WO2017216620A1 - Conjugués de facteur de croissance des fibroblastes 2 humain (fgf2) et de médicament cytotoxique pour le traitement ciblé de cancers liés au fgfr - Google Patents

Conjugués de facteur de croissance des fibroblastes 2 humain (fgf2) et de médicament cytotoxique pour le traitement ciblé de cancers liés au fgfr Download PDF

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WO2017216620A1
WO2017216620A1 PCT/IB2016/058083 IB2016058083W WO2017216620A1 WO 2017216620 A1 WO2017216620 A1 WO 2017216620A1 IB 2016058083 W IB2016058083 W IB 2016058083W WO 2017216620 A1 WO2017216620 A1 WO 2017216620A1
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fgf2
kck
protein variant
cancer
amino acid
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PCT/IB2016/058083
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Mateusz KRZYŚCIK
Michał ŁOBOCKI
Aleksandra SOKOŁOWSKA-WĘDZINA
Karolina ŚWIDERSKA
Piotr JAKIMOWICZ
Małgorzata ZAKRZEWSKA
Daniel KROWARSCH
Jacek OTLEWSKI
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Uniwersytet Wrocławski
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Priority to EP16831758.4A priority Critical patent/EP3468611A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/642—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a cytokine, e.g. IL2, chemokine, growth factors or interferons being the inactive part of the conjugate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • C07K14/503—Fibroblast growth factor [FGF] basic FGF [bFGF]

Definitions

  • FGF2 Human fibroblast growth factor 2
  • the field of the invention is cancer treatment and targeted therapies. More specifically, the present invention is directed to the new generation of therapeutics, being conjugates of a potent cytotoxic drug and a targeting delivery molecule, a human fibroblast growth factor 2 (FGF2), that are effective in killing cancer cells of FGFR-related cancers.
  • FGF2 human fibroblast growth factor 2
  • FGFs fibroblast growth factors
  • FGFRs fibroblast growth factor receptors
  • FGFs are signalling molecules of high importance throughout all stages of development as well as adult life of metazoan organisms (Ornitz, 2001; Itoh 2004; Powers, 2000). FGFs are responsible for a wide spectrum of biological effects induced in numerous different cell types and a wide variety of tissues (Powers, 2000; Eswarakumar, 2005). FGFs through their receptors regulate many physiological processes such as cell proliferation, morphogenesis, differentiation, migration and cell survival (Dailey, 2005). They act in a paracrine or autocrine fashion and are responsible for angiogenesis, osteogenesis, neurogenesis and wound healing (Dailey, 2005).
  • deregulations and abnormalities of FGFs or FGFRs such as mutations, protein overexpression or imbalanced signalling may lead to diverse pathological conditions including skeleton disorders, diabetic retinopathy, atherosclerosis and cancer (Wiedlocha, 2004; Wesche 2011).
  • FGF2 Human fibroblast growth factor 2
  • FGF2 is one of the best characterized members of the FGF superfamily.
  • FGF2 is a powerful mitogen involved in diverse physiological processes such as cell migration, angiogenesis, osteogenesis and wound healing (Bikfalvi, 1997). Five FGF2 isoforms (18, 22, 22.5, 24 and 34 kDa) in human have been described.
  • FGF2 consists of 12 ⁇ -strands, arranged antiparallel in a ⁇ -trefoil motif with a pseudo-threefold internal symmetry (Zhu, 1991).
  • Wild-type of FGF2 contains 4 cysteine residues: two of them (at positions 34 and 101) are buried within the protein core and inert, and two are solvent accessible (at position 78 and 96) and are highly reactive.
  • FGF2 The biological action of FGF s, excluding FFIFs, is exerted through binding to and activation of four high-affinity cell-surface FGF receptors (FGFRl-4) that have intrinstic tyrosine kinase activity.
  • FGF2 is more selective in binding and activation of FGFRs, compared with FGF1.
  • FGF2 exhibits high affinity and ability to stimulate the following receptors: FGFRlb, FGFRlc, FGFR3c, FGFR2c and FGFR4, but does not activate FGFR2b and FGFR3b (Ornitz, 1996).
  • the specificity of FGF2 to target tumor cells has been shown for several different cancer types, including neuroblastoma (Xu, 2010).
  • FGFRs overexpression has been found in different types of human cancer, including breast, bladder, prostate, endometrial, lung and hematologic cancers (Haugsten, 2010). This makes them promising candidates as potential targets for anticancer therapy.
  • FGFR1 The most common alteration of FGFRs in breast cancers involves amplification of FGFR1 (app. 10% of human breast cancers) and is associated with poor prognosis.
  • Cell culture studies confirmed the oncogenic potential of FGFR1 and showed that activation of FGFR1 resulted in increased cell proliferation and invasion. It has been found that amplification of FGFR1 gene plays a role in resistance to endocrine therapy and it is a very promising therapeutic target in breast cancer.
  • amplification of FGFR2 is also related to breast tumors, especially to triple-negative breast cancers, which are aggressive breast tumors negative for the oestrogen receptor, progesterone receptor and HER2, and not efficiently treated with current therapies (Schneider, 2008).
  • FGFR4 proliferation potential of cell lines derived from these tumors were effectively decreased in the presence of FGFR inhibitor and siRNA (Wesche, 2011).
  • anticancer therapy targeted to FGFR2 appears to be possible.
  • overexpression of FGFR4 has been observed in human breast cancers and was reported to be associated with resistance to chemotherapy.
  • breast cancer cell line with upregulated expression of FGFR4 was treated with specific FGFR4 antibody, blocking signaling from the receptor, enhanced chemosensitivity of cancer cells was observed (Roidl, 2009). Therefore FGFR4 is also a very good potential therapeutic target.
  • FGFR1 is also often overexpressed in prostate cancer and is believed to disrupt the interplay between mesenchymal and epithelial cells of the prostate (Sahadevan, 2007; Giri, 1999). It was clearly showed that inhibition of FGFR1 led to reduced tumor cells proliferation and progression (Wesche, 2011). The same effects were observed in endometrial carcinomas cell lines, in which mutations enhancing receptor activation were identified in FGFR2 (Dutt, 2008). Similarly, the role of FGFRs has been indicated in both types of lung cancer: SCLC (small cell lung carcinoma) and NSCLC (non-small cell lung carcinoma).
  • Tasigna® (AMN107, Nilotinib) is a recently approved BCR-ABL kinase inhibitor for the treatment of drug-resistant chronic myelogenous leukemia (CML).
  • AZD4547 FGFRl and 2 inhibitor against advanced solid malignancies (AstraZeneca)
  • Dovitinib is an inhibitor of both VEGFR and FGFRs against renal cell carcinoma and advanced breast cancer, relapsed MM and bladder cancer
  • BGJ398 is a selective inhibitor of FGFRs (Novartis).
  • these inhibitors also inhibit VEGFR, due to the high structural similarity of the kinase domains many of these multiple targeting inhibitors may be relatively less potent as inhibitors against FGFRs. This also increases the side effect profile, limiting the deliverability of the drug at doses necessary for inhibition of FGF signaling (Ahmad, 2012).
  • Off-target toxicities and induction of rapid resistance are major drawbacks that highly compromise successful long term administration.
  • FGF ligand traps are soluble fusion proteins consisting of the extracellular FGFRl domain fused to the Fc region of IgGl . Such protein fusion prevents FGFs from binding to their receptors and therefore decreases overall FGFR activation (Ellsworth, 2002).
  • Five Prime drug FP-1039 is thought to bind to FGF ligands circulating in the extracellular space, thereby preventing these signaling proteins from reaching FGFR1 on the surface of tumor cells where they would otherwise stimulate cancer cell division and/or angiogenesis. They exhibit antiproliferative and anti-angiogenic effects (Ahmad, 2012).
  • WO2014179448 discloses a method of treating breast cancer in a subject comprising administering a therapeutically effective amount of a fibroblast growth factor receptor 1 (FGFR1) extracellular domain (ECD) or an FGFR1 ECD fusion molecule to the subject, wherein prior to administration at least a portion of the cells of the breast cancer were determined to have FGFR1 gene amplification, FGFR1 overexpression, FGFR3 overexpression, or FGF2 overexpression; and to be estrogen receptor (ER) positive, progesterone (PR) positive, or ER positive and PR positive.
  • FGFR1 fibroblast growth factor receptor 1
  • ECD extracellular domain
  • FGF2 FGF2 overexpression
  • ER estrogen receptor
  • PR progesterone
  • a R3Mab antibody which targets FGFR3, demonstrating antiproliferative effects on xenografts of bladder cancer and t(4; 14) myeloma cells (Qing, 2009).
  • WO 2011143318 provides monoclonal antibodies that bind and inhibit biological activities of human FGFR2.
  • the antibodies can be used to treat cell proliferative diseases and disorders, including certain forms of cancer, associated with activation or overexpression of FGFR2 in particular breast cancer, ovarian cancer, prostate cancer, cervical cancer, lung cancer, some forms of brain cancer, melanomas, and gastrointestinal cancers (e.g., colorectal, pancreatic, gastric, head and neck).
  • FGFR2-IIIb-specific antibody has been used in vitro and in vivo to suppress the Illb isoform in FGFR2 amplified human breast and gastric cancer cell lines (Nord, 2009).
  • mAb monoclonal antibodies
  • the anti-FGF2 mAb completely inhibit binding of FGF2 to each of the FGF receptors FGFR1, FGFR2, FGFR3 and FGFR4.
  • Exemplary antibodies are GAL-F2 and its chimeric or humanized forms, and those having the same epitope as GAL-F2 or competitive in binding with GAL-F2.
  • the FGFly-coated Au Ps conjugates were used for photothermal therapy in cancer treatment (Szlachcic, 2012).
  • FGFR-targeted gold nanoconjugates were designed for infrared-induced thermal ablation (localized heating lead to cancer cell death) based on gold nanoparticles (AuNPs). It was shown that a recombinant ligand of all FGFRs, human fibroblast growth factor 1 (FGF1), can be used as an agent targeting covalently bound AuNPs to cancer cells overexpressing FGFRs.
  • FGF1 human fibroblast growth factor 1
  • ADCs Antibody-Drug Conjugates
  • DAR parameter drug to antibody ratio
  • Conjugation of cytotoxic compounds to lysine residues in antibody leads to formation of mixture, in which the cytotoxic molecules are randomly distributed within the antibody resulting in different DAR values (Kaur, 2013). It was calculated that conjugation of cytotoxic compounds via lysine residues occurring in forty different locations, may give 106 different variants of ADC (Wang, 2005). Despite the large number of lysines available for conjugation within single IgGl, the average DAR value obtained for Kadcyla, one of the two ADC drugs on the market, is about 3.5 of cytotoxic compound molecules per antibody and the actual number of substitutions ranges from 1 to 7 (Wakankar, 2010; Junutula, 2010).
  • cysteine residues require the reduction of disulfide bonds within the hinge region of the antibody and leads to a conjugate with maximal 8 molecules of a cytotoxic compound per one antibody (for IgGl and IgG2). Conjugation via cysteine residues provides more homogeneous product, but still may generate over 100 different variants of the ADC (Hamblett, 2004).
  • the internal disulfide bridges located within immunoglobulin domains are not exposed to solvent and exhibit high stability, thus they are not affected during conjugation reaction (Liu et al. 2010; Liu & May, 2012).
  • Adcetris® prepared using cysteines conjugation technology, there is about four molecules of the cytotoxic compound per single antibody (Sun, 2005; van de Donk & Dhimolea, 2012). Substitution of all eight cysteines is not desirable, because it leads to higher toxicity and increase removal of ADC from the body, without a corresponding improvement in the therapeutic efficiency. In addition, it was observed that a high number of cytotoxic drug has a negative impact on the structure and properties of antibodies, leading to the loss of their functionality (Singh, 2015). High DAR value increases the level of aggregation of the final product, which has a negative impact on overall pharmacokinetic properties and affinity for antigen. Conjugation process also alters the biological half-life of the antibody, usually reducing it from several weeks to several days (Chan, 2007; Sun, 2005; Hamblett, 2004).
  • a disadvantage of the strategies used for conjugation of cytotoxic compounds to antibodies is the heterogeneity of the final formulation of ADC.
  • the main technology of site-specific conjugation includes the use of a modified cysteine residue (technology Thiomab, Genentech), the use of unnatural amino acids (Sutro Biopharma and Ambryx) and the application of the enzyme transglutaminase (Innate Pharma) (Junutula, 2008; Panowski, 2014; Zaro, 2015).
  • cytostatic suitable for use in the ADC should meet several conditions, including high cytotoxicity and stability in the bloodstream. Furthermore, it should be small, to reduce the risk of immunogenicity and provide good solubility in aqueous solutions, and also have a specific group for conjugation to the antibody.
  • the aim of the invention is a provision of a new anticancer therapeutic suitable for targeted therapy of FGFR-related cancers that would be devoid of reported drawbacks.
  • the subject invention provides strategy for destroying cancer cells using targeting molecules fused with highly cytotoxic agents.
  • a natural ligand of FGF receptor, the fibroblast growth factor 2 (FGF2) or engineered variants thereof are used for bioconjugation with a potent cytotoxic compound such as monomethyl auristatin E (MMAE) or auristatin Y to establish a delivery technique for effective killing of cancer cells overexpressing FGFR.
  • a potent cytotoxic compound such as monomethyl auristatin E (MMAE) or auristatin Y
  • the FGF2 molecule and its engineered variants act as a delivery molecules specifically directing cytotoxic drug to cancer cell by binding to FGF receptors. Obtained conjugates showed a prominent cytotoxic effect toward FGFR - related cancers.
  • the therapeutics of the invention prevent or inhibit the activation of (i.e.
  • the conjugates of the invention can be used to inhibit the proliferation of tumor cells in vitro or in vivo. When administered to a human cancer patient (or an animal model), the conjugates have a potential to inhibit or reduce tumor growth in the human patient (or animal model).
  • the subject of the invention is a conjugate for targeted therapy of FGFR-related cancers comprising at least one molecule of a cytotoxic drug and a delivery molecule being a recombinant human fibroblast growth factor 2 (FGF2) protein or its variant, wherein the cytotoxic drug molecule binds at least one cysteine residue of the FGF2 or its variant.
  • FGF2 human fibroblast growth factor 2
  • FGF2 protein variant is a polypeptide comprising amino acid sequence having at least 84% of homology or higher with the amino acid sequence of SEQ ID NO: 1.
  • FGF2 protein variant comprises amino acid sequence wherein one or multiple amino acids are deleted, substituted, inserted, and/or added in the amino acid sequence of SEQ ID NO: 1.
  • FGF2 variants are peptides selected from a group consisting of :
  • KCK-FGF2[C78S/C96S] protein variant having KCKSGG sequence on the N- terminus, and substitutions of two surface cysteine residues to serine at positions 78 and 96;
  • the human fibroblast growth factor 2 (FGF2) term relates to all isoforms of FGF2 protein.
  • the human fibroblast growth factor 2 (FGF2) protein is isoform 3 of FGF2.
  • FGF2 is 155 aa lenght protein.
  • FGF2 (FGF2 WT) is preferably identified by a SEQ ID: NO 1.
  • KCK-FGF2 variant is identified by a SEQ ID NO: 2
  • FGF2-KCK variant is identified by a SEQ ID NO:3
  • KCK-FGF2[C78S/C96S] variant is identified by a SEQ ID NO:4
  • FGF2[C78S/C96S]-KCK variant is identified by a SEQ ID NO:5
  • sFGF2 WT variant is identified by a SEQ ID NO:6
  • sFGF2[C78S] variant is identified by a SEQ ID NO:7
  • sFGF2[C96S] is variant is identified by a SEQ ID NO:8.
  • FGF2 proteins are identified by any of the amino acid sequences selected from SEQ ID NO: 1-8.
  • the cytotoxic drugs are derivatives of dolastatin, A-amanitin, PBD dimers.
  • the drug is monom ethyl auri statin E or auri statin Y.
  • the conjugate comprises PEG molecule.
  • Another object of the invention is a recombinant human fibroblast growth factor 2 (FGF2) protein variant suitable for targeted anticancer therapy of FGFR- related cancers selected from:
  • KCK-FGF2[C78S/C96S] protein variant having inserted KCKSGG sequence on the N-terminus, and a substitution of two surface cysteine residues to serine at positions 78 and 96;
  • FGF2[C78S/C96S]-KCK protein variant having inserted GGSKCK sequence on the C-terminus, and a substitution of two surface cysteine residues to serine at positions 78 and 96;
  • KCK-FGF2 variant is identified by a SEQ ID NO: 2
  • FGF2-KCK variant is identified by a SEQ ID NO:3
  • KCK-FGF2[C78S/C96S] variant is identified by a SEQ ID NO:4
  • FGF2[C78S/C96S]-KCK variant is identified by a SEQ ID NO:5
  • sFGF2 WT variant is identified by a SEQ ID NO:6
  • sFGF2[C78S] variant is identified by a SEQ ID NO:7
  • sFGF2 [C96S] is variant is identified by a SEQ ID NO:8.
  • a variant sFGF2[C78S/C96S] is identified by Met-Ala-25-155 of FGF2 amino acid sequence having a substitution of cysteine residues to serine at position 78 and 96 (SEQ ID NO:9).
  • proteins having identity to FGF2 or relevant FGF2 mutant of at least 84%, 85% 90%, 95%, 98%, 99% and that remain capable of binding to FGFRs are proteins having identity to FGF2 or relevant FGF2 mutant of at least 84%, 85% 90%, 95%, 98%, 99% and that remain capable of binding to FGFRs.
  • the % identity of two polypeptides can be measured by a similarity score determined by comparing the amino acid sequences of the two polypeptides using routine programs with the default settings for determining similarity (i.e. local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981)) to find the best segment of similarity between two sequences.
  • the FGF2 protein variant is a peptide comprising amino acids having at least 84% of homology or higher with the amino acid sequence of SEQ ID NO: 1.
  • the FGF2 protein variant comprises amino acids wherein one or multiple amino acids are deleted, substituted, inserted, and/or added in the amino acid sequence of SEQ ID NO: 1.
  • Another object of the invention is a method of obtaining the defined above conjugate of a cytotoxic drug and a human fibroblast growth factor 2 (FGF2) protein or its variant comprising performing conjugation reaction at the temperature range 4 - 40°C and pH 6.0 - 8.0.
  • Another object of the invention is the conjugate as defined above for use in targeted treatment of cancers.
  • the cancers are selected from cancers overexpressing FGFRs, particularly FGFR3 or FGFR1, more particularly III isoform of FGFR1.
  • the cancer is selected from breast cancer, ovarian cancer, prostate cancer, cervical cancer, lung cancer, brain cancer, melanomas, colorectal, pancreatic, gastric, bladder, endometrial, hematologic cancer, head and neck cancer.
  • FGF2 and FGF2 variants conjugates show high specificity and potency in killing FGFR1 expressed tumor cells.
  • the conjugates according to the invention show also many advantages over known therapies.
  • the selectivity of FGF2 may cause less severe side-effects and lower the overall toxicity of cytotoxic FGF2-conjugates in comparison to FGF1 -conjugates.
  • FGF1 is moderately stable protein with denaturation temperature close to physiological, and contains three reactive cystienes localized within protein interior (Copeland, 1991; Blaber, 1999; Lee, 2009), so combination of low protein stability with reactivity of cysteine thiol group leads to irreversible denaturation, protein degradation or aggregation (Alsenaidy, 2011).
  • FGF2 shows higher denaturation temperature (Vemuri, 1994) and therefore is less prone to degradation (Chen, 2012). Increased resistance to degradation or to thermally induced unfolding leads to longer biological activity of FGF2 as compared to FGF1.
  • FGF2 has a high specificity for selected isoforms of FGFRs. It exhibits high affinity to IIIc isoform of FGFR1 or FGFR3 and lower to FGFR2-IIIc, FGFRl-IIIb and FGFR4 (Zhang, 2006).
  • FGF2 is effectively internalized into a cell via receptor-depended pathway (Wiedlocha & Sorensen, 2004).
  • FGF2 WT 2) KCK-FGF2 variant having KCKSGG sequence (abbreviated as KCK) on the N- terminus of FGF2;
  • FGF2-KCK variant having GGSKCK sequence (abbreviated as KCK) on the C- terminus of FGF2;
  • KCK-FGF2[C78S/C96S] variant having KCKSGG sequence (abbreviated as KCK) on the N-terminus of FGF2 and a substitution of two surface cysteine residues to serine at positions 78 and 96 in FGF2 sequence;
  • a “cell with FGFR overexpression” or a “cell that overexpresses FGFR” refers to a cell that has at least a 2-fold greater level of FGFR mRNA or protein than a reference cell.
  • a “cancer with FGFR overexpression” or a “cancer that overexpresses FGFR” refers to a cancer in which at least a portion of the cells have at least a 2-fold greater level of FGFR mRNA or protein than a reference cell.
  • FIG. 2 Conjugation reaction of FGF2 variants with vcMMAE.
  • A Electrophoretic separation and
  • B mass spectra of FGF2 variants before and after the reaction performed for 1 h at 25°C in 50 mM monosodium phosphate buffer, pH 7.0, in the presence of 10 mM Na 2 S0 4 , 10 mM methionine, 1 mM EDTA.
  • Fig. 3 Conjugation reaction of sFGF2 (truncated form of FGF2) variants with vcMMAE. Electrophoretic separation and mass spectra of sFGF2 variants and their conjugates. Fig. 4 Functional competence of FGF2 conjugates.
  • FIG. 5 Specific internalization of FGF2 WT, KCK-FGF2[C78S/C96S]-(vcMMAE)i and KCK- FGF2-(vcMMAE) 3 conjugates into cells expressing FGFR1. Representative images of specific internalization of FGF2 WT, KCK-FGF2[C78S/C96S]-(vcMMAE)i or KCK-FGF2- (vcMMAE) 3 into U20S-R1 cells versus U20S cells.
  • Fig. 7 Conjugation reaction of FGF2 variants with vcMMAE and YCP. Electrophoretic separation of FGF2 variants before and after the reaction performed for 1 h at 25°C in 50 mM monosodium phosphate buffer, pH 7.0, in the presence of 10 mM Na 2 S0 4 , 10 mM methionine, 1 mM EDTA.
  • Fig. 8 Dependence of number of cytotoxic molecules per FGF2 molecule on the cytotoxicity of conjugate towards cells overexpressing FGFR1.
  • Fig. 9 Dependence of number of cytotoxic molecules per FGF2 molecule on the cytotoxicity of conjugate towards cells non-producing the FGFRl .
  • FIG. 10 Fluorescence emission spectra of FGF2 variants and their conjugates. Measurements were performed at a protein concentration 4xl0 "6 M upon excitation at 280 nm. Curves were normalized to tyrosine emission at 303 nm.
  • Fig. 11 Viability of U20S and U20S-R1 cells treated with KCK-FGF2-YCP 3 and FGF2- vcMMAE 3 for 96 h, assessed with AlamarBlue assay. Results shown are mean values from three experiments ⁇ SD
  • Example 1 Cell lines and bacterial strains
  • BJ cells (CRL-2522) were grown in Eagle's Minimum Essential Medium.
  • U20S (HTB-96TM) and U20S stably transfected with FGFR1 (U20S-R1) were grown in McCoy's 5A Modified Medium. All media were supplemented with 10% fetal bovine and 1% penicillin/streptomycin mix. Additionally, U20S-R1 cell culture contained 50 ⁇ g/mL gentamicin sulfate. All cell lines were cultured in a humidified incubator at 37 °C in 5% C02 atmosphere.
  • BJ and U20S cell lines were obtained from American Type Culture Collection ATCC (Manassas, VA, USA).
  • the U20S cells stably expressing FGFR1 (U20S-R1) was a kind gift from Dr. Ellen M. Haugsten from The Norwegian Radium Hospital (Haugsten, 2008).
  • E. coli strains Rosetta 2(DE3)pLysS was from Novagen-EMD Biosciences (Madison, WI, USA).
  • FGF2 human fibroblast growth factor 2
  • Method-Ala-residues 25-155 The sequence encoding human fibroblast growth factor 2 (FGF2) (residues 1-155) or its truncated form (Met-Ala-residues 25-155) was cloned into the pET-3c expression vector from Stratagene (La Jolla, CA, USA). Insertion of KCKSGG N- and GGSKCK C-terminal linker and the point mutations C78S and C96S were introduced using QuikChange Site-Directed Mutagenesis Kit from Agilent Technologies (Santa Clara, CA, USA) according to manufacturer's protocol.
  • protein expression was induced by the addition of IPTG to the final concentrations of 0.3 mM and the culture was incubated at 25 °C for 12 h.
  • vcMMAE Maleimidocaproyl-Val-Cit-PABC-monomethylauristatin E
  • FGF2 Maleimidocaproyl-Val-Cit-PABC-monomethylauristatin E
  • MMAE is a highly cytotoxic derivative of dolastatin containing a maleimide moiety suitable for conjugation to cysteines residue and a protease-sensitive valine-citrulline dipeptide sequence designed for optimal stability in human plasma and effective cleavage by human cathepsin B.
  • the conjugation reaction was optimized to provide high yield and optimal conditions, avoiding protein unfolding and a loss of receptor binding activity. For optimal reaction conditions few temperature points between 4 and 40°C were tested in several time points. The conjugation reaction occurs in every temperature in this range with different speeds and efficiencies, but the reaction conducted by 1 h at 20 °C leads to the best results. Moreover, wide range of buffers pH can be used in reaction (from 6 to 8). Purified proteins were desalted using Zeba Spin Desalting column into reaction buffer (50 mM monosodium phosphate, 10 mM Na 2 S0 4 , 10 mM methionine, 1 mM EDTA, pH 7.0).
  • the yield of protein expression was between 8 and 40 mg per liter of culture.
  • the variants exhibited a very similar elution profile during purification on a heparin-Sepharose column.
  • the excess of unconjugated vcMMAE was removed from reaction mixture by buffer exchange to Dulbecco's PBS using Zeba Spin Desalting column.
  • the fluorescence spectrum of properly folded wild-type FGF2 showed very low emission at around at 353 nm, since the signal from the single tryptophan residue is completely quenched and the spectrum is dominated by emission of tyrosine residues (maximum at around 303 nm). Upon unfolding, the quenching effect is abolished, resulting in a significant increase in the fluorescence at 353 nm. All fluorescence emission spectra were similar to the spectrum of native FGF2 WT, showing no changes in the tertiary structure of proteins conjugated to vcMMAE.
  • Total cell lysates was separated by SDS-PAGE (12%) and analyzed by Western blotting using following antibodies: anti-phospho-Erkl/2 , anti-Erkl/2 and anti-y-tubulin. Specific protein bands were visualized using HRP-conjugated secondary antibodies and an enhanced chemoluminescence substrate in a ChemiDoc station (BioRad, Hercules, CA, USA ).
  • U20S cells were pre-stained with CellTraceTMViolet fluorescent dye and co-cultured with equal number of non-stained U20S-R1 cells that allowed us to discriminate between two cell lines on the same coverslip.
  • U20S cells stained with CellTraceTM Violet according the manufacturer's protocol were seeded on coverslips with equal number of non-stained U20S-R1 cells and grown together up to 70% of confluence.
  • the cells were incubated with 100 ng/mL of labeled samples in the presence of 10 U/ml heparin at 37 °C for 15 min. Then, the cells were washed with PBS, fixed in 4% formaldehyde for 15 min at RT, permeabilized in 0.5% Triton X-100 for 10 min at 4 °C and blocked with blocking buffer (1% BSA, 10% Normal Goat Serum, 0.2% Tween-20 and 0.3 M glycine in PBS) for 1 h at RT.
  • blocking buffer 1% BSA, 10% Normal Goat Serum, 0.2% Tween-20 and 0.3 M glycine in PBS
  • the cells were incubated with primary rabbit anti-EEAl antibody overnight at 4°C, and then incubated with an AlexaFluor488 goat anti-rabbit secondary antibody at RT for 1 h.
  • Nuclei were stained with DAPI and cover slips were mounted with ProLongGold Antifade Mountant.
  • the cell staining was analyzed using Cell Observer SD confocal system (Zeiss, Germany) equipped with EMCCD Qlmaging Rolera EM-C2 camera with a 63 x oil immersion objective. All images data were processed in Fiji software (Schindelin et al. 2012).
  • BJ non- malignant cells naturally expressing relatively high level of FGFRl
  • U20S cells that show hardly detectable level of FGFRl and serve as a negative control
  • U20S-R1 U20S cells stably expressing very high level of FGFRl
  • U20S (HTB-96TM) and U20S stably transfected with FGFRl (U20S-R1) were grown in McCoy's 5A Modified Medium. All media were supplemented with 10% fetal bovine and 1% penicillin/streptomycin mix. Additionally, U20S-R1 cell culture contained 50 ⁇ g/mL gentamicin sulfate. All cell lines were cultured in a humidified incubator at 37 °C in 5% C02 atmosphere. U20S cell lines was obtained from American Type Culture Collection ATCC (Manassas, VA, USA). The U20S cells stably expressing FGFRl (U20S-R1) was a kind gift from Dr. Ellen M. Haugsten from The Norwegian Radium Hospital (Haugsten, 2008). E. coli strains Rosetta 2(DE3)pLysS was from Novagen-EMD Biosciences (Madison, WI, USA).
  • FGF2 human fibroblast growth factor 2
  • Total cell extract was centrifuged at 50 000xg at 4 °C for 1 h.
  • YCP Maleimid-PEG-Val-Thr-auristatin Y
  • Auristatin Y is a highly cytotoxic derivative of dolastatin, more hydrophilic than MMAE (Doronina, 2012). It contains a maleimide moiety suitable for conjugation to cysteines residue.
  • PEG molecule was added to auristatin Y. Any of PEG 513, PEG 1570 or PEG 3000 was suitable for this use.
  • the conjugation reaction was optimized, as in example 1, to provide high yield and optimal conditions, protecting against protein unfolding and a loss of biological activity.
  • Purified proteins were desalted using Zeba Spin Desalting column into reaction buffer (50 mM monosodium phosphate, 10 mM Na2S04, 10 mM methionine, 1 mM EDTA, pH 7.0).
  • Maleimide derivative of PEGylated auristatin Y (YCP) dissolved in DMAc (N,N- Dimethylacetamide) at concentration of 50 mg/mL was added to 1.5 mg/mL protein solutions. There was a two-fold molar excess of the drug over protein SH-groups.
  • the conjugation reaction mixture was incubated for 1 h at 20 °C.
  • the fluorescence spectrum of properly folded wild-type FGF2 showed very low emission at around at 353 nm, since the signal from the single tryptophan residue is completely quenched and the spectrum is dominated by emission of tyrosine residues (maximum at around 303 nm). Upon unfolding, the quenching effect is abolished, resulting in a significant increase in the fluorescence at 353 nm. All fluorescence emission spectra were similar to the spectrum of native FGF2 WT, showing no changes in the tertiary structure of proteins conjugated to YCP.
  • U20S cells that show hardly detectable level of FGFR1 and serve as a negative control
  • U20S- Rl U20S cells stably expressing very high level of FGFR1
  • Junutula JR Junutula JR, Flagella KM, Graham RA, Parsons KL, Ha E, Raab H, Bhakta S, Nguyen T, Dugger DL, Li G, Mai E, Lewis Phillips GD, Hiraragi H, Fuji RN, Tibbitts J, Vandlen R, Spencer SD, Scheller RH, Polakis P,

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Abstract

La présente invention concerne une stratégie visant à détruire des cellules cancéreuses en utilisant des molécules de ciblage fusionnées avec des agents hautement cytotoxiques. Un ligand naturel du récepteur du FGF, le facteur de croissance de fibroblaste 2 (FGF2), ou des variants modifiés de ce dernier, sont utilisés pour être bioconjugués avec un composé cytotoxique puissant tel que la monométhyl auristatine E (MMAE) ou l'auristatine Y afin de créer une technique d'administration visant à la destruction efficace des cellules cancéreuses présentant une surexpression du FGFR. La molécule de FGF2 et ses variants modifiés agissent comme des molécules d'administration qui dirigent de façon spécifique un médicament cytotoxique vers une cellule cancéreuse en se liant aux récepteurs du FGF et en étant par la suite internalisées dans la cellule. Les conjugués obtenus présentent un effet cytotoxique important vis-à-vis des cellules de cancers associés au FGFR.
PCT/IB2016/058083 2016-06-13 2016-12-29 Conjugués de facteur de croissance des fibroblastes 2 humain (fgf2) et de médicament cytotoxique pour le traitement ciblé de cancers liés au fgfr WO2017216620A1 (fr)

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Cited By (3)

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WO2020080656A1 (fr) * 2018-10-15 2020-04-23 국립암센터 Peptide dérivé de fgf2 ou de api5 et utilisation associée
WO2021025352A1 (fr) * 2019-08-08 2021-02-11 국립암센터 Virus anticancéreux exprimant le peptide dérivé du fgf2 ou de l'api5, et son utilisation
CN116650679A (zh) * 2023-04-14 2023-08-29 河北医科大学第四医院 一种靶向fgfr1的影像探针及其应用

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WO2020080656A1 (fr) * 2018-10-15 2020-04-23 국립암센터 Peptide dérivé de fgf2 ou de api5 et utilisation associée
KR20200042389A (ko) * 2018-10-15 2020-04-23 국립암센터 Fgf2 또는 api5 유래 펩타이드 및 그의 용도
KR102252881B1 (ko) 2018-10-15 2021-05-17 국립암센터 Fgf2 또는 api5 유래 펩타이드 및 그의 용도
WO2021025352A1 (fr) * 2019-08-08 2021-02-11 국립암센터 Virus anticancéreux exprimant le peptide dérivé du fgf2 ou de l'api5, et son utilisation
CN116650679A (zh) * 2023-04-14 2023-08-29 河北医科大学第四医院 一种靶向fgfr1的影像探针及其应用

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