FIG. 16 WO 2015/006555 A2 Llll II II 11III II
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(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 W O 2015/006555 A2 15 January 2015 (15.01.2015) P O P C T (51) International Patent Classification: nia 94080 (US). GILL, Avinash; 310 Utah Avenue, Suite C07K 16/00 (2006.01) 150, South San Francisco, California 94080 (US). (21) International Application Number: (74) Agents: PATHAK, Rahul et al; Squire Patton Boggs PCT/US2014/046141 (US) LLP, 275 Battery Street, Suite 2600, San Francisco, California 941 11 (US). (22) International Filing Date: 10 July 2014 (10.07.2014) (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (25) Filing Language: English AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, (26) Publication Language: English BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (30) Priority Data: HN, HR, HU, ID, IL, EST, IR, IS, JP, KE, KG, KN, KP, KR, 61/844,771 10 July 2013 (10.07.2013) US KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, 61/890,121 11 October 2013 ( 11. 10.2013) US MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, (71) Applicant: SUTRO BIOPHARMA, INC. [US/US]; 310 OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, Utah Avenue, Suite 150, South San Francisco, California SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, 94080 (US). TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (72) Inventors: THANOS, Christopher D.; 310 Utah Avenue, Suite 150, South San Francisco, California 94080 (US). (84) Designated States (unless otherwise indicated, for every BALIGA, Ramesh; 310 Utah Avenue, Suite 150, South kind of regional protection available): ARIPO (BW, GH, San Francisco, California 94080 (US). PENTA, Kalyani; GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, 310 Utah Avenue, Suite 150, South San Francisco, Califor UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, [Continued on nextpage] (54) Title: ANTIBODIES COMPRISING MULTIPLE SITE-SPECIFIC NON-NATURAL AMINO ACID RESIDUES, METHODS OF THEIR PREPARATION AND METHODS OF THEIR USE (57) Abstract: Provided herein are antibodies comprising multiple non-natural amino acid nnAA1 residues at site-specific positions, compositions nnAA1 was comprising the antibodies, methods of their pro - incorporated to LC duction and methods of their use. The antibodies are useful for methods of treatment and preven tion, methods of detection and methods of dia gnosis. nnAA2 was incorporated to HC in the presence of LC IgG was conjugated to two drugs by two orthogonal < chemistries Drug2 o FIG. 16 WO 2015/006555 A2 llll II II 11III II EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, — as to the applicant's entitlement to claim the priority of LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, the earlier application (Rule 4.1 ?'(in)) SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG). Published: Declarations under Rule 4.17: without international search report and to be republished upon receipt of that report (Rule 48.2(g)) — as to applicant's entitlement to apply for and be granted apatent (Rule 4.1 7(H)) ANTIBODIES COMPRISING MULTIPLE SITE-SPECIFIC NON-NATURAL AMINO ACID RESIDUES, METHODS OF THEIR PREPARATION AND METHODS OF THEIR USE CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No. 61/844,771, filed July 10, 2013, and U.S. Provisional Application No. 61/890,121, filed October 11, 2013, each of which is incorporated by reference in its entirety. FIELD [0002] Provided herein are antibodies comprising multiple non-natural amino acid residues at site-specific positions, compositions comprising the antibodies, methods of their production and methods of their use. BACKGROUND [0003] Antibodies are biological molecules with remarkable affinities for their target antigens. Nature provides antibodies as part of a defense system in certain vertebrates for the elimination or destruction of foreign proteins, cells and organisms. If a certain vertebrate is presented with a foreign protein on, for example, an infected cell or an infectious bacterium, an antibody can bind its target foreign protein to direct the foreign entity to its elimination or destruction. [0004] The selective affinity of antibodies can be used by man to target nearly any antigen desired. The antigen can be a protein on an infected cell or infectious microorganism. It can also be, for example, a protein on a cancer cell, a protein on a cell of a target tissue, a protein in the bloodstream, a protein on an inflamed or inflammatory cell or any other protein whose selective binding is useful. Antibodies have thus found use in therapy for conditions such as cancer, inflammatory diseases, autoimmune diseases and transplant rejection. The antibody can signal the immune system to destroy or eliminate a diseased cell, or an engineered antibody can carry a molecular payload to destroy the target. In certain applications therapeutic antibodies are linked to molecular shields to increase their lifetime within an organism. Antibodies have also found use as diagnostics. These antibodies can carry a label to indicate the presence of a target antigen on a cell or in a tissue. These labels are typically linked to the antibodies by covalent bonds. [0005] To date, techniques for linking antibodies molecular entities such as molecular payloads, molecular shields and labels have been limited by their heterogeneity in degree and location of linking to the antibodies, by their low yields and by losses in activity. Typical conjugation sites include random locations on antibody chains, e.g. random amines on amino acid side chains, and the N-terminus of certain antibody chains. In such techniques, some antibodies might be linked to the conjugate at one location while some antibodies are linked to the same conjugate at another location, and some antibodies might not be linked at all. [0006] There is a need for antibodies modified at site-specific positions optimized for uniformity, yield and/or activity to further the promising use of antibodies in, for example, therapy and diagnostics. SUMMARY [0007] Provided herein are antibodies modified at two or more site-specific positions with 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 antibodies that are uniform in substitution, i.e. that are substantially modified in the selected position. In certain embodiments, an antibody substituted at these site-specific positions has advantageous production yield, advantageous solubility, advantageous binding and/or advantageous activity. The properties of these antibodies are described in detail in the sections below. [0008] In one aspect, provided herein are antibodies comprising one or more polypeptide chains, together having two or more non-natural amino acid residues at positions in the polypeptide chains that are optimally substitutable. The antibody can be any polypeptide or multimeric polypeptide recognized as an antibody to those of skill in the art. The polypeptide chain can be any polypeptide chain of the antibody, including any heavy chain and any light chain. Each position in the polypeptide chain that is 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 antibodies comprising non-natural amino acids. [0009] In another aspect, provided herein are compositions comprising said antibodies. Advantageously, such compositions can have high uniformity because of the uniformity of the substitution of the antibodies provided herein. In certain embodiments, the compositions comprise a substantial amount of the antibody when measured by total weight of protein or when measured by total weight of antibody. In certain embodiments, the compositions comprise at least 80 % of the antibody, at least 85 % of the antibody, at least 90 % of the antibody or at least 95 % of the antibody by weight. [0010] In another aspect, provided herein are methods of making the antibodies. The antibodies 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 antibody chains. In certain embodiments, the antibodies are made by solid phase synthesis, semi-synthesis, in vivo translation, in vitro translation or cell-free translation. [0011] In another aspect, provided herein are methods of using the antibodies for therapy. Antibodies directed to a therapeutic target can incorporate one or more site-specific non- natural amino acids according to the description herein. These antibodies can be used for treating or preventing a disease or condition associated with the therapeutic target. Advantageously, a site-specific non-natural amino acid can be used to link the antibody to a therapeutic payload to facilitate efficacy. Exemplary antibodies, therapeutic targets and diseases or conditions are described herein. [0012] In another aspect, provided herein are methods of using the antibodies for detection. Antibodies directed to a detection target can incorporate one or more site-specific non-natural amino acids according to the description herein. These antibodies can be used with a label to signal binding to the detection target. Advantageously, a site-specific non- natural amino acid can be used to link the antibody to a label to facilitate detection.