Novel Uses of PEG-Phospholipid Molecules

Abstract

The present invention relates to the use of PEG-phospholipid molecules to selectively mask surface antigens on red blood cells, platelets and / or endothelial cells, whereby blood products treated with PEG-phospholipid molecules can be infused into cross-incompatible or non-compatible recipients with reduced risk of antibody binding to surface antigens and aggregation. Correspondingly, organ grafts treated with PEG-phospholipid molecules can be transplanted into cross-incompatible or non-compatible recipients with reduced risk of antibody binding and antibody-mediated rejection.

Description

[Technical Field]

[0001] The present invention relates generally to poly(ethylene glycol) phospholipid (PEG-phospholipid) molecules and their use in the selective masking of cell surface antigens. [Background technology]

[0002] Donor-recipient testing, also known as crossmatching or cross-matching, is a pre-transfusion test performed as part of blood compatibility testing. This typically involves adding the recipient's plasma to a sample of the donor's red blood cells. If the donor's blood is incompatible with the recipient's, antibodies in the recipient's blood will bind to antigens on the donor's red blood cells. This antibody-antigen reaction causes the red blood cells to clump together, known as agglutination, or break down.

[0003] In addition to blood typing of the donor and recipient and screening for unexpected blood group antibodies, cross-matching is one of several steps in pre-transfusion testing. The complete cross-matching process can take up to an hour, so it is not always used in emergency situations. In such emergency situations, O Rh-negative blood may be given to patients who do not have preformed antibodies. However, this type of blood is rare and cannot be given in large quantities.

[0004] ABO crossmatch and complement-dependent cytotoxicity (CDC) assays are also used to determine compatibility between donors and recipients in organ transplants, where preexisting anti-ABO or anti-class I human leukocyte antigen (HLA) antibodies may be present. If a recipient has received multiple transfusions or given birth to a child, there is a risk of immunization against one or several of the more than 50 blood group antigens. These preexisting antibodies, called "irregular antibodies," make it difficult to select compatible blood. Furthermore, preexisting anti-ABO and anti-class I HLA antibodies can also shorten the circulation time of transfused platelets administered to patients to prevent bleeding due to thrombocytopenia.

[0005] WO 2004 / 050897 discloses a method for preparing an RBC composition with reduced antigenicity and a reduced level of hemolysis. The method involves reacting an activated antigen-masking compound having a molecular weight of approximately 20-40 kDa, and the resulting red blood cells are not easily hemolyzed by any serum or plasma sample, for example, by complement lysis. The RBC composition is particularly useful for introduction into individuals where there is a high potential for an immune response, such as in alloimmunized blood recipients or in trauma situations where the possibility of transfusing incompatible blood units is higher.

[0006] Advanced Drug Delivery Reviews 62:827-840 (2010) reviews various attempts to encapsulate islets of Langerhans or to create a bioartificial pancreas to isolate the islets from the recipient's immune system.

[0007] Nevertheless, there is a need for blood product treatments that can be used in emergency situations, for example, when a complete cross-matching process between the donor and recipient is not possible. There is also a need for transplant treatments to treat antibody-mediated rejection in connection with transplantation. Summary of the Invention

[0008] A common goal is to inhibit antibody binding and agglutination in the context of transfusion.

[0009] Inhibiting antibody binding and antibody-mediated rejection in the context of organ transplantation is another common goal.

[0010] These and other objectives are met by the invention as defined herein.

[0011] The invention is defined in the independent claims. Further embodiments of the invention are defined in the dependent claims.

[0012] One aspect of the present invention relates to poly(ethylene glycol) phospholipid (PEG-phospholipid) molecules for use in selectively masking surface antigens of red blood cells and / or platelets in blood products from a donor to inhibit antibody binding to the surface antigens in connection with transfusion of the blood product into a cross-incompatible or incompatible recipient. The PEG chains of the PEG-phospholipid molecules have an average molecular weight selected within the range of 3,000 to 10,000 Da.

[0013] Another aspect of the present invention relates to PEG-phospholipid molecules for use in selectively masking surface antigens of organ grafts, including red blood cells, platelets, and / or endothelial cells from a donor, to inhibit antibody binding to the surface antigens and antibody-mediated rejection (AMR) associated with transplantation of the organ graft into a cross-incompatible or incompatible recipient.

[0014] A further aspect of the present invention relates to a blood product comprising red blood cells and / or platelets and PEG-phospholipid molecules immobilized within the cell membrane of the red blood cells and / or platelets, the PEG chains of which have an average molecular weight selected within the range of 3,000 to 10,000 Da.

[0015] Yet another aspect of the present invention relates to an in vitro method for treating red blood cells and / or platelets, comprising selectively masking surface antigens of the red blood cells and / or platelets by adding PEG-phospholipid molecules to the red blood cells and / or platelets in vitro, wherein the PEG chains of the PEG-phospholipid molecules have an average molecular weight selected within the range of 3,000 to 10,000 Da.

[0016] Another aspect of the present invention relates to a method of transfusion comprising adding PEG-phospholipid molecules to a blood product from a donor to selectively mask surface antigens on red blood cells and / or platelets present in the blood product. The PEG chains of the PEG-phospholipid molecules have an average molecular weight selected within the range of 3,000 to 10,000 Da. The method also includes transfusing the blood product into a cross-incompatible or incompatible recipient.

[0017] A further aspect of the present invention relates to a method for screening for irregular antibodies. The method comprises adding PEG-phospholipid molecules to a blood sample from a subject to selectively mask surface antigens on red blood cells present in the blood sample. The PEG chains of the PEG-phospholipid molecules have an average molecular weight selected within the range of 3,000 to 10,000 Da. The method also comprises screening for irregular antibodies present in the blood sample or bound to red blood cells obtained from the blood sample.

[0018] PEG-phospholipid molecules of the embodiments can selectively mask surface antigens on red blood cells, platelets, and / or endothelial cells. Thus, blood products treated with PEG-phospholipid molecules can be infused into cross-incompatible or non-compatible recipients with reduced risk of antibody binding to surface antigens and agglutination. Correspondingly, organ grafts treated with PEG-phospholipid molecules can be transplanted into cross-incompatible or non-compatible recipients with reduced risk of antibody binding and AMR.

[0019] The embodiments, together with further objects and advantages thereof, may best be understood by reference to the following description taken in conjunction with the accompanying drawings, in which: [Brief explanation of the drawings]

[0020] [Figure 1] The mechanism of hydrophobic interaction of PEG-phospholipid molecules to the lipid bilayer of the cell membrane is explained. [Figure 2] 1 shows flow cytometry analysis of the binding of FITC-labeled PEG-phospholipids to CCRF-CEM cells. [Figure 3A] In vitro results illustrate the mechanism of action (MOA) of PEG-phospholipids, showing inhibition of binding to surface antigens when cells are coated with PEG-phospholipids. [Figure 3B] In vitro results illustrate the mechanism of action (MOA) of PEG-phospholipids, showing inhibition of binding to blood group antigens when RBCs are coated with PEG-phospholipids. [Figure 3C] In vitro results illustrate the mechanism of action (MOA) of PEG-phospholipids: inhibition of complement-mediated lysis of RBCs. [Figure 4] Figure 1 shows binding to surface antigens using antibodies directed against large and small surface antigens (CD52 and CD4, respectively). Coating the cells with PEG-phospholipid almost completely inhibits binding to small surface antigens. [Figure 5] Figure 1 shows the agglutination test card for Rh phenotyping. A is an O Rh+ blood sample treated with PEG-phospholipid molecules. B is an O Rh+ control blood sample. [Figure 6] Figure 1 shows the agglutination test card for Rh phenotyping. A is an A Rh+ blood sample treated with PEG-phospholipid molecules. B is an A Rh+ control blood sample. [Figure 7] Figure 1 shows the agglutination test card for Rh phenotyping. A is an A Rh+ blood sample treated with PEG-phospholipid molecules. B is an A Rh+ control blood sample. [Figure 8] Binding of the LP recognition molecule CL-11 (n = 5) to native and coated HUVECs after exposure to human plasma, and binding of concanavalin A (a broad-specificity lectin) (n = 3) to unexposed HUVECs was analyzed by flow cytometry. CL-11 binding values ​​are shown after subtracting the binding of CL-11 in the presence of 10 mM EDTA. It was shown that PEG-phospholipid coating can mask the acceptor molecules of both concanavalin A and (to a lesser extent) CL-11. Statistical evaluation was performed by one-sample t-test and one-way analysis of variance (ANOVA). [Figure 9]The binding of the LP recognition molecule MBL on native and coated HUVECs after exposure to human plasma was analyzed by flow cytometry. [Figure 10] PEG-phospholipid release from cells was analyzed by incubating 3 million CCRF-CEM cells with 50 μL of FAM-PEG-phospholipid (2 mg / mL) for 30 minutes at room temperature (n=3). After washing, the cells were cultured for 7 days, and the amount of bound PEG-phospholipid was assessed by flow cytometry (left panel) and confocal microscopy (right panel). [Figure 11] Two donor pigs underwent bilateral nephrectomy. The kidneys were then harvested and refrigerated for 24 hours. Biotin-labeled PEG-phospholipid was administered and incubated in both kidneys for 40 minutes. After 40 minutes, unbound biotin-PEG-phospholipid was flushed from the system, and the kidneys were transplanted into recipient pigs. Initial biopsies were taken from the treated and native kidneys at the time of transplantation and 60 minutes later (n=4). The pigs were euthanized consecutively at 12, 24, 48, and 72 hours, and biopsies were taken from the transplanted and native kidneys. Fluorescence was detected using secondary FITC-streptavidin and confocal microscopy. Fluorescence in the native kidney was compared with that of the transplanted kidney. The half-life of PEG-phospholipid was calculated from the fluorescence images, and a one-stage exponential decay model was applied to fit a curve to the data points, resulting in a t of 14 h (95% confidence interval 8–26). [Figure 12] Detection of fluorescence in urine from PEG-phospholipid-treated kidneys and four of the native kidneys in ANSM confirms that PEG-phospholipids are excreted by the kidneys. Two-way repeated measures ANOVA (coated / uncoated); p=0.0013. [Figure 13] Wedge biopsies were taken at 1, 60, and 360 minutes and immunohistochemically quantified by confocal microscopy for protein expression of nitrotyrosine, HO-1, and INOS. No significant differences were found between treated and untreated tissues. Two-way repeated measures ANOVA (coated / uncoated); ns. [Figure 14] Complement (C3a, sC5b-9), coagulation (TAT), and contact system (FXIIa-C1INH) markers were sampled continuously from the renal vein of PEG-phospholipid-treated kidneys and their controls for the first 60 min, then systemically for up to 360 min. Two-way repeated measures ANOVA (coated / uncoated); TAT p = 0.0042; C3 a = 0.0002; sC5b-9 p = 0.0042; FXIIa / C1INH p = 0.0029). [Figure 15] Wedge biopsies were taken at 1, 60, and 360 minutes and analyzed by immunohistochemistry for C4d and C3b. Two-way repeated measures ANOVA (coated / uncoated); C4d ns; C3b p=0.013. [Figure 16] Complement (C3a, sC5b-9) and coagulation (TAT) markers as indicators of thromboinflammation were serially sampled from the renal vein of kidneys treated with PEG-phospholipid and their controls at 60 min, followed by systemic sampling at 24, 48, 72, and 96 h. Two-way repeated measures ANOVA (coated / uncoated); TAT p = 0.006; C3 a = 0.0059; sC5b-9 p = 0.0023) (n = 6 (treated) + 4 (control)). [Figure 17] Wedge biopsies were taken at 5 minutes and 96 hours and analyzed by immunohistochemistry for C3b and MAC. Two-way repeated measures ANOVA (coated / uncoated); C3b ns, sC5b-9ns (n=6 (treated) + 4 (control)). [Figure 18]NET analysis was performed on biopsies from ANSM by electron microscopy imaging, demonstrating intense deposition of NETs in untreated controls. In untreated kidneys (top row), dense reticular structures (NETosis) were already observed in biopsies 1 min after reperfusion, primarily located within Bowman's space (BS), tubules, and peritubular capillaries (PTC). In PEG-phospholipid-treated kidneys (bottom panel), NET formation was significantly reduced, with a larger area of ​​kidney tissue free of NETs. Resting neutrophilic granulocytes were visualized in PEG-phospholipid-treated kidneys (representative images of six analyzed). [Figure 19] Kidneys treated with PEG-phospholipid showed a highly significant reduction in NET deposition at all locations (n=6): Two-way repeated measures ANOVA (coated / uncoated); glomeruli p≦0.0001; tubules p=0.0010; vessels p=0.0011. [Figure 20] Cytokine mRNA expression of proinflammatory cytokines in ANSM: RT-PCR of proinflammatory cytokines and TF in wedge biopsies from individual kidneys, shown to be suppressed throughout the observation period. Expressed as a percentage of individual values ​​at the time of reperfusion (5 min). Two-way repeated measures ANOVA (coated / uncoated); IL-1β = p = 0.0021; IL-6 p < 0.0001, TNF p = 0.0077; TF = 0.0019) (n = 6). [Figure 21] Protein levels of proinflammatory cytokines in ANSM: Serial samples from the renal vein of PEG-phospholipid-treated kidneys and their controls were collected systemically at the first 60 min and then up to 360 min. Two-way repeated measures ANOVA (coated / uncoated); IL-1β ns; IL-6 p=0.0005; TNF p=0.0328; TF p=0.0103) (n=6). [Figure 22]Cytokine protein levels in SM: Cytokines were serially sampled from the renal vein of PEG-phospholipid-treated kidneys and their controls at 5 and 60 minutes, and then systemically at 96 hours. IL-1α and IL-12 were suppressed by PEG-phospholipid treatment in most cases already at 5 minutes. After 60 minutes, all cytokines stabilized at low steady states representing control values. After 96 hours, all cytokines were still suppressed to baseline levels in treated animals, except for IL-1RA and IL-8, which were unaffected. Two-way repeated measures ANOVA (coated vs. uncoated): IL-1α, IL-β, IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-12, and IL-18 p<0.01, IL-1RA, TNF, and IL-8 ns (n=6 (treated) + 4 (control)). [Figure 23] Renal function was monitored by pre- and post-transplant plasma creatinine levels in SM (including CLSM and in vivo separation studies). Baseline pre-transplant plasma creatinine was estimated daily, then at 24, 48, 72, and 96 hours. Two-way repeated measures ANOVA (coated / uncoated); p = 0.0179 (n = 6 (treated) + 4 (control)). [Figure 24] Corresponding data in CLSM. Two-way repeated measures ANOVA (coated / uncoated); p=0.0252 (n=5 (treated) + 5 (control)). [Figure 25] PEG-phospholipids inhibited the binding of anti-HLA class I antibody W6 / 32 to PBMCs. [Figure 26] PEG-phospholipid inhibited the binding of anti-HLA-ABC G46-2.6 to PBMCs. DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention relates generally to poly(ethylene glycol) phospholipid (PEG-phospholipid) molecules and their use in the selective masking of cell surface antigens.

[0022] Traditionally, cross-matching has been performed on blood products used in transfusions and organs transplanted from donors to recipients. Such cross-matching is used to prevent agglutination associated with antibody-mediated rejection (AMR) of transfused blood or transplanted organs. Both of these processes are mediated by the recipient's antibodies against surface antigens present on cells within the donor's blood or organ. However, in emergency situations, there may not be enough time available to perform a complete cross-matching process to save the recipient's life. Therefore, there is a need for blood product and / or organ treatments that can be used to suppress adverse reactions, such as agglutination or AMR, that would otherwise occur when transfusing cross-incompatible blood or transplanting cross-incompatible organs.

[0023] The present invention relates to the use of poly(ethylene glycol) phospholipid (PEG-phospholipid) molecules to create a transient, non-toxic artificial layer on the surface of cells within blood products or organs. This shielding PEG layer effectively masks surface antigens on the surface of cells from the recipient's antibodies against the surface antigens. As a result, treating blood products or organs with PEG-phospholipids prior to infusion or transplantation reduces the risk of antibodies binding to the surface antigens, thereby reducing the risk of agglutination or AMR by masking the surface antigens from antibodies within the recipient's body.

[0024] PEG-phospholipid molecules, together with their phospholipid domains, have the ability to uniformly anchor themselves to the lipid membrane of cells through hydrophobic interactions, creating a non-static artificial layer (see Figure 1). The PEG domains of PEG-phospholipid molecules are negatively charged, which has the ability to shield the surface from close interactions with macromolecules such as antibodies. Furthermore, cell surface polymer coating (CSPC) with PEG-phospholipid molecules prevents nonspecific adsorption of macromolecules such as albumin. This is an important finding, as intracellular adsorption of proteins is known to induce biological responses such as coagulation and activation of the complement system.

[0025] Experimental data presented herein demonstrate that PEG-phospholipid molecules of the embodiments can mask blood group antigens, such as A and B antigens, and Rhesus factors, such as RhD, RhC, Rhc, RhE, and Rhe, present on endothelial cells, i.e., red blood cells. As a result of this surface antigen masking, agglutination and cell lysis were inhibited by masking the surface antigens from antibodies, thereby preventing or at least significantly inhibiting antibody binding to the surface antigens on red blood cells.

[0026] Furthermore, the experimental data presented herein demonstrate that the masking of surface antigens achieved by the PEG-phospholipid molecules of the embodiments is selective, with the masking effect depending on the length of the PEG polymer and the size of the surface antigen on the cell surface. For example, a PEG-phospholipid molecule having a PEG domain or chain with a molecular weight of approximately 5 kDa has a length of approximately 6 nm, resulting in a 6 nm PEG layer when anchored to a cell membrane. This allows such PEG-phospholipid molecules to selectively mask surface antigens extending less than or slightly more than 6 nm from the cell surface. For example, such PEG-phospholipid molecules were able to effectively mask blood group B antigen and the cluster of differentiation antigen (CD52), also known as the CAMPATH-1 antigen. Both of these antigens are approximately 1 nm in size from the cell surface. Furthermore, partial masking was achieved for CD8, but not for CD4. While CD4 extends approximately 15 nm from the cell surface, the stalk region of CD8 causes this molecule to protrude less from the cell membrane, despite the similar size of CD8 and CD4.

[0027] One aspect of the present invention relates to PEG-phospholipid molecules for use in selectively masking surface antigens of red blood cells and / or platelets in blood products from a donor to inhibit antibody binding to the surface antigens in connection with transfusion of the blood product into a cross-incompatible or incompatible recipient. The PEG chains of the PEG-phospholipid molecules have an average molecular weight selected within the range of 3,000 to 10,000 Da.

[0028] A related aspect of the present invention provides the use of PEG-phospholipid molecules for the manufacture of a medicament for inhibiting antibody binding to surface antigens in connection with the transfusion of a blood product containing red blood cells and / or platelets having surface antigens selectively masked by PEG-phospholipid molecules into a cross-incompatible or incompatible recipient, wherein the PEG chains of the PEG-phospholipid molecules have an average molecular weight selected within the range of 3,000 to 10,000 Da.

[0029] Selective masking of surface antigens of red blood cells and / or platelets by PEG-phospholipid molecules of the embodiments thereby inhibits agglutination by inhibiting antibody binding to the surface antigens in connection with transfusion of the blood product to a cross-incompatible or incompatible recipient. Thus, this aspect of the invention relates to PEG-phospholipid molecules for use in selectively masking surface antigens of red blood cells and / or platelets in a blood product from a donor to inhibit agglutination in connection with transfusion of the blood product to a cross-incompatible or incompatible recipient.

[0030] As described in the disclosure of WO 2004 / 050897, it was quite surprising to find that PEG-phospholipid molecules with PEG chains or domains having average molecular weights selected within the range of 3 to 10 kDa could be used to selectively mask surface antigens on red blood cells and / or platelets. Herein, activated PEG compounds with molecular weights of 20 to 40 kDa were reacted to covalently bind the PEG compounds to the surface of red blood cells. Experimental data presented herein demonstrated that methoxy-PEG (mPEG) must have a molecular weight of at least 20 kDa to efficiently mask antigens on red blood cells and thereby inhibit antibody binding to these cells. When smaller mPEG molecules (5 kDa and 10 kDa) were tested, it was concluded that they caused red blood cell lysis at the levels required to mask the antigens.

[0031] Compared with WO2004 / 050897 (20-40 kDa), the PEG-phospholipid molecules of the present invention, which have smaller PEG chains (3-10 kDa), did not induce any cell lysis at levels that could effectively mask surface antigens on erythrocytes. Therefore, the use of PEG-phospholipid molecules with phospholipid domains to anchor PEG-phospholipid molecules in the cell membrane offers significant advantages over chemically reacting mPEG molecules with proteins and other macromolecules anchored in the cell membrane of erythrocytes.

[0032] The blood product is typically a whole blood product, but may alternatively be a treated and / or processed blood product comprising red blood cells (erythrocytes), platelets (thrombocytes), or red blood cells and platelets. Examples of processed blood products are packed red blood cells, packed platelets, or concentrated blood products comprising packed red blood cells and platelets. For example, a red blood cell sample obtained after centrifugation of whole blood is a packed red blood cell blood product, a buffy coat obtained after centrifugation of whole blood is a packed platelet blood product, and removal of plasma after centrifugation of whole blood leaves a combined packed red blood cell blood product and a platelet blood product.

[0033] Another aspect of the present invention relates to PEG-phospholipid molecules for use in selectively masking surface antigens of organ grafts, including red blood cells, platelets, and / or endothelial cells from a donor, to inhibit antibody binding to the surface antigens and / or antibody-mediated rejection (AMR) in connection with transplantation of the organ graft into a cross-incompatible or incompatible recipient.

[0034] A related aspect of the present invention defines the use of PEG-phospholipid molecules for the manufacture of a medicament for inhibiting antibody binding to surface antigens and / or antibody-mediated rejection (AMR) in connection with the transplantation of organ grafts comprising red blood cells, platelets, and / or endothelial cells having surface antigens selectively masked by the PEG-phospholipid molecules into cross-incompatible or incompatible recipients.

[0035] Selective masking of surface antigens of red blood cells, platelets, and / or endothelial cells by the PEG-phospholipid molecules of the embodiments thereby inhibits agglutination and AMR by inhibiting antibody binding to the surface antigens in association with transplantation of the organ graft into a cross-incompatible or incompatible recipient. Accordingly, this aspect of the invention relates to PEG-phospholipid molecules for use in selectively masking surface antigens of an organ graft comprising red blood cells, platelets, and / or endothelial cells from a donor to inhibit agglutination and / or antibody-mediated rejection (AMR) in association with transplantation of the cell or organ graft into a cross-incompatible or incompatible recipient.

[0036] Antibody-mediated rejection (AMR), also known as B cell-mediated or humoral rejection, is a serious complication after organ transplantation, such as kidney transplantation. Fewer than 10% of kidney transplant patients experience AMR, but as many as 30% of these patients experience graft loss as a result. AMR is mediated by antibodies against the allograft, particularly anti-HLA antibodies and A / B blood group antibodies, and leads to histological changes in the allograft vasculature that differ from cellular rejection (T cell-mediated rejection). Therefore, AMR is a distinct disease process compared to cellular rejection. AMR is initially characterized by microvascular inflammation, endothelial injury, and serological evidence of donor-specific antibodies (DSA). Symptoms of AMR can progress to transplant glomerulopathy, a form of advanced glomerular injury and remodeling.

[0037] Standard care for AMR includes plasmapheresis and intravenous immunoglobulin, which remove and neutralize antibodies, respectively. Agents that target B cells (rituximab and alemtuzumab), plasma cells (bortezomib), and the complement system (eculizumab) have also been used to treat AMR in kidney transplant recipients.

[0038] Therefore, AMR is a rejection process distinct from the initial cellular rejection caused by the innate immune system and occurs immediately after organ graft transplantation. Therefore, AMR is associated with the early cellular rejection process. The PEG-phospholipids of the present invention have a half-life of approximately 14 hours in pigs, well before the AMR process begins. However, as shown in the experimental section, the PEG-phospholipid molecules of the present invention were able to suppress cytokines even 4 days after transplantation. For example, as shown in Figure 22, at 60 minutes after transplantation, there was no significant difference in the levels of several cytokines, such as IL-1α, IL-1β, IFNγ, IL-2, IL-4, IL-6, IL-01, IL-12, and IL-18, between untreated control kidneys and kidneys treated with PEG-phospholipid. However, 4 days after kidney transplantation, recipients who received PEG-phospholipid-treated kidneys had significantly lower levels of these cytokines than recipients who received untreated control kidneys. This was quite surprising, as the PEG-phospholipid molecule, which has a half-life of only 14 hours, was cleared from the treated kidney 4 days after implantation, yet still had residual effects.

[0039] These long-term effects observed by treating organ transplants with PEG-phospholipids suggest that PEG-phospholipids provide protection against the adaptive immune system and AMR. Because inhibition of IL-6 / IL-6R signaling is considered a novel therapeutic option for preventing and treating allograft injury and AMR, the consistent and significant inhibition of IL-6 supports this concept. Clinical trial evidence supports the use of IL-6 blockade for desensitization and treatment of AMR in kidney transplants (Curr Transplant Reports 8:1-14 (2021), J Am Soc Nephrol 20:1032-1040 (2009), Transplantation 101:32-44 (2017)). As shown in Figure 22 and Figures 20-21, PEG-phospholipid molecules provided significant inhibition of IL-6, which is important for targeting and treating AMR.

[0040] As used herein, a cross-incompatible recipient refers to a recipient in whom a cross-match has not been made between the donor and recipient of a blood product or organ transplant. As used herein, a cross-incompatible recipient refers to a recipient who is incompatible with at least one surface antigen on red blood cells and / or platelets in a blood product, i.e., a recipient who has antibodies to at least one surface antigen on red blood cells and / or platelets, or antibodies to at least one surface antigen on red blood cells, platelets, and / or endothelial cells in an organ graft.

[0041] The present invention is not limited to the allogeneic transplantation of organ transplants, i.e., organ allografts.In sharp contrast, the present invention also provides the advantages related to the xenogeneic transplantation of organ transplants, i.e., organ xenografts.For example, the organ transplant from one mammalian species, typically non-human mammalian species donor, can be treated with the PEG-phospholipid molecule of the present invention before transplantation into another mammalian species, typically human recipient, so as to selectively mask the surface antigen of the organ transplant.

[0042] In one embodiment, the recipient is a cross-mismatched recipient. In another embodiment, the recipient is a non-matched recipient.

[0043] As used herein, an organ transplant includes an entire organ or tissue or a portion of an organ that is transplanted into the body of a recipient. Illustrative, non-limiting examples of organ transplants include kidney, liver, pancreas, heart, lung, uterus, bladder, thymus, and intestine, portions thereof, and the like.

[0044] As used herein, agglutination refers to the clumping of cells that occurs when antigens are mixed with corresponding antibodies. For example, when a cross-incompatible or incompatible recipient receives a blood transfusion, antibodies in the recipient may react with surface antigens on red blood cells and / or platelets, causing the red blood cells and / or platelets to clump together, adhere together, and aggregate.

[0045] In embodiments, the selective masking of surface antigens on red blood cells by PEG-phospholipid molecules includes selective masking of blood group antigens. Such blood group antigens are preferably selected from the group consisting of A and B antigens. For example, treating a blood product containing red blood cells from a donor with blood type A to selectively mask the A antigen on the red blood cells would result in a blood product that can be administered not only to type A recipients, but also to type B, AB, or O recipients, with no or at least a significantly reduced risk of having any initial agglutination reaction associated with the transfusion. Thus, in one embodiment, the surface antigen is a blood group antigen. In a specific embodiment, the surface antigen is selected from the group consisting of A and B antigens.

[0046] In another embodiment, the surface antigen is Rhesus factor. In a particular embodiment, the surface antigen is selected from the group consisting of RhD, RhC, Rhc, RhE, and Rhe.

[0047] Thus, in one embodiment, the PEG-phospholipid molecules selectively mask blood group antigens, such as A antigen and / or B antigen. In another embodiment, the PEG-phospholipid molecules selectively mask Rhesus factors, such as RhD, RhC, Rhc, RhE, and / or Rhe. In a further embodiment, the PEG-phospholipid molecules selectively mask blood group antigens, such as A antigen and / or B antigen, and selectively mask Rhesus factors, such as RhD, RhC, Rhc, RhE, and / or Rhe.

[0048] In a preferred embodiment, the PEG-phospholipid molecules of the present embodiments selectively mask any blood group antigens and any Rhesus factors present on the cell surface of red blood cells in a blood product.

[0049] Correspondingly, endothelial cells of organ transplants contain surface antigens in the form of blood group antigens. Thus, the PEG-phospholipid molecules of the embodiments can selectively mask any blood group antigens present on the cell surface of endothelial cells in organ transplants. Furthermore, endothelial cells contain surface antigens in the form of human leukocyte antigens (HLA). Thus, in one embodiment, the PEG-phospholipid molecules of the embodiments can selectively mask any HLA present on the cell surface of endothelial cells in organ transplants. In certain embodiments, the HLA is selected from the group consisting of HLA-A, HLA-B, and HLA-C, which are the major antigens of major histocompatibility complex (MHC) class I. This MHC class I also contains a minority of HLA antigens in the form of HLA-E, HLA-F, and HLA-G. While these MHC class I proteins form functional receptors on most nucleated cells in the body, MHC class II proteins, such as HLA-DP, HLA-DG, HLA-DR, HLA-DM, and HLA-DO, occur only on antigen-presenting cells, B cells, and T cells.

[0050] Furthermore, the PEG-phospholipid molecules of the embodiments can selectively mask CL-11. During transplantation, CL-11 is expressed in organ graft transplants, such as kidney grafts, to recognize ischemic cells and contribute to complement activation via the lectin (LP) pathway. Other recognition molecules of LP, such as MBL, are also involved in complement activation associated with organ transplantation. Therefore, selectively masking these cell membrane proteins on the endothelium by shielding the endothelial membrane of organ transplants with a PEG-phospholipid coating will reduce complement activation associated with organ transplantation.

[0051] Platelets contain surface antigens called human platelet antigens (HPAs). In one embodiment, the surface antigen is HPA. In a specific embodiment, the surface antigen is selected from the group consisting of HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, HPA6, HPA-9, and HPA-15. In another specific embodiment, the surface antigen is selected from the group consisting of HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, HPA6, and HPA-15. In a further specific embodiment, the surface antigen is HPA-1.

[0052] By treating blood products containing both red blood cells and platelets, or cells or organ grafts containing platelets in addition to red blood cells and / or endothelial cells, the PEG-phospholipid molecules selectively mask both blood group antigens, Resus factor, and HPA.

[0053] In one embodiment, the PEG-phospholipid molecules, when immobilized in a cell membrane, have an average extracellular length selected within the interval of 4 nm to 8 nm. In a particular embodiment, the PEG-phospholipid molecules, when immobilized in a cell membrane, have an average extracellular length selected within the interval of 5 nm to 7 nm, preferably about 6 nm.

[0054] As used herein, the average extracellular length indicates that individual PEG-phospholipid molecules may have lengths extending from the cell surface when anchored within the cell membrane that are longer or shorter than the average extracellular length, however, the average extracellular length is the average or median of the individual lengths of the PEG-phospholipid molecules.

[0055] The above average extracellular length is long enough to mask blood group antigens, Rhesus factors, HLA and HPA, but is not long enough to effectively mask extracellular or transmembrane molecules and proteins within the cell membrane that have extracellular domains or portions significantly longer or larger than this average extracellular length of the PEG-phospholipid molecule.

[0056] In one embodiment, the PEG-phospholipid molecule has the formula (I): [ka]

[0057] In one embodiment, n and m are independently selected integers in the range of 10 to 16. The parameters n and m are preferably independently 10, 12, 14 or 16, more preferably n=m=14.

[0058] In one embodiment, p is selected so that the PEG chains or domains have an average molecular weight selected within the range of 1000 Da to 40000 Da. The parameter p is preferably selected so that the PEG chains have an average molecular weight of 3000 to 10000 Da, more preferably about 5000 Da, e.g., 4500 to 5500 Da, 4600 to 5400 Da, 4700 to 5300 Da, 4800 to 5200 Da, or 4900 to 5100 Da.

[0059] The average molecular weights defined herein indicate that individual PEG-phospholipid molecules may have average molecular weights that differ from this average molecular weight, but the average molecular weight represents the average molecular weight of the PEG-phospholipid molecules, which further implies that for a PEG-phospholipid sample, there is a natural distribution of molecular weights centered around this average molecular weight.

[0060] The terminal group R, in one embodiment, is selected from the group consisting of H, methyl (CH), and a C-C alkylamine. In one embodiment, the C-C alkylamine is n-propylamine. In a preferred embodiment, the terminal group R is methyl.

[0061] In one embodiment, the PEG-phospholipid molecule is N-(methylpolyoxyethyleneoxycarbonyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine.

[0062] In another embodiment, the terminal group R may comprise a maleimide group, a biotin group, a streptavidin group, an avidin group, or a functionalizing molecule, which in the case of a functionalizing molecule is preferably selected from the group consisting of a complement inhibitor, a coagulation inhibitor, a platelet inhibitor, a molecule capable of binding to a complement inhibitor, a molecule capable of binding to a coagulation inhibitor, a molecule capable of binding to a platelet inhibitor, and mixtures thereof.

[0063] In such cases, the maleimide group, biotin group, streptavidin group, avidin group, or functionalized molecule is preferably attached to the PEG-phospholipid molecule of formula (I) by a linker. An illustrative, but non-limiting example of such a linker is -CH2CH2CH2NHC(O)CH2CH2-. For example, the terminal group R comprising such a linker and a maleimide group is represented by formula (II): [ka]

[0064] The functionalizing molecule can also be attached to the PEG-phospholipid molecule of formula (I) via a linker. Thus, in this embodiment, the terminal group R has the general formula L-(FM), where L represents the linker and (FM) represents the functionalizing molecule.

[0065] Illustrative, but non-limiting examples of complement inhibitors include factor H; C4b-binding protein (C4BP); the N-terminal 4-6 short consensus repeats (SCR) of complement receptor 1 (CR1), also known as C3b / C4b receptor or cluster of differentiation 35 (CD35); CD46 complement regulatory protein (CD46), also known as membrane cofactor protein (MCP); and complement decay-accelerating factor (DAF), also known as CD55.

[0066] Illustrative, but non-limiting examples of coagulation inhibitors include heparin.

[0067] Illustrative, but non-limiting examples of platelet inhibitors are adenosine diphosphate (ADP) degrading enzymes such as apyrase and ectonucleoside triphosphate diphosphohydrolase-1 (NTPDase1, also known as CD39).

[0068] Illustrative, but non-limiting examples of molecules capable of binding complement inhibitors are factor H-binding peptides such as 5C6 (Nilsson et al., Autoregulation of thromboinflammation on biomaterial surfaces by a multicomponent therapeutic coating. Biomaterials. 2013, 34(4):985-994), and C4BP-binding peptides, such as Streptococcus M protein-derived peptides M2-N, M4-N, or M22-N (Engberg et al., Inhibition of complement activation on a model biomaterial surface by streptococcal M protein-derived peptides. Biomaterials. 2009, 30(13):2653-2659).

[0069] An illustrative, but non-limiting example, of a molecule that can be conjugated to a coagulation inhibitor is a heparin-binding peptide (Asif et al., Heparinization of cell surfaces with short peptide-conjugated PEG-phospholipid regulates thromboinflammation in transplantation of human MSCs and hepatocytes. Acta Biomateriala. 2016, 35:194-205).

[0070] Thus, in one embodiment, the functionalizing molecule is selected from the group consisting of heparin-binding peptides, the N-terminal 4-6 SCR of CR1, CD46, DAF, FI factor-binding molecules, ADP-degrading enzymes, and mixtures thereof.

[0071] The fatty acid chains of the PEG-phospholipid molecule may be saturated. Alternatively, at least one or both fatty acid chains may be unsaturated, i.e., contain at least one -CH=CH- group, ... [ka] or a combination thereof. Each fatty acid chain may be straight or branched.

[0072] In one embodiment, the PEG-phospholipid molecules have an average molecular weight selected from the interval 5-7 kDa, as determined by gel permeation chromatography. In a particular embodiment, the PEG-phospholipid molecules have an average molecular weight selected from the interval 5.5-6.6 kDa, as determined by gel permeation chromatography. In a preferred embodiment, the PEG-phospholipid molecules have an average molecular weight selected from the interval 5.8-6.0 kDa, e.g., about 5.9 kDa, as determined by gel permeation chromatography.

[0073] In one embodiment, the lipid moiety or domain of the PEG-phospholipid molecule is a phospholipid moiety or domain.

[0074] In one embodiment, the lipid moiety or domain of the PEG-phospholipid molecule is 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine (DPPE). Other non-limiting examples of lipid moieties or domains of the PEG-phospholipid molecule include 1,2-dimyristoyl-sn-glycero-3-phosphorylethanolamine (DMPE), (also referred to as 1,2-ditetradecanoyl-sn-glycero-3-phosphoethanolamine), [(2R)-3-[2-aminoethoxy(hydroxy)phosphoryl]oxy-2-tetradecanoyloxypropyl]tetradecanoate, and 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE).

[0075] In one embodiment, the PEG moieties or domains of the PEG-phospholipid molecule have an average degree of polymerization (DP) selected within the interval of 105 to 125. In certain embodiments, the PEG moieties or domains of the PEG-phospholipid molecule have an average DP selected within the interval of 110 to 120. In preferred embodiments, the PEG moieties or domains of the PEG-phospholipid molecule have an average DP selected within the interval of 114 to 116, such as about 115.

[0076] In one embodiment, the PEG-phospholipid molecule comprises at least one sulfated glycosaminoglycan. In certain embodiments, the PEG-phospholipid molecule comprises a K group interconnecting at least one sulfated glycosaminoglycan and the PEG-phospholipid. n C and / or CK n In this particular embodiment, C is cysteine, K is lysine, and n is zero or a positive integer less than or equal to 20, preferably n is selected within the interval of 0 to 15, and more preferably n is selected within the interval of 0 to 10.

[0077] In one embodiment, the sulfated glycosaminoglycan is a fragmented heparin. In a specific embodiment, the fragmented heparin has an average molecular weight selected within the interval of 2.5 kDa to 15 kDa, preferably within the interval of 5 kDa to 10 kDa, and more preferably within the interval of 7 kDa to 9 kDa.

[0078] In one embodiment, any free amino groups in the sulfated glycosaminoglycan-PEG-phospholipid are converted to carboxyl groups.

[0079] Such PEG-phospholipid molecules can be produced by combining a cationic-PEG-phospholipid containing at least one amino group with a sulfated glycosaminoglycan containing at least one carbonyl group, preferably at least one aldehyde group, to form a Schiff base intermediate, to which a reducing agent is added to form the sulfated glycosaminoglycan-PEG-phospholipid.

[0080] For example, α-N-hydroxysuccinimidyl-ω-maleimidyl PEG (NHS-PEG-Mal), triethylamine, and 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine (DPPE) are mixed in dichloromethane. The next step involves precipitating the resulting maleimide-conjugated PEG-phospholipid by adding diethyl ether to the dichloromethane containing the NHS-PEG-Mal and DPPE mixture.

[0081] The preferred sulfated glycosaminoglycan, i.e., fragmented heparin, preferably contains at least one carbonyl group, more preferably at least one aldehyde group. Such fragmented heparin can be obtained by mixing an acidic solution with an aqueous sodium nitrite (NaNO) solution to form a mixed solution. The pH of the mixed solution is adjusted to 2 to 6, preferably within the interval of 3 to 5, more preferably 4. Heparin, preferably sodium heparin, is added to the mixed solution to form a heparin solution, and the pH of the heparin solution is adjusted to 6 to 8, preferably within the interval of 6.5 to 7.5, more preferably 7, to form fragmented heparin containing at least one carbonyl group. The fragmented heparin containing at least one carbonyl group is optionally dialyzed against water, and the fragmented heparin containing at least one carbonyl group is lyophilized.

[0082] An illustrative example of a reducing agent added to a Schiff base is sodium cyanoborohydride.

[0083] In one embodiment, the PEG-phospholipid molecules, when immobilized within the cell membrane of red blood cells and / or platelets, or organ grafts, are separate or individual PEG-phospholipid molecules, meaning that the PEG-phospholipid molecules are not interconnected or linked to one another with any interconnecting molecule or linker, such as poly(vinyl alcohol) (PVA) or multi-arm PEG-SH, for the purpose of interconnecting the individual PEG-phospholipid molecules and forming a lamellar surface mask or pseudolayer on the cell surface.

[0084] A further aspect of the present invention relates to a blood product comprising red blood cells and / or platelets and PEG-phospholipid molecules immobilized within the cell membrane of the red blood cells and / or platelets, the PEG chains of which have an average molecular weight selected within the range of 3,000 to 10,000 Da.

[0085] Yet another aspect of the present invention relates to an in vitro method for treating red blood cells and / or platelets, which method comprises selectively masking surface antigens of red blood cells and / or platelets by adding PEG-phospholipid molecules to the red blood cells and / or platelets in vitro, wherein the PEG chains of the PEG-phospholipid molecules have an average molecular weight selected within the range of 3,000 to 10,000 Da.

[0086] In transfusion services, recipients are traditionally screened for irregular antibodies so that an appropriately compatible blood can be selected. These irregular antibodies are pre-existing antibodies that the recipient may have already developed after a previous transfusion and / or a previous birth. Generally, irregular antibodies are all non-ABO antibodies, but the primary application of the term refers to non-ABO isoantibodies that can cause transfusion incompatibilities. Irregular antibodies are most commonly of the IgG type, and they first appear after exposure to foreign antigens. Illustrative, but non-limiting, examples of such irregular antibodies include anti-c, anti-Cw, anti-D, anti-E, anti-Fya, anti-Jka, anti-Kell, anti-Kpb, anti-Lea, anti-Leb (Lewis), anti-Lua, anti-Lub, anti-M, anti-N, anti-P1, anti-Public, anti-S, anti-s, and auto-Pap antibodies. Because patients may have multiple of these irregular antibodies, antibody identification can be difficult. At least a portion of the surface antigens present on the surface of red blood cells can be masked and hidden by the PEG-phospholipid molecules of the embodiments, making identification easier by selectively masking the surface antigens to simplify identification.

[0087] Thus, a further aspect of the present invention relates to a method for screening for irregular antibodies. The method comprises adding PEG-phospholipid molecules to a blood sample from a subject to selectively mask surface antigens on red blood cells present in the blood sample. The method also comprises screening for irregular antibodies present in the blood sample or bound to red blood cells obtained from the blood sample. The PEG chains of the PEG-phospholipid molecules have an average molecular weight selected within the range of 3,000 to 10,000 Da.

[0088] The first step of this method selectively masks A and / or B antigens present on the surface of red blood cells, but does not selectively mask all surface antigens to which irregular antibodies can arise and bind. The treated blood sample can then be subjected to a screening test for irregular antibodies. Antibody screening is often performed by performing a Coombs test, also known as an antiglobulin test or red blood cell antibody screening. There are two types of Coombs tests: direct and indirect. The direct Coombs test involves collecting erythrocytes (red blood cells) from a blood sample taken from a subject, preferably washing the red blood cells. The collected and optionally washed red blood cells are then incubated with an anti-human antibody (Coombs reagent), which binds to the Fc region of any irregular antibodies present in the blood sample and binds to the surface antigens on the red blood cells. If any irregular antibodies are present in the blood sample, the anti-human antibody will bind to the human irregular antibodies on the red blood cells, causing red blood cell agglutination, thereby forming links between the red blood cells. In a direct Coombs test, the PEG-phospholipid molecules of the embodiments are added to the blood sample before the red blood cells are collected and / or preferably added to the red blood cells collected from the blood sample.

[0089] The indirect Coombs test involves mixing a blood sample from a donor with a serum sample from a recipient. Any irregular antibodies present in the recipient-derived serum sample can bind to the surface agent present on red blood cells in the donor-derived blood sample, thereby forming an antibody-antigen complex. If the recipient-derived serum sample contains any irregular antibodies that can bind to the surface antigens on red blood cells in the donor-derived blood sample, an anti-human antibody (Coombs reagent) is added to the mixture to cause red blood cell agglutination, similar to the direct Coombs test. In the direct Coombs test, the PEG-phospholipid molecule of the embodiment may be added to the donor's blood sample, preferably before mixing with the recipient's serum sample.

[0090] The screening method for irregular antibodies is an in vitro screening method.

[0091] Another aspect of the present invention relates to a method for transfusion, which comprises adding PEG-phospholipid molecules to a donor-derived blood product to selectively mask surface antigens on red blood cells and / or platelets present in the blood product. The method also comprises transfusing the blood product into a cross-incompatible or incompatible recipient. The PEG chains of the PEG-phospholipid molecules have an average molecular weight selected within the range of 3,000 to 10,000 Da.

[0092] The PEG-phospholipid molecules of the embodiments can be added to blood products or cells in vitro at a concentration selected to efficiently and selectively mask surface antigens on red blood cells and / or platelets, such as in blood products. Thus, in one embodiment, the final concentration of the PEG-phospholipid molecules in the blood product can depend on the amount of red blood cells and / or platelets in the blood product.

[0093] In typical embodiments, PEG-phospholipid molecules can be added to cells, blood products, or cell grafts to achieve a final concentration of PEG-phospholipid molecules in the cells, blood products, or cell grafts at a selected concentration within the interval of 0.25 mg / ml to 25 mg / ml, preferably 0.25 mg / ml to 10 mg / ml. In certain embodiments, the final concentration of PEG-phospholipid molecules in the cells, blood products, or cell grafts can be 0.25 mg / ml to 5 mg / ml, preferably 0.5 mg / ml to 4 mg / ml, or 1 to 3 mg / ml. In preferred embodiments, the final concentration of PEG-phospholipid molecules in the cells, blood products, or cell grafts is 1.5 mg / ml to 2.5 mg / ml, e.g., about 2 mg / ml.

[0094] The PEG-phospholipid molecules can be added in the form of a solution of the PEG-phospholipid molecules in a solvent, preferably an aqueous solvent, illustrative but non-limiting examples of which include saline, buffer solutions, and organ preservation solutions.

[0095] The above embodiments of surface antigens and PEG-phospholipids also apply to these aspects of the embodiment.

[0096] In transplantation, particularly in living donor kidney transplantation, ABO incompatibility hinders effective engraftment. To perform transplantation under these conditions, anti-ABO antibodies are conventionally removed over several weeks using plasmapheresis and anti-CD20 antibodies. This pretreatment over several weeks is very time-consuming and cumbersome. Therefore, there is a need for treatment of transplants and grafts, for example, to mask ABO antigens, thereby facilitating engraftment.

[0097] Organ transplants can be treated with the PEG-phospholipid molecules of the embodiments by administering the PEG-phospholipid molecules to the vasculature of the organ transplant and / or by immersing the organ transplant in a solution comprising the PEG-phospholipid molecules, preferably an organ preservation solution.

[0098] For example, a solution containing PEG-phospholipid molecules is injected into the vasculature and, optionally, into the parenchyma of an organ graft ex vivo. The solution containing PEG-phospholipid molecules is preferably incubated in the vasculature and, optionally, into the parenchyma ex vivo, thereby allowing at least a portion of the endothelium of the vasculature, preferably the parenchyma, to be coated with the PEG-phospholipid molecules.

[0099] In one embodiment, the ex vivo incubating step comprises incubating the solution comprising the PEG-phospholipid molecules ex vivo within the vasculature, and optionally the parenchyma, thereby allowing at least a portion of the endothelial lining of the vasculature, preferably the parenchyma, to be coated with the PEG-phospholipid molecules while the organ graft remains immersed in the organ preservation solution, preferably the organ preservation solution comprising the PEG-phospholipid molecules.

[0100] Thus, ex vivo treatment of organ transplants involves introducing PEG-phospholipid molecules into the vasculature of the organ graft, where they are able to interact with and bind to endothelial and parenchymal cell membranes.

[0101] The interaction of the PEG-phospholipid molecules with the lipid bilayer membrane of the endothelium, and optionally with the parenchyma, such as the renal parenchyma in the case of the kidney, preferably occurs ex vivo, while the organ graft is immersed or submerged in an organ preservation solution, preferably an organ preservation solution comprising the PEG-phospholipid molecules.

[0102] In certain embodiments, the organ transplant is first ex vivo injected into the vasculature and, optionally, into the parenchyma of the organ transplant with a solution containing PEG-phospholipid molecules. This ex vivo injection is advantageously performed as soon as possible after explantation and removal of the organ from the donor's body. The perfused organ is then preferably immersed in an organ preservation solution containing PEG-phospholipid molecules and maintained therein at a low temperature, preferably about 4°C.

[0103] In another specific embodiment, the organ is first immersed in an organ preservation solution, preferably an organ preservation solution containing PEG-phospholipid molecules, and then the solution containing PEG-phospholipid molecules is injected ex vivo into the organ's vasculature, optionally intraparenchyma. This ex vivo injection can be performed while the organ is preferably kept immersed in the organ preservation solution containing PEG-phospholipid molecules. Alternatively, the organ is temporarily removed from the organ preservation solution for ex vivo injection, and then preferably returned to the organ preservation solution containing PEG-phospholipid molecules.

[0104] In one embodiment, the method also includes injecting an organ preservation solution into the vascular system ex vivo to flush unbound PEG-phospholipid molecules from the vascular system. Thus, unbound PEG-phospholipid molecules are preferably washed away using the organ preservation solution in one or multiple washing steps, i.e., at least two washing steps.

[0105] In one embodiment, injecting the solution comprising the PEG-phospholipid molecules ex vivo comprises clamping one of an artery and a vein of the vascular system ex vivo, This embodiment also comprises injecting the solution comprising the PEG-phospholipid molecules ex vivo into the other of the artery and vein, and clamping the other of the artery and vein ex vivo.

[0106] In another embodiment, a solution containing PEG-phospholipid molecules is infused into an artery (or vein) of the organ transplant's vasculature until the solution appears in the organ transplant's vein (or artery), confirming that the vasculature has been filled with the solution containing PEG-phospholipid molecules. At that point, the artery and vein are clamped.

[0107] The solution containing PEG-phospholipid molecules can be added either through a vein or through an artery.In certain embodiments, the solution is injected into an artery.In such certain embodiments, then, any initial clamping is preferably performed on the vein of the vascular system.

[0108] The solution containing the PEG-phospholipid molecules is preferably incubated ex vivo in the vasculature for a period of from 10 minutes to 48 hours, allowing the PEG-phospholipid molecules to hydrophobically interact with the endothelial cell membrane, thereby coating at least a portion of the vasculature of the organ graft. Ex vivo incubation is preferably carried out for from 20 minutes to 36 hours, more preferably from 30 minutes to 24 hours, e.g., from 30 minutes to 12 hours, up to 8 hours, up to 4 hours, or up to 1 hour.

[0109] The amount of solution containing PEG-phospholipid molecules injected into the vasculature depends on the type of organ and the size of the organ (adult vs. pediatric). Generally, the volume of solution should be sufficient to fill the vasculature of the organ. In most practical applications, 5 ml to 500 ml of solution containing PEG-phospholipid molecules is injected into the ex vivo vasculature. In a preferred embodiment, 5 ml to 300 mL, preferably 10 mL to 250 mL of solution containing PEG-phospholipid molecules is injected into the ex vivo vasculature.

[0110] In one embodiment, the solution contains 0.25 mg / ml to 25 mg / ml, preferably 0.25 mg / ml to 10 mg / ml, of PEG-phospholipid molecules. In certain embodiments, the solution contains 0.25 mg / ml to 5 mg / ml, preferably 0.5 mg / ml to 4 mg / ml, or 1 mg / ml to 3 mg / ml, of PEG-phospholipid molecules. In preferred embodiments, the solution contains 1.5 mg / ml to 2.5 mg / ml, e.g., about 2 mg / ml, of PEG-phospholipid molecules.

[0111] The above concentrations of PEG-phospholipid molecules can also be used for organ preservation solutions containing PEG-phospholipid molecules.

[0112] The PEG-phospholipid molecules are preferably administered in the form of a PEG-phospholipid solution. The solution containing the PEG-phospholipid molecules can be, for example, saline, an aqueous buffer solution, or an organ preservation solution. Illustrative, but non-limiting, examples of aqueous buffer solutions that can be used include phosphate-buffered saline (PBS) and citrate solution.

[0113] Illustrative, but non-limiting, examples of organ preservation solutions that can be used in accordance with embodiments include histidine-tryptophan-ketoglutarate (HTK) solution, citrate solution, University of Wisconsin (UW) solution, Collins solution, Celsiol solution, Kyoto University solution, and Institute Georges Lopez-1 (IGL-1) solution. In certain embodiments, the organ preservation solution is HTK solution.

[0114] The above embodiments of surface antigens and PEG-phospholipids also apply to this aspect of the embodiment. [Example]

[0115] Example 1 In this example, the protective effect of specific PEG-phospholipids was investigated by shielding different cell types with PEG-phospholipid coatings and assessing the blocking effect this has on antibody binding to surface antigens.

[0116] Materials and Methods Manufacturing Procedure PEG-containing polyethylene glycol-phospholipid (PEG-phospholipid) 5 kDa or free PEG 5 kDa was used in this experiment (NOF Corporation, Japan). Unconjugated PEG-phospholipid (SUNBRIGHT® PP-050CN, N-(methylpolyoxyethyleneoxycarbonyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, sodium salt, NOF Corporation, Japan) was used as is. Free PEG (5 kDa) was prepared from α-N-hydroxysuccinimidyl-ω-maleimidyl poly(ethylene glycol) (NHS-PEG-Mal, Mw: 5,000, NOF), which was reacted with glycine and cysteine ​​to deactivate the NHS and maleimide groups, followed by spin column purification.

[0117] cell type Human red blood cells were isolated from healthy human donors. They were preselected for specific blood types, and only individuals exposing the blood type B antigen were used. Human blood was collected from donors using vacuum blood collection tubes (5 mL, EDTA-2Na treated, TERUMO Co., Tokyo, Japan). After removing the plasma and buffy coat by centrifugation (15 min, 2500 × g three times), the red blood cells were resuspended in cold PBS.

[0118] Human acute lymphoblastic leukemia cell line (CCRF-CEM) was purchased from the Health Science Research Resources Bank (Health Science Research Resources Bank, Tokyo, Japan). CCRF-CEM cells were cultured in RPMI-1640 medium (containing 10% heat-inactivated fetal bovine serum, 50 IU / mL penicillin, and 50 μg / mL streptomycin) at 37°C and 5% CO.

[0119] Coating effect of PEG-phospholipid on antibody reaction using human red blood cells This example was approved by the Ethics Committee of the University of Tokyo (KE17-13). Human red blood cells (RBCs) were isolated from whole blood of type B donors and suspended in phosphate-buffered saline (PBS). RBC cell pellets (5x10 7 The RBCs were incubated with PEG-phospholipid (20 μl, 2 mg / ml in PBS) or PBS (50 μl, as a control) for 30 minutes at room temperature (RT, 20–25°C) with gentle mixing. After washing twice with PBS by centrifugation (120 × g, 4°C, 1 minute), Alexa488-labeled anti-blood group B antigen antibody (10 μl, used undiluted; blood group B antigen antibody [HEB-29] GeneTex) or PBS (10 μl) was mixed with the treated RBCs for 10 minutes at room temperature. After washing with PBS by centrifugation (120 × g, 4°C, 1 minute), the RBCs were resuspended in PBS (1 ml) for flow cytometry analysis. All samples were analyzed on an Accuri C6 flow cytometer (BD). Additional controls in the form of isotype controls and unstained controls were run in parallel. Post-analysis processing was performed using FlowJo (BD).

[0120] PEG-phospholipid coating effect from antibody reaction using CCRF-CEM Add CCRF-CEM to cell culture medium (1x10 6 The CCRF-CEM cell pellet (1 x 10 cells) was collected from the spleen and washed with PBS by centrifugation (250 x g, 3 min, RT). 6The cells were incubated with PEG-phospholipid (50 μl, 2 mg / ml in PBS), 5 kDa unconjugated PEG (50 μl, 2 mg / ml in PBS), or PBS (50 μl, as a control) for 30 min at room temperature with gentle mixing. After washing twice with PBS by centrifugation (200 × g, 4 °C, 3 min), three FITC-labeled antibodies, FITC-anti-human CD52 (400 μg / ml, 40 μl) (BioLegend), FITC-anti-human CD4 (200 μg / ml, 40 μl) (BioLegend), or Alexa488-labeled anti-human CD8 (200 μg / ml, 40 μl, Alexa488 labeled with a labeling kit) (GeneTex), or PBS (40 μl) were mixed with the treated CCRF-CEM for 15 min at room temperature. As a control, CCRF-CEMs were treated with 2 mg / ml PEG (5 kDa, in PBS) and further incubated with FITC- or Alexa488-labeled antibodies. After washing with PBS by centrifugation (200 × g, 4 °C, 3 min), CCRF-CEMs were resuspended in PBS (1 ml) for flow cytometry analysis. Samples were analyzed using a flow cytometer (BD LSR II, BD Biosciences). Post-analysis processing was performed using FlowJo (BD).

[0121] Inhibition of complement-mediated lysis by PEG-phospholipid coating To investigate whether PEG-phospholipids could protect against complement-mediated lysis, we used RBCs from 13 different donors typed for Rh complex and ABO antigens. RBCs were isolated and preincubated with various concentrations of PEG-phospholipids, free PEG, or PBS as described above. The cells (and the supernatants saved for analysis) were then washed with PBS (800 g × 5 min, RT) and incubated with human serum (in which the complement system is active) for 20 min at 37°C. Afterwards, they were again centrifuged (800 g × 5 min), and the supernatants were collected for hemolysis analysis. RBCs were analyzed for complement fixation (cells were incubated with FITC-labeled anti-C3c antibody for 10 min). For flow cytometry measurements, RBCs were resuspended in PBS (1 mL). All samples were analyzed on an Accuri C6 flow cytometer, with additional controls in the form of isotype controls and unstained controls run in parallel. Post-analysis processing was performed using FlowJo (BD). The supernatant was analyzed for absorbance at 540 nm using a Synergy HTX variable wavelength plate reader. Osmotically lysed RBCs were run in parallel as a positive control. The background absorbance at 540 nm from PBS without cells was subtracted, and the difference in absorbance between cells coated with PEG-phospholipid or PBS was then calculated.

[0122] result The cell surface was modified with FITC-labeled PEG-phospholipid and CCRF-CEM cells (5x10 6) were investigated. Cells were incubated with different concentrations of FITC-labeled PEG-phospholipid (0.1, 0.5, 1.0, and 2.0 mg / ml in PBS) or PBS without PEG-phospholipid (as a control). Cells were incubated at room temperature for 10, 20, 30, or 60 minutes with gentle mixing. After removing excess FITC-labeled PEG-phospholipid, the remaining PEG-phospholipid on the cell surface was quantified by flow cytometry. Binding of FITC-labeled PEG-phospholipid to the cell surface was dependent on the incubation time and concentration (see Figure 2). Binding of FITC-labeled PEG-phospholipid reached a plateau after 30 minutes of incubation. Furthermore, no difference in FITC-labeled PEG-phospholipid binding was observed at concentrations above 0.5 mg / ml. In conclusion, these results demonstrate that FITC-labeled PEG-phospholipid rapidly coats the cell membrane and that the reaction is saturable.

[0123] CCRF-CEM cells were incubated with PEG-phospholipid (2 mg / ml in PBS), free PEG (5 kDa, equivalent molecular weight used in the synthesis of PEG-phospholipid), or PBS (vehicle control) for 30 min at room temperature. After removing excess PEG-phospholipid, FITC-labeled antibodies directed against surface antigens of different sizes, CD52, CD4, and CD8, were added. Samples were analyzed by flow cytometry. Antibodies directed against CD52, a small surface antigen (approximately 1 nm), were completely blocked by PEG-phospholipid (Figure 3A). The binding ability of CD8 antibodies was reduced but not completely blocked by PEG-phospholipid (Figure 3A). The size of CD8 is estimated to be approximately 5-10 nm based on the crystal structure of the nonmembranous portion. Finally, CD4 antibodies were not blocked by PEG-phospholipid (Figure 3A), as expected, given that the size of the antigen (15 nm) is larger than the size of the PEG-phospholipid (estimated to be approximately 6 nm). Free PEG did not significantly affect the binding capacity of the antibody to any of the surface antigens.

[0124] Blood group antigens are important surface antigens in the field of transplantation. Flow cytometry was used to assess the binding capacity of fluorescently labeled anti-blood group B antigen antibodies after coating with PEG-phospholipid. Red blood cells (RBCs) were isolated from healthy human donors with blood group B, and the RBCs were incubated with either PEG-phospholipid (2 mg / ml in PBS) or PBS (control) for 30 minutes at room temperature. Binding capacity was effectively reduced by the PEG-phospholipid coating on the RBCs and assessed by incubating the coated RBCs with plasma containing complement factors and anti-blood group antigen B antibodies at 37°C for 20 minutes (see Figure 3B). Complement fixation (using anti-C3c antibody) and lysis were examined using flow cytometry. PEG-phospholipid dose-dependently reduced complement-mediated RBC lysis in ABO-mismatched donors compared with untreated controls (Figure 3C).

[0125] PEG is a linear polymeric substance, and its molecular weight correlates with the length of the polymer, allowing it to protrude from the cell membrane. The PEG in PEG-phospholipids has a molecular weight of 5 kDa, resulting in a size of approximately 6 nm, which, when immobilized within the membrane, results in a 6 nm PEG layer. The sizes of blood group B antigens, CD52, CD8, and CD4, from the cell surface are approximately 1 nm, 1 nm, 5-10 nm, and 15 nm, respectively. Therefore, this conforms to size-dependent inhibition. A more pronounced inhibition of antibody binding was observed for CD8 than for CD4. Despite the similar size of CD8 and CD4, the stalk region of CD8 causes this molecule to protrude less from the membrane. See Figure 4.

[0126] Example 2 In this example, the effect of PEG-phospholipids in masking Rhesus factors D, C, c, E and e on red blood cells was investigated.

[0127] Materials and Methods Whole blood samples (one O, Rh+ blood sample and two ARh+ blood samples) from the blood center were centrifuged in Eppendorf tubes in an Eppendorf 5810R centrifuge at 2500 rpm for 10 minutes at room temperature (20-25°C), and the plasma was removed and discarded. The cell fraction was subjected to three washing steps in 50 ml Falcon tubes. Each washing step involved adding 45 ml of phosphate-buffered saline (PBS) to 1 ml of the cell fraction, followed by centrifugation at 2500 rpm for 10 minutes at room temperature.

[0128] 100 μl of cell suspension was mixed with 900 μl of PBS and 1000 μl of PEG-phospholipid (PBS containing 4 mg / ml polyethylene glycol-phospholipid and PEG 5 kDa (PEG-phospholipid), SUNBRIGHT® PP-050CN, N-(methylpolyoxyethyleneoxycarbonyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, sodium salt, NOFCorp., Japan), and erythrocytes were coated with 2 mg / ml PEG-phospholipid (final concentration) or 1000 μl of PBS (control). The erythrocyte samples were incubated at 37°C for 30 minutes in a POL-EKO laboratory incubator under rotation. The erythrocyte samples were washed three times with 2 ml of PBS and centrifuged at 2500 rpm at room temperature for 5 minutes in a Heraeus Biofuge Pico centrifuge.

[0129] The washed red blood cell samples were then subjected to agglutination testing and Rh phenotyping. More specifically, EDTA blood was drawn, and RBCs were collected and washed three times in PBS (45 mL of PBS containing 1 mL of blood). A 100 μL aliquot of RBCs was then incubated with 1.9 mL of PEG-phospholipid in PBS or with PBS alone (control) for 30 minutes at 37°C in a rotating incubator. The RBCs were washed three times with PBS (2 mL) and suspended at 50% hematocrit. The treated and control RBCs were then subjected to blood typing using Diaclon ABO / Rh ID cards (Biorad) according to the manufacturer's instructions.

[0130] result The results of the agglutination tests are presented in Figures 5A-7B, with Figures 5A and 5B for the O Rh+ blood sample and Figures 6A-6B and 7A-7B for the two A Rh+ blood samples. As can be clearly seen in Figures 5B, 6B, and 7B, agglutination occurred in the untreated control. However, treatment of red blood cells with PEG-phospholipid masked the Rhesus factor antigen on the cell surface, thereby inhibiting agglutination (Figures 5A, 6A, and 7A).

[0131] Thus, PEG-phospholipids could mask the Rhesus factor antigen on the cell surface of red blood cells, thereby preventing red blood cell agglutination.

[0132] Experimental Example 3 In this example, the effect of PEG-phospholipids on masking cell membrane proteins on human umbilical vein endothelial (HUVEC) cells was investigated.

[0133] Materials and Methods Preparation of HUVECs HUVEC cells were cultured in cell culture flasks (Thermofisher Scientific) and Petri dishes (NUNC) in endothelial cell culture medium containing supplements (Promocell). For flow cytometry analysis, cells were detached using trypsin-EDTA buffer (BD Accuri C6 serial number 5173). Cells were rinsed once with PBS (10 mL) by centrifugation (120 × g, 4 °C, 3 min), and fresh medium was added to the detached cells.

[0134] Masking antigens on HUVECs by coating with PEG-phospholipids 100 μL of HUVEC cell pellet (5 × 10 4The cells were incubated with 0.5 mg / mL PEG-phospholipid solution (final concentration: 5 kDa PEG in polyethylene glycol-phospholipid (PEG-phospholipid), SUNBRIGHT® PP-050CN, N-(methylpolyoxyethyleneoxycarbonyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, sodium salt, NOF Corporation, Japan) or PBS for 45 min at room temperature. Next, the cells were incubated with 5 μg / mL FITC-labeled antibodies: CD31 antibody (5 μg / mL) or isotype control Ab-FITC (BD Biosciences), or rabbit anti-von Willebrand factor (vWF) rabbit IgG as a control, for 15 min at room temperature. Both were visualized with Alexa488 goat anti-rabbit antibody (Table 1).

[0135] Additionally, HUVEC cells were incubated with PEG-phospholipid (0.5 mg / mL), 10 mM free PEG (5 kDa), or PBS (control) for 45 min at room temperature, then washed with PBS and incubated with EDTA-plasma (control) in human serum for 30 min at 37°C. Both were diluted 1:50 in PBS. After washing, the lectin pathway recognition molecules collectin-11 (CL-11) and mannan-binding lectin (MBL) were detected using FITC-conjugated antibodies and isotype control antibodies listed in Table 1. For both analytes, values ​​represent the level of binding subtracted from that in EDTA-plasma. Additionally, FITC-conjugated concavalin A (a broad-specificity lectin) was incubated at a concentration of 50 μg / mL with coated HUVECs and native HUVECs (not pre-exposed to plasma).

[0136] After incubation with the detection antibody, cells were washed with PBS. All samples were analyzed on an AccuriC6 flow cytometer, and unstained cells were used to gate on the endothelial cell population. 10,000 events were recorded and run in parallel against additional controls in the form of isotype and unstained controls. Post-analysis processing was performed in FlowJo. [Table 1]

[0137] result PEG-phospholipid-treated HUVEC cells were incubated with human plasma, and then binding of an FITC-labeled antibody directed against CL-11 or binding by the lectin concanavalin A was assessed without exposure to plasma. In both cases, binding was significantly reduced by PEG-lipid (Figure 8).

[0138] PEG-phospholipid coating induced modest masking of CD31 (<20%) and resulted in a significant reduction (25-30%) of MBL binding from human serum without diminishing vWF detection (see Figure 9).

[0139] Although the mechanism by which ischemia-reperfusion injury (IRI) is induced has not yet been established, it has been suggested that the binding of MBL, CL-11, MASP-2, and natural IgM antibodies binds to ligands on ischemic cells, thereby causing IRI. We hypothesize that after reperfusion, the PEG-phospholipid coating shields the endothelial and tubular cell membranes by preventing cell membrane proteins from reaching and binding to plasma proteins in the blood in kidney graft grafts. Our data support this concept in that PEG-phospholipid effectively shields small surface antigens such as CD52 and Rh antigens. See Examples 1 and 2, where they extend approximately 1 nm from the surface. Furthermore, CD8, which is in the 5-10 nm range based on the crystal structure of the non-membrane portion, is partially blocked by the PEG-phospholipid coating, but the PEG-phospholipid layer does not extend far enough to encompass longer antigens such as CD4, which has an estimated molecular extension of 15 nm from the membrane. See Example 1. Antibodies to CD31 and vWF (extending 15 nm and 30 nm, respectively, from the surface) were also unaffected. These results are consistent with the PEG-phospholipids having 5 kDa PEG chains that theoretically extend 6 nm from the cell membrane (Figure 4).

[0140] Example 4 In this example, the pharmacokinetics of PEG-phospholipids in the porcine kidney in vivo was investigated.

[0141] Materials and Methods Preparation of PEG-phospholipid derivatives PEG-phospholipid was purchased from NOF Corporation, Tokyo, Japan (SUNBRIGHT® PP-050CN, N-(methylpolyoxyethyleneoxycarbonyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, sodium salt). Fluorescein labeling was performed by conjugating 5FAM-GC-OH (5-carboxyfluorescein) to Mal-PEG-phospholipid. Mal-PEG(5 kDa)-DPPE was first synthesized by combining α-N-hydroxysuccinimidyl-ω-maleimidyl poly(ethylene glycol) (180 mg, NHS-PEG-Mal, Mw: 5 kDa, NOF), triethylamine (50 μL), and DPPE (26 mg) with dichloromethane and stirring at room temperature (RT) for 36 hours. Precipitation with diethyl ether afforded Mal-PEG-phospholipid as a white powder (190 mg, 95% yield). 5FAM-GC-OH (FAM-GC) was conjugated to Mal-PEG-phospholipid. Briefly, 107 μL of FAM-GC (10 mg / mL) in DMSO was mixed with 1 mL of Mal-PEG-phospholipid (10 mg / mL in PBS) at room temperature for 24 hours. Fluorescein-GC-conjugated PEG-phospholipid (FAM-PEG-phospholipid) was purified using a spin column (Sephadex G-25, GE Healthcare, Buckinghamshire, UK) before application to cell modification. The resulting label was approximately 0.7 mol FAM / mol PEG-phospholipid.

[0142] Biotin-PEG-DPPE (biotin-PEG-phospholipid) was synthesized by combining poly(ethylene glycol)(N-hydroxysuccinimide 5-pentanoic acid) ether 2-(biotinamino)ethane (biotin-PEG-NHS, Mw: 5000 Da (NOF) 180 mg) and DPPE (20 mg) with triethylamine (50 μL) and dichloromethane (4 mL) and stirred at room temperature for 48 h. Precipitation with diethyl ether afforded biotin-PEG-lipid as a white powder (165 mg; 80% yield). All preparations of PEG-phospholipid were used at a final concentration of 2 mg / mL.

[0143] Culturing CCRF-CEM cells The CCRF-CEM cell line was applied to this experimental setup due to its suspension properties, thus avoiding the use of trypsin during the splitting process, which is beneficial because trypsin can affect the PEG-phospholipid coating on the cell surface. The CCRF-CEM cell line was purchased from the Japan Health Science Research Resources Bank (Tokyo, Japan). CCRF-CEM cells were cultured in RPMI-1640 medium (containing 10% heat-inactivated fetal bovine serum, 50 IU / mL penicillin, and 50 μg / mL streptomycin) at 37°C and 5% CO2.

[0144] Retention time, cell growth, and viability after PEG-phospholipid modification CCRF-CEM cells (3 x 10 6 The cells were harvested and rinsed once with PBS (3 mL) by centrifugation (120 × g, 4 °C, 3 min). The cell pellet was suspended in FAM-PEG-phospholipid (50 μl, 2 mg / mL in PBS) or PBS (50 μL, control) at room temperature for 30 min and gently stirred. The cells were rinsed once with PBS (10 mL) by centrifugation (120 × g, 4 °C, 3 min), and the cell pellet was suspended in RPMI 1640 (1 mL). The coated cells were seeded into 6-well plates (5 × 10 cells in 5 mL of RPMI 1640). 5Viability and cell number (cells / mL) were calculated using a cell counter on days 0, 1, 2, 4, and 7 of incubation (dead cells were stained with trypan blue). Binding of cell surface-bound FAM-PEG-phospholipid was assessed by confocal laser scanning microscopy and flow cytometry at the same time points.

[0145] In vivo vascular and tubular distribution and segregation of PEG-phospholipids Complete information about the donor and recipient pigs is provided in Example 5. Donor kidneys were treated with 10–15 mL of a 2 mg / mL biotin-PEG-phospholipid solution. Four recipient pigs were transplanted with the biotin-PEG-phospholipid-treated kidneys. Their native kidneys were not removed but remained within the animals throughout the study. The four recipient pigs were euthanized after 12, 24, 48, and 72 hours, respectively. Biopsies were taken from one of the biotin-PEG-phospholipid-treated and native kidneys at the time of euthanasia. Frozen sections (4 μm thick) were incubated in Alexa488-streptavidin (GE Healthcare, 1:500) at room temperature for 10 minutes. The sections were analyzed using a laser scanning confocal microscope (LSM510, META, Carlzeiss, Germany). Slides were visualized using a Zaiss Axio Imacher A1 microscope.

[0146] result Dose-response binding of PEG-phospholipids to cell surfaces To optimize the binding of PEG-phospholipid to cells, a nonadherent cell line (CCRF-CEM) was used. CCRF-CEM cells were incubated with FAM-PEG-phospholipid (up to 2.0 mg / mL), and the amount of bound PEG-phospholipid was determined by flow cytometry. Binding of PEG-phospholipid to the cell surface depended on both the incubation time and the concentration (Figure 2). Binding of PEG-phospholipid at all concentrations reached a plateau after 30 minutes, and no clear increase in FAM-PEG-phospholipid binding was observed at concentrations above 0.5 mg / mL, confirming a rapid and saturable coating of the cell membrane.

[0147] In vitro and in vivo dissociation and half-life of PEG-phospholipids The release of FAM-PEG-phospholipid from CCRF-CEM cells in vitro was examined over time by flow cytometry and confocal microscopy (Figure 10). The fluorescent signal was minimal after 2 days and was not detectable after 4 days of culture. The PEG-phospholipid conjugate was restricted to the cell membrane and was not internalized by the cells.

[0148] The kinetics of PEG-phospholipids attached to the renal parenchyma in vivo were examined in a porcine survival model in which ex vivo biotin-PEG-phospholipid-treated grafts were implanted in pigs (see Example 5). Animals were subsequently euthanized. The half-life of PEG-phospholipids attached to the kidney was calculated from fluorescence analysis by confocal microscopy using Alexa488-streptavidin. A one-stage exponential decay model was applied to fit the data points, and the half-life of PEG-phospholipids bound to the renal parenchyma was calculated to be approximately 14 hours (SD: 8–26) (Figure 11). Autologous recipient kidneys were also preserved and analyzed to estimate systemic leakage of PEG-phospholipids, which was shown to be very low and reliably detected only after 1 hour. In contrast, FAM-PEG-phospholipids were consistently found in the urine of an acute non-survival model (ANSM, see Example 5) (Figure 12).

[0149] When the fluorescence of cell-bound PEG-phospholipids was monitored in vitro, it was concluded that the intensity decreased over the observation period, indicating that the PEG-phospholipids had detached from the endothelium. In vitro, the PEG-phospholipids completely detached after 48 hours. In vivo ANSM demonstrated that the fluorescence appeared to migrate from the glomerulus to the tubular system within 6 hours after implantation. These data were supported by the fact that in survival studies, FAM-PEG-phospholipid fluorescence was detectable for 12 to 24 hours after reperfusion and had completely disappeared by 48 hours. At 14 hours, the fluorescence was completely detached from the glomerulus to the tubular system. 1 / 2 was calculated, confirming that the PEG-phospholipid constructs were excreted via the kidney and fluorescence was found in the urine during the observation period of the non-viability study.

[0150] Example 5 In this example, PEG-phospholipid was tested for up to 4 days without immunosuppression in three different porcine transplant models: an acute non-survival model (ANSM), an allogeneic survival model (SM), and a clinical-like allogeneic survival model (CLSM).

[0151] Materials and Methods Preparation of PEG-phospholipid derivatives PEG-phospholipids were prepared as described in Example 4.

[0152] Routine Clinical Chemistry Markers Before surgery for both long-term survival studies (SM and CLSM), samples were collected in EDTA tubes from both donors and recipients for analysis of total and differential white blood cell counts and hematology (EPK, Hb, EVF, MCV, MCHC, reticulocytes, platelets). Plasma samples were analyzed for aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), γ-glutamyltransferase (GT), glutamate dehydrogenase (GLDH), and creatinine at the Section of Clinical Chemistry, SLU, Uppsala.

[0153] Systemic blood was collected during and after surgery for up to 4 days and analyzed for creatinine, sodium, potassium, and phosphate (Section for Clinical Chemistry, Upsala University Hospital).

[0154] Complement and coagulation activation markers in EDTA plasma The levels of complement activation products C3a and soluble C5b-9 (sC5b-9) in pig plasma were assessed using commercially available kits (ABIN2543272 and ABIN6202276, Antibodies-online.com) according to the manufacturer's instructions. Human C3a was measured using anti-human C3a mAb 4SD17.1 for capture and biotinylated polyclonal rabbit anti-C3a antibody for detection. Human sC5b-9 was measured using mAb anti-human neoC9 aEII (BioportoDiagnostics A / S, Hellerup, Denmark) for capture and biotinylated polyclonal sheep anti-Hu-C5 antibody (BP373, OriGene, Herford, Germany) for detection.

[0155] TAT was measured in both human and porcine plasma using an anti-human thrombin monoclonal antibody for capture and an HRP-conjugated anti-human antithrombin (AT) antibody for detection (Enzyme Research Laboratories, South Bend, IN, USA). FXIIa-C1INH complexes were analyzed by sandwich ELISA essentially as described in 6, using a goat anti-human FXII polyclonal antibody for capture (Enzyme Laboratories) and a goat anti-C1INH antibody (Enzyme Research Laboratories) for detection. Note that the assays used here for human and porcine TAT and FXIIa-C1INH complexes are fully cross-reactive, whereas the assays for C3a and sC5b-9 are not cross-reactive.

[0156] Cytokines and Chemokines Multiplex protein analysis was performed using pig-specific kits for 12 cytokines / chemokines: interferon-gamma (INFγ), interleukins IL-1b, IL-2, IL-1a, IL-1RA, IL-4, IL-6, IL-10, IL-18, IL-8, IL-12, and TNF using Multiplex Luminex xMAP Technology (Millipore Corporation, Billerica, MA, USA). Plasma was incubated with agent-specific colored magnetic beads followed by incubation with detection antibodies and streptavidin-phycoerythrin (Millipore Corporation). Plates were measured using a MAGPIX instrument (Luminex Corporation, Austin, TX, USA). Raw data [median fluorescence intensity (MFI)] were converted to protein concentration (ng / mL) using a standard curve.

[0157] Furthermore, TNF, IL-1b, IL-6, and tissue factor (TF) were quantified in plasma by immunoassay using a GyroLab workstation (Gyrus, Uppsala, Sweden), and the presence of these compounds and IFNγ in biopsies (below) was verified by PCR.

[0158] Kidney biopsy Detection of PEG-phospholipids by histofluorescence Frozen sections (4 μm thick) from biotin-PEG-phospholipid-treated and control kidneys were incubated in Alexa488-streptavidin (GE Healthcare, 1:500) at room temperature for 10 minutes and analyzed by laser scanning confocal microscopy (LSM510, META, Carlzeiss, Germany).

[0159] immunohistochemistry Paraffin sections of porcine kidney tissue were cut (3-5 μm), deparaffinized in xylene, and rehydrated in a graded series of ethanol and distilled water. For IHC staining, heat-induced antigen retrieval was performed by boiling sections in sodium citrate buffer (pH 6). The sections were blocked with 10% normal goat serum (Jackson Immunoresearch) followed by primary antibody incubation: anti-MAC (Abcam ab66768); anti-C3b alpha chain (Bioss bs-4873R); anti-C4d (Abcam ab64157); anti-C5aR (Acris AP06509PU-N); anti-HO1 (Abcam ab52947); anti-CSE (Abnova H00001491-M01); anti-nitrotyrosine (Merck Millipore AB5411); and anti-NOS2 (Santa Cruz sc-651). Detection was performed using the DAKO Real Detection System alkaline phosphatase red (Dako). Sections were counterstained with Mayer's hematoxylin (Sigma #51275). Control staining was performed with nonspecific rabbit IgG (Dako #X0936). Slides were visualized using a Zeiss Axio Imager A1 microscope equipped with a 10x objective (100x magnification). Staining intensity was assessed using AxioVision 4.8 software (Zeiss) at multiple randomly selected 800,000 μm 2 Quantification was performed on sections and data are presented as mean densitometric sum red blood cells.

[0160] Quantitative RT-PCR of cytokines in tissue samples Snap-frozen kidney wedge biopsies were minced using a QIA shredder (Qiagen, Uppsala, Sweden) and stored at -70°C. Total RNA extraction and cDNA synthesis were performed using the RNAeasy Mini kit (Qiagen, Holden, Germany) and the Superscript II reverse transcriptase kit (Invitrogen, Stockholm, Sweden), respectively. All cDNA sequences for porcine genes were obtained from Genbank. All primers were provided by Invitrogen. Gene expression was determined by RT-PCR. The specificity of PCR products was assessed by electrophoresis on a 2% agarose gel. mRNA expression was normalized to beta-actin, and quantification values ​​were obtained from threshold cycles (Ct). Fold changes in expression were evaluated using the ΔΔCt method. Analytes selected included the proinflammatory cytokines IL-1b, IL-6, IFNγ, and TNF, as well as TF.

[0161] NET analysis Deparaffinized kidney tissue samples were fixed with 2.5% glutaraldehyde in 0.15 mol / L sodium cacodylate, pH 7.4 cacodylate buffer for 30 minutes at room temperature and used for NET detection. After fixation, the samples were washed with cacodylate buffer and dehydrated in an ethanol series (10 min / step) ascending from 50% (vol / vol) to absolute ethanol. The specimens were subjected to critical point staining in carbon dioxide using absolute ethanol as the intermediate solvent. The specimens were examined using a HITACHI SU3500 scanning electron microscope at the Bio-Microscopy Facility, Department of Biology. The location of individual target molecules was analyzed at high resolution by ultrathin sectioning and transmission immunoelectron microscopy. Coverslips containing the specimens were embedded in Epon 812 and sectioned into 50 nm-thick ultrathin sections using a diamond knife on an ultramicrotome. To quantify NET area, characteristic reticular fibrous structures were first identified at high magnification. Quantitative NET surface area assessment was performed using Adobe Photoshop CS5. Briefly, pixels per square micrometer were determined using the ruler tool. NET area was then converted to pixel count using the Magic Wand selection tool. Thus, the percentage of NET area relative to the total area of ​​a given electron micrograph was calculated.

[0162] urine In the acute non-survival model, the ureters from both kidneys were catheterized separately, and continuous urine samples were collected for 6 hours. The amount of fluorescein-labeled FAM-PEG-phospholipid in urine was quantified in samples added to black microwell plates (Thermo Scientific, 96F Maxisorb, black microwell plates; 43711) and measured using a fluorescent plate reader, Synergy HTX, at an excitation wavelength of 485 nm and an emission wavelength of 528 nm.

[0163] Experimental design - in vivo study in pigs Pigs were chosen as a large animal model for allogeneic transplantation because their anatomy and physiology closely resemble those of humans. Specifically, the structure and function of the kidney are highly similar to those of primates. Pig kidneys have been described as truly multirenaculate, multipapillate, with a calyceal system similar to that of humans. The blood supply is divided transversely between the cranial and caudal poles, rather than longitudinally as in most other species. Pigs have similar nephron types to humans, and renal cytochrome enzymes (P450 3A, 2A, and 2C) exhibit similar activity to their human homologs. Furthermore, there are many examples of similar or identical genes encoding enzymes present in both human and porcine kidneys, such as fucosidases, galactosidases, prostaglandin 9-ketoreductase, and dipeptidyl-aminopeptidase IV. Furthermore, there is considerable similarity between the human and porcine immune systems, with over 80% similarity in the parameters tested. The corresponding similarity between the human and the parameters is only approximately 10%.

[0164] Three porcine allotransplantation models In addition to in vivo studies to examine tissue distribution and segregation (Example 4), one acute non-survival model (ANSM), one allo-survival model (SM), and one clinical-like allo-survival model (CLSM) were used to test the effect of PEG-phospholipid coating in porcine allografts. Samples: Plasma, biopsies, and urine were collected as described below in the sections describing each model and analyzed as described above. [Table 2]

[0165] Porcine Acute Non-Survival Transplant Model (ANSM) This study was approved by the Ethics Committee for Animal Experimentation, Upsala, Sweden, Dnr C175 / 12.

[0166] Twelve pigs (Yorkshire × Landrace × Hampshire), 10–12 weeks old and weighing 30–35 kg, were purchased from a conventional farm containing pigs in good health. The farm is supervised by a swine specialist at the Veterinary Faculty, SLU. The animals were transported to the animal laboratory at Uppsala University Hospital, where they immediately underwent non-survival transplant surgery.

[0167] anesthesia Upon arrival at the facility, animals were given an intramuscular (IM) injection of 2.2 mg / kg xylazine (Rompun®, Bayer, Leverkusen, Germany) combined with 6 mg / kg zolazepam-tiltamine (Zoletil 100®, Virbac, Carros, France) for induction purposes, after which they were placed in a prone position. Peripheral venous catheters were inserted into both ears, and TIVA anesthesia was administered: midazolam 0.105 mg / kg / h (Midazolam Actavis 5 mg / mL, Actavis AB, Sweden), ketamine 28 mg / kg / h (Ketaminol®vet, 100 mg / mL, Intervet AB, Sweden), and fentanyl 3.5 μg / kg / h (Fentanyl B. Braun 50 μg / mL, B. Braun Medical AB, Sweden). Fluid administration was performed with lactated Ringer's solution (Ringer acetate, Fresenus Kabi AB, Sweden) and succinylated gelatin (Gelofusine®-B. 40 mg / mL, Braun Mesungen AG, Germany) at 10 mL / kg / h. A small supratracheal incision was made for tracheal exposure and transtracheal intubation. Animals were mechanically ventilated with 30% oxygen in air to obtain 5.0–5.5 kPa CO (Servo-I mechanical ventilator, MAQUET, Medical Systems, US). A mean arterial pressure (MAP) above 60 mmHg was targeted to ensure adequate organ perfusion.

[0168] transplant surgery We have developed a porcine transplantation model that allows for the acquisition and transplantation of both kidneys en bloc from a donor to a recipient animal. By incubating isolated ex vivo PEG-phospholipid with only one randomly selected kidney in an en bloc package, this technique allows for the simultaneous transplantation of the treated organ along with its perfectly matched control organ into a single recipient pig, thereby not only reducing the number of experimental pigs but also minimizing confounding variables in the experiment.

[0169] Both kidneys, the suprarenal and infrarenal aortic trunks, and the inferior vena cava were mobilized to create an en bloc package consisting of two kidneys, the corresponding ureters, and the renal vasculature. The en bloc package was immediately removed and cold flushed ex situ on the back table with cold HTK solution. Here, the time between in situ clamping of the donor and ex situ perfusion with cold HTK solution was less than 5 minutes, resulting in the initial warm ischemia in this model. The en bloc package was then cold-stored in HTK solution at 4°C for 24 hours. After retrieval, the donor pig was euthanized by an IV overdose of sodium pentobarbital (Euthasol® vet, 400 mg / mL, Virbac).

[0170] After storage, one kidney in each EnBloc package was randomly selected for PEG-phospholipid incubation. The vein and artery of the contralateral kidney (control) and the vein of the selected kidney were clamped. A total of 3–5 mL of 2 mg / mL PEG-phospholipid solution was slowly injected into the selected kidney. The artery of the treated kidney was then clamped, and the EnBloc package was cold-stored in HTK at 4°C for an additional 40 minutes. After incubation and before transplantation, excess PEG-phospholipid was flushed from the HTK-treated kidney without removing the clamp from the control kidney.

[0171] The recipient pig was handled and anesthetized as described above. The enbloc package was placed horizontally in the abdominal cavity. The distal ends of the graft's vena cava and aorta were anastomosed to the recipient's vena cava and aorta, respectively, via end-to-side anastomosis. Upon release of the clamps, the treated and control kidneys were sequentially reperfused (by delayed release of the control kidney clamp), while flushing out the effluent from the treated kidney. This was done to prevent contamination of the control kidney from residual PEG-phospholipid solution in the aortic conduit. Both transplanted ureters were catheterized separately to record urine output.

[0172] sampling EDTA blood was collected from both allograft veins at 1, 5, 15, 30, 60, 120, 240, and 360 minutes after reperfusion. Two wedge biopsies were obtained from each graft at 1, 60, and 360 minutes after reperfusion. One biopsy was snap-frozen in liquid nitrogen for cytokine analysis and PEG-phospholipid measurement, while the other biopsy was preserved in 4% PFA and used for immunohistochemistry. A total volume of urine was collected at 360 minutes after reperfusion.

[0173] Porcine survival transplant model SM and CLSM These studies were approved by the Ethics Committee for Animal Experimentation, Uppsala, Sweden, Dnr C123 / 14. For each of these studies (SM and CLSM), five donor pigs and ten recipient pigs of both sexes were used. All pigs were certified SPF (Yorkshire x Hampshire) and were bred at Lovsta University herd, SLU, Sweden. Pigs were 8 weeks old when they arrived at SLU.

[0174] For SM testing, pigs were selected after swine leukocyte antigen (SLA) class 1 typing in the following manner: donor pigs were from two litters and unrelated to either recipient. Recipient pigs were siblings and pairwise matched according to SLA class 1 (see below). For CLSM testing, all recipient pigs were siblings and unrelated to the donors, but these pigs were not SLA typed.

[0175] SLA typing by PCR-SSP SLA typing was performed using complete primer sets specific for alleles at three SLA class I loci, SLA-1, SLA-2, and SLA-3, and three SLA class II loci, DRB1, DQB1, and DQA8-10. Total genomic DNA was isolated from whole blood samples of 27 purebred pigs using the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma, St. Louis, MO, USA) according to the manufacturer's instructions. The typing PCR reaction contained 1x TopTaq™ Master Mix (Qiagen GmbH, Hilden, Germany), 1x CoralLoad Loading Buffer (Qiagen), 0.2 pmol / μl α-actin positive control primer, 0.2 pmol / μl allele-specific primers (Eurofins Genomics, Ebersberg, Germany), and 30 ng of DNA in a total volume of 10 μl. Each pig typing included a negative control containing no DNA to check for reagent contamination. Electrophoresis was performed in a standard 96-well format setup. Thermal cycling conditions in a T Gradient thermal cycler (Biometra, Goetttingen, Germany) consisted of an initial incubation at 95°C for 2 minutes, followed by 30 cycles of 95°C for 15 seconds, 65°C for 20 seconds, and 72°C for 20 seconds. PCR products were electrophoresed at 150V for 5 minutes in 2.5% DNA-grade agarose gels containing 1x TAE buffer using a Micro SSP Gel System (One Lambda, Canoga Park, CA, USA) and visualized after staining with GelStar™ (Lonza, Rockland, ME, USA). Interpretation of results was based on the presence of allele-specific PCR products of the expected size in each lane.

[0176] Habituation and training The recipients were placed within visual and hearing range of each other, approximately 2.5 m apart. 2The donors were housed together in a single pen. A 12:12-hour light / dark schedule was used, with infrared lamps (24 hours a day) installed in the corners of each pen. Straw and wood shavings were used as bedding. The pigs were fed twice daily with commercial pig feed (SOLO330, Lantmannen) in amounts consistent with the SLU regimen for growth. Water was available ad libitum. Pens were cleaned twice daily. Recipients underwent a 14-day socialization and training program. The pigs were trained to accept ear contact and palpation in preparation for stress-free blood sampling from the auricular vein. Additionally, the animals were accustomed to a dummy ultrasound transducer placed on their abdomen to tolerate postoperative ultrasound examination of the bladder and transplanted kidney. The pigs were also trained to ride a spring scale, accept free-flow urine sampling, and undergo clinical examinations, including auscultation of the heart and lungs. After 14 days of acclimation and training, the recipients weighed 32.4±2.2 kg (mean±SD) and were ready for the transplant procedure. In total, the experiment lasted 3 weeks.

[0177] Prior to surgery, blood samples were analyzed for blood counts and hematology as described above to ensure that donors and recipients in both studies were healthy.

[0178] Anesthesia and Analgesia Anesthesia was induced in the pig pen with tiletamine-zolazepam (Zoletil 100® vet, Virbac, Carro, France) mixed with medetomidine (Domitor® vet 1 mg / mL) IM according to Ryden et al. Nursing and training of pigs used in renal transplantation studies, Laboratory Animals (2020) 54(5):469-478. Before surgery, buprenorphine was administered at a dose of 0.01 mg / kg body weight IM and benzylpenicillin procaine (Penovet® vet 300 mg / mL, BeOHringer Ingelheim, Ingelheim, Germany) was administered at 20 mg / kg body weight IM. The animal was covered with a blanket and transferred to the preparation room, where oxygen saturation and pulse rate measurements were initiated. Socks were placed on the claws and distal legs to prevent heat loss. An intravenous (IV) catheter (BD Venflon™ 20G, 32 mm, BD Medical, Franklin Lakes, US) was placed in an auricular vein, and blood samples were collected for complete blood count (CBC) and enzyme activity analysis. Endotracheal intubation was facilitated using a laryngoscope. Oxygen (4 L / min) was delivered via a face mask until the animals were connected to an anesthesia circuit. Anesthesia was maintained with an oxygen / air mixture (FiO2 0.3) containing isoflurane (IsoFlo vet, Orion Pharma Animal Health, Sweden) from a rebreathing circuit, and all pigs received mechanical ventilation (FLOW-i Anesthesia Delivery System, MAQUET Medical Systems, US). Epidural morphine (Morfin Epidral Meda 2 mg / mL, Meda AB, Sweden) 0.1–0.12 mg / kg was administered according to the protocol of Malavasi et al. (2006).The pigs received an IV infusion of lactated Ringer's solution (Ringer-acetate, Fresenius Kabi AB, Sweden) at approximately 10 mL / kg / h, and succinylated gelatin (Gelofusine®-B, 40 mg / mL, Braun Melsungen AG, Germany) as a bolus of 3 mL / kg for 10 min, followed by an infusion of 3 mL / kg / h. To achieve adequate arterial blood pressure (≥60 mmHg), dobutamine (Dobutamine Carino® 250 mg / 50 mL, Carinopharm, Germany) was given as an infusion (5–15 μg / kg / min). During anesthesia, circulatory and respiratory parameters were continuously monitored (AS / 3 anesthesia monitor, Datex-Ohmeda, Finland). Arterial blood pressure was measured intermittently by oscillometric methods using inflatable cuffs placed around the forelimbs and connected to a monitor.

[0179] To allow repeated blood sampling in the recipient pigs, a polyurethane catheter (BD Careflow™ 3 Fr 200 mm, BD Medical, US) was introduced into the jugular vein via the ear vein using the sterile Seldinger technique. The catheter was sutured above the ear with a monofil-coated polyamide (Supramid 2-0, B Braun Medical, Sweden) and covered with a bandage (Snogg AS, Norway).

[0180] Donor surgery and graft treatment SM study: Using the same procedure as in the ANSM study, kidneys were harvested en bloc. Again, kidneys were exposed to an initial warm ischemia of less than 5 minutes. After harvest, donor pigs were euthanized by an IV overdose of sodium pentobarbital (Euthasol® vet, 400 mg / mL, Virbac).

[0181] After storage, kidneys were randomized and, after clamping the renal vein, received either PEG-phospholipid (15 mL, 2 mg / mL) or HTK solution at +4°C via injection through the renal artery. The renal artery and vein were kept clamped during a 40-60 min incubation. The SM study included six PEG-phospholipid-treated kidneys and four control kidneys.

[0182] CLSM Study: In this study, kidney retrieval resembled standard surgical procedures for human organ procurement. Each donor was randomized to either PEG-phospholipid or HTK treatment. After warming, dissecting, and mobilizing the kidneys, the supra-iliac aorta was clamped near the iliac bone, and a 12-French aortic cannula was inserted. Immediately after clamping the supra-celiac aorta, the kidney was irrigated in situ through the aortic cannula by gravity infusion of either PEG-phospholipid (250 mL, 2 mg / mL) or HTK (250 mL) solution. Blood was withdrawn through an incision in the distal vena cava, and the kidney was cooled in situ by surrounding it with slush ice. After retrieval, the kidney was immersed in either PEG-phospholipid or HTK solution and cold-stored at +4°C for 24 hours. In contrast to the ANSM and SM studies, kidneys were not exposed to initial warm ischemia. The CLSM study included five PEG-phospholipid-treated kidneys and five control kidneys (Table 2).

[0183] transplant surgery Recipient pigs underwent allogeneic single kidney transplants. Through a 15-20 cm abdominal midline incision, the iliac vessels in the right iliac fossa were identified and carefully mobilized. The distal segment of the vena cava and iliac artery (as they emerge from the aorta) were mobilized, and the surrounding lymphatic tissue was sealed. The renal graft was then positioned in the right iliac fossa proximal to the iliac vessels. The renal vein and artery were excised and then anastomosed end-to-side to the recipient's right iliac artery and distal vena cava using a polypropylene 7 / 0 running suture (PROLENE®, Ethicon, US). The ureter was transplanted to the top of the bladder via an extravesical ureterocystomy using a 6-0 polydioxanone suture (PDS®, Ethicon, US). Bilateral nephrectomies of the native kidneys were then performed. The midline incision was closed with passing 2 / 0 polyglactin (VICRYL®, Ethicon, US) fascial sutures and skin clips. Recipients were not administered anticoagulants. After completion of the inguinal transplantation, urine was collected directly from the bladder by cystocentesis, which showed that all of the transplanted kidneys began to produce urine immediately after reperfusion. Samples were analyzed for proteinuria and hematuria using a urine stick.

[0184] The average operative time was 2.5 hours (1.75-3.25 hours).

[0185] Postoperative care Toward the end of surgery, isoflurane administration was discontinued, the fraction of inspired oxygen was increased (FiO2 1.0), and the pig was weaned from the mechanical ventilation system. Animals were kept in their cages and continuously monitored by staff. Supplemental oxygen was administered via face mask. Heart rate, respiratory rate, oxygen saturation, and body temperature were monitored until the pig regained consciousness. At the end of anesthesia and throughout the remainder of the postoperative period, additional buprenorphine (0.03 mg / kg) was administered IV after assessment of general condition and behavior. Antibiotic treatment with enrofloxacin (Baytril® vet, 100 mg / mL, Bayer) at a dose of 2.6 mg / kg BW IM and benzylpenicillin procaine (Penovet® vet, 300 mg / mL, Boehringer Ingelheim) at a dose of 20 mg / kg BW IM was administered the day after surgery. If necessary, the pigs were hand-fed fruit to stimulate appetite, assisted to drink water, and assisted to stand and walk post-operatively.

[0186] Sampling for SM and CLSM tests During and after transplantation, EDTA blood was collected from the local renal vein at 0, 15, 30, and 60 min after reperfusion, and systemic blood samples were obtained 1, 2, 3, and 4 days after surgery.

[0187] Two wedge biopsies were taken from each graft before reperfusion, 15 min after reperfusion, and immediately after euthanasia: one biopsy was snap-frozen in liquid nitrogen for cytokine analysis, and the other biopsy was preserved in 4% PFA and used for immunohistochemistry.

[0188] Clinical examination, including ultrasound evaluation of the bladder and kidney graft Clinical appearance was assessed several times daily after surgery, and a thorough clinical examination was performed once daily. The bladder was examined at 9:00 AM and 6:00 AM daily to determine whether the transplanted kidney was producing urine. When possible, free-flow urine was collected and evaluated visually, and concentration, pH, blood, and protein were measured.

[0189] Each kidney was examined once after transplantation by ultrasound (LogiqeR6, GE Healthcare, Wauwatosa, USA) using linear (10 MHz) and curved (4 MHz) probes. Renal length and echogenicity, as well as corticomedullary definition, were estimated. In addition, the pelvic region was assessed for distension. Intrarenal blood flow was assessed using color Doppler.

[0190] autopsy Four days after transplantation, the pigs were sacrificed in their pig pens by an IV overdose of sodium pentobarbital (Euthasolvet, 400 mg / mL, Virbac).

[0191] All pigs, both donor and recipient, were examined post-mortem by veterinary pathologists at the Department of Biomedical Sciences and Veterinary Public Health (Section of Pathology, SLU, Uppsala).

[0192] statistical analysis Statistical analyses were performed using Prism macOS version 9.4.1 (458). Single-sample, unpaired and paired, and multiple unpaired t-tests were used as indicated in the legends. One-way ANOVA and two-way repeated measures ANOVA followed by multiple comparison tests without and with Bonferroni correction were also employed. ANOVA results are shown in the figure legends. t-test and multiple comparison test results are indicated with the appropriate data plots as ns, *p<=0.05, **p<0.01, ***p<0.001, ****, and p<0.0001. Specific statistical methods are described in the figure legends.

[0193] result PEG-phospholipid-mediated attenuation of ischemia-reperfusion-induced thromboinflammation in three porcine transplantation models To assess the ischemic state of the PEG-phospholipid treated compared to untreated kidneys, biopsies were collected after 1, 60, and 360 minutes in ANSM and stained for nitrotyrosine, HO-1, and INOS. No differences were observed between treated and untreated kidneys, indicating that the tissues were in a similar ischemic state (FIG. 13).

[0194] In the ANSM, thromboinflammatory biomarkers were analyzed in blood samples collected from the renal vein of each kidney. Over the 6-hour observation period, complement (C3a, sC5b-9), coagulation (TAT), and contact system (FXIIa-C1INH) markers were found to be substantially lower in treated kidneys compared with untreated kidneys (Figure 14). Biopsies at 1 minute revealed local deposition of complement fragments C4d and C3b. PEG-phospholipid-treated kidneys showed significantly less C3b binding, and there was a trend toward less C4d binding in treated kidneys (Figure 15). This pattern was confirmed in the SM, where levels of C3a, sC5b-9, and TAT in blood samples were significantly lower in PEG-phospholipid-treated kidneys throughout the entire observation period (up to 4 days posttransplant) (Figure 16). Renal biopsies taken at 5 min and on post-mortem day 4 also showed decreased C3b binding (two-way ANOVA) (Figure 17). Results from CLSM confirmed these results, although showing lower values ​​(not shown).

[0195] Furthermore, neutrophil extracellular traps (NETs), one driving force of thromboinflammation, were analyzed by scanning electron microscopy in kidney explant biopsies from ANSM (SEM; Figure 18). NET deposition was observed already 1 minute after reperfusion, primarily in the glomerular and tubular lumen. Quantification of renal NET deposition demonstrated a significant reduction in both glomerular and tubular NETosis in PEG-phospholipid-treated kidneys compared with their matched individual controls (Figure 19).

[0196] PEG-phospholipid-mediated reduction of ischemia-reperfusion-induced systemic inflammation in three porcine transplant models In ANSM, both mRNA expression in renal biopsies (RT-PCR, Figure 20) and renal vein plasma concentrations of proinflammatory cytokines (IL-1β, IL-6, TNF) and TF (Figure 21) were lower in PEG-phospholipid-treated animals compared with matched controls. In particular, IL-6 was clearly suppressed at most time points.

[0197] Proinflammatory cytokines, reflecting local inflammation at 5 and 60 minutes and systemic inflammation at 4 days after transplantation, were also assessed by SM (Figure 22). Although not significant, an inflammatory response was already evident at 5 minutes and was observed only in the untreated kidney. After 60 minutes, this inflammatory response disappeared and reached a steady state. After 4 days, a robust response involving most of the analyzed cytokines, except for IL-8 and IL-1RA, was observed particularly in pigs transplanted with untreated kidneys. Therefore, when examining the sustained effect of ex vivo graft coating with PEG-phospholipids, a reduction in systemic inflammation was clearly observed 4 days after transplantation. Considering the short duration of the PEG-phospholipid coating, this indicates that PEG-phospholipid-mediated inhibition of the instantaneous thromboinflammation caused by IRI also impacts downstream systemic inflammation over time. CLSM results confirmed these results (not shown).

[0198] No toxicity or safety concerns regarding clinical, laboratory, or pathological parameters were observed in any of the transplant models, including no differences in general histology (hematoxylin staining) or gross appearance between treated and untreated kidneys.

[0199] Functional effects of ex vivo PEG-phospholipid treatment in a porcine transplant model Renal function was assessed by monitoring post-reperfusion diuresis in ANSM and creatinine levels in blood samples collected from recipients in SM over time, and it was found that, despite very severe reperfusion injury with progressive renal failure, pigs with PEG-phospholipid-coated grafts had significantly lower creatinine levels post-transplant than those in control grafts (Figure 23). Consistent with this finding was a similar effect of the coating in CLSM, which was less pronounced in IRI (Figure 24).

[0200] Although the mechanism by which IRI is induced has not yet been established, it has been suggested that the binding of MBL, CL-11, MASP-2, and natural IgM antibodies binds to ligands on ischemic cells, thereby causing IRI. The PEG-phospholipid coating of the present invention shields endothelial and tubular cell membranes by preventing cell membrane proteins from reaching and binding to plasma proteins in the blood in kidney graft grafts after reperfusion. Data support this concept in that PEG-phospholipids effectively shield small surface antigens such as CD52 and Rh antigens, which extend approximately 1 nm from the surface (see Example 1). Furthermore, CD8, which has a range of 5–10 nm based on the crystal structure of its non-membrane portion, is partially blocked by the PEG-phospholipid coat, but the PEG-phospholipid layer does not reach far enough to encompass longer antigens such as CD4, which has an estimated molecular extension of 15 nm from the membrane. Furthermore, antibodies to CD3135 and vWF (extending 15 nm and 30 nm from the surface, respectively) were unaffected (see Example 3). These results are consistent with the PEG-phospholipids having 5 kDa PEG chains that theoretically extend 6 nm from the cell membrane (Figure 4).

[0201] To demonstrate the effects of PEG-phospholipids on IRI, we used non-survival and survival porcine transplant models in which recipients were not immunosuppressed and followed for up to 4 days. In these in vivo transplant models, kidneys treated with PEG-phospholipids showed significantly less innate immune activation compared with untreated kidneys, as reflected by reduced plasma levels of TAT, FXIIa-C1INH, TF, C3a, and sC5b-9, as well as reduced plasma levels of C3b deposition in renal biopsies. This demonstrated that PEG-phospholipid constructs can inhibit activation of the coagulation, contact, and complement systems, all hallmarks of IRI, and protect cell membranes from C3b deposition after reperfusion with blood from the recipient. Supporting these findings, the ANSM study showed that PEG-phospholipid-treated kidneys exhibited reduced expression of proinflammatory cytokines, such as IL-1β, IL-6, TNF, and INFγ, as well as reduced NET formation, indicating a significantly lower thromboinflammatory response in the treated group. This pattern was even more pronounced after 4 days in the survival model, where the expression of all tested cytokines was found to be very low in the treated group, while most cytokines, except for IL-8 and IL-1RA, were increased in the untreated group. The large effect indicates that PEG-phospholipids provide broad protection against post-transplant IRI and systemic inflammation, which is transmitted to the adaptive immune response and leads to humoral and cellular rejection. PEG-phospholipids may also protect against the adaptive immune system and antibody-mediated rejection (AMR). Because inhibition of IL-6 / IL-6R signaling is considered a novel therapeutic option for preventing and treating allograft injury, the consistent and significant inhibition of IL-6 in all three transplant models supports this concept. Clinical trial evidence supports the use of IL-6 blockade for desensitization and treatment of AMR in kidney transplantation (Curr Transplant Reports 8:1-14(2021), J Am Soc Nephrol 20:1032-1040(2009), Transplantation 101:32-44(2017)).

[0202] Example 6 In this example, we evaluated the ability of PEG-phospholipids to block the binding of anti-human leukocyte antigen (HLA) antibodies to freshly isolated human peripheral blood mononuclear cells (PBMCs) expressing HLA antigens. HLA antigens are proteins found on the surface of all nuclear cells and present foreign peptides to T cell receptors on T cells during cellular immune responses. Antibodies against these structures are formed during allogeneic immune responses, such as blood transfusions and transplants, and constitute the majority of all so-called donor-specific antibodies (DSA). These antibodies are the primary cause of antibody-mediated rejection (AMR). To avoid such incompatibility reactions, crossmatching is performed before transplantation, in which patient serum, which may contain DSA, is mixed with PBMCs from the donor. If the DSA does not recognize the HLA antigen and lysis of donor PBMC cells does not occur, the crossmatch is considered negative and the transplant can proceed. On the other hand, if the crossmatch is positive, the transplanted organ must be transferred to another patient and matched (selected) with the other patient for HLA antigens. If the crossmatch combination is negative for the donor and recipient, the transplant can proceed.

[0203] Materials and Methods To test the effectiveness of PEG-phospholipid (SUNBRIGHT® PP-050CN, N-(methylpolyoxyethyleneoxycarbonyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, sodium salt, NOFCorp., Japan) in blocking the binding of anti-HLA antibodies to HLA antigens, we used human PBMCs from healthy donors to block HLA antigens, similar to a potential clinical setting.

[0204] material FITC Mouse Anti-Human HLA-ABC, Monoclonal (G46-2.6) (BD Pharmingen) Catalog No. 555552 Biotin Mouse Anti-Human HLA Class I Antibody [W6 / 32] (Abcam) Catalog No. ab110665 PE-Streptavidin (BD Pharmingen) Catalog No. 554061

[0205] method PBMCs were isolated from whole blood using Cytiva™ Ficoll-Paque™ Plus Medium (Cytiva 17-1440-02) according to standard protocols. Briefly, whole blood was diluted with an equal volume of PBS, carefully layered on top of Ficoll-Paque, and centrifuged at 800 × g for 25 minutes. The cloudy layer was separated from the top of the Ficoll and washed twice again with PBS.

[0206] Cells were stained with FITC mouse anti-human HLA-ABC monoclonal antibody and biotin mouse anti-human HLA class I antibody [W6 / 32] (Abcam), followed by PE-streptavidin. Stained cells were analyzed using a Beckman Coulter Cytoflex S (Biovis, facility IGP, Rudbeck laboratory).

[0207] result The results shown in Figures 25 and 26 demonstrate the potent inhibition of both anti-HLA antibodies on PBMCs treated with PEG-phospholipids, demonstrating a strong masking effect mediated by PEG-phospholipids. Thus, PEG-phospholipids can mask HLA antigens on PBMCs, thereby blocking DSA binding to such HLA antigens and can be used to suppress AMR in organ transplants.

[0208] The above-described embodiments should be understood as some illustrative examples of the present invention. Those skilled in the art will understand that various modifications, combinations, and changes can be made to the embodiments without departing from the scope of the present invention. In particular, different part solutions in different embodiments can be combined in other configurations, if technically possible. However, the scope of the present invention is defined by the appended claims.

Claims

1. 1. A poly(ethylene glycol) phospholipid (PEG-phospholipid) molecule for use in selectively masking surface antigens of red blood cells and / or platelets in a blood product from a donor to inhibit antibody binding to the surface antigens in connection with transfusion of the blood product into a cross-incompatible or incompatible recipient, wherein the PEG chains of the PEG-phospholipid molecule have an average molecular weight selected within the range of 3,000 to 10,000 Da.

2. Poly(ethylene glycol) phospholipids (PEG-phospholipid molecules) for use in selectively masking the surface antigens of organ grafts comprising red blood cells, platelets and / or endothelial cells from a donor to inhibit antibody binding to said surface antigens and antibody-mediated rejection (AMR) associated with transplantation of said organ graft into a cross-incompatible or incompatible recipient.

3. 3. The PEG-phospholipid molecule for use according to claim 2, wherein the organ transplant is selected from the group consisting of kidney, liver, pancreas, heart, lung, uterus, bladder, thymus and intestine.

4. 4. The PEG-phospholipid molecule for use according to claim 2 or 3, wherein the surface antigen is a human leukocyte antigen (HLA), preferably selected from the group consisting of HLA A, HLA B, and HLA C.

5. 4. The PEG-phospholipid molecule for use according to any one of claims 1 to 3, wherein the surface antigen is a Rhesus factor, preferably selected from the group consisting of RhD, RhC, Rhc, RhE, and Rhe.

6. 4. The PEG-phospholipid molecule for use according to any one of claims 1 to 3, wherein the surface antigen is a blood group antigen, preferably selected from the group consisting of A antigen and B antigen.

7. 4. The PEG-phospholipid molecule for use according to any one of claims 1 to 3, wherein the surface antigen is a human platelet antigen (HPA), preferably selected from the group consisting of HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, HPA6, HPA-9 and HPA-15.

8. 4. The PEG-phospholipid molecule for use according to any one of claims 1 to 3, wherein said PEG-phospholipid molecule, when anchored in a cell membrane, has an average extracellular length chosen within the interval between 4 nm and 8 nm, preferably between 5 nm and 7 nm, more preferably 6 nm.

9. The PEG-phospholipid molecule has the formula (I): 【Chemistry 1】 (In the formula, n and m are independently selected integers in the range of 10 to 16, preferably n and m are independently 10, 12, 14, or 16, more preferably n=m=14; p is selected so that the PEG chain has an average molecular weight selected in the range of 3000 to 10000 Da, more preferably 5000 Da; and R is selected from the group consisting of H, methyl, and C1-C4 alkylamine.

4. A PEG-phospholipid molecule for use according to any one of claims 1 to 3, comprising:

10. 4. The PEG-phospholipid molecule for use according to any one of claims 1 to 3, wherein the PEG-phospholipid molecule has an average molecular weight selected within the interval of 5 to 7 kDa, preferably 5.5 to 6.6 kDa, more preferably 5.8 to 6.0 kDa, as determined by gel permeation chromatography.

11. 4. The PEG-phospholipid molecule for use according to any one of claims 1 to 3, wherein the lipid portion of the PEG-phospholipid molecule is 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine (DPPE).

12. 4. The PEG-phospholipid molecule for use according to any one of claims 1 to 3, wherein the PEG moiety of the PEG-phospholipid molecule has an average degree of polymerization selected within the interval of 105 to 125, preferably within the interval of 110 to 120, more preferably within the interval of 114 to 116.

13. 4. The PEG-phospholipid molecule for use according to any one of claims 1 to 3, wherein the PEG-phospholipid molecule is N-(methylpolyoxyethyleneoxycarbonyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine.

14. 4. The PEG-phospholipid molecule for use according to any one of claims 1 to 3, wherein said PEG-phospholipid molecule is not interconnected with other PEG-phospholipid molecules by any linker.

15. A blood product comprising red blood cells and / or platelets and poly(ethylene glycol) phospholipid (PEG-phospholipid) molecules immobilized within the cell membranes of the red blood cells and / or platelets, the blood product masking surface antigens of the red blood cells and / or platelets, wherein the PEG chains of the PEG-phospholipid molecules have an average molecular weight selected within the range of 3,000 to 10,000 Da.

16. 16. The blood product of claim 15, wherein the surface antigen is a Rhesus factor, preferably selected from the group consisting of RhD, RhC, Rhc, RhE, and Rhe.

17. 17. The blood product according to claim 15 or 16, wherein the surface antigen is a blood group antigen, preferably selected from the group consisting of A antigen and B antigen.

18. The blood product according to claim 15 or 16, wherein the surface antigen is a human platelet antigen (HPA), preferably selected from the group consisting of HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, HPA6, HPA-9 and HPA-15.

19. 17. The blood product according to claim 15, wherein the PEG-phospholipid molecules, when fixed in the cell membrane of red blood cells, have an average extracellular length selected within the interval of 4 nm to 8 nm, preferably 5 nm to 7 nm, more preferably 6 nm.

20. The PEG-phospholipid molecule has the formula (I): 【Chemistry 2】 (In the formula, n and m are independently selected integers in the range of 10 to 16, preferably n and m are independently 10, 12, 14, or 16, more preferably n=m=14; p is selected so that the PEG chain has an average molecular weight selected in the range of 3000 to 10000 Da, more preferably 5000 Da; and R is selected from the group consisting of H, methyl, and C1-C4 alkylamine.

17. The blood product of claim 15 or 16, having the formula:

21. 17. The blood product of claim 15, wherein the PEG-phospholipid molecules have an average molecular weight selected within the interval of 5 to 7 kDa, preferably 5.5 to 6.6 kDa, more preferably 5.8 to 6.0 kDa, as determined by gel permeation chromatography.

22. 17. The blood product of claim 15, wherein the lipid portion of the PEG-phospholipid molecule is 1,2-dipalmitoyl-sn-glycerol-3-phosphatidylethanolamine (DPPE).

23. 17. The blood product of claim 15, wherein the PEG moieties of the PEG-phospholipid molecules have an average degree of polymerization selected within the interval of 105 to 125, preferably within the interval of 110 to 120, more preferably within the interval of 114 to 116.

24. The blood product according to claim 15 or 16, wherein the PEG-phospholipid molecule is N-(methylpolyoxyethyleneoxycarbonyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine.

25. 17. The blood product of claim 15 or 16, wherein the PEG-phospholipid molecules are not interconnected with other PEG-phospholipid molecules by any linker.

26. 1. An in vitro method for treating red blood cells and / or platelets, the method comprising selectively masking surface antigens of the red blood cells and / or platelets by adding poly(ethylene glycol)-lipid (PEG-phospholipid) molecules to the red blood cells and / or platelets in vitro, wherein the PEG chains of the PEG-phospholipid molecules have an average molecular weight selected within the range of 3,000 to 10,000 Da.

27. A blood transfusion method comprising: adding poly(ethylene glycol)-lipid (PEG-phospholipid) molecules to a blood product from a donor to selectively mask surface antigens on red blood cells and / or platelets present in said blood product, wherein the PEG chains of said PEG-phospholipid molecules have an average molecular weight selected within the range of 3,000 to 10,000 Da; transfusing the blood product into a cross-incompatible or incompatible recipient.

28. A method for screening irregular antibodies, comprising: adding PEG-phospholipid molecules to a blood sample from a subject to selectively mask surface antigens on red blood cells present in said blood sample, wherein the PEG chains of said PEG-phospholipid molecules have an average molecular weight selected within the range of 3,000 to 10,000 Da; screening for irregular antibodies present in the blood sample or bound to red blood cells obtained from the blood sample.

29. The PEG-phospholipid molecule has the formula (I): 【Transformation 3】 (In the formula, n and m are independently selected integers in the range of 10 to 16, preferably n and m are independently 10, 12, 14, or 16, more preferably n=m=14; p is selected so that the PEG chain has an average molecular weight selected in the range of 3000 to 10000 Da, more preferably 5000 Da; and R is selected from the group consisting of H, methyl, and C1-C4 alkylamine. The method according to any one of claims 26 to 28, comprising:

30. 30. The method of claim 29, wherein the PEG-phospholipid molecule is N-(methylpolyoxyethyleneoxycarbonyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine.

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