• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    The present invention relates to a modified α-helical bundle cytokine, with reduced activity via a α-helical bundle cytokine receptor, wherein said α-helical bundle cytokine is specifically delivered to target cells. Preferably, said α-helical bundle cytokine is a mutant, more preferably it is a mutant interferon, with low affinity to the interferon receptor, wherein said mutant interferon is specifically delivered to target cells. The targeting is realized by fusion of the modified α-helical bundle cytokine to a targeting moiety, preferably an antibody. The invention relates further to the use of such targeted modified α-helical bundle cytokine to treat diseases. A preferred embodiment is the use of a targeted mutant interferon, to treat diseases, preferably viral diseases and tumors.
    公开号:AU2013211059A1
    申请号:U2013211059
    申请日:2013-01-17
    公开日:2014-08-21
    发明作者:Guillaume Cartron；Franciane PAUL；Jacob Piehler；Jan Tavernier；Gilles Uze
    申请人:UNI OSNABRUCK；Universiteit Gent；Vlaams Instituut voor Biotechnologie VIB；Universitat Osnabruck；Centre National de la Recherche Scientifique CNRS；Centre Hospitalier Universitaire de Montpellier CHUM；
    IPC主号:C07K16-28
    专利说明:
    WO 2013/107791 PCT/EP2013/050787 TARGETED MUTANT ALPHA-HELICAL BUNDLE CYTOKINES The present invention relates to a modified a-helical bundle cytokine, with reduced activity via a a-helical bundle cytokine receptor, wherein said a-helical bundle cytokine is specifically 5 delivered to target cells. Preferably, said a-helical bundle cytokine is a mutant, more preferably it is a mutant interferon, with low affinity to the interferon receptor, wherein said mutant interferon is specifically delivered to target cells. The targeting is realized by fusion of the modified a-helical bundle cytokine to a targeting moiety, preferably an antibody. The invention relates further to the use of such targeted modified a-helical bundle cytokine to treat diseases. 10 A preferred embodiment is the use of a targeted mutant interferon, to treat diseases, preferably viral diseases and tumors. Cytokines are small proteins that play an important role in intercellular communication. Cytokines can be classified based on their structure, the largest group being the four-a-helix 15 bundle family. This family can, based on the use of receptors, further be divided in the interferon (IFN) and interleukin (IL)-2, -3, -10 and -12 subfamilies. The a helical bundle cytokines are important as possible biopharmaceutical for treatment of human diseases; as a non-limiting example erythropoietin is used for treatment of anemia or red blood cell deficiency, somatotropin for treatment of growth hormone deficiency and interleukin-2 in the treatment of 20 cancer. Within the a helical bundle cytokines, type I IFNs belong to a cytokine family having important biological functions. In human, there are 17 different type I IFNs (13a, P, E, K, w) which signal through an ubiquitously expressed cell surface receptor composed of two chains IFNAR1 and IFNAR2. The assembling of the IFN-receptor complex initiates the activation of several signal 25 transduction pathways that, depending of the cell type, modify cellular differentiation and/or functions. By acting on virtually every cell type, type I IFN is able to prevent productive viral infection. In addition, it exhibits marked antiangiogenic and proapoptotic effects. Type I IFNs are also deeply implicated in the regulation of several functions of the innate and adaptive immunity as 30 well as on bone homeostasis. It acts particularly on the activation/differentiation of dendritic cells and osteoclasts. The type I IFN system is in fact critically important for the health of mammals. Preclinical studies in mice have established a remarkable efficacy of type I IFN for the treatment of both viral or tumor diseases. Noteworthy, mice cured of an experimental tumor by 35 IFN treatment have been found immunized against the initial tumor, suggesting that IFN acts not only to engage the processes of tumor rejection but also to break the immune tolerance 1 WO 2013/107791 PCT/EP2013/050787 against the tumor. Based on these studies, IFNa was approved in clinic for the treatment of both viral infection and cancer. More recently, IFNp was shown to be effective in relapsing remitting multiple sclerosis and was also approved for this pathology. Unfortunately, the clinical efficacy of IFN was often found disappointing and today other therapeutic strategies such as 5 specific antiviral compounds, chemotherapies and monoclonal antibodies have, when possible, largely supplanted IFN broad application. Today, IFN is the first line therapeutic choice for only HBV and HCV chronic infections and for a limited number of tumors. The efficacy of type I IFN in clinical practice is limited by ineffective dosing due to significant systemic toxicity and side effects, including flu-like syndrome, depression, hepatotoxicity, 10 autoimmune disease, thyroid dysfunction and weight loss. It would thus be highly worthwhile to target IFN activity toward only the cellular population which should be treated with IFN (e.g. infected organ or tumor mass) or activated by IFN (e.g. subsets of immune cells). In order to solve or limit the systemic toxicity of cytokines, specific targeting of cytokines by antibody-cytokine fusion proteins has been proposed (Ortiz-Sanchez et al., 2008). Rossi et al. 15 (2009) specifically discloses CD20-targeted tetrameric IFNa, and its use in B-cell lymphoma therapy. However, the fusion maintains its biological activity, and is even more active than commercial pegylated IFN, which means that the unwanted side effects in human treatment would still be present, or would even be more severe. W02009039409 discloses targeted IFN and its apoptotic and anti-tumor activities. The patent application discloses the fusion of an 20 antibody as targeting moiety with wild type IFN, but also with mutated IFN. However, it is stated that the IFN fragment should retain its endogeneous activity at a level of at least 80%, or even at a higher level than wild type IFN. Also in this case, the fusion is retaining the unwanted side effects of the wild type. Surprisingly we found that a modified a-helical bundle cytokine, with a decreased affinity for 25 the a-helical bundle cytokine receptor and a consequent decreased specific bioactivity can be fused to a targeting moiety, wherein the bioactivity is restored towards the targeted cells, but not towards cells that are not targeted by the construct. Such construct has the advantage over the art of having less side effects, especially a lower systemic toxicity, while retaining the bioactivity against the target cells. 30 A first aspect of the invention is a targeting construct, comprising modified a-helical bundle cytokine, characterized by a reduced affinity for the a-helical bundle cytokine receptor, and a targeting moiety. a-helical bundle cytokines are known to the person skilled in the art, and include, but are not limited Cardiotrophin-like cytokine NNT-1, Ciliary neurothrophic factor, 35 Macrophage colony stimulating factor, Granulocyte-macrophage colony stimulating factor, Granulocyte colony stimulating factor, Cardiotrophin-1, Erythropoietin, FLT3 ligand, 2 WO 2013/107791 PCT/EP2013/050787 Somatotropin, Interferon a-1, 2, 4, 5, 6, 7, 8, 10, 13, 14, 16, 17, 21,, Interferon p, Interferon y, Interferon K, Interferon E, Interferon c-1, Interferon o-1, Interleukin 2, 3, 4, 5, 6, 7, 9, 10, 11, 12 a chain, 13, 15, 19, 20, 21, 22, 23, 24, 26, 27, 28A, 29, 31, Stem cell factor, Leptin, Leukemia inhibitor factor, Oncostatin M,Prolactin, and Thrombopoietin. For a review on a-helical bundle 5 cytokines, see Conklin (2004). A modified a-helical bundle cytokine means that the a-helical bundle cytokine has been changed to alter the affinity to the receptor, with as final result that the modified a-helical bundle cytokine has a reduced affinity for the receptor and a consequent reduced biological activity, as compared to the endogenous wild type cytokine that binds normally to the receptor. Such a modification can be a modification that decreases the activity 10 of the normal wild type cytokine, or it can be a modification that increases the affinity of a homologous, non-endogenous a-helical bundle cytokine (such as, but not limited to a mouse a-helical bundle cytokine, binding to a human a-helical bundle cytokine receptor). Modifications can be any modification reducing or increasing the activity, known to the person skilled in the art, including but not limited to chemical and/or enzymatic modifications such as 15 pegylation and glycosylation, fusion to other proteins and mutations. Preferably said modification is a mutation, even more preferably it is a mutation decreasing the affinity of the a-helical bundle cytokine. A reduced affinity and a consequent reduced biological activity as used here means that the modified a-helical bundle cytokine has a biological activity of less than 70% of the biological activity of the a-helical bundle cytokine, even more preferably less 20 than 60% of the biological activity of the a-helical bundle cytokine, more preferably less than 50% of the biological activity of the a-helical bundle cytokine, more preferably less than 40% of the biological activity of the a-helical bundle cytokine, more preferably less than 30% of the biological activity of the a-helical bundle cytokine, more preferably less than 20% of the biological activity of the a-helical bundle cytokine, most preferably less than 10% of the 25 biological activity of the a-helical bundle cytokine as compared to the a-helical bundle cytokine that normally binds to the receptor. Preferably, the modified a-helical bundle cytokine is a mutant of the wild type a-helical bundle cytokine and the activity is compared with the wild type a-helical bundle cytokine. The affinity and/or the activity can be measured by any method known to the person skilled in the art. Preferably, the activity is measured by measuring and 30 quantifying STAT phosphorylation. A preferred embodiment of the invention is a targeting construct, comprising a mutant IFN characterized by reduced affinity for the IFN receptor, and a targeting moiety. IFN can be any IFN, including but not limited to IFNa, IFNp and o). A mutant IFN as used here can be any mutant form that has a lower affinity for the receptor and as a consequence a lower 35 antiproliferative activity and/or a lower antiviral activity. Indeed, as shown by Piehler et al. 3 WO 2013/107791 PCT/EP2013/050787 (2000), the relative affinity correlates directly with the relative antiproliferative activity and with the relative antiviral activity. The affinity of the mutant IFN to the receptor, in comparison to the affinity of the wild type IFN to the receptor can be measured by reflectometric interference spectroscopy under flow through conditions, as described by Brecht et al. (1993). The mutant 5 may be a point mutant, a deletion or an insertion mutant, or a combination thereof. Preferably, said mutant IFN is obtained by active mutagenesis, such as, but not limited to site directed mutagenesis by polymerase chain reaction amplification. Preferably, said mutant IFN has a biological activity of less than 70% of the biological activity of the wild type IFN, even more preferably less than 60% of the biological activity of the wild type IFN, more preferably less 10 than 50% of the biological activity of the wild IFN, more preferably less than 40% of the biological activity of the wild IFN, more preferably less than 30% of the biological activity of the wild IFN, more preferably less than 20% of the biological activity of the wild IFN, most preferably less than 10% of the biological activity of the wild type of which it is deduced (i.e. the wild type IFN of which the coding sequence has been mutated to obtain the mutant IFN). 15 Mutant forms of IFN are known to the person skilled in the art. As a non-limiting example, IFNa2 mutants have been listed in Piehler et al. (2000). Preferably, said IFN is a type I IFN. Even more preferably, said mutant is an IFNa, even more preferably said mutant is an IFNa2. More preferably, said IFNa2 mutant is mutated in one or more amino acids of the region 144 154, preferably at position 148, 149 and/or 153, even more preferably, said mutant IFNa2 is 20 selected from the group consisting of IFNa2 L153A, IFNa2 R149A and IFNa2 M148A. Most preferably said mutant is selected from the group consisting of IFNa2 L153A and IFNa2 R149A. Preferably, said receptor is IFNAR2. Preferably, said targeting moiety is targeting to a marker expressed on an IFN receptor 25 expressing cell, preferably a cell expressing IFNAR2. In one preferred embodiment, said targeting moiety is directed to a tissue specific marker. Preferably, said tissue is a cancer tissue. Said cancer can be any cancer, including but not limited to B cell lymphoma, lung cancer, breast cancer, colorectal cancer or prostate cancer. In another preferred embodiment, said targeting moiety is directed to a marker selected from the group consisting of Her2 and 30 CD20. In still another preferred embodiment, said targeting moiety is directed to a cell surface marker specific for viral infected cells such as but not limited to influenza M2 protein, LMP1 and EBV proteins). In still another embodiment said targeting moiety is directed towards an osteoclast marker such as DC-STAMP or RANK. Indeed, it is know that IFN-p plays an important role in bone homeostasis, regulated by RANK and IFNAR coexpressing cells 35 (Abraham et al., 2009). In still another embodiment said targeting moiety is directed towards a marker specifically expressed on the surface of an immune cell type on which IFN may 4 WO 2013/107791 PCT/EP2013/050787 regulates activity and/or differentiation. The marker PDL2 specifically expressed on dendritic cells and some immune cells is an example. A targeting moiety, as used here, can be a protein as a part of a specifically binding protein 5 complex, or any specifically binding protein or protein fragment, known to the person skilled in the art. It includes, but is not limited to carbohydrate binding domains (CBD) (Blake et al, 2006), lectin binding proteins, heavy chain antibodies (hcAb), single domain antibodies (sdAb), minibodies (Tramontano et al., 1994), the variable domain of camelid heavy chain antibodies (VHH), the variable domain of the new antigen receptors (VNAR), affibodies (Nygren et al., 10 2008), alphabodies (W02010066740), designed ankyrin-repeat domains (DARPins) (Stumpp et al., 2008), anticalins (Skerra et al., 2008), knottins (Kolmar et al., 2008) and engineered CH2 domains (nanoantibodies; Dimitrov, 2009). Preferably, said targeting moiety consists of a single polypeptide chain and is not post-translationally modified. Even more preferably, said targeting moiety is a nanobody. 15 The targeting construct can be any targeting construct known to the person skilled in the art. As a non-limiting example, the targeting moiety may be chemically linked to the mutant interferon, or it may be a recombinant fusion protein. Preferably, said targeting construct is a recombinant fusion protein. The targeting moiety may be fused directly to the mutant IFN, or it may be fused with the help of a linker fragment. The targeting moiety may be fused at the 20 aminoterminal or at the carboxyterminal end of the mutated IFN; preferably said targeting moiety is fused at the amino-terminal extremity of the mutated IFN molecule. Another aspect of the invention is a targeting construct according to the invention for use as a medicament. Still another aspect of the invention is the use of a targeting construct according to the 25 invention for the manufacture of a medicament to treat cancer. Still another aspect of the invention is the use of a targeting construct according to the invention for the manufacture of a medicament to treat a viral disease. As a non-limiting example said viral disease may be HIV infection, HBV infection or HCV infection. Another aspect of the invention is a targeting construct according to the invention for use in 30 treatment of cancer. Still another aspect of the invention is a targeting construct according to the invention for use in treatment of a viral disease. As a non-limiting example said viral disease may be HIV infection, HBV infection or HCV infection. Still another aspect of the invention is a targeting construct according to the invention for use in 35 treatment of diseases involving bone degradation, such as, but not limited to osteoporosis. Still another aspect of the invention is a pharmaceutical composition, comprising a targeting construct according to the invention and a suitable excipient. It is clear for the person skilled in 5 WO 2013/107791 PCT/EP2013/050787 the art that such a pharmaceutical composition can be used alone, or in a combination treatment, such as, but not limited to a combination with chemotherapy. BRIEF DESCRIPTION OF THE FIGURES 5 Figure 1: Representation of the structural elements of the nanobody-IFN fusion protein. Figure 2: Firefly luciferase activity induced by the indicated IFN preparation on HL1 16 cells (panels A and B) or HL116 cells expressing the murine leptin receptor (mLR) (panels C and D). Panels A and C on the one hand and panels B and D on the other hand were generated in 10 two separate experiments. Consequently only vertical comparison (panel A versus panel C or panel B versus panel D) is possible. Figure3: Renilla (light grey) and Firefly (dark grey) luciferase activity induced by the nanobody IFNa2R149A or by the IFNa2 (7pM) in a 1:1 coculture of cells expressing the leptin receptor 15 and an IFN-inducible firefly luciferase or in cells expressing an IFN-inducible renilla luciferase but devoid of leptin receptor. Luciferase activities are expressed as a percentage of the luciferase activities induced by 3 nM IFN a2. Figure 4: Activity of the purified constructs targeting the mLR: A. Quantification of their specific 20 activities on cells expressing the target (HL1 16-mLR) or on cells lacking the target (HL116). B. Calculation of the targeting efficiencies of the different constructs. Figure 5: Activity of the construct 4-11-IFNA2-R149A in presence and absence of the unconjugated leptin receptor binding nanobody. HL116 cells expressing the mLR were 25 incubated for 6 hours with either the IFN-a2 (IFNA2) or the IFNA2-R149A fused to the nanobody 4-11 (Nanobody-IFNA2-R149A) at their respective EC50 concentration in the presence or absence (control) of a 100-fold molar excess of free 4-11 nanobody. Figure 6: Targeting the mutant IFN using the leptin binding nanobody 4-10. 30 Figure 7: Firefly luciferase activity induced in HL116 cells expressing the mLR by the nanobody-IFNa2R149A in the presence of anti IFNAR1 monoclonal antibody 64G12 (Benoit et al. J. Immunol. 150, 707-716. 1993) or anti IFNAR2 monoclonal antibody MMHAR2 (PBL Interferon Source) 35 Figure 8: Specificity of the targeting to of 4-11-IFNA2-R149A to cells expressing the mLR. A: Cytopathic effect of the EMCV on HL1 16 cells (dark gray symbols) or on HL1 16-mLR (light 6 WO 2013/107791 PCT/EP2013/050787 grey symbols) of parental IFNA2 (upper left panel) or of the 4-11-IFNA2-R149A (lower left panel). B: Upper panel: calculated EC50 for antiviral activity; lower panel: calculated targeting efficiencies 5 Figure 9: Specific activities (EC50) of IFNa2 (panel A) and the nanobody-IFNa2R149A (2R5A; panel B) on BXPC3 and BT474 cell lines which express different number of Her2 molecule at their surface (10.9 x 10 3 and 478 x 10 3 , respectively). The ordinate scale of panel A cannot be compared to the ordinate scale of panel B. 10 Figure 10: Targeting of the 1R59B-IFNA2-Q124R to human Her2 expressing mouse cells. Quantification of the OASL2 mRNA expression in BTG9A cells with and without Her2 expression. Figure 11: Targeting of mutant IFNA2 to human Her2 expressing mouse cells, using a single 15 chain antibody. Quantification of the ISG15 mRNA expression in BTG9A cells with and without Her2 expression. Figure 12: Control of the activation of Her2 phosphorylation: Lane 1 3 to 76: no phosphorylated Her2 in extract of BTG9A cells expressing human Her2 treated with different concentration 20 (200 pM for lane 3 to 5, 2 nM for lane 6) and time (lane 3: 5 min, lane 4 and 6: 10 min, lane 5: 30 min.) with the construct 1 R59B-IFNA2-Q124R. Lane 7 and 8: control for the detection of phosphorylated Her2 in the human BT474 cell line. Lane 1 extract of BTG9A cells. Lane2: extract of BTG9A cells expressing human Her2. 25 Figure 13: Targeting the anti PD-L2 122-IFNA2-Q124R to mouse primary cells endogenously expressing PD-L2. The activation is measured as STAT phosphorylation. The light gray area represents the PD-L2 negative cell population; the dark gray area represents the PD-L2 positive population. 30 Figure 14: In vivo targeting of 122-IFNA2-Q124R to PD-L2 expressing cells. Mice were injected intraperitoneally (IP) or intravenous (IV) with either PBS, a control construct (nanobody against GFP fused to mutant IFNA2-Q124R, indicated as control) or a targeted mutant IFN (targeted to PD-L2, Nb122-IFN2-Q124R, indicated as 122-Q124R. The light gray 35 area represents the PD-L2 negative cell population; the dark gray area represents the PD-L2 positive population. 7 WO 2013/107791 PCT/EP2013/050787 Figure 15: Dose response curve after IV injection of 122-IFN-Q124R in mice. The light gray area represents the PD-L2 negative cell population; the dark gray area represents the PD-L2 positive population. 5 Figure 16: Leptin-dependent growth induced by targeted mutant leptin: the loss in activity of a mutant leptin can be rescued in Ba/F3 cells expressing the human TNFR1. Upper panels: experiment using the H6-leptin construct; lower panel, experiment using the mleptin construct. H6 indicated the his tag (6 xhis). 10 Figure 17: construction of the targeted leptin constructs EXAMPLES 15 Materials & Methods to the examples Nanobodies and ScFv The nanobody 4-11 directed against the murine leptin receptor was described in Zabeau et al. (2012), and in the patent WO 2006/053883. Its coding sequence is cloned into the mammalian 20 expression vector pMET7 (Takebe et al., 1988) in fusion with the Slgk leader peptide, the HA tag and albumin. Plasmid name: pMET7 SlgK-HA-4.1 1-Albumin. The nanobody 4-10 is also described in Zabeau et al. (2012). The anti Her2 nanobodies 1R59B and 2R5A are described in Vaneycken et al. (2011). They were fused to the human IFNA2-Q124R and to the human IFNA2-R149A in the pMET7 vector. 25 Fusion protein was produced by transfection of 293T cells. The anti PD-L2 nanobody 122 was from Johan Grooten (VIB). It was fused to the human IFNA2-Q124R in the pMET7 vector. The fusion protein was produced by transfection of 293T cells and purified using the HisPur Ni-NTA purification kit (Pierce, Thermo Scientific). The anti TNF nanobody was obtained from Claude Libert (VIB). 30 The anti Her2 ScFv was obtained from Andrea PlOckthun (W6rn et al., 1998) It was fused to the human IFNA2-Q124R in the pMET7 vector. The fusion protein was produced by transfection of 293T cells. 35 Control nanobody against GFP was obtained from Katrien Van Impe (University Ghent) Interferons 8 WO 2013/107791 PCT/EP2013/050787 The IFNa2 and the mutants L153A and R149A which show an IFNAR2 affinity reduced by a factor 10 and 100, respectively, have been described in Roisman et al., (2001). IFN coding sequences are cloned in the pT3T7 vector (Stratagene) in fusion with the ybbR tag. Plasmid names: pT7T3ybbR-IFNa2, pT7T3ybbR-IFNa2-L153A, pT7T3ybbR-IFNa2-R149A. 5 The human IFNA2 Q124R has a high affinity for the murine IFNAR1 chain and a low affinity for the murine IFNAR2 chain. (Weber et al., 1987) Nanobody-IFN fusion construction The coding sequence of the IFNa2, wild type, L153A and R149A were synthesized by PCR 10 from the corresponding pT3T7ybbR IFNa2 plasmids using the Expand High Fidelity PCR system from Roche Diagnostics and the following primers: Forward: 5'GGGGGGTCCGGACCATCACCATCACCATCACCATCACCATCACCCTGCTTCTCCCGCC TCCCCAGCATCACCTGCCAGCCCAGCAAGTGATAGCCTGGAATTTATTGC3', Reverse: 5'CGTCTAGATCATTCCTTACTTCTTAAAC3'. This PCR introduces a His tag and a series of 5 15 Proline - Alanine - Serine (PAS) repeats at the amino terminal extremity of the IFNs. The PCR products were digested with BspEl and Xbal and cloned into BspEl-Xbal digested pMET7 SlgK-HA-4.11-Albumin vector to obtain pMET7 SlgK-HA-4.11-His-PAS-ybbr-IFNA2, pMET7 SlgK-HA-4.11-His-PAS-ybbr-IFNA2-L153A and pMET7 SlgK-HA-4.11-His-PAS-ybbr-IFNA2 R149A. 20 In a similar way the human mutant Q124R was fused to the 1 R59B nanobody and to the anti PD-L2 nanobody. Production of the nanobody-IFN fusion protein HEK293T cells were grown in DMEM supplemented with 10% FCS. They were transfected 25 with pMET7 SlgK-HA-4.11-His-PAS-ybbr-IFNA2, pMET7 SlgK-HA-4.11-His-PAS-ybbr-IFNA2 L153A pMET7 SlgK-HA-4.11-His-PAS-ybbr-IFNA2-R149A, pMET7 SlgK-HA-2R5A-His-PAS ybbr-IFNA2-R149A, pMET7 SlgK-HA-1R59B-His-PAS-ybbr-IFNA2-Q124R, pMET7 SIgK-HA 4D5-His-PAS-ybbr-IFNA2-Q124R or pMET7 SlgK-HA-122-His-PAS-ybbr-IFNA2-Q124R using lipofectamin (Invitrogen). 48 hours after the transfection culture mediums were harvested and 30 stored at -20'C. Alternatively, sequences encoding the different nanobody-IFN fusions where subcloned into the baculovirus transfer plasmid pBAC-3 (Novagen). Proteins were produced by insect cells using the BacVector kit (Novagen) and purified to homogeneity using the HisPur Ni-NTA purification kit (Pierce, Thermo Scientific) and gel filtration. Protein concentrations were 35 measured by absorbance at 280 nm. 9 WO 2013/107791 PCT/EP2013/050787 IFN reporter cell lines The HL1 16 clone (Uze et al. 1994) is derived from the human HT1080 cell line. It contains the firefly luciferase gene controlled by the IFN-inducible 6-16 promoter. The HL1 16 cells were co transfected with an expression vector encoding the short isoform of the murine leptin receptor 5 (pMET7 mLRsh-FLAG, Eyckerman et al., 1999) and pSV2neo (Southern and Berg 1982). Stable transfected clones were isolated in G418 containing medium. The clone 10 was selected after analysis of the surface expression level of the murine leptin receptor by FACS, using the the biotinylated anti-mouse leptin receptor antibody BAF497 from R&D and streptavidin-APC (BD bioscience) 10 HT1080 cells were cotransfected with p6-16-RL, a plasmid encoding the Renilla luciferase (from pRL-null, Promega) controlled by the IFN-inducible 6-16 promoter (from pl.8gpt-5, Pellegrini et al. 1989), pBB3 (Bourachot et al. 1982) and salmon sperm DNA (Sigma). Stable transfected clones were isolated in HAT containing medium. The clone 4 was selected for a high level of renilla luciferase activity induction upon IFN induction. 15 The human pancreatic carcinoma BXPC3 (Tan et al., 1986; ATCC: CRL 1687) and breast cancer BT474 (Lasfargues et al., 1979; ATCC: HTB-20) cell lines were obtained from ATCC. The mouse BTG9A cells were described in Uze et al. (1990). Measurement of the luciferase activities 20 IFN specific activities were measured by quantifying the luciferase activity induced in HL116 cells and on the HL116 clone 10 expressing the mLR. The EC50 were calculated using non linear data regression with GraphPad Prism software. Luciferase activities were determined on a Berthold centro LB960 luminometer using either the Firefly Luciferase Assay System or the Dual-Luciferase Reporter Assay System from Promega 25 after 6hr IFN stimulation. Quantitative RT-PCR The expression of the interferon inducible gene 6-16 was quantified by RT-PCR relatively to GAPDH or p-actin. Cells were treated with targeted or control IFN for 4 hr. Total RNA was 30 purified with RNeasy columns (Qiagen). Reverse transcriptions were primed with random primers and performed using Moloney murine leukemia virus reverse transcriptase (Invitrogen). Quantitative real-time PCR (qRT-PCR) was performed using a Light Cycler as described (Coccia et al. 2004). For Her2, the transfection culture medium was assayed on murine BTG9A and BTG9A cells 35 expressing human Her2 for expression of the OASL2 gene relatively to the expression of the P actin gene by quantitative RT-PCR using a Light Cycler (Roche) and the following primers: OASL2 forward: CAC-GAC-TGT-AGG-CCC-CAG-CGA; OASL2 reverse: AGC-AGC-TGT 10 WO 2013/107791 PCT/EP2013/050787 CTC-TCC-CCT-CCG; Pactin forward: AGA-GGG-AAA-TCG-TGC-GTG-AC; pactin reverse: CAA-TAG-TGA-TGA-CCT-GGC-CGT. In a similar way the ISG expression in Her2 targeted cells was measured using the same pactin primers and the following primer ISG15 primers: ISG15 forward: GAG-CTA-GAG-CCT-GCA-GCA-AT; ISG15 reverse: TTC-TGG-GCA-ATC 5 TGC-TTC-TT. Antiviral Assay The antiviral assay was performed using the EMC virus and scoring the virus replication dependent cytopathic effect as described in Stewart (1979) 10 Measurement of Her2 phosphorylation BTG9A cells expressing human Her2 were treated with 200 pM to 2 nM of 1R59B-IFNA2 Q124R for 10 to 30 min. Cells were lysed in RIPA, and analysed by western blot on an Odyssey Fc (Licor Bioscience) after 7% SDS-PAGE (40 pg/lane). Phopho-Her2 was detected 15 with the anti Her2 Y-P 1248 (Upstate #06-229) and the Goat anti rabbit secondary antibody IRDye 680 (Licor Bioscience #926-32221). Measurement of STA T1 phosphorylation STAT1 phosporylated on Y701 were detected by FACS using the STAT1-PY701 (PE) 20 (Beckton Dickinston #612564) and the manufacturer instruction for the PhosFlow technology. Targeted leptin constructs The sequence of the targeted leptin constructs is given in Figure 17. The L86 which is indicated is the amino acid that is mutated either to S or N. 25 Example 1: The nanobody-interferon fusion proteins Figure 1 shows a schematic representation of the nanobody-IFN fusion proteins constructed with either I FNa2 wild type or the Li 53A and R1 49A mutants. 30 Example 2: IFN activity of the nanobody-IFN fusion proteins is targeted toward murine leptin receptor expressing cells The three nanobody fusion proteins with IFNa2 WT, IFNa2 L153A or R149A were assayed on both HL116 and HL116-mLR-clone 10 cells which express the murine leptin receptor. The IFNa2 alone was also assayed in this assay system in order to check that the two cell clones 35 do not differ in their IFN responsiveness. Indeed both HL116 and HL116-mLR-clone 10 cells are equally sensitive to this IFN (Fig 2A and 2C, black symbols). The IFN activity of the three nanobody-IFN fusion proteins is however dramatically increased in cells expressing the leptin 11 WO 2013/107791 PCT/EP2013/050787 receptor compared to parental HL1 16 cells (compare Fig. 2A with Fig. 2C and Fig. 2B with Fig. 2D). We estimated that cells expressing the leptin receptor are 10, 100 and 1000-fold more sensitive than parental HL116 cells to the nanobody-IFN WT, L153A and R149A, respectively. 5 Since the affinities for IFNAR2 of the IFN mutant L153A and R149A are 0.1 and 0.01 relatively to the WT, there is a correlation between the loss of activity caused by mutations in the IFNAR2 binding site and the targeting efficiency by the nanobody. In order to determine whether the IFN activity of the nanobody-IFN fusion proteins is delivered only on cells expressing the nanobody target or also on neighboring cells, the nanobody 10 IFNa2R149A was assayed on a coculture of HL116-mLR-clonel0 and HT1080-6-16 renilla luciferase clone4. Both cell types will express luciferase activity in response to IFN stimulation but cells expressing the target of the nanobody will display a firefly luciferase activity whereas cells devoid of leptin receptor will display a renilla luciferase activity. The dilution of the nanobody-IFNa2R149A protein was chosen at 1/30, a dilution which induces a maximal 15 response in cells carrying the leptin receptor and a minimal response on cells devoid of the nanobody target (see Fig.2B and D, black curves). Fig. 3 shows clearly that the renilla luciferase activity is not induced upon stimulation of the co-culture with the nanobody IFNa2R149A, indicating that the targeted IFN activity is delivered only on cells expressing the antigen recognized by the nanobody. 20 The efficacy of the targeting is further illustrated by comparing the activity of wild type and two types of mutant IFN (L153A and R149A) when added to HL116 expressing or not expressing the murine leptin receptor that is used for the targeting. The results clearly show that the activity of the mutants is higher when the construct is targeted, and that the effect of targeting for the mutant is bigger than for wild type. (Figure 4A and B) 25 In order to prove that the targeting was nanobody specific, HL116 cells expressing the mLR were incubated for 6 hours with either the IFN-a2 (indicated as IFNA2) or the IFNA2-R149A fused to the nanobody 4-11 (Nanobody-IFNA2-R149A) at their respective EC50 concentration in the presence or absence (control) of a 100-fold molar excess of free 4-11 nanobody. Cells were lysed and the IFN-induced luciferase activities were measured. As shown in Figure 5, the 30 non-targeted IFN is not inhibited by the free nanobody, while the targeted construct is strongly inhibited, showing the specific effect of the targeting. The targeting to the leptin receptor is independent of the epitope on the receptor: using the anti-leptin receptor nanobody 4-10 (Zabeau et al., 2012) which recognizes a different domain on the receptor than the nanobody 4-11, a similar activation can be obtained using a targeted 35 mutant IFN (Figure 6). 12 WO 2013/107791 PCT/EP2013/050787 Example 3: The IFN activity of the nanobody-IFN fusion proteins on cells expressing the leptin receptor is mediated by both IFN receptor chains In order to determine whether the IFN activity of the nanobody-IFN fusion proteins needs the activation of the IFN receptor, HL116 cells expressing the murine leptin receptor were 5 pretreated with neutralizing antibodies against IFNAR1 or IFNAR2, and then stimulated with the nanobody-IFNA2-R149A fusion protein. The activity of the IFN-induced luciferase was measured. The figure 7 shows that both anti IFNAR1 and anti IFNAR2 neutralizing antibodies inhibit the IFN activity of the nanobody-IFNA2-R149A. 10 Example 4: Target-specific induction of antiviral activity by 4-11-IFNA2-R149A in cells expressing the murine leptin receptor Antiviral activity is an integrated part of the IFN response, implying the expression of several genes. Therefore, the antiviral activity on mLR expressing cell was controlled, after targeting the mutant R149A IFN using the anti-leptin receptor antibody 4-11. The results are 15 summarized in Figure 8. The activity was measured as the cytopathic effect on HL116 cells, with or without leptin receptor expression. The specific antiviral activity of the 4-11-IFNA2 R149A nanobody-IFN fusion protein is 716-fold higher when assayed on leptin receptor expressing cells compared to HL116 cells. 20 Example 5: Targeting of IFN activity on Her2 expressing cells In order to demonstrate that the concept is not restricted to cytokine receptor targeting, we generated similar fusion protein using the nanobody 2R5A against Her2 (Vaneycken et al., 2011) and the mutant IFN alpha2 R149A (2R5A-IFNA2-R149A). This molecule was assayed on BXPC3 (Pancreatic cancer, from ATCC) and BT474 (Breast cancer, from ATCC) cell lines 25 and compared with the activity of IFN-a2 (IFNA2) for the induction of the 6-16 IFN-inducible gene as determined relatively to GAPDH by quantitative RT-PCR. The BXPC3 and BT474 cells lines differ by their number of Her2 molecules expressed at their surface (10.9 x 10 3 and 478 x 103 , respectively as reported by Gaborit et al. (2011)). Figure 9 shows the EC 50 determination of IFNA2 activity (Panel A) and 2R5A-IFNA2-R149A 30 activity (Panel B) for the induction of the IFN-inducible gene 6-16 on BXPC3 and BT474 cell lines. Panel A shows that BXPC3 and BT474 cell lines exhibit the same sensitivity to IFN-a2. Panel B shows that the 2R5A-IFNA2-R149A nanobody-IFN fusion protein is much more potent on the BT474 cell line which expresses 40-fold more Her2 molecule than BXPC3. In conclusion, the concept which consists to target type I IFN activity on cells expressing a 35 specific cell surface antigen, as shown on human cells expressing the mouse leptin receptor, 13 WO 2013/107791 PCT/EP2013/050787 can be extended to untransfected human cells expressing another cell surface molecule from a different structural family, at a level naturally found in several types of breast carcinoma. Example 6: Targeting of mutant IFNA2-Q149R to mouse cells expressing human Her2 5 Mutant human IFNA2 Q149R was targeted to murine cells, expressing the human Her2, using the nanobody 1R59B in the 1R59B-IFNA2-Q124R. The IFNA2 Q124R has a high affinity for the murine IFNAR1 chain and a low activity for the murine IFNAR2 chain (Weber et al., 1987). The induction by IFN was measured as expression of the OASL2 messenger RNA, by RT QPCR. The results are shown in Figure 10. There is clearly a targeting-specific induction in the 10 Her2 expressing cells, whereas there is no significant expression detected in untransfected BTG9A cells. Similar results were obtained when the Her2 specific ScFv against Her2 was used to target the mutant IFN Q124R. In this case, the IFN induction was measured using the ISG15 messenger RNA expression. The results are shown in Figure 11. Again, a specific induction of ISG15 is 15 seen in the cells expressing Her2, while there is little effect of the mutant IFN on the cells that do not express Her2. Example 7: The construct 1R59B-IFNA2-Q124R does not activate the phosphorylation of Her2 20 To check whether targeting of Her2 is resulting in a Her2 activation, Her2 phosphorylation was controlled in targeted cells. The results are shown in Figure 12, clearly demonstrating that no phosphorylated Her2 could be detected in 1R59B-IFNA2-Q124R targeted cells, irrespective of the concentration or time of treatment. 25 Example 8: The anti PD-L2 Nb122-IFNA2-Q124R construct activity is targeted on mouse primary cells expressing PD-L2 Cells from a mouse peritoneal cavity were isolated and treated in vitro with Nb122-IFNA2 Q124R or natural mlFNa/P for 30 min. Cells were, fixed, permeabilized, labelled with antibodies against PD-L2 (APC) (BD #560086) and STAT1-PY701 (PE) (BD #612564) and 30 analysed by FACS. The PD-L2 positive cell population represents 20% of the total cell population present in the mouse peritoneal cavity. The results are shown in Figure 13. It is clear from this figure that in untreated cells, or in non targeted, murine IFN treated cells the peaks of STAT1-P for PD-L2 expressing and non 35 expressing cells coincide. Moreover, a clear induction in STAT1-P can be seen by murine IFN treatment. Treatment with the targeted mutant IFN however results in a specific shift in the STAT1-P only for the PD-L2 expressing cells. 14 WO 2013/107791 PCT/EP2013/050787 A same result is obtained if the IFN response of splenocytes is analysed in a similar experiment. The PD-L2 positive cell population represents 1% of the total cell population present in mouse spleen, indicating that also a minor cell population can be targeted in an efficient way. 5 Example 9: In vivo injection of 122-IFNA2-Q124R construct induces an IFN response only in PD-L2-expressing cells Mice were injected (IP or IV) with either PBS, Nb122-IFNA2-Q124R or a control Nb (against GFP) fused to IFNA2-Q124R. 30 min post injection, mice were killed, cells from the peritoneal 10 cavity were recovered by washing the peritoneal cavity with PBS, fixed (PhosLow Fix buffer I BD # 557870), permeabilized (PhosFlow Perm buffer Ill, BD #558050), labelled with Abs against PD-L2 (APC) (BD #560086) and STAT1-PY701 (PE) (BD #612564) and analysed by FACS. The results are shown in Figure 14. STAT1-P coincides in PD-L2 positive and negative cells treated with either PBS or control nanobody. However, a clear induction in STAT1-P (only 15 in the PDL2 positive cell population) can be seen when the mice are injected with the targeted mutant IFN. As a control, STAT1-P was checked in mice, iv injected with different doses of natural mouse IFN (10 000, 100 000 or 1 000 000 units), and no difference in STAT1-P could be detected between the PD-L2 positive and PD-L2 negative cells. 20 Figure 15 shows a similar dose response curve after iv injection of the Nb122-IFNA2-Q124R construct. A shift in STAT1-P in the PD-L2 expressing cells can be noticed even at the lowest dose of 64ng. Example 10: Targeting of mutant leptin to the leptin receptor, using a truncated 25 TNFa receptor Ba/F3 cells are growth-dependent on IL-3. After transfection with the mLR, Ba/F3 cells also proliferate with leptin. Leptin mutants with reduced affinity for their receptor are less potent in inducing and sustaining proliferation of Ba/F3-mLR cells. Leptin mutant L86S has a moderate and mutant L86N has a strong reduction in affinity and hence a moderate and strong reduced 30 capacity to induce proliferation respectively. Additional transfection of Ba/F3-mLR cells with the human TNFa Receptor 1 (hTNFR1) lacking its intracellular domain introduces a non-functional receptor which can function as a membrane bound extracellular marker. Chimeric proteins consisting of leptin and a nanobody against human TNFR1 (here nb96) will 35 bind to cells carrying the mLR and to cells carrying the hTNFR1. Chimeric proteins with leptin 15 WO 2013/107791 PCT/EP2013/050787 mutants L86S and L86N have reduced affinity for the LR but retain their affinity for the hTNFR1. Chimeric proteins were produced by transient transfection of Hek293T cells with expression plasmids. Supernatant was 0.45 pm filtered and serially diluted in 96-well plates for the assay. 5 A serial dilution of purified recombinant leptin was used as a reference. 3000 to 10000 cells were plated per well and proliferation was measured by staining with XTT four or five days later. OD was measured at 450 nm. The results are shown in Figure 16, for two experiments using a different leptin construct (see Figure 17). For both constructs, a hTNFR depending growth stimulation can be seen for the mutant constructs, whereas the hTNFR expression 10 does not affect the growth of the cells treated with wt (non-targeted) leptin. It is clear from these results that the targeting can compensate for the negative effect of the mutation. 16 WO 2013/107791 PCT/EP2013/050787 REFERENCES - Abraham, A.K., Ramanathan, M. Weinstock-Guttman, B. and Mager, D.E. (2009). Biochemical pharmacology 77, 1757-1762. - Benoit, P., Maguire, D., Plavec, I., Kocher, H., Tovey, M. and Meyer, F. (1993). J. Immunol., 150,707-716 - Blake, A.W., McCartney, L., Flint,J., Bolam, D.N., Boraston, A.B., Gilbert, H.J., and Knox, J.P. (2006). J. Biol. Chem., 281, 29321-29329. - Bourachot, B., Jouanneau, J., Giri, I., Katinka, M., Cereghini, S., and Yaniv, M. (1982). EMBO J., 1, 895-900. - Brecht, A., Gauglitz, G. And Polster, J. (1993). Biosens. Bioelectron.. 8, 387-392. - Coccia, E.M., Severa, M, Giacomini, E., Monneron, D., Remoli, M-E., Julkunen, I., Cella, M., Lande, R. and Uze, G. (2004). Eur. J. Immunol. 34. 796-805. - Conklin, D. (2004). J. Computiational Biol. 11, 1189-1200. - Dimitrov, D.S. (2009). mAbs 1, 26-28. - Eyckerman, S., Waelput, W., Verhee, A., Broekaert, D., Vendekerckhove, J., and Tavernier, J. (1999). Eur. Cytok. Netw. 10, 549-559. - Gaborit, N., Labouret, C., Vallaghe, J., Peyrusson, F., Bascoul-Mollevi, C. Crapez, E., Azria, D., Chardes, T., Poul, M.A., Mathis, G., Bazin, H., Pblegrin, A. (2011). J. Biol. Chem. 286, 11337-11345. - Ortiz-Sanchez, E., Helguera, G., Daniels, T.R. and Penichet, M.L. (2008). Expert Opin. Biol. Ther., 8, 609-632. - Pellegrini, S., John, J., Shearer, M., Kerr, I.M. and Stark, G.R. (1989). Mol. Cell. Biol. 9, 4605-4012. - Piehler, J., Roisman, L.C. and Schreiber, G. (2000). J. Biol. Chem. 275, 40425-40433. - Lasfargues, E.Y., Coutino, W.G. and Dion, A.S. (1979). In Vitro, 15, 723-729. - Nygren, P.A. (2008). FEBS J., 275, 2668-2676. - Roisman, L.C., Piehler, J., Trosset, J.Y., Scheraga, H.A. and Schreiber, G. (2001). Proc. NatI. Acad. Sci. YSA, 98, 13231-13236. - Rossi, E.A., Goldenberg, D.M., Cardillo, T.M., Stein, R. And Chang, C.H. (2009) Blood, 114, 3964-3871. - Southern, P.J. and Berg, P. (1982) J. Mol. Apple. Genet. 1, 327-341. Skerra, A. (2008). FEBS J., 275, 2677-2683. - Stewart II W.E. The interferon system. Springer-Verlag, Wien, New York. 1979. - Stumpp, M.T., Binz, H.K. and Amstutz, P (2008). Drug Discov. Today 13, 695-701. 17 WO 2013/107791 PCT/EP2013/050787 - Takabe, Y., Seiki, M., Fujisawa, J., Hoy, P., Yokata, K. and Arai, N. (1988). Mol. Cell. Biol. 8, 466-472. - Tan, M.H., Nowak, N.J., Loor, R., Ochi, H., Sandberg, A.A., Lopez, C., Pickren, J.W., Berjian, R., Douglass, H.O. jr. and Chu, T.M. (1986) Cancer Invest. 4, 15-23. - Tramontano, A., Bianchi, E., Venturini, S., Martin, F., Pessi, A and Sollazzo, M. (1994). J. Mol. Recognition 7, 9-24. - Uze et al. Cell 60, 225-234 (1990). - Uze, G., Di Marco, S., Mouchel-Veilh, E., Monneron, D., Bandu, M.T., Horisberger, M.A., Dorques, A., Lutfalla, G., and Mogensen, K.E. (1994). J. Mol. Biol. 243, 245-257. - Vaneycken, I., Devoogdt, N., Van Gassen, N., Vincke, C., Xavier, C., Wernery, U., Muyldermans, S., Lahoutte, T. And Caveliers, V. (2011). FASEB J. 25, 2433-2446. - Weber et al. (1987) EMBO J. 6, 591-598. - W6rn et al., FEBS Lett. 427, 357-361 (1998) - Zabeau, L., Verhee, A., Catteeuw, D., Faes, L., Seeuws, S., Decruy, T., Elewaut, D., Peelman, F. And Tavernier, J. (2012). Biochem. J. . 441, 425-434. 18
    权利要求:
    Claims (19)
    [1] 1. A targeting construct, comprising a modified a-helical bundle cytokine characterized by a reduced affinity for the a-helical bundle cytokine receptor, and a targeting moiety.
    [2] 2. The targeting construct, according to claim 1, wherein said modified a-helical bundle cytokine is a mutant a-helical bundle cytokine.
    [3] 3. The targeting construct according to claim 1 or 2, wherein said targeting moiety is targeting to a marker expressed on a a-helical bundle cytokine receptor expressing cell.
    [4] 4. The targeting construct, according to claim 2, wherein said mutant a-helical bundle cytokine is a mutant interferon.
    [5] 5. The targeting construct, according to claim 4, wherein said targeting moiety is targeting to a marker expressed on an interferon receptor expressing cell.
    [6] 6. The targeting construct according to claim 5, wherein said interferon receptor is IFNAR2.
    [7] 7. The targeting construct according to any of the preceding claims, wherein said targeting moiety is directed to a tissue specific marker
    [8] 8. The targeting construct according to any of the claims 1-6, wherein said targeting moiety is directed to a marker selected from the group consisting of Her2, DC-STAMP and CD20.
    [9] 9. The targeting construct according to any of the claims 1-6, wherein said targeting moiety is directed to a cell surface marker specific for viral infected cells
    [10] 10. The targeting construct according to any of the previous claims, wherein said targeting moiety is an antibody.
    [11] 11. The targeting construct according to claim 10, wherein said antibody is a nanobody.
    [12] 12. The targeting construct according to claim 4, wherein the mutant interferon is a mutant interferon alpha 2.
    [13] 13. The targeting construct according to claim 12, wherein said mutant interferon alpha 2 is mutated in one or more amino acids of the region 144-154.
    [14] 14. The targeting construct according to claim 12 , wherein said mutant interferon alpha 2 is selected from the group consisting of lFNa2 L153A, lFNa2 R149A and lFNa2 M148A.
    [15] 15. A targeting construct according to any of the claims 1-14 for use as a medicament.
    [16] 16. The targeting construct according to any of the claims 1-14 for use in treatment of cancer.
    [17] 17. The targeting construct according to any of the claims 1-14 for use in treatment of a viral disease.
    [18] 18. The targeting construct according to any of the claims 1-14 for use in treatment of diseases involving bone degradation.
    [19] 19. A pharmaceutical composition, comprising a targeting construct according to any of the claims 1-14, and a suitable excipient. 19
    类似技术:
    公开号 | 公开日 | 专利标题
    US10946070B2|2021-03-16|Targeted and mutated human-interferon fusion proteins
    US10072059B2|2018-09-11|Targeting of human interferon antagonists
    Vincent et al.2013|The BAFF/APRIL system: emerging functions beyond B cell biology and autoimmunity
    Wolk et al.2010|Biology of interleukin-22
    US20190016793A1|2019-01-17|Albumin binding domain fusion proteins
    WO2009039409A1|2009-03-26|Targeted interferon demonstrates potent apoptotic and anti-tumor activities
    JP2021522786A|2021-09-02|Interleukin 15 fusion protein, its composition and therapeutic method
    Spitzer et al.2010|A genetically encoded multifunctional TRAIL trimer facilitates cell-specific targeting and tumor cell killing
    CN112996805A|2021-06-18|Multi-chain chimeric polypeptides and uses thereof
    EP2941439B1|2018-01-31|Human serum albumin binding compounds and fusion proteins thereof
    KR20210145254A|2021-12-01|FC modified biologics for local delivery to compartments, particularly the CNS
    Class et al.2014|Patent application title: TARGETED MUTANT ALPHA-HELICAL BUNDLE CYTOKINES Inventors: Jan Tavernier | Jan Tavernier | Gilles Uze | Guillaume Cartron | Franciane Paul | Jacob Piehler |
    CN114195901A|2022-03-18|Bispecific recombinant proteins and uses thereof
    WO2021262985A1|2021-12-30|Cytokine conjugates
    同族专利:
    公开号 | 公开日
    US20180028616A1|2018-02-01|
    EP2804877B1|2018-08-22|
    KR20140138129A|2014-12-03|
    US9492562B2|2016-11-15|
    US10946070B2|2021-03-16|
    CA2861927A1|2013-07-25|
    CN104245734B|2017-09-05|
    US20170072020A1|2017-03-16|
    US20180333465A1|2018-11-22|
    US10034919B2|2018-07-31|
    US9878014B2|2018-01-30|
    US20210162010A1|2021-06-03|
    KR102050119B1|2019-11-28|
    JP6416628B2|2018-10-31|
    AU2013211059A8|2017-12-21|
    WO2013107791A1|2013-07-25|
    EP2804877A1|2014-11-26|
    JP2015511219A|2015-04-16|
    CN104245734A|2014-12-24|
    CA2861927C|2021-01-26|
    BR112014017876A2|2017-06-27|
    ES2694180T3|2018-12-18|
    AU2013211059B2|2017-11-02|
    HK1205134A1|2015-12-11|
    AU2013211059B8|2017-12-21|
    US20140348789A1|2014-11-27|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    DE69007975T2|1989-08-22|1994-07-21|Immunex Corp|FUSION PROTEIN CONSISTING OF GM-CSF AND IL-3.|
    US6277969B1|1991-03-18|2001-08-21|New York University|Anti-TNF antibodies and peptides of human tumor necrosis factor|
    US5914254A|1993-08-02|1999-06-22|Celtrix Pharmaceuticals, Inc.|Expression of fusion polypeptides transported out of the cytoplasm without leader sequences|
    IL108584A|1994-02-07|2008-03-20|Yeda Res & Dev|Cloning of interferon -alpha/beta binding protein|
    US20020193569A1|2001-06-04|2002-12-19|Idec Pharmaceuticals Corporation|Bispecific fusion protein and method of use for enhancing effector cell killing of target cells|
    PT1537878E|2002-07-03|2010-11-18|Ono Pharmaceutical Co|Immunopotentiating compositions|
    US7575878B2|2004-11-18|2009-08-18|Vib Vzw|Methods of inhibiting leptin-induced signaling with fibronectin III domain antibodies|
    WO2006115800A2|2005-04-15|2006-11-02|The Regents Of The University Of California|Enhanced wound healing utilizing an anti-her2 antibody coupled to a tnf alpha|
    EP2052081A1|2006-08-02|2009-04-29|McGill University|Fusion proteins and methods for modulation of immune response|
    CA2672595A1|2006-12-15|2008-06-19|Guy Hermans|Amino acid sequences that modulate the interaction between cells of the immune system|
    US20110274658A1|2007-04-05|2011-11-10|President And Fellows Of Harvard College|Chimeric activators: quantitatively designed protein therapeutics and uses thereof|
    EP2173377B1|2007-06-26|2017-11-29|University of Miami|Antibody-endostatin fusion protein and its variants|
    WO2009039409A1|2007-09-21|2009-03-26|The Regents Of The University Of Californina|Targeted interferon demonstrates potent apoptotic and anti-tumor activities|
    US8334101B2|2008-09-26|2012-12-18|University Of Massachusetts|Intracellular DNA receptor|
    JP2012510806A|2008-12-08|2012-05-17|コンプリクスエンヴェー|Single-chain antiparallel coiled-coil protein|
    EP2467165B1|2009-08-17|2015-01-07|Roche Glycart AG|Targeted immunoconjugates|
    EP2475397A1|2009-09-10|2012-07-18|Cytos Biotechnology AG|Use of interleukin-1 beta mutein conjugates in the treatment of diabetes|
    US9534056B2|2011-06-06|2017-01-03|Immungene Inc|Engineered TAA antibody-TNFSF member ligand fusion molecules|
    JP6184965B2|2011-10-28|2017-08-23|テバ・ファーマシューティカルズ・オーストラリア・ピーティワイ・リミテッド|Polypeptide constructs and uses thereof|
    EP2804877B1|2012-01-20|2018-08-22|VIB vzw|Targeted mutant alpha-helical bundle cytokines|
    HUE049945T2|2012-03-03|2020-11-30|Immungene Inc|Engineered antibody-interferon mutant fusion molecules|
    JP6475712B2|2013-07-19|2019-02-27|ヴィブ ブイゼットダブリュー|Targeting cytokine antagonists|JP6184965B2|2011-10-28|2017-08-23|テバ・ファーマシューティカルズ・オーストラリア・ピーティワイ・リミテッド|Polypeptide constructs and uses thereof|
    EP2804877B1|2012-01-20|2018-08-22|VIB vzw|Targeted mutant alpha-helical bundle cytokines|
    HUE049945T2|2012-03-03|2020-11-30|Immungene Inc|Engineered antibody-interferon mutant fusion molecules|
    US11117975B2|2013-04-29|2021-09-14|Teva Pharmaceuticals Australia Pty Ltd|Anti-CD38 antibodies and fusions to attenuated interferon alpha-2B|
    NZ713641A|2013-04-29|2019-08-30|Teva Pharmaceuticals Australia Pty Ltd|Anti-cd38 antibodies and fusions to attenuated interferon alpha-2b|
    ES2757501T3|2013-07-18|2020-04-29|Vib Vzw|Fusocins involving cytokines with strongly reduced receptor binding affinities|
    JP6595988B2|2013-07-19|2019-10-23|ヴィブブイゼットダブリュー|Targeted modified TNF family members|
    SG11201600167SA|2013-07-19|2016-02-26|Vib Vzw|Targeted modified il-1 family members|
    JP6475712B2|2013-07-19|2019-02-27|ヴィブ ブイゼットダブリュー|Targeting cytokine antagonists|
    MX2016013863A|2014-05-01|2017-07-24|Teva Pharmaceuticals Australia Pty Ltd|Combination of lenalidomide or pomalidomide and cd38 antibody-attenuated interferon-alpha constructs, and the use thereof.|
    EP3212216A4|2014-10-29|2018-04-18|Teva Pharmaceuticals Australia Pty Ltd|Interferonalpha2b variants|
    GB201504859D0|2015-03-23|2015-05-06|Vib Vzw|Viral particle based small molecule-protein interaction trap|
    CA3003969A1|2015-11-06|2017-05-11|Orionis Biosciences Nv|Bi-functional chimeric proteins and uses thereof|
    EP3411407A1|2016-02-05|2018-12-12|Orionis Biosciences NV|Bispecific signaling agents and uses thereof|
    US11248057B2|2016-03-07|2022-02-15|Vib Vzw|CD20 binding single domain antibodies|
    US11236141B2|2016-05-13|2022-02-01|Orionis Biosciences BV|Targeted mutant interferon-beta and uses thereof|
    EP3455245A2|2016-05-13|2019-03-20|Orionis Biosciences NV|Therapeutic targeting of non-cellular structures|
    CA3040802A1|2016-10-24|2018-05-03|Orionis Biosciences Nv|Targeted mutant interferon-gamma and uses thereof|
    CA3052523A1|2017-02-06|2018-08-09|Orionis Biosciences Nv|Targeted chimeric proteins and uses thereof|
    EP3576765A4|2017-02-06|2020-12-02|Orionis Biosciences, Inc.|Targeted engineered interferon and uses thereof|
    EP3580230A1|2017-02-07|2019-12-18|VIB vzw|Immune-cell targeted bispecific chimeric proteins and uses thereof|
    BR112020016859A2|2018-02-28|2020-12-29|Pfizer Inc.|IL-15 VARIANTS AND USES OF THE SAME|
    EP3946409A1|2019-03-28|2022-02-09|Orionis Biosciences, Inc.|Fms-like tyrosine kinase 3 ligand -based chimeric proteins|
    CA3139291A1|2019-07-18|2021-01-21|Jacky Vonderscher|Method for decreasing adverse-effects of interferon|
    法律状态:
    2017-12-21| TH| Corrigenda|Free format text: IN VOL 31 , NO 43 , PAGE(S) 6317 UNDER THE HEADING APPLICATIONS ACCEPTED - NAME INDEX UNDER THE NAME VIB VZW; CENTRE HOSPITALIER REGIONAL UNIVERSITAIRE DE MONTPELLIER; UNIVERSITEIT GENT; UNIVERSITAT OSNABRUCK; CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE, APPLICATION NO. 2013211059 UNDER INID (71) ADD CO-APPLICANT UNIVERSITE MONTPELLIER 2 Free format text: IN VOL 28 , NO 33 , PAGE(S) 4452 UNDER THE HEADING PCT APPLICATIONS THAT HAVE ENTERED THE NATIONAL PHASE - NAME INDEX UNDER THE NAME VIB VZW; CENTRE HOSPITALIER REGIONAL UNIVERSITAIRE DE MONTPELLIER; UNIVERSITEIT GENT; UNIVERSITAT OSNABRUCK; CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE, APPLICATION NO. 2013211059 UNDER INID (71) ADD CO-APPLICANT UNIVERSITE MONTPELLIER 2 |
    2018-03-01| FGA| Letters patent sealed or granted (standard patent)|
    2018-06-14| HB| Alteration of name in register|Owner name: UNIVERSITEIT GENT Free format text: FORMER NAME(S): VIB VZW; UNIVERSITEIT GENT; CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE; CENTRE HOSPITALIER REGIONAL UNIVERSITAIRE DE MONTPELLIER; UNIVERSITE MONTPELLIER 2 Owner name: VIB VZW Free format text: FORMER NAME(S): VIB VZW; UNIVERSITEIT GENT; CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE; CENTRE HOSPITALIER REGIONAL UNIVERSITAIRE DE MONTPELLIER; UNIVERSITE MONTPELLIER 2 Owner name: CENTRE HOSPITALIER REGIONAL UNIVERSITAIRE DE MONTP Free format text: FORMER NAME(S): VIB VZW; UNIVERSITEIT GENT; CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE; CENTRE HOSPITALIER REGIONAL UNIVERSITAIRE DE MONTPELLIER; UNIVERSITE MONTPELLIER 2 Owner name: UNIVERSITE DE MONTPELLIER Free format text: FORMER NAME(S): VIB VZW; UNIVERSITEIT GENT; CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE; CENTRE HOSPITALIER REGIONAL UNIVERSITAIRE DE MONTPELLIER; UNIVERSITE MONTPELLIER 2 Owner name: CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE Free format text: FORMER NAME(S): VIB VZW; UNIVERSITEIT GENT; CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE; CENTRE HOSPITALIER REGIONAL UNIVERSITAIRE DE MONTPELLIER; UNIVERSITE MONTPELLIER 2 Owner name: UNIVERSITAT OSNABRUCK Free format text: FORMER NAME(S): VIB VZW; UNIVERSITEIT GENT; CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE; CENTRE HOSPITALIER REGIONAL UNIVERSITAIRE DE MONTPELLIER; UNIVERSITE MONTPELLIER 2 |
    优先权:
    申请号 | 申请日 | 专利标题
    EP12305075.9||2012-01-20||
    EP12305075||2012-01-20||
    PCT/EP2013/050787|WO2013107791A1|2012-01-20|2013-01-17|Targeted mutant alpha-helical bundle cytokines|
    [返回顶部]