Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/521—Chemokines
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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  • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
  • A01N1/10—Preservation of living parts
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  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/42—Oxazoles
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  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
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  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
  • A61K31/52—Purines, e.g. adenine
  • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/66—Phosphorus compounds
  • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/14—Blood; Artificial blood
  • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
  • A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/20—Antivirals for DNA viruses
  • A61P31/22—Antivirals for DNA viruses for herpes viruses
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
  • C07K14/7158—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/62—DNA sequences coding for fusion proteins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
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  • C07K2319/00—Fusion polypeptide
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  • C07K2319/50—Fusion polypeptide containing protease site
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  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin

Definitions

• the present invention relates to a fusion protein which comprises a first and a second peptide.
• the first peptide enables the fusion protein to bind to a receptor expressed on a cell, which may then internalize, and the second peptide having a cleavage site, with optimized selective properties, which may thus be called an optimized or selective cleavage site that enables the fusion protein to kill said cell.
• the fusion protein is thus useful for the prevention or treatment of an infection caused by a pathogen, such as a virus, where CMV is an example of such virus.
• Nucleic acids encoding the fusion protein and methods of making and using the fusion protein are also provided.
• Immunotoxins are proteins having a targeting portion linked to a toxin.
• An immunotoxin is an example of a fusion protein.
• Immunotoxins can be used for the treatment of infections caused by a range of pathogens.
• toxins linked to antibodies have previously been utilized for targeting CMV-infected cells, albeit with limited success.
• HCMV Human cytomegalovirus
• W008/003327 discloses immunotoxins useful in treating diseases related to CMV infection.
• the immunotoxins disclosed therein comprise a variant of the chemokine domain of human CX3CL1 and a toxin.
• a fusion protein comprising a first peptide providing target selectivity and a second peptide providing a toxin and further providing for internal cleavage of the toxin due to a cleavage site and resulting effector toxicity in the targeted cells.
• the fusion protein provided herein is useful for the treatment of pathogen-induced infections, including treatment of latent infections.
• the present inventors have generated a fusion protein that effectively targets and kills infected cells, such as CMV infected cells, including latently infected cells.
• This fusion protein comprises a first peptide, which is capable of binding to at least one receptor, which may be a virus-encoded receptor, such as US28, and a second peptide, which comprises an optimized cleavage site and a toxin, such as selected domains of Exotoxin A, e.g. domains II and III.
• the receptor is preferably internalized and the second peptide is at least partly cleaved at the cleavage site, thus releasing the toxin part, which then in turn kills the infected cells.
• a certain cleavage site motif (ArgX1X2Arg, wherein X2 is basic such as Arg) corresponding to mutations in the native furin cleavage site of Exotoxin A increase selectivity towards cells expressing pathogen encoded receptors, e.g. US28.
• This finding allows for improved targeting and killing of infected cells, e.g. CMV infected cells.
• the optimized cleavage site described herein may thus be utilized for fusion proteins, and in particular immunotoxins, to increase toxicity/cell killing potency and especially selectivity.
• the present invention provides a fusion protein comprising: a) a first peptide which binds at least one receptor expressed on a cell; and b) a second peptide comprising a cleavage site having an amino acid sequence ArgX1X2Arg, wherein X2 is Arg or Lys, and wherein the second peptide comprises a toxin.
• the present invention provides an isolated nucleic acid molecule encoding a fusion protein according to the above aspect.
• the present invention provides a vector comprising a nucleic acid molecule according to the above aspect.
• the present invention provides a recombinant host cell comprising a nucleic acid molecule or a vector according to the above aspects.
• the present invention provides a pharmaceutical composition
• a pharmaceutical composition comprising a fusion protein, a nucleic acid, a vector or a recombinant host cell according to the above aspects and a pharmaceutically acceptable carrier, , diluent and/or excipient.
• the present invention provides a fusion protein, a nucleic acid, a vector, a recombinant host cell or a pharmaceutical composition according to the above aspects for use as a medicament.
• the present invention provides a fusion protein, a nucleic acid, a vector, a recombinant host cell or a pharmaceutical composition according to the above aspects for use in the prevention or treatment of an infection caused by a pathogen and/or pathogen associated disorders.
• the present invention provides a fusion protein, a nucleic acid, a vector, a recombinant host cell or a pharmaceutical composition according to the above aspects for use in the prevention or treatment of CMV infections and/or CMV-associated disorders.
• the present invention provides a method of treating or preventing infection caused by a pathogen such as CMV infections in an individual in need thereof, the method comprising administering a therapeutically effective amount of a fusion protein, a nucleic acid, a vector, a recombinant host cell or a pharmaceutical composition according to any one of the above aspects to the individual.
• the present invention provides an ex vivo use of a fusion protein or a pharmaceutical composition according to any one of the above aspects for treatment of solid organs for transplantation and/or hematopoietic stem cells for transplantation.
• the present invention provides a fusion protein, a nucleic acid, a vector, a host cell or a pharmaceutical composition according to the above aspects for use in the manufacture of a medicament for the prevention or treatment of an infection caused by a pathogen and/or pathogen associated disorders, such as for the prevention or treatment of CMV infections and/or CMV-associated disorders.
• the present invention provides a method for producing a fusion protein according to any one of the above aspects, the method comprising culturing a host cell as defined in any of the above aspects under conditions which permit expression of the encoded fusion protein.
• Figure 1 Domain structures of an exemplary first peptide being human CX3CL1, Pseudomonas Exotoxin A and fusion protein SYN002.
• B Schematic diagram of SYN002.
• a mutation in a given domain is written in single letter code of the amino acid involved along with its number.
• P303R means that proline at position number 303 has been substituted with arginine.
• Single letter code is also used at the N- and C-terminus of the constructs and between domains. A dashed line between two domains indicates that the amino acids are connected. Furin cleaves between amino acids 304 and 305 of domain II of Exotoxin A.
• SYN000 is the wild type human chemokine (C-X3-C motif) ligand 1 (CX3CL1) (native chemokine sequence) while SYN001 has a single mutation (F49A) in the receptor binding part of the protein.
• FIG. 3 Introducing a Furin cleavage site (in this case full length translocation domain of Exotoxin A) increases potency.
• Figure 4 In vitro cleavage by Furin.
• in vitro cleavage by Furin of the SYN002-construct which has the optimized cleavage site (RQRR), compared to a fusion protein construct SYN016 with the native furin cleavage site sequence (RQPR) does not yield an improvement.
• In vitro Furin digest of SYNx Lane 1; Mark 12 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 has native CX3CL1 chemokine sequence, while SYN001, SYN016 and SYN002 has F49A mutation in chemokine part.
• SYN000, SYN017, SYN001 and SYN016 has native Furin cleavage site (RQPR) while SYN014 and SYN002 have optimized cleavage site (RQRR).
• FIG. 5 SYN002 is cleaved by Furin over a wide pH range.
• SYN002 was cleaved in vitro over a wide pH range.
• Lane 1 Mark 12 protein standard, lane 2; No Furin added, lane 3; + 8 mM HCI (approx. pH 5.2), lane 4; + 6 mM HCI (approx. pH 6.3), lane 5; + 4 mM HCI (approx. pH 6.6), lane 6; + 2 mM HCI (approx. pH 7.0), lane 7; no titrant added (approx. pH 7.4), lane 8; 2 mM NaOH (approx. pH 7.7) and lane 9; + 4 mM NaOH (approx. pH 9.0).
• the present disclosure relates to a fusion protein comprising a first peptide providing target recognition and target selectivity and a second peptide providing a toxin and further providing for intracellular cleavage and release of the toxin and resulting effector toxicity in the targeted cells.
• the fusion protein provided herein is useful for the treatment of pathogen-induced infections, including treatment of latent infections.
• the present inventors have generated a fusion protein that effectively targets and kills infected cells, such as CMV infected cells including latently infected cells, upon binding to a receptor, e.g. a receptor encoded by a virus, such as a US28, which may be advantageously constitutively internalized.
• a receptor e.g. a receptor encoded by a virus, such as a US28
• US28 is a G protein coupled receptor encoded by human cytomegalovirus open reading frame US28. US28 is a constitutively internalizing receptor. Thus, chemokines or other compounds that binds US28 are internalized into the cell that express the receptor. US28 is expressed on CMV infected cells including cells latently infected with CMV.
• the fusion protein comprises a first peptide, which is capable of binding to a receptor, especially receptors encoded by a pathogen, such as US28, and a second peptide which comprises an optimized cleavage site and a toxin, such as selected domains of Exotoxin A.
• the selectivity towards pathogen encoded receptors, such as US28, of the fusion protein is surprisingly enhanced following mutations in the cleavage site.
• the optimized cleavage site described herein may be utilized for a variety of fusion proteins, and in particular immunotoxins, to increase cell killing selectivity, by altering cell killing potency in a target receptor selective manner.
• the present invention provides a fusion protein comprising: a) a first peptide which binds at least one receptor expressed on a cell; and b) a second peptide comprising a cleavage site having an amino acid sequence ArgX1X2Arg, wherein X2 is Arg or Lys, and wherein the second peptide comprises a toxin.
• amino acid as used herein includes the standard twenty genetically-encoded amino acids and their corresponding stereoisomers in the ⁇ ’ form (as compared to the natural ‘L’ form), omega-amino acids and other naturally-occurring amino acids, unconventional amino acids (e.g. a,a-disubstituted amino acids, N-alkyl amino acids, etc.) and chemically derivatised amino acids.
• omega-amino acids e.g. a,a-disubstituted amino acids, N-alkyl amino acids, etc.
• chemically derivatised amino acids e.g. a,a-disubstituted amino acids, N-alkyl amino acids, etc.
• the fusion protein only consists of naturally occurring amino acids.
• the second peptide of the present disclosure comprises a cleavage site having the amino acid sequence
• X1 may be any amino acid. In one embodiment, X1 is selected from the group consisting of Gin, Ser, Thr and Asn. In one embodiment X1 is Gin.
• X2 is selected from Arg and Lys.
• X2 is Arg.
• X2 is Lys.
• a basic amino acid such as Arg at amino acid position X2
• the cell killing selectivity is enhanced.
• virus infected cells expressing a receptor encoded by the virus and binding the first peptide are killed more efficiently than e.g. cells expressing an endogenous receptor, which may also bind the first peptide.
• the cleavage site comprises or consists of the amino acid sequence ArgGInArgArg (RQRR). In one embodiment, the cleavage site comprises or consists of the amino acid sequence ArgGInLysArg. In one embodiment, the cleavage site comprises or consists of the amino acid sequence ArgSerLysArg. In one embodiment, the cleavage site comprises or consists of the amino acid sequence ArgSerArgArg. In one embodiment, the cleavage site comprises or consists of the amino acid sequence ArgThrLysArg. In one embodiment, the cleavage site comprises or consists of the amino acid sequence ArgThrArgArg. In one embodiment, the cleavage site comprises or consists of the amino acid sequence ArgAsnLysArg. In one embodiment, the cleavage site comprises or consists of the amino acid sequence ArgAsnArgArg.
• cleavage is occurring at the C-terminal end of the cleavage site.
• the cleavage site is an enzymatic cleavage site.
• the enzymatic cleavage site is a furin cleavage site.
• the first peptide binds to at least one receptor expressed on a cell. In one embodiment, the first peptide binds to at least two receptors expressed on a cell.
• one receptor binding the first peptide is a receptor encoded by a pathogen and a further receptor binding the first peptide is a human encoded receptor and/or endogenous receptor for the first peptide or a variant thereof.
• the receptor is a G-protein coupled receptor (GPCR), such as US28 of SEQ ID NO: 10.
• the receptor is a chemokine receptor.
• the chemokine receptor is a CC chemokine receptor and/or a CX3C chemokine receptor.
• the CC chemokine receptor is US28.
• the CX3C chemokine receptor is CX3CR1.
• the receptor is encoded by a pathogen.
• the pathogen is a bacteria.
• the pathogen is a virus.
• the virus is a DNA virus.
• the virus is a RNA virus.
• the virus is a herpesvirus.
• the virus is cytomegalovirus.
• the receptor is capable of internalizing. 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 a pathogen is advantageously constitutively internalized as this will ensure efficient uptake of the fusion protein by the pathogen infected cell and thereby the death of the infected cell with a minimum of unwanted toxicity and side effects.
• Internalization refers to the process of a receptor being moved into the cell that it is expressed on. For example, the receptor might enter the cell by endocytosis or phagocytosis.
• the present invention provides a fusion protein that targets a specific cell by binding in a selective manner to a receptor leading to the internalization of said receptor. The toxin part of the fusion protein is then cleaved and said cell is killed.
• the fusion protein is an immunotoxin.
• An “immunotoxin” refers to a protein consisting of a targeting portion linked to a toxin.
• the targeting portion may also be called a ligand.
• the toxin may be different kinds of toxins.
• the second peptide comprises a toxin selected from the group consisting of Pseudomonas Exotoxin A, gelonin, bouganin, saporin, ricin, ricin A chain, bryodin, restrictocin, diphteria toxin, diphteria toxin A chain and variants and fragments thereof.
• the toxin part of the fusion protein enables the killing of the cells.
• the toxin is cytotoxic.
• the second peptide comprises one or more domains of Pseudomonas Exotoxin A. In one embodiment, the second peptide comprises at least a part of the Exotoxin A of SEQ ID NO: 9.
• the fusion protein is a targeting moiety, which allows for binding to a receptor expressed on a cell.
• first and the second peptide are operably linked.
• operably linked means that the sequences being linked are contiguous and/or placed into a functional relationship with each other, such as covalently linked.
• the first peptide comprises or consists of a. an amino acid sequence of SEQ ID NO: 1 ; b. a variant of SEQ ID NO: 1 comprising or consisting of an amino acid sequence with 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.
• SEQ ID NO: 1 being more than 50 amino acids in length, such as more than 60, 70, or 75 amino acids in length, ora variant thereof with 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.
• the first peptide comprises or consists of a variant of SEQ ID NO: 1 comprising or consisting of an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1. In one embodiment the first peptide comprises or consists of a variant of SEQ ID NO: 1 comprising or consisting of an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1. In one embodiment the first peptide comprises or consists of a variant of SEQ ID NO: 1 comprising or consisting of an amino acid sequence with at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 1.
• the first peptide comprises or consists of a variant of SEQ ID NO: 1 comprising or consisting of an amino acid sequence with at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 1. In one embodiment the first peptide comprises or consists of a variant of SEQ ID NO: 1 comprising or consisting of an amino acid sequence with at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1. In one embodiment the first peptide comprises or consists of a variant of SEQ ID NO: 1 comprising or consisting of an amino acid sequence with at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1.
• the first peptide comprises or consist of a fragment of SEQ ID NO: 1 being more than 50 amino acids in length. In one embodiment the first peptide comprises or consist of a fragment of SEQ ID NO: 1 being more than 60 amino acids in length. In one embodiment the first peptide comprises or consist of a fragment of SEQ ID NO: 1 being more than 70 amino acids in length. In one embodiment the first peptide comprises or consist of a fragment of SEQ ID NO: 1 being more than 75 amino acids in length.
• the first peptide comprises or consist of a fragment of SEQ ID NO: 1 , or a variant thereof with at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 1. In one embodiment the first peptide comprises or consist of a fragment of SEQ ID NO: 1 , or a variant thereof with at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 1. In one embodiment the first peptide comprises or consist of a fragment of SEQ ID NO: 1 , or a variant thereof with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1. In one embodiment the first peptide comprises or consist of a fragment of SEQ ID NO: 1, or a variant thereof with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 1.
• the first peptide comprises or consist of a fragment of SEQ ID NO: 1 , or a variant thereof with at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 1. In one embodiment the first peptide comprises or consist of a fragment of SEQ ID NO: 1, or a variant thereof with at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 1. In one embodiment the first peptide comprises or consist of a fragment of SEQ ID NO: 1, or a variant thereof with at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 1. In one embodiment the first peptide comprises or consist of a fragment of SEQ ID NO: 1, or a variant thereof with at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1.
• the amino acid residue of SEQ ID NO:1 in position 49 is mutated to an Ala, a Lys or an Asp, preferably Ala. Theses substitutions may lead to increased cell killing selectivity towards cells expressing a pathogen encoded receptor binding the first peptide compared to a human or endogenous encoded receptor also binding the first peptide.
• the first peptide further comprises a Methionine (M) at the N- terminus.
• M Methionine
• the first peptide comprises or consists of one or more additional amino acids, inserted at the N- and/or C-terminus and/or internally within the amino acid sequence of SEQ ID NO:1.
• one or more amino acids are deleted at the N- and/or C-terminus and/or internally within the amino acid sequence of SEQ ID NO:1.
• the first peptide is less than 100 amino acids in length.
• the first peptide is less than 90 amino acids in length.
• the first peptide is less than 85 amino acids in length.
• the first peptide is less than 80 amino acids in length.
• the second peptide comprises a domain A, which may be a translocation domain, such as a domain A having an amino acid sequence according to SEQ ID NO:3 or a fragment or variant thereof, and/or a domain B, such as a domain B having an amino acid sequence according to SEQ ID NO:4 or a fragment or variant thereof, and/or a domain C, which may be a cytotoxic domain, such as a domain C having an amino acid sequence according to SEQ ID NO:5 or a fragment or variant thereof.
• the second peptide comprises a domain A, such as a domain A having an amino acid sequence according to SEQ ID NO:3 or a fragment or variant thereof.
• the second peptide comprises a domain A, such as a domain A having an amino acid sequence according to SEQ ID NO:3 or a fragment or variant thereof, and a domain B, such as a domain B having an amino acid sequence according to SEQ ID NO:4 or a fragment or variant thereof.
• the second peptide comprises a domain A, such as a domain A having an amino acid sequence according to SEQ ID NO:3 or a fragment or variant thereof, and a domain B, such as a domain B having an amino acid sequence according to SEQ ID NO:4 or a fragment or variant thereof, and a domain C such as a domain C having an amino acid sequence according to SEQ ID NO:5 or a fragment or variant thereof.
• the second peptide comprises a domain B having an amino acid sequence according to SEQ ID NO:4 or a fragment or variant thereof. In one embodiment, the second peptide comprises a domain C, such as a domain C having an amino acid sequence according to SEQ ID NO:5 or a fragment or variant thereof.
• the second peptide comprises a. an amino acid sequence of SEQ ID NO: 3; b. a variant of SEQ ID NO: 3 comprising or consisting of an amino acid sequence with 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 being more than 80 amino acids in length, such as more than 90, 100, or 110 amino acids in length, or a variant thereof with 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.
• the second peptide comprises a. an amino acid sequence of SEQ ID NO: 4; b. a variant of SEQ ID NO: 4 comprising or consisting of an amino acid sequence with 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.
• SEQ ID NO: 4 being more than 6 amino acids in length, such as more than 8, 10, or 12 amino acids in length, or a variant thereof with 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.
• the second peptide comprises a. an amino acid sequence of SEQ ID NO: 5; b. a variant of SEQ ID NO: 5 comprising or consisting of an amino acid sequence with at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 5, more preferably at least 85% or 90% sequence identity to SEQ ID NO: 5, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 5; c.
• a fragment of SEQ ID NO: 5 being more than 180 amino acids in length, such as more than 190, 200, or 210 amino acids in length, or a variant thereof with 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.
• the second peptide comprises a. an amino acid sequence of SEQ ID NO: 3; b. a variant of SEQ ID NO: 3 comprising or consisting of an amino acid sequence with 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 being more than 80 amino acids in length, such as more than 90, 100, or 110 amino acids in length, or a variant thereof with 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. an amino acid sequence of SEQ ID NO: 4; e.
• a variant of SEQ ID NO: 4 comprising or consisting of an amino acid sequence with at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 4, more preferably at least 85% or 90% sequence identity to SEQ ID NO: 4, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 4; f. a fragment of SEQ ID NO: 4 being more than 6 amino acids in length, such as more than 8, 10, or 12 amino acids in length, or a variant thereof with 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.
• the second peptide comprises a. an amino acid sequence of SEQ ID NO: 3; b. a variant of SEQ ID NO: 3 comprising or consisting of an amino acid sequence with 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 being more than 80 amino acids in length, such as more than 90, 100, or 110 amino acids in length, or a variant thereof with 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. an amino acid sequence of SEQ ID NO: 4; e.
• SEQ ID NO: 4 a variant of SEQ ID NO: 4 comprising or consisting of an amino acid sequence with at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 4, more preferably at least 85% or 90% sequence identity to SEQ ID NO: 4, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 4; f.
• a fragment of SEQ ID NO: 4 being more than 6 amino acids in length, such as more than 8, 10, or 12 amino acids in length, or a variant thereof with 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 g. an amino acid sequence of SEQ ID NO: 5; h.
• variant of SEQ ID NO: 5 comprising or consisting of an amino acid sequence with at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 5, more preferably at least 85% or 90% sequence identity to SEQ ID NO: 5, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 5; i.
• a fragment of SEQ ID NO: 5 being more than 180 amino acids in length, such as more than 190, 200, or 210 amino acids in length, or a variant thereof with 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.
• the second peptide comprises a. an amino acid sequence of SEQ ID NO: 3; b. a variant of SEQ ID NO: 3 comprising or consisting of an amino acid sequence with 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 being more than 80 amino acids in length, such as more than 90, 100, or 110 amino acids in length, or a variant thereof with 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. an amino acid sequence of SEQ ID NO: 5; e.
• SEQ ID NO: 5 comprising or consisting of an amino acid sequence with at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 5, more preferably at least 85% or 90% sequence identity to SEQ ID NO: 5, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 5; f.
• a fragment of SEQ ID NO: 5 being more than 180 amino acids in length, such as more than 190, 200, or 210 amino acids in length, or a variant thereof with 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.
• the second peptide comprises a. an amino acid sequence of SEQ ID NO: 4; b. a variant of SEQ ID NO: 4 comprising or consisting of an amino acid sequence with at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 4, more preferably at least 85% or 90% sequence identity to SEQ ID NO: 4, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 4; c.
• a fragment of SEQ ID NO: 4 being more than 6 amino acids in length, such as more than 8, 10, or 12 amino acids in length, or a variant thereof with 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 d. an amino acid sequence of SEQ ID NO: 5; e.
• SEQ ID NO: 5 comprising or consisting of an amino acid sequence with at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 5, more preferably at least 85% or 90% sequence identity to SEQ ID NO: 5, and most preferably at least 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 5; f.
• the domain A is a translocation domain and the domain C is a cytotoxic domain, such as an ADP-ribosylating domain.
• the second peptide comprises the amino acid sequence KDEL of SEQ ID NO:8 in the C-terminus. In one embodiment, the 5 last amino acids of the second peptide are replaced with the amino acid sequence of SEQ ID NO: 8. In one embodiment, the second peptide comprises or consists of a. an amino acid sequence of SEQ ID NO: 2; b. a variant of SEQ ID NO: 2 comprising or consisting of an amino acid sequence with 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 being more than 300 amino acids in length, such as more than 310, 330, or 340 amino acids in length, or a variant thereof with 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.
• the second peptide comprises or consists of a variant of SEQ ID NO: 2 comprising or consisting of an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2. In one embodiment the second peptide comprises or consists of a variant of SEQ ID NO: 2 comprising or consisting of an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2. In one embodiment the second peptide comprises or consists of a variant of SEQ ID NO: 2 comprising or consisting of an amino acid sequence with at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 2.
• the second peptide comprises or consists of a variant of SEQ ID NO: 2 comprising or consisting of an amino acid sequence with at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 2. In one embodiment the second peptide comprises or consists of a variant of SEQ ID NO: 2 comprising or consisting of an amino acid sequence with at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 2. In one embodiment the second peptide comprises or consists of a variant of SEQ ID NO: 2 comprising or consisting of an amino acid sequence with at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 2.
• the second peptide comprises or consist of a fragment of SEQ ID NO: 2 being more than 300 amino acids in length. In one embodiment the second peptide comprises or consist of a fragment of SEQ ID NO: 2 being more than 310 amino acids in length. In one embodiment the second peptide comprises or consist of a fragment of SEQ ID NO: 2 being more than 330 amino acids in length. In one embodiment the second peptide comprises or consist of a fragment of SEQ ID NO: 2 being more than 340 amino acids in length.
• the second peptide comprises or consist of a fragment of SEQ ID NO: 2, or a variant thereof with at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 2. In one embodiment the second peptide comprises or consist of a fragment of SEQ ID NO: 2, or a variant thereof with at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 2. In one embodiment the second peptide comprises or consist of a fragment of SEQ ID NO: 2, or a variant thereof with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 2. In one embodiment the second peptide comprises or consist of a fragment of SEQ ID NO: 2, or a variant thereof with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2.
• the second peptide comprises or consist of a fragment of SEQ ID NO: 2, or a variant thereof with at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 2. In one embodiment the second peptide comprises or consist of a fragment of SEQ ID NO: 2, or a variant thereof with at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 2. In one embodiment the second peptide comprises or consist of a fragment of SEQ ID NO: 2, or a variant thereof with at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 2. In one embodiment the second peptide comprises or consist of a fragment of SEQ ID NO: 2, or a variant thereof with at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 2.
• the second peptide is less than 400 amino acids in length, for example less than 380, 370, 360, 350 or 345 amino acids in length. In one embodiment, the second peptide is less than 400 amino acids in length.
• the second peptide is less than 380 amino acids in length.
• the second peptide is less than 370 amino acids in length. In one embodiment, the second peptide is less than 360 amino acids in length. In one embodiment, the second peptide is less than 350 amino acids in length.
• the second peptide is less than 345 amino acids in length.
• the fusion protein comprises or consists of a. an amino acid sequence of SEQ ID NO: 6; b. a variant of SEQ ID NO: 6 comprising or consisting of an amino acid sequence with 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.
• the fusion protein comprises or consists of a variant of SEQ ID NO: 6 comprising or consisting of an amino acid sequence with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 6.
• the fusion protein comprises or consists of a variant of SEQ ID NO: 6 comprising or consisting of an amino acid sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 6. In one embodiment the fusion protein comprises or consists of a variant of SEQ ID NO: 6 comprising or consisting of an amino acid sequence with at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 6. In one embodiment the fusion protein comprises or consists of a variant of SEQ ID NO: 6 comprising or consisting of an amino acid sequence with at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 6.
• the fusion protein comprises or consists of a variant of SEQ ID NO: 6 comprising or consisting of an amino acid sequence with at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 6. In one embodiment the fusion protein comprises or consists of a variant of SEQ ID NO: 6 comprising or consisting of an amino acid sequence with at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 6.
• the fusion protein comprises or consist of a fragment of SEQ ID NO: 6 being more than 360 amino acids in length. In one embodiment the fusion protein comprises or consist of a fragment of SEQ ID NO: 6 being more than 380 amino acids in length. In one embodiment the fusion protein comprises or consist of a fragment of SEQ ID NO: 6 being more than 400 amino acids in length. In one embodiment the fusion protein comprises or consist of a fragment of SEQ ID NO: 6 being more than 420 amino acids in length.
• the fusion protein comprises or consist of a fragment of SEQ ID NO: 6, or a variant thereof with at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 6. In one embodiment the fusion protein comprises or consist of a fragment of SEQ ID NO: 6, or a variant thereof with at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 6. In one embodiment the fusion protein comprises or consist of a fragment of SEQ ID NO: 6, or a variant thereof with at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 6. In one embodiment the fusion protein comprises or consist of a fragment of SEQ ID NO: 6, or a variant thereof with at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 6.
• the fusion protein comprises or consist of a fragment of SEQ ID NO: 6, or a variant thereof with at least 96% sequence identity to the amino acid sequence of SEQ ID NO: 6. In one embodiment the fusion protein comprises or consist of a fragment of SEQ ID NO: 6, or a variant thereof with at least 97% sequence identity to the amino acid sequence of SEQ ID NO: 6. In one embodiment the fusion protein comprises or consist of a fragment of SEQ ID NO: 6, or a variant thereof with at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 6. In one embodiment the fusion protein comprises or consist of a fragment of SEQ ID NO: 6, or a variant thereof with at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 6.
• the fusion protein is less than 500 amino acids in length, for example less than 490, 480, 470, 460, 450, 440, 430, 425 or less amino acids in length. In one embodiment, the fusion protein is less than 500 amino acids in length. In one embodiment, the fusion protein is less than 490 amino acids in length. In one embodiment, the fusion protein is less than 480 amino acids in length. In one embodiment, the fusion protein is less than 470 amino acids in length. In one embodiment, the fusion protein is less than 460 amino acids in length. In one embodiment, the fusion protein is less than 450 amino acids in length. In one embodiment, the fusion protein is less than 440amino acids in length. In one embodiment, the fusion protein is less than 430 amino acids in length. In one embodiment, the fusion protein is less than 420 amino acids in length.
• the fusion protein kills cells infected by a pathogen, such as cells latently infected by a pathogen.
• the pathogen is a virus such as DNA virus such as a herpesvirus, such as cytomegalovirus, or a RNA virus.
• the fusion protein induces cell death of cells expressing the receptor.
• the cell death is selected from the group consisting of apoptosis, necrosis, autophagic cell death and mitosis associated cell death.
• apoptosis and “apoptotic activity” are used in a broad sense and refer to the orderly or controlled form of cell death in mammals that is typically accompanied by one or more characteristic cell changes, including condensation of cytoplasm, loss of plasma membrane microvilli, segmentation of the nucleus, degradation of chromosomal DNA or loss of mitochondrial function.
• the fusion protein induces a direct or indirect effect on a pathogen resulting in inhibition of pathogen growth, replication, genome stability, maturation, packaging, latency, reactivation, dissemination and/or immune inhibition.
• the fusion protein selectively kills cells that express a pathogen encoded receptor.
• the fusion protein kills cells that express US28.
• the fusion protein has an IC50 value 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 for a receptor encoded by a virus such as US28.
• the fusion protein has increased potency against cells expressing a receptor encoded by a virus, such as US28, as compared to the potency against cells expressing an endogenous receptor or human encoded receptor, such as CX3CR1, such as at least 100-fold 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.
• the fusion protein has increased potency against cells expressing US28 as compared to the potency against cells expressing CX3CR1, such as at least 100-fold increased potency.
• the fusion protein has increased potency against cells expressing US28 as compared to the potency against cells expressing CX3CR1, such as at least 300-fold increased potency In one embodiment, the fusion protein has increased potency against cells expressing US28 as compared to the potency against cells expressing CX3CR1 , such as at least 400-fold increased potency. In one embodiment, the fusion protein has increased potency against cells expressing US28 as compared to the potency against cells expressing CX3CR1 , such as at least 500-fold increased potency.
• the fusion protein has increased affinity for a receptor encoded by a virus such as US28 as compared to the affinity for an endogenous receptor or human encoded receptor such as CX3CR1 , such as at least 50-fold increased affinity, such as at least 100-fold increased affinity. In one embodiment, the fusion protein has increased affinity for US28 as compared to the affinity for CX3CR1, such as at least 100-fold increased affinity.
• the selectivity ratio US28/CX3CR1 of the fusion protein of the present invention is at least 750, such as at least 800, such as at least 850, such as at least 900. In one embodiment, the selectivity ratio US28/CX3CR1 of the fusion protein of the 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.
• the present invention provides an isolated nucleic acid molecule encoding a fusion protein as described herein.
• nucleic acid molecule we include DNA (e.g. genomic DNA or complementary DNA) and mRNA molecules, which may be single- or double-stranded.
• isolated we mean that the nucleic acid molecule is not located or otherwise provided within a cell.
• the present invention provides a vector comprising a nucleic acid molecule as described herein.
• the vector is an expression vector.
• the present invention provides a recombinant host cell comprising a nucleic acid molecule or a vector as described herein.
• the host cell is a bacterial cell, a yeast cell, a mammalian cell such as a human cell or an insect cell.
• the present invention provides a pharmaceutical composition
• a pharmaceutical composition comprising a fusion protein, a nucleic acid, a vector or a recombinant host cell as described herein and a pharmaceutically acceptable carrier, diluent and/or excipient.
• the pharmaceutical composition further comprises one or more agents.
• the agent is selected from the group consisting of immunosuppressive agents, anti-viral agents and immunotherapy.
• the anti-viral agent is selected from the group consisting of valganciclovir, ganciclovir, cidofovir, leflunomide, prevymis, maribavir and brincidofovir.
• the immunotherapy is cell-based therapy. In one embodiment, the immunotherapy is T cell therapy.
• the present invention provides a fusion protein, a nucleic acid, a vector, a recombinant host cell or a pharmaceutical composition as described herein for use as a medicament.
• the present invention provides a fusion protein, a nucleic acid, a vector, a recombinant host cell or a pharmaceutical composition as described herein for use in the prevention or treatment of an infection caused by a pathogen and/or a pathogen associated disorder.
• the pathogen is a bacteria or a virus.
• the virus is a DNA virus, such as a herpesvirus, such as cytomegalovirus, or a RNA virus.
• the present invention provides a fusion protein, a nucleic acid, a vector, a recombinant host cell ora pharmaceutical composition as described herein for use in the prevention or treatment of CMV infections and/or CMV-associated disorders.
• the CMV infection is a latent and/or lytic CMV infection.
• the CMV infection can be detected in: i) a tissue selected from one or more of the group consisting of retina, cornea, heart, liver, kidney, lung, gastro-intestinal tissue, thymus, spleen, skin and muscle, and/or ii) a body fluid selected from one or more of the group consisting of saliva, blood, urine, semen and breast milk.
• the CMV infection is an infection in an immuno-compromised patient selected from the group consisting of HIV-patients, neonates and immunosuppressive patients, bone marrow transplant patients, solid organ transplant patients, immune therapy patients, cancer patients, intensive care patients, trauma patients, stem cell patients, gene therapy patients, cell therapy patients, geriatric patients and multimorbid patients.
• an immuno-compromised patient selected from the group consisting of HIV-patients, neonates and immunosuppressive patients, bone marrow transplant patients, solid organ transplant patients, immune therapy patients, cancer patients, intensive care patients, trauma patients, stem cell patients, gene therapy patients, cell therapy patients, geriatric patients and multimorbid patients.
• the CMV infection is an infection in a patient suffering from a coronary disease and/or a vascular disease.
• the fusion protein, the nucleic acid, the vector, the host cell or the composition is administered intravenously, intratumorously, intraperitoneally, intrathecally and/or intralymphatically.
• the fusion protein, the nucleic acid, the vector, the host cell or the composition is administered one or more times to the individual.
• the individual is a human.
• the human is an immunocompromised patient.
• immunocompromised patient we refer to a patient not having the ability to respond normally to an infection due to an impaired or weakened immune system.
• an immunocompromised patient may be diagnosed with a disease that affect the immune system such as diabetes and HIV.
• An immunocompromised patient may have a suppressed immune response following treatment such as chemotherapy.
• the human is a child. In one embodiment, the human is an adult.
• the human is in need of a solid organ transplantation and/or a hematopoietic stem cell transplantation.
• Hematopoietic stem cell transplantation is the transplantation of multipotent hematopoietic stem cells, usually derived from one marrow, peripheral blood or umbilical cord blood.
• the transplantation may be autologous (stem cells are isolated from the same patient) or allogeneic (stem cells are isolated from a different patient).
• the CMV-associated disorder is selected from the group consisting of cytomegaloviral pneumonitis, cytomegaloviral hepatitis, cytomegaloviral pancreatitis, cytomegaloviral mononucleosis, CMV polyradiculomyelopathy, cytomegalic 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).
• GVHD graft versus host disease
• SOT- GVHD solid organ transplant graft versus host disease
• the present invention provides a fusion protein, a nucleic acid, a vector, a host cell or a pharmaceutical composition as described herein for use in the manufacture of a medicament for the prevention or treatment of an infection caused by a pathogen and/or pathogen associated disorders, such as for the prevention or treatment of CMV infections and/or CMV-associated disorders.
• the present invention provides an ex vivo use of a fusion protein or a pharmaceutical composition as described herein for treatment of solid organs for transplantation and/or hematopoietic stem cells for transplantation.
• treating refers to curative therapy, prophylactic therapy, ameliorative and palliative therapy.
• the treatment is curative.
• the present invention provides a method of treating or preventing infection caused by a pathogen such as CMV infections in an individual in need thereof, the method comprising administering a therapeutically effective amount of a fusion protein, a nucleic acid, a vector, a recombinant host cell or a pharmaceutical composition as described herein to the individual.
• an “effective amount” or “therapeutically effective amount” of a compound is that amount of compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered.
• the phrase “therapeutically effective amount,” as used herein, may refer to an amount of a fusion protein that is sufficient or effective to treat (delay or prevent the onset of, prevent the progression of, inhibit, decrease or reverse) an infection.
• the present invention provides a method for producing a fusion protein as described herein, the method comprising culturing a host cell as defined herein under conditions which permit expression of the encoded fusion protein.
• SEQ ID NO: 1 (exemplary first peptide) QHHGVTKCNITCSKMTSKI PVALLIHYQQNQASCGKRAIILETRQHRLFCADPKEQWV KDAMQHLDRQAAALTRNG
• SEQ ID NO: 2 (exemplary second peptide)
• SEQ ID NO: 6 (SYN002) MQHHGVTKCNITCSKMTSKI PVALLIHYQQNQASCGKRAIILETRQHRLACADPKEQW VKDAMQHLDRQAAALTRNGGGSLAALTAHQACHLPLETFTRHRQRRGWEQLEQCG YPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAE SERFVRQGTGNDEAGAASGPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWT VERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDP ALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGH PLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQ
• SEQ ID NO: 7 (human CX3CL1) MAPISLSWLLRLATFCHLTVLLAGQHHGVTKCNITCSKMTSKIPVALLIHYQQNQASCG
• a fusion protein comprising: a) a first peptide which binds to at least one receptor expressed on a cell; and b) a second peptide comprising a cleavage site having an amino acid sequence
• ArgX1X2Arg wherein X2 is Arg or Lys, and wherein the second peptide comprises a toxin.
• fusion protein according to item 1 wherein the fusion protein is an immunotoxin.
• the cleavage site comprises or consists of the amino acid sequence ArgGInArgArg.
• fusion protein according to any one of the preceding items, wherein one receptor binding the first peptide is a receptor encoded by a pathogen and a further receptor binding the first peptide is a human encoded receptor and/or endogenous receptor for the first peptide or a variant thereof.
• GPCR G-protein coupled receptor
• the second peptide comprises a toxin selected from the group consisting of Pseudomonas exotoxin A, gelonin, bouganin, saporin, ricin, ricin A chain, bryodin, restrictocin, diphteria toxin, diphteria toxin A chain and variants and fragments thereof.
• the first peptide comprises or consists of a. an amino acid sequence of SEQ ID NO: 1 ; b. a variant of SEQ ID NO: 1 comprising or consisting of an amino acid sequence with 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%,
• SEQ ID NO: 1 96%, 97%, 98% or 99% identity to SEQ ID NO: 1; c. a fragment of SEQ ID NO: 1 being more than 50 amino acids in length, such as more than 60, 70, or 75 amino acids in length, or a variant thereof with 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%,
• the second peptide comprises or consists of a. an amino acid sequence of SEQ ID NO: 2; b. a variant of SEQ ID NO: 2 comprising or consisting of an amino acid sequence with 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 being more than 300 amino acids in length, such as more than 310, 330, or 340 amino acids in length, or a variant thereof with 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.
• the fusion protein according to any one of the preceding items, wherein the fusion protein comprises or consists of a. an amino acid sequence of SEQ ID NO: 6; b.
• SEQ ID NO: 6 a variant of SEQ ID NO: 6 comprising or consisting of an amino acid sequence with 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 being more than 360 amino acids in length, such as more than 380, 400, or 420 amino acids in length, or a variant thereof with 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. 13.
• the fusion protein according to any one of the preceding items, wherein the fusion protein kills cells infected by a pathogen, such as cells latently infected by a pathogen.
• fusion protein has increased potency against cells expressing a receptor encoded by a virus such as US28 as compared to the potency against cells expressing an endogenous receptor or human encoded receptor such as CX3CR1, 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.
• a fusion protein according to any one of items 1 to 14 for use in the prevention or treatment of an infection caused by a pathogen and/or pathogen associated disorders.
• Example 1 Mutation in the first peptide of the fusion protein SYNx leads to selectivity in cell killing.
• HA-CX3CR1- and HA-US28-expressing cells were constructed as described in Hjorto GM et al., 2013. Briefly, stable and inducible clones of HA-CX3CR1- and HA-US28- expressing cells were generated by co-transfecting Flp-ln T-Rex-293 cells with the Flp- recombinase expression vector, pOG44 and either of the pcDNA5/FRT/TO receptor constructs. This targeted cloning at the FRT site brings the receptor gene under the control of the Tetracycline repressor/operator system. The expression of the receptors was confirmed by Western blotting.
• the PDL-solution was aspirated from the plate and each well was washed with 100 pL 1x PBS. After aspiration of the wash buffer, the 96-well plate was seeded with 2,000 to 10,000 cells/well in 100 pL and placed in a C02-incubator (37 °C, 10% C02) overnight.
• a fusion toxin protein aliquot was thawed on ice and used to make a dilution series in 1 mM Acetic acid and 5 g/L BSA .
• the receptor expression growth media was aspirated and replaced with 100 pL of fresh growth media.
• 5 pL of the fusion toxin protein dilution series was added to each well and 5 pL of 1 g/L Cycloheximide was added as a positive control to one well.
• the plate was placed back in the C02-incubator (37 °C, 10% C02) overnight.
• a 1 :10 dilution of AlamarBlue in assay growth medium was prepared.
• the solution was covered with tinfoil and heated for 10 min. in a 37 °C water bath and then filtered through a 0.2 pm filter.
• the well solutions were aspirated and 100 pL of the AlamarBlue solution was added to each well and the plate was placed back in the C02-incubator (37 °C, 10% C02) for 4 hours. Fluorescence data was collected on a FlexStation 3 using excitation at 540 nm and emission at 585 nm.
• Cell killing efficiency of SYNx was determined using tetracycline-induced HEK 293 cells expressing either a receptor encoded by a virus such as US28 or an endogenous receptor such as CX3CR1 together with non-induced cells with no receptor expression (negative controls). The data were normalized to the maximum number of living cells. Substituting a single amino acid in the first peptide (here chemokine part) of SYNx leads to selectivity towards the virus encoded receptor US28 as shown in figure 2.
• the single amino acid F49A substitution in the first peptide (e.g a chemokine part) of the fusion protein construct induces selectivity of the fusion protein (SYNx) towards the virus encoded receptor US28.
• Example 2 Increased potency obtained by adding a furin cleavage site through the full length translocation domain of Exotoxin A.
• SYNx Cell killing efficiency of SYNx was determined using tetracycline-induced HEK 293 cells expressing either the virus encoded receptor US28 or the endogenous receptor CX3CR1 together with non-induced cells with no receptor expression (negative controls). The data were normalized to the maximum number of living cells. Adding a furin cleavage site, such as the full length translocation domain, to the SYN001 construct, yielding SYN016, increases the potency on both the endogenous/human CX3CR1- and the pathogen encoded US28-receptor expressing cells as shown in figure 3 and table 1.
• Domain II of Exotoxin A has been identified as the translocation domain responsible for the transfer of the catalytic domain, domain III, from an endocytic vesicle into the cytosol (Hwang J et al., 1987; Siegall CB etal., 1989).
• Example 3 In vitro cleavage of SYNx by human Furin.
• the purified SYNx construct was thawed on the lab bench. When thawed, the sample was then spun down. The concentration of the sample was measured and a digest was set up with 20 mM SYNx in 50 mI_ in a new tube in 1x PBS containing 5 mM CaCI2. 5 mg was removed for SDS-PAGE analysis. 1 mI_ of human Furin (NEB, 2 units/pL) was added to the mixture and was placed in a water bath at 37 °C. After 1.5 hours 5 pg of SYNx was removed for SDS-PAGE analysis. The pH of the reaction mixture is adjusted with either 1 M HCL or 1 M NaOH prior to addition of SYNx.
• Purified SYNx constructs were treated with purified human Furin in vitro to determine the cleavage efficiency of the different SYNx constructs by Furin. Other cleavage mechanisms than by Furin may possibly take place. The results were analysed by SDS- PAGE. Addition of a full translocation domain, such as in this case a full ExoA translocation domain, to SYN000 or SYN001, yielding SYN017 and SYN016 respectively, improves in vitro cleavage by Furin. Optimization of the Furin cleavage site in SYN017 and SYN016, yielding SYN014 and SYN002 respectively, does not appear to improve in vitro cleavage by Furin further as shown in figure 4.
• Purified SYN002 was treated with purified human Furin in vitro at different pH values to determine the cleavage efficiency and the results were analysed by SDS-PAGE. SYN002 is cleaved by Furin over a wide pH range as shown in figure 5.
• SYNx constructs (here the SYN002-construct also comprising the translocation domain) does not yield an improvement in the in vitro cleavage by Furin, compared to a construct with the native sequence (ArgGInProArg) as in SYN016.
• SYN002 could be cleaved in vitro over a broad pH range, suggesting that cleavage by Furin could occur at any cellular location and not only in endosomes.
• Example 4 Cell killing selectivity between endogenous and virus encoded receptor-expressing cells is also enhanced and mediated by the optimized cleavage site.
• Cell killing efficiency of SYNx was determined using tetracycline-induced HEK 293 cells expressing either US28 or CX3CR1 together with non-induced cells with no receptor expression (negative controls). The data was normalized to the maximum number of living cells.
• the surprising effect on cell-killing selectivity is obtained by optimization of the cleavage site. Without being bound by any theory, this may be due to a difference in internalization and intracellular trafficking for a receptor encoded by a virus, such as the US28 receptor, versus an endogenous receptor, such as the CX3CR1 receptor.

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