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(12) United States Patent (10) Patent No.: US 9.260,522 B2 Kufer Et Al

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(12) United States Patent (10) Patent No.: US 9.260,522 B2 Kufer Et Al US009260522B2 (12) United States Patent (10) Patent No.: US 9.260,522 B2 Kufer et al. (45) Date of Patent: Feb. 16, 2016 (54) BISPECIFIC SINGLE CHAIN ANTIBODIES WO WO 2008, 119565 A2 10/2008 WITH SPECIFICITY FOR HIGH WO WO 2008, 119566 A2 10/2008 MOLECULAR WEIGHT TARGET ANTIGENS WO WO 20089567 A2 102008 OTHER PUBLICATIONS (75) Inventors: Peter Kufer, Munich (DE); Claudia Blimel, Munich (DE); Roman Kischel, Sist etal (r. NA i. S. 2. Munich (DE) 139-159).*ariuZZa et al. eV. Ophy S. Ophy S. e. : (73) Assignee: AMGEN RESEARCH (MUNICH) syst al. (Proc. Natl. Acad. Sci. USA. May 1987; 84 (9): 2926 GMBH, Munich (DE) Chien et al. (Proc. Natl. Acad. Sci. USA. Jul. 1989: 86 (14): 5532 5536).* (*) Notice: Subject to any disclaimer, the term of this Caldas et al. (Mol. Immunol. May 2003; 39 (15): 941-952).* patent is extended or adjusted under 35 Wils a systs, lig,...si:18): U.S.C. 154(b) by 553 days. 5.adoSeal. Elia? J. VTOl. (J. Immunol.S1Ol. Jul. 2002;, 169 (6): 3076-3084).*: (21) Appl. No.: 13/122,271 WuCasset et al. et (J.t Mol.(Biochem. Biol. Nov.Biophys. 19, 1999;Res. &R294 (1): 151-162).*Jul. 2003; 307 (1): 198-205).* (22) PCT Filed: Oct. 1, 2009 MacCallum et al. (J. Mol. Biol. Oct. 11, 1996; 262 (5): 732-745).* Holmetal. (Mol. Immunol. Feb. 2007; 44 (6): 1075-1084).* (86) PCT NO.: PCT/EP2009/062794 ClinicalTrials.gov archive, "Phase II Study of the BiTE(R) Blinatumomab (MT103) in Patients With Minimal Residual Disease S371 (c)(1), of B-Precursor Acute ALL.” View of NCT00560794 on Aug. 11, (2), (4) Date: Jul. 14, 2011 2008, pp. 1-3, DXP-002572438. Maletz, K., et al., “Bispecific Single-Chain Antibodies as Effective (87) PCT Pub. No.: WO2010/037837 Tools for Eliminating Epithelial Cancer Cells From Human Stem Cell Preparations by Redirected Cell Cytotoxicity.” Int. J. Cancer, PCT Pub. Date: Apr. 8, 2010 (2001), pp. 409-416, vol. 93 DXP-00230 1955. Wolf, E., et al., “BiTEs: bispecific antibody constructs with unique (65) Prior Publication Data anti-tumor activity.” DDT. (2005), pp. 1237-1244, vol. 10 n. 18. www.drugdiscoverytoday.com. US 2011 FO262439 A1 Oct. 27, 2011 Bihler, et al. "A bispecific diabody directed against prostate-specific membrane antigen and CD3 induces T-cell mediated lysis of prostate cancer cells.” Cancer Immunol Immunother 57:43-52 (2008). Related U.S. ApplicationO O Data E.Kipriyanov, et MairAal., "Bispecific Human CD3xCD19 B Cells.” Int. Diabody J. C for 77,763-771T Cell-Me (60) Provisional application No. 61/101,933, filed on Oct. (1998). 1, 2008. Dreier et al., T cell costimulus-independent and very efficacious s inhibition of tumor growth in mice bearing Subcutaneous or leukemic (51) Int. Cl. human B cell lymphoma xenografts by a CD19-/CD3-bispecific C07K 6/30 (2006.01) isshain antibody construct, J. Immunol. 170(8):4397-402 C07K 6/28 (2006.01) Kipriyanov et al., Bispecific tandem diabody for tumor therapy with A61 K39/00 (2006.01) improved antigen binding and pharmacokinetics, J. Molec. Biol. (52) U.S. Cl. 293:41-56 (1999). CPC ......... C07K 16/2809 (2013.01); C07K 16/3069 (Continued) (2013.01); A61 K 2039/505 (2013.01); C07K 2317/31 (2013.01); C07K 23.17/34 (2013.01) Primary Examiner — Stephen Rawlings (58) Field of Classification Search (74) Attorney, Agent, or Firm — Marshall, Gerstein & Borun CPC. G01N33/505; C12N5/0636; C12N 5/0693; LLP C12N5/0093: C07K 16/28: C07K 16/2809; C07K 16/30; A61K 2039/5152 (57) ABSTRACT USPC ................... 435/4, 7.1, 325, 352,366,372.3: The present invention provides a method for the selection of 436/501; 530/387.1, 387.3, 388.2, bispecific single chain antibodies comprising a first binding 530/388.22,388.75 domain capable of binding to an epitope of CD3 and a second See application file for complete search history. binding domain capable of binding to the extracellular domain cell Surface antigens with a high molecular weight (56) References Cited extracellular domain. Moreover, the invention provides bispecific single chain antibodies produced by the use of the U.S. PATENT DOCUMENTS method of the invention, nucleic acid molecules encoding these antibodies, vectors comprising Such nucleic acid mol 5,726,044 A 3, 1998 Lo et al. ecules and methods for the production of the antibodies. Furthermore, the invention provides pharmaceutical compo FOREIGN PATENT DOCUMENTS sitions comprising bispecific single chain antibodies of the EP 1293514 A1 3, 2003 invention, medical uses of the same and methods for the WO WO-99.54440 A1 10, 1999 treatment of diseases comprising the administration of bispe WO WO 2004/106381 A1 12/2004 cific single chain antibodies of the invention. WO WO-2006, 125481 A1 11, 2006 WO WO 2007/042261 A2 4, 2007 4 Claims, 36 Drawing Sheets US 9.260,522 B2 Page 2 (56) References Cited Loeffler et al. Effect elimination of chronic lymphocyte leukaemia B cells by autologous T cells with a bispecific anti-CD 19, anti-CD3 OTHER PUBLICATIONS single-chain antibody construct, Leukemia 17(5):900-9 (2003). Kipriyanov et al., Effect of domain order on the activity ofbacterially International Search Report and Written Opinion of the International produced bispecific single-chain Fv antibodies, J. Molec. Biol. Searching Authority, European Patent Office, PCT/EP2004/005685, 330(1):99-111 (2003). dated Nov. 12, 2004. Loeffler et al. A recombinant bispecific single-chain antibody, CD19xCD3, induces rapid and highlymphoma-directed cytotoxicity by unstimulated T lymphocytes, Blood 95(6):2098-103 (2000). * cited by examiner U.S. Patent Feb. 16, 2016 Sheet 1 of 36 US 9.260,522 B2 Figure 1 MAb 5-10 EpCAM-D1-hNG2-CHO EpCAM-D3-hNG2-CHO EpCAM-D1D3-hNG2-CHO EpCAM-D1D2-hNG2-CHO EpCAM-hNG2-CHO EpCAM-CHO Fluorescence intensity U.S. Patent Feb. 16, 2016 Sheet 2 of 36 US 9.260,522 B2 Figure 2 0 EpCAM-CHO o EpCAM-D1-hNG2-CHO - - - O EpCAM-D3-hNG2-CHO x EpCAM-D1D3-hNG2-CHO 110- -D EpCAM-D1D2-hNG2-CHO 100 EpCAM-hNG2-CHO 90 80 ess 70 60 50 40 30 20 10 10-4 10-3 10-2 10-1 100 1 0 1 102 dilution of supernatant (%) U.S. Patent Feb. 16, 2016 Sheet 3 of 36 US 9.260,522 B2 U.S. Patent Feb. 16, 2016 Sheet 4 of 36 US 9.260,522 B2 Figure 4 human mutated unmutated PSMA rat PSMA rat PSMA La P1 x 2C La W W P2 x 2C La W A P3 x 2C La W A P4 x 12C La W W P5 x 12C La La W D1 X 2C La F-1 D2 X 2C amanmmanummbFluorescence intensity U.S. Patent Feb. 16, 2016 Sheet 5 of 36 US 9.260,522 B2 Figure 5 90 80 as pse - - - P1 x 2C 60 Arsyfait ksoal P2 x 2C s 4. M 4 - - - - P3 x 2C S. 50 % / ^ P4 x 12C 1. ? M g 40 27 A P5 x 2C 27 A eam D1 x 12C 30 --Y-42 / M M 20 traig2-1 - ? a o o D2x2C 1. -Negative control 10 -------- 10-1 100 101 102 dilution of supernatant % U.S. Patent Feb. 16, 2016 Sheet 6 of 36 US 9.260,522 B2 Figure 6 human mutated unmutaed PSMA rat PSMA rat PSMA C O r P6 X 2C U y n t S D3 x 12C Fluorescence Intensity Figure 7 100 a 90 fi 80 -21 f - 70 fa SS y - - - A D3 x 12C d 60 f M / - - - - P6 X 2C g 50 M A. negative control - 40 A 30 20 10 O 10-4 10-3 10-2 10-1 100 101 102 dilution of supernatant % U.S. Patent Feb. 16, 2016 Sheet 7 of 36 US 9.260,522 B2 Sunod 8eun61-I KOZIX7d U.S. Patent Feb. 16, 2016 Sheet 8 of 36 US 9.260,522 B2 Z[9TOiz LTi790Z U.S. Patent Feb. 16, 2016 Sheet 9 of 36 US 9.260,522 B2 Anti-FLAG CHO transfected with a mutated human FAPA antigen with murine membrane-distal epitopes CHO transfected With murine FAPA Figure 10 U.S. Patent Feb. 16, 2016 Sheet 10 of 36 US 9.260,522 B2 O O S C -- to U.S. Patent US 9.260,522 B2 U.S. Patent Feb. 16, 2016 Sheet 12 of 36 US 9.260,522 B2 Effector T cells: stimulated human CD4/CD56 depleted human PBMC Target cells: CHO transfected with human FAPA E.T ratio: 10:1 FA2OH3HLx2CHL 9 8 FA22A9HL)d2CHL 8.- : RA22C11HL}{2CHL 70 x FA22D8HLd2CHL SO 5 A. 3. 2 -1) 10-8 - 1-8 1-5 10-4 1-3 1-2 1-1 dilution of supernatant Effector T cells: stimulated human CD4/CD56 depleted human PBMC Target cells: CHO transfected with human FAPA E:T ratio: 10:1 g)- 8 FA 19D9HL}d2CHL B x FA22E8HLd2CHL & 70 A 50 " A. 20 ------- 1-I - 10-G 1-5 1-4 - 1-2 1-1 dilution of supernatant Figure 12a U.S. Patent Feb. 16, 2016 Sheet 13 of 36 US 9.260,522 B2 Effector T cells: stimulated CD4/CD56 depleted human PBMC Target cells: CHO transfected with a mutated human FAPA antigen with murine membrane-distal epitopes E.T ratio: 10:1 110 1OO 90 8O TO SO SO 40 O o FA22E8HLX2CHL O ------mm O-3 10-7 10-6 10-5 10-4 10-3 10-2 10-1 dilution of supernatant 6 Effector T cells: stimulated CD4/CD56 depleted human PBMC Target cells: CHO transfected with a mutated human FAPA antigen with murine membrane-distal epitopes E:T ratio: 10:1 110 TF 1OO 5 O 3 O FA2CHL2CHL 10-8 O-7 O-6 10-5 10-4 10-3 10-2 10-1 dilution of supernatant 6 Figure 12b U.S.
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