US 20090175867A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0175867 A1 Thompson et al. (43) Pub. Date: Jul. 9, 2009 (54) SINGLE-CHAIN MULTIVALENT BINDING Publication Classification PROTEINS WITH EFFECTOR FUNCTION (51) Int. Cl. (75) Inventors: Peter A. Thompson, Bellevue, WA g Bis CR (US); Jeffrey A. Ledbetter, ( .01) Shoreline, WA (US); Martha S. EN fo C Hayden-Ledbetter, Shoreline, WA CI2N 5/06 :08: S.US); WilliamIIIlam Bradv,5rady, Bothell,Sounell, WA CI2P 2L/04 (2006.01) C7H I/00 (2006.01) Correspondence Address: GOIN 33/566 (2006.01) Seed Intelectuall Property Law Group PLLC GOIN 33/53 (2006.01) 701 Fifth Avenue, Suite 5400 A6IP 43/00 (2006.01) Seatle, WA 98104 (US) (73) Assignee: TRUBION (52) U.S. Cl. ................ 424/135.1; 530/387.3; 536/23.53; PHARMACEUTICALS, INC. 435/320.1; 435/325; 435/69.6; 536/25.3: Seattle, WA (US) s 435/375; 436/501; 435/7.21 (21) Appl. No.: 12/041590 (22) Filed: Mar. 3, 2008 (57)57 ABSTRACT Multivalent binding peptides, including bi-specific binding Related U.S. Application Data peptides, having immunoglobulin effector function are pro (63) Continuation-in-part of application No. PCT/US07/ vided, along with encoding nucleic acids, vectors and host 71052, filed on Jun. 12, 2007. cells as well as methods for making Such peptides and meth s s ods for using Such peptides to treat or prevent a variety of (60) Provisional application No. 60/813.261, filed on Jun. diseases, disorders or conditions, as well as to ameliorate at 12, 2006, provisional application No. 60/853,287, least one symptom associated with such a disease, disorder or filed on Oct. 20, 2006. condition. Binding of Purified Protein from COS Cells to WIL2-S: TRU015 vs. 2H7-SSSIgG-STD1-2e 12 Multispecific 256 Fusion Proteins Goat anti-human FTC 1:100 2 IE TRUO155ug/mL g Eo 2H7-2e 12 LH 5ug/mL 2H7-2e 12 HL 5ug/mL Patent Application Publication Jul. 9, 2009 Sheet 1 of 58 US 2009/0175867 A1 Figure 1. Schematic Structure of a Multispecific Binding Protein AKA "Scorpion" Patent Application Publication Jul. 9, 2009 Sheet 2 of 58 US 2009/0175867 A1 Figure 2. Western Blot Of Non-Reduced SDS PAGE COS Supernatants, 30 ul/Well 1 2 3 4 5 6 7 8 191-x 64 51 - S3 Patent Application Publication Jul. 9, 2009 Sheet 3 of 58 US 2009/0175867 A1 Figure 3. Binding of Protein Expressed in COS Supernatants to Cells Expressing Target Antigens a. Binding of COS Supernatants to WIL-2S Cells 2e 12 SMIPs vs. 2H7-IgG-2e 12 Scorpions 350 cc 3OO 250 200 150 N 100 50 neat 1 to 1 1 to 2 1 to 4 1 to 8 1 to 16 D2h7-SSS-hCG-STD1-2e 12 LH 2h/-Sss-hlgG-STD1-2e 12 HL 2h/IgG SMP mOCK b. Binding of COS Supernatants to CD28 CHO Cells 2e 12 SMIPs vs. 2H7-2e 12 Scorpions 350 250 2OO Š 150 1OO 50 O s SS S. y & S S& X W SS S & neat 1 to 1 1 to 2 1 to 4 1 to 8 1 to 16 D2H7-sss-hlgG-STD1-2e 12 LH & 2H7-sss-higG-STD1-2e 12 HL S2e 12 LHSMP 32e 12 HL SMIP Smock Patent Application Publication Jul. 9, 2009 Sheet 4 of 58 US 2009/0175867 A1 Figure 4. Binding of Purified Protein from COS Cells to WIL2-S: TRUO15 vs. 2H7-SSSIgG-STD1-2e 12 Multispecific 256 Fusion Proteins GOat anti-human FTC 1:100 II TRU015 5ug/mL C- 2H7-2e 12 LH 5ug/mL i 2H7-2e 12 HL 5ug/mL 104 Patent Application Publication Jul. 9, 2009 Sheet 5 of 58 US 2009/0175867 A1 Figure 5. 256 A. Binding of Purified Protein from COS Cells to CD28CHO: 2e 12 HL SMIP vs.2H7-SSSIgG-STD1-2e 12 HL Multispecific Fusion Protein o Goat anti-human FTC 1:100 1. m 2e 12 HL SMIP 5ug/mL i - 2h7-2e 12 HL Scorpion 5ug/mL. O /(4...twis-St. 100 101 102 1O3 104 FL-H B. Binding of Purified Protein from COS Cells to CD28CHO 256 2H7-SSSIgG-STD1-2e 12 LH vs. 2H7-SSSIgG-STD1-2e 12 HL Multispecific Fusion Protein I Goat anti-human FITC 1:100 i m 2h7-2e 12 LH 5ug/mL c= 2h7-2e 12 HL 5ug/mL i. E. Eliyas. 101 102 103 104 FL-H Patent Application Publication Jul. 9, 2009 Sheet 6 of 58 US 2009/0175867 A1 Figure 6. Table lodentifying Functional Elements For Multispecific Fusion Proteins BD EED Linker 2 sequence fusion ED2 Binding Effector (- restriction junction Binding Domain function site) with BD2 Domain 2 (L2 RS-- 1. domain WQ 2e12) Modified hIgG1- NYGGGGSGGGGSGGGGSG hIgG1 CH2-CH3 w w hinge CCC-YSSS Modified hIgG1- NGGGGSGGGGSGGGGSG hIgG1 CH2-CH3 NYGGGGSGGGGSGGGGSG w w hinge CCC-SSS Modified hIgG1 hIgG1 CH2-CH3 V y hinge CCC-9sss Modified hIgG1 hIgG1 CH2-CH3 w y hinge CCC-Gdsss Modified hIgGl hIgG1 CH2-CH3 hinge CCC-Gdsss se ww. Modified hIgGl hIgG1 CH2-CH3 w y hinge CCC-Gdsss Modified hIgGl- NYGGGGSGGGGSG hIgG1 CH2-CH3 w y hinge CCC-Gdsss Modified hIgG1- GGGGSGGGGSGGGGSG hIgG1 CH2-CH3 w y hinge CCC-Ysss Modified hIgG1- GCPPCP hIgG1 CH2-CH3 w y hinge CCC-Ysss Patent Application Publication Jul. 9, 2009 Sheet 7 of 58 US 2009/0175867 A1 Figure 6B (1/5) Constructs one epi Ina Fe Itine so Patent Application Publication Jul. 9, 2009 Sheet 10 of 58 US 2009/0175867 A1 Figure 6B (4/5) name of Inge EF Ener Bo ) ) higG1-SSS higG or higG it is My ) higG1 -CSC higG or higG e is viv) ) higG1-SSS higG or higG e is My ) higG1 -CSC higG or higG e is vivo ) higG1-SSS higG or higG is is v-vi ) higG1-SSS higG or higG is is My Patent Application Publication Jul. 9, 2009 Sheet 11 of 58 US 2009/0175867 A1 one of Inge EFD Inker Bo h P238 IgG H6 2H7-SSS-higG-H6-G19-4HL at Vivi is -SSS higG or higG H6 VH-VL) h IgG h C IgG H7 VL-VH) E. VH-VL) Patent Application Publication Jul. 9, 2009 Sheet 12 of 58 US 2009/0175867 A1 Figure 7. Altering the Linker after the EFD and the orientation of V regions in BD2 Affects Binding Efficiency of Multispecific Fusion Proteins Binding of 2H7-SSShlgG-L/HX-2e 12 Multispecific Fusion ProteinS with Various Linkers to WIL2-S and CD28 CHO 2H7SSS SMP 2e12 HLSMIP is mock H6 H5 H4 H3 H2 WIL 2-S H1 CD28CHO L6 L5 L4 L3 L2 L1 HL-STD2 old O 2O 40 60 8O MFI *"All fusion proteins used at 0.72 ug/ml, Protein A purified, generated from transient COS Supernatants. Patent Application Publication Jul. 9, 2009 Sheet 13 of 58 US 2009/0175867 A1 Figure 8. Effect of Gly, Serlinker length on 2H7-ssslgG-HX-2e 12 HL Protein Increased Cleavage of Linker Region with increasing Linker Length c c s c N Reduced Non-reduced Patent Application Publication Jul. 9, 2009 Sheet 14 of 58 US 2009/0175867 A1 Figure 9. Western Blots of Multispecific Fusion Proteins With H6 Linker A. Absence Of SMP Or Smaller CD28 detectable forms B. Presence of a SMIP sized form using CD20 anti-id Fab A. Detection of 2e 12 BD2 B. Detection Of 2H7 CD2O by CD28-murinelgG BD1 by Fab: AbyD02429.2 CN SN SN Sn ad & & & CO (b cab 5 I. I. CD C 2, 2, CD 2 i T SCD - 2 3 S 2 %s it CD to : m GN N3 ob m GN CD NP OO in C\ 5 5 m CN 5 OO 35 Stm &5 35 S Pm & CDSN 353 S G edišS& 32G - (5S. SS RS S CD28 murine IgG | GAM-HRP only GAM-HRP Patent Application Publication Jul. 9, 2009 Sheet 15 of 58 US 2009/0175867 A1 Figure 10. A. Binding of Multispecific Fusion Proteins With Variant Linkers to WIL-2S Cells Binding of TRU015ssslgG2e12HLH1-H6 to WIL2S Cells 300 250 150 50 OO O O. O.O. OOO Concentration (ug/ml) --TRU015 -- H1 -A-H2-e-H3-3-H4 -&-H5 -- H6 B. Binding of Multispecific Fusion Protein With Variant Linkers to CD28 CHO Cells 250 Binding of TRUO15ssslgG2e 12HL H1-H6 to CD28CHO Cells 2OO 150 50 1OO 1 O 1 O.1 concentration (ug/ml) Patent Application Publication Jul. 9, 2009 Sheet 16 of 58 US 2009/0175867 A1 Figure 11. Summary of SEC Fractionation of 2H7-SsslgG-2e 12 HL Multispecific Fusion Proteins with Variant Linkers Linker ID Retention Time % Other Forms SDS-PAGE for POI Analysis of Cleavage at EFD/BD2 junction H1 H 2 H 4 7.622 (shoulder) 6667 8.8.7.O. O759 H 5 7.933/7,680 32.42/33.94 (doublet with shoulder) H 6 7.901/7.69 34.5/29.3 (doublet with shoulder) H 7 15.8 (HMW) Patent Application Publication Jul. 9, 2009 Sheet 17 of 58 US 2009/0175867 A1 Figure 12. Binding of 2H7-sss-hlgG-H-2e 12 HL). Fusion Proteins with Different Linkers to Cells Expressing Target Antigen for BD1 or BD2 250 cy 2OO - 150 1OO – 50 10 0.1 O.O1 O.OO1 concentration (ug/ml) -0- H3-HH6 - A - H7 ->e-gah FITC 10 1 O. OO1 OOO1 concentration (ug/ml) -0- H3-HH6 -A-H7 -ee-gah FITC Patent Application Publication Jul.