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. Author manuscript; available in PMC: 2023 Oct 20.
Published in final edited form as: J Heart Lung Transplant. 2021 Jul 12;40(10):1112–1121. doi: 10.1016/j.healun.2021.07.004

Increasing the efficacy and safety of a human complement inhibitor for treating post-transplant cardiac ischemia reperfusion injury by targeting to a graft-specific neoepitope

Chaowen Zheng 1,2, Mohamad Mahdi Sleiman 1, Xiaofeng Yang 1, Songqing He 2, Carl Atkinson 1,3,4,5, Stephen Tomlinson 1,6
PMCID: PMC10587835  NIHMSID: NIHMS1936721  PMID: 34334299

Abstract

Background:

Post-transplant ischemia reperfusion injury (IRI) is a recognized risk factor for subsequent organ dysfunction, alloresponsiveness, and rejection. Complement is known to play a role in IRI and represents a therapeutic target. Complement is activated in transplanted grafts when circulating IgM antibodies bind to exposed ischemia-induced neoepitopes upon reperfusion, and we investigated the targeting of a human complement inhibitor, CR1, to a post-transplant ischemia-induced neoepitope.

Methods.

A fragment of human CR1 was linked to a single chain antibody construct (C2 scFv) recognizing an injury-specific neoepitope to yield C2-CR1. This construct, along with a soluble untargeted counterpart, was characterized in a cardiac allograft transplantation model of IRI in terms of efficacy and safety.

Results:

CR1 was similarly effective against mouse and human complement. C2-CR1 provided effective protection against cardiac IRI at a lower dose than untargeted CR1. The increased efficacy of C2-CR1 relative to CR1 correlated with decreased C3 deposition, and C2-CR1, but not CR1, targeted to cardiac allografts. At a dose necessary to reduce IRI, C2-CR1 had minimal impact on serum complement activity, in contrast to CR1 which resulted in a high level of systemic inhibition. The circulatory half-life of CR1 was markedly longer than that of C2-CR1, and whereas a minimum therapeutic dose of CR1 severely impaired host susceptibility to infection, C2-CR1 had no impact.

Conclusion:

We show the translational potential of a human complement inhibitor targeted to a universal ischemia-induced graft-specific epitope, and demonstrate advantages compared to an untargeted counterpart in terms of efficacy and safety.

Introduction

While cardiac graft rejection is principally dependent on T cells, other immune factors can increase graft antigenicity leading to a strengthening of the rejection response. Of these other factors, ischemia reperfusion injury (IRI) is thought to be a major risk factor for subsequent organ dysfunction, alloresponsiveness, and the development of acute and chronic rejection. Complement is known to play a key role in IRI, and here we use a mouse model of cardiac transplantation to characterize a translationally relevant approach to reduce IRI using a graft-targeted human complement inhibitor.

An accepted paradigm is that following ischemia and reperfusion (IR), complement is activated by natural IgM antibodies that bind danger associated molecular patterns (neoepitopes) that become exposed on ischemia-stressed cells(1). We previously demonstrated this mechanism of IgM and complement dependent IRI in a mouse model of heart transplantation(2). Cardiac grafts were protected from IRI when transplanted into antibody-deficient Rag1−/− recipients, and IRI was restored when recipients were reconstituted with either B4 or C2 IgM mAb(2). The B4 mAb recognizes an unidentified epitope expressed on post-translationally modified mouse annexin IV(2–4), and the C2 mAb recognizes an identified subset of phospholipids(2–4). While the B4 mAb also recognizes an injury-specific neoepitope expressed on human tissue, its human target remains unidentified. On the other hand, the phospholipid targets of the C2 mAb occur universally, and have been shown to be expressed in an injury (ischemia)-specific manner in both mouse and human tissues(5).

We have previously investigated complement inhibition in murine models using Crry, a murine structural and functional equivalent of human CR1. Here we investigate site-specific targeting of a human CR1 in a murine model of post-transplant cardiac IRI. We linked a fragment of human CR1 to C2 scFv derived from the C2 mAb, which recognizes identified injury-specific epitopes in mouse and human tissue. We compared efficacy and safety aspects of targeted C2-CR1 with its untargeted counterpart, CR1, a version of which has been investigated in transplant clinical trials(6).

Materials and Methods

Construction of expression plasmids, protein expression, and protein purification.

A C2 scFv expressing plasmid was prepared using isolated variable heavy (VH) and variable light (VL) chain domains from C2 IgM mAb hybridoma as described(2). To construct the C2-Crry (C2 scFv-Crry) expression plasmid, the C2 scFv sequence was linked to the extracellular region of mouse Crry (residues 1–319 of mature protein, GenBank accession number NM013499). See Supplementary Material.

Binding and complement inhibitory assays of recombinant proteins

Anti-his tag enzyme-linked immunosorbent assays (ELISA) were used to determine binding of recombinant proteins to plate-bound antigens.

Cardiac transplantation

BALB/c hearts were transplanted into abdomen of age-matched C57BL/6 recipients (male, 9–11 weeks old) as described(7). Following procurement, donor hearts were perfused and stored in PBS on ice for 2 hours prior to implantation. Groups of recipient mice were treated with PBS vehicle or with different recombinant proteins by intravenous injection immediately following reperfusion. Transplanted hearts were explanted at 48h post-transplantation and formalin fixed prior to processing to paraffin. For assay of serum troponin levels, blood (50 ul) was collected 24h post-implantation and serum isolated as previously described(8).

Histology

Hematoxylin and eosin (H&E) stained sections were assessed for evidence of injury as previously described(9), and scored on a scale of 0–3, with results expressed as cumulative scores from 0 to 12 (Supplementary Material)

Troponin, creatinine and alanine transaminase (ALT) assays

Serum concentration of cardiac troponin I was measured in serum derived from recipients 24 h after transplantation as a marker of cardiac damage using ELISA (Life Diagnostics, USA). Serum creatinine concentration and ALT activity in recipients was measured 24 h after transplantation as markers of kidney and liver damage, respectively, using assay kits (Abcam, USA).

Immunohistochemical and immunofluorescence microscopy

Paraffin-embedded graft sections (4 um) were immuno-stained to detect the presence of the complement activation product, C3d, and for analysis of human CR1 binding/deposition.

In vivo kinetics of CR1 and C2-CR1 and systemic complement inhibition.

Blood was collected at various time points after construct administration, and serum concentration of constructs determined by ELISA. Systemic complement activity was determined pre and 2 hr-post construct administration by zymosan assay (Supplementary Material).

Graft viability and survival

Was assessed by direct abdominal palpation of the heterotopically transplanted heart

Cecal Ligation and puncture (CLP) sepsis model.

Cecal ligation and puncture was performed as described by Maier et al.,(10), with slight modification as described by our group(11).

Results

In vitro characterization of C2 neoepitope-targeted mouse and human complement inhibitors.

A C2 scFv targeting moiety derived from the parent C2 IgM mAb hybridoma was recombinantly linked to the first 10 SCR’s of human CR1. Based on the previous identification of phosphatidylcholine (PC) as one of a subset of phospholipids recognized by C2 IgM(3), we confirmed that C2 scFv and C2-CR1 bound specifically to PC-BSA coated plates (Fig. 1A), and demonstrated that soluble CR1 did not bind to PC-BSA. Complement inhibitory activity of the C2-CR1 construct against both mouse and human serum was also demonstrated (Fig. 1B, C). Of note, C2-CR1 and C2-Crry had similar complement inhibitory activities against mouse complement, whereas C2-Crry did not inhibit human complement.

Figure 1. In vitro characterization of constructs.

Figure 1.

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A, Binding of constructs to C2 phospholipid antigen. Increasing doses of construct were assayed for binding to phosphatidylcholine-BSA or BSA (control) coated wells of ELISA plates. Mean +/− SD, n = 3. B, Human complement inhibitory activity of C2-CR1 and C2-Crry measured using human serum as determined by zymosan assay. Mean +/− SD, n = 2 C, Mouse complement inhibitory activity of C2-CR1 and C2-Crry measured using mouse serum as determined by zymosan assay. Mean +/− SD, n =2–3.

Soluble (untargeted) CR1 confers dose-dependent protection against early allograft injury following heart transplantation.

A goal of these studies was to determine benefits associated with C2 epitope-targeted complement inhibition versus systemic complement inhibition with the human inhibitor, CR1. We investigated this in the context of allograft IRI following the abdominal heterotopic transplantation of hearts from Balb/C donors to C57BL/6 recipients. In order to compare the relative efficacy of targeted vs. untargeted CR1, we first performed a dose response experiment with CR1 in our model of cardiac IRI, with assessment of injury at 48 h post transplantation. A dose of 180 nM and higher, administered immediately after reperfusion, provided a significant level of protection in terms of histopathological analysis and serum troponin I levels (index of cardiac cell damage) when compared to vehicle treated controls. In this dose range, CR1 reduced myocyte damage in the epicardium (outer protective layer of the heart), endocardium (inner layer of the heart), and myocardium (muscular middle layer wall of the heart), and reduced inflammatory cell infiltration and endothelial activation as denoted by endothelial swelling. A dose of 90 nM and lower did not significantly improve either measure of injury (Fig. 2A, B, & C). The measured levels of graft injury also correlated with the level of C3d deposition in grafts from recipients treated with different doses of CR1 (Fig. 2D & E).

Figure 2. Dose response of soluble (untargeted) CR1 in post-transplant cardiac allograft injury.

Figure 2.

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Recipient mice were treated with indicated dose of CR1 immediately following allograft reperfusion, and grafts isolated and analyzed 48 h later. A, Representative H&E stained cardiac sections from grafts showing epicardial and endocardial inflammation and injury is reduced with increasing concentrations of CR1. Scale bars, 100μm. B, Semiquantitative histology injury scores. Non-parametric One-Way ANOVA (Kruskal-Wallis) with Dunn’s correction (comparing each group to PBS control). C, Serum cardiac troponin levels, as indicator of cardiac injury. Parametric One-Way ANOVA with Dunnett’s correction (comparing each group to PBS control). D, Representative images of sections stained for C3d deposition. Scale bars, 100μm. E, quantification of C3d deposition. Parametric One-Way ANOVA with Dunnett’s correction (comparing each group to PBS control). Quantification data expressed as Mean ± SD, n = 4–8 recipients; *p≤0.05,** p≤0.01, ***p≤0.001, ****p≤0.0001, ns = not significant.

Comparison of C2-CR1 and CR1 in a cardiac allograft IRI model

We compared the efficacies of C2-CR1 and CR1 at a dose of 90 nM, since this was the highest dose of CR1 tested that did not provide significant protection against cardiac IRI. In contrast to CR1, and compared to vehicle treated controls, a 90 nM dose of C2-CR1 provided a significant level of protection against cardiac IRI, as assessed by histopathological scoring (Fig. 3A) and serum troponin I levels (Fig 3B). As a control, we demonstrated that serum creatinine and ALT levels, which are markers for kidney and liver injury, respectively, were not different between control and treatment groups (Fig. 3C,D). There was also significantly less injury in allografts from recipients treated with C2-Crry compared to CR1 treated recipients (Fig. 3A & B) (Crry is the murine structural and functional analog of human CR1). We additionally demonstrated that the targeting vehicle alone, C2 scFv, did not provide protection against cardiac IRI, and that C2-CR1 and C2-Crry were similarly protective. Furthermore, the improved outcome measures observed with C2-CR1 compared to CR1, correlated with significantly lower relative levels of C3d deposition in allografts from C2-CR1 treated mice (Fig. 3E & F).

Figure 3. Comparison of C2-CR1 and CR1 on post-transplant cardiac allograft injury.

Figure 3.

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Recipient mice were treated with a 90 nM dose of C2-CR1 or CR1, which corresponds to a dose of CR1 that does not provide a significant level of protection from early allograft injury. Treatments were administered immediately after reperfusion, and grafts isolated for analysis 48 h later. C2 scFv, the targeting vehicle alone, and Crry, a murine structural and functional analogue of human CR1, were included in the analyses. A, Semiquantitative histology injury scores. Non-parametric One-Way ANOVA (Kruskal-Wallis) with Dunn’s correction (comparing each group to PBS control). B, Serum concentration of cardiac troponin, as a maker of cardiac injury. Parametric One-Way ANOVA with Dunnett’s correction (comparing each group to PBS control). C, Serum creatinine concentration. There is no significant difference between each group. Parametric One-way ANOVA with Tukey’s correction (comparing each group to every other group). D, Serum ALT activity. There is no significant difference between any group. Parametric One-way ANOVA with Tukey’s correction (comparing each group to every other group). E, Representative images of sections stained for C3d deposition. Scale bars, 100μm. F, quantification of C3d deposition. Parametric One-Way ANOVA with Tukey’s correction (comparing each group to every other group). Quantification data expressed as Mean ± SD, n = 4 recipients; *p≤0.05, ** p≤0.01, ns = not significant.

To further explore the protective mechanism, we investigated the effect of C2-CR1 and CR1 on neutrophil infiltration, as measured by MPO staining of graft tissue. C2-CR1 at 90 nM significantly reduced neutrophil infiltration whereas the lower dose of C2-CR1 (45nM) and both doses of CR1 (90 and 180 nM) failed to do so (Fig. 4A, B). Finally, to demonstrate target specificity of C2-CR1, we demonstrated that the construct did not induce any histological changes in native hearts of recipients (Fig. 4C & D).

Figure 4. Immune cell infiltration and histological comparison of transplanted (abdominal) and native heart.

Figure 4.

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A, Representative images of sections from indicated treatment groups, stained for MPO. Scale bars, 50μm. B. Quantification of MPO positive cells per field. Parametric One-way ANOVA with Dunnett’s correction (comparing each group to PBS control). C, Representative H&E stained sections from transplanted cardiac grafts and recipient’s native hearts from mice treated with C2-CR1 or PBS. Scale bars, 100μm. D, Semiquantitative histologic injury scores. Non-parametric One-Way ANOVA (Kruskal-Wallis) with Dunn’s correction (comparing graft vs native heart within each treatment and C2-CR1 vs PBS native hearts). Results expressed as Mean ± SD, n = 4; * p≤0.05.

Systemic complement inhibition, graft localization and pharmacokinetics

A major potential advantage of targeted vs. untargeted complement inhibition is the ability to inhibit complement locally at sites of complement-dependent pathology, while minimizing systemic effects that could interfere with normal physiological functions of complement. We therefore examined serum complement activity in recipient mice 2 hours after transplantation and administration of a minimum protective dose of C2-Crry (90 nM) or CR1 (180 nM). Compared to PBS treated recipients, C2-Crry treatment reduced serum complement activity by less than 10%, whereas CR1 reduced serum complement activity by 60% (Fig. 5A). The CR1 data is consistent with studies in other models of disease and injury that showed a high level of systemic complement inhibition is required for CR1 efficacy(12).

Figure 5. Systemic complement inhibition in treated recipients and CR1 localization to cardiac allografts.

Figure 5.

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A, Effect of C2-CR1 and CR1 on serum complement activity level. The minimum protective dose of C2-CR1 (90 nM) or CR1 (180 nM) was administered to recipients immediately following graft reperfusion, and serum isolated 2 h later for analysis of complement activity. Data shown as percent complement inhibition relative to serum sample isolated from recipient just prior to surgery. One-way ANOVA with Tukey’s correction (comparing each group to every other group). N = 4. B, Immunodetection of CR1 in allografts of recipients treated with either C2-CR1 or CR1 at minimum protective doses. Representative anti-CR1 immunofluorescence images of sections prepared from grafts isolated 24 h after transplantation and treatment. Scale bars: 100μm high-power inset, 200μm low-power inset. C, Quantitative analysis of CR1 staining in grafts. One-way ANOVA with Tukey’s correction (comparing each group to every other group). Results expressed as Mean ± SD; *p≤0.05, ** p≤0.01, **** p≤0.0001

To support the concept that the enhanced efficacy of C2-CR1 compared to CR1 was due to its ability to target and localize at sites of complement activation, we examined C2-CR1 and CR1 binding in allografts, as well as in the native heart of recipients. The recipients were treated with the minimum effective dose of either C2-CR1 or CR1, and immunofluorescence microscopy was used to determine CR1 deposition in transplanted and native hearts. CR1 is not expressed in rodents and thus immunolocalization of CR1 is specific for the injected C2-CR1. C2-CR1 was localized throughout the heart, with localization prominent in the epicardium and endocardium. Sparse staining was noted in myocardium (Fig. 5B,C). This is in keeping with the injury profile seen in this heterotopic heart transplant model where IRI injury predominates in the epicardium and endocardium and spreads into the myocardium with increasing injury. While complement C3d staining appeared to focus on the endothelium of microvessels within the heart, C2-CR1 binding was similarly seen in the microvasculature in a pattern consistent with C3d deposition. Interestingly, C2-CR1 could also be seen on myocytes within the epi and endocardium.

We additionally investigated the pharmacokinetics of each inhibitor using blood samples taken at various times after administration. The data was fitted to two-phase decay curves using non-linear regression and the results showed a good fit (r2=0.94–0.98). The model used to estimate pharmacokinetics assumes the presence of two phases of decline in serum concentration. The first fast phase corresponds to redistribution of the drug from the circulation into peripheral tissue, and a second slow phase which corresponds to drug elimination. C2-CR1 had a T1/2 fast of 0.59 hours, and a T1/2 slow of 19.97 hours. However, CR1 had slower half-lives with a T1/2 fast of 3.47 hours, and a T1/2 slow of 29.13 hours (Fig. 6). This shows that C2-CR1 exhibits a faster redistribution phase where it moves from the blood into peripheral tissue. Of note, most C2-CR1 is removed from the circulation in a short period of time, thus limiting any systemic effects. On the other hand, CR1 did not exhibit a substantial fast redistribution phase and had longer half-lives, and thus higher systemic levels of complement inhibition.

Figure 6. Serum circulatory half-life of C2-CR1 and CR1.

Figure 6.

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C2-CR1 or CR1 at minimum protective dose was administered i.v., and blood samples collected at indicated times for analysis of protein construct levels by anti-CR1 ELISA. Two-phase exponential decay curves were fitted to data for both constructs and pharmacokinetic parameters were calculated, A, C2-CR1 with fast half-life of 0.59 h and slow half-life of 19.97 h B, CR1 with fast half-life of 3.47 h and slow half-life of 29.13 h. Mean ± SD, n=3.

Effect of C2-CR1 on graft survival

To demonstrate that differences in acute histological and immunological measures of inflammation translate to improved long-term outcome, we assessed graft survival along with palpable abdominal heartbeat. There was a modest, but significant increase in allograft survival in C2-CR1 treated recipients compared to PBS treated controls (Fig. 7A). There was also improved palpable abdominal heartbeat scores with C2-CR1 treatment (Fig. 7B). Thus, C2-CR1 mediated protection against IRI translates to improved functional outcome and increased graft survival.

Figure 7. Effect of C2-CR1 on graft survival in allograft transplantation model, and effect of C2-CR1 and CR1 on animal survival in a model of polymicrobial septic peritonitis.

Figure 7.

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A, Cardiac graft survival in a model acute rejection. Following cardiac transplantation, mice were given a minimum protective dose of C2-CR1 (against cardiac IRI) (n = 8). A control group was treated with PBS (n =7). C2-CR1 treatment significantly extended graft survival. Mantel-Cox (log-rank) test. B, Cardiac graft contraction was monitored by palpation and graded on a scale from 0 to 4. Two-way ANOVA. C, Animal survival following cecal ligation and puncture (CLP). Mice were treated with a minimum protective dose (against cardiac IRI) of either C2-CR1 (n = 15) or CR1 (n = 10). A control group was treated with PBS (n = 15), and a C3-deficient group (n = 6) was also included. Unlike C3 deficiency or CR1 treatment, C2-CR1 treatment did not significantly affect mortality. Mantel-Cox (log-rank) test. Results expressed as Mean ± SD; * p≤0.05, ** p≤0.01, ***p<0.001, ****p<0.0001.

Immunity to infection.

A concern recognized by the FDA, and thus also by companies developing complement inhibitors, is increased susceptibility to infection of patients receiving complement inhibitors. The above data shows that a therapeutic dose of C2-CR1 in the context of cardiac IRI, has minimal impact on serum complement activity, which suggests it would also have minimal impact on complement-mediated resistance to infection. We formally examined this supposition using a cecal ligation and puncture (CLP) model of polymicrobial sepsis. Immediately following CLP, groups of mice were treated with PBS, 90 nM C2-CR1, or 180 nM CR1 (minimum effective doses). A C3 deficient mouse cohort was also included. C3 deficient mice and mice treated with CR1 all died within 48 hours. On the other hand, C2-CR1 treated mice had a significantly slower mortality rate with about 30% survival at day 10. Furthermore, there was no significant difference in mortality between control (PBS treated) and C2-CR1 treated mice (Fig. 7C).

Discussion

Ischemia reperfusion injury has been shown to promote early graft injury, increase graft allogenicity, and pre-dispose to the development of chronic rejection(13). Thus, there is an urgent need to develop pharmacotherapeutics that ameliorate IRI as a means to promote improved graft outcomes. We (2, 9), and others (14–16), have shown that complement plays a significant role in IRI, and complement inhibitors have been successfully applied to reduce IRI in various preclinical models of IRI (2, 9, 17, 18). Nearly all complement inhibitors that have been investigated clinically, including soluble human CR1 (TP10) and the vast majority of complement inhibitors currently in development, rely on systemic inhibition of the complement system (6, 19–23). There are significant potential drawbacks with this approach, particularly with regard to transplant patients, who will be heavily immunocompromised. Although infection in cardiac transplant recipients is not common, complement has important roles in innate, humoral and cell-mediated immunity, and it is not clear how the additional immunosuppressive effect of complement inhibition would affect outcomes in patients heavily T cell immunosuppressed. Complement also has important physiological roles other than immunity to infection, such as clearance of immune complexes and apoptotic cells, and tissue repair and regeneration (24–27).

Soluble systemic delivery of CR1 (sCR1) constructs have been used in pre-clinical and clinical models of IRI in transplant and non-transplant settings. In both settings, systemic complement inhibition was associated with amelioration of injury, and while encouraging, it is recognized that systemic inhibition of complement functions is likely not an optimum approach for application in clinical transplantation. In light of this, Pratt et al. (28), using a rat kidney transplantation model, investigated an approach to target a membrane inserting CR1-based inhibitor to the donor kidney ex-vivo by including it as a constituent of the organ preservation solution(28). Pre-treatment of grafts with a lipid-tagged CR1, which non-specifically inserts into cell membranes, resulted in a reduction in early graft damage and improved graft survival. More recently, Yu et al., (29), also using a rat kidney transplantation model, investigated the therapeutic potential of ex-vivo graft treatment with an anti-C5 monoclonal antibody, which reduced delayed graft function and improved graft survival. The former approach (lipid-tagged sCR1) is limited by only being able to deliver the complement therapeutic at the time of organ procurement, whereas the second approach (anti-C5) would require systemic complement inhibition if administered to the recipient.

An approach to lessen the concerns of systemic inhibition is to target a complement inhibitor to the site of complement-dependent pathology. This approach has been investigated in terms of targeting complement inhibitors to sites of complement activation by means of linking a complement inhibitor to a fragment of complement receptor 2 (CR2), which binds C3 fragments deposited at sites of complement activation(30). A more recently investigated targeting approach involves linking a complement inhibitor to an scFv that binds injury-specific epitopes. This approach takes advantage of an innate immune injury sensing mechanism that involves the expression of neoepitopes on injured/stressed cell membranes that are recognized by natural circulating self-reactive IgM antibodies. Once bound, these antibodies activate complement which, under normal physiological conditions, tag the cell for removal (1, 4, 31–33). We previously linked the murine complement inhibitor, Crry, to a B4 scFv construct that recognizes an injury-specific, but unidentified neoepitope expressed on annexin IV in mice, but on an unknown protein in humans (2). Here, by means of a C2 scFv construct, we target a complement inhibitor to an identified injury-specific neoepitope that consists of a subset of phospholipids that occur across species (5). For the first time, we investigated neoepitope-targeting of a human complement inhibitor, and we undertook the first formal comparison of a neoepitope targeted versus untargeted complement inhibition.

The CR1 constructs used herein contained the first 10 N-terminal SCRs of the native protein. This region contains two active complement inhibitory sites with co-factor activity for C3b and C4b, and decay accelerating activity for C3 convertases(34). Of note, a soluble form of CR1 consisting of 17 N-terminal SCR’s (TP-10), was previously shown to be well tolerated humans, but it is noteworthy in the context of the demonstrated benefits of targeted CR1 shown here, that 90% complement inhibition was measured at 24 hours following its administration to lung transplant patients(6). At a dose necessary to reduce IRI, C2-CR1 was shown to have minimal impact on serum complement activity, in contrast to CR1. This relates to a desired design feature of targeted complement inhibitors in that they should be taken up and retained at the target site, with a short circulatory half-life. In this regard, the circulatory half-life of CR1 was four times longer than that of C2-CR1. We also show that C2-CR1 mediated protection against IRI translates to improved functional outcome and increased graft survival. Of note, only a single dose was administered immediately after reperfusion. Multiple doses of C2-CR1 may well improve graft survival further, and previous work suggests that if administered as an adjuvant therapy, C2-CR1 could favorably impact immunosuppressive protocols and dosing(35, 36).

Finally, and as a likely consequence of systemic complement inhibition, we demonstrated that a minimum therapeutic dose of CR1, but not of C2-CR1, severely impaired host susceptibility to infection, an important consideration for a heavily immuno-compromised transplant recipient.

In summary, we investigated the translational potential of a human complement inhibitor targeted to a universally expressed ischemia-induced graft-specific epitope, and demonstrated significant advantages compared to an untargeted counterpart in terms of efficacy and safety.

Supplementary Material

supplement

Acknowledgments

These studies were supported by the NIH (U01AI32894 and R56AI56383 to ST and CA), the Dept. Veteran’s Affairs (BX005235 to ST), the Dept. of Defense (RT190030 to ST), and an award from Enduring Hearts and the American Heart Association (18PRE34070023 to MS) and 111 Project from Ministry of Education and State Administration of Foreign Experts Affairs of the Peoples Republic of China (D17011 to ST, CA and SH).

List of non-standard abbreviations:

IRI

Ischemia reperfusion injury

IR

Ischemia and reperfusion

ScFv

A single chain antibody

PC

Phosphatidylcholine

CR2

Complement receptor 2

MPO

Myeloperoxidase

ALT

Alanine transaminase

Footnotes

Financial conflict of interest statement

ST is co-founder of a company that is developing complement inhibitors, and ST and CA are inventors on licensed patents for complement inhibitors.

References

  • 1.Holers VM, Tomlinson S, Kulik L, et al. : New therapeutic and diagnostic opportunities for injured tissue-specific targeting of complement inhibitors and imaging modalities. Semin Immunol 2016;28:260–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Atkinson C, Qiao F, Yang X, et al. : Targeting pathogenic postischemic self-recognition by natural IgM to protect against posttransplantation cardiac reperfusion injury. Circulation 2015;131:1171–80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Elvington A, Atkinson C, Kulik L, et al. : Pathogenic natural antibodies propagate cerebral injury following ischemic stroke in mice. J Immunol 2012;188:1460–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Kulik L, Fleming SD, Moratz C, et al. : Pathogenic natural antibodies recognizing annexin IV are required to develop intestinal ischemia-reperfusion injury. J Immunol 2009;182:5363–73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Li C, Patel K, Tu Z, et al. : A novel injury site-natural antibody targeted complement inhibitor protects against lung transplant injury. Am J Transplant 2020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Keshavjee S, Davis RD, Zamora MR, de Perrot M, Patterson GA: A randomized, placebo-controlled trial of complement inhibition in ischemia-reperfusion injury after lung transplantation in human beings. J Thorac Cardiovasc Surg 2005;129:423–8. [DOI] [PubMed] [Google Scholar]
  • 7.Corry RJ, Winn HJ, Russell PS: Primarily vascularized allografts of hearts in mice. The role of H-2D, H-2K, and non-H-2 antigens in rejection. Transplantation 1973;16:343–50. [DOI] [PubMed] [Google Scholar]
  • 8.Tuck MK, Chan DW, Chia D, et al. : Standard operating procedures for serum and plasma collection: early detection research network consensus statement standard operating procedure integration working group. J Proteome Res 2009;8:113–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Atkinson C, He S, Morris K, et al. : Targeted complement inhibitors protect against posttransplant cardiac ischemia and reperfusion injury and reveal an important role for the alternative pathway of complement activation. J Immunol 2010;185:7007–13. [DOI] [PubMed] [Google Scholar]
  • 10.Maier S, Traeger T, Entleutner M, et al. : Cecal ligation and puncture versus colon ascendens stent peritonitis: two distinct animal models for polymicrobial sepsis. Shock 2004;21:505–11. [DOI] [PubMed] [Google Scholar]
  • 11.Atkinson C, Song H, Lu B, et al. : Targeted complement inhibition by C3d recognition ameliorates tissue injury without apparent increase in susceptibility to infection. J Clin Invest 2005;115:2444–53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Zhang Y, Nester CM, Holanda DG, et al. : Soluble CR1 therapy improves complement regulation in C3 glomerulopathy. J Am Soc Nephrol 2013;24:1820–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Minasian SM, Galagudza MM, Dmitriev YV, Karpov AA, Vlasov TD: Preservation of the donor heart: from basic science to clinical studies. Interact Cardiovasc Thorac Surg 2015;20:510–9. [DOI] [PubMed] [Google Scholar]
  • 14.Chun N, Haddadin AS, Liu J, et al. : Activation of complement factor B contributes to murine and human myocardial ischemia/reperfusion injury. PLoS One 2017;12:e0179450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Sharif-Paghaleh E, Yap ML, Meader LL, et al. : Noninvasive Imaging of Activated Complement in Ischemia-Reperfusion Injury Post-Cardiac Transplant. Am J Transplant 2015;15:2483–90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Homeister JW, Lucchesi BR: Complement activation and inhibition in myocardial ischemia and reperfusion injury. Annu Rev Pharmacol Toxicol 1994;34:17–40. [DOI] [PubMed] [Google Scholar]
  • 17.Sihag S, Haas MS, Kim KM, et al. : Natural IgM Blockade Limits Infarct Expansion and Left Ventricular Dysfunction in a Swine Myocardial Infarct Model. Circ Cardiovasc Interv 2016;9:e002547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Haas MS, Alicot EM, Schuerpf F, et al. : Blockade of self-reactive IgM significantly reduces injury in a murine model of acute myocardial infarction. Cardiovasc Res 2010;87:618–27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Sommer W, Tudorache I, Kuhn C, et al. : C1-esterase-inhibitor for primary graft dysfunction in lung transplantation. Transplantation 2014;97:1185–91. [DOI] [PubMed] [Google Scholar]
  • 20.Wan SS, Ying TD, Wyburn K, Roberts DM, Wyld M, Chadban SJ: The Treatment of Antibody-Mediated Rejection in Kidney Transplantation: An Updated Systematic Review and Meta-Analysis. Transplantation 2018;102:557–68. [DOI] [PubMed] [Google Scholar]
  • 21.Park S, Lee JG, Jang JY, et al. : Induction of Accommodation by Anti-complement Component 5 Antibody-based Immunosuppression in ABO-incompatible Heart Transplantation. Transplantation 2019;103:e248–e55. [DOI] [PubMed] [Google Scholar]
  • 22.Bohmig GA, Eskandary F, Doberer K, Halloran PF: The therapeutic challenge of late antibody-mediated kidney allograft rejection. Transpl Int 2019;32:775–88. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Stegall MD, Diwan T, Raghavaiah S, et al. : Terminal complement inhibition decreases antibody-mediated rejection in sensitized renal transplant recipients. Am J Transplant 2011;11:2405–13. [DOI] [PubMed] [Google Scholar]
  • 24.Medof ME, Iida K, Mold C, Nussenzweig V: Unique role of the complement receptor CR1 in the degradation of C3b associated with immune complexes. J Exp Med 1982;156:1739–54. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Clynes R, Dumitru C, Ravetch JV: Uncoupling of immune complex formation and kidney damage in autoimmune glomerulonephritis. Science 1998;279:1052–4. [DOI] [PubMed] [Google Scholar]
  • 26.Pickering MC, Botto M, Taylor PR, Lachmann PJ, Walport MJ: Systemic lupus erythematosus, complement deficiency, and apoptosis. Adv Immunol 2000;76:227–324. [DOI] [PubMed] [Google Scholar]
  • 27.Jones J, Morgan BP: Apoptosis is associated with reduced expression of complement regulatory molecules, adhesion molecules and other receptors on polymorphonuclear leucocytes: functional relevance and role in inflammation. Immunology 1995;86:651–60. [PMC free article] [PubMed] [Google Scholar]
  • 28.Pratt JR, Jones ME, Dong J, et al. : Nontransgenic hyperexpression of a complement regulator in donor kidney modulates transplant ischemia/reperfusion damage, acute rejection, and chronic nephropathy. Am J Pathol 2003;163:1457–65. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Yu ZX, Qi S, Lasaro MA, et al. : Targeting Complement Pathways During Cold Ischemia and Reperfusion Prevents Delayed Graft Function. Am J Transplant 2016;16:2589–97. [DOI] [PubMed] [Google Scholar]
  • 30.Song H, He C, Knaak C, Guthridge JM, Holers VM, Tomlinson S: Complement receptor 2-mediated targeting of complement inhibitors to sites of complement activation. J Clin Invest 2003;111:1875–85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Zhang M, Michael LH, Grosjean SA, Kelly RA, Carroll MC, Entman ML: The role of natural IgM in myocardial ischemia-reperfusion injury. J Mol Cell Cardiol 2006;41:62–7. [DOI] [PubMed] [Google Scholar]
  • 32.Marshall K, Jin J, Atkinson C, et al. : Natural immunoglobulin M initiates an inflammatory response important for both hepatic ischemia reperfusion injury and regeneration in mice. Hepatology 2018;67:721–35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Haas MS, Alicot EM, Schuerpf F, et al. : Blockade of self-reactive IgM significantly reduces injury in a murine model of acute myocardial infarction. Cardiovasc Res 2010;87:618–27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Krych-Goldberg M, Atkinson JP: Structure-function relationships of complement receptor type 1. Immunol Rev 2001;180:112–22. [DOI] [PubMed] [Google Scholar]
  • 35.Chun N, Fairchild RL, Li Y, et al. : Complement Dependence of Murine Costimulatory Blockade-Resistant Cellular Cardiac Allograft Rejection. Am J Transplant 2017;17:2810–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Zhu P, Bailey SR, Lei B, Paulos CM, Atkinson C, Tomlinson S: Targeted Complement Inhibition Protects Vascularized Composite Allografts From Acute Graft Injury and Prolongs Graft Survival When Combined With Subtherapeutic Cyclosporine A Therapy. Transplantation 2017;101:e75–e85. [DOI] [PMC free article] [PubMed] [Google Scholar]

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