WO2023104794A1 - Technologie à lieur pour réduire la rétention rénale - Google Patents

Technologie à lieur pour réduire la rétention rénale Download PDF

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Publication number
WO2023104794A1
WO2023104794A1 PCT/EP2022/084593 EP2022084593W WO2023104794A1 WO 2023104794 A1 WO2023104794 A1 WO 2023104794A1 EP 2022084593 W EP2022084593 W EP 2022084593W WO 2023104794 A1 WO2023104794 A1 WO 2023104794A1
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alkyl
payload
alkylene
protein
conjugate
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PCT/EP2022/084593
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English (en)
Inventor
Giulia VALPREDA
Linjing Mu
Martin Behe
Belinda TRACHSEL
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Paul Scherrer Institut
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Publication of WO2023104794A1 publication Critical patent/WO2023104794A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins

Definitions

  • the present invention relates to a linker of the present invention, a protein-payload conjugate of the present invention, payload-linker conjugate of the present invention and their respective therapeutic and diagnostic uses.
  • the linkers, protein-payload conjugates and payload-linker conjugates of the present invention as particularly useful due to their reduced renal retention.
  • LMW-Abs low-molecular-weight antibody constructs
  • peptide-based pharmaceuticals undergoing renal clearance thus posing limitations to their in vivo applications.
  • the undesired renal activity mainly lies in the long renal residence time of metabolites (including radiometabolites) generated upon proteolytic degradation of the parent molecule.
  • NEP neprilysin
  • a metalloendopeptidase abundantly expressed on the BBM proximal convoluted tubules
  • NEP leads to the generation of the radiometabolite [ 67/68 Ga]Ga-NOTA-Bn-M, displaying a fast elimination rate from the coated vesicle of the renal cells into the urinary tract (Uehara et al., 2014).
  • Abs-based constructs the same approach was found to be also effective in the case of peptides, such as Exendin-4, as demonstrated by Zheng et al. in a subsequent study (Zhang et al., 2019).
  • the present inventors have found that surprisingly a repetition of two sequence motifs that sensitize the linker to the cleavage at the renal brush border, in particular the cleavage by neprilysin protease, reduce the renal retention of the payload, in particular of a radiopharmaceutical payload.
  • the present inventors have further found that surprisingly if the N-terminal motif that sensitizes the linker to the cleavage at the renal brush border does not comprise lysin or ornithine residue, the renal retention is even further reduced.
  • the present invention relates to a linker comprising the sequence motif:
  • (AA1) is an amino acid residue selected from methionine and cysteine
  • (AA2) is an amino acid residue selected from valine, glycine, proline, leucine and isoleucine;
  • (AA3) is absent or
  • (AA3) is an amino acid residue selected from lysine or ornithine, wherein the side chain amino group of said lysine or ornithine residue may be optionally substituted with one or two substituents selected from C1-3 alkyl and -CO-(Ci-3 alkyl);
  • X1 is selected from -O-, -S-, -NH- and -N(CI-6 alkyl)- or X1 is absent,
  • X2 is selected from C1-6 alkylene, C2-6 alkenylene, C2-6 alkynylene, -(C1-6 alkylene)-Y-(Ci 6 alkylene)-, - (Co-6 alkylene)-Y-carbocyclylene-Y-(Co-6 alkylene)-, and -(Co-6 alkylene)-Y-heterocyclylene-Y-(Co 6 alkylene)-, wherein each Y is independently selected from a covalent bond, -O-, -S-, -NH-, -N(CI-6 alkyl)- , -CO-, -CONH-, -CON(CI- 6 alkyl)-, -NHCO- and -N(CI-6 alkyl)CO-, and
  • X3 is selected from -CO- and -CS- or X3 is absent;
  • -X1-X2-X3- is replaced by a peptidylene moiety comprising 1 to 6 amino acid residues, wherein the N- terminal amino group of said peptidylene moiety forms a peptide bond with the carboxyl group of (AA3), and wherein the C-terminal carboxyl group of said peptidylene moiety forms the peptide bond with the amino group of (AA4);
  • (AA4) is an amino acid residue selected from methionine and cysteine;
  • (AA5) is an amino acid residue selected from valine, glycine, proline, leucine and isoleucine;
  • (AA6) is an amino acid residue selected from lysine or ornithine, wherein the side chain amino group of said lysine or ornithine residue may be optionally substituted with one or two substituents selected from C1-3 alkyl and -CO-(Ci-3 alkyl); wherein said alkylene, alkenylene, and alkynylene are each optionally substituted with one or more optional substituents selected from halogen, -CN, -OH, -0(Ci-6 alkyl), -NH2, -NH(CI-6 alkyl), and -N(CI-6 alkyl)(Ci-6 alkyl), and wherein said carbocyclylene and heterocyclylene are each optionally substituted with one or more optional substituents selected from halogen, -CN, C1-6 alkyl, -OH, -0(Ci-6 alkyl), -NH2, - NH(CI-6 alkyl), and -N(CI-6 alkyl)(Ci-6 al
  • the present invention relates to a protein-payload conjugate comprising the linker of the following sequence motif: (AA1 )-(AA2)-(AA3)-X1 -X2-X3-(AA4)-(AA5)-(AA6) wherein:
  • (AA1) is an amino acid residue selected from methionine and cysteine
  • AA2 is an amino acid residue selected from valine, glycine, proline, leucine and isoleucine;
  • (AA3) is absent or (AA3) is an amino acid residue selected from lysine or ornithine, wherein the side chain amino group of said lysine or ornithine residue may be optionally substituted with one or two substituents selected from C1-3 alkyl and -CO-(Ci-3 alkyl);
  • X1 is selected from -0-, -S-, -NH- and -N(CI-6 alkyl)- or X1 is absent,
  • X2 is selected from C1-6 alkylene, C2-6 alkenylene, C2-6 alkynylene, -(C1-6 alkylene)-Y-(Ci 6 alkylene)-, - (Co-6 alkylene)-Y-carbocyclylene-Y-(Co-6 alkylene)-, and -(Co-6 alkylene)-Y-heterocyclylene-Y-(Co-6 alkylene)-, wherein Y is selected from a covalent bond, -0-, -S-, -NH-, -N(CI-6 alkyl)-, -CO-, -CONH-, - CON(CI- 6 alkyl)-, -NHCO- and -N(CI-6 alkyl)CO-, and
  • X3 is selected from -CO- and -CS- or X3 is absent;
  • -X1-X2-X3- is replaced by a peptide moiety comprising 1 to 6 amino acid residues, wherein the N-terminal amino group of said peptide moiety forms a peptide bond with the carboxyl group of (AA3), and wherein the C-terminal carboxyl group of said peptide moiety forms the peptide bond with the amino group of (AA4);
  • (AA4) is an amino acid residue selected from methionine and cysteine;
  • (AA5) is an amino acid residue selected from valine, glycine, proline, leucine and isoleucine;
  • (AA6) is an amino acid residue selected from lysine or ornithine, wherein the side chain amino group of said lysine or ornithine residue may be optionally substituted with one or two substituents selected from C1-3 alkyl and -CO-(Ci-3 alkyl); wherein said alkylene, alkenylene, and alkynylene are each optionally substituted with one or more optional substituents selected from halogen, -CN, -OH, -O(Ci-6 alkyl), -NH2, -NH(CI-6 alkyl), and -N(CI-6 alkyl)(Ci-6 alkyl), and wherein said carbocyclylene and heterocyclylene are each optionally substituted with one or more optional substituents selected from halogen, -CN, C1-6 alkyl, -OH, -O(Ci-6 alkyl), -NH2, - NH(CI-6 alkyl), and -N(CI-6 alkyl)(Ci- 6
  • the present invention relates to a compound of the formula
  • B is a payload
  • (AA1) is an amino acid residue selected from methionine and cysteine; wherein B is attached to the N- terminal amino group of (AA1);
  • AA2 is an amino acid residue selected from valine, glycine, proline, leucine and isoleucine;
  • (AA3) is absent or (AA3) is an amino acid residue selected from lysine or ornithine, wherein the side chain amino group of said lysine or ornithine residue may be optionally substituted with one or two substituents selected from C1-3 alkyl and -CO-(Ci-3 alkyl);
  • X1 is selected from -0-, -S-, -NH- and -N(CI-6 alkyl)- or X1 is absent,
  • X2 is selected from C1-6 alkylene, C2-6 alkenylene, C2-6 alkynylene, -(C1-6 alkylene)-Y-(Ci 6 alkylene)-, - (Co-6 alkylene)-Y-carbocyclylene-Y-(Co-6 alkylene)-, and -(Co-6 alkylene)-Y-heterocyclylene-Y-(Co-6 alkylene)-, wherein Y is selected from a covalent bond, -0-, -S-, -NH-, -N(CI-6 alkyl)-, -CO-, -CONH-, - CON(CI- 6 alkyl)-, -NHCO- and -N(CI-6 alkyl)CO-, and
  • X3 is selected from -CO- and -CS- or X3 is absent;
  • -X1-X2-X3- is replaced by a peptide moiety comprising 1 to 6 amino acid residues, wherein the N-terminal amino group of said peptide moiety forms a peptide bond with the carboxyl group of (AA3), and wherein the C-terminal carboxyl group of said peptide moiety forms the peptide bond with the amino group of (AA4);
  • AA4 is an amino acid residue selected from methionine and cysteine
  • AA5 is an amino acid residue selected from valine, glycine, proline, leucine and isoleucine
  • (AA6) is an amino acid residue selected from lysine or ornithine, wherein the side chain amino group of said lysine or ornithine residue may be optionally substituted with one or two substituents selected from C1-3 alkyl and -CO-(Ci-3 alkyl); wherein said alkylene, alkenylene, and alkynylene are each optionally substituted with one or more optional substituents selected from halogen, -CN, -OH, -O(Ci-6 alkyl), -NH2, -NH(CI-6 alkyl), and -N(CI-6 alkyl)(Ci-6 alkyl), and wherein said carbocyclylene and heterocyclylene are each optionally substituted with one or more optional substituents selected from halogen, -CN, C1-6 alkyl, -OH, -0(Ci-6 alkyl), -NH2, - NH(CI-6 alkyl), and -N(CI- 6 alkyl)(Ci-
  • the present invention relates to a compound of the present invention or the protein-payload conjugate of the present invention for use in therapy or diagnosis.
  • the present invention relates to a compound of the present invention or the proteinpayload conjugate of the present invention for use in therapy or diagnosis of a disease selected from cancer, fibrosis, lymphedema, immune disease, autoimmune disease and atherosclerosis.
  • the present invention relates to a compound of the present invention or the proteinpayload conjugate of the present invention for use in therapy or diagnosis of cancer, wherein the therapeutic application requires that the payload is not retained in the kidneys.
  • the present invention relates to use of the linker of the present invention for linking a payload to a protein.
  • hydrocarbon group refers to a group consisting of carbon atoms and hydrogen atoms.
  • alkyl refers to a monovalent saturated acyclic (i.e., non-cyclic) hydrocarbon group which may be linear or branched. Accordingly, an “alkyl” group does not comprise any carbon-to- carbon double bond or any carbon-to-carbon triple bond.
  • a “C1-8 alkyl” denotes an alkyl group having 1 to 8 carbon atoms.
  • Preferred exemplary alkyl groups are methyl, ethyl, propyl (e.g., n-propyl or isopropyl), or butyl (e.g., n-butyl, isobutyl, sec-butyl, or tert-butyl).
  • alkyl preferably refers to Ci-4 alkyl, more preferably to methyl or ethyl, and even more preferably to methyl.
  • alkylene refers to an alkanediyl group, i.e. a divalent saturated acyclic hydrocarbon group which may be linear or branched.
  • a “C1-6 alkylene” denotes an alkylene group having 1 to 6 carbon atoms, and the term “Co-6 alkylene” indicates that a covalent bond (corresponding to the option “Co alkylene”) or a C1-6 alkylene is present.
  • Preferred exemplary alkylene groups are methylene (- CH2-), ethylene (e.g., -CH2-CH2- or -CH(-CH 3 )-), propylene (e.g., -CH2-CH2-CH2-, -CH(-CH 2 -CH 3 )-, -CH2- CH(-CH 3 )-, or -CH(-CH 3 )-CH2-), or butylene (e.g., -CH2-CH2-CH2-CH2-).
  • alkylene preferably refers to C1-4 alkylene (including, in particular, linear C1-4 alkylene), more preferably to methylene or ethylene, and even more preferably to methylene.
  • alkenylene refers to an alkenediyl group, i.e. a divalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon- to-carbon double bonds while it does not comprise any carbon-to-carbon triple bond.
  • a “C2-5 alkenylene” denotes an alkenylene group having 2 to 5 carbon atoms.
  • alkenylene preferably refers to C2-4 alkenylene (including, in particular, linear C2-4 alkenylene).
  • alkynylene refers to an alkynediyl group, i.e. a divalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon- to-carbon triple bonds and optionally one or more (e.g., one or two) carbon-to-carbon double bonds.
  • a “C2-5 alkynylene” denotes an alkynylene group having 2 to 5 carbon atoms.
  • alkynylene preferably refers to C2-4 alkynylene (including, in particular, linear C2-4 alkynylene).
  • carbocyclyl refers to a hydrocarbon ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings), wherein said ring group may be saturated, partially unsaturated (i.e., unsaturated but not aromatic) or aromatic.
  • “carbocyclyl” preferably refers to aryl, cycloalkyl or cycloalkenyl.
  • heterocyclyl refers to a ring group, including monocyclic rings as well as bridged ring, spiro ring and/or fused ring systems (which may be composed, e.g., of two or three rings), wherein said ring group comprises one or more (such as, e.g., one, two, three, or four) ring heteroatoms independently selected from 0, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and/or one or more N ring atoms (if present) may optionally be oxidized, wherein one or more carbon ring atoms may optionally be oxidized (i.e., to form an oxo group), and further wherein said ring group may be saturated, partially unsaturated (i.e., unsaturated but not aromatic) or aromatic.
  • each heteroatom-containing ring comprised in said ring group may contain one or two 0 atoms and/or one or two S atoms (which may optionally be oxidized) and/or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4 and that there is at least one carbon ring atom (which may optionally be oxidized) in the corresponding heteroatom-containing ring.
  • heterocyclyl preferably refers to heteroaryl, heterocycloalkyl or heterocycloalkenyl.
  • carbocyclic group has the same meaning as “carbocyclyl”, and the term “heterocyclic group” has the same meaning as “heterocyclyl”.
  • carbocyclylene refers to a carbocyclyl group, as defined herein above, but having two points of attachment (i.e., a divalent carbocyclyl group). Unless defined otherwise, “carbocyclylene” preferably refers to cycloalkylene or arylene.
  • heterocyclylene refers to a heterocyclyl group, as defined herein above, but having two points of attachment (i.e., a divalent heterocyclyl group). Unless defined otherwise, “heterocyclylene” preferably refers to heterocycloalkylene or heteroarylene.
  • aryl refers to an aromatic hydrocarbon ring group, including monocyclic aromatic rings as well as bridged ring and/or fused ring systems containing at least one aromatic ring (e.g., ring systems composed of two or three fused rings, wherein at least one of these fused rings is aromatic; or bridged ring systems composed of two or three rings, wherein at least one of these bridged rings is aromatic).
  • Aryl may, e.g., refer to phenyl, naphthyl, dialinyl (i.e., 1 ,2-dihydronaphthyl), tetralinyl (i.e., 1 ,2,3,4-tetrahydronaphthyl), indanyl, indenyl (e.g., 1 H-indenyl), anthracenyl, phenanthrenyl, 9H- fluorenyl, or azulenyl.
  • an “aryl” preferably has 6 to 14 ring atoms, more preferably 6 to 10 ring atoms, even more preferably refers to phenyl or naphthyl, and most preferably refers to phenyl.
  • arylene refers to an aryl group, as defined herein above, but having two points of attachment, i.e. a divalent aromatic hydrocarbon ring group, including monocyclic aromatic rings as well as bridged ring and/or fused ring systems containing at least one aromatic ring (e.g., ring systems composed of two or three fused rings, wherein at least one of these fused rings is aromatic; or bridged ring systems composed of two or three rings, wherein at least one of these bridged rings is aromatic).
  • “Arylene” may, e.g., refer to phenylene (e.g., phen-1 ,2-diyl, phen-1 , 3-diyl, or phen-1 ,4-diyl), naphthylene (e.g., naphthalen-1 ,2-diyl, naphthalen-1 ,3-diyl, naphthalen-1 ,4-diyl, naphthalen-1 ,5-diyl, naphthalen-1 ,6- diyl, naphthalen-1 , 7-diyl, naphthalen-2, 3-diyl, naphthalen-2, 5-diyl, naphthalen-2, 6-diyl, naphthalen-2, 7- diyl, or naphthalen-2, 8-diyl), 1 ,2-dihydronaphthylene, 1 ,2,3,4-tetrahydronaph
  • an “arylene” preferably has 6 to 14 ring atoms, more preferably 6 to 10 ring atoms, even more preferably refers to phenylene or naphthylene, and most preferably refers to phenylene (particularly phen- 1 ,4-diyl).
  • substituents such as, e.g., one, two, three or four substituents. It will be understood that the maximum number of substituents is limited by the number of attachment sites available on the substituted moiety.
  • the “optionally substituted” groups referred to in this specification carry preferably not more than two substituents and may, in particular, carry only one substituent.
  • the optional substituents are absent, i.e. that the corresponding groups are unsubstituted.
  • substituent groups comprised in the compounds of the present invention may be attached to the remainder of the respective compound via a number of different positions of the corresponding specific substituent group. Unless defined otherwise, the preferred attachment positions for the various specific substituent groups are as illustrated in the examples.
  • sequences involving amino acid residues as shown in the present application are preferably to be understood as presented from left to right as being presented from the N-terminus to the C-terminus.
  • the notation (AA1 )-(AA2) wherein (AA1) and (AA2) are each an amino acid residue is preferably understood as the amino acid residue (AA1) being connected to the amino acid residue (AA2) by a peptide bond formed between the carboxy group of (AA1) and the amino group of (AA2).
  • an amino acid residue if attached in the sequence to two other chemical moieties/groups, is herein understood as bivalent radical wherein carboxy and amino group(s) of the main chain of said amino acids are used as the points of attachment.
  • an alanine residue is to be understood as -NH-CH(CH3)-CO-.
  • an amino acid residue is attached in the sequence to only one other chemical moiety, it is preferably understood as a monovalent radical wherein either carboxy or amino group of said amino acid is used as a point of attachment.
  • an amino acid residue is connected in the sequence to a residue or moiety placed on its left side, it will be understood that it is attached via its amino group as an attachment point.
  • an amino acid residue is connected in the sequence to a residue or moiety placed on its right side, it will be understood that it is attached via its carboxy group as an attachment point.
  • an amino acid residue in the sequence has no further moiety on its left side, it will preferably be characterized by a free amino group (NH2- ).
  • a substitution with an acyl group e.g. with (C1-3 alkyl)- C0-, preferably with CH3CO- is also envisaged.
  • an amino acid residue in the sequence has no further moiety on its rights side, it will preferably be characterized by a free carboxy group (-COOH).
  • an amide group (-CONH2, -CONH(CI-3 alkyl), -CON(CI-3 alkyl)(Ci-3 alkyl)), an ester group (-COO-(Ci-3 alkyl)) or a salt (-C00-) are also envisaged.
  • amBn refers to the chemical moiety of formula -NH-CH2-P-C6H4-CH2-CO-.
  • Figure 1 Cleavage efficiency of (a) [ 111 ln]ln-NOTA-Bn-MVK(Ac), (b) [ 111 ln]ln-NOTA-Bn-MV-amBn- MVK(Ac), (c) [ 111 ln]ln-NOTA-Bn-MVK(Me) 2 -amBn-MVK(Ac) and their respective FnBPA5.1 conjugates (A) [ 111 ln]ln-NOTA-Bn-MVK(hex-FnBPA5.1), (B) [ 111 ln]ln-NOTA-Bn-MV-amBn- MVK(hex-FnBPA5.1), (C) [ 111 ln]ln-NOTA-Bn-MVK(Me) 2 -amBn-MVK(hex-FnBPA5.1 ) in the presence of BBMVs (1 st bar), or buffer only (2 nd bar). Results are shown as
  • Figure 3 In vivo metabolism study showing representative radio-chromatograms of (A) [ 111 ln]ln- NOTA-Bn-MVK(hex-FnBPA5.1), (B) [ 111 ln]ln-NOTA-Bn-MV-amBn-MVK(hex-FnBPA5.1) and (C) [ 111 ln]ln-NOTA-Bn-MVK(Me) 2 -amBn-MVK(hex-FnBPA5.1) from kidney homogenates and urine samples (24 h) in comparison to in vitro enzymatic cleavage assay (4 h).
  • Figure 5 Comparison between theoretical and experimental isotopic pattern of (A) 111 ln-NOTA-Bn-M (sum formula: C 2 5H34lnN5O8S 2 , [M + H] + ) and (B) 111 ln-NOTA-Bn-M - H 2 O (sum formula: C 2 5H3 2 lnN5O/S 2 , [M + H] + ) obtained upon BBM enzymes mediated cleavage from 111 ln- NOTA-Bn-MVK(Ac).
  • Figure 6 Comparison between theoretical and experimental isotopic pattern of 111 ln-NOTA-Bn-MV- amBn-M (Sum formula: C44H611 nNsOi 1 S3, [M + H] + ) obtained upon BBM enzymes mediated cleavage from 111 ln-NOTA-Bn-MV-amBn-MVK(Ac).
  • Figure 7 Comparison between theoretical and experimental isotopic pattern of 111 ln-NOTA-Bn-M - H 2 O (sum formula: C 2 5H3 2 lnN5O/S 2 , [M + H] + ) obtained upon BBM enzymes mediated cleavage from 111 ln-NOTA-Bn-MVK(Me) 2 -amBn-MVK(Ac).
  • the present invention relates to a linker comprising the sequence motif:
  • (AA1) is an amino acid residue selected from methionine and cysteine.
  • AA2 is an amino acid residue selected from valine, glycine, proline, leucine and isoleucine.
  • (AA3) is absent or (AA3) is an amino acid residue selected from lysine or ornithine, wherein the side chain amino group of said lysine or ornithine residue may be optionally substituted with one or two substituents selected from C1-3 alkyl and -CO-(Ci-3 alkyl).
  • X1 is selected from -0-, -S-, -NH- and -N(CI-6 alkyl)- or X1 is absent
  • X2 is selected from C1-6 alkylene, C2-6 alkenylene, C2-6 alkynylene, -(C1-6 alkylene)-Y-(Ci 6 alkylene)-, -(Co-6 alkylene)-Y-carbocyclylene-Y- (Co-6 alkylene)-, and -(Co-6 alkylene)-Y-heterocyclylene-Y-(Co-6 alkylene)-, wherein each Y is independently selected from a covalent bond, -0-, -S-, -NH-, -N(CI-6 alkyl)-, -CO-, -CONH-, -CON(CI-6 alkyl)-, -NHCO- and -N(CI-6 alkyl)CO-, and X3 is selected from -CO- and -CS- or X3
  • (AA4) is an amino acid residue selected from methionine and cysteine.
  • (AA5) is an amino acid residue selected from valine, glycine, proline, leucine and isoleucine.
  • (AA6) is an amino acid residue selected from lysine or ornithine, wherein the side chain amino group of said lysine or ornithine residue may be optionally substituted with one or two substituents selected from C1-3 alkyl and -CO-(Ci-3 alkyl).
  • said alkylene, alkenylene, and alkynylene are each optionally substituted with one or more optional substituents selected from halogen, -CN, -OH, -0(Ci-6 alkyl), -NH2, -NH(CI-6 alkyl), and -N(CI-6 alkyl)(Ci-6 alkyl), and said carbocyclylene and heterocyclylene are each optionally substituted with one or more optional substituents selected from halogen, -CN, C1-6 alkyl, -OH, -0(Ci-6 alkyl), -NH2, -NH(CI-6 alkyl), and -N(CI-6 alkyl)(Ci- 6 alkyl).
  • the linker of the present invention two enzymatic cleavage sites are present, separated by the spacer.
  • the first enzymatic cleavage site is defined as (AA1)-(AA2)-(AA3).
  • the second enzymatic cleavage site is depicted as (AA4)-(AA5)-(AA6).
  • (AA1), (AA2), (AA3), (AA4), (AA5), and (AA6) are preferably as defined herein.
  • the first and the second enzymatic cleavage site are separated by the spacer moiety -X1-X2-X3- .
  • the spacer moiety is preferably as defined herein. However, it will be conceivable to the skilled person that the spacer moiety is not particularly limited and other spacer moieties, for example involving PEG chain, can also be encompassed in the linkers of the present invention.
  • (AA1) is a methionine residue.
  • (AA2) is an amino acid residue selected from valine and glycine. More preferably, (AA2) is a valine residue.
  • (AA3) is absent.
  • X1 is selected from -0-, -S-, -NH- and -N(CI-6 alkyl)- or X1 is absent
  • X2 is selected from C1-6 alkylene, C2-6 alkenylene, C2-6 alkynylene, -(C1-6 alkylene)-Y-(Ci 6 alkylene)-, -(Co-6 alkylene)-Y- carbocyclylene-Y-(Co-6 alkylene)-, and -(Co-6 alkylene)-Y-heterocyclylene-Y-(Co-6 alkylene)-, wherein each Y is independently selected from a covalent bond, -0-, -S-, -NH-, -N(CI-6 alkyl)-, -CO-, -CONH-, -CON(Ci- 6 alkyl)-, -NHCO- and -N(CI-6 alkyl)CO-, and X3 is selected from -CO- and -CS- or
  • X1 is NH.
  • X2 is -(Co-6 alkylene)-Y-carbocyclylene-Y-(Co-6 alkylene)-, preferably X2 is -(Co-6 alkylene)-carbocyclylene-(Co-6 alkylene)-, more preferably X2 is -(C0-3 alkylene)- carbocyclylene-(Co-3 alkylene)-, even more preferably X2 is -(C0-3 alky lene)-arylene-(Co 3 alkylene)-, even more preferably X2 is -(C1-3 alkylene)-arylene-(C 1 3 alkylene)-, even more preferably X2 is -(C1-3 alkylene)- phenylene-(Ci-3 alkylene)-, most preferably X2 is -CH2-phenylene-CH2-. More preferably, X1 is NH, X2 is -(Co-6 alkylene)-Y-(Co-6 alky
  • X1 is NH
  • X2 is -(Co-6 alkylene)-carbocyclylene-(Co-6 alkylene)- and X3 is -CO-.
  • X1 is NH
  • X2 is -(Co-3 alkylene)-carbocyclylene-(Co-3 alkylene)-
  • X3 is -CO-.
  • X1 is NH
  • X2 is -(C1-3 alkylene)-phenylene-(Ci 3 alkylene)-
  • X3 is -CO-.
  • X1 is NH
  • X2 is -CH2-phenylene-CH2-
  • X3 is -CO-.
  • (AA4) is a methionine residue.
  • (AA5) is an amino acid residue selected from valine and glycine. More preferably, (AA5) is a valine residue.
  • (AA6) is a lysine residue.
  • (AA1) is a methionine residue and (AA2) is a valine or a proline.
  • (AA2) is a valine residue.
  • (AA1) is a cysteine residue and (AA2) is a glycine residue.
  • (AA3) is an amino acid residue selected from lysine or ornithine, wherein the side chain amino group of said lysine or ornithine residue may be optionally substituted with one or two substituents selected from C1-3 alkyl and -CO-(Ci-3 alkyl).
  • C1-3 alkyl groups are methyl and ethyl.
  • the side chain amino group of said lysine residue or said ornithine residue is optionally substituted with one methyl group, with two methyl groups or with one acetyl group.
  • -X1-X2-X3- is replaced by a peptidylene moiety comprising 1 to 6 amino acid residues, wherein the N-terminal amino group of said peptidylene moiety forms a peptide bond with the carboxyl group of (AA3), and wherein the C-terminal carboxyl group of said peptidylene moiety forms the peptide bond with the amino group of (AA4).
  • the peptidylene moiety comprises glycine residue(s) and/or serine residue(s).
  • (AA4) is a methionine and (AA5) is a valine residue or a proline residue, preferably (AA5) is a valine residue.
  • (AA4) is a cysteine residue and (AA5) is a glycine residue.
  • (AA1) is a methionine residue
  • (AA2) is a valine residue
  • (AA3) is absent.
  • (AA1) is a methionine residue
  • (AA2) is a valine residue
  • (AA3) is a lysine residue
  • the side chain amino group of said lysine residue may be optionally substituted with one or two substituents selected from C1-3 alkyl and -CO-(Ci-3 alkyl).
  • Preferred C1-3 alkyl groups are methyl and ethyl.
  • the side chain amino group of said lysine residue or said ornithine residue is optionally substituted with one methyl group, with two methyl groups or with one acetyl group.
  • (AA4) is a methionine residue
  • (AA5) is a valine residue
  • (AA6) is a lysine residue.
  • the first enzymatic cleavage site is defined as follows: (AA1) is a methionine residue, (AA2) is a valine residue, and (AA6) is as defined hereinabove, preferably (AA6) is a lysine residue.
  • (AA4)- (AA5) sequence can also be replaced with -glycyl-phenylalanyl- sequence.
  • (AA4) is a methionine residue and (AA5) is a valine residue, or wherein (AA4) is a cysteine residue and (AA5) is a glycine residue, or wherein (AA4) is a glycine residue and (AA5) is a phenylalanyl residue.
  • the linker of the present invention can be comprised in a larger molecule, for example a protein conjugate, a protein-payload conjugate or a payload-conjugate.
  • the skilled person will then consider the linker as a divalent moiety linking two conjugated moieties (for example a payload or a protein).
  • the linker may also be conjugated to one conjugated moiety only.
  • linker as a monovalent moiety attached to one conjugated moiety
  • the linker and said conjugated moieties are linked in a manner that is sufficiently stable under physiological conditions, preferably in blood plasma, to physically and/or chemically co n nect/allocate/bi nd the components together until they reach the target site.
  • the linker is to be so connected to the conjugated moieties that the enzymatic cleavage at the renal brush border, i.e.
  • enzymatic cleavage with neprilysin at the first enzymatic cleavage site and/or at the second enzymatic cleavage site is substantially not affected by the way the conjugated moieties are connected to the linker.
  • the linker can be conjugated to one or two conjugated moieties.
  • each conjugated moiety can be directly conjugated to any amino acid residue within the linker of the invention (or to any suitable chemical group therein), using any chemical coupling known in the art or described herein suitable for the conjugation of the payload moiety preferably to an amino acid residue (e.g., an amino acid side chain). Accordingly, said coupling may result in one or more chemical groups spaced between the conjugated moiety and the amino acid (e.g., amino acid side chain) of the linker of the invention, which groups form as a result of the coupling reaction, as known in the art.
  • the linker is to be connected to the first conjugated moiety through (AA1), preferably through the amino group of (AA1), and/or the linker is to be connected to the second conjugated moiety through (AA6), preferably through the side chain amino group of (AA6) or through carboxy group of (AA6), more preferably through the side chain amino group of (AA6).
  • the linker of the present invention may also encompass more than two enzymatic cleavage sites.
  • the present invention relates to a linker comprising the sequence motif:
  • AA1, (AA2), (AA3), (AA4), (AA5), (AA6), and X1-X2-X3 are as defined herein, m is selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, and 10, preferably from 0, 1 , 2, and 3.
  • Each moiety (AA1’), (AA2’), (AA3’), and -XT- X2’-X3’- are independently defined as for (AA4), (AA5), (AA6), and -X1-X2-X3-, respectively.
  • (AA1) is a methionine residue
  • (AA2) is a valine residue
  • (AA3) is a lysine residue
  • the present invention further relates to an embodiment, wherein (AA1’) is a methionine residue, (AA2’) is a valine residue, and (AA3’) is a lysine residue, or wherein (AA1’) is a cysteine residue, (AA2’) is a glycine residue, and (AA3’) is a lysine residue, or wherein (AA1’) is a glycine residue, (AA2’) is a phenylalanine residue, and (AA3’) is a lysine residue.
  • the linker as defined herein is conjugated to a first conjugated moiety, the first conjugated moiety being the payload moiety, and to a second conjugated moiety, the second conjugated moiety being the protein.
  • the present invention relates to a protein-payload conjugate comprising the linker of the following sequence motif:
  • (AA1) is an amino acid residue selected from methionine and cysteine.
  • AA2 is an amino acid residue selected from valine, glycine, proline, leucine and isoleucine.
  • (AA3) is absent or (AA3) is an amino acid residue selected from lysine or ornithine, wherein the side chain amino group of said lysine or ornithine residue may be optionally substituted with one or two substituents selected from C1-3 alkyl and -CO-(Ci-3 alkyl).
  • X1 is selected from -0-, -S-, -NH- and -N(CI-6 alkyl)- or X1 is absent
  • X2 is selected from C1-6 alkylene, C2-6 alkenylene, C2-6 alkynylene, -(C1-6 alkylene)-Y-(Ci 6 alkylene)-, -(Co-6 alkylene)-Y-carbocyclylene-Y- (Co-6 alkylene)-, and -(Co-6 alkylene)-Y-heterocyclylene-Y-(Co 6 alkylene)-, wherein each Y is independently selected from a covalent bond, -0-, -S-, -NH-, -N(CI-6 alkyl)-, -CO-, -CONH-, -CON(CI-6 alkyl)-, -NHCO- and -N(CI-6 alkyl)CO-, and X3 is selected from -CO- and -CS- or X3
  • (AA5) is an amino acid residue selected from valine, glycine, proline, leucine and isoleucine.
  • (AA6) is an amino acid residue selected from lysine or ornithine, wherein the side chain amino group of said lysine or ornithine residue may be optionally substituted with one or two substituents selected from C1-3 alkyl and -CO-(Ci-3 alkyl).
  • said alkylene, alkenylene, and alkynylene are each optionally substituted with one or more optional substituents selected from halogen, -CN, -OH, - 0(Ci-6 alkyl), -NH2, -NH(CI-6 alkyl), and -N(CI-6 alkyl)(Ci-6 alkyl), and said carbocyclylene and heterocyclylene are each optionally substituted with one or more optional substituents selected from halogen, -CN, C1-6 alkyl, -OH, -0(Ci-6 alkyl), -NH2, -NH(CI-6 alkyl), and -N(CI-6 alkyl)(Ci-6 alkyl).
  • (AA1) is a methionine residue.
  • (AA2) is an amino acid residue selected from valine and glycine. More preferably, (AA2) is a valine residue.
  • (AA3) is absent.
  • X1 is selected from -0-, -S-, -NH- and -N(CI-6 alkyl)- or X1 is absent
  • X2 is selected from C1-6 alkylene, C2-6 alkenylene, C2-6 alkynylene, -(C1-6 alkylene)-Y-(Ci 6 alkylene)-, -(Co-6 alkylene)-Y- carbocyclylene-Y-(Co-6 alkylene)-, and -(Co-6 alkylene)-Y-heterocyclylene-Y-(Co 6 alkylene)-, wherein each Y is independently selected from a covalent bond, -0-, -S-, -NH-, -N(CI-6 alkyl)-, -CO-, -CONH-, -CON(Ci- 6 alkyl)-, -NHCO- and -N(CI-6 alkyl)CO-, and X3 is selected from -CO- and -CS- or
  • X1 is NH.
  • X2 is -(Co-6 alkylene)-Y-carbocyclylene-Y-(Co-6 alkylene)-, preferably X2 is -(Co-6 alkylene)-carbocyclylene-(Co 6 alkylene)-, more preferably X2 is -(C0-3 alkylene)- carbocyclylene-(Co-3 alkylene)-, even more preferably X2 is -(C0-3 alky lene)-arylene-(Co 3 alkylene)-, even more preferably X2 is -(C1-3 alkylene)-arylene-(C 1 3 alkylene)-, even more preferably X2 is -(C1-3 alkylene)- phenylene-(Ci-3 alkylene)-, most preferably X2 is -CH2-phenylene-CH2-. More preferably, X1 is NH, X2 is -(Co-6 alkylene)-Y-(Co-6 alky
  • X1 is NH
  • X2 is -(Co-6 alkylene)-carbocyclylene-(Co 6 alkylene)- and X3 is -CO-.
  • X1 is NH
  • X2 is -(Co-3 alkylene)-carbocyclylene-(Co 3 alkylene)-
  • X3 is -CO-.
  • X1 is NH
  • X2 is -(C1-3 alkylene)-phenylene-(Ci 3 alkylene)-
  • X3 is -CO-.
  • X1 is NH
  • X2 is -CH2-phenylene-CH2-
  • X3 is -CO-.
  • (AA4) is a methionine residue.
  • (AA5) is an amino acid residue selected from valine and glycine. More preferably, (AA5) is a valine residue
  • (AA6) is a lysine residue.
  • (AA1) is a methionine and (AA2) is a valine or a proline.
  • (AA2) is a valine.
  • (AA1) is a cysteine and (AA2) is a glycine.
  • (AA3) is an amino acid residue selected from lysine or ornithine, wherein the side chain amino group of said lysine or ornithine residue may be optionally substituted with one or two substituents selected from C1-3 alkyl and -CO-(Ci-3 alkyl).
  • C1-3 alkyl groups are methyl and ethyl.
  • the side chain amino group of said lysine residue or said ornithine residue is optionally substituted with one methyl group, with two methyl groups or with one acetyl group.
  • -X1-X2-X3- is replaced by a peptidylene moiety comprising 1 to 6 amino acid residues, wherein the N-terminal amino group of said peptidylene moiety forms a peptide bond with the carboxyl group of (AA3), and wherein the C-terminal carboxyl group of said peptidylene moiety forms the peptide bond with the amino group of (AA4).
  • the peptidylene moiety comprises glycine residue(s) and/or serine residue(s).
  • (AA4) is a methionine and (AA5) is a valine residue or a proline residue, preferably (AA5) is a valine residue.
  • (AA4) is a cysteine residue and (AA5) is a glycine residue.
  • (AA1) is a methionine residue
  • (AA2) is a valine residue
  • (AA3) is absent.
  • (AA1) is a methionine residue
  • (AA2) is a valine residue
  • (AA3) is a lysine residue
  • the side chain amino group of said lysine residue may be optionally substituted with one or two substituents selected from Ci- 3 alkyl and -CO-(Ci-3 alkyl).
  • Preferred C1-3 alkyl groups are methyl and ethyl.
  • the side chain amino group of said lysine residue or said ornithine residue is optionally substituted with one methyl group, with two methyl groups or with one acetyl group.
  • (AA4) is a methionine residue
  • (AA5) is a valine residue
  • (AA6) is a lysine residue.
  • the first enzymatic cleavage site is defined as follows: (AA1) is a methionine residue, (AA2) is a valine residue, and (AA6) is as defined hereinabove, preferably (AA6) is a lysine residue.
  • (AA4)- (AA5) sequence can also be replaced with -glycyl-phenylalanyl- sequence.
  • (AA4) is a methionine residue and (AA5) is a valine residue, or wherein (AA4) is a cysteine residue and (AA5) is a glycine residue, or wherein (AA4) is a glycine residue and (AA5) is a phenylalanyl residue.
  • the conjugated moiety (a payload or a protein) can be directly conjugated to any amino acid residue within the linker comprised therein (or to any suitable chemical group therein), using any chemical coupling known in the art or described herein suitable for the conjugation of the payload moiety preferably to an amino acid residue (e.g., an amino acid side chain).
  • said coupling may result in one or more chemical groups spaced between the conjugated moiety and the amino acid (e.g., amino acid side chain) of the linker of the invention, which groups form as a result of the coupling reaction, as known in the art.
  • the linker is to be connected to the first conjugated moiety, herein the payload moiety, through (AA1), preferably through the amino group of (AA1), and/or the linker is to be connected to the second conjugated moiety, in particular the protein, through (AA6), preferably through the side chain amino group of (AA6) or through carboxy group of (AA6), more preferably through the side chain amino group of (AA6).
  • each conjugated moiety may be conjugated to any amino acid residue within the linker of the invention indirectly, that is, via an additional group.
  • the payload is conjugated to an additional group, which additional group is conjugated to an amino acid residue within the linker of the invention.
  • the conjugation between the payload and the linker group and between the linker group and an amino acid residue or chemical group of the protein of the invention may be any conjugation method and/or compound suitable for effecting such conjugation as described herein or as otherwise known in the art.
  • the conjugation of the payload moiety may be directed to any amino acid residue within the linker of the invention.
  • the payload moiety may be directly or indirectly conjugated to an amino acid residue within the linker of the invention.
  • the payload moiety may be directly or indirectly conjugated to an amino acid residue that is at an N-terminal end of the linker of the invention (i.e., to the amino group of (AA1) amino acid residue) or C-terminal end of the linker of the invention (i.e., to (AA6) amino acid residue of the invention, preferably to the carboxy group thereof or to the side chain amino group thereof, more preferably to the side chain amino group thereof).
  • the payload moiety may be directly or indirectly conjugated to an amino acid residue that is at an N-terminal end of the linker of the invention (i.e., to the amino group of (AA1) amino acid residue).
  • the payload moiety is conjugated to the amino group of (AA1) amino acid residue via amide bond.
  • the payload moiety is conjugated to the amino group of (AA1) via a moiety derived from thiourea.
  • the payload moiety may be directly or indirectly conjugated to an internal amino acid residue within the linker of the invention.
  • an internal residue or internal chemical group references an amino acid residue or chemical group of the linker of the invention that is not at the terminus of a peptide chain.
  • conjugation methods may require the chemical modification of one or both sites of conjugation (e.g., modification of an amino acid residue and/or modification of the payload moiety). Accordingly, the present invention also encompasses chemical modification of the linker of the invention.
  • the conjugation of the protein may be directed to any amino acid residue within the linker of the invention.
  • the payload moiety may be directly or indirectly conjugated to an amino acid residue within the linker of the invention.
  • the protein (the protein moiety) may be directly or indirectly conjugated to an amino acid residue that is at an N-terminal end of the linker of the invention (i.e., to the amino group of (AA1) amino acid residue) or C-terminal end of the linker of the invention (i.e., to (AA6) amino acid residue of the invention, preferably to the carboxy group thereof or to the side chain amino group thereof, more preferably to the side chain amino group thereof).
  • the protein may be directly or indirectly conjugated to an amino acid residue that is at an C- terminal end of the linker of the invention (i.e., to (AA6) amino acid residue of the invention, preferably to the carboxy group thereof or to the side chain amino group thereof, more preferably to the side chain amino group thereof).
  • the payload is attached to the N-terminal amino group of (AA1).
  • the protein is attached to the side chain amino group of (AA6).
  • the protein is attached to the side chain amino group of (AA6) through the following moiety: wherein P is a protein, and x is an integer from 2 to 10, preferably wherein x is 5.
  • the protein can be comprised in said moiety through the attachment through the side chain of the cysteine residue.
  • the attachment of the protein can be performed through maleimide-thiol chemistry, known to the skilled person, as shown in the following:
  • the (AA6) residue for the purpose of conjugation is so modified that the side chain amino group of the lysine residue or the ornithine residue is replaced with an azido moiety (-N3).
  • said azido moiety can be coupled to an alkyne moiety or cyanate moiety affording a five-membered ring through a reaction referred to as click chemistry.
  • the so modified payload-linker conjugate can be coupled to a protein modified to include an alkyne or a cyanate moiety in order to afford a protein-payload conjugate of the invention.
  • cysteine in the protein sequence may be included at the terminus of the protein sequence, preferably at the N-terminus. Further preferably, the cysteine residue may be included with a further spacer, preferably comprising glycine and/or lysine residue(s).
  • a further spacer preferably comprising glycine and/or lysine residue(s).
  • One preferred sequence motif that can be appended to the N-terminus of the protein sequence is Cys-Gly-Gly-Gly (SEQ ID NO: 1).
  • the protein-payload conjugate of the present invention is of the formula:
  • P is a protein.
  • the attachment of the payload and/or of the protein is as described herein.
  • the present invention relates to a payload-linker conjugate of the formula
  • B is a payload.
  • (AA1) is an amino acid residue selected from methionine and cysteine; wherein B is attached to the N- terminal amino group of (AA1).
  • AA2 is an amino acid residue selected from valine, glycine, proline, leucine and isoleucine.
  • (AA3) is absent or (AA3) is an amino acid residue selected from lysine or ornithine, wherein the side chain amino group of said lysine or ornithine residue may be optionally substituted with one or two substituents selected from C1-3 alkyl and -CO-(Ci-3 alkyl).
  • X1 is selected from -0-, -S-, -NH- and -N(CI-6 alkyl)- or X1 is absent
  • X2 is selected from C1-6 alkylene, C2-6 alkenylene, C2-6 alkynylene, -(C1-6 alkylene)-Y-(Ci 6 alkylene)-, -(Co-6 alkylene)-Y-carbocyclylene-Y- (Co-6 alkylene)-, and -(Co-6 alkylene)-Y-heterocyclylene-Y-(Co 6 alkylene)-, wherein Y is selected from a covalent bond, -0-, -S-, -NH-, -N(CI-6 alkyl)-, -CO-, -CONH-, -CON(CI-6 alkyl)-, -NHCO- and -N(CI-6 alkyl)CO-, and X3 is selected from -CO- and -CS- or X3 is absent
  • (AA4) is an amino acid residue selected from methionine and cysteine.
  • (AA5) is an amino acid residue selected from valine, glycine, proline, leucine and isoleucine;
  • (AA6) is an amino acid residue selected from lysine or ornithine, wherein the side chain amino group of said lysine or ornithine residue may be optionally substituted with one or two substituents selected from C1-3 alkyl and -CO-(Ci-3 alkyl).
  • said alkylene, alkenylene, and alkynylene are each optionally substituted with one or more optional substituents selected from halogen, -CN, -OH, -0(Ci-6 alkyl), -NH2, -NH(CI-6 alkyl), and -N(CI-6 alkyl)(Ci-6 alkyl), and said carbocyclylene and heterocyclylene are each optionally substituted with one or more optional substituents selected from halogen, -CN, C1-6 alkyl, -OH, -0(Ci-6 alkyl), -NH 2 , -NH(CI-6 alkyl), and -N(CI-6 alkyl)(Ci-6 alkyl).
  • (AA1) is a methionine residue.
  • (AA2) is an amino acid residue selected from valine and glycine. More preferably, (AA2) is a valine residue.
  • (AA3) is absent.
  • X1 is selected from -0-, -S-, -NH- and -N(CI-6 alkyl)- or X1 is absent
  • X2 is selected from C1-6 alkylene, C2-6 alkenylene, C2-6 alkynylene, -(C1-6 alkylene)-Y-(Ci 6 alkylene)-, -(Co-6 alkylene)-Y- carbocyclylene-Y-(Co-6 alkylene)-, and -(Co-6 alkylene)-Y-heterocyclylene-Y-(Co 6 alkylene)-, wherein each Y is independently selected from a covalent bond, -0-, -S-, -NH-, -N(CI-6 alkyl)-, -CO-, -CONH-, -CON(Ci- 6 alkyl)-, -NHCO- and -N(CI-6 alkyl)CO-, and X3 is selected from -CO- and -CS- or
  • X1 is NH.
  • X2 is -(Co-6 alkylene)-Y-carbocyclylene-Y-(Co-6 alkylene)-, preferably X2 is -(Co-6 alkylene)-carbocyclylene-(Co 6 alkylene)-, more preferably X2 is -(Co-3 alkylene)- carbocyclylene-(Co-3 alkylene)-, even more preferably X2 is -(Co-3 alky lene)-arylene-(Co 3 alkylene)-, even more preferably X2 is -(C1-3 alkylene)-arylene-(C 1 3 alkylene)-, even more preferably X2 is -(C1-3 alkylene)- phenylene-(Ci-3 alkylene)-, most preferably X2 is -CH2-phenylene-CH2-.
  • X1 is NH
  • X2 is -(Co-6 alkylene)-Y-carbocyclylene-Y-(Co-6 alkylene)-
  • X3 is -CO-.
  • X1 is NH
  • X2 is -(Co-6 alkylene)-carbocyclylene-(Co 6 alkylene)- and X3 is -CO-.
  • X1 is NH
  • X2 is -(C0-3 alkylene)-carbocyclylene-(Co 3 alkylene)-
  • X3 is -CO-.
  • X1 is NH
  • X2 is -(C1-3 alkylene)-phenylene-(Ci 3 alkylene)-
  • X3 is -CO-.
  • X1 is NH
  • X2 is -CH2-phenylene-CH2-
  • X3 is -CO-.
  • (AA4) is a methionine residue.
  • (AA5) is an amino acid residue selected from valine and glycine. More preferably, (AA5) is a valine residue
  • (AA6) is a lysine residue.
  • (AA1) is a methionine and (AA2) is a valine or a proline.
  • (AA2) is a valine.
  • (AA1) is a cysteine and (AA2) is a glycine.
  • (AA3) is an amino acid residue selected from lysine or ornithine, wherein the side chain amino group of said lysine or ornithine residue may be optionally substituted with one or two substituents selected from C1-3 alkyl and -CO-(Ci-3 alkyl).
  • Preferred C1-3 alkyl groups are methyl and ethyl.
  • the side chain amino group of said lysine residue or said ornithine residue is optionally substituted with one methyl group, with two methyl groups or with one acetyl group.
  • -X1-X2-X3- is replaced by a peptidylene moiety comprising 1 to 6 amino acid residues, wherein the N-terminal amino group of said peptidylene moiety forms a peptide bond with the carboxyl group of (AA3), and wherein the C-terminal carboxyl group of said peptidylene moiety forms the peptide bond with the amino group of (AA4).
  • the peptidylene moiety comprises glycine residue(s) and/or serine residue(s).
  • (AA4) is a methionine and (AA5) is a valine residue or a proline residue, preferably (AA5) is a valine residue.
  • (AA4) is a cysteine residue and (AA5) is a glycine residue.
  • (AA1) is a methionine residue
  • (AA2) is a valine residue
  • (AA3) is absent.
  • (AA1) is a methionine residue
  • (AA2) is a valine residue
  • (AA3) is a lysine residue
  • the side chain amino group of said lysine residue may be optionally substituted with one or two substituents selected from Ci- 3 alkyl and -CO-(Ci-3 alkyl).
  • Preferred C1-3 alkyl groups are methyl and ethyl.
  • the side chain amino group of said lysine residue or said ornithine residue is optionally substituted with one methyl group, with two methyl groups or with one acetyl group.
  • (AA4) is a methionine residue
  • (AA5) is a valine residue
  • (AA6) is a lysine residue.
  • the first enzymatic cleavage site is defined as follows: (AA1) is a methionine residue, (AA2) is a valine residue, and (AA6) is as defined hereinabove, preferably (AA6) is a lysine residue.
  • (AA4)-(AA5) sequence can also be replaced with -glycylphenylalanyl- sequence.
  • (AA4) is a methionine residue and (AA5) is a valine residue, or wherein (AA4) is a cysteine residue and (AA5) is a glycine residue, or wherein (AA4) is a glycine residue and (AA5) is a phenylalanyl residue.
  • the payload B as in the protein-payload conjugate of the present invention or in payload-linker conjugate of the present invention is not particularly limited.
  • the payload is a moiety not exceeding 5 kDa. More preferably, the payload is a moiety not exceeding 2 kDa.
  • the linker of the present invention is cleavable at the renal brush border by the enzymes present therein, in particular by neprilysin.
  • the protein-payload conjugate of the present invention is cleavable at the renal brush border by the enzymes present therein, in particular by neprilysin, affording (at least) a cleavage product comprising the payload, and a cleavage product comprising the protein.
  • the payload is to be so selected that, upon enzymatic cleavage of the linker by the enzymes of the renal brush border, the generated product of cleavage comprising the payload (the payload moiety) can be excreted from the kidneys.
  • said product of cleavage comprising the payload (the payload moiety) must preferably be characterized by faster excretion kinetics from the kidneys when compared to the protein-payload conjugate that gives raise to it upon enzymatic cleavage.
  • the cleavage of the payload-protein conjugate at the renal brush border must lead to a reduced renal retention of the payload-containing species when compared to the renal retention of the protein-payload conjugate that cannot be cleaved at the renal brush border by the enzymes present therein, in particular by neprilysin.
  • payloads that include a radionuclide, preferably selected from 225 Ac, 213 Bi, 67 Cu, 90 Y, 111 ln, 131 l, 161 Tb, 169 Er, and 177 Lu. Illustrative examples of such metabolites are shown in Figures 5 to 7.
  • the payload is a biologically active molecule (BAM) or an imaging agent.
  • BAM biologically active molecule
  • the payload is a biologically active molecule (BAM).
  • BAM biologically active molecule
  • the biologically active molecule as encompassed by the present invention is selected from the group consisting of cytostatic agent, cytotoxic agent, cytokine, transcription factor inhibitor, proteasome and protease inhibitor, apoptosis modulator, cell cycle modulator, angiogenesis inhibitor, hormone or hormone derivative, photodynamic therapy molecule, nano- and microparticle for thermoablation therapy, radionuclide, miRNA, siRNA and immunomodulatory antigen molecule.
  • the preferably cytostatic agent is selected from Doxorubicin, Paclitaxel, Chlorambucil, Topotecan and Vincristine.
  • cytokine is selected from lnterleukin-2, lnterleukin-7, Interferon-y and tumor necrosis factors.
  • cytokines for use as payload according to the present invention are, for example, interleukin 2, interleukin 7, interferon a-2a, interferon a-2b, interferon-la, interferon-ip, interferon y-ip, tumor necrosis factor, and any derivatives thereof.
  • transcription factor inhibitor is preferably selected from Curcumin, Ribavirin and Genistein.
  • Further suitable transcription factor inhibitors for use as payload according to the present invention are, for example compounds that inhibit activation of N F-KB such as curcumin (diferuloylmethane) epigallocatechin-3-gallate (EGCG; green tea polyphenols), phenanthrolines, pyrrolinedithiocarba- mate (PDTC), quercetin, tepoxaline (5-(4- chlorophenyl)-N-hydroxy-(4- methoxyphenyl)-N-m ethyl-1 H-pyrazole-3-propan- amide), PMC (2,2,5,7,8-pentamethyl-6- hydroxychromane), benzyisocyanate, resveratol, genistein, lupeol, lycopene, panepoxydone, epoxyquinomicin C, dehydroxymethylepoxy-
  • proteasome and protease inhibitors are preferably selected from peptide aldehydes: ALLnL (N-acetyl-leucinyl-leucynil-norleucynal, MG101), LLM (N-acetyl-leucinyl-leucynil- methional), Z-LLnV (carbobenzoxyl-leucinyl-leucyni I- norvalinal, MG115), Z-LLL (carbobenzoxyl- leucinyl-leucynil-leucynal, MG132), boronic acid derivatives, e.g.
  • APNE N-acetyl-DL-phenylalanine-beta-
  • apoptosis modulator is preferably selected from Imatinib, Erlotinib and Bryostatin.
  • apoptosis modulators for use as payload according to the present invention are, for example, farnesyl transferase inhibitors, e.g. R115777, SCH66336, BMS214662, Imatinib, 17- AAG, EGFR inhibitors, e.g. ZD1839, ZD647, BIBW 2992, or erlotinib, MEK inhibitors, e.g. PD 032590, RAF inhibitors e.g. BAY43-9006, PKG inhibitors, e.g.
  • Especially preferred cell cycle modulators for use accord ing to the present invention are, for example, flavopiridol, bryostain-1 , roscovitine, BMS-387032, perifosine, or lovastatin.
  • cell cycle modulator is selected from Flavopiridol and Roscovitine.
  • angiogenesis inhibitor is preferably selected from Endostatin, Celexocib, ADH-1 (exherin) and Sunitinib.
  • suitable angiogenesis inhibitors for use as payload according to the present invention are, for example thalidomide, endostatin, celecoxib, ABT-510, combrestatin A4, dalteparin, dimethyl-xanthenone acetic acid, lenalidomide, LY317615 (enzastaurin), PPI-2458, ADH-1 (exherin), AG- 013736, AMG-706, AZD2171 , Bay 43-9006 (sorafenib), BMS-582664, CH IR-265, GW786034 (pazopanib), PI-88, PTK787/ZK 222584 (vatalanib), RAD001 (everolimus), SU 11248 (sunitinib), suramin, XL184, ZD6474, ATM-16
  • hormone and hormone derivative is preferably selected from Flutamide, Fosfestrol, Tamoxifen and Relaxin.
  • suitable hormones or hormone derivatives for use as payload according to the present invention are, for example, aminogluthemid, buserilin, cyproteronacetate, droloxifen, ethinylestradiol, flutamid, formesta, fosfestrol, gestonoroncaproate, goserilin, leuprolein, lynestrenol, medrogeston, medroxyprogesteronacetate, megestrolactetate, octreotid, relaxin, tamoxifen, toremifin, triptorelin, anastrazole, exemestane, or letrozole.
  • suitable miRNAs and siRNAs are, for example, those that are specific for CD40, CD80 and CD86, and also any agents that target clustered regularly interspaced short palindromic repeat (CRISPR) components for gene-editing purposes, or antigens that modulate the immune system, for example, insulin-associated antigens, P31 , whole gliadin, myelin oligodendrocyte glycoprotein (preferably amino acids 35-55), proteolipid protein 1 (preferably amino acids 139-151 and 178-191), Factor V (preferably amino acids 75-89, 1723-1737 and 2191-2210).
  • CRISPR clustered regularly interspaced short palindromic repeat
  • the payload may be a radionuclide.
  • the protein-payload conjugates wherein the payload is a radionuclide may be characterized by undesirable renal retention.
  • a cleavage product comprising radionuclide is generated that may be characterized by the reduced renal retention compared to the protein-payload conjugate wherein the payload is radionuclide.
  • radionuclides are preferably not bound directly to the linker of the present invention but are comprised in a complexing agent or chelator which can be conjugated as payload to the fibronectin binding peptide of the invention.
  • a reference to radionuclide as payload is meant to be understood as a chelator charged with a radionuclide.
  • Chelators that may be conjugated to the fibronectin binding peptide of the invention include, but are not limited to, 1,4,7,10- tetraazacyclododecane-1 ,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic (DTPA), desferrioxamine (DFO) and triethylenetetramine (TETA), 1 ,4,8,11 -tetraazabicyclo[6.6.2]hexadecane- 4,11 -diacetic acid (CB-TE2A); ethylenediaminetetraacetic acid (EDTA); ethylene glycolbis(2-aminoethyl)- N,N,N',N'-tetraacetic acid (EGTA); 1 ,4, 8,11-tetraazacyclotetradecane-1 ,4,8,11 -tetraacetic acid (TETA); ethylenebis-(2-4 hydroxy-phenylglycine) (EHPG); 5-CI-
  • TETMA 1 .4.7.10-tetra(methyl tetraacetic acid), benzo-TETMA, where TETMA is 1 ,4,8,11- tetraazacyclotetradecane-1 ,4,8,11-(methyl tetraacetic acid); derivatives of 1 ,3- propylenediaminetetraacetic acid (PDTA); triethylenetetraaminehexaacetic acid (TTHA); derivatives of
  • the payload may comprise more than one chelator.
  • chelators can be selected from the group consisting of cyclic DPT A (diethylene triaminepentaacetic acid ) anhydride, ethylenediaminetetraacetic acid (EDTA), DOTA (1 ,4,7,10- tetraazacyclododecane-1 ,4,7,10- tetraacetic acid), and OTA (1 ,4,7-triazonane-l,4,7-triacetic acid).
  • EDTA ethylenediaminetetraacetic acid
  • OTA 1,4,7,10- tetraazacyclododecane-1 ,4,7,10- tetraacetic acid
  • OTA 1,4,7-triazonane-l,4,7-triacetic acid
  • radionuclide is preferably selected from 225 Ac, 213 Bi,
  • radionuclide for use in the present invention is 177 Lu or 225 Ac.
  • the radionuclide can be selected from the group consisting of 161 Tb and 213 Bi. Further suitable radionuclides include those emitting p +
  • the biologically active molecule is selected from the group consisting of Paclitaxel, Chlorambucil, Endostatin, Sunitinib, lnterleukin-7, 177 Lu, and 225 Ac.
  • the payload can be an imaging agent.
  • the imaging agent may be any imaging agent known in the art.
  • the protein-payload conjugate of the invention, wherein the payload is an imaging agent may be used in the visualization of cells and/or tissues in vitro and/or in vivo.
  • an imaging agent is selected that is functional under the imaging conditions. Similar criteria apply for imaging agents that are used for the visualization of target cells and/or tissues in vivo.
  • additional care has to be taken that the imaging agent is biocompatible, e.g. has no toxic or otherwise detrimental effects on the subject that compound of the invention comprising the imaging agent is administered to.
  • imaging agent refers to any element, molecule, functional group, compound, fragments thereof or moiety that facilitates detection of an agent (e.g., the compound of the invention) to which it is conjugated.
  • imaging agents include, but are not limited to: various ligands, radionuclides, fluorescent dyes (for specific exemplary fluorescent dyes, see below), chemiluminescent agents (such as, for example, acridinum esters, stabilized dioxetanes, and the like), bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper, platinum, etc.) nanoclusters, paramagnetic metal ions, enzymes (for specific examples of enzymes, see below), colorimetric labels (such as, for example, dyes, colloidal gold, and the like), biotin, dioxigenin, haptens, and proteins for which antis
  • the imaging agent may be conjugated to the linker of the proteinpayload conjugate of the invention directly or indirectly via an additional group, or that the imaging agent may be comprised in a molecule that is conjugated to the linker of the invention.
  • the skilled person would understand which imaging agents may be conjugated directly to the linker of the invention and which imaging agents need to be embedded in a molecule that can be conjugated to the compound of the invention or a linker.
  • the imaging agent is a radionuclide
  • the radionuclide is preferably embedded in a molecule (e.g. chelated by a chelator being part of the said molecule) that can be conjugated to the linker of the invention or comprised in the protein-payload conjugate of the invention.
  • the imaging agent comprises a radionuclide, a fluorescent dye, a chemiluminescent agent, a bioluminescent agent, a spectrally resolvable inorganic fluorescent semiconductor nanocrystal, a metal nanoparticle, a nanocluster, a paramagnetic metal ion, an enzyme, a colorimetric label, biotin, dioxigenin, a hapten or a protein.
  • the imaging agent comprises a radionuclide, fluorescent dye, a chemiluminescent agent, or a bioluminescent agent.
  • the imaging agent is selected from the group consisting of radionuclide, MRI active compound, ultrasound contrast agent, fluorophore, marker for PET and SPECT, preferably selected from 44 Sc, 64 Cu, 67/68 Ga 99m Tc, 111 ln, fluorophore in the far red/near-IR spectral region, and Gd-based and Fe-oxide particle based MRI contrast agent.
  • the present invention relates to the protein-payload conjugate of the present invention or payload-linker conjugate of the present invention, wherein the imaging agent is selected from 99m Tc, 111 ln, 44 Sc and 64 Cu.
  • the present invention relates to the protein-payload conjugate of the present invention or payload-linker conjugate of the present invention, wherein the payload comprises a radionuclide, preferably selected from 64 Cu, 90 Y, 111 ln, 131 l, 161 Tb, 169 Er and 177 Lu or preferably selected from 44 Sc, 64 Cu, 67/68 Ga 99m Tc, and 111 ln.
  • the payload comprises a radionuclide, preferably selected from 64 Cu, 90 Y, 111 ln, 131 l, 161 Tb, 169 Er and 177 Lu or preferably selected from 44 Sc, 64 Cu, 67/68 Ga 99m Tc, and 111 ln.
  • the protein-payload conjugate of the invention may comprise more than one payload molecule.
  • said protein may be conjugated to one or more contrast/imaging agents (e.g., fluorescent dyes, (chelated) radionuclides (SPECT, PET), MR-active agents, CT-agents).
  • contrast/imaging agents e.g., fluorescent dyes, (chelated) radionuclides (SPECT, PET), MR-active agents, CT-agents).
  • a contrast/imaging agent may be incorporated in the protein-payload conjugate of the invention for medical or biological imaging.
  • the protein-payload conjugates of the present invention may be useful in certain imaging techniques, which may include positron emission tomography (PET), single photon emission computed tomography (SPECT), computerized tomography (CT), magnetic resonance imaging (MRI), optical bioluminescence imaging, optical fluorescence imaging, and combinations thereof.
  • the contrast/imaging agent may be any molecule, substance or compound known in the art for PET, SPECT, CT, MRI, and optical imaging.
  • the contrast agent may be radionuclides, radiometals, positron emitters, beta emitters, gamma emitters, alpha emitters, paramagnetic metal ions, and supraparamagnetic metal ions.
  • the contrast agents include, but are not limited to, iodine, fluorine, Cu, Zr, Lu, At, Yt, Ga, In, Tc, Gd, Dy, Fe, Mn, Ba and BaSO4.
  • the payload-linker conjugate wherein the payload is as described herein is also encompassed.
  • the imaging agent may be a fluorescent reporter.
  • the fluorescent reporter may be a near infrared or far red dye.
  • the fluorescent reporter may be selected from the group consisting of a fluorophore, fluorochrome, dye, pigment, fluorescent transition metal, and fluorescent protein.
  • the fluorescent reporter is selected from the group consisting of Cy5, Cy5.5, Cy2, FITC, TRITC, Cy7, FAM, Cy3, Cy3.5, Texas Red, ROX, HEX, JA133, AlexaFluor 488, AlexaFluor 546, AlexaFluor 633, AlexaFluor 555, AlexaFluor 647, DAPI, TMR, R6G, GFP, enhanced GFP, CFP, ECFP, YFP, Citrine, Venus, YPet, CyPet, AMCA, Spectrum Green, Spectrum Orange, Spectrum Aqua, Lissamine and Europium.
  • imaging agents include, for example, the following: Cy5.5, Cy5, Cy7.5 and Cy7 (GE® Healthcare); AlexaFluor660, AlexaFluor680, AlexaFluor790, and AlexaFluor750 (Invitrogen); VivoTagTM680, VivoTagTM-S680, VivoTag TM-S750 (VISEN Medical); Dy677, Dy682, Dy752 and Dy780 (Dyomics ®); DyLight ® 547, and/or DyLight ® 647 (Pierce); HiLyte FluorTM 647, HiLyte FluorTM 680, and HiLyte FluorTM 750 (AnaSpec ®); IRDye ® 800CW, IRDye ® 800RS, and IRDye ® 700DX (Li-Cor ®); ADS780WS, ADS830WS, and ADS832WS (American Dye Source); XenoLight CFTM 680, X
  • Said payload can be charged with a radionuclide, as described hereinabove.
  • Preferred radionuclides are
  • the protein-payload conjugate of the present invention comprises a protein (or a protein moiety).
  • protein is also understood to include the protein complexes (preferably wherein at least one polypeptide chain of said protein complex is conjugated according to the present invention to the payload) and peptides that lack well-defined secondary or tertiary structures.
  • protein includes at least 5 amino acid residues, more preferably at least 10 amino acid residues, even more preferably at least 20 amino acid residues.
  • the definition of the protein further includes posttranslational modifications of the protein, which are known to the skilled person.
  • the protein as defined herein may also be referred to as targeting molecule.
  • the protein as recited herein is not meant to be particularly limited and in principle any protein can be used in the protein-payload conjugate of the present invention.
  • the protein of the present invention can localize to the organ or part of the body affected by a disease.
  • the disease as recited herein, is preferably selected from cancer, fibrosis, lymphedema, immune disease, autoimmune disease and atherosclerosis.
  • the protein in the protein-peptide conjugate of the present invention is selected so that it can, when administered to a subject, localize to cancer tissue, fibrotic tissue, tissue affected by lymphedema (lymphatic edema), tissue affected by immune disease, tissue affected by autoimmune disease, tissue affected by inflammation, or tissue affected by artherosclerosis.
  • the linker of the present invention and as comprised in the protein-payload conjugate of the present invention is cleavable at the renal brush border by the enzymes present therein, in particular by neprilysin. Therefore, renal retention of the payload comprised in such a protein-payload conjugate of the invention may be reduced, if in the protein-payload conjugate of the present invention the protein may be cleaved off from the payload.
  • the present invention provides protein-payload conjugates comprising the linker of the present invention, comprising protein that is retained in kidneys, the administration of which leads to reduced renal retention of the payload when compared to the protein-payload conjugates not comprising the linker of the present invention.
  • the protein of the protein-payload conjugate of the present invention is a fibronectin binding peptide.
  • Fibronectin binding peptide is herein understood as a peptide that binds to fibronectin.
  • binding to fibronectin is determined in the affinity determination experiment, using fluorescence polarization and measuring the complex formation between the fibronectin binding peptide and Fib1 - N-terminal 30 kDa fragment of fibronectin according to SEQ ID NO.: 2.
  • the fibronectin binding peptide is considered to bind to fibronectin when the affinity as determined according to the method described herein is better than 1000 nM, more preferably wherein said affinity is better than 100 nM.
  • the measurement is performed using the fibronectin binding peptide N-terminally labelled with Cy5.
  • the fibronectin binding peptide of the invention comprises a polypeptide of sequence of at least 80% identity to SEQ ID NO: 3 (Gln-Val-Thr-Thr-Gly-Ser-Asn-Leu-Val-Glu-Phe-Thr-Glu-Glu-Ser-Leu- Gly-lle-Val-Thr-Gly-Ala-Val-Ser-Asp-His-Thr-Thr-Val-Glu-Asp-Thr), more preferably of at least 90% identity to SEQ ID NO: 3, even more preferably of at least 95% sequence identity to SEQ ID NO.:3, most preferably the sequence according to SEQ ID NO.:3.
  • the sequence as in SEQ ID NO.: 3 is also referred to as FnBPA5.1.
  • the present invention relates to the protein-payload conjugate of the present invention, wherein the protein comprises the polypeptide according to SEQ ID NO: 3. In one embodiment, the present invention relates to the protein-payload conjugate of the present invention, wherein the protein comprises the polypeptide according to SEQ ID NO: 4.
  • Fibronectin-binding peptides consist of bacterial adhesins-derived peptides capable of specifically distinguishing between differential tensional conformations of fibronectin (Fn) and preferentially binding to relaxed Fn fibrils, therefore constituting highly attractive strain-sensitive nanotools for visualizing altered ECM tensional architecture within pathological tissues (Arnoldini et al., 2017, Fonta et al., 2020).
  • the protein is an antibody or a fragment thereof.
  • the antibody or the fragment thereof preferably can localize to the organ or part of the body affected by a disease.
  • the disease as recited herein, is preferably selected from cancer, fibrosis, lymphedema, immune disease, autoimmune disease and atherosclerosis.
  • the protein in the protein- peptide conjugate of the present invention is selected so that it can, when administered to a subject, localize to cancer tissue, fibrotic tissue, tissue affected by lymphedema (lymphatic edema), tissue affected by immune disease, tissue affected by autoimmune disease, tissue affected by inflammation, or tissue affected by artherosclerosis.
  • the protein is a nanobody or exendin4.
  • the protein is a darpin, a nanobody, another engineered protein or an antibody fragment with molecular weight preferably in the range from 10 to 70 kDa, preferably a darpin, a nanobody or an antibody fragment with molecular weight preferably in the range from 10 to 70 kDa.
  • the protein of the present invention is conjugated to the side chain amino group of (AA6) of the linker comprised in the protein-payload conjugate of the present invention.
  • the conjugation may occur via amide bond formation or maleimide-thiol conjugation.
  • any conjugation method known to the skilled person can be used, and said method is not particularly limited, preferably as long as it does not interfere with the enzymatic cleavage of the linker at the renal brush border.
  • the present invention further relates to use of the linker of the present invention, as described hereinabove, for linking the payload, as described herein, to the protein, as described herein, affording the protein-payload conjugate of the present invention, as described herein.
  • the linker of the present invention may also encompass more than two enzymatic cleavage sites.
  • the present invention relates to a protein-payload conjugate comprising the following sequence motif:
  • P, B (AA1), (AA2), (AA3), (AA4), (AA5), (AA6), and X1-X2-X3 are as defined herein, m is selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, and 10, preferably from 0, 1 , 2, and 3.
  • Each (AA1 ’), (AA2’), (AA3’), and -XT -X2’- X3’- are independently defined as (AA4), (AA5), (AA6), and -X1-X2-X3-, respectively.
  • particularly preferred protein-payload conjugates are the compounds selected from the compounds 4, 5 and 6 and their radionuclide complexes, wherein preferably the radionuclide is selected from 225 Ac, 213 Bi, 67 Cu, 90 Y, 111 ln, 131 l, 161 Tb, 169 Er, and 177 Lu.
  • linker of the present invention the protein-payload conjugate of the present invention and the payload-linker conjugate of the present invention are obtainable analogously to the methods shown in the Preparative Examples hereinbelow.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the protein-payload conjugate as described herein or a payload-linker conjugate and a pharmaceutically acceptable carrier.
  • the protein-payload conjugate of the invention including a payload (/.e., a biologically active molecule or an imaging agent conjugated to the protein via the linker of the invention) may affect targeting of the payload to the target cell or tissue.
  • the protein-payload conjugate may also affect intracellular transport of the payload into a target cell.
  • the protein-payload conjugate may also affect the renal clearance of the payload.
  • the invention encompasses a pharmaceutical composition comprising a protein-payload conjugate, and a pharmaceutically acceptable carrier or excipient.
  • Such a carrier or excipient includes, but is not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and/or combinations thereof.
  • the pharmaceutical compositions also may include additional therapeutic agents for the treatment of the given disease being treated.
  • the formulation is made to suit the mode of administration. In general, methods of administering polypeptides are well known in the art and can be applied to administration of the conjugates of the invention.
  • Administration is by any of the routes normally used for introducing a protein-payload conjugate into ultimate contact with blood. Suitable methods of administering such protein-payload conjugate in the context of the present invention to a patient are available including oral and parenteral routes. Although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective action or reaction than another route.
  • the protein-payload conjugates or payload-linker conjugates of the invention are administered by parenteral modes of administration, in particular by intravenous, intraperitoneal, intramuscular, intradermal, subcutaneous intrathecal, intraocular, retrobulbar, intrapulmonary or intraarticular means.
  • parenteral modes of administration in particular by intravenous, intraperitoneal, intramuscular, intradermal, subcutaneous intrathecal, intraocular, retrobulbar, intrapulmonary or intraarticular means.
  • parenteral modes of administration in particular by intravenous, intraperitoneal, intramuscular, intradermal, subcutaneous intrathecal, intraocular, retrobulbar, intrapulmonary or intraarticular means.
  • parenteral modes of administration in particular by intravenous, intraperitoneal, intramuscular, intradermal, subcutaneous intrathecal, intraocular, retrobulbar, intrapulmonary or intraarticular means.
  • Such administration routes and appropriate formulations are generally known to those of skill in the art.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilisers, thickening agents, stabilizers, and preservatives. Protein-payload conjugates can also be administered via liposomes.
  • the protein-payload conjugate of the invention can also be made into aerosol formulations (/.e., they can be “nebulised”) to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • the pharmaceutical compositions of the invention are provided in lyophilized form to be reconstituted prior to administration.
  • Buffers and solutions for the reconstitution of the pharmaceutical compositions may be provided along with the pharmaceutical formulation to produce aqueous compositions of the present invention for administration.
  • Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions of the present invention.
  • Pharmaceutically acceptable carriers and excipients are well known in the art, and one or more conjugates of the invention can be formulated into pharmaceutical compositions by well-known methods (see, e.g., Remington: The Science and Practice of Pharmacy, 21st edition, A. R. Gennaro, Ed., Mack Publishing Company (2005); Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis (2000); and Handbook of Pharmaceutical Excipients, 3rd edition, A.
  • compositions comprising one or more protein-payload conjugate(s) of the invention are optionally tested in one or more appropriate in vitro and/or in vivo animal models of disease, to confirm efficacy, tissue metabolism, and to estimate dosages, according to methods well known in the art.
  • suitable dose of a composition according to the present invention will depend upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • the dosage is tailored to the individual subject, as is determinable by one of skill in the art, without undue experimentation.
  • the total dose of therapeutic agent may be administered in multiple doses or in a single dose.
  • the compositions are administered alone, in other embodiments the compositions are administered in conjunction with other therapeutics directed to the disease or directed to other symptoms thereof.
  • the protein-payload conjugate in the present invention may be administered alone or in combination with adjuvants that enhance stability, facilitate administration of pharmaceutical compositions containing them, provide increased dissolution or dispersion, increase inhibitory activity, provide adjunct therapy, and the like, including other active ingredients.
  • adjuvants that enhance stability, facilitate administration of pharmaceutical compositions containing them, provide increased dissolution or dispersion, increase inhibitory activity, provide adjunct therapy, and the like, including other active ingredients.
  • combination therapies utilize lower dosages of the conventional therapeutics, thus avoiding possible toxicity and adverse side effects incurred when those agents are used as monotherapies.
  • the above described protein-payload conjugates may be physically combined with the conventional therapeutics or other adjuvants into a single pharmaceutical composition.
  • the dose administered to a patient is sufficient to affect a beneficial therapeutic response in the patient over time, or, e.g., to inhibit infection by a pathogen, to reduce or prevent the symptoms of a disease state, or other appropriate activity, depending on the application.
  • the dose is determined by the efficacy of a particular composition/formulation, and the activity, stability or serum half-life of the BAM polypeptide conjugate employed and the condition of the patient, as well as the body weight or surface area of the patient to be treated.
  • the size of the dose is also determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular composition/formulation, or the like in a particular patient.
  • dosage levels range from about 0,5 pg - 100 mg/dose for a 70 kg patient. Although one dose per day may be sufficient, up to 5 doses per day may be given. For oral doses, up to 2000 mg/day may be required. For radionuclide therapy a dose every 4 to 8 week for 2 to 8 times may be applicable. As the person skilled in the art will appreciate, lower or higher doses may be required depending on particular factors. For instance, specific doses and treatment regimens will depend on factors such as the patient's general health profile, the severity and course of the patient's disorder or disposition thereto, and the judgment of the treating physician. For example, protein-payload conjugates of the present invention can be administered the same way as other peptide-based medicaments.
  • the protein-payload conjugates in the present invention may be formulated into capsules the same way other peptide-based medicaments are formulated.
  • Each capsule may contain 100 to 500, preferably 150 to 300, more preferably 200 to 250 mg of a compound of the invention.
  • non- medicinal ingredients in capsules for the compounds of the present invention are - capsule shell: D&C yellow No. 10, FD&C blue No. 1 , FD&C red No. 3, FD&C yellow No. 6, gelatin and titanium dioxide. Bottles of 100. (see also Martindale: the complete drug reference, 34 Edition, 2005, Pharmaceutical Press, p 612.).
  • the present invention relates to the protein-payload conjugate of the present invention as described hereinabove or the pharmaceutical composition of the present invention as described hereinabove for use in therapy.
  • Therapy is herein preferably understood as treatment and/or prevention, more preferably as treatment.
  • the payload-linker conjugate of the present invention and the protein-payload conjugate of the present invention is suitable for use in therapy or diagnosis of a disease selected from cancer, fibrosis, lymphedema, immune disease, autoimmune disease and atherosclerosis.
  • the protein is fibronectin binding peptide
  • the protein-payload conjugate of the present invention is suitable for use in therapy or diagnosis of a disease selected from cancer, fibrosis, lymphedema, immune disease, autoimmune disease and atherosclerosis, preferably wherein the disease is associated with pathological fibronectin accumulation.
  • the disease associated with pathogenic fibronectin accumulation is a disease associated with abnormal accumulation of soluble plasma fibronectin and/or insoluble ECM fibronectin.
  • cancer refers to an uncontrolled growth of cells which interferes with the normal functioning of the bodily organs and systems.
  • examples of cancer include, but are not limited to B-cell lymphomas (Hodgkin's lymphomas and/or non-Hodgkin's lymphomas), brain tumor, breast cancer, colon cancer, lung cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma and other skin cancers, head and neck cancer (preferably head and neck squamous cell carcinoma), brain cancer, and prostate cancer, including but not limited to androgendependent prostate cancer and androgen-independent prostate cancer.
  • B-cell lymphomas Hodgkin's lymphomas and/or non-Hodgkin's lymphomas
  • gastric cancer pancreatic cancer
  • cervical cancer ovarian cancer
  • liver cancer bladder cancer
  • cancer of the urinary tract thyroid cancer
  • renal cancer carcinoma
  • the fibrosis is selected from pulmonary fibrosis, liver fibrosis, and kidney fibrosis.
  • the present invention relates to the protein-payload conjugate of the present invention as described hereinabove or the pharmaceutical composition of the present invention as described hereinabove for use in the treatment and/or prevention of a disease selected from pulmonary fibrosis, liver fibrosis, and kidney fibrosis.
  • fibrosis can refer to any disease characterized by fibrosis, including but not limited to systemic sclerosis, multifocal fibrosclerosis, sclerodermatous graft-vs-host- disease, nephrogenic systemic fibrosis, organ specific fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, Crohn's Disease, Keloid, arthrofibrosis, Peyronie's Disease, Dupuytren's Contracture, adhesive capsulitis, and the like.
  • Illustrative organ specific fibrosis include, but are not limited to, pulmonary fibrosis, pulmonary hypertension, cystic fibrosis, asthma, chronic obstructive pulmonary disease, liver fibrosis, kidney fibrosis, fibrosis of the pancreas, non-alcoholic steatohepatitis (NASH), lymph node fibrosis, corneal fibrosis, fibrous cartilage, endometriosis, and the like.
  • NASH non-alcoholic steatohepatitis
  • lymph node fibrosis corneal fibrosis
  • fibrous cartilage endometriosis
  • endometriosis and the like.
  • Many fibrosis diseases, disorders or conditions have disordered and/or exaggerated deposition of extracellular matrix in affected tissues. Fibrosis may be associated with inflammation, occur as a symptom of underlying disease, and/or caused by surgical procedure or injuries with limited wound healing capacities.
  • the cancer is preferably selected from breast cancer, head and neck squamous cell carcinoma, prostate cancer, renal cancer, pancreatic cancer and lung cancer.
  • the present invention relates to the protein-payload conjugate of the present invention as described hereinabove or the pharmaceutical composition of the present invention as described hereinabove for use in the treatment and/or prevention of a disease selected from breast cancer, head and neck squamous cell carcinoma, prostate cancer, renal cancer, pancreatic cancer and lung cancer.
  • the autoimmune diseases is preferably selected from systemic sclerosis, diabetes type 1 , Graves’ disease, multiple sclerosis and rheumatoid arthritis.
  • the present invention relates to the protein-payload conjugate of the present invention as described hereinabove or the pharmaceutical composition of the present invention as described hereinabove for use in the treatment and/or prevention of a disease selected from systemic sclerosis, diabetes type 1 , Graves’ disease, multiple sclerosis and rheumatoid arthritis.
  • the present invention further relates to the protein-payload conjugate of the present invention for use in therapy or diagnosis of cancer, wherein the therapeutic application requires that the payload is not retained in the kidneys.
  • Particularly suitable are protein-payload conjugates wherein the payload comprises a radionuclide.
  • the present invention relates to the protein-payload conjugate (in particular wherein the protein is fibronectin binding peptide) of the present invention as described hereinabove, or the pharmaceutical composition of the present invention for use in the treatmentor prevention of a disease associated with pathological fibronectin accumulation.
  • pathological fibronectin accumulation refers to any disease or condition in which the amount of fibronectin deposited at a given site is higher than in a healthy state. For example, pathological fibronectin accumulation is found regularly in fibrosis or cancer.
  • the present invention further relates to use of the protein-payload conjugate of the present invention as described hereinabove, or the pharmaceutical composition of the present invention for the manufacture of a medicament for use in the treatment or prevention of a disease associated with pathologic fibronectin accumulation.
  • the disease associated with pathologic fibronectin accumulation is as described hereinabove.
  • the present invention further relates to use of the protein-payload conjugate of the present invention as described hereinabove, or the pharmaceutical composition of the present invention for the manufacture of a medicament for use in the treatment or prevention of a disease selected from fibrosis, cancer, lymphedema, immune disease, autoimmune disease, and atherosclerosis.
  • the present invention further relates to use of the protein-payload conjugate of the present invention as described hereinabove, or the pharmaceutical composition of the present invention for the manufacture of a medicament for use in the treatment or prevention of a disease selected from systemic sclerosis, diabetes type 1 , Graves’ disease, multiple sclerosis and rheumatoid arthritis. Further accordingly, the present invention further relates to use of the protein-payload conjugate of the present invention as described hereinabove, or the pharmaceutical composition of the present invention for the manufacture of a medicament for use in the treatment or prevention of a disease selected from pulmonary fibrosis, liver fibrosis, and kidney fibrosis.
  • the present invention further relates to use of the protein-payload conjugate of the present invention as described hereinabove, or the pharmaceutical composition of the present invention for the manufacture of a medicament for use in the treatment or prevention of a disease selected from breast cancer, head and neck squamous cell carcinoma, prostate cancer, renal cancer, pancreatic cancer and lung cancer.
  • the present invention further relates to use of the protein-payload conjugate of the present invention as described hereinabove, or the pharmaceutical composition of the present invention for the manufacture of a medicament for use in the treatment or prevention of non-small cell lung cell cancer.
  • the present invention relates to a method of treatment or prevention (preferably to a method of treatment) of a disease associated with pathologic fibronectin accumulation.
  • the said method comprises the step of administering the therapeutically effective amount of the protein-payload conjugate of the present invention or the pharmaceutical composition of the present invention to the subject in need thereof.
  • the subject herein is preferably defined as a human subject, preferably suffering from a disease associated with pathologic fibronectin accumulation.
  • the disease associated with pathologic fibronectin accumulation is as defined hereinabove.
  • the present invention further relates to a method of treatment or prevention (preferably to a method of treatment) of a disease selected from fibrosis, cancer, lymphedema, immune disease, autoimmune disease, and atherosclerosis.
  • the said method comprises the step of administering the therapeutically effective amount of the fibronectin binding peptide of the present invention or the pharmaceutical composition of the present invention to the subject in need thereof.
  • the present invention further relates to a method of treatment or prevention (preferably to a method of treatment) of a disease selected from systemic sclerosis, diabetes type 1 , Graves’ disease, multiple sclerosis and rheumatoid arthritis.
  • the said method comprises the step of administering the therapeutically effective amount of the protein-payload conjugate of the present invention or the pharmaceutical composition of the present invention to the subject in need thereof.
  • the present invention further relates to a method of treatment or prevention (preferably to a method of treatment) of a disease selected from pulmonary fibrosis, liver fibrosis, and kidney fibrosis.
  • the said method comprises the step of administering the therapeutically effective amount of the protein-payload conjugate of the present invention or the pharmaceutical composition of the present invention to the subject in need thereof.
  • the present invention further relates to a method of treatment or prevention (preferably to a method of treatment) of a disease selected from breast cancer, head and neck squamous cell carcinoma, prostate cancer, renal cancer, pancreatic cancer and lung cancer.
  • the said method comprises the step of administering the therapeutically effective amount of the protein-payload conjugate of the present invention or the pharmaceutical composition of the present invention to the subject in need thereof.
  • the present invention further relates to a method of treatment or prevention (preferably to a method of treatment) of non-small cell lung cell cancer.
  • the said method comprises the step of administering the therapeutically effective amount of the protein-payload conjugate of the present invention or the pharmaceutical composition of the present invention to the subject in need thereof.
  • Particularly useful protein-payload conjugate of the present invention are those bearing a biologically active molecule as a payload, as described hereinabove.
  • the present invention relates to the protein-payload conjugate of the present invention or the pharmaceutical composition of the present invention for use in diagnosis.
  • the present invention relates to the protein-payload conjugate of the present invention or the pharmaceutical composition of the present invention for use in diagnosis of a disease associated with pathologic fibronectin accumulation.
  • the disease associated with pathologic fibronectin accumulation is as described hereinabove.
  • the protein-payload conjugate of the present invention or the pharmaceutical composition of the present invention are useful in diagnosis of a disease selected from the group consisting of fibrosis, cancer, lymphedema, immune disease, autoimmune disease, and atherosclerosis.
  • the present invention relates to the protein-payload conjugate of the present invention or the pharmaceutical composition of the present invention for use in diagnosis of a disease selected from the group consisting of fibrosis, cancer, lymphedema, immune disease, autoimmune disease, and atherosclerosis.
  • the present invention relates to the protein-payload conjugate of the present invention or the pharmaceutical composition of the present invention for use in diagnosis of autoimmune diseases, preferably selected from systemic sclerosis, diabetes type 1 , Graves’ disease, multiple sclerosis and rheumatoid arthritis.
  • autoimmune diseases preferably selected from systemic sclerosis, diabetes type 1 , Graves’ disease, multiple sclerosis and rheumatoid arthritis.
  • the present invention relates to the protein-payload conjugate of the present invention or the pharmaceutical composition of the present invention for use in diagnosis of fibrosis, preferably selected from pulmonary fibrosis, liver fibrosis, and kidney fibrosis.
  • the present invention relates to the protein-payload conjugate of the present invention or the pharmaceutical composition of the present invention for use in diagnosis of cancer, preferably selected from breast cancer, head and neck squamous cell carcinoma, prostate cancer, renal cancer, pancreatic cancer and lung cancer.
  • the present invention relates to the protein-payload conjugate of the present invention or the pharmaceutical composition of the present invention for use in in diagnosis of non-small lung cell cancer.
  • Particularly useful protein-payload conjugates of the present invention are those bearing an imaging agent as a payload, as described hereinabove.
  • Reagents and solvents were purchased from Fluorochem Ltd (UK), MilliporeSigma (MO, USA) or Thermo Fisher Scientific (MA, USA) and employed without any further purification or drying.
  • Fmoc- and Boc- protected amino acids, 2-Chlorotrityl chloride (2-CTC) resin and 4-Alkoxybenzyl alcohol resin (Wang) resin were purchased from abcr GmbH (Karlsruhe, Germany), Bachem (Bubendorf, Switzerland) or MilliporeSigma (MO, USA).
  • Fmoc-4-aminomethyl-phenylacetic acid was bought from Combi-Blocks Inc (CA, USA), and p-SCN-Bn-NOTA from Macrocyclics (TX, USA).
  • Cys-FnBPA5.1 , NODAGA-FnBPA5.1 and Cy5-FnBPA5.1 were purchased from PSL GmbH (Heidelberg, Germany), their synthesis is given in reference preparative examples below.
  • [ 111 ln]lnCl3 was obtained from Mallinckrodt (Woll Vogel, Switzerland).
  • RP-HPLC conjugates non-labeled with radionuclide was performed on a Merck-Hitachi LaChrom HPLC system equipped with a D-7000 interface, an L-7400 UV detector, and an L-7100 pump.
  • RP C18 analytical column with a gradient ranging from 10 to 80 % A in B over 10 min, at a flow rate of 1 mL per min.
  • Plasma stability studies and radiometabolite studies were conducted on a Reprosil Gold, 200 A, 3 pm, 200 x 10 mm (Dr. Maisch GmbH, Germany) RP C4 analytical column, that was eluted with 10 to 80% A in B over 25 min (plasma stability) or with 20% A in B over 10 min, followed by a 5 min gradient up to 95% A in B (radiometabolite profiling) at a flow rate of 1 mL per min.
  • R depicts a protein moiety, the protein moiety characterized by the polypeptide sequence according to SEQ ID NO.: 4 (Cys-Gly-Gly-Gly-GIn-Val-Thr-Thr-Gly-Ser-Asn-Leu- Val-Glu-Phe-Thr-Glu-Glu-Ser-Leu-Gly-lle-Val-Thr-Gly-Ala-Val-Ser-Asp-His-Thr-Thr-Val-Glu-Asp-Thr) (it is noted that the sequence according to SEQ ID NO: 3 is comprised in the sequence according to SEQ ID NO.: 4), attached to the rest of molecules 1 to 6 through the side chain thiol group of N-terminal Cys residue.:
  • the synthetic routes to access the final NOTA-MVK-based FnBPA5.1 conjugates 4-6 are depicted in Scheme 1.
  • the maleimide-activated MVK-based scaffolds (7-9) were synthetized via manual SPPS employing a standard Fmoc/tBu proto-col. Briefly, the first orthogonally protected Lys was loaded onto Wang or, alternatively, 2-CTC resin. Sequence elonga-tion proceeded linearly until the attachment of the last N-terminal protected amino acid Boc-Met-OH. 6-Maleimido hexanoic acid was then coupled at the Lys side chain, following selective cleavage of the Lys orthogonal protecting group.
  • the maleimide- activated constructs 7-9 were obtained in satisfying overall yields of 28-30%. These entities were then con-jugated at the N-terminus with p-SCN-Bn-NOTA, to yield the correspondent NOTA-conjugates 1-3, which were iso-lated in yields ranging from 50% to 80%, after HPLC purification.
  • a final conjugation step with Cys-FnBPA5.1 led to the desired NOTA-MVK-based FnBPA5.1 conjugates 4 NOTA-Bn-MVK(hex- FnBPA5.1), 5 NOTA-Bn-MV-amBn-MVK(hex-FnBPA5.1) and 6 NOTA-Bn-MVK(Me)2-amBn-MVK(hex- FnBPA5.1 ).
  • the number of steps performed in liquid phase for the syntheses of the maleimide-activated BFCAs was reduced from 3 to 1 , in favor of a nearly total employment of the more advantageous SPPS.
  • 2-Chlorotrityl chloride (2-CTC) resin (100-200 mesh, 1 % DVB, 0.8-1.2 mmol/g) or Wang resin (100-200 mesh, 0.8-1.2 mmol/g) were placed into a polypropylene syringe equipped with a polyethylene frit and a Teflon plunge, and extensively swollen in both DCM and DMF.
  • 2- CTC resin was loaded by treatment with a solution of Fmoc-Lys(Dde)-OH (2 equiv.) and DI PEA (5 equiv.) in DCM for 30 min.
  • the Fmoc deprotected peptidyl resin was obtained by treatment with a solution of 20% piperidine in DMF (3 min, 3 times).
  • Cys-FnBPA5.1 is herein understood as polypeptide according to the SEQ ID NO: 4.
  • An appropriate volume of the correspondent NOTA-MVK-based maleimide-activated conjugate at a concentration of 1 mg/mL in metal-free PBS (50 pM, pH 6.5) was added to a solution of Cys-FnBPA5.1 at a concentration of 2 mg/mL in in metal-free PBS (50 pM, pH 6.5), in a molar ratio 1 :2. The mixture was gently stirred up to 4 h at rt.
  • the conjugation reaction was monitored by means of analytical HPLC and LRMS until the complete consumption of the NOTA-conjugated MVK-based maleimide-activated entity was recorded.
  • the obtained NOTA-conjugated MVK-based-FnBPA5.1 proceeded to radiolabeling without any further purification step.
  • H-MVK(hex-Maleimide)-OH was prepared according to General Procedures starting from Wang resin. After loading Fmoc-Lys(Mtt)-OH to the solid support, sequence elongation was conducted coupling Fmoc-Val-OH and Boc-Met-OH, in the order. Subsequently, the Mtt protecting group was removed by treating the resin with a solution of TFA:DCM:TIS in ratio 1 :94:5 v/v/v (30 min, 3 times), and 6-maleimidohexanoic acid was coupled at the Lys side chain.
  • H-MV-amBn-MVK(hex-Maleimide)-OH (SEQ ID NO: 5) was prepared according to General Procedures starting from Wang resin. After loading Fmoc- Lys(Mtt)-OH to the solid support, sequence elongation was conducted coupling Fmoc-Val-OH, Fmoc-Met- OH, Fmoc-4-aminomethyl-phenylacetic acid, Fmoc-Val-OH and Boc-Met-OH in the order.
  • the Mtt protecting group was removed by treating the resin with a solution of TFA:DCM:TIS in a ratio of 1 :94:5 v/v/v (30 min, 3 times), and 6-maleimidohexanoic acid was coupled at the Lys side chain.
  • H-MVK(Me)2-amBn-MVK(hex-Maleimide)-0H 9
  • H-MVK(Me) 2 -amBn-MVK(hex- Maleimide)-OH (SEQ ID NO: 6) was prepared according to General Procedures starting from 2-CTC resin. After loading Fmoc-Lys(Dde)-OH to the solid support, sequence elongation was conducted coupling Fmoc-Val-OH, Fmoc-Met-OH, Fmoc-Lys(Me)2-OH, Fmoc-4-aminomethyl-phenylacetic acid, Fmoc-Val- OH and Boc-Met-OH in the order.
  • MALDI-FTICR- MS m/z calculated [M+H] + for NOTA-Bn-MVK(hex-Maleimide)-OH C46H70N9O13S2: 1020.4529; m/z found: 1020.4538.
  • MALDI-FTICR-MS m/z calculated [M+H] + for NOTA-Bn-MVK(Me)2-amBn-MVK(hex-Maleimide)-OH C73H113N14O17S3: 1553.7565; m/z found: 1553.7566.
  • NOTA-Bn-MVK(hexFnBPA5.1)-OH was prepared according to General Procedures starting from NOTA-Bn-MVK(hex-Maleimide)-OH (71 pL, 0.07 pmol) and Cys-FnBPA5.1 (250 pL, 0.14 pmol).
  • MALDI-FTICR-MS m/z calculated [M+H] + for NOTA-Bn- MVK(hexFnBPA5.1)-OH C196H310N49O74S3: 4630.1157; m/z found: 4630.1202.
  • NOTA-Bn-MV-amBn-MVK(hexFnBPA5.1)-OH was prepared according to General Procedures starting from NOTA-Bn-MV- amBn-MVK(hex-Maleimide)-OH (39 pL, 0.03 pmol) and Cys-FnBPA5.1 (100 pL, 0.06 pmol).
  • MALDI- FTICR-MS m/z calculated [M+H] + for NOTA-Bn-MV-amBn-MVK(hexFnBPA5.1)-OH C215H337N52O77S4: 5007.2930; m/z found: 5007.2910.
  • NOTA-Bn-MVK(Me)2-amBn-MVK(hexFnBPA5.1)-OH was prepared according to General Procedures starting from NOTA-Bn- MVK(Me)2-amBn-MVK(hex-Maleimide)-OH (43 pL, 0.03 pmol) and Cys-FnBPA5.1 (100 pL, 0.06 pmol).
  • MALDI-FTICR-MS m/z calculated [M+H] + for NOTA-Bn-MVK(Me) 2 -amBn-MVK(hexFnBPA5.1)-OH C223H353N54O78S4: 5163.4193; m/z found: 5163.4226.
  • TFA DOM IS 1 :94:5 v/v/v (30 min, 3 times);
  • b Ac2O:pyridine:DMF 20:18:62 v/v/v (5 min, 3 times);
  • c TFA:TIS:H2O 95:2.5:2.5 v/v/v, 4 h;
  • d p-SCN-Bn-NOTA, TEA in DMF, 4h;
  • e 2% hydrazine in DMF (15 min, 3 times).
  • H-Met-Val-Lys(Ac)-OH 10
  • H-Met-Val-Lys(Ac)-OH synthesis proceeded on the same solid support as H-Met-Val-Lys(hexMaleimide)-OH (7) until the removal of the Mtt protecting group, following which the resin was split in two halves in order to allow both syntheses.
  • the Lys side chain was acetylated with a mixture of Ac20:pyridine:DMF in ratio 20:18:62 (5 min, 3 times).
  • H-Met-Val-amBn-Met-Val-Lys(Ac)-OH (SEQ ID NO.: 10) was prepared according to General Procedures starting from 2-CTC resin. After loading Fmoc- Lys(Dde)-OH to the solid support, sequence elongation was conducted coupling Fmoc-Val-OH, Fmoc- Met-OH, Fmoc-4-aminomethyl-phenylacetic acid, Fmoc-Val-OH and Boc-Met-OH in the order, employing standard Fmoc/tBu chemistry.
  • the Dde protecting group was removed with 2% hydrazine in DMF (15 min, 3 times), the Lys side chain was acetylated with a mixture of Ac2O:pyridine:DMF in ratio 20:18:62 (5 min, 3 times).
  • the isolated yield was 34.5%.
  • MALDI-FTICR-MS m/z calculated [M+H] + for H-Met-Val-amBn-Met-Val-Lys(Ac)-OH C37H62N7O8S2: 796.4096; m/z found: 796.4103.
  • Analytical HPLC: (10 to 90 A in B over 30 min) tr 13.98 min.
  • H-Met-Val-Lys(Me)2-amBn-Met-Val-Lys(Ac)-OH 14
  • H-Met-Val-Lys(Me)2-amBn-Met-Val- Lys(Ac)-OH SEQ ID NO: 11
  • synthesis proceeded on the same solid support as H-Met-Val-Lys(Me)2- amBn-Met-Val-Lys(hexMaleimide)-OH (9) until the removal of the Dde protecting group, following which the resin was split in two halves in order to allow both syntheses.
  • the Lys side chain was acetylated with a mixture of Ac2O:pyridine:DMF in ratio 20:18:62 (5 min, 3 times).
  • the isolated yield was 16% and the analytical purity >99%.
  • NOTA-Bn-Met-Val-Lys(Ac)-OH (11).
  • NOTA-Bn-Met-Val-Lys(Ac)-OH was prepared according to General Procedures (Main Article) starting from H-Met-Val-Lys(Ac)-OH (10) (4 mg, 9.56 mol).
  • MALDI-FTICR-MS m/zcalculated [M+H] + for NOTA-Bn-Met-Val-Lys(Ac)-OH C38H6iN8OnS2: 869.3896; m/z found: 869.3894.
  • NOTA-Bn-Met-Val-amBn-Met-Val-Lys(Ac)-OH (13).
  • NOTA-Bn-Met-Val-amBn-Met-Val- Lys(Ac)-OH SEQ ID NO: 12 was prepared according to General Procedures starting from H-Met-Val- amBn-Met-Val-Lys(Ac)-OH (12) (4 mg, 5.02 mol).
  • MALDI-FTICR-MS m/z calculated [M+H] + for NOTA-Bn-Met-Val-amBn- Met-Val-Lys(Ac)-OH C57H88N11O14S3: 1246.5669; m/z found: 1246.5663.
  • NOTA-Bn-Met-Val-Lys(Me)2-amBn-Met-Val-Lys(Ac)-OH (15).
  • NOTA-Bn-Met-Val-Lys(Me)2- amBn-Met-Val-Lys(Ac)-OH (SEQ ID NO.: 13) was prepared according to starting from H-Met-Val- Lys(Me)2-amBn-Met-Val-Lys(Ac)-OH (14) (3 mg, 3.15 pmol).
  • SP-HPLC purification with a gradient ranging from 23% to 40% A in B over 15 min (tr 11 .05 min) yielded the desired conjugate in 97% purity and 54% yield (2.40 mg).
  • MALDI-FTICR-MS m/z calculated [M+H] + for NOTA-Bn-Met-Val-Lys(Me) 2 - amBn-Met-Val-Lys(Ac)-OH C65H104N13O15S3: 1402.6931 ; m/z found: 1402.6940.
  • Analytical HPLC: (10 to 90 A in B over 30 min) tr 15.08 min.
  • Fibronectin-binding peptides were either synthesized or outsourced in HPLC purity greater than 95%.
  • the peptide-conjugates were obtained via introduction of the desired moiety (e.g. NODAGA and/or Cy5) at the N-terminus of the fibronectin-binding sequence via amide bond formation maleimide-thiol conjugation.
  • a short spacer of three glycines (and an additional cysteine residue in the case of conjugates obtained via maleimide-thiol chemistry) between the last N- terminal amino-acid of the fibronectin-binding sequence and the functionalization (chelator, dye) was added, resulting in the following structure: chelator/dye-GGG-fibronectin-binding sequence.
  • NODAGA- and/or Cyanine5-conjugated FnBPA5.1 were either synthesized and/or outsourced. In either case, the peptide-conjugates were obtained as lyophilized powders in HPLC purity greater than 95%.
  • Linear peptide sequences were synthesized via automatized solid-phase peptide synthesis employing standard Fmoc/tBu protocols, following which a final conjugation step via maleimide-thiol conjugation at the N-terminus of the fibronectin-binding sequence provided the final NODAGA and/or Cy5-conjugates.
  • a short spacer of three glycines and an additional cysteine residue between the last N-terminal aminoacid of the fibronectin-binding sequence and the functionalization (chelator, dye) was introduced.
  • NODAGA-FnBPA5.1 (sequence: Ac-C(Mal-NODAGA)-GGG-QVTTGSNLVEFTEESLGIVTGAVSDHTT VEDT) (SEQ ID NO.: 14) was obtained from Bachem (Bubendorf, Switzerland) upon conjugation of the N-terminal Cys-activated sequence with Maleimide-NODA-GA (Chematech, Dijion, France).
  • Cy5-FnBPA5.1 (sequence: C(Mal-Cy5)-GGG-QVTTGSNLVEFTEESLGIVTGAVSDHTTVEDT) (SEQ ID NO.: 15) was obtained upon conjugation of the N-terminal Cys-activated sequence (Cys-FnBPA5.1 , PSL GmbH, Heidelberg, Germany) with Cy5-Maleimide (Lumiprobe, Hannover, Germany). Cys-FnBPA5.1 was dissolved in PBS (50 mM, pH 5.5) to a cone, of 30 mM. Following Cy5-Maleimide addition (1 equiv.), the solution was stirred up to 2h.
  • the NOTA-MVK-based FnBPA5.1 conjugates solutions were diluted with TraceSELECT® Water (Sigma-Aldrich, Buchs, Switzerland) to a final concentration of 0.1 mM.
  • the conjugates were labeled with [ 111 1 n] I nCb in metal-free ammonium acetate (0.5 M, pH 5.5) in the presence of Met(Se) (50-fold molar excess) at a molar activity of 6 MBq/nmol, followed by a 15 min incubation step at 50 °C.
  • An Eppendorf Thermomixer comfort (Eppendorf, Hamburg, Germany) was used for heating and simultaneous shaking of the labelling mixture.
  • NODAGA-FnBPA5.1 the radiolabeling was performed without addition of additives.
  • an aliquot of the reaction mixture was subjected to quality control by means of RP-yHPLC.
  • Radio-Metal-labelling was conducted in 1.5 ml protein low-binding Eppendorf tubes.
  • An Eppendorf Thermomixer comfort (Eppendorf, Hamburg, Germany) was used for heating and simultaneous shaking of the labelling mixture.
  • Quantitative y-counting was performed by a Packard COBRA-II Auto-Gamma counter (Perkin Elmer, Waltham, MA, USA) by measuring energy windows A (50-400 keV) and B (190— 230 keV) for 1 min.
  • Plasma Stability Studies Plasma Stability of peptides was assessed incubating 7.5 MBq of each radiotracer in human blood plasma (Kantonsspital Aarau, Switzerland) at 37 °C under gentle agitation. At different time points (0, 0.5, 1 , 4 and 24 h) aliquots were drawn and proteins were precipitated by the addition of an equal volume of MeCN. Upon centrifugation (14000 rpm, 10 min), the supernatant was filtered through a Mini-UniPrep Filter into a MiniPrep Whatman tube (Whatman Inc., NJ, USA) and analyzed by means of RP-yHPLC. Data are represented as percentages of recovered test compound at the individual time-points normalized to the reference sample at time 0 min.
  • the BBM-enzymes mediated cleavage of the 111 ln-NOTA-MVK-based acetylated linkers and their respective FnBPA5.1 conjugates was evaluated based on a published procedure (Uehara et al., 2018). Briefly, to a pre-incubated (37 °C, 15 min) solution of BBMVs (9 pL, 50 pg/pL), 150 pmol of the substrate (ca. 1 MBq) were added and diluted with PBS to a final BBMVs cone, of 15 pg/pL. The control vials did not contain any BBMVs solution, but an equal volume of buffer instead.
  • Human prostate cancer cell line PC-3 was purchased from ATCC and cultured using Roswell Park Memorial Institute (RPMI) 1640 medium, while human embryonic kidney cells (HEK-293, ATCC) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM). Both media were supplemented with 10 % (v/v) fetal calf serum (Bio Concept Ltd., Allschwil, Switzerland) and 2 mM L- glutamine as well as antibiotics (100 lU/mL penicillin, 0.1 mg/mL streptomycin, 0.25 pg/mL fungizone). For the selection of plasmid expressing HEK-293 cells, DMEM was further supplemented with 0.5 mg/mL Geneticin. Cells were cultured under standard conditions at 37 °C in a humified atmosphere containing 5 % CO2
  • mice were euthanized using CO2.
  • mice were kept under anesthesia with isoflurane (Attane, Piramal Enterpises, India) 1.5%. Data were reconstructed with HiSPECT software (version 1.4.3049, SciVis GmbH, Gottingen, Germany) and analyzed using VivoQuant (version 3.0, inviCRO Imaging Services and Software, Boston USA). Scale of activity was set to 0 - 20 Bq/voxel. Radiometabolites analysis. For the profiling of the urine metabolites, urines samples from SPECT mice were collected at 24 h p.i. after anesthesia. Upon collection, samples were treated with an equal V of MeCN and subjected to centrifugation (14000 rpm, 10 min) to precipitate the proteins.
  • BBM brush border membrane
  • BBMVs brush border membrane vesicles
  • BFCA(s) bi-functional chelating agent(s)
  • 2-CTC 2-Chlorotrityl chloride
  • Cy5 Cyanine 5; Dde, 1-(4,4-dimethyl-2,6- dioxocyclohexylidene)ethyl; DI PEA, /V,/V-diisopropylethylamine; Et20, diethyl ether; Fmoc, 9- fluorenylmethoxycarbonyl; Fn, fibronectin; FnBP(s), fibronectin-binding peptide(s); HATU, 1- [bis(d i methylami no) methylene]- 1 H-1 ,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; Mtt, methyltrityl; NEP, neprilysin;
  • Example 1 In Vitro Enzyme Mediated Cleavage Study.
  • the renal cleavage efficiency of the novel designed 111 1 n-labelled conjugates was at first evaluated with the linkers only, by replacing the thiol-reactive maleimide hexanoyl chain with an acetyl moiety.
  • Example 2 SPECT/CT Studies and Radiometabolite Analysis.
  • the radioconjugate equipped with a single MVK sequence [ 111 ln]ln-NOTA-Bn-MVK(hex-FnBPA5.1) revealed the poorest reduction of the signal intensity with respect to the control ( Figure 2A).
  • radiometabolites in kidneys and urine were analyzed at 24 h p.i. by means of radio-HPLC.
  • Example 3 Biodistribution Study in PC-3 Tumor-bearing Xenografts.
  • the renal accumulation of [ 111 ln]ln-NOTA- Bn-MVK(hex-FnBPA5.1) (compound 6 labelled with 111 1 n) featuring one single cleavable linker was 52.75 ⁇ 9.79 %iA/g, which corresponded to a statistically significant reduction of -43% compared to the reference [ 111 ln]ln-NODAGA-FnBPA5.1 (set at 100%).
  • Renal brush border strategy A developing procedure to reduce renal radioactivity levels of radiolabeled polypeptides, Nuclear Medicine and Biology.
  • Fibronectin fibers are highly tensed in healthy organs in contrast to tumors and virus-infected lymph nodes, Matrix Biology Plus.
  • NEP neutral metalloendopeptidase
  • a linker comprising the sequence motif:
  • (AA1) is an amino acid residue selected from methionine and cysteine
  • AA2 is an amino acid residue selected from valine, glycine, proline, leucine and isoleucine;
  • (AA3) is absent or (AA3) is an amino acid residue selected from lysine or ornithine, wherein the side chain amino group of said lysine or ornithine residue may be optionally substituted with one or two substituents selected from C1-3 alkyl and -CO-(Ci-3 alkyl);
  • X1 is selected from -O-, -S-, -NH- and -N(CI-6 alkyl)- or X1 is absent,
  • X2 is selected from C1-6 alkylene, C2-6 alkenylene, C2-6 alkynylene, -(C1-6 alkylene)-Y-(Ci 6 alkylene)-, -(Co-6 alkylene)-Y-carbocyclylene-Y-(Co-6 alkylene)-, and -(Co-6 alkylene)-Y- heterocyclylene-Y-(Co-6 alkylene)-, wherein each Y is independently selected from a covalent bond, -O-, -S-, -NH-, -N(CI- 6 alkyl)-, -CO-, -CONH-, -CON(CI-6 alkyl)-, -NHCO- and -N(CI-6 alkyl)CO-, and X3 is selected from -CO- and -CS- or X3 is absent; or
  • -X1-X2-X3- is replaced by a peptidylene moiety comprising 1 to 6 amino acid residues, wherein the N-terminal amino group of said peptidylene moiety forms a peptide bond with the carboxyl group of (AA3), and wherein the C-terminal carboxyl group of said peptidylene moiety forms the peptide bond with the amino group of (AA4);
  • (AA4) is an amino acid residue selected from methionine and cysteine;
  • (AA5) is an amino acid residue selected from valine, glycine, proline, leucine and isoleucine;
  • (AA6) is an amino acid residue selected from lysine or ornithine, wherein the side chain amino group of said lysine or ornithine residue may be optionally substituted with one or two substituents selected from C1-3 alkyl and -CO-(Ci-3 alkyl); wherein said alkylene, alkenylene, and alkynylene are each optionally substituted with one or more optional substituents selected from halogen, -CN, -OH, -0(Ci-6 alkyl), -NH2, -NH(CI-6 alkyl), and - N(CI-6 alkyl)(Ci-6 alkyl), and wherein said carbocyclylene and heterocyclylene are each optionally substituted with one or more optional substituents selected from halogen, -CN, C1-6 alkyl, -OH, - O(Ci- 6 alkyl), -NH2, -NH(CI-6 alkyl), and -N(CI-6 alkyl)(Ci- 6
  • the linker of item 1 wherein (AA1) is a methionine and (AA2) is a valine or a proline, preferably (AA2) is a valine, or wherein (AA1) is a cysteine and (AA2) is a glycine.
  • the linker of item 1 wherein (AA2) is an amino acid residue selected from valine and glycine, preferably wherein (AA2) is a valine residue.
  • X1 is NH
  • X2 is -(Co-6 alkylene)-Y-carbocyclylene-Y- (Co-6 alkylene)-, preferably -(Co-6 alkylene)-carbocyclylene-(Co-6 alkylene)-, more preferably -(C0-3 alkylene)-carbocyclylene-(Co-3 alkylene)-, even more preferably -(C1-3 alkylene)-phenylene-(Ci-3 alkylene)-, and X3 is -CO-.
  • a protein-payload conjugate comprising the linker of the following sequence motif:
  • (AA1) is an amino acid residue selected from methionine and cysteine
  • AA2 is an amino acid residue selected from valine, glycine, proline, leucine and isoleucine;
  • (AA3) is absent or (AA3) is an amino acid residue selected from lysine or ornithine, wherein the side chain amino group of said lysine or ornithine residue may be optionally substituted with one or two substituents selected from C1-3 alkyl and -CO-(Ci-3 alkyl);
  • X1 is selected from -O-, -S-, -NH- and -N(CI-6 alkyl)- or X1 is absent,
  • X2 is selected from C1-6 alkylene, C2-6 alkenylene, C2-6 alkynylene, -(C1-6 alkylene)-Y-(Ci 6 alkylene)-, -(Co-6 alkylene)-Y-carbocyclylene-Y-(Co-6 alkylene)-, and -(Co-6 alkylene)-Y- heterocyclylene-Y-(Co-6 alkylene)-, wherein Y is selected from a covalent bond, -O-, -S-, -NH-, - N(CI- 6 alkyl)-, -CO-, -CONH-, -CON(CI-6 alkyl)-, -NHCO- and -N(CI-6 alkyl)CO-, and
  • X3 is selected from -CO- and -CS- or X3 is absent;
  • -X1-X2-X3- is replaced by a peptide moiety comprising 1 to 6 amino acid residues, wherein the N- terminal amino group of said peptide moiety forms a peptide bond with the carboxyl group of (AA3), and wherein the C-terminal carboxyl group of said peptide moiety forms the peptide bond with the amino group of (AA4);
  • (AA4) is an amino acid residue selected from methionine and cysteine;
  • (AA5) is an amino acid residue selected from valine, glycine, proline, leucine and isoleucine;
  • (AA6) is an amino acid residue selected from lysine or ornithine, wherein the side chain amino group of said lysine or ornithine residue may be optionally substituted with one or two substituents selected from C1-3 alkyl and -CO-(Ci-3 alkyl); wherein said alkylene, alkenylene, and alkynylene are each optionally substituted with one or more optional substituents selected from halogen, -CN, -OH, -0(Ci-6 alkyl), -NH2, -NH(CI-6 alkyl), and - N(CI-6 alkyl)(Ci-6 alkyl), and wherein said carbocyclylene and heterocyclylene are each optionally substituted with one or more optional substituents selected from halogen, -CN, C1-6 alkyl, -OH, - O(Ci- 6 alkyl), -NH2, -NH(CI-6 alkyl), and -N(CI-6 alkyl)(Ci- 6
  • the protein-payload conjugate of item 10 wherein the payload is attached to the N-terminal amino group of (AA1), and/or wherein the protein is attached to the side chain amino group of (AA6).
  • the protein-payload conjugate of item 10 or 11 wherein (AA1 ) is a methionine and (AA2) is a valine or a proline, preferably (AA2) is a valine, or wherein (AA1) is a cysteine and (AA2) is a glycine.
  • the protein-payload conjugate of item 10 or 11 wherein (AA2) is an amino acid residue selected from valine and glycine, preferably wherein (AA2) is a valine residue.
  • the protein-payload conjugate of item 21 wherein the biologically active molecule is selected from the group consisting of cytostatic agent, cytotoxic agent, cytokine, transcription factor inhibitor, proteasome and protease inhibitor, apoptosis modulator, cell cycle modulator, angiogenesis inhibitor, hormone or hormone derivative, photodynamic therapy molecule, nano- and microparticle for thermoablation therapy, radionuclide, miRNA, siRNA and immunomodulatory antigen molecule, preferably wherein the cytostatic agent is selected from Doxorubicin, Paclitaxel, chlorambucil, Topotecan and Vincristine; preferably wherein the cytokine is selected from lnterleukin-2, lnterleukin-7, Interferon
  • the protein-payload conjugate of item 21 wherein the biologically active molecule is selected from the group consisting of Paclitaxel, Chlorambucil, Endostatin, Sunitinib, lnterleukin-7, 177 Lu, and 111 ln.
  • the imaging agent comprises a radionuclide, a fluorescent dye, a chemiluminescent agent, a bioluminescent agent, a spectrally resolvable inorganic fluorescent semiconductor nanocrystal, a metal nanoparticle, a nanocluster, a paramagnetic metal ion, an enzyme, a colorimetric label, biotin, dioxigenin, a hapten or a protein.
  • the imaging agent is selected from 68 Ga, 99m Tc, 111 ln, 44 Sc and 64 Cu.
  • the protein-payload conjugate of any one of items 10 to 28, wherein the protein is an antibody or a fragment thereof.
  • the protein-payload conjugate of any one of items 10 to 28, wherein the protein is a nanobody or exendin.
  • B is a payload
  • (AA1) is an amino acid residue selected from methionine and cysteine; wherein B is attached to the N-terminal amino group of (AA1);
  • (AA2) is an amino acid residue selected from valine, valine, proline, leucine and isoleucine;
  • (AA3) is absent or (AA3) is an amino acid residue selected from lysine or ornithine, wherein the side chain amino group of said lysine or ornithine residue may be optionally substituted with one or two substituents selected from C1-3 alkyl and -CO-(Ci-3 alkyl);
  • X1 is selected from -O-, -S-, -NH- and -N(CI-6 alkyl)- or X1 is absent,
  • X2 is selected from C1-6 alkylene, C2-6 alkenylene, C2-6 alkynylene, -(C1-6 alkylene)-Y-(Ci 6 alkylene)-, -(Co-6 alkylene)-Y-carbocyclylene-Y-(Co-6 alkylene)-, and -(Co-6 alkylene)-Y- heterocyclylene-Y-(Co-6 alkylene)-, wherein Y is selected from a covalent bond, -O-, -S-, -NH-, - N(CI- 6 alkyl)-, -CO-, -CONH-, -CON(CI-6 alkyl)-, -NHCO- and -N(CI-6 alkyl)CO-, and
  • X3 is selected from -CO- and -CS- or X3 is absent; or -X1-X2-X3- is replaced by a peptide moiety comprising 1 to 6 amino acid residues, wherein the N- terminal amino group of said peptide moiety forms a peptide bond with the carboxyl group of (AA3), and wherein the C-terminal carboxyl group of said peptide moiety forms the peptide bond with the amino group of (AA4);
  • (AA4) is an amino acid residue selected from methionine and cysteine;
  • (AA5) is an amino acid residue selected from valine, glycine, proline, leucine and isoleucine;
  • (AA6) is an amino acid residue selected from lysine or ornithine, wherein the side chain amino group of said lysine or ornithine residue may be optionally substituted with one or two substituents selected from C1-3 alkyl and -CO-(Ci-3 alkyl); wherein said alkyl, alkenyl, alkynyl, and alkylene are each optionally substituted with one or more optional substituents selected from halogen, -CN, -OH, -0(Ci-6 alkyl), -NH2, -NH(CI-6 alkyl), and - N(CI-6 alkyl)(Ci-6 alkyl), and wherein said carbocyclylene and heterocyclylene are each optionally substituted with one or more optional substituents selected from halogen, -CN, C1-6 alkyl, -OH, - O(Ci- 6 alkyl), -NH2, -NH(CI-6 alkyl), and -N(CI-6 alkyl)(
  • the payload-linker conjugate of item 32 wherein (AA1) is a methionine and (AA2) is a valine or a proline, preferably (AA2) is a valine, or wherein (AA1) is a cysteine and (AA2) is a glycine.
  • the use of item 45 or 46, wherein the payload is as defined in any one of items 20 to 28.

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Abstract

La présente invention concerne un lieur de la présente invention, un conjugué protéine-charge utile de la présente invention, un conjugué charge utile-lieur de la présente invention et leurs utilisations thérapeutiques et diagnostiques respectives. Les lieurs, les conjugués protéine-charge utile et les conjugués charge utile-lieur de la présente invention sont particulièrement utiles en raison de leur effect réducteur de rétention rénale.
PCT/EP2022/084593 2021-12-06 2022-12-06 Technologie à lieur pour réduire la rétention rénale WO2023104794A1 (fr)

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Citations (2)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150320825A1 (en) 2011-12-22 2015-11-12 Children's Medical Center Corporation Saposin-a derived peptides and uses thereof
WO2017150549A1 (fr) 2016-03-01 2017-09-08 国立大学法人 千葉大学 Médicament radiomarqué

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