(19) TZZ¥_¥Z_T (11) EP 3 135 690 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 01.03.2017 Bulletin 2017/09 C07K 16/28 (2006.01) A61K 47/50 (2017.01) (21) Application number: 16188712.0 (22) Date of filing: 26.06.2013 (84) Designated Contracting States: • BALIGA, Ramesh AL AT BE BG CH CY CZ DE DK EE ES FI FR GB Foster City, CA 94404 (US) GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO • BAJAD, Sunil PL PT RO RS SE SI SK SM TR Fremont, CA 94538-3219 (US) • POLLITT, Sonia (30) Priority: 26.06.2012 US 201261664686 P San Mateo, CA 94402 (US) 12.11.2012 US 201261725439 P • MURRAY, Chris Soquel, CA 95073 (US) (62) Document number(s) of the earlier application(s) in •STEINER,Alex accordance with Art. 76 EPC: San Francisco, CA 94107 (US) 13737046.6 / 2 863 955 • GILL, Avinash Emeryville, CA 94608 (US) (71) Applicant: Sutro Biopharma, Inc. South San Francisco, California 94080 (US) (74) Representative: Hutter, Anton et al Venner Shipley LLP (72) Inventors: 200 Aldersgate • THANOS, Christopher London EC1A 4HD (GB) Tiburon, CA 94920 (US) • MCEVOY, Leslie Remarks: Mountain View, CA 94043 (US) This applicationwas filed on 14.9.2016 as a divisional • YIN, Gang application to the application mentioned under INID South San Francisco, CA 94080 (US) code 62. • PENTA, Kalyani Palo Alto, CA 94303 (US) (54) MODIFIED FC PROTEINS COMPRISING SITE-SPECIFIC NON-NATURAL AMINO ACID RESIDUES, CONJUGATES OF THE SAME, METHODS OF THEIR PREPARATION AND METHODS OF THEIR USE (57) Provided herein are modified Fc proteins com- duction and methods of their use. The modified Fc pro- prising non-natural amino acid residues at site-specific teins and conjugates are useful for methods of treatment positions, conjugates of the modified Fc proteins for ther- and prevention, methods of detection and methods of apy or diagnosis, compositions comprising the modified diagnosis. Fc proteins and conjugates thereof, methods of their pro- EP 3 135 690 A1 Printed by Jouve, 75001 PARIS (FR) EP 3 135 690 A1 Description FIELD 5 [0001] Provided herein are modified Fc proteins comprising non-natural amino acid residues at site-specific positions, compositions comprising the modified Fc proteins and conjugates thereof, methods of their production and methods of their use. BACKGROUND 10 [0002] Antibodies or immunoglobulins comprise two functionally independent parts, a variable domain known as "Fab," which binds antigen, and a constant domain known as "Fc," which links to such effector functions as complement activation and attack by phagocytic cells. An immunoglobulin Fc domain has a long serum half-life, whereas a Fab domain is short-lived. See, for example, Capon et al., 1989, Nature 337: 525-531, which is hereby incorporated by 15 reference herein in its entirety. [0003] Immunoglobulin Fc domains and fragments thereof have found widespread use as carrier or conjugate proteins for a variety of therapeutic and diagnostic molecules. When constructed together with, for example, a therapeutic protein or peptide, an immunoglobulin Fc domain can provide longer half-life, or can incorporate such functions as Fc receptor binding, protein A binding, complement fixation and perhaps even placental transfer. As a carrier or conjugate, an Fc 20 domain or fragment can be superior to other conjugates, e.g., albumin and PEG: an Fc domain or fragment provides more stability, longer half-life, and reduced immunogenicity to the molecules attached thereto. For example, attachment of a drug to an Fc domain can increase the serum half-life of the drug and reduce the risk of inducing immune responses. [0004] Various methods have been used to attach therapeutic and/or diagnostic molecules to an Fc domain or fragment. For example, conventional approaches for chemical conjugation to the immunoglobulin Fc domain include random 25 coupling to naturally occurring primary amines such as lysine and the amino-terminus or carboxylic acids such as glutamic acid, aspartic acid and the carboxy terminus. Alternatively, semi-selective site-specific coupling may be achieved through N-terminal conjugation under appropriate conditions, or derivatized carbohydrates as found on Fc proteins isolated from eukaryotic sources, or by partial reduction and coupling of native cysteine residues. ( E.g., Kim et al., A pharmaceutical composition comprising an immunoglobulin Fc region as a carrier, WO 2005/047337). While each of these approaches 30 has been applied successfully, they typically suffer from varying degrees of conjugate heterogeneity, relatively low yields and sometimes, significant losses in functional activity. [0005] In addition, modifications have been made to Fc domains and/or fragments to optimize their function as carrier or conjugate proteins. For example, numerous fusions of proteins and peptides have been engineered at either the amino- or carboxy-terminus of an Fc domain and/or fragment thereof. Also, a variety of enzymes and synthetic reporter 35 molecules have been chemically conjugated to the side chains of non-terminal amino acids as well as the derivatized carbohydrate moieties of the Fc domain. Further, polymers such as polyethylene glycol (PEG) have been conjugated to the Fc domain for the purpose of improved half-life in vivo and reduced immunogenicity. [0006] However, there are problems associated with existing Fc-based conjugates, including adverse or less optimal effects on the specificity, efficiency, yield, solubility, and activity of the therapeutic or diagnostic molecules. There is a 40 need for better Fc-based carrier proteins to further improve the properties of the therapeutic or diagnostic molecules conjugated thereto; in particular, to further increase their half-life in serum. SUMMARY 45 [0007] Provided herein are modified Fc proteins modified at one or more site-specific positions with one or more non- natural amino acid residues. These site-specific positions are optimal for substitution of a natural amino acid residue with a non-natural amino acid residue. In certain embodiments, substitution at these site-specific positions yields Fc proteins that areuniform in substitution, i.e. thatare substantially modifiedin the selected position. In certain embodiments, a modified Fc protein substituted at one or more of these site-specific positions has advantageous production yield, 50 advantageous solubility, advantageous binding and/or advantageous activity. The properties of these modified Fc pro- teins are described in detail in the sections below. [0008] In one aspect, provided herein are Fc proteins comprising a polypeptide chain having at least one non-natural amino acid residue at a position in the polypeptide chain that is optimally substitutable. The modified Fc protein can be a monomer or dimer. Said dimers can be homodimers or heterodimers. The position in the polypeptide chain that is 55 optimally substitutable is any position in the polypeptide chain that can provide a substitution with optimal yield, uniformity, solubility, binding and/or activity. The sections below describe in detail the optimally substitutable positions of such polypeptide chains. Also described below are useful Fc proteins containing useful non-natural amino acids. [0009] In a further aspect, provide herein are conjugates of the Fc proteins with one or more payload molecules. The 2 EP 3 135 690 A1 payload molecule can be any molecule deemed useful for conjugating to a modified Fc protein. In certain embodiments, the payload molecule can be a therapeutic molecule or a diagnostic molecule. The payload molecule can be linked to the Fc protein directly via a covalent bond or indirectly via a linker. Advantageously, in certain embodiments, the non- natural amino acids of the modified Fc proteins provide sites useful for linking to the linker or to the payload molecule. 5 Accordingly, provided herein are conjugates comprising a modified Fc protein linked to a payload moiety through a non- natural amino acid at an optimally substitutable site of the Fc protein. [0010] In another aspect, provided herein are compositions comprising said modified Fc proteins or conjugates thereof. Advantageously, such compositions can have high uniformity because of the uniformity of the substitution of the modified Fc proteins provided herein. In certain embodiments, the compositions comprise a substantial amount of the modified 10 Fc protein or conjugate thereof when measured by total weight of protein or when measured by total weight of Fc protein or conjugate. In certain embodiments, the compositions comprise at least 80 % of the modified Fc protein or conjugate thereof, at least 85 % of the Fc protein or conjugate, at least 90 % of the modified Fc protein or conjugate thereof, or at least 95 % of the Fc protein or conjugate by weight. [0011] In another aspect, provided herein are methods of making the modified Fc proteins. The modified Fc proteins 15 can be made by any technique apparent to those of skill in the art for incorporating non-natural amino acids into site- specific positions of Fc protein chains. In certain embodiments, the modified Fc proteins are made by solid phase synthesis, semi-synthesis, in vivo translation, in vitro translation or cell-free translation. [0012] In another aspect, provided herein are methods of making the conjugates of the modified Fc protein (also referred to as the Fc protein conjugates). The Fc protein conjugates can be made by any technique apparent to those 20 of skill in the art for incorporating non-natural amino acids into site-specific positions of Fc protein chains and for linking the Fc proteins to payload molecules. In certain embodiments, the modified Fc proteins are made by solid phase synthesis, semi-synthesis, in vivo translation, in vitro translation or cell-free translation. [0013] In another aspect, provided herein are methods of using the Fc protein conjugates for therapy. Modified Fc proteins directed to a therapeutic target can incorporate one or more site-specific non-natural amino acids according to 25 the description herein.