Fusion proteins and uses thereof

Abstract

Fusion proteins are provided that include a first and a second peptide. The first peptide enables the fusion protein to bind to a receptor expressed on a cell, and the second peptide has a cleavage site that enables the fusion protein to kill the cell. Thus, the fusion protein is useful for preventing or treating infections caused by pathogens. Nucleic acids encoding the fusion proteins and methods for producing and using the fusion proteins are also provided.

Description

Technical Field

[0001] The present invention relates to a fusion protein comprising a first and a second peptide. The first peptide enables the fusion protein to bind to a receptor expressed on a cell and then to internalize, and the second peptide has a cleavage site with optimized selective properties, thus potentially referred to as an optimized or selective cleavage site, whereby the fusion protein can kill said cell. Thus, the fusion protein is useful for preventing or treating infections caused by pathogens such as viruses, and CMV is an example of such a virus. Nucleic acids encoding the fusion protein as well as methods for manufacturing and using the fusion protein are also provided.

Background Art

[0002] Immunotoxins are proteins having a targeting moiety conjugated to a toxin. Immunotoxins are an example of fusion proteins. Immunotoxins can be used for the treatment of infections caused by a range of pathogens. For example, toxins conjugated to antibodies have been previously used to target CMV-infected cells, with little success.

[0003] Human cytomegalovirus (HCMV) is a species-specific herpesvirus and is an important pathogen particularly in immunocompromised individuals, neonates, and transplant recipients.

[0004] Currently, all drugs used for the clinical treatment of HCMV infection are associated with significant adverse side effects. Moreover, the emergence of drug resistance often results in treatment failure.

[0005] In addition to the pharmacological problems and insufficient cell-targeting selectivity of existing immunotoxins, another cause of treatment failure is often insufficient internalization leading to insufficient killing of infected cells.

[0006] These limitations support the value of developing novel drug therapies for pathogen-induced infections such as HCMV.

[0007] Issue WO08 / 003327 discloses immunotoxins useful in the treatment of CMV infection and related diseases. The immunotoxins disclosed therein include variants and toxins of the chemokine domain of human CX3CL1. [Overview of the project]

[0008] The present invention is as defined in the claims.

[0009] This specification provides a fusion protein comprising a first peptide that provides target selectivity, and a second peptide that provides a toxin and further provides internal cleavage of the toxin by a cleavage site and consequently effector toxicity in target cells. The fusion protein provided herein is useful for treating pathogen-induced infections, for example, for treating latent infections.

[0010] The inventors have created a fusion protein that effectively targets and kills infected cells, such as CMV-infected cells, including latent infected cells. This fusion protein comprises a first peptide capable of binding to at least one receptor (which may be a virus-encoded receptor, e.g., US28), and a second peptide comprising an optimized cleavage site and a toxin, e.g., selected domains of exotoxin A, e.g., domains II and III. Upon target binding, the receptor is preferably internalized, and the second peptide is cleaved at least partially at the cleavage site, thus releasing the toxin portion, which then kills the infected cells. Surprisingly, the inventors have found that a certain cleavage site motif (ArgX1X2Arg, where X2 is basic, e.g., Arg) corresponding to a mutation in the native furin cleavage site of exotoxin A increases selectivity for cells expressing the pathogen-encoded receptor, e.g., US28. This finding enables improved targeting and killing of infected cells, e.g., CMV-infected cells. Therefore, the optimized cleavage sites described herein can be used for fusion proteins, particularly immunotoxins, to increase toxicity / cell-killing efficacy, and especially selectivity.

[0011] In one embodiment, the present invention provides a fusion protein comprising: a) A first peptide that binds to at least one receptor expressed on a cell; and b) A second peptide containing a cleavage site having the amino acid sequence ArgX1X2Arg, X2 is either Arg or Lys, and the second peptide contains a toxin.

[0012] In another embodiment, the present invention provides an isolated nucleic acid molecule encoding a fusion protein according to the above embodiment.

[0013] In one embodiment, the present invention provides a vector comprising a nucleic acid molecule according to the above embodiment.

[0014] In another embodiment, the present invention provides recombinant host cells comprising nucleic acid molecules or vectors according to the above embodiment.

[0015] In one embodiment, the present invention provides a pharmaceutical composition comprising a fusion protein, nucleic acid, vector or recombinant host cell according to the above embodiment, and a pharmaceutically acceptable carrier, diluent and / or excipient.

[0016] In another embodiment, the present invention provides fusion proteins, nucleic acids, vectors, recombinant host cells, or pharmaceutical compositions according to the above embodiment for use as pharmaceuticals.

[0017] In one embodiment, the present invention provides a fusion protein, nucleic acid, vector, recombinant host cell, or pharmaceutical composition according to the above embodiment for use in the prevention or treatment of infection and / or pathogen-related disorders caused by a pathogen.

[0018] In another embodiment, the present invention provides fusion proteins, nucleic acids, vectors, recombinant host cells, or pharmaceutical compositions according to the above embodiment for use in the prevention or treatment of CMV infection and / or CMV-related disorders.

[0019] In one embodiment, the present invention provides a method for treating or preventing an infection caused by a pathogen, such as CMV infection, in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of a fusion protein, nucleic acid, vector, recombinant host cell, or pharmaceutical composition according to any one of the above embodiments.

[0020] In another embodiment, the present invention provides ex vivo use of a fusion protein or pharmaceutical composition according to any one of the above embodiments for the treatment of solid organs and / or hematopoietic stem cells for transplantation.

[0021] In one embodiment, the present invention provides a fusion protein, nucleic acid, vector, host cell, or pharmaceutical composition according to the above embodiment for use in the manufacture of a pharmaceutical for the prevention or treatment of infection and / or pathogen-related disorders caused by a pathogen, for example, for the prevention or treatment of CMV infection and / or CMV-related disorders.

[0022] In another embodiment, the present invention provides a method for generating a fusion protein according to any one of the above embodiments, the method comprising culturing a host cell as defined in any of the above embodiments under conditions that enable the expression of the coding fusion protein. [Brief explanation of the drawing]

[0023] [Figure 1] The domain structure of the first exemplary peptide, which is human CX3CL1, Pseudomonas exotoxin A, and fusion protein SYN002, is shown below. A. Schematic diagram of the domain structure below: Human CX3CL1 (S=signal sequence, CX3CL1=chemokine domain, Stalk=mucin-like stalk, M=transmembrane and C=cytoplasmic domain), Pseudomonas erginosa exotoxin A (S=signal sequence, domain I=receptor-binding domain, domain II=translocation domain, Ib=domain Ib with unknown function and domain III=ADP-ribosylated domain), amino acid numbering for the precursor protein is given on each protein. Disulfide crosslinks are present on each tan The protein is shown below in square brackets, along with the numbering of the amino acids involved. Schematic diagram of B.SYN002. Mutations in a given domain are indicated by a single letter notation of the amino acid involved, along with its number. For example, P303R means that proline at position number 303 is substituted with arginine. Single letter notation is also used at the N and C-terminuses of constructs and between domains. A dashed line between two domains indicates that amino acids are linked. Furin cleaves between amino acids 304 and 305 in domain II of exotoxin A. [Figure 2]Selectivity is exhibited due to the F49A mutation. A single amino acid substitution in the chemokine portion of SYNx results in selectivity to the US28 receptor (A) compared to the CXCR1 receptor (B). SYN000 is the wild-type human chemokine (C-X3-C motif) ligand 1 (CX3CL1) (natural chemokine sequence), while SYN001 has a single mutation (F49A) in the receptor-binding portion of the protein. [Figure 3] This study demonstrates that introducing a furin cleavage site (in this case, the full-length translocation domain of exotoxin A) increases potency. Adding the full-length translocation domain of exotoxin A, including the furin cleavage site, to the SYN001 construct yields SYN016, which exhibits increased potency on both endogenous CX3CR1- and virus-encoded US28- receptor-expressing cells. [Figure 4] This shows in vitro cleavage by furin. In vitro cleavage by furin of the SYN002 construct with an optimized cleavage site (RQRR) does not result in improvement compared to the fusion protein construct SYN016 with a native furin cleavage site sequence (RQPR). In vitro, furin digests SYNx. Lane 1: Mark12 protein standard, Lane 2: SYN000, Lane 3: SYN017, Lane 4: SYN014, Lane 5: SYN001, Lane 6: SYN016, and Lane 7: SYN002. SYN000, SYN017, and SYN014 have the native CX3CL1 chemokine sequence, while SYN001, SYN016, and SYN002 have the F49A mutation in the chemokine portion. SYN000, SYN017, SYN001, and SYN016 have a natural furin cleavage region (RQPR), while SYN014 and SYN002 have an optimized cleavage region (RQRR). [Figure 5]SYN002 is shown to be cleaved by furin over a broad pH range. SYN002 was cleaved in vitro over a broad pH range. Lane 1; Mark12 protein standard, Lane 2; without furin addition, Lane 3; +8 mM HCl (approx. pH 5.2), Lane 4; +6 mM HCl (approx. pH 6.3), Lane 5; +4 mM HCl (approx. pH 6.6), Lane 6; +2 mM HCl (approx. pH 7.0), Lane 7; without titrant addition (approx. pH 7.4), Lane 8; 2 mM NaOH (approx. pH 7.7) and Lane 9; +4 mM NaOH (approx. pH 9.0). [Figure 6] The optimized cleavage site shows an increase in selectivity. When the optimized cleavage site ArgX1X2Arg, in this particular case the optimized cleavage site ArgGlnArgArg, is introduced into the second peptide, SYN016, to obtain SYN002, an increase in selectivity for the receptor encoded by the virus is obtained. In SYN002, for cells expressing the receptor encoded by the virus, such as US28, the cell killing efficacy is maintained approximately compared to SYN016 which has the cleavage site ArgGlnProArg (thus, X2 = Pro), however, the efficacy against cells expressing the endogenous / human receptor, such as CX3CR1, is decreased compared to SYN016. Both selectivity and efficacy increase compared to a second peptide that does not contain a furin cleavage site, such as SYN000 (not shown in the figure). **Modes for Carrying Out the Invention**

[0024] The present disclosure relates to a fusion protein comprising a first peptide that provides target recognition and target selectivity and a toxin, and further comprising a second peptide that provides intracellular cleavage and release of the toxin and resultant effector toxicity in target cells. The fusion proteins provided herein are useful for the treatment of infections induced by pathogens, including the treatment of latent infections.

[0025] The inventors have created a fusion protein that, when bound to a receptor, such as a virus-encoded receptor, such as US28 (which can be constitutively internalized for convenience), effectively targets and kills infected cells, such as CMV-infected cells, including latent infected cells.

[0026] US28 is a G protein-coupled receptor encoded by the human cytomegalovirus open reading frame US28. US28 is a constitutively internalizing receptor; therefore, chemokines or other compounds that bind to US28 are internalized within the cells expressing the receptor. US28 is expressed on CMV-infected cells, including cells latently infected with CMV.

[0027] The fusion protein comprises a first peptide capable of binding to receptors, particularly those encoded by pathogens, such as US28, and a second peptide containing an optimized cleavage site and a selected domain of a toxin, such as exotoxin A. Surprisingly, the selectivity of the fusion protein to pathogen-encoded receptors, such as US28, is enhanced by mutations at the cleavage site. The optimized cleavage sites described herein can be used for various fusion proteins, particularly immunotoxins, to increase cell death selectivity by altering the cell death efficacy in a target receptor-selective manner.

[0028] In one embodiment, the present invention provides a fusion protein comprising: a) A first peptide that binds to at least one receptor expressed on a cell; and b) A second peptide containing a cleavage site having the amino acid sequence ArgX1X2Arg, X2 is either Arg or Lys, and the second peptide contains a toxin.

[0029] The term “amino acid” as used herein includes the 20 standard genetically encoded amino acids and their corresponding “D” type stereoisomers (compared to the natural “L” type), ω-amino acids and other naturally occurring amino acids, special amino acids (e.g., α,α-disubstituted amino acids, N-alkyl amino acids, etc.), and chemically derivatized amino acids. Where an amino acid is specifically listed, for example, “alanine” or “Ala” or “A”, the term refers to both l-alanine and d-alanine unless otherwise explicitly stated.

[0030] In one embodiment of the invention, the fusion protein is composed solely of naturally occurring amino acids.

[0031] Cutting site The second peptide of this disclosure has an amino acid sequence ArgX1X2Arg Includes a cutting portion having 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.

[0032] X2 is selected from Arg and Lys. In a favorable embodiment, X2 is Arg. In one embodiment, X2 is Lys. By introducing a basic amino acid, such as Arg, to amino acid position X2, cell death selectivity is enhanced. Thus, for example, virus-infected cells expressing a virus-encoded receptor and binding to the first peptide are killed more efficiently than, for example, cells expressing an endogenous receptor (which can also bind to the first peptide).

[0033] In a preferred embodiment, the cleavage site includes or is composed of the amino acid sequence ArgGlnArgArg(RQRR). In one embodiment, the cleavage site includes or is composed of the amino acid sequence ArgGlnLysArg. In one embodiment, the cleavage site includes or is composed of the amino acid sequence ArgSerLysArg. In one embodiment, the cleavage site includes or is composed of the amino acid sequence ArgSerArgArg. In one embodiment, the cleavage site includes or is composed of the amino acid sequence ArgThrLysArg. In one embodiment, the cleavage site includes or is composed of the amino acid sequence ArgThrArgArg. In one embodiment, the cleavage site includes or is composed of the amino acid sequence ArgAsnLysArg. In one embodiment, the cleavage site includes or is composed of the amino acid sequence ArgAsnArgArg.

[0034] In one embodiment, the cleavage occurs at the C-terminus of the cleavage site.

[0035] While it may be possible to cleave fusion proteins with furin, other enzymes or proteases may also be involved in cleavage at the cleavage site.

[0036] Therefore, in one embodiment, the cleavage site is the enzyme cleavage site.

[0037] In one embodiment, the enzyme cleavage site is a furin cleavage site.

[0038] 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.

[0039] In one embodiment, one receptor that binds to the first peptide is a receptor encoded by a pathogen, and further receptors that bind to the first peptide are human-coded receptors and / or endogenous receptors for the first peptide or a variant thereof.

[0040] In one embodiment, the receptor is a G protein-coupled receptor (GPCR), for example, US28 with SEQ ID NO:10.

[0041] 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. In one embodiment, the CC chemokine receptor is US28. In one embodiment, the CX3C chemokine receptor is CX3CR1.

[0042] 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 herpesvirus. In one embodiment, the virus is a cytomegalovirus.

[0043] 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. The receptor encoded by the pathogen is conveniently and constitutively internalized, thereby ensuring efficient uptake of the fusion protein by pathogen-infected cells, and thus ensuring the death of infected cells with minimal undesirable toxicity and side effects. "Internalization" refers to the process by which the receptor is moved into the cell in which it is expressed. For example, the receptor can enter the cell by endocytosis or phagocytosis.

[0044] In some embodiments, the present invention provides a fusion protein that targets specific cells by selectively binding to a receptor and leading to the internalization of the receptor. The toxic portion of the fusion protein is then cleaved, causing the cells to die.

[0045] toxin In one embodiment, the fusion protein is an immunotoxin. “Immunotoxin” refers to a protein consisting of a targeting moiety bound to a toxin. The targeting moiety is also called a ligand.

[0046] The toxins may be of different types. In one embodiment, the second peptide contains a toxin selected from the group consisting of Pseudomonas exotoxin A, geronin, bouganin, saporin, lysine, lysine A chain, bryodin, restrictosin, diphtheria toxin, diphtheria toxin A chain, and its variants and fragments. The toxic portion of the fusion protein enables cell death. The toxins are cytotoxic.

[0047] In one embodiment, the second peptide comprises one or more domains of Pseudomonas exotoxin A. In one embodiment, the second peptide comprises at least a portion of exotoxin A of SEQ ID NO:9.

[0048] Fusion protein The first peptide is the targeting portion, which enables it to bind to receptors expressed on the cell.

[0049] In one embodiment, the first and second peptides are operably linked. Generally, "operably linked" means that the linked sequences are in close proximity to each other and / or are arranged to have a functional relationship, and are linked, for example, by a covalent bond.

[0050] In one embodiment, the first peptide comprises or consists of: a. Amino acid sequence of SEQ ID NO:1; b. A variant of SEQ ID NO:1 comprising, or composed of, an amino acid sequence having at least 80% sequence identity with respect to SEQ ID NO:1, more preferably at least 85% or 90% sequence identity with respect to SEQ ID NO:1, and most preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO:1; c. Fragments of SEQ ID NO:1 having an amino acid length greater than 50, for example, 60, 70, or 75 amino acid lengths, or variants 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.

[0051] In one embodiment, the first peptide includes, or is composed of, an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:1, or a variant of SEQ ID NO:1 composed of or composed of such a sequence. In one embodiment, the first peptide includes, or is composed of, an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:1, or a variant of SEQ ID NO:1 composed of such a sequence. In one embodiment, the first peptide includes, or is composed of, an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO:1, or a variant of SEQ ID NO:1 composed of such a sequence. In one embodiment, the first peptide includes, or is composed of, an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO:1, or a variant of SEQ ID NO:1 composed of such a sequence. In one embodiment, the first peptide includes, or is composed of, a variant of SEQ ID NO:1 having at least 98% sequence identity with respect to the amino acid sequence of SEQ ID NO:1. In one embodiment, the first peptide includes, or is composed of, a variant of SEQ ID NO:1 having at least 99% sequence identity with respect to the amino acid sequence of SEQ ID NO:1.

[0052] In one embodiment, the first peptide comprises or is composed of a SEQ ID NO:1 fragment having a length of more than 50 amino acids. In one embodiment, the first peptide comprises or is composed of a SEQ ID NO:1 fragment having a length of more than 60 amino acids. In one embodiment, the first peptide comprises or is composed of a SEQ ID NO:1 fragment having a length of more than 70 amino acids. In one embodiment, the first peptide comprises or is composed of a SEQ ID NO:1 fragment having a length of more than 75 amino acids.

[0053] In one embodiment, the first peptide comprises or is composed of a fragment of SEQ ID NO:1, or a variant thereof having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO:1. In one embodiment, the first peptide comprises or is composed of a fragment of SEQ ID NO:1, or a variant thereof having at least 85% sequence identity with respect to the amino acid sequence of SEQ ID NO:1. In one embodiment, the first peptide comprises or is composed of a fragment of SEQ ID NO:1, or a variant thereof having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO:1. In one embodiment, the first peptide comprises or is composed of a fragment of SEQ ID NO:1, or a variant thereof having at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO:1. In one embodiment, the first peptide comprises or is composed of a fragment of SEQ ID NO:1, or a variant thereof having at least 96% sequence identity with respect to the amino acid sequence of SEQ ID NO:1. In one embodiment, the first peptide comprises or is composed of a fragment of SEQ ID NO:1, or a variant thereof having at least 97% sequence identity with respect to the amino acid sequence of SEQ ID NO:1. In one embodiment, the first peptide comprises or is composed of a fragment of SEQ ID NO:1, or a variant thereof having at least 98% sequence identity with respect to the amino acid sequence of SEQ ID NO:1. In one embodiment, the first peptide comprises or is composed of a fragment of SEQ ID NO:1, or a variant thereof having at least 99% sequence identity with respect to the amino acid sequence of SEQ ID NO:1.

[0054] In one embodiment, the amino acid residue of SEQ ID NO:1 at position 49 is mutated to Ala, Lys, or Asp, preferably Ala. These substitutions can lead to increased cell death selectivity towards cells expressing pathogen-encoded receptors that bind to the first peptide, compared to human or endogenous coding receptors that bind to the first peptide.

[0055] In one embodiment, the first peptide further comprises methionine (M) at its N-terminus.

[0056] In one embodiment, the first peptide comprises or is composed of one or more additional amino acids inserted at the N and / or C-terminus of SEQ ID NO:1 and / or within the amino acid sequence.

[0057] In one embodiment, one or more amino acids are deleted at the N and / or C terminus of SEQ ID NO:1 and / or within the amino acid sequence.

[0058] In one embodiment, the first peptide has a length of less than 100 amino acids.

[0059] In one embodiment, the first peptide has a length of less than 90 amino acids.

[0060] In one embodiment, the first peptide has a length of less than 85 amino acids.

[0061] In one embodiment, the first peptide has a length of less than 80 amino acids.

[0062] In one embodiment, the second peptide includes domain A (which may be a translocation domain), for example, domain A having an amino acid sequence of SEQ ID NO:3 or a fragment or variant thereof, and / or domain B, for example, domain B having an amino acid sequence of SEQ ID NO:4 or a fragment or variant thereof, and / or domain C (which may be a cytotoxic domain), for example, domain C having an amino acid sequence of SEQ ID NO:5 or a fragment or variant thereof.

[0063] In one embodiment, the second peptide includes domain A, for example, domain A having an amino acid sequence of SEQ ID NO:3 or a fragment or variant thereof. In one embodiment, the second peptide includes domain A, for example, domain A having an amino acid sequence of SEQ ID NO:3 or a fragment or variant thereof, and domain B, for example, domain B having an amino acid sequence of SEQ ID NO:4 or a fragment or variant thereof. In one embodiment, the second peptide includes domain A, for example, domain A having an amino acid sequence of SEQ ID NO:3 or a fragment or variant thereof, and domain B, for example, domain B having an amino acid sequence of SEQ ID NO:4 or a fragment or variant thereof, and domain C, for example, domain C having an amino acid sequence of SEQ ID NO:5 or a fragment or variant thereof. In one embodiment, the second peptide includes domain B having an amino acid sequence of SEQ ID NO:4 or a fragment or variant thereof. In one embodiment, the second peptide includes domain C, for example, domain C having an amino acid sequence of SEQ ID NO:5 or a fragment or variant thereof.

[0064] In one embodiment, the second peptide comprises: a. Amino acid sequence of SEQ ID NO:3; b. A variant of SEQ ID NO:3 comprising, or composed of, an amino acid sequence having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO:3, more preferably at least 85% or 90% sequence identity with respect to SEQ ID NO:3, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity with respect to SEQ ID NO:3; c. Fragments of SEQ ID NO:3 having an amino acid length greater than 80, for example, 90, 100, or 110 amino acid lengths, 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.

[0065] In one embodiment, the second peptide comprises: a. Amino acid sequence of SEQ ID NO:4; b. A variant of SEQ ID NO:4 comprising or composed of an amino acid sequence having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO:4, more preferably at least 85% or 90% sequence identity with respect to SEQ ID NO:4, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity with respect to SEQ ID NO:4; c. Fragments of SEQ ID NO:4 having an amino acid length greater than 6, for example, 8, 10, or 12 amino acid lengths, 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.

[0066] In one embodiment, the second peptide comprises: a. Amino acid sequence of SEQ ID NO: 5; b. A variant of SEQ ID NO:5 comprising or composed of an amino acid sequence having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO:5, more preferably at least 85% or 90% sequence identity with respect to SEQ ID NO:5, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity with respect to SEQ ID NO:5; c. Fragments of SEQ ID NO:5 having a length of more than 180 amino acids, for example, more than 190, 200, or 210 amino acids, or variants 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.

[0067] In one embodiment, the second peptide comprises: a. Amino acid sequence of SEQ ID NO:3; b. A variant of SEQ ID NO:3 comprising, or composed of, an amino acid sequence having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO:3, more preferably at least 85% or 90% sequence identity with respect to SEQ ID NO:3, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity with respect to SEQ ID NO:3; c. Fragments of SEQ ID NO:3 having an amino acid length greater than 80, for example, 90, 100, or 110 amino acid lengths, 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; and d. Amino acid sequence of SEQ ID NO:4; e. A variant of SEQ ID NO:4 comprising or composed of an amino acid sequence having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO:4, more preferably at least 85% or 90% sequence identity with respect to SEQ ID NO:4, and most preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO:4; A fragment of SEQ ID NO:4 having an amino acid length greater than f.6, for example, a length of 8, 10, or 12 amino acids, 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.

[0068] In one embodiment, the second peptide comprises: a. Amino acid sequence of SEQ ID NO:3; b. A variant of SEQ ID NO:3 comprising, or composed of, an amino acid sequence having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO:3, more preferably at least 85% or 90% sequence identity with respect to SEQ ID NO:3, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity with respect to SEQ ID NO:3; c. Fragments of SEQ ID NO:3 having an amino acid length greater than 80, for example, 90, 100, or 110 amino acid lengths, 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; and d. Amino acid sequence of SEQ ID NO:4; e. A variant of SEQ ID NO:4 comprising or composed of an amino acid sequence having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO:4, more preferably at least 85% or 90% sequence identity with respect to SEQ ID NO:4, and most preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO:4; Fragments of SEQ ID NO:4 having an amino acid length greater than f.6, for example, 8, 10, or 12 amino acid lengths, 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; and Amino acid sequence of g.SEQ ID NO:5; h. A variant of SEQ ID NO:5 comprising, or composed of, an amino acid sequence having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO:5, more preferably at least 85% or 90% sequence identity with respect to SEQ ID NO:5, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity with respect to SEQ ID NO:5; i. Fragments of SEQ ID NO:5 having a length of more than 180 amino acids, for example, more than 190, 200, or 210 amino acids, or variants 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.

[0069] In one embodiment, the second peptide comprises: a. Amino acid sequence of SEQ ID NO:3; b. A variant of SEQ ID NO:3 comprising, or composed of, an amino acid sequence having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO:3, more preferably at least 85% or 90% sequence identity with respect to SEQ ID NO:3, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity with respect to SEQ ID NO:3; c. Fragments of SEQ ID NO:3 having an amino acid length greater than 80, for example, 90, 100, or 110 amino acid lengths, 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; and d. Amino acid sequence of SEQ ID NO: 5; e. A variant of SEQ ID NO:5 comprising or composed of an amino acid sequence having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO:5, more preferably at least 85% or 90% sequence identity with respect to SEQ ID NO:5, and most preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO:5; f. Fragments of SEQ ID NO:5 having an amino acid length greater than 180, for example, 190, 200, or 210 amino acid lengths, or variants 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.

[0070] In one embodiment, the second peptide comprises: a. Amino acid sequence of SEQ ID NO:4; b. A variant of SEQ ID NO:4 comprising or composed of an amino acid sequence having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO:4, more preferably at least 85% or 90% sequence identity with respect to SEQ ID NO:4, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity with respect to SEQ ID NO:4; c. Fragments of SEQ ID NO:4 having an amino acid length greater than 6, for example, 8, 10, or 12 amino acid lengths, 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, most preferably at least 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:4; and d. Amino acid sequence of SEQ ID NO: 5; e. A variant of SEQ ID NO:5 comprising or composed of an amino acid sequence having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO:5, more preferably at least 85% or 90% sequence identity with respect to SEQ ID NO:5, and most preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO:5; f. Fragments of SEQ ID NO:5 having an amino acid length greater than 180, for example, 190, 200, or 210 amino acid lengths, or variants 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.

[0071] In one embodiment, domain A is a translocation domain, and domain C is a cytotoxic domain, such as an ADP-ribosylation domain.

[0072] In one embodiment, the second peptide contains the amino acid sequence KDEL of SEQ ID NO:8 at its C-terminus. In one embodiment, the last five amino acids of the second peptide are replaced with the amino acid sequence of SEQ ID NO:8.

[0073] In one embodiment, the second peptide comprises or consists of: a. Amino acid sequence of SEQ ID NO:2; b. A variant of SEQ ID NO:2 comprising or composed of an amino acid sequence having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO:2, more preferably at least 85% or 90% sequence identity with respect to SEQ ID NO:2, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity with respect to SEQ ID NO:2; c. Fragments of SEQ ID NO:2 having a length of more than 300 amino acids, for example, more than 310, 330, or 340 amino acids, or variants 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.

[0074] In one embodiment, the second peptide includes, or is composed of, an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:2, or a variant of SEQ ID NO:2 composed of, or derived from, an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:2, or a variant of SEQ ID NO:2 composed of, or derived from, an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO:2, or a variant of SEQ ID NO:2 composed of, or derived from, an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO:2, or a variant of SEQ ID NO:2 composed of, or derived from, an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO:2. In one embodiment, the second peptide includes, or is composed of, an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO:2, or a variant of SEQ ID NO:2 composed of, or is composed of, an amino acid sequence having at least 99% sequence identity with the amino acid sequence of SEQ ID NO:2, or is composed of, or is composed of, a variant of SEQ ID NO:2 composed of, or is composed of, an amino acid sequence having at least 99% sequence identity with, or is composed of, or is composed of

[0075] In one embodiment, the second peptide comprises or is composed of a SEQ ID NO:2 fragment having a length of more than 300 amino acids. In one embodiment, the second peptide comprises or is composed of a SEQ ID NO:2 fragment having a length of more than 310 amino acids. In one embodiment, the second peptide comprises or is composed of a SEQ ID NO:2 fragment having a length of more than 330 amino acids. In one embodiment, the second peptide comprises or is composed of a SEQ ID NO:2 fragment having a length of more than 340 amino acids.

[0076] In one embodiment, the second peptide comprises or is composed of a fragment of SEQ ID NO:2, or a variant thereof having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO:2. In one embodiment, the second peptide comprises or is composed of a fragment of SEQ ID NO:2, or a variant thereof having at least 85% sequence identity with respect to the amino acid sequence of SEQ ID NO:2. In one embodiment, the second peptide comprises or is composed of a fragment of SEQ ID NO:2, or a variant thereof having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO:2. In one embodiment, the second peptide comprises or is composed of a fragment of SEQ ID NO:2, or a variant thereof having at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO:2. In one embodiment, the second peptide comprises or is composed of a fragment of SEQ ID NO:2, or a variant thereof having at least 96% sequence identity with respect to the amino acid sequence of SEQ ID NO:2. In one embodiment, the second peptide comprises or is composed of a fragment of SEQ ID NO:2, or a variant thereof having at least 97% sequence identity with respect to the amino acid sequence of SEQ ID NO:2. In one embodiment, the second peptide comprises or is composed of a fragment of SEQ ID NO:2, or a variant thereof having at least 98% sequence identity with respect to the amino acid sequence of SEQ ID NO:2. In one embodiment, the second peptide comprises or is composed of a fragment of SEQ ID NO:2, or a variant thereof having at least 99% sequence identity with respect to the amino acid sequence of SEQ ID NO:2.

[0077] In one embodiment, the second peptide has an amino acid length of less than 400, for example, less than 380, 370, 360, 350, or 345 amino acids.

[0078] In one embodiment, the second peptide has a length of less than 400 amino acids.

[0079] In one embodiment, the second peptide has a length of less than 380 amino acids.

[0080] In one embodiment, the second peptide has a length of less than 370 amino acids.

[0081] In one embodiment, the second peptide has a length of less than 360 amino acids.

[0082] In one embodiment, the second peptide has a length of less than 350 amino acids.

[0083] In one embodiment, the second peptide has a length of less than 345 amino acids.

[0084] In one embodiment, the fusion protein comprises or is composed of: a. Amino acid sequence of SEQ ID NO:6; b. A variant of SEQ ID NO:6 comprising or composed of an amino acid sequence having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO:6, more preferably at least 85% or 90% sequence identity with respect to SEQ ID NO:6, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity with respect to SEQ ID NO:6; c. Fragments of SEQ ID NO:6 having a length of more than 360 amino acids, for example, more than 380, 400, or 420 amino acids, or variants 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.

[0085] In one embodiment, the fusion protein includes, or is composed of, a variant of SEQ ID NO:6 that includes, or is composed of, an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:6. In one embodiment, the fusion protein includes, or is composed of, an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:6 that includes, or is composed of, a variant of SEQ ID NO:6 that includes, or is composed of, an amino acid sequence having at least 96% sequence identity with the amino acid sequence of SEQ ID NO:6. In one embodiment, the fusion protein includes, or is composed of, an amino acid sequence having at least 97% sequence identity with the amino acid sequence of SEQ ID NO:6 that includes, or is composed of, a variant of SEQ ID NO:6 that includes, or is composed of, an amino acid sequence having at least 98% sequence identity with the amino acid sequence of SEQ ID NO:6. In one embodiment, the fusion protein comprises or comprises a variant of SEQ ID NO:6 having at least 99% sequence identity with respect to the amino acid sequence of SEQ ID NO:6.

[0086] In one embodiment, the fusion protein comprises or is composed of a SEQ ID NO:6 fragment having a length of more than 360 amino acids. In one embodiment, the fusion protein comprises or is composed of a SEQ ID NO:6 fragment having a length of more than 380 amino acids. In one embodiment, the fusion protein comprises or is composed of a SEQ ID NO:6 fragment having a length of more than 400 amino acids. In one embodiment, the fusion protein comprises or is composed of a SEQ ID NO:6 fragment having a length of more than 420 amino acids.

[0087] In one embodiment, the fusion protein comprises or is composed of a fragment of SEQ ID NO:6, or a variant thereof having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO:6. In one embodiment, the fusion protein comprises or is composed of a fragment of SEQ ID NO:6, or a variant thereof having at least 85% sequence identity with respect to the amino acid sequence of SEQ ID NO:6. In one embodiment, the fusion protein comprises or is composed of a fragment of SEQ ID NO:6, or a variant thereof having at least 90% sequence identity with respect to the amino acid sequence of SEQ ID NO:6. In one embodiment, the fusion protein comprises or is composed of a fragment of SEQ ID NO:6, or a variant thereof having at least 95% sequence identity with respect to the amino acid sequence of SEQ ID NO:6. In one embodiment, the fusion protein comprises or is composed of a fragment of SEQ ID NO:6, or a variant thereof having at least 96% sequence identity with respect to the amino acid sequence of SEQ ID NO:6. In one embodiment, the fusion protein comprises or is composed of a fragment of SEQ ID NO:6, or a variant thereof having at least 97% sequence identity with respect to the amino acid sequence of SEQ ID NO:6. In one embodiment, the fusion protein comprises or is composed of a fragment of SEQ ID NO:6, or a variant thereof having at least 98% sequence identity with respect to the amino acid sequence of SEQ ID NO:6. In one embodiment, the fusion protein comprises or is composed of a fragment of SEQ ID NO:6, or a variant thereof having at least 99% sequence identity with respect to the amino acid sequence of SEQ ID NO:6.

[0088] In one embodiment, the fusion protein has an amino acid length of less than 500, for example, less than 490, 480, 470, 460, 450, 440, 430, or 425 amino acids.

[0089] In one embodiment, the fusion protein has an amino acid length of less than 500. In one embodiment, the fusion protein has an amino acid length of less than 490. In one embodiment, the fusion protein has an amino acid length of less than 480. In one embodiment, the fusion protein has an amino acid length of less than 470. In one embodiment, the fusion protein has an amino acid length of less than 460. In one embodiment, the fusion protein has an amino acid length of less than 450. In one embodiment, the fusion protein has an amino acid length of less than 440. In one embodiment, the fusion protein has an amino acid length of less than 430. In one embodiment, the fusion protein has an amino acid length of less than 420.

[0090] In one embodiment, the fusion protein kills cells infected with a pathogen, such as cells that are latently infected with a pathogen.

[0091] In one embodiment, the pathogen is a virus, such as a DNA virus, such as a herpesvirus, such as a cytomegalovirus, or an RNA virus.

[0092] In one embodiment, the fusion protein induces cell death in cells expressing the receptor.

[0093] In one embodiment, cell death is selected from the group consisting of apoptosis, necrosis, autophagy, and mitosis-related cell death. The terms “apoptosis” and “apoptotic activity” are used in a broad sense and typically refer to an ordered or controlled form of cell death in mammals, involving one or more characteristic cellular changes, including cytoplasmic enrichment, loss of cell membrane microvilli, nuclear splintering, degradation of chromosomal DNA, or loss of mitochondrial function.

[0094] In one embodiment, the fusion protein induces direct or indirect effects on the pathogen, resulting in inhibition of pathogen growth, replication, genomic stability, maturation, packaging, latency, reactivation, dissemination, and / or immunosuppression.

[0095] In one embodiment, the fusion protein selectively kills cells that express a receptor encoded by a pathogen.

[0096] In one embodiment, the fusion protein kills cells that express US28.

[0097] In one embodiment, the fusion protein has an IC50 value of less than 10 nM, e.g., less than 5 nM, e.g., less than 1 nM, e.g., less than 0.5 nM, e.g., less than 0.1 nM, or less than 0.001 nM, for a receptor encoded by the virus, e.g., US28.

[0098] In one embodiment, the fusion protein has increased potency against cells expressing a virus-encoded receptor, such as US28, compared to its potency against cells expressing an endogenous receptor or human-coded receptor, such as CX3CR1, for example, an increased potency of at least 100 times, for example, an increased potency of at least 300 times, for example, an increased potency of at least 400 times, for example, an increased potency of at least 500 times. In one embodiment, the fusion protein has increased potency against cells expressing US28, for example, an increased potency of at least 100 times, compared to its potency against cells expressing CX3CR1. In one embodiment, the fusion protein has increased potency against cells expressing US28, for example, an increased potency of at least 300 times, compared to its potency against cells expressing CX3CR1. In one embodiment, the fusion protein has increased potency against cells expressing US28, for example, an increased potency of at least 400 times, compared to its potency against cells expressing CX3CR1. In one embodiment, the fusion protein has increased potency against cells expressing US28 compared to cells expressing CX3CR1, for example, an increased potency of at least 500 times.

[0099] In one embodiment, the fusion protein has an increased affinity for a virus-encoded receptor, such as US28, compared to its affinity for an endogenous receptor or human-coded receptor, such as CX3CR1, such as an increased affinity of at least 50 times, such as an increased affinity of at least 100 times.

[0100] In one embodiment, the selectivity ratio US28 / CX3CR1 of the fusion protein of the present invention is at least 750, for example, at least 800, for example, at least 850, for example, at least 900. In one embodiment, the selectivity ratio US28 / CX3CR1 of the fusion protein of the present invention is at least 880. In one embodiment, the selectivity ratio US28 / CX3CR1 of the fusion protein of the present invention is at least 750. In one embodiment, the selectivity ratio US28 / CX3CR1 of the fusion protein of the present invention is at least 800. In one embodiment, the selectivity ratio US28 / CX3CR1 of the fusion protein of the present invention is at least 850. In one embodiment, the selectivity ratio US28 / CX3CR1 of the fusion protein of the present invention is at least 900.

[0101] nucleic acid In one embodiment, the present invention provides an isolated nucleic acid molecule encoding a fusion protein as described herein.

[0102] The term "nucleic acid molecule" includes DNA (e.g., genomic DNA or complementary DNA) and mRNA molecules, which can be single-stranded or double-stranded. "Isolated" means that the nucleic acid molecule is not located within a cell and is not supplied within a cell.

[0103] vector In one embodiment, the present invention provides a vector comprising nucleic acid molecules as described herein.

[0104] In one embodiment, the vector is an expression vector.

[0105] host cell In one embodiment, the present invention provides recombinant host cells comprising nucleic acid molecules or vectors described herein.

[0106] In one embodiment, the host cell is a bacterial cell, a yeast cell, a mammalian cell, such as a human cell or an insect cell.

[0107] Pharmaceutical composition In one embodiment, the present invention provides a pharmaceutical composition comprising a fusion protein, nucleic acid, vector or recombinant host cell as described herein, and a pharmaceutically acceptable carrier, diluent and / or excipient.

[0108] In one embodiment, the pharmaceutical composition further comprises one or more agents. In one embodiment, the agents are selected from the group consisting of immunosuppressants, antiviral agents and immunotherapeutic agents.

[0109] In one embodiment, the antiviral agent is selected from the group consisting of valganciclovir, ganciclovir, cidofovir, leflunomide, prevaimis, maribavir, and brincidofovir.

[0110] In one embodiment, immunotherapy is cell therapy. In one embodiment, immunotherapy is T-cell therapy.

[0111] disease In one embodiment, the present invention provides a fusion protein, nucleic acid, vector, recombinant host cell, or pharmaceutical composition described herein for use as a pharmaceutical.

[0112] In another aspect, the present invention provides fusion proteins, nucleic acids, vectors, recombinant host cells, or pharmaceutical compositions described herein for use in the prevention or treatment of infections and / or pathogen-related disorders caused by pathogens.

[0113] In one embodiment, the pathogen is a bacterium or a virus. In one embodiment, the virus is a DNA virus, such as a herpesvirus, such as a cytomegalovirus, or an RNA virus.

[0114] In one embodiment, the present invention provides fusion proteins, nucleic acids, vectors, recombinant host cells, or pharmaceutical compositions described herein for use in the prevention or treatment of CMV infection and / or CMV-related disorders.

[0115] In one embodiment, CMV infection is latent and / or lytic CMV infection.

[0116] In one embodiment, CMV infection may be detected as follows: i) One or more tissues selected from the group consisting of the retina, cornea, heart, liver, kidney, lung, gastrointestinal tissue, thymus, spleen, skin and muscle, and / or ii) A bodily fluid selected from one or more of the following groups: saliva, blood, urine, semen, and breast milk.

[0117] In one embodiment, CMV infection is an infection in immunocompromised patients selected from a group consisting of HIV-positive patients, neonates 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 patients with multiple morbidities.

[0118] In one embodiment, CMV infection is an infection in patients with coronary and / or vascular disease.

[0119] In one embodiment, the fusion protein, nucleic acid, vector, host cell, or composition is administered intravenously, intratumorally, intraperitoneally, intraarachnoidally, and / or intralymphatically.

[0120] In one embodiment, the fusion protein, nucleic acid, vector, host cell, or composition is administered to an individual one or more times.

[0121] In one embodiment, the individual is a human.

[0122] In one embodiment, the human being is an immunocompromised patient. An immunocompromised patient is a patient who is unable to respond normally to infection due to a disorder or weakening of the immune system. For example, an immunocompromised patient may be 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.

[0123] In one embodiment, the human is a child. In another embodiment, the human is an adult.

[0124] In one embodiment, a human requires solid organ transplantation and / or hematopoietic stem cell transplantation. Hematopoietic stem cell transplantation is typically the transplantation of pluripotent hematopoietic stem cells derived from a single bone marrow, peripheral blood, or umbilical cord blood. The transplantation can be autologous (stem cells are isolated from the same patient) or allogeneic (stem cells are isolated from different patients).

[0125] In one embodiment, CMV-related disorders are selected from the group consisting of cytomegalovirus interstitial pneumonia, cytomegalovirus hepatitis, cytomegalovirus pancreatitis, cytomegalovirus mononucleosis, CMV polyradiculomyelopathy, giant cell inclusion body disease, cytomegalovirus colitis, cytomegalovirus esophagitis, cytomegalovirus retinitis, Guillain-Barré syndrome, mucoepidermoid carcinoma and ulcerative colitis, graft-versus-host disease (GVHD), and solid organ graft-versus-host disease (SOT-GVHD).

[0126] In one embodiment, the present invention provides fusion proteins, nucleic acids, vectors, host cells, or pharmaceutical compositions described herein for use in the manufacture of pharmaceuticals for the prevention or treatment of infections and / or pathogen-related disorders caused by pathogens, for example, CMV infection and / or CMV-related disorders.

[0127] In another aspect, the present invention provides ex vivo use of the fusion proteins or pharmaceutical compositions described herein for the treatment of solid organs and / or hematopoietic stem cells for transplantation.

[0128] The terms “treating,” “treatment,” and “therapy” refer herein to curative treatment, preventive therapy, remission, and symptomatic treatment. Preferably, treatment is curative.

[0129] Treatment method In one embodiment, the present invention provides a method for treating or preventing an infection caused by a pathogen, such as CMV infection, in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of a fusion protein, nucleic acid, vector, recombinant host cell, or pharmaceutical composition described herein.

[0130] The “effective dose” or “therapeutic effective dose” of a compound is the amount of the compound sufficient to provide a beneficial effect to a subject to which the compound is administered. The phrase “therapeutic effective dose” as used herein may refer to the amount of fusion protein sufficient or effective to treat an infection (to delay or prevent its onset, to prevent, inhibit, reduce or reverse its progression).

[0131] Method for generating In one aspect, the present invention provides a method for generating a fusion protein as described herein, the method comprising culturing a host cell as defined herein under conditions that enable the expression of the coding fusion protein.

[0132] array SEQ ID NO: 1 (Example Peptide 1) QHHGVTKCNITCSKMTSKIPVALLIHYQQNQASCGKRAIILETRQHRLFCADPKEQWVKDAMQHLDRQAAALTRNG SEQ ID NO:2 (Example second peptide) GGSLAALTAHQACHLPLETFTRHRQRRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASGPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVG YHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPP SEQ ID NO:3 (Domain A of exotoxin A) GGSLAALTAHQACHLPLETFTRHRQRRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAAS SEQ ID NO:4 (Exotoxin A, Domain B) GPADSGDALLERNYP SEQ ID NO: 5 (Exotoxin A, Domain C) TGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRV YVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPP SEQ ID NO:6(SYN002) MQHHGVTKCNITCSKMTSKIPVALLIHYQQNQASCGKRAIILETRQHRLACADPKEQWVKDAMQHLDRQAAALTRNGGGSLAALTAHQACHLPLETFTRHRQRRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASGPADSGDALLERNYPTGAEFL GDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRS SLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPKDEL SEQ ID NO:7 (Human CX3CL1) MAPISLSWLLRLATFCHLTVLLAGQHHGVTKCNITCSKMTSKIPVALLIHYQQNQASCGKRAIILETRQHRLFCADPKEQWVKDAMQHLDRQAAALTRNGGTFEKQIGEVKPRTTPAAGGMDESVVLEPEATGESSSLEPTPSSQEAQRALGTSPELPTGVTGSSGTRLPPTPKAQDGGPVGTELFRVPPVSTAATWQ SSAPHQPGPSLWAEAKTSEAPSTQDPSTQASTASSPAPEENAPSEGQRVWGQGQSPRPENSLEREEMGPVPAHTDAFQDWGPGSMAHVVPVSSEGTPSREPVASGSWTPKAEEPIHATMDPQRLGVLITPVPDAQAATRRQAVGLLAFLGLLFCLGVAMFTYQSLQGCPRKMAGEMAEGLRYIPRSCGSNSYVLVPV SEQ ID NO:8 (a sequence that ensures binding to the KDEL receptor for retrograde transport within cells) KDEL SEQ ID NO:9 (Full-length Exotoxin A) MHLTPHWIPLVASLGLLAGGSFASAAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK SEQ ID NO:10 (US28) MTPTTTTAELTTEFDYDEAATPCVFTDVLNQSKPVTLFLYGVVFLFGSIGNFLVIFTITWRRRIQCSGDVYFINLAAADLLFVCTLPLWMQYLLDHNSLASVPCTLLTACFYVAMFASLCFITEIALDRYYAIVYMRYRPVKQACLFSIFWWIFAVIIAIPHFMVVTKKDNQCMTDYDYLEVSYPIILNVELMLGAFVIPLSVISYCYYRISRIVAVSQSRHKGRIVRVLIAVVLVFIIFWLPYHLTLFVDTLKLLKWISSSCEFERSLKRALILTESLAFCHCCLNPLLYVFVGTKFRQELHCLLAEFRQRLFSRDVSWYHSMSFSRRSSPSRRETSSDTLSDEVCRVSQIIP SEQ ID NO:11(SYN000) MQHHGVTKCNITCSKMTSKIPVALLIHYQQNQASCGKRAIILETRQHRLFCADPKEQWVKDAMQHLDRQAAALTRNRQPRGWEQLEQSGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASGPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPKDEL SEQ ID NO:12(SYN001) MQHHGVTKCNITCSKMTSKIPVALLIHYQQNQASCGKRAIILETRQHRLACADPKEQWVKDAMQHLDRQAAALTRNRQPRGWEQLEQSGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASGPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPKDE SEQ ID NO:13(SYN014) MQHHGVTKCNITCSKMTSKIPVALLIHYQQNQASCGKRAIILETRQHRLFCADPKEQWVKDAMQHLDRQAAALTRNGGGSLAALTAHQACHLPLETFTRHRQRRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASGPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPKDEL SEQ ID NO:14(SYN016) MQHHGVTKCNITCSKMTSKIPVALLIHYQQNQASCGKRAIILETRQHRLACADPKEQWVKDAMQHLDRQAAALTRNGGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASGPADSGDALLERNYPTGAEFL GDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRS SLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPKDEL SEQ ID NO:15(SYN017) MQHHGVTKCNITCSKMTSKIPVALLIHYQQNQASCGKRAIILETRQHRLFCADPKEQWVKDAMQHLDRQAAALTRNGGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASGPADSGDALLERNYPTGAEFL GDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRS SLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPKDEL

[0133] item 1. A fusion protein comprising the following: a) A first peptide that binds to at least one receptor expressed on a cell; and b) A second peptide containing a cleavage site having the amino acid sequence ArgX1X2Arg, X2 is either Arg or Lys, and the second peptide is a fusion protein containing a toxin.

[0134] 2. The fusion protein is an immunotoxin, as defined in item 1.

[0135] 3. A fusion protein comprising any one of the above items, wherein the cleavage site contains or is composed of the amino acid sequence ArgGlnArgArg.

[0136] 4. A fusion protein comprising any one of the above items, wherein the first peptide binds to at least two different receptors expressed on a cell.

[0137] 5. A fusion protein comprising any one of the above items, wherein one receptor that binds to the first peptide is a receptor encoded by a pathogen, and a further receptor that binds to the first peptide is a human-coded receptor and / or endogenous receptor for the first peptide or a variant thereof.

[0138] 6. The receptor is a G protein-coupled receptor (GPCR), for example, US28 with SEQ ID NO:10, a fusion protein of any one of the above items.

[0139] 7. A fusion protein comprising any one of the above items, wherein the second peptide contains a toxin selected from the group consisting of Pseudomonas exotoxin A, geronin, bouganin, saporin, lysine, lysine A chain, bryodin, restrictosin, diphtheria toxin, diphtheria toxin A chain, and its variants and fragments.

[0140] 8. A fusion protein comprising any one of the above items, in which the receptor is internalized upon binding to the first peptide.

[0141] 9. The receptor is a fusion protein constitutively internalized by any one of the above items.

[0142] 10. The first peptide is a fusion protein comprising or composed of any one of the following items: a. Amino acid sequence of SEQ ID NO:1; b. A variant of SEQ ID NO:1 comprising, or composed of, an amino acid sequence having at least 80% sequence identity with respect to SEQ ID NO:1, more preferably at least 85% or 90% sequence identity with respect to SEQ ID NO:1, and most preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO:1; c. Fragments of SEQ ID NO:1 having an amino acid length greater than 50, for example, 60, 70, or 75 amino acid lengths, or variants 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.

[0143] 11. The second peptide is a fusion protein comprising or composed of any one of the following: a. Amino acid sequence of SEQ ID NO:2; b. A variant of SEQ ID NO:2 comprising or composed of an amino acid sequence having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO:2, more preferably at least 85% or 90% sequence identity with respect to SEQ ID NO:2, and most preferably at least 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO:2; c. Fragments of SEQ ID NO:2 having a length of more than 300 amino acids, for example, more than 310, 330, or 340 amino acids, or variants 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.

[0144] 12. A fusion protein comprising any one of the following items: a. Amino acid sequence of SEQ ID NO:6; b. A variant of SEQ ID NO:6 comprising or composed of an amino acid sequence having at least 80% sequence identity with respect to the amino acid sequence of SEQ ID NO:6, more preferably at least 85% or 90% sequence identity with respect to SEQ ID NO:6, and most preferably at least 95%, 96%, 97%, 98%, or 99% identity with respect to SEQ ID NO:6; c. Fragments of SEQ ID NO:6 having a length of more than 360 amino acids, for example, more than 380, 400, or 420 amino acids, or variants 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.

[0145] 13. A fusion protein that kills cells infected with a pathogen, such as cells latently infected with a pathogen, and comprises any one of the above items.

[0146] 14. A fusion protein having increased efficacy against cells expressing a virus-encoded receptor, such as US28, compared to its efficacy against cells expressing an endogenous receptor or a human-encoded receptor, such as CX3CR1, for example, by at least 300 times, for example, by at least 400 times, for example, by at least 500 times, for example, by at least 700 times, according to any one of the above items.

[0147] 15. A fusion protein according to any one of items 1-14, for use in the prevention or treatment of infection and / or pathogen-related disorders caused by a pathogen.

[0148] 16. A fusion protein according to any one of items 1-14, for use in the prevention or treatment of CMV infection and / or CMV-related disorders.

[0149] Examples Example 1: Mutation in the first peptide of the fusion protein SYNx leads to selectivity in cell death. material and method Inducible US28 or CX3CR1 expressing HEK293 cells For binding and cell death assays, cells were constructed as described in Hjorto GM et al., 2013. Briefly, stable inducible clones of HA-CX3CR1- and HA-US28-expressing cells were generated by cotransfecting Flp-In T-Rex-293 cells with either the Flp-recombinase expression vector, pOG44, or the pcDNA5 / FRT / TO receptor construct. This targeted cloning at the FRT site places the receptor gene under the control of a 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, 45 μg / mL streptomycin, 13.5 μg / mL blastosidine, and 1.32 mg / mL hygromycin B) in a humidified incubator (37°C, 5% CO2).

[0150] Cell death assay 80 μL of 1x PBS containing 20 μg / mL poly-D-lysine (PDL) was added to each well of a white 96-well plate and incubated at room temperature for 30 minutes. Inducible US28 or CX3CR1-expressing HEK293 cells were washed in 10 mL of 1x PBS and released for 2 minutes using 2 mL of 0.05% (w / v) trypsin-EDTA. Cells were resuspended in 10 mL of growth medium (1x DMEM, 9% (v / v) FBS, 180 U / mL penicillin, and 45 μg / mL streptomycin), and the cell density was then determined. The PDL solution was aspirated from the plate, and each well was washed with 100 μL of 1x PBS. After aspirating the wash buffer, the 96-well plates were seeded at 2,000–10,000 cells / well in 100 μL and incubated overnight in a CO2 incubator (37°C, 10% CO2).

[0151] For receptor expression, growth medium containing 10 μL of tetracycline, 1.375 μg / mL US28, and 5.5 μg / mL CX3CR1)) was added to each well in the plate without selection, and the plate was incubated overnight in a CO2 incubator (37°C, 10% CO2).

[0152] Fusion toxin protein aliquots were thawed on ice and used to prepare dilution series in 1 mM acetate and 5 g / L BSA. The 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. The plates were returned to a CO2 incubator (37°C, 10% CO2) overnight.

[0153] Assay growth medium containing a 1:10 dilution of AlamarBlue was prepared. The solution was covered with tin 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, 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 were collected on a FlexStation3 using excitation at 540 nm and emission at 585 nm.

[0154] result The cell death efficiency of SYNx was determined using tetracycline-inducible HEK293 cells expressing either a virus-encoded receptor, e.g., US28, or an endogenous receptor, e.g., CX3CR1, together with non-inducible cells (negative control) that did not express the receptor. Data were normalized to the maximum number of viable cells. A single amino acid substitution in the first peptide of SYNx (here, the chemokine portion) yields selectivity to the virus-encoded receptor US28, as shown in Figure 2. Both constructs bind to US28 with similar selectivity (Figure 2A), however, SYN001, which has a single mutation in the first peptide of the protein (receptor-binding portion) (F49A, corresponding to F73A using precursor numbering), exhibits lower selectivity to the endogenous receptor, here CXC3R1 (Figure 2B).

[0155] conclusion A single amino acid F49A substitution in the first peptide of the fusion protein construct (e.g., the chemokine portion) induces selectivity of the fusion protein (SYNx) to the virus-encoded receptor US28.

[0156] Example 2: Increased potency obtained by adding a furin cleavage site to exotoxin A using the full-length translocation domain. material and method Please refer to Example 1.

[0157] result The cell death efficiency of SYNx was determined using tetracycline-inducible HEK293 cells expressing either the virus-encoded receptor US28 or the endogenous receptor CX3CR1, together with non-inducible cells (negative control) that did not express the receptors. Data were normalized to the maximum number of viable cells. Adding a furin cleavage site, e.g., the full-length translocation domain, to the SYN001 construct to obtain SYN016 increased efficacy against both endogenous / human CX3CR1- and pathogen-encoded US28- receptor-expressing cells, as shown in Figure 3 and Table 1.

[0158] conclusion Domain II of exotoxin A has been identified as a catalytic domain and a translocation domain that contributes to the transfer of domain III from intracellular vesicles into the cytosol (Hwang J et al., 1987; Siegall CB et al., 1989). By adding a furin cleavage site, such as the translocation domain containing domain II of exotoxin A, to SYN001, SYN016 was obtained, resulting in a fusion protein construct with increased potency against both the endogenous receptor CX3CR1 and the virus-encoded receptor US28. [Table 1]

[0159] Example 3: In vitro cleavage of SYNx with human furin material and method In vitro cleavage of SYNx by human furin The purified SYNx construct was thawed on a lab bench. After thawing, the sample was allowed to settle. The sample concentration was measured, and digestion was started in a new tube using 50 μL of 20 μM SYNx in 1x PBS containing 5 mM CaCl2. 5 μg was taken out 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 taken out for SDS-PAGE analysis. The pH of the reaction mixture was adjusted with either 1 M HCl or 1 M NaOH, and then SYNx was added.

[0160] Reduced SDS-PAGE analysis Samples were prepared and analyzed along with protein marker standards on a NuPAGE Bis-Tris 4-12% gel using 1x MES electrophoresis buffer containing 2 mM DTT, according to the manufacturer's protocol. The gels were run at a constant voltage of 125 V for 75 minutes. The gels were stained according to the SimplyBlue® SafeStain Manual (Novex).

[0161] result Purified SYNx constructs were treated in vitro with purified human furin to determine the furin-mediated cleavage efficiency of different SYNx constructs. Other cleavage mechanisms besides furin may occur. The results were analyzed by SDS-PAGE. Addition of the entire translocation domain, for example, the entire ExoA translocation domain in this case, to SYN000 or SYN001 yielded SYN017 and SYN016, respectively, improving in vitro furin-mediated cleavage. Optimization of the furin-mediated cleavage site in SYN017 and SYN016 yielded SYN014 and SYN002, respectively, and as shown in Figure 4, further improvement in in vitro furin-mediated cleavage does not appear to be achieved.

[0162] Purified SYN002 was treated with purified human furin in vitro at different pH values ​​to determine cleavage efficiency, and the results were analyzed by SDS-PAGE. As shown in Figure 5, SYN002 is cleaved by furin over a wide pH range.

[0163] conclusion Adding an optimized cleavage site (in this case, ArgGlnArgArg) to the SYNx construct (here, the SYN002 construct also includes a translocation domain) did not result in any improvement in in vitro cleavage by furin compared to constructs with the native sequence (ArgGlnProArg), such as SYN016. SYN002 could be cleaved in vitro across a wide pH range, suggesting that furin-mediated cleavage can occur not only within endosomes but also in any cellular region.

[0164] Example 4: Cell death selectivity between receptor-expressing cells encoded by endogenous receptors and viruses is also enhanced and mediated by optimized cleavage sites. material and method Please refer to Example 1.

[0165] result The cell death efficiency of SYNx was determined using tetracycline-induced HEK293 cells expressing either US28 or CX3CR1, along with uninducible cells (negative control) that did not express the receptor. Data were normalized to the maximum number of viable cells.

[0166] Introducing the optimized cleavage site ArgX1X2Arg into the second peptide, and in this special case, the optimized cleavage site ArgGlnArgArg into SYN016 to obtain SYN002, yields a surprising increase in selectivity. For SYN002 on cells expressing virus-encoded receptors, e.g., US28, the cell-killing efficacy is roughly maintained compared to SYN016 with the cleavage site ArgGlnProArg (hence X2=Pro) (Figure 6A, Table 1). However, the efficacy on cells expressing endogenous receptors, e.g., CX3CR1, is reduced (approximately 0.9log(EC50); see Figure 6B), thus providing an effect that supports a drug with fewer side effects and greater efficacy. Compared to the second peptide without the optimized cleavage site, both selectivity and efficacy are increased.

[0167] conclusion The surprising effect on cell death selectivity is obtained by optimizing the cleavage site. While not bound by any particular theory, this may be due to differences in internalization and intracellular transport for virus-encoded receptors, such as the US28 receptor, and endogenous receptors, such as the CX3CR1 receptor.

[0168] References 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. a) a first peptide that binds to at least one receptor expressed on a cell; and b) a second peptide containing a cleavage site having the amino acid sequence ArgX1X2Arg; Including, A fusion protein, wherein X2 is Arg or Lys and said second peptide comprises a toxin.

2. 2. The fusion protein of claim 1, wherein X1 is Gln, Ser, Thr or Asn.

3. 10. The fusion protein of claim 9, wherein X1 is Gln.

4. The fusion protein of any preceding claim, wherein the fusion protein is an immunotoxin.

5. 10. A fusion protein according to any preceding claim, wherein the cleavage site comprises or consists of the amino acid sequence ArgGlnArgArg.

6. 10. The fusion protein of claim 1, wherein the first peptide binds to at least two different receptors expressed on a cell.

7. 10. The fusion protein of any of the preceding claims, wherein one receptor that binds to the first peptide is a receptor encoded by a pathogen 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.

8. 10. The fusion protein of any of the preceding claims, wherein the receptor is a G protein-coupled receptor (GPCR), such as US28 of SEQ ID NO:

10.

9. 10. The fusion protein of any preceding claim, wherein the second peptide comprises a toxin selected from the group consisting of Pseudomonas exotoxin A, gelonin, bouganin, saporin, ricin, ricin A chain, bryodin, restrictocin, diphtheria toxin, diphtheria toxin A chain, and variants and fragments thereof.

10. A fusion protein according to any preceding claim, wherein the receptor is a chemokine receptor.

11. The fusion protein of claim 10, wherein the chemokine receptor is a CC chemokine receptor and / or a CX3C chemokine receptor.

12. The fusion protein of claim 11, wherein the CC chemokine receptor is US28.

13. The fusion protein of claim 11, wherein the CX3C chemokine receptor is CX3CR1.

14. A fusion protein according to any preceding claim, wherein the receptor is encoded by a pathogen.

15. The fusion protein of claim 14, wherein the pathogen is a bacterium.

16. The fusion protein of claim 14, wherein the pathogen is a virus.

17. The fusion protein of claim 16, wherein the virus is a DNA virus.

18. The fusion protein of claim 16, wherein the virus is an RNA virus.

19. The fusion protein of claim 16, wherein the virus is a herpes virus.

20. The fusion protein of claim 16, wherein the virus is a cytomegalovirus.

21. A fusion protein according to any preceding claim, wherein the receptor is capable of being internalised.

22. A fusion protein according to any preceding claim, wherein the receptor is internalised upon binding to the first peptide.

23. A fusion protein according to any preceding claim, wherein the receptor is constitutively internalised.

24. A fusion protein according to any preceding claim, wherein the cleavage site is an enzymatic cleavage site.

25. 25. The fusion protein of claim 24, wherein the enzyme cleavage site is a furin cleavage site.

26. 10. The fusion protein of claim 1, wherein the first peptide is a targeting moiety.

27. A fusion protein according to any preceding claim, wherein the first and second peptides are operably linked.

28. The first peptide comprises: 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 having a length of more than 50 amino acids, for example, more than 60, 70, or 75 amino acids, 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.

10. A fusion protein according to any preceding claim, comprising or consisting of:

29. 10. The fusion protein according to any of the preceding claims, wherein the amino acid residue of SEQ ID NO: 1 at position 49 is mutated to Ala, Lys or Asp, preferably Ala.

30. The fusion protein of any preceding claim, wherein the first peptide further comprises a methionine (M) at the N-terminus.

31. 10. The fusion protein of claim 1, wherein the first peptide comprises or consists of one or more additional amino acids inserted at the N- and / or C-terminus of SEQ ID NO: 1 and / or within the amino acid sequence.

32. 10. The fusion protein according to any one of the preceding claims, wherein one or more amino acids are deleted at the N- and / or C-terminus of SEQ ID NO: 1 and / or within the amino acid sequence.

33. 10. The fusion protein of claim 1, wherein the first peptide has a length of less than 100 amino acids, such as a length of less than 90, 85, or 80 amino acids.

34. 10. The fusion protein of any preceding claim, wherein the second peptide comprises Domain A, e.g., Domain A having an amino acid sequence according to SEQ ID NO: 3 or a fragment or variant thereof, and / or Domain B, e.g., Domain B having an amino acid sequence according to SEQ ID NO: 4 or a fragment or variant thereof, and / or Domain C, e.g., Domain C having an amino acid sequence according to SEQ ID NO: 5 or a fragment or variant thereof.

35. 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. A fragment of SEQ ID NO:3 having a length of more than 80 amino acids, for example, a length of more than 90, 100, or 110 amino acids, 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.

10. The fusion protein of any preceding claim, comprising:

36. 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:3 having a length of more than 6 amino acids, for example, a length of more than 8, 10, or 12 amino acids, 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.

10. The fusion protein of any preceding claim, comprising:

37. 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:3 having a length of more than 180 amino acids, for example, more than 190, 200, or 210 amino acids, 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.

10. The fusion protein of any preceding claim, comprising:

38. The fusion protein of claim 34, wherein domain A is a translocation domain and domain C is a cytotoxic domain, such as an ADP-ribosylation domain.

39. 2. The fusion protein of claim 1, wherein the second peptide comprises the amino acid sequence KDEL of SEQ ID NO: 8 at the C-terminus.

40. The second peptide comprises: 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 having a length of more than 300 amino acids, for example, more than 310, 330, or 340 amino acids, 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.

10. A fusion protein according to any preceding claim, comprising or consisting of:

41. 10. The fusion protein of claim 9, wherein the second peptide has a length of less than 400 amino acids, such as less than 380, 370, 360, 350 or 345 amino acids.

42. The fusion protein comprises: 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; c. A fragment of SEQ ID NO:6 having a length of more than 360 amino acids, for example, more than 380, 400, or 420 amino acids, 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.

10. A fusion protein according to any preceding claim, comprising or consisting of:

43. 10. The fusion protein of any one of the preceding claims, wherein the fusion protein has a length of less than 500 amino acids, such as less than 490, 480, 470, 460, 450, 440, 430, 425 amino acids.

44. 10. The fusion protein of any one of the preceding claims, wherein the fusion protein kills cells infected with a pathogen, such as cells latently infected with a pathogen.

45. 45. The fusion protein of claim 44, wherein the pathogen is a virus, such as a DNA virus, such as a herpes virus, such as a cytomegalovirus, or an RNA virus.

46. A fusion protein according to any preceding claim, wherein the fusion protein induces cell death in cells expressing the receptor.

47. 47. The fusion protein of claim 46, wherein the cell death is selected from the group consisting of apoptosis, necrosis, autophagic cell death and mitosis-associated cell death.

48. 10. The fusion protein of any one of the preceding claims, wherein the fusion protein induces a direct or indirect effect on a pathogen, resulting in the prevention of pathogen growth, replication, genome stability, maturation, packaging, latency, reactivation, dissemination and / or immunosuppression.

49. 10. The fusion protein of any one of the preceding claims, wherein the fusion protein kills cells expressing US28.

50. 10. The fusion protein of any one of the preceding claims, wherein 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, 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.

51. 10. A fusion protein according to any one of the preceding claims, wherein the fusion protein has increased potency, such as at least 300-fold increased potency, such as at least 400-fold increased potency, such as at least 500-fold increased potency, such as at least 700-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.

52. A fusion protein described in any one of the preceding claims, wherein the fusion protein has increased affinity, for example at least 50-fold increased affinity, for example at least 100-fold increased affinity, for a virally encoded receptor, such as US28, compared to its affinity for an endogenous receptor or a human encoded receptor, such as CX3CR1.

53. An isolated nucleic acid molecule encoding the fusion protein of any one of claims 1 to 52.

54. A vector comprising the nucleic acid molecule of claim 53.

55. 55. The vector of claim 54, wherein the vector is an expression vector.

56. A recombinant host cell comprising the nucleic acid molecule of claim 53 or the vector of claims 54-55.

57. 57. The recombinant host cell of claim 56, wherein the host cell is a bacterial cell, a yeast cell, a mammalian cell, such as a human cell, or an insect cell.

58. A pharmaceutical composition comprising a fusion protein according to claims 1 to 52, a nucleic acid according to claim 53, a vector according to claims 54 to 55 or a recombinant host cell according to claims 56 to 57 and a pharmaceutically acceptable carrier, diluent and / or excipient.

59. 59. The pharmaceutical composition of claim 58, further comprising one or more additional pharmaceutical agents.

60. 60. The pharmaceutical composition of claim 59, wherein the agent is selected from the group consisting of an immunosuppressant, an antiviral agent, and an immunotherapy.

61. 61. The pharmaceutical composition of claim 60, wherein the antiviral agent is selected from the group consisting of valganciclovir, ganciclovir, cidofovir, leflunomide, prebymis, maribavir and brincidofovir.

62. 61. The pharmaceutical composition of claim 60, wherein the immunotherapy is T cell therapy.

63. A fusion protein according to claims 1 to 52, a nucleic acid according to claim 53, a vector according to claims 54 to 55, a recombinant host cell according to claims 56 to 57 or a pharmaceutical composition according to claims 58 to 62 for use as a medicament.

64. A fusion protein according to claims 1 to 52, a nucleic acid according to claim 53, a vector according to claims 54 to 55, a recombinant host cell according to claims 56 to 57 or a pharmaceutical composition according to claims 58 to 62 for use in the prevention or treatment of infections caused by pathogens and / or pathogen-related disorders.

65. 65. The fusion protein, nucleic acid, vector, host cell or composition for use according to claim 64, wherein the pathogen is a bacterium or a virus.

66. 66. The fusion protein, nucleic acid, vector, host cell or composition for use according to claim 65, wherein the virus is a DNA virus, such as a herpes virus, such as a cytomegalovirus, or an RNA virus.

67. A fusion protein according to claims 1 to 52, a nucleic acid according to claim 53, a vector according to claims 54 to 55, a recombinant host cell according to claims 56 to 57 or a pharmaceutical composition according to claims 58 to 62 for use in the prevention or treatment of CMV infection and / or CMV-associated disorders.

68. 68. The fusion protein, nucleic acid, vector, host cell or composition for use according to claim 67, wherein the CMV infection is a latent and / or lytic CMV infection.

69. The CMV infection is 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) a bodily fluid selected from one or more of the group consisting of saliva, blood, urine, semen, and breast milk 69. A fusion protein, nucleic acid, vector, host cell or composition for use according to claims 67 to 68, which can be detected in

70. 70. The fusion protein, nucleic acid, vector, host cell or composition for use according to any one of claims 67 to 69, wherein the CMV infection is an infection in an immunocompromised patient 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 multimorbid patients.

71. The fusion protein, nucleic acid, vector, host cell or composition for use according to any one of claims 67 to 70, wherein said CMV infection is an infection in a patient suffering from coronary and / or vascular disease.

72. 69. The fusion protein, nucleic acid, vector, host cell or composition for use according to any one of claims 63 to 68, wherein the fusion protein, nucleic acid, vector, host cell or composition is administered intravenously, intratumorally, intraperitoneally, intrathecally and / or intralymphatically.

73. 73. The fusion protein, nucleic acid, vector, host cell or composition for use according to any one of claims 63 to 72, wherein the fusion protein, nucleic acid, vector, host cell or composition is administered to the individual one or more times.

74. 74. The fusion protein, nucleic acid, vector, host cell or composition for use according to claim 73, wherein the individual is a human.

75. 75. The fusion protein, nucleic acid, vector, host cell or composition for use of claim 74, wherein the human is an immunocompromised patient.

76. The fusion protein, nucleic acid, vector, host cell or composition for use according to any one of claims 74 to 75, wherein the human is a child.

77. The fusion protein, nucleic acid, vector, host cell or composition for use according to any one of claims 74 to 75, wherein the human is an adult.

78. 78. The fusion protein, nucleic acid, vector, host cell or composition for use according to any one of claims 74 to 77, wherein the human is in need of a solid organ transplant and / or a hematopoietic stem cell transplant.

79. 79. The fusion protein, nucleic acid, vector, host cell or composition for use according to any one of claims 64 to 78, wherein the CMV-associated disorder is selected from the group consisting of cytomegalovirus interstitial pneumonia, cytomegalovirus hepatitis, cytomegalovirus pancreatitis, cytomegalovirus mononucleosis, CMV polyradiculomyelopathy, 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).

80. A method for treating or preventing an infection caused by a pathogen, such as a CMV infection, in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a fusion protein, nucleic acid, vector, recombinant host cell or pharmaceutical composition described in any one of the preceding claims.

81. 10. Ex vivo use of a fusion protein or a pharmaceutical composition according to any of the preceding claims for the treatment of solid organs for transplantation and / or hematopoietic stem cells for transplantation.

82. 62. A fusion protein according to claims 1 to 52, a nucleic acid according to claim 53, a vector according to claims 54 to 55, a host cell according to claims 56 to 57 or a pharmaceutical composition according to claims 58 to 62 for use in the manufacture of a medicament for the prevention or treatment of infection caused by a pathogen and / or a pathogen-related disorder, such as for the prevention or treatment of CMV infection and / or a CMV-related disorder.

83. 58. A method for producing a fusion protein according to any one of claims 1 to 52, comprising culturing a host cell as defined in any one of claims 56 to 57 under conditions allowing expression of the encoded fusion protein.

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