Treatment or prevention of ischemia reperfusion injury in organ tranplantation

Abstract

The present invention relates to methods of treating or preventing ischemia reperfusion injury (IRI) in a subject receiving a donor organ transplant, wherein the methods involve administration of a complement factor 2 (C2) inhibitor to the subject. The present invention also relates to methods of improving allograft function and also to methods of reducing the risk of allograft loss in a subject receiving a donor organ transplant, wherein the methods involve administration of a C2 inhibitor to the subject. Furthermore, the present invention relates to methods of treating or preventing delayed graft function (DGF) in a subject receiving a kidney transplant, wherein the methods involve administration of a C2 inhibitor to the subject. The C2 inhibitor may be empasiprubart.

Description

[0001] TREATMENT OR PREVENTION OF ISCHEMIA REPERFUSION INJURY IN ORGAN TRANPLANTATION

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to methods of treating or preventing ischemia reperfusion injury (IRI) in a subject receiving a donor organ transplant, wherein the methods involve administration of a complement factor 2 (C2) inhibitor to the subject. The present invention also relates to methods of improving allograft function and also to methods of reducing the risk of allograft loss in a subject receiving a donor organ transplant, wherein the methods involve administration of a C2 inhibitor to the subject. Furthermore, the present invention relates to methods of treating or preventing delayed graft function (DGF) in a subject receiving a kidney transplant, wherein the methods involve administration of a C2 inhibitor to the subject. The C2 inhibitor may be empasiprubart (also referred to herein as ARGX-117).

[0004] BACKGROUND OF THE INVENTION

[0005] Ischemia reperfusion injury (IRI) remains one of the biggest challenges in organ transplantation. During the transplant procedure, the donor organ is cut-off from a blood supply and starved of oxygen for a period of time (i.e. ischemia) before connection to a recipient blood circulation resulting in rapid re-oxygenation (i.e. reperfusion). Re-establishment of blood flow is essential to salvage ischaemic tissues. However, reperfusion itself paradoxically causes further damage, threatening function and viability of the organ. IRI occurs in a wide range of organ transplants including heart, lung, kidney, liver, pancreas and bowel, and it is a major factor affecting donor graft function and the success of organ transplantation (Tang et al. 2022. Inflamm Res; 71 (12): 1463-1476; Fernandez et al. 2020. IntJ Mol Scr, 21 (22): 8549).

[0006] Delayed graft function (DGF) is a frequent complication associated with kidney transplantation whereby the transplanted organ fails to work immediately. DGF often correlates with poor outcomes such as long-term allograft dysfunction and in the worst-case scenario, organ rejection. DGF may be defined in different ways but is most commonly diagnosed at a clinical level as the requirement for at least one life-sustaining dialysis treatment within the first week post-transplantation, prior to the onset of graft function (Ponticelli et al. 2022. J Pers Med. 12(10): 1557). DGF is influenced by a variety of factors including the primary disease and physical condition of the donor, organ procurement and preservation procedures, the warm- and / or cold-ischemia time of the organ prior to transplant, reperfusion during transplantation surgery, and the posttransplant immune response. Each of these factors can potentially damage the graft. However, major insults are caused by ischemia and subsequent reperfusion and thus ischemia-reperfusion injury (IRI) is thought to be a primary contributor to DGF (Nieuwenhuijs-Moeke et al. 2020. J Clin Med. 9(1 ): 253). In particular, it is observed that DGF is more prevalent in allografts from deceased donors such as deceased brain death (DBD) donors and deceased cardiac death (DCD) donors, where it occurs in up to 40% of transplants (Irish et al. 2003. J Am Soc Nephrol. 14: 2967-2974; Hollmen et al. 2011 . Critical Care\ 15: R121 ). This is predominantly because deceased donor transplantations are difficult to plan and synchronise in advance and are therefore associated with substantially longer warm- and / or cold-ischemia times than for living donor transplantation.

[0007] Current immunosuppression and organ preservation techniques have not substantially mitigated the risk of ischemia reperfusion injury and delayed graft function following organ transplant. Proposed therapies for DGF in particular are varied and include: donor therapy with dopamine; hypothermic machine perfusion of deceased donor kidneys; intra-operative rabbit anti-thymocyte globulin administered before reperfusion; minimisation of calcineurin inhibitors; epoetin-a treatment; leukocyte adhesion blockade; hepatocyte growth factor mimetics; siRNA targeting the p53 gene; and C5 inhibition with eculizumab (Lim & Bloom; 2020; Clinical Journal of the American Society of Nephrology, 15: 13-15). However, the majority of proposed clinical studies have been disappointing in that they have shown minimal or no difference in DGF rates or no eventual positive effects on allograft function or survival. For example, peri-transplant administration of eculizumab was tested in pilot trials in deceased donor kidney transplants but disappointingly failed to reduce the incidence of DGF in kidney transplant recipients (Lim & Bloom; 2020; Clinical Journal of the American Society of Nephrology, 15: 13-15). Accordingly, there is still an unmet need for the prevention of ischemia reperfusion injury following organ transplantation, for example the prevention of DGF following kidney transplantation.

[0008] SUMMARY OF INVENTION

[0009] Evidence suggests that the complement system may be involved at various stages of the transplantation procedure and in particular, in ischemia-reperfusion injury. During preservation and transportation of the donor organ, the tissue is deprived of oxygen that is normally transported to the organ by the circulation. The resulting ischemia activates complement by several mechanisms, including by lowering the pH of the blood as a result of anaerobic metabolism. The resulting acidic conditions interfere with the complement system, for example by facilitating activation of the alternative pathway. Ischemia also induces the expression of heparinase and metalloproteinases in endothelial cells, which results in the cleavage and breakdown of the glycocalyx (a dense gel-like physical barrier that surrounds and protects the cell). This breakdown results in the loss of complement regulators such as C4b-binding protein, factor H and C1 INH, such that the endothelial cell surface is no longer protected. Reperfusion of the ischemic organ induces a process that is necessary to enable repair of the tissue but the rapid oxygenation also causes injury. During reperfusion the unprotected endothelial surface and intravascular cell debris together with local acidosis and hypoxia from the preceding ischemia trigger and amplify the complement cascade eliciting widespread inflammation (Biglarnia et al. 2018; Nature Reviews I Nephrology, 14: 4767-781 ).

[0010] The present inventors have found that an anti-C2b antibody, empasiprubart (also referred to herein as ARGX-117), is able to inhibit complement activity induced by ischemia-reperfusion injury. In particular, the data herein shows that complement is activated in primary kidney and lung-derived endothelial cells that have been exposed to hypoxia and reoxygenation, but that empasiprubart (ARGX-117) is able to inhibit complement in these cells. Moreover, the inventors have shown similar effects using a human kidney chip model of IRI . Again, empasiprubart (ARGX- 117) is shown to inhibit IR-induced complement activation by reducing free 02 levels and inhibiting 03 deposition on the endothelial cells, confirming that binding to 02 by empasiprubart (ARGX-117) inhibits complement activation downstream of 02. In addition, the examples herein demonstrate that complement is activated in the early days post-lung transplant, as shown in both mouse and human patient samples. Using an in vivo mouse single lung transplant model to study the effects of lung-IRI, the inventors have shown that treatment with an anti-mouse 02 antibody causes complete blocking of 02 and a clear reduction in complement activation, with early findings indicating improvements in lung function / reduced tissue damage. Taken together, these findings provide a strong rationale for a therapeutic strategy involving 02 inhibitors to treat or prevent ischemia reperfusion injury following organ transplantation.

[0011] Accordingly, in a first aspect, there is provided a method of treating or preventing ischemia reperfusion injury (IRI) in a subject receiving a donor organ transplant, the method comprising administering an effective amount of a complement factor 2 (02) inhibitor to the subject.

[0012] In certain embodiments, the donor organ is a solid organ and the subject is receiving a solid organ transplant. In certain embodiments, the donor organ is selected from: kidney, lung, heart, pancreas, bowel and liver. In preferred embodiments, the donor organ is a kidney. For embodiments wherein the donor organ is a kidney, the subject may be at risk of delayed graft function. For such embodiments, the methods may be for the treatment or prevention of delayed graft function.

[0013] In a further example described below, using an in vivo rat model, it is shown that targeting the complement pathway at the level of C2 (with an anti-rat C2 antibody) during syngeneic kidney transplant is effective to improve allograft function. In this model, the donor kidney is subjected to an extended cold ischemia time prior to reperfusion to mimic the damaging effects of IRL Treatment with an anti-rat C2 antibody prior to reperfusion of the organ reduced urea and creatinine levels compared to control, indicating improved kidney function in the presence of a C2 inhibitor. In a further example described below, using an in vivo renal clamping mouse model, the mouse kidney was subjected to an extended warm ischemia time to mimic IRI and subsequent acute kidney injury (AKI). Complement inhibition at the level of C2 (using an anti-C2 antibody) early after ischemia was shown to have a protective effect on AKI development.

[0014] Accordingly, in a second aspect, there is provided a method of improving allograft function in a subject receiving a donor organ transplant, the method comprising administering an effective amount of a complement factor 2 (C2) inhibitor to the subject.

[0015] In a third aspect, there is provided a method of reducing the risk of allograft loss in a subject receiving a donor organ transplant, the method comprising administering an effective amount of a complement factor 2 (C2) inhibitor to the subject.

[0016] In a fourth aspect, there is provided a method of treating or preventing delayed graft function in a subject receiving a kidney transplant, the method comprising administering an effective amount of a complement factor 2 (C2) inhibitor to the subject.

[0017] In certain embodiments, the subject is receiving a donor organ from a deceased donor, such as a brain-death donor (DBD or a circulatory-death donor (DCD). Typically, cold-ischemia times (CIT) are longer during deceased donor organ transplants compared to living donor organ transplants. In some embodiments, the donor organ has been subjected to cold-ischemia. In some embodiments, the cold-ischemia time of the donor organ is at least 12 hours.

[0018] In certain embodiments, the donor organ is a kidney and the subject has been diagnosed with end stage renal disease (ESRD). In certain embodiments, the donor organ is a kidney and the subject has been stable on chronic dialysis for at least 3 months. In certain embodiments, the donor organ is a kidney and the subject has not received a previous kidney transplant. In certain embodiments, the donor organ is a kidney and the subject has received at least one previous kidney transplant. In certain embodiments, the donor organ is a kidney and the subject does not require post-transplant dialysis within the first 7 days post-transplant.

[0019] In certain embodiments, a first dose of the C2 inhibitor is administered to the subject on the day of the transplant procedure. In certain embodiments, a first dose of the C2 inhibitor is administered to the subject prior to or during the transplant procedure. In certain embodiments, a first dose of the C2 inhibitor is administered to the subject prior to or during reperfusion of the organ. In certain embodiments, the C2 inhibitor is administered to the subject intravenously. In certain embodiments, the C2 inhibitor is administered to the subject as a continuous intravenous infusion, and administration is initiated prior to reperfusion of the organ. In certain embodiments, administration is initiated and completed before reperfusion of the organ begins.

[0020] In certain embodiments, the C2 inhibitor is administered to the subject at a dose of 0.1 mg / kg to 100 mg / kg. In certain embodiments, the C2 inhibitor is administered to the subject at a dose of 0.1 mg / kg, 0.5 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg or 100 mg / kg. In certain embodiments, the C2 inhibitor is administered to the subject at a dose of 40 mg / kg to 80 mg / kg. In certain embodiments, the C2 inhibitor is administered to the subject at a dose of 60 mg / kg. In certain embodiments, one or more further doses of the C2 inhibitor are administered to the subject post- operatively. In certain embodiments, a post-operative dose is administered to the subject 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14 days post-transplant. In certain embodiments, a postoperative dose is administered to the subject 7-9 days post-transplant. In certain embodiments, the one or more further dose(s) is / are administered to the subject intravenously or subcutaneously. In certain embodiments, the one or more further dose(s) is / are administered to the subject at a dose of 0.1 mg / kg to 100 mg / kg. In certain embodiments, the one or more further dose(s) is / are administered to the subject at a dose of 0.1 mg / kg, 0.5 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg or 100 mg / kg.

[0021] In certain embodiments, the one or more further dose(s) is / are administered to the subject at a dose of 40 mg / kg to 80 mg / kg. In certain embodiments, the one or more further dose(s) is / are administered to the subject at a dose of 60 mg / kg. In certain embodiments, a first dose of the C2 inhibitor is administered to the subject prior to and / or during reperfusion of the organ, and a second dose of the C2 inhibitor is administered to the subject post-operatively. In certain embodiments, the C2 inhibitor is selected from: inhibitory RNA species, for example siRNAs or shRNAs; small molecule inhibitors; biological antagonists including inhibitory peptides, antibody mimetics such as affibodies, affilins, affitins, adnectins, atrimers, evasins, DARPins, anticalins, avimers, fynomers, versabodies and duocalins; antibodies and antigen-binding fragments thereof. In certain embodiments, the C2 inhibitor is an antibody or antigen-binding fragment thereof that specifically binds to C2. In certain embodiments, the antibody or antigen binding fragment thereof specifically binds to C2b. In certain embodiments, the antigen-binding fragment is selected from: an antibody light chain variable domain (VL), an antibody heavy chain variable domain (VH), a single chain antibody (scFv), a F(ab’)2 fragment, a Fab fragment, an Fd fragment, an Fv fragment, a one-armed (monovalent) antibody, a diabody, a triabody, a tetrabody, a unibody, a domain antibody and a nanobody.

[0022] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a variable heavy chain domain (VH) and a variable light chain domain (VL) wherein the VH and VL domains comprise the CDR sequences:

[0023] - HCDR3 comprising or consisting of SEQ ID NO: 4;

[0024] - HCDR2 comprising or consisting of SEQ ID NO: 3;

[0025] - HCDR1 comprising or consisting of SEQ ID NO: 2;

[0026] - LCDR3 comprising or consisting of SEQ ID NO: 7;

[0027] - LCDR2 comprising or consisting of SEQ ID NO: 6; and

[0028] - LCDR1 comprising or consisting of SEQ ID NO: 5.

[0029] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 8 and a VL domain comprising or consisting of the amino acid sequence of SEQ ID NO: 9.

[0030] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a human IgG heavy chain constant domain. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 10 and a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 11 . In certain embodiments, the C2 inhibitor is empasiprubart (also referred to herein as ARGX-117).

[0031] In certain embodiments, the 02 inhibitor reduces complement activity in the serum, plasma and / or urine of the subject. In certain embodiments, the 02 inhibitor does not affect alternative complement pathway activity. In certain embodiments, the 02 inhibitor reduces the level of free C2 in the serum, plasma and / or urine of the subject. As used herein, free C2 (or active C2) means C2 which is not bound by empasiprubart (ARGX-117).

[0032] In certain embodiments, the method further comprises administering to the subject one or more additional therapeutic agents, such as one or more immunosuppressive agents. In certain embodiments, the one or more immunosuppressive agents is selected from: antithymocyte globulin (ATG), tacrolimus, mycophenolate mofetil (MMF), enteric-coated mycophenolic acid (EC-MPA), enteric-coated mycophenolate sodium (EC-MPS), cyclosporine, corticosteroids, azathioprine, everolimus, sirolimus, rapamycin, belatacept, basiliximab, alemtuzumab, and any combination thereof. In certain embodiments, the method comprises the administration of an induction immunosuppressive therapy, preferably antithymocyte globulin (ATG) and corticosteroids. In certain embodiments, the method comprises the administration of a maintenance immunosuppressive therapy, preferably tacrolimus, and MMF or EC-MPA, and optionally corticosteroids.

[0033] In a further aspect, there is provided a C2 inhibitor for use in the treatment or prevention of ischemia reperfusion injury (IRI) in a subject receiving a donor organ transplant according to the methods disclosed herein.

[0034] In a further aspect, there is provided a C2 inhibitor for use in improving allograft function in a subject receiving a donor organ transplant according to the methods disclosed herein.

[0035] In a further aspect, there is provided a C2 inhibitor for use in reducing the risk of allograft loss in a subject receiving a donor organ transplant according to the methods disclosed herein.

[0036] In a further aspect, there is provided a C2 inhibitor for use in the treatment or prevention of delayed graft function in a subject receiving a kidney transplant according to the methods disclosed herein.

[0037] In a further aspect, there is provided a use of a 02 inhibitor in the manufacture of a medicament for the treatment or prevention of ischemia reperfusion injury (IRI) in a subject receiving a donor organ transplant, wherein the treatment or prevention is carried out according to the methods disclosed herein.

[0038] In a further aspect, there is provided a use of a 02 inhibitor in the manufacture of a medicament for improving allograft function in a subject receiving a donor organ transplant, wherein the treatment or prevention is carried out according to the methods disclosed herein. In a further aspect, there is provided a use of a C2 inhibitor in the manufacture of a medicament for reducing the risk of allograft loss in a subject receiving a donor organ transplant, wherein the treatment or prevention is carried out according to the methods disclosed herein.

[0039] In a further aspect, there is provided a use of a C2 inhibitor in the manufacture of a medicament for the treatment or prevention of delayed graft function in a subject receiving a kidney transplant, wherein the treatment or prevention is carried out according to the methods disclosed herein.

[0040] BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 shows IgM binding to and complement activation on primary lung-derived endothelial cells (ECs) upon hypoxia and reoxygenation. A Lower ATP levels are measured in cells after 24h culture in hypoxic conditions (0.1 % O2). B 24hexposure to hypoxia increases the number of AnnexinV (AnV)+ and AnV+ / 7 -Aminoactinomycin (7AAD)+ cells. C Reoxygenation of hypoxic cells in the presence of 30% serum results in increased IgM binding, which is not affected by heat inactivation of the serum, or preincubation of the serum with anti-complement antibodies. Reoxygenation in the presence of IgG / IgM-depleted serum completely abrogates IgM binding. D After hypoxia, reoxygenation in the presence of 30% human serum increased C3 fixation. Heat inactivated (HI) serum was included as a negative control for C3 fixation. ARGX-117 dose- dependently inhibited C3 fixation, whereas no inhibition was seen when serum was preincubated with an isotype control. Limited inhibition of C3 fixation was observed when serum was preincubated with an anti-01 s antibody. In line with the abrogated IgM binding, no C3 fixation was observed when IgG / IgM-depleted serum was used as a source of complement. Both IgM binding (E) and C3 fixation (F) were predominantly observed on AnV+ and AnV+ / 7AAD+ cells and not on living cells. APC: allophycocyanin; MFI: mean fluorescent intensity.

[0042] Figure 2 shows IgM binding and complement activation on primary lung-derived ECs after hypoxia and reoxygenation in human serum. A IgM binding to primary lung-derived ECs cultured for 24h in 0.1 % O2 was increased in multiple experiments (n=3 / 4) compared to the normoxic serum control (depicted as fold change (FC) with the normoxia serum control from the respective experiment set at 1 ). The mean IgM binding for all serum conditions was comparable. No IgM binding was observed when IgM / IgG-depleted serum was used as source of complement. B ARGX-117 dose-dependently inhibited complement activation on hypoxic primary lung-derived ECs, with a maximum inhibitory capacity of approximately 95% at a concentration of 2 mg / ml. An anti-C1 s antibody inhibited C3 fixation up to 60% for the concentrations analyzed. No inhibitory effect was observed upon pre-incubation of complement active serum with an isotype control. Each depicted single bullet represents data from an independent experiment. Mean + SEM from a maximum of 4 individual experiments.

[0043] Figure 3 shows IgM binding to and complement activation on primary kidney-derived ECs upon hypoxia and reoxygenation. A Lower ATP levels are measured in cells after 24h culture in hypoxic conditions (0.1% O2). B 24h exposure to hypoxia resulted in increased AnnexinV (AnV)+ and AnV+ / 7-Aminoactinomycin (7AAD)+ cells. C Reoxygenation of hypoxic cells in the presence of 30% serum increased IgM binding, which was not affected by preincubation of serum with anticomplement antibodies or heat inactivation. Equal IgM binding was observed using (reconstituted) C2-depleted serum as a source of complement D After hypoxia, reoxygenation in the presence of 30% human serum increased C3 fixation. Heat inactivated (HI) serum was included as a negative control for C3 fixation. ARGX-117 inhibited C3 fixation, whereas no inhibition was seen when serum was pre-incubated with an isotype control (isotype ARGX-117). Limited inhibition of C3 fixation was observed when serum was preincubated with an anti-C1 s antibody. Upon incubation with C2-depleted serum, 03 fixation was largely inhibited. C2-depleted serum reconstituted with human C2 restored C3 fixation to the level of the serum control. Both IgM binding (E) and C3 fixation (F) were predominantly observed on AnV+ and AnV+ / 7AAD+ cells and not on living cells.

[0044] Figure 4 shows IgM binding and complement activation on primary kidney-derived ECs after hypoxia and reoxygenation in human serum. A Increased IgM binding to primary kidney-derived ECs cultured for 24h in 0.1 % O2 (n=5) compared to normoxic serum control (depicted as fold change (FC) with the normoxia serum control from the respective experiment set at 1 ). B ARGX- 117 dose-dependently inhibited complement activation on hypoxic kidney ECs, with a maximum inhibitory capacity of 75% at a concentration of 2 mg / mL. An anti-01 s antibody inhibited 50% of 03 fixation for the maximum concentration analyzed. No inhibitory effect was observed upon preincubation of complement active serum with an isotype control or an anti-05 antibody. C Complement activation on hypoxic kidney ECs is shown to be 02 dependent. Setting the mean fluorescent intensities (MFIs) for C2-depleted serum at 100% and C2-depleted serum reconstituted with 02 at 0% inhibition respectively, 03 fixation was restored at a 02 concentration above 1 .97 pg / mL. Fully reconstituted C2-depleted serum (30 pg / mL 02) preincubated with ARGX-117 completely abrogated 03 fixation above the level of C2-depleted serum, presumably due to residual 02 in the C2-depleted serum source. Each depicted single bullet represents the data from an independent experiment. Mean + SEM from a maximum of 4 individual experiments. Depl: depleted. Figure 5 shows a 3D human proximal tubule / endothelial vessel on-a-chip co-culture model.

[0045] A Photograph of the microfluidic culture platform, the OrganoPlate 3-lane 40 and a zoom-in on 1 of the 40 chips. B Schematic representation of a single chip indicating three channels that join in the center and are separated by two phaseguides. C Three-dimensional (3D) schematic of the co-culture model comprising renal proximal tubule epithelial cells (RPTEC) in the top channel, human umbilical vein endothelial cells (HUVEC) in the bottom channel, and in the middle a collagen I extracellular matrix (ECM) gel. D Phase contrast image of the co-culture at day 6 of culture (left) and zoom-in images of both tubules (right). E 3D immunofluorescent reconstruction showing both tubules and a visible lumen of the tubules. DNA was stained with Hoechst to indicate the cell nuclei. Scalebars in white = 100 pm.

[0046] Figure 6 shows quantification of immunostaining for IR-induced C3 and IgM deposition on HUVECs, in the presence or absence of ARGX-117. A Schematic depicting the culture conditions for the cells in the co-culture model. Cultures were exposed to 48h ischemia (1% O2, static) (or normoxia as control) followed by 24h reperfusion (21 % O2, perfusion). During reperfusion, medium supplemented with 30% human complement-active serum (= serum) + / - ARGX-117 or isotype control, medium supplemented with 30% heat-inactivated (HI) serum, or medium-only, was added to the HUVEC vessel. B Quantification of C3 deposition. In the presence of complement-active serum, IR increased C3 deposition as compared to normoxia- reperfusion conditions. ARGX-117 dose-dependently blocked C3 deposition. Data of three independent experiments (step 4 iterations 1 , 2, and 3) are shown. n=2-15 depending on the condition and the iteration. C Pooled C3 data were analyzed by Kruskal-Wallis test. D Quantification of IgM deposition. In the presence of HI serum or serum, IR increased IgM deposition as compared to normoxia-reperfusion conditions. IgM deposition was not affected by ARGX-117. Data of three independent experiments (step 4 iterations 1 , 2, and 3) are shown. n=2-8 depending on the condition and the iteration. E Pooled IgM data by One-way ANOVA. Statistics shown compare all IR conditions to the “IR condition supplemented with serum”. Graphs show mean ± standard deviation, ns = not significant, * P<0.05, **** P<0.0001 . norm. + rep. = normoxia + reperfusion, isch. + rep. = ischemia + reperfusion. Step 4.1 , 4.2 and 4.3 = step 4 iterations 1 , 2 and 3, respectively.

[0047] Figure 7 shows the results from the human free C2 ELISA assay: samples derived from cell culture under normoxic or ischemia conditions and treated with ARGX-117 or controls. A Results obtained for the supernatant samples of HUVECs. B Results obtained for the supernatant samples of RPTECs. HI serum: heat-inactivated serum. LOQ: limit of quantification. RPTEC: human renal proximal tubular epithelial cells. HUVEC: human umbilical vein endothelial cells. ARGX-117 / isotype conditions: medium + 30% complement-active serum was supplemented with indicated concentration of antibody (depicted concentration = actual concentration on the plate). Lower limit of quantification: 39.06 ng / mL in 100% supernatant. Samples below the quantification limit were plotted as limit of quantification / 2. ns: non-significant. *p-value = 0.00144, **p-value < 0.00015.

[0048] Figure 8 shows quantification of immunostaining for ICAM-1 on RPTECS following an IR insult in the presence or absence of human serum + / - ARGX-117. Cultures were exposed to 48h ischemia (1 % O2, static) (or normoxia as control) followed by 24h reperfusion (21 % O2, perfusion). During reperfusion, medium supplemented with 30% human complement-active serum (= serum) + / - ARGX-117 or isotype control, medium supplemented with 30% heat- inactivated (HI) serum, or medium-only, was added to the HUVEC vessel. ICAM-1 expression is quantified. Addition of ARGX-117 dose-dependently reduced IR-induced ICAM-1 expression. n=5-12 depending on the condition.

[0049] Figure 9 is a schematic of the experimental design for the rat kidney transplant model in which rats received a kidney exposed to a long cold ischemia time (CIT). Donor kidneys from syngeneic donors were perfused and stored in UW solution at 4°C (cold storage duration of 32 hours). Subsequently these donor kidneys were transplanted into syngeneic recipients. The recipient rats received a bolus IV injection of anti-C2 mAb, anti-C5 mAb or isotype control, immediately (30-60s) before reperfusion, and an additional injection 72 hours later (day 4 of the study). Serum samples were collected on day 3, 5, 7 and / or 14. Animals were euthanized when humane endpoints were reached or on day 14 (or on day 3, 5 or 7 for histology cohorts).

[0050] Figure 10 compares serum creatinine concentrations in male Lewis rats following kidney transplantation and treatment with anti-C2 mAb or isotype control. Blood sampling was performed on day 3 (with surgery day = day 1 ). Serum creatinine was quantified from the blood samples to determine kidney function after transplantation. Serum creatinine concentrations were significantly reduced following treatment with the anti-02 mAb compared to the isotype control.

[0051] Figure 11 compares A urea (UREA) and B serum creatinine (GREAT) concentrations over time in male Lewis rats following kidney transplantation and treatment with an anti-C2 mAb (Group 10), an anti-C5 mAb (Group 11 ), or an isotype control (Group 9). Blood sampling was performed on day 3, 5, 7 and 14 (with surgery day = day 1 ). Serum creatinine and urea were quantified from the blood samples to determine kidney function after transplantation. Serum creatinine (on day 3 and on average across time-points) and urea (on day 3) concentrations were significantly reduced following treatment with either an anti-C2 mAb or an anti-C5 mAb compared to the isotype control. Shown are mean ± SEM. Figure 12 shows rat serum free C2 levels measured from serum samples at baseline (pretransplant donor values) and after transplantation in recipient rats treated with an anti-C2 mAb (Groups 7 and 8) or an isotype control (Group 6). The lower limit of quantification is 0.9375 pg / mL in 100% rat serum which corresponds to >95% reduction of free 02. Samples below the quantification limit were plotted as limit of quantification / 2. Surgery day = day 1 . The first dose of anti-C2 mAb (or isotype control) was infused on day 1 , immediately prior to anastomoses.

[0052] Figure 13 shows rat serum free C2 levels measured from serum samples at baseline (Day -1 pre-transplant donor values) and after transplantation in recipient rats treated with an anti-02 mAb, an anti-05 mAb or an isotype control. The lower limit of quantification is 0.9375 pg / mL in 100% rat serum which corresponds to >95% reduction of free C2. Samples below quantification limit were plotted as limit of quantification / 2. Surgery day = day 1 . First dose of the anti-02 antibody, the anti-05 antibody or the isotype control antibody was infused on day 1 , immediately prior to anastomoses.

[0053] Figure 14 provides an overview for the clinical study described in Example 4. The study duration is approximately 64 weeks, comprising the following study periods: Screening period: <24 hours; Treatment and evaluation period: 52 weeks; and Follow-up period: 12 weeks. D=day; IMP=investigational medicinal product; IV=intravenous; n=number of participants; W=week.

[0054] Figure 15 provides A A graphical representation of the experiment 1 set-up. Two groups (isotype and anti-02 antibody treated, 6 animals per group) of allograft (BALB / c to C57BL / 6) transplantation were performed and followed up for 3 weeks. At different timepoints blood samples were collected and micro-CT was performed (indicated respectively with a drop and microscope icon). At sacrifice, bronchoalveolar lavage (BAL) samples were collected, and left lung cells were used for FACS analysis. The right lungs and spleens were collected and snap frozen. IP: intraperitoneal, SC: subcutaneous, BAL: bronchoalveolar lavage, Tx: transplant, PFA: paraformaldehyde. B Cold ischemic time in experiment 1 . C Total time of transplantation in experiment 1 .

[0055] Figure 16 provides a graphical representation of the experiment 2 set-up. Two groups (isotype and anti-C2 antibody treated, 6 animals per group) of allograft (BALB / c to C57BL / 6) transplantation were performed and followed up for 7 days. At different timepoints blood samples were collected and micro-CT was performed (indicated respectively with a drop and microscope icon). At sacrifice, BAL samples were collected and left lung was either used for FACS analysis (3 / 6 animals) or for immunohistochemistry (3 / 6 animals). The liver, thymus and spleen were collected and snap frozen. The right lung was either snap frozen (3 / 6 animals) or fixed and embedded (3 / 6 animals). IP: intraperitoneal, SC: subcutaneous, BAL: bronchoalveolar lavage, Tx: transplant, PFA: paraformaldehyde

[0056] Figure 17 shows A Free “unbound” C2 analysis via ELISA of complement preserved serum samples from experiment 1 . Complete blocking of C2 was observed in all anti-C2 treated animals except for A9 and A11 which exhibit C2 values similar to isotype control treated at POD 3, suggesting misdosing of these animals. Isotype treated animals exhibited a sustained decrease in serum C2 levels throughout the experimental period. B C3a analysis via ELISA of complement preserved serum samples from experiment 1 . A reduction of C3a levels was observed in all anti- 02 treated animals except for A9 and A1 . Isotype treated animals exhibited an increase in serum C3a levels throughout the experimental period. POD: post operative day, AB: antibody

[0057] Figure 18 shows A Free “unbound” C2 analysis via ELISA of BAL samples from experiment 1 . No 02 levels were detected in anti-02 treated animals. 02 levels in isotype treated animals vary between 14 and 257 ng / mL. B 03a analysis via ELISA of BAL samples from experiment 1 . No difference in 03a levels were observed between isotype versus anti-02 treated animals in BAL. POD: post operative day

[0058] Figure 19 shows the micro-CT density analysis of experiment 1 . A slight improvement of pCT density was observed for the anti-02 treated animals as compared to the isotype treated group. micro-CT: micro computed tomography, WP: work package, POD: post operative day, HU: Hounsfield Units

[0059] Figure 20 shows A Free “unbound”C2 analysis via ELISA of complement preserved serum samples from experiment 2. Complete blocking of 02 was observed in all anti-02 treated animals. Isotype treated animals exhibited a sustained decrease in serum 02 levels throughout the experimental period. B 03a analysis via ELISA of complement preserved serum samples from experiment 2. A reduction of C3a levels was observed in all anti-02 treated animals. Isotype treated animals exhibited an increase in serum 03a levels throughout the experimental period. One isotype treated animal B4 was misdosed at POD 3 with anti-02 treatment and also illustrates low 03a levels at POD3. POD: post operative day

[0060] Figure 21 shows A Free 02 analysis via ELISA of BAL samples from experiment 2. No 02 levels were detected in anti-02 treated animals. 02 levels in isotype treated animals vary between 2.5 and 264 ng / mL. B 03a analysis via ELISA of BAL samples from experiment 2. No difference in C3a levels were observed between isotype versus anti-02 treated animals in BAL. Figure 22 shows the results from the experiment 2 proinflammatory panel serum sample assay. POD 0 was a pre-treatment sample and is therefore a baseline level. A IFN- y serum levels; B IL- 1 P serum levels; C IL-5 serum levels; D IL-6 serum levels; E KC GRO serum levels; F IL-10 serum levels; G TNF-aserum levels; H IL-12p70 serum levels; I IL-2 serum levels; J IL-4 serum levels. Dotted line = LLOQ value

[0061] Figure 23 shows A Visual representation of histopathology scoring of TUNEL, MPO, C5b9, C4d and C3d (0: no staining, 1 : weak (+), 2: moderate (++), 3: strong (+++)). A reduction of C5b9 and C3d was observed for anti 02 treated animals as compared to isotype treated. (Perivascular inflammation:- 0: no perivascular inflammation, 1 : minimal acute rejection, 2: mild acute rejection, 3: moderate acute rejection, 4: severe acute rejection). B Visual representation of the histopathology scoring of H&E. (Lymphocytic bronchitis:- 0: no airway inflammation, 1 : low- grade airway inflammation, 2: high-grade airway inflammation). A beneficial effect on lymphocytic bronchitis was observed for anti-02 treated animals as compared to isotype treated ones.

[0062] Isotype treated animal B5 is indicated with a ‘x’ on the figure given this animal presented as a normal healthy lung but appeared not functional (no inflation).

[0063] Figure 24 shows C2 analysis of a subset of lung transplant plasma EDTA samples as measured using ELISA. EDTA: ethylenediaminetetraacetic acid, LTx: lung transplant; NHP: normal human plasma, POD: post operative day; Tx: transplant

[0064] Figure 25 shows 03a analysis of a subset of lung transplant plasma EDTA samples as measured using ELISA. EDTA: ethylenediaminetetraacetic acid, LTx: lung transplant; LLOQ: lower limit of quantification, ULOQ: upper limit of quantification; NHP: normal human plasma, POD: post operative day; Tx: transplant

[0065] Figure 26 shows C3bc analysis of a subset of lung transplant plasma EDTA samples as measured using ELISA. EDTA: ethylenediaminetetraacetic acid, LTx: lung transplant; LLOQ: lower limit of quantification; NHP: normal human plasma, POD: post operative day; Tx: transplant

[0066] Figure 27 shows human plasma complement dynamics in lung transplantation. All analytes (C1 q, C2, C3, 04, 05 and 09) in 100% EDTA plasma / BCA (ng / mg) as measured using Luminex analysis. The black line connects the median across time points. BCA: bicinchoninic acid, POD: Figure 28 shows human plasma complement dynamics in lung transplantation. All analytes (C3b / iC3b, MBL, Factor D, and Factor B) in 100% EDTA plasma / BCA (ng / mg) as measured using Luminex analysis. The black line connects the median across time points. BCA: bicinchoninic acid, MBL: mannose-binding lectin, POD: post operative day, preTx: pre transplant, p-value (comparison to preTx): ns = not significant, * < 0.05, ** <0.01, *** < 0.001

[0067] Figure 29 shows a schematic overview of the relative abundance of each complement factor in BAL / BCA compared to plasma / BCA. BAL: broncho alveolar lavage; BCA: bicinchoninic acid; MBL: mannose binding lectin; POD: post operative day

[0068] Figure 30 shows human plasma inflammatory analyte dynamics in lung transplantation. All analytes (neutrophil elastase, CRP, IL-6, IL-8, IL-10, MCP-1 , IP10 and VCAM-1 ) in 100% EDTA plasma / BCA (ng / mg) as measured using MSD multiplex analysis. The black line connects the median across time points. BCA: bicinchoninic acid, CRP: C-reactive protein, IL: interleukin, IP-

[0069] Figure 31 shows human plasma inflammatory analyte dynamics in lung transplantation. All analytes (ICAM-1 , VEGF-A, VEGF-C and VEGF-D) in 100% EDTA plasma / BCA (ng / mg) as measured using MSD multiplex analysis. The black line connects the median across time points.

[0070] Figure 32 shows the optimisation of ischemia reperfusion time in Balb / C humanized C2 transgenic mice. Eighteen minutes (A) and 21 minutes (B) ischemia time. Kidney injury is indicated by staining for KIM1 in grey (early injury marker), SOX9 in red (repair response marker), and aSMA in green (fibrosis marker). C Fold-change to baseline of Havcrl (KIM1 ) gene expression over-time (48 hours and 21 days) after 18 min and 21 min ischemia time. Error bars indicate mean + SD. aSMA: alpha-smooth muscle actin, KIM1: kidney injury molecule 1, min: minutes

[0071] Figure 33 shows serum analysis of free C2, classical pathway complement activation and NGAL levels after different ischemia times. A Free C2 levels measured in sham control, and animals exposed to different warm ischemia times. B Classical pathway activation depicted as fold change to MgEGTA. C NGAL levels measured in sham control, and animals exposed to different warm ischemia times. In all panels Tg mouse serum was used as control. Error bars indicate mean + SD. h: hour, min: minutes, MgEGTA: magnesium ethylene glycol tetraacetic acid, NGAL: neutrophil gelatinase-associated lipocalin, Tg: transgenic, ULOQ: upper limit of quantification

[0072] Figure 34 shows kidney injury response and complement markers after ischemia-induced kidney injury in presence of a complement inhibitor targeting 02. A Area positive (%) for KIM1 (early damage marker) staining in the kidney cortex or outermedulla. hC2 indicates dosing with anti human C2 antibody BRO-02, control indicates dosing with the isotype control antibody. B Area positive (%) for KIM1 staining 21 days post-ischemia (18 min) after dosing with control or hC2 antibody. C Area positive (%) for aSMA (fibrosis marker) 21 days post-ischemia (18 min) after dosing with control or hC2 antibody. D Classical pathway activation depicted as 03c deposition after incubation with serum samples from different warm ischemia conditions. E Free 02 levels measured in sham control, and animals exposed to different warm ischemia times. F NGAL levels measured in untreated healthy, sham control, and animals exposed to different warm ischemia times. Error bars indicate mean + SD. * P<0.05. aSMA: alpha-smooth muscle actin, d: day, h: hour, IRI: ischemia reperfusion injury, KIMI: kidney injury molecule 1, nm: nano meter

[0073] Figure 35 shows kidney injury response and complement markers after ischemia-induced kidney injury in presence of a complement inhibitor targeting 05. A Area positive (%) for KIM1 staining 21 days post-ischemia (18 min) after dosing with control or hC5 antibody (anti-human 05 antibody BB5.1 ). B Area positive (%) for aSMA (fibrosis marker) 21 days post-ischemia (18 min) after dosing with control or hC5 antibody. C Classical pathway activation depicted as C5b9 deposition after incubation with serum samples from different warm ischemia conditions. Error bars indicate mean + SD. aSMA: alpha-smooth muscle actin, d: day, h: hour, IRI: ischemia reperfusion injury, KIM1: kidney injury molecule 1, MgEGTA: magnesium ethylene glycol tetraacetic acid, nm: nano meter, ns: not significant

[0074] DETAILED DESCRIPTION

[0075] A. Definitions

[0076] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by the ordinary person skilled in the art to which the invention pertains. Without limiting any term, further clarifications of some of the terms used herein are provided below. As used herein, the term “transplantation” or “transplant” is intended to refer to the medical procedure whereby an organ is obtained from a “donor” and transferred into the body of a “recipient”. The donor and recipient are typically of the same species e.g. both human, but may be different in some cases. “Allotransplantation” refers to the transplantation of an organ (or cells or tissue) to a recipient from a genetically non-identical donor of the same species. In “syngeneic transplantation”, the donor is an identical twin, making the graft genetically identical to the recipient. Solid organ transplantation or solid organ transplant typically involves transfer of a kidney, lung, heart, pancreas, bowel or liver. If the organ is taken from a deceased donor, this can be referred to as “deceased donor transplantation”.

[0077] As used herein, the term “ischemia reperfusion injury” or “IRI” refers to the situation that can arise when an initial reduction in blood flow to an organ or tissue (ischemia) is followed by restoration of blood flow (reperfusion) causing damage or “injury” to the organ or tissue. Increased production of reactive oxygen species, necrosis, vascular injury, and increase in mucosal permeability are some of the prominent features of IRI. In the context of the present invention, “ischemia reperfusion injury (IRI) in a subject receiving a donor organ transplant” refers to the IRI occurring specifically as a consequence of organ transplantation. During organ transplantation, the donor organ suffers from ischemia when the blood supply to the organ is interrupted. Once transplanted into the recipient, blood supply to the donor organ is restored, resulting in rapid reoxygenation of the organ. This re-establishment of blood flow to previously ischemic tissue results in significant injury to the organ. Accordingly, the present invention is concerned with IRI associated with (i.e. occurring as a result of) solid organ transplant. This situation is to be distinguished from IRI that occurs in other disease states or conditions such as the ischemic injury involved in stroke, myocardial infarction or peripheral vascular disease, for instance.

[0078] As used herein, “warm-ischemia time” is defined as the time a tissue, organ, or body part remains at body temperature after its blood supply has been reduced or cutoff but before it is cooled or reconnected to a blood supply.

[0079] As used herein, “cold-ischemia time” or “CIT” is defined as the time between the chilling of a tissue, organ, or body part after its blood supply has been reduced or cut off and the time it is warmed by having its blood supply restored. Chilling can occur while the organ is still in the body or after it is removed from the body if the organ is to be used for transplantation. Acceptable cold ischemia time limits vary from organ to organ. Prolonged cold ischemia time is an independent risk factor for the development of primary nonfunction (PNF) of the allograft and delayed graft function (DGF) in kidney or renal transplant recipients. In particular, a cold ischemia time of >12 hours in renal transplant recipients of deceased donors increases the risk of delayed graft function.

[0080] As used herein, the term “reperfusion of the organ” refers to the process whereby blood supply is restored to the donor organ following transplantation into the recipient. As discussed above, reperfusion of a donor organ is often associated with tissue injury, referred to as “ischaemiareperfusion injury” or “IRI”.

[0081] As used herein, the term "graft" or "allograft” may be used to refer to the transplanted material i.e. the tissue of the donated organ. Allografts, by definition, comprise tissue from a genetically non-identical donor of the same species as the recipient. “Allograft injury” is common in transplant recipients as a result of the recipient’s immune system mounting a response against the donor organ. Allograft injury or allograft damage typically s results in donor organ dysfunction and can, ultimately, lead to failure of the transplanted organ.

[0082] As used herein, “allograft dysfunction” is the term used to describe the reduced organ function that may be observed in transplant recipients as a result of allograft injury or allograft damage, for example the damage caused following ischemia-reperfusion. Allograft dysfunction may be categorised in various ways. Early dysfunction typically occurs within <6 months post-transplant, whereas late dysfunction typically occurs >6 months post-transplant. Early and late dysfunction may also be referred to as acute and chronic dysfunction, respectively. The invention herein provides methods for improving allograft function in a subject receiving a donor organ transplant. Improved allograft function may include improvements in early (acute) allograft function and / or improvements in late (chronic) allograft function.

[0083] In some case, allograft dysfunction may progress to “allograft loss”, a situation wherein the transplanted organ fails altogether. The invention herein also provides methods for reducing the risk of allograft loss in a subject receiving a donor organ transplant. The term “allograft loss” is used herein interchangeably with “allograft failure” and encompasses “allograft rejection”.

[0084] As used herein, “delayed graft function” or “DGF”, refers to a situation occurring in kidney transplant recipients - whereby the donor kidney fails to work immediately upon transplantation. DGF may be regarded as a form of acute kidney allograft dysfunction. The most common clinical definition of DGF, also known as “dialysed-DGF” is the requirement for one or more lifesustaining dialysis treatments within the first 7 days post-transplantation, prior to the onset of graft function. Note that the term “life-sustaining dialysis treatment” is used herein to differentiate life-sustaining dialysis required as a result of graft dysfunction or failure, from supportive dialysis required for other reasons such as for the management of fluid or electrolyte disturbances (e.g. hyperkalemia). Other criteria may be used to classify a subject as having DGF. An alternative definition of DGF, often termed, “functional delayed graft function (fDGF)” or “non-dialysed- DGF” is a failure of the serum creatinine level to decrease by at least 10% daily on 3 successive days during the first week post-transplantation (regardless of the need for dialysis). As discussed elsewhere herein, persistent elevated serum creatinine levels are indicative of graft failure. DGF remains one of the main predictors of poor graft survival, particularly after deceased donor transplantation. DGF has been shown to be a risk factor for chronic kidney allograft dysfunction and allograft failure (Yarlagadda et al. 2009. Nephrol Dial Transplant, 24: 1039-1047).

[0085] Serum creatinine levels are commonly used as measure of kidney function. As used herein, the term “creatinine reduction ratio (CRR)” is a measure of the reduction in serum creatinine levels on any given day post-transplant. CRR2% (wherein CRR2 is the CRR on post-transplant day 2) may be calculated as follows: CRR2% = ([Cr1 — Cr2]x 100) / Cr1 , where Cr1 and Cr2 are serum creatinine on post-transplant day 1 and day 2, respectively. The CRR may be used as a prognostic marker for DGF and / or as a criterion for diagnosis of DGF at earlier than 1 week. The CRR may also predict longer-term transplant outcomes. CRR2 for example correlates with renal function throughout the first year. Early diagnosis of DGF may be defined as CRR2% < 30.

[0086] As used herein, “estimated glomerular filtration rate” or “eGFR” refers to a measure of kidney function. Estimated glomerular filtration rate (eGFR) is the best overall index of kidney function. Normal eGFR varies according to age, sex, and body size, and declines with age. The National Kidney Foundation recommends using the CKD-EPI Creatinine Equation (2021 ) to estimate eGFR (see equation below): eGFR = 142 x min(Scr / K, 1 )a x max(Scr / K, 1 )-1 .200 x 0.9938Age x 1.012 [if female], where: Scr is serum creatinine in mg / dL, K is 0.7 for females and 0.9 for males, a is -0.241 for females and -0.302 for males, min indicates the minimum of Scr / K or 1 , and max indicates the maximum of Scr / K or 1 .

[0087] Age (years)

[0088] Normal eGFR is usually >90 ml / min / 1 ,73m2. Kidney damage is graded into 5 stages based on the eGFR:

[0089] Stage 2 - Mild damage: 60-89 ml / min / 1 ,73m2 Stage 3a - Mild-moderate damage: 45-59 ml / min / 1 ,73m2

[0090] Stage 3b - Moderate-severe damage: 30-44 ml / min / 1 ,73m2

[0091] Stage 4 - Severe damage: 15-29 ml / min / 1 ,73m2

[0092] Stage 5 - Kidney failure / ESRD: <15 ml / min / 1 ,73m2.

[0093] As used herein, the terms “preoperative” or “pre-transplant” are used to refer to an event, e.g. administration of a dose of a C2 inhibitor, occurring before the surgical transplant operation in the recipient.

[0094] As used herein, “perioperative”, “peritransplant”, or “intraoperative” are used to refer to an event occurring during the surgical transplant operation in the recipient.

[0095] As used herein, “postoperative” or “post-transplant” are used to refer to an event occurring after the surgical transplant operation in the recipient.

[0096] As used herein, “deceased brain-death donation (DBD)” refers to organ donation that follows the confirmation of death using neurological criteria. A deceased brain-death donor has been confirmed dead using neurological criteria. The timing of withdrawal of life-sustaining treatment is important in determining the potential for DBD. Thus, the potential for DBD varies from country to country and appears to be related to the end-of-life care practices in that country. DBD is the most common form of deceased organ donation in the UK.

[0097] As used herein, “deceased circulatory-death donation (DCD)” refers to organ donation that follows the confirmation of death using cardiorespiratory criteria. A deceased circulatory-death donor has been confirmed dead using cardiorespiratory criteria. Donation after circulatory death may follow unsuccessful cardiopulmonary resuscitation (uncontrolled DCD); the planned withdrawal of life-sustaining treatments (controlled DCD); or after euthanasia (in countries where this has been legalised).

[0098] As used herein, “recipient” may be used synonymously with “subject” or “patient” to refer to the individual receiving a donated organ. In preferred embodiments described herein, the recipient / subject / patient is a human.

[0099] As used herein, the term “baseline” may refer to a measurement (e.g., free C2 level) in a recipient (e.g., in a recipient’s plasma, serum, or urine) prior to administration of a C2 inhibitor. A second measurement may be taken after administration of the C2 inhibitor and compared to baseline to confirm the inhibitory effects of the C2 inhibitor. The second measurement may be presented as a percentage (e.g. a percent reduction or percent increase) of the baseline value. Repeat measurements may be taken over an extended time period, e.g. weeks or months, in order to monitor effects of the C2 inhibitor over time. In this scenario, a baseline measurement may refer to any earlier measurement taken in the recipient, and does not necessarily need to have been taken prior to administration of a C2 inhibitor. For example, a C2 inhibitor may be administered to a recipient one week after transplantation; the level of free C2 may be measured at 2 months post-transplant and compared to a baseline level of free C2 taken at 2 weeks posttransplant.

[0100] As used herein, the “complement system” is an important branch of the innate immune system. The complement system is made up of a closely regulated cascade system of more than 30 soluble and surface-expressed proteins. Activation of the system leads to an activation cascade where one factor activates the subsequent one by specific proteolysis of a complement protein further downstream in the cascade. This cascade can ultimately result in: the production of anaphylatoxins that attract and activate macrophages and leukocytes; formation of the lytic membrane attack complex (MAC); and opsonization of targets for phagocytosis and destruction. Activation of the complement system can occur via three pathways: the classical pathway; the lectin pathway; and the alternative pathway. Each pathway activates a central component, C3, which results in the activation of a common termination pathway leading to the formation of the membrane attack complex (MAC).

[0101] The classical pathway is often referred to as antibody-dependent because it is strongly initiated by IgM or IgG clusters. This pathway is usually activated when the hexameric C1 q binds to the Fc regions of IgG or IgM molecules that are present in an antibody / antigen complex. Upon binding, C1 s cleaves C4 to produce C4a and C4b. Next, C2 binds to surface-bound C4b (in the presence of Mg2+) to form a C4bC2 complex, which is then cleaved by activated C1 s into two fragments: a smaller 30 kDa fragment C2b, and a larger 70 kDa fragment C2a, which remains attached to C4b to form the C4bC2a classical pathway C3 convertase. The C3 convertase is capable of cleaving C3 into C3a (an anaphylatoxin; enhances inflammation) and C3b, thereby initiating amplification and downstream effector functions.

[0102] Activation of the lectin pathway is mediated by binding of mannose-binding lectins (MBLs), or ficolins, to bacterial carbohydrate motifs expressed on the surface of pathogens or microbes. MBL binding subsequently stimulates the activation of MBL-associated serine proteinase-1 (MASP-1 ) and MASP-2 leading to cleavage of 04 and C2 leading to generation of the C4bC2a lectin pathway C3 convertase. The alternative pathway may be considered an amplification loop that is engaged regardless of the initial trigger. C3b directly binds to targets on cell surfaces of microbes, foreign material or damaged tissue. The surface-bound C3b can then bind factor B to form C3bB. This complex in the presence of factor D is cleaved into Ba and Bb. Bb remains associated with C3b to form C3bBb, which is the alternative pathway C3 convertase.

[0103] The three pathways converge at the central C3 convertases. The C3 convertases, C4bC2a or C3bBb, form multimeric complexes with additional C3b molecules, yielding the C5 convertases, C4bC2aC3b and C3bBbC3b, respectively. These enzymes preferentially cleave complement factor C5, releasing C5a (an anaphylatoxin; enhances inflammation) and the fragment C5b. C5b recruits and associates with C6 and C7; the complex becomes inserted into cell membranes and interacts with C8; which induces the binding of multiple units of C9 molecules to form the C5b-9 membrane attack complex (MAC) or soluble Terminal Complement Complex (TCC).

[0104] As used herein, “CH50 assay” or “50% haemolytic complement assay” or “total complement activity assay” is the most commonly used method of screening patient sera for functional activity of the classical complement pathway. The assay is also suited to assess the effects of pharmaceuticals on inhibition or consumption of complement components. The CH50 tests the functional capability of serum complement components of the classical pathway to lyse sheep red blood cells (SRBC) pre-coated with rabbit anti-sheep red blood cell antibody (haemolysin). When antibody-coated SRBC are incubated with test serum, the classical pathway of complement is activated and haemolysis results. A fixed volume of optimally sensitised SRBC is added to various dilutions of test serum. After incubation, the mixture is centrifuged and the degree of haemolysis is quantified by measuring the absorbance of the haemoglobin released into the supernatant at 540nm. The amount of complement activity is determined by examining the capacity of each dilution of test serum to lyse antibody coated SRBC. The titer at which 50% hemolysis occurs (CH50 unit) is proportional to the functional activity of the classical pathway in the serum.

[0105] As used herein, “complement component 2” or “C2” is a 90-100 kDa glycoprotein which participates in the classical and lectin pathways of complement activation. C2 can be activated by C1 s of the classical pathway or activated by MASP2 of the lectin pathway. C2 binds to surface-bound C4b (in the presence of Mg2+) to form a C4bC2 complex, which then is cleaved by activated C1 s or MASP2 into two fragments: a larger 70 kDa fragment C2a, which remains attached to C4b to form a C3-convertase (C4bC2a), and a smaller 30 kDa N-terminal fragment C2b, which is released into the fluid phase. Once activated and bound to C4b, C2a constitutes the catalytic subunit of the C3 and 05 convertases which are able to cleave 03 and 05, respectively.

[0106] The amino acid sequence of human 02 is known (GenBank Accession No. NM 000063) and shown as SEQ ID NO: 1 below.

[0107] Amino Acid Sequence of human C2 (SEQ ID NO: 1 ):

[0108] 1 MGPLMVLFCL LFLYPGLADS AP SCPQNVNI SGGTFTLSHG WAPGSLLTYS CPQGLYP SPA

[0109] 61 SRLCKS SGQW QTPGATRSLS KAVCKPVRCP APVSFENGIY TPRLGSYPVG GNVSFECEDG

[0110] 121 FILRGSPVRQ CRPNGMWDGE TAVCDNGAGH CPNPGI SLGA VRTGFRFGHG DKVRYRCS SN

[0111] 181 LVLTGS SERE CQGNGVWSGT EP ICRQPYSY DFPEDVAPAL GTSFSHMLGA TNPTQKTKES

[0112] 241 LGRKIQIQRS GHLNLYLLLD CSQSVSENDF LIFKESASLM VDRIFSFEIN VSVAI I TFAS

[0113] 301 EPKVLMSVLN DNSRDMTEVI S SLENANYKD HENGTGTNTY AALNSVYLMM NNQMRLLGME

[0114] 361 TMAWQEIRHA I ILLTDGKSN MGGSPKTAVD HIREILNINQ KRNDYLDIYA IGVGKLDVDW

[0115] 421 RELNELGSKK DGERHAFILQ DTKALHQVFE HMLDVSKLTD TICGVGNMSA NASDQERTPW

[0116] 481 HVTIKPKSQE TCRGALI SDQ WVLTAAHCFR DGNDHSLWRV NVGDPKSQWG KEFLIEKAVI

[0117] 541 SPGFDVFAKK NQGILEFYGD DIALLKLAQK VKMSTHARP I CLPCTMEANL ALRRPQGSTC

[0118] 601 RDHENELLNK QSVPAHFVAL NGSKLNINLK MGVEWTSCAE WSQEKTMFP NLTDVREWT

[0119] 661 DQFLCSGTQE DESPCKGESG GAVFLERRFR FFQVGLVSWG LYNPCLGSAD KNSRKRAPRS

[0120] 721 KVPPPRDFHI NLFRMQPWLR QHLGDVLNFL PL

[0121] As used herein, “C2a”, “C2a subunit” or “C2a domain” is used to refer to the larger 70 kDa fragment of complement factor 2. The C2a domain spans residues 244-752 of SEQ ID NO: 1 .

[0122] As used herein, “C2b”, “C2b subunit”, or “C2b domain” is used to refer to the smaller 30 kDa N- terminal fragment of complement factor 2. The C2b domain spans residues 21 -243 of SEQ ID NO: 1.

[0123] As used herein, the term “C2 inhibitor” refers to any molecule capable of binding specifically to C2 and inhibiting 02 function within the complement cascade. In preferred embodiments, as described herein, the 02 inhibitor for use in accordance with the methods described herein binds specifically to 02b. Inhibition of 02 will directly prevent or reduce formation of the 03 convertase and thus inhibit activation of other complement products downstream in the pathway. The 02 inhibitor may directly inhibit the larger 02a subunit preventing formation of the 03 convertase (C4bC2a). Alternatively, the 02 inhibitor may inhibit the smaller 02b subunit preventing the initial binding of 02 to surface-bound 04b. An inhibitor of 02b may leave the binding of 02a to 04b intact. 02 activity may nevertheless be significantly inhibited by an inhibitor of 02b.

[0124] As used herein, the term “immunoglobulin” includes a polypeptide having a combination of two heavy and two light chains whether or not it possesses any relevant specific immunoreactivity. “Antibodies” refer to such assemblies which have significant known specific immunoreactive activity to an antigen of interest. Antibodies and immunoglobulins comprise light and heavy chains, with or without an interchain covalent linkage between them. Basic immunoglobulin structures in vertebrate systems are relatively well understood.

[0125] The generic term “immunoglobulin” comprises five distinct classes of antibody that can be distinguished biochemically. With regard to IgG, immunoglobulins comprise two identical light polypeptide chains of molecular weight approximately 23,000 Daltons, and two identical heavy chains of molecular weight 53,000-70,000. The four chains are joined by disulfide bonds in a "Y" configuration wherein the light chains bracket the heavy chains starting at the mouth of the "Y" and continuing through the variable region. The light chains of an antibody are classified as either kappa or lambda (K,X). Each heavy chain class may be bound with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon, (y, JLL, a, 8, e) with some subclasses among them (e.g., yl- y4). It is the nature of this chain that determines the "class" of the antibody as IgG, IgM, IgA, IgD or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 , etc. are well characterised and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernible to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant invention.

[0126] As indicated above, the variable region of an antibody allows the antibody to selectively recognize and specifically bind epitopes on antigens. That is, the VL domain and VH domain of an antibody combine to form the variable region that defines a three-dimensional antigen binding site. This quaternary antibody structure forms the antigen binding site present at the end of each arm of the Y. More specifically, the antigen binding site is defined by three complementary determining regions (CDRs) on each of the VH and VL chains.

[0127] The term “antibody” as used herein is also intended to encompass “VHH antibodies” or “Heavychain only antibodies”.

[0128] The terms “variable region” and “variable domain” are used herein interchangeably and are intended to have equivalent meaning. The term "variable" refers to the fact that certain portions of the variable domains VH and VL differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its target antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called "hypervariable loops" in each of the VL domain and the VH domain which form part of the antigen binding site. The first, second and third hypervariable loops of the VLambda light chain domain are referred to herein as L1 (A), L2(A) and L3(A) and may be defined as comprising residues 24-33 (L1 (A), consisting of 9, 10 or 11 amino acid residues), 49-53 (L2(A), consisting of 3 residues) and 90-96 (L3(A), consisting of 5 residues) in the VL domain (Morea et al., Methods 20:267-279 (2000)). The first, second and third hypervariable loops of the VKappa light chain domain are referred to herein as L1 (K), L2(K) and L3(K) and may be defined as comprising residues 25-33 (L1 (K), consisting of 6, 7, 8, 11 , 12 or 13 residues), 49-53 (L2(K), consisting of 3 residues) and 90-97 (L3(K), consisting of 6 residues) in the VL domain (Morea et aL, Methods 20:267-279 (2000)). The first, second and third hypervariable loops of the VH domain are referred to herein as H1 , H2 and H3 and may be defined as comprising residues 25-33 (H1 , consisting of 7, 8 or 9 residues), 52-56 (H2, consisting of 3 or 4 residues) and 91 -105 (H3, highly variable in length) in the VH domain (Morea et aL, Methods 20:267-279 (2000)).

[0129] Unless otherwise indicated, the terms L1 , L2 and L3 respectively refer to the first, second and third hypervariable loops of a VL domain, and encompass hypervariable loops obtained from both Vkappa and Vlambda isotypes. The terms H1 , H2 and H3 respectively refer to the first, second and third hypervariable loops of the VH domain, and encompass hypervariable loops obtained from any of the known heavy chain isotypes, including y, E, 5, a or p.

[0130] The hypervariable loops L1 , L2, L3, H1 , H2 and H3 may each comprise part of a "complementarity determining region" or "CDR", as defined below. The terms "hypervariable loop" and "complementarity determining region" are not strictly synonymous, since the hypervariable loops (HVs) are defined on the basis of structure, whereas complementarity determining regions (CDRs) are defined based on sequence variability (Kabat et aL, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD., 1983) and the limits of the HVs and the CDRs may be different in some VH and VL domains.

[0131] The CDRs of the VL and VH domains can typically be defined as comprising the following amino acids: residues 24-34 (LCDR1 ), 50-56 (LCDR2) and 89-97 (LCDR3) in the light chain variable domain, and residues 31 -35 or 31 -35b (HCDR1 ), 50-65 (HCDR2) and 95-102 (HCDR3) in the heavy chain variable domain; (Kabat et aL, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991 )). Thus, the HVs may be comprised within the corresponding CDRs and references herein to the "hypervariable loops" of VH and VL domains should be interpreted as also encompassing the corresponding CDRs, and vice versa, unless otherwise indicated.

[0132] The more highly conserved portions of variable domains are called the framework region (FR), as defined below. The variable domains of native heavy and light chains each comprise four FRs (FR1 , FR2, FR3 and FR4, respectively), largely adopting a -sheet configuration, connected by the three hypervariable loops. The hypervariable loops in each chain are held together in close proximity by the FRs and, with the hypervariable loops from the other chain, contribute to the formation of the antigen binding site of antibodies. Structural analysis of antibodies revealed the relationship between the sequence and the shape of the binding site formed by the complementarity determining regions (Chothia et al., J. Mol. Biol. 227: 799-817 (1992));

[0133] Tramontane et al., J. Mol. Biol, 215:175-182 (1990)). Despite their high sequence variability, five of the six loops adopt just a small repertoire of main-chain conformations, called “canonical structures”. These conformations are first of all determined by the length of the loops and secondly by the presence of key residues at certain positions in the loops and in the framework regions that determine the conformation through their packing, hydrogen bonding or the ability to assume unusual main-chain conformations.

[0134] As used herein, the term “CDR” or “complementarity determining region” means the noncontiguous antigen binding sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991 ), and by Chothia et al., J. Mol. Biol. 196:901 -917 (1987) and by MacCallum et al., J. Mol. Biol. 262:732- 745 (1996) where the definitions include overlapping or subsets of amino acid residues when compared against each other. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth for comparison. Preferably, the term “CDR” is a CDR as defined by Kabat based on sequence comparisons.

[0135] Table 1 : CDR definitions

[0136] 1Residue numbering follows the nomenclature of Kabat etal., supra2Residue numbering follows the nomenclature of Chothia etal., supra3Residue numbering follows the nomenclature of MacCallum etal., supra

[0137] The term “fragment” or “antigen-binding fragment” refers to a part or portion of an antibody or antibody chain comprising fewer amino acid residues than an intact or complete antibody or antibody chain. The term “antigen-binding fragment” refers to a polypeptide fragment of an immunoglobulin or antibody that binds antigen or competes with intact antibody (i.e., with the intact antibody from which they were derived) for antigen binding. As used herein, the term “fragment” of an antibody includes antigen binding fragments of antibodies, for example, an antibody light chain variable domain (VL), an antibody heavy chain variable domain (VH), a single chain antibody (scFv), a F(ab’)2 fragment, a Fab fragment, an Fd fragment, an Fv fragment, a one-armed (monovalent) antibody, diabodies, triabodies, tetrabodies or any antigen binding molecule formed by combination, assembly or conjugation of such antigen binding fragments. The term “antigen-binding fragment” as used herein is further intended to encompass antibody fragments selected from the group consisting of unibodies, domain antibodies and nanobodies. Fragments can be obtained, e.g., via chemical or enzymatic treatment of an intact or complete antibody or antibody chain or by recombinant means.

[0138] The C2 inhibitors, specifically the anti-C2 antibodies and antigen-binding fragments thereof, bind to the target antigen C2 within the complement system. It is preferred that the inhibitors (e.g. antibodies) “specifically bind” to C2, wherein the term “specifically bind” refers to the ability of any inhibitor (e.g. antibody) to preferentially bind (immunoreact) to / with C2. C2 antibodies for use in accordance with the methods described herein may be monospecific and contain one or more binding sites which specifically bind C2. The antibodies may be incorporated into “multispecific antibody” formats, for example bispecific antibodies, wherein the multispecific antibody binds to two or more target antigens, at least one of which is C2. In order to achieve multiple specificities, “multispecific antibodies” are typically engineered to include different combinations or pairings of heavy and light chain polypeptides with different VH-VL pairs. Multispecific, notably bispecific antibodies, may be engineered so as to adopt the overall conformation of a native antibody, for example a Y-shaped antibody having Fab arms of different specificities conjugated to an Fc region. Alternatively, multispecific antibodies, for example bispecific antibodies, may be engineered so as to adopt a non-native conformation, for example wherein the variable domains or variable domain pairs having different specificities are positioned at opposite ends of the Fc region. Unless otherwise stated in the present application, “% sequence identity” between two amino acid sequences may be determined by comparing these two sequences aligned in an optimum manner and in which the amino acid sequence to be compared can comprise additions or deletions with respect to the reference sequence for an optimum alignment between these two sequences. The percentage of identity is calculated by determining the number of identical positions for which the amino acid residue is identical between the two sequences, by dividing this number of identical positions by the total number of positions in the comparison window and by multiplying the result obtained by 100 in order to obtain the percentage of identity between these two sequences. For example, it is possible to use the BLAST program, “BLAST 2 sequences” (Tatusova et al, "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250), the parameters used being those given by default (in particular for the parameters "open gap penalty": 5, and "extension gap penalty": 2; the matrix chosen being, for example, the matrix "BLOSUM 62" proposed by the program), the percentage of identity between the two sequences to be compared being calculated directly by the program.

[0139] As used herein, the terms “treat”, “treating” and “treatment” refer to the prevention, curing, delaying, reducing the severity of, or amelioration of one or more symptoms of the condition to be treated - in this instance the symptoms linked to IRI in a subject receiving a donor organ transplant and / or the symptoms linked to DGF in a subject receiving a kidney transplant and / or the symptoms linked to allograft dysfunction (poor allograft function) in a subject receiving a donor organ transplant and / or the symptoms linked to allograft loss in a subject receiving a donor organ transplant. The term “treatment”, in the context of the present invention, encompasses prophylactic treatment.

[0140] As used herein, the terms “prevent”, “preventing” or “prevention”, in the context of the present invention, mean “reducing the risk” of a subject developing the conditions or one or more symptoms associated with the conditions described herein. For example, “preventing IRI in a subject receiving a donor organ transplant” means reducing the risk of a subject receiving a donor organ transplant experiencing graft injury as a result of ischemia reperfusion. “Preventing DGF” means reducing the risk of a subject receiving a kidney transplant experiencing DGF, particularly for a subject at increased risk of DGF. “Reducing the risk of allograft loss in a subject receiving a donor organ transplant” is synonymous with preventing premature loss of allograft function or preventing allograft failure. B. Methods of treatment or prevention

[0141] The present invention is directed to methods of treating or preventing ischemia reperfusion injury (IRI) in subjects receiving a donor organ transplant i.e. transplant recipients. Subjects may be receiving a donor organ via allotransplantation.

[0142] The success of organ transplantation is affected by multiple factors. On the basis that there is rarely a perfect genetic match between donor and recipient, a major challenge in transplant surgery is management of the immune system and the immune response that is inevitably triggered when the donor organ is transferred into the recipient body.

[0143] Post-transplant immunosuppression regimens are used to prevent graft rejection and minimise damage occurring to the transplanted organ by the recipient’s immune system.

[0144] Immunosuppressive drugs can be classified as induction therapies, maintenance therapies, and anti-rejection therapies. For instance, the monoclonal antibodies, basiliximab (an anti-IL-2R antibody) and alemtuzumab (an anti-CD52 antibody) are often used as induction agents. Maintenance therapies typically include some combination of calcineurin inhibitors, such as cyclosporine or tacrolimus; anti-proliferative agents, such as mycophenolate mofetil or azathioprine; mTOR inhibitors, such as sirolimus and everolimus; or corticosteroids, such as prednisolone or methylprednisolone.

[0145] As mentioned above, ischemia reperfusion injury (IRI) remains one of the biggest challenges in organ transplantation, particularly during deceased organ donor transplants. Donated organs are frequently subjected to significant ischemic challenge prior to transplant into the recipient and upon restoration of blood flow, significant damage to the organ can occur. This damage can result in poor allograft function both in the short and longer term.

[0146] It is an object of the present invention to provide new methods for the treatment or prevention of IRI in transplant recipients. The methods described herein are intended to reduce injury to allografts, particularly injury caused by components of the immune system. It is a further object of the invention to provide new methods for improving allograft function in transplant recipients. It is a further object of the present invention to provide new methods for reducing the risk of allograft loss in transplant recipients.

[0147] The complement system has been reported as playing a role in tissue damage associated with IRI. The present application demonstrates activation of the complement system under conditions of ischemia / hypoxia followed by reperfusion, particularly in the context of experimental models relevant to organ transplantation. Moreover, the present application reports, for the first time, that inhibition, specifically at the level of complement factor C2, can inhibit downstream complement activation in ischemia-reperfusion models. In particular, the present application reports the ability of anti-C2 antibodies to inhibit ischemia-reperfusion-induced complement activation in an organ transplant setting.

[0148] The methods described herein involve the administration of a complement factor 2 (C2) inhibitor to a subject receiving a donor organ transplant. The inhibition of C2 serves as an effective means to block particular arms of the complement cascade and thereby prevent or limit the damage to the transplanted organ.

[0149] In a first aspect, the present invention provides a method of treating or preventing ischemia reperfusion injury in a subject receiving a donor organ transplant, the method comprising administering an effective amount, i.e. a therapeutically effective amount, of a complement factor 2 (C2) inhibitor to the subject. The present invention also provides a C2 inhibitor for use in the treatment or prevention of ischemia reperfusion injury in a subject receiving a donor organ transplant. The invention further provides use of a C2 inhibitor in the manufacture of a medicament for the treatment or prevention of IRI in a subject receiving a donor organ transplant.

[0150] In a second aspect, the present invention provides a method for improving allograft function in a subject receiving a donor organ transplant, the method comprising administering an effective amount, i.e. a therapeutically effective amount, of a complement factor 2 (C2) inhibitor to the subject. The present invention also provides a C2 inhibitor for use in improving allograft function in a subject receiving a donor organ transplant. The invention further provides use of a C2 inhibitor in the manufacture of a medicament for improving allograft function in a subject receiving a donor organ transplant. In a third aspect, the present invention provides a method for reducing the risk of allograft loss in a subject receiving a donor organ transplant, the method comprising administering an effective amount, i.e. a therapeutically effective amount, of a complement factor 2 (C2) inhibitor to the subject. The present invention also provides a C2 inhibitor for use in reducing the risk of allograft loss in a subject receiving a donor organ transplant. The invention further provides use of a C2 inhibitor in the manufacture of a medicament for reducing the risk of allograft loss in a subject receiving a donor organ transplant. On the basis that IRI occurs following transplant of all types of solid organs, C2 inhibition has the potential to treat or prevent ischemia-reperfusion-mediated complement activation in recipients receiving different donor organs. Accordingly, in certain embodiments, the methods are for the treatment or prevention of IRI in a subject receiving any solid organ transplant. Solid organ transplants refer to transplants of the solid organs i.e. the kidney, liver, heart, lung, pancreas and bowel. Therefore, in certain embodiments, the donor organ is selected from: kidney, liver, heart, lung, pancreas and bowel. In preferred embodiments, the donor organ is a kidney. In other embodiments, the donor organ is a lung.

[0151] For embodiments wherein the donor organ is a kidney, the subject may be at risk of delayed graft function (DGF). In certain embodiments, the subject receiving the kidney transplant may be at increased risk of DGF. Factors that would lead to a subject / recipient being classified as “at increased risk” of DGF include, but are not limited to: (i) receiving a donor organ from a deceased donor; (ii) receiving a donor organ from a donor aged > 40 years old; (iii) receiving a donor organ from a donor having a terminal serum creatinine value of > 1 .5 mg / dL; (iv) a recipient that is obese; (v) a recipient that has been on dialysis for a prolonged period prior to transplant; (vi) a recipient who is male; and / or (vii) a recipient who is African-American. In certain embodiments, there is provided a method for treating or preventing ischemia reperfusion injury in a subject receiving a donor organ transplant, particularly a subject classified as “at increased risk” of DGF. In certain embodiments, there is provided a method for improving allograft function in a subject receiving a donor organ transplant, particularly a subject classified as “at increased risk” of DGF. In certain embodiments, there is provided a method for reducing the risk of allograft loss in a subject receiving a donor organ transplant, particularly a subject classified as “at increased risk” of DGF.

[0152] The methods described herein may be employed for the purposes of treating or preventing delayed graft function in a subject receiving a kidney transplant, particularly a subject classified as “at increased risk” of DGF.

[0153] In a fourth aspect, the present invention provides a method of treating or preventing delayed graft function in a subject receiving a kidney transplant. The method involves administration of an effective amount, i.e. a therapeutically effective amount, of a complement factor 2 (02) inhibitor to the subject. The present invention also provides a 02 inhibitor for use in treating or preventing delayed graft function in a subject receiving a kidney transplant. The invention also provides use of a 02 inhibitor in the manufacture of a medicament for the treatment or prevention of delayed graft function in a subject receiving a kidney transplant.

[0154] Delayed graft function (DGF) is defined elsewhere herein and describes a form of early graft dysfunction whereby a donated kidney fails to work immediately following transplantation to the recipient. There are various different ways of defining or diagnosing DGF and different clinicians may use different methods. For example (and as described herein), DGF encompasses “dialysed DGF” wherein DGF is defined according to a transplant recipient’s need for life-sustaining dialysis treatment in the 7 days post-transplant, and “functional DGF” (also known as nondialysed DGF”) wherein DGF is defined according to serum creatinine levels. The methods of the present invention are for the treatment or prevention of DGF defined or diagnosed by any suitable method.

[0155] The methods of treating or preventing DGF described herein may result in a situation wherein life-sustaining dialysis is not required within the first 7 days post-transplant. In certain embodiments, (life-sustaining) dialysis is not required within the first 10 days post-transplant. In certain embodiments, (life-sustaining) dialysis is not required within the first 14 days posttransplant. In certain embodiments, (life-sustaining) dialysis is not required within the first 21 days post-transplant. In certain embodiments, (life-sustaining) dialysis is not required within the first 30 days post-transplant.

[0156] Alternatively or in addition, the methods of treating or preventing DGF described herein may result in a situation wherein serum creatinine levels in the subject decrease by at least 10% daily on 3 successive days during the first week post-transplantation.

[0157] Further assessments may be carried out within the first few days following transplantation. These tests may be useful as early prognostic markers of DGF and of longer-term graft outcomes. The creatinine reduction ratio on the second day (CRR2) after kidney transplantation measures kidney impairment and predicts renal function in the future. Calculation of CRR2% is provided elsewhere herein. Patients with a CRR2% of less than or equal to 30% may be classified as having “slow graft function” (often correlating with a subsequent diagnosis of DGF by posttransplant day 7). Patients with a CRR2 of greater than 30% may be classified as having “immediate graft function”. Accordingly, in certain embodiments following administration of the C2 inhibitor, the creatinine reduction ratio on post-transplant day 2 (CRR2) is higher than 30%.

[0158] Donor Orc / an

[0159] The present invention provides methods of treating or preventing IRI in subjects receiving a donor organ transplant. Several factors relating to the donor organ itself can impact the likelihood of IRI and poor graft function post-transplant and, in the instance of kidney transplant, the likelihood of delayed graft function.

[0160] IRI is often more frequent and severe in organs obtained from deceased donors than in those obtained from living donors. Deceased donor organ transplants are often associated with much longer warm and / or cold-ischemia times, which has a significant impact on the development of IRI. Consequently, the risk of IRI, allograft dysfunction or loss, and / or DGF is higher when a recipient is receiving a donor organ from a deceased donor (although notably some risk is still present in living-organ donation).

[0161] It follows, that in certain embodiments the donor organ is from a deceased donor. In certain embodiments, the donor organ is from a deceased donor that is a brain-death donor (DBD). In other embodiments, the donor organ is from a deceased donor that is a circulatory-death donor (DCD). In alternative embodiments, the donor organ is from a living donor.

[0162] Additional factors relating to the donor can also affect the likelihood and / or severity of IRI, allograft dysfunction or loss, and / or DGF following transplant of a donor organ into a recipient. For instance, the risk of IRI, allograft dysfunction or loss, and / or DGF can increase with older donor age, hypertension in the donor and / or terminal creatinine levels in the donor.

[0163] DGF is typically more prevalent following transplantation of an organ from an older donor, particularly from donors over the age of 60. In certain embodiments, the donor organ is from a donor aged >30. In certain embodiments, the donor organ is from a donor aged >40. In certain embodiments, the donor organ is from a donor aged >50. In certain embodiments, the donor organ is from a donor aged >60. In certain embodiments, the donor organ is from a donor aged >65. In certain embodiments, the donor organ is from a donor aged >70. In certain embodiments, the donor organ is from a donor aged >80. Preferably, the donor organ is from a donor aged between 40 and 70 years old.

[0164] The donor may exhibit high terminal serum creatinine levels. This can place the recipient at increased risk of IRI, allograft dysfunction or loss, and / or DGF. A high serum creatine level is typically a level over 1 .3 mg / dL, although this can vary depending on age, race, gender and body size. In certain embodiments, the donor’s terminal serum creatinine level is >1 .0 mg / dL. In certain embodiments, the donor’s terminal serum creatinine level is >1 .5 mg / dL. In certain embodiments, the donor’s terminal serum creatinine level is >2 mg / dL.

[0165] Factors relating to the procurement, transport and / or storage of the organ can also affect the likelihood of IRI, allograft dysfunction or loss, and / or DGF following transplant of the donor organ into the recipient. For example, prolonged cold ischemia time (CIT) is one of the most important factors contributing to IRI, allograft dysfunction or loss, and / or DGF after organ transplant. Cold ischemia time (CIT) is defined elsewhere herein and refers to the amount of time that an organ is chilled or cold and not receiving a blood supply. Recommended cold ischemia time varies by organ, but in general, the sooner an organ can be transplanted, the better. Deceased-donor organ transplants tend to be associated with significantly longer CIT than living-donor organ transplants due to the unpredictable nature of the donor’s death, and the fact that the distance between the donor and recipient may be substantial. The current accepted standard for organ preservation during transplants is static cold storage (SCS) whereby the organ is stored on ice after removal from the donor and then removed from the ice box at the time of implantation to the recipient. As a result, the donor organ may be on ice during procurement and transportation for a number of hours. The longer the CIT, the greater the risk for IRI, allograft dysfunction or loss, and / or DGF. Accordingly, in certain embodiments, the donor organ has undergone static cold storage. In certain embodiments, the donor organ is preserved via static cold storage. In certain embodiments, the donor organ is preserved in a static cold storage solution. In certain embodiments, the donor organ is preserved in a static cold storage solution prior to implantation to the recipient. The donor organ may be preserved in any suitable static cold storage solution, which would be known to the skilled person. Common cold preservation solutions include, but are not limited to: EC (Euro Collins), UW (University of Wisconsin), HTK (histidine tryptophan ketoglutarate), Custodial-N, Celsior, LPDG (low-potassium dextran glucose), Ep4, ET-Kyoto (extracellular-type trehalose-containing Kyoto), and IGL-1 (Institut Georges Lopez-1 ).

[0166] As an alternative, machine perfusion preservation techniques have been employed in an attempt to reduce the damage caused during storage and transport. Unlike static cold storage, machine perfusion preservation is dynamic and provides a continuous supply of oxygen and other metabolic precursors during the ex situ phase of solid organ transplantation. There are two main methods of machine perfusion: hypothermic machine perfusion (HMP), which involves the use of temperatures lower than the physiological range; and normothermic machine perfusion (NMP), which employs temperatures within the physiological range. Evidence shows that hypothermic machine perfusion reduces the rate of DGF in kidneys from deceased donors, compared with standard static cold storage (Tingle et al, 2019; Cochrane Database of Systematic Reviews (3): CD011671 ). As such, the combination of machine perfusion together with administration of a 02 inhibitor may lead to a further decreased rate of DGF. Accordingly, in certain embodiments, the donor organ has undergone machine perfusion. In certain embodiments, the donor organ is preserved via machine perfusion prior to implantation to the recipient. In certain embodiments, the donor organ has been machine-perfused between the time of organ procurement and the time of transplant.

[0167] Static cold storage remains the standard, based on both improved convenience and cost. Therefore, it is particularly advantageous that the present application reports the ability of 02 inhibition to inhibit complement activation induced by ischemia reperfusion in models that mimic static cold storage. The data herein demonstrates, in particular, that allograft dysfunction, such as DGF, can be prevented despite the donor kidney having been subjected to extensive cold ischemia times. Accordingly, in certain embodiments of the methods described herein, the donor organ has not been machine-perfused between the time of organ procurement and the time of transplant.

[0168] In certain embodiments, the donor organ has been preserved on ice between the time of organ procurement and the time of transplant. In certain embodiments, the cold ischemia time of the donor organ is between 1 and 12 hours. In certain embodiments, the cold ischemia time of the donor organ is at least 1 hour. In certain embodiments, the cold ischemia time of the donor organ is at least 2 hours. In certain embodiments, the cold ischemia time of the donor organ is at least 3 hours. In certain embodiments, the cold ischemia time of the donor organ is at least 4 hours. In certain embodiments, the cold ischemia time of the donor organ is at least 5 hours. In certain embodiments, the cold ischemia time of the donor organ is at least 6 hours. In certain embodiments, the cold ischemia time of the donor organ is at least 7 hours. In certain embodiments, the cold ischemia time of the donor organ is at least 8 hours. In certain embodiments, the cold ischemia time of the donor organ is at least 9 hours. In certain embodiments, the cold ischemia time of the donor organ is at least 10 hours. In certain embodiments, the cold ischemia time of the donor organ is at least 11 hours. In certain embodiments, the cold ischemia time of the donor organ is at least 12 hours.

[0169] A subject receiving a donor kidney is considered to be at greater risk of DGF if the cold ischemia time for the donor organ exceeds 12 hours. Accordingly, in certain embodiments, the cold ischemia time of the donor organ is between 12 and 48 hours. In certain embodiments, the cold ischemia time of the donor organ is at least 16 hours. In certain embodiments, the cold ischemia time of the donor organ is at least 18 hours. In certain embodiments, the cold ischemia time of the donor organ is at least 20 hours. In certain embodiments, the cold ischemia time of the donor organ is at least 22 hours. In certain embodiments, the cold ischemia time of the donor organ is at least 24 hours. In certain embodiments, the cold ischemia time of the donor organ is at least 26 hours. In certain embodiments, the cold ischemia time of the donor organ is at least 28 hours. In certain embodiments, the cold ischemia time of the donor organ is at least 30 hours. In certain embodiments, the cold ischemia time of the donor organ is at least 32 hours. In certain embodiments, the cold ischemia time of the donor organ is at least 34 hours. In certain embodiments, the cold ischemia time of the donor organ is at least 36 hours. In certain embodiments, the cold ischemia time of the donor organ is at least 38 hours. In certain embodiments, the cold ischemia time of the donor organ is at least 40 hours. In certain embodiments, the cold ischemia time of the donor organ is at least 42 hours. In certain embodiments, the cold ischemia time of the donor organ is at least 44 hours. In certain embodiments, the cold ischemia time of the donor organ is at least 46 hours. In certain embodiments, the cold ischemia time of the donor organ is at least 48 hours.

[0170] Organ Recipient

[0171] The recipient of the donor organ may be a human or non-human subject. Preferably, the recipient is human.

[0172] In certain embodiments, the recipient is identified as at increased risk of IRI prior to transplantation. For embodiments wherein the subject is undergoing a kidney transplant, the recipient may be identified as at increased risk of IRI and / or DGF prior to transplantation. There are numerous recipient-specific factors that can increase the risk of DGF. For instance, DGF has been observed more commonly if the recipient is male and / or African-American. The risk of DGF also tends to be higher in obese recipients, and also in those recipients who have been on dialysis for a prolonged period of time prior to receiving a donor kidney.

[0173] In certain embodiments, the donor organ is a kidney and the recipient is diagnosed with end stage renal disease (ESRD). In certain embodiments, the recipient has been stable on chronic dialysis for at least 3 months. In certain embodiments, the recipient has been stable on chronic dialysis for at least 4 months. In certain embodiments, the recipient has been stable on chronic dialysis for at least 5 months. In certain embodiments, the recipient has been stable on chronic dialysis for at least 6 months. In certain embodiments, the recipient has been stable on chronic dialysis for at least 8 months. In certain embodiments, the recipient has been stable on chronic dialysis for at least 12 months.

[0174] In certain embodiments, the recipient is a candidate for de novo kidney transplantation i.e. has not received a previous kidney transplant. In certain embodiments, the recipient is a candidate for second-time kidney transplantation i.e. has received a previous kidney transplant.

[0175] Dosing Reaimens

[0176] The 02 inhibitors for use in accordance with the methods of the invention, particularly the anti-C2 antibodies and antigen-binding fragments thereof, may be administered according to any suitable dosage regimen as described herein.

[0177] The present invention contemplates methods whereby the 02 inhibitor is administered to the recipient of a donor organ transplant, as opposed to administration to the organ donor or the donor organ perse. The dosage regimens described herein below are thus “recipient-treatment regimens”. In certain embodiments, a first dose of the C2 inhibitor is administered to the recipient prior to transplantation. Preferably, the C2 inhibitor is administered to the recipient prior to initiation of the transplant procedure i.e. pre-operatively. In certain embodiments, a first dose of the C2 inhibitor is administered to the recipient on the day of the transplant. The C2 inhibitor may be administered 10 mins prior to initiation of the transplant procedure. The C2 inhibitor may be administered 20 mins prior to initiation of the transplant procedure. The C2 inhibitor may be administered 30 mins prior to initiation of the transplant procedure. The C2 inhibitor may be administered 1 hour prior to initiation of the transplant procedure. The C2 inhibitor may be administered 2 hours prior to initiation of the transplant procedure. The C2 inhibitor may be administered 4 hours prior to initiation of the transplant procedure. The C2 inhibitor may be administered 6 hours prior to initiation of the transplant procedure. The C2 inhibitor may be administered 8 hours prior to initiation of the transplant procedure. The C2 inhibitor may be administered 10 hours prior to initiation of the transplant procedure. The C2 inhibitor may be administered 12 hours prior to initiation of the transplant procedure.

[0178] In certain embodiments, the C2 inhibitor is administered to the recipient prior to reperfusion of the organ. Preferably, the C2 inhibitor is administered to the recipient prior to initiation of reperfusion of the organ. The C2 inhibitor may be administered 10 mins prior to initiation of reperfusion of the organ. The C2 inhibitor may be administered 20 mins prior to initiation of reperfusion of the organ. The C2 inhibitor may be administered 30 mins prior to initiation of reperfusion of the organ. The C2 inhibitor may be administered 1 hour prior to initiation of reperfusion of the organ. The C2 inhibitor may be administered 2 hours prior to initiation of reperfusion of the organ. The C2 inhibitor may be administered 4 hours prior to initiation of reperfusion of the organ. The C2 inhibitor may be administered 6 hours prior to initiation of reperfusion of the organ. The C2 inhibitor may be administered 8 hours prior to initiation of reperfusion of the organ. The C2 inhibitor may be administered 10 hours prior to initiation of reperfusion of the organ. The C2 inhibitor may be administered 12 hours prior to initiation of reperfusion of the organ.

[0179] In certain embodiments, the C2 inhibitor is administered to the recipient during transplantation i.e. during the transplant procedure. In certain embodiments, the C2 inhibitor is administered to the recipient peri-operatively. In certain embodiments, the C2 inhibitor is administered to the recipient during reperfusion of the organ.

[0180] In certain embodiments, the C2 inhibitor is administered to the recipient prior to and during the transplant procedure. In certain embodiments, the C2 inhibitor is administered to the recipient prior to and during reperfusion of the organ. In certain embodiments, the C2 inhibitor is administered to the recipient intravenously. In other embodiments, the C2 inhibitor is administered to the recipient subcutaneously. In certain embodiments, the C2 inhibitor is administered to the recipient as a bolus injection. In certain embodiments, the C2 inhibitor is administered as a continuous infusion. In certain embodiments, the C2 inhibitor is administered to the recipient via intravenous infusion over a period of approximately 120 minutes. In certain embodiments, the C2 inhibitor is administered to the recipient via intravenous infusion over a period of approximately 90 minutes. In certain embodiments, the C2 inhibitor is administered to the recipient via intravenous infusion over a period of approximately 60 minutes. In certain embodiments, the C2 inhibitor is administered to the recipient via intravenous infusion over a period of approximately 30 minutes.

[0181] In certain preferred embodiments, the C2 inhibitor is administered to the recipient as a continuous intravenous infusion. The intravenous infusion may initiate and terminate prior to initiation of reperfusion of the organ. In alternative embodiments, the intravenous infusion may initiate prior to initiation of reperfusion of the organ, continue during reperfusion of the organ and terminate before reperfusion of the organ is completed. In such embodiments, the intravenous infusion may be carried out for a period of approximately 90 minutes.

[0182] In certain embodiments, the C2 inhibitor is administered to the recipient at a dose of 0.1 mg / kg to 100 mg / kg. In certain embodiments, the C2 inhibitor is administered to the recipient at a dose of 0.1 mg / kg, 0.5 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg or 100 mg / kg. In certain embodiments, the C2 inhibitor is administered to the recipient at a dose of 40 mg / kg to 80 mg / kg. In certain embodiments, the C2 inhibitor is administered to the recipient at a dose of 60 mg / kg.

[0183] In certain embodiments, the C2 inhibitor is administered to the recipient at a fixed dose of 500 mg to 6000 mg. In certain embodiments, the C2 inhibitor is administered to the recipient at a fixed dose of 500 mg, 1000 mg, 1200 mg, 1500 mg, 1600 mg, 1800 mg, 2000 mg, 2500 mg, 3000 mg, 4000 mg, 5000 mg, or 6000 mg.

[0184] The methods of the present invention also contemplate administering one or more further doses of the C2 inhibitor to the recipient after transplantation in order to prevent further injury to the organ and / or improve longer-term outcomes. These one or more further doses may also be referred to as “post-operative doses”. Accordingly, in certain embodiments, one or more further doses are administered to the recipient post-operatively. In certain embodiments, one further dose (i.e. one post-operative dose) of the C2 inhibitor is administered to the recipient. In certain embodiments, one post-operative dose is administered to the recipient. In certain embodiments, two post-operative doses are administered to the recipient. In certain embodiments, three post-operative doses are administered to the recipient. In certain embodiments, four post-operative doses are administered to the recipient. In certain embodiments, five post-operative doses are administered to the recipient.

[0185] Preferably, one further dose (i.e. one post-operative dose) of the C2 inhibitor is administered to the recipient within the week after the transplant procedure. In Example 4 below, study day 1 (0- 24hrs) refers to the day in which the transplant operation takes place, whereby hour zero refers to the time that reperfusion begins. Study day 2 (48hrs) refers to post-transplant day 1 ; study day 3 (72hrs) refers to post-transplant day 2; study day 4 refers to post-transplant day 3; study day 5 refers to post-transplant day 4, and so on.

[0186] In certain embodiments, a post-operative dose is administered 1 day post-transplant (i.e. equivalent to study day 2). In certain embodiments, a post-operative dose is administered 2 days post-transplant (i.e. equivalent to study day 3). In certain embodiments, a post-operative dose is administered 3 days post-transplant (i.e. equivalent to study day 4). In certain embodiments, a post-operative dose is administered 4 days post-transplant (i.e. equivalent to study day 5). In certain embodiments, a post-operative dose is administered 5 days post-transplant (i.e. equivalent to study day 6). In certain embodiments, a post-operative dose is administered 6 days post-transplant (i.e. equivalent to study day 7). In certain preferred embodiments, a postoperative dose is administered 7 days post-transplant (i.e. equivalent to study day 8). In certain preferred embodiments, a post-operative dose is administered 1 week post-transplant. In certain embodiments, a post-operative dose is administered 8 days post-transplant (i.e. equivalent to study day 9). In certain embodiments, a post-operative dose is administered 9 days posttransplant (i.e. equivalent to study day 10). In certain embodiments, a post-operative dose is administered 10 days post-transplant (i.e. equivalent to study day 11 ). In certain embodiments, a post-operative dose is administered 11 days post-transplant (i.e. equivalent to study day 12). In certain embodiments, a post-operative dose is administered 12 days post-transplant (i.e. equivalent to study day 13). In certain embodiments, a post-operative dose is administered 13 days post-transplant (i.e. equivalent to study day 14). In certain embodiments, a post-operative dose is administered 14 days post-transplant (i.e. equivalent to study day 15). In certain embodiments, a post-operative dose is administered 7-9 days post-transplant. In certain embodiments, a post-operative dose is administered 7-10 days post-transplant. In certain embodiments, a post-operative dose is administered 7-11 days post-transplant. In certain embodiments, a post-operative dose is administered 7-12 days post-transplant. In certain embodiments, a post-operative dose is administered 7-13 days post-transplant. In certain embodiments, a post-operative dose is administered 7-14 days post-transplant.

[0187] In certain embodiments, a post-operative dose is administered to the recipient 2 weeks posttransplant. In certain embodiments, a post-operative dose is administered to the recipient 3 weeks post-transplant. In certain embodiments, a post-operative dose is administered to the recipient 4 weeks post-transplant. In certain embodiments, a post-operative dose is administered to the recipient 1 month post-transplant. In certain embodiments, a post-operative dose is administered to the recipient 2 months post-transplant. In certain embodiments, a post-operative dose is administered to the recipient 3 months post-transplant. In certain embodiments, a postoperative dose is administered to the recipient 4 months post-transplant. In certain embodiments, a post-operative dose is administered to the recipient 5 months post-transplant. In certain embodiments, a post-operative dose is administered to the recipient 6 months post-transplant.

[0188] In certain embodiments, one or more further doses (i.e. one or more post-operative doses) are administered to the recipient once weekly. In certain embodiments, one or more post-operative doses are administered to the recipient once every 2 weeks. In certain embodiments, one or more post-operative doses are administered to the recipient once every 4 weeks. In certain embodiments, one or more post-operative doses are administered to the recipient once every 2 months. In certain embodiments, one or more post-operative doses are administered to the recipient once every 6 months.

[0189] The one or more further doses (i.e. one or more post-operative doses) of the C2 inhibitor may be administered to the recipient via the same route of administration as the initial pre- and / or perioperative doses discussed above. Alternatively, the one or more further doses may be administered to the recipient via an alternative route of administration.

[0190] In certain embodiments, the one or more post-operative dose(s) is / are administered to the recipient intravenously. In certain embodiments, the one or more post-operative dose(s) is / are administered to the recipient subcutaneously. In certain embodiments, the one or more postoperative dose(s) is administered to the recipient as a bolus injection. In certain embodiments, the one or more post-operative dose(s) is administered to the recipient as a continuous infusion. In certain embodiments, the one or more post-operative dose(s) is / are administered to the recipient via intravenous infusion over a period of approximately 120 minutes. In certain embodiments, the one or more post-operative dose(s) is / are administered to the recipient via intravenous infusion over a period of approximately 90 minutes. In certain embodiments, the one or more post-operative dose(s) is / are administered to the recipient via intravenous infusion over a period of 60 minutes. In certain embodiments, the one or more post-operative dose(s) is / are administered to the recipient via intravenous infusion over a period of 30 minutes.

[0191] The one or more further doses (i.e. one or more post-operative doses) of the C2 inhibitor may be administered to the recipient at the same amount as the initial pre- and / or peri-operative doses discussed above. Alternatively, the one or more further doses may be administered to the recipient in an alternative amount. In certain embodiments, the one or more post-operative dose(s) is / are administered to the recipient at a dose of 0.1 mg / kg to 100 mg / kg. In certain embodiments, the one or more post-operative dose(s) is / are administered to the recipient at a dose 0.1 mg / kg, 0.5 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg or 100 mg / kg. In certain embodiments, the one or more post-operative dose(s) is / are administered to the recipient at a dose of 40 mg / kg to 80 mg / kg. In certain embodiments, the one or more post-operative dose(s) is / are administered to the recipient at a dose of 60 mg / kg.

[0192] In certain embodiments, the one or more post-operative dose(s) is / are administered to the recipient at a fixed dose of 500 mg to 6000 mg. In certain embodiments, the one or more postoperative dose(s) is / are administered to the recipient at a fixed dose of 500 mg, 1000 mg, 1200 mg, 1500 mg, 1600 mg, 1800 mg, 2000 mg, 2500 mg, 3000 mg, 4000 mg, 5000 mg, or 6000 mg.

[0193] In preferred embodiments, a first dose of the C2 inhibitor is administered to the recipient prior to and / or during the transplant operation, and then a second dose is administered to the recipient after the transplant operation. Accordingly, in certain embodiments, a first dose of the C2 inhibitor is administered to the recipient prior to and / or during transplantation, and a second dose of the C2 inhibitor is administered to the recipient after transplantation. In certain embodiments, a first dose of the C2 inhibitor is administered to the recipient pre-operatively and / or peri-operatively, and a second dose of the C2 inhibitor is administered to the recipient post-operatively. In certain embodiments, a first dose of the C2 inhibitor is administered to the recipient prior to and / or during reperfusion of the donor organ, and a second dose of the C2 inhibitor is administered to the recipient after reperfusion of the donor organ.

[0194] In certain embodiments, a first dose of the C2 inhibitor is administered to the recipient prior to and / or during reperfusion of the donor organ; and a second dose of the C2 inhibitor is administered to the recipient between 1 day and 6 months post-transplant. In certain preferred embodiments, the second dose is administered to the recipient 7 days post-transplant. In certain preferred embodiments, the second dose is administered to the recipient 7-9 days posttransplant. In certain embodiments, the second dose is administered to the recipient 14 days post-transplant. In certain embodiments, the second dose is administered to the recipient 1 month post-transplant. In certain embodiments, the second dose is administered to the recipient 6 months post-transplant.

[0195] In certain embodiments, both the first and second doses are administered intravenously. In certain embodiments, both the first and second doses are administered subcutaneously. In certain embodiments, the first dose is administered intravenously, and the second dose is administered subcutaneously. In certain embodiments, the first dose is administered subcutaneously, and the second dose is administered intravenously. In certain embodiments, the first dose is administered intravenously, and the second dose is administered intravenously or subcutaneously.

[0196] In certain embodiments, the first dose is administered at a dose of 0.1 mg / kg to 100 mg / kg, and the second dose is administered at a dose of 0.1 mg / kg to 100 mg / kg, wherein the first and second dose amounts may be the same or different. In certain embodiments, the first dose is administered at a dose of 40 mg / kg to 80 mg / kg, and the second dose is administered at a dose of 40 mg / kg to 80 mg / kg, wherein the first and second dose amounts may be the same or different. In certain preferred embodiments, the first dose is administered to the recipient at a dose of 60 mg / kg and the second dose is administered to the recipient at a dose of 60 mg / kg. In certain embodiments, the second dose is administered to the recipient at a lower amount than the first dose. For example, in certain embodiments, the first dose is administered to the recipient at a dose of 60 mg / kg and the second dose is administered at a lower amount than 60 mg / kg. In certain embodiments, the second dose is administered to the recipient at a higher amount than the first dose. For example, in certain embodiments, the first dose is administered to the recipient at a dose of 60 mg / kg and the second dose is administered at a higher amount than 60 mg / kg.

[0197] In certain preferred embodiments of the invention, a first dose of the C2 inhibitor is administered to the recipient on the day of the transplant prior to and / or during reperfusion of the donor organ (e.g. kidney); and a second dose of the C2 inhibitor is administered to the recipient 7-9 days post-transplant; wherein both the first and the second doses are administered intravenously, and wherein both the first and second doses are administered at a dose of 60 mg / kg. Preferably, the C2 inhibitor is an anti-C2b antibody, particularly preferably wherein the anti-C2b antibody is empasiprubart.

[0198] C2 inhibitors

[0199] The methods of the present invention involve the administration of a “C2 inhibitor”. As explained elsewhere herein, C2 inhibitors for use in accordance with the present invention inhibit complement factor 2 (C2) and thereby disrupt the proper functioning of C2 within the classical and lectin complement pathways. Thus, the C2 inhibitors described herein can reduce classical complement pathway activity and lectin complement pathway activity but do not affect the alternative complement pathway. In some instances, it may be beneficial to leave one of the complement pathways intact such that this important arm of the innate immune system is not disabled entirely. Alternatively or in addition, the C2 inhibitor may only need to bring about partial inhibition of complement activity in order to achieve a therapeutic effect.

[0200] In certain embodiments, the C2 inhibitor reduces activity of the classical pathway. In certain embodiments, the C2 inhibitor reduces activity of the lectin pathway. In certain embodiments, the C2 inhibitor reduces activity of both the classical pathway and the lectin pathway. In certain embodiments, the C2 inhibitor does not affect the activity of the alternative pathway.

[0201] The C2 inhibitor may prevent, at least in part, damaging effects of complement-derived peptides on cells and tissues. The C2 inhibitor may inhibit or reduce complement activity by reducing deposition of anaphylatoxins, by reducing deposition of opsonins, by reducing cytokine and / or chemokine production or secretion, by reducing phagocytosis, by reducing formation of the membrane attack complex (MAC), by reducing cell lysis, by reducing the recruitment of immune cells, or any combination thereof. In certain embodiments, the C2 inhibitor prevents or reduces the deposition of anaphylatoxins. In certain embodiments, the C2 inhibitor prevents or reduces the deposition of C3a or C5a. In certain embodiments, the C2 inhibitor prevents or reduces the deposition of opsonins. In certain embodiments, the C2 inhibitor prevents or reduces the deposition of C3b or C4b. In certain embodiments, the C2 inhibitor prevents or reduces production and / or secretion of inflammatory cytokines and / or chemokines. In some embodiments, the C2 inhibitor prevents or reduces the production and / or secretion of MCP-1 . In some embodiments, the C2 inhibitor prevents or reduces formation of the membrane attack complex (MAC). In some embodiments, the C2 inhibitor prevents or reduces MAC-dependent cell lysis.

[0202] In some embodiments, the C2 inhibitor is selected from: inhibitory RNA species, for example siRNAs or shRNAs; small molecule inhibitors; biological antagonists including inhibitory peptides, antibody mimetics such as affibodies, affilins, affitins, adnectins, atrimers, evasins, DARPins, anticalins, avimers, fynomers, versabodies and duocalins; antibodies and antigen-binding fragments thereof. Anti-C2 antibodies and antigen-binding fragments thereof

[0203] In preferred embodiments, the C2 inhibitor is an antibody or antigen-binding fragment thereof that specifically binds to C2. The terms “C2 antibody or antigen-binding fragment” and “anti-C2 antibody or antigen binding fragment” are used interchangeably herein.

[0204] In certain embodiments, the C2 inhibitor is an antibody or antigen-binding fragment thereof that specifically binds to C2a. In certain embodiments, the C2 inhibitor is an antibody or antigenbinding fragment thereof that specifically binds to C2b. In certain embodiments, the C2 inhibitor is an antibody or antigen-binding fragment thereof that specifically binds to C2a and C2b. In certain embodiments, the C2 inhibitor is an antibody or antigen-binding fragment thereof that binds to C2b in a pH- and Ca2+-dependent manner. In certain embodiments, the C2 inhibitor is an antibody or antigen-binding fragment thereof that inhibits the function of C2 and blocks downstream complement activation.

[0205] The anti-C2 antibodies and antigen-binding fragments thereof for use in the methods described herein are intended for human therapeutic use and therefore, will typically be of the IgA, IgD, IgE, IgG, IgM type, often of the IgG type, in which case they can belong to any of the four sub-classes IgG 1 , lgG2a and b, lgG3 or lgG4. In preferred embodiments, the antibodies are IgG antibodies, optionally lgG1 antibodies. The antibodies may be monoclonal, polyclonal, multispecific (e.g. bispecific antibodies) antibodies, provided that they exhibit the appropriate immunological specificity for the target. Monoclonal antibodies are preferred since they are highly specific, being directed against a single antigenic site.

[0206] The antigen-binding fragments described herein will typically comprise a portion of a full-length antibody, generally the antigen binding or variable domain thereof. In certain embodiments, the antigen-binding fragment is selected from: an antibody light chain variable domain (VL); an antibody heavy chain variable domain (VH); a single chain antibody (scFv); a F(ab’)2 fragment; a Fab fragment; an Fd fragment; an Fv fragment; a one-armed (monovalent) antibody; a diabody; a triabody; a tetrabody; a unibody; a domain antibody; and a nanobody.

[0207] The antibodies or antigen-binding fragments for use in accordance with the methods described herein may exhibit high human homology. Such antibody molecules having high human homology may include antibodies comprising VH and VL domains of native non-human antibodies which exhibit sufficiently high % sequence identity to human germline sequences. In certain embodiments, the antibody molecules are humanised or germlined variants of non- human antibodies. In non-limiting embodiments, the antibodies may comprise CH1 domains and / or CL domains (from the heavy chain and light chain, respectively), the amino acid sequence of which is fully or substantially human. For antibody molecules intended for human therapeutic use, it is typical for the entire constant region of the antibody, or at least a part thereof, to have a fully or substantially human amino acid sequence. Therefore, one or more or any combination of the CH1 domain, hinge region, CH2 domain, CH3 domain and CL domain (and CH4 domain if present) may be fully or substantially human with respect to its amino acid sequence. The CH1 domain, hinge region, CH2 domain, CH3 domain and / or CL domain (and / or CH4 domain if present) may be derived from a human antibody, preferably a human IgG antibody, more preferably a human lgG1 antibody of subtype lgG1 , lgG2, lgG3 or lgG4.

[0208] Advantageously, the CH1 domain, hinge region, CH2 domain, CH3 domain and CL domain (and CH4 domain if present) may all have fully or substantially human amino acid sequences. In the context of the constant region of a humanised or chimeric antibody, or an antibody fragment, the term "substantially human" refers to an amino acid sequence identity of at least 90%, or at least 92%, or at least 95%, or at least 97%, or at least 99% with a human constant region. The term “human amino acid sequence” in this context refers to an amino acid sequence which is encoded by a human immunoglobulin gene, which includes germline, rearranged and somatically mutated genes.

[0209] In some embodiments, the antibody or antigen-binding fragment comprises a human Fc domain, which comprises one or more mutations designed to increase the serum half-life of the antibody or antibody molecule. Such optimization efforts aim to improve antibody circulation in vivo. Examples of half-life affecting mutations in the human IgG Fc domain include His433Lys + Asn434Phe (NHance); Arg435His; Asn434Ala; Met252Tyr + Ser254Thr + Thr256Glu (YTE); Met428Leu +Asn434Ser (LS); Thr252Leu + Thr253Ser + Thr254Phe (LSF); Glu294delta + Thr307Pro + Asn434Tyr (C6A-66); Thr256Asn + Ala378Val + Ser383Asn + Asn434Tyr (C6A-78); and Glu294delta (del). Depending on the serum half-life of the 02 antibody, it may be administered as a single dose, or it may be administered as multiple doses with an interval of from 1 day to 1 month between subsequent doses.

[0210] In further embodiments, the Fc domain may comprise one or more mutations designed to impair the effector function of the Fc domain. Such mutations are well known to those skilled in the art.

[0211] In the case of human lgG1 , the antibody may comprise a human Fc domain that has been modified to abrogate or impair its effector function. Such Fc domain mutations generally comprise changing at least 1 amino acid from the heavy chain constant region on position 234, 235, 236, 237, 297, 318, 320 or 322, thereby causing an alteration in effector function while retaining binding to antigen. Examples of effector-impairing mutations in the human IgG Fc domain include Leu234Ala + Leu235Ala (referred to as LALA); Leu234Ala + Leu235Ala + Pro329Gly (referred to as LALA-PG); Ser228Pro + Leu235Glu in lgG4; Pro331 Ser + Leu234Glu + Leu235Phe; and Pro331 Ser + Leu234Ala + Leu235Ala.

[0212] Anti-02 antibodies and antigen-binding fragments thereof for use in the methods described herein include the antibodies and antigen-binding fragments identified in International patent application no. WO2014 / 189378 and W02020 / 121282, the contents of which are incorporated herein in their entirety.

[0213] In certain embodiments, the C2 inhibitor is an anti-C2b antibody, preferably empasiprubart (also referred to herein as ARGX-117). In certain embodiments, the 02 inhibitor is an antigen-binding fragment of empasiprubart (ARGX-117). In certain embodiments, the 02 inhibitor is an anti-C2b antibody having the same six CDRs as empasiprubart (ARGX-117). The CDR, VH and VL sequences of empasiprubart (ARGX-117) are shown in Table 2 below.

[0214] Table 2: ARGX-117 amino acid sequences

[0215] Empasiprubart binds human C2 and cynomolgus monkey C2 in a pH- and Ca2+-dependent manner with affinity to human C2 ranging from 0.109 nM to 2.02 nM and affinity to cynomolgus monkey C2 from 0.056 nM to 0.13 nM, respectively. In an embodiment of any one of the methods disclosed herein, the anti-C2 antibody binds human C2 and cynomolgus monkey C2. Empasiprubart is not cross-reactive to C2 from rat, rabbit, hamster, mouse, and guinea pig.

[0216] In certain embodiments, the anti-C2b antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein the VH and VL domains comprise the CDR sequences:

[0217] - HCDR3 comprising or consisting of SEQ ID NO: 4 [EDDHDAFAY];

[0218] - HCDR2 comprising or consisting of SEQ ID NO: 3 [DINPNYESTGYNQKFKG];

[0219] - HCDR1 comprising or consisting of SEQ ID NO: 2 [DYNMD];

[0220] - LCDR3 comprising or consisting of SEQ ID NO: 7 [QHSRELPYT];

[0221] - LCDR2 comprising or consisting of SEQ ID NO: 6 [LASNLKS]; and

[0222] - LCDR1 comprising or consisting of SEQ ID NO: 5 [RASKSVRTSGYNYMH],

[0223] In certain embodiments, the anti-C2b antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) domain comprising or consisting of a sequence at least 70%, at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 8 and a variable light chain (VL) domain comprising or consisting of a sequence at least 70%, at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 9. In certain embodiments, the anti- C2b antibody or antigen-binding fragment comprises a variable heavy chain domain (VH domain) comprising or consisting of SEQ ID NO: 8 and a variable light chain domain (VL domain) comprising or consisting of SEQ ID NO: 9.

[0224] In certain embodiments, the anti-C2b antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein the VH domain comprises or consists of a sequence at least 70%, at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 8 and the VL domain comprises the CDR sequences: - LCDR3 comprising or consisting of SEQ ID NO: 7 [QHSRELPYT];

[0225] - LCDR2 comprising or consisting of SEQ ID NO: 6 [LASNLKS]; and

[0226] - LCDR1 comprising or consisting of SEQ ID NO: 5 [RASKSVRTSGYNYMH],

[0227] In certain embodiments, the anti-C2b antibody or antigen-binding fragment comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein the VH domain comprises or consists of the amino acid sequence of SEQ ID NO: 8 and the VL domain comprises the CDR sequences:

[0228] - LCDR3 comprising or consisting of SEQ ID NO: 7 [QHSRELPYT];

[0229] - LCDR2 comprising or consisting of SEQ ID NO: 6 [LASNLKS]; and

[0230] - LCDR1 comprising or consisting of SEQ ID NO: 5 [RASKSVRTSGYNYMH],

[0231] In certain embodiments, the anti-C2b antibody or antigen-binding fragment comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein the VH domain comprises the CDR sequences:

[0232] - HCDR3 comprising or consisting of SEQ ID NO: 4 [EDDHDAFAY];

[0233] - HCDR2 comprising or consisting of SEQ ID NO: 3 [DINPNYESTGYNQKFKG];

[0234] - HCDR1 comprising or consisting of SEQ ID NO: 2 [DYNMD], and the VL domain comprises or consists of a sequence at least 70%, at least 80%, at least 85%, at least 90% or at least 95% identical to SEQ ID NO: 9.

[0235] In certain embodiments, the anti-C2b antibody or antigen-binding fragment comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein the VH domain comprises the CDR sequences:

[0236] - HCDR3 comprising or consisting of SEQ ID NO: 4 [EDDHDAFAY];

[0237] - HCDR2 comprising or consisting of SEQ ID NO: 3 [DINPNYESTGYNQKFKG];

[0238] - HCDR1 comprising or consisting of SEQ ID NO: 2 [DYNMD], and the VL domain comprises or consists of the amino acid sequence of SEQ ID NO: 9.

[0239] For embodiments wherein the domains of the antibodies or antigen binding fragments are defined by a particular percentage sequence identity to a reference sequence, the VH and / or VL domains may retain identical CDR sequences to those present in the reference sequence such that the variation is present only within the framework regions.

[0240] In certain embodiments, the anti-C2 or anti-C2b antibodies include the CH1 domain, hinge domain, CH2 domain, and / or CH3 domain of a human antibody, in particular human lgG1 , lgG2, lgG3 or lgG4. In certain embodiments, the anti-C2 or anti-C2b antibody includes the CH1 domain, hinge domain, CH2 domain, and CH3 domain of a human lgG1 and includes the substitutions L234A and L235A in the CH2 domain, wherein the positions are defined in accordance with EU numbering. Alternatively or in addition, the anti-C2 or anti-C2b antibody includes the CH1 domain, hinge domain, CH2 domain, and CH3 domain of a human lgG1 and includes the substitutions H433K and N434F in the CH3 domain, wherein the positions are defined in accordance with EU numbering. EU numbering refers to the convention for the Fc region described in Edelman, G.M. et al., Proc. Natl. Acad. Sci. USA, 63: 78-85 (1969); and Kabat et al., in "Sequences of Proteins of Immunological Interest", U.S. Dept. Health and Human Services, 5th edition, 1991 .

[0241] In certain embodiments, the anti-02 or anti-C2b antibody includes the CH1 domain, hinge domain, CH2 domain, and CH3 domain of a human lgG4. In certain embodiments, the antibody includes the CH1 domain, hinge domain, CH2 domain, and CH3 domain of a human lgG4 and includes the substitution S228P in the hinge domain.

[0242] In certain embodiments, the anti-C2 or anti-C2b antibody includes the CH1 domain, hinge domain, CH2 domain, and CH3 domain of a human lgG4 and includes the substitution L445P in the CH3 domain.

[0243] In certain embodiments, the anti-02 or anti-C2b antibody includes the CH1 domain, hinge domain, CH2 domain, and CH3 domain of a human lgG4 and includes both the substitution S228P in the hinge domain and the substitution L445P in the CH3 domain.

[0244] In certain embodiments, the anti-C2 or anti-C2b antibody includes the CH1 domain, hinge domain, CH2 domain, and CH3 domain of a human lgG4 and includes the substitutions H433K and N434F in the CH3 domain.

[0245] In certain embodiments, the anti-C2 or anti-C2b antibody includes the CH1 domain, hinge domain, CH2 domain, and CH3 domain of a human lgG4 and includes the substitution S228P in the hinge domain, and the substitutions H433K and N434F in the CH3 domain.

[0246] In certain embodiments, the anti-02 or anti-C2b antibody comprises a human IgG heavy chain constant domain. In certain embodiments, the heavy chain constant domain comprises a human lgG1 heavy chain constant domain. In certain embodiments, the heavy chain constant domain consists of a human lgG1 heavy chain constant domain. In certain embodiments, the heavy chain constant domain comprises a human lgG4 heavy chain constant domain. In certain embodiments, the heavy chain constant domain consists of a human lgG4 heavy chain constant domain.

[0247] In certain embodiments, the anti-C2b antibody comprises or consists of a heavy chain with at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence shown as SEQ ID NO: 10, and a light chain with at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence shown as SEQ ID NO: 11 .

[0248] In a preferred embodiment, the anti-C2b antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 10, and a light chain having the amino acid sequence of SEQ ID NO: 11.

[0249] In a preferred embodiment, the anti-C2 or anti-C2b antibody is a monoclonal IgG antibody.

[0250] For embodiments wherein the heavy and / or light chains of the antibodies are defined by a particular percentage sequence identity to a reference sequence, the heavy chain and / or light chain may retain identical CDR sequences to those present in the reference sequence such that the variation is present only outside the CDR regions.

[0251] The anti-C2 or anti-C2b antibodies may be modified within the Fc region to increase binding affinity for the neonatal receptor FcRn, preferably human FcRn. The increased binding affinity may be measurable at acidic pH (for example from about approximately pH 5.5 to approximately pH 6.0). The increased binding affinity may also be measurable at neutral pH (for example from approximately pH 6.9 to approximately pH 7.4). By “increased binding affinity” is meant increased binding affinity to FcRn relative to binding affinity of unmodified Fc region. Typically the unmodified Fc region will possess the wild-type amino acid sequence of human IgG 1 , lgG2, lgG3 or lgG4. In such embodiments, the increased binding affinity to FcRn of the antibody molecule having the modified Fc region will be measured relative to the binding affinity of wild-type IgG 1 , lgG2, lgG3 or lgG4 for FcRn, preferably human FcRn.

[0252] In preferred embodiments of all aspects of the invention described herein, the anti-C2b antibody is empasiprubart. Pharmaceutical Compositions

[0253] The C2 inhibitors, particularly the antibodies and antigen-binding fragments thereof, for use in the methods described herein may be formulated as pharmaceutical compositions for administration to the recipient.

[0254] Pharmaceutical compositions may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 1995. The term “pharmaceutically acceptable carrier” relates to carriers or excipients, which are inherently non-toxic. Examples of such excipients are, but are not limited to, saline, Ringer’s solution, dextrose solution and Hanks’ solution. Non-aqueous excipients such as fixed oils and ethyl oleate may also be used.

[0255] Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, micro-emulsion, liposome, or other ordered structure suitable to high drug concentration. Examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutical compositions include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0256] The pharmaceutical compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonicity agents, such as sugars, polyalcohols such as mannitol, sorbitol, glycerol or sodium chloride in the compositions. Pharmaceutically-acceptable antioxidants may also be included, for example (1 ) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Pharmaceutical compositions may be administered via any suitable mode of administration. For example, administration may be parenteral, preferably by intravenous (i.v.) or subcutaneous (s.c.) injection or infusion. The phrases “parenteral administration” and “administered parenterally” as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intraperitoneal, subcutaneous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0257] C. T eatment Outcomes

[0258] Clinical Outcomes and Treatment Biomarkers

[0259] The methods of the present invention are for the treatment or prevention of IRI in a subject receiving a donor organ transplant. The present invention also provides methods for improving allograft function in a subject receiving a donor organ transplant and methods of reducing the risk of allograft loss in a subject receiving a donor organ transplant. In subjects receiving a kidney transplant, the methods may treat or prevent delayed graft function (DGF) as defined herein.

[0260] In certain embodiments of the methods described herein, allograft function is preserved or improved following administration of the C2 inhibitor. In certain embodiments, the risk of allograft loss is reduced following administration of the C2 inhibitor. Suitable methods for measuring allograft function and / or allograft loss, depending on the type of organ transplanted into the recipient, would be known to those skilled in the art. As noted elsewhere herein, allograft loss constitutes the complete failure of the transplanted organ. In the case of kidney transplants there are various means of assessing kidney function that are known to the skilled person.

[0261] A primary measure of kidney function following organ transplantation is the estimated glomerular filtration rate (eGFR). The estimated glomerular filtration rate (eGFR) is a calculation that indicates the level of kidney function, based on the results of a creatinine serum test and other factors — such as age, gender, muscle mass, and ethnicity. The recommended method for calculating the eGFR is the CKD-EPI Creatinine Equation (2021 ): eGFR = 142 x min(Scr / K, 1 )ax max(Scr / K, 1 )1-200x 0.9938A9ex 1 .012 [if female], where:

[0262] ■ SCr is serum creatinine in mg / dL,

[0263] ■ K is 0.7 for females and 0.9 for males,

[0264] ■ a is -0.241 for females and -0.302 for males, min indicates the minimum of Scr / K or 1 , and max indicates the maximum of Scr / K or 1 . Age (years)

[0265] An eGFR below 90 may mean that the kidneys are not functioning as well as they should be, whilst an eGFR below 45 may indicate severe damage to the organ. The eGFR can be calculated following a simple blood test which may be carried out at various time points posttransplantation to confirm the effectiveness of the C2 inhibitor.

[0266] The degree of allograft injury following administration of a 02 inhibitor may be assessed by measuring one or more biomarkers. Suitable biomarkers depending on whether the allograft is kidney, heart, lung, pancreas, bowel or liver would be known to those skilled in the art. Accordingly, the level of one or more biomarkers of allograft injury, preferably serum, blood and / or urine biomarkers of allograft injury, may be assessed in the subject following administration of the C2 inhibitor. Evaluation of certain biomarkers may be used to determine whether a subject would benefit from administration of one or more post-operative doses of the 02 inhibitor.

[0267] For embodiments wherein the subject is receiving a kidney transplant, kidney function can be evaluated based on the levels of certain biomarkers known to be associated with kidney injury. Accordingly, in certain embodiments wherein the donor organ is a kidney, the level of one or more biomarkers of kidney injury in the serum, blood and / or urine of the recipient may be assessed following administration of the C2 inhibitor. In certain embodiments, the one or more biomarkers of kidney injury is selected from: neutrophil gelatinase-associated lipocalin; albuminuria; cell debris; extracellular DNA; creatinine; cystatin C; B2 microglobulin; kidney injury molecule-1 ; urinary retinol binding protein 4; fibroblast growth factor-23; urinary connective tissue growth factor; apolipoprotein A-IV; MCP-1 ; liver-type fatty acid-binding protein; asymmetric dimethylarginine; and uromodulin.

[0268] The success of the 02 inhibitor treatment may also be measured using a patient reported outcome (PRO). A patient-reported outcome is any report of the status of a patient's health condition that comes directly from the patient without interpretation of the patient's response by a clinician or anyone else. Examples of PROs include but are not limited to: EQ-5D-5L, PROMIS and KDQOL-36.

[0269] The C2 inhibitors described herein are effective for the treatment or prevention of IRI, allograft dysfunction and / or DGF, due to their direct action in inhibiting complement and thus preventing complement-mediated damage to the donor organ. Accordingly, any C2 inhibitor and any dosing regimen can be used in the methods of the invention provided that said C2 inhibitor or dosing regimen has the desired effect of inhibiting complement and preventing complement-mediated damage to the organ. There are various different means of quantifying complement activity and these would be known to the skilled person.

[0270] Hemolytic assays have traditionally been used to assess the functional activity of the classical pathway of the complement system. They provide insight into the integrity of the entire cascade reaction. In brief, serial dilutions of the sample to be analysed are incubated with antibody- sensitized sheep erythrocytes at a defined temperature. Hemolytic assays are performed either in tubes or in agarose plates. The results are usually expressed as reciprocal dilutions of the sample required to produce 50 or 100% lysis (CH50 assay or CH100 assay, respectively). Hemolytic assays evaluating the functional activity of the alternative pathway (AH50) use guinea pig, rabbit, or chicken erythrocytes as target cells. Here, activation of the classical pathway has to be blocked by adding EGTA to chelate Ca2+, and an optimal concentration of Mg2+is required.

[0271] Newer automated methods include the liposome immunoassay (LIA). The LIA relies on liposomes encapsulating glucose-6-phosphate dehydrogenase (G6PDH), to mimic an invading microorganism. On addition of sample, antibodies in the reagent combine with dinitrophenyl groups on the surface of the liposomes. The resultant complex activates complement in the sample, which lyses the liposomes, releasing G6PDH to react with glucose-6-phosphate and NAD in the reagent. The change in absorbance can be measured and is proportional to the complement activity in the sample. Comparison to a calibration curve gives a value for the unknown patient sample.

[0272] Other known tests for determining complement activity include the enzyme immunoassay (EIA). The EIA combines the principles of the hemolytic assay with the use of a monoclonal antibody specific for neoantigen (C5b-9 complex) produced as a result of complement activation (Incstar, Stillwater, Minn.). The amount of polymerized C5b-9 (final product) is proportional to the functional activity of C1 through C9.

[0273] It is expected that complement activity will increase in a subject receiving an organ transplant. In the methods of the invention, the C2 inhibitor reduces complement activity in the subject following organ transplantation compared to the complement activity of a control subject that has not been administered the C2 inhibitor. Accordingly, in the methods of the invention, the C2 inhibitor inhibits or prevents an increase in complement activity following organ transplantation. In certain embodiments, the C2 inhibitor reduces classical pathway complement activity in the subject following organ transplantation compared to the classical pathway complement activity in a control subject that has not been administered the C2 inhibitor. In certain embodiments, the C2 inhibitor reduces lectin pathway complement activity in the subject following organ transplantation compared to the lectin pathway complement activity in a control subject that has not been administered the C2 inhibitor. In certain embodiments, the C2 inhibitor reduces classical and lectin pathway complement activity in the subject following organ transplantation compared to a control subject that has not been administered the C2 inhibitor. In certain embodiments, the C2 inhibitor does not affect the alternative pathway complement activity in the subject.

[0274] The levels and / or activity of individual complement proteins can also be assessed. In particular, it is shown in the examples herein that an anti-rat C2 antibody is effective to reduce serum free C2 levels by as much as 95% in a rat kidney transplant model. Accordingly, in certain embodiments, the C2 inhibitor reduces the level of free C2 in the subject following organ transplantation compared to the level of free C2 in a control subject that has not been administered the C2 inhibitor. More specifically, the C2 inhibitor reduces the levels of free C2 in the serum, plasma and / or urine of the subject.

[0275] In certain embodiments, the level of free C2 in the subject is reduced to or maintained at or below a threshold level. In certain embodiments, the level of free C2 in the serum, plasma and / or urine of the subject is reduced to or maintained at or below a threshold level. In certain embodiments, a C2 inhibitor is administered if the level of free C2 in the subject is above a threshold level. In certain embodiments, a C2 inhibitor is administered if the level of free C2 in the serum, plasma and / or urine of the subject is above a threshold level. In certain embodiments, one or more post-operative dose(s) of a C2 inhibitor is administered if the level of free C2 in the subject is above a threshold level. In certain embodiments, one or more post-operative dose(s) of a C2 inhibitor is administered if the level of free C2 in the serum, plasma and / or urine of the subject is above a threshold level. In certain embodiments, the threshold level is 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 pg / mL.

[0276] The level and / or activity of other complement proteins may also be assessed, such as any of the downstream proteins C3-C9. In certain embodiments, a C2 inhibitor is administered if the level of C3, C4, C5, C6, C7, C8 and / or C9 in the subject is above a threshold level. In certain embodiments, following administration of the C2 inhibitor, the level of C3, C4, C5, C6, C7, C8 and / or C9 in the subject is reduced compared to a control subject that has not been administered the C2 inhibitor. In certain embodiments, following administration of the C2 inhibitor, the level of C3, C4, C5, C6, C7, C8 and / or C9 in the serum, plasma and / or urine of the subject is reduced compared to a control subject that has not been administered the C2 inhibitor.

[0277] D. Additional treatments

[0278] The methods of the present invention may involve the use of a C2 inhibitor in conjunction with one or more additional therapeutic agents, for example one or more immunosuppressive therapies.

[0279] Accordingly, in certain embodiments, the method further comprises administration to the subject of one or more immunosuppressive agents. The skilled person is aware of known immunosuppressive therapies and regimens suitable for organ transplantation. Examples of immunosuppressive therapies include but are not limited to: antithymocyte globulin (ATG), tacrolimus, mycophenolate mofetil (MMF), enteric-coated mycophenolic acid (EC-MPA), enteric- coated mycophenolate sodium (EC-MPS), cyclosporine, corticosteroids, azathioprine, everolimus, sirolimus, rapamycin, belatacept, basiliximab, alemtuzumab, and any combination thereof. In certain embodiments, the method further comprises administration to the subject of antithymocyte globulin (ATG) and corticosteroids. In certain embodiments, the method further comprises administration to the subject of tacrolimus and MMF or EC-MPA and optionally corticosteroids.

[0280] In certain embodiments, the subject receiving the donor organ transplant, particularly a kidney transplant, receives, in addition to the C2 inhibitor an immunosuppressive treatment regimen comprising or consisting of: (i) induction therapy with antithymocyte globulin (ATG) and IV corticosteroids; or (ii) maintenance therapy with tacrolimus, MMF or EC-MPA and optionally oral corticosteroids. In certain embodiments, the subject receiving the donor organ transplant, particularly a kidney transplant, receives, in addition to the C2 inhibitor an immunosuppressive treatment regimen comprising or consisting of: (i) induction therapy with antithymocyte globulin (ATG) and IV corticosteroids; and (ii) maintenance therapy with tacrolimus, MMF or EC-MPA and optionally oral corticosteroids. The induction therapy may involve IV infusion of corticosteroids to be completed 30 minutes before reperfusion of the kidney allograft and before initiation of the ATG. The total dose of ATG may be 4mg / kg to 6 mg / kg, preferably divided into once daily doses to be administered over a 3-4 day period. The co-administration of ATG and the C2 inhibitor is typically not permitted. The maintenance therapy may involve oral administration of tacrolimus, preferably for a 24-week period post-transplant. Tacrolimus may be administered per standard of care with dose adjustments based on trough measurements. Trough levels may be 6 to 12 ng / mL through week 4 and 4 to 8 ng / mL from weeks 4 through 52. MMF or EC-MPA may be administered post-transplant as per standard of care. MMF may be administered at a dose of 500 mg to 1000 mg twice a day. EC-MPA may be administered at a dose of up to 720 mg twice a day. In some embodiments, the maintenance therapy involves the administration of oral corticosteroids (e.g. prednisone). Dosing of the oral corticosteroids (e.g. prednisone) may be tapered such that a daily dosage of up to 5 mg is administered by 3 months post-transplant.

[0281] The invention will now be further understood with reference to the following non-limiting examples.

[0282] EXAMPLES

[0283] EXAMPLE 1 : Complement inhibition in in vitro models of ischemia reperfusion injury

[0284] The complement inhibitory effect of a monoclonal antibody blocking complement factor C2b (ARGX-117, also known as empasiprubart) was evaluated in an in vitro model for ischemia reperfusion injury (IRI) using primary lung-derived and primary kidney-derived endothelial cells. An objective of the study was to investigate the C2 dependency of IRI-mediated complement activation.

[0285] The results of this study demonstrate that primary lung-derived endothelial cells and primary kidney-derived endothelial cells cultured at low oxygen levels exhibit a decrease in ATP levels. Subsequent reoxygenation in the presence of human serum increased early apoptosis (Annexin V+) and apoptotic (Annexin V+ / 7AAD+) cells that were opsonized by IgM antibodies. The IgM binding resulted in C2-dependent complement activation which could effectively be inhibited by ARGX-117 in a dose-dependent manner.

[0286] 1.1 MATERIALS AND METHODS

[0287] 1.1.1 Protocol for culturing primary endothelial cells

[0288] Primary lung endothelial cells (ECs) and primary kidney ECs were obtained during lung or kidney transplantation and cultured as described previously (Budding etal., 2017). To reduce the risk of thromboembolic complications shortly after transplantation, grafted organs were flushed antegrade using perfadex solution. Subsequently, lungs were flushed via the pulmonary or renal vein respectively, until outlet fluid was clear and free of blood clots upon visual inspection. The perfusion fluid was collected, centrifuged (125 x g, 10 min), and the cell pellet was resuspended in PBS, and subjected to Ficoll-Paque cell density gradient centrifugation. Cells were isolated from the interphase and stored in 1 mL aliquots (RPMI-1640, supplemented with 10%, v / v, DMSO and 20%, v / v, FCS) in liquid nitrogen. For cell culturing, frozen aliquots were thawed at 37°C and washed with RPMI-1640 20%, v / v, FCS. After centrifugation (125 x g, 10 min), the pellet was resuspended in endothelial cell specific medium and cultured at 37°C and 5%, v / v, CO2. ECs were characterized for the expression of EC markers CD13, CD31 , and VWF. ECs were negative for CD45, and morphology was assessed using light microscopy. 1.1.2 Protocol for hypoxia-induction and measurement of complement activation

[0289] One day before the start of the experiment, endothelial cells were seeded in a 96-well flat-bottom plate at a density of 50,000 cells / well. Prior to culture under hypoxic conditions, medium was refreshed. Cells were kept under hypoxic conditions for 16hrs overnight (O / N). Depending on the experimental set-up, hypoxic plates were kept on ice or at 37°C. A normoxia control plate was kept under normal culture conditions, and when indicated, kept on ice O / N. Next, medium was discarded and cells were incubated with fresh human pooled serum (as a source of complement) at different dilutions in 100 pL volume. Serum samples were pre-incubated with respective anticomplement antibodies or complement controls (EDTA as negative control for complement activation and MgEGTA as control for AP-mediated complement activation) for 20 min on ice. Next, cells were reoxygenated for 2 hrs at 37°C by culturing at normal oxygen levels. Subsequently, cells were washed with 100 pL PBS, following detachment using accutase (25 pL / well). After detachment, 75 pL PBS was added, cells were centrifuged (5 min at 125 x g) and resuspended in 100 pL PBS. Next, cells were transferred to a v-bottom plate, and stained with respective IgM or C3 detection antibodies diluted in FACS buffer (PBS, 1% BSA, 0.01% sodium azide) for 45 min incubation on ice in the dark. Then, cells were washed once by adding 100 pL FACS buffer followed by 5 min centrifugation at 125 x g and incubated with a secondary antibody for 45 min on ice in the dark. After incubation, 100 pL FACS buffer was added and cells were centrifuged (5 min at 125 x g). When applicable, cells were stained with Annexin V / 7AAD to discriminate between viable and (early / late) apoptotic cells, prior to measurement. For staining with AnV / 7AAD, the cells were washed with ice-cold PBS after the secondary antibody staining and incubated with AnV / 7AAD in Annexin V binding buffer for 15 min at RT in the dark. Prior to measurement, 100 pL Annexin V binding buffer was added. When AnV / 7AAD staining was not done, the cell pellet was resuspended in 100 pL FACS buffer, and the cells were analyzed using a FACS Canto II and accompanying software.

[0290] 1.2 RESULTS

[0291] 1.2.1 IgM binding to and complement activation on primary lung-derived endothelial cells exposed to hypoxia and reoxygenation in the presence of fresh human serum

[0292] Primary lung-derived endothelial cells were cultured as described above. These cells were quite sensitive to hypoxia as shown in a representative experiment depicted in Fig. 1. Overnight exposure to 0.1% O2 resulted in a strong decrease of intracellular ATP concentrations (Fig. 1A). This coincided with increased numbers of AnV+ / 7AAD+ cells (Fig. 1 B). After O / N culture under normoxic or hypoxic conditions, cells were reoxygenated for 2 hrs in the presence of 30% human serum. Various serum conditions were analyzed, in which the respective serum sample was preincubated with ARGX-117, an isotype control, or an anti-C1 s antibody. Heat-inactivated (HI) serum was included as a negative control as heat inactivation destroys the functional activity of complement. Also, IgG / IgM-depleted serum was used as a negative control for IgM binding. Next, cells were analyzed for IgM binding and C3 fixation. Increased IgM binding to hypoxic cells was observed as compared to cells cultured under normoxic conditions. Importantly, this was not observed when using IgG / IgM-depleted complement active serum (Fig. 1C). After reoxygenation, increased 03 fixation was detected in the 30% serum control, as well as in the serum condition preincubated with an isotype control antibody. Both HI serum and IgG / IgM-depleted serum only showed a small increase in C3 fixation.

[0293] Moreover, preincubation of the complement active serum with ARGX-117 dose-dependently abrogated 03 fixation to the level of the negative controls (serum HI and IgG / IgM-depleted serum). Pre-incubation with anti-01 s resulted in approximately 40-50% decrease of 03 fixation (Fig. 1 D). Both IgM binding and 03 fixation occurred predominantly on early apoptotic (AnV+) and late apoptotic cells (AnV+ / 7AAD+), and not on living cells (AnV- / 7AAD-) (Fig. 1E and F).

[0294] The results discussed above were confirmed in follow-up independent experiments (n=4, for the majority of conditions). Although variation was observed between individual experiments, the fold change for IgM (FCigwi) within a single experiment was equal for each individual serum condition (Fig. 2A). Also, the inhibitory effect of ARGX-117 was found to be reproducible, with maximal inhibition of C3 fixation of about 95% at 2 mg / mL. Anti-C1 s maximally inhibited C3 fixation by 60%. As expected, no effect on overall C3 fixation was measured when the complement active serum was pre-incubated with an isotype antibody (Fig. 2B).

[0295] 1.2.2 IgM binding to and complement activation on primary kidney-derived endothelial cells exposed to hypoxia and reoxygenation in the presence of fresh human serum

[0296] In order to link the biological relevance of the IRI model to the delayed graft function associated with kidney transplant, primary kidney-derived ECs were used to investigate the effect of hypoxia, IgM binding, and subsequent complement activation. Like the primary lung-derived ECs, kidney-derived ECs cultured O / N in hypoxic conditions (0.1 % O2) showed a marked decrease of intracellular ATP (Fig. 3A) and an increase in AnV+ and AnV+ / 7AAD+ cells (Fig. 3B). Subsequent reoxygenation in the presence of 30% serum, either pre-incubated with or without complement control antibodies, HI serum, or 30% C2-depleted serum either reconstituted or not with C2, resulted in all cases in increased IgM binding. This increase in IgM binding was not observed when ECs were cultured in normoxic conditions (Fig. 3C).

[0297] Next, complement activation was analyzed by assessing C3 fixation. C3 fixation was not increased on kidney ECs cultured in normoxic conditions and subsequently incubated with serum. However, the same cells cultured in hypoxic conditions and then incubated with 30% human serum, showed a marked increase of C3 fixation. Exposing the cells during reoxygenation with HI serum instead of human serum abrogated C3 fixation indicating the increased C3 fixation was due to complement activation on the cells.

[0298] Pre-incubation of human serum with ARGX-117 inhibited C3 fixation to the level of the C2- depleted serum control, while no inhibitory effect was observed when an isotype control antibody instead of ARGX-117 was added to the serum. Reconstitution of C2-depleted serum with physiological concentrations of human 02 (30 pg / mL) restored C3 fixation to the level of the serum control. A limited decrease in C3 fixation was observed in the presence of the anti-01 s antibody (Fig. 3D). Both IgM binding (Fig. 3E) and 03 fixation (Fig. 3F) were predominantly present on AnV+ and AnV+ / 7AAD+ cells, and not on living cells.

[0299] The results described above were obtained in a single experiment. Therefore, the experiments with kidney-derived endothelial cells were repeated to confirm the results seen. Increased IgM binding to hypoxic cells was detected for all serum conditions relative to the normoxia serum control. In Fig. 4 this binding is quantified as fold change (FC) using the normoxia human serum control set at 1 . Importantly, IgM binding to hypoxic cells was similar upon reoxygenation with HI serum instead of human serum. Furthermore, using C2-depleted serum as a source of complement also resulted in a comparable increased IgM binding (Fig. 4A). Pre-incubation of complement active serum with ARGX-117 dose-dependently inhibited 03 fixation. At a concentration of ARGX-117 of 2 mg / mL, 03 fixation was reduced by 75%. Eculizumab showed no inhibitory effect, similar to the isotype control. Pre-incubation of serum with anti-01 s resulted in a maximum of 50% reduction of 03 fixation. In Fig. 4C, the signal of 03 fixation using 02- depleted serum was set at 100% inhibition of 03 fixation and that of the C2-depleted serum reconstituted with 30 pg / mL 02 at 0% inhibition of 03 fixation. 03 fixation to primary kidney ECs could be dose-dependently restored by supplementation of C2-depleted serum with purified 02. Increased 03 fixation compared to that of cells incubated with 02 depleted serum, was already detected at a 02 concentration of 1 .11 pg / mL, suggesting that hypoxia-induced IgM-mediated complement activation on kidney ECs occurs at relatively low 02 concentrations, when compared to lung-derived ECs. Finally, the C2-depleted serum used in the experiments described above, may have contained residual 02, since pre-incubation of reconstituted 02- depleted serum with ARGX-117 resulted in 130% inhibition compared to the C2-depleted serum control at 100%.

[0300] CONCLUSIONS

[0301] The complement inhibitory effect of ARGX-117, targeting human 02, was evaluated in an in vitro model for hypoxia using primary lung-derived endothelial cells and primary kidney-derived endothelial cells as a disease model for ischemia reperfusion injury. A model for ischemiareperfusion injury was set up involving O / N culture of endothelial cells under hypoxic conditions. The hypoxic conditions resulted in a decrease in ATP levels confirming that exposure to low oxygen levels had been sufficient to induce ischemic stress of these cells. Subsequent reoxygenation in the presence of complement active serum resulted in an increase of AnnexinV positive and AnnexinV / 7AAD double positive cells, a marker for membrane damage and apoptosis. Incubation with human serum of these cells resulted in increased IgM binding, and complement activation, shown by increased C3 fixation. In contrast to increased IgM fixation to the cells, increased C3 fixation was not observed with heat-inactivated serum, which contains no active complement system, neither with C2 depleted serum, which does not contain functional classical pathway (CP) or lectin pathway (LP) activity. Thus, C3 fixation to ECs cultured in ischemic conditions and reoxygenated in presence of human serum, induced C3 fixation due to complement activation via the CP and / or LP. Importantly, this IgM-mediated complement activation was dependent on C2 and could be inhibited dose-dependently by ARGX-117.

[0302] Taken together, these data demonstrate exposure of ECs to ischemic stress and subsequent reoxygenation results in complement activation and C3 fixation to these cells. This activation is triggered by IgM fixed to these cells due to the ischemic stress, and occurs via the CP and / or LP. In agreement, absence of C2 in serum during reoxygenation of ECs, markedly decreases C3 fixation to these cells. ARGX-117 is an antibody, which upon addition to serum, inhibits complement activation. Addition of ARGX-117 to human serum dose-dependently prevented C3 fixation during reoxygenation. Therefore, C2 inhibition has therapeutic potential to attenuate the effects of ischemia-reperfusion-mediated complement activation in various human organs. inhibition in an in vitro chip model of human renal ischemia

[0303] In this study, a microfluidic 3D co-culture of human renal proximal tubule epithelial cells (RPTECs) and human umbilical vein endothelial cells (HUVECs) was used to model human renal ischemia reperfusion injury (IRI). This model was used to evaluate inhibition of ischemia reperfusion-induced complement activation using ARGX-117, an antibody that binds human complement factor C2 (at the level of C2b) and blocks its activation.

[0304] The results of this study showed that when cultures were exposed to ischemia, followed by reperfusion in the presence of complement-active human serum, complement activation was induced in the endothelial compartment, as measured by IgM and 03 deposition. ARGX-117 dose-dependently inhibited 03 deposition on HUVECs during reperfusion. 2.1 Establishment of the microfluidic co-culture model of renal ischemia reperfusion injury (IRI)

[0305] In this study, a microfluidic 3D co-culture of renal proximal tubule epithelial cells (RPTECs) and human umbilical vein endothelial cells (HUVECs) was used. To guide cells to grow in a tubular conformation, the RPTECs and HUVECs were plated in the OrganoPlate® 3-Lane 40 against a collagen I extracellular matrix gel (Fig. 5). On day 6 or 7 of culture, fully grown and polarized tubular structures were established, as indicated by phase contrast images per experiment (Fig. 5D). Fig. 5E shows a published immunofluorescent 3D reconstruction of this model.

[0306] The microfluidic co-culture model has previously been used for studying renal ischemia reperfusion injury as described by Vormann et al. (2022). In this previous study, ischemia was introduced by exposing the cultures to low oxygen (5% O2) and / or low glucose and nutrient availability and / or lack of perfusion flow. Following ischemia, medium was replaced, and the plates were cultured on an interval rocker platform under normoxic conditions for 24 hours to mimic reperfusion flow. This was compared with a normoxic culture comprising atmospheric O2 levels of 21%, glucose and nutrient rich medium and perfusion flow. Ischemic conditions followed by reperfusion induced cell damage, reminiscent of ischemia reperfusion injury. The combination of low oxygen, glucose absence, and lack of perfusion flow was the most potent condition in terms of damage. Morphological assessment of the cultures revealed rounded-up cells and clustered morphology of the RPTECs after the ischemic period which was more severe after reperfusion where parts of the tubule were gone. The HUVECs were shown to be less sensitive to ischemia reperfusion conditions than the RPTECs.

[0307] The co-culture model was optimized so as to study potential damaging effects of complement activation induced by ischemia reperfusion. Various times and conditions of ischemia were tested. To this end, cultures were first exposed to hypoxia (1 % O2) (or normoxia as a control) in a hypoxic incubator, under (1 ) perfusion flow (5h, 20h or 24h), (2) perfusion flow followed by static incubation (20h flow + 4h static, 24h flow + 24h static, or 44h flow + 4h static), or (3) completely static conditions (24h or 48h). The cultures were then reperfused for 24 hours, with medium containing 30% human serum in the HUVEC channel, as a source of complement proteins. Control cultures were reperfused with medium only or with medium containing heat- inactivated (HI) serum (in which the complement system is inactivated).

[0308] Exposure of the chips to 48h ischemia (i.e. static hypoxia), followed by 24h reperfusion with or without (HI) serum, induced IRI to the cultures, in particular to the RPTECs. Both HUVECs and RPTECs showed visual damage and caspase-3 / 7 activity after ischemia, which was further exacerbated upon reperfusion. In addition, RPTECs exposed to ischemia, but not normoxia, also had reduced barrier integrity, which was not further exacerbated upon reperfusion. The 48h static hypoxic conditions were selected for further tests as this not only reproducibly induced ischemic damage to the cells in the experimental co-culture model, but also better reflects the in vivo situation following occlusion or clamping of a renal afferent vessel.

[0309] 2.2 Ischemia reperfusion induced complement activation on HUVECs in the co-culture system, which was blocked by ARGX-117

[0310] The co-culture model was exposed to ischemia reperfusion conditions (selected in 2.1 ), in the presence or absence of complement-active, or heat-inactivated, human serum in the HUVEC channel during reperfusion. Conditions were added where human complement-active serum was pre-incubated with different concentrations of ARGX-117. Cultures exposed to normoxia instead of ischemia were also added as controls (Fig 6A). The experiment was performed three times and the aim of these experiments was to test whether 1 ) ischemia reperfusion induced IgM and C3 deposition in the co-culture system and 2) whether ARGX-117 inhibited complement deposition.

[0311] 2.2.1 Ischemia reperfusion induced IgM and C3 deposition on HUVECs

[0312] Cultures exposed as explained above were fixed after the reperfusion period, and stained for C3 and IgM. In cultures with 30% human serum as the complement source, IR increased C3 deposition in the HUVEC channel as compared to normoxia-reperfusion cultures (Fig. 6B and 6C). Under normoxic conditions, only limited C3 deposition was observed but mostly at the ECM-HUVEC interface, which was excluded from quantification. (Non-specific C3 binding to the ECM was detected, which partially overlaps with the HUVEC channel in maximum projection images due to the meniscus shape of the ECM). C3 deposition on HUVECs was not observed when only medium was added (in the absence of a complement source) ruling out non-specific binding of anti-C3 antibodies. It was also not observed when 30% HI serum was added, indicating that C3 deposition was an active process dependent on IR-induced complement activation.

[0313] Ischemia also increased IgM deposition on HUVECs as compared to normoxia, both in chips supplemented with human serum and HI human serum (Fig. 6D and 6E). In the absence of serum, IgM was not detected, as expected.

[0314] IgM and C3 deposition were also observed on the RPTEC tubules in some chips exposed to IR and supplemented with complement-active human serum in the HUVEC channel. In line with the findings for HUVECs, IgM deposition on RPTECs was also observed when human serum was heat inactivated. These findings were not further quantified.

[0315] In conclusion, ischemia reperfusion induced complement activation in the 3D co-culture model, as shown by C3 and IgM deposition. These depositions were not seen in normoxia-reperfusion cultures, confirming complement activation by ischemia reperfusion specifically. Whereas IgM deposited in cultures supplemented with HI serum, C3 did not, confirming that C3 deposition was an active process dependent on the presence of an active complement system in the serum used as a complement source.

[0316] 2.2.2 ARGX-117 reduced free C2 levels in cultures exposed to complement-active human serum

[0317] The efficacy of ARGX-117 to inhibit IR-induced complement activation in this renal co-culture model was evaluated by adding different concentrations of ARGX-117 to the HUVEC channel during the reperfusion phase (pre-incubated in the human serum). First, to test whether ARGX- 117 effectively bound all 02 in the test system, free 02 levels (not bound by ARGX-117) in the culture supernatants at the end of the experiments were measured (Fig. 7).

[0318] In chips supplemented with only medium, or with medium supplemented with HI serum, almost all free 02 levels were below the quantification limit, both in the HUVEC and the RPTEC compartments. Therefore, the data suggests that HUVECs and RPTECs do not produce detectable levels of endogenous C2 in this test system under these experimental conditions.

[0319] In the complement-active serum conditions without ARGX-117, free C2 was detected, as expected. Levels were highest in the HUVEC supernatants, as expected since the serum was added to the HUVEC channel. For HUVECs, free 02 levels were similar to slightly higher in chips exposed to IR than in chips exposed to normoxia-reperfusion. Free 02 levels were dose- dependently reduced by ARGX-117, with the highest ARGX-117 dose (2 mg / mL, end concentration) resulting in free 02 levels below the quantification limit, thus showing that ARGX- 117 effectively bound 02 in the test system.

[0320] Free C2 was also detected to a smaller extent in RPTEC supernatants from chips exposed to complement-active human serum in the HUVEC channel. Free 02 levels were variable and low in normoxia-reperfusion conditions but increased in IR conditions. Also in the RPTEC channels, free 02 levels were dose-dependently reduced by ARGX-117, with the highest ARGX-117 dose (2 mg / mL, end concentration) resulting in free 02 levels below the quantification limit. It is hypothesized that the C2 present in the RPTEC tubule diffused from the HUVEC channel, with the channel permeability being higher in the ischemic conditions as a result of damage to the tubules. In line with this, human albumin levels, originating from the human serum supplemented to the HUVEC channel, were also drastically increased in RPTEC channels upon ischemia reperfusion as compared to normoxia-reperfusion conditions (data not shown).

[0321] 2.2.3 ARGX-117 reduced IR-induced IgM and C3 deposition

[0322] Addition of ARGX-117 decreased IR-induced C3 deposition on HUVECs. Such reduced deposition was observed in all three separate experiments (Fig. 6B). By pooling the data of the independent experiments, a significant reduction in C3 deposition on HUVECs was observed after addition of 2 mg / mL ARGX-117 (end concentration) (P=0.016) (Fig. 6C). Several concentrations of ARGX-117 were tested in experiment 2 and 3. This indicated that ARGX-117 dose-dependently reduced C3 deposition on the HUVECs.

[0323] IgM deposition on HUVECs was not affected by ARGX-117, as expected since IgM deposition is upstream of C2 activation (Fig. 6D and 6E). This was observed in all three experiments.

[0324] IgM and C3 depositions were also observed on the RPTECs in some chips exposed to IR and supplemented with (complement-active) human serum in the HUVEC channel. Upon addition of ARGX-117, decreased C3 deposition was observed in the RPTEC channel which was most pronounced with 2 mg / mL ARGX-117 (highest concentration). It is hypothesized that these complement proteins diffused from the HUVEC channel, followed by activation in the RPTEC compartment. The findings on RPTECs were not further quantified.

[0325] To summarise, the addition of ARGX-117 to 30% complement-active human serum significantly decreased IR-induced C3 deposition in the HUVEC tubule, without affecting IgM deposition, confirming that C2 binding by ARGX-117 inhibited complement activation downstream of C2 in this model for human renal IRL

[0326] 2.3 IR-induced complement activation increased ICAM-1 expression on RPTECs, which was reduced by ARGX-117

[0327] ICAM-1 expression by the HUVECs and RPTEC tubules was determined by immunofluorescent staining. Expression of ICAM-1 , a major adhesion molecule involved in leukocyte migration by the renal proximal tubule occurs during various types of renal injury and is linked to increased leukocyte and monocyte infiltration and adhesion in vivo (Wu et al., 2007). In the test system used, expression of ICAM-1 by HUVEC and RPTEC was low to absent under normoxia- reperfusion conditions but increased after IR in the HUVEC channel, independently of the presence of complement-active or HI serum. ICAM-1 expression by HUVEC was not further quantified.

[0328] In contrast, ICAM-1 upregulation by RPTEC after IR was only observed upon reperfusion with complement-active serum, but not upon reperfusion with HI serum or medium-only, indicating that this expression reflected a complement-dependent IR response by RPTEC (Fig. 8).

[0329] Specifically, ICAM-1 upregulation was observed in the RPTEC-ECM interface. 3D reconstructions confirmed that the staining was specifically located on the RPTEC, and not due to an artificial background staining of the ECM, as seen for C3 and IgM. Therefore, ICAM-1 staining of the whole RPTEC tubule was used for quantification (Fig. 8). Addition of ARGX-117 during reperfusion with complement-active serum dose-dependently reduced ICAM-1 expression by RPTEC after IR exposure, indicating a potentially beneficial effect of ARGX-117 on RPTEC IRI (Fig. 8). IR-induced ICAM-1 staining of the RPTEC channel was somewhat variable; however, in all cases where substantial staining was observed, this staining was reduced by ARGX-117.

[0330] ICAM-1 expression by RPTECs can occur in vivo and is an indicator of renal damage. It may increase injury by recruiting and activating several immune cells such as neutrophils and monocytes. Indeed, treatment of animals undergoing renal IR with anti-ICAM-1 antibodies or anti-sense oligonucleotides reduces renal IRI and improves kidney function (Rabb et al. 1995; Kelly et al. 1994; and Dragun et al. 1998)). Therefore, the finding that ARGX-117 reduced IR- induced expression of ICAM-1 by RPTECs in the in vitro model for human renal IRI supports the potential of ARGX-117 as a treatment for DGF and / or other forms of renal IRI.

[0331] CONCLUSIONS

[0332] A 3D human renal proximal tubule / endothelial vessel co-culture model was optimized so as to study IR-induced complement deposition. An ischemic event was induced by exposing the cultures for 48 hours to a combination of two parameters: low oxygen (1 % O2) and lack of perfusion flow (static). Reperfusion was mimicked by culturing the cells in 21% O2 under flow conditions for 24 hours. Under these conditions, IR induced morphological alterations in both the RPTEC and HUVEC tubules, indicative of IR-induced injury (IRI).

[0333] When 30% complement-active human serum was added to the reperfusion medium for the HUVECs, increased C3 and IgM deposition on the cells was observed upon an IR event, as compared to normoxia-reperfusion. Reperfusion with heat-inactivated serum, which does not have a functional complement system, did not result in C3 deposition, whereas IgM deposition was not affected, indicating that in this experimental model, IR induces complement activation, most likely triggered by natural IgM binding to ischemia-induced neoepitopes on the HUVECs. The addition of ARGX-117 to 30% complement-active human serum significantly decreased C3 deposition in the HUVEC tubule, without affecting IgM deposition, confirming that blockade of C2 by ARGX-117 inhibited complement activation downstream of C2 in this model for human renal IRI.

[0334] EXAMPLE 3: Complement inhibition and its functional consequences in an in vivo kidney transplant model in male Lewis rats, where the transplanted organ was exposed to cold ischemia and subsequent reperfusion leading to IRI

[0335] The objective of this study was to determine the efficacy of an anti-rat 02 monoclonal antibody in a syngeneic kidney transplant model in male Lewis rats. The organ was exposed to cold ischemia and subsequent reperfusion, thereby inducing IRI, and the rats were administered the anti-C2 mAb twice intravenously. Blocking complement at the level of 05 using an anti-05 mAb was also evaluated in the same model. Read-outs of the study were mortality beyond 24 hours of surgery (NB mortality in the first 24 hours was considered to be procedure-related), serum creatinine and urea levels, free 02 levels (unbound to anti-rat 02 mAb), and histopathology.

[0336] 3.1 Study Design

[0337] The Lewis rat was chosen as the animal model for this study as the mAbs tested in this study were shown to bind to and block activity of rat 02 and the disease model studied - the syngeneic kidney transplant model - was available in this rat strain. Donor kidneys were obtained by surgical resection of the left kidney from donor rats. Immediately after removal, the left kidney was placed in a sterile container with at least 50 mL of Belzer UW cold preservation solution. The duration of kidney preservation in cold storage solution with the temperature set at 4°C is defined herein as the Cold Ischemia Time (CIT). For transplant into the recipient rat, the left kidney of the recipient was resected and the donor kidney artery and venal cuff were connected to the recipient abdominal aorta and vena cava respectively by anastomosis. Immediately (30-60 seconds) before renal reperfusion, the animal received a bolus IV injection of 50 mg / kg of the anti-02 mAb test item, anti-05 mAb test item, or isotype control reference item. This was followed with a 1 mL flush of saline for the recipient animals that received the test item. The ureter was then connected. After the left kidney transplant, the recipient right kidney was removed and discarded (as depicted in Fig. 9). 72 hours after the first dose, a second dose of 25 mg / kg of the same test / reference item was given. Serum samples were collected on various days post-surgery (days 3, 5, 7 and / or 14) as shown in Fig. 9 (with surgery day = day 1 ). Baseline samples were taken for comparison purposes from a subset of donors 1 to 2 days prior to kidney removal. Animals were euthanized when humane endpoints were reached, at day 3, 5 or 7 for histology cohorts in phase 4 (see below), or at day 14 (terminal euthanasia).

[0338] The study described herein consisted of two optimization phases to determine the CIT in the harvested donor kidneys that would lead to significant graft failure in recipient animals (Phases 1 and 2), and two phases where anti-rat C2 mAb was tested (Phases 3 and 4):

[0339] Phase 3 - The primary objective of phase 3 was to determine if anti-rat C2 mAb would prevent DGF and reduce free C2 levels in serum in syngeneic recipient rats. Based on Phases 1 and 2, a CIT of 32h was considered sensitive enough to achieve the anticipated mortality. Three groups of rats were tested: group 6 - treated with an isotype control Ab; group 7 - treated with the anti- 02 mAb; and group 8 - treated with the anti-C2 mAb and a longer CIT of 34h (NB. Only added for optimization purposes; there is no control for this longer CIT). In this phase of the study, recipient rats received 50 mg / kg intravenous bolus injection of antibody immediately before renal reperfusion and an additional 25 mg / kg injection of the same test material 72 hours after the first dose. Serum samples for creatinine and urea measurements were taken on Day 3 for all animals, with surgery day = Day 1 .

[0340] Phase 4 - The primary objective of phase 4 was equivalent to that of phase 3. Phase 4 was performed to determine if the findings of phase 3 could be repeated in an independent experiment. Furthermore, animal number was increased to increase statistical power, serum creatinine and urea measurements were performed at additional time-points and an additional group of recipient animals treated with the reference complement blocker anti-C5 mAb was added. Histology cohorts were also added.

[0341] The experimental study design of Phase 3 and Phase 4, including the different treatment groups in each phase, is summarised in Table 3. Phase 3 and 4 of the study were carried out to determine if targeting the complement pathway at the level of C2 during kidney transplant of 10 to 11 -week old male Lewis rats, at the time of reperfusion may prevent delayed graft function (DGF) after cold ischemia time of the donor kidney.

[0342] Table 3. Phase 3 and 4 test groups of the rat kidney transplant model

[0343] 3.2 RESULTS

[0344] 3.2.1 Mortality, clinical observations, body weights, body weight gains, clinical signs

[0345] There were no anti-C2 mAb or anti-C5 mAb-related effects on mortality, clinical observations, body weights, body weight gains and clinical signs. In Phase 3, there was one preterminal death in group 7. In Phase 4, mortality occurred in all dose groups between days 2 and 8: three in the isotype control, three in the anti-C2 mAb, and five in the anti-C5 mAb treated animals. Mortality that could be assigned to kidney ischemia reperfusion injury based on results from histopathology and creatinine levels was slightly less: two in the isotype control, two in the anti- C2 mAb and three in the anti-C5 mAb treated animals. Remaining mortality was considered likely secondary to surgical procedures. Thus, no differences were found in mortality rate (total or kidney ischemia reperfusion injury related) between the different dosing groups.

[0346] 3.2.2 Serum creatinine and urea levels in Phase 3 and Phase 4 group rats

[0347] Serum creatinine and urea levels were used to assess kidney function post-transplant. The rationale for the use of creatinine or urea measurement to assess renal function (i.e. to assess DGF) is that plasma / serum levels of both reflect glomerular filtration rate (GFR), the parameter that defines kidney function. High creatinine and / or urea levels can be an indicator that the kidneys are not functioning properly. In Phases 1 and 2, the increases in urea and creatinine concentration at day 3 were observed to be proportional to the length of cold-ischemia time (CIT), with longer ischemia time resulting in higher urea and creatinine concentrations. Phase 3 Results

[0348] The creatinine results for individual rats within Groups 6 and 7 are shown in Table 4 below and in

[0349] Fig. 10.

[0350] Table 4. Individual serum creatinine concentrations (pmol / L) at day 3 for groups 6 and 7 in phase 3

[0351] The p-value of the exact Mann-Whitney test was 0.010 and confirmed a significant difference in serum creatinine concentrations between the isotype control and anti-C2 mAb treated animals. The average ratio of serum creatinine concentrations for anti-C2 to isotype control was 0.537 indicating serum creatinine concentrations were nearly 50% reduced, on average, compared to the isotype control.

[0352] Table 5 shows the mean urea and creatinine levels per group as expressed relative to mean donor levels or isotype-control levels (Group 6), as indicated.

[0353] Table 5. Clinical Chemistry Changes (Phase 3)

[0354] M= Male; CIT = Cold Ischemia time hours; NA= Not availableaNumerical values indicate fold changes of mean recipient group value relative to pre-transplant donor group mean value.bNumerical values indicate fold changes of mean test-item-treated group value relative to Isotype control group mean value (Group 6).

[0355] Urea and creatinine concentrations on day 3 were increased in both groups of recipients that received a kidney exposed to CIT 32 hours (Group 6 and Group 7), as compared to values in donor animals prior to transplantation. Increases in urea and creatinine concentrations on day 3 were of a significantly lesser magnitude in animals administered the anti-02 mAb (Group 7) than in those administered the isotype control (Group 6). Phase 4 Results

[0356] The creatinine results for individual rats within Groups 9, 10 and 11 are shown in Table 6.

[0357] Table 6. Individual serum creatinine concentrations (pmol / L) at days 3, 5, 7 and 14 in phase 4 Mean creatinine and urea levels ±SEM are shown in Fig. 11. In anti-02 mAb-treated animals, creatinine concentrations on day 3 were 40.3% (p = 0.008) lower as compared to the isotype control. Urea concentrations on day 3 were also significantly lower in anti-C2 mAb-treated animals than in isotype control animals (p = 0.0434). Creatinine and urea concentrations on day 3 in anti-C5 mAb treated animals were lower than in isotype control animals (p = 0.0048 and p = 0.0037, respectively). On average across time-points, creatinine values were significantly lower in anti-C2 mAb and anti-C5 mAb treated animals than in the isotype control animals (49.4%, p=0.014 for anti-C2 mAb, and 58.1%, p=0.002 for anti-C5 mAb).

[0358] Table 7 shows the mean urea and creatinine levels per group as expressed relative to mean donor levels or isotype-control levels (Group 9), as indicated.

[0359] Table 7. Clinical Chemistry Changes (Phase 4)

[0360] M= Male; CIT = Cold Ischemia time hours

[0361] A dash ( — ) indicates absence of change.aFor one animal, data are included for Day 3, but excluded from the means of Days 5 and 7 because it received the wrong test-item at d4.bNumerical values indicate fold changes of mean recipient group value relative to mean pre-transplant donor group valuecNumerical values indicate fold changes of mean test item-treated group value relative to mean Isotype control group mean value (Group 9)dNumerical values indicate fold changes of mean Anti-C5 mAb treated group value relative to mean Anti-C2 mAb treated group value (Group 10).eIncrease mainly driven by two animals

[0362] Mean urea and creatinine concentrations were markedly increased in all recipients on days 3, 5, and 7 when compared to pre-transplant donors’ mean values.

[0363] In animals administered the Isotype control (Group 9), mean increases in creatinine concentrations were highest on day 3 and progressively decreased on days 5 and 7. Mean increases in urea concentration were comparable on days 3, 5 and 7. Urea and creatinine concentrations reached comparable donors’ values on day 14 for most animals.

[0364] In animals administered the anti-C2 mAb (Group 10) or anti-05 mAb (Group 11 ), mean increases in urea and creatinine concentrations also peaked on day 3, and progressively decreased on days 5 and 7 and were comparable to donors’ values on day 14. Increases noted in anti-C2 mAb or anti-C5 mAb treated animals were of a significantly lesser magnitude than in isotype control treated animals (Group 9).

[0365] 3.2.3 Evaluation of free C2 levels

[0366] During phase 3 and phase 4 of the study described above, serum free C2 levels (= 02 unbound by anti-rat 02 mAb) were determined to confirm free 02 reduction in animals treated with anti-02 mAb.

[0367] Free 02 levels in rat serum samples were determined using an enzyme-linked immunosorbent assay (ELISA) based on a sandwich principle. Anti-02 mAb was coated overnight at a concentration of 10 pg / mL in 1x PBS on a 96-well high binding half-area plate (50 pL / well). The next day, the assay plate was washed 4 times with 1 x PBS-T (1 x PBS with 0.05% Tween20) using a plate washer and subsequently blocked with 3% BSA diluted in 1x PBS for 1 hour at room temperature (RT) (120 pL / well).

[0368] Dilutions of recombinant rat 02 (=standard curve, starting at 12 pg / mL and V2 serial diluted until 0.012 pg / mL), QC (= pooled Lewis rat complement preserved serum, diluted 1 :20) and samples (diluted 1 :20) were prepared on ice in 1 x TBS- CaCh buffer (1 OX TBS buffer diluted to 1 x in MilliQ with 2mM CaCh).

[0369] After blocking, the plate was washed 4 times with 1x PBS-T. Standard curve and samples were added to the plate (50 pL / well) immediately after dilution, to prevent prolonged incubation time of diluted samples which might have resulted in disturbance of the equilibrium of C2 / anti-C2 mAb complexes in serum. Standards and samples were incubated on the assay plate for exactly 15 minutes on ice. Free 02, i.e. 02 not already bound by the anti-02 mAb present in the sample itself, was captured by the coated anti-02 mAb antibody.

[0370] After washing, the biotinylated detection antibody 3H07 (an antibody binding rat 02, but on an epitope that is distinct from the anti-02 mAb binding site) was added (2.5 pg / mL in TBS- CaCh buffer) and incubated for 15 minutes on ice. The plate was washed again and streptavidin-HRP diluted 1 :1000 in TBS- CaCI2 buffer, was added to the plate. After 15 minutes of incubation on ice, the plate was washed and s(HS)TMB diluted 1 :1 with MQ was added to the plate. The colouring reaction was allowed to proceed for 15 minutes at RT, shielded from light, and was stopped by the addition of 0.5 mol / L sulfuric acid.

[0371] OD values were measured at 450 nm (ref 620 nm) using a spectrophotometer (Infinite M Nano Tecan). Estimated free C2 levels were obtained by back-calculating the OD responses against the 5PL fitted calibrator curve prepared with recombinant rat C2.

[0372] Phase 3 Results

[0373] The free C2 levels in Group 6-8 rats are shown in Fig. 12.

[0374] The free 02 levels in anti-C2 mAb-treated animals were below the quantification limit (0.046875 pg / mL C2 on plate, meaning levels below 0.9375 pg / mL in 100% rat serum as a 1 / 20 dilution was performed of the 100% rat serum samples). This corresponds with a reduction of free C2 values > 95% compared to baseline. Only 2 samples from group 7 day 3 and 1 sample from group 7 day 14, showed free C2 levels just above quantification limit (still a reduction of >91%). These data confirm that free C2 levels were reduced in rats treated with anti-02 mAb during the rat kidney transplant experiments of phase 3.

[0375] No change from baseline was observed for free C2 levels for the isotype control group (group 6).

[0376] Phase 4 Results

[0377] The free C2 levels in Group 9-11 rats are shown in Fig. 13.

[0378] The free 02 levels in anti-02 mAb-treated animals were below the quantification limit (0.046875 pg / mL 02 on plate, meaning levels below 0.9375 pg / mL in 100% rat serum as a 1 / 20 dilution was performed of the 100% rat serum samples). This corresponds to a reduction of free C2 values > 95% compared to baseline and this effect was observed for up to 14 days. These data confirm that anti-02 mAb was able to reduce rat free C2 levels over the course of the rat kidney transplant experiments of phase 4.

[0379] No change from baseline (day -1 , donor values) was observed for free 02 levels in the isotype control or anti-05 mAb-treated rats.

[0380] 3.2.4 Histopathology Phase 4

[0381] Gross pathology findings of the transplanted kidney are summarized in Table 8.

[0382] Microscopic findings are summarized in Table 9. Table 8. Gross pathology

[0383] = non-applicablebkidney IRI-related findingc= Procedure-related finding

[0384] Table 9. Microscopic findings - = not applicablea= numbers in parentheses represent the number of animals with the findingb= includes one animal with granulomatous inflammation without necrosisd= kidney ischemia reperfusion injury-related findinge= procedure-related finding

[0385] At terminal euthanasia day 14, the gross pathology findings were similar in nature among all groups, but had slightly lower incidences in animals given anti-C2 mAb (Group 10) or anti-05 mAb (Group 11 ) compared to isotype control animals (Group 9). On terminal euthanasia day 14, there was also a slight decrease in severity and / or incidence of all microscopic findings considered associated with ischemia reperfusion injury in animals that received anti-C2 mAb or anti-C5 mAb compared to isotype control animals. This observation suggested protective effects of anti-C2 mAb and anti-C5 mAb on ischemia reperfusion injury-induced microscopic changes in the transplanted kidney when examined on Day 14; evidence of protective effects was similar between both test items.

[0386] Gross findings noted in the transplanted kidney at day 14 considered associated to ischemia reperfusion injury included enlargement and an abnormal consistency, correlating microscopically with tubular regeneration, as well as pale / dark / mottled discoloration correlating microscopically with tubular regeneration and / or necrosis. Other findings were considered related or likely related to the surgical procedure.

[0387] The microscopic findings observed in the transplanted kidney on day 14 were predominantly regenerative and inflammatory (subacute / chronic) and were similar in nature among all 3 groups. Ischemia reperfusion injury-related findings that showed a slight decrease in severity and / or incidence in animals that received anti-02 mAb (Group 10) or anti-C5 mAb (Group 11 ) compared to control animals (Group 9) included mild to marked (Group 10) or minimal to severe (Group 11 ) tubular regeneration compared with moderate to severe severities in all controls; minimal to mild hyaline / granular casts (predominantly minimal in Groups 10 and 11 ); minimal mineralization in Groups 10 and 11 compared to minimal to moderate in controls; and minimal to mild mononuclear cell inflammation in Groups 10 and 11 compared to mild in all controls.

[0388] On Terminal Euthanasia Day 14, the mean absolute weights for the transplanted kidney were lower in animals that received anti-02 mAb (-22.91%, Group 10) or anti-C5 mAb (-22.94%, Group 11 ) compared to the mean absolute weight of the transplanted kidney in isotype control animals (Group 9). The lower kidney weights in animals that received the test items compared to control animals correlated with the marginally reduced incidence and / or severity of kidney microscopic findings (notably tubular regeneration and inflammation) observed on Day 14 in animals of Groups 10 and 11 compared to Group 9 control animals. At Terminal Euthanasia on Days 3, 5 and 7, gross findings noted in the transplanted kidney that were considered to be related to ischemia reperfusion injury were similar in nature between animals given anti-C2 mAb (Group 10) or anti-05 mAb (Group 11 ). Ischemia reperfusion injury- related microscopic findings noted in the transplanted kidney of animals given anti-C2 mAb or anti-C5 mAb on Terminal Euthanasia Days 3, 5 and 7 corresponded mainly to acute / subacute ischemia-related tubular changes. Kidney microscopic findings were associated with impairment of renal functions on Terminal Euthanasia Days 3, 5 and 7 (including marked increases in urea and creatinine concentrations). There were no clear differences in the severity, incidence and nature of ischemia reperfusion injury-related microscopic findings noted in the transplanted kidney of animals that received anti-02 mAb (Group 10) or anti-C5 mAb (Group 11 ); However, the transplanted kidney of control animals (Group 9) was not examined on these time-points.

[0389] In conclusion, on Terminal Euthanasia Day 14, there was a decrease in severity and / or incidence of all microscopic and most gross ischemia reperfusion injury-related kidney findings in animals given anti-C2 mAb or anti-C5 mAb compared to isotype control animals, correlating with approximately 20% decrease in transplanted kidney weight means in animals given the test items compared to isotype control values. These results suggested comparable protective effects of anti-02 mAb and anti-C5 mAb on ischemia reperfusion injury.

[0390] EXAMPLE 4 - Safety, Efficacy, and Tolerability of ARGX-117 in Improving Allograft Function in Recipients of a Deceased Donor Renal Allograft at Risk for Delayed Graft Function

[0391] This example describes a phase 2, multicentre, randomized, double-blinded, placebo-controlled study to assess the safety, efficacy, tolerability, pharmacokinetics, pharmacodynamics and immunogenicity of ARGX-117 in improving allograft function in recipients of a deceased donor renal allograft at risk for delayed graft function (DGF). The study will evaluate the efficacy of ARGX-117 compared to placebo to reduce the risk of DGF, promote early recovery, and improve overall allograft function in deceased donor kidney transplants.

[0392] Renal transplantation is the optimal treatment for patients with early stage renal disease (ESRD). The imbalance between donor organ availability and patients with ESRD awaiting a kidney transplant is rising exponentially. One strategy is to allocate deceased donor kidneys at risk for DGF, to patients already at risk for DGF. Despite sophisticated immunosuppressive regimens and advances in transplant diagnostics, the risk of DGF has not been substantially mitigated, and the average lifespan of a deceased donor kidney allograft has not improved significantly. Advancements in developing new treatments to prevent DGF in kidney transplantation have been limited by failure to demonstrate substantial improvement in the pathophysiology of ischemia-reperfusion injury (IRI), which is the primary contributor to the development of DGF.

[0393] The objective of this trial is to assess the therapeutic value of ARGX-117 to mitigate the effects of complement-mediated injury associated with IRI following organ transplant. ARGX-117 is a monoclonal antibody that binds to the C2b component of complement 02, functioning as a complement inhibitor. Specifically, complement inhibition by ARGX-117 impacts activation of the classical pathway and lectin pathway of the complement system. By targeting the 02 component, ARGX-117 leaves the alternative pathway intact.

[0394] The mechanism of action of ARGX-117 positions this drug as a novel agent to target the primary early pathogenic mechanisms of DGF, subsequent allograft dysfunction and premature allograft failure. The pH-dependent binding and recycling properties of ARGX-117 are designed to prolong the drug’s half-life (approximately 88 days), and enhance 02 clearance from the bloodstream, making ARGX-117 a desirable drug for use as an induction agent in kidney transplantation.

[0395] 4.1 Study Design

[0396] Overall Desic / n

[0397] The study population includes adults with ESRD who are stable on dialysis. Participants must be candidates for de novo or second-time, single kidney transplantation with deceased donor renal allografts who are at risk for DGF and allograft dysfunction. Participants will be randomized in a 1 :1 ratio to investigative medicinal product (IMP) - ARGX-117 or placebo. Randomization will be stratified by donor type (i.e. , DBD and DCD).

[0398] Participants will receive:

[0399] • Initial infusion of IMP (ARGX-117 IV 60 mg / kg) or placebo with dose calculated using dry body weight at screening, administered preoperatively, infused over 90 minutes, and completed before organ reperfusion.

[0400] • Second infusion of IMP (ARGX-117 IV 60 mg / kg) or placebo with dose calculated using the same dry body weight at screening, administered on study day 8, and infused over 90 minutes.

[0401] • Induction immunosuppressive therapy with antithymocyte globulin + corticosteroids.

[0402] • Maintenance immunosuppressive therapy with tacrolimus and MMF / EC-MPA ± corticosteroids.

[0403] • Prophylactic antimicrobial therapy during the study.

[0404] • Vaccinations, if not received before transplant. The study duration is approximately 64 weeks (see Fig. 14), comprising the following study periods: Screening period: <24 hours Treatment and evaluation period: 52 weeks Follow-up period: 12 weeks

[0405] Table 10: Study Arms

[0406] Participants will be screened upon availability of a deceased donor kidney, coinciding with hospital admission. Eligibility assessments will include data obtained from the pretransplant workup. Eligible participants will be randomized to receive either ARGX-117 IV 60 mg / kg or placebo.

[0407] An unblinded analysis will be performed after all randomized participants have completed the scheduled week 24 assessment or have discontinued from the study. Sites, participants, and key study personnel will remain blinded. Full analysis including analysis of week 52 efficacy data will be performed after all randomized participants have completed the scheduled week 64 follow-up assessment or have discontinued from the study.

[0408] Justification for dose

[0409] The 2-dose regimen (ARGX-117 60 mg / kg IV on day 1 + 60 mg / kg IV on day 8) aims to provide a PD effect with a magnitude and duration proportional to the robust and sustained level of complement activation associated with renal transplantation. The selected dosing regimen of ARGX-117 is predicted to reduce free 02 concentrations by approximately 99% in approximately 90% of participants over the first 4 weeks post-transplant. The initial and subsequent complement activation throughout the process of acute kidney injury (AKI) are key in propagating the inflammatory cascade that leads to further injury, allograft damage, DGF, and a predisposition for acute and chronic rejection.

[0410] The 2-dose regimen of ARGX-117 selected for this indication is intended to maintain ARGX-117 levels expected to provide complement blockade throughout the duration described below:

[0411] • Early stages of AKI (<4 weeks post-transplant): Early inhibition of complement activity is expected to prevent molecular signals presented by damaged epithelium / endothelium from triggering complement activation at any time during this critical period.

[0412] • Late stages of AKI (>4 weeks post-transplant): Continued inhibition of other complement activity after the first 4 weeks post-transplant is expected to mitigate the propagation of downstream events from the initial reperfusion injury and the overall risk of developing alloimmunity to the renal allograft.

[0413] Post-transplant immunosuppression

[0414] IMP will be administered as part of induction therapy. IMP administration can be started as soon as a negative crossmatch is verified, the organ has been inspected, and the kidney is determined to be transplantable by the supervising surgeon. The order of administration of other induction medications will be at the discretion of the participating centres’ standard of care, provided that antithymocyte globulin is not co-administered concurrently with IMP.

[0415] All baseline blood samples will be taken before initiation of IMP administration and other induction medications.

[0416] The first dose of IMP (ARGX-117 60 mg / kg IV or placebo, infused over 90 minutes) will be initiated pre-operatively and completed before organ reperfusion. The IMP dose may be coadministered with other medications. Biologic therapies, including antithymocyte globulin, must not be administered concurrently. The second dose of IMP (ARGX-117 60 mg / kg IV or placebo, infused over 90 minutes) will be administered on day 8, with a window up to day 10 to accommodate outpatient administration, if needed.

[0417] Planned immunosuppressive regimens for all participants regardless of treatment assignment must include:

[0418] • Induction therapy with antithymocyte globulin and IV corticosteroids

[0419] • Maintenance therapy with tacrolimus, MMF / EC-MPA + / - oral corticosteroids

[0420] Participants should maintain planned treatment regimen during study participation, particularly through week 24, unless unsafe in the opinion of the investigator. The medical monitor will be informed of any changes to immunosuppressive regimen within 24 hours. All participants are expected to receive standard anti-infective prophylaxis.

[0421] Induction Immunosuppression: It is recommended that infusion of corticosteroids be completed 30 minutes before reperfusion of the kidney allograft and before initiation of antithymocyte globulin, as dictated by institutional protocol. The recommended cumulative dosage of antithymocyte globulin is 4 mg / kg divided into once daily doses to be administered over 3-4 days, not to exceed 6 mg / kg. Coadministration with IMP is not allowed.

[0422] Maintenance Immunosuppression: Tacrolimus by mouth will be initiated post-transplant per institutional standard of care with dose adjustments based on trough measurements. Suggested trough levels: 6 to 12 ng / mL through week 4, and 4 to 8 ng / mL from weeks 4 through 52. If dosing outside of these targets is clinically indicated, it is at the discretion of the investigator. Tacrolimus-based immunosuppression should be maintained throughout the study, especially during the first 24 weeks post-transplant. MMF (CellCept) or EC-MPA will be initiated posttransplant with dosing per institutional standard of care. Suggested maintenance doses: MMF:- 500 to1000 mg twice a day; EC-MPA:- up to 720 mg twice a day. Clinically indicated dose changes (e.g., due to participant intolerance, infectious sequelae) are acceptable at the discretion of the investigator. Oral corticosteroids (e.g., prednisone) will be initiated and administered at the discretion of the participating site’s protocol, with recommendation to taper the medication to have a daily dosage of up to 5 mg by 3 months post-transplant. Steroid avoidance protocols are allowed following completion of induction.

[0423] End of Study Definition

[0424] The end of the study is defined as the date of the last participant’s last visit. 4.2 Study Population

[0425] Inclusion and exclusion criteria have been carefully selected to enrich the population with participants at risk for DGF.

[0426] Inclusion Criteria

[0427] Kidneys from deceased donors are eligible for transplant to study participants if the following criteria are met:

[0428] A. Cold ischemia time (CIT) >12 hours, and

[0429] B. At least 1 of the following:

[0430] B1 . Donor aged >40 and <70 years

[0431] B2. Donor terminal serum creatinine >1 .5 mg / dL

[0432] Participants can be included in the study only if all of the following criteria apply:

[0433] 1 . Is at least the local legal age of consent for clinical studies and at least aged 18 years and less than 70 years when signing the IGF

[0434] 2. Is capable of providing signed informed consent, and complying with protocol requirements

[0435] 3. Agree to use contraceptive measures consistent with local regulations and the following: a. Male participants:- Males cannot donate sperm while receiving IMP and for at least

[0436] 15 months after the last dose of IMP. b. Women of child-bearing potential (WOCBP) must have a negative serum pregnancy test at screening

[0437] 4. Have dry body weight less than 120 kg and body mass index less than 40 kg / m2at screening

[0438] 5. Are diagnosed with ESRD and have been stable on chronic dialysis for at least 3 months

[0439] 6. Are recipients of de novo or second-time, single kidney transplant from a deceased donor, either DCD or DBD

[0440] 7. Are ABO compatible with donor allograft, except for type A2 donor to type B recipient kidneys

[0441] 8. Have a negative cross match

[0442] 9. Have received pretransplant vaccinations for: Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae, or are willing to receive the vaccinations approximately 3 to 4 months posttransplant

[0443] 10. Have received SARS-CoV-2 vaccinations consistent with participating site’s requirements Exclusion criteria

[0444] Kidneys from deceased donors will be excluded if any of the following criteria apply:

[0445] A. Any history of participant anti-HLA DSA to the current donor

[0446] B. Donor kidney that is continuously machine perfused from the time of organ procurement to the time of transplant

[0447] C. Donor kidney from an HIV positive donor

[0448] Participants will be excluded from the study if any of the following criteria apply:

[0449] 1. Any history of prothrombotic disorder (eg, factor V Leiden deficiency), or history of thrombosis or hypercoagulable state, excluding vascular access clotting

[0450] 2. Any known history of complement deficiency

[0451] 3. Evidence of peritonitis in participants on peritoneal dialysis

[0452] 4. Received any solid organ, bone marrow, or hematopoietic stem cell transplant, with the exception of prior first kidney transplant

[0453] 5. Any of the following laboratory abnormalities: a) Alanine aminotransferase or aspartate aminotransferase >2 x ULN b) Total bilirubin >1 .5 x ULN c) Absolute neutrophil count <1000 cells / pL d) Hemoglobin <8.0 g / dL e) White blood cell count <3000 cells / pL f) Platelet count <100 000 / pL

[0454] 6. Planned administration of any prohibited or restricted medication within the first 6 months posttransplant

[0455] 7. High risk within the study period for recurrence of underlying renal disease, including but not limited to focal segmental glomerulosclerosis, atypical hemolytic-uremic syndrome, C3 nephropathy, antiglomerular basement membrane disease, or lupus nephritis, in the opinion of the investigator.

[0456] 8. Any autoimmune disease requiring treatment to control systemic disease activity (eg, SLE, antineutrophil cytoplasmic antibody-associated vasculitis, etc) that would pose a significant safety risk or put the participant at undue harm in the opinion of the investigator.

[0457] 9. Any history of clinically significant arrythmia, known long QT syndrome, or clinically significant ECG abnormality at screening, including QTcF >450 ms for male participants and QTcF >470ms for female participants

[0458] 10. Any history of malignancy unless considered cured by adequate treatment with no evidence of recurrence for more than 5 years before the first administration of IMP. Adequately treated participants with the following cancers can be included at any time: a) Basal cell or squamous cell skin cancer b) Carcinoma in situ of the cervix c) Carcinoma in situ of the breast d) Incidental histological finding of prostate cancer (TNM stage T1 a or T1 b)

[0459] 11 . Unwillingness to receive vaccinations consistent with protocol-mandated and participating site requirements

[0460] 12. Clinically significant active bacterial, viral, or fungal infection

[0461] 13. Infection with any of the following, as specified below and confirmed by the investigator: a) HBV that is indicative of an acute or chronic infection, unless associated with a negative hepatitis B surface antigen test or negative HBV DNA test b) HCV within 12 months based on HCV antibody assay, confirmed by HCV RNA test is available and documentation of curative treatment c) Any history of HIV diagnosis d) Any history or evidence of tuberculosis with the requirement of negative chest x-ray within 6 months before randomization. A repeat chest x-ray is recommended during screening at the discretion of the investigator for known tuberculosis exposure or recent travel to endemic regions.

[0462] Note: Participants with no documentation of any prior HBV, HCV, and HIV screening will be excluded

[0463] 14. Clinically significant comorbidity, recent major surgery (within 3 months of screening), history of any treatment nonadherence, or intention to have surgery during the study other than kidney transplantation; or any other medical condition that, in the investigator’s opinion, would confound the results of the study or put the participant at undue risk

[0464] 15. Received a different IMP in another clinical study less than 12 weeks or 5 half-lives (whichever is longer) before screening

[0465] 16. Currently participating in another interventional clinical study

[0466] 17. Previously participated in an ARGX-117 clinical study and received at least 1 dose of IMP

[0467] 18. Known hypersensitivity to ARGX-117 or any of its excipients

[0468] 19. Known hypersensitivity to tacrolimus, MMF or mycophenolic acid, or antithymocyte globulin or allergy to Leporidae (e.g., rabbit)

[0469] 20. History (within 12 months before screening) of current alcohol, drug, or medication abuse as assessed by the investigator

[0470] 21 . Pregnant or lactating state or intention to become pregnant during the study

[0471] 22. Received any prior desensitization therapies or any pretransplant immunosuppressive therapy within 5 half-lives or twice the duration of the biological effect, whichever is longer: a. DNA alkylating agents (eg, cyclophosphamide) b. Any therapeutic antibody, including the following: i. Anti-B-cell therapies (eg, rituximab, belimumab) ii. Anti-IL-6 therapies (eg, clazakizumab, tocilizumab) iii. Anti-IL-2 receptor antagonists (eg, basiliximab, daclizumab) iv. Anti-CD-52 therapy (eg, alemtuzumab) v. Anti-CTLA-4 mediated co-stimulatory antagonists (eg, abatacept, belatacept) vi. Complement inhibitors (eg, C1 esterase inhibitors, eculizumab) vii. FcRn receptor antagonists (eg, efgartigimod, nipocalimab) viii. IgG endopeptidase therapy (eg, imlifidase) ix. Proteasome inhibitors (eg, bortezomib) c. IVIg and / or plasmapheresis d. Any immunoadsorption therapy e. Any nontraditional medications with known immunomodulatory action 4.3 Study Assessments and Procedures

[0472] Study procedures and their timing are summarized in the Schedule of Activities (SoA).

[0473] Table 11: Schedule of Activities

[0474]

[0475]

[0476]

[0477] ADA=antidrug antibodies; ABO=blood group testing; AE=adverse event; AESI=adverse events of special interest; ANA=antinuclear antibody; AP=alternative pathway;

[0478] BL=baseline; C2=complement component 2; CIT=cold ischemia time; CKD-EPI=Chronic Kidney Disease Epidemiology Collaboration; CMV=cytomegalovirus; CNI=calcineurin inhibitor; cPRA=calculated panel reactive antibody; D=day; DSA=donor-specific antibodies; EBV=Epstein-Barr virus; ECG=electrocardiogram; EDV=early discontinuation visit; eGFR=estimated glomerular filtration rate; h=hours; hCG=human chorionic gonadotropin; HLA=human leukocyte antigen; iBox=integrative box; ICF=informed consent form;

[0479] IFA=indirect immunofluorescence assay; IMP=investigational medicinal product; INR=international normalized ratio; IO=intraoperative; IV=intravenous; NGAL=neutrophil gelatinase-associated lipocalin; PD=pharmacodynamics; PK=pharmacokinetics; PO=preoperative; PT=prothrombin time; PTT=partial thromboplastin time; SAE = serious adverse event; SCR=screening; SFV=safety follow-up visit; SLE=systemic lupus erythematosus; USV=unscheduled visit; W=week; WOCBP=women of childbearing potential a Screening information for the ABO blood group, cPRA, crossmatch verification, and viral serology review will be obtained from the pretransplant evaluation. Missing eligibility data will result in screen failure. Chest x-ray for exclusion of active tuberculosis infection will be performed only if a negative chest x-ray within 6 months before randomization is not available. If negative HIV test results are older than 6 months at the time of screening, local HIV standard-of-care test results must be negative before randomization to confirm eligibility. The local pregnancy test, and other screening blood sample laboratory tests will be performed during screening with the expectation that results are available prior to randomization.

[0480] bAt unscheduled visits, participants may be evaluated for infection, kidney dysfunction, transplant rejection, medication adherence, and CNI toxicity, as clinically indicated. Additional study-related assessments (e.g. biopsy) will be completed at the discretion of the investigator.cHour zero refers to the time of reperfusion of the renal allograft on day 1 .dThese baseline activities (blood sample collection, biopsy, and IMP administration) occur up to the time of reperfusion of the renal transplant. Baseline assessments will be performed predose to IMP administration. Baseline laboratory samples may be obtained simultaneously as screening samples unless hemodialysis is performed during the screening period. However, baseline samples should not be sent for analysis if a participant screen fails.eVital signs, urine output, kidney transplantation, and IMP administration activities occur after reperfusion of the renal transplant. Timing may be rounded to the nearest hour. ’ Chemistry and hematology assessments should be performed at all specified time points if the participant remains hospitalized.

[0481] 0 Home study visits are optional at the week 64 visit, as permitted per local regulations.hDemographic characteristics for participants and donors include year of birth, age, sex, race, and ethnicity (per local regulations). Race and ethnicity data will be source verified.

[0482] ' Donor medical history includes HLA data, comorbid conditions, relevant or significant medical history related to donor cause of death, CIT, and cause of death due to cardiovascular disease or brain death. i Participant medical / surgical history includes all comorbid conditions, relevant or significant medical history (including all major hospitalizations and surgeries) and substance abuse. Any relevant renal history (dialysis, transplant, etc.) and / or the underlying aetiology of the disease should be included.kParticipants who have not received pretransplant vaccinations for S. pneumoniae, N. meningitidis, and H. influenzae at least 4 weeks before screening must agree to receive these vaccinations approximately 3-4 months posttransplant. Vaccination for SARS-CoV2 will be administered per participating site’s protocol.

[0483] 1All attempts should be made to collect protocol-specified laboratory tests before dialysis.mResults of viral evaluations will be obtained from medical history to include evidence on transplant evaluation of negative results for HIV and HBV / HCV. Participants with prior history of HCV will be eligible provided documentation of posttreatment cure. If historical data are not available for any of the specified viral infections, the participant will be considered a screen failure.nPregnancy testing will use serum beta hCG at screening and a urine test at subsequent visits. A pregnancy test should be performed before IMP administration, if applicable. Serum beta hCG will be used if the participant is anuric or eGFR is less than 30 mL / min / 1 .73 m2. Local regulations will be followed if they require more stringent or frequent testing.

[0484] 0Complete PE is only required at SCR, day 8, and USV. Brief PE for this study includes assessments of cardiovascular system, respiratory system, gastrointestinal system (to include examination for graft tenderness), extremities, and general appearance. Height will be measured at screening only.peGFR will be calculated using the 2021 CKD-EPI formula.qThe ANA panel will include ANA by IFA with reflex to titer. The remainder of the SLE autoantibodies should be checked if the ANA titer is more than 1 :80.rA review of cPRA includes the most recent PRA value (%), max / peak PRA, list of unacceptable antigens, and current and historical HLA.

[0485] 3PT, PTT, and D-dimer will be measured during initial hospitalization for transplant, unless clinically indicated.

[0486] * The preoperative immunogenicity screening sample after randomization and will be completed for validation purposes.uConsidered optional if not collected at the previous DNA sample timepoint.vAdditional markers related to complement biology (e.g., complement factors and / or regulators), kidney biology (e.g., markers for kidney damage) and / or related to ARGX-117wUrine samples will only be collected if the participant can produce 10 mL or greater of urine.

[0487] * The IMP will be administered as a 90-minute IV infusion, with the first dose initiated preoperatively and completed before organ reperfusion. IMP administration can be started as soon as a negative crossmatch is verified, the organ has been inspected, and the kidney is determined to be transplantable by the supervising surgeon. Participants will be monitored for safety for at least 1 hour after IMP infusion. Vital signs will be measured before each infusion of IMP.yAn abbreviated iBox will be used, because a biopsy does not occur at this visit.zAdverse events, AESIs, SAEs, and use of concomitant therapies will be collected from the time the IGF is signed until the last study-related activity. However, any existing or reported medical condition that is present at the time the participant is screened is considered baseline and not considered an AE. After signing of the IGF, any new or worsening medical condition will be reported as AE or SAE.

[0488] Efficacy Assessments: Primary Endpoint eGFR at 24 weeks

[0489] DGF and DGF-related events are correlated with estimated glomerular filtration rate (eGFR) at both 24 and 52 weeks post-transplant. The primary endpoint, eGFR at 24 weeks, was selected in combination with carefully determined secondary endpoints so that the totality of data will inform on the potential effectiveness of ARGX-117 in the study population, noting that the ability to control confounders declines after week 24.

[0490] Kidney allograft function at week 24, assessed by eGFR (mL / min / 1 ,73m2), will be evaluated based on serum creatinine measurement. Calculation of eGFR will use the 2021 CKD-EPI equation below: eGFR = 142 x min(Scr / K, 1 )ax max(Scr / K, 1 )1-200x 0.9938A9ex 1 .012 [if female], where:

[0491] ■ SCr is serum creatinine in mg / dL,

[0492] ■ K is 0.7 for females and 0.9 for males,

[0493] ■ a is -0.241 for females and -0.302 for males,

[0494] ■ min indicates the minimum of Scr / K or 1 , and

[0495] ■ max indicates the maximum of Scr / K or 1 .

[0496] ■ Age (years)

[0497] Efficacy Assessments: Secondary Endpoints

[0498] Other assessments of graft recovery include the following:

[0499] Proportion of participants with DGF

[0500] DGF is defined as requiring one or more hemodialysis treatments following transplantation within the first 7 days before the onset of graft function. Single dialysis treatments during the first 7 days post-transplant for indications other than renal clearance (e.g., fluid overload, hyperkalemia) do not qualify as DGF. The reason(s) for initiating dialysis will be recorded, and all episodes of single-treatment dialysis will be reviewed individually for confirmation of DGF. All participants who start dialysis during the first 7 days and receive > 2 dialysis sessions will be classified as having DGF, regardless of treatment indication or timing of subsequent dialysis sessions.

[0501] Proportion of participants with fDGF

[0502] Participants exhibiting a decrease in serum creatinine level <10% per day over a 3 consecutive day period during the first 7 days post-transplant, regardless of dialysis requirement, will be recorded as having fDGF. The proportions of participants with DGF and fDGF will be compared between treatment groups using a Cochran-Mantel-Haenszel test stratified by donor type. The common odds ratio will be provided, along with the 90% Cl and 2-sided p-value.

[0503] Creatinine reduction ratio (CRR)

[0504] CRR at 72 hours and 7 days post-transplant: the CRR will be calculated from serum creatinine levels using the following equation:

[0505] CRR = [baseline Scr(mg / dL) - current Scr(mg / dL)] I baseline Scr(mg / dL)

[0506] Serum creatinine concentration at screening should be used as the baseline value for CRR calculations. If dialysis is done during screening, all assessments to be completed before dialysis sessions must be obtained before initiating dialysis.

[0507] Table 12: Creatinine-based assessment time points aThese timepoints are relative to allograft reperfusion.

[0508] The CRR at 72 hours (study day 3) and day 7 (study day 8) post-transplant will be analyzed using analysis of variance with factors in the model for treatment group and donor type. The estimate of the treatment effect, together with its standard error and 90% Cl will be provided.

[0509] Duration of dialysis treatment for DGF

[0510] Duration of dialysis treatment for DGF within the first 30 days post-transplant (i.e. , date of last dialysis treatment).

[0511] Proportion of participants not requiring dialysis at 30 days post-transplant

[0512] Proportion of participants not requiring dialysis at 30 days post-transplant: The number of participants who have ongoing dialysis requirement at day 30. iBox score at 52 weeks post-transplant iBox score at 52 weeks post-transplant: The iBox risk calculation predicts long-term risk of renal allograft failure. Its use at 1 -year post-transplant has been qualified by EMA as a secondary endpoint in clinical studies for death-censored allograft failure at 5 years. The iBox consists of 4 pillars: eGFR

[0513] Proteinuria, as assessed by spot urine protein / creatinine ratio

[0514] Biopsy histology

[0515] Presence or absence of DSAs

[0516] Dialysis-free survival and eGFR at 52 weeks

[0517] Dialysis-free survival through 52 weeks posttransplant and eGFR at 52 weeks posttransplant, calculated using the 2021 CKD-EPI equation.

[0518] Incidence of PNF

[0519] Incidence of PNF: lack of allograft recovery at week 12 post-transplant with requirement for chronic renal replacement therapy.

[0520] Efficacy Assessments: Exploratory Endpoints

[0521] Incidence of DSA

[0522] Incidence of de novo donor specific antibodies (DSAs) or DSA development.

[0523] Urine output

[0524] Urine output (mL / hr) during the first 72 hours post-transplant: Calculation of urine output will be based on measured urine output over 8-12 hour intervals (i.e., 0-12 hours, 12-24 hours, etc.). All diuretic use will be captured to determine accurate interpretation of urine output data.

[0525] Early graft function analysis

[0526] Additional analyses of early graft function recovery may include:

[0527] ■ Incidence of slow graft function: Serum creatinine >3 mg / dL at day 5 without requirement for dialysis

[0528] ■ Number of days to achieve a calculated eGFR >10 mL / min / 1 .73 m2

[0529] Renal allograft biopsies

[0530] Biopsies should not be performed, if - in the judgment of the investigator, the transplant surgeon, or other designated individual(s) conducting the procedure - the procedure poses an unacceptable risk to the participant and / or allograft. Kidney biopsies will be obtained under the following scenarios:

[0531] Protocol biopsies will be performed at the following time points:

[0532] ■ Time-zero: intraoperatively, before reperfusion, and may be a core or wedge biopsy at the discretion of the operating surgeon Week 52 post-transplant: Results from the week 52 biopsy will be used as a component for iBox scoring

[0533] Indication allograft biopsies may be performed at any time at the discretion of the investigator if there are clinical signs of allograft dysfunction. Unscheduled assessments for PD and PK may be collected at the time of any indication biopsy.

[0534] All biopsies, including protocol and indication renal biopsies, will be read at the site’s local pathology laboratory and can be used for clinical management. Independent pathologists will review processed slides and 4 paraffin-embedded unstained slides from the same sample at the central pathology laboratory. An optional frozen section and 2 optional 18-pm paraffin scrolls from the same sample will also be sent to a central pathology laboratory for potential future analyses (e.g., complement staining or GEP analysis) if informed consent is obtained from the participant.

[0535] Biopsies performed during the study, including protocol and indication renal biopsies, will include assessments by light microscopy, immunohistochemical staining, and electron microscopy. Evidence of transplant glomerulopathy, interstitial fibrosis and tubular atrophy, and inflammation (e.g., tubulitis, glomerulitis, peritubular capillaritis) will be assessed using ultrastructural evaluation. Standard assessments, including grading of antibody-mediated rejection, will be performed following the 2019 revised Banff criteria.

[0536] Time points for all PRO questionnaires are provided in the SoA.

[0537] EQ-5D 5L is a standardized measure of health status developed by the EuroQol Group to provide a simple, generic measure of health status for clinical and economic appraisal. The descriptive system comprises 5 dimensions:

[0538] • Mobility

[0539] • Self-care

[0540] • Usual activities

[0541] • Pain / discomfort

[0542] • Anxiety / depression

[0543] Each dimension has 5 levels:

[0544] • No problem

[0545] • Slight problem

[0546] • Moderate problem Severe problem

[0547] Extreme problem

[0548] The participant will indicate their health state by selecting the box next to the most appropriate statement in each of the 5 dimensions using a recall period of today. This decision results in a 1 digit number expressing the level selected for that dimension. The digits for 5 dimensions will be combined in a 5-digit number describing the respondent’s health state. A unique health state is defined by combining 1 level from each of the 5 dimensions. A total of 3125 possible health states could be defined in this way. Each state is referred to in terms of a 5-digit code. For example, state 11111 would indicate no problems in any of the 5 dimensions, and 12345 would indicate no problem with mobility, slight problems with washing or dressing, moderate problems with doing usual activities, severe pain or discomfort, and extreme anxiety or depression.

[0549] A visual analog scale (VAS) is included in the questionnaire. Participants will be asked to mark their health status from 0 to 100 on the day the interview is conducted, with a score of 0 corresponding to “the worst health you can imagine” and 100 corresponding to “the best health you can imagine.”

[0550] PROMIS-29 is a reliable and valid measure in kidney transplant recipients, and higher scores represent higher quality of life. PROMIS-29 is a collection of 4-item short forms that assesses 7 domains. The 7 domains include physical function, anxiety, depression, fatigue, pain (interference and intensity), sleep disturbance, and the ability to participate in social roles and activities. For each question, participants will be asked to score the question across 5-10 levels of severity.

[0551] KDQOL-36 has been validated in patients with kidney disease who require dialysis. The KDQOL- 36 questionnaire assesses 5 dimensions, including physical composite, mental composite, symptoms / problems, effects of kidney disease, and burden of kidney disease. Higher scores represent improved quality of life. The KDQOL-36 questionnaire is a patient reported measure that the patients will complete. The reliability and validity of the KDQOL-36 has been demonstrated in a variety of studies in patients with various stages of kidney disease.

[0552] 4.4 Pharmacokinetics

[0553] ARGX-117 serum concentrations will be determined using a validated method. PK samples may also be used for methodology validation and / or for future research purposes. Such use of these samples is permitted only after obtaining consent from the participant. PK blood samples will be collected in all participants. PK sampling is optional for participants who have been discharged from the hospital. In a sub-study of consenting participants, approximately 12 to 20 participants at select sites will consent to extra PK sampling through the end of the study. Additional PK samples may be obtained in the event of an AESL

[0554] PK parameters include, but are not limited to, the following:

[0555] Day 1 : AUCo-i68h, C 168h j Cmax] and Tmax

[0556] Day 8: AUCo-i68h, AUCo-int, C 168h , Cmax, and Tmax

[0557] 4.5 Pharmacodynamics

[0558] PD samples will be collected in all participants. PD sampling is optional for participants who have been discharged from the hospital. In a sub-study of consenting participants, approximately 12 to 20 participants at select sites will consent to collect extra PD sampling through the end of the study. Additional PD samples for CH50, total and free C2 may be obtained in the event of an AESL

[0559] Blood samples will be collected for the determination of total C2 concentrations, free C2 concentrations and CH50 as indicated in the SoA. These PD markers will be determined using assays that are validated for their intended use. PD samples may also be used for methodology validation and / or for future research purposes. Such use of these samples is permitted only after obtaining consent from the participant.

[0560] 4.6 Biomarkers

[0561] The exploratory endpoints listed as follows will be reported separately from the clinical study report. Samples will be collected according to the schedule described in the SoA.

[0562] • Blood (serum and EDTA-plasma) and urine samples will be collected to assess the impact of ARGX-117 treatment on components and split products of the complement cascade (e.g., C3, C4, C5, and serum terminal complement complex). Additionally, alternative complement pathway activity will be evaluated in the collected serum samples.

[0563] • For participants who have consented to optional sampling, 2 parts of any protocol or indication biopsy tissue (one frozen portion of 3 to 5 mm and two 18-pm paraffin scrolls) will be stored for possible future analyses. The analyses may comprise: o the extraction of RNA and measurement of gene expression profiles (GEPs) related to clinically relevant processes o staining for complement split products to evaluate the impact of ARGX-117 on complement deposition within the kidney biopsy

[0564] • An abbreviated iBox score at 24 weeks will be calculated in the absence of biopsy histology, with full iBox scoring when a biopsy is available, as described in the SoA.

[0565] • Blood samples will be collected at early time points to assess the impact of ARGX- 117 on kidney damage markers, including, but not limited to, neutrophil gelatinase- associated lipocalin levels, as a marker of early tubular damage and a risk predictor of DGF.

[0566] • Blood samples will be collected to assess the impact of ARGX-117 on the development of donor-specific antibodies (DSA), including but not limited to de novo DSAs and non- HLA DSA. Participants routinely undergo post-transplant monitoring for development of circulating DSAs. DSA monitoring will be performed as specified in the SoA, and will be used for research purposes only.

[0567] • Blood and urine samples will be collected and stored for possible future analyses. Such use of these samples is only permitted after obtaining informed consent from the participant. This is described further in the IGF. The analyses may include the extraction of RNA and measurement of GEPs related to clinically relevant processes, including markers related to complement biology (eg, complement factors and / or regulators), kidney biology (eg, markers for kidney damage) and / or related to ARGX-117.

[0568] Samples may be stored for up to 15 years (or according to local regulations) after the end of the study.

[0569] 4.7 Immunogenicity assessments

[0570] Assessments of Anti-ARGX-117 Antibodies Blood samples will be collected at the time points indicated in the SoA and predose on IMP administration visits (within <2 hours before administration), to evaluate serum levels of ADA against ARGX-117. Samples will be analyzed by the designated laboratory in a tiered approach using validated immunogenicity assays. Initially, samples will be screened for a positive assay response (tier 1 ). Screened positive samples will then be tested in a confirmation assay (tier 2). Finally, a titration of the ADA response will be performed on positive tier 2 samples to characterize the magnitude of the antibody response (tier 3).

[0571] Assessment of Anti-ARGX-117 Neutralizing Antibodies Neutralizing antibodies to ARGX-117 can be evaluated in the collected serum samples and banked from all participants according to the SoA. Samples will be stored for up to 15 years (or according to local regulations). Immunogenicity blood samples may be used for methodology validation and / or for future research purposes. Such use of these samples is permitted only after obtaining informed consent from the participant.

[0572] 4.8 Objectives and Endpoints Table 13: Objectives and Endpoints

[0573] EXAMPLE 5: Complement inhibition and its functional consequences in an in vivo lung transplant model in C57BI / 6 mice, where the transplanted organ was exposed to cold ischemia and subseouent reperfusion leading to IRI

[0574] In this study, anti-C2 treatment was evaluated in mice which underwent a left-lung allograft (BALB / c donor to C57BL / 6 recipient) transplantation and compared with isotype treatment. The results reveal a role of complement in the early post-transplant period.

[0575] 5.1 MATERIALS AND METHODS

[0576] 5.1.1 Anti-mouse C2 antibody

[0577] 12E08 (anti-mouse C2 antibody) and Mota (isotype control) were used in the study. The antibodies were expressed in HEK293 cells and were produced in the mlgG2b_FcD backbone format. The FcD format ensures that backbone effector functions were diminished by introduction of the DANA mutations (D265A, N297A) in CH2 domain of the heavy chain.

[0578] 5.1.2 Mouse orthotopic left lung transplantation

[0579] All animals were purchased from Janvier Labs (Le Genest-Saint-lsle, France) and subjected to allograft transplantation between ten to twelve weeks of age. Recipients were male C57BL / 6N (H-2Kb ) and donors were male BALB / c (H-2Kd ). Orthotopic left lung transplantation was performed as described by Heigl et al. (2024; Front Immunol 15: 1369536). Briefly, after dissection of the donor mice left lungs, lungs were stored at 4°C until implantation. After positioning the transplanted lung in the recipient thorax, the native lung was excised, the thoracic wall was closed and the orotracheal tube removed when spontaneous respiration had resumed. All animals received daily subcutaneous immunosuppression (10 mg / kg cyclosporin A and 1 .6 mg / kg methylprednisolone) until the end of the experiment, unless otherwise indicated. Animals were dosed through intraperitoneal (IP) injection with 25 mg / kg 12E08 mlgG2b_FcD (n=6) or isotype control (Mota mlgG2_FcD) (n=6) every 3 days during the full course of the experiment. At different timepoints (indicated below), blood was sampled and in-vivo micro-CT imaging was performed. Mice were sacrificed through terminal ocular bleeding and injection with ketamine / xylazine (350 pL). Lungs were ventilated followed by perfusion through pulmonary artery with 1x 700 pL saline for bronchoalveolar lavage (BAL) sampling, whenever lungs turned white. After BAL collection, lungs were perfused with 4% PFA (in PBS), still on ventilation. Afterwards, the trachea was closed with a suture, at full inspiration (300 pL). Heart-lung block was placed in 4% PFA (in PBS) at 4°C for 48 hours before switching to 1 x PBS or preparation for FACS.

[0580] 5.1.3 Blood sampling

[0581] Blood was sampled via heparin coated capillaries (Hirschmann cat n° 9030208) in Eppendorf tubes. Samples were allowed to clot for 30 min at room temperature, followed by immediate centrifugation at 4°C, 1300-2000 g for 10 min. Centrifugation was performed maximum 1 h after sampling. After centrifugation, the supernatant is serum, which was collected, snap frozen and immediately stored at -80°C.

[0582] 5.1.4 Mouse free C2 assay

[0583] The mouse free C2 assay is an enzyme-linked immunosorbent assay (ELISA) based on a sandwich principle intended for the pharmacodynamic (PD) analysis of free “unbound”C2 in mouse serum. Briefly, anti-mouse sushi 2 antibody mab117_014 (“12E08”) was coated (10 pg / ml) overnight in 1x PBS on a 96-well Nunc MaxiSorp flat bottom plate (100 pL / well) at 4°C. Next day, the assay plate was washed 5 times with 200 pL PBS-T (1x PBS with 0.05% tween20) using a plate washer and subsequently blocked with 3% BSA diluted in 1 x PBS for 1 hour at room temperature (RT) while shaking gently (450 rpm). Dilutions of recombinant mouse C2 (1 / 2 dilution series from 300 ng / mL to 0.293 ng / mL), detection antibody and samples (1 / 180 diluted) were prepared on ice in 1x TBS supplemented with 2 mM CaCh After blocking, plates were washed 5 times with 1 x PBS-T directly followed by addition of standard and samples to the plate (100 pL / well). Standard and samples were incubated on the plate for exactly 15 min on ice before washing the plate again 5 times with 1 x PBS-T. Next, mab117 016 (anti-mouse C2) biotinylated detection antibody was added to each well (100 pL / well at 1 pg / mL) and incubated on ice for exactly 15 min. After incubation, the plate was washed 5 times with 1 x PBS-T before loading streptavidin HRP antibody (100 pL / well, 1 / 10 000 diluted in 1xPBS-T) onto the plate and incubating for 15 min on ice. Last, the plate was washed 5 times with 1 xPBS-T and s(HS)TMB diluted 1 :1 with MQ and put at RT before use was added to the plate (100 pL / well). The colouring reaction was allowed to proceed for 15 min at RT, shielded from light, and was stopped by the addition of 0.5 mol / L sulfuric acid (100 pL / well). OD values were measured at 450 nm (ref 620 nm) using a spectrophotometer (Infinite M Nano Tecan). Data was analysed with GraphPad Prism 9.5.1 software.

[0584] 5.1.5 Mouse C3a assay

[0585] The kit’s protocol (TECO medical) was followed according to manufactures instructions without deviations. Serum and BAL samples were diluted respectively 1 / 100 and 1 / 5 into the kit’s dilution buffer. Data was analysed with GraphPad Prism 9.5.1 software.

[0586] 5.1.6 Mouse pro-inflammatory panel 1 kit

[0587] The kit’s protocol (MesoScale Discovery; MSD) was followed according to manufactures instructions without deviations. Serum samples were diluted 1 / 10 and into the kit’s dilution buffer, BAL samples were measured undiluted for the first experiment samples and 1 / 2 diluted for the second experiment samples. Results were analysed with GraphPad Prism 9.5.1 software. The different measured analytes are summarized in Table 14.

[0588] Table 14: Overview of different measured analytes

[0589] 5.1.7 FACS analysis

[0590] Right before isolation, lung digestion buffer was prepared. Lungs were cut into small pieces and the tissue was placed in a gentleMACS C tube containing 5 mL lung digestion buffer and kept on ice. Tubes were placed in a MACS™ dissociator and the m_lung_01 protocol was performed (pre-programmed by manufacturer). Samples were incubated in digestion buffer in a 37°C water bath for 30 minutes. The tube was shaken vigorously by hand every 5 minutes for faster tissue dissociation. Next, the m_lung_02 protocol was run (pre-programmed by manufacturer) followed by addition of 20 mL MACS buffer. The sample was filtered through a 70 pm cell strainer into a 50 mL tube, the tube and strainer were rinsed with 20 mL MACS buffer and added to the new tube. Cell suspension was centrifuged (350g, 8 min) and supernatant was removed. Pellet was vortexed and 1 mL RBC lysing buffer Hybri-max was added. After vortexing again and incubating 1 minute at room temperature (RT), 10 mL MACS buffer was added. The sample was filtered through a 70 pm cell strainer into a 50 mL tube. The tube and strainer were again rinsed with 10 mL MACS buffer and added to the new tube. Cell suspension was centrifuged (350g, 5 min) and supernatant was removed. Pellet was vortexed and resuspended in 500 pL MACS buffer.

[0591] For live / dead staining, 50 pL of the single cell suspension was added to an Eppendorf tube and kept for 15-20 minutes in warm water bath (42°C) to prepare dead cells. Next, 200 pL of each sample was added to a 96-well round bottom plate (one well per panel). One extra well was applied as unstained control. 50 pL of fresh cells were added to the Eppendorf with dead cells and transferred into a well of the round bottom plate as well. The mastermix of antibodies was prepared and the 96-well plate was spun down at 350 g for 5 minutes (4°C). Pellet was resuspended in master mix (50pL / sample) and incubated for 30 minutes at 4°C in a dark room. 200 pL MACS buffer was added to each well and the plate was centrifuged for 5 minutes (350 g, 4°C). One additional wash was performed with 200 pL MACS buffer, supernatant was taken off and pellet was resuspended again in 200 pL MACS buffer and transferred to FACS tubes. Read out was performed at 50000 events.

[0592] 5.2 STUDY DESIGN

[0593] 5.2.1 Experiment 1

[0594] In the first experiment, two groups (isotype (Mota_mlgG2b) and anti-C2 antibody (12E08_mlgG2b), 6 animals per group) underwent allograft transplantation (BALB / c donor to C57BL / 6 recipient) (Figure 15A). The donor lung was subjected to a cold ischemia time of at least 1 hour at 4°C (Figure 15B). Antibody was dosed (25 mg / kg every 3 days) based on previous PK / PD data to obtain full blocking of C2 during the entire experiment (3 weeks). At different indicated timepoints blood samples were collected and micro-CT imaging of the lungs was performed. At sacrifice, BAL samples of both left and right lungs were collected and cells of left lungs were used for flow cytometry analysis. 5.2.2 Experiment 2

[0595] In a second experiment (Figure 16), the set-up of experiment 1 was performed with the following adaptions:

[0596] ■ No immunosuppressive agents were administered.

[0597] ■ Each treatment group (N=6) was divided in two for analysis, 3 animals were used for histology and 3 for FACS analysis.

[0598] ■ The animals were followed up for 1 week after transplantation, compared to 3 weeks in the first experiment.

[0599] ■ Immunohistochemistry staining on mice lung tissue were performed by C-path. Tissues were sectioned at 4 pm and stained with hematoxylin and eosin staining (H&E), myeloperoxidase (MPO), C3d, C4d, TUNEL and C5b-9.

[0600] 5.3 RESULTS

[0601] 5.3.1 Experiment 1

[0602] Free C2 analysis of blood samples confirmed complete blocking of complement C2

[0603] (Figure 17A) following anti-mouse C2 antibody treatment, which resulted in a clear reduction of complement activation, as measured by the amount of produced C3a (Figure 17B). Interestingly, control isotype-treated animals exhibited a sustained decrease in serum C2 levels throughout the experimental period (up to POD21 ) suggesting complement consumption. In line with this finding, a marked elevation in C3a levels for isotype-treated animals was observed on POD7 compared to baseline. This increase was transient, as C3a levels returned to baseline by POD21 .

[0604] C2 analysis of BAL samples taken at sacrifice (POD21 ) demonstrated a complete blocking of local C2 in all animals dosed with anti-mouse C2 antibody. In isotype treated mice, C2 levels varied between 14 to 257 ng / mL (Figure 18A). No difference in C3a level was observed in BAL samples between isotype or anti-C2 treated animals (Figure 18B). Nevertheless, analysis of serum samples (Figure 17) indicated that the disparity in complement levels might be more pronounced during the initial week post-transplantation.

[0605] Density of the lungs was determined via micro-CT scan (Figure 19) and suggested a slight improvement at POD3 for the anti-C2 treated animals compared to isotype treated animals (lower density represents more air in the lung).

[0606] Flow cytometry data of left lung cell suspension was generated at POD21 . A significant higher percentage of dendritic cells (DCs) and natural killer (NK) cells were found in the anti-C2 treated group as compared to isotype treated animals (data not shown). Different inflammation markers were measured in serum samples. On the initial post-operative days an increase of IFN-y, IL-5, IL-6, KC GRO and IL-10 was observed in serum. The clear upregulation of inflammatory cytokines confirmed that a systemic inflammation was present (data not shown).

[0607] 5.3.2 Experiment 2

[0608] Free 02 analysis of blood samples confirmed complete blocking of complement C2

[0609] (Figure 20A) following anti-mouse 02 antibody treatment, which resulted in clear reduction of complement activation, as measured by the amount of produced 03a (Figure 20B). Isotype treated animals showed a sustained decrease in serum 02 levels throughout the experimental period (up to POD7) suggesting complement consumption. Additionally, a marked elevation in 03a levels for the isotype treated animals compared to baseline was observed, which was in line with the observation in experiment 1 . Noteworthy, 03a levels reached a maximum on POD3 in this experiment, whereas a peak was observed on POD7 in experiment 1 .

[0610] 02 analysis of BAL samples taken at sacrifice (POD 7) confirmed complete blocking of 02 in all animals dosed with anti-mouse 02 antibody. For the isotype control, 02 levels varied from 2.5 and 264 ng / mL (Figure 21 A). For 03a most values felt below the LLOQ (63 ng / ml) except for two isotype treated animals where elevated BAL 03a was seen (Figure 21 B). Overall, 02 and 03a measurements were similar to experiment 1 . These data suggested that administration of immunosuppression had no impact on systemic or BAL complement levels.

[0611] In contrast to experiment 1 , no difference between isotype or anti-02 treated animals was observed on lung density determined via micro-CT scan (data not shown). This discrepancy might be related to experimental variation or to the removal of immunosuppression.

[0612] Flow cytometry data of left lung cell suspension was generated at POD7. A significant lower percentage of neutrophils and higher percentage of NK cells were found in the anti-02 treated group as compared to isotype treated animals (data not shown).

[0613] Ten different analytes were measured in serum (Figure 22). On POD3, an increase was noted for IFN-y, IL-1 p, IL-5, IL-6 and KC GRO in serum. All these analytes returned to baseline at POD7, except for IFN-y which remained elevated. These results align with the results of experiment 1 .

[0614] Left lungs of three isotype-treated animals and three anti-C2-treated animals were prepared for immunohistochemistry (IHC) and stained for damage and inflammation markers (H&E, TUNEL, and MPO) as well as complement markers (C3d, C4d and C5b9). No differences between anti- 02 and isotype-treated animals was observed for TUNEL, MPO and C3d, whereas lower expression of C5b9 and C3d was observed in the anti-C2 blocking group as compared to isotype treated lungs (Figure 23A).

[0615] Further, no difference was observed between anti-C2 and isotype treatment for perivascular inflammation but slightly reduced staining for lymphocytic bronchitis was observed for the anti-C2 treated group (H&E staining, Figure 23B).

[0616] A decrease in C3d, C5b9 and lymphocytic bronchitis following anti-C2 treatment could be indicative of reduced complement-mediated IRI tissue damage.

[0617] CONCLUSION

[0618] Serum C2 levels of the isotype treated animals decreased over time and remained low up to day 21 post lung transplant. The combined reduction in C2 levels and the increases C3a levels in the isotype treated group supported a role for complement in the early post-lung transplant period.

[0619] In both experiments, analysis of blood and BAL samples revealed a complete inhibition of complement C2 in the anti-C2 treated group. This inhibition was measured by the absence of detectable unbound C2. Consequently, a reduction in complement activation was observed, as evidenced by a decrease in C3a production relative to the isotype treated control group.

[0620] No discernible effect of immunosuppression was observed on complement levels of C2 or C3a when comparing both experiments indicating that systemic complement is not affected by the administration of immunosuppression.

[0621] EXAMPLE 6: Characterisation of C2 levels in plasma and BAL samples of human lung transplant patients

[0622] The objective of this study was to provide a comprehensive analysis of systemic and bronchoalveolar lavage (BAL) complement levels and inflammatory dynamics during early posttransplantation period in human transplant patient samples.

[0623] 6.1 MATERIALS AND METHODS

[0624] 6.1.1 BCA assay

[0625] Total protein levels in all plasma and BAL samples were determined using the Pierce BCA protein assay kit (Thermo Scientific, Cat N°:23225,). BAL samples were diluted 1 / 5 and plasma samples 1 / 100 in 1x PBS. 6.1.2 Total C2 analysis

[0626] Total C2 levels in plasma and BAL samples were determined using the Abeam human complement C2 ELISA kit (Cat N°: ab154132) according to manufacturer’s recommendations. Plasma samples were diluted 1 / 200 and BAL samples 1 / 2 in kit buffer.

[0627] 6.1.3 C3a analysis

[0628] Patient samples were screened for complement activation in plasma and BAL using the human C3a assay (Tecomedical group, Cat N°: TE1071 ) assay according to manufacturers’ recommendations. Plasma samples were diluted 4000-fold and BAL samples 20-fold in dilution buffer.

[0629] 6.1.4 C3bc analysis

[0630] Patient samples were screened for complement activation in plasma using an in-house developed C3bc assay. Briefly, wells of a MaxiSorp 96-well plate were coated with anti-human C3bc mlgG2a clone 9 (ImmunoPrecise Antibodies Ltd, The Netherlands) overnight at RT and incubated with sample diluted 25 000-fold in PBS-0.1%Tween20 supplemented with 10mM EDTA. After washing, wells were incubated with biotinylated polyclonal goat anti-human C3 and subsequently with streptavidin HRP. Afterwards, wells were washed and incubated with TMB solution. The substrate reaction was stopped with sulfuric acid and C)D450 / 600nm was measured with a spectrophotometer.

[0631] 6.1.5 sTCC analysis

[0632] Patient samples were screened for complement activation in plasma and BAL using the sTCC assay (Hycult Biotech, Cat N°: HK328-01 ) according to manufacturers’ recommendations. Plasma samples were diluted 500-fold and BAL samples 20-fold in dilution buffer.

[0633] 6.1.6 MSD multiplex kit

[0634] Individual inflammation analytes in plasma and BAL were evaluated using Mesoscale U-plex custom biomarker group 3 (ICAM-1 , VCAM-1 , CRP) (Mesoscale, Cat N°: K151 AGM-1 ) and group 1 (GM-CSF, IL-6, IL-8, IL-10, IL-17A, IP-10, MCP-1 , VEGF-A) (Mesoscale, Cat N°: K15067M-1 ). Individual angiogenesis analytes in plasma and BAL were evaluated using custom Mesoscale V-plex angiogenesis panel 1 (VEGF-C, VEGF-D) (Mesoscale, Cat N°: K151A9H-1 ).

[0635] 6.1.7 Luminex analysis

[0636] Individual complement analytes in plasma and BAL were evaluated using the human complement expanded panel 1 (Merck, Cat N°: HCMPEX1 -19K-06) (C2, MBL, C5, C9 and factor D) and human complement panel 2 (Merck, Cat N°: HCMP2MAG-19K-05) bead panel (iC3b, C3, C1q, C4 and factor B) according to manufacturers’ instructions. For complement panel 1 , plasma and BAL were respectively diluted 1000- and 10-fold. For complement panel 2, plasma and BAL were respectively diluted 40000- and 20-fold.

[0637] 6.1.8 Neutrophil elastase

[0638] Participants were screened for neutrophil elastase in plasma and BAL using the Neutrophil Elastase assay (abeam, Cat N°: ab270204) according to manufacturers’ recommendations. Plasma samples were diluted 1 / 1600 or 1 / 12800.

[0639] 6.1.9 Statistical analysis

[0640] Statistical analysis was performed with SAS v5.4. All analyte concentrations were normalized for BCA concentrations measured in the same matrix, except for C-reactive protein (CRP) which was quantified in whole blood and corrected for plasma BCA concentrations. Visualizations of the BCA normalized analyte concentrations and cell profiles were created over time. A Welch t-test was performed to compare the normalized values in plasma or the cell numbers at each post operative day (POD) pairwise to pre transplantation (pre-Tx) to test for differences following lungtransplantation. A log-transformation was applied to the data to satisfy the normality assumption. Due to the presence of zeroes for certain cell types, 0.1 was added to all cell type results (incl. CRP) before applying a log-transformation. A ‘Welch’ test was performed to accommodate for possible heterogeneous variances between two time points. Due to the exploratory nature of the analysis, a correction for multiple testing was not performed. All tests were performed at a 5% significance level with results indicated on the graphs with stars: *** p < 0.001 , ** p < 0.01 , * p < 0.05, ns if p > 0.05. Due to the observational nature of the study, longitudinal models could not be used for the analysis as patients were not followed up longitudinally. Similarly, pre-Tx concentrations were not available for BAL, so pre- and post-transplantation results could not be compared.

[0641] 6.2 STUDY DESIGN

[0642] Samples from human lung transplanted patients were collected from an observational study. Plasma samples were collected from the day of admission, on day 1 , day 2, day 3, day 4, day 5, day 6, and day 7 post-transplantation (post-Tx), and on day 90 post-Tx. Additionally, bronchoalveolar lavage (BAL) samples were collected on the first 3 post-Tx days and cryopreserved (at -20°C). For whole blood samples only, each subject was sampled pre-Tx, at each of the first 7 post-Tx days and at day 90 post-Tx. 6.3 RESULTS

[0643] 6.3.1 Exploratory complement screening of plasma and BAL samples

[0644] An initial exploratory screening was performed on a subset of samples for complement factors C2, C3a and C3bc. C2 analysis (Figure 24) revealed a substantial decrease of C2 on the initial days post lung transplant as compared to pre-transplant or post-operative day (POD) 90. Samples were also analysed for C3a and C3bc which are both complement split factors and, when elevated, indicative of complement activation. High levels of both C3a (Figure 25) and C3bc (Figure 26) as compared to healthy complement preserved human plasma pool (NHP) were observed at the initial 7 days post lung transplant. Remarkably, pre-transplant and POD90 samples illustrated even higher levels of both complement split products.

[0645] A limited number of bronchoalveolar lavage (BAL) samples were exploratory screened for C2, C3a and C3bc. All 3 complement components were found to be present in the BAL, with no observed temporal changes (data not shown).

[0646] 6.3.2 Temporal changes in complement and inflammatory components

[0647] Based on the temporal changes observed in complement level profiles following lung transplantation (section 6.3.1 ), a larger cohort of plasma samples (total of #266) and BAL samples (total of #118) were selected for further analysis. This expanded investigation included additional complement components and inflammatory markers (Table 15) to gain a more comprehensive understanding of the immunological processes involved after lung transplantation. Importantly, all measured analytes were corrected for total protein concentration (BCA) to ensure correct comparison between the samples.

[0648] Table 15: Overview of measured analytes in both BAL and plasma of lung transplant patients

[0649] 6.3.2.1 Temporal complement analyte profiles

[0650] Temporal plasma profiles of the different measured complement analytes (corrected for total protein) are depicted in Figure 27 and Figure 28. A summary of the profiles can be found in Table 16. The data illustrate a clear decrease for most complement factors (C1q, Factor B, C2, C4, C5 and C9) on the first day post-transplant. For C2, a significant decrease was observed which was sustained until POD7 and returned to pre-Tx levels at POD90. For C1q, C4 and C5, pretransplant levels were obtained as from POD2 or 3 onwards, after an initial decrease. For Factor B and C9, significant increases were obtained starting at POD3 until POD7. For mannose binding lectin (MBL), a high variability in levels was observed, as expected. C3 showed a significant increase from POD2 to 7, while Factor D and C3b / iC3b did not change compared to pre-transplant levels.

[0651] Table 16: Schematic representation of human plasma complement factors and dynamics in lung transplantation relative to BCA.

[0652] Bold boxed arrows represent a maximum or minimum in the temporal profile of that complement analyte;

[0653] BCA: bicinchoninic acid; BM: biomarker; MBL: mannose binding lectin, POD: post operative day; Tx: transplant

[0654] All measured complement components were found to be present in BAL on the three initial days post-lung transplant but with no observed temporal changes (data not shown).

[0655] Figure 29 represents a schematic overview of the relative abundance of each complement factor in BAL / BCA compared to plasma / BCA, facilitating a comparative analysis of their levels. Remarkably, C2 and C9 are respectively 5- and 7-fold higher in BAL as compared to plasma, whereas C3 was 12 times lower.

[0656] 6.3.2.2 Temporal inflammation analyte profiles

[0657] Temporal profiles of the different measured systemic inflammation analytes in the initial days post lung transplant were compared to pre transplant day -1 and POD90. Results are depicted in Figure 30 and Figure 31 and a summary of these profiles can be found in Table 17. Data are excluded from graphical representation for IL-17A and GM-CSF due to values approaching detection limits. CRP, IL-6, IL-8, IL-10, MCP-1 , IP-10, VCAM-1 , ICAM-1 and NE are clearly elevated at the first day post lung transplant. Vascular endothelial growth factors (VEGFs), VEGF-A, VEGF-C, and VEGF-D showed a clear decrease. Most analytes remained impacted for at least 7 days post lung transplant and restored over time (POD90).

[0658] Table 17: Schematic representation of human plasma inflammatory dynamics in lung transplantation relative to BCA.

[0659] Bold boxed arrows represent a maximum or minimum in the temporal profile of that analyte;

[0660] Tx: transplant, IL: interleukin, NE: neutrophil elastase, MCP: monocyte chemoattractant protein-1 , VEGF: vascular endothelial growth factors, VCAM: vascular cell adhesion molecule, IP-10: interferon gamma-induced protein 10, GRP: C-reactive protein , ICAM: intercellular adhesion molecule, Tx: Transplant, BCA: bicinchoninic acid, BM: biomarker

[0661] 6.2.3.2 Temporal blood cell profiles

[0662] Blood cell counts of all patient samples were used to generate temporal profiles (Table 18). Eosinophils, lymphocytes, basophils and monocytes demonstrated a decrease at the first day post lung transplant while neutrophils peaked at POD1 . These results are in line with a typical immune response seen after trauma.

[0663] Table 18: Schematic representation of blood cell dynamics after lung transplantation.

[0664] Bold boxed arrows represent a maximum or minimum in the temporal profile of that cell type; preTx: pre transplantation, POD: post operative day CONCLUSION

[0665] This comprehensive overview on systemic and intrapulmonary complement dynamics, in the context of lung transplantation, provides evidence of early complement activation, with classical and alternative pathway being activated.

[0666] In particular, the study demonstrated:

[0667] • A clear decrease for complement factors C1q, Factor D, Factor B, C2, C4, C5 and C9 in human plasma on the first day post-transplant.

[0668] • Presence of all measured complement analytes in BAL of lung transplanted patients, during the first 3 days post-transplant.

[0669] • A rapid elevation in the initial days post lung transplantation of: o Cytokines, either pro-inflammatory (IL-6, IL-8, IL-17A, GM-CSF) or antiinflammatory (IL-10) in the first days post-transplant o Chemokines such as monocyte chemoattractant protein-1 (MCP-1 ) and interferon gamma-induced protein 10 (IP-10) in the first days post-transplant o Neutrophil Elastase, as marker for neutrophil activation o Endothelial injury markers ICAM-1 and VCAM-1 and acute phase protein C- reactive protein (CRP)

[0670] • A decrease in the initial days post lung transplantation was observed for VEGF-A, VEGF- C and VEGF-D.

[0671] EXAMPLE 7: Complement inhibition and its functional consequences in an in v / 'vowarm ischemia kidney model in humanized C2 transgenic mice (Balb / C)

[0672] In this study, anti-C2 treatment was evaluated in mice which were exposed to renal clamping to mimic warm ischemia reperfusion injury (IRI) and subsequent acute kidney injury (AKI) and compared with isotype or anti-C5 treatment. In this model, both renal pedicles were clamped to induce ischemic conditions and subsequent acute kidney injury. The results suggest a role for early complement activation upstream of 05 in the development of AKI.

[0673] 7.1 MATERIALS & METHODS

[0674] 7.1.1 Anti-human C2 antibody

[0675] BRO-2 (anti-human C2 antibody, hlgG4 backbone) and Mota (isotype control, hlgG4 backbone) were used in this study. The antibodies were expressed in HEK293 cells and were produced in the hlgG4 backbone format. BRO-2 is an anti-C2 neutralizing antibody targeting human C2b and shares its six CDR sequences with the anti-C2b antibody referred to herein as ARGX-117.

[0676] 7.1.2 Anti-mouse C5 antibody

[0677] Anti-mouse 05 antibody clone BB5.1 was used in this study.

[0678] 7.1.3 Mouse warm ischemia model

[0679] Balb / C humanized 02 (hC2) transgenic mice (mice expressing mouse 02 where the mouse sushi-2 domain is replaced by its human counterpart) were purchased from Taconic (Lille Skensved, Denmark) and subjected to warm ischemia between 9 to 11 weeks of age, weighing 25-28 g. Only male mice were used in this study. Exposure to warm ischemia was performed as described by Liu et al. (2017; JCI / ns / g / 7t; 2(18):e94716). Briefly, midline abdominal incision was performed for the exposure of both renal pedicles, which were clamped for 18 or 21 min, to identify the optimal ischemia time. Occlusion of blood flow was monitored by color change from normal to dark purple immediately after the clamping. Blood returned to its normal original color after removal of the clamps. All mice underwent the same procedure. Sham-operated mice underwent the same procedure except for clamping of the pedicles. Animals were dosed once immediately after ischemia through intraperitoneal (IP) injection with 25 mg / kg BRO-2-hlgG4 or isotype control, or 40 mg / kg BB5.1 . Mice were sacrificed at 48 h or 21 days post-surgery and blood was sampled. Paraformaldehyde-fixed kidney tissue samples were used for immunofluorescence, equilibrated in 30% sucrose / PBS overnight and then embedded in optimal cutting temperature within a dry ice ethanol bath. Eight to ten-pm frozen sections were washed in PBT (PBS + 2.5% Triton-X), blocked in 5% normal donkey serum in PBT, and incubated overnight at 4’0 with primary antibodies (kidney injury molecule-1 (KIM1 ), goat; aSMA, mouse monoclonal; Sox9, rabbit) before incubation with species-specific secondary antibodies coupled to Alexa Fluor dyes.

[0680] 7.1.4 Blood sampling

[0681] Samples were allowed to clot for 30 min at room temperature, followed by immediate centrifugation at 4°C, 1300-2000 g for 10 min. Centrifugation was performed maximum 1 h after sampling. After centrifugation, the supernatant is serum, which was collected, snap frozen and immediately stored at -80°C.

[0682] 7.1.5 Mouse free C2 assay

[0683] Microtiter wells (Greiner Bio-one, Cat N°: 675061 ) were coated with anti-mouse sushi 2 antibody BRO-2 and incubated at 4°C ON. After blocking Superblock and washing, wells were incubated with mouse serum samples diluted in Low cross buffer diluted 1 -to-1 with 1 xTBS supplemented with 1 .25 mM CaCh. After washing, wells were incubated with biotinylated mab117 016 (antimouse C2), followed by streptavidin-HRP (Pharmingen, Cat N°: 554066). Afterwards, wells were washed and incubated with s(HS)TMB solution diluted 1 -to-1 with MQ. The substrate reaction was stopped with sulfuric acid and OD450 / 620nm was measured with a spectrophotometer (Infinite M Nano, Tecan).

[0684] 7.1.6 Mouse classical pathway (CP) complement assay (C3c detection)

[0685] Microtiter wells (Greiner Bio-one, Cat N°: 675061 ) were coated with immunoglobulin (lg)G and incubated overnight at 4°C. After blocking with 1% w / v casein (Bio-Rad Laboratories, Cat N°: 1610783) and washing, wells were incubated with mouse serum samples diluted in veronal buffered saline at 37°C. As controls, MgEGTA (inhibits CP and LP) or EDTA (inhibits all pathways) were added to the buffer. After washing, wells were incubated with biotinylated antimouse complement component C3 (TebuBio, Cat N°: CL7503B), followed by streptavidin-HRP (Pharmingen, Cat N°: 554066). Afterwards, wells were washed and incubated with TMB solution. The substrate reaction was stopped with sulfuric acid and OD450 / 620nm was measured with a spectrophotometer (Infinite M Nano, Tecan).

[0686] 7.1.7 Mouse CP complement assay (C5b9 detection)

[0687] Mouse CP activation levels were assessed at the level of C5b9 formation using a commercially available ELISA kit (Hycult Biotech, HIT420, Uden, the Netherlands). Serum was diluted to 5% v / v with kit provided sample dilution buffer. The manufacturer’s instructions were followed, and absorbance was measured at 450 nm. In addition to the kit’s positive and negative control, healthy pooled transgenic mouse serum was added as positive control (Taconic).

[0688] 7.1.8 Mouse neutrophil gelatinase-associated lipocalin (NGAL) assay

[0689] Mouse NGAL levels were assessed using a commercially available ELISA kit (Cat nr 443707, Biolegend MAX). Serum samples were diluted 250X or 750X with kit provided sample dilution buffer. The manufacturer’s instructions were followed, and absorbance was measured at 450 nm. Healthy pooled transgenic mouse serum was added as positive control (Taconic).

[0690] 7.2 STUDY DESIGN

[0691] 7.2.1 Experiment 1

[0692] In the first experiment the optimal ischemia time was identified for hC2 Balb / C mice. Twenty-five mice were enrolled and exposed to IRI for 18 min (n=10), 21 min (n=12) or used as normal control (n=3). Samples were collected 48 h or 21 days post-surgery and analyzed for markers of AKI (KIM1 / SOX9 / aSMA), NGAL, free-C2, and overall CP activity. 7.2.2 Experiment 2

[0693] In the second experiment complement inhibition at the level of C2 and the effect on IRI-induced AKI was investigated. A total of 29 animals were enrolled in this experiment. Eight animals were not exposed to any surgical procedure [untreated (n=4), isotype treated (n=2) BRO-2 treated (n=2)]. Two sham-surgery animals were included [isotype treated (n=1 ), BRO-2 treated (n=1 )] and 19 animals were subjected to IRI and analyzed 48 h post-surgery [isotype treated (n=4) , BRO-2 treated (n=4)], or 21 days post-surgery [isotype treated (n=5), BRO-2 treated (n=5)]. Samples were collected for the 48 h and 21 day timepoints and analyzed for markers of AKI (KIM1 , aSMA), NGAL, free C2, and overall CP activity.

[0694] 7.2.3 Experiment 3

[0695] In the third experiment complement inhibition at the level of C5 and the effect on IRI-induced AKI was investigated. A total of 18 animals were enrolled in this experiment. Three sham controls 48 h n=2; 21 days n=1 (all isotype treated) were included. Fifteen animals were exposed to 18 min ischemia; 48 h n=6 [isotype treated (n=3), BB5.1 treated (n=3)]; 21 d n=9 (isotype treated (n=5), BB5.1 treated (n=4)). Samples were collected for both time-points and analyzed for markers of AKI (KIM1 ), and overall CP activity.

[0696] 7.3 RESULTS

[0697] 7.3.1 Experiment 1

[0698] A 10% (1 / 10 animals) mortality rate was observed in the hC2 animals exposed to 18 min ischemia, whereas 21 min ischemia resulted in 33.3% (4 / 12 animals) mortality rate post-surgery. Both ischemia times led to significant and extensive acute injury / repair responses as indicated by KIM1 / SOX9 / aSMA response (Figure 32A / B). Additionally, IRI-induced AKI resulted in increased Havcr (KIM1 gene) expression 48 h post-surgery in both groups. These levels returned to baseline after 21 days (Figure 32C).

[0699] Serum analysis indicated stable free C2 levels between sham surgery treated and the different ischemia time treated groups (Figure 33A). Additionally, the overall complement activity of the collected sera was equal between groups (Figure 33B). NGAL levels increased 48 h post-surgery in an ischemia time-dependent manner. For both ischemia times NGAL levels returned to baseline levels 21 days post-surgery (Figure 33C).

[0700] Based on these findings an ischemia time of 18 min was selected for subsequent experiments. 7.3.2 Experiment 2

[0701] In this experiment hC2 transgenic mice were subjected to 18 min ischemia either in the presence or not in the presence of anti-human C2 antibody BRO-2. In the ischemia treated animals, at 48h post-surgery, no difference in KIM1 expression was observed in either the kidney cortex or outer medulla between the BRO-2 or isotype dosed groups (Figure 34A). In contrast, BRO-2 dosed animals displayed a significant reduction in the extent of KIM1 and SMA expression at day 21 post-ischemia compared to the isotype dosed animals (Figure 34B,C).

[0702] Free C2 levels were reduced in all BRO-2 animals, but were restored to baseline levels 21 days after surgery. No effect on free 02 levels was observed in the isotype-treated animals (Figure 34E). BRO-2 treatment also reduced complement activation in a mouse CP assay, in a similar pattern as the free 02 results (Figure 34D). NGAL levels increased after ischemia, but no effect of BRO-2 dosing was observed (Figure 34F).

[0703] Overall these results suggest that complement inhibition at the level of 02 early after ischemia has a protective effect on the development of AKI, here assessed at day 21 post-surgery.

[0704] 7.3.3 Experiment 3

[0705] Next, the effect of complement inhibition at the level of 05 on AKI development was investigated. At 48 h post-surgery, there was no difference in KIM1 staining between anti-05 or isotype dosed animals (Figure 35A). No protective effect on the development of AKI was observed at 21 days post-surgery after complement inhibition at the level of 05 (Figure 35B), in contrast to the results obtained for 02 inhibition (Figure 34B). Despite the absence of a protective effect on AKI, complement was completely inhibited in the BB5.1 dosed animals early after surgery, as indicated by the absence of C5b9 deposition in the mouse CP assay. Complement activity was restored 21 days post-ischemia (Figure 35C).

[0706] CONCLUSION

[0707] In this study hC2 transgenic Balb / c mice were exposed to renal clamping to mimic warm ischemia reperfusion injury (IRI) and subsequent acute kidney injury (AKI). Eighteen min ischemia resulted in significant and extensive acute injury / repair responses as indicated by early and late markers for kidney damage. Serum analysis indicated stable free 02 levels between sham surgery treated and the different ischemia time treated groups. Complement inhibition at the level of 02 using BRO-2 early after ischemia had a protective effect on AKI development. This was not observed when complement was inhibited downstream at the level of 05. These results suggest classical and lectin pathway complement involvement in ischemia-induced AKI and identify 02 as a target for future therapeutic strategies. The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. Moreover, all aspects and embodiments of the invention described herein are considered to be broadly applicable and combinable with any and all other consistent embodiments, including those taken from other aspects of the invention (including in isolation) as appropriate.

[0708] Various publications and patent applications are cited herein, the disclosures of which are incorporated by reference in their entireties.

[0709] REFERENCES

[0710] Biglarnia AR et al. The multifaceted role of complement in kidney transplantation. Nature Reviews I Nephrology. 2018. 14: 767-781 .

[0711] Budding K., van de Graaf EA., Kardol-Hoefnagel, T, Oudijk, E-J.D., Kwakkel-van Erp, JM., Hack, GE., et al. Antibodies against apoptotic cells present in end-stage lung disease patients do not correlate with clinical outcome after lung transplantation. Front. Immunol. 2017, Mar 21 ;8:322.

[0712] Dragun D, Tullius SG, Park JK, et al. ICAM-1 antisense oligodesoxynucleotides prevent reperfusion injury and enhance immediate graft function in renal transplantation. Kidney Int. 1998;54(2):590-602.

[0713] Fernandez AR et al. Review: Ischemia reperfusion injury - a translational perspective in organ transplantation. IntJ Mol Scr, 2020. 21 (22): 8549

[0714] Heigl et al. The nature of chronic rejection after lung transplantation: a murine orthotopic lung transplant study. Front Immunol. 2024.15: 1369536.

[0715] Hollmen ME et al. Deceased donor neutrophil gelatinase-associated lipocalin and delayed graft function after kidney transplantation: a prospective study. Critical Care. 2011 . 15:R121

[0716] Irish WD et al. Nomogram for predicting the likelihood of delayed graft function in adult- cadaveric renal transplant recipients. J Am Soc Nephol. 2003. 14: 2967-2974. Kelly KJ, Williams WW, Colvin RB, Bonventre J V. Antibody to intercellular adhesion molecule 1 protects the kidney against ischemic injury. Proceedings of the National Academy of Sciences. 1994;91 (2):812-816.

[0717] Liu et al. Molecular characterization of the transition from acute to chronic kidney injury following ischemia / reperfusion. JCI Insight. 2017 Sep 21 ;2(18):e94716.

[0718] Lim MA and Bloom RD. Medical therapies to reduce delayed graft function and improve longterm graft survival. Am Soc Nephrol. 2020. 15: 13-15.

[0719] Nieuwenhuijs-Moeke GJ at al. Ischemia and reperfusion injury in kidney transplantation: relevant mechanism in injury and repair. J Clin Med. 2020; 9; 253

[0720] Ponticelli C, Reggiani F, and Moroni G. Delayed graft function in kidney transplants: risj factors, consequences and prevention strategies. J Pers Med. 2022; 12;1557

[0721] Rabb H, Mendiola CC, Saba SR, et al. Antibodies to ICAM-1 Protect Kidneys in Severe Ischemic Reperfusion Injury. Biochem Biophys Res Commun. 1995; 211 (1 ):67-73. doi:10.1006 / bbrc.1995.1779

[0722] Tang Q, Dong C, and Sun Q. Immune response associated with ischemia and reperfusion injury during organ transplantation. Inflamm Res; 2022; 71 (12): 1463-1476

[0723] Tingle SJ et al. Machine perfusion preservation versus static cold storage for deceased donor kidney transplantation (Review). Cochrane Database of Systematic Reviews. 2019. Issue 3. CD011671

[0724] Vormann K, Tool L, et al. Modelling and Prevention of Acute Kidney Injury through Ischemia and Reperfusion in a Combined Human Renal Proximal Tubule / Blood Vessel-on-a-Chip. Kidney360 2021 Nov 4;3(2):217-231.

[0725] Wu X, Guo R, Wang Y, Cunningham PN. The role of ICAM-1 in endotoxin-induced acute renal failure. Am J Physiol Renal Physiol. 2007 Oct;293(4):F1262-71 .

[0726] Yarlagadda SG et al. Association between delayed graft function and allograft and patient survival: a systematic review and meta-analysis. Nephrol Dial Transplant. 2009. 24: 1039-1047.

Claims

CLAIMS1 . A method of treating or preventing ischemia reperfusion injury (IRI) in a subject receiving a donor organ transplant, the method comprising administering an effective amount of a complement factor 2 (C2) inhibitor to the subject.

2. A method of improving allograft function in a subject receiving a donor organ transplant, the method comprising administering an effective amount of a complement factor 2 (C2) inhibitor to the subject.

3. A method of reducing the risk of allograft loss in a subject receiving a donor organ transplant, the method comprising administering an effective amount of a complement factor 2 (C2) inhibitor to the subject.

4. The method of any one of claims 1 -3, wherein the donor organ is selected from: kidney, lung, heart, pancreas, bowel and liver.

5. The method of claim 4, wherein the donor organ is a kidney.

6. The method of claim 5, wherein the subject is at risk of delayed graft function.

7. The method of claim 5 or claim 6, wherein the method treats or prevents delayed graft function.

8. A method of treating or preventing delayed graft function in a subject receiving a kidney transplant, the method comprising administering an effective amount of a complement factor 2 (C2) inhibitor to the subject.

9. The method of any one of the preceding claims, wherein the subject is receiving a donor organ from a deceased donor.

10. The method of claim 9, wherein the deceased donor is a brain-death donor (DBD).11 . The method of claim 9, wherein the deceased donor is a circulatory-death donor (DCD).

12. The method of any one of the preceding claims, wherein the donor organ has not been machine-perfused between the time of organ procurement and the time of transplant.

13. The method of any one of the preceding claims, wherein the donor organ has been preserved on ice between the time of organ procurement and the time of transplant.

14. The method of any one of the preceding claims, wherein the donor organ has been subjected to cold-ischemia and the cold-ischemia time of the donor organ is at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 14 hours, at least 16 hours, least 18 hours, at least 20 hours, at least 22 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, or at least 48 hours.

15. The method of any one of the preceding claims, wherein the donor organ has been subjected to cold-ischemia and the cold-ischemia time of the donor organ is at least 12 hours.

16. The method of any one of the preceding claims, wherein the donor from whom the donor organ is obtained is aged > 40 and <70 years old.

17. The method of any one of the preceding claims, wherein the donor from whom the donor organ is obtained has a terminal serum creatinine value of > 1 .5 mg / dL.

18. The method of any one of claims 5-17, wherein the donor organ is a kidney and the subject has been diagnosed with end stage renal disease (ESRD).

19. The method of any one of claims 5-18, wherein the donor organ is a kidney and the subject has been stable on chronic dialysis for at least 3 months.

20. The method of any one of claims 5-19, wherein the donor organ is a kidney and the subject has not received a previous kidney transplant.21 . The method of any one of claims 5-19, wherein the donor organ is a kidney and the subject has received at least one previous kidney transplant.

22. The method of any one of claims 5-21 , wherein the donor organ is a kidney and the subject does not require post-transplant dialysis within the first 7 days post-transplant.

23. The method of any one of the preceding claims, wherein a first dose of the C2 inhibitor is administered to the subject on the day of the transplant procedure.

24. The method of any one of the preceding claims, wherein a first dose of the C2 inhibitor is administered to the subject prior to or during the transplant procedure.

25. The method of any one of the preceding claims, wherein a first dose of the C2 inhibitor is administered to the subject prior to or during reperfusion of the organ.

26. The method of any one of the preceding claims, wherein a first dose of the C2 inhibitor is administered to the subject intravenously.

27. The method of claim 26, wherein the C2 inhibitor is administered to the subject as a continuous intravenous infusion, and administration is initiated prior to reperfusion of the organ.

28. The method of any one of claims 23-27, wherein administration of the first dose is initiated and completed before reperfusion of the organ begins.

29. The method of any one of the preceding claims, wherein the C2 inhibitor is administered to the subject at a dose of 0.1 mg / kg to 100 mg / kg.

30. The method of claim 29, wherein the C2 inhibitor is administered to the subject at a dose of 0.1 mg / kg, 0.5 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg or 100 mg / kg.31 . The method of claim 29, wherein the C2 inhibitor is administered to the subject at a dose of 40 mg / kg to 80 mg / kg.

32. The method of claim 31 , wherein the C2 inhibitor is administered to the subject at a dose of 60 mg / kg.

33. The method of any one of claims 23-32, wherein one or more further doses of the C2 inhibitor are administered to the subject post-operatively.

34. The method of claim 33, wherein one further dose is administered to the subject post- operatively.

35. The method of claim 33 or claim 34, wherein a post-operative dose is administered to the subject 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13 or 14 days post-transplant.

36. The method of claim 33 or claim 34, wherein a post-operative dose is administered to the subject 7-9 days post-transplant.

37. The method of any one of claims 33-36, wherein the one or more further dose(s) is / are administered to the subject intravenously.

38. The method of any one of claims 33-36, wherein the one or more further dose(s) is / are administered to the subject subcutaneously.

39. The method of any one of claims 33-38, wherein the one or more further dose(s) is / are administered to the subject at a dose of 0.1 mg / kg to 100 mg / kg.

40. The method of claim 39, wherein the one or more further dose(s) is / are administered to the subject at a dose of 0.1 mg / kg, 0.5 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg, 60 mg / kg, 70 mg / kg, 80 mg / kg, 90 mg / kg or 100 mg / kg.41 . The method of claim 39, wherein the one or more further dose(s) is / are administered to the subject at a dose of 40 mg / kg to 80 mg / kg.

42. The method of claim 40, wherein the one or more further dose(s) is / are administered to the subject at a dose of 60 mg / kg.

43. The method of any one of the preceding claims, wherein a first dose of the C2 inhibitor is administered to the subject on the day of transplant prior to and / or during reperfusion of the organ, and a second dose of the C2 inhibitor is administered to the subject post-operatively.

44. The method of claim 43, wherein the second dose is administered to the subject 7-9 days post-transplant.

45. The method of claim 44, wherein the second dose is administered to the subject 7 days post-transplant.

46. The method of any one of claims 43-45, wherein the first dose is administered to the subject at a dose of 60 mg / kg and / or the second dose is administered to the subject at a dose of 60 mg / kg.

47. The method of any one of claims 43-46, wherein the first dose is administered to the subject intravenously, and the second dose is administered to the subject intravenously or subcutaneously.

48. The method of any one of the preceding claims, wherein the C2 inhibitor is selected from: inhibitory RNA species, for example siRNAs or shRNAs; small molecule inhibitors; biological antagonists including inhibitory peptides, antibody mimetics such as affibodies, affilins, affitins, adnectins, atrimers, evasins, DARPins, anticalins, avimers, fynomers, versabodies and duocalins; antibodies and antigen-binding fragments thereof.

49. The method of any one of the preceding claims, wherein the C2 inhibitor is an antibody or antigen-binding fragment thereof that specifically binds to C2.

50. The method of claim 49, wherein the antibody or antigen-binding fragment thereof specifically binds to C2b.51 . The method of any one of claims 48-50, wherein the antigen-binding fragment is selected from: an antibody light chain variable domain (VL), an antibody heavy chain variable domain (VH), a single chain antibody (scFv), a F(ab’)2 fragment, a Fab fragment, an Fd fragment, an Fv fragment, a one-armed (monovalent) antibody, a diabody, a triabody, a tetrabody, a unibody, a domain antibody and a nanobody.

52. The method of any one of claims 48-51 , wherein the antibody or antigen-binding fragment thereof comprises a variable heavy chain domain (VH) and a variable light chain domain (VL) wherein the VH and VL domains comprise the CDR sequences:- HCDR3 comprising or consisting of SEQ ID NO: 4;- HCDR2 comprising or consisting of SEQ ID NO: 3;- HCDR1 comprising or consisting of SEQ ID NO: 2;- LCDR3 comprising or consisting of SEQ ID NO: 7;- LCDR2 comprising or consisting of SEQ ID NO: 6; and- LCDR1 comprising or consisting of SEQ ID NO: 5.

53. The method of any one of claims 48-52, wherein the antibody or antigen-binding fragment thereof comprises a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having at least 70% identity thereto and a VL domain comprising or consisting of the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence having at least 70% identity thereto.

54. The method of claim 53, wherein the antibody or antigen-binding fragment thereof comprises a VH domain comprising or consisting of the amino acid sequence of SEQ ID NO: 8 and a VL domain comprising or consisting of the amino acid sequence of SEQ ID NO: 9.

55. The method of any one of claims 48-54, wherein the antibody or antigen-binding fragment thereof comprises a human IgG heavy chain constant domain.

56. The method of any one of claims 48-55, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising or consisting of the amino acid sequence of SEQ ID NO: 10 and a light chain comprising or consisting of the amino acid sequence of SEQ ID NO: 11.

57. The method of any one of the preceding claims wherein the C2 inhibitor is empasiprubart.

58. The method of any one of the preceding claims, wherein the C2 inhibitor reduces complement activity in the serum, plasma and / or urine of the subject.

59. The method of any one of the preceding claims, wherein the C2 inhibitor does not affect alternative complement pathway activity.

60. The method of any one of the preceding claims, wherein the C2 inhibitor reduces the level of free C2 in the serum, plasma and / or urine of the subject.61 . The method according to any one of the preceding claims, further comprising administering to the subject one or more additional therapeutic agents.

62. The method of claim 61 , wherein the method further comprises the administration of one or more immunosuppressive agents.

63. The method of claim 62, wherein the one or more immunosuppressive agents is selected from: antithymocyte globulin (ATG), tacrolimus, mycophenolate mofetil (MMF), enteric-coated mycophenolic acid (EC-MPA), enteric-coated mycophenolate sodium (EC-MPS), cyclosporine, corticosteroids, azathioprine, everolimus, sirolimus, rapamycin, belatacept, basiliximab, alemtuzumab, and any combination thereof.

64. The method of claim 62 or claim 63, wherein the method comprises the administration of an induction immunosuppressive therapy, preferably antithymocyte globulin (ATG) and corticosteroids.

65. The method of any one of claims 62-64, wherein the method comprises the administration of a maintenance immunosuppressive therapy, preferably tacrolimus, and MMF or EC-MPA, and optionally corticosteroids.

66. A C2 inhibitor for use in the treatment or prevention of ischemia reperfusion injury (IRI) in a subject receiving a donor organ transplant according to the method of any one of claims 1 -65.

67. A C2 inhibitor for use in improving allograft function in a subject receiving a donor organ transplant according to the method of any one of claims 1 -65.

68. A C2 inhibitor for use in reducing the risk of allograft loss in a subject receiving a donor organ transplant according to the method of any one of claims 1 -65.

69. A C2 inhibitor for use in the treatment or prevention of delayed graft function in a subject receiving a kidney transplant according to the method of any one of claims 5-65.

70. Use of a C2 inhibitor in the manufacture of a medicament for the treatment or prevention of ischemia reperfusion injury (IRI) in a subject receiving a donor organ transplant, wherein the treatment or prevention is carried out according to the method of any one of claims 1 -65.71 . Use of a C2 inhibitor in the manufacture of a medicament for improving allograft function in a subject receiving a donor organ transplant, wherein the treatment or prevention is carried out according to the method of any one of claims 1 -65.

72. Use of a C2 inhibitor in the manufacture of a medicament for reducing the risk of allograft loss in a subject receiving a donor organ transplant, wherein the treatment or prevention is carried out according to the method of any one of claims 1 -65.

73. Use of a C2 inhibitor in the manufacture of a medicament for the treatment or prevention of delayed graft function in a subject receiving a kidney transplant, wherein the treatment or prevention is carried out according to the method of any one of claims 5-65.

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