WO2024052333A1 - Ligands multivalents de protéine d'activation des fibroblastes pour applications d'administration ciblée - Google Patents

Ligands multivalents de protéine d'activation des fibroblastes pour applications d'administration ciblée Download PDF

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WO2024052333A1
WO2024052333A1 PCT/EP2023/074310 EP2023074310W WO2024052333A1 WO 2024052333 A1 WO2024052333 A1 WO 2024052333A1 EP 2023074310 W EP2023074310 W EP 2023074310W WO 2024052333 A1 WO2024052333 A1 WO 2024052333A1
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independently
moiety
compound according
cooh
cancer
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Matilde BOCCI
Samuele CAZZAMALLI
Andrea Galbiati
Dario Neri
Aureliano ZANA
Ettore GILARDONI
Lucrezia PRINCIPI
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Philochem Ag
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings

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  • the present invention relates to ligands of Fibroblast Activation Protein (FAP) for the active delivery of various therapeutic payloads (e.g., cytotoxic drugs, therapeutic radionuclides, proteins and immunomodulators) at the site of disease.
  • FAP Fibroblast Activation Protein
  • the present invention relates to the development of multivalent FAP ligands for therapeutic applications, in relation to a disease or disorder, such as cancer, inflammation or another disease characterized by overexpression of FAP.
  • a disease or disorder such as cancer, inflammation or another disease characterized by overexpression of FAP.
  • Chemotherapy is still widely applied for the treatment of cancer patients and of other diseases.
  • Conventional anti-cancer chemotherapeutic agents act on basic mechanisms of cell survival and cannot distinguish between healthy cells and malignant cells.
  • therapeutic drugs do not accumulate efficiently to the site of the disease upon systemic administration. Unspecific mechanism of actions and inefficient localization at the tumor site account for unsustainable side-effects and poor therapeutic efficacy of conventional chemotherapy.
  • a strategy to generate such therapeutic drugs is represented by the chemical conjugation of a therapeutic payload, like cytotoxic drugs or therapeutic radionuclides, to a ligand specific to a marker of a disease.
  • a therapeutic payload like cytotoxic drugs or therapeutic radionuclides
  • small ligands for therapeutic applications has several advantages compared to bigger molecules like peptides and antibodies: more rapid and efficient tumor penetration, lower immunogenicity and lower manufacturing costs.
  • Small organic ligands specific to prostate-specific membrane antigen, folate receptor and carbonic anhydrase IX have shown excellent biodistribution profiles in preclinical models of cancer and in patients. These ligands have been conjugated to cytotoxic drugs and to radionuclides to generate small molecule- drug conjugate and small molecule-radio conjugate products (SMDCs and SMRCs) for the treatment of cancer.
  • SMDCs and SMRCs small molecule- drug conjugate and small molecule-radio conjugate products
  • 177-Lutetium-PSMA-617 represents an example of a late stage SMRC which is now being investigated in a phase III trial for the treatment of metastatic castrate-resistant prostate cancer (mCRPC) patients (VISION trial).
  • Fibroblast activation protein (FAP) is a membrane-bound gelatinase which promotes tumor growth and progression and is overexpressed in cancer-associated fibroblasts.
  • FAP represents an ideal target for the development of therapeutic SMDCs and SMRCs due to its low expression in normal organs.
  • WO2019154886 and WO2019154859 describe heterocyclic compounds as fibroblast activation protein- alpha inhibitors used to treat different cancer types.
  • WO2019118932 describes substituted N-containing cyclic compounds as fibroblast activation protein alpha inhibitors used to treat different pathological conditions.
  • WO2019083990 describes imaging and radiotherapeutic targeting fibroblast-activation protein- alpha (FAP-alpha) compounds as FAP-alpha inhibitors used for imaging disease associated with FAP-alpha and to treat proliferative diseases, and notes that the 4-isoquinolinoyl and 8-quinolinoyl derivatives described therein are characterized by very low FAP-affinity.
  • WO2013107820 describes substituted pyrrolidine derivatives used in the treatment of proliferative disorders such as cancers and diseases indicated by tissue remodeling or chronic inflammation such as osteoarthritis.
  • WO2005087235 describes pyrrolidine derivatives as dipeptidyl peptidase IV inhibitors to treat Type II diabetes.
  • WO2018111989 describes conjugates comprising fibroblast activation protein (FAP) inhibitor, bivalent linker and e.g. near infrared (NIR) dye, useful for removing cancer-associated fibroblasts, imaging population of cells in vitro, and treating cancer.
  • FAP fibroblast activation protein
  • NIR near infrared
  • WO2021/160825 A1 WO2022/171811 A1
  • WO2021/016392 A1 describe ligands of FAP for the active delivery of various payloads.
  • FAP-2286 (a FAP-binding peptide coupled to a radionuclide chelator), and report that radiolabeled FAP-2286 demonstrated high tumor uptake and retention, as well as potent efficacy in FAP-positive tumors. Nevertheless, there remains a demand for further improved FAP binders for the above and further applications.
  • the present invention thus aims at the problem of providing improved therapeutic binders (ligands) of fibroblast activation protein (FAP) suitable for therapeutic applications.
  • the binders should be suitable for inhibition of FAP and/or targeted delivery of a therapeutic payload, to a site afflicted by or at risk of disease or disorder characterized by overexpression of FAP, such as FAP-positive tumors.
  • the binders should exhibit improved binding parameter(s) (e.g., FAP inhibitory activity, FAP binding affinity and/or prolonged binding to FAP-positive cells), while showing advantageous tumor uptake or tumor-to-organ ratio (e.g., tumor-to-kidney ratio).
  • FAP fibroblast activation protein
  • the compounds according to the present invention comprise more than two (e.g., three) small binding moieties A having the following structure:
  • a compound according to the present invention may be represented by following general Formula I, its individual diastereoisomers, its hydrates, its solvates, its crystal forms, its individual tautomers or a pharmaceutically acceptable salt thereof, wherein A is a binding moiety; B is a multifunctional moiety comprising a ramification point, and covalently connects the moieties A to C; and C is an atom, a molecule or a particle, and is a therapeutic agent.
  • An exemplary structure is represented by general Formula II as defined hereinbelow.
  • moiety C comprises a ramification point, such that three moieties A are attached to C, and B is absent, i.e., moiety C can also serve as a ramification point therefore substituting B, preferably when C is a chelator.
  • An exemplary structure is represented by general Formula III as defined hereinbelow.
  • the present invention further provides a pharmaceutical composition comprising said compound and a pharmaceutically acceptable excipient.
  • the present invention further provides said compound or pharmaceutical composition for use in a method for treatment of the human or animal body therapy; as well as a method for treatment of the human or animal body by therapy comprising administering a therapeutically effective amount of said compound or pharmaceutical composition to a subject in need thereof.
  • the present invention further provides said compound or pharmaceutical composition for use in a method for therapy of a subject suffering from or having risk for a disease or disorder; as well as a method for treatment therapy of a disease or disorder comprising administering a therapeutically effective amount of said compound or pharmaceutical composition to a subject suffering from or having risk for said disease or disorder.
  • the present invention further provides said compound or pharmaceutical composition for use in a method for targeted delivery of a therapeutic agent to a subject suffering from or having risk for a disease or disorder; as well as a method for targeted delivery of a therapeutically effective amount of said compound or pharmaceutical composition to a subject suffering from or having risk for a disease or disorder.
  • the aforementioned disease or disorder is characterized by overexpression of FAP and is independently selected from cancer, inflammation, atherosclerosis, fibrosis, tissue remodeling and keloid disorder, preferably wherein the cancer is selected from the group consisting of breast cancer, pancreatic cancer, small intestine cancer, colon cancer, multi-drug resistant colon cancer, rectal cancer, colorectal cancer, metastatic colorectal cancer, lung cancer, non-small cell lung cancer, head and neck cancer, ovarian cancer, hepatocellular cancer, esophageal cancer, hypopharynx cancer, nasopharynx cancer, larynx cancer, myeloma cells, bladder cancer, cholangiocarcinoma, clear cell renal carcinoma, neuroendocrine tumor, oncogenic osteomalacia, sarcoma, CUP (carcinoma of unknown primary), thymus cancer, desmoid tumors, glioma, astrocytoma, cervix cancer, skin cancer, kidney cancer and
  • FIG.1 Inhibition assay with recombinant hFAP at a concentration of 66 pM. Tri-ESV6-DOTAGA exhibits a remarkably lower IC 50 compared to ESV6-DOTAGA, Bi-ESV6-DOTAGA and FAP-2286.
  • FIG.2 Fluorescence Polarization assay with recombinant hFAP, with fluorescent ligands at a concentration of 1 nM. Both ESV6-PEG 2 -FITC and Tri-ESV6-PEG 2 -FITC exhibit a remarkably lower K D compared to FAP-2286-PEG2-FITC.
  • FIG.1 Inhibition assay with recombinant hFAP at a concentration of 66 pM. Tri-ESV6-DOTAGA exhibits a remarkably lower IC 50 compared to ESV6-DOTAGA, Bi-ESV6-DOTAGA and FAP-2286.
  • FIG.2 Fluorescence Polarization assay with recombin
  • 177 Lu-Tri-ESV6 exhibits the longer residence time in FAP-positive tumor cells, followed by 177 Lu-Bi-ESV6, 177 Lu-ESV6 and 177 Lu- FAP2286.
  • FIG.4 Quantitative biodistribution analysis with 177 Lu-ESV6, 177 Lu-Bi-ESV6, 177 Lu-Tri-ESV6, 177 Lu- Tetra-ESV6, 177 Lu-Hexa-ESV6, 177 Lu-Octa-ESV6 and 177 Lu-FAP-2286 (each 250 nmol/kg; 50 MBq/kg) on SK-RC-52.hFAP tumor bearing mice.
  • Tri-ESV6-DOTAGA exhibits a remarkably high tumor uptake even after 48, 72 and 96 hours post injection. At the same dosage, the Tri-ESV6 binder exhibits the best biodistribution profile, when considering both tumor uptake over time and uptake in healthy organs.
  • FIG.5 Therapeutic activity of 177 Lu-ESV6-DOTAGA, 177 Lu-Bi-ESV6-DOTAGA and 177 Lu-Tri-ESV6- DOTAGA or saline as single agents (single administration on day 8, 250 nmol/kg, 250 MBq/kg) in BALB/c nu/nu mice bearing SK-RC-52.hFAP tumors.
  • Tri-ESV6-DOTAGA exhibits a remarkable therapeutic activity.
  • FIG.7 The tolerability of the different treatments with 177 Lu-ESV6-DOTAGA, 177 Lu-Bi-ESV6-DOTAGA and 177 Lu-Tri-ESV6-DOTAGA as assessed by the evaluation of changes (%) in body weight during the experiment. All conjugates were very well tolerated in a comparable manner.
  • FIG. 8 Inhibition assay with recombinant hFAP at a concentration of 66 pM. Surprisingly, Tri-ESV6- DOTAGA exhibits a ⁇ 1000-fold lower IC50 compared to its derivative (Tri-ESV6-PEG12-DOTAGA) with longer spacers between the ESV6 moiety and the ramification.
  • FIG. 8 Inhibition assay with recombinant hFAP at a concentration of 66 pM.
  • Tri-ESV6- DOTAGA exhibits a ⁇ 1000-fold lower IC50 compared to its derivative (Tri-ESV6-PEG12-DOTAGA) with longer
  • FIG. 10 Inhibition assay with recombinant hFAP at a concentration of 66 pM.
  • the compound with the shortest distance between the targeting moiety and the ramification point (PEG Unit 0) was surprisingly found to have the lowest IC50.
  • FIG. 11 Inhibition assay with recombinant hFAP at a concentration of 66 pM. The inhibitory activity is directly proportional to the valency until Tetra-ESV6-DOTAGA (tetravalent compound).
  • Hexa-ESV6-DOTAGA hexavalent compound
  • Octa-ESV6-DOTAGA octavalent compound
  • Compounds with alternative linker present IC50 comparable to the ones of original derivatives (ESV6-DOTAGA and Bi-ESV6-DOTAGA in FIG.1).
  • FIG. 1 Compounds with alternative linker
  • Preferred immunocytokine for use in the present invention namely an L19-IL2 conjugate immunocytokine, in which, in each polypeptide chain, an IL2 polypeptide sequence is fused, at its N- terminus via a linker, to the C-terminus of the VL domain of a single-chain variable fragment (scFv) sequence comprising the VH and VL domains of L19, and the scFv unit of one L19-IL2 polypeptide chain forms a homodimer with an scFv unit of another L19-IL2 polypeptide chain (“scFv2-format immunocytokine”).
  • scFv2-format immunocytokine a single-chain variable fragment
  • FIG. 14 Quantitative in vivo MMAE released in tumor and healthy organs by ESV6-GlyPro-MMAE (Conjugate 58a of EP3891138B1), Bi-ESV6-GlyPro-MMAE (Conjugate 11 of WO2022/171811) and Tri- ESV6-GlyPro-MMAE (Conjugate 9 of the present invention).
  • Each conjugate was injected at a dose of 250 nmol/Kg and the MMAE release recorded at different timepoints after administration. Values are expressed as percentage of injected dose per gram of tissue (%ID/g).
  • the quantification of MMAE released after administration of Tri-ESV6-GlyPro-MMAE revealed a cleaner biodistribution profile, with high and prolonged accumulation at the tumor site, and lower release in the healthy organs as compared to ESV6- GlyPro-MMAE and Bi-ESV6-GlyPro-MMAE.
  • FIG. 15 To identify the best dose for radiotherapeutic applications, 177 Lu-Tri-ESV6-DOTAGA was injected in tumour bearing mice at eight different doses ranging from 3 nmol/Kg to 2250/Kg.
  • FIG. 16 Therapeutic anti-cancer efficacy evaluation of ESV6-GlyPro-MMAE and Tri-ESV6-GlyPro- MMAE in HT-1080.hFAP tumor bearing mice, in terms of tumor volume over time (A) and percent survival ( ⁇ ). The tolerability was assessed by the evaluation of changes (%) in body weight during the experiment, indicating that all conjugates were well tolerated (C).
  • FIG.17 Data sets (single plots; one curve for each animal) underlying the results shown in FIG.16: ESV6- GlyPro-MMAE at 50 nmol/kg (A) or 125 nmol/kg (B); Tri-ESV6-GlyPro-MMAE at 50 nmol/kg (D) or 125 nmol/kg (E); and saline (C).
  • FIG. 18 Comparative hFAP inhibition assays with trivalent binders 11, 12, 13 and 14 having, inter alia, different linker groups B and/or different payload groups C.
  • FIG.19 177 Lu biodistribution results at different radioisotope molar activities (MBq of 177 Lu per nmol of ligand): low (0.2 MBq/nmol) and high (4.8 MBq/nmol, preferably used in clinical practice) with binder compound 177 Lu-Tri-ESV6-DOTAGA at 24 h post injection, suggesting that varying the molar activity does not significantly interfere with the advantageous biodistribution of the binder.
  • FIG.20 Comparative hFAP inhibition assays with tetravalent binders 10 and 29. DETAILED DESCRIPTION OF THE INVENTION The present inventors have identified small molecule binders of fibroblast activation protein (FAP) which are suitable for therapeutic applications.
  • FAP fibroblast activation protein
  • therapeutic binders according to the invention can provide high FAP inhibitory activity, high FAP binding affinity and prolonged binding to FAP-positive cells, and are therefore suitable candidates for targeted delivery of a therapeutic payload, to a site afflicted by or at risk of disease or disorder characterized by overexpression of FAP.
  • therapeutic binders according to the invention can provide high and prolonged tumor uptake, and potent antitumor effect. At the same time, remarkably high tumor- to-organ ratios (in particular: tumor-to-kidney) can be achieved.
  • the therapeutic binders according to the invention are surprisingly advantageous in terms of one or more of the above-mentioned and further effects as compared to related prior art FAP-binders, such as FAP- 2286.
  • binders according to the invention exhibit improved FAP inhibitory activity, improved FAP-binding affinity, prolonged binding to FAP-positive cells, higher tumor uptake and better tumor-to-organ ratios than FAP-2286.
  • the therapeutic binders according to the invention are surprisingly advantageous in terms of one or more of the above-mentioned and further effects as compared to monovalent FAP-binders, e.g., ESV6-DOTAGA having only one FAP-binding moiety A. As evident from the results in FIG.
  • Tri-ESV6-DOTAGA shows a 43-fold improvement in IC50 as compared to the monovalent binder ESV6-DOTAGA, which is surprising and exceeds by far a 3-fold improvement which would normally be expected when assuming additive behavior.
  • the trivalent binder according to the invention also shows a 10-fold improvement in IC50 as compared to the bivalent binder Bi-ESV6-DOTAGA, which is surprising and exceeds by far a 1.5-fold improvement which would normally be expected when assuming additive behavior.
  • binders according to the present invention can provide unexpected synergistic improvement in FAP inhibitory activity.
  • the trivalent binder according to the invention shows surprisingly improved efflux duration in FAP-positive tumor cells when compared to the bivalent binder, which is even further improved when compared to monovalent binders.
  • binders according to the present invention can thus provide unexpected improvement in terms of prolonged tumor uptake duration.
  • the trivalent binder according to the invention shows surprisingly improved in vivo tumor uptake in FAP-positive tumors when compared to monovalent, bivalent, hexavalent and octavalent binders (see also Tables 1 to 3 and Tables 11 to 12), while maintaining a surprisingly high tumor-to-organ ratios, in particular tumor-to-kidney (see also Tables 5 to 7 and Tables 13 to 15).
  • This is surprising, since increasing the binder valency would normally be expected to lead to higher organ accumulation and deterioration of the tumor-to-organ ratios.
  • the Tri-ESV6 binder exhibits much better (cleaner) biodistribution, i.e., more advantageous tumor-to-organ ratio, as compared to other tested binders (e.g., hexavalent and octavalent).
  • Lu-TriESV6-DOTAGA presents the best biodistribution profile, considering both tumor uptake over time (i.e., ⁇ 49 % ID/g, 6 h post injection) and uptake in healthy organs (e.g., spleen uptake of ⁇ 0.3 % ID/g and liver uptake of ⁇ 0.7 % ID/g, 6 h post injection), thereby highlighting the trivalent TriESV6-based binders as best-in-class tumor-targeting ligands.
  • the trivalent compounds are smaller (lower molecular weight), present a higher atom economy, and are easier and cheaper to manufacture compared to their, e.g., tetravalent counterparts.
  • binders according to the present invention can provide unexpected improvement in tumor uptake while at the same time providing advantageous biodistribution, thus having an improved therapeutic potential.
  • the trivalent binder according to the invention shows surprisingly improved in vivo therapeutic activity on FAP-positive tumors when compared to mono- and bivalent binders, while at the same time maintaining the tolerability, as evident from FIG.7.
  • binders according to the present invention can provide unexpected improvement in therapeutic efficacy and/or targeting specificity. As evident from the results in FIG.
  • a short spacer (L) a between the trivalent ESV6 moiety and the ramification point confers a superior inhibitory activity as compared to longer spacers (see also Table 16). This is surprising, considering that spacers in multivalent compounds with enough length to reach multiple adjacent FAP’s on the target cell would have rather been expected to be advantageous.
  • the present invention provides particularly advantageous therapeutic compounds which can be administered at lower dosages (due to their improved binding properties), and are expected provide advantageous side effect profile (due to the lower required dosage, on the one hand, and their improved biodistribution, particularly high tumor:kidney ratio and tolerability profile, on the other hand).
  • the compound of the present invention is represented by the following Formula II: wherein each occurrence of L, BS and BL is independently a moiety comprising or consisting of a structural unit selected from alkylene, cycloalkylene, arylalkylene, heteroarylalkylene, heteroalkylene, heterocycloalkylene, alkenylene, cycloalkenylene, arylalkenylene, heteroarylalkenylene, heteroalkenylene, heterocycloalenkylene, alkynylene, heteroalkynylene, arylene, heteroarylene, aminoacyl, oxyalkylene, oxoalkylene, dioxoalkylene, aminoalkylene, diaminoalkylene, diacid ester, dialkylsiloxane, amide, thioamide, thioether, thioester, ester, carbamate, hydrazone, thiazolidine, methylene alkoxy carbamate, disul
  • moiety C comprises a ramification point, such that three moieties A are attached to C, and B is absent, i.e., moiety C serves as a ramification point therefore substituting B, preferably when C is a chelator (e.g., a chelating agent group suitable for radiolabeling with therapeutic nuclides.
  • a chelator e.g., a chelating agent group suitable for radiolabeling with therapeutic nuclides.
  • An exemplary structure is represented by general Formula III below, wherein all groups and variables are as defined in the claims, and wherein preferably each occurrence of a is 0: III
  • C is a chelator bearing multiple –COOH groups
  • each A–(L) a – arm is attached to a –C(O)– groups in the chelator structure derived from the respective –COOH group.
  • the arm may be attached to a respective –COOH group of the chelator, thereby forming an ester (–C(O)O–R–A);
  • the terminal atom of A–(L) a – is an amino nitrogen (i.e., the arm has the general structure A–R–NH–)
  • this arm may be attached to a respective –COOH group of the chelator, thereby forming an amide (–CONH–R–A), wherein R is the particular structure of (L) a in the respective arm excluding the terminal atom.
  • a molecule comprising three moieties A can comprise, e.g., one or two ramification points, and preferably comprises only one ramification point.
  • the number of covalent bonds between each ramification point and the closest ramification point is preferably 7 or less, more preferably 5 or less, most preferably 3 or less.
  • the terms “distance” or “length” between moieties or groups denote, unless specified otherwise, the longest interatomic (through space) distance in the most expanded conformation between the two non-hydrogen atoms belonging to the respective groups which atoms are connected through the path comprising the smallest number of covalent bonds.
  • the multiple FAP-binding moieties A present in the molecule of a binder according to the present invention preferably up to 4, more preferably 3, engage in binding and rebinding to the very same FAP target molecule and/or establishing additional synergistic interactions that a single moiety cannot.
  • Tri-ESV6-DOTAGA (having a distance between each moiety A and the closest ramification point of approx.17 ⁇ ) exhibits a much stronger FAP inhibition ( ⁇ 1000-fold lower IC50) as compared to Tri-ESV6-PEG12-DOTAGA with a longer distance (approx. 60 ⁇ ) between each moiety A and the point of ramification.
  • particularly suitable distances (d 1 + d 2 ) between each moiety A and the closest ramification point are considered to be 30 ⁇ or less, preferably 24 ⁇ or less, more preferably 17 ⁇ or less.
  • the distance between each moiety A and J is 24 ⁇ or less, preferably 18 ⁇ or less, more preferably 11 ⁇ or less.
  • This distance represents the length of the spacer that connects a ligand A (that binds to fibroblast activation protein (FAP) on a target cell) with a multipoint template J to which the multiple arms of the compound connect, and may also be denoted as d1, as exemplified in the below schematic representation. In other words, is the distance between the two atoms directly bound to each end of moiety (L) a .
  • lowering distance d1 is considered to contribute to improving the binding properties.
  • the distance between each moiety L and the closest ramification point is 19 ⁇ or less, preferably 12 ⁇ or less, more preferably 6 ⁇ or less.
  • This distance represents the length from the ramification point to the “first” atom belonging to moiety L along the chain which represents the shortest path between the ramification point and the respective moiety A, and circumscribes the part of the distance between each moiety A and the closest ramification point which belongs to the multipoint template J.
  • This distance may also be denoted as d2, as exemplified in the below schematic representation.
  • d2 is the distance between the atom belonging to group moiety (L) a (or A, if a is 0) directly bound to moiety J, and the ramification point. Without wishing to be bound by theory, lowering distance d2 is considered to contribute to improving the binding properties.
  • the highest enrichment is achieved after 5 min, 10 min, 20 min, 30 min, 45 min, 1 h, 2 h, 3 h, 4 h, 5 h or 6 h; and/or enrichment in the disease site is maintained at a therapeutically relevant level, over a period of or at least for 5 min, 10 min, 20 min, 30 min, 45 min, 1 h, 2 h, 3 h, 4 h, 5 h or 6 h, 24 h, 48 h, 72h , 96 h, more preferably beyond 6 h, even more preferably beyond 24 h post injection.
  • Moiety B is a covalent bond or a moiety comprising a chain of atoms that covalently connects the moieties A to the payload C.
  • the moiety B may be cleavable or non-cleavable, multifunctional moiety which can be used to link one or more payload and/or binder moieties to form the targeted conjugate of the invention.
  • moiety B is a multifunctional moiety linking one or more moieties C and/or moieties A.
  • the structure of the compound can comprise 3 moieties A per molecule.
  • the structure of the compound may comprise more than one moiety C, preferably 2, 3, 4, 5, 6, 7, 8, 9 or 10 moieties C per molecule.
  • the structure of the compound comprises 3 moieties A and 1 moiety C per molecule.
  • release mechanisms can be identical to those specific to antibodies linked to cytotoxic payloads.
  • the nature of the binding moieties is independent in that respect. Therefore, there is envisaged pH-dependent [Leamon, C.P. et al (2006) Bioconjugate Chem., 17, 1226; Casi, G. et al (2012) J. Am. Chem. Soc., 134, 5887], reductive [Bernardes, G.J. et al (2012) Angew. Chem. Int. Ed. Engl., 51.941; Yang, J. et al (2006) Proc.
  • Moiety B can comprise or consist of a unit shown in Table A below wherein the substituents R and R n shown in the formulae may suitably be independently selected from H, halogen, substituted or unsubstituted (hetero)alkyl, (hetero)alkenyl, (hetero)alkynyl, (hetero)aryl, (hetero)arylalkyl, (hetero)cycloalkyl, (hetero)cycloalkylaryl, heterocyclylalkyl, a peptide, an oligosaccharide or a steroid group.
  • substituents R and R n shown in the formulae may suitably be independently selected from H, halogen, substituted or unsubstituted (hetero)alkyl, (hetero)alkenyl, (hetero)alkynyl, (hetero)aryl, (hetero)arylalkyl, (hetero)cycloalkyl, (
  • each of R, R1, R2 and R3 is independently selected from H, OH, SH, NH2, halogen, cyano, carboxy, alkyl, cycloalkyl, aryl and heteroaryl, each of which is substituted or unsubstituted.
  • R and R n are independently selected from H, or C1-C7 alkyl or heteroalkyl. More suitably, R and R n are independently selected from H, methyl or ethyl.
  • Moiety B, unit(s) B L and/or unit(s) B S may suitably comprise as a cleavable bond a disulfide linkage since these linkages are stable to hydrolysis, while giving suitable drug release kinetics at the target in vivo, and can provide traceless cleavage of drug moieties including a thiol group.
  • Moiety B, unit(s) BL and/or unit(s) BS may be polar or charged in order to improve water solubility of the conjugate.
  • the linker may comprise from about 1 to about 20, suitably from about 2 to about 10, residues of one or more known water-soluble oligomers such as peptides, oligosaccharides, glycosaminoglycans, polyacrylic acid or salts thereof, polyethylene glycol, polyhydroxyethyl (meth) acrylates, polysulfonates, etc.
  • the linker may comprise a polar or charged peptide moiety comprising e.g. from 2 to 10 amino acid residues.
  • Amino acids may refer to any natural or non-natural amino acid.
  • the peptide linker suitably includes a free thiol group, preferably a N-terminal cysteine, for forming the said cleavable disulfide linkage with a thiol group on the drug moiety.
  • a free thiol group preferably a N-terminal cysteine
  • Any peptide containing L- or D-aminoacids can be suitable; particularly suitable peptide linkers of this type are Asp-Arg-Asp-Cys and/or Asp-Lys-Asp-Cys.
  • moiety B, unit(s) BL and/or unit(s) BS may comprise a cleavable or non- cleavable peptide unit that is specifically tailored so that it will be selectively enzymatically cleaved from the drug moiety by one or more proteases on the cell surface or the extracellular regions of the target tissue.
  • the amino acid residue chain length of the peptide unit suitably ranges from that of a single amino acid to about eight amino acid residues.
  • Numerous specific cleavable peptide sequences suitable for use in the present invention can be designed and optimized in their selectivity for enzymatic cleavage by a particular tumor-associated enzyme e.g. a protease.
  • Cleavable peptides for use in the present invention include those which are optimized toward the proteases MMP-1, 2 or 3, or cathepsin B, C or D. Especially suitable are peptides cleavable by Cathepsin B.
  • Cathepsin B is a ubiquitous cysteine protease. It is an intracellular enzyme, except in pathological conditions, such as metastatic tumors or rheumatoid arthritis.
  • An example for a peptide cleavable by Cathepsin B is containing the sequence Val-Cit.
  • the moiety B and in particular, unit(s) B L suitably further comprise(s) self-immolative moiety can or cannot be present after the linker.
  • the self-immolative linkers are also known as electronic cascade linkers. These linkers undergo elimination and fragmentation upon enzymatic cleavage of the peptide to release the drug in active, preferably free form.
  • the conjugate is stable extracellularly in the absence of an enzyme capable of cleaving the linker. However, upon exposure to a suitable enzyme, the linker is cleaved initiating a spontaneous self-immolative reaction resulting in the cleavage of the bond covalently linking the self-immolative moiety to the drug, to thereby effect release of the drug in its underivatized or pharmacologically active form.
  • the self-immolative linker is coupled to the binding moiety through an enzymatically cleavable peptide sequence that provides a substrate for an enzyme to cleave the amide bond to initiate the self-immolative reaction.
  • the drug moiety is connected to the self-immolative moiety of the linker via a chemically reactive functional group pending from the drug such as a primary or secondary amine, hydroxyl, sulfhydryl or carboxyl group.
  • self-immolative linkers are PABC or PAB (para-aminobenzyloxycarbonyl), attaching the drug moiety to the binding moiety in the conjugate (Carl et al (1981) J. Med. Chem.
  • the amide bond linking the carboxy terminus of a peptide unit and the para-aminobenzyl of PAB may be a substrate and cleavable by certain proteases.
  • the aromatic amine becomes electron-donating and initiates an electronic cascade that leads to the expulsion of the leaving group, which releases the free drug after elimination of carbon dioxide (de Groot, et al (2001) Journal of Organic Chemistry 66 (26): 8815-8830). Further self-immolating linkers are described in WO2005/082023.
  • the linker comprises a glucuronyl group that is cleavable by glucoronidase present on the cell surface or the extracellular region of the target tissue. It has been shown that lysosomal beta-glucuronidase is liberated extracellularly in high local concentrations in necrotic areas in human cancers, and that this provides a route to targeted chemotherapy (Bosslet, K. et al. Cancer Res. 58, 1195- 1201 (1998)).
  • the moiety ⁇ suitably further comprises a spacer unit.
  • a spacer unit can be the unit BS, which may be linked to the binding moiety A, for example via an amide, amine or thioether bond.
  • the spacer unit is of a length that enables e.g. the cleavable peptide sequence to be contacted by the cleaving enzyme (e. g. cathepsin B) and suitably also the hydrolysis of the amide bond coupling the cleavable peptide to the self-immolative moiety X.
  • Spacer units may for example comprise a divalent radical such as alkylene, arylene, a heteroarylene, repeating units of alkyloxy (e.g. polyethylenoxy, PEG, polymethyleneoxy) and alkylamino (e.g. polyethyleneamino), or diacid ester and amides including succinate, succinamide, diglycolate, malonate, and caproamide.
  • * represents a point of attachment to moiety A or a point of attachment for which the shortest path to moiety A comprises less atoms than that for •, as the case may be; and • represents a point of attachment a point of attachment to therapeutic moiety C or a point of attachment to therapeutic moiety C for which the shortest path to therapeutic moiety C comprises less atoms than that for *, as the case may be.
  • a reactive moiety L is present rather than the therapeutic payload moiety C.
  • each * represents a point of attachment for which the shortest path to moiety A comprises less atoms than that for •; and each • represents a point of attachment for which the shortest path to therapeutic moiety C comprises less atoms than that for *, with the proviso that when n is > 1 and a respective point of attachment is indicated on any one of R a , R b and R c , then it can be independently present in one or more of the peptide monomeric units, preferably in one peptide monomeric unit most distant from the other point of attachment indicated in the respective structure.
  • peptide refers to peptide mono- or oligomers having a backbone formed by proteinogenic and/or a non- proteinogenic amino acids.
  • aminoacyl or “aminoacid” generally refer to any proteinogenic or a non-proteinogenic amino acid.
  • the side-chain residues of a proteinogenic or a non-proteinogenic amino acid are represented by any of R a , R b and R c , each of which is selected from the following list: wherein each of R, R 1 , R 2 and R 3 is independently selected from H, OH, SH, NH2, halogen, cyano, carboxy, alkyl, cycloalkyl, aryl and heteroaryl, each of which is substituted or unsubstituted; each X is independently selected from NH, NR, S, O and CH 2 , preferably NH; and each n and m is independently an integer preferably selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20.
  • side-chain residues of a proteinogenic or a non- proteinogenic amino acid are represented by any of R a , R b and R c , each of which may be part of a 3-, 4-, 5-, 6- or 7-membered ring.
  • the side chain alpha, beta and/or gamma position of said proteinogenic or non-proteinogenic amino acid can be part of a cyclic structure selected from an azetidine ring, pyrrolidine ring and a piperidine ring, such as in the following aminoacids (proline and hydroxyproline): each of which may independently be part of an unsaturated structure (i.e.
  • the H atom geminal to the respective group R a , R b and R c is absent), e.g.: .
  • the following notation of peptide sequences refers to a sequence from N to C terminus, and attachment of group through a horizontal bond (here: moiety C) means covalent attachment to the peptide backbone via amide bond to the respective terminal amino acid (here: AA3):
  • the following notation of peptide sequences refers to a sequence from N to C terminus, and attachment of group through a vertical bond (here: moiety C) means covalent attachment via the sidechain of the respective amino acid (here: AA3): .
  • B comprises the following structure wherein each x is an integer independently selected from the range of 0 to 100, preferably 0 to 50, more preferably 0 to 30, yet more preferably selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20; each y is an integer independently selected from the range of 0 to 30, preferably selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20; each z is an integer independently selected from the range of 0 to 5, preferably selected from selected from 0, 1, 2, 3 and 4 * represents a point of attachment to a moiety A; and • represents a point of attachment to therapeutic moiety C.
  • Each of L, BS and BL can be independently a moiety comprising or consisting of a structural unit selected from alkylene, cycloalkylene, arylalkylene, heteroarylalkylene, heteroalkylene, heterocycloalkylene, alkenylene, cycloalkenylene, arylalkenylene, heteroarylalkenylene, heteroalkenylene, heterocycloalenkylene, alkynylene, heteroalkynylene, arylene, heteroarylene, aminoacyl, oxyalkylene, oxoalkylene, dioxoalkylene, aminoalkylene, diaminoalkylene, diacid ester, dialkylsiloxane, amide, thioamide, thioether, thioester, ester, carbamate, hydrazone, thiazolidine, methylene alkoxy carbamate, disulfide, vinylene, imine, imidamide, phosphoramide, saccharide
  • Moiety J forms part of the linking moiety B, and is characterized in that it comprises a ramification point, i.e., a central atom bound to at least three non-hydrogen atoms, wherein at least two of said non-hydrogen atoms are located on a covalent chain which represents the shortest path between a moiety A and said central atom. It can thus be regarded as a multipoint template to which the multiple arms bearing moieties A are joined together.
  • the moiety J together with the groups (L)a are herein collectively denoted as moiety K.
  • J can be independently a moiety comprising a ramification point, comprising or consisting of a structural unit independently selected from the group consisting of alkylene, cycloalkylene, arylalkylene, heteroarylalkylene, heteroalkylene, heterocycloalkylene, alkenylene, cycloalkenylene, arylalkenylene, heteroarylalkenylene, heteroalkenylene, heterocycloalenkylene, alkynylene, heteroalkynylene, arylene, heteroarylene, aminoacyl, oxyalkylene, (oxo)alkylene, dioxoalkylene, aminoalkylene, diaminoalkylene, diacid ester, dialkylsiloxane, amide, thioamide, thioether, thioester, ester, carbamate, hydrazone, thiazolidine, methylene alkoxy carbamate, disulfide, vinylene, imine, imidamide,
  • Moiety C in the present invention represents a therapeutic payload, which can be generally any atom (including H), molecule or particle.
  • moiety C is not a hydrogen atom.
  • the payload may be a chelator for radiolabeling a therapeutic conjugate with a therapeutic nuclide.
  • the radionuclide is not released.
  • Chelators are well known to those skilled in the art, and for example, include chelators such as sulfur colloid, diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), 1,4,7,10-tetraazacyclododececane, N-(glutaric acid)-N',N'',N'''-triacetic acid (DOTAGA), 1,4,7- triazacyclononane-N,N',N''-triacetic acid (NOTA), 1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''- tetraacetic acid (TETA), or any of the preferred chelator structures recited in the in the items further below or in the appended claims.
  • chelators such as sulfur colloid
  • the therapeutic payload may be a radioactive group comprising or consisting of a therapeutic radioisotope, including isotopes such as 223 Ra, 89 Sr, 90 Y, 121 Sn, 177 Lu, 131 I, 211 At, 225 Ac, 188 Re, 149 Tb, 161 Tb and 227 Th, preferably 90 Y, 225 Ac or 177 Lu, more preferably 177 Lu; which may not be used for diagnostic applications.
  • a therapeutic radioisotope including isotopes such as 223 Ra, 89 Sr, 90 Y, 121 Sn, 177 Lu, 131 I, 211 At, 225 Ac, 188 Re, 149 Tb, 161 Tb and 227 Th, preferably 90 Y, 225 Ac or 177 Lu, more preferably 177 Lu; which may not be used for diagnostic applications.
  • the therapeutic payload may be a chelate of a therapeutic radioactive isotope, preferably of an isotope listed under above, with a chelating agent, preferably a chelating agent listed above or any of the preferred chelator structures recited further below; or a group selected from the structures listed further below.
  • the payload may be a cytotoxic and/or cytostatic agent. Such agents can inhibit or prevent the function of cells and/or cause destruction of cells. Examples of cytotoxic agents include radioactive therapeutic isotopes, chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including synthetic analogues and derivatives thereof.
  • the cytotoxic agent may be selected from the group consisting of an auristatin, a DNA minor groove binding agent, a DNA minor groove alkylating agent, an enediyne, a lexitropsin, a duocarmycin, a taxane, a puromycin, a dolastatin, a maytansinoid and a vinca alkaloid or a combination of two or more thereof.
  • Preferred cytotoxic and/or cytostatic payload moieties are listed in item 8 (e) further below.
  • the payload is a therapeutic chemotherapeutic agent selected from the group consisting of a topoisomerase inhibitor, an alkylating agent (e.g., nitrogen mustards; ethylenimes; alkylsulfonates; triazenes; piperazines; and nitrosureas), an antimetabolite (e.g., mercaptopurine, thioguanine, 5- fluorouracil), an antibiotics (e.g., anthracyclines, dactinomycin, bleomycin, adriamycin, mithramycin.
  • a topoisomerase inhibitor e.g., an alkylating agent (e.g., nitrogen mustards; ethylenimes; alkylsulfonates; triazenes; piperazines; and nitrosureas)
  • an antimetabolite e.g., mercaptopurine, thioguanine, 5- fluorouracil
  • dactinomycin a mitotic disrupter (e.g., plant alkaloids – such as vincristine and/or microtubule antagonists – such as paclitaxel), a DNA methylating agent, a DNA intercalating agent (e.g., carboplatin and/or cisplatin, daunomycin and/or doxorubicin and/or bleomycin and/or thalidomide), a DNA synthesis inhibitor, a DNA-RNA transcription regulator, an enzyme inhibitor, a gene regulator, a hormone response modifier, a hypoxia-selective cytotoxin (e.g., tirapazamine), an epidermal growth factor inhibitor, an anti- vascular agent (e.g., xanthenone 5,6-dimethylxanthenone-4-acetic acid), a radiation-activated prodrug (e.g., nitroarylmethyl quaternary (NMQ) salts) or a bioreductive drug or a combination of two or more
  • the payload (i.e., moiety C) is not derived from an anthracycline, preferably not derived from PNU 159682.
  • the therapeutic chemotherapeutic agent may selected from the group consisting of Erlotinib (TARCEVA®), Bortezomib (VELCADE®), Fulvestrant (FASLODEX®), Sutent (SU11248), Letrozole (FEMARA®), Imatinib mesylate (GLEEVEC®), PTK787/ZK 222584, Oxaliplatin (Eloxatin®.), 5-FU (5- fluorouracil), Leucovorin, Rapamycin (Sirolimus, RAPAMUNE®.), Lapatinib (GSK572016), Lonafarnib (SCH 66336), Sorafenib (BAY43-9006), and Gefitinib (IRESSA®.), AG1478, AG1571 (SU 5271; Sugen) or a combination of two or
  • the therapeutic chemotherapeutic agent may be an alkylating agent – such as thiotepa, CYTOXAN® and/or cyclosphosphamide; an alkyl sulfonate – such as busulfan, improsulfan and/or piposulfan; an aziridine - such as benzodopa, carboquone, meturedopa and/or uredopa; ethylenimines and/or methylamelamines – such as altretamine, triethylenemelamine, triethylenepbosphoramide, triethylenethiophosphoramide and/or trimethylomelamine; acetogenin – such as bullatacin and/or bullatacinone; camptothecin; bryostatin; callystatin; cryptophycins; dolastatin; duocarmycin; eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustard
  • doxorubicin such as morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and/or deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins - such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti- metabolites - such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues - such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues - such as fludarabine, 6-mercaptopurine, thiamiprine,
  • paclitaxel paclitaxel, abraxane, and/or TAXOTERE®, doxetaxel; chloranbucil; GEMZAR®.
  • gemcitabine 6- thioguanine; mercaptopurine; methotrexate; platinum analogues - such as cisplatin and carboplatin; vinblastine; platinum; etoposide; ifosfamide; mitoxantrone; vincristine; NAVELBINE®, vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids - such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids, derivatives or combinations of two or more of any of the above.
  • platinum analogues - such as
  • the therapeutic payload may be a tubulin disruptor including but are not limited to: taxanes - such as paclitaxel and docetaxel, vinca alkaloids, discodermolide, epothilones A and B, desoxyepothilone, cryptophycins, curacin A, combretastatin A-4-phosphate, BMS 247550, BMS 184476, BMS 188791; LEP, RPR 109881A, EPO 906, TXD 258, ZD 6126, vinflunine, LU 103793, dolastatin 10, E7010, T138067 and T900607, colchicine, phenstatin, chalcones, indanocine, T138067, oncocidin, vincristine, vinblastine, vinorelbine, vinflunine, halichondrin B, isohomohalichondrin B, ER-86526, pironetin, spongistatin 1, spiket P, crypto
  • the therapeutic payload may be a DNA intercalator including but are not limited to: acridines, actinomycins, anthracyclines, benzothiopyranoindazoles, pixantrone, crisnatol, brostallicin, CI-958, doxorubicin (adriamycin), actinomycin D, daunorubicin (daunomycin), bleomycin, idarubicin, mitoxantrone, cyclophosphamide, melphalan, mitomycin C, bizelesin, etoposide, mitoxantrone, SN-38, carboplatin, cis-platin, actinomycin D, amsacrine, DACA, pyrazoloacridine, irinotecan and topotecan and pharmaceutically acceptable salts, acids, derivatives or combinations of two or more of any of the above.
  • a DNA intercalator including but are not limited to:
  • the therapeutic payload may be an anti-hormonal agent that acts to regulate or inhibit hormone action on tumors - such as anti-estrogens and selective estrogen receptor modulators, including, but not limited to, tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and/or fareston toremifene and pharmaceutically acceptable salts, acids, derivatives or combinations of two or more of any of the above.
  • an anti-hormonal agent that acts to regulate or inhibit hormone action on tumors -
  • selective estrogen receptor modulators including, but not limited to, tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and/or fareston toremifene and pharmaceutically acceptable salts, acids, derivatives or combinations of two or more
  • the therapeutic payload may be an aromatase inhibitor that inhibits the enzyme aromatase, which regulates estrogen production in the adrenal glands - such as, for example, 4(5)- imidazoles, aminoglutethimide, megestrol acetate, AROMASIN®. exemestane, formestanie, fadrozole, RIVISOR®. vorozole, FEMARA®. letrozole, and ARIMIDEX® and/or anastrozole and pharmaceutically acceptable salts, acids, derivatives or combinations of two or more of any of the above.
  • an aromatase inhibitor that inhibits the enzyme aromatase, which regulates estrogen production in the adrenal glands - such as, for example, 4(5)- imidazoles, aminoglutethimide, megestrol acetate, AROMASIN®. exemestane, formestanie, fadrozole, RIVISOR®. vorozole, FEMARA®. letrozole, and AR
  • the therapeutic payload may be an anti-androgen such as flutamide, nilutamide, bicalutamide, leuprolide, goserelin and/or troxacitabine and pharmaceutically acceptable salts, acids, derivatives or combinations of two or more of any of the above.
  • the therapeutic payload may be a protein or an antibody.
  • the payload is a cytokine (e.g., an interleukin such as IL2, IL10, IL12, IL15; a member of the TNF superfamily; or an interferon such as interferon gamma.). Any therapeutic payload may be used in unmodified or modified form. Combinations of therapeutic payloads in which some are unmodified and some are modified may be used.
  • the therapeutic payload may be chemically modified.
  • One form of chemical modification is the derivatisation of a carbonyl group – such as an aldehyde.
  • the payload moiety C is a topoisomerase inhibitor; preferably camptothecin (CPT) or a derivative thereof; more preferably derived (e.g., by replacing a hydrogen atom) from topotecan, irinotecan, silatecan, cositecan, exatecan, lurtotecan, gimatecan, belotecan, rubitecan, deruxtecan, DXd; even more preferably exatecan; even more preferably or , wherein each n is 0, 1, 2, 3, 4, 5 or 6; and most preferably .
  • therapeutic moiety C is an auristatin (i.e., having a structure derived from an auristatin compound family member) or an auristatin derivative. More preferably, therapeutic moiety C has a structure according to the following formula: wherein: R 1d is independently H or C 1 -C 6 alkyl; preferably H or CH 3 ; is independently C 1 -C 6 alkyl; preferably CH 3 or iPr; R 3d is independently H or C 1 -C 6 alkyl; preferably H or CH 3 ; R 4d is independently H, C 1 -C 6 alkyl, COO(C 1 -C 6 alkyl), CON(H or C 1 -C 6 alkyl), C 3 -C 10 aryl or C 3 - C 10 heteroaryl; preferably H, CH 3 , COOH, COOCH 3 or thiazolyl; R 5d is independently H, OH, C 1 -C 6 alkyl; preferably H or OH;
  • therapeutic moiety C is derived from MMAE or MMAF.
  • therapeutic moiety C has a structure according to the following formula: wherein: n is 0, 1, 2, 3, 4 or 5; preferably 1; R 1e is independently H, COOH, aryl-COOH or heteroaryl-COOH; preferably COOH; R 2e is independently H, COOH, aryl-COOH or heteroaryl-COOH; preferably COOH; each R 3e is independently H, COOH, aryl-COOH or heteroaryl-COOH; preferably COOH; R 4e is independently H, COOH, aryl-COOH or heteroaryl-COOH; preferably COOH; and X is O, NH or S; preferably O.
  • therapeutic moiety C has a structure according to the following formula: wherein: n is 0, 1, 2, 3, 4 or 5; preferably 1 R 1f is independently H, COOH, aryl-COOH or heteroaryl-COOH; preferably COOH; R 2f is independently H, COOH, aryl-COOH or heteroaryl-COOH; preferably COOH; R 3f is independently H, COOH, aryl-COOH or heteroaryl-COOH; preferably COOH; and X is O, NH or S; preferably O.
  • Particularly preferred embodiments for the therapeutic moiety C as well as the compound according to the present invention are shown in the items further below and the appended claims.
  • C is a chelator
  • An exemplary, particularly preferred structure of this type is represented by Formula IIIa, wherein all variables and groups are as defined in the claims, and which may be present in the form of a complex with any of the therapeutic radioactive isotopes disclosed herein, preferably 90-Yttrium, 225-Actinium or 177-Lutetium, more preferably 177-Lutetium: IIIa
  • the fragment ((BS)x(BL)y)z can be represented by one of the following structures:
  • each of AA 3 , AA 4 , AA 5 , AA 6 , AA 7 , and AA 8 represents a proteinogenic or non-proteinogenic amino acid, or is absent; wherein preferably: each proteinogenic or non-proteinogenic amino acid is preferably independently represented by one of the following structures: and/or AA 4 is an amino acid with a charged sidechain, and AA 7 is an amino acid with an aliphatic sidechain; wherein more preferably: AA 3 is selected from Asp, Glu, and Lys, or is absent; preferably Asp; AA 4 is selected from Arg, HomoArg, Lys, Asp, and Glu, or is absent; preferably Lys or Arg; AA 5 is selected from Asp, Glu, and Lys; preferably Asp; AA 6 is selected from Cys, Lys, Gly and Val; preferably Cys or Lys; AA 7 is selected from Gly, Ala, Val, Arg, Ile, Pro; and AA 8 is selected from
  • the compound may be represented by one of the following structures:
  • Preferred compounds according to the present invention may be represented by: , wherein BS, BL, x, y and n, and the remaining groups are as defined elsewhere herein, wherein each of AA3, AA4, AA5, AA6, AA7 and AA8 represents a proteinogenic or non-proteinogenic amino acid, or is absent; preferably wherein each (B S ) x and (B L ) y is independently represented by bond, —NHC(O)(CH 2 ) n C(O)–, –NH(CH 2 ) n C(O)–, –NHC(O)(CH 2 CH 2 O) m (CH 2 ) n –, –C(O)(CH 2 CH 2 O) m (CH 2 ) n –, – C(O)(CH2CH2O)m(CH2)nNH–, –(CH2CH2O)m(CH2)n–, –(CH2CH2O)m(CH2)nNH–, –(CH
  • Preferred compounds according to the present invention may also be represented by : more preferably:
  • each (B S ) x and (B L ) y is independently represented by bond, —NHC(O)(CH 2 ) n C(O)–, —NH(CH 2 ) n C(O)–,–NHC(O)(CH 2 CH 2 O) m (CH 2 ) n –, –C(O)(CH 2 CH 2 O) m (CH 2 ) n –, – C(O)(CH2CH2O)m(CH2)nNH–, –(CH2CH2O)m(CH2)n–, –(CH2CH2O)m(CH2)nNH–, – (CH2CH2O)m(CH2)nNHC(O)–, –(CH2)nO(CH(CH2CH2O)m(CH2)nNHC(O)–, –(CH2)nO(CH(CH2CH2O)m(CH2)nNHC(O)–, –(CH2)nO(CH(CH2CH2O
  • the moiety C is a therapeutic agent which may be: (a) a chelating agent group suitable for radiolabelling with therapeutic nuclides; (b) a therapeutic radioactive group comprising a therapeutic radioisotope; (c) a chelate of a therapeutic radioactive isotope with a chelating agent; (d) a cytotoxic and/or cytostatic therapeutic agent; (f) immunomodulator agent; or (g) a protein, preferably: (a) the chelating agent group suitable for radiolabeling with therapeutic nuclides is selected from sulfur colloid, diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), 1,4,7,10- tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA), 1,4,8, l l-t
  • the cytotoxic and/or cytostatic therapeutic agent is selected from chemotherapeutic agent selected from the group consisting of topoisomerase inhibitors, alkylating agents, antimetabolites, antibiotics, mitotic disrupters, DNA intercalating agents, DNA synthesis inhibitors, DNA-RNA transcription regulator, enzyme inhibitors, gene regulators, hormone response modifiers, hypoxia- selective cytotoxins, epidermal growth factor inhibitors, anti-vascular agents and a combination of two or more thereof, preferably selected from the following structures:
  • STING agonist 1 STING agonist 2 moiety C is an auristatin, preferably having a structure according to the following formula: wherein: independently H or C 1 -C 6 alkyl; preferably H or CH 3 ; independently C 1 -C 6 alkyl; preferably CH 3 or iPr; R 3d is independently H or C 1 -C 6 alkyl; preferably H or CH 3 ; R 4d is independently H, C 1 -C 6 alkyl, COO(C 1 -C 6 alkyl), CON(H or C 1 -C 6 alkyl), C 3 -C 10 aryl or C 3 -C 10 heteroaryl; preferably H, CH 3 , COOH, COOCH 3 or thiazolyl; R 5d is independently H, OH, C 1 -C 6 alkyl; preferably H or OH; and R 6d is independently C 3 -C 10 aryl or C 3 -C 10 heteroaryl; preferably optionally substituted phenyl or
  • the compounds described herein may be used to treat disease.
  • the treatment may be therapeutic treatment, with the aim being to prevent, reduce or stop an undesired physiological change or disorder.
  • the treatment may prolong survival as compared to expected survival if not receiving treatment.
  • the disease that is treated by the compound may be any disease that might benefit from treatment. This includes chronic and acute disorders or diseases including those pathological conditions which predispose to the disorder.
  • cancer and "cancerous” is used in its broadest sense as meaning the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • a tumor comprises one or more cancerous cells.
  • the therapeutically effect that is observed may be a reduction in the number of cancer cells; a reduction in tumor size; inhibition or retardation of cancer cell infiltration into peripheral organs; inhibition of tumor growth; and/or relief of one or more of the symptoms associated with the cancer.
  • efficacy may be assessed by physical measurements of the tumor during the treatment, and/or by determining partial and complete remission of the cancer.
  • efficacy can, for example, be measured by assessing the time to disease progression (TTP) and/or determining the response rate (RR).
  • said disease or disorder may be independently selected from cancer, inflammation, atherosclerosis, fibrosis, tissue remodelling and keloid disorder, preferably wherein the cancer is selected from the group consisting of breast cancer, pancreatic cancer, small intestine cancer, colon cancer, multi-drug resistant colon cancer, rectal cancer, colorectal cancer, metastatic colorectal cancer, lung cancer, non-small cell lung cancer, head and neck cancer, ovarian cancer, hepatocellular cancer, oesophageal cancer, hypopharynx cancer, nasopharynx cancer, larynx cancer, myeloma cells, bladder cancer, cholangiocarcinoma, clear cell renal carcinoma, neuroendocrine tumor, oncogenic osteomalacia, sarcoma, CUP (carcinoma of unknown primary), thymus cancer, desmoid tumors, glioma, astrocytoma, cervix cancer, skin cancer, kidney cancer and prostate cancer.
  • the cancer is selected from the group consist
  • the compound When used in the methods disclosed herein, the compound has a prolonged residence at the disease site at a therapeutically relevant level, e.g., for at least 5 min, 10 min, 20 min, 30 min, 45 min, 1 h, 2 h, 3 h, 4 h, 5 h or 6 h, 24 h, 48 h, 72h, 96 h, preferably beyond 1 h, more preferably beyond 6 h, even more preferably beyond 24 h post injection.
  • a therapeutically relevant level e.g., for at least 5 min, 10 min, 20 min, 30 min, 45 min, 1 h, 2 h, 3 h, 4 h, 5 h or 6 h, 24 h, 48 h, 72h, 96 h, preferably beyond 1 h, more preferably beyond 6 h, even more preferably beyond 24 h post injection.
  • compositions or combinations for use in any methods for treatment of the human or animal body disclosed herein is also intended to extend to corresponding methods for treating a disease or disorder in subject in need thereof comprising administration of a therapeutically effective amount of the compound, composition or combination to the subject; and to corresponding uses of the compounds, compositions or combinations in the manufacture of a medicament for treating the respective disease or disorder.
  • the present inventors have surprisingly found that the compounds, when given at certain doses, can provide optimal tumor uptake and/or tumor-to-organ ratio, thus showing minimal trapping in other organs (i.e., particularly clean biodistribution profde).
  • the compounds, pharmaceutical compositions or combinations described herein may be administered to a subject at a dose of the compound of general Formula I, II, III, IV, V, VI or VII of 10-500 nmol/kg, preferably 30-250 nmol/kg, more preferably 90-250 nmol/kg, even more preferably 90-160 nmol/kg, most preferably 90-125 nmol/kg, expressed as a mouse dose; or a corresponding human equivalent dose.
  • the compounds, pharmaceutical compositions or combinations described herein may be administered to a human subject at a dose of said compound of 0.8-40 nmol/kg, preferably 2-20 nmol/kg, more preferably 7-20 nmol/kg, even more preferably 7-13 nmol/kg, most preferably 7-10 nmol/kg.
  • mice it was found that at doses in the range of preferably 10 to 500 nmol/kg, in particular at 90 nmol/kg or more, a particularly clean biodistribution profde and optimal tumor uptake can be achieved. Above 250 nmol/kg, saturation effects are observed. Accordingly, the range of 90 nmol/kg or more, preferably 90 to 250 nmol/kg (expressed as mouse dose) is particularly preferred. The range of 7 nmol/kg or more, preferably 7-20 nmol/kg (expressed as human dose) is particularly preferred.
  • Mouse doses can be converted to human equivalent doses (HED), e.g., considering the recommendation reported by FDA in the USFDA. Guidance for Industry: Estimating the Maximum Safe Starting Dose in Adult Healthy Volunteer. Rockville, MD: US Food and Drug Administration; 2005, for which the mouse dose (nmol/kg) has to be divided by a factor 12.3 to obtain the corresponding HED (nmol/kg).
  • HED human equivalent dose
  • a human reference body weight 60, 70 or 80 kg, preferably 70 kg can be used. The conversion takes into consideration the differences in surface area between the two different species (Mus musculus vs. Homo sapiens sapiens).
  • the range of 90 nmol/kg to 250 nmol/kg, expressed as mouse dose corresponds to a HED of 0.016 mg/kg to 0.046 mg/kg or 1.1 mg to 3.2 mg, considering an average body weight of 70 kg.
  • a human dose of 1 mg or more, preferably 1 to 3 mg per administration is particularly advantageous.
  • the compounds described herein may be in the form of pharmaceutical compositions which may be for human or animal therapeutic use in human and/or veterinary medicine, and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
  • the choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
  • lubricant(s) e.g., talc, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, kaolin, sorbic acid, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.
  • the therapeutic pharmaceutical composition may be formulated to be administered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestable solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route.
  • the therapeutic formulation may be designed to be administered by a number of routes.
  • the agent If the agent is to be administered mucosally through the gastrointestinal mucosa, it should be able to remain stable during transit though the gastrointestinal tract; for example, it should be resistant to proteolytic degradation, stable at acid pH and resistant to the detergent effects of bile.
  • the therapeutic pharmaceutical compositions may be administered by inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or the pharmaceutical compositions can be injected parenterally, for example, intravenously, intramuscularly or subcutaneously.
  • the therapeutic compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or monosaccharides to make the solution isotonic with blood.
  • the therapeutic compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
  • the therapeutic compound of the present invention may be administered in the form of a pharmaceutically acceptable or active salt.
  • Pharmaceutically-acceptable salts are well known to those skilled in the art, and for example, include those mentioned by Berge et al, in J.Pharm.Sci., 66, 1-19 (1977).
  • Salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., l,l'-methylene-bis-(2-hydroxy-3-naphthoate)) salts.
  • pamoate i.e., l,l'-methylene-bis-(
  • the routes for administration may include, but are not limited to, one or more of oral (e.g. as a tablet, capsule, or as an ingestable solution), topical, mucosal (e.g. as a nasal spray or aerosol for inhalation), nasal, parenteral (e.g. by an injectable form), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intracerebroventricular, intracerebral, subcutaneous, ophthalmic (including intravitreal or intracameral), transdermal, rectal, buccal, vaginal, epidural, sublingual.
  • oral e.g. as a tablet, capsule, or as an ingestable solution
  • mucosal e.g. as a nasal spray or aerosol for inhalation
  • nasal parenteral (e.g. by an injectable form)
  • gastrointestinal intraspinal, intraperitoneal
  • a physician will determine the actual therapeutic dosage which will be most suitable for an individual subject.
  • the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy.
  • the therapeutic formulations may be packaged in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water, for administration. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described.
  • Exemplary unit dosage formulations contain a daily dose or unit daily sub-dose, or an appropriate fraction thereof, of the active ingredient.
  • the term “pharmaceutical combination” means, e.g., that the individual components comprised therein can be administered in the same dosage form or as separate dosage forms, concomitantly or sequentially; preferably, the subject is exposed, at a point in time, to a therapeutically or prophylactically effective amount of each of the individual components of the combination.
  • the pharmaceutical combination may comprise the two or more individual components within the same formulation, or as separate formulations intended (e.g., as a part of a therapeutic dosage regimen/scheme, or by means of instructions for use) or arranged (e.g., as a kit of parts) for concomitant or sequential administration.
  • the compounds described herein may be administered to a patient in combination with a therapeutic agent, such as an immunoconjugate, preferably an immunocytokine, most preferably an immunocytokine comprising a human IL2 polypeptide conjugated to an scFv polypeptide sequence that is capable of binding to the epitope represented by SEQ ID NO:40 (and is further preferably an L19-IL2 immunocytokine further comprising linked VL and VH domains derived from the antibody LI 9, optionally capable of homodimerization in the scFv2 format), as described herein below.
  • a therapeutic agent such as an immunoconjugate, preferably an immunocytokine, most preferably an immunocytokine comprising a human IL2 polypeptide conjugated to an scFv polypeptide sequence that is capable of binding to the epitope represented by SEQ ID NO:40 (and is further preferably an L19-IL2 immunocytokine further comprising linked VL and VH domains derived from the antibody LI 9, optionally capable
  • a pharmaceutical combination comprising a compound or pharmaceutical composition as disclosed herein and an immunocytokine.
  • immunocytokine refers to a conjugate protein or fusion protein comprising a cytokine and an antibody, antibody fragment or antibody derivative.
  • a fusion protein is a polypeptide that is a translation product resulting from the fusion of two or more genes or nucleic acid coding sequences into one open reading frame (ORF). The fused expression products of the two genes or ORFs may be conjugated by a linker.
  • conjugate protein or fusion protein are generally used interchangeably.
  • the fusion protein may further comprise a signal peptide sequence, normally located upstream (5’) of the specific binding member and subunit.
  • Immunocytokine comprising IL2
  • the immunocytokine comprises a sequence having IL2 activity, i.e., an IL2 polypeptide, i.e., the cytokine IL2 or a functional fragment thereof.
  • the immunocytokine comprises only one (i.e., a single) IL2 polypeptide per polypeptide chain.
  • IL2 and “IL2 polypeptide” are used interchangeably.
  • the IL2 may be derived from any animal, e.g. human, rodent (e.g. rat, mouse), horse, cow, pig, sheep, dog, etc. Human IL2 is preferred in conjugates for administration to humans.
  • the amino acid sequence of human IL2 is set out in SEQ ID NO: 21.
  • the immunocytokine conjugate preferably comprises a single IL2 polypeptide.
  • An IL2 polypeptide in an immunocytokine of the invention retains a biological activity of IL2, e.g., an ability to promote proliferation and/or differentiation of activated T and B lymphocytes and natural killer (NK) cells, and/or to induce cytotoxic T cell (CTL) activity, and/or to induce NK/lymphokine- activated killer (LAK) cell antitumor cytotoxicity.
  • a biological activity of IL2 e.g., an ability to promote proliferation and/or differentiation of activated T and B lymphocytes and natural killer (NK) cells, and/or to induce cytotoxic T cell (CTL) activity, and/or to induce NK/lymphokine- activated killer (LAK) cell antitumor cytotoxicity.
  • the immunocytokine comprises an antibody or an antibody fragment or an antibody derivative.
  • the immunocytokine herein comprises an antibody derivative.
  • the antibody derivative may, e.g., comprise a single-chain variable fragment (scFv), a diabody or a single chain diabody (scDb) or a Fab or a Fab2 or a nanobody or an “SIP” (W02003/076469) or a “Crab” (Neri et al., (1995) J Mol Biol, 246, 367-73).
  • the immunocytokine may comprise an IgG antibody or an IgG derivative.
  • the antibody derivate comprises an scFv.
  • an scFv comprises a VH domain and a VL domain, wherein the domains are linked by a linker that allows association of VH and VL domains to form an antigen binding site.
  • the scFv may be stabilized by the incorporation of disulphide bridges linking the VH and VL domains [48]
  • Single chain Fv (scFv) antibody polypeptide sequences are particularly preferred for incorporation in the immunocytokine (e.g., with a further polypeptide sequence having IL2 activity), owing to their small size of the scFv format, which provides physiological and therapeutic advantages for in vivo use of the immunocytokine conjugates.
  • scFv lack an Fc region, potentially reducing anti-idiotypic reactions and also minimizing undesirable properties relating to activation of complement and interaction with Fc receptors that may hinder tumour targeting and cause non-specific cell activation.
  • the linker joining the VH and VL domains within an scFv chain may be a peptide linker sequence that is not long enough to allow pairing of the VH and VL domains within the same scFv polypeptide chain.
  • a homodimer of scFvs may form instead, in which the VH of one scFv chain pairs with the VL of the other scFv chain (and vice versa).
  • This general format may be referred to as an “scFv2” format, or, alternatively, in some cases, as a “diabody”.
  • suitable short linker sequences are GSSGG (SEQ ID NO: 26) and GGSGG (SEQ ID NO: 27).
  • the linker is such as the 12-residue linker SEQ ID NO: 22.
  • scDb linker a peptide linker
  • the scDb linker sequence is sufficiently long and/or flexible to allow pairing of the VH domain of one scFv unit (i.e., the first VH and VL-containing polypeptide) with the VL domain of the other, complementary scFv unit (i.e., the second VH and VL-containing polypeptide), and vice versa, within a single polypeptide chain.
  • a long and/or flexible linker that allows two complementary VH and VL-containing polypeptides to dimerize within a single polypeptide chain in this manner is 10 to 20 amino acids in length.
  • the antibody, antibody fragment or antibody derivative suitably binds specifically to an extra-cellular matrix (ECM) component associated with neoplastic growth and/or angiogenesis.
  • ECM extra-cellular matrix
  • the antibody, fragment or derivative thereof e.g., scFv, diabody or single-chain diabody
  • CDRs complementarity determining regions
  • VH and/or VL domains of an antibody capable of specifically binding to an antigen of interest may comprise one or more CD Rs or VH and/or VL domains of an antibody capable of specifically binding to an antigen of the ECM.
  • the antigen-binding sites of the antibody, antibody fragment or antibody derivative may be identical or different, but preferably are identical (e.g., the L19 antigen-binding sites of antibody LI 9, see below).
  • Each of the antigen-binding sites may bind the same antigen or epitope. This can be achieved by providing two identical antigen-binding sites such as two identical VH-VL domain pairs, or by providing two different antigen-binding sites, for example comprising different VH and VL domains, which nevertheless both bind the same antigen or epitope.
  • the antibody, antibody fragment or antibody derivative may be bispecific.
  • each of the antigen-binding sites binds a different antigen.
  • two antigen-binding sites may bind two different antigens mentioned herein, e.g. two different antigens of the extracellular matrix, or two different domains of a particular antigen (e.g. fibronectin or tenascin-C).
  • the antigen may be an antigen preferentially expressed by cells of a tumour or tumour neovasculature or associated with the ECM.
  • antigens include fibronectin and tenascin C, as described above.
  • the term "specific binding” means that one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner(s).
  • the term is also applicable where, e.g., an antigen-binding site is specific for a particular epitope that is present on a number of different antigens, in which case the antibody, antibody fragment or antibody derivative carrying the antigen-binding site will be able to bind to the various antigens carrying the epitope.
  • the antibody, fragment or derivative thereof may specifically bind fibronectin.
  • Fibronectin is an antigen subject to alternative splicing, and a number of alternative isoforms of fibronectin are known, including alternatively spliced isoforms A-FN and B-FN, comprising domains ED-A or ED-B, respectively, which are known markers of angiogenesis.
  • the antibody, fragment or derivative thereof binds to fibronectin isoform B-FN, e.g., most preferably it binds to the ED-B domain (extra domain B) of fibronectin isoform B-FN.
  • the amino acid sequence of the ED-B domain of B-FN is provided by residues 1266-1356 of the UniProt database entry P02751 (human Fibronectin), and herein as SEQ ID NO: 39.
  • the antibody, fragment or derivative portion of the immunocytokine binds to an epitope of SEQ ID NO:39.
  • the antibody, fragment or derivative e.g., an scFv polypeptide sequence
  • Fibronectin isoform B-FN is one ofthe best-known markers for angiogenesis (W01997/045544).
  • the extra domain “ED-B” of 91 amino acids is found in the B-FN isoform and is identical in mouse, rat, rabbit, dog and man.
  • B-FN accumulates around neovascular structures in aggressive tumours and other tissues undergoing angiogenesis, such as the endometrium in the proliferative phase and some ocular structures in pathological conditions, but is otherwise undetectable in normal adult tissues.
  • the antibody derivative is a human monoclonal scFv sequence that specifically binds to alternatively spliced ED-B domain of fibronectin isoform B-FN, wherein preferably the scFv sequence binds to an epitope of SEQ ID NO:39, e.g., the epitope represented by SEQ ID NO: 40.
  • the antibody L19 binds specifically to the alternatively spliced ED-B domain of fibronectin isoform B-FN, wherein the epitope represented by SEQ ID NO: 40.
  • the sequence of antibody L19 is disclosed in Pini et al. (1998) J. Biol. Chem. 273: 21769-21776 or US patent 8,097,254.
  • L19 scFv which has been described previously (WO1999/058570; WO2006/119897 or W02003/076469); see also the L19 scFv-comprising immunocytokine of SEQ ID NO: 15 and Fig IB in W02020/070150).
  • the immunocytokine comprises (as the antibody derivative) a human monoclonal scFv polypeptide sequence specific for alternatively spliced ED-B domain of fibronectin isoform B-FN (e.g. the epitope represented by SEQ ID NO: 40) as described above and (as the cytokine) IL2 (see, e.g., SEQ ID NO: 15 and Fig IB in W02020/070150, and SEQ ID NO: 12 herein).
  • the antigen-binding site may, e.g., comprise one, two, three, four, five or six CDRs of antibody L19.
  • Amino acid sequences of the CDRs of L19 are: SEQ ID NO: 13 (CDR1 VH); SEQ ID NO: 14 (CDR2 VH); SEQ ID NO: 15 (CDR3 VH); SEQ ID NO: 16 (CDR1 VL); SEQ ID NO: 17 (CDR2 VL), and/or SEQ ID NO: 18 (CDR3 VL).
  • SEQ ID Nos 13-15 are the amino acid sequences of the VH CDR regions (1-3, respectively) of the human monoclonal antibody LI 9.
  • SEQ ID Nos 16-18 are the amino acid of the VL CDR regions (1-3, respectively) of the human monoclonal antibody LI 9.
  • the antigen-binding site may be flanked by one, two, three, four, five, six, seven, or eight of the framework regions of antibody L19.
  • the amino acid sequences of the framework regions of L19 are: SEQ ID NO: 30 (Framework region 1 VH); SEQ ID NO: 31 (Framework region 1 VH); SEQ ID NO: 32 (Framework region 1 VH); SEQ ID NO: 33 (Framework region 1 VH); SEQ ID NO: 34 (Framework region 1 VL); SEQ ID NO: 35 (Framework region 1 VL); SEQ ID NO: 36 (Framework region 1 VL); and/or SEQ ID NO: 37 (Framework region 1 VL).
  • amino acid sequence of the VH and VL domains of antibody L19 correspond to SEQ ID NOs 19 and 20, respectively.
  • the antibody derivative comprises a VH domain with an amino acid sequence comprising VH CDR1, VH CDR2, and/or VH CDR3 of LI 9, and a VL domain with an amino acid sequence comprising VL CDR1, VL CDR2, and/or VL CDR3 of L19.
  • the antibody derivative as described above may comprise a VH domain having an amino acid sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% (preferably 80%) sequence identity with the amino acid sequence of the L19 VH domain as set out in SEQ ID NO: 22, and/or may comprise a VL domain having an amino acid sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% % (preferably 80%) sequence identity with the amino acid sequence of the L19 VL domain as set out in SEQ ID NO: 23.
  • the antibody derivative as described above is an L19 scFv (i.e., an scFv derived from and comprising one or more antigen-specific portions of antibody LI 9; interchangeably referred to as “scFv(L19)”).
  • An L19 scFv may thus comprise one, two, three, four, five or six CDRs of antibody L19 (preferably all six CDRs).
  • the L19 scFv may comprise an L19 VH domain (SEQ ID NO: 22) and/or an L19 VL domain (SEQ ID NO: 23), e.g., both the L19 VH and the L19 VL.
  • the VH and VL domains are joined by a linker.
  • the linker may be a peptide linker sequence that is not long enough to allow pairing of the VH and VL domains.
  • a homodimer of scFvs may form instead, in which the VH of one scFv chain pairs with the VL of the other scFv chain (and vice versa).
  • This general format may be referred to as an “scFv2” format, or, alternatively, in some cases, as a “diabody”.
  • suitable short linker sequences are GSSGG (SEQ ID NO: 26) and GGSGG (SEQ ID NO: 27).
  • the linker is the 12-residue linker SEQ ID NO: 22.
  • the antibody derivative may be an L 19 diabody having an amino acid sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% (preferably 80%) sequence identity with the amino acid sequence of as set out in SEQ ID NO: 28 or an L19 scDb having an amino acid sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% (preferably 80%) sequence identity with the amino acid sequence of as set out in SEQ ID NO: 29
  • the antibody derivative is an scFv(L19) having an amino acid sequence with at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% (preferably 80%) sequence identity with the amino acid sequence of as set out in SEQ ID NO: 38.
  • the immunocytokine comprises a cytokine (e.g., an IL2 polypeptide) and is conjugated to an antibody, an antibody fragment or an antibody derivative.
  • This conjugation may be effected through any suitable covalent bond or linker moiety, e.g., a disulphide or peptide bond, most preferably a peptide linker sequence.
  • a peptide linker sequence may be a short (2-30, preferably 10-20) residue stretch of amino acids. Suitable examples of peptide linker sequences are known in the art. Examples of suitable linker sequences are (648)3 (SEQ ID NO:24) or GSLDGAGGSAGADGG (SEQ ID NO: 25). One or more different linkers may be used.
  • the linker may be the 17-residue linker of SEQ ID NO: 23.
  • the antibody, antibody fragment, or antibody derivative and IL2 may be produced and/or secreted as a single -chain polypeptide.
  • the IL2 is preferably linked to the C-terminus of the antibody or the antibody fragment or the antibody derivative. Preferably, that linkage may be via a peptide linker sequence, as disclosed herein. Where the IL2 is conjugated to the C-terminus, the N-terminus of the antibody or the antibody fragment or the antibody derivative is preferably free. “Free” in this context refers to the N-terminus not being linked or otherwise conjugated to another moiety, such as IL2.
  • the immunocytokine comprises an L19-derived scFv unit linked to an IL2 polypeptide in a conjugate polypeptide chain that that forms a homodimer due to complementary pairing between the VH and VL domains of the scFv unit in two polypeptide chains (in accordance with the scFv2 or diabody format).
  • the immunocytokine comprises a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% (preferably 80%) sequence identity with the amino acid sequence of the “L19-IL2” immunocytokine represented by SEQ ID NO: 12, which comprises an L19-derived scFv unit linked to IL2, possesses such functionality and forms a homodimer via the scFv units in this manner.
  • the immunocytokine comprises the sequence of SEQ ID NO: 12 (i.e., an amino acid sequence of an scFv-format L19-IL2 polypeptide).
  • the production and purification of L19-IL2 constructs may be performed as described in WOO 1/062298.
  • a schematic representation of the scFv-format L19-IL2 conjugate is shown in FIG. 12.
  • GAP Garnier GCG package, Accelerys Inc, San Diego USA.
  • Use of GAP may be preferred but other algorithms may be used, e.g. BUAST (which uses the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Uipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981 ) J. Mol Biol.
  • VH and VU domains and CDRs may also be employed in antibody molecules for use in conjugates as described herein. Suitable variants can be obtained by means of methods of sequence alteration, or mutation, and screening.
  • Particular variants for use as described herein may include one or more amino acid sequence alterations (addition, deletion, substitution and/or insertion of an amino acid residue), maybe less than about 20 alterations, less than about 15 alterations, less than about 10 alterations or less than about 5 alterations, 4, 3, 2 or 1.
  • herein disclosed is a compound, its individual diastereoisomers, its hydrates, its solvates, its crystal forms, its individual tautomers or a salt thereof, wherein the compound (precursor compound) comprises three moieties A and a reactive moiety G capable of reacting and forming a covalent bond with a conjugation partner.
  • the former precursor compound Upon conjugation (i.e., reacting and forming a covalent bond), the former precursor compound is bound to the former conjugation partner, which in turn to a therapeutic payload moiety C.
  • the conjugation partner can be an atom, a molecule, a particle, a therapeutic agent.
  • the conjugation is a therapeutic agent, and can correspond to the payload moieties already described in detail above with respect to the therapeutic conjugates according to the invention.
  • Each moiety A preferably has the structure A 1 or A 2 as previously defined.
  • the precursor compound is represented by the following formula: wherein B is a covalent bond or a multifunctional moiety covalently attaching the moieties A to G.
  • G is capable of forming, upon reacting, an amide, ester, carbamate, hydrazone, thiazolidine, methylene alkoxy carbamate, disulphide, alkylene, cycloalkylene, arylalkylene, heteroarylalkylene, heteroalkylene, heterocycloalkylene, alkenylene, cycloalkenylene, arylalkenylene, heteroarylalkenylene, heteroalkenylene, heterocycloalenkylene, alkynylene, heteroalkynylene, arylene, heteroarylene, aminoacyl, oxyalkylene, aminoalkylene, diacid ester, dialkylsiloxane, amide, thioamide, thioether, thioester, ester, carbamate, hydrazone, thiazolidine, methylene alkoxy carbamate, disulfide, vinylene, imine, imidamide, phosphoramide, saccharide,
  • Moiety B preferably has a structure as described in detail above with respect to the conjugates according to the invention.
  • Moiety G is preferably capable of forming, upon reacting, an amide, ester, carbamate, hydrazone, thiazolidine, methylene alkoxy carbamate, disulphide, alkylene, cycloalkylene, arylalkylene, heteroarylalkylene, heteroalkylene, heterocycloalkylene, alkenylene, cycloalkenylene, arylalkenylene, heteroarylalkenylene, heteroalkenylene, heterocycloalenkylene, alkynylene, heteroalkynylene, arylene, heteroarylene, aminoacyl, oxyalkylene, aminoalkylene, diacid ester, dialkylsiloxane, amide, thioamide, thioether, thioester, ester, carbamate, hydrazone, thiazolidine, methylene alkoxy carbamate, disulfide, vinylene, imine, imidamide, phosphoramide, saccharide,
  • the moiety B may be cleavable or non-cleavable, multifunctional moiety which can be used to link one or more reactive and/or binder moieties to form the conjugate precursor of the invention.
  • the structure of the compound comprises, independently, more than one moiety A, preferably 2, 3, 4, 5, 6, 7, 8, 9 or 10 moieties A; and/or more than one moiety G, preferably 2, 3, 4, 5, 6, 7, 8, 9 or 10 moieties G per molecule.
  • the structure of the compound comprises 3 moieties A and 1 moiety G; or 3 moieties A and 2 moieties G per molecule.
  • Moiety G is preferably selected from: H, NH2, OH, N3, COOH, SH, Hal,
  • Tetra- ESV6 novel tetravalent organic ligands of fibroblast activation protein (FAP)
  • FAP fibroblast activation protein
  • a compound according to the present invention may be represented by following general Formula IV, its individual diastereoisomers, its hydrates, its solvates, its crystal forms, its individual tautomers or a pharmaceutically acceptable salt thereof, wherein A is a binding moiety; B is a multifunctional moiety comprising a ramification point, and covalently connects the moieties A to C; and C is an atom, a molecule or a particle, and is a therapeutic agent.
  • moiety C comprises a ramification point, such that four moieties A are attached to C, and B is absent, i.e., moiety C can also serve as a ramification point therefore substituting B, preferably when C is a chelator.
  • An exemplary structure is represented by general Formula V: All exemplary structures, aspects, embodiments and definitions disclosed forthetrivalent binders of general Formulae I and III disclosed in the present specification apply mutatis mutandis for general Formulae IV and V.
  • the tetravalent binder can be represented by Formulae VI or VII, wherein all groups and variables are otherwise the same as those defined for Formulae II or III: wherein all groups and variables are otherwise the same as defined herein.
  • each i and j is an integer independently selected from 0, 1, 2, 3 and 4; preferably wherein each i is 1 or 2, and each j is 1, 2 or 3.
  • Preferred structures for J’ include: .
  • Particularly preferred structures for fragment ((L)a)4J’ include:
  • herein disclosed is a method for preparing a therapeutic conjugate comprising the step of conjugating with a precursor compound as described above with a conjugation partner.
  • the precursor compound is conjugated to the conjugation partner by reacting therewith to form a covalent bond.
  • the thus obtained conjugate is a therapeutic conjugate compound as described elsewhere in the present specification.
  • the conjugation partner can be an atom, a molecule or a particle which is a therapeutic agent, and can correspond to the therapeutic payload moieties already described in detail above with respect to the conjugates according to the invention.
  • the method further comprises formulating the conjugate as a therapeutic pharmaceutical composition.
  • the pharmaceutical compositions may be for human or animal therapy in human and veterinary medicine and will typically comprise any one or more of a pharmaceutically acceptable diluent, carrier, or excipient.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). The choice of carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s). All formulation details and aspects disclosed above in the section “Pharmaceutical compositions” fully apply here too.
  • the compounds described herein may be prepared by chemical synthesis techniques. It will be apparent to those skilled in the art that sensitive functional groups may need to be protected and deprotected during synthesis of a compound. This may be achieved by conventional techniques, for example as described in "Protective Groups in Organic Synthesis” by T W Greene and P G M Wuts, John Wiley and Sons Inc. (1991), and by P.J. Kocienski, in “Protecting Groups", Georg Thieme Verlag (1994). It is possible during some of the reactions that any stereocentres present could, under certain conditions, be epimerised, for example if a base is used in a reaction with a substrate having an optical centre comprising a base-sensitive group. It should be possible to circumvent potential problems such as this by choice of reaction sequence, conditions, reagents, protection/deprotection regimes, etc. as is well-known in the art.
  • Antibody is used in its broadest sense and covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), veneered antibodies, antibody fragments and small immune proteins (SIPs) (see Int. J. Cancer (2002) 102, 75-85).
  • An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen.
  • a target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody.
  • An antibody includes a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e. a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof.
  • the antibodies may be of any type - such as IgG, IgE, IgM, IgD, and IgA - any class - such as IgGl, IgG2, IgG3, IgG4, IgAl and IgA2 - or subclass thereof.
  • the antibody may be or may be derived from murine, human, rabbit or from other species.
  • antibody fragments refers to a portion of a full length antibody, generally the antigen binding or variable region thereof.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single domain antibodies, including dAbs, camelid VHH antibodies and the IgNAR antibodies of cartilaginous fish.
  • Antibodies and their fragments may be replaced by binding molecules based on alternative non-immunoglobulin scaffolds, peptide aptamers, nucleic acid aptamers, structured polypeptides comprising polypeptide loops subtended on a non-peptide backbone, natural receptors or domains thereof.
  • a derivative includes the chemical modification of a compound. Examples of such modifications include the replacement of a hydrogen by a halo group, an alkyl group, an acyl group or an amino group and the like. The modification may increase or decrease one or more hydrogen bonding interactions, charge interactions, hydrophobic interactions, van der Waals interactions and/or dipole interactions. Analog. This term encompasses any enantiomers, racemates and stereoisomers, as well as all pharmaceutically acceptable salts and hydrates of such compounds.
  • Alkyl refers to a branched or unbranched saturated hydrocarbyl radical.
  • the alkyl group comprises from 1 to 100, preferably 3 to 30, carbon atoms, more preferably from 5 to 25 carbon atoms.
  • alkyl refers to methyl, ethyl, propyl, butyl, pentyl, or hexyl.
  • Alkenyl refers to a branched or unbranched hydrocarbyl radical containing one or more carbon-carbon double bonds.
  • the alkenyl group comprises from 2 to 30 carbon atoms, preferably from 5 to about 25 carbon atoms.
  • Alkynyl refers to a branched or unbranched hydrocarbyl radical containing one or more carbon-carbon triple bonds.
  • the alkynyl group comprises from about 3 to about 30 carbon atoms, for example from about 5 to about 25 carbon atoms.
  • Halogen refers to fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine.
  • Cycloalkyl refers to an alicyclic moiety, suitably having 3, 4, 5, 6, 7 or 8 carbon atoms.
  • the group may be a bridged or polycyclic ring system. More often cycloalkyl groups are monocyclic. This term includes reference to groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbomyl, bicyclo [2.2.2] octyl and the like.
  • Aryl refers to an aromatic carbocyclic ring system, suitably comprising 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring carbon atoms.
  • Aryl may be a polycyclic ring system, having two or more rings, at least one of which is aromatic. This term includes reference to groups such as phenyl, naphthyl fluorenyl, azulenyl, indenyl, anthryl and the like.
  • Hetero signifies that one or more of the carbon atoms of the group may be substituted by nitrogen, oxygen, phosphorus, silicon or sulfur.
  • Heteroalkyl groups include for example, alkyloxy groups and alkythio groups.
  • Heterocycloalkyl or heteroaryl groups herein may have from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 ring atoms, at least one of which is selected from nitrogen, oxygen, phosphorus, silicon and sulfur.
  • a 3 - to 10-membered ring or ring system and more particularly a 5- or 6-membered ring which may be saturated or unsaturated.
  • oxiranyl selected from oxiranyl, azirinyl, 1,2-oxathiolanyl, imidazolyl, thienyl, furyl, tetrahydrofuryl, pyranyl, thiopyranyl, thianthrenyl, isobenzofuranyl, benzofuranyl, chromenyl, 2H-pyrrolyl, pyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl, imidazolidinyl, benzimidazolyl, pyrazolyl, pyrazinyl, pyrazolidinyl, thiazolyl, isothiazolyl, dithiazolyl, oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, piperidyl, piperazinyl, pyridazinyl, morpholinyl, thiomorpholinyl, especially
  • “Substituted” signifies that one or more, especially up to 5, more especially 1, 2 or 3, of the hydrogen atoms in said moiety are replaced independently of each other by the corresponding number of substituents.
  • the term “optionally substituted” as used herein includes substituted or unsubstituted. It will, of course, be understood that substituents are only at positions where they are chemically possible, the person skilled in the art being able to decide (either experimentally or theoretically) without inappropriate effort whether a particular substitution is possible. For example, amino or hydroxy groups with free hydrogen may be unstable if bound to carbon atoms with unsaturated (e.g. olefinic) bonds.
  • the term “substituted” signifies one or more, especially up to 5, more especially 1, 2 or 3, of the hydrogen atoms in said moiety are replaced independently of each other by the corresponding number of substituents selected from OH, SH, NH2, halogen, cyano, carboxy, alkyl, cycloalkyl, aryl and heteroaryl.
  • substituents described herein may themselves be substituted by any substituent, subject to the aforementioned restriction to appropriate substitutions as recognised by the skilled person.
  • any of the aforementioned substituents may be further substituted by any of the aforementioned substituents, each of which may be further substituted by any of the aforementioned substituents.
  • Substituents may suitably include halogen atoms and halomethyl groups such as CF3 and CCF; oxygen containing groups such as oxo, hydroxy, carboxy, carboxyalkyl, alkoxy, alkoyl, alkoyloxy, aryloxy, aryloyl and aryloyloxy; nitrogen containing groups such as amino, alkylamino, dialkylamino, cyano, azide and nitro; sulfur containing groups such as thiol, alkylthiol, sulfonyl and sulfoxide; heterocyclic groups which may themselves be substituted; alkyl groups, which may themselves be substituted; and aryl groups, which may themselves be substituted, such as phenyl and substituted phenyl.
  • Alkyl includes substituted and unsubstituted benzyl.
  • RP-HPLC reversed-phase high-pressure liquid chromatography
  • Step 2 (S)-4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8- yl)amino)4-oxobutanoic acid (P4).
  • Step 1 8-(4-(tert-butoxy)-4-oxobutanamido)quinoline-4-carboxylic acid
  • Step 2 tert-butyl (S)-4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-l-yl)-2-oxoethyl)carbamoyl)quinolin- 8-yl)amino)-4-oxobutanoate.
  • Step 3 (S)-4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-l-yl)-2-oxoethyl)carbamoyl)quinolin-8- yl)amino)-4-oxobutanoic acid (P4).
  • the resin was then treated with a solution of ESV6-Succinic-COOH (P4, 137 mg, 0.300 mmol, 2.0 eq.), HATU (86 mg, 0.22 mmol, 1.5 eq.) and DIPEA (97 ⁇ L, 0.75 mmol, 5.0 eq.) in DMF (5 mL) for 1 h. After multiple washing with DMF, the resin was submitted to the cleavage with 30% solution of TFA in DCM (10 mL) for 1 h. The cleaved solution was recovered, concentrated under vacuo and purified by RP-Chromatography (gradient: water/acetonitrile + 0.1% FA 98:2 to 0:100 in 45 min).
  • Bi-ESV6-DOTAGA- 175 Lu for therapeutic use.
  • a solution of LuCl3 hexahydrate (2.0 mg, 5.2 ⁇ mol, 2 eq.) dissolved in 0.05 N HCl (1.50 mL) was added.
  • the reaction was stirred at 90°C for 20 min, then cooled down to r. t.
  • Step 1) tert-butyl (A)-(2-(4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-l-yl)-2- oxoethyl)carbamoyl)quinolin-8-yl)amino)-4-oxobutanamido)ethyl) carbamate (2) (S)-4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-4- oxobutanoic acid (ESV6-COOH, 1, 100 mg, 217 ⁇ mol, 1 eq.), tert-butyl (2-aminoethyl)carbamate (42 mg, 261 ⁇ mol, 1.2 eq.) and HATU (100 mg, 261 ⁇ mol, 1.2 eq.)
  • Step 2 (S)-N 1 -(2-aminoethyl)-N 4 -(4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2- oxoethyl)carbamoyl)quinolin-8-yl)succinimide (3) tert-butyl (S)-(2-(4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl) quinolin-8- yl)amino)-4-oxobutanamido)ethyl)carbamate (104 mg, 176 ⁇ mol) was dissolved in 2 mL of a 30% v/v solution of TFA in DCM and stirred at room temperature for 1 h.
  • the crude was purified via CombiFlash Nextgen 300+ (parameters: flow 30 mL/min, 24gr C18 column, Water/Acetonitrile + 0.1% Formic Acid 98:2 to 0:100 in 30 minutes) and the collected fractions were lyophilized to obtain a white solid (66 mg, 133 ⁇ mol, 76 % yield).
  • Step 4) N 1 ,N 1 '-(9-amino-9-((3-((2-(4-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2- oxoethyl)carbamoyl)quinolin-8-yl)amino)-4-oxobutanamido)ethyl)amino)-3- oxopropoxy)methyl)-4,14-dioxo-7,11-dioxa-3,15-diazaheptadecane-1,17-diyl)bis(N 4 -(4-((2- ((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)succinamide) (5, Tri-ESV6-NH 2 ) tert-butyl (1,27
  • the crude was purified via CombiFlash Nextgen 300+ (parameters: flow 15 mL/min, 4gr C18 column, Water/Acetonitrile + 0.1% Formic Acid 98:2 to 0:100 in 30 minutes) and the collected fractions were lyophilized to obtain a white solid (13 mg, 7.2 ⁇ mol, 85 % yield).
  • Step 2 (S)-44-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8- yl)amino)-41,44-dioxo-4,7,10,13,16,19,22,25,28,31,34,37-dodecaoxa-40- azatetratetracontanoic acid 2,5-dioxopyrrolidin-1-yl (S)-4-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl) quinolin-8-yl)amino)-4-oxobutanoate (ESV6-NHS, 90 mg, 162 ⁇ mol) and H2N-PEG12-COOH (CAS: 1415408-69-3, 60 mg, 97
  • Step 4 (S)-N 1 -(42-amino-39-oxo-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxa-40-azadotetracontyl)- N4-(4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8- yl)succinamide tert-butyl (S)-(47-((4-((2-(2-cyano-4,4-difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8- yl)amino)-4,44,47-trioxo-7,10,13,16,19,22,25,28,31,34,37,40-dodecaoxa-3,43- diazaheptatetracontyl)
  • the crude was purified via CombiFlash Nextgen 300+ (parameters: flow 30 mL/min, 24gr C18 column, Water/Acetonitrile + 0.1% Formic Acid 98:2 to 0:100 in 30 minutes) and the collected fractions were lyophilized to obtain a yellow oil (67 mg, 61 ⁇ mol, 64 % yield).
  • Step 5 tert-butyl (1,107-bis((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2- oxoethyl)carbamoyl)quinolin-8-yl)amino)-54-(53-((4-((2-((S)-2-cyano-4,4- difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-5,10,50,53-tetraoxo- 2,13,16,19,22,25,28,31,34,37,40,43,46-tridecaoxa-6,9,49-triazatripentacontyl)- 1,4,44,49,59,64,104,107-octaoxo- 8,11,14,17,20,23,26,29,32,35,38,41,52,
  • Step 6 N 1 ,N 1 '-(49-amino-49-(53-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2- oxoethyl)carbamoyl)quinolin-8-yl)amino)-5,10,50,53-tetraoxo- 2,13,16,19,22,25,28,31,34,37,40,43,46-tridecaoxa-6,9,49-triazatripentacontyl)-39,44,54,59- tetraoxo-3,6,9,12,15,18,21,24,27,30,33,36,47,51,62,65,68,71,74,77,80,83,86,89,92,95- hexacosaoxa-40,43,55,58-tetraazaheptanonacontane-1,97-diyl
  • the crude was purified via CombiFlash Nextgen 300+ (parameters: flow 15 mL/min, 4gr C18 column, Water/Acetonitrile + 0.1% Formic Acid 98:2 to 0:100 in 30 minutes) and the collected fractions were lyophilized to obtain a white solid (15 mg, 4.2 ⁇ mol, 52 % yield).
  • Step 7) 2,2',2''-(10-(1-carboxy-26-((4-((2-((S)-2-cyano-4,4-difluoropyrrolidin-1-yl)-2- oxoethyl)carbamoyl)quinolin-8-yl)amino)-6,6-bis(20-((4-((2-((S)-2-cyano-4,4- difluoropyrrolidin-1-yl)-2-oxoethyl)carbamoyl)quinolin-8-yl)amino)-5,10,17,20-tetraoxo- 2,13-dioxa-6,9,16-triazaicosyl)-4,11,16,23,26-pentaoxo-8,19-dioxa-5,12,15,22- tetraazahexacosyl)-1,4,7,10-tetraazacyclododecane-1,
  • the value is defined as the concentration of inhibitor required to reduce the enzyme activity by 50% after addition of the substrate [FIG.1 and FIG.8].
  • further comparative inhibition assays with trimeric binders having inter alia different lengths of linker group B and/or different payload groups C were performed with compounds Tri-ESV6-ValCit-MMAE (14), Tri-ESV6-linker-DOTAGA (13) and diastereoisomers of Tri-ESV6- DOTAGA (11, 12) [FIG.18].
  • Comparative hFAP inhibition assays with tetravalent binders 10 and 29 were also performed [FIG.20].
  • SK-RC-52.hFAP were grown to 80% confluence in RPMI-1640 with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic and detached with Trypsin-EDTA (ethylenediaminetetraacetic acid) 0.05%. Tumor cells were resuspended in Hanks’ Balanced Salt Solution medium. Aliquots of 5 million cells (100 ⁇ L of suspension) were injected subcutaneously in the right flank of female athymic Balb/c AnNRj-Foxn1 mice (6 to 8 wk of age).
  • FBS fetal bovine serum
  • Trypsin-EDTA ethylenediaminetetraacetic acid
  • precursors (25 nmol) were dissolved in 25 ⁇ L of milliQ water, then sodium acetate buffer (75 ⁇ L, 1 M in water) and 25, 75 or 150 MBq of 177Lu solution were added.
  • 177 Lu solution (4 or 96 MBq) were added and the mixture was heated at 90°C for 10 minutes, followed by dilution with PBS to achieve final volume of 400 ⁇ L (4 doses of 100 ⁇ L each, corresponding to 0.2 MBq/nmol and 4.8MBq/nmol, respectively).
  • ESV6-DOTAGA, Bi-ESV6-DOTAGA, Tri-ESV6-DOTAGA, Tetra-ESV6-DOTAGA, Hexa-ESV6- DOTAGA, Octa-ESV6-DOTAGA and FAP-2286 were radiolabeled with 177 Lu as described above. Tumors were allowed to grow to an average volume of 300 mm 3 .
  • mice were randomized and injected intravenously with radiolabeled preparations of 177 Lu-ESV6, 177 Lu-Bi-ESV6, 177 Lu-Tri-ESV6, Tetra-ESV6-DOTAGA, Hexa-ESV6-DOTAGA, Octa-ESV6-DOTAGA or 177 Lu-FAP-2286 (250 nmol/kg; 50 MBq/kg).
  • Mice were euthanized at different time-points after the intravenous injection by CO2 asphyxiation. Tumors, organs, and blood were collected, weighted, and radioactivity was measured with a Packard Cobra Gamma Counter. Values are expressed as percent ID/g ⁇ SD [FIG.4].
  • 177 Lu-ESV6, 177 Lu-Bi-ESV6 and 177 Lu-Tri-ESV6 were assessed in athymic Balb/c AnNRj-Foxn1 mice bearing SK-RC-52.hFAP tumor in the right flank.
  • 177 Lu-ESV6, 177 Lu-Bi-ESV6 or 177 Lu-Tri-ESV6 were intravenously administered at a dose of 250 nmol/kg, 250 MBq/kg (single administration on day 9 after tumor implantation). Therapy experiments started when the average volume of established tumors had reached 100-150 mm 3 .
  • mice bearing subcutis SK-RC-52.hFAP tumors were injected intravenously with eight different doses of 177 Lu-Tri-ESV6-DOTAGA ranging from 3 nmol/kg to 2250 nmol/kg. The mice were sacrificed after 24h and tumor and healthy organs were harvested and measured with a gamma counter. Results reported as %ID/g are shown in FIG. 15A. Results reported as tumor to organ ratio are shown in FIG.15B.
  • the L19-IL2 immunocytokine conjugate used in the present Example comprises a human IL2 polypeptide having the sequence of SEQ ID NO: 21 fused at its N-terminus via a 17 amino acid linker (SEQ ID NO: 23) to the C- terminus of the VL domain of a single-chain variable fragment (scFv) molecule comprising the VH (SEQ ID NO: 19) and VL (SEQ ID NO: 20) domains of antibody LI 9, which specifically bind the extra-domain B of fibronectin (ED-B), as described in WO2001/062298.
  • the epitope bound is the sequence of SEQ ID NO: 40 (cf. Fig 4C in Fattorusso et al., Structure 7:381-390).
  • the pairing of the VH domain of one L19-IL2 molecule with the VL domain of another L19-IL2 molecule permits the formation of homodimers via the scFv portion of the immunocytokine, essentially in an “scFv2” or “diabody” format, as shown in FIG. 12.
  • amino acid sequence of the L19-IL2 immunocytokine conjugate is set forth in SEQ ID NO 12:
  • the anti -cancer efficacy was assessed in athymic Balb/c AnNRj-Foxnl mice bearing SK-RC-52.hFAP tumor in the right flank. Therapy experiments started when the average volume of established tumors had reached 100-150 mm 3 . Body weight of the animals and tumor volume were daily measured and recorded. Tumor dimensions were measured with an electronic caliper and tumor volume was calculated with the formula (long side, mm) x (short side, mm) x (short side, mm) x 0.5. Animals were euthanized when one or more termination criterium indicated by the experimental license was reached. Prism 7 software (GraphPad Software) was used for data analysis.
  • the ESV6 binding moiety in monomeric, dimeric or trimeric form was conjugated to an identical linkerpayload structure based on a Glycine-Proline linker and an MMAE cytotoxic moiety.
  • HT-1080.hFAP tumors were implanted into the right flank of athymic Balb/c AnNRj-Foxnl mice and allowed to grow to an average volume of approximately 200 mm 3 . Mice were injected with the three conjugates at a dose of 250 nmol/kg and sacrificed at different timepoints after administration. Fresh blood was collected in lithium heparin tubes (BD Microcontainer LH Tubes), vortexed, and centrifuged (15,000 g, 15 minutes). Plasma was frozen and stored at -80°C. Healthy organs and tumors were subsequently excised, frozen with dry ice, and stored at -80°C.
  • Frozen plasma 50 pL and mouse tissues ( ⁇ 50 mg) were thawed, and 500 pL of PBS was added. Samples were kept on ice, and 50 pL solution of internal standard (d8-MMAE, 50 nM) was added. Samples were then homogenized at 4°C with a tissue lyser for 2 minutes at 30 Hz for 4 cycles. After homogenization, samples were centrifuged (21’000 g, 10 min). Subsequently, 100 pL of supernatants were collected and added to 900 pL of acetonitrile (ACN) to induce protein precipitation.
  • ACN acetonitrile
  • the LC system was coupled to a Q-Exactive mass spectrometer via an Ion Max HESI Source. Ionization was carried out with a spray voltage of 3.5 kV; Sheath gas 40 units; Aux gas 10 units; capillary temperature of 380°C; Aux gas temperature 450°C; S-lens RF level 60.
  • the mass spectrometer was operating in targeted Single Ion Monitoring mode (t-SIM) following the molecular ion 718.5113 m/z.
  • the detector was working in positive ionization mode with the following parameters: resolution 70’000 (FWHM at 200 m/z); AGC target of 5 * 104; maximum injection time of 200 ms; isolation window 14 m/z; isolation offset 5 m/z. Peak areas of analytes and internal standards were integrated, and corresponding ratios were calculated. The ratios were then transformed into pmol/g of wet tissue using singleconcentration external calibration points and corrected by the total weight of the sample analysed. The percentage of injected dose per gram (%ID/g) was finally calculated by normalizing the value based on the total dose injected into the mouse. Data analysis was performed with Skyline v22.2.0.351. Results are shown in FIG. 14. h) Therapeutic efficacy evaluation of ESV6-GlyPro-MMAE and Tri-ESV6-GlyPro-MMAE in HT- 1080. hFAP tumor hearing mice (schedule optimization)
  • ESV6-GlyPro-MMAE 50 or 125 nmol/kg - corresponding to Conjugate 58a of EP3891138B1 and
  • Tri-ESV6-GlyPro-MMAE 50 or 125 nmol/kg according to the present invention was assessed in athymic Balb/c AnNRj-Foxnl mice bearing HT-1080.hFAP tumor in the right flank. Therapy experiments started when the average volume of established tumors had reached 80-100 mm 3 . Body weight of the animals and tumor volume were daily measured and recorded.
  • Tumor dimensions were measured with an electronic caliper and tumor volume was calculated with the formula (long side, mm) x (short side, mm) x (short side, mm) x 0.5. Animals were euthanized when one or more termination criterium indicated by the experimental license was reached. Prism 7 software (GraphPad Software) was used for data analysis. Results are shown in FIG. 16 and FIG. 17.
  • Enzymatic activity of hFAP on the Z-Gly-Pro-AMC substrate was measured at room temperature on a microtiter plate reader, monitoring the fluorescence at an excitation wavelength of 360 nm and an emission wavelength of 465 nm.
  • the value is defined as the concentration of inhibitor required to reduce the enzyme activity by 50% after addition of the substrate.
  • the results are shown in FIG. 10.
  • Enzymatic activity of hFAP on the Z- Gly-Pro-AMC substrate was measured at room temperature on a microtiter plate reader, monitoring the fluorescence at an excitation wavelength of 360 nm and an emission wavelength of 465 nm.
  • Tri-ESV6-D0TAGA was used as its (.S'./?)-diastcrcomcr, Chemical structure of Tri-ESV6-linker-DOTAGA (13)

Abstract

La présente invention concerne des ligands de la protéine d'activation des fibroblastes (FAP) pour l'administration active de diverses charges thérapeutiques (par exemple, des médicaments cytotoxiques, des radionucléides thérapeutiques, des protéines et des immunomodulateurs) au site de maladie. En particulier, la présente invention concerne le développement de ligands de FAP multivalents pour des applications thérapeutiques, en relation avec une maladie ou un trouble, tel qu'un cancer, une inflammation ou une autre maladie caractérisée par une surexpression de FAP.
PCT/EP2023/074310 2022-09-06 2023-09-05 Ligands multivalents de protéine d'activation des fibroblastes pour applications d'administration ciblée WO2024052333A1 (fr)

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Citations (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997045544A1 (fr) 1996-05-24 1997-12-04 Philogen S.R.L. Anticorps contre le domaine ed-b de la fibronectine, leur construction et leurs utilisations
WO1999058570A2 (fr) 1998-05-11 1999-11-18 Eidgenössische Technische Hochschule Zürich Molecules de liaison specifiques pour scintigraphie, conjugues contenant ces molecules et traitement de l'angiogenese
WO2001062298A2 (fr) 2000-02-24 2001-08-30 Philogen S.R.L. Compositions et procedes de traitement de l'angiogenese dans des lesions pathologiques
WO2003076469A2 (fr) 2002-03-11 2003-09-18 Philogen S.P.A. Ciblage selectif d'un systeme vasculaire tumoral au moyen de molecules d'anticorps
WO2005082023A2 (fr) 2004-02-23 2005-09-09 Genentech, Inc. Liants et conjugues heterocycliques auto-immolateurs
WO2005087235A1 (fr) 2004-03-09 2005-09-22 National Health Research Institutes Composes de pyrrolidine
WO2006119897A2 (fr) 2005-05-11 2006-11-16 Philogen S.P.A Conjugue de ciblage de medicament
US8097254B2 (en) 1998-05-11 2012-01-17 Eidgenossische Technische Hochschule Zurich Specific binding molecules for scintigraphy, conjugates containing them and therapeutic method for treatment of angiogenesis
WO2013107820A1 (fr) 2012-01-17 2013-07-25 Universiteit Antwerpen Nouveaux inhibiteurs de fap
WO2018111989A1 (fr) 2016-12-14 2018-06-21 Purdue Research Foundation Imagerie et thérapie ciblées par une protéine d'activation des fibroblastes (fap)
WO2019083990A2 (fr) 2017-10-23 2019-05-02 The Johns Hopkins University AGENTS D'IMAGERIE ET DE RADIOTHÉRAPIE CIBLANT LA PROTÉINE-α D'ACTIVATION DES FIBROBLASTES (FAP-α)
WO2019118932A1 (fr) 2017-12-15 2019-06-20 Praxis Biotech LLC Inhibiteurs de la protéine d'activation des fibroblastes
WO2019154886A1 (fr) 2018-02-06 2019-08-15 Universität Heidelberg Inhibiteur de fap
WO2019154859A1 (fr) 2018-02-06 2019-08-15 Universität Heidelberg Inhibiteur de fap
WO2020070150A1 (fr) 2018-10-02 2020-04-09 Philogen S.P.A Immunoconjugués d'il2
WO2021016392A1 (fr) 2019-07-22 2021-01-28 Purdue Research Foundation Agents ciblés sur des fibroblastes multivalents et procédés d'utilisation
WO2021160825A1 (fr) 2020-02-12 2021-08-19 Philochem Ag Ligands de protéine d'activation des fibroblastes pour applications d'administration ciblée
EP4043452A1 (fr) * 2021-02-12 2022-08-17 Philochem AG Ligands de protéines d'activation de fibroblastes bivalents pour des applications d'administration ciblées
WO2022171811A1 (fr) 2021-02-12 2022-08-18 Philochem Ag Ligands de protéine d'activation des fibroblastes bivalents pour applications d'administration ciblée

Patent Citations (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997045544A1 (fr) 1996-05-24 1997-12-04 Philogen S.R.L. Anticorps contre le domaine ed-b de la fibronectine, leur construction et leurs utilisations
WO1999058570A2 (fr) 1998-05-11 1999-11-18 Eidgenössische Technische Hochschule Zürich Molecules de liaison specifiques pour scintigraphie, conjugues contenant ces molecules et traitement de l'angiogenese
US8097254B2 (en) 1998-05-11 2012-01-17 Eidgenossische Technische Hochschule Zurich Specific binding molecules for scintigraphy, conjugates containing them and therapeutic method for treatment of angiogenesis
WO2001062298A2 (fr) 2000-02-24 2001-08-30 Philogen S.R.L. Compositions et procedes de traitement de l'angiogenese dans des lesions pathologiques
WO2003076469A2 (fr) 2002-03-11 2003-09-18 Philogen S.P.A. Ciblage selectif d'un systeme vasculaire tumoral au moyen de molecules d'anticorps
WO2005082023A2 (fr) 2004-02-23 2005-09-09 Genentech, Inc. Liants et conjugues heterocycliques auto-immolateurs
WO2005087235A1 (fr) 2004-03-09 2005-09-22 National Health Research Institutes Composes de pyrrolidine
WO2006119897A2 (fr) 2005-05-11 2006-11-16 Philogen S.P.A Conjugue de ciblage de medicament
WO2013107820A1 (fr) 2012-01-17 2013-07-25 Universiteit Antwerpen Nouveaux inhibiteurs de fap
WO2018111989A1 (fr) 2016-12-14 2018-06-21 Purdue Research Foundation Imagerie et thérapie ciblées par une protéine d'activation des fibroblastes (fap)
WO2019083990A2 (fr) 2017-10-23 2019-05-02 The Johns Hopkins University AGENTS D'IMAGERIE ET DE RADIOTHÉRAPIE CIBLANT LA PROTÉINE-α D'ACTIVATION DES FIBROBLASTES (FAP-α)
WO2019118932A1 (fr) 2017-12-15 2019-06-20 Praxis Biotech LLC Inhibiteurs de la protéine d'activation des fibroblastes
WO2019154886A1 (fr) 2018-02-06 2019-08-15 Universität Heidelberg Inhibiteur de fap
WO2019154859A1 (fr) 2018-02-06 2019-08-15 Universität Heidelberg Inhibiteur de fap
WO2020070150A1 (fr) 2018-10-02 2020-04-09 Philogen S.P.A Immunoconjugués d'il2
WO2021016392A1 (fr) 2019-07-22 2021-01-28 Purdue Research Foundation Agents ciblés sur des fibroblastes multivalents et procédés d'utilisation
WO2021160825A1 (fr) 2020-02-12 2021-08-19 Philochem Ag Ligands de protéine d'activation des fibroblastes pour applications d'administration ciblée
EP3891138B1 (fr) 2020-02-12 2022-01-12 Philochem AG Ligands de protéines d'activation de fibroblastes pour des applications d'administration ciblées
EP4043452A1 (fr) * 2021-02-12 2022-08-17 Philochem AG Ligands de protéines d'activation de fibroblastes bivalents pour des applications d'administration ciblées
WO2022171811A1 (fr) 2021-02-12 2022-08-18 Philochem Ag Ligands de protéine d'activation des fibroblastes bivalents pour applications d'administration ciblée

Non-Patent Citations (32)

* Cited by examiner, † Cited by third party
Title
"Handbook of Experimental Immunology", vol. I-IV, 1986, SCIENTIFIC PUBLICATIONS
"Molecular Biology Techniques: An Intensive Laboratory Course", 1998, ACADEMIC PRESS
"Molecular Cloning: A Laboratory Manual", vol. I-III, 1989, COLD SPRING HARBOR LABORATORY PRESS
"PCR (Introduction to Biotechniques Series", 1997, SPRINGER VERLAG
"Remington's Pharmaceutical Sciences", 1990, MACK PUBLISHING CO.
"Short Protocols in Molecular Biology", 1999, JOHN WILEY & SONS
"The Pharmacological Basis of Therapeutics", 2001, MCGRAW-HILL CO.
"UniProt", Database accession no. P0275 I
ALTSCHUL ET AL., MOL. BIOL., vol. 215, 1990, pages 405 - 410
BERGE ET AL., J.PHARM.SCI., vol. 66, 1977, pages 1 - 19
BERNARDES, G.J. ET AL., ANGEW. CHEM. INT. ED. ENGL., vol. 51, 2012, pages 941
BOSSLET, K. ET AL., CANCER RES., vol. 58, 1998, pages 1195 - 1201
CARL ET AL., J. MED. CHEM., vol. 24, 1981, pages 479 - 480
CASI, G. ET AL., J. AM. CHEM. SOC., vol. 134, 2012, pages 5887
CHAKRAVARTY ET AL., J. MED. CHEM., vol. 26, 1983, pages 638 - 644
DE GROOT ET AL., JOURNAL OF ORGANIC CHEMISTRY, vol. 66, no. 26, 2001, pages 8815 - 8830
DORONINA S.O. ET AL., BIOCONJUGATE CHEM, vol. 19, 2008, pages 1960
FATTORUSSO ET AL., STRUCTURE, vol. 7, pages 381 - 390
INT. J. CANCER, vol. 102, 2002, pages 75 - 85
LEAMON, C.P. ET AL., BIOCONJUGATE CHEM., vol. 17, 2006, pages 1226
NERI ET AL., J MOL BIOL, vol. 246, 1995, pages 367 - 73
NUCL. ACIDS RES., vol. 25, 1997, pages 3389 - 3402
P.J. KOCIENSKI: "Protecting Groups", 1994, GEORG THIEME VERLAG
PEARSONLIPMAN, PNAS USA, vol. 85, 1988, pages 2444 - 2448
PINI ET AL., J. BIOL. CHEM., vol. 273, 1998, pages 21769 - 21776
ROCKVILLE, MD: "Guidance for Industry: Estimating the Maximum Safe Starting Dose in Adult Healthy Volunteer", US FOOD AND DRUG ADMINISTRATION, 2005
SMITHWATERMAN, J. MOL BIOL., vol. 147, 1981, pages 195 - 197
SUTHERLAND, M.S.K., J. BIOL. CHEM, vol. 281, 2006, pages 10540
T W GREENEP G M WUTS: "Protective Groups in Organic Synthesis", 1991, JOHN WILEY AND SONS INC.
WICHERT ET AL., NATURE CHEMISTRY, vol. 7, 2015, pages 241 - 249
YANG, J. ET AL., PROC. NATL. ACAD. SCI. USA, vol. 103, 2006, pages 13872
ZBORALSKI, D.HOEHNE, A.BREDENBECK, A. ET AL., EUR J NUCL MED MOL IMAGING, vol. 49, 2022, pages 3651 - 3667

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