Use of lipid-binding protein-based complexes in organ preservation solutions

Abstract

A lipid-binding protein-based complex for use in an organ preservation solution, an organ preservation solution comprising a lipid-binding protein-based complex, a kit for making the organ preservation solution, a process for preserving an organ using the organ preservation solution, organs preserved thereby, a system for preserving an organ comprising the organ preservation solution, and a method for transplanting an organ obtained by the organ preservation process.

Description

[Technical field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 175,330, filed April 15, 2021, the contents of which are incorporated by reference in their entirety herein.

[0002] 2. Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on April 11, 2022, is named CRN-043WO_SL.txt and is 4,326 bytes in size. [Background technology]

[0003] 3.Background Kidney transplantation is a life-saving procedure for patients with end-stage renal disease (ESRD). The latter represents about 10% of the world's population of patients with chronic kidney disease, which is expected to rise to 7.64 million by 2030 (Liyanage et al., 2015, Lancet, 385(9981):1975-82; Levin et al., 2017, Lancet, 390(10105):1888-917). However, despite the fact that kidney transplantation can dramatically affect the quality of life and save expensive dialysis costs, there are still unsolved problems. First, the number of available donor kidneys is insufficient, and more than 20 people die every day while waiting for a transplanted organ. This organ shortage forces transplantation professionals to accept “marginal” organs from cardiac death or elderly (over 60 years old) donors, i.e. DCD (Donation After Circulatory Death) and ECD (Expanded Criteria Donors). However, the use of these kidneys is correlated with poorer survival rates, incidence of rejection and delayed graft function (DGF). Secondly, regardless of the donor used, the transport and transplantation surgery itself carries an unavoidable risk of ischemia / reperfusion injury (IRI), which can greatly affect the quality of the organ. Besides kidneys, other organs such as the heart and lungs are also prone to IRI (Fernandez et al., 2020, Int. J. Mol. Sci. 21:8549).

[0004] Organ preservation solutions have been developed to reduce the injury caused to donor organs during storage and transportation to improve graft survival after organ transplantation. The use of these organ preservation solutions with machine perfusion has shown superior outcomes in early allograft dysfunction compared to static cold storage (CS). Recent studies have shown that ex vivo normothermic machine perfusion (NMP) results in lower rates of DGF, improved renal metabolism, and reduced renal IRI (Kataria et al., 2019, Curr Opin Organ Transplant. 24(4):378-384). NMP appears to be a superior preservation method when compared to CS (Hosgood et al., 2018, Br J Surg 105(4), 388-394), thus potentially increasing the donor pool by improving transplant outcomes of grafts derived from expanded eligible donation (ECD) as well as from post-cardiac arrest donation (DCD). Renal grafts preserved by NMP suffer less ischemia-reperfusion injury after warm ischemia, even when compared to immediately transplanted non-preserved kidneys (Kaths et al., 2017, Transplantation 101(4), 754-763). In addition to allowing for the retention of function, NMP can be used to maintain organs in controlled conditions, allowing for close observation and viability assessment to allow for successful transplantation (Hamar et al., 2018, Transplantation 102(8), 1262-1270). Although various organ preservation solutions have been developed to reduce donor organ injury prior to transplantation, organ injury prior to transplantation remains a problem.

[0005] Therefore, there is an urgent need to develop new approaches in organ preservation. Summary of the Invention

[0006] 4. Overview The present disclosure provides, in various aspects, a lipid-binding protein-based complex for use in an organ preservation solution, an organ preservation solution comprising a lipid-binding protein-based complex, a kit comprising a lipid-binding protein-based complex and one or more components of an organ preservation solution, a process for preparing an organ preservation solution comprising a lipid-binding protein-based complex, a system comprising the organ preservation solution of the present disclosure and a perfusion machine and / or an organ, a process for ex-vivo organ preservation, an organ obtained by the organ preservation process of the present disclosure, and a method for transplanting the organ of the present disclosure into a subject in need of an organ transplant. The organ preservation solution described herein can be used to preserve both organs and tissues (e.g., cornea). Thus, in various aspects, the present disclosure provides a system comprising the organ preservation solution of the present disclosure and a perfusion machine and / or a tissue, a process for ex-vivo tissue preservation, a tissue obtained by the tissue preservation process of the present disclosure, and a method for transplanting the tissue of the present disclosure into a subject in need of a tissue transplant.

[0007] Several studies have demonstrated that high-density lipoprotein (HDL) particles, the central transporter of cholesterol from peripheral tissues to the liver, are involved in important cytoprotective functions. HDL complexes exert anti-inflammatory, anti-atherogenic, and anti-fibrotic functions, preventing LDL oxidation by ROS with vasoprotective effects, and inhibiting coagulation and platelet aggregation. HDL particles can carry antioxidant enzymes (e.g., serum paraoxonase / arylesterase 1 (PON1), lecithin-cholesterol acyltransferase LCAT, and lipoprotein-associated phospholipase A2 LpPLA2), which can prevent lipid peroxidation (Rysz et al., 2020, Int J Mol Sci. 21(2):601).

[0008] Administration of HDL or ApoA-I in a rat model of renal IRI was found to significantly improve renal function, reduce renal and tubular dysfunction, and decrease the number of polymorphonuclear leukocytes (PMNs) infiltrating into renal tissue during reperfusion, reflected in the attenuation of the increase in renal myeloperoxidase activity caused by I / R (Kaths et al., 2017, Transplantation 101(4), 754-763;Rysz et al., 2020, Int J Mol Sci. 21(2):601). Moreover, HDL significantly reduced the expression of adhesion molecules, intercellular adhesion molecule-1 (ICAM-1), and P-selectin during reperfusion (Thiemermann et al., 2003, J Am Soc Nephrol. 14(7):1833-1843; Lee et al., 2005, Atherosclerosis. 183(2):251-258). In particular, administration of ApoA-I significantly reduced serum creatinine levels, serum TNF-alpha and IL-1beta levels, and tissue myeloperoxidase (MPO) activity compared to IRI controls. Furthermore, ApoA-I treatment suppressed the expression of intercellular adhesion molecule-1 (ICAM-1) and P-selectin on the endothelium, thus reducing neutrophil adhesion and subsequent tissue injury (Shi., 2008, J Biomed Sci. 15(5):577-583).

[0009] Considering the protective function of HDL and its ability to reduce systemic inflammation and oxidative stress, it is believed that lipid-binding protein-based complexes such as HDL mimetic CER-001 can be suitably used ex vivo in organ preservation solutions to preserve kidney function and combat endothelial and tubular dysfunction, without being bound by theory. Again, without being bound by theory, it is believed that the ex vivo use of lipid-binding protein-based complexes such as CER-001 can protect graft endothelial cells and improve kidney function by reducing adhesion molecules that control the recruitment of potentially harmful pro-inflammatory mononuclear cells to the graft, thus resulting in a reduced risk of DGF and acute rejection of the donor kidney. Furthermore, lipid-binding protein-based complexes such as HDL mimetic CER-001 can similarly protect other organs, e.g., heart and lungs, during storage and transportation.

[0010] Thus, in one aspect, the present disclosure provides a lipid-binding protein-based complex (e.g., CER-001) for use in organ preservation solutions. Exemplary features of lipid-binding protein-based complexes are described in Section 6.1 below and in specific embodiments 1-21 and 24-43.

[0011] In another aspect, the present disclosure provides an organ preservation solution comprising a lipid-binding protein-based complex (e.g., CER-001). Exemplary features of the organ preservation solution are described in Section 6.2 below as well as in specific embodiments 22 to 63.

[0012] In another aspect, the present disclosure provides a kit comprising a lipid-binding protein-based complex and one or more components of an organ preservation solution. Exemplary features of the kit are described in Section 6.3 below as well as in specific embodiments 64 to 78.

[0013] In certain aspects, the present disclosure provides a process for preparing an organ preservation solution and the organ preservation solution prepared thereby. Exemplary features of the process for preparing an organ preservation solution of the present disclosure and the organ preservation solution prepared by such process are described in Section 6.2 below and in specific embodiments 79 to 95.

[0014] In certain aspects, the present disclosure provides a system comprising an organ preservation solution and a perfusion machine and / or an organ. In certain aspects, the present disclosure provides a system comprising an organ preservation solution and a tissue. Exemplary features of the systems of the present disclosure are described in Section 6.3 below and in specific embodiments 96 to 112.

[0015] In certain aspects, the present disclosure provides a process for ex-vivo organ preservation and the organs obtained thereby. In certain aspects, the present disclosure provides a process for ex-vivo tissue preservation and the tissues obtained thereby. Exemplary features of ex-vivo organ and tissue preservation processes and the organs and tissues obtained thereby are described in Section 6.4 below and in specific embodiments 113-154 and 156-179.

[0016] In a further aspect, the present disclosure provides a method for transplanting an organ into a subject that needs an organ transplant.In a further aspect, the present disclosure provides a method for transplanting a tissue into a subject that needs a tissue transplant.The exemplary features of the transplantation method of the present disclosure are described in the following section 6.4 and numbered embodiments 155 and 180 to 186. [Brief description of the drawings]

[0017] [Figure 1] Vascular resistance (FIG. 1A) and flow rate (FIG. 1B) for pig kidneys HMP-perfused for 4 hours with PumpProtect® solution (●) or PumpProtect® solution supplemented with CER-001 (□) (Example 4). n=5 for each group. [Figure 2-1]Histological analysis performed by Periodic Acid Schiff (PAS) staining from porcine kidneys perfused with HMP using PumpProtect® solution (control) or PumpProtect® solution supplemented with CER-001 (CER-001) (Figure 2A); tubular injury score (Figure 2B); [Figure 2-2] the level of MCP-1 in the perfusate (Figure 2C);the level of TNF-α in the perfusate (Figure 2D); [Figure 2-3] Aspartate aminotransferase levels in the perfusate (FIG. 2E) (Example 4). In FIGS. 2C-2E, control data are indicated by ● and CER-001 data are indicated by □. [Figure 3-1] CCL2 (MCP-1) in kidneys perfused with PumpProtect® solution (control) or PumpProtect® solution supplemented with CER-001 (CER-001) or maintained in static cold storage (SCS) (Figure 3A) [Figure 3-2] IL-6 (FIG. 3B) and ET-1 (FIG. 3C) gene expression (Example 4). Results are mean values ​​± SD. n=5. *<0.05. In FIG. 3B-FIG. 3C, SCS data are indicated with △, control data with ●, and CER-001 data with □. [Figure 4] FACS analysis showing Ser 1177-eNOS phosphorylation in endothelial cells (Example 5). [Figure 5-1] Renal perfusion parameters of kidneys perfused with NMP using conventional preservation solution (control) or conventional preservation solution supplemented with CER-001 (Example 6). Figure 5A: Vascular resistance; [Figure 5-2] Figures 5B-C: Vascular resistance; [Figure 5-3] Figure 5D: flow rate; Figure 5E: urine volume. In Figures 5A-5E, control data are indicated by ● and CER-001 data are indicated by □. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] 6. Detailed Description In various aspects, the present disclosure provides a lipid-binding protein-based complex for use in an organ preservation solution, an organ preservation solution comprising a lipid-binding protein-based complex, a kit comprising a lipid-binding protein-based complex and one or more components of an organ preservation solution, a process for preparing an organ preservation solution comprising a lipid-binding protein-based complex, a system comprising the organ preservation solution of the present disclosure and a perfusion machine and / or an organ, a process for ex-vivo organ preservation, an organ obtained by the process of the present disclosure, and a method for transplanting the organ of the present disclosure into a subject in need of an organ transplant. The organ preservation solution of the present disclosure can be used to preserve both organs and tissues (e.g., eyes (e.g., cornea or sclera), skin, fat, muscle, bone, cartilage, fetal thymus, and nervous system). Thus, the present disclosure further provides a system comprising the organ preservation solution of the present disclosure and a tissue, a process for ex-vivo tissue preservation, a tissue disclosed by the process of the present disclosure, and a method for transplanting the tissue of the present disclosure into a subject in need of a tissue transplant.

[0019] Exemplary features of lipid-binding protein-based complexes are described in Section 6.1. Exemplary features of organ preservation solutions and processes for their production are described in Section 6.2. Exemplary features of kits and systems are described in Section 6.3. Exemplary features of ex-vivo organ and tissue preservation processes, the organs and tissues obtained thereby, and transplantation methods are described in Section 6.4.

[0020] 6.1. Lipid-binding protein-based complexes 6.1.1. HDL and HDL mimetic-based complexes In one embodiment, the lipid-binding protein-based complex comprises a HDL or HDL mimetic-based complex.For example, the complex may comprise a lipoprotein complex as described in U.S. Pat. No. 8,206,750, PCT International Publication No. WO 2012 / 109162, PCT International Publication No. WO 2015 / 173633 (e.g., CER-001) or U.S. Patent Application Publication No. 2004 / 0229794, the contents of each of which are incorporated herein by reference in their entirety.The terms "lipoprotein" and "apolipoprotein" are used interchangeably herein, and unless otherwise required by context, the term "lipoprotein" encompasses lipoprotein mimetics.The terms "lipid-binding protein" and "lipid-binding polypeptide" are also used interchangeably herein, and unless otherwise required by context, the terms do not imply a specific length of amino acid sequence.

[0021] Lipoprotein complexes can include a protein fraction (e.g., an apolipoprotein fraction) and a lipid fraction (e.g., a phospholipid fraction). The protein fraction includes one or more lipid-binding protein molecules, such as an apolipoprotein, peptide, or an analog or mimetic of an apolipoprotein peptide, e.g., one or more lipid-binding protein molecules described in Section 6.1.4.

[0022] A lipid fraction typically contains one or more phospholipids, which may be neutral, negatively charged, positively charged, or a combination thereof. Exemplary phospholipids and other amphipathic molecules that may be included in a lipid fraction are described in Section 6.1.5.

[0023] In certain embodiments, the lipid fraction contains at least one neutral phospholipid (e.g., sphingomyelin (SM)) and optionally one or more negatively charged phospholipids. In lipoprotein complexes that include both neutral and negatively charged phospholipids, the neutral and negatively charged phospholipids may have fatty acid chains with the same or different numbers of carbons and the same or different degrees of saturation. In some cases, the neutral and negatively charged phospholipids have the same acyl tail, e.g., C16:0, or palmitoyl, acyl chains. In certain embodiments, particularly those in which egg SM is used as the neutral lipid, the weight ratio of apolipoprotein fraction:lipid fraction ranges from about 1:2.7 to about 1:3 (e.g., 1:2.7).

[0024] Any phospholipid that carries at least a partial negative charge at physiological pH can be used as negatively charged phospholipid.Non-limiting examples include the negatively charged forms, such as salts, of phosphatidylinositol, phosphatidylserine, phosphatidylglycerol and phosphatidic acid.In a particular embodiment, the negatively charged phospholipid is 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], or DPPG, phosphatidylglycerol.Preferred salts include potassium salt and sodium salt.

[0025] In some embodiments, lipoprotein complexes used in the compositions and methods of the present disclosure are lipoprotein complexes as described in U.S. Pat. No. 8,206,750 or WO 2012 / 109162 (and its U.S. counterpart, U.S. Patent Application Publication No. 2012 / 0232005), the contents of each of which are incorporated herein by reference in their entireties. In certain embodiments, the protein components of the lipoprotein complex are as described in Section 6.1, preferably Section 6.1.1, of WO 2012 / 109162 (and US 2012 / 0232005), and the lipid components are as described in Section 6.2 of WO 2012 / 109162 (and US 2012 / 0232005), which may optionally be complexed together in the amounts described in Section 6.3 of WO 2012 / 109162 (and US 2012 / 0232005). The contents of each of these sections are incorporated herein by reference. In certain embodiments, the lipoprotein complexes of the present disclosure are present in a population of complexes that are at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% homogeneous, as described in Section 6.4 of WO 2012 / 109162 (and U.S. Patent Application Publication No. 2012 / 0232005), the contents of which are incorporated herein by reference.

[0026] In certain embodiments, a lipoprotein complex that can be used in the compositions and methods of the present disclosure comprises 2-4 ApoA-I equivalents, 2 molecules of charged phospholipid, 50-80 molecules of lecithin, and 20-50 molecules of SM.

[0027] In another specific embodiment, a lipoprotein complex that can be used in the compositions and methods of the present disclosure comprises 2-4 ApoA-I equivalents, 2 molecules of charged phospholipid, 50 molecules of lecithin, and 50 molecules of SM50.

[0028] In yet another specific embodiment, a lipoprotein complex that can be used in the compositions and methods of the present disclosure comprises 2-4 ApoA-I equivalents, 2 molecules of charged phospholipid, a molecule of lecithin 80, and a molecule of SM20.

[0029] In yet another specific embodiment, a lipoprotein complex that can be used in the compositions and methods of the present disclosure comprises 2-4 ApoA-I equivalents, 2 molecules of charged phospholipid, lecithin 70 molecules, and SM30 molecules.

[0030] In yet another specific embodiment, a lipoprotein complex that can be used in the compositions and methods of the present disclosure comprises 2-4 ApoA-I equivalents, 2 molecules of charged phospholipid, a molecule of lecithin 60, and a molecule of SM40.

[0031] In certain embodiments, lipoprotein complexes that can be used in the compositions and methods of the present disclosure consist essentially of 2-4 ApoA-I equivalents, 2 molecules of charged phospholipid, 50-80 molecules of lecithin, and 20-50 molecules of SM.

[0032] In another specific embodiment, a lipoprotein complex that can be used in the compositions and methods of the present disclosure consists essentially of 2-4 ApoA-I equivalents, 2 molecules of charged phospholipid, 50 molecules of lecithin, and 50 molecules of SM50.

[0033] In yet another specific embodiment, a lipoprotein complex that can be used in the compositions and methods of the present disclosure consists essentially of 2-4 ApoA-I equivalents, 2 molecules of charged phospholipid, lecithin 80 molecules, and SM20 molecules.

[0034] In yet another specific embodiment, a lipoprotein complex that can be used in the compositions and methods of the present disclosure consists essentially of 2-4 ApoA-I equivalents, 2 molecules of charged phospholipid, lecithin 70 molecules, and SM30 molecules.

[0035] In yet another specific embodiment, the lipoprotein complex that can be used in the compositions and methods of the present disclosure consists essentially of 2-4 ApoA-I equivalents, 2 molecules of charged phospholipid, lecithin 60 molecules, and SM40 molecules.

[0036] In certain embodiments, lipoprotein complexes that can be used in the compositions and methods of the present disclosure comprise a lipid component that includes about 90 to 99.8% SM by weight and about 0.2 to 10% negatively charged phospholipids by weight, e.g., about 0.2-1%, 0.2-2%, 0.2-3%, 0.2-4%, 0.2-5%, 0.2-6%, 0.2-7%, 0.2-8%, 0.2-9%, or 0.2-10% total negatively charged phospholipids (which can be complexed) by weight. In another specific embodiment, a lipoprotein complex that can be used in the methods of the present disclosure comprises about 90 to 99.8% by weight lecithin and about 0.2 to 10% by weight negatively charged phospholipids, e.g., about 0.2-1%, 0.2-2%, 0.2-3%, 0.2-4%, 0.2-5%, 0.2-6%, 0.2-7%, 0.2-8%, 0.2-9%, or 0.2-10% by weight total negatively charged phospholipids (which can be complexed).

[0037] In certain embodiments, lipoprotein complexes that can be used in the compositions and methods of the disclosure comprise a liquid component consisting essentially of about 90 to 99.8% SM and about 0.2 to 10% negatively charged phospholipids by weight, e.g., about 0.2-1%, 0.2-2%, 0.2-3%, 0.2-4%, 0.2-5%, 0.2-6%, 0.2-7%, 0.2-8%, 0.2-9%, or 0.2-10% total negatively charged phospholipids (which may be complexed) by weight. In another specific embodiment, the lipoprotein complex that can be used in the methods of the present disclosure consists essentially of about 90 to 99.8% by weight lecithin and about 0.2 to 10% by weight negatively charged phospholipids, e.g., about 0.2-1%, 0.2-2%, 0.2-3%, 0.2-4%, 0.2-5%, 0.2-6%, 0.2-7%, 0.2-8%, 0.2-9%, or 0.2-10% by weight total negatively charged phospholipids (which can be complexed).

[0038] In yet another specific embodiment, lipoprotein complexes that can be used in the compositions and methods of the present disclosure comprise a liquid fraction that comprises about 9.8 to 90% by weight SM, about 9.8 to 90% by weight lecithin, and about 0.2-10% by weight negatively charged phospholipids, e.g., about 0.2-1%, 0.2-2%, 0.2-3%, 0.2-4%, 0.2-5%, 0.2-6%, 0.2-7%, 0.2-8%, 0.2-9% to 0.2-10% by weight total negatively charged phospholipids (which can be complexed).

[0039] In yet another embodiment, a lipoprotein complex that can be used in the compositions and methods of the present disclosure comprises a liquid fraction consisting essentially of about 9.8 to 90% SM, about 9.8 to 90% lecithin, and about 0.2-10% negatively charged phospholipids, e.g., about 0.2-1%, 0.2-2%, 0.2-3%, 0.2-4%, 0.2-5%, 0.2-6%, 0.2-7%, 0.2-8%, 0.2-9% to 0.2-10% total negatively charged phospholipids (which may be complexed).

[0040] In another specific embodiment, lipoprotein complexes that can be used in the compositions and methods of the present disclosure comprise ApoA-I apolipoprotein and a lipid fraction, wherein the lipid fraction comprises sphingomyelin and about 3% by weight of negatively charged phospholipids, the molar ratio of lipid fraction to ApoA-I apolipoprotein is about 2:1 to 200:1, and the complexes are small or large discoid particles containing 2-4 ApoA-I equivalents.

[0041] In another specific embodiment, lipoprotein complexes that can be used in the compositions and methods of the present disclosure comprise ApoA-I apolipoprotein and a lipid fraction, wherein the lipid fraction consists essentially of sphingomyelin and about 3% by weight negatively charged phospholipids, the molar ratio of lipid fraction to ApoA-I apolipoprotein is about 2:1 to 200:1, and the complexes are small or large discoid particles containing 2-4 ApoA-I equivalents.

[0042] HDL-based or HDL mimetic-based complexes may contain a single type of lipid-binding protein or a mixture of two or more different lipid-binding proteins that can be derived from the same or different species.Although not essential, in order to avoid inducing immune response against therapy, the complexes preferably contain lipid-binding proteins that are derived from or correspond in amino acid to the animal species that is treated or the organ species that is preserved.Therefore, for the treatment of human patients and / or the preservation of human organs, it is preferred that lipid-binding proteins of human origin are used.The use of peptide mimetic apolipoproteins can also reduce or avoid immune response.

[0043] In some embodiments, the lipid component comprises two types of phospholipids: sphingomyelin (SM) and negatively charged phospholipids. Exemplary SM and negatively charged phospholipids are described in Section 6.1.5.1.

[0044] The lipid component comprising the SM may optionally contain small amounts of additional lipids. Virtually any type of lipid may be used, including, but not limited to, lysophospholipids, galactocerebrosides, gangliosides, cerebrosides, glycerides, triglycerides, and cholesterol and its derivatives.

[0045] When included, such optional lipids usually account for less than about 15% by weight of lipid fraction, but in some cases, more optional lipids may be included.In some embodiments, optional lipids account for less than about 10% by weight, less than about 5% by weight, or less than about 2% by weight.In some embodiments, lipid fraction does not include optional lipids.

[0046] In particular embodiments, the phospholipid fraction contains egg SM or palmitoyl SM or phytosphingomyelin and DPPG in a weight ratio (SM:negatively charged phospholipid) ranging from 90:10 to 99:1, more preferably ranging from 95:5 to 98:2. In one embodiment, the weight ratio is 97:3.

[0047] The molar ratio of lipid to protein components of the complexes of the present disclosure varies and depends, among other factors, on the identity(ies) of the apolipoprotein(s) that comprise the protein component, the identity and amount of the lipid(s) that comprise the lipid component, and the desired size of the complex. Because the biological activity of apolipoproteins such as ApoA-I is believed to be mediated by the amphipathic helices that comprise the apolipoprotein, it is convenient to express the apolipoprotein fraction of the lipid:apolipoprotein molar ratio using ApoA-I protein equivalents. It is generally accepted that ApoA-I contains 6-10 amphipathic helices, depending on the method used to calculate the helices. Other apolipoproteins can be expressed in terms of ApoA-I equivalents based on the number of amphipathic helices they contain. For example, ApoA-I, which normally exists as a disulfide-bridged dimer, is a M ApoA-I MEach molecule of apoA-I contains twice as many amphipathic helices as a molecule of ApoA-I, and therefore may be expressed as 2 ApoA-I equivalents. Conversely, a peptide apolipoprotein containing a single amphipathic helix may be expressed as 1 / 10 to 1 / 6 ApoA-I equivalents, and therefore each molecule of apoA-I contains 1 / 10 to 1 / 6 as many amphipathic helices as a molecule of ApoA-I. Generally, the lipid:ApoA-I equivalent molar ratio (defined herein as "Ri") of the lipoprotein complex ranges from about 105:1 to 110:1. In some embodiments, Ri is about 108:1. Weight ratios can be obtained using MWs of approximately 650 to 800 for phospholipids.

[0048] In some embodiments, the lipid:ApoA-I equivalent molar ratio ("RSM") ranges from about 80:1 to about 110:1, such as from about 80:1 to about 100:1. In a specific example, the RSM for the complex can be about 82:1.

[0049] In some embodiments, the lipoprotein complexes used in the compositions and methods of the disclosure are negatively charged complexes comprising a protein fraction, preferably mature, full-length ApoA-I, and a lipid fraction comprising neutral phospholipids, sphingomyelin (SM), and negatively charged phospholipids.

[0050] In certain embodiments, the lipid component contains SM (e.g., egg SM, palmitoyl SM, phyto SM, or a combination thereof) and a negatively charged phospholipid (e.g., DPPG) in a weight ratio (SM:negatively charged phospholipid) ranging from 90:10 to 99:1, more preferably ranging from 95:5 to 98:2, e.g., 97:3.

[0051] In certain embodiments, the ratio of protein component to lipid component may range from about 1:2.7 to about 1:3, with 1:2.7 being preferred. This corresponds to a molar ratio of ApoA-I protein to lipid ranging from approximately 1:90 to 1:140. In some embodiments, the molar ratio of protein to lipid in the complex is from about 1:90 to about 1:120, from about 1:100 to about 1:140, or from about 1:95 to about 1:125.

[0052] In certain embodiments, the complex comprises CER-001, CSL-111, CSL-112, CER-522 or ETC-216. In a preferred embodiment, the complex is CER-001.

[0053] CER-001 as used in the literature and in the following examples refers to the complex described in Example 4 of WO 2012 / 109162. WO 2012 / 109162 refers to CER-001 as a complex having a SM:DPPG weight:weight ratio of 97:3 and a lipoprotein weight:total phospholipid weight ratio of 1:2.7. Example 4 of WO 2012 / 109162 also describes its method of manufacture.

[0054] When used in the context of the CER-001 dosing regimen or composition of the present disclosure, CER-001 refers to a lipoprotein complex whose individual components may differ from CER-001 as described in Example 4 of WO 2012 / 109162 by up to 20%. In certain embodiments, the components of the lipoprotein complex differ from CER-001 as described in Example 4 of WO 2012 / 109162 by up to 10%. Preferably, the components of the lipoprotein complex are those described in Example 4 of WO 2012 / 109162 (+ / - acceptable manufacturing tolerance variation). The SM in CER-001 can be natural or synthetic. In some embodiments, the SM is a natural SM, such as a natural SM described in WO 2012 / 109162, such as chicken egg SM. In some embodiments, the SM is a synthetic SM, such as a synthetic SM described in WO 2012 / 109162, such as a synthetic palmitoyl sphingomyelin as described in WO 2012 / 109162. Methods for synthesizing palmitoyl sphingomyelin are known in the art, for example, as described in WO 2014 / 140787. The lipoprotein in CER-001, apolipoprotein AI (ApoA-I), preferably has an amino acid sequence corresponding to amino acids 25 to 267 of SEQ ID NO: 1 of WO 2012 / 109162 (said SEQ ID NO: 1 of WO 2012 / 109162 begins herein as SEQ ID NO: 2). ApoA-I can be purified from an animal source, particularly from a human source, or can be recombinantly produced. In a preferred embodiment, the ApoA-I in CER-001 is recombinant ApoA-I. CER-001 used in the dosing regimens of the present disclosure is preferably highly homogenous, e.g., at least 80%, at least 85%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% homogenous, as reflected by a single peak in gel permeation chromatography.See, for example, Section 6.4 of WO 2012 / 109162.

[0055] CSL-111 is reconstituted ApoA-I purified from plasma complexed with soybean phosphatidylcholine (SBPC) (Tardif et al., 2007, JAMA 297:1675-1682).

[0056] CSL-112 is a formulation of ApoA-I purified from plasma and reconstituted to form HDL suitable for intravenous infusion (Diditchenko et al., 2013, DOI 10.1161 / ATVBAHA.113.301981).

[0057] ETC-216 (also known as MDCO-216) is a recombinant ApoA-I Milano It is a lipid-depleted form of HDL that contains. See Nicholls et al., 2011, Expert Opin Biol Ther. 11(3):387-94. doi: 10.1517 / 14712598.2011.557061.

[0058] In another embodiment, the complex that can be used in the compositions and methods of the present disclosure is CER-522. CER-522 is a lipoprotein complex, CT80522, that contains a combination of three phospholipids and a 22 amino acid peptide:

[0059] [ka]

[0060] The phospholipid components of CER-522 consist of egg sphingomyelin, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (dipalmitoylphosphatidylcholine, DPPC) and 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (dipalmitoylphosphatidyl-glycerol, DPPG) in a weight ratio of 48.5:48.5:3. The peptide to total phospholipid ratio in the CER-522 complex is 1:2.5 (w / w).

[0061] In some embodiments, the lipoprotein complex is delipidated HDL. Most HDL in plasma is rich in cholesterol. Lipids in HDL can be depleted, for example partially and / or selectively, for example to reduce its cholesterol content. In some embodiments, delipidated HDL can be like small alpha, pre-beta-1, and other pre-beta forms of HDL. The process of selective depletion of HDL is described in Sacks et al., 2009, J Lipid Res. 50(5): 894-907.

[0062] In certain embodiments, the lipoprotein complex comprises a bioactive agent delivery particle as described in US 2004 / 0229794.

[0063] The bioactive agent delivery particle can include a lipid-binding polypeptide (e.g., an apolipoprotein as previously described in this section or in Section 6.1.4), a lipid bilayer (e.g., including one or more phospholipids as previously described in this section or in Section 6.1.5.1), and a bioactive agent (e.g., an anticancer agent), where the interior of the lipid bilayer includes a hydrophobic region and the bioactive agent is associated with the hydrophobic region of the lipid bilayer. In some embodiments, the bioactive agent delivery particle is described in US 2004 / 0229794.

[0064] In some embodiments, the bioactive agent delivery particles do not include a hydrophilic core.

[0065] In some embodiments, the bioactive agent delivery particles are disc-shaped (eg, having a diameter of about 7 to about 29 nm).

[0066] The bioactive agent delivery particle comprises a lipid bilayer-forming lipid, e.g., a phospholipid (e.g., as previously described in this section or in Section 6.1.5.1). In some embodiments, the bioactive agent delivery particle comprises both bilayer-forming and non-bilayer-forming lipids. In some embodiments, the lipid bilayer of the bioactive agent delivery particle comprises a phospholipid. In one embodiment, the phospholipid incorporated in the delivery particle comprises dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG). In one embodiment, the phospholipid layer comprises DMPC and DMPG in a molar ratio of 7:3.

[0067] In some embodiments, the lipid-binding polypeptide is an apolipoprotein (e.g., as described in this section or in section 6.1.4). The main interaction between the lipid-binding polypeptide, e.g., apolipoprotein molecule, and the lipid bilayer is generally a hydrophobic interaction between the residues of the hydrophobic face of the lipid-binding polypeptide's amphipathic structure, the α-helix, and the fatty acyl residues of the lipid on the outer face at the periphery of the particle. The bioactive agent delivery particle may include exchangeable and / or non-exchangeable apolipoproteins. In some embodiments, the lipid-binding polypeptide is ApoA-I.

[0068] In some embodiments, the bioactive agent delivery particles include lipid-binding polypeptide molecules, such as apolipoprotein molecules, that have been modified to enhance the stability of the particles. In one embodiment, the modification includes the introduction of cysteine ​​residues to form intra- and / or intermolecular disulfide bonds.

[0069] In another embodiment, the bioactive agent delivery particle comprises a chimeric lipid-binding polypeptide molecule, e.g., a chimeric apolipoprotein molecule, with one or more binding functional moieties, e.g., one or more targeting moieties and / or one or more moieties having a desired biological activity, e.g., antimicrobial activity, that can enhance or function synergistically with the activity of the bioactive agent incorporated into the delivery particle.

[0070] 6.1.2.Apomer-based complexes In one aspect, the lipid-binding protein-based complex that can be used in the method and composition of the present disclosure comprises Apomer.The features of Apomer that can be included in Apomer-based complex are described in International Publication No. WO2019 / 030575, the contents of which are incorporated herein by reference in their entirety.

[0071] Apomers generally include apolipoproteins in monomeric or multimeric form complexed with amphiphilic molecules. Generally, apomers include one or more apolipoprotein molecules, each complexed with one or more amphiphilic molecules. In certain embodiments, the amphiphilic molecules together impart a net charge of at least +1 or -1 per apolipoprotein molecule in the apomer. Exemplary apolipoprotein molecules that can be used in apomers are described in Section 6.1.4.1. Exemplary amphiphilic molecules are described in Section 6.1.5.

[0072] Cargomer-based Complexes In one embodiment, the lipid-binding protein-based complex that can be used in the methods and compositions of the present disclosure comprises Cargomer, which is a lipid-binding protein-based complex having one or more cargo moieties.The characteristics of Cargomer that can be included in Cargomer-based complexes are described in WO2019 / 030574, the contents of which are incorporated herein by reference in their entirety.

[0073] Cargomers generally include a monomeric or multimeric form of apolipoprotein (e.g., 2, 4, or 8 apolipoprotein molecules) and one or more cargo moieties. The cargo moieties may be amphipathic or non-amphipathic. An amphipathic cargo moiety may solubilize the apolipoprotein and prevent it from aggregating. If the cargo moiety is not amphipathic or is insufficient to solubilize the apolipoprotein molecule(s), the Cargomer may also include one or more additional amphipathic molecules to solubilize the apolipoprotein. Thus, reference to an amphipathic molecule in the context of a Cargomer includes an amphipathic molecule that is a cargo moiety, an amphipathic molecule that is not a cargo moiety, or some combination thereof. Preferably, the Cargomer is not discoid, for example, as determined using NMR spectroscopy.

[0074] Cargo moieties may include biologically active molecules (e.g., drugs, biologics, and / or immunogens) or other agents, such as agents used in diagnostics. As used herein, the terms "molecule" and "agent" also include complexes and conjugates (e.g., antibody-drug conjugates). Terms such as "biologically active," "diagnostically useful," and the like are not limited to substances with direct pharmacological or biological activity, but may include substances that become active after administration, for example, due to metabolism of a prodrug or cleavage of a linker. Thus, the terms "biologically active" and "diagnostically useful" also include substances that become biologically active or diagnostically useful after administration through the generation or metabolic or other cleavage products that exert a pharmacological or biological effect and / or are detectable in a diagnostic test.

[0075] The amphipathic molecule in the Cargomer can solubilize the apolipoprotein and / or reduce or minimize apolipoprotein aggregation, and can have other functions in the Cargomer. For example, the amphipathic molecule can have a therapeutic utility and thus be a cargo moiety intended for delivery by the Cargomer upon administration to a subject. Additionally, as discussed in Section 6.1.5 below, the amphipathic molecule can be used to anchor a non-amphipathic cargo moiety in the Cargomer to an apolipoprotein. Thus, in some embodiments, the cargo moiety and the amphipathic molecule in the Cargomer are the same. In other embodiments, the anchor moiety and the amphipathic molecule in the Cargomer are the same. In still other embodiments, the cargo moiety, anchor moiety and the amphipathic molecule in the Cargomer are the same (e.g., when the amphipathic molecule has a therapeutic activity and also anchors another biologically active molecule to the apolipoprotein molecule(s)).

[0076] The anchor and / or linker moieties are particularly useful for Cargomers that have cargo moieties that are not amphipathic molecules.

[0077] In some embodiments, at least one of the cargo moieties, a majority of the cargo moieties, or all of the cargo moieties in a Cargomer of the present disclosure are coupled to the Cargomer via an anchor. In some embodiments, at least one of the cargo moieties in a Cargomer are coupled to the Cargomer via an anchor. In some embodiments, a majority of the cargo moieties in a Cargomer are coupled to the Cargomer via an anchor. In some embodiments, all of the cargo moieties in a Cargomer are coupled to the Cargomer via an anchor. Each of the anchors in a Cargomer may be the same, or different types of anchors may be included in a single Cargomer (e.g., one type of cargo moiety may be coupled to the Cargomer via one type of anchor and a second type of cargo moiety may be coupled to the Cargomer via a second type of anchor).

[0078] In certain aspects, the amphipathic molecule, the cargo, and, if present, the anchor and / or linker together contribute a net charge of at least +1 or -1 (e.g., +1, +2, +3, -1, -2, or -3) per apolipoprotein molecule in the Cargomer. In some embodiments, the net charge is a negative charge. In other embodiments, the net charge is a positive charge. Charges are measured at physiological pH, unless the context requires otherwise.

[0079] The molar ratio of apolipoprotein molecules to amphipathic molecules in a Cargomer can be an integer or can reflect a 1:1 relationship between apolipoproteins and amphipathic molecules, but need not necessarily be. By way of example and without limitation, a Cargomer can have a molar ratio of apolipoprotein to amphipathic molecules of 2:5, 8:7, 3:2, or 4:7.

[0080] In some embodiments, the Cargomer is 8:1 to 1:15 (e.g., 8:1 to 1:15, 7:1 to 1:15, 6:1 to 1:15, 5:1 to 1:15, 4:1 to 1:15, 3:1 to 1:15, 2:1 to 1:15, 1:1 to 1:15, 8:1 to 1:14, 7:1 to 1:14, 6:1 to 1:14, 5:1 to 1:14, 4:1 to 1:14, 3:1 to 1:14, 2:1 to 1:14, 1:1 to 1:14, 8:1 to 1:13, 7:1 to 1:13, 6:1 to 1:13, 5:1 to 1:13, 4:1 to 1:13, 3:1 to 1 :13, 2:1 to 1:13, 1:1 to 1:13, 8:1 to 1:12, 7:1 to 1:12, 6:1 to 1:12, 5:1 to 1:12, 4:1 to 1:12, 3:1 to 1:12, 2:1 to 1:12, 1:1 to 1:12, 8:1 to 1:11, 7:1 to 1:11, 6:1 to 1:11, 5:1 to 1:11, 4:1 to 1:11, 3:1 to 1:11, 2:1 to 1:11, 1:1 to 1:11, 8:1 to 1:10, 7:1 to 1:10, 6:1 to 1:10, 5:1 to 1:10, 4:1 to 1:10, 3:1 to 1:10, 2:1 to 1 :10, 1:1 to 1:10, 8:1 to 1:9, 7:1 to 1:9, 6:1 to 1:9, 5:1 to 1:9, 4:1 to 1:9, 3:1 to 1:9, 2:1 to 1:9, 1:1 to 1:9, 8:1 to 1:8, 7:1 to 1:8, 6:1 to 1:8, 5:1 to 1:8, 4:1 to 1:8, 3:1 to 1:8, 2:1 to 1:8, 1:1 to 1:8, 8:1 to 1:7, 7:1 to 1:7, 6:1 to 1:7, 5:1 to 1:7, 4:1 to 1:7, 3:1 to 1:7, 2:1 to 1:7, 1:1 to 1:7, 8:1 to 1:6, 7:1 to 1:6, 6: 1 to 1:6, 5:1 to 1:6, 4:1 to 1:6, 3:1 to 1:6, 2:1 to 1:6, 1:1 to 1:6, 8:1 to 1:5, 7:1 to 1:5, 6:1 to 1:5, 5:1 to 1:5, 4:1 to 1:5, 3:1 to 1:5, 2:1 to 1:5, 1:1 to 1:5, 8:1 to 1:4, 7:1 to 1:4, 6:1 to 1:4, 5:1 to 1:4, 4:1 to 1:4, 3:1 to 1:4, 2:1 to 1:4, 1:1 to 1:4, 8:1 to 1:3, 7:1 to 1:3, 6:1 to 1:3, 5:1 to 1:3, 4:1 to 1:3, 3:1 to 1:3,the apolipoprotein molecule complexed with the amphiphilic molecule in a molar ratio of apolipoprotein:amphiphilic molecule ranging from 2:1 to 1:3, 1:1 to 1:3, 8:1 to 1:2, 7:1 to 1:2, 6:1 to 1:2, 5:1 to 1:2, 4:1 to 1:2, 3:1 to 1:2, 2:1 to 1:2, 1:1 to 1:2, 8:1 to 1:1, 7:1 to 1:1, 6:1 to 1:1, 5:1 to 1:1, 4:1 to 1:1, 3:1 to 1:1, or 2:1 to 1:1;

[0081] In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule in the Cargomer ranges from 6:1 to 1:6. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 5:1 to 1:6. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 4:1 to 1:6. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 3:1 to 1:6. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 2:1 to 1:6. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 5:1 to 1:5. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 4:1 to 1:5. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 3:1 to 1:5. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 2:1 to 1:5. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 5:1 to 1:4. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 4:1 to 1:4. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 3:1 to 1:4. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 2:1 to 1:4. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 5:1 to 1:3. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 4:1 to 1:3. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 3:1 to 1:3. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 2:1 to 1:3. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 5:1 to 1:2. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 4:1 to 1:2. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 3:1 to 1:2. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 2:1 to 1:2.In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 5:1 to 1:1. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 4:1 to 1:1. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 3:1 to 1:1. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 2:1 to 1:1. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 1:1 to 1:6. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 1:1 to 1:5. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 1:1 to 1:4. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 1:1 to 1:3. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 1:1 to 1:2. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 1:2 to 1:6. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 1:2 to 1:5. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 1:2 to 1:4. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 1:2 to 1:3. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 1:3 to 1:6. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 1:3 to 1:5. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 1:3 to 1:4. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 1:4 to 1:6. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 1:4 to 1:5. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 1:5 to 1:6. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 1.5:1 to 1:2. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 5:4 to 4:5.In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 5:3 to 3:5. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 5:2 to 2:5. In some embodiments, the molar ratio of apolipoprotein to amphipathic molecule ranges from 3:2 to 2:3.

[0082] In some embodiments, the ratio of apolipoprotein molecules to amphipathic molecules is about 1:1. In other embodiments, the ratio of apolipoprotein molecules to amphipathic molecules is about 1:2. In still other embodiments, the ratio of apolipoprotein molecules to amphipathic molecules is about 1:3. In still other embodiments, the ratio of apolipoprotein molecules to amphipathic molecules is about 1:4. In still other embodiments, the ratio of apolipoprotein molecules to amphipathic molecules is about 1:5. In still other embodiments, the ratio of apolipoprotein molecules to amphipathic molecules is about 1:6.

[0083] In some embodiments, the Cargomer comprises one apolipoprotein molecule.

[0084] In other embodiments, Cargomer comprises two apolipoprotein molecules. Cargomer comprising two apolipoprotein molecules preferably has a Stokes radius of 3 nm or less. In some embodiments, Cargomer may comprise two apolipoprotein molecules and one, two, or three negatively charged amphiphilic molecules (e.g., negatively charged phospholipid molecules) per apolipoprotein molecule.

[0085] In other embodiments, Cargomer comprises 4 apolipoprotein molecules. Cargomer comprising 4 apolipoprotein molecules preferably has a Stokes radius of 4 nm or less. In some embodiments, Cargomer may comprise 4 apolipoprotein molecules and 1, 2, or 3 negatively charged amphiphilic molecules (e.g., negatively charged phospholipid molecules) per apolipoprotein molecule.

[0086] In other embodiments, Cargomer comprises 8 apolipoprotein molecules. Cargomer comprising 4 apolipoprotein molecules preferably has a Stokes radius of 5 nm or less. In some embodiments, Cargomer may comprise 8 apolipoprotein molecules and 1, 2, or 3 negatively charged amphiphilic molecules (e.g., negatively charged phospholipid molecules) per apolipoprotein molecule. In certain embodiments, Cargomer of the present disclosure does not contain cholesterol and / or cholesterol derivatives (e.g., cholesterol esters).

[0087] In some embodiments, the Cargomer comprises a weight ratio of apolipoprotein to phospholipid ranging from about 1:2 to about 1:3.

[0088] In some embodiments, the Cargomer comprises a weight ratio of apolipoprotein to phospholipid in the range of 1:2.7.

[0089] The Cargomer may be soluble in one or more of biological fluids, such as lymph, cerebrospinal fluid, vitreous fluid, aqueous humor, and blood or a blood fraction (eg, serum or plasma).

[0090] Cargomers may contain targeting functionality, for example, to target Cargomers to specific cell or tissue types. In some embodiments, Cargomers contain targeting moieties attached to apolipoprotein molecules or amphiphilic molecules. In some embodiments, one or more cargo moieties incorporated into Cargomers have targeting capabilities.

[0091] 6.1.4. Lipid-binding protein molecules Lipid-binding protein molecules that can be used in the complexes described herein include apolipoproteins, such as those described in Section 6.1.4.1, and apolipoprotein mimetic peptides, such as those described in Section 6.1.4.2. In some embodiments, the complexes include a mixture of lipid-binding protein molecules. In some embodiments, the complexes include a mixture of one or more lipid-binding protein molecules and one or more apolipoprotein mimetic peptides.

[0092] In some embodiments, the complex comprises 1 to 8 ApoA-I equivalents (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 8, 2 to 6, 2 to 4, 4 to 6, or 4 to 8 ApoA-I equivalents). Lipid binding proteins can be expressed in terms of ApoA-I equivalents based on the number of amphipathic helices they contain. For example, ApoA-I, which normally exists as a disulfide-bridged dimer, can be expressed in terms of ApoA-I equivalents based on the number of amphipathic helices it contains. M ApoA-I M Each molecule of a peptidomimetic contains twice as many amphipathic helices as a molecule of ApoA-I and therefore can be expressed as 2 ApoA-I equivalents. Conversely, a peptidomimetic containing a single amphipathic helix can be expressed as 1 / 10 to 1 / 6 ApoA-I equivalents, since each molecule contains 1 / 10 to 1 / 6 as many amphipathic helices as a molecule of ApoA-I.

[0093] Apolipoproteins Suitable apolipoproteins that may be included in the lipid-binding protein-based complex include apolipoproteins ApoA-I, ApoA-II, ApoA-IV, ApoA-V, ApoB, ApoC-I, ApoC-II, ApoC-III, ApoD, ApoE, ApoJ, ApoH, and any combination of two or more of the foregoing. Polymorphic forms, isoforms, variants and mutants, as well as truncated forms of the foregoing apolipoproteins, the most common of which is apolipoprotein AI. Milano(ApoA-I M ), apolipoprotein AI Paris (ApoA-I P ), and apolipoprotein AI Zaragoza (ApoA-I Z )) can also be used. Apolipoprotein mutants containing cysteine ​​residues are also known and can be used similarly (see, e.g., U.S. Patent Application Publication No. 2003 / 0181372). Apolipoproteins can exist in the form of monomers or dimers, which can be homodimers or heterodimers. For example, ApoA-I (Duverger et al., 1996, Arterioscler. Thromb. Vasc. Biol. 16(12):1424-29), ApoA-I M (Franceschini et al., 1985, J. Biol. Chem. 260:1632-35), ApoA-I p (Daum et al., 1999, J. Mol. Med. 77:614-22), ApoA-II (Shelness et al., 1985, J. Biol. Chem. 260(14):8637-46;Shelness et al., 1984, J. Biol. Chem. 259(15):9929-35), ApoA-IV (Duverger et al., 1991, Euro. J. Biochem. 201(2):373-83), ApoE (McLean et al., 1983, J. Biol. Chem. 258(14):8993-9000), ApoJ and homo- and heterodimers (where feasible) of ApoH may be used.

[0094] Apolipoproteins can be modified in their primary sequence to make them less susceptible to oxidation, for example, as described in US Patent Application Publication Nos. 2008 / 0234192 and 2013 / 0137628, and US Patent Nos. 8,143,224 and 8,541,236. Apolipoproteins can contain residues that correspond to elements that facilitate their isolation, such as His tags, or other elements designed for other purposes. Preferably, the apolipoproteins in the complex are soluble in biological fluids, such as lymph, cerebrospinal fluid, vitreous humor, aqueous humor, and blood, or blood fractions, such as serum or plasma.

[0095] In some embodiments, the complex comprises a covalently linked lipid-binding protein monomer, such as a dimeric apolipoprotein ApoA-I, which is a mutant form of ApoA-I that contains cysteines. Milano The cysteines allow the formation of disulfide bridges that can lead to the formation of homodimers or heterodimers (e.g., ApoA-I Milano-ApoA-II).

[0096] In some embodiments, the apolipoprotein molecule comprises an ApoA-I, ApoA-II, ApoA-IV, ApoA-V, ApoB, ApoC-I, ApoC-II, ApoC-III, ApoD, ApoE, ApoJ, or ApoH molecule or a combination thereof.

[0097] In some embodiments, the apolipoprotein molecule comprises or consists of an ApoA-I molecule. In some embodiments, the ApoA-I molecule is a human ApoA-I molecule. In some embodiments, the ApoA-I molecule is recombinant. In some embodiments, the ApoA-I molecule is an ApoA-I Milano isn't it.

[0098] In some embodiments, the ApoA-I molecule is apolipoprotein AI Milano (ApoA-IM), apolipoprotein AI Paris(ApoA-IP), or apolipoprotein AI Zaragoza (ApoA-IZ) molecule.

[0099] Apolipoproteins can be purified from animal sources (particularly from human sources) or recombinantly produced as known to those skilled in the art. See, e.g., Chung et al., 1980, J. Lipid Res. 21(3):284-91; Cheung et al., 1987, J. Lipid Res. 28(8):913-29. See also U.S. Patent Nos. 5,059,528, 5,128,318, 6,617,134; U.S. Patent Application Publication Nos. 2002 / 0156007, 2004 / 0067873, 2004 / 0077541, and 2004 / 0266660; and PCT Publication Nos. WO 2008 / 104890 and WO 2007 / 023476. Other purification methods are also possible, for example as described in PCT Publication No. WO 2012 / 109162, the disclosure of which is incorporated herein by reference in its entirety.

[0100] Apolipoproteins can be present in prepro, pro, or mature forms. For example, the complex can include ApoA-I (e.g., human ApoA-I) where ApoA-I is preproApoA-I, proApoA-I, or mature ApoA-I. In some embodiments, the complex includes ApoA-I with at least 90% sequence identity to SEQ ID NO:1.

[0101] [ka]

[0102] In other embodiments, the complex comprises an ApoA-I having at least 95% sequence identity to SEQ ID NO: 1. In other embodiments, the complex comprises an ApoA-I having at least 98% sequence identity to SEQ ID NO: 1. In other embodiments, the complex comprises an ApoA-I having at least 99% sequence identity to SEQ ID NO: 1. In other embodiments, the complex comprises an ApoA-I having 100% sequence identity to SEQ ID NO: 1.

[0103] In other embodiments, the complex comprises an ApoA-I having at least 95% sequence identity to amino acids 25 to 267 of SEQ ID NO:2. In other embodiments, the complex comprises an ApoA-I having at least 98% sequence identity to amino acids 25 to 267 of SEQ ID NO:2. In other embodiments, the complex comprises an ApoA-I having at least 99% sequence identity to amino acids 25 to 267 of SEQ ID NO:2. In other embodiments, the complex comprises an ApoA-I having at least 100% sequence identity to amino acids 25 to 267 of SEQ ID NO:2.

[0104] In some embodiments, the complex comprises 1 to 8 apolipoprotein molecules (e.g., 1 to 6, 1 to 4, 1 to 2, 2 to 8, 2 to 6, 2 to 4, 4 to 8, 4 to 6, or 6 to 8 apolipoprotein molecules). In some embodiments, the complex comprises 1 apolipoprotein molecule. In some embodiments, the complex comprises 2 apolipoprotein molecules. In some embodiments, the complex comprises 3 apolipoprotein molecules. In some embodiments, the complex comprises 4 apolipoprotein molecules. In some embodiments, the complex comprises 5 apolipoprotein molecules. In some embodiments, the complex comprises 6 apolipoprotein molecules. In some embodiments, the complex comprises 7 apolipoprotein molecules. In some embodiments, the complex comprises 8 apolipoprotein molecules.

[0105] The apolipoprotein molecule(s) may include chimeric apolipoproteins that include an apolipoprotein and one or more attached functional moieties, such as, for example, one or more CRN-001 conjugate(s), one or more targeting moieties, a moiety with a desired biological activity, an affinity tag to aid in purification, and / or a reporter molecule for characterization or localization studies. The attached moiety with biological activity may have an activity that can enhance and / or synergize with the biological activity of the compound or cargo moiety incorporated into the conjugate of the present disclosure. For example, the moiety with biological activity may have antimicrobial (e.g., antifungal, antibacterial, antiprotozoal, bacteriostatic, fungistatic, or antiviral) activity. In one embodiment, the attached functional moiety of the chimeric apolipoprotein does not contact a hydrophobic surface of the conjugate. In another embodiment, the attached functional moiety contacts a hydrophobic surface of the conjugate. In some embodiments, the functional moiety of the chimeric apolipoprotein may be inherent to the native protein. In some embodiments, the chimeric apolipoprotein comprises a ligand or sequence capable of being recognized by or interacting with a cell surface receptor or other cell surface moiety.

[0106] In one embodiment, the chimeric apolipoprotein comprises a targeting moiety that is not native to the natural apolipoprotein, such as, for example, S. cerevisiae α-mating factor peptide, folic acid, transferrin, or lactoferrin. In another embodiment, the chimeric apolipoprotein comprises a moiety that has a desired biological activity that enhances and / or synergizes with the activity of the compound or cargo moiety incorporated into the complex of the present disclosure. In one embodiment, the chimeric apolipoprotein may comprise an apolipoprotein native functional moiety. One example of an apolipoprotein native functional moiety is the native targeting moiety formed by approximately amino acids 130-150 of human ApoE, which contains the receptor binding region recognized by members of the low density lipoprotein receptor family. Other examples of apolipoprotein native functional moieties include the region of ApoB-100 that interacts with the low density lipoprotein receptor and the region of ApoA-I that interacts with the scavenger receptor type B1. In other embodiments, functional moieties can be synthetically or recombinantly added to produce chimeric apolipoproteins. Another example is an apolipoprotein that has a prepro or pro sequence from another preproapolipoprotein (e.g., a prepro sequence from preproapoA-II is substituted for the prepro sequence of preproapoA-I). Another example is an apolipoprotein in which some of the amphipathic sequence segments are replaced by other amphipathic sequence segments from another apolipoprotein.

[0107] As used herein, "chimera" refers to two or more molecules that can exist individually and are linked together to form a single molecule that has all the desired functionality of the component molecules. The component molecules of a chimeric molecule can be synthetically linked by chimeric conjugation, or, if the component molecules are all polypeptides or analogs thereof, the polynucleotides encoding the polypeptides can be recombinantly fused together so that a single continuous polypeptide is expressed. Such chimeric molecules are referred to as fusion proteins. A "fusion protein" is a chimeric molecule in which the component molecules are all polypeptides and are linked (fused) together so that the chimeric molecule forms a continuous single chain. The various components can be directly linked together or coupled through one or more linkers. One or more segments of the various components can be inserted, for example, in the sequence of the apolipoprotein, or in another example, added at the N-terminus or C-terminus to the sequence of the apolipoprotein. For example, the fusion protein can include an antibody light chain, an antibody fragment, a heavy chain antibody, or a single domain antibody.

[0108] In some embodiments, the chimeric apolipoprotein is prepared by chemically conjugating the apolipoprotein and the functional moiety to be attached. Means for chemically conjugating molecules are well known to those skilled in the art. Such means vary according to the structure of the moiety to be attached, but are easily ascertainable by those skilled in the art. Polypeptides usually contain a variety of functional groups, such as carboxylic acid (--COOH), free amino (--NH2), or sulfhydryl (--SH group), that are available for reaction with the appropriate functional group on the functional moiety or on the linker to attach the moiety to it. The functional moiety can be attached to a functional group at the N-terminus, C-terminus, or on an internal residue (i.e., a residue located midway between the N-terminus and C-terminus) of the apolipoprotein molecule. Alternatively, the apolipoprotein and / or the moiety to be tagged can be derivatized to expose or attach additional reactive functional groups.

[0109] In some embodiments, fusion proteins that contain polypeptide functional moieties are synthesized using recombinant expression systems. Typically, this involves creating a nucleic acid (e.g., DNA) sequence that codes for an apolipoprotein and a functional moiety such that the two polypeptides are in frame when expressed, placing the DNA under the control of a promoter, expressing the protein in a host cell, and isolating the expressed protein.

[0110] The nucleic acid encoding the chimeric apolipoprotein can be incorporated into a recombinant expression vector in a form suitable for expression in a host cell. As used herein, an "expression vector" is a nucleic acid that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. The vector can also contain regulatory sequences such as promoters, enhancers, or other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are known to those skilled in the art (see, for example, Goeddel, 1990, Gene Expression Technology: Meth. Enzymol. 185, Academic Press, San Diego, Calif.; Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology 152 Academic Press, Inc., San Diego, Calif.; Sambrook et al., 1989, Molecular Cloning--A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY, etc.).

[0111] In some embodiments, the apolipoprotein is modified such that when the apolipoprotein is incorporated into the complex of the present disclosure, the modification enhances the stability of the complex, confers targeting ability, or increases the capacity. In one embodiment, the modification includes the introduction of a cysteine ​​residue into the apolipoprotein molecule to allow the formation of intramolecular or intermolecular disulfide bonds, for example, by site-directed mutagenesis. In another embodiment, a chimeric crosslinker is used to form an intercellular link between apolipoprotein molecules to enhance the stability of the complex. The intermolecular crosslink prevents or reduces the dissociation of the apolipoprotein molecule from the complex and / or prevents replacement by endogenous apolipoprotein molecules in the individual to whom the complex is administered. In other embodiments, the apolipoprotein is modified by chemical derivatization of one or more amino acid residues or by site mutagenesis to confer targeting ability to or recognition by a cell surface receptor.

[0112] The complex can be targeted to a specific cell surface receptor by engineering receptor recognition properties into the apolipoprotein. For example, the complex can be targeted to a specific cell type known to carry a specific type of infectious agent by modifying the apolipoprotein to enable it to interact with a receptor on the surface of the targeted cell type. For example, the complex can be targeted to macrophages by altering the apolipoprotein to confer recognition by macrophage endocytic class A scavenger receptor (SR-A). SR-A binding ability can be conferred to the complex by modifying the apolipoprotein to replace one or more positively charged amino acids with neutral or negatively charged amino acids by site-directed mutagenesis. SR-A recognition can also be conferred by preparing chimeric apolipoproteins that contain N- or C-terminal extensions with ligands recognized by SR-A or amino acid sequences with a high concentration of negatively charged residues. Complexes containing apolipoproteins can also interact with apolipoprotein receptors, such as, but not limited to, the ABCA1 receptor, the ABCG1 receptor, megalin, cubulin, and HDL receptors, such as SR-B1.

[0113] The conjugate may include a lipid binding protein (e.g., an apolipoprotein molecule) that anchors the cargo moiety to the Cargomer. In some embodiments, the apolipoprotein molecule is coupled to the cargo moiety by a direct bond. In other embodiments, the apolipoprotein molecule is coupled to the cargo moiety by a linker, e.g., as described in Section 6.1.7.

[0114] 6.1.4.2. Apolipoprotein Mimetics Peptides, peptide analogs, and agonists that mimic the activity of apolipoproteins (collectively referred to herein as "apolipoprotein peptidomimetics") can also be used in the conjugates described herein, either alone or in combination with one or more other lipid-binding proteins. Peptides and peptide analogs corresponding to apolipoproteins suitable for inclusion in the conjugates and compositions described herein include ApoA-I, ApoA-I, and ApoA-I. M Non-limiting examples of agonists that mimic the activity of ApoA-II, ApoA-IV, and ApoE are described in U.S. Pat. Nos. 6,004,925, 6,037,323, and 6,046,166 (Dasseux et al.), U.S. Pat. No. 5,840,688 (issued to Tso), U.S. Pat. No. 6,743,778 (issued to Kohno), U.S. Patent Application Publication Nos. 2004 / 0266671, 2004 / 0254120, and 2003 / 0171277. and US Patent Publication No. 2003 / 0045460 (to Fogelman), US Patent Publication No. 2006 / 0069030 (to Bachovchin), US Patent Publication No. 2003 / 0087819 (to Bielicki), US Patent Publication No. 2009 / 0081293 (to Murase et al.), and PCT Publication No. WO 2010 / 093918 (to Dasseux et al.), the disclosures of which are incorporated herein by reference in their entirety. These peptides and peptide analogs may be composed of L- or D-amino acids or mixtures of L- and D-amino acids. These peptides and peptide analogs may also contain one or more non-peptide or amide bonds, such as one or more well-known peptide / amide isosteres. Such apolipoprotein peptide mimetics can be synthesized or produced using any technique for peptide synthesis known in the art, including, for example, the techniques described in U.S. Pat. Nos. 6,004,925, 6,037,323 and 6,046,166.

[0115] In some embodiments, the lipid binding protein molecule comprises an apolipoprotein peptide mimetic molecule and optionally one or more apolipoprotein molecules, such as those described above.

[0116] In some embodiments, the apolipoprotein peptidomimetic molecule comprises an ApoA-I peptidomimetic, an ApoA-II peptidomimetic, an ApoA-IV peptidomimetic, an ApoA-I peptidomimetic, or an ApoE peptidomimetic, or a combination thereof.

[0117] The conjugates of the present disclosure may include an apolipoprotein peptide mimetic molecule that anchors a cargo moiety to the conjugate. In some embodiments, the apolipoprotein peptide mimetic is coupled to the cargo moiety by a direct bond. In other embodiments, the apolipoprotein peptide mimetic molecule is coupled to the cargo moiety by a linker, e.g., as described in Section 6.1.7.

[0118] 6.1.5. Amphiphilic molecules Amphipathic molecules are molecules that possess both hydrophobic (non-polar) and hydrophilic (polar) elements. Amphipathic molecules that can be used in the complexes described herein include lipids (e.g., as described in Section 6.1.5.1), detergents (e.g., as described in 6.1.5.2.), fatty acids (e.g., as described in Section 6.1.5.3), and non-polar molecules covalently bound to polar molecules, such as, but not limited to, sugars or nucleic acids, and sterols (e.g., as described in Section 6.1.5.4).

[0119] The complexes can contain a single type of amphipathic molecule (e.g., a single type of phospholipid or a mixture of phospholipids) or can contain a combination of types of amphipathic molecules (e.g., phospholipids and detergents). The complexes can contain one type of amphipathic molecule or a combination of amphipathic molecules designed to facilitate solubilization of the lipid-binding protein molecule(s).

[0120] In some embodiments, the Apomer and / or Cargomer-based complexes contain only a sufficient amount of amphipathic molecules to solubilize the lipid-binding protein molecule. In other words, the Apomer and / or Cargomer-based complexes may contain the minimum amount of one or more amphipathic molecules necessary to solubilize the lipid-binding protein molecule.

[0121] In some embodiments, the amphiphilic molecules included include phospholipids, detergents, fatty acids, apolar moieties covalently attached to sugars or sterols, or combinations thereof (e.g., selected from the types of amphiphilic molecules discussed above).

[0122] In some embodiments, the amphipathic molecules comprise or consist of phospholipid molecules. In some embodiments, the phospholipid molecules comprise negatively charged phospholipids, neutral phospholipids, positively charged phospholipids, or combinations thereof. In some embodiments, the phospholipid molecules contribute 1-3 net charges per apolipoprotein molecule in the complex. In some embodiments, the net charge is a negative net charge. In some embodiments, the net charge is a positive net charge. In some embodiments, the phospholipid molecules consist of a combination of negatively charged phospholipids and neutral phospholipids. In some embodiments, the molar ratio of negatively charged phospholipids to neutral phospholipids ranges from 1:1 to 1:3. In some embodiments, the molar ratio of negatively charged phospholipids to neutral phospholipids ranges from about 1:1 to about 1:2.

[0123] In some embodiments, the complex comprises at least one amphipathic molecule that is an anchor.

[0124] In some embodiments, the amphipathic molecules comprise neutral phospholipids and negatively charged phospholipids in a weight ratio of 95:5 to 99:1.

[0125] Lipids A lipid-binding protein-based complex may include one or more lipids. In various embodiments, the one or more lipids may be saturated and / or unsaturated, natural and / or synthetic, charged or uncharged zwitterionic, or not. In some embodiments, lipid molecules (e.g., phospholipid molecules) together may contribute 1-3 (e.g., 1-3, 1-2, 2-3, 1, 2, or 3) net charges per lipid-binding protein molecule in the complex. In some embodiments, the net charge is negative. In other embodiments, the net charge is positive.

[0126] In some embodiments, the lipid comprises a phospholipid. The phospholipid can have two acyl chains that are the same or different (e.g., chains with different numbers of carbon atoms, different degrees of saturation between acyl chains, different branching of acyl chains, or combinations thereof). The lipid can also be modified to contain a fluorescent probe (e.g., as described in avantilipids.com / product-category / products / fluorescent-lipids / ). Preferably, the lipid comprises at least one phospholipid.

[0127] Phospholipids can have unsaturated or saturated acyl chains ranging from about 6 to about 24 carbon atoms (e.g., 6-20, 6-16, 6-12, 12-24, 12-20, 12-16, 16-24, 16-20, or 20-24). In some embodiments, phospholipids used in the conjugates of the present disclosure have one or two acyl chains (e.g., two acyl chains of the same length or two acyl chains of different lengths) of 12, 14, 16, 18, 20, 22, or 24 carbon atoms.

[0128] Non-limiting examples of acyl chains present in commonly occurring fatty acids that may be included in phospholipids are provided in Table 1 below:

[0129] [Table 1]

[0130] Lipids that may be present in the complexes of the present disclosure include small alkyl chain phospholipids, egg phosphatidylcholine, soy phosphatidylcholine, dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidylcholine, distearyl phosphatidylcholine, 1-myristoyl-2-palmitoyl phosphatidylcholine, 1-palmitoyl-2-myristoyl phosphatidylcholine, 1-palmitoyl-2-stearoyl phosphatidylcholine, 1-stearoyl-2-palmito ... phosphatidylcholine, dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, dilauroylphosphatidylglycerol phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerols, diphosphatidylglycerols, e.g. dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, di Oleoylphosphatidylglycerol, Dimyristoylphosphatidic acid, Dipalmitoylphosphatidic acid, Dimyristoylphosphatidylethanolamine, Dipalmitoylphosphatidylethanolamine, Dimyristoylphosphatidylserine, Dipalmitoylphosphatidylserine, Brain phosphatidylserine, Brain sphingomyelin, Palmitoyl sphingomyelin, Dipalmitoyl sphingomyelin, Egg sphingomyelin, Milk sphingomyelin, F These lipids include, but are not limited to, phytosphingomyelin, distearoylsphingomyelin, dipalmitoylphosphatidylglycerol salts, phosphatidic acid, galactocerebroside, gangliosides, cerebrosides, dilaurylphosphatidylcholine, (1,3)-D-mannosyl-(1,3) diglyceride, aminophenylglycosides, 3-cholesteryl-6'-(glycosylthio)hexyl ether glycolipids, and cholesterol and its derivatives. Synthetic lipids such as synthetic palmitoylsphingomyelin or N-palmitoyl-4-hydroxysphinganine-1-phosphocholine (a form of phytosphingomyelin) can be used to minimize lipid oxidation.

[0131] In some embodiments, the lipid-binding protein-based complex comprises two types of phospholipids: a neutral lipid, such as lecithin and / or sphingomyelin (abbreviated as SM), and a charged phospholipid (e.g., a negatively charged phospholipid). A "neutral" phospholipid has a net charge of approximately zero at physiological pH. In many embodiments, the neutral phospholipid is zwitterionic, although other types of net neutral phospholipids are known and can be used. In some embodiments, the molar ratio of charged phospholipid (e.g., negatively charged phospholipid) to neutral phospholipid ranges from 1:1 to 1:3, e.g., about 1:1, about 1:2, or about 1:3.

[0132] The neutral phospholipids can include, for example, one or both of lecithin and / or SM, and can optionally include other neutral phospholipids. In some embodiments, the neutral phospholipids include lecithin but do not include SM. In other embodiments, the neutral phospholipids include SM but do not include lecithin. In still other embodiments, the neutral phospholipids include both lecithin and SM. All of these specific exemplary embodiments can include neutral phospholipids in addition to lecithin and / or SM, although many embodiments do not include such additional neutral phospholipids.

[0133] As used herein, the term "SM" includes sphingomyelin derived from or obtained from natural sources, as well as analogs and derivatives of naturally occurring SM that are not susceptible to hydrolysis by LCAT as occurs with naturally occurring SM. SM is a phospholipid that is very similar in structure to lecithin, but unlike lecithin, SM does not have a glycerol backbone and therefore does not have ester bonds linking the acyl chains. Rather, SM has a ceramide backbone with amide bonds linking the acyl chains. SM can be obtained, for example, from milk, eggs, or brain. SM analogs or derivatives can also be used. Non-limiting examples of useful analogs and derivatives include, but are not limited to, palmitoyl sphingomyelin, N-palmitoyl-4-hydroxysphinganine-1-phosphocholine (a form of phytosphingomyelin), palmitoyl sphingomyelin, stearoyl sphingomyelin, D-erythro-N-16:0-sphingomyelin and its dihydro isomer, D-erythro-N-16:0-dihydro-sphingomyelin. To produce a more homogeneous complex with fewer contaminants and / or oxidation products than sphingolipids of animal origin, synthetic SMs such as synthetic palmitoyl sphingomyelin or N-palmitoyl-4-hydroxysphinganine-1-phosphocholine (phytosphingomyelin) can be used. Methods for synthesizing SMs are described in US Patent Publication No. 2016 / 0075634.

[0134] Sphingomyelin isolated from natural sources can be artificially enriched in one particular saturated or unsaturated acyl chain. For example, milk sphingomyelin (Avanti Phospholipid, Alabaster, Ala.) is characterized by a long saturated acyl chain (i.e., an acyl chain having 20 or more carbon atoms). In contrast, egg sphingomyelin is characterized by a short saturated acyl chain (i.e., an acyl chain having fewer than 20 carbon atoms). For example, only about 20% of milk sphingomyelin accounts for C16:0 (16 carbons, saturated) acyl chains, whereas about 80% of egg sphingomyelin accounts for C16:0 acyl chains. Using solvent extraction, the composition of milk sphingomyelin can be enriched to have an acyl chain composition comparable to that of egg sphingomyelin, or vice versa.

[0135] SM may be semi-synthetic so that it has a specific acyl chain. For example, milk sphingomyelin may first be purified from milk, after which one specific acyl group, e.g., the C16:0 acyl chain, is cleaved and replaced by another acyl chain. SM may also be fully synthesized, e.g., by large-scale synthesis. See, e.g., U.S. Patent No. 5,220,043 to Dong et al., entitled Synthesis of D-erythro-sphingomyelins, issued June 15, 1993; Weis, 1999, Chem. Phys. Lipids 102 (1-2):3-12. SM may be fully synthetic, e.g., as described in U.S. Patent Application Publication No. 2014 / 0275590.

[0136] The length and saturation level of the acyl chains comprising the semi-synthetic or synthetic SM can be selectively varied. The acyl chains can be saturated or unsaturated and can contain from about 6 to about 24 carbon atoms. Each chain can contain the same number of carbon atoms, or each chain can contain a different number of carbon atoms. In some embodiments, the semi-synthetic or synthetic SM comprises mixed acyl chains, where one chain is saturated and one chain is unsaturated. In such mixed acyl chain SM, the chain lengths can be the same or different. In other embodiments, the acyl chains of the semi-synthetic or synthetic SM are both saturated or both unsaturated. Furthermore, the chains can contain the same or different number of carbon atoms. In some embodiments, both acyl chains comprising the semi-synthetic or synthetic SM are identical. In certain embodiments, the chains correspond to the acyl chains of naturally occurring fatty acids, such as oleic acid, palmitic acid, or stearic acid. In another embodiment, SM with saturated or unsaturated functionalized chains is used. In another particular embodiment, the acyl chains are both saturated and contain 6 to 24 carbon atoms. Non-limiting examples of acyl chains present in commonly occurring fatty acids that can be included in semi-synthetic and synthetic SMs are provided in Table 1 above.

[0137] In some embodiments, the SM is palmitoyl SM, such as synthetic palmitoyl SM having a C16:0 acyl chain, or egg SM that contains palmitoyl SM as a major component.

[0138] In certain embodiments, a functionalized SM, such as phytosphingomyelin, is used.

[0139] Lecithin can be derived from or isolated from natural sources, or lecithin can be obtained synthetically.The examples of suitable lecithin isolated from natural sources include, but are not limited to, egg phosphatidylcholine and soybean phosphatidylcholine.Further non-limiting examples of suitable lecithin include, but are not limited to, dipalmitoyl phosphatidylcholine, dimyristoyl phosphatidylcholine, distearoyl phosphatidylcholine, 1-myristoyl-2-palmitoyl phosphatidylcholine, 1-palmitoyl-2-myristoyl phosphatidylcholine, 1-palmitoyl-2-stearoyl phosphatidylcholine, 1-stearoyl-2-palmitoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine, 1-oleoyl-2-palmitoyl phosphatidylcholine, dioleoyl phosphatidylcholine and their ether derivatives or analogues.

[0140] Lecithin derived from or isolated from natural sources can be enriched to contain specific acyl chains. In embodiments using semi-synthetic or synthetic lecithin, the identity(ies) of the acyl chains can be selectively varied as discussed above with respect to SM. In some embodiments of the complexes described herein, both acyl chains on the lecithin are identical. In some embodiments of complexes containing both SM and lecithin, the acyl chains of the SM and lecithin are all identical. In particular embodiments, the acyl chains correspond to the acyl chains of myristic acid, palmitic acid, oleic acid, or stearic acid.

[0141] The complexes of the present disclosure may include one or more negatively charged phospholipids (e.g., alone or in combination with one or more neutral phospholipids). As used herein, a "negatively charged phospholipid" is a phospholipid that has a net negative charge at physiological pH. A negatively charged phospholipid may include a single type of negatively charged phospholipid or a mixture of two or more different negatively charged phospholipids. In some embodiments, the charged phospholipid is a negatively charged glycerophospholipid. Specific examples of suitable negatively charged phospholipids include, but are not limited to, 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, phosphatidic acid, and salts thereof (e.g., sodium or potassium salts). In some embodiments, the negatively charged phospholipid comprises one or more of phosphatidylinositol, phosphatidylserine, phosphatidylglycerol and / or phosphatidic acid.In a particular embodiment, the negatively charged phospholipid comprises or consists of a salt of phosphatidylglycerol or a salt of phosphatidylinositol.In another particular embodiment, the negatively charged phospholipid comprises or consists of 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], or DPPG, or a salt thereof.

[0142] Negatively charged phospholipids may be obtained from natural sources or prepared by chemical synthesis. In embodiments using synthetic negatively charged phospholipids, the identity of the acyl chains may be selectively varied as discussed above in connection with SM. In some embodiments of the complexes of the present disclosure, both acyl chains on the negatively charged phospholipids are identical. In some embodiments, the acyl chains of all types of phospholipids included in the complexes of the present disclosure are all identical. In particular embodiments, the complexes include negatively charged phospholipid(s) and / or SMs that all have C16:0 or C16:1 acyl chains. In particular embodiments, the fatty acid moiety of the SM is primarily C16:1 palmitoyl. In one particular embodiment, the acyl chains of the charged phospholipid(s), lecithin and / or SM correspond to the acyl chains of palmitic acid. In yet another particular embodiment, the acyl chains of the charged phospholipid(s), lecithin and / or SM correspond to the acyl chains of oleic acid.

[0143] Examples of positively charged phospholipids that can be included in the complexes of the present disclosure include N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide, 1,2-di-O-octadecenyl-3-trimethylammonium propane, 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero- 3-Ethylphosphocholine, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, 1,2-distearoyl-sn-glycero-3-ethylphosphocholine, 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine, 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine, 1,2-dioleoyl-3-dimethyl 1,2-Dimyristoyl-3-dimethylammonium-propane, 1,2-Dipalmitoyl-3-dimethylammonium-propane, N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium, 1,2-Dioleoyl-3-trimethylammonium-propane, 1,2-Dioleoyl-3-trimethylammonium-propane, 1,2-Stearo ... ,2-dipalmitoyl-3-trimethylammonium-propane, 1,2-dimyristoyl-3-trimethylammonium-propane, N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide, N,N,N-trimethyl-2-bis[(1-oxo-9-octadecenyl)oxy]-(Z,Z)-1-propanaminium methylsulfonate, and salts thereof (e.g., chloride or bromide salts).

[0144] The lipids used are preferably at least 95% pure and / or have reduced levels of oxidizing agents, such as but not limited to peroxides. Lipids obtained from natural sources preferably have less polyunsaturated fatty acid moieties and fatty acid moieties that are not susceptible to oxidation. The level of oxidation in a sample can be determined using iodometric titration, which provides a peroxide value expressed as milliequivalents of isolated iodine per kg of sample, abbreviated as meqO / kg. See, for example, Gray, 1978, Measurement of Lipid Oxidation: A Review, Journal of the American Oil Chemists Society 55:539-545;Heaton, FW and Ur, Improved Iodometric Methods for the Determination of Lipid Peroxides, 1958, Journal of the Science of Food and Agriculture 9:781-786. Preferably, the level of oxidation, or peroxide levels, is low, for example, less than 5 meqO / kg, less than 4 meqO / kg, less than 3 meqO / kg, or less than 2 meqO / kg.

[0145] The complex may, in some embodiments, contain a small amount of additional lipid. Virtually any type of lipid may be used, including but not limited to lysophospholipids, galactocerebrosides, gangliosides, cerebrosides, glycerides, triglycerides, and sterols and sterol derivatives (e.g., plant sterols, animal sterols, such as cholesterol, or sterol derivatives, such as cholesterol derivatives). For example, the complex of the present disclosure may contain cholesterol or cholesterol derivatives, such as cholesterol esters. The cholesterol derivatives may also be substituted cholesterol or substituted cholesterol esters. The complex of the present disclosure may also contain oxidized sterols, such as but not limited to oxidized cholesterol or oxidized sterol derivatives (e.g., but not limited to oxidized cholesterol esters). In some embodiments, the complex does not contain cholesterol and / or its derivatives (e.g., cholesterol esters or oxidized cholesterol esters).

[0146] Cleaning agents The complex may contain one or more detergents. The detergents may be zwitterionic, nonionic, cationic, anionic, or a combination thereof. Exemplary zwitterionic detergents include 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), and N,N-dimethyldodecylamine N-oxide (LDAO). Exemplary non-ionic detergents include 6-monooleic acid D-(+)-trehalose, N-octanoyl-N-methylglucamine, N-nonanoyl-N-methylglucamine, N-decanoyl-N-methylglucamine, 1-(7Z-hexadecenoyl)-rac-glycerol, 1-(8Z-hexadecenoyl)-rac-glycerol, 1-(8Z-heptadecenoyl)-rac-glycerol, 1-(9Z-hexadecenoyl)-rac-glycerol, 1-decanoyl-rac-glycerol. Exemplary cationic detergents include (S)-O-methyl-serine dodecylamide hydrochloride, dodecylammonium chloride, dodecyltrimethylammonium bromide, and cetyltrimethylammonium sulfate. Exemplary anionic detergents include cholesteryl hemisuccinate, cholate, alkyl sulfate, and alkyl sulfonate.

[0147] 6.1.5.3.Fatty acids The complex may contain one or more fatty acids, which may include short chain fatty acids with an aliphatic tail of 5 or less carbons (e.g., butyric acid, isobutyric acid, valeric acid, or isovaleric acid), medium chain fatty acids with an aliphatic tail of 6 to 12 carbons (e.g., caproic acid, caprylic acid, capric acid, or lauric acid), long chain fatty acids with a long chain fatty acid tail of 13 to 21 carbons (e.g., myristic acid, palmitic acid, stearic acid, or arachidonic acid), very long chain fatty acids with an aliphatic tail of 22 or more carbons (e.g., behenic acid, lignoceric acid, or cerotic acid), or combinations thereof. The one or more fatty acids may be saturated (e.g., caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidonic acid, behenic acid, lignoceric acid, or cerotic acid), unsaturated (e.g., myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, alpha-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, or docosahexaenoic acid), or a combination thereof. The unsaturated fatty acid may be a cis or trans fatty acid. In some embodiments, the unsaturated fatty acid used in the complex of the present disclosure is a cis fatty acid.

[0148] 6.1.5.4. Nonpolar molecules and sterols bound to sugars The conjugates may contain one or more amphiphilic molecules, including a nonpolar molecule or moiety (e.g., a hydrocarbon chain, an acyl or diacyl chain) or a sterol (e.g., cholesterol) attached to a sugar (e.g., a monosaccharide such as glucose or galactose, or a disaccharide such as maltose or trehalose). The sugar may be a modified or substituted sugar. Exemplary amphiphilic molecules comprising a nonpolar molecule attached to a sugar include dodecan-2-yloxy-β-D-maltoside, tridecan-3-yloxy-β-D-maltoside, tridecan-2-yloxy-β-D-maltoside, n-dodecyl-β-D-maltoside (DDM), n-octyl-β-D-glucoside, n-nonyl-β-D-glucoside, n-decyl-β-D-maltoside, n-dodecyl-β-D-maltopyranoside, 4-n-dodecyl-α,α-trehalose, 6-n-dodecyl-α,α-trehalose, and 3-n-dodecyl-α,α-trehalose.

[0149] In some embodiments, the non-polar moiety is an acyl or diacyl chain.

[0150] In some embodiments, the sugar is a modified or substituted sugar.

[0151] Anchor The cargo moiety can be covalently attached to the amphipathic or apolar moiety to facilitate coupling of the cargo moiety to the lipid-binding protein-based complex. The amphipathic and apolar moieties can interact with apolar regions in the lipid-binding protein-based complex, thereby anchoring the cargo moiety attached to the amphipathic and apolar moieties to the complex.

[0152] Amphiphilic moieties that can be used as anchors include lipids (e.g., as described in Section 6.1.5.1) and fatty acids (e.g., as described in Section 6.1.5.3). In some embodiments, the anchor comprises a sterol or a sterol derivative, such as a phytosterol, an animal sterol, or a sterol derivative, such as a vitamin. For example, a sterol, such as cholesterol, can be covalently attached to a cargo moiety (e.g., via the hydroxyl group at the 3-position of the A ring of the sterol) and used to anchor the cargo moiety to the complex. Nonpolar moieties that can be used as anchors include alkyl chains, acyl chains, and diacyl chains. The cargo moiety can be covalently attached to the anchor moiety directly or indirectly through a linker (e.g., via a bifunctional peptide or other linker described in Section 6.1.7). Biologically active cargo moieties may retain their biological activity while covalently attached to the anchor (or to a linker attached to the anchor), while other moieties may require cleavage (e.g., by hydrolysis) of the covalent bond that connects the cargo moiety to the anchor (or to a linker attached to the anchor).

[0153] In some embodiments, at least one cargo moiety is coupled to the anchor. In some embodiments, the anchor comprises an aliphatic and / or apolar moiety. In some embodiments, the anchor comprises an amphiphilic moiety. In some embodiments, the amphiphilic moiety comprises one of the amphiphilic molecules in the complex. In some embodiments, the amphiphilic moiety comprises a lipid, a detergent, a fatty acid, an apolar molecule bound to a sugar, or a sterol bound to a sugar.

[0154] In some embodiments, the amphiphilic moiety comprises a sterol. In some embodiments, the sterol comprises an animal sterol or a plant sterol. In some embodiments, the sterol comprises cholesterol.

[0155] In other embodiments, the anchor comprises a non-polar moiety, hi some embodiments, the non-polar moiety comprises an alkyl chain, an acyl chain, or a diacyl chain.

[0156] In some embodiments, the cargo moiety is coupled to the anchor by a direct bond.

[0157] In some embodiments, the cargo moiety is coupled to the anchor by a linker.

[0158] Linker Linker refers to the chain of atoms that covalently connects cargo moiety to other moieties in cargo-carrying complexes such as Cargomer, for example, apolipoprotein molecules, amphiphilic molecules, and anchors.Some linker molecules are commercially available, for example, from ThermoFisher Scientific.Suitable linkers are well known to those skilled in the art, and include, but are not limited to, straight or branched carbon linkers, heterocyclic carbon linkers, and peptide linkers.Linker can be a bifunctional linker, which is either homobifunctional or heterobifunctional.

[0159] Suitable linkers include cleavable and non-cleavable linkers.

[0160] The linker may be a cleavable linker, facilitating the release of the cargo moiety in vivo. Cleavable linkers include acid-labile linkers (e.g., including hydrazine or cis-aconityl), protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, or disulfide-containing linkers (Chari et al., 1992, Cancer Research 52:127-131; U.S. Pat. No. 5,208,020). Cleavable linkers are usually susceptible to cleavage under intracellular conditions. Suitable cleavable linkers include, for example, peptide linkers that are cleavable by intracellular proteases, such as lysosomal or endosomal proteases. In an exemplary embodiment, the linker may be a dipeptide linker, such as a valine-citrulline (val-cit) or phenylalanine-lysine (phe-lys) linker.

[0161] Cleavable linker may be pH sensitive, i.e., sensitive to hydrolysis at a certain pH value. Usually, pH sensitive linker is hydrolyzable under acidic conditions. For example, lysosomal hydrolyzable acid labile linker (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic acid amide, orthoester, acetal, ketal, etc.) may be used. (See, e.g., U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions, such as those in blood, but are unstable below pH 5.5 or 5.0, the approximate pH of the lysosome. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether attached to the cargo moiety via an acylhydrazone bond (see, e.g., U.S. Pat. No. 5,622,929).

[0162] In some embodiments, the linker is cleavable under reducing conditions (e.g., a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-2-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridylthio)butyrate) and SMPT (N-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene), which can be formed using SPDB and SMPT (see, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer, C.W. Vogel ed., Oxford U. Press, 1987; see also U.S. Pat. No. 4,880,935).

[0163] In some embodiments, the linker is cleavable by a cleavage agent, e.g., an enzyme, present in the intracellular environment (e.g., in a lysosome or endosome or caveolae). The linker may be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long. The cleavage agent may include cathepsin B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of active drugs inside target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). In some embodiments, the peptidyl linker that is cleavable by an intracellular protease is a Val-Cit linker or a Phe-Lys linker.

[0164] In some embodiments, the linker is a malonate linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10): 1305-12).

[0165] In other embodiments, the linker unit is non-cleavable and the cargo moiety is released, for example, by complex degradation. Exemplary non-cleavable linkers include maleimidocaproyl, N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC) and N-succinimidyl 4-(iodoacetyl)aminobenzoate (SIAB).

[0166] In some embodiments, the cargo moiety is coupled to the anchor by a linker (e.g., as described in Section 6.1.6). In some embodiments, the linker coupling the cargo moiety to the anchor is a bifunctional linker. In some embodiments, the linker coupling the cargo moiety to the anchor is a cleavable linker. In some embodiments, the cleavable linker is a dipeptide linker, such as a valine-citrulline (val-cit) or phenylalanine-lysine (phe-lys) linker. In some embodiments, the linker coupling the cargo moiety to the anchor is a non-cleavable linker. Exemplary non-cleavable linkers include maleimidocaproyl, N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC) and N-succinimidyl 4-(iodoacetyl)aminobenzoate (SIAB).

[0167] 6.2. Organ preservation solutions There are several commercially available organ preservation solutions. Many of these organ preservation solutions contain components that minimize damage caused to explanted organs and tissues during storage and transportation. Organ preservation solutions are also formulated to reduce the likelihood of graft rejection. Organ preservation solutions may contain various components, such as components selected from colloids, impermeants, gases, electrolytes, antioxidants, nutrients and / or metabolic substrates, buffers, and combinations thereof.

[0168] Some commercially available kidney preservation solutions include Collins solution, EC solution, University of Wisconsin solution (UW solution), histidine-tryptophan-ketoglutarate solution (HTK solution), Celsior® solution, hypertonic adenine citrate solution (HC-A solution and HC-A II solution), phosphate buffered sucrose (PBS) 140, HP16, HBS, B2, Lifor, Ecosol, Biolasol, renal preservation solution 2 (RPS-2), FM, AQIXRS-I, WMO-II, Institute Georges Lopez-1 (IGL-1®), and CZ-1 solution (Chen et al., 2019, Cell Transplantation, 28(12): 1472-1489). Various components may be included in the organ preservation solution to minimize ischemic / hypoxic injury and maximize organ function after transplantation.

[0169] Exemplary components of commercially available organ preservation solutions are provided below in Table 2:

[0170] [Table 2-1]

[0171] [Table 2-2]

[0172] The organ preservation solution of the present disclosure may include, for example, a lipid-binding protein-based complex and one or more components listed in Table 2. For example, the organ preservation solution may include a lipid-binding protein-based complex (e.g., CER-001) and one or more components of Celsior® solution, EC solution, UW solution, HTK solution, IGL-1® solution, or HC-A solution. In some embodiments, the organ preservation solution of the present disclosure includes a lipid-binding protein-based complex (e.g., CER-001) and a component of Celsior® solution, EC solution, UW solution, HTK solution, IGL-1® solution, or HC-A solution, optionally, the component of Celsior® solution, EC solution, UW solution, HTK solution, IGL-1® solution, or HC-A solution is present at the concentration shown in Table 2 or at ±20%, ±15%, ±10%, or ±5% of the concentration shown in Table 2. In some embodiments, the organ preservation solution of the present disclosure comprises CER-001 and components of Celsior® solution. In some embodiments, CER-001 is present at a concentration of 0.1 mg / ml to 5 mg / ml (e.g., 0.3 mg / ml to 0.5 mg / ml, 0.2 mg / ml to 0.6 mg / ml, 0.1 mg / ml to 1 mg / ml, 1 mg / ml to 2 mg / ml, or 2 mg / ml to 5 mg / ml) on a protein basis. In some embodiments, CER-001 is present at a concentration of 0.4 mg / ml on a protein basis. As used herein, the term "protein basis" means that the amount of lipid-binding protein-based complex (e.g., CER-001) is calculated based on the amount of lipid-binding protein (e.g., ApoA-I) in the lipid-binding protein-based complex (e.g., CER-001).

[0173] Another exemplary organ preservation solution that can be used is PumpProtect® solution, which contains 0.5 mM calcium chloride (dihydrate), 10 mM HEPES (free acid), 25 mM potassium phosphate (monobasic), 30 mM mannitol, 10 mM glucose (anhydrous), 80 mM sodium gluconate, 5 mM magnesium gluconate, 5 mM D-ribose, 50 g / L pentafraction (HES), 3 mM glutathione (reduced), and 3 mM adenine (free base). In some embodiments, the organ preservation solution of the present disclosure includes a lipid-binding protein-based complex (e.g., CER-001) and a component of the PumpProtect® solution. In some embodiments, the components of the PumpProtect® solution are present at the concentrations listed in this paragraph or at ±20%, ±15%, ±10%, or ±5%.

[0174] Solutions containing chondroitin sulfate and dextran (e.g., Optisol™, Optisol GS™) can be used in solutions for preserving corneal tissue. McCarey-Kaufman (MK) medium, Chen medium, and Cornisol can also be used. For example, commercially available corneal preservation solutions can be supplemented with lipid-binding protein-based complexes (e.g., CER-001). In some embodiments, solutions for preserving corneal tissue include lipid-binding protein-based complexes (e.g., CER-001) and one or more (e.g., any one, two, three, four, five, six, seven, or eight) of the following: chondroitin sulfate, dextran, sodium bicarbonate, antibiotics (e.g., gentamicin and / or streptomycin), a mixture of amino acids, sodium pyruvate, L-glutamic acid, and 2-mercaptoethanol.

[0175] The organ preservation solution of the present disclosure can be prepared, for example, by combining the lipid-binding protein-based complex with other components of the solution.For example, the lipid-binding protein-based complex can be combined with a pre-prepared (e.g., commercially available) organ preservation solution.Alternatively, the components of the organ preservation solution can be combined in any other manner, for example, added and mixed sequentially.

[0176] In some embodiments, the present disclosure provides an organ preservation solution product. Such a product may include the organ preservation solution of the present disclosure in a sealed container, such as a bag (e.g., containing 1 L of solution) or a bottle (e.g., containing 1 L of solution).

[0177] Colloids Colloids, particularly high molecular weight colloids, can be included in organ preservation solution.In organ preservation solution, the addition of high molecular weight colloids can maintain intravascular oncotic pressure and prevent interstitial edema.Exemplary colloids that can be included in organ preservation solution include, but are not limited to, hydroxyethyl starch (HES) (e.g., 50 kDa), dextran (e.g., 40 kDa), polyethylene glycol (PEG), such as PEG35 (35 kDa) or PEG20 (20 kDa), and combinations thereof.

[0178] 6.2.2. Non-permeable substances Impermeable substances can be included in organ preservation solutions to limit cell edema. The effectiveness of impermeable substances in preventing cell swelling is generally determined by their molecular weight. In general, larger molecules are better at preventing cell swelling. Examples of impermeable substances include, but are not limited to, glucose (molecular weight 180 kDa), mannitol (molecular weight 182 kDa), sucrose (molecular weight 342 Da), raffinose (molecular weight 504 kDa), lactobionate, and combinations thereof.

[0179] Gas Several gases, including oxygen (O2), hydrogen (H2), carbon monoxide (CO), nitric oxide (NO), hydrogen sulfide (HS) and argon (Ar), have been used to reduce ischemic / hypoxic injury in organ transplantation. Gases can be added to organ preservation solutions.

[0180] 6.2.4. Electrolytes Electrolytes, for example from salts, can be included in the organ preservation solution to help maintain electrolyte homeostasis in the donor organ. Exemplary electrolytes include Na + , K + , Mg 2+ , Ca 2+ , Cl - , SO4 2- , PO4 3- , HCO 3- , citrate and combinations thereof.

[0181] Antioxidants Organ preservation solutions may contain antioxidants and / or radical scavengers to help limit ischemic / hypoxic injury to explanted organs. Additives that impede ROS generation pathways and scavenge existing ROS may help prevent or reduce ischemic / hypoxic injury during organ preservation. Exemplary antioxidants and / or radical scavengers include allopurinol (xanthan oxidase inhibitor), reduced glutathione (thiol-containing amino acid), ROS-scavenging amino acids such as tryptophan or L-arginine and histidine, lecithinized superoxide dismutase (lec-SOD), H2S, N-acetylcysteine, propofol, TMZ, rh-BMP-7, trolox, edaravone, selenium, nicaraven, prostaglandin E1, tanshinone IIA, and combinations thereof.

[0182] 6.2.6. Nutrients and / or metabolic substrates Amino acids may be included in the organ preservation solution to provide nutrients and / or act as metabolic substrates. In some embodiments, the amino acids are selected from one or more of tryptophan, glutamic acid, histidine, L-arginine, N-acetylcysteine, and D-cysteine. In some embodiments, nutrients included in the organ preservation solution include, but are not limited to, cyclic helix B peptide trophic factors, such as bovine neutrophil peptide-1 (BNP-1), substance P (SP), nerve growth factor-β (NGF-β), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGE), hepatocyte growth factor (HGF), recombinant human bone morphogenetic protein-7 (rh BMP-7), lecithinized superoxide dismutase (lec-SOD), TNF-receptor fusion protein (TNF-RFP), and combinations thereof.

[0183] 6.2.7.Buffer A buffering agent may be included in the organ preservation solution to control cell pH. In some embodiments, the pH of the organ preservation solution of the present disclosure is about 7.0 to about 7.4. In some embodiments, the buffering agent is selected from borate, borate-polyol complex, succinate, phosphate buffer, citrate buffer, acetate buffer, carbonate buffer, organic buffer, amino acid buffer such as histidine, and combinations thereof.

[0184] 6.2.8. Other components Mitochondrial dysfunction is a critical event during ischemia, which can lead to the impairment of ATP synthesis and possible ATP deficiency.Maintaining mitochondrial integrity and protecting mitochondrial function are the important requirements of organ preservation solution.In some embodiments, the organ preservation solution of the present disclosure contains one or more mitochondrial protection reagents.Suitable mitochondrial protection reagents include but are not limited to H2S, MitoQ, quinacrine, TMZ, and AP39.

[0185] Multiple inflammatory pathways and factors may be activated during reperfusion of organs, which may result in post-ischemic injury. In some embodiments, specific antibodies or pathway inhibitors may be included in organ preservation solutions to regulate inflammatory response and attenuate post-ischemic injury. Suitable compounds include endothelial receptor antagonists, TNF-receptor fusion proteins (TNF-RFPs), ICAM-1 antisense deoxynucleotides, endothelial receptor antagonists, p38MAPK inhibitors, such as FR167653, exogenous CO or CO-releasing molecules, H2S, O2, Ar, melagatran, cyclic helix B peptide, TMZ, lec-SOD, Rho-kinase inhibitor HA1077, thrombin inhibitors, such as melagatran, platelet activating factor (PAF) receptor antagonists, proteasome inhibitors, and combinations thereof.

[0186] Ischemia / hypoxia followed by reperfusion injury leads to activation of cell death programs such as apoptosis, necrosis and autophagy-related cell death. In some embodiments, the organ preservation solution contains additives that block activation of cell death programs such as glucocorticoids, e.g., dexamethasone, naked caspase 3 siRNA, matrix metalloproteinase (MMP)-2 siRNA, AMP-activated protein kinase (AMPK) activators and combinations thereof.

[0187] In some embodiments, energetic substances such as adenosine may be added to the organ preservation solution to allow rapid ATP regeneration during preservation.

[0188] Other useful additions to organ preservation solutions include calcium channel blockers such as verapamil. 2+ Homeostatic drugs or PI3K / Akt / PKG-dependent pathways can increase Na + / H + Other pharmacological agents that can prevent calcium overload that can block exchanger activity include H2S.

[0189] In certain embodiments, one or more additives are included in the organ preservation solution, including, but not limited to, prostaglandin E1, taurine, ranolazine, and combinations thereof.

[0190] 6.3. Kits and Systems In certain aspects, the present disclosure provides a kit comprising a lipid-binding protein-based complex, for example as described in section 6.1, and one or more components of an organ preservation solution, for example as described in section 6.2. In certain embodiments, the lipid-binding protein-based complex is provided in the kit in the form of a solution. In certain embodiments, the lipid-binding protein-based complex is provided in the kit in lyophilized form.

[0191] In certain embodiments, one or more components of the kit are provided in a sealed container. In some embodiments, the sealed container is a bag.

[0192] In certain embodiments, one or more components of the organ preservation solution are present in the kit in the form of a solution.

[0193] In certain embodiments, one or more components of the organ preservation solution complex are present in a sealed container. In some embodiments, the sealed container is a bag.

[0194] In some embodiments, the kit includes a lipid-binding protein-based complex (e.g., CER-001) in one container and the remaining components of the organ preservation solution in one or more additional containers. For example, the kit may include a lipid-binding protein-based complex (e.g., CER-001) in one container and a Celsior® solution, an EC solution, a UW solution, a HTK solution, an IGL-1® solution, or an HC-A solution in a second container. In some embodiments, the kit includes CER-001 in one container and a Celsior® solution in yet another container. A finished organ preservation solution can be made from such a kit by combining the lipid-binding protein-based complex with other components.

[0195] In another aspect, the disclosure provides a system comprising (a) an organ preservation solution or organ preservation solution product of the disclosure, and (b) a perfusion machine and / or an organ (e.g., kidney, liver, heart, lung, pancreas, intestine, or trachea, which may be from, for example, a mammal, such as a human or a pig). In some embodiments, the system comprises a perfusion machine. In other embodiments, the system comprises an organ. In yet another embodiment, the system comprises a perfusion machine and an organ. Exemplary perfusion machines include, but are not limited to, heart-lung machines, normothermic perfusion machines, and subnormothermic perfusion machines.

[0196] In another aspect, the present disclosure provides a system comprising (a) an organ preservation solution or organ preservation solution product of the present disclosure, and (b) tissue (e.g., eye (e.g., cornea or sclera), skin, fat, muscle, bone, cartilage, fetal thymus, or neural tissue), which may be from, for example, a mammal, such as a human or a pig. In some embodiments, the system further comprises a perfusion machine.

[0197] 6.4. Organs, tissues, organ and tissue preservation processes and transplantation methods In some aspects, the present disclosure provides a process of ex vivo organ preservation using the organ preservation solution of the present disclosure.The organ can be a mammalian organ, such as a human or porcine organ.Exemplary organs include, but are not limited to, kidney, liver, heart, lung, pancreas, intestine, and trachea.In some embodiments, the organ is a kidney.In some embodiments, the organ is an eye.

[0198] The process may include, for example, performing machine perfusion of an organ using an organ preservation solution of the present disclosure. The organ preservation solution may, in some embodiments, be diluted with blood, e.g., whole blood. For example, the organ preservation solution may be diluted with whole blood at a volume:volume ratio of organ preservation solution to whole blood of 1:1 to 1:3. In some embodiments, the ratio is 1:1. In other embodiments, the ratio is 1:3. Alternatively, the organ preservation solution may be used without dilution.

[0199] Machine perfusion can be, for example, at normal temperatures, e.g., 30° C. to 38° C., or below normal temperatures, e.g., 2° C. to 8° C. In some embodiments, machine perfusion is performed at 30° C. to 38° C. In other embodiments, machine perfusion is performed at 2° C. to 8° C. In other embodiments, machine perfusion is performed at a temperature between 8° C. and 30° C. (e.g., 20° C. to 25° C.). Machine perfusion can, in some embodiments, be preceded by flushing the organ with an organ preservation solution, e.g., a cold organ preservation solution (e.g., 2° C. to 8° C.).

[0200] Also provided is a process for preserving an organ using cold storage (CS). In some embodiments, cold storage comprises storing the organ in an organ preservation solution in the absence of machine perfusion, e.g., at 2° C. to 6° C. Cold storage may in some embodiments be preceded by flushing the organ with an organ preservation solution, e.g., a cold organ preservation solution (e.g., 2° C. to 8° C.).

[0201] Machine perfusion and cold storage can be performed for any suitable time, for example, from the time the organ is harvested from the donor (or shortly thereafter) to the time of transplantation into the recipient (or shortly thereafter). In some embodiments, machine perfusion or cold storage is performed on the organ for at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, and / or for up to 1 week, up to 5 days, up to 4 days, up to 3 days, up to 2 days, up to 36 hours, up to 1 day, up to 12 hours, or any range bracketed by any two of the foregoing values, for example, 2 to 4 hours, 2 to 6 hours, 4 to 6 hours, 6 to 12 hours, 12 hours to 1 day, 1 to 2 days. etc.

[0202] In some embodiments, the kidney is subjected to machine perfusion or cold storage for up to 24 hours or up to 36 hours.

[0203] In some embodiments, the liver is subjected to machine perfusion or cold storage for up to 12 hours.

[0204] In some embodiments, the lungs are subjected to machine perfusion or cold storage for up to 6 hours or up to 8 hours.

[0205] In some embodiments, the heart is subjected to machine perfusion or cold storage for up to 4 hours or up to 6 hours.

[0206] In some embodiments, the pancreas is subjected to machine perfusion or cold storage for up to 12 hours or up to 1 day.

[0207] In some embodiments, the intestine is subjected to machine perfusion or cold storage for up to 12 hours or up to 1 day.

[0208] In some embodiments, the trachea is subjected to machine perfusion or cold storage for up to 12 hours or up to 1 day.

[0209] The process of organ preservation may further include removing the organ from the organ donor. In some embodiments, the organ donor is a living donor (e.g., a kidney donor). In other embodiments, the donor is deceased.

[0210] The present disclosure further provides a method of organ transplantation comprising transplanting an organ preserved by the organ preservation process of the present disclosure into a subject in need of an organ transplant. Subjects that may be treated according to the methods described herein are preferably mammals, most preferably humans.

[0211] In some aspects, the present disclosure provides a process of ex vivo tissue preservation using the organ preservation solution of the present disclosure.The tissue can be mammalian tissue, such as human or porcine tissue.Exemplary tissues include, but are not limited to, eye (e.g., cornea or sclera), skin, fat, muscle, bone, cartilage, fetal thymus, and nerve tissue.In some embodiments, the tissue is corneal tissue.

[0212] The process may, for example, include storing the tissue in an organ preservation solution of the present disclosure (e.g., cold storage (CS)). In some embodiments, cold storage includes storing the tissue in the organ preservation solution, for example, at 2°C to 6°C.

[0213] Storage of tissue in organ preservation solution can be performed for any suitable time, for example, from the time the tissue is collected from the donor (or shortly thereafter) to the time of transplantation into the recipient (or shortly thereafter). In some embodiments, storage is performed for at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, and / or up to 4 weeks, up to 2 weeks, up to 1 week, up to 5 days, up to 4 days, up to 3 days, up to 2 days, up to 36 hours, up to 1 day, up to 12 hours, or any range bracketed by any two of the preceding values, such as 2 to 4 hours, 2 to 6 hours, 4 to 6 hours, 6 hours to 12 hours, 12 hours to 1 day, 1 day to 2 days, 1 day to 1 week, 1 week to 2 weeks, 2 weeks to 4 weeks, etc. In some embodiments, corneal tissue is stored in organ preservation solution for up to 4 weeks.

[0214] The process of organ preservation may further include removing tissue from a tissue donor. In some embodiments, the tissue donor is a living donor. In other embodiments, the donor is deceased.

[0215] The present disclosure further provides a method of tissue transplantation comprising transplanting tissue preserved by the organ preservation process of the present disclosure into a subject in need of an organ transplant. The subjects that may be treated according to the methods described herein are preferably mammals, most preferably humans. EXAMPLES

[0216] 7. Working Example Despite recent improvements in immunosuppressive therapy and in the field of transplantation surgery techniques, ischemia / reperfusion injury remains one of the major causes of functional loss of transplanted organs.

[0217] Interest in new strategies capable of limiting IRI damage has received enormous force in recent decades due to the growing technical improvements of perfusion machines required for organ preservation. In addition to being an excellent opportunity to preserve the quality of the organ during the transportation or preparation of the recipient, the perfusion machine allows the organ to be pharmacologically treated before transplantation. In the context of renal IRI damage, several pharmacological approaches have been tried in mouse models, such as the monoclonal antibody αCD47Ab, the soluble complement receptor factor sCR1, and the recombinant protein thrombomodulin (rTM), which has an anticoagulant role, capable of modulating oxidative stress. However, many of these drugs were not effective when transferred to the clinical situation (Hameed et al., 2020, Sci Rep. 10(1):6930). Without being bound by theory, it is believed that HDL and HDL mimetics such as CER-001 are capable of limiting IRI damage ex vivo in organ preservation solutions by acting on the same mechanisms of oxidative stress, inflammation and coagulation. Furthermore, HDL and HDL mimetics such as CER-001 contain endogenous proteins that would not be expected to cause the same secondary effects of monoclonal antibodies.

[0218] Example 1 describes an organ preservation study in a porcine model of IRI kidney injury. The IRI porcine model is a good animal model of what occurs in the human system of kidney transplantation because it mimics the reduction in serum creatinine, interstitial fibrosis, tubular atrophy, and infiltration of circulating leukocytes (Delpech et al 2016, J Transl Med 14, 277).

[0219] Another advantage of the porcine model is the ability to control the entire procedure, characterized by clamping of the renal artery from warm and cold ischemia to reperfusion, which allows the processes modulated by the drug to be clearly highlighted: reduction of intracellular ATP, increase of lactate, Ca ++ The metabolic changes of intracellular accumulation of O and the formation of free radicals of O at the mitochondrial level can be precisely studied in the model.

[0220] Pig and human kidneys are anatomically similar (characterized by a multilobular structure versus rodent and monolobular mouse kidneys). The porcine body size allows for similar surgical procedures as the human body size, repeated collection of peripheral blood or biopsies for evaluation and optimization of preperfusion techniques. Finally, the close similarity to the physiology of the immune system allows for the evaluation of the efficacy of HDL and HDL mimetics such as CER-001 in organ preservation solutions.

[0221] 7.1. Example 1: Evaluation of the efficacy of CER-001 to reduce ischemia / reperfusion injury in a porcine ex vivo perfusion model A total of 28 pigs are used in this study. The pigs weighing 45-60 kg are fasted for 24 h before the study. All animals are premedicated with an intramuscular mixture of azaperone (8 mg / kg) and atropine (0.03 mg / kg) to reduce pharyngeal and tracheal secretions and prevent bradycardia after intubation. After cannulation of the femoral vein, 600 mL of venous blood for ex vivo perfusion of the kidneys is collected into sterile blood bags filled with 5,000 IU of heparin, respectively (activated clotting time, ACT, up to 480 seconds). After anesthesia, both kidneys are accessed through a midline ventral incision. The renal artery and vein are then isolated and a vascular loop is placed around the renal artery using a right-angle clamp. Warm ischemia is induced for 60 min by retracting the vascular loop, followed by perfusion for 3 h. The animals are then euthanized by IV administration of 1 mL / kg (body weight) of pentobarbital. After organ explantation, kidneys were weighed and flushed with Celsiory solution at 4°C. For each pig, one kidney is statically stored at 4°C (cold static storage, CS) whereas the other kidney is inserted into the machine perfusion system. For the perfused kidney, the renal artery is cannulated (retrofluorimetric cardioplegia catheter), the renal vein (1 / 4" tubing connector, 1 / 4" tubing) and the ureter (14Fr. catheter). Heart rate, oxyhemoglobin saturation, respiratory gas composition, respiratory rate, tidal volume, airway pressure, systolic blood pressure and central venous pressure are continuously monitored and automatically recorded (Ohmeda Modulus CD; Datex Ohmeda, Helsinki, Finland).

[0222] 7.1.1 Research design After organ explantation, kidneys are randomized into the following groups:

[0223] Group 1: Kidneys cryopreserved at 4° C. for 6 hours using Celsior® as preservation solution (CS), N=7.

[0224] Group 2: Kidneys cryopreserved with Celsior® as preservation solution supplemented with CER-001 (0.4 mg / ml) at 4° C. for 6 hours (CS+CER-001), N=7.

[0225] Group 3: kidneys perfused in a normothermic perfusion machine with Celsior® solution + whole blood (1:1 ratio) at 32° C. for 6 hours (NMP), N=7.

[0226] Group 4: Kidneys perfused in a normothermic perfusion machine with Celsior® solution supplemented with CER-001 (0.4 mg / ml) plus whole blood (1:1 ratio) at 32° C. for 6 hours (NMP+CER-001).

[0227] After flushing all kidneys through the renal artery with Celsior® solution, each kidney is cannulated. NMP is performed by an S3 heart-lung machine (HLM) (Stockert GmbH, Germany) equipped with a 3T Heater-Cooler device (Stockert GmbH, Germany) that allows for precise temperature control. In addition, the S3 HLM is equipped with a Sechrist Model 3500CP-G low-flow gas blender (Sechrist, USA) that ensures precise gas delivery in terms of sweep flow rate and fraction of inspired oxygen (FiO2). The perfusion circuit includes several consumables: a pediatric oxygenator Lilliput2 with phosphorylcholine (PC) coating (LivaNova, Italy); a centrifugal pump (LivaNova, Italy), a cardiotomy (LivaNova, Italy), PVC1 / 4 in the tubing (LivaNova, Italy). A target mean arterial pressure (MAP) of 75 mmHg is maintained by manually adjusting a custom-designed pump controller and is continuously monitored. Renal blood flow (RBF) is monitored via an ultrasonic flow sensor.

[0228] 7.1.2. Perfusate and urine sampling and analysis Arterial and venous perfusate and urine samples are collected at separate time points. Arterial and venous pO2 and pH levels are measured using a blood gas analyzer. Renal metabolic activity is calculated using arterial and venous oxygen content, arterial and venous SO2 and pO2 values, and hemoglobin concentration to calculate oxygen consumption ((caO2-cvO2) * It is estimated by RBF / kidney weight (ca / vO2 = Sa / vO2 * 1.34 * c(Hb)+pa / vO2 * Urine is collected separately and urine volume is recorded.

[0229] Perfusate plasma samples and urine samples are stored at -80°C for subsequent analysis. Perfusate samples are analyzed for sodium and creatinine levels. Protein, sodium and creatinine concentrations in urine samples are determined. Arterial perfusate and urine levels are used to calculate creatinine clearance (urinary creatinine * urine flow / plasma creatinine / kidney weight) and sodium excretion rate (urinary sodium * Calculate plasma creatinine / plasma sodium / urine creatinine).

[0230] Perfusion parameters Perfusate solution: Celsior® solution + whole blood (1:1 / 1:3 ratio based on hematocrit HCT); · Hb: 9-11 mg / dL (below this parameter, leukocyte-depleted plasma-free blood obtained via the Cell Saver device is added during the collection procedure); · UI of unfractionated heparin (UFH); · Duration of perfusion: 6 hours; · Perfusion pressure: >75mmHg; · Renal venous pressure: 0-3mmHg; · Flow rate: adjusted based on metabolic parameters and vascular resistance; · Perfusion temperature: 32℃.

[0231] monitoring · Flow rate (mL / min): Continuous monitoring; · Pressure (mmHg): Continuous monitoring; Intrarenal resistance (mmHg / mL / min): checked at 10 minutes; Blood-Gas Analysis (Acid-Base Homeostasis, Electrolytes, Hb, PaO2, PaCO2, etc.): Check in 30 minutes; Metabolic parameters (DO2, VO2, O2ER): checked at 30 minutes; · Perfusion temperature: Continuous monitoring.

[0232] analysis Pre- and post-perfusion: · Weight Blood gas measurements: Arterial: pH, pOs, pCO2, HCO3-, base excess, lactate, Na+, K+, Cl-, Ca 2+ Venous: pH, pOs, pCO2, HCO3-, base excess, lactate, Na+, K+, Cl-, Ca 2+ Renal function: · Urine volume (ml / h) Serum creatinine level (umol / l) Creatinine clearance (ml / min / 100g) Renal oxygen consumption (ml / min / g (pO2 arterial - pO2 venous) x (flow rate) / body weight (gewicht)), sodium excretion rate (FENa = [Na+]urine x creatine concentration plasma / [Na+]plasma x creatine concentration urine) Damage Markers: AST · LDH · Cytokines: IL-6, MCP-1, CRP, IL-8, TNF-α, CXCL-10, PAI-1

[0233] Renal biopsies were performed at T0 and T end to measure: ATP Complement · Histology Staining H&E morphology, PMN infiltrate, ICAM-1, P-selectin, MPO

[0234] 7.1.3.Results In preliminary results, improvements in renal fluid mechanics, inflammatory cytokine levels, and histology were observed in explanted kidneys preserved with Celsior® solution supplemented with CER-001 versus explanted kidneys preserved with Celsior® solution not supplemented with CER-001.

[0235] 7.2. Example 2: In vitro evaluation of the efficacy of CER-001 to protect human endothelial and renal tubular epithelial cells The aim of this in vitro study is to evaluate the molecular mechanism of CER-001 protection on endothelial and tubular epithelial cells. The effect of CER-001 (50μg / ml and 500μg / ml) on human endothelial and tubular epithelial cells after C5a and H202 stimulation followed by exposure to CER-001 is evaluated in vitro. The C5a complement component is the most potent complement anaphylatoxin capable of inducing strong inflammation in cell culture and is used to mimic ischemia / reperfusion-associated immune activation (Peng et al., 2012, J Am Soc Nephrol. 23(9):1474-1485;Curci et al., 2014, Nephrol Dial Transplant. 29(4):799-808;Franzin et al., 2020, Front Immunol. 11:734). Pretreatment with CER-001 followed by C5a and H202 stimulation will also be evaluated.

[0236] The following analyses will be carried out: Vitality test (MTT, Annexin V / PI) ELISA tests for cytokines (IL-6, as MCP-1) Tests related to oxidative stress analysis (i.e. AOPP ASSAY KIT Oxiselect) VCAM, ICAM-1 Signaling pathway analysis by SR-BI in endothelial cells and Western blot for phosphorylation of Ser 1177 in eNOS protein to detect HDL-mediated signaling (Uittenbogaard et al., 2000, J Biol Chem, 275:11278-11283;Adelheid Kratzer et al., 2014, Cardiovascular Research, Vol. 103(3): 350-361).

[0237] CER-001 reduces immune activation following C5a and H202 stimulation.

[0238] 7.3. Example 3: Use of CER-001 in ex-vivo normothermic machine perfusion to improve the quality of discarded kidneys from ECD and DCD donors The purpose of this example is to compare the levels of renal function, endothelial dysfunction, cytokine release and histological damage in the context of a new subnormothermic preservation strategy based on supplementation of Celsior® solution with CER-001. Renal function, inflammation, apoptosis, endothelial dysfunction and graft vasculopathy are evaluated during ex-vivo perfusion of discarded DCD and ECD kidneys. One of the goals of this study is to optimize a commercially available perfusion system for organ transplantation by delivery of CER-001 to improve graft survival.

[0239] 7.3.1. Research design The study has two arms. In both arms, kidneys are from uncontrolled donation after cardiac arrest (uDCD) donors and / or expanded criteria donors (ECD) or declared not transplantable based on histological scores (Karpinsky score) and indicators that can predict graft outcome such as the Kidney Donor Risk Index (KDRI) and Kidney Donor Profile Index (KDPI).

[0240] The KDRI was developed for graft evaluation and decision making using donor factors including age, prevalence of hypertension and diabetes, cause of death, and serum creatinine (sCr) level. Following the KDRI, the KDPI has been widely used in the prediction of postoperative graft function and allocation process.

[0241] The KDRI and KDPI scores are based on several clinical factors, however age is the most important factor in calculating these scores.

[0242] The two groups are: A control group undergoing standard kidney procurement by in-situ cryoflush with conventional Celsior® solution followed by 6 hours of normothermic machine perfusion. · CER-001 group undergoing standard kidney acquisition with in-situ hypothermic flush using conventional Celsior® solution supplemented with CER-001 at 0.4 mg / ml, followed by 6 hours of normothermic machine perfusion.

[0243] 7.3.2.Analysis The main output analyzed is renal function, including urine production:

[0244] Kidney function: · Urine volume (ml / h) Serum creatine level (umol / l) Creatinine clearance (ml / min / 100g) Renal oxygen consumption (ml / min / g (pO2 arterial - pO2 venous) x (flow rate) / body weight), sodium excretion rate (FENa = [Na+]urine x creatine concentration plasma / [Na+]plasma x creatine concentration urine)

[0245] Pre- and post-perfusion: Weight

[0246] Blood gas measurements: Arterial: pH, pOs, pCO2, HCO3-, base excess, lactate, Na+, K+, CL-, Ca2+ Venous pH, pOs, pCO2, HCO3-, base excess, lactate, Na+, K+, CL-, Ca2+

[0247] Renal biopsies were performed at T0 and T end to measure: ATP Complement · Histology · Stained H&E morphology, PMN infiltrate ICAM-1, P-selectin, MPO

[0248] Perfusion parameters: Perfusate solution Celsior® solution + whole blood (1:1 / 1:3 ratio based on hematocrit HCT) Hb: >7mg / dL (if less than this, leukocyte-depleted plasma-free blood obtained via the Cell Saver device is added during the collection procedure) UI of Unfractionated Heparin (UFH) Perfusion pressure: >75mmHg Renal vein pressure: 0~3mmHg Flow rate: adjusted based on metabolic parameters and vascular resistance · Perfusion temperature: 32℃

[0249] monitoring Flow rate (mL / min): Continuous monitoring Pressure (mmHg): Continuous monitoring Intrarenal resistance (mmHg / mL / min): Check in 10 minutes Blood-Gas Analysis (Acid-Base Homeostasis, Electrolytes, Hb, PaO2, PaCO2, etc.): Check in 30 minutes Metabolic parameters (DO2, VO2, O2ER): Check in 30 minutes Perfusion temperature: Continuous monitoring

[0250] Damage Markers: AST · LDH Cytokines: IL-6, MCP-1, CRP, IL-8, TNF-alpha, CXCL-10, PAI-1

[0251] 7.3.3.Results Kidneys subjected to NMP in the presence of CER-001 show reduced kidney damage compared to kidneys subjected to NMP in the absence of CER-001. Without being bound by theory, it is believed that CER-001, as well as other lipid-binding protein-based complexes, when used in the organ preservation solutions described herein, can help preserve organ function and limit organ damage, for example, in the kidney, liver, heart, lung, pancreas, intestine, and trachea.

[0252] 7.4. Example 4: Evaluation of the efficacy of CER-001 to reduce ischemia / reperfusion injury in a porcine ex vivo perfusion model Materials and Methods A total of 10 pigs were stunned with bilateral electric shock and subsequently bled according to normal slaughterhouse procedures (UNI En ISO 9001). After 60 min of warm ischemia, the kidneys were flushed and cooled with 500 ml of lactated Ringer's solution at 4° C., which marked the beginning of cold ischemia (T-1). The kidneys were then kept cold overnight to increase the level of injury. The next day, the blood vessels from the organ were exposed and connected to the kidney perfusion device (T0). Oxygenated pulsatile cold machine perfusion (HMP) was performed at a mean arterial pressure of 25 mmHg for 4 h (T4 or Tend) using PumpProtect® solution supplemented with and without CER-001.

[0253] Histological analysis was performed at T0, T2, and Tend by Periodic Acid Schiff (PAS) staining. Digital slides were acquired and analyzed by using an AperioScanScope CS2 device (Aperio, Vista, CA, USA). Tubular injury scoring was performed using the Aperio Scan score software. Tubular damage was scored semiquantitatively by two blinded observers. The score index in each animal was expressed as the mean value of all scores obtained and was also expressed as the median ± IQR.

[0254] CCL2 (MCP-1) and TNF-α levels, as well as aspartate aminotransferase levels, were measured in the perfusate at T-1, T0, T2, and Tend.

[0255] CCL2 (MCP-1), IL-6 and endothelin-1 (ET-1) gene expression levels were measured by qRT-PCR at T0 and Tend in kidney biopsies.

[0256] 7.4.2.Results Improved renal vascular resistance parameters were observed in explanted kidneys preserved with PumpProtect® solution supplemented with CER-001 versus explanted kidneys preserved with PumpProtect® solution not supplemented with CER-001 (Figures 1A-1B).

[0257] Histological analysis showed typical changes in renal morphology in porcine kidneys after cardiac death. These changes included loss of tubular brush borders, vacuolation and dilation of tubular cells, which became more evident after overnight static cold storage (SCS) (Figure 2A, T0). HMP treatment with conventional PumpProtect® solution partially preserved renal physiological morphology and reduced injury (Figure 2A, control). However, HMP treatment with CER-001 replacement solution significantly improved renal tissue, with reduced swelling and necrosis / apoptosis of tubular epithelial cells, reduced squamous epithelium, reduced edema and overall improvement of renal tissue (Figure 2A, "CER001").

[0258] Tubular injury was reduced in explanted kidneys preserved with PumpProtect® solution supplemented with CER-001 versus explanted kidneys preserved with PumpProtect® solution not supplemented with CER-001 (Figure 2B). Perfusate analysis of inflammatory cytokines revealed reduced MCP-1 and TNF-α levels after HMP treatment in CER-001-supplemented preservation solution compared to non-supplemented solution (Figures 2C-2D). Aspartate transferase levels (assessed as a marker of renal injury) were reduced at T2 and Tend for kidneys preserved with PumpProtect® solution supplemented with CER-001 versus explanted kidneys preserved with PumpProtect® solution not supplemented with CER-001 (Figure 2E).

[0259] CCL2 (MCP-1), IL-6, and ET-1 gene expression was decreased in kidneys perfused with CER-001-supplemented PumpProtect® solution compared to kidneys perfused with non-supplemented solution (Figures 3A-3C).

[0260] 7.5. Example 5: In vitro evaluation of the efficacy of CER-001 to protect human endothelial cells The aim of this in vitro study was to evaluate the molecular mechanisms of CER-001 protection on endothelial cells.

[0261] In endothelial cells, phosphorylation of eNOS at Ser-1177 regulates in vivo NO production, altering both the enzyme's Ca2+ sensitivity and the rate of NO formation, with protective antiapoptotic, antioxidant, and anti-inflammatory effects. Normally, phosphorylation of eNOS at Ser-1177 is stimulated by vascular endothelial growth factor (VEGF). Phosphorylation of Thr-495 indirectly affects this process through regulation of calmodulin and caveolin interactions (Chen et al., 2008, J Biol Chem. 283(40):27038-27047).

[0262] In this study, human endothelial cells (HUVECs) were grown in EndoGro medium and then: (i) 10 -7 (ii) 4 μg / ml LPS for 60 min; (iii) CER-001 (range 5–100 μg / l) for 60 min; or (iv) 10 -7 HUVEC cells were incubated with 50 ng / ml C5a for 30 min followed by CER-001 for 30 min. HUVEC cells incubated with 50 ng / ml VEGF for 60 min were used as a positive control for eNOS phosphorylation at Ser-1177. Ser 1177-eNOS phosphorylation was analyzed by FACS.

[0263] CER-001 reduced endothelial cell dysfunction after C5a stimulation (Figure 4). Compared to untreated conditions (basal), both C5a and LPS induced a significant decrease in phospho-Ser1177-eNOS, whereas CER-001 increased phospho-Ser1177-eNOS levels. Endothelial cells exposed to C5a for 30 min followed by CER-001 for another 30 min showed a restoration of phospho-Ser1177-eNOS levels compared to C5a conditions, indicating a protective role of CER-001.

[0264] 7.6. Example 6: Use of CER-001 in ex-vivo normothermic machine perfusion to improve renal quality Kidneys were subjected to normothermic machine perfusion (NMP) with conventional organ preservation solution or with conventional organ preservation solution supplemented with CER-001.

[0265] The results are shown in Figures 5A-5E. Significant improvements were observed in vascular renal resistance (Figures 5A-5C) and renal flow (Figure 5D) in NMP-perfused kidneys perfused with conventional solution supplemented with CER-001 compared to NMP-perfused kidneys perfused with conventional solution not supplemented with CER-001. Urine volume showed increased levels in kidneys perfused with CER-001-supplemented solution (Figure 5E).

[0266] 8. Specific embodiments Various aspects of the disclosure are described in the embodiments set forth in the numbered paragraphs below. 1. Lipid-binding protein-based complexes used in organ preservation solutions. 2. A lipid-binding protein-based complex for use according to embodiment 1, which is a reconstituted HDL or HDL mimetic. 3. A lipid-binding protein-based complex for use according to embodiment 1 or embodiment 2, comprising sphingomyelin. 4. A lipid-binding protein-based complex for use according to any one of embodiments 1 to 3, comprising a negatively charged lipid. 5. The lipid-binding protein-based complex for use according to embodiment 4, wherein said negatively charged lipid is 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG) or a salt thereof. 6. A lipid-binding protein-based complex for use according to embodiment 2, which is CER-001, CSL-111, CSL-112, CER-522 or ETC-216. 7. A lipid-binding protein-based complex for use according to embodiment 6, which is CER-001. 8. The lipid-binding protein-based complex for use according to embodiment 7, wherein said CER-001 is a lipoprotein complex comprising ApoA-I and phospholipids in an ApoA-I weight:total phospholipids weight ratio of 1:2.7+ / -20% and said phospholipids sphingomyelin and DPPG in a sphingomyelin:DPPG weight:weight ratio of 97:3+ / -20%. 9. The lipid-binding protein-based complex for use according to embodiment 7, wherein said CER-001 is a lipoprotein complex comprising ApoA-I and phospholipids in an ApoA-I weight:total phospholipids weight ratio of 1:2.7+ / -10% and said phospholipids sphingomyelin and DPPG in a sphingomyelin:DPPG weight:weight ratio of 97:3+ / -10%. 10. The lipid-binding protein-based complex for use according to embodiment 7, wherein said CER-001 is a lipoprotein complex comprising ApoA-I and phospholipids in an ApoA-I weight:total phospholipids weight ratio of 1:2.7 and said phospholipids sphingomyelin and DPPG in a sphingomyelin:DPPG weight:weight ratio of 97:3. 11. A lipid-binding protein-based complex for use according to any one of embodiments 7 to 10, wherein said ApoA-I has the amino acid sequence of amino acids 25 to 267 of SEQ ID NO: 1 of WO 2012 / 109162. 12. The lipid-binding protein-based complex for use according to any one of embodiments 7 to 11, wherein said ApoA-I is recombinantly expressed. 13. The lipid-binding protein-based complex for use according to any one of embodiments 7 to 12, wherein said CER-001 comprises natural sphingomyelin. 14. A lipid-binding protein-based complex for use according to embodiment 13, wherein said native sphingomyelin is hen egg sphingomyelin. 15. The lipid-binding protein-based complex for use according to any one of embodiments 7 to 14, wherein said CER-001 comprises a synthetic sphingomyelin. 16. The lipid-binding protein-based complex for use according to embodiment 15, wherein said synthetic sphingomyelin is palmitoyl sphingomyelin. 17. A lipid-binding protein-based complex for use according to any one of embodiments 7 to 16, wherein CER-001 is at least 95% homogeneous. 18. The lipid-binding protein-based complex for use according to any one of embodiments 7 to 17, wherein CER-001 is at least 97% homogeneous. 19. A lipid-binding protein-based complex for use according to any one of embodiments 7 to 18, wherein CER-001 is at least 98% homogeneous. 20. A lipid-binding protein-based complex for use according to any one of embodiments 7 to 19, wherein CER-001 is at least 99% homogeneous. 21. A lipid-binding protein-based complex for use according to any one of embodiments 1 to 5, which is an Apomer or a Cargomer. 22. An organ preservation solution comprising a lipid-binding protein-based complex according to any one of embodiments 1 to 21. 23. Organ preservation solution containing lipid-binding protein-based complex. 24. The organ preservation solution of embodiment 23, wherein said lipid-binding protein-based complex is a reconstituted HDL or HDL mimic. 25. The organ preservation solution of embodiment 23 or embodiment 24, wherein the lipid-binding protein-based complex comprises sphingomyelin. 26. The organ preservation solution of any one of embodiments 23 to 25, wherein the lipid-binding protein-based complex comprises a negatively charged lipid. 27. The organ preservation solution of embodiment 26, wherein said negatively charged lipid is 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG) or a salt thereof. 28. The organ preservation solution of embodiment 24, wherein said lipid-binding protein-based complex is CER-001, CSL-111, CSL-112, CER-522 or ETC-216. 29. The organ preservation solution of embodiment 28, wherein said lipid-binding protein-based complex is CER-001. 30. The organ preservation solution of embodiment 29, wherein said CER-001 is a lipoprotein complex comprising ApoA-I and phospholipids in an ApoA-I weight:total phospholipids weight ratio of 1:2.7+ / -20% and said phospholipids sphingomyelin and DPPG in a sphingomyelin:DPPG weight:weight ratio of 97:3+ / -20%. 31. The organ preservation solution of embodiment 29, wherein said CER-001 is a lipoprotein complex comprising ApoA-I and phospholipids in an ApoA-I weight:total phospholipids weight ratio of 1:2.7+ / -10% and said phospholipids sphingomyelin and DPPG in a sphingomyelin:DPPG weight:weight ratio of 97:3+ / -10%. 32. The organ preservation solution of embodiment 29, wherein said CER-001 is a lipoprotein complex comprising ApoA-I and phospholipids in an ApoA-I weight:total phospholipids weight ratio of 1:2.7 and said phospholipids sphingomyelin and DPPG in a sphingomyelin:DPPG weight:weight ratio of 97:3. 33. The organ preservation solution of any one of embodiments 29 to 32, wherein said ApoA-I has the amino acid sequence of amino acids 25 to 267 of SEQ ID NO: 1 of WO 2012 / 109162. 34. The organ preservation solution of any one of embodiments 29 to 33, wherein said ApoA-I is recombinantly expressed. 35. The organ preservation solution of any one of embodiments 29 to 34, wherein said CER-001 comprises natural sphingomyelin. 36. The organ preservation solution of embodiment 35, wherein said natural sphingomyelin is hen egg sphingomyelin. 37. The organ preservation solution of any one of embodiments 29 to 36, wherein said CER-001 comprises synthetic sphingomyelin. 38. The organ preservation solution of embodiment 37, wherein said synthetic sphingomyelin is palmitoyl sphingomyelin. 39. The organ preservation solution of any one of embodiments 29 to 38, wherein CER-001 is at least 95% homogeneous. 40. The organ preservation solution of any one of embodiments 29 to 39, wherein CER-001 is at least 97% homogeneous. 41. The organ preservation solution of any one of embodiments 29 to 40, wherein CER-001 is at least 98% homogeneous. 42. The organ preservation solution of any one of embodiments 29 to 41, wherein CER-001 is at least 99% homogeneous. 43. The organ preservation solution of any one of embodiments 23 to 27, wherein said lipid-binding protein-based complex is an Apomer or a Cargomer. 44. The organ preservation solution of any one of embodiments 22 to 43, comprising a buffer, an antioxidant, a nutrient and / or metabolic substrate, an electrolyte, a colloid, an impermeable substance, a gas, or a combination thereof. 45. The organ preservation solution of embodiment 44, comprising a buffer, optionally wherein the buffer comprises borate, a borate-polyol complex, a succinate, a phosphate buffer, a citrate buffer, an acetate buffer, a carbonate buffer, an organic buffer, an amino acid buffer such as histidine, or a combination thereof. 46. ​​The organ preservation solution of embodiment 44 or embodiment 45, comprising an antioxidant, optionally wherein the antioxidant comprises allopurinol, reduced glutathione, tryptophan or an ROS scavenging amino acid such as l-arginine or histidine, lecithinized superoxide dismutase (lec-SOD), H2S, N-acetylcysteine, propofol, TMZ, rh-BMP-7, trolox, edaravone, selenium, nicaraven, prostaglandin E1, tanshinone IIA, or a combination thereof. 47. The organ preservation solution of any one of embodiments 44 to 46, comprising nutrients and / or metabolic substrates, optionally wherein the nutrients and / or metabolic substrates comprise amino acids such as tryptophan, glutamic acid, histidine, l-arginine, N-acetylcysteine, d-cysteine, or combinations thereof. 48. The organ preservation solution of any one of embodiments 44 to 47, comprising an electrolyte, optionally wherein the electrolyte is Na + , K + , Mg 2+ , Ca 2+ , Cl - , SO4 2- , PO4 3- , HCO 3- , citrate or a combination thereof. 49. The organ preservation solution of any one of embodiments 44 to 48, comprising a colloid, optionally wherein the colloid is hydroxyethyl starch (HES) (e.g., 50 kDa), dextran (e.g., 40 kDa), polyethylene glycol (PEG), such as PEG35 (35 kDa) or PEG20 (20 kDa), or a combination thereof. 50. The organ preservation solution of any one of embodiments 44 to 49, comprising a non-permeable substance, optionally wherein the non-permeable substance is glucose, mannitol, sucrose, raffinose, lactobionate or a combination thereof. 51. The organ preservation solution of any one of embodiments 44 to 50, comprising a gas, optionally wherein the gas is oxygen (O2), hydrogen (H2), carbon monoxide (CO), nitric oxide (NO), hydrogen sulfide (H2S), argon (Ar), or a combination thereof. 52. The organ preservation solution of embodiment 44, comprising one or more components (as described in Table 2) of Celsior® solution, EC solution, UW solution, HTK solution, IGL-1® solution, or HC-A solution. 53. The organ preservation solution of embodiment 45, comprising components of Celsior® solution, EC solution, UW solution, HTK solution, IGL-1® solution, or HC-A solution (as described in Table 2). 54. The organ preservation solution of embodiment 53, comprising the components of Celsior® solution, EC solution, UW solution, HTK solution, IGL-1® solution, or HC-A solution (as described in Table 2) at the concentrations described in Table 2 ± 20%. 55. The organ preservation solution of embodiment 53, comprising the components of Celsior® solution, EC solution, UW solution, HTK solution, IGL-1® solution, or HC-A solution (as described in Table 2) at the concentrations described in Table 2 ± 15%. 56. The organ preservation solution of embodiment 53, comprising the components of Celsior® solution, EC solution, UW solution, HTK solution, IGL-1® solution, or HC-A solution (as described in Table 2) at the concentrations described in Table 2 ± 10%. 57. The organ preservation solution of embodiment 53, comprising the components of Celsior® solution, EC solution, UW solution, HTK solution, IGL-1® solution, or HC-A solution (as described in Table 2) at the concentrations listed in Table 2 ± 5%. 58. The organ preservation solution of any one of embodiments 53 to 57, comprising the components of Celsior® solution (as described in Table 2). 59. The organ preservation solution of embodiment 44, comprising one or more components of PumpProtect® solution (as described in section 6.2). 60. The organ preservation solution of embodiment 59, comprising components of PumpProtect® solution (as described in section 6.2). 61. The organ preservation solution of embodiment 60, comprising components of PumpProtect® solution (as described in Section 6.2) at concentrations ±20%, ±15%, ±10%, or ±5% of those described in Section 6.2. 62. The organ preservation solution of any one of embodiments 22 to 61, comprising the lipid-binding protein-based complex at a concentration of 0.1 mg / ml to 5 mg / ml (e.g., 0.3 mg / ml to 0.5 mg / ml, 0.2 mg / ml to 0.6 mg / ml, 0.1 mg / ml to 1 mg / ml, 1 mg / ml to 2 mg / ml, or 2 mg / ml to 5 mg / ml). 63. The organ preservation solution of any of embodiments 22 to 61, comprising the lipid-binding protein-based complex at a concentration of 0.4 mg / ml. 64. A kit comprising a lipid-binding protein-based complex and one or more components of an organ preservation solution, optionally wherein the lipid-binding protein-based complex is described in any one of embodiments 1 to 21. 65. The kit of embodiment 64, wherein one or more components of the organ preservation solution include a buffer, an antioxidant, a nutrient and / or metabolic substrate, an electrolyte, a colloid, an impermeable substance, a gas, or a combination thereof. 66. The kit of embodiment 65, wherein the one or more components of the organ preservation solution comprise one or more components of Celsior® solution, EC solution, UW solution, HTK solution, IGL-1® solution, or HC-A solution (as described in Table 2). 67. The kit of embodiment 66, wherein the one or more components of the organ preservation solution comprise components of Celsior® solution, EC solution, UW solution, HTK solution, IGL-1® solution, or HC-A solution (as described in Table 2). 68. The kit of embodiment 67, comprising the components of Celsior® solution, EC solution, UW solution, HTK solution, IGL-1® solution, or HC-A solution (as described in Table 2) at the concentrations listed in Table 2 ±20%. 69. The kit of embodiment 67, comprising the components of Celsior® solution, EC solution, UW solution, HTK solution, IGL-1® solution, or HC-A solution (as described in Table 2) at the concentrations listed in Table 2 ±15%. 70. The kit of embodiment 67, comprising the components of Celsior® solution, EC solution, UW solution, HTK solution, IGL-1® solution, or HC-A solution (as described in Table 2) at the concentrations listed in Table 2 ±10%. 71. The kit of embodiment 67, comprising the components of Celsior® solution, EC solution, UW solution, HTK solution, IGL-1® solution, or HC-A solution (as described in Table 2) at the concentrations listed in Table 2 ± 5%. 72. The kit of any one of embodiments 67 to 71, wherein the one or more components of the organ preservation solution comprise the components of Celsior® solution (as described in Table 2). 73. The kit of embodiment 65, wherein the one or more components of the organ preservation solution comprise one or more components of PumpProtect® solution (as described in section 6.2). 74. The kit of embodiment 73, comprising components of PumpProtect® solution (as described in section 6.2). 75. The kit of embodiment 74, comprising components of PumpProtect® solution (as described in Section 6.2) at concentrations ±20%, ±15%, ±10%, or ±5% of those described in Section 6.2. 76. The kit of any one of embodiments 64 to 75, comprising a solution containing one or more components of the organ preservation solution. 77. The kit of any one of embodiments 64 to 76, comprising a solution of said lipid-binding protein-based complex. 78. The kit of any one of embodiments 64 to 76, comprising said lipid-binding protein-based complex in lyophilized form. 79. A process for preparing an organ preservation solution from the kit of any one of embodiments 64 to 78, comprising combining the lipid-binding protein-based complex and one or more components of the organ preservation solution. 80. A process for preparing an organ preservation solution, comprising combining a lipid-binding protein-based complex and one or more components of an organ preservation solution, optionally wherein the lipid-binding protein-based complex is described in any one of embodiments 1 to 21. 81. The process of embodiment 80, wherein the one or more components of the organ preservation solution include a buffer, an antioxidant, a nutrient and / or metabolic substrate, an electrolyte, a colloid, an impermeable substance, a gas, or a combination thereof. 82. The process of embodiment 81, wherein the one or more components of the organ preservation solution comprise one or more components of Celsior® solution, EC solution, UW solution, HTK solution, IGL-1® solution, or HC-A solution (as described in Table 2). 83. The process of embodiment 82, wherein the one or more components of the organ preservation solution comprise components of Celsior® solution, EC solution, UW solution, HTK solution, IGL-1® solution, or HC-A solution (as described in Table 2). 84. The process of embodiment 83, wherein the components of the Celsior® solution, EC solution, UW solution, HTK solution, IGL-1® solution, or HC-A solution (as described in Table 2) are present at the concentrations described in Table 2 ± 20%. 85. The process of embodiment 83, wherein the components of the Celsior® solution, EC solution, UW solution, HTK solution, IGL-1® solution, or HC-A solution (as described in Table 2) are present at the concentrations described in Table 2 ± 15%. 86. The process of embodiment 83, wherein the components of the Celsior® solution, EC solution, UW solution, HTK solution, IGL-1® solution, or HC-A solution (as described in Table 2) are present at the concentrations described in Table 2 ± 10%. 87. The process of embodiment 83, wherein the components of the Celsior® solution, EC solution, UW solution, HTK solution, IGL-1® solution, or HC-A solution (as described in Table 2) are present at the concentrations described in Table 2 ± 5%. 88. The process of any one of embodiments 83 to 87, wherein the one or more components of the organ preservation solution comprise the components of Celsior® solution (as described in Table 2). 89. The process of embodiment 81, wherein the one or more components of the organ preservation solution include one or more components of PumpProtect® solution (as described in section 6.2). 90. The process of embodiment 89, wherein the one or more components of the organ preservation solution include components of PumpProtect® solution (as described in section 6.2). 91. The process of embodiment 90, wherein one or more components of the organ preservation solution comprise components of PumpProtect® solution (as described in Section 6.2) at concentrations ±20%, ±15%, ±10%, or ±5% of those described in Section 6.2. 92. An organ preservation solution produced by the process of any one of embodiments 80 to 91. 93. An organ preservation solution product comprising the organ preservation solution of any one of embodiments 22 to 63 and 92 in a sealed container. 94. The organ preservation solution product of embodiment 93, wherein the container is a bag. 95. The organ preservation solution product of embodiment 93 or embodiment 94, wherein the container contains 1 L of the preservation solution. 96. A system comprising: (a) the organ preservation solution of any one of embodiments 22 to 63 and 92 or the organ preservation solution product of any one of embodiments 93 to 95; and (b) a perfusion machine and / or an organ. 97. The system of embodiment 96, including a perfusion machine. 98. The system of embodiment 97, wherein the perfusion machine is a heart-lung machine. 99. The system of any one of embodiments 96 to 98, comprising an organ. 100. The system of embodiment 99, wherein the organ is a kidney, liver, heart, lung, pancreas, intestine, or trachea. 101. The system of embodiment 100, wherein the organ is a kidney. 102. The system of any one of embodiments 96 to 101, wherein the organ is from a mammal. 103. The system of embodiment 102, wherein the mammal is a human or a pig. 104. The system of embodiment 103, wherein the mammal is a human. 105. The system of embodiment 103, wherein the mammal is a pig. 106. A system comprising: (a) the organ preservation solution of any one of embodiments 22 to 63 and 92 or the organ preservation solution product of any one of embodiments 93 to 95; and (b) tissue. 107. The system of embodiment 106, wherein the tissue is eye, skin, fat, muscle, bone, cartilage, fetal thymus, or neural tissue. 108. The system of embodiment 107, wherein the tissue is corneal tissue. 109. The system of any one of embodiments 106 to 108, wherein the tissue is from a mammal. 110. The system of embodiment 109, wherein the mammal is a human or a pig. 111. The system of embodiment 110, wherein the mammal is a human. 112. The system of embodiment 110, wherein the mammal is a pig. 113. A process for ex-vivo organ preservation, comprising contacting a donor organ with the organ preservation solution of any one of embodiments 22 to 63 and 92. 114. The process of embodiment 113, comprising subjecting the organ to machine perfusion with the organ preservation solution. 115. The process of embodiment 114, comprising subjecting the organ to machine perfusion with the organ preservation solution for up to 2 days. 116. The process of embodiment 114, comprising subjecting the organ to machine perfusion with the organ preservation solution for up to 36 hours. 117. The process of embodiment 114, comprising subjecting the organ to machine perfusion with the organ preservation solution for up to 1 day. 118. The process of embodiment 114, comprising subjecting the organ to machine perfusion with the organ preservation solution for up to 12 hours. 119. The process of any one of embodiments 114 to 118, comprising subjecting the organ to machine perfusion with the organ preservation solution for at least 1 hour. 120. The process of any one of embodiments 114 to 118, comprising subjecting the organ to machine perfusion with the organ preservation solution for at least 2 hours. 121. The process of any one of embodiments 114 to 118, comprising subjecting the organ to machine perfusion with the organ preservation solution for at least 4 hours. 122. The process of any one of embodiments 114 to 118, comprising subjecting the organ to machine perfusion with the organ preservation solution for at least 6 hours. 123. The process of any one of embodiments 114 to 122, wherein the machine perfusion is performed using a system of any one of embodiments 96 to 105. 124. The process of any one of embodiments 113 to 123, wherein the organ preservation solution is diluted with whole blood. 125. The process of embodiment 124, wherein the volume:volume ratio of organ preservation solution to whole blood is 1:1 to 1:3. 126. The process of any one of embodiments 113 to 123, wherein the organ preservation solution is not diluted. 127. The process of any one of embodiments 114 to 126, wherein the machine perfusion is normothermic, optionally between 30°C and 38°C. 128. The process of any one of embodiments 114 to 126, wherein the machine perfusion is at 8°C to 25°C, optionally 20°C to 25°C. 129. The process of any one of embodiments 114 to 126, wherein the machine perfusion is at a temperature below normothermic, optionally between 2°C and 8°C. 130. The process of any one of embodiments 114 to 129, comprising flushing the organ with the preservation solution prior to the machine perfusion, and optionally, the organ preservation solution is at 2°C to 8°C prior to flushing the organ. 131. The process of any one of embodiments 114 to 130, further comprising low temperature storage of the organ before and / or after the machine perfusion, optionally at 2 ° C to 6 ° C. 132. The process of embodiment 113, optionally comprising low temperature storage of the organ at 2°C to 6°C in the absence of machine perfusion. 133. The process of embodiment 131 or 132, comprising cold storage of the organ in the organ preservation solution for up to one week. 134. The process of embodiment 131 or 132, comprising cold storage of the organ in the organ preservation solution for up to 5 days. 135. The process of embodiment 131 or 132, comprising cold storage of the organ in the organ preservation solution for up to 4 days. 136. The process of embodiment 131 or 132, comprising cold storage of the organ in the organ preservation solution for up to 3 days. 137. The process of embodiment 131 or 132, comprising cold storage of the organ in the organ preservation solution for up to 2 days. 138. The process of embodiment 131 or 132, comprising cold storage of the organ in the organ preservation solution for up to 36 hours. 139. The process of embodiment 131 or 132, comprising cold storage of the organ in the organ preservation solution for up to 1 day. 140. The process of embodiment 131 or 132, comprising cold storage of the organ in the organ preservation solution for up to 12 hours. 141. The process of any one of embodiments 131 to 140, comprising cold storage of the organ in the organ preservation solution for at least 1 hour. 142. The process of any one of embodiments 131 to 140, comprising cold storage of the organ in the organ preservation solution for at least 2 hours. 143. The process of any one of embodiments 131 to 140, comprising cold storage of the organ in the organ preservation solution for at least 4 hours. 144. The process of any one of embodiments 131 to 140, comprising cold storage of the organ in the organ preservation solution for at least 6 hours. 145. The process of any one of embodiments 113 to 144, comprising flushing the organ with the preservation solution, optionally at 2°C to 8°C or 2°C to 6°C, before and / or after removal of the organ from the donor. 146. The process of embodiment 145, wherein the preservation solution is left in the vasculature of the organ during cryogenic storage and / or transportation. 147. The process of any one of embodiments 113 to 146, wherein the organ is a kidney, liver, heart, lung, pancreas, intestine, or trachea. 148. The process of embodiment 147, wherein the organ is a kidney. 149. The process of any one of embodiments 113 to 148, wherein the organ is from a mammal. 150. The process of embodiment 149, wherein the mammal is a human or a pig. 151. The process of embodiment 150, wherein the mammal is a human. 152. The process of embodiment 150, wherein the mammal is a pig. 153. The process of any one of embodiments 113 to 152, further comprising removing the organ from the organ donor. 154. An organ obtained by the process of any one of embodiments 113 to 153. 155. A method for transplanting an organ, comprising transplanting the organ of embodiment 154 into a subject in need of an organ transplant. 156. A process for ex-vivo tissue preservation, comprising contacting donor tissue with the organ preservation solution of any one of embodiments 22 to 63 and 92. 157. The process of embodiment 156, comprising storing the tissue in the organ preservation solution. 158. The process of embodiment 156, optionally comprising normothermic storage of said tissue in said organ preservation solution at 30°C to 38°C. 159. The process of embodiment 156, optionally comprising low-temperature storage of the tissue in the organ preservation solution at 2°C to 6°C. 160. The process of any one of embodiments 157 to 159, comprising storing the tissue in the organ preservation solution for up to 4 weeks. 161. The process of any one of embodiments 157 to 159, comprising storing the tissue in the organ preservation solution for up to 2 weeks. 162. The process of any one of embodiments 157 to 159, comprising storing the tissue in the organ preservation solution for up to 1 week. 163. The process of any one of embodiments 157 to 159, comprising storing the tissue in the organ preservation solution for up to 4 days. 164. The process of any one of embodiments 157 to 159, comprising storing the tissue in the organ preservation solution for up to 2 days. 165. The process of any one of embodiments 157 to 159, comprising storing the tissue in the organ preservation solution for up to 36 hours. 166. The process of any one of embodiments 157 to 159, comprising storing the tissue in the organ preservation solution for up to 1 day. 167. The process of any one of embodiments 157 to 159, comprising storing the tissue in the organ preservation solution for up to 12 hours. 168. The process of any one of embodiments 157 to 167, comprising storing the tissue in the organ preservation solution for at least 1 hour. 169. The process of any one of embodiments 157 to 167, comprising storing the tissue in the organ preservation solution for at least 2 hours. 170. The process of any one of embodiments 157 to 167, comprising storing the tissue in the organ preservation solution for at least 4 hours. 171. The process of any one of embodiments 157 to 167, comprising storing the tissue in the organ preservation solution for at least 6 hours. 172. The process of any one of embodiments 156 to 171, wherein the tissue is eye, skin, fat, muscle, bone, cartilage, fetal thymus, or neural tissue. 173. The process of embodiment 172, wherein the tissue is corneal tissue. 174. The process of any one of embodiments 156 to 173, wherein the tissue is from a mammal. 175. The process of embodiment 174, wherein the mammal is a human or a pig. 176. The process of embodiment 175, wherein the mammal is a human. 177. The process of embodiment 175, wherein the mammal is a pig. 178. The process of any one of embodiments 156 to 177, further comprising removing the tissue from the tissue donor. 179. A tissue obtained by the process of any one of embodiments 156 to 179. 180. A method for transplanting tissue, comprising transplanting the tissue of embodiment 179 into a subject in need of tissue transplantation. 181.a. Obtaining a donor organ; b. Contacting the donor organ with the organ preservation solution of any one of embodiments 22 to 63 and 92, The contacting step comprises: i. machine perfusion of said organ with said organ preservation solution; or ii. Hygrostoring said organ in said organ preservation solution. and c. Transplanting the organ into a subject in need of an organ transplant. and a transplantation method comprising: 182. The method of embodiment 181, wherein the donor organ is a kidney, liver, heart, lung, pancreas, intestine, or trachea. 183. The method according to embodiment 181 or embodiment 182, wherein the contacting step comprises machine perfusion or cold storage of the organ with the organ preservation solution for at least 1 hour and / or up to 1 week. 184.a. Obtaining donor tissue; b. storing the donor tissue in an organ preservation solution of any one of embodiments 22 to 63 and 92; c. Transplanting the tissue into a subject in need of a tissue transplant. and a transplantation method comprising: 185. The method of embodiment 184, wherein the donor tissue is eye, skin, fat, muscle, bone, cartilage, fetal thymus, or neural tissue. 186. The method of embodiment 184 or embodiment 185, wherein the storing comprises storing the tissue with the organ preservation solution for at least 1 hour and / or up to 4 weeks.

[0267] While various specific embodiments have been illustrated and described, it will be understood that various modifications can be made without departing from the spirit and scope of the disclosure(s).

[0268] 9. INCORPORATION BY REFERENCE All publications, patents, patent applications, and other documents cited in this application are herein incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes.

[0269] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present disclosure and should not be construed as an admission that any or all of such matter formed part of the prior art or was common general knowledge in the field relevant to the present disclosure, if any, prior to the priority date of this application.

Claims

1. Lipid-binding protein-based complexes used in organ preservation solutions.

2. 2. The lipid binding protein-based complex of claim 1, which is a reconstituted HDL or HDL mimic.

3. The lipid-binding protein-based complex of claim 2, which is CER-001, CSL-111, CSL-112, CER-522 or ETC-216.

4. The lipid-binding protein-based complex of claim 3, which is CER-001.

5. An organ preservation solution comprising a lipid-binding protein-based complex.

6. 6. The organ preservation solution of claim 5, wherein said lipid binding protein-based complex is a reconstituted HDL or HDL mimic.

7. 7. The organ preservation solution of claim 6, wherein the lipid-binding protein-based complex is CER-001, CSL-111, CSL-112, CER-522 or ETC-216.

8. 8. The organ preservation solution of claim 7, wherein the lipid-binding protein-based complex is CER-001.

9. 6. The organ preservation solution of claim 5, comprising a buffer, an antioxidant, a nutrient and / or metabolic substrate, an electrolyte, a colloid, an impermeable substance, a gas, or a combination thereof.

10. 6. The organ preservation solution of claim 5, comprising said lipid-binding protein-based complex at a concentration of 0.1 mg / ml to 5 mg / ml.

11. A kit comprising a lipid-binding protein-based complex and one or more components of an organ preservation solution, wherein the lipid-binding protein-based complex may be as described in claim 1.

12. 12. The kit of claim 11, wherein the one or more components of the organ preservation solution include a buffer, an antioxidant, a nutrient and / or metabolic substrate, an electrolyte, a colloid, an impermeable substance, a gas, or a combination thereof.

13. 12. A process for preparing an organ preservation solution from the kit of claim 11, comprising combining the lipid binding protein-based complex and one or more components of the organ preservation solution.

14. 6. An organ preservation solution product comprising the organ preservation solution of claim 5 in a sealed container.

15. 15. The organ preservation solution product of claim 14, wherein the container is a bag.

16. 15. The organ preservation solution product of claim 14, wherein said container contains 1 L of said organ preservation solution.

17. 15. A system comprising: (a) the organ preservation solution of claim 5 or the organ preservation solution product of claim 14; and (b) a perfusion machine and / or an organ.

18. 20. The system of claim 17 including a perfusion machine.

19. 20. The system of claim 17, comprising an organ.

20. 20. The system of claim 19, wherein the organ is a kidney, liver, heart, lung, pancreas, intestine, or trachea.

21. A process of ex-vivo organ preservation comprising contacting a donor organ with the organ preservation solution of claim 5.

22. 22. The process of claim 21, comprising subjecting the organ to machine perfusion with the organ preservation solution (for at least 1 hour and / or optionally up to 1 week).

23. 22. A process according to claim 21, comprising cold storage of the organ (which may be between 2°C and 6°C) in the absence of machine perfusion.

24. 10. A process of ex-vivo tissue preservation comprising contacting a donor tissue with the organ preservation solution of claim 5, optionally wherein the donor tissue is stored in the organ preservation solution for at least 1 hour and / or up to 4 weeks.

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