Ex vivo organ care compositions

Abstract

A composition is provided that includes a fusion protein in an aqueous medium. The fusion protein can bind to a receptor expressed on a cell and kill the cell. The aqueous medium includes one or more components that allow for the treatment of ex vivo organs, tissues, and / or stem cell cultures before transplantation. Thus, the composition is useful for preventing or treating infection caused by pathogens in ex vivo organs, tissues, and / or stem cell cultures.

Description

[Technical field]

[0001] The present invention relates to a composition comprising a fusion protein in an aqueous medium. The fusion protein can bind to a receptor expressed on a particular cell, and then be taken up into the cell and kill the cell. The aqueous medium comprises one or more components that allow the treatment of ex vivo organ, tissue and / or stem cell cultures before transplantation. Thus, the composition is useful for the prevention or treatment of infectious diseases caused by pathogens such as viruses, CMV being one example of such a virus, in ex vivo organ, tissue and / or stem cell cultures. [Background technology]

[0002] Immunotoxins are proteins with a targeting moiety linked to a toxin. Immunotoxins are an example of a fusion protein because they are artificial proteins consisting of a targeting moiety linked or fused to a toxin. Immunotoxins can be used to treat infections caused by a variety of pathogens. For example, toxins conjugated to antibodies have previously been utilized to target CMV-infected cells, but with limited success.

[0003] Human cytomegalovirus (HCMV) is a species-specific herpesvirus that is an important pathogen, especially in immunocompromised individuals, neonates, and transplant recipients. HCMV has a life cycle consisting of latent and lytic phases, and during both phases it constitutively expresses the internalizing receptor US28.

[0004] For example, the presence of CMV antibodies in lungs for transplant was found to be approximately 62% (Chambers et al., 2020). As a result, transplant recipients are at higher risk of developing CMV infection due to the immunosuppressant medications they must take to avoid rejecting the transplant.

[0005] Organ transplantation is a well-established treatment and is now recognised as the best, and often only, life-saving treatment for end-stage organ failure. According to the latest data from the WHO Global Observatory on Donation and Transplantation, it is estimated that over 130,000 solid organ transplants have been performed worldwide, which represents less than 10% of the global need.

[0006] One of the reasons for the under-utilization of organ transplantation is that out of 10 donated thoracic organs, only 2-3 are suitable for transplantation. Cold storage methods limit the utilization of donor organs and may negatively affect outcomes. Organ Care Systems (OCS), on the other hand, mimic the physiological state of the organ. OCS allows organs to survive longer periods outside the body and be less susceptible to damage during transport by pumping warm, oxygenated, nutrient-rich blood or fluids to the organ from the time it is harvested until it is transplanted. OCS is becoming increasingly popular to preserve organs before transplantation, as opposed to traditional cold storage, because optimization of donor organs and evaluation of organ viability by OCS may allow more organs to be utilized, resulting in better outcomes for transplant patients and shorter waiting times.

[0007] HCMV infection is also one of the most significant complications after allogeneic hematopoietic stem cell transplantation (HSCT) and remains one of the leading causes of morbidity and mortality after this procedure. The virus can cause multisystem disease in stem cell transplant recipients, including pneumonia, hepatitis, gastroenteritis, retinitis, and encephalitis, and this disease can develop both early and late after the transplant procedure.

[0008] Hematopoietic stem cells (HSCs) represent a heterogeneous population of primitive blood progenitor cells. In the human body, they are primarily present in the bone marrow in adults, but also in various fetal tissues, such as umbilical cord blood, placenta, and fetal liver. HSCs are functionally defined by their self-renewal capacity and by their multipotency, which allows the replenishment of all types of blood cells. The myeloid branch of their progeny is represented by monocytes / macrophages, granulocytes (neutrophils, basophils, eosinophils), erythrocytes, megakaryocytes (platelet-producing cells), and dendritic cells. The lymphoid branch is composed of T lymphocytes, B lymphocytes, and NK cells.

[0009] An example of HSCT is the treatment of cancer. Some of the most effective treatments for cancer, such as chemotherapy and radiation therapy, are toxic to the bone marrow. In HSCT, very high doses of chemotherapy or radiation therapy are administered. This is aimed at killing cancer cells that may be resistant to more standard doses of chemotherapy. Unfortunately, this also destroys normal cells in the bone marrow, including stem cells. After treatment, a healthy supply of reintroduced or transplanted stem cells is needed. The transplanted cells then re-establish the process of blood cell production in the bone marrow.

[0010] All drugs currently used in the clinical treatment of HCMV infection are associated with significant side effects, and the emergence of drug resistance frequently leads to treatment failure.

[0011] In addition to the pharmacological challenges and poor cell targeting selectivity of existing immunotoxins, another cause of therapeutic failure is insufficient internalization, which often leads to inadequate killing of infected cells.

[0012] These limitations underscore the value of developing new drug treatments for pathogen-induced infections such as HCMV that can be used, for example, to treat stem cell cultures, tissues or organs ex vivo before they are transplanted into patients. Summary of the Invention

[0013] Provided herein is a composition comprising a fusion protein in an aqueous medium. The fusion protein can selectively bind to a receptor expressed on a specific cell, which can then be internalized, and the fusion protein can kill the target cell. The aqueous medium comprises one or more components that allow for the treatment of ex vivo organs, tissues, and / or stem cell cultures before transplantation. Thus, the composition is useful for the prevention or treatment of pathogen-induced infections, including latent infections, in ex vivo organs, tissues, and / or stem cell cultures.

[0014] The inventors have generated a fusion protein that effectively targets and kills infected cells, such as CMV-infected cells, including latently infected cells. The fusion protein comprises a first peptide capable of binding to at least one receptor, which may be a virally encoded receptor, such as US28, and a second peptide that comprises an optimized cleavage site and a toxin, such as a selected domain of exotoxin A, e.g., domains II and III. Upon target binding, the receptor is preferably internalized and the second peptide is at least partially cleaved at the cleavage site, thus releasing the toxin moiety and then killing the infected cell. Surprisingly, the inventors have found that a specific cleavage site motif (ArgX1X2Arg, where X2 is basic, such as Arg), corresponding to a mutation in the native furin cleavage site of exotoxin A, increases selectivity for cells expressing a pathogen-encoded receptor, such as US28. This discovery allows for improved targeting and killing of infected cells, e.g., CMV-infected cells. Thus, the optimized cleavage sites described herein can be utilized in fusion proteins, particularly immunotoxins, to enhance toxicity / cell killing potency, particularly selectivity.The inventors further combined this fusion protein with an aqueous medium containing one or more components that allow for ex vivo treatment of organs, tissues, and / or stem cell cultures prior to transplantation.

[0015] In one aspect, the disclosure provides a composition for preventing or treating infection by a pathogen in a pre-transplant ex vivo organ, tissue, and / or stem cell culture, the composition comprising in an aqueous medium: A fusion protein comprising: i. a first peptide that binds to at least one receptor expressed on a cell; ii. a second peptide comprising a cleavage site having the amino acid sequence ArgX1X2Arg; X2 is Arg or Lys, and the second peptide comprises a toxin; The aqueous medium further comprises one or more of the following: i. Buffer component; ii. amino acids; iii. carbohydrates; or iv. Growth factors or cytokines.

[0016] In one aspect, the disclosure provides a composition as described herein for use in preventing or treating CMV infection and / or CMV-associated disorders in ex vivo organ, tissue, and / or stem cell cultures prior to transplantation.

[0017] In one aspect, the invention provides for the use of a composition as described herein for the treatment or prevention of infection by a pathogen in ex vivo organ, tissue, and / or stem cell cultures for transplantation.

[0018] In some aspects of the disclosure, methods are provided for the treatment or prevention of infection by a pathogen in ex vivo organ, tissue, and / or stem cell cultures prior to transplantation, the methods comprising immersing, perfusing, preserving, conditioning, and / or flushing the ex vivo organ, tissue, and / or stem cell culture in a composition described herein.

[0019] Some aspects also provide for the use of the compositions described herein for the treatment or prevention of infection by a pathogen in ex vivo organ, tissue, and / or stem cell cultures for transplantation.

[0020] In some aspects of the disclosure, a kit is provided for preventing or treating infection by a pathogen in an ex vivo organ, tissue, and / or stem cell culture, the kit comprising: a. a composition described herein; b. Optionally, instructions for use.

[0021] In some aspects of the present disclosure, a perfusion system configured to maintain an ex vivo organ in a functional state under physiological or near-physiological conditions is provided, the perfusion system comprising one or more chambers configured to maintain the ex vivo organ in a functional state, at least one chamber comprising a composition described herein. [Brief description of the drawings]

[0022] [Figure 1] Domain structure of human CX3CL1, Pseudomonas aeruginosa exotoxin A, and an exemplary first peptide, which is a fusion protein SYN002. A. Schematic of the domain structure of human CX3CL1 (S=signal sequence, CX3CL1=chemokine domain, stalk=mucin-like stalk, M=transmembrane portion, C=cytoplasmic domain), Pseudomonas aeruginosa exotoxin A (S=signal sequence, domain I=receptor binding domain, domain II=translocation domain, Ib=domain Ib with unknown function, and domain III=ADP-ribosylation domain). Amino acid numbering of the precursor proteins is shown above each protein. Disulfide bridges are shown below each protein in brackets along with the numbering of the amino acids involved. A. Schematic diagram of SYN002. Mutations in specific domains are described by the one-letter code of the amino acid involved and its number. For example, P303R means that a proline at position 303 has been replaced by an arginine. One-letter codes are also used at the N- and C-termini of the constructs and between domains. A dashed line between two domains indicates that the amino acids are connected. Furin cleaves between amino acids 304 and 305 in domain II of exotoxin A. The five C-terminal residues of exotoxin A are replaced by the endoplasmic reticulum retention signal KDEL, which has been optimized in SYN002. [Diagram 2]Selectivity due to the F49A mutation. A single amino acid substitution in the chemokine portion of SYNx confers selectivity for the US28 receptor (A) compared with the CXCR1 receptor (B). SYN000 is wild-type human chemokine (C-X3-C motif) ligand 1 (CX3CL1) (native chemokine sequence), whereas SYN001 has a single mutation (F49A) in the receptor-binding portion of the protein. [Diagram 3] Introduction of a furin cleavage site (in this case the full-length translocation domain of exotoxin A) increases potency. Addition of the full-length translocation domain of exotoxin A containing a furin cleavage site to the SYN001 construct to generate SYN016 increases potency in cells expressing both endogenous CX3CR1 and the virally encoded US28 receptor. [Figure 4] In vitro cleavage by furin. In vitro cleavage by furin of the SYN002 construct with the optimized cleavage site (RQRR) does not result in any improvement compared to the fusion protein construct SYN016 with the native furin cleavage site sequence (RQPR). In vitro furin digestion of SYNx. Lane 1; Mark 12 protein standard, lane 2; SYN000, lane 3; SYN017, lane 4; SYN014, lane 5; SYN001, lane 6; SYN016, lane 7; SYN002. SYN000, SYN017, SYN014 have the native CX3CL1 chemokine sequence, while SYN001, SYN016, SYN002 have the F73A mutation in the chemokine portion. SYN000, SYN017, SYN001, and SYN016 have a natural furin cleavage site (RQPR), while SYN014 and SYN002 have an optimized cleavage site (RQRR). [Diagram 5]SYN002 is cleaved by furin over a wide pH range. SYN002 was cleaved in vitro over a wide pH range. Lane 1: Mark 12 protein standard, lane 2: no furin, lane 3: +8 mM HCl (~pH 5.2), lane 4: +6 mM HCl (~pH 6.3), lane 5: +4 mM HCl (~pH 6.6), lane 6: +2 mM HCl (~pH 7.0), lane 7: no titrant added (~pH 7.4), lane 8: 2 mM NaOH (~pH 7.7), lane 9: +4 mM NaOH (~pH 9.0). [Figure 6] Optimized cleavage site improves selectivity When an optimized cleavage site ArgX1X2Arg is introduced into a second peptide, in this particular case the optimized cleavage site ArgGlnArgArg is introduced into SYN016 to give SYN002, resulting in increased selectivity against the virally encoded receptor. In the case of SYN002 on cells expressing a virally encoded receptor such as US28, the cell killing potency is almost maintained compared to SYN016 with the cleavage site ArgGlnProArg (hence X2=Pro), but the potency against cells expressing endogenous / human receptors such as CX3CR1 is reduced compared to SYN016. When compared to a second peptide not containing a furin cleavage site, e.g. SYN000 (not shown), both selectivity and potency are increased. [Figure 7] Potency assay with SYN001 from 1 mg / L sample. Potency assay with SYN001 dilution series made from 1 mg / L (1000 ng / mL or 23.1 nM) sample after 0, 15, 180, and 300 minutes of exposure to perfusate at 37° C. (sample A). SYN002 samples represented as dots correspond to SYN002 not exposed to perfusate. Cycloheximide is the positive control for the assay and inhibits protein synthesis. (Data from two different plates are combined in one figure). Results are further described in Example 5. [Figure 8]Potency assay with SYN002 from 1.7 mg / L sample. Potency assay with SYN002 dilution series made from 1.7 mg / L (1700 ng / mL or 37.1 nM) sample after 0, 15, 180, and 300 minutes of exposure to perfusate at 37° C. (sample A). SYN002 samples represented as dots correspond to SYN002 not exposed to perfusate. Cycloheximide is the positive control for the assay and inhibits protein synthesis. (Data from two different plates are combined in one figure). Results are further described in Example 5. [Figure 9] Potency assay with SYN002 from a 0.85 mg / L sample. Potency assay with a SYN002 dilution series made from a 0.85 mg / L (850 ng / mL or 18.6 nM) sample after 0, 15, 180, and 300 minutes of exposure to perfusate at 37° C. (sample C). SYN002 samples represented as dots correspond to SYN002 not exposed to perfusate. Cycloheximide is the positive control for the assay and inhibits protein synthesis. (Data from two different plates are combined in one figure). Results are further described in Example 5. [Figure 10] Potency assay with SYN002 from a 0.085 mg / L sample. Potency assay with a SYN002 dilution series made from a 0.085 mg / L (85 ng / mL or 1.86 nM) sample after 0, 15, 180, and 300 minutes of exposure to perfusate at 37° C. (sample D). SYN002 samples represented as dots correspond to SYN002 not exposed to perfusate. Cycloheximide is the positive control for the assay and inhibits protein synthesis. (Data from two different plates are combined in one figure). Results are further described in Example 5. [Figure 11]Potency assay with SYN001 from a 0.085 mg / L sample at 30 and 60 minutes. Potency assay with a SYN002 dilution series made from a 0.085 mg / L (85 ng / mL or 1.86 nM) sample after 30 and 300 minutes of exposure to perfusate at 37° C. (Sample D). SYN002 samples represented as dots correspond to SYN002 not exposed to perfusate. Cycloheximide is the positive control for the assay and inhibits protein synthesis. Results are further described in Example 5. [Figure 12] Potency assay with SYN001 from a 0.085 mg / L sample at 1140 min. Potency assay with a SYN002 dilution series made from a 0.085 mg / L (85 ng / mL or 1.86 nM) sample after 1140 min of exposure to perfusate at 37° C. (Sample D). SYN002 samples represented as dots correspond to SYN002 not exposed to perfusate. Cycloheximide is the positive control for the assay and inhibits protein synthesis. Results are further described in Example 5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present disclosure relates to compositions comprising a fusion protein in an aqueous medium with one or more additional components that provide stability and efficacy of the fusion protein, thus enabling efficient treatment or prevention of infection in ex vivo organs, tissues, and / or stem cell cultures. The additional components may further help to maintain or preserve the function of the ex vivo organs, tissues, and / or stem cell cultures. The fusion protein comprises a first peptide that provides target recognition and target selectivity, and a second peptide that provides a toxin, and further provides intracellular cleavage and release of the toxin, resulting in effector toxicity in the target cell. Thus, the compositions provided herein are useful for the prevention or treatment of pathogen-induced infection, including the treatment of latent infection in ex vivo organs, tissues, and / or stem cell cultures.

[0024] The present inventors have generated fusion proteins that, upon binding to a receptor, e.g., a virally encoded receptor such as US28, that can advantageously be constitutively internalized, effectively target and kill infected cells, such as CMV-infected cells, including latently infected cells.

[0025] US28 is a G protein-coupled receptor encoded by the human cytomegalovirus open reading frame US28. US28 is a constitutively internalized receptor. Thus, chemokines or other compounds that bind to US28 are internalized in cells that express the receptor. US28 is expressed on the surface of CMV-infected cells that are lytically or latently infected with CMV.

[0026] The fusion protein comprises a first peptide capable of binding to a receptor, in particular a pathogen-encoded receptor such as US28 encoded by HCMV, and a second peptide comprising an optimized cleavage site and a selected domain of a toxin, e.g., exotoxin A. The selectivity of the fusion protein for a pathogen-encoded receptor such as US28 is surprisingly enhanced after mutation in the cleavage site.

[0027] The optimized cleavage sites described herein can be utilized in a variety of fusion proteins, particularly immunotoxins, to enhance cell killing selectivity by modifying cell killing capacity in a target receptor selective manner.

[0028] definition As used herein, "a" can mean one or more, depending on the context in which it is used.

[0029] As used herein, the term "about" means ±10%, preferably ±5%, and even more preferably ±2%.

[0030] As used herein, the term "amino acid" includes the standard 20 genetically encoded amino acids and their corresponding stereoisomers of the "D" form (as compared to the naturally occurring "I" form), the omega amino acids and other naturally occurring amino acids, unconventional amino acids (e.g., α,α-disubstituted amino acids, N-alkyl amino acids, etc.), and chemically derivatized amino acids. When an amino acid such as "alanine" or "Ala" or "A" is specifically recited, the term refers to both l-alanine and d-alanine unless otherwise indicated.

[0031] In one embodiment of the invention, the fusion protein consists only of naturally occurring amino acids.

[0032] As used herein, the term "sequence identity" refers to the relationship between two amino acid sequences or two nucleotide sequences, i.e., between a candidate sequence (e.g., a mutant sequence) and a reference sequence (such as a wild-type sequence) based on their pairwise alignment. For the purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mo / . Biol. 48: 443-453), as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later (available at https: / / www.ebi.ac.uk / Tools / psa / emboss_needle / ). The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of 30BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity, calculated as follows: (identical residues x 100) / (length of alignment - total number of gaps in the alignment). The Needleman-Wunsch algorithm is also used to determine whether a particular amino acid in a sequence other than the reference sequence corresponds to a particular position in the reference sequence. For the purposes of the present invention, sequence identity between two nucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra), preferably version 5.0.0 or later, as implemented in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277).The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the DNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity, calculated as follows: (identical deoxyribonucleotides × 100) / (length of alignment - total number of gaps in the alignment).

[0033] As used herein, the terms "treat", "treatment" and "therapy" refer to curative, prophylactic, ameliorative and palliative therapy. Preferably, the treatment is curative.

[0034] composition One aspect of the present disclosure provides a composition for preventing or treating infection by a pathogen in ex vivo organ, tissue, and / or stem cell cultures prior to transplantation, the composition comprising in an aqueous medium: A fusion protein comprising: i. a first peptide that binds to at least one receptor expressed on a cell; ii. a second peptide comprising a cleavage site having the amino acid sequence ArgX1X2Arg; X2 is Arg or Lys, and the second peptide comprises a toxin; The aqueous medium further comprises one or more of the following: i. Buffer component; ii. amino acids; iii. carbohydrates; or iv. Growth factors or cytokines.

[0035] In some embodiments, the aqueous medium comprises: i. Buffer component; ii. Amino acids.

[0036] In some embodiments, the aqueous medium comprises: i. Buffer component; ii. Carbohydrates.

[0037] In some embodiments, the aqueous medium comprises: i. Buffer component; ii. Growth factors or cytokines.

[0038] In some embodiments, the aqueous medium comprises: i. Amino acids: ii. Carbohydrates.

[0039] In some embodiments, the aqueous medium comprises: i. Amino acids: ii. Growth factors or cytokines.

[0040] In some embodiments, the aqueous medium comprises: i. carbohydrates; ii. Growth factors or cytokines.

[0041] In some embodiments, the aqueous medium comprises: i. Buffer component; ii. Amino acids: iii. Carbohydrates.

[0042] In some embodiments, the aqueous medium comprises: i. Buffer component; ii. Amino acids: iii. Growth factors or cytokines.

[0043] In some embodiments, the aqueous medium comprises: i. Buffer component; ii. carbohydrates; iii. Growth factors or cytokines.

[0044] In some embodiments, the aqueous medium comprises: i. amino acids; ii. carbohydrates; or iii. Growth factors or cytokines.

[0045] In some embodiments, the aqueous medium comprises: i. Buffer component; ii. amino acids; iii. carbohydrates; iv. Growth factors or cytokines.

[0046] In some embodiments, the aqueous medium further comprises an antibiotic. In some embodiments, the aqueous medium further comprises a glucocorticoid. In some embodiments, the aqueous medium further comprises an anticoagulant. In some embodiments, the aqueous medium further comprises a prostaglandin.

[0047] In some embodiments, the compositions disclosed herein are suitable for maintaining the viability of said ex vivo organ, tissue, and / or stem cell culture prior to transplantation.

[0048] In some embodiments, the compositions disclosed herein are suitable for maintaining said ex vivo organ, tissue, and / or stem cell cultures in a functional state.

[0049] In some embodiments, the compositions disclosed herein are suitable for maintaining the ex vivo organ, tissue, and / or stem cell cultures under physiological or near-physiological conditions.

[0050] In some embodiments, the compositions disclosed herein are suitable for preserving the ex vivo organ in an organ care system.

[0051] In some embodiments, the compositions disclosed herein further comprise one or more additional agents. In some embodiments, the additional agents are selected from the group consisting of immunosuppressants and antiviral agents. In some embodiments, the antiviral agents are selected from the group consisting of valganciclovir, ganciclovir, cidofovir, leflunomide, prevmis, ​​maribavir, and brincidofovir.

[0052] Cutting site The second peptide of the disclosure has the amino acid sequence: ArgX1X2Arg (X1 can be any amino acid). In one embodiment, X1 is selected from the group consisting of Gln, Ser, Thr, and Asn. In one embodiment, X1 is Gln.

[0053] X2 is selected from Arg and Lys. In an advantageous embodiment, X2 is Arg. In one embodiment, X2 is Lys. Introducing a basic amino acid, such as Arg, into amino acid position X2 enhances cell killing selectivity. For example, virus-infected cells that express a virus-encoded receptor and bind to the first peptide are killed more efficiently than cells that express an endogenous receptor that can also bind to the first peptide.

[0054] In a preferred embodiment, the cleavage site comprises or consists of the amino acid sequence ArgGlnArgArg (RQRR; SEQ ID NO: 16). In one embodiment, the cleavage site comprises or consists of the amino acid sequence ArgGlnLysArg (RQKR; SEQ ID NO: 17). In one embodiment, the cleavage site comprises or consists of the amino acid sequence ArgSerLysArg (RSKR; SEQ ID NO: 18). In one embodiment, the cleavage site comprises or consists of the amino acid sequence ArgSerArgArg (RSRR; SEQ ID NO: 19). In one embodiment, the cleavage site comprises or consists of the amino acid sequence ArgThrLysArg (RTKR; SEQ ID NO: 20). In one embodiment, the cleavage site comprises or consists of the amino acid sequence ArgThrArgArg (RTRR; SEQ ID NO: 21). In one embodiment, the cleavage site comprises or consists of the amino acid sequence ArgAsnLysArg (RNKR; SEQ ID NO: 22). In one embodiment, the cleavage site comprises or consists of the amino acid sequence ArgAsnArgArg (RNRR; SEQ ID NO:23).

[0055] In one embodiment, cleavage occurs C-terminal to the cleavage site.

[0056] It may be possible to cleave the fusion protein with furin, although other enzymes or proteases may also be involved in cleavage at the cleavage site. Thus, in one embodiment, the cleavage site is an enzymatic cleavage site.

[0057] In one embodiment, the enzymatic cleavage site is a furin cleavage site.

[0058] Receptor In one embodiment, the first peptide binds to at least one receptor expressed on the cell. In one embodiment, the first peptide binds to at least two receptors expressed on the cell.

[0059] In one embodiment, one receptor that binds to the first peptide is a pathogen-encoded receptor and a further receptor that binds to the first peptide is a human-encoded and / or endogenous receptor for the first peptide or a variant thereof.

[0060] In one embodiment, the receptor is a G protein-coupled receptor (GPCR), such as US28 of SEQ ID NO:10.

[0061] In one embodiment, the receptor is a chemokine receptor. In one embodiment, the chemokine receptor is a CC chemokine receptor and / or a CX3C chemokine receptor.

[0062] In one embodiment, the CC chemokine receptor is US28. In one embodiment, the CX3C chemokine receptor is CX3CR1.

[0063] In one embodiment, the receptor is encoded by a pathogen. In one embodiment, the pathogen is a bacterium. In one embodiment, the pathogen is a virus. In one embodiment, the virus is a DNA virus. In one embodiment, the virus is an RNA virus. In one embodiment, the virus is a herpes virus. In one embodiment, the virus is a cytomegalovirus.

[0064] In one embodiment, the receptor can be internalized. In one embodiment, the receptor is internalized upon binding to the first peptide. In one embodiment, the receptor is constitutively internalized. It is advantageous for the pathogen-encoded receptor to be constitutively internalized, since it ensures efficient uptake of the fusion protein by the pathogen-infected cell, thereby killing the infected cell with minimal undesirable toxicity and side effects. "Internalization" refers to the process by which the receptor moves into the cell in which it is expressed. For example, the receptor may enter the cell by endocytosis or phagocytosis.

[0065] In some embodiments, the invention provides fusion proteins that target specific cells by selectively binding to a receptor, resulting in internalization of the receptor, after which the toxin portion of the fusion protein is cleaved, killing the cell.

[0066] toxin In one embodiment, the fusion protein is an immunotoxin. "Immunotoxin" refers to a protein consisting of a targeting moiety linked to a toxin. The targeting moiety is sometimes called a ligand.

[0067] The toxin can be of various types of toxins. In one embodiment, the second peptide comprises a toxin selected from the group consisting of Pseudomonas aeruginosa exotoxin A, gelonin, bouganin, saporin, ricin, ricin A chain, bryodin, restrictocin, diphtheria toxin, diphtheria toxin A chain, and variants and fragments thereof. The toxin portion of the fusion protein allows for cell killing. The toxin is cytotoxic.

[0068] In one embodiment, the second peptide comprises one or more domains of Pseudomonas aeruginosa exotoxin A. In one embodiment, the second peptide comprises at least a portion of exotoxin A of SEQ ID NO:9.

[0069] In some embodiments, the second peptide contains a radioisotope or emitter, e.g., 211 At, 225 Ac,227 Th, 224 Ra, 213 Bi, 67 Cu, 90 Y, 131 I, 177 Lu, 186 Re, 188 Re, and 125 I further includes those selected from the group consisting of:

[0070] In some embodiments, the radioisotope or emitter emits alpha particles upon radioactive decay. In some embodiments, the radioisotope or emitter emits beta particles upon radioactive decay. In some embodiments, the radioisotope or emitter emits electrons (also known as Auger emission).

[0071] The radioisotope or emitter further enables killing of the target cell: the ionizing radiation emitted by the radioisotope or emitter is cytotoxic to the cell.

[0072] Fusion proteins The first peptide is a targeting moiety that allows for binding to a receptor expressed on a cell.

[0073] In one embodiment, the first and second peptides are operably linked. Generally, "operably linked" means that the sequences being linked are contiguous and / or are in a functional relationship with each other, such as by a covalent bond.

[0074] In one embodiment, the first peptide comprises or consists of: a. the amino acid sequence of SEQ ID NO:1; b. a variant of SEQ ID NO:1 comprising or consisting of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:1, more preferably at least 85% or 90% sequence identity to SEQ ID NO:1, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:1; c. A fragment of SEQ ID NO:1 that is more than 50 amino acids in length, such as more than 60, 70, or 75 amino acids in length, or a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:1, more preferably at least 85% or 90% sequence identity to SEQ ID NO:1, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:1.

[0075] In one embodiment, the first peptide comprises or consists of a variant of SEQ ID NO: 1 comprising or consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1. In one embodiment, the first peptide comprises or consists of a variant of SEQ ID NO: 1 comprising or consisting of an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 1. In one embodiment, the first peptide comprises or consists of a variant of SEQ ID NO: 1 comprising or consisting of an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO: 1. In one embodiment, the first peptide comprises or consists of a variant of SEQ ID NO: 1 comprising or consisting of an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO: 1. In one embodiment, the first peptide comprises or consists of a variant of SEQ ID NO: 1 comprising or consisting of an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 1. In one embodiment, the first peptide comprises or consists of a variant of SEQ ID NO:1 comprising or consisting of an amino acid sequence having at least 99% sequence identity thereto.

[0076] In one embodiment, the first peptide comprises or consists of a fragment of SEQ ID NO:1 that is greater than 50 amino acids in length. In one embodiment, the first peptide comprises or consists of a fragment of SEQ ID NO:1 that is greater than 60 amino acids in length. In one embodiment, the first peptide comprises or consists of a fragment of SEQ ID NO:1 that is greater than 70 amino acids in length. In one embodiment, the first peptide comprises or consists of a fragment of SEQ ID NO:1 that is greater than 75 amino acids in length.

[0077] In one embodiment, the first peptide comprises or consists of a fragment of SEQ ID NO:1 or a variant thereof having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:1. In one embodiment, the first peptide comprises or consists of a fragment of SEQ ID NO:1 or a variant thereof having at least 85% sequence identity with the amino acid sequence of SEQ ID NO:1. In one embodiment, the first peptide comprises or consists of a fragment of SEQ ID NO:1 or a variant thereof having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:1. In one embodiment, the first peptide comprises or consists of a fragment of SEQ ID NO:1 or a variant thereof having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:1. In one embodiment, the first peptide comprises or consists of a fragment of SEQ ID NO:1 or a variant thereof having at least 96% sequence identity with the amino acid sequence of SEQ ID NO:1. In one embodiment, the first peptide comprises or consists of a fragment of SEQ ID NO:1 or a variant thereof having at least 97% sequence identity with the amino acid sequence of SEQ ID NO:1. In one embodiment, the first peptide comprises or consists of a fragment of SEQ ID NO: 1, or a variant thereof having at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 1. In one embodiment, the first peptide comprises or consists of a fragment of SEQ ID NO: 1, or a variant thereof having at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 1.

[0078] In one embodiment, the amino acid residue at position 49 of SEQ ID NO:1 is mutated to Ala, Lys, or Asp, preferably Ala. These substitutions may result in increased cell killing selectivity against cells expressing a pathogen-encoded receptor that binds to the first peptide compared to a human or endogenously encoded receptor that also binds to the first peptide.

[0079] In one embodiment, the first peptide further comprises a methionine (M) at the N-terminus.

[0080] In one embodiment, the first peptide comprises or consists of one or more additional amino acids inserted at the N-terminus and / or C-terminus and / or within the amino acid sequence of SEQ ID NO:1.

[0081] In one embodiment, one or more amino acids are deleted at the N-terminus and / or C-terminus and / or within the amino acid sequence of SEQ ID NO:1.

[0082] In one embodiment, the first peptide is less than 100 amino acids in length.

[0083] In one embodiment, the first peptide is less than 90 amino acids in length.

[0084] In one embodiment, the first peptide is less than 85 amino acids in length.

[0085] In one embodiment, the first peptide is less than 80 amino acids in length.

[0086] In one embodiment, the second peptide comprises Domain A, which may be a translocation domain, such as Domain A having an amino acid sequence according to SEQ ID NO: 3 or a fragment or variant thereof, and / or Domain B, such as Domain B having an amino acid sequence according to SEQ ID NO: 4 or a fragment or variant thereof, and / or Domain C, which may be a cytotoxic domain, such as Domain C having an amino acid sequence according to SEQ ID NO: 5 or a fragment or variant thereof.

[0087] In one embodiment, the second peptide comprises a domain A, such as domain A having an amino acid sequence according to SEQ ID NO: 3 or a fragment or variant thereof. In one embodiment, the second peptide comprises a domain A, such as domain A having an amino acid sequence according to SEQ ID NO: 3 or a fragment or variant thereof, and a domain B, such as domain B having an amino acid sequence according to SEQ ID NO: 4 or a fragment or variant thereof. In one embodiment, the second peptide comprises a domain A, such as domain A having an amino acid sequence according to SEQ ID NO: 3 or a fragment or variant thereof, and a domain B, such as domain B having an amino acid sequence according to SEQ ID NO: 4 or a fragment or variant thereof, and a domain C, such as domain C having an amino acid sequence according to SEQ ID NO: 5 or a fragment or variant thereof. In one embodiment, the second peptide comprises a domain B having an amino acid sequence according to SEQ ID NO: 4 or a fragment or variant thereof. In one embodiment, the second peptide comprises a domain C, such as domain C having an amino acid sequence according to SEQ ID NO: 5 or a fragment or variant thereof.

[0088] In one embodiment, the second peptide comprises: a. the amino acid sequence of SEQ ID NO:3; b. a variant of SEQ ID NO:3 comprising or consisting of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:3, more preferably at least 85% or 90% sequence identity to SEQ ID NO:4, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:3; c. A fragment of SEQ ID NO:3 that is more than 80 amino acids in length, such as more than 90, 100, or 110 amino acids in length, or a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:3, more preferably at least 85% or 90% sequence identity to SEQ ID NO:3, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:3.

[0089] In one embodiment, the second peptide comprises: a. the amino acid sequence of SEQ ID NO:4; b. a variant of SEQ ID NO:4 comprising or consisting of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:4, more preferably at least 85% or 90% sequence identity to SEQ ID NO:4, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:4; c. A fragment of SEQ ID NO:4 that is more than 6 amino acids in length, such as more than 8, 10, or 12 amino acids in length, or a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:4, more preferably at least 85% or 90% sequence identity to SEQ ID NO:4, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:4.

[0090] In one embodiment, the second peptide comprises: a. the amino acid sequence of SEQ ID NO:5; b. a variant of SEQ ID NO:5 comprising or consisting of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:5, more preferably at least 85% or 90% sequence identity to SEQ ID NO:5, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:5; c. A fragment of SEQ ID NO:5 that is more than 180 amino acids in length, such as more than 190, 200, or 210 amino acids in length, or a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:5, more preferably at least 85% or 90% sequence identity to SEQ ID NO:5, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:5.

[0091] In one embodiment, the second peptide comprises: a. the amino acid sequence of SEQ ID NO:3; b. a variant of SEQ ID NO:3 comprising or consisting of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:3, more preferably at least 85% or 90% sequence identity to SEQ ID NO:3, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:3; c. fragments of SEQ ID NO:3 that are more than 80 amino acids in length, such as more than 90, 100, or 110 amino acids in length, or variants thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:3, more preferably at least 85% or 90% sequence identity to SEQ ID NO:3, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:3; d. the amino acid sequence of SEQ ID NO:4; e. a variant of SEQ ID NO:4 comprising or consisting of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:4, more preferably at least 85% or 90% sequence identity to SEQ ID NO:4, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:4; f. A fragment of SEQ ID NO:4 that is more than 6 amino acids in length, such as more than 8, 10, or 12 amino acids in length, or a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:4, more preferably at least 85% or 90% sequence identity to SEQ ID NO:4, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:4.

[0092] In one embodiment, the second peptide comprises: a. the amino acid sequence of SEQ ID NO:3; b. a variant of SEQ ID NO:3 comprising or consisting of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:3, more preferably at least 85% or 90% sequence identity to SEQ ID NO:3, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:3; c. fragments of SEQ ID NO:3 that are more than 80 amino acids in length, such as more than 90, 100, or 110 amino acids in length, or variants thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:3, more preferably at least 85% or 90% sequence identity to SEQ ID NO:3, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:3; d. the amino acid sequence of SEQ ID NO:4; e. a variant of SEQ ID NO:4 comprising or consisting of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:4, more preferably at least 85% or 90% sequence identity to SEQ ID NO:4, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:4; f. fragments of SEQ ID NO:4 that are more than 6 amino acids in length, such as more than 8, 10, or 12 amino acids in length, or variants thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:4, more preferably at least 85% or 90% sequence identity to SEQ ID NO:4, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:4; g. the amino acid sequence of SEQ ID NO:5; h. a variant of SEQ ID NO:5 comprising or consisting of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:5, more preferably at least 85% or 90% sequence identity to SEQ ID NO:5, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:5; i. A fragment of SEQ ID NO:5 that is more than 180 amino acids in length, such as more than 190, 200, or 210 amino acids in length, or a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:5, more preferably at least 85% or 90% sequence identity to SEQ ID NO:5, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:5.

[0093] In one embodiment, the second peptide comprises: a. the amino acid sequence of SEQ ID NO:3; b. a variant of SEQ ID NO:3 comprising or consisting of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:3, more preferably at least 85% or 90% sequence identity to SEQ ID NO:3, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:3; c. fragments of SEQ ID NO:3 that are more than 80 amino acids in length, such as more than 90, 100, or 110 amino acids in length, or variants thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:3, more preferably at least 85% or 90% sequence identity to SEQ ID NO:3, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:3; d. the amino acid sequence of SEQ ID NO:5; e. a variant of SEQ ID NO:5 comprising or consisting of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:5, more preferably at least 85% or 90% sequence identity to SEQ ID NO:5, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:5; f. A fragment of SEQ ID NO:5 that is more than 180 amino acids in length, such as more than 190, 200, or 210 amino acids in length, or a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:5, more preferably at least 85% or 90% sequence identity to SEQ ID NO:5, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:5.

[0094] In one embodiment, the second peptide comprises: a. the amino acid sequence of SEQ ID NO:4; b. a variant of SEQ ID NO:4 comprising or consisting of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:4, more preferably at least 85% or 90% sequence identity to SEQ ID NO:4, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:4; c. a fragment of SEQ ID NO:4 that is more than 6 amino acids in length, such as more than 8, 10, or 12 amino acids in length, or a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:4, more preferably at least 85% or 90% sequence identity to SEQ ID NO:4, most preferably at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:4; d. the amino acid sequence of SEQ ID NO:5; e. a variant of SEQ ID NO:5 comprising or consisting of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:5, more preferably at least 85% or 90% sequence identity to SEQ ID NO:5, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:5; f. A fragment of SEQ ID NO:5 that is more than 180 amino acids in length, such as more than 190, 200, or 210 amino acids in length, or a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:5, more preferably at least 85% or 90% sequence identity to SEQ ID NO:5, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:5.

[0095] In one embodiment, domain A is a translocation domain and domain C is a cytotoxicity domain, such as an ADP-ribosylation domain.

[0096] In one embodiment, the second peptide comprises at its C-terminus the amino acid sequence KDEL of SEQ ID NO: 8. In one embodiment, the last five amino acids of the second peptide are replaced with the amino acid sequence of SEQ ID NO: 8.

[0097] In one embodiment, the second peptide comprises or consists of: a. the amino acid sequence of SEQ ID NO:2; b. a variant of SEQ ID NO:2 comprising or consisting of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:2, more preferably at least 85% or 90% sequence identity to SEQ ID NO:2, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:2; c. A fragment of SEQ ID NO:2 that is more than 300 amino acids in length, such as more than 310, 330, or 340 amino acids in length, or a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:2, more preferably at least 85% or 90% sequence identity to SEQ ID NO:2, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:2.

[0098] In one embodiment, the second peptide comprises or consists of a variant of SEQ ID NO:2 comprising or consisting of an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:2. In one embodiment, the second peptide comprises or consists of a variant of SEQ ID NO:2 comprising or consisting of an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:2. In one embodiment, the second peptide comprises or consists of a variant of SEQ ID NO:2 comprising or consisting of an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO:2. In one embodiment, the second peptide comprises or consists of a variant of SEQ ID NO:2 comprising or consisting of an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:2. In one embodiment, the second peptide comprises or consists of a variant of SEQ ID NO:2 comprising or consisting of an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO:2. In one embodiment, the second peptide comprises or consists of a variant of SEQ ID NO:2 comprising or consisting of an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO:2.

[0099] In one embodiment, the second peptide comprises or consists of a fragment of SEQ ID NO:2 that is greater than 300 amino acids in length. In one embodiment, the second peptide comprises or consists of a fragment of SEQ ID NO:2 that is greater than 310 amino acids in length. In one embodiment, the second peptide comprises or consists of a fragment of SEQ ID NO:2 that is greater than 330 amino acids in length. In one embodiment, the second peptide comprises or consists of a fragment of SEQ ID NO:2 that is greater than 340 amino acids in length.

[0100] In one embodiment, the second peptide comprises or consists of a fragment of SEQ ID NO:2 or a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:2. In one embodiment, the second peptide comprises or consists of a fragment of SEQ ID NO:2 or a variant thereof having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:2. In one embodiment, the second peptide comprises or consists of a fragment of SEQ ID NO:2 or a variant thereof having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:2. In one embodiment, the second peptide comprises or consists of a fragment of SEQ ID NO:2 or a variant thereof having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:2. In one embodiment, the second peptide comprises or consists of a fragment of SEQ ID NO:2 or a variant thereof having at least 96% sequence identity to the amino acid sequence of SEQ ID NO:2. In one embodiment, the second peptide comprises or consists of a fragment of SEQ ID NO:2 or a variant thereof having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:2. In one embodiment, the second peptide comprises or consists of a fragment of SEQ ID NO:2, or a variant thereof having at least 98% sequence identity to the amino acid sequence of SEQ ID NO:2. In one embodiment, the second peptide comprises or consists of a fragment of SEQ ID NO:2, or a variant thereof having at least 99% sequence identity to the amino acid sequence of SEQ ID NO:2.

[0101] In one embodiment, the second peptide is less than 400 amino acids in length, eg, less than 380, 370, 360, 350, or 345 amino acids in length.

[0102] In one embodiment, the second peptide is less than 400 amino acids in length.

[0103] In one embodiment, the second peptide is less than 380 amino acids in length.

[0104] In one embodiment, the second peptide is less than 370 amino acids in length.

[0105] In one embodiment, the second peptide is less than 360 amino acids in length.

[0106] In one embodiment, the second peptide is less than 350 amino acids in length.

[0107] In one embodiment, the second peptide is less than 345 amino acids in length.

[0108] In one embodiment, the fusion protein is SYN002 or a functional variant thereof comprising or consisting of: 1. the amino acid sequence of SEQ ID NO:6; 2. A variant of SEQ ID NO:6 comprising or consisting of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:6, more preferably at least 85% or 90% sequence identity to SEQ ID NO:6, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:6; 3. A fragment of SEQ ID NO:6 that is more than 360 amino acids in length, such as more than 380, 400, or 420 amino acids in length, or a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:6, more preferably at least 85% or 90% sequence identity to SEQ ID NO:6, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:6.

[0109] The term "functional variant" refers to a variant that exhibits similar, identical or improved selectivity and potency compared to the original sequence. That is, a functional variant of SEQ ID NO: 6 exhibits similar, identical or improved selectivity and potency compared to SEQ ID NO: 6. Preferably, the functional variant exhibits the same or improved selectivity and potency compared to the original sequence.

[0110] In one embodiment, the fusion protein comprises or consists of a variant of SEQ ID NO:6 comprising or consisting of an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:6. In one embodiment, the fusion protein comprises or consists of a variant of SEQ ID NO:6 comprising or consisting of an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:6. In one embodiment, the fusion protein comprises or consists of a variant of SEQ ID NO:6 comprising or consisting of an amino acid sequence having at least 96% sequence identity to the amino acid sequence of SEQ ID NO:6. In one embodiment, the fusion protein comprises or consists of a variant of SEQ ID NO:6 comprising or consisting of an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:6. In one embodiment, the fusion protein comprises or consists of a variant of SEQ ID NO:6 comprising or consisting of an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO:6. In one embodiment, the fusion protein comprises or consists of a variant of SEQ ID NO:6 comprising or consisting of an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO:6.

[0111] In one embodiment, the fusion protein comprises or consists of a fragment of SEQ ID NO:6 that is greater than 360 amino acids in length. In one embodiment, the fusion protein comprises or consists of a fragment of SEQ ID NO:6 that is greater than SEQ ID NO:380 amino acids in length. In one embodiment, the fusion protein comprises or consists of a fragment of SEQ ID NO:6 that is greater than SEQ ID NO:400 amino acids in length. In one embodiment, the fusion protein comprises or consists of a fragment of SEQ ID NO:6 that is greater than SEQ ID NO:420 amino acids in length.

[0112] In one embodiment, the fusion protein comprises or consists of a fragment of SEQ ID NO:6 or a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:6. In one embodiment, the fusion protein comprises or consists of a fragment of SEQ ID NO:6 or a variant thereof having at least 85% sequence identity to the amino acid sequence of SEQ ID NO:6. In one embodiment, the fusion protein comprises or consists of a fragment of SEQ ID NO:6 or a variant thereof having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:6. In one embodiment, the fusion protein comprises or consists of a fragment of SEQ ID NO:6 or a variant thereof having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:6. In one embodiment, the fusion protein comprises or consists of a fragment of SEQ ID NO:6 or a variant thereof having at least 96% sequence identity to the amino acid sequence of SEQ ID NO:6. In one embodiment, the fusion protein comprises or consists of a fragment of SEQ ID NO:6 or a variant thereof having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:6. In one embodiment, the fusion protein comprises or consists of a fragment of SEQ ID NO: 6, or a variant thereof having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 6. In one embodiment, the fusion protein comprises or consists of a fragment of SEQ ID NO: 6, or a variant thereof having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 6.

[0113] In one embodiment, the fusion protein is less than 500 amino acids in length, e.g., 490, 480, 470, 460, 450, 440, 430, 425, or fewer amino acids in length.

[0114] In one embodiment, the fusion protein is less than 500 amino acids in length. In one embodiment, the fusion protein is less than 490 amino acids in length. In one embodiment, the fusion protein is less than 480 amino acids in length. In one embodiment, the fusion protein is less than 470 amino acids in length. In one embodiment, the fusion protein is less than 460 amino acids in length. In one embodiment, the fusion protein is less than 450 amino acids in length. In one embodiment, the fusion protein is less than 440 amino acids in length. In one embodiment, the fusion protein is less than 430 amino acids in length. In one embodiment, the fusion protein is less than 420 amino acids in length.

[0115] In one embodiment, the fusion protein kills cells infected with a pathogen, such as cells latently infected with a pathogen.

[0116] In one embodiment, the pathogen is a virus, such as a DNA virus, such as a herpes virus, such as a cytomegalovirus, or an RNA virus.

[0117] In one embodiment, the fusion protein induces cell death in cells expressing the receptor.

[0118] In one embodiment, the cell death is selected from the group consisting of apoptosis, necrosis, autophagic cell death, and mitosis-associated cell death. The terms "apoptosis" and "apoptotic activity" are used broadly to refer to an orderly or controlled form of cell death in mammals that typically involves one or more characteristic cellular changes, including cytoplasmic condensation, loss of plasma membrane microvilli, nuclear segmentation, degradation of chromosomal DNA, or loss of mitochondrial function.

[0119] In one embodiment, the fusion protein induces a direct or indirect effect on the pathogen that results in inhibition of pathogen growth, replication, genomic stability, maturation, packaging, latency, reactivation, dissemination and / or immune inhibition.

[0120] In one embodiment, the fusion protein selectively kills cells that express a pathogen-encoded receptor.

[0121] In one embodiment, the fusion protein kills cells that express US28.

[0122] In one embodiment the fusion protein has an IC50 value against a virally encoded receptor such as US28 of less than 10 nM, such as less than 5 nM, such as less than 1 nM, such as less than 0.5 nM, such as less than 0.1 nM or less than 0.001 nM.

[0123] In one embodiment, the fusion protein has increased potency, such as at least 100-fold increased potency, at least 300-fold increased potency, at least 400-fold increased potency, at least 500-fold increased potency, against cells expressing a virally encoded receptor, such as US28, compared to its potency against cells expressing an endogenous receptor or a human encoded receptor, such as CX3CR1. In one embodiment, the fusion protein has increased potency, such as at least 100-fold increased potency, against cells expressing US28, compared to its potency against cells expressing CX3CR1. In one embodiment, the fusion protein has increased potency, such as at least 300-fold increased potency, against cells expressing US28, compared to its potency against cells expressing CX3CR1. In one embodiment, the fusion protein has increased potency, such as at least 400-fold increased potency, against cells expressing US28, compared to its potency against cells expressing CX3CR1. In one embodiment, the fusion protein has increased potency, eg, at least a 500-fold increased potency, against cells expressing US28 compared to its potency against cells expressing CX3CR1.

[0124] In one embodiment, the fusion protein has an increased affinity, such as at least a 50-fold increased affinity, such as at least a 100-fold increased affinity, for a virally encoded receptor, such as US28, compared to the affinity for an endogenous receptor or a human encoded receptor, such as CX3CR1. In one embodiment, the fusion protein has an increased affinity, such as at least a 100-fold increased affinity, for US28 compared to the affinity for CX3CR1.

[0125] In one embodiment, the selectivity ratio US28 / CX3CR1 of the fusion protein of the invention is at least 750, such as at least 800, such as at least 850, such as at least 900. In one embodiment, the selectivity ratio US28 / CX3CR1 of the fusion protein of the invention is at least 880. In one embodiment, the selectivity ratio US28 / CX3CR1 of the fusion protein of the invention is at least 750. In one embodiment, the selectivity ratio US28 / CX3CR1 of the fusion protein of the invention is at least 800. In one embodiment, the selectivity ratio US28 / CX3CR1 of the fusion protein of the invention is at least 850. In one embodiment, the selectivity ratio US28 / CX3CR1 of the fusion protein of the invention is at least 900.

[0126] aqueous medium In one aspect of the disclosure, the aqueous medium of the compositions described herein comprises, in addition to the fusion protein, a buffer component, an amino acid, a carbohydrate, or a growth factor or cytokine, making the composition suitable for preventing or treating infection by a pathogen in ex vivo organ, tissue, and / or stem cell cultures prior to transplantation, and optionally for maintaining the viability of said ex vivo organ, tissue, and / or stem cell cultures.

[0127] In some embodiments, the buffer component is a bicarbonate buffer (HCO - / CO2), ammonium buffer solution (NH3 / NH4 + ), phosphate buffer (H2PO4 - / HPO4 2-), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 3-(N-morpholino)propanesulfonic acid (MOPS), and 2-(N-morpholino)ethanesulfonic acid (MES).

[0128] In some embodiments, the amino acid is one or more amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0129] In some embodiments, the amino acid is one or more amino acids selected from the group consisting of alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0130] In some embodiments, the carbohydrate is one or more carbohydrates selected from the group consisting of a sugar, such as a monosaccharide, disaccharide, oligosaccharide, or polysaccharide, and a sugar alcohol.

[0131] In some embodiments, the carbohydrate is one or more carbohydrates selected from the group consisting of dextran, dextrose (D-glucose), glucose, glucose monohydrate, mannitol, raffinose, and ribose.

[0132] In some embodiments, the growth factor or cytokine is one or more growth factors or cytokines selected from the group consisting of thrombopoietin, insulin, fibroblast growth factor 1, human stem cell factor, IL-3, IL-6, Flt-3 ligand, and granulocyte colony-stimulating factor (G-CSF).

[0133] In some embodiments, the aqueous medium further comprises one or more additional components selected from the group consisting of: i. Vitamins; ii. Electrolytes; iii. Transcription factors such as OAC1; iv. Diterpenes such as forskolin; v. Low density lipoprotein; vi. Cardiac stimulants; vii. Immunosuppressants; viii. serum albumin, such as bovine serum albumin (BSA) or human serum albumin (HAS); ix. Heparin, such as unfractionated heparin; x. Human transferrin; xi. Pentafraction; xii. 2-mercaptoethanol; xiii. Glutathione; xiv. Lactobionic acid; xv. glutamic acid; xvi. Allopurinol; xvii. Adenosine; xviii. adenine; xix. Hydroxyethyl starch; xx. Perfluorocarbons; xxi. Polyethylene glycol-modified superoxide dismutase (PEG-modified SOD); xxii. Dexamethasone; xxiii. Lysine acetate; xxiv. Methylprednisolone sodium succinate; xxv. CHIR99021; xxvi. Antibiotics; xxvii. Glucocorticoids; xxviii. Anticoagulants; xxix. Prostaglandins.

[0134] In some embodiments, the electrolyte is one or more electrolytes selected from the group consisting of calcium salts, potassium salts, sodium salts, magnesium salts, chloride salts, sulfate salts, and phosphate salts.

[0135] In some embodiments, the cardiac stimulant is one or more cardiac stimulants selected from the group consisting of catecholamines, peptides, polypeptides, beta1 / beta2 adrenergic receptor blockers, buprinalol, pindolol, alprenolol, cardiac glycosides, digitalis, palustrine, ferulic acid, and epinephrine.

[0136] In some embodiments, the antibiotic is one or more antibiotics selected from the group consisting of imipenem, meropenem, and cefuroxime.

[0137] In some embodiments, the glucocorticoid is one or more glucocorticoids selected from the group consisting of methylprednisolone and dexamethasone.

[0138] In some embodiments, the electrolytes are sodium chloride, sodium lactate, potassium chloride, and calcium chloride, such as Ringer's lactate solution (RL), also known as sodium lactate solution and Hartmann's solution.

[0139] In some embodiments, the anticoagulant is a heparin, such as unfractionated heparin.

[0140] In some embodiments, the prostaglandin is prostacyclin.

[0141] In some embodiments, the aqueous medium further comprises blood or a blood component such as red blood cells.

[0142] In some embodiments, the aqueous medium comprises: - one or more cardiac stimulants selected from the group consisting of catecholamines, peptides, polypeptides, beta1 / beta2 adrenergic receptor blockers, buprinalol, pindolol, alprenolol, cardiac glycosides, digitalis, palustrine, ferulic acid, and epinephrine; - a plurality of amino acids selected from the group consisting of alanine, arginine, aspartic acid, glutamic acid, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and lysine, but not including asparagine, glutamine, and cysteine; - Optionally, adenosine, insulin, an immunosuppressant, a multivitamin composition, and / or one or more electrolytes, such as calcium.

[0143] In some embodiments, the aqueous medium comprises: - Adenosine; - Calcium chloride dehydrate; - Glycine; - L-Alanine; - L-arginine; - L-aspartic acid; - L-glutamic acid; - L-Histidine; - L-Isoleucine; - L-Leucine; - L-Methionine; - L-Phenylalanine; - L-Proline; - L-serine; - L-Threonine; - L-tryptophan; - L-Tyrosine; - L-valine; - Lysine acetate; - Magnesium sulfate heptahydrate; - Potassium chloride; - Sodium chloride; - Dextrose; - epinephrine; - Insulin; - one or more vitamins; - Methylprednisolone sodium succinate; - Sodium bicarbonate; - Mannitol; - Sodium glycerophosphate.

[0144] In some embodiments, the aqueous medium comprises: - Mannitol; - Sodium chloride; - Potassium chloride; - Magnesium sulfate heptahydrate; - Sodium glycerophosphate.

[0145] In certain embodiments, the composition is particularly suitable for treating and preserving ex vivo organs in an organ care system, wherein the aqueous medium comprises: - adenosine in a concentration of about 675 mg / 1500 mL to about 825 mg / 1500 mL, calcium chloride dehydrate in an amount of about 2100 mg / 1500 mL to about 2600 mg / 1500 mL, glycine in an amount of about 315 mg / 1500 mL to about 385 mg / 1500 mL; - L-alanine in an amount of about 150 mg / 1500 mL to about 200 mg / 1500 mL; - L-arginine in an amount of about 600 mg / 1500 mL to about 800 mg / 1500 mL; - L-aspartic acid in an amount of about 220 mg / 1500 mL to about 270 mg / 1500 mL; - L-glutamic acid in an amount of about 230 mg / 1500 mL to about 8290 mg / 1500 mL; L-histidine in an amount of about 200 mg / 1500 mL to about 250 mg / 1500 mL; - L-isoleucine in an amount of about 100 mg / 1500 mL to about 130 mg / 1500 mL; - L-leucine in an amount of about 300 mg / 1500 mL to about 380 mg / 1500 mL; - L-methionine in an amount of about 50 mg / 1500 mL to about 65 mg / 1500 mL; - L-phenylalanine in an amount of about 45 mg / 1500 mL to about 60 mg / 1500 mL; - L-proline in an amount of about 110 mg / 1500 mL to about 140 mg / 1500 mL, - L-serine in an amount of about 80 mg / 1500 mL to about 105 mg / 1500 mL; - L-threonine in an amount of about 60 mg / 1500 mL to about 80 mg / 1500 mL; - L-tryptophan in an amount of about 30 mg / 1500 mL to about 40 mg / 1500 mL; - L-tyrosine in an amount of about 80 mg / 1500 mL to about 110 mg / 1500 mL; - L-valine in an amount of about 150 mg / 1500 mL to about 190 mg / 1500 mL; lysine acetate in an amount of about 200 mg / 1500 mL to about 250 mg / 1500 mL; - magnesium sulfate heptahydrate in an amount of about 535 mg / 1500 mL to about 635 mg / 1500 mL; - potassium chloride in an amount of about 200 mg / 1500 mL to about 210 mg / 1500 mL; - sodium chloride in an amount of about 11.1 g / 1500 mL to about 11.6 g / 1500 mL; - dextrose in an amount of about 25 g / 1500 mL to about 120 g / 1500 mL; epinephrine in an amount of about 0.25 mg / 1500 mL to about 1.0 mg / 1500 mL; - insulin in an amount of about 75 IU / 1500 mL to about 150 IU / 1500 mL; - one or more vitamins in an amount of about 1 mg / 1500 mL to about 30 mg / 1500 mL; - methylprednisolone sodium succinate in an amount of about 200 mg / 1500 mL to 500 mg / 1500 mL; - Sodium bicarbonate at approximately 10 mEq / 1500 mL to 25 mEq / 1500 mL mannitol in an amount of about 12.5 g / 1500 mL, and - Sodium glycerophosphate in an amount of approximately 900 mg / 1500 mL.

[0146] In some embodiments, the aqueous medium comprises: - dextran; - glucose monohydrate; - Magnesium sulfate heptahydrate; - Potassium chloride; - Sodium chloride - Sodium phosphate dibasic dihydrate; - Monopotassium phosphate.

[0147] In certain embodiments, the composition is particularly suitable for treating and preserving ex vivo organs in an organ care system, wherein the aqueous medium comprises: - Dextran 40 in an amount of approximately 50 g / L; - glucose monohydrate in an amount of about 2 g / L; - magnesium sulfate heptahydrate in an amount of approximately 0.201 g / L; - Potassium chloride in an amount of approximately 0.4 g / L; - sodium chloride in an amount of approximately 8 g / L; - Sodium phosphate dibasic dihydrate in an amount of about 0.058 g / L; - Monopotassium phosphate in an amount of about 0.063 g / L.

[0148] In some embodiments, the aqueous medium comprises: - Human serum albumin (HSA); - Dextran.

[0149] In certain embodiments, the composition is particularly suitable for treating and preserving ex vivo organs in an organ care system, wherein the aqueous medium comprises: - Human serum albumin (HSA) in an amount of about 25%; - Dextran 40.

[0150] In some embodiments, the aqueous medium comprises: - dextran; - Na + ; -K + ; - Mg 2+ ; - Cl - ; - SO4 2- ; - H2PO4 - ; - Glucose.

[0151] In certain embodiments, the composition is particularly suitable for treating and preserving ex vivo organs during cold ischemic storage or perfusion in organ care systems, wherein the aqueous medium comprises: - Dextran 40 in an amount of about 5%; - Na in an amount of about 138 mmol + ; - K in an amount of about 6 mmol + ; - Mg in an amount of about 0.8 mmol 2+ ; - Cl in an amount of about 142 mmol - ; - SO4 in an amount of about 0.8 mmol 2- ; - H2PO4 in an amount of about 0.8 mmol - and HPO4 2- ; - glucose in an amount of about 5 mmol.

[0152] In some embodiments, the aqueous medium comprises: - Sodium; - Potassium; - Magnesium; - Calcium; - Ketoglutarate / Glutamic Acid; - Histidine; - Mannitol; - Tryptophan.

[0153] In a particular embodiment, the composition is particularly suitable for treating and preserving ex vivo organs during cryogenic storage, wherein the aqueous medium comprises: - sodium in an amount of about 15 mM; - Potassium in an amount of about 9 mM; - Magnesium in an amount of about 4mM; - calcium in an amount of about 0.015 mM; - Ketoglutarate / glutamic acid in an amount of about 1 mM; - histidine in an amount of about 198 mM; - mannitol in an amount of about 30 mM; - Tryptophan in an amount of about 2mM.

[0154] In some embodiments, the aqueous medium comprises: - Glutathione; - Mannitol; - Lactobionic acid; - Glutamic acid; - Sodium hydroxide; - Calcium chloride dihydrate; - Potassium chloride; - Magnesium chloride hexahydrate; - Histidine.

[0155] In a particular embodiment, the composition is particularly suitable for treating and preserving ex vivo organs during cryogenic storage, wherein the aqueous medium comprises: - Glutathione in an amount of approximately 0.921 g / L; - mannitol in an amount of about 10.930 g / L; - lactobionic acid in an amount of about 28.664 g / L; - glutamic acid in an amount of about 2.942 g / L; - sodium hydroxide in an amount of approximately 4.000 g / L; - calcium chloride dihydrate in an amount of about 0.037 g / L; - Potassium chloride in an amount of approximately 1.118 g / L; - magnesium chloride hexahydrate in an amount of about 2.642 g / L; - Histidine in an amount of about 4.650 g / L.

[0156] In some embodiments, the aqueous medium comprises: - Pentafraction; - Allopurinol; - Lactobionic acid; - Glutathione; - Potassium dihydrogen phosphate; - Potassium hydroxide; - Magnesium sulfate heptahydrate; - Raffinose pentahydrate; - Adenosine.

[0157] In a particular embodiment, the composition is particularly suitable for treating and preserving ex vivo organs during cryogenic storage, wherein the aqueous medium comprises: - Pentafraction of about 50 g / L; - Allopurinol in an amount of approximately 0.136 g / L (1 mM); - lactobionic acid in an amount of about 35.83 g / L (105 mM); - glutathione in an amount of about 0.922 g / L (3 mM); - potassium dihydrogen phosphate in an amount of about 3.4 g / L (25 mM); - potassium hydroxide in an amount of about 5.61 g / L (100 mM); - magnesium sulfate heptahydrate in an amount of about 1.23 g / L (5 mM); - raffinose pentahydrate in an amount of about 17.83 g / L (30 mM); - Adenosine in an amount of about 1.34 g / L (5 mM).

[0158] In some embodiments, the aqueous medium comprises: - Adenine; - Calcium chloride; - Dextrose; - Glutathione; - HEPES; - Hydroxyethyl starch; - Magnesium Gluconate; - Mannitol; - Potassium phosphate; - Ribose; - Sodium gluconate; - Sodium hydroxide.

[0159] In certain embodiments, the composition is particularly suitable for treating and preserving ex vivo organs during temporary continuous machine perfusion, wherein the aqueous medium comprises: - adenine (free base) in an amount of about 0.68 g / L (5 mM); - Calcium chloride (dihydrate) in an amount of approximately 0.068 g / L (0.5 mM); - dextrose (+) in an amount of about 1.80 g / L (10 mM); - Glutathione (reduced) in an amount of about 0.92 g / L (3 mM); - hepes (free acid) in an amount of about 2.38 g / L (10 mM); - hydroxyethyl starch in an amount of about 50.0 g / L; - magnesium gluconate in an amount of about 1.13 g / L (5 mM); - mannitol in an amount of about 5.4 g / L (30 mM); - potassium dihydrogen phosphate in an amount of about 3.4 g / L (25 mM); - Ribose, D(-), in an amount of about 0.75 g / L (5 mM); - sodium gluconate in an amount of about 17.45 g / L (80 mM); - Sodium hydroxide in an amount of about 0.70 g / L.

[0160] In some embodiments, the aqueous medium comprises: - hydroxyethyl starch, - Lactobionic acid; - Potassium dihydrogen phosphate; - Magnesium sulfate heptahydrate; - Raffinose pentahydrate; - Adenosine; - Allopurinol; - Glutathione; - Potassium hydroxide.

[0161] In a particular embodiment, the composition is particularly suitable for treating and preserving ex vivo organs during cryogenic storage, wherein the aqueous medium comprises: - hydroxyethyl starch in an amount of about 50 g / L; - lactobionic acid in an amount of about 35.83 g / L; - potassium dihydrogen phosphate in an amount of approximately 3.4 g / L; - magnesium sulfate heptahydrate in an amount of about 1.23 g / L; - raffinose pentahydrate in an amount of about 17.83 g / L; - Adenosine in an amount of about 1.34 g / L; - Allopurinol in an amount of approximately 0.136 g / L; - Glutathione (reduced form) in an amount of approximately 0.922 g / L; - Potassium hydroxide in an amount of about 5.61 g / L.

[0162] In some embodiments, the aqueous medium comprises: - Perfluorocarbons; - glucose; - Insulin; - Allopurinol; - PEG-modified SOD; - Adenosine; - Dexamethasone; - Hydroxyethyl starch; - sodium ions; - potassium ions; - Chloride.

[0163] In a particular embodiment, the composition is particularly suitable for treating and preserving ex vivo organs during storage at room temperature, and the aqueous medium comprises: - perfluorocarbons in an amount of about 0.1-10% (w / v); - glucose in an amount of about 1-20 mmol / L; - insulin in amounts of about 10-200 U / L; - Allopurinol in amounts of approximately 0.1-5 mmol / L; - PEG-modified SOD in an amount of about 1-10 mg / L; - Adenosine in amounts of about 1-10 mmol / L; - Dexamethasone in amounts of approximately 1-20 mg / L; - hydroxyethyl starch in an amount of about 1-5% (w / v); - sodium ions in an amount of about 140-145 mEq / L; - Potassium ions in an amount of about 2-6 mEq / L; - Chloride in an amount of about 90-95 mEq / L.

[0164] In some embodiments, the aqueous medium comprises: - Iscove's Modified Dulbecco's Medium (IMDM); - Human Stem Cell Factor; - Thrombopoietin; - CHIR99021 - Forskolin; - OAC1.

[0165] In certain embodiments, the composition comprises CD34 + Particularly suitable for treating and culturing ex vivo stem cell cultures, such as stem cell cultures, the aqueous medium comprises: - IMDM; - Human stem cell factor in an amount of about 100ng / mL; - Thrombopoietin in an amount of about 50 ng / mL; - CHIR99021; - Forskolin; - OAC1.

[0166] In some embodiments, the aqueous medium comprises: - IMDM; - Bovine serum albumin; - Human insulin; - Human transferrin (iron saturated); - β-mercaptoethanol.

[0167] In some embodiments, the aqueous medium comprises: - Serum-free medium; - Heparin; - Thrombopoietin; - Human Stem Cell Factor; - Fibroblast growth factor 1.

[0168] In certain embodiments, the composition is particularly suitable for treating and culturing ex vivo stem cell cultures, such as hematopoietic stem cell cultures, wherein the aqueous medium comprises: - Serum-free medium; - heparin in an amount of about 10 μg / mL; - Thrombopoietin in an amount of about 20 ng / mL; - Human stem cell factor in an amount of about 10ng / mL; - Fibroblast growth factor 1 in an amount of about 10ng / mL.

[0169] In some embodiments, the aqueous medium comprises: - Serum-free medium; - human IL-3; - human IL-6; - Thrombopoietin; - Flt-3 ligand; - Human Stem Cell Factor.

[0170] In certain embodiments, the composition is particularly suitable for treating and culturing ex vivo stem cell cultures, such as hematopoietic stem cell cultures, wherein the aqueous medium comprises: - Serum-free medium; - recombinant human IL-3 in an amount of about 10 ng / ml; - IL-6 in an amount of about 100 ng / mL; - thrombopoietin in an amount of about 100 ng / mL; - Flt-3 ligand in an amount of about 300 ng / mL; - Human stem cell factor, such as "5GF" human stem cell factor, in an amount of about 300ng / mL.

[0171] In some embodiments, the aqueous medium comprises: - IMDM; - BSA; - Human insulin; - Human transferrin; - low density lipoproteins; - β-mercaptoethanol; - Human Stem Cell Factor; - Flt-3 ligand; - G-CSF; - IL-3; - IL6.

[0172] In certain embodiments, the composition is particularly suitable for treating and culturing ex vivo stem cell cultures, such as hematopoietic stem cell cultures, wherein the aqueous medium comprises: - IMDM; - Approximately 1% BSA; - Approximately 5 μg / mL of human insulin; - Approximately 100 μg / mL human transferrin; - Low density lipoprotein of about 10 μg / mL; - about 10 -4 M, β-mercaptoethanol; - Approximately 300ng / mL human stem cell factor; - Flt-3 at about 300 ng / mL; - Granulocytic CSF of about 50 ng / mL; - IL-3 at about 10 ng / mL; - Approximately 10ng / mL of IL-6.

[0173] In certain embodiments, the composition is particularly suitable for treating and preserving ex vivo organs, such as ex vivo lungs, and the aqueous medium comprises: 2 L of saline solution containing human serum albumin and dextran 40, for example in the form of Steen Solution™; 500mg methylprednisolone; · 3000IU unfractionated heparin; 500mg of imipenem; 500mg of Cilastatin.

[0174] In certain embodiments, the composition is particularly suitable for treating and preserving ex vivo organs, such as ex vivo kidneys, and the aqueous medium comprises: · 100mL human serum albumin 20%; · 400mL of Ringer's lactate solution; · 5mL of mannitol 10%; · 5mL calcium gluconate 10%; 0.5mg meropenem; · 12mL sodium bicarbonate 8.4%; 6mg of dexamethasone.

[0175] In certain embodiments, the composition is particularly suitable for treating and preserving ex vivo organs, such as ex vivo kidneys, and the aqueous medium comprises: · 100mL human serum albumin 20%; · 400mL of Ringer's lactate solution; · 1 unit of red blood cells; · 5mL of mannitol 10%; · 5mL calcium gluconate 10%; 0.5mg meropenem; · 12mL sodium bicarbonate 8.4%; 6mg of dexamethasone.

[0176] In certain embodiments, the composition is particularly suitable for treating and preserving ex vivo organs, such as ex vivo liver, and the aqueous medium comprises: A saline solution containing human serum albumin and dextran 40, for example in the form of Steen Solution™; · Red blood cells; Cefuroxime; Heparin, such as unfractionated heparin; Calcium gluconate; Insulin; Prostacyclin.

[0177] Ex vivo organ, tissue or stem cell cultures The present invention is useful for the prevention and / or treatment of infectious diseases, such as HCMV, in a wide range of ex vivo organ, tissue, or stem cell cultures prior to transplantation.

[0178] In some embodiments, the ex vivo organ is a heart. In some embodiments, the ex vivo organ is a lung. In some embodiments, the ex vivo organ is a liver. In some embodiments, the ex vivo organ is a kidney. In some embodiments, the ex vivo organ is a pancreas. In some embodiments, the ex vivo organ is a small intestine. In some embodiments, the ex vivo organ is a large intestine. In some embodiments, the ex vivo organ is a stomach.

[0179] In some embodiments, the tissue is skin, hi some embodiments, the tissue is corneal tissue.

[0180] The organs and / or tissues can be harvested from a donor, such as a human donor. The organs and / or tissues can also be produced from human tissue or cellular components through advanced biomedical engineering.

[0181] In some embodiments, the stem cell culture is an adult stem cell culture. In some embodiments, the stem cell culture is a mesenchymal stem cell culture. In some embodiments, the stem cell culture is an induced pluripotent stem cell (IPSC) culture. In some embodiments, the stem cell culture is a progenitor cell culture. In some embodiments, the stem cell culture is a precursor cell culture.

[0182] In some embodiments, the stem cell cultures are embryonic stem cell cultures obtained by methods that do not involve the destruction of human embryos.

[0183] In some embodiments, the stem cell culture is a hematopoietic stem cell culture.

[0184] In some embodiments, the stem cell culture comprises CD34 positive cells (CD34 + In some embodiments, all cells in the stem cell culture are CD34 + In some embodiments, only a subset of cells in the stem cell culture are CD34 + It is a cell.

[0185] disease The present invention is useful for preventing or treating infection by pathogens in ex vivo organ, tissue and / or stem cell cultures prior to transplantation.

[0186] In one embodiment, the pathogen is a bacterium or a virus. In one embodiment, the virus is a DNA virus, such as a herpes virus, such as a cytomegalovirus, or an RNA virus.

[0187] In one aspect, the disclosure provides a composition as described herein for use in preventing or treating CMV infection and / or CMV-associated disorders in ex vivo organ, tissue, and / or stem cell cultures prior to transplantation.

[0188] In one embodiment, the CMV infection is a latent and / or lytic CMV infection.

[0189] In some embodiments, CMV infection can be detected in ex vivo organs, tissues or stem cell cultures. In some embodiments, CMV infection can be detected in ex vivo organs. In some embodiments, CMV infection can be detected in ex vivo tissues. In some embodiments, CMV infection can be detected in ex vivo stem cell cultures.

[0190] In some embodiments, the CMV infection occurs in a donor of an organ, tissue or stem cell culture, e.g., i) tissue selected from one or more of the group consisting of retina, cornea, heart, liver, kidney, lung, gastrointestinal tissue, thymus, spleen, skin and muscle; and / or ii) detectable in a bodily fluid selected from one or more of the group consisting of saliva, blood, urine, semen, and breast milk.

[0191] In one embodiment, the CMV infection is in ex vivo organ, tissue, and / or stem cell cultures for transplant into immunocompromised patients selected from the group consisting of HIV patients, newborns and immunosuppressed patients, bone marrow transplant patients, solid organ transplant patients, immunotherapy patients, cancer patients, intensive care patients, trauma patients, stem cell patients, gene therapy patients, cell therapy patients, elderly patients, and multi-morbidity patients.

[0192] In one embodiment, the CMV infection is in ex vivo organ, tissue, and / or stem cell cultures for transplant into patients suffering from coronary artery disease and / or vascular disease.

[0193] In some embodiments, the patient receiving the transplant organ, tissue, and / or stem cell culture is a human. In one embodiment, the human is a child. In one embodiment, the human is an adult.

[0194] In one embodiment, the human receiving the transplanted organ, tissue, and / or stem cell culture is an immunocompromised patient. An immunocompromised patient refers to a patient who does not have the ability to respond normally to infection due to an impaired or weakened immune system. For example, an immunocompromised patient may have been diagnosed with a disease that affects the immune system, such as diabetes and HIV. An immunocompromised patient may have a suppressed immune response after treatment, such as chemotherapy.

[0195] In one embodiment, the human is in need of a solid organ transplant and / or a hematopoietic stem cell transplant. Hematopoietic stem cell transplant is the transplantation of multipotent hematopoietic stem cells, usually derived from one bone marrow, peripheral blood, or umbilical cord blood. The transplant may be an autologous transplant (stem cells are isolated from the same patient) or an allogeneic transplant (stem cells are isolated from another patient).

[0196] In one embodiment, the CMV-associated disorder is selected from the group consisting of cytomegalovirus pneumonia, cytomegalovirus hepatitis, cytomegalovirus pancreatitis, cytomegalovirus mononucleosis, CMV radiculopathy, cytomegalovirus inclusion body disease, cytomegalovirus colitis, cytomegalovirus esophagitis, cytomegalovirus retinitis, Guillain-Barre syndrome, mucoepidermoid carcinoma, and ulcerative colitis, graft-versus-host disease (GVHD), solid organ transplant graft-versus-host disease (SOT-GVHD).

[0197] In some embodiments, the compositions described herein prevent or reduce the severity of graft-versus-host disease (GVHD) and / or solid organ transplant graft-versus-host disease (SOT-GVHD). In some embodiments, the compositions described herein prevent or reduce the severity of GVHD. In some embodiments, the compositions described herein prevent or reduce the severity of SOT-GVHD.

[0198] In one aspect, the disclosure provides for the use of a composition described herein for the treatment or prevention of infection by a pathogen in ex vivo organ, tissue, and / or stem cell cultures for transplantation.

[0199] Treatment method In some aspects of the invention, methods are provided for the treatment or prevention of infection by a pathogen in ex vivo organ, tissue, and / or stem cell cultures prior to transplantation, the methods comprising immersing, perfusing, preserving, conditioning, and / or flushing the ex vivo organ, tissue, and / or stem cell culture in a composition described herein.

[0200] In some embodiments, a method for treating or preventing infection by a pathogen in an ex vivo organ, tissue, and / or stem cell culture prior to transplantation comprises perfusing the ex vivo organ, tissue, and / or stem cell culture with a composition described herein. The perfusion may be performed for about 1 hour to about 24 hours, such as about 2 hours to 21 hours, such as about 3 hours to 18 hours, such as about 4 hours to 15 hours, such as about 5 hours to 12 hours, such as about 6 hours to 10 hours, such as about 1 hour to 10 hours, such as about 4, 5, 6, 7 or 8 hours. In some embodiments, the perfusion is performed for about 6 hours.

[0201] In some embodiments, the treatment, such as perfusion, is at a temperature between about 36.5°C and about 37.5°C, such as about 37°C. In some embodiments, the treatment, such as perfusion, is at a temperature between about 30°C and about 36.5°C, such as about 30°C, such as about 31°C, such as about 32°C, such as about 33°C, such as about 34°C, such as about 35°C, or about 36°C, which is near normal body temperature. In some embodiments, the treatment, such as perfusion, is at a temperature below about 30°C, such as about 2°C to about 30°C, such as about 2°C, such as about 4°C, such as about 6°C, such as about 8°C, such as about 10°C, such as about 12°C, such as about 14°C, such as about 16°C, such as about 18°C, such as about 20°C, such as about 22°C, such as about 24°C, such as about 26°C, such as about 28°C, or such as about 30°C. In some embodiments, the treatment is at a low temperature, such as a temperature between about 2°C and about 8°C.

[0202] In some embodiments, the methods are further suitable for maintaining the viability of said ex vivo organ, tissue, and / or stem cell culture prior to transplantation.

[0203] Some aspects also provide for the use of the compositions described herein for the treatment or prevention of infection by a pathogen in ex vivo organ, tissue, and / or stem cell cultures for transplantation.

[0204] In some embodiments, ex vivo organ, tissue, and / or stem cell cultures are flushed with a composition described herein prior to refrigerated storage of the ex vivo organ, tissue, and / or stem cell cultures.

[0205] Kits and Perfusion Systems In some aspects of the disclosure, a kit is provided for preventing or treating infection by a pathogen in an ex vivo organ, tissue, and / or stem cell culture, the kit comprising: a. a composition described herein; b. Optionally, instructions for use.

[0206] In some embodiments, the kit is further suitable for maintaining the viability of organs, tissues, and / or stem cell cultures prior to transplantation.

[0207] In some aspects of the present disclosure, a perfusion system configured to maintain an ex vivo organ in a functional state under physiological or near-physiological conditions is provided, the perfusion system comprising one or more chambers configured to maintain the ex vivo organ in a functional state, at least one chamber comprising a composition described herein.

[0208] In some embodiments, the perfusion system is an organ care system (OCS).

[0209] In some embodiments, the fusion protein described herein is stable for at least 1 hour in a perfusate at 37° C., such as an ex vivo lung perfusion perfusate at 37° C., and the fusion protein is present at a concentration of at least 0.0850 mg / L, such as at least 0.100 mg / L, such as at least 0.250 mg / L, such as at least 0.500 mg / L, such as at least 0.750 mg / L, such as at least 1.00 mg / L, such as at least 1.25 mg / L, such as at least 1.50 mg / L, such as at least 1.70 mg / L. In some embodiments, the fusion protein is a protein according to SEQ ID NO: 6 (SYN002).

[0210] In some embodiments, the fusion protein described herein is stable for at least 2 hours in a perfusate at 37° C., such as an ex vivo lung perfusate at 37° C., and the fusion protein is present at a concentration of at least 0.0850 mg / L, such as at least 0.100 mg / L, such as at least 0.250 mg / L, such as at least 0.500 mg / L, such as at least 0.750 mg / L, such as at least 1.00 mg / L, such as at least 1.25 mg / L, such as at least 1.50 mg / L, such as at least 1.70 mg / L. In some embodiments, the fusion protein is a protein according to SEQ ID NO:6.

[0211] In some embodiments, the fusion protein described herein is stable for at least 3 hours in a perfusate at 37° C., such as an ex vivo lung perfusate at 37° C., and the fusion protein is present at a concentration of at least 0.0850 mg / L, such as at least 0.100 mg / L, such as at least 0.250 mg / L, such as at least 0.500 mg / L, such as at least 0.750 mg / L, such as at least 1.00 mg / L, such as at least 1.25 mg / L, such as at least 1.50 mg / L, such as at least 1.70 mg / L. In some embodiments, the fusion protein is a protein according to SEQ ID NO:6.

[0212] In some embodiments, the fusion protein described herein is stable for at least 4 hours in a perfusate at 37° C., such as an ex vivo lung perfusate at 37° C., and the fusion protein is present at a concentration of at least 0.0850 mg / L, such as at least 0.100 mg / L, such as at least 0.250 mg / L, such as at least 0.500 mg / L, such as at least 0.750 mg / L, such as at least 1.00 mg / L, such as at least 1.25 mg / L, such as at least 1.50 mg / L, such as at least 1.70 mg / L. In some embodiments, the fusion protein is a protein according to SEQ ID NO:6.

[0213] In some embodiments, the fusion protein described herein is stable for at least 5 hours in a perfusate at 37° C., such as an ex vivo lung perfusate at 37° C., and the fusion protein is present at a concentration of at least 0.0850 mg / L, such as at least 0.100 mg / L, such as at least 0.250 mg / L, such as at least 0.500 mg / L, such as at least 0.750 mg / L, such as at least 1.00 mg / L, such as at least 1.25 mg / L, such as at least 1.50 mg / L, such as at least 1.70 mg / L. In some embodiments, the fusion protein is a protein according to SEQ ID NO:6.

[0214] item 1. A composition for preventing or treating infection by a pathogen in ex vivo organ, tissue, and / or stem cell cultures prior to transplantation, said composition comprising in an aqueous medium: A fusion protein comprising: i. a first peptide that binds to at least one receptor expressed on a cell; ii. a second peptide comprising a cleavage site having the amino acid sequence ArgX1X2Arg; X2 is Arg or Lys, and the second peptide comprises a toxin; The aqueous medium further comprises one or more of the following: i. Buffer component; ii. amino acids; iii. carbohydrates; or iv. Growth factors or cytokines.

[0215] 2. The composition of item 1, wherein X1 is Gln, Ser, Thr or Asn.

[0216] 3. The compound according to any one of the preceding items, wherein X1 is Gln.

[0217] 4. The composition of any one of the preceding items, wherein the fusion protein is an immunotoxin.

[0218] 5. The composition of any one of the preceding items, wherein the cleavage site comprises or consists of the amino acid sequence ArgGlnArgArg.

[0219] 6. The composition of any one of the preceding items, wherein the first peptide binds to at least two different receptors expressed on a cell.

[0220] 7. The composition of any one of the preceding items, wherein one receptor that binds to the first peptide is a pathogen-encoded receptor and a further receptor that binds to the first peptide is a human-encoded and / or endogenous receptor for the first peptide or a variant thereof.

[0221] 8. The composition of any one of the preceding items, wherein the receptor is a G protein-coupled receptor (GPCR), such as US28 of SEQ ID NO: 10.

[0222] 9. The composition of any one of the preceding items, wherein the second peptide comprises a toxin selected from the group consisting of Pseudomonas aeruginosa exotoxin A, gelonin, bouganin, saporin, ricin, ricin A chain, bryodin, restrictocin, diphtheria toxin, diphtheria toxin A chain, and variants and fragments thereof.

[0223] 10. The second peptide comprises: 211 At, 225 Ac, 227 Th, 224 Ra, 213 Bi, 67 Cu, 90 Y, 131 I, 177 Lu, 186 Re, 188 Re, and 125 13. The composition of any one of the preceding items, comprising a radioisotope or emitter selected from the group consisting of I.

[0224] 11. The composition of any one of the preceding items, wherein the receptor is a chemokine receptor.

[0225] 12. The composition described in item 10, wherein the chemokine receptor is a CC chemokine receptor and / or a CX3C chemokine receptor.

[0226] 13. The composition described in item 12, wherein the CC chemokine receptor is US28.

[0227] 14. The composition described in item 12, wherein the CX3C chemokine receptor is CX3CR1.

[0228] 15. The composition of any one of the preceding items, wherein the receptor is encoded by a pathogen.

[0229] 16. The composition of any one of the preceding items, wherein the pathogen is a bacterium.

[0230] 17. The composition according to any one of items 1 to 15, wherein the pathogen is a virus.

[0231] 18. The composition described in item 17, wherein the virus is a DNA virus.

[0232] 19. The composition described in item 17, wherein the virus is an RNA virus.

[0233] 20. The composition according to item 17, wherein the virus is a herpes virus.

[0234] 21. The composition described in item 17, wherein the virus is a cytomegalovirus.

[0235] 22. The composition of any one of the preceding items, wherein the receptor is capable of internalization.

[0236] 23. The composition of any one of the preceding items, wherein the composition is internalized upon binding of the receptor to the first peptide.

[0237] 24. The composition of any one of the preceding items, wherein the receptor is constitutively internalized.

[0238] 25. The composition of any one of the preceding items, wherein the cleavage site is an enzymatic cleavage site.

[0239] 26. The composition described in item 25, wherein the enzyme cleavage site is a furin cleavage site.

[0240] 27. The composition of any one of the preceding items, wherein the first peptide is a targeting moiety.

[0241] 28. The composition of any one of the preceding items, wherein the first peptide and the second peptide are operably linked.

[0242] 29. The composition of any one of the preceding items, wherein the first peptide comprises or consists of: a. the amino acid sequence of SEQ ID NO:1; b. a variant of SEQ ID NO: 1 comprising or consisting of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 1, more preferably at least 85% or 90% sequence identity to SEQ ID NO: 1, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1; and / or c. A fragment of SEQ ID NO:1 that is more than 50 amino acids in length, such as more than 60, 70, or 75 amino acids in length, or a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:1, more preferably at least 85% or 90% sequence identity to SEQ ID NO:1, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:1.

[0243] 30. The composition of any one of the preceding items, wherein the amino acid residue at position 49 of SEQ ID NO:1 is mutated to Ala, Lys, or Asp, preferably Ala.

[0244] 31. The composition of any one of the preceding items, wherein the first peptide further comprises a methionine (M) at the N-terminus.

[0245] 32. The composition of any one of the preceding items, wherein the first peptide comprises or consists of one or more additional amino acids inserted at the N-terminus and / or C-terminus and / or within the amino acid sequence of the SEQ ID NO:

[0246] 33. The composition of any one of the preceding items, wherein one or more amino acids are deleted at the N-terminus and / or C-terminus and / or within the amino acid sequence of SEQ ID NO:1.

[0247] 34. The composition of any one of the preceding items, wherein the first peptide is less than 100 amino acids in length, e.g., less than 90, 85, or 80 amino acids in length.

[0248] 35. The composition of any one of the preceding items, wherein the second peptide comprises domain A, such as domain A having an amino acid sequence according to SEQ ID NO: 3 or a fragment or variant thereof, and / or domain B, such as domain B having an amino acid sequence according to SEQ ID NO: 4 or a fragment or variant thereof, and / or domain C, which may be a cytotoxic domain, such as domain C having an amino acid sequence according to SEQ ID NO: 5 or a fragment or variant thereof.

[0249] 36. The composition of any one of the preceding items, wherein the second peptide comprises: a. the amino acid sequence of SEQ ID NO:3; b. a variant of SEQ ID NO:3 comprising or consisting of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:3, more preferably at least 85% or 90% sequence identity to SEQ ID NO:3, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:3; and / or c. A fragment of SEQ ID NO:3 that is more than 80 amino acids in length, such as more than 90, 100, or 110 amino acids in length, or a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:3, more preferably at least 85% or 90% sequence identity to SEQ ID NO:3, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:3.

[0250] 37. The composition of any one of the preceding items, wherein the second peptide comprises: a. the amino acid sequence of SEQ ID NO:4; b. a variant of SEQ ID NO:4 comprising or consisting of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:4, more preferably at least 85% or 90% sequence identity to SEQ ID NO:4, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:4; and / or c. A fragment of SEQ ID NO:3 that is more than 6 amino acids in length, such as more than 8, 10, or 12 amino acids in length, or a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:3, more preferably at least 85% or 90% sequence identity to SEQ ID NO:4, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:4.

[0251] 38. The composition of any one of the preceding items, wherein the second peptide comprises: a. the amino acid sequence of SEQ ID NO:5; b. a variant of SEQ ID NO:5 comprising or consisting of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:5, more preferably at least 85% or 90% sequence identity to SEQ ID NO:5, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:5; and / or c. A fragment of SEQ ID NO:3 that is more than 180 amino acids in length, such as more than 190, 200, or 210 amino acids in length, or a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:5, more preferably at least 85% or 90% sequence identity to SEQ ID NO:5, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:5.

[0252] 39. The composition described in item 35, wherein domain A is a translocation domain and domain C is a cytotoxic domain such as an ADP-ribosylation domain.

[0253] 40. The composition of any one of the preceding items, wherein the second peptide comprises the amino acid sequence KDEL of SEQ ID NO:8 at its C-terminus.

[0254] 41. The composition of any one of the preceding items, wherein the second peptide comprises or consists of: a. the amino acid sequence of SEQ ID NO:2; b. a variant of SEQ ID NO:2 comprising or consisting of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:2, more preferably at least 85% or 90% sequence identity to SEQ ID NO:2, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:2; and / or c. A fragment of SEQ ID NO:2 that is more than 300 amino acids in length, such as more than 310, 330, or 340 amino acids in length, or a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:5, more preferably at least 85% or 90% sequence identity to SEQ ID NO:2, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:2.

[0255] 42. The composition of any one of the preceding items, wherein the second peptide is less than 400 amino acids in length, e.g., less than 380, 370, 360, 350, or 345 amino acids in length.

[0256] 43. The composition of any one of the preceding items, wherein the fusion protein comprises or consists of: a. the amino acid sequence of SEQ ID NO:6; b. a variant of SEQ ID NO:6 comprising or consisting of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:6, more preferably at least 85% or 90% sequence identity to SEQ ID NO:6, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:6; and / or c. A fragment of SEQ ID NO:6 that is more than 360 amino acids in length, such as more than 380, 400, or 420 amino acids in length, or a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:6, more preferably at least 85% or 90% sequence identity to SEQ ID NO:6, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:6.

[0257] 44. The composition of any one of the preceding items, wherein the fusion protein is less than 500 amino acids in length, e.g., 490, 480, 470, 460, 450, 440, 430, 425, or fewer amino acids in length.

[0258] 45. The composition of any one of the preceding items, wherein the fusion protein kills a cell infected with a pathogen, e.g., a cell latently infected with a pathogen.

[0259] 46. ​​The composition of item 45, wherein the pathogen is a virus, such as a DNA virus, such as a herpes virus, such as a cytomegalovirus, or an RNA virus.

[0260] 47. The composition of any one of the preceding items, wherein the fusion protein induces cell death of a cell expressing the receptor.

[0261] 48. The composition described in item 47, wherein the cell death is selected from the group consisting of apoptosis, necrosis, autophagic cell death, and mitosis-associated cell death.

[0262] 49. The composition of any one of the preceding items, wherein the fusion protein induces a direct or indirect effect on the pathogen that results in inhibition of pathogen growth, replication, genomic stability, maturation, packaging, latency, reactivation, dissemination and / or immune inhibition.

[0263] 50. The composition of any one of the preceding items, wherein the fusion protein kills cells that express US28.

[0264] 51. A composition described in any one of the preceding items, wherein the fusion protein has an IC50 value against a receptor encoded by a virus, such as US28, of less than 10 nM, such as less than 5 nM, for example less than 1 nM, such as less than 0.5 nM, for example less than 0.1 nM or less than 0.001 nM.

[0265] 52. The composition of any one of the preceding items, wherein the fusion protein has increased potency, such as at least 300-fold increased potency, at least 400-fold increased potency, at least 500-fold increased potency, at least 700-fold increased potency, against cells expressing a virally encoded receptor, such as US28, compared to potency against cells expressing an endogenous receptor or a human encoded receptor, such as CX3CR1.

[0266] 53. The composition of any one of the preceding items, wherein the fusion protein has increased affinity, such as at least a 50-fold increased affinity, at least a 100-fold increased affinity, for a virally encoded receptor, such as US28, compared to affinity for an endogenous receptor or a human encoded receptor, such as CX3CR1.

[0267] 54. The buffer component is a bicarbonate buffer (HCO3 - / CO2), ammonium buffer solution (NH3 / NH4 + ), phosphate buffer (H2PO4 - / HPO4 2- ), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 3-(N-morpholino)propanesulfonic acid (MOPS), and 2-(N-morpholino)ethanesulfonic acid (MES).

[0268] 55. The composition of any one of the preceding items, wherein the amino acid is one or more amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0269] 56. The composition according to item 55, wherein the amino acid is one or more amino acids selected from the group consisting of alanine, arginine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0270] 57. The composition of any one of the preceding items, wherein the carbohydrate is one or more carbohydrates selected from the group consisting of sugars, such as monosaccharides, disaccharides, oligosaccharides, or polysaccharides, and sugar alcohols.

[0271] 58. The composition of any one of the preceding items, wherein the carbohydrate is one or more carbohydrates selected from the group consisting of dextran, dextrose (D-glucose), glucose, glucose monohydrate, mannitol, raffinose, and ribose.

[0272] 59. The composition of any one of the preceding items, wherein the growth factor or cytokine is one or more growth factors or cytokines selected from the group consisting of thrombopoietin, insulin, fibroblast growth factor 1, human stem cell factor, IL-3, IL-6, Flt-3 ligand, and granulocyte colony-stimulating factor (G-CSF).

[0273] 60. The composition of any one of the preceding items, wherein the aqueous medium further comprises one or more additional ingredients selected from the group consisting of: i. Vitamins; ii. Electrolytes; iii. Transcription factors such as OAC1; iv. Diterpenes such as forskolin; v. Low density lipoprotein; vi. Cardiac stimulants; vii. Immunosuppressants; viii. serum albumin, such as bovine serum albumin (BSA) or human serum albumin (HAS); ix. Heparin, such as unfractionated heparin; x. Human transferrin; xi. Pentafraction; xii. 2-mercaptoethanol; xiii. Glutathione; xiv. Lactobionic acid; xv. glutamic acid; xvi. Allopurinol; xvii. Adenosine; xviii. adenine; xix. Hydroxyethyl starch; xx. Perfluorocarbons; xxi. Polyethylene glycol-modified superoxide dismutase (PEG-modified SOD); xxii. Dexamethasone; xxiii. Lysine acetate; xxiv. Methylprednisolone sodium succinate; xxv. CHIR99021; xxvi. Antibiotics; xxvii. Glucocorticoids; xxviii. Anticoagulants; xxix. Prostaglandins.

[0274] 61. The composition according to item 60, wherein the electrolyte is one or more electrolytes selected from the group consisting of calcium salts, potassium salts, sodium salts, magnesium salts, chloride salts, sulfate salts, and phosphate salts.

[0275] 62. The composition of claim 60, wherein the cardiac stimulant is one or more cardiac stimulants selected from the group consisting of catecholamines, peptides, polypeptides, beta1 / beta2 adrenergic receptor blockers, buprinalol, pindolol, alprenolol, cardiac glycosides, digitalis, palustrine, ferulic acid, and epinephrine.

[0276] 63. The composition of any one of the preceding items, wherein the aqueous medium comprises: - one or more cardiac stimulants selected from the group consisting of catecholamines, peptides, polypeptides, beta1 / beta2 adrenergic receptor blockers, buprinalol, pindolol, alprenolol, cardiac glycosides, digitalis, palustrine, ferulic acid, and epinephrine; - a plurality of amino acids selected from the group consisting of alanine, arginine, aspartic acid, glutamic acid, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and lysine, but not including asparagine, glutamine, and cysteine; - Optionally, adenosine, insulin, an immunosuppressant, a multivitamin composition, and / or one or more electrolytes, such as calcium.

[0277] 64. The composition of any one of the preceding items, wherein the aqueous medium comprises: - Mannitol; - Sodium chloride; - Potassium chloride; - Magnesium sulfate heptahydrate; - Sodium glycerophosphate.

[0278] 65. The composition of any one of the preceding items, wherein the aqueous medium comprises: - Adenosine; - Calcium chloride dehydrate; - Glycine; - L-Alanine; - L-arginine; - L-aspartic acid; - L-glutamic acid; - L-Histidine; - L-Isoleucine; - L-Leucine; - L-Methionine; - L-Phenylalanine; - L-Proline; - L-serine; - L-Threonine; - L-tryptophan; - L-Tyrosine; - L-valine; - Lysine acetate; - Magnesium sulfate heptahydrate; - Potassium chloride; - Sodium chloride; - Dextrose; - epinephrine; - Insulin; - one or more vitamins; - Methylprednisolone sodium succinate; - Sodium bicarbonate; - Mannitol; - Sodium glycerophosphate.

[0279] 66. The composition of any one of the preceding items, wherein the aqueous medium comprises: - dextran; - glucose monohydrate; - Magnesium sulfate heptahydrate; - Potassium chloride; - Sodium chloride; - Sodium phosphate dibasic dihydrate; - Monopotassium phosphate.

[0280] 67. The composition of any one of the preceding items, wherein the aqueous medium comprises: - Human serum albumin (HSA); - Dextran.

[0281] 68. The composition of any one of the preceding items, wherein the aqueous medium comprises: - dextran; - Na + ; -K + ; - Mg 2+ ; - Cl - ; - SO4 2- ; - H2PO4 - ; - Glucose.

[0282] 69. The composition of any one of the preceding items, wherein the aqueous medium comprises: - Sodium; - Potassium; - Magnesium; - Calcium; - Ketoglutarate / Glutamic Acid; - Histidine; - Mannitol; - Tryptophan.

[0283] 70. The composition of any one of the preceding items, wherein the aqueous medium comprises: - Glutathione; - Mannitol; - Lactobionic acid; - Glutamic acid; - Sodium hydroxide; - Calcium chloride dihydrate; - Potassium chloride; - Magnesium chloride hexahydrate; - Histidine.

[0284] 71. The composition of any one of the preceding items, wherein the aqueous medium comprises: - Pentafraction; - Allopurinol; - Lactobionic acid; - Glutathione; - Potassium dihydrogen phosphate; - Potassium hydroxide; - Magnesium sulfate heptahydrate; - Raffinose pentahydrate; - Adenosine.

[0285] 72. The composition of any one of the preceding items, wherein the aqueous medium comprises: - Adenine; - Calcium chloride; - Dextrose; - Glutathione; - HEPES; - Hydroxyethyl starch; - Magnesium Gluconate; - Mannitol; - Potassium phosphate; - Ribose; - Sodium gluconate; - Sodium hydroxide.

[0286] 73. The composition of any one of the preceding items, wherein the aqueous medium comprises: - hydroxyethyl starch, - Lactobionic acid; - Potassium dihydrogen phosphate; - Magnesium sulfate heptahydrate; - Raffinose pentahydrate; - Adenosine; - Allopurinol; - Glutathione; - Potassium hydroxide.

[0287] 74. The composition of any one of the preceding items, wherein the aqueous medium comprises: - Perfluorocarbons; - glucose; - Insulin; - Allopurinol; - PEG-modified SOD; - Adenosine; - Dexamethasone; - Hydroxyethyl starch; - sodium ions; - potassium ions; - Chloride.

[0288] 75. The composition of any one of the preceding items, wherein the aqueous medium comprises: - Iscove's Modified Dulbecco's Medium (IMDM); - Human Stem Cell Factor; - Thrombopoietin; - CHIR99021 - Forskolin; - OAC1.

[0289] 76. The composition of any one of the preceding items, wherein the aqueous medium comprises: - IMDM; - Bovine serum albumin; - Human insulin; - Human transferrin (iron saturated); - β-mercaptoethanol.

[0290] 77. The composition of any one of the preceding items, wherein the aqueous medium comprises: - Serum-free medium; - Heparin; - Thrombopoietin; - Human Stem Cell Factor; - Fibroblast growth factor 1.

[0291] 78. The composition of any one of the preceding items, wherein the aqueous medium comprises: - Serum-free medium; - human IL-3; - human IL-6; - Thrombopoietin; - Flt-3 ligand; - Human stem cell factors, such as "5GF" human stem cell factor.

[0292] 79. The composition of any one of the preceding items, wherein the aqueous medium comprises: - IMDM; - BSA; - Human insulin; - Human transferrin; - low density lipoproteins; - β-mercaptoethanol; - Human Stem Cell Factor; - Flt-3 ligand; - G-CSF; - IL-3; - IL6.

[0293] 80. The composition of any one of the preceding items, wherein the aqueous medium comprises: - Human serum albumin and dextran 40; - Methylprednisolone; - Heparin, such as unfractionated heparin; - Imipenem and optionally cilastatin.

[0294] 81. The composition of any one of the preceding items, wherein the aqueous medium comprises: - Human serum albumin; - Ringer's lactate solution; - Mannitol; - Calcium gluconate; - Meropenem; - Sodium bicarbonate; - Dexamethasone.

[0295] 82. The composition of any one of the preceding items, wherein the aqueous medium comprises: - Human serum albumin; - Ringer's lactate solution; - red blood cells, - Mannitol; - Calcium gluconate; - Meropenem; - Sodium bicarbonate; - Dexamethasone.

[0296] 83. The composition of any one of the preceding items, wherein the aqueous medium comprises: - Human serum albumin and dextran 40; - red blood cells; - Cefuroxime; - Heparin, such as unfractionated heparin; - Calcium gluconate; - Insulin; - Prostacyclin.

[0297] 84. The composition of any one of the preceding items, wherein the ex vivo organ is selected from the group consisting of heart, lung, liver, kidney, pancreas, small intestine, large intestine, and stomach.

[0298] 85. The composition according to any one of items 1 to 83, wherein the tissue is skin or corneal tissue.

[0299] 86. The composition according to any one of items 1 to 83, wherein the stem cell culture is an adult stem cell culture, such as a mesenchymal stem cell culture or an induced pluripotent stem cell (IPSC) culture, a precursor cell culture or a progenitor cell culture.

[0300] 87. The composition of any one of items 1 to 83, wherein the stem cell culture is an embryonic stem cell culture.

[0301] 88. The composition described in any one of items 1 to 83, wherein the stem cell culture is a hematopoietic stem cell culture.

[0302] 89. The stem cell culture is a CD34 positive cell (CD34 + 84. The composition according to any one of items 1 to 83, comprising a medicament for treating or preventing the infection of a patient with atopic dermatitis (cell).

[0303] 90. The composition of any one of the preceding items, wherein the composition is suitable for maintaining the viability of the ex vivo organ, tissue, and / or stem cell culture prior to transplantation.

[0304] 91. The composition of any one of the preceding items, wherein the composition is suitable for maintaining the ex vivo organ, tissue, and / or stem cell culture in a functional state.

[0305] 92. The composition described in item 91, wherein the composition is suitable for maintaining the ex vivo organ, tissue, and / or stem cell culture under physiological or near-physiological conditions.

[0306] 93. The composition according to any one of items 91 to 92, wherein the composition is suitable for preserving the ex vivo organ in an organ care system.

[0307] 94. The composition of any one of the preceding items, further comprising one or more additional agents.

[0308] 95. The composition described in item 94, wherein the additional agent is selected from the group consisting of immunosuppressants and antiviral agents.

[0309] 96. The composition of item 95, wherein the antiviral agent is selected from the group consisting of valganciclovir, ganciclovir, cidofovir, leflunomide, prevmis, ​​maribavir, and brincidofovir.

[0310] 97. A composition according to any one of the preceding items for use in the prevention or treatment of pathogen infection and / or pathogen-associated disorders in said ex vivo organ, tissue, and / or stem cell cultures.

[0311] 98. The composition for use according to item 97, wherein the infection by the pathogen is a CMV infection, such as a latent and / or lytic CMV infection.

[0312] 99. The composition for use according to item 98, wherein the CMV infection can be detected in ex vivo organ, tissue, and / or stem cell cultures.

[0313] 100. A composition for use according to any one of items 97 to 99, wherein the composition prevents or reduces the severity of graft-versus-host disease (GVHD) and / or solid organ transplant graft-versus-host disease (SOT-GVHD).

[0314] 101. A kit for the prevention or treatment of infection by a pathogen in ex vivo organ, tissue, and / or stem cell cultures, said kit comprising: a. the composition according to any one of items 1 to 96; b. Optionally, with instructions for use.

[0315] 102. The kit according to item 101, wherein the kit is further suitable for maintaining the viability of the organ, tissue, and / or stem cell culture prior to transplantation.

[0316] 103. A perfusion system configured to maintain an ex vivo organ in a functional state under physiological or near-physiological conditions, comprising one or more chambers configured to maintain the ex vivo organ in a functional state under physiological or near-physiological conditions, at least one chamber comprising a composition described in any one of items 1 to 96.

[0317] 104. The perfusion system of item 103, wherein the perfusion system is an organ care system.

[0318] 105. A method for the treatment or prevention of infection by a pathogen in an ex vivo organ, tissue and / or stem cell culture prior to transplantation, comprising immersing, perfusing, storing, conditioning and / or rinsing said ex vivo organ, tissue and / or stem cell culture in a composition as defined in any one of items 1 to 96.

[0319] 106. The method of claim 105, wherein the method is for maintaining the viability of the ex vivo organ, tissue, and / or stem cell culture prior to transplantation.

[0320] 107. The method according to any one of items 105 to 106, wherein the perfusion is carried out for about 1 hour to about 10 hours, for example for about 6 hours.

[0321] 108. Use of a composition according to any one of items 1 to 96 for the treatment or prevention of infection by a pathogen in ex vivo organ, tissue and / or stem cell cultures for transplantation. EXAMPLES

[0322] Example 1: Mutations in the first peptide of the fusion protein SYNx result in selectivity of cell killing Materials and Methods HEK293 cells expressing inducible US28 or CX3CR1 For binding and cell killing assays, cells were constructed as described in Hjortoe GM et al., 2013. Briefly, stable inducible clones of cells expressing HA-CX3CR1 and HA-US28 were generated by co-transfecting Flp-In T-Rex-293 cells with either the Flp-recombinase expression vector, pOG44, and the pcDNA5 / FRT / TO receptor construct. This targeted cloning at the FRT site places the receptor gene under the control of the tetracycline repressor / operator system. Receptor expression was confirmed by Western blotting. For cell culture, cells were grown in 1x DMEM (containing 9% (v / v) FBS, 180 U / mL penicillin and 45 μg / mL streptomycin, 13.5 μg / mL blasticidin and 1.32 mg / mL hygromycin B) in a humidified incubator (37°C, 5% CO2).

[0323] Cell killing assay 80 μL of 20 μg / mL poly-D-lysine (PDL) in 1× PBS was added to each well of a white 96-well plate and incubated at room temperature for 30 min. HEK293 cells expressing inducible US28 or CX3CR1 were washed with 10 mL of 1× PBS and released with 2 mL of 0.05% (w / v) trypsin-EDTA for 2 min. Cells were resuspended in 10 mL of growth medium (1× DMEM, 9% (v / v) FBS, 180 U / mL penicillin and 45 μg / mL streptomycin) and then cell density was determined. PDL solution was aspirated from the plate and each well was washed with 100 μL of 1× PBS. After aspiration of the wash buffer, 96-well plates were seeded at 2,000–10,000 cells / well in 100 μL and placed in a CO2 incubator (37°C, 10% CO2) overnight.

[0324] For receptor expression, 10 μL of tetracycline, 1.375 μg / mL of US28 and 5.5 μg / mL of CX3CR1 in growth medium without selection were added to each well of the plate and the plate was returned to a CO2 incubator (37° C., 10% CO2) overnight.

[0325] Fusion toxin protein aliquots were thawed on ice and used to make a dilution series in 1 mM acetic acid and 5 g / L BSA. Receptor expression growth medium was aspirated and replaced with 100 μL of fresh growth medium. 5 μL of the fusion toxin protein dilution series was added to each well, and 5 μL of 1 g / L cycloheximide was added to one well as a positive control. Plates were returned to a CO2 incubator (37°C, 10% CO2) overnight.

[0326] A 1:10 dilution of AlamarBlue in assay growth medium was prepared. The solution was covered with aluminum foil and heated in a 37°C water bath for 10 minutes, then filtered through a 0.2 μm filter. The well solution was aspirated and 100 μL of AlamarBlue solution was added to each well, and the plate was returned to a CO2 incubator (37°C, 10% CO2) for 4 hours. Fluorescence data was collected on a FlexStation3 using an excitation of 540 nm and an emission of 585 nm.

[0327] result The cell killing efficiency of SYNx was determined using tetracycline-induced HEK293 cells expressing either a virally encoded receptor such as US28 or an endogenous receptor such as CX3CR1, and non-induced cells with no receptor expression (negative control). Data were normalized to the maximum number of viable cells.

[0328] Substitution of a single amino acid in the first peptide of SYNx (here the chemokine portion) results in selectivity for the virus-encoded receptor US28, as shown in Figure 2. A single mutation in the first peptide (receptor-binding portion) of the protein (F49A, corresponding to F73A using precursor numbering) results in less selectivity for the endogenous receptor, here CXC3R1 (Figure 2B).

[0329] conclusion A single amino acid F49A substitution in the first peptide (eg, the chemokine portion) of the fusion protein construct induces selectivity of the fusion protein (SYNx) for the virally encoded receptor US28.

[0330] Example 2: Increased potency obtained by adding a furin cleavage site through the full-length translocation domain of exotoxin A Materials and Methods See Example 1.

[0331] result The cell killing efficiency of SYNx was determined using tetracycline-induced HEK293 cells expressing either the virus-encoded receptor US28 or the endogenous receptor CX3CR1, and non-induced cells with no receptor expression (negative control). Data were normalized to the maximum number of viable cells. Addition of a furin cleavage site, such as a full-length translocation domain, to the SYN001 construct generating SYN016 increases potency in both cells expressing the endogenous / human CX3CR1 and pathogen-encoded US28 receptors, as shown in Figure 3 and Table 1.

[0332] conclusion Domain II of exotoxin A has been identified as a translocation domain involved in the translocation of the catalytic domain, domain III, from endocytic vesicles to the cytosol (Hwang J et al., 1987; Siegall CB et al., 1989). Addition of a translocation domain containing a furin cleavage site, such as domain II of exotoxin A, to SYN001 to generate SYN016 resulted in a fusion protein construct with increased potency at both the endogenous receptor CX3CR1 and the virally encoded receptor US28.

[0333] [Table 1]

[0334] Example 3: In vitro cleavage of SYNx by human furin Materials and Methods In vitro cleavage of SYNx by human furin Purified SYNx constructs were thawed on the bench. Once thawed, samples were spun down. Sample concentration was measured and digests were set up with 20 μM SYNx in 50 μL in a new tube in 1×PBS with 5 mM CaCl2. 5 μg were removed for SDS-PAGE analysis. 1 μL of human furin (NEB, 2 units / μL) was added to the mixture and placed in a 37°C water bath. After 1.5 hours, 5 μg of SYNx was removed for SDS-PAGE analysis. The pH of the reaction mixture is adjusted with either 1 M HCL or 1 M NaOH prior to adding SYNx.

[0335] Reduction of SDS-PAGE analysis Samples were prepared and analyzed on NuPAGE Bis-Tris 4-12% gels along with protein marker standards using 1x MES running buffer with 2mM DTT according to the manufacturer's protocol. Gels were run at a constant voltage of 125V for 75 minutes. Gels were stained according to the SimplyBlue™ SafeStain manual (Novex).

[0336] result Purified SYNx constructs were treated in vitro with purified human furin to determine the cleavage efficiency of the various SYNx constructs by furin. Cleavage mechanisms other than furin may occur. Results were analyzed by SDS-PAGE. Addition of a complete translocation domain, such as the complete ExoA translocation domain, to SYN000 or SYN001, generating SYN017 and SYN016, respectively, improves in vitro cleavage by furin. Optimization of the furin cleavage site in SYN017 and SYN016, generating SYN014 and SYN002, respectively, does not appear to further improve in vitro cleavage by furin, as shown in Figure 4.

[0337] Purified SYN002 was treated in vitro with purified human furin at different pH values ​​to determine the cleavage efficiency, and the results were analyzed by SDS-PAGE. SYN002 is cleaved by furin over a wide pH range, as shown in Figure 5.

[0338] conclusion As with SYN016, the addition of an optimized cleavage site (in this case ArgGlnArgArg) to a SYNx construct (here the SYN002 construct that also contains the translocation domain) did not improve in vitro cleavage by furin compared to a construct with the native sequence (ArgGlnProArg). SYN002 can be cleaved in vitro over a wide pH range, suggesting that cleavage by furin can occur in any cellular location, not just endosomes.

[0339] Example 4: Cell killing selectivity between cells expressing endogenous receptors and cells expressing virally encoded receptors is also enhanced and mediated by the optimized cleavage site. Materials and Methods See Example 1.

[0340] result The cell killing efficiency of SYNx was determined using tetracycline-induced HEK293 cells expressing either US28 or CX3CR1, and non-induced cells with no receptor expression (negative control). Data were normalized to the maximum number of viable cells.

[0341] When the optimized cleavage site ArgX1X2Arg is introduced into the second peptide, in this particular case the optimized cleavage site ArgGlnArgArg is introduced into SYN016 to obtain SYN002, a surprising increase in selectivity is obtained. In the case of SYN002 on cells expressing a virus-encoded receptor such as US28, the cell killing potency is almost maintained compared to SYN016 with the cleavage site ArgGlnProArg (hence X2=Pro) (Figure 6A, Table 1). However, the potency against cells expressing an endogenous receptor such as CX3CR1 is reduced (approximately 0.9 log(EC50)), see Figure 6B), an effect that can support a more effective drug with fewer side effects. When compared to the second peptide that does not contain the optimized cleavage site, both selectivity and potency are increased.

[0342] conclusion Surprising effects on cell killing selectivity are obtained by optimizing the cleavage site, and without being bound by any theory, this may be due to differences in internalization and intracellular trafficking of virally encoded receptors such as the US28 receptor versus endogenous receptors such as the CX3CR1 receptor.

[0343] Example 5: Stability of Use of SYN002 in Lung Ex Vivo Studies Background and Summary Prior to testing SYN002 (SEQ ID NO: 6) in an ex vivo lung perfusion (EVLP) system, its stability in the perfusate (perfusate) was investigated. During EVLP, the lungs are placed in an incubator connected to a ventilator and perfusion circuit, and SYN002 may be added to remove cytomegalovirus (CMV) from the organ before transplantation. The lungs are gradually heated to 37°C over the first hour after which SYN002 can be added to the perfusate.

[0344] The SYN002 reference material used for the ex vivo study is formulated in 10 mM citric acid and 300 mM L-arginine pH 6.5. Due to the high potency of SYN002, ex vivo studies for efficacy and toxicity are performed at very low concentrations. In this study, SYN002 was tested at approximately 1.9-37 nM (0.085-1.7 mg / L, respectively).

[0345] The concentrations of SYN002 in this study were too low to allow for typical analytical methods, therefore potency assays were used as a measure of stability.

[0346] The stability of SYN002 was demonstrated in EVLP perfusate at concentrations ranging from 0.085 to 1.7 mg / L when exposed to polypropylene containers at 37°C for 5 hours.

[0347] The stability of SYN001 (SEQ ID NO: 12) was also demonstrated in EVLP perfusate at a concentration of 1 mg / L over a 5 hour period at 37° C. exposed to polypropylene containers.

[0348] Materials and Methods The perfusion solution in this example consisted of the following:

[0349] 2L of Steen solution (XVIVO, Sweden). STEEN Solution™ is a clear, sterile, nonpyrogenic, nontoxic saline solution containing human serum albumin (HSA) and dextran 40, designed for use as a temporary continuous machine perfusion solution for evaluation of explanted lungs following removal from a donor. Dextran is a heterogeneous glucose polymer produced by bacteria and is uncharged.

[0350] 500 mg of methylprednisolone (Solu-Medrol, Sandoz Canada, Boucherville, Canada) Methylprednisolone is a corticosteroid used to treat inflammation or immune responses in various organ systems, endocrine disorders, and neoplastic diseases.

[0351] 3,000 IU unfractionated heparin (Leo Pharma, Thornhill, Canada) Heparin is an anticoagulant indicated for the prevention of blood clots and for the treatment of thrombosis associated with various conditions such as pulmonary embolism and atrial fibrillation. · Unfractionated heparin is a heterogeneous preparation of anionic sulfated glycosaminoglycan polymers ranging in weight from 3,000 to 30,000 Da.

[0352] 500 mg each of imipenem / cilastatin (Primaxin, Merck, Whitehouse Station, NJ) · Imipenem is a carbapenem antibiotic that is usually given with cilastatin to treat a variety of infections, with broad spectrum antibacterial activity against gram-negative and gram-positive aerobic and anaerobic bacteria. Cilastatin is a renal dehydropeptidase inhibitor used to prevent the breakdown of imipenem.

[0353] SYN001 (SEQ ID NO: 12) and SYN002 (SEQ ID NO: 6) were tested at the concentrations shown below and sampled at the indicated time points: [Table 2]

[0354] Briefly, the potency of SYN001 and SYN002 was determined in US28-expressing HEK293 cells. US28 is a viral GPCR receptor that recognizes and internalizes SYN002. Potency is determined by measuring the cytotoxic effect of SYN001 or SYN002 on cell viability at increasing concentrations of SYN001 or SYN002. The level of viable cells after a period of SYN001 or SYN002 treatment is assessed by a fluorescent assay and plotted as a function of SYN001 or SYN002 concentration, respectively. The higher the fluorescence, the higher the measured cell viability and therefore the lower the potency of the SYN001 or SYN002 sample.

[0355] Containers used for stability: Polypropylene low protein binding tubes (Sarstedtk catalogue number 72.706.600).

[0356] result Selected time pull points were analyzed in a potency assay to determine whether the potency of SYN001 or SYN002 was lost over time upon exposure to perfusate at 37°C.

[0357] Potency assays of a SYN001 dilution series made from a 1 mg / L sample after perfusate exposure for 0, 15, 180, and 300 minutes at 37° C. are shown in FIG.

[0358] Potency assays for SYN002 dilution series generated from 1.7 mg / L, 0.85 mg / L, and 0.085 mg / L samples after perfusate exposure for 0, 15, 180, and 300 minutes at 37° C. are shown in FIGS. 8, 9, and 10, respectively.

[0359] The lowest concentration of SYN002 was also evaluated in the potency assay at 30, 60, and 1140 minutes (see Figures 11 and 12).

[0360] All ECs in Figures 7 to 12 50 The values ​​are summarized in the table below.

[0361] [Table 3]

[0362] conclusion The stability of SYN001 and SYN002 was investigated using a potency assay as a readout.

[0363] The stability of SYN001 was demonstrated in EVLP perfusate at a concentration of 1 mg / L exposed to polypropylene containers for 5 hours at 37°C.

[0364] The stability of SYN002 was demonstrated in EVLP perfusate at concentrations ranging from 0.085 to 1.7 mg / L when exposed in polypropylene containers for 5 hours at 37 °C. Although the lowest concentration of SYN002 appears to have lost some of its potency after 19 hours, this time point was not considered appropriate for clinical EVLP and was chosen to see if longer exposures would induce negative effects. Five hours of exposure to SYN002 perfusate is considered a sufficient test to confirm stability in human lungs and early preclinical studies with SYN002.

[0365] Example 6: Ex vivo organ care using SYN002 lung Normothermic Ex Vivo Lung Perfusion Protocol Immediately after the donor lungs are retrieved, they are flushed with a low potassium solution and stored at 4°C in an inflated state. The left atrium (LA) and main pulmonary artery (PA) are cannulated and an endotracheal tube is placed. The circuit is primed with 2.0 L of EVLP perfusion solution (Steen solution, XVIVO, Sweden) containing 500 mg methylprednisolone (Solu-Medrol, Sandoz Canada, Boucherville, Canada), 3,000 IU unfractionated heparin (Leo Pharma, Thornhill, Canada) and 500 mg imipenem / cilastatin (Primaxin, Merck, White-house Station, NJ). The lungs are connected to the EVLP circuit via the cannula and normothermia is slowly achieved during the first hour of perfusion with a target flow rate of 40% of the donor predicted cardiac output. Apply protective ventilation during perfusion using an ICU ventilator (7 cc per kg donor ideal body weight, inspired oxygen fraction 21%, end-expiratory pressure 5 cm HO).

[0366] Pulmonary assessment: Every hour important physiological parameters such as graft oxygenation, lung compliance, airway pressure, perfusate consumption, and perfusate metabolic products are recorded.

[0367] Perfusion solution (irrigation fluid): 2 L of Steen's solution containing 500 mg methylprednisolone, 3000 IU unfractionated heparin, 500 mg imipenem, and 500 mg cilastatin.

[0368] Perfusion solution with SYN002: Add the required amount of diluted SYN002 to the perfusion buffer (see: Preparation of SYN002) and start the perfusion with perfusion solution + / - SYN002 immediately, but within 15 minutes after preparation of the solution.

[0369] Perfusion temperature: slowly increase temperature to 37°C during the first hour, then keep at 37°C for the duration of treatment (5 hours).

[0370] Perfusion time: 6 hours (1 hour lung priming followed by 5 hours perfusion with SYN002).

[0371] Adding perfusate to the system: Add 500 mL of fresh perfusate during the first hour (1 h), followed by 200 mL of perfusate every hour (2-6 h). Perfusate added to the circuit should contain SYN002 at the concentration for each dose group being evaluated.

[0372] Washout: At the end of the perfusion, add 2 L of Perfadex solution (for 2 min) to wash out the perfusion buffer and cool the lungs.

[0373] kidney Acellular normothermic mechanical perfusion (NMP) After transport on ice and arrival at the transplant laboratory, the kidney undergoes a period of NMP perfusion (6 h). The kidney is connected to an NMP circuit and perfused with 500 mL of a cell-free solution containing Ringer's solution and human serum albumin at 35-37 °C for 4-6 h.

[0374] The perfusate flows into the kidney and intrarenal resistance and temperature are monitored throughout the perfusion.

[0375] SYN002 is added to the perfusate to treat CMV.

[0376] Overview of Arrays SEQ ID NO:1 (Exemplary First Peptide) QHHGVTKCNITCSKMTSKIPVALLIHYQQNQASCGKRAIILETRQHRLFCADPKEQWVKDAMQHLDRQAAALTRNG SEQ ID NO:2 (Exemplary second peptide) GGSLAALTAHQACHLPLETFTRHRQRRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASGPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVG YHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPP SEQ ID NO:3 (Domain A of Exotoxin A) GGSLAALTAHQACHLPLETFTRHRQRRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAAS SEQ ID NO: 4 (Domain B of Exotoxin A) GPADSGDALLERNYP SEQ ID NO:5 (Domain C of Exotoxin A) TGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRV YVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPP SEQ ID NO:6 (SYN002) MQHHGVTKCNITCSKMTSKIPVALLIHYQQNQASCGKRAIILETRQHRLACADPKEQWVKDAMQHLDRQAAALTRNGGGSLAALTAHQACHLPLETFTRHRQRRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASGPADSGDALLERNYPTGAEFL GDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRS SLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPKDEL SEQ ID NO: 7 (human CX3CL1) MAPISLSWLLRLATFCHLTVLLAGQHHGVTKCNITCSKMTSKIPVALLIHYQQNQASCGKRAIILETRQHRLFCADPKEQWVKDAMQHLDRQAAALTRNGGTFEKQIGEVKPRTTPAAGGMDESVVLEPEATGESSSLEPTPSSQEAQRALGTSPELPTGVTGSSGTRLPPTPKAQDGGPVGTELFRVPPVSTAATWQ SSAPHQPGPSLWAEAKTSEAPSTQDPSTQASTASSPAPEENAPSEGQRVWGQGQSPRPENSLEREEMGPVPAHTDAFQDWGPGSMAHVVPVSSEGTPSREPVASGSWTPKAEEPIHATMDPQRLGVLITPVPDAQAATRRQAVGLLAFLGLLFCLGVAMFTYQSLQGCPRKMAGEMAEGLRYIPRSCGSNSYVLVPV SEQ ID NO: 8 (sequence ensuring binding to the KDEL receptor for intracellular retrograde transport) KDEL SEQ ID NO:9 (full length exotoxin A) MHLTPHWIPLVASLGLLAGGSFASAAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYT IEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRL VALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFG GVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK SEQ ID NO: 10 (US28) MTPTTTTAELTTEFDYDEAATPCVFTDVLNQSKPVTLFLYGVVFLFGSIGNFLVIFTITW RRRIQCSGDVYFINLAADLLFVCTLPLWMQYLLDHNSLASVPCTLLTACFYVAMFASLCFITEIALDRYYAIVYMRYRPVKQACLFSIFWWIFAVIIAIPHFMVKKVTDNQCMTDYDYLEVSYPIILNVELMLGAFVIPLSVISYCYYRISRIVAVSQSRHKGRIVRVLIAVVLVFIIF WLPYHLTLFVDTLKLLKWISSSCEFERSLKRALILTESLAFCHCCLNPLLYVFVGTKFRQELHCLLAEFRQRLFSRDVSWYHSMSFSRRSSPSRRETSSDTLSDEVCRVSQIIP SEQ ID NO:11 (SYN000) MQHHGVTKCNITCSKMTSKIPVALLIHYQQNQASCGKRAIILETRQHRLFCADPKEQWVKDAMQHLDRQAAALTRNRQPRGWEQLEQSGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASGPADSGDALLERNYPTGAEFLGDGGGDISFSTRG TQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPKDEL SEQ ID NO:12 (SYN001) MQHHGVTKCNITCSKMTSKIPVALLIHYQQNQASCGKRAIILETRQHRLACADPKEQWVKDAMQHLDRQAAALTRNRQPRGWEQLEQSGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASGPADSGDALLERNYPTGAEFLGDGGGDISFSTRG TQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPKDE SEQ ID NO:13 (SYN014) MQHHGVTKCNITCSKMTSKIPVALLIHYQQNQASCGKRAIILETRQHRLFCADPKEQWVKDAMQHLDRQAAALTRNGGGSLAALTAHQACHLPLETFTRHRQRRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASGPADSGDALLERNYPTGAEFL GDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRS SLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPKDEL SEQ ID NO:14 (SYN016) MQHHGVTKCNITCSKMTSKIPVALLIHYQQNQASCGKRAIILETRQHRLACADPKEQWVKDAMQHLDRQAAALTRNGGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASGPADSGDALLERNYPTGAEFL GDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRS SLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPKDEL SEQ ID NO:15 (SYN017) MQHHGVTKCNITCSKMTSKIPVALLIHYQQNQASCGKRAIILETRQHRLFCADPKEQWVKDAMQHLDRQAAALTRNGGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASGPADSGDALLERNYPTGAEFL GDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRS SLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPKDEL SEQ ID NO: 16 (cleavage site) RQRR SEQ ID NO: 17 (cleavage site) RQKR SEQ ID NO: 18 (cleavage site) RSKR SEQ ID NO: 19 (cleavage site) RSRR SEQ ID NO: 20 (cleavage site) RTKR SEQ ID NO:21 (cleavage site) RTRR SEQ ID NO:22 (cleavage site) RNKR SEQ ID NO:23 (cleavage site) RNRR

[0377] References Chambers, Daniel C et al. “The International Thoracic Organ Transplant Registry of the International Society for Heart and Lung Transplantation:37th adult lung transplantation report - 2020;focus on deceased donor characteristics.”The Journal of heart and lung transplantation :the official publication of the International Society for Heart Transplantationvol.39,10(2020):1016-1027.doi:10.1016 / j.healun.2020.07.009 Hwang J et al.,Cell.1987 Jan 16;48(1):129-36; Siegall CB et al., J Biol Chem.1989 Aug 25;264(24):14256-61).

Claims

1. 1. A composition for preventing or treating infection by a pathogen in ex vivo organ, tissue, and / or stem cell cultures prior to transplantation, said composition comprising a fusion protein in an aqueous medium comprising: i. a first peptide that binds to at least one receptor expressed on a cell, such as a receptor encoded by a pathogen; and ii. a second peptide comprising a cleavage site having the amino acid sequence ArgX1X2Arg; wherein X2 is Arg or Lys and the second peptide comprises a toxin; The aqueous medium may be one or more of the following: i. Buffer component; ii. amino acids; iii. carbohydrates; or iv. Growth factors or cytokines The composition further comprises:

2. 2. The composition of claim 1, wherein the fusion protein comprises or consists of: a. the amino acid sequence of SEQ ID NO:6; b. a variant of SEQ ID NO: 6 comprising or consisting of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 6, more preferably at least 85% or 90% sequence identity to SEQ ID NO: 6, most preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 6; and / or c. A fragment of SEQ ID NO:6 that is more than 360 amino acids in length, such as more than 380, 400, or 420 amino acids in length, or a variant thereof having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:6, more preferably at least 85% or 90% sequence identity to SEQ ID NO:6, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:

6.

3. The composition of claim 1 or 2, wherein the fusion protein kills a cell infected with a pathogen, for example a cell latently infected with a pathogen.

4. The buffer component is a bicarbonate buffer (HCO ３ － / CO ２ ), ammonium buffer (NH ３ / NH ４ ＋ ), phosphate buffer (H ２ P.O. ４ － / H.P.O. ４ ２－ 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 3-(N-morpholino)propanesulfonic acid (MOPS), and 2-(N-morpholino)ethanesulfonic acid (MES).

5. The composition according to any one of claims 1 to 4, wherein the amino acid is one or more amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

6. 6. The composition of claim 1, wherein the carbohydrate is one or more carbohydrates selected from the group consisting of sugars, such as monosaccharides, disaccharides, oligosaccharides, or polysaccharides, and sugar alcohols.

7. The composition according to any one of claims 1 to 6, wherein the growth factor or cytokine is one or more growth factors or cytokines selected from the group consisting of thrombopoietin, insulin, fibroblast growth factor 1, human stem cell factor, IL-3, IL-6, Flt-3 ligand, and granulocyte colony-stimulating factor (G-CSF).

8. The composition of any one of claims 1 to 7, wherein the aqueous medium further comprises one or more additional components selected from the group consisting of: i. vitamins; ii. an electrolyte, such as one or more electrolytes selected from the group consisting of calcium salts, potassium salts, sodium salts, magnesium salts, chloride salts, sulfate salts, and phosphate salts; iii. Transcription factors such as OAC1; iv. diterpenes such as forskolin; v. low density lipoprotein; vi. a cardiac stimulant such as one or more cardiac stimulants selected from the group consisting of catecholamines, peptides, polypeptides, beta1 / beta2 adrenergic receptor blockers, buprinalol, pindolol, alprenolol, cardiac glycosides, digitalis, palustrine, ferulic acid, and epinephrine; vii. immunosuppressants; viii. serum albumin, such as bovine serum albumin (BSA) or human serum albumin (HAS); ix. heparin, such as unfractionated heparin; x. human transferrin; xi. Pentafraction; xii. 2-mercaptoethanol; xiii. glutathione; xiv. lactobionic acid; xv. glutamic acid; xvi. Allopurinol; xvii. adenosine; xviii. adenine; xix. hydroxyethyl starch; xx. perfluorocarbon; xxi. polyethylene glycol-modified superoxide dismutase (PEG-modified SOD); xxii. dexamethasone; xxiii. Lysine acetate; xxiv. methylprednisolone sodium succinate; xxv. CHIR99021; xxvi.An antiviral agent such as an antiviral agent selected from the group consisting of valganciclovir, ganciclovir, cidofovir, leflunomide, prevmis, ​​maribavir, and brincidofovir; xxvii. antibiotics; xxviii. glucocorticoids; xxix. anticoagulants; and xxx. Prostaglandins.

9. The aqueous medium is one or more cardiac stimulants selected from the group consisting of catecholamines, peptides, polypeptides, beta1 / beta2 adrenergic receptor blockers, buprinalol, pindolol, alprenolol, cardiac glycosides, digitalis, palustrine, ferulic acid, and epinephrine; - a plurality of amino acids selected from the group consisting of alanine, arginine, aspartic acid, glutamic acid, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and lysine, excluding asparagine, glutamine, and cysteine; A composition according to any one of claims 1 to 8, optionally comprising adenosine, insulin, an immunosuppressant, a multivitamin composition and / or one or more electrolytes, such as calcium.

10. The aqueous medium is - mannitol; - sodium chloride; - potassium chloride; - magnesium sulfate heptahydrate; and - containing sodium glycerophosphate, and / or The aqueous medium is - adenosine; - calcium chloride dehydrate; - glycine; - L-alanine; - L-arginine; - L-aspartic acid; - L-glutamic acid; - L-histidine; - L-isoleucine; - L-leucine; - L-methionine; - L-phenylalanine; - L-proline; - L-serine; - L-Threonine; - L-tryptophan; - L-tyrosine; - L-valine; - lysine acetate; - magnesium sulfate heptahydrate; - potassium chloride; - sodium chloride; - dextrose; - epinephrine; - insulin; - one or more vitamins; - methylprednisolone sodium succinate; - sodium bicarbonate; - mannitol; and - containing sodium glycerophosphate, and / or The aqueous medium is - dextran; glucose monohydrate; - magnesium sulfate heptahydrate; - potassium chloride; - sodium chloride; - dibasic sodium phosphate dihydrate; and - contains monopotassium phosphate and / or The aqueous medium is - dextran; -Na ＋ ; - K ＋ ; - Mg ２＋ ; - Cl － ; - SO4 ２－ ; - H ２ PO ４ － ; and - contains glucose, and / or The aqueous medium is - sodium; - potassium; - magnesium; - calcium; - ketoglutarate / glutamic acid; - histidine; - mannitol; and - contains tryptophan, and / or The aqueous medium is - Glutathione; - mannitol; - lactobionic acid; - glutamic acid; - sodium hydroxide; - calcium chloride dihydrate; - potassium chloride; - magnesium chloride hexahydrate; and - contains histidine, and / or The aqueous medium is - pentafraction; - Allopurinol; - lactobionic acid; - Glutathione; - potassium dihydrogen phosphate; - potassium hydroxide; - magnesium sulfate heptahydrate; raffinose pentahydrate; and - contains adenosine, and / or The aqueous medium is - adenine; - calcium chloride; - glucose; - Glutathione; - HEPES; - hydroxyethyl starch; - magnesium gluconate; - mannitol; - potassium phosphate; - ribose; - sodium gluconate; and - contains sodium hydroxide and / or The aqueous medium is - hydroxyethyl starch, - lactobionic acid; - potassium dihydrogen phosphate; - magnesium sulfate heptahydrate; - raffinose pentahydrate; - adenosine; - Allopurinol; - Glutathione; and - contains potassium hydroxide and / or The aqueous medium is - perfluorocarbons; - glucose; - insulin; - Allopurinol; - PEG-modified SOD; - adenosine; - dexamethasone; - hydroxyethyl starch; - sodium ions; potassium ions; and - contains chloride and / or The aqueous medium is - Iscove's Modified Dulbecco's Medium (IMDM); - human stem cell factor; - thrombopoietin; - CHIR99021 - Forskolin; and - includes OAC1, and / or The aqueous medium is - IMD; - bovine serum albumin; - human insulin; - human transferrin (iron saturated); and - containing β-mercaptoethanol, and / or The aqueous medium is - serum-free medium; - heparin; - thrombopoietin; - human stem cell factor; and - containing fibroblast growth factor 1, and / or The aqueous medium is - serum-free medium; - human IL-3; - human IL-6; - thrombopoietin; - Flt-3 ligand; and - comprises human stem cell factor, such as the "5GF" human stem cell factor; and / or The aqueous medium is - IMD; - BSA; - human insulin; - human transferrin; - low density lipoproteins; β-mercaptoethanol; - human stem cell factor; - Flt-3 ligand; - G-CSF - IL-3; and - contains IL6, and / or The aqueous medium is - human serum albumin and dextran 40; - methylprednisolone; heparin, such as unfractionated heparin; and - containing imipenem and optionally cilastatin, and / or The aqueous medium is - human serum albumin; - Lactated Ringer's solution; - mannitol; - calcium gluconate; - meropenem; - sodium bicarbonate; and - contains dexamethasone and / or The aqueous medium is - human serum albumin; - Lactated Ringer's solution; - red blood cells, - mannitol; - calcium gluconate; - meropenem; - sodium bicarbonate; and - contains dexamethasone and / or The aqueous medium is - human serum albumin and dextran 40; - red blood cells; - cefuroxime; - heparin, such as unfractionated heparin; - calcium gluconate; - insulin; and A composition according to any one of claims 1 to 9, comprising prostacyclin.

11. The composition according to any one of claims 1 to 10, wherein the ex vivo organ is selected from the group consisting of heart, lung, liver, kidney, pancreas, small intestine, large intestine, and stomach.

12. The composition according to any one of claims 1 to 11, wherein said composition is suitable for maintaining the viability of said ex vivo organ, tissue and / or stem cell culture prior to transplantation.

13. 13. A composition according to any one of claims 1 to 12 for use in the prevention or treatment of pathogen infection and / or pathogen-associated disorders in said ex vivo organ, tissue and / or stem cell cultures.

14. 1. A kit for the prevention or treatment of infection by a pathogen in ex vivo organ, tissue and / or stem cell cultures, comprising: a. the composition of any one of claims 1 to 12; b. Optionally, instructions for use. The kit comprising:

15. 13. A perfusion system configured to maintain an ex vivo organ in a functional state under physiological or near-physiological conditions, the perfusion system such as an organ care system comprising one or more chambers configured to maintain the ex vivo organ in a functional state under physiological or near-physiological conditions, at least one chamber comprising a composition according to any one of claims 1 to 12.

Images