with eculizumab was not associated with a significant reduction in proteinuria, indicating that complement inhibition may not be of benefit in the disease. It is possible, though, that the lack of benefit was due to the short duration of treatment. It is also noteworthy that the doses of eculizumab used in the study may not have completely blocked terminal complement activation (76). Many new complement inhibitors are currently in development (13, 77), and several ongoing clinical trials are including patients with MN. Omeros has developed an inhibitory antibody to MASP2 (OMS721). OMS721 and APL-2 (a C3 activation inhibitor) are currently being tested in Phase 2 studies that include patients with MN (13).

# Myasthenia Gravis

Myasthenia gravis (MG) is an autoimmune disease caused by antibodies to proteins expressed at the neuromuscular junction, most commonly the AChR (78). The antibodies can block the function of the receptor. Complement activation on the muscle membrane surface also decreases expression of AChRs on the muscle cell. This results in weakness of skeletal muscles, and almost always includes eye muscles (79).

#### The Role of Complement in MG

Immunohistologic examination of the post-synaptic membranes from patients with MG demonstrates deposition of immunoglobulin and C3 in nearly all cases (80). C5b-9 has also been detected at this location (81). C5 deficiency was protective in a passive model of the disease in rats. In a mouse model of MG, C4 and C5 deficiency were both similarly protective, indicating that disease is caused through activation of the classical pathway by pathologic antibodies and is mediated by C5a or C5b-9 (82, 83). Conversely, deficiency of CD55 or CD59 exacerbates disease, indicating a role for these complement regulators in attenuating injury (84).

Several therapeutic complement inhibitors have shown efficacy in pre-clinical models. A soluble CR1 construct was protective in the rat models (85), and complement blockade with monoclonal antibodies to C5 or C6 has also proven effective in rodent models (86, 87).

#### Therapeutic Complement Inhibitors in MG

Based on the promising pre-clinical data, eculizumab has studied in several clinical trials of patients with relapsing or refractory MG. A phase 2 placebo controlled study showed that treatment with the drug was associated with improved muscle strength (88). This was followed by a randomized, double-blind, phase 3 study (REGAIN) (89). In this study, 126 patients were randomized to eculizumab or placebo for 26 weeks. The primary endpoint for the study was the Myasthenia Gravis-Activities of Daily Living (MG-ADL) score. This endpoint was not significantly different in the eculizumab group when evaluated by worstrank ANCOVA. There was a significant improvement when prespecified secondary endpoints were evaluated, however. Based on these clinical studies, eculizumab has been approved in the United States, Europe, and in Japan for treatment of MG (90). As more MG patients are treated with complement inhibitors the benefits and limitations of this approach will become clearer.

#### Neuromyelitis Optica

Neuromyelitis optica (NMO) is an autoimmune disease of the nervous system caused by autoantibodies, usually to aquaporin 4 expressed on astrocytes (91). Antibody deposition on the cells leads to demyelination. NMO primarily affects the optic nerve and spinal cord, but it can also affect the cerebral cortex. Immunosuppressive drugs are beneficial in some cases, and therapeutic plasma exchange ameliorates disease by removing the pathogenic autoantibodies.

#### The Role of Complement in NMO

Complement proteins, including C1q, C3, C4, and C5b-9, are deposited in nearly all cases of active NMO (92, 93). In animal models, the injection of AQP4-IgG alone is insufficient to initiate demyelination, but intracerebral co-injection of the antibodies with human complement sufficient serum induces lesions with the seminal histopathologic features of NMO (94). In the absence of complement or in the presence of complement inhibition, on the other hand, there is no evidence astrocyte or oligodendrocyte loss. In a rat model, peripherally administered AQP4-IgG caused NMO-like lesions in rats that received an intracerebral needle injury, to permit passage of the antibodies into the central nervous system (95). Injury was prevented, however, by depletion of complement with cobra venom factor (95). Similarly, in mice, it has been shown that lesions induced by injection of anti AQP4 autoantibodies require complement activation by the immunoglobulin (96).

#### Therapeutic Complement Inhibitors in NMO

Eculizumab successfully reduced the number of disease flares in an open label study that included 14 patients with NMO who had previously had relapsing disease (97). Patients were treated with the drug for 1 year. During that period 12 of the patients did not have any disease flares, and the other two patients had only single, minor flares. A phase 3 randomized controlled double-blind trial to test the efficacy of eculizumab in patients with relapsing disease (PREVENT study) recently completed its recruitment phase. This study enrolled 143 seropositive patients, who had had active disease within the previous 2 years.

# CONCLUSIONS

The complement system is activated in almost all antibodymediated autoimmune diseases. Therefore, drugs that block complement activation may block downstream mediators of injury that are common to most, if not all, of these diseases. The complement cascade plays a central role in the immune response. Complement inhibition may rapidly reduce tissue inflammation and damage, and it may also attenuate T cell and B cell activation, thereby reducing autoimmunity (11). Most ongoing trials are using complement inhibitors to block acute tissue inflammation. As these studies are analyzed, however, it will be interesting to see whether an effect on autoantibody titers can also be detected.

The complement system is activated by antibodies in the autoimmune diseases discussed above, and activation usually occurs through the classical pathway. In some of these diseases, such as MN, activation may occur through the alternative or lectin pathways. Furthermore, the classical pathway may help to eliminate nuclear antigens in diseases such as lupus. Thus, there is a rationale to develop and test inhibitors that can specifically block these other pathways or specific downstream complement activation fragments, such as C5a. Many new complement inhibitory drugs are currently in development (12, 13). As these drugs enter the clinic, it will be fascinating to test whether the benefits of complement inhibition are similar among the various autoimmune diseases, or whether disease specific approaches will be needed.

# AUTHOR CONTRIBUTIONS

JT contributed to the conception, writing, and editing of this manuscript. RY contributed to the conception and writing of this manuscript.

#### REFERENCES

# FUNDING

JT has received support from National Institutes of Health Grants R01DK076690 and R01DK113586, and RY received support from T32 DK007135.

in human systemic autoimmune diseases. Front Immunol. (2016) 7:36. doi: 10.3389/fimmu.2016.00036

with a specific receptor antagonist. Eur J Immunol. (2005) 35:2496–506. doi: 10.1002/eji.200526327

**Conflict of Interest Statement:** JT receives royalties from Alexion Pharmaceuticals, Inc. JT is also a consultant for AdMIRx, Inc., a company developing complement inhibitors. He holds stocks and will receive royalty income from AdMIRx.

The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Thurman and Yapa. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Complementing the Cancer-Immunity Cycle

#### Ruben Pio1,2,3 \*, Daniel Ajona1,2,3, Sergio Ortiz-Espinosa<sup>1</sup> , Alberto Mantovani 4,5 and John D. Lambris <sup>6</sup>

<sup>1</sup> Program in Solid Tumors (CIMA) and Department of Biochemistry and Genetics (School of Medicine), University of Navarra, Pamplona, Spain, <sup>2</sup> Navarra Institute for Health Research (IDISNA), Pamplona, Spain, <sup>3</sup> Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid, Spain, <sup>4</sup> Humanitas Clinical and Research Center, Humanitas University, Milan, Italy, <sup>5</sup> William Harvey Research Institute, Queen Mary University of London, London, United Kingdom, <sup>6</sup> Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, United States

Reactivation of cytotoxic CD8<sup>+</sup> T-cell responses has set a new direction for cancer immunotherapy. Neutralizing antibodies targeting immune checkpoint programmed cell death protein 1 (PD-1) or its ligand (PD-L1) have been particularly successful for tumor types with limited therapeutic options such as melanoma and lung cancer. However, reactivation of T cells is only one step toward tumor elimination, and a substantial fraction of patients fails to respond to these therapies. In this context, combination therapies targeting more than one of the steps of the cancer-immune cycle may provide significant benefits. To find the best combinations, it is of upmost importance to understand the interplay between cancer cells and all the components of the immune response. This review focuses on the elements of the complement system that come into play in the cancer-immunity cycle. The complement system, an essential part of innate immunity, has emerged as a major regulator of cancer immunity. Complement effectors such as C1q, anaphylatoxins C3a and C5a, and their receptors C3aR and C5aR1, have been associated with tolerogenic cell death and inhibition of antitumor T-cell responses through the recruitment and/or activation of immunosuppressive cell subpopulations such as myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), or M2 tumor-associated macrophages (TAMs). Evidence is provided to support the idea that complement blocks many of the effector routes associated with the cancer-immunity cycle, providing the rationale for new therapeutic combinations aimed to enhance the antitumor efficacy of anti-PD-1/PD-L1 checkpoint inhibitors.

Keywords: cancer immunity, immunotherapy, complement system, C3a, C5a, C1q, PD-1, PD-L1

# INTRODUCTION

Profound advances in our understanding of the interactions between tumors and the immune system have allowed the development of therapeutic approaches that boost the body's natural defenses against cancer. These therapies are aimed to mount effective antitumor immune responses and include immunomodulators, vaccines, and adoptive transfer of immune cells (1). Some of the most clinically effective immunotherapies to date target the programmed cell death protein 1 (PD-1) immune checkpoint. PD-1 is expressed by T cells during priming or expansion and binds to one of its two ligands PD-L1 or PD-L2 (2). Tumor cells upregulate PD-L1 in response to cytokines

Edited by: Carlo Pucillo, University of Udine, Italy

#### Reviewed by:

Martin V. Kolev, GlaxoSmithKline, United Kingdom Peter Monk, University of Sheffield, United Kingdom Viktor Umansky, German Cancer Research Center (DKFZ), Germany

> \*Correspondence: Ruben Pio<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology

Received: 01 February 2019 Accepted: 25 March 2019 Published: 12 April 2019

#### Citation:

Pio R, Ajona D, Ortiz-Espinosa S, Mantovani A and Lambris JD (2019) Complementing the Cancer-Immunity Cycle. Front. Immunol. 10:774. doi: 10.3389/fimmu.2019.00774 such as interferon (IFN)-γ (3). Interaction of PD-L1 with PD-1 on T cells causes T-cell apoptosis, anergy, and exhaustion, protecting tumor cells from CD8<sup>+</sup> T cell-mediated cytolysis (3). PD-L1 can also deliver intrinsic intracellular signals that enhance cancer cell survival, regulate stress responses, and confer resistance toward apoptotic stimuli (4, 5). PD-1 and other checkpoints are also expressed by NK cells which may contribute to the antitumor activity of some therapeutic strategies under development (6, 7).

Neutralizing monoclonal antibodies against PD-1 or PD-L1 have transformed the therapeutic landscape of a wide range of cancers (**Table 1**), being particularly successful for tumors with limited therapeutic options such as melanoma or lung cancer (3). Notably, these antibodies generate durable responses without causing serious side effects. However, a significant fraction of patients manifests innate or acquired resistance to these therapies. Immune escape mechanisms stem from different cell interactions within the tumor microenvironment, and emphasize the need of developing rational combination strategies to obtain more potent anticancer responses (8).

In 2013, Daniel Chen and Ira Mellman described a series of self-sustaining stepwise events, referred as the cancer-immunity cycle, by which the anti-cancer immune responses lead to an effective elimination of cancer cells (9). The existence of negative feedback mechanisms developed by tumors hinders this cycle of

\*For patients with mismatch repair deficiency (dMMR) or microsatellite instability high (MSI-H).

cancer immunity and may pose a barrier to the development of effective clinical responses. Anticancer immunotherapies should be aimed to reactivate all the steps of the cycle, which include immunogenic cell death, maturation of antigen-presenting cells, T-cell priming and activation, promotion of immune infiltration, blockade of immunosuppression, and enhancement of effector T-cell activity. In this context, combination therapies would provide synergistic effects for the maintenance of the cancerimmunity cycle.

 We, and others, have actively contributed to this field, leading to the proposal that modulation of complement activation can improve the antitumor efficacy of inhibitors targeting the PD-1/PD-L1 pathway. In 2008, making a paradigm shift in tumor immunology, we demonstrated that complement activation, followed by C5a signaling, has a tumor-promoting role in cancer (11). In 2012, using a lung cancer model, we first demonstrated an association between the inhibition of C5a receptor 1 (C5aR1) and the expression of PD-L1 within the tumor microenvironment (12). These results suggested the possibility of blocking complement factors to increase the efficacy of other immune therapeutic strategies (12). Following this line, we demonstrated that inhibition of PD-1/PD-L1 synergizes with the inhibition of C5a/C5aR1 in various preclinical models of lung cancer (13). This rationale provided the basis for a clinical trial in which the anti-C5aR1 antibody IPH5401 is being evaluated in combination with the anti-PD-L1 antibody durvalumab in patients with solid tumors (STELLAR-001).

In this review we describe the participation of complement elements in the steps of the cancer-immunity cycle. We propose that a combinational therapy using anti-PD-1/PD-L1 antibodies together with modulators of the complement system may open new therapeutic opportunities for tumors resistant to PD-1/PD-L1 blockade.

### THE CANCER-IMMUNITY CYCLE

The cancer-immunity cycle is defined as a series of functional stepwise events needed to obtain an efficient control of cancer growth by the immune system (9). The process is initiated by the release of neo-antigens generated as a result of genomic instability. Cancer-associated antigens are captured by dendritic cells which, upon migration to lymph nodes, prime and activate tumor-specific cytolytic CD8<sup>+</sup> T cells. These effector cells migrate and infiltrate the tumor stroma, where potentially are able to recognize and eliminate cancer cells. T cell-mediated cytotoxic responses release new tumor antigens, fueling the cancer-immunity cycle. Interestingly, this model provides the rationale for targeting different steps of the cycle in order to maintain its functionality. An effective cancer immunotherapy should be designed based on the specific resistance mechanisms underlining the rate-limiting steps in each particular patient (14, 15). In the case of anti-PD-1/PD-L1 therapies, a variety of biological factors contribute to treatment resistance, including lack of cancer antigens recognizable by T cells, impaired cancer-antigen presentation, impaired activation of cancer-specific T cells, poor infiltration of T cells into tumors, and accumulation of immunosuppressive factors and cells in the tumor microenvironment (15). The evading strategies present in a given tumor would determine whether this tumor shows an inflamed or a noninflamed phenotype (16). Clinical evidence suggests that anti-PD-1/PD-L1 inhibitors are most effective in inflamed tumors characterized by high tumor PD-L1 expression, CD8<sup>+</sup> T-cell infiltration or mutational burden (17–20). Jerby-Arnon et al. recently analyzed the relationship between malignant cell states and CD8<sup>+</sup> T-cell infiltration and identified a T-cell exclusion program that predicts responses to PD-1/PD-L1 blockade (21). This program was enriched for genes involved in predictable processes, such as antigen processing and presentation, IFN-γ signaling, and immune modulation; but also identified genes associated with activation and modulation of the complement system (21). Elements of the complement cascade are also present in a signature of serum proteins that predicts survival in patients receiving PD-1 blocking antibodies, suggesting that complement activation may inhibit the efficacy of adaptive antitumor immunity (22).

# THE COMPLEMENT SYSTEM

The complement system, a central element of innate immunity, represents a first line of defense against unwanted non-self and host elements, and orchestrates many immunological and inflammatory processes that substantially contribute to body homeostasis (23, 24). Complement activities are mediated by more than 50 circulating, cell surface-bound and intracellular proteins. There are three main mechanisms of complement activation, known as classical, lectin, and alternative pathways. The classical pathway is commonly initiated by the binding of C1q to complement-fixing antibodies (mostly IgM and IgG types); although C1q can also recognize non-immunoglobulin ligands such as C-reactive protein (CRP), pentraxin 3 (PTX3), or apoptotic cells (25). The lectin pathway is activated by homologous proteins to C1q (mannose-binding lectin, collectins, and ficolins) that recognize repetitive carbohydrate patterns (26). Lastly, the alternative pathway is initiated by spontaneous cleavage of C3 on activating surfaces (27). Although the three complement pathways differ in their mechanisms of target recognition, in all cases, initiation of the complement cascade leads to the formation of C3 convertases and the activation of the central component C3. After this activation, C5 convertases are formed, C5 is cleaved, and the assembly of the pore-like membrane attack complex (MAC) is initiated. The enzymatic cleavage of complement elements leads to the release of proteolytic fragments such as C3a and C5a, and the deposition of other fragments such as C3b and iC3b. These molecules modulate a diverse set of processes (23), including the initiation and regulation of effector Tcell responses (28). Prevention of inappropriate activation by complement regulators takes place at three main levels: inhibition of protease activities in the activation cascade, decay and destruction of convertases, and control of MAC formation (29). Finally, recent experimental and clinical evidences suggest that intracellular complement components have important roles in cell physiology (30).

Complement has been traditionally regarded as playing a role in the elimination of tumor cells. Accordingly, an effective control of tumor growth may be achieved by complementfixing antibodies (31). However, growing evidence, starting for the initial observation in a model of cervical cancer (11), strongly supports a tumor-promoting role of complement in several tumor types. This topic has been extensively reviewed elsewhere (10, 32–35). Briefly, complement establishes an immunosuppressive microenvironment, promotes angiogenesis, sustains cellular proliferation, and participates in tumor cell invasion and migration. In light of the various contributions of complement to cancer progression, it is not surprising that expression of complement effectors and receptors is associated with disease progression and poor prognosis (36–42). Among all the complement elements with potential pro-cancer activities, C1q, C3-derived fragments, and C5a are recognized as major modulators of tumor progression (43–45).

# COMPLEMENT IN THE REGULATION OF THE CANCER-IMMUNITY CYCLE

In this section we will discuss the potential implication of effectors and regulators of the complement system in the steps of the cancer-immunity cycle. In light of the breadth and complexity of the immune response, we will focus our review on the specific aspects of the regulation of CD8<sup>+</sup> cytotoxic T cells, which are in large part the mediators of anti-PD-1/PD-L1 therapies. We will also examine evidence supporting the participation of complement in the regulation of the type 1 T helper (Th1) response, as it has a profound influence on the quality and extension of cytotoxic T-cell responses (46). Recent reviews have extensively addressed other complement-mediated immune functions not covered in the present review (10, 28, 47–50).

# Modulation of the Initiation of T-Cell Immunity by the Complement System

The cancer-immunity cycle is initiated by tumor-specific neoantigens generated by somatic mutations (51). Dying cancer cells release these antigens to the tumor microenvironment, where are captured and processed by dendritic cells, the principal cell type responsible for instructing naïve T cells to undergo antigenspecific effector functions. Depending on the stimuli provided by dying cancer cells, their interaction with dendritic cells can have immunogenic or tolerogenic consequences (52). The generation of an immunogenic or a tolerogenic cell death is mainly regulated by damage-associated molecular patterns (DAMPs). DAMPs are endogenous co-stimulatory signals secreted or presented on the cell surface of dying cells that interact with pattern-recognition receptors (PRRs) alerting the host of danger. Complement is required for efficient sensing of DAMPs (53–55). The specific interactions of danger sensors with complement elements allow to differentiate between physiological and pathological danger, shaping the maturation of dendritic cells (23). This activity depends mainly on the classical complement pathway. Direct binding of complement C1q to apoptotic cells promotes a phagocytic-mediated uptake of dying cells, which sustains an anti-inflammatory innate immune response through the expression of cytokines such as transforming growth factor (TGF)-β (56). In fact, genetic deficiencies in C1q, as well as other elements of the classical complement pathway, can compromise the induction of self-tolerance and result in systemic autoimmune diseases (57, 58). Another complement element involved in the recognition of danger signals is factor H, a soluble complement inhibitor produced and secreted by cancer cells (59, 60). Upon opsonization of apoptotic cells, factor H induces an anti-inflammatory cytokine profile (61, 62) and a tolerogenic stage (63). CD46, a membrane-bound complement regulatory protein able to interact with C3 activation fragments and found at high levels in some cancer types (64, 65), has also been proposed as a negative regulator of immune recognition (66). Complement proteins are easily detectable in various types of cancer, consistent with complement activation by these tumors (32). Therefore, upregulation of complement components in the surface of dying cancer cells may be associated with a tolerogenic cell death, in contrast to the immunogenic cell death required for an effective anticancer immune response (67, 68).

# Modulation of Priming and Activation of T Cells by the Complement System

Progress of the cancer-immunity cycle requires the presence of activation signals that allow dendritic cells to mature, migrate to the lymph nodes, and present the neo-antigens to naïve T cells. Efficient priming also relies on the contextual information provided by the microenvironment. Mature dendritic cells in the presence of suitable signals are able to induce T-cell effector functions; whereas in the absence of appropriate conditions, antigen presentation leads to T-cell anergy or generation of regulatory T cells (Tregs) that suppress effector responses.

Locally-produced complement elements determine the state of dendritic cell activation (69), and are critical in the regulation of T-cell responses (28). Production of C1q and C3 by dendritic cells induces their maturation and their capacity to stimulate Th1-cell responses (70, 71). C3 may also facilitate intracellular antigen processing and presentation (72). In agreement with these observations, optimal priming and expansion of CD4<sup>+</sup> and CD8<sup>+</sup> T cells in infection models is dampened by C3 deficiency (73, 74), and the complement fragment C3d amplifies antitumor T-cell responses (75). In the case of C3a and C5a, through activation of their respective receptors C3aR and C5aR1, these anaphylatoxins enhance the capacity of human monocytederived dendritic cells to stimulate T cells (76). In accordance, C3aR pathway inhibition in dendritic cells results in defective T-cell priming, associated with a reduced surface expression of major histocompatibility complex (MHC) and costimulatory molecules (77). Finally, downregulation of the expression of the complement regulator CD55 in antigen-presenting cells during T-cell activation increases the local production of C3a and C5a, providing costimulatory signals to induce T-cell proliferation and differentiation (78, 79).

Complement elements can also exert a direct influence on T cells. Activation of human CD4<sup>+</sup> T cells by CD46 stimulates the effector potential of Th1 cells (80–82). As on dendritic cells, paracrine and autocrine interactions of C3a and C5a with their respective receptors C3aR and C5aR1 mediate Th1 cytokine production and T-cell induction (78, 83). It has also been suggested that Tregs express C3aR and C5aR1, and that signaling through these receptors inhibits Treg function (84, 85).

A central role in T-cell homeostasis has been recently assigned to intracellular elements of the complement system (30). Activation of lysosomal C3aR by intracellularly generated C3a contributes to the survival of resting CD4<sup>+</sup> T cells. Upon activation, the intracellular stores of the C3 system translocate to the cell surface triggering the upregulation of IFN-γ and Th1 cell responses in conjunction with the extracellular engagement of C3b to CD46 (86). Intracellular C5a can also be generated from endogenous C5. Upon T-cell activation, C5a binds to intracellular C5aR1, inducing the activation of the NLRP3 inflammasome and, consequently, the initiation of a Th1 response (87).

All these activities underline the importance of complement effectors and regulators in the initiation of T-cell responses. In contrast, the complement system has also been associated with the prevention of T-cell priming and the induction of tolerance; probably as a regulatory mechanism to facilitate the timely resolution of the immune response. Thus, C1q can suppress macrophage-mediated inflammation and dendritic cell-mediated Th1-cell proliferation (88, 89). Binding of the C3 fragment iC3b to complement receptor type 3 (CR3) on antigen-presenting cells results in the production of TGF-β2 and interleukin (IL)-10, and the induction of antigen-specific tolerance (90). C3a and C3b also participate in the contraction phase of human Th1 responses (80, 82). CD46 promotes the switching of CD4<sup>+</sup> T cells toward IL-10 producing cells with a regulatory phenotype (80, 91), and negatively regulates Th1 activity through the binding of endogenous C5a to surface-expressed C5aR2 (87). C5aR2 was first proposed as a negative regulator of C5aR1, but some specific functions have been ascribed to this C5a receptor (92). CD55 may also have a role in the suppression of adaptive immune responses. Mice lacking CD55 experience enhanced T-cell responses to active immunization, characterized by an increased production of INF-γ and IL-2, as well as downregulation of IL-10 (93).

All these studies point to the dual role played by complement in the activation of effector T-cell responses. On the one hand, complement elements are central in the primary phase of effector expansion. On the other hand, complement can mediate a suboptimal T-cell activation associated with the contraction phase or the establishment of tolerance. With this duality in mind, it is interesting to analyze the relative role played by complement in the context of well-established tumors. It has been suggested that C5a affects T-cell responses in a concentrationdependent manner (94). Tumor-bearing mice with low C5aproducing tumor cells exhibit a reduced tumor burden with increased IFN-γ-producing CD4<sup>+</sup> and CD8<sup>+</sup> T cells in the spleen and tumor-draining lymph nodes. In contrast, tumorbearing mice with high C5a-producing cancer cells have an

accelerated tumor progression with less CD4<sup>+</sup> and CD8<sup>+</sup> T cells in the tumor, tumor-draining lymph nodes, and the spleen (94). This effect was associated with the presence of more myeloidderived suppressor cells (MDSCs) in the spleen. Interestingly, other studies have found elevated levels of C5a in cancer patients (12), which have been implicated in the recruitment of MDSCs to tumors (11). MDSCs are immunosuppressive immature myeloid cells able to disrupt major mechanisms of antitumor immune responses (95–97). In models of breast cancer, C5aR1 signaling in MDSCs induces the production of immunosuppressive cytokines, such as TGF-β, and reduces Th1 immune responses (98, 99). Treatment of mouse squamous cell carcinomas with paclitaxel and PMX-53, a C5aR1 inhibitor, results in peripheral priming and expansion of antigen-specific clones (100). Therefore, we can conclude that complementmediated effects may have evolved at established tumors to interfere with the generation of antitumor T-cell responses.

# Modulation of T-Cell Trafficking by the Complement System

A range of tumors escape from antitumor immune responses even after activation and expansion of T cells. In some cases, this can be attributed to the ability of the tumor endothelium to prevent T-cell trafficking. A complex network of endothelial adhesion molecules, Th1 cytokines, and surface receptors regulates T-cell homing and infiltration (101). Tumor cells, in concert with the endothelium, interfere with T-cell infiltration through a variety of molecular mechanisms, including the downregulation of endothelial adhesion molecules and the expression of T-cell inhibitory ligands (102).

Although complement elements can directly act on the endothelium (103–105), little is known about its contribution to the biology of tumor-associated endothelial cells. In the context of adoptive T-cell transfer, the capacity of tumor-reactive CD4<sup>+</sup> and CD8<sup>+</sup> T cells to infiltrate tumors requires the local production of C3, complement activation and release of C5a (106). Accordingly, the abrogation of CD55 expression enhances tumor T-cell infiltration (106). Radiotherapy has been found to upregulate the release of C3a and C5a within the irradiated tumors, leading to pronounced infiltration of CD8<sup>+</sup> effector T cells (107). The presence of C3d on melanoma cells yields greater infiltration by CD4<sup>+</sup> and CD8<sup>+</sup> lymphocytes (75). However, in apparent contradiction to these findings, blocking of C3aR or C5aR1 in most cancer models has been associated with an increased infiltration of T cells both in the primary tumor (11, 13, 42, 99, 100, 108, 109) and the metastatic niche (98, 109). The mechanisms underlying this outcome are not fully understood but may be related to the downregulation of immunosuppressive cell populations, such as MDSCs, able to impair T-cell trafficking (11, 12). Complement inhibition also reduces the levels of VEGF (110), which may normalize the tumor vasculature, increasing the infiltration of lymphocytes into tumors (111).

# Modulation of Cytotoxic T-Cell Activity by Complement

An immunosuppressive microenvironment hampers the killing capacity of cytotoxic CD8<sup>+</sup> T-cells. The pro-tumorigenic effect of complement activity is mediated, in an important way, by promoting immunosuppressing responses within the tumor microenvironment. The critical contribution of complement to regulating immunosuppressive cell populations, such as TAMs,

MDSCs, or Tregs, has been recently reviewed (47). C5a acts as a potent chemoattractant for polymorphonuclear MDSCs and stimulates the production of immunosuppressive reactive oxygen and nitrogen species by tumor infiltrating monocytic MDSCs (11). Accordingly, pharmacological blockade of C5aR1 decreases the frequency of MDSCs and impairs tumor growth (11–13, 112). C5aR1 inhibition also downregulates the expression of immunosuppression-related genes within the tumor milieu (12). In addition, C5a contributes to conditioning the premetastatic niche through TGF-β and IL-10-mediated accumulation of Tregs, proliferation of resident alveolar macrophages, and decrease in number and maturation of dendritic cells (98). As a consequence, effector CD4<sup>+</sup> T-cell responses skew toward a Th2 phenotype, limiting Th1 responses (98, 113).

C5a also affects the biology of macrophages. C5a skews macrophage polarization toward an M2 phenotype via C5aR1 signaling upon Leishmania infection (114). After ex vivo challenge of human whole blood with heat-killed Pseudomonas aeruginosa, C5a induces PD-L1 expression on monocytes, and the production of IL-10 and TGF-β (115). Elevation of PD-L1 expression has also been reported after C1q-mediated polarization of macrophages (89). M2 tumor-associated macrophages (TAMs) are an essential component of the tumor microenvironment that contribute to tumor progression by blocking CD8<sup>+</sup> T-cell responses (116). Recruitment of tumorpromoting TAMs with a M2-like phenotype is also observed in mouse sarcomas induced in a PTX3-deficient context and characterized by an increase in C5a and CCL2 (44). C5a also promotes hepatic metastases of colon cancer associated with an increase of monocyte chemoattractant protein-1 (MCP1), antiinflammatory modulators such as arginase-1, IL-10, or TGF-β,

and M2-like macrophages (117, 118). Similarly, in a model of squamous carcinogenesis, C5a regulates the protumorogenic properties of C5aR1-expressing mast cells and macrophages, leading to hampered antitumor CD8<sup>+</sup> T-cell responses (100). A combined treatment with cytotoxic chemotherapy and the blockade of C5aR1 synergistically inhibits the recruitment of effector memory CD8<sup>+</sup> T cells by both the modification of macrophage- and IFNγ-dependent mechanisms (100). Interestingly, this study suggests that C5a is not generated in the tumors through C3 activation, although further studies are needed to rule out this possibility (119).

Complement C3 activation fragments can also precondition the tumor microenvironment toward immunosuppression. The C3 degradation product iC3b promotes the development of MDSCs in vitro (120). Inhibition of complement C3 abrogates the suppressor phenotype of polymorphonuclear MDSCs in the ovarian tumor microenvironment (121). Deletion of C3 in tumor cells also inhibits M2 polarization (122). Signaling mediated by C3a contributes to melanoma tumorigenesis by inhibiting neutrophil and CD4<sup>+</sup> T-cell responses (108). Interestingly, some studies have suggested a direct effect of complement effectors in the functionality of T cells. C3 inhibits IL-10 mediated cytotoxic properties of tumor-infiltrating CD8<sup>+</sup> T lymphocytes in an autocrine manner, enhancing melanoma and breast cancer growth (123). Alterations in CD4<sup>+</sup> T cells by C3/C5-dependent pathways may also have a major role in lung cancer progression (109).

Finally, complement can also slow down the feeding of the cancer-immunity cycle by dying cancer cells. Ribosomal protein S19 (RPS19), upon release from dying tumor cells, interacts with C5aR1 expressed on MDSCs, promoting its recruitment to tumors, the generation of Tregs, the production of immunosuppressive cytokines (including TGF-β), and the reduction of CD8<sup>+</sup> T-cell tumor infiltration (99).

Overall, tumor-associated complement activation deeply influences the tumor microenvironment, leading to an immunosuppressive state and the attenuation of tumor-specific cytotoxic T-cell responses.

# COMPLEMENTING THE CANCER-IMMUNITY CYCLE

As reviewed in the previous section, a growing body of evidence supports the notion that complement activities support cancer growth and metastasis in the context of established tumors (124). Many mechanisms related to immune escape and resistance to checkpoint inhibitors can be modulated by elements of the complement system (summarized in **Figure 1**). The non-immunology-related effects of complement on cancer cell biology, including cancer cell proliferation, survival and invasion capacity (42, 43, 117, 125–137), further reinforces the impact of complement activation in cancer progression.

Based on the regulatory functions of complement in the cancer-immunity cycle, we sought to evaluate whether complement inhibition may represent an effective target for combined immunotherapies in preclinical syngeneic models of

cancer. Clinical successes and limitations of anti-PD-1/PD-L1 monotherapy prompted us to use this target as the primary building block for the combination. The C5a/C5aR1 axis was selected as the complement-related target based on the abundant evidence supporting the role of this pathway in the establishment of an immunosuppressive microenvironment (**Table 2**) (45). Using different lung cancer models, we observed a remarkable synergistic control of lung tumor burden and metastatic progression in animals simultaneously treated with an aptamer against C5a (AON-D21) and an anti-PD-1 monoclonal antibody (13). This effect is accompanied by a negative association between the frequency of CD8<sup>+</sup> T cells and the presence of MDSCs within tumors, and by a reduction of CD8<sup>+</sup> T-cell exhaustion markers (13). The synergistic benefit of this combination was later confirmed in models of melanoma and colon cancer (138). Interestingly, PD-1/PD-L1 antibodies induce the production of C5a (138), establishing a regulatory loop between both pathways.

Other complement elements, such as C1q, C3, or C3a, may be also targeted to re-educate the tumor microenvironment and sensitize it to the subsequent administration of immune checkpoint blockers (**Table 2**). A multifaceted repertoire of therapeutic inhibitors targeting these complement elements has been developed, and are currently in preclinical or clinical development (139, 140). **Figure 2** shows examples of compounds that may be used to target complement in the context of cancer immunotherapy. Complement C3, the centerpiece of complement activation, represents a particularly attractive target for therapeutic complement inhibition (141). Nonresponsive patients to PD-1/PD-L1 blockade frequently have noninflamed tumors with a defect in the early stages of the cancer-immunity cycle (15). Opsonization of dying cells with C3 fragments induces the production of anti-inflammatory cytokines and reduces the costimulatory molecules needed for the maturation of dendritic cells, resulting in T-cell tolerance (90). Therefore, C3 blockade may have a beneficial impact in the early stages of the cancer-immunity cycle, converting a noninflamed tumor into an inflamed tumor susceptible to PD-1/PD-L1 blockade. Moreover, both C3a and C5a production would be impaired (at least in the case of inhibitors, such as compstatin, that blocks C3 activation by all pathways). Interestingly, a simultaneous blockade of C3aR and C5aR1 has been reported to enhance the efficacy of anti-PD-1 therapy against melanoma cells (123). Deletion of C3 in tumor cells that had high C3 expression enhanced efficacy of anti–PD-L1 treatment (122). Additionally, complement C3 inhibition may have antitumor potential in the context of other immune combinations. For example, C3 inhibition by complement depletion or the use of the inhibitor compstatin enhances the antitumor efficacy of oncolytic virus (142, 143) and induces natural killer (NK) mediated antitumoral responses (144). Inhibition of complement activation upstream of all complement effectors also appears to be a rational approach. In this sense, combinatorial therapies involving inhibitors of C1q (e.g., C1-INH), which presents both complement-dependent and -independent tumor promoting activities, merit further investigation. Finally, it has to be noted that most complement inhibitors target the extracellular complement system, preserving its intracellular activity. This may be of upmost importance, since intracellular C3aR and C5aR1 signaling pathways seem to be required for T-cell survival (28).

Finally, it is interesting to point that the preclinical findings showing the feasibility and value of blocking C5a/C5aR1 to increase tumor-killing efficacy of checkpoint inhibitors have been the basis for the design of a phase I/II study (STELLAR-001). In this trial, the safety and efficacy of durvalumab (an anti-PD-L1 monoclonal antibody) is being tested in combination with IPH5401 (an anti-C5aR1 monoclonal antibody) in patients with selected solid tumors, including non-small cell lung cancer and hepatocellular carcinoma (NCT03665129). We are looking forward to the outcome of this trial, as well as to the clinical evaluation of novel combinations involving complement inhibitors.

#### CONCLUDING REMARKS

This is an exciting time for the complement field, in which new biological concepts have brought new therapeutic opportunities. Based on the extensive literature associating complement activation and cancer progression, we propose here that substantial clinical benefits can be achieved by multi-modal anticancer immunotherapies targeting both complementmediated mechanisms (to reverse immunosuppression), and PD-1/PD-L1 immune checkpoints (to re-activate Tcell functionality). Our preclinical studies supporting the idea that C5a/C5aR1 inhibition creates a "window of opportunity" for the administration of anti-PD-1/PD-L1 checkpoint inhibitors pave the way for the evaluation of other complement-based combinations. The challenge being that many potential combinations can be evaluated. Insights into how complement switches from tumor suppressing to tumor promoting activities at the onset of disease, as well as how to manage this dichotomy should be important research areas in order to establish the best therapeutic strategies. The differences between mice and humans in

#### REFERENCES

complement-mediated T-cell responses should also be considered (28, 87). To overcome this limitation, faithful mouse models that recapitulate the complexity of the human immune context in the tumor microenvironment are urgently needed (145).

#### AUTHOR CONTRIBUTIONS

RP and JL designed the concept. All authors wrote the manuscript. SO-E prepared the figures. All authors read and approved the final version of the manuscript.

# FUNDING

Authors' research was supported by FIMA, CIBERONC, Fundación Científica de la Asociación Española Contra el Cáncer, Fundación Ramón Areces, Juan Serrano and Fondo de Investigación Sanitaria-Fondo Europeo de Desarrollo Regional Una manera de hacer Europa (PI17/00411) to RP and DA; U.S. National Institutes of Health (AI068730 and AI030040) to JL; AIRC IG, AIRC 5x1000, and Ministero della Salute to AM. SO-E was supported from fellowships from Amigos de la Universidad de Navarra and Formación de Personal Universitario (FPU 17/00032).

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immune humans and cynomolgus macaques. Mol Ther. (2015) 23:1066–76. doi: 10.1038/mt.2015.49

**Conflict of Interest Statement:** JL is the founder of Amyndas Pharmaceuticals, which is developing complement inhibitors (including third-generation compstatin analogs such as AMY-101) and inventor of patents or patent applications that describe the use of complement inhibitors for therapeutic purposes, some of which are developed by Amyndas Pharmaceuticals. JL is also the inventor of the compstatin technology licensed to Apellis Pharmaceuticals (i.e., 4(1MeW)7W/POT-4/APL-1 and PEGylated derivatives). AM is inventor of patents related to PTX3 and gets royalties from related immunoassays. RP and DA are inventors of patents related to the use of complement C4 fragments as diagnostic and prognostic cancer biomarkers.

The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Pio, Ajona, Ortiz-Espinosa, Mantovani and Lambris. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The Challenges and Promise of Complement Therapeutics for Ocular Diseases

#### Dong Ho Park <sup>1</sup> , Kip M. Connor 2,3 \* and John D. Lambris <sup>4</sup>

<sup>1</sup> Department of Ophthalmology, School of Medicine, Kyungpook National University, Kyungpook National University Hospital, Daegu, South Korea, <sup>2</sup> Angiogenesis Laboratory, Department of Ophthalmology, Massachusetts Eye & Ear Infirmary, Boston, MA, United States, <sup>3</sup> Department of Ophthalmology, Harvard Medical School, Boston, MA, United States, <sup>4</sup> Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Stellar Chance Laboratories, Philadelphia, PA, United States

Ocular inflammation is a defining feature of sight threating diseases and its dysregulation can catalyze and or propagate ocular neurodegenerative maladies such as age-related macular degeneration (AMD). The complement system, an intrinsic component of the innate immunity, has an integral role in maintaining immune-surveillance and homeostasis in the ocular microenvironment; however, overstimulation can drive ocular inflammatory diseases. The mechanism for complement disease propagation in AMD is not fully understood, although there is accumulating evidence showing that targeted modulation of complement-specific proteins has the potential to become a viable therapeutic approach. To date, a major focus of complement therapeutics has been on targeting the alternative complement system in AMD. Recent studies have outlined potential complement cascade inhibitors that might mitigate AMD disease progression. First-in-class complement inhibitors target the modulation of complement proteins C3, C5, factor B, factor D, and properdin. Herein, we will summarize ocular inflammation in the context of AMD disease progression, current clinical outcomes and complications of complement-mediated therapeutics. Given the need for additional therapeutic approaches for ocular inflammatory diseases, targeted complement modulation has emerged as a leading candidate for eliminating inflammation-driven ocular maladies.

#### Edited by:

Robert Braidwood Sim, University of Oxford, United Kingdom

#### Reviewed by:

Michael Kirschfink, Universität Heidelberg, Germany Simon John Clark, University of Manchester, United Kingdom

#### \*Correspondence:

Kip M. Connor<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology

Received: 18 February 2019 Accepted: 18 April 2019 Published: 15 May 2019

#### Citation:

Park DH, Connor KM and Lambris JD (2019) The Challenges and Promise of Complement Therapeutics for Ocular Diseases. Front. Immunol. 10:1007. doi: 10.3389/fimmu.2019.01007 Keywords: age-related macular degeneration, complement system, immune modulation, ocular inflammation, therapeutics

Age-related macular degeneration (AMD) is the leading cause of blindness of the elderly in the Western world (1). Its prevalence is expected to rise as aging populations rise and ∼288 million people will be affected by AMD by 2040 (2). The buildup of debris within retinal pigment epithelial (RPE) cells and under the RPE layer is considered a hallmark of AMD development and progression (3).  The RPE are specialized cells that phagocytize the shed outer segments of photoreceptors, and are the most active phagocytizing cells of the body (4). As aging progresses, it is thought that the machinery of these cells deteriorates, along with their capacity to degrade and recycle photoreceptor metabolic waste. During the lifetime of a person, the choroidal capillary area becomes thinner, and Bruch's membrane may also accumulate lipoprotein material from the choroidal capillaries, which adds to the incompletely digested material deposited by RPE cells. Collectively, this accumulating material is referred to as drusen, which is a major pathological feature of AMD.

**102**

Drusen size can predict the development of AMD. Accordingly, a new clinical classification scheme for AMD using drusen size and presence of pigmentary abnormalities has been introduced by the Beckman Initiative for Macular Research Classification Committee (5). In this scheme, individuals over 55 years of age with small (<63µm) drusen were considered to have normal aging. Early AMD is characterized by the presence of medium-sized drusen (≥63 and <125µm) with no pigmentary abnormalities, and is accompanied by a mild loss of vision. Intermediate AMD is characterized by larger drusen deposits (≥125µm) and/or presence of pigmentary abnormalities, and is associated with a moderate loss of vision. Late/Advanced AMD is characterized by the presence of any choroidal neovascularization (CNV), known as neovascular AMD, or gross pigment abnormalities and cellular degeneration such as geographic atrophy (GA). Collectively, advanced AMD causes severe loss of vision and can lead to blindness. The incidence of late-stage AMD increases exponentially with age, with the two forms of late AMD, neovascular AMD and GA, occurring with roughly equal prevalence (2, 6). An estimated 1.22 million and 973,000 people in the United States have neovascular AMD and GA in at least one eye, respectively (7). As the population ages, the prevalence of late AMD is expected to rise from 9.6 million to 11.3 million in 2020 and 18.6 million in 2040 (2).

Currently, there is no effective prevention, individual risk estimation, or reliable prognostic evaluation available for the clinical management of AMD. Further, there are no therapeutics approved for the treatment of GA. The development of AMD depends on a complex interplay of risk factors such as age, genetics (8), and behavior including; smoking (9), diet (10), and sunlight exposure (11). Genetic variations in genes involved in the complement system, as well as others, are associated with risk for developing AMD, or risk of progression from early to late AMD (12). Overall, these findings suggest that AMD is a progressive neurodegenerative disease involving inflammation (13), and in particular an inflammatory immune response (14).

# THE COMPLEMENT SYSTEM IS A VITAL COMPONENT OF INNATE IMMUNITY

The immune system is divided into two distinct types, the innate and adaptive systems. The complement system, as part of the innate immune system, plays an integral role in maintaining immune-surveillance and homeostasis in the ocular microenvironment (15, 16). The complement system consists of three systems classical, lectin, and alternative (17). The classical system is mediated by the binding of the complement component C1q of C1 protein to antigen–antibody complexes. The lectin system is activated by mannose-binding lectin recognition of the polysaccharide or glycoprotein motifs on the cell surface of damaged host and non-host cells (17, 18). Lastly, the alternative complement system is constitutively active through the spontaneous cleavage of an internal C3 thioester bond (17, 19) (**Figure 1**). The spontaneous hydrolysis of this internal thioester bond within the complement protein C3 forms C3(H2O), and while not cleaved, C3(H2O) can function in the same manner as C3b in a C3 convertase. This "tick-over" of C3 to C3 (H2O) enables it to complex with complement factor B, following by its cleavage by complement factor D, a serine protease, into Ba and Bb fragments. The resulting C3(H2O)Bb complex is a C3 convertase, which can efficiently cleave C3 into C3a and C3b. Factor B enables further downstream activation of the alternative system through its binding to C3b (17–21). These components form the C3 convertase enzyme, C3bBb, promoting the cleavage of C3 and creating a positive feedback loop (19, 21). Subsequently, C5 convertase is created by the combination of the C3 convertase with an additional C3b molecule. The C5 convertase then cleaves C5 into C5a and C5b (17–19). C5b recruits C6, − 7, −8, and−9, forming the membrane attack complex (MAC), which forms a pore in the cell membrane causing cell lysis and death, which can be down-regulated by complement inhibitors such as CD59 (22, 23). However, according to a previous immunohistochemical study from human donor eyes with drusen, the antibody against C5b-9 indicating MAC showed a restricted pattern, while the C3 or C5 antibody showed a diffuse pattern throughout the drusen and choroid (24). Thus, it is unclear if MAC formation is linked to AMD disease progression. It may be complement's upstream opsonization functions that are perturbed in this pathology.

# ALTERNATIVE COMPLEMENT CASCADE DYSFUNCTION IN AGE-RELATED MACULAR DEGENERATION

#### Complement Factor H and Related Proteins

Over a lifetime, the photoreceptors and RPE cells of the retina encounter a number of innate immune activators, such that tight regulation of immunity is necessary to prevent deleterious inflammatory events. Dysregulation of the complement system may potentially drive ocular inflammation, which contributes to vision loss in AMD. Complement byproducts such as complement factor H (FH) in the drusen were the first indication that the complement system is involved in AMD progression (25). In 2005, genome-wide association studies (GWAS) suggested that the complement cascade was involved in AMD disease progression. **Table 1** summarizes the genes for the alternative complement pathway that are known to be involved in AMD. Early GWAS and targeted sequencing studies identified a common single nucleotide polymorphism (SNP) in the CFH gene (rs1061170) that is associated with an increased risk of developing AMD (26–29). This SNP replaces a tyrosine residue with a histidine residue at position 402 (Y402H) (49). Heterozygous individuals and homozygotes for the Y402H polymorphism have a 2.3 and 5.2-fold increased risk of developing AMD, respectively (30). Approximately 30% of individuals of European descents are carriers of at least one of the Y402H risk alleles (30).

FH is an important regulator of the complement system (50). Increasingly, it has been found that complement activation is linked with AMD associated with genetic variants on chromosome 1 locus (1q32) that contains the CFH and

CHFR genes, rather than chromosome 10 (10q31) around the ARMS2/HTRA1 genes (51). AMD patients with chromosome 1 mutations have significant complement over-activation in the extracellular matrix of the choriocapillaris, underlying Bruch's membrane. Complement activation and turnover have been identified in the choriocapillaris layer as well, and increased levels are found in individuals with AMD (52, 53). This is also found in donor eyes from individuals genetically at risk for AMD, but who do not yet have clinical manifestations of the disease (54). The activation of the alternative complement pathway is initiated by the formation of the C3bBb complex, also known as C3 convertase. Formation of this complex leads to the amplification of complement signaling and immune response. FH accelerates dissociation of this C3bBb complex, inhibiting alternative complement system activation. Furthermore, FH, as a cofactor, facilitates factor I-mediated C3b inactivation. FH is anchored to the extracellular matrix and the cell surface through interactions with glycosaminoglycans (GAGs) (55). The Y402H polymorphism does not alter the overall protein structure (56). However, the Y402H polymorphism disrupts binding of TABLE 1 | Genes in the alternative complement pathway that are involved in age-related macular degeneration.

the complement control region to GAG chains in the Bruch's membrane (55). Because the Y402H variant has decreased binding affinity to numerous components of the damaged retina (57–59), the inhibitory effect of FH on the complement system is thought to be decreased. This could result in poorly controlled complement turnover and excessive chronic local inflammation. The FH and factor H-like protein (FHL-1), components of the alternative system, are capable of suppressing complement activation on the extracellular matrix (51). The shortened splice variant of FH and FHL-1 appears to prevail in the ECM in/around Bruch's membrane (60–63). Because FHL-1 only has the one GAG-mediated anchoring site in its CCP7 domain which anchors FHL-1 to Bruch's membrane and the intercapillary septa, this GAG-binding site is affected by the Y402H polymorphism. In contrast, FH has two anchoring sites and is not particularly affected by the Y402H polymorphism (51). This may explain why the Y402H polymorphism has a disproportionate effect on protein anchoring via GAGs.

Other rare variants in CFH SNPs have also been reported to affect AMD, including the R1210C, R53C and D90G polymorphisms (31, 32). R1210C is extremely rare, with a minor allele frequency of 0.0173 %, but has an even stronger association with AMD than Y402H, potentially by acting as a functionally null allele (31). The highly penetrant R1210C variant is associated with a 6-year earlier onset of AMD with drusen phenotype (33). Advanced age and decreased FH induced sub-RPE deposit formation leading to complement activation, which contributed to RPE damage and visual function impairment (64).

A number of other SNPs further downstream on chromosome 1 are associated with AMD, suggesting involvement of the five factor H-related (FHR) proteins in disease pathogenesis (34). Although the exact function of FHR proteins is unclear at present, some of them may bind competitively to ligands involved in FH binding and may facilitate formation of a novel C3 convertase by binding C3b (65). Furthermore, a haplotype of complete deletion of the genes for FHR-1 and FHR-3 was associated with a decreased risk of AMD in human studies (34–36).

#### C3

C3 is the central component of all three complement systems, and functional changes in C3 directly affect the downstream cascade (66). A common SNP, rs2230199 (R80G), is associated with increased risk of AMD (odds ratio 2.6) (37, 38). The R102G polymorphism of C3 results in reduced FH binding to the 102G variant. As FH acts as a cofactor for factor I-mediated cleavage of C3b, R102G polymorphism of C3 decreased the factor I-mediated cofactor activity. By extending the convertase lifetime, activation of the alternative complement system is enhanced (39). A single rare variant in C3, K155Q, is positively associated with AMD risk (odds ratio 3.8) (41). K155 in C3 is in close proximity to the FH binding site (40). In surface plasmon resonance experiments, binding of this C3 mutant to FH was reduced compared to wild type C3. The K155Q allele in C3 results in resistance to proteolytic inactivation by FH and CFI. Taken together, these findings implicate that loss of C3 functionality contributes to AMD pathogenesis (41). Increased plasma complement protein is also associated with advanced stages of AMD, further supporting the hypothesis that complement activation may be correlated with disease progression as well (67, 68). C3a was significantly increased in AMD patients, ranging from 4.6 to 87.2 % (69). Furthermore, C3a and C5a are markers of acute activation of the alternative complement system. Thus, the plasma concentrations of activation peptides including C3a and C5a are positively correlated biomarkers of AMD (69).

#### Factor B

Both haplotypes, L9H variant of factor B and the E318D variant in C2, as well as a variant in intron 10 of C2 and the R32Q variant of factor B, are considered highly protective against AMD development (odds ratio 0.45 and 0.36, respectively), although the protection is likely mediated by factor B mutations (42, 43). Factor B fragments are found in the drusen at similar levels to that of factor H, and convertase formation, which strengthens complement activation, is decreased by the R32Q mutation (44). Furthermore, studies in mouse models of complement dysregulation support a causative role for the complement cascade in multiple retinal pathologies. Factor B is up-regulated in the retinal detachment mouse model, and in the vitreous from patients with retinal detachment (70). In addition, factor B knockout mice are protected from retinal detachment associated photoreceptor death which further emphasizes a role for this system in photoreceptor degeneration.

# Factor D

Factor D is one of the serine proteases that regulates the activation of the alternative complement system (71). Factor D becomes transiently active and is able to cleave factor B within this complex into Ba and Bb fragments. This is a rate limiting step in the formation of C3bBb which strengthens the signal and consequently activates the complement system (21, 71). A small case-control series showed that factor D gene SNP rs3826945 is positively associated with AMD risk (odds ratio 1.44) (45).

#### Factor I

The factor I gene spans 63 kb and contains 13 exons, the first 8 of which encode the heavy chain and the last 5 the light chain, which contains a serine protease domain (72). The serine protease domain is responsible for cleaving and inactivating C4b and C3b (73). Factor I-mediated C3b inactivation is assisted by FH. The FH acts as a cofactor for factor I-mediated cleavage of C3b (74), and thus accelerating breakdown of the alternative system C3 convertase, C3bBb. A common polymorphism near the factor I gene, rs10033900, has been described (46). A cohort study of 2,493 advanced AMD cases and controls showed that 7.8% of AMD cases compared to 2.3% of controls are carriers of rare missense factor I variants (odds ratio 3.6) (41). Another cohort study reported that two variants of factor I, G119R and G188A remained significant (47). The rare factor I G119R variant conferred a particularly high risk of AMD (odds ratio 22.2). Functional studies on the G119R variant showed that the secretion of the mutant protein was lower than that of the wild type protein, which led to a decrease in factor I-mediated cleavage of C3b.

# Complement 9

Complement component 9 (C9) is the most downstream component of the terminal complement complex cascade, acting as the terminal effector molecule of all three complement activation systems. Because the ultrastructure terminal complement complex is partially dependent on the amount of C9 molecules relative to C5b-8 molecules, a significant reduction in C9 can alter the terminal complement complex stoichiometry, reducing cytolytic activity (75, 76). Genotyping in 5,115 independent samples confirmed associations to AMD with a P167S allele in C9 (41). When compared to the controls, a two-fold increase was observed for this variant in the AMD cases. Another R95X variant was negatively associated with AMD risk in a small study (48), but has not been confirmed. Together, these findings suggest that the terminal complement pathway has a potential role in AMD pathogenesis.

## THERAPEUTIC APPROACHES AND CLINICAL TRIALS USING COMPLEMENT-MEDIATED DRUGS

In addition to strong genetic and basic research studies supporting a pathological role for the complement system in AMD, many clinical trials for therapeutics targeting alternative complement cascade are at various stages or completed (**Table 2**). Clinical trials for eight drugs that target the alternative complement cascade include two targeting C3, one targeting factor D, one targeting properdin, three targeting C5, and one targeting CD59.

# C3-Mediated Therapeutics

Inhibiting the C3 protein, including the C3 convertase, may be a desirable therapeutic approach. C3 modulates amplification of all initiation pathways, the generation of anaphylatoxins (C3a, C5a), and the membrane attack complex (MAC), while C3 inhibition attenuates these events. C3 functions as a central hub that mediates and controls the upstream activation and downstream effector functions of the complement cascade. Initial attempts in developing small molecules that inhibit conversion of C3 failed due to lack of potency and specificity (85).

TABLE 2 | Complement-mediated therapeutics in clinical trials for the treatment of age-related macular degeneration.

(Continued)

#### TABLE 2 | Continued

Ab, antibody; GA, geographic atrophy; IV, intravenous; IVT, intravitreal; nAMD, neovascular age-related macular degeneration; PCV, polypoidal choroidal vasculopathy; SAD, single ascending dose; STGD1, autosomal recessive Stargardt disease 1.

In contrast, the peptidic inhibitor compstatin exerts its function via direct binding to native C3. Compstatin was discovered by screening phage-display libraries searching for C3b-binding peptides (86). Compstatin is a 13 amino acid cyclic peptide that inhibits complement activation by binding C3 and interfering with convertase formation and C3 cleavage (and the subsequent C3b opsonization, amplification, and generation of effectors) (85). Thus, compstatin could be a potent inhibitor, blocking all three complement systems (87).