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WO 2017/211278 Al 14 December 2017 (14.12.2017) W !P O PCT

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WO 2017/211278 Al 14 December 2017 (14.12.2017) W !P O PCT (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2017/211278 Al 14 December 2017 (14.12.2017) W !P O PCT (51) International Patent Classification: Pei-Yi; 45 Sec 1, Ling-Yun Road, Wugu, New Taipei City C12N 15/87 (2006.0 1) A61P 35/00 (2006.0 1) 248, Taiwan (CN). C07K 16/28 (2006.01) (74) Agent: LEE AND LI - LEAVEN IPR AGENCY LTD.; (21) International Application Number: Unit 2202, Tower A, Beijing Marriott Center, No.7, Jian PCT/CN2017/087350 Guo Men South Avenue, Beijing 100005 (CN). (22) International Filing Date: (81) Designated States (unless otherwise indicated, for every 06 June 2017 (06.06.2017) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, (25) Filing Language: English CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, (26) Publication Language: English DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KH, KN, KP, KR, (30) Priority Data: KW,KZ, LA, LC, LK, LR, LS, LU, LY,MA, MD, ME, MG, 62/346,386 06 June 2016 (06.06.2016) US MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, (71) Applicant: ASCLEPIUMM TAIWAN CO., LTD PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, [CN/CN]; 45 Sec 1, Ling-Yun Road, Wugu, New Taipei SD, SE, SG, SK, SL, SM, ST, SV, SY,TH, TJ, TM, TN, TR, City 248, Taiwan (CN). TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (72) Inventors: CHEN, Min-Che; 45 Sec 1, Ling-Yun Road, (84) Designated States (unless otherwise indicated, for every Wugu, New Taipei City 248, Taiwan (CN). LIU, Ya- kind of regional protection available): ARIPO (BW, GH, Chuan; 45 Sec 1, Ling-Yun Road, Wugu, New Taipei City GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, 248, Taiwan (CN). CHANG, Po-Hao; 45 Sec 1, Ling-Yun UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, Road, Wugu, New Taipei City 248, Taiwan (CN). TSAI, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, Ya-Ping; 45 Sec 1, Ling-Yun Road, Wugu, New Taipei City EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, 248, Taiwan (CN). CHEN, Chun-Wei; 45 Sec 1, Ling-Yun MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, Road, Wugu, New Taipei City 248, Taiwan (CN). LEE, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). (54) Title: ANTIBODY FUSION PROTEINS FOR DRUG DELIVERY VIC 7 H1299 A B 45 E 30 3 C ε> 15 o o 5 µ / π \ 10 µ / η Ι 00 Fig. 3 (57) Abstract: Disclosed herein relates to antibody fusion proteins. Particularly, it relates to antibody fusion proteins for intra-cellular and intra-nucleus drugs delivery. The fusion protein can be used as a peptide penetration system that specifically binds to various targets for the delivery of effector peptides across a biological barrier. o [Continued on nextpage] WO 2017/2 11278 Al |III III || III IIII 11||| 11|||| ||||| 111III IIII |||| 11||| Published: — with international search report (Art. 21(3)) — with sequence listing part of description (Rule 5.2(a)) ANTIBODY FUSION PROTEINS FOR DRUG DELIVERY Field of the Invention [0001 ] The invention relates to antibody fusion proteins. Particularly, the invention relates to antibody fusion proteins for intra-cellular and intra-nucleus drugs delivery. The antibody fusion proteins of the invention have dual functional targets to bind the extracellular surface markers and achieve intracellular targets regulation. Background of the Invention [0002 ] Among targeted therapeutic options, antibody-drug conjugates (ADCs) have emerged as the most promising. ADCs are monoclonal antibodies designed to selectively deliver potent cytotoxic drugs into antigen expressing cells. Several components of an ADC including the selection of the antibody, the linker, the cytotoxic drug payload and the site of attachment used to attach the drug to the antibody are critical to the activity of the ADC. The cytotoxic drugs (or payloads) used to make ADCs are typically conjugated to the antibody through cysteine or lysine residues. This results in ADCs that have a heterogeneous number of drugs per antibody. The number of drugs per antibody commonly referred to as the drug to antibody ratio (DAR), can vary between 0 and 8 drugs for an IgGl antibody. Antibodies with 0 drugs are ineffective and compete with the ADC for binding to the antigen expressing cells. Antibodies with 8 drugs per antibody have reduced in vivo stability, which may contribute to non-target related toxicities. Even a purified ADC with a uniform stoichiometry would still carry drugs conjugated to multiple sites and therefore be a complex mixture of unique entities. Early heterogeneous ADCs suffered from stability, pharmacokinetic, and efficacy issues that hindered clinical development. Though recent advances in chemical site-selective antibody conjugation, including use of engineered cysteine residues, unnatural amino acids, and enzymatic conjugation through glycotransferases and transglutaminases have led to the creation of more-homogenous ADCs, several differences between the methods exist, including the requirement for genetic modification of antibodies, use of enzymes for conjugation, and conjugation site number/location. [0003] WO 2009/025846 provides activatable binding polypeptides (ABPs), which contain a target binding moiety (TBM), a masking moiety (MM), and a cleavable moiety (CM) and suggests that the ABPs exhibit an activatable conformation such that at least one of the TBMs is less accessible to target when uncleaved than after cleavage of the CM in the presence of a cleaving agent capable of cleaving the CM. US 2016/0185875 discloses a hinge antibody capable of being selectively activated in a target cell or tissue to treat a condition therein. The hinge antibody includes a functional antibody, two inhibitory domains and four cleavable linkers. However, the payloads of the two patent publications cannot achieve a satisfied effect, so there is a need to develop a drug delivery system with higher payload. Summary of the Invention [000 ] The invention uses antibody fusion proteins comprising an antibody and one or more cell penetrating effector peptide (CPEP), which simultaneously targets to a target of interest and deliver peptide payloads for intra-cellular and intra-nucleus treatment. The antibody fusion proteins of the invention are not only simplify the homogenous production than using chemical modification strategies, but also does not contain any chemical drugs as payloads or modified chemical linkers to conjugate. [0005 ] The invention provides a fusion protein comprising: (a) an antibody or an antigen binding fragment thereof (Ab), which is capable of targeting an extracellular surface marker; and (b) one or more cell penetrating effector peptide (CPEP) fused to the Ab of (a) or fused inside of the Ab of (a), wherein one CPEP comprises, an optional polyanionic domain (PAD), one or two cleavable linkers (CLs), a polycation domain (PCD) and an effector peptide (EP) in an arrangement of (EP-PCD-CL), (PCD-EP-CL), (EP-PCD-CL-PAD), (PAD-CL-PCD-EP-CL), (CL-PCD-EP), (CL-EP-PCD), (PAD-CL-PCD-EP) or (CL-EP-PCD-CL-PAD), from N-terminus to C-terminus, when the CPEP is fused to the terminus of the Ab of (a), or one CPEP comprises, an optional polyanionic domain (PAD), two cleavable linkers (CLs), a polycation domain (PCD) and an effector peptide (EP) in an arrangement of (CL-PCD-EP-CL), (CL-EP-PCD-CL), (PAD- CL-PCD-EP-CL), (PAD-CL-EP-PCD-CL), (CL-EP-PCD-CL-PAD) or (CL-PCD-EP-CL-PAD), from N-terminus to C-terminus, when the CPEP is fused inside of the Ab with its two terminuses. [0006 ] In one embodiment, one CPEP comprises an optional polyanionic domain (PAD), one or two cleavable linkers (CLs), a polycation domain (PCD) and an effector peptide binding to an intracellular target (EP) in an arrangement of (EP-PCD-CL), (PCD-EP-CL), (EP-PCD-CL-PAD), (PAD-CL-PCD-EP-CL), (CL-PCD-EP), (CL-EP-PCD), (PAD-CL-PCD-EP) or (CL-EP-PCD- CL-PAD), from N-terminus to C-terminus, when the CPEP is fused to the terminus of the antibody or an antigen binding fragment thereof. [0007 ] In another embodiment, one CPEP comprises an optional polyanionic domain (PAD), two cleavable linkers (CLs), a polycation domain (PCD) and an effector peptide binding to an intracellular target (EP) in an arrangement of (CL-PCD-EP-CL), (CL-EP-PCD-CL), (PAD-CL- PCD-EP-CL), (PAD-CL-EP-PCD-CL), (CL-EP-PCD-CL-PAD) or (CL-PCD-EP-CL-PAD), from N-terminus to C-terminus, when the CPEP is fused inside of the antibody or an antigen binding fragment thereof with its two terminuses. That is, the CPEP integrates into the antibody or an antigen binding fragment thereof (Ab) in any one of the above-mentioned arrangements. In one embodiment, the CPEP is fused inside of the heavy chain of an Ab. [0008 ] In a further embodiment, the CPEP is fused to the terminus or inside of the heavy chain of the Ab. [0009 ] In exemplary embodiments, the preferred antibodies are described in paragraph [0053] herein. In one embodiment, the antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 4 and a light chain sequence having the amino acid sequence of SEQ ID NO: 11.
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