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Therapeutic Mechanism and Efficacy of the Antibody–Drug Conjugate

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Therapeutic Mechanism and Efficacy of the Antibody–Drug Conjugate Published OnlineFirst December 6, 2011; DOI: 10.1158/1535-7163.MCT-11-0523 Molecular Cancer Therapeutic Discovery Therapeutics Therapeutic Mechanism and Efficacy of the Antibody–Drug Conjugate BAY 79-4620 Targeting Human Carbonic Anhydrase 9 Heike M. Petrul, Christoph A. Schatz, Charlotte C. Kopitz, Lila Adnane, Timothy J. McCabe, Pamela Trail, Sha Ha, Yong S. Chang, Andrei Voznesensky, Gerald Ranges, and Paul P. Tamburini Abstract Carbonic anhydrase IX (CAIX) is a cell surface glycoprotein that is expressed in many different tumors and yet restricted in normal tissues to the gastrointestinal tract. It is upregulated by hypoxia and correlates with tumor grade and poor survival in several tumor indications. Monoclonal antibodies (mAb) with single digit nanomolar binding affinity for CAIX were derived by panning with the recombinant ectodomain of CAIX against the MorphoSys HUCAL Gold library of human Fabs. Highest affinity Fabs were converted to full-length IgGs and subjected to further characterization based upon their avidity and selectivity for CAIX, their capacity to undergo internalization in CAIX-expressing cell lines, and their selective localization to CAIX-positive human xenografted tumors when administered to mice as fluorescent conjugates. Through this selection process, the 3ee9 mAb was identified, which upon conjugation to monomethyl auristatin E through a self-immolative enzyme-cleavable linker yielded the potent and selective CAIX antibody–drug conjugate CAIX-ADC (BAY 79-4620). In preclinical human xenograft models in mice representing several tumor indications, BAY 79-4620 showed potent antitumor efficacy and in some models showed partial and complete tumor shrinkage even following a single dose. The mechanism of action was shown by histology to involve the sequelae of events typical of antitubulin agents. Efficacy in murine preclinical models correlated semiquantitatively, with CAIX expression levels as determined by immunohistochemistry and ELISA. These preclinical data collectively support the development of BAY 79-4620 for the treatment of cancer patients with CAIX overexpressing tumors. Mol Cancer Ther; 11(2); 340–9. Ó2011 AACR. Introduction to the tumor marker G250 (named after the antibody with which it was identified) published by Oosterwijk Carbonic anhydrase IX (CAIX) was first identified and colleagues (3). by Pastorekova as an endogenous HeLa cell antigen CAIX is a cell surface glycoprotein that is expressed in that was recognized by the antibody M75 raised carcinomas of several histologic types, including a strik- against this cervix carcinoma cell line (1). Although ingly high proportion of renal cell carcinomas (4, 5), initially referred to as MN antigen, it was shortly carcinomas of the esophagus (4, 6), cervical carcinomas thereafter identified as a new carbonic anhydrase by (4, 7), malignant colon carcinomas (4, 8), non–small cell thesamegroup(2)andwasconfirmedtobeidentical lung carcinomas (NSCLC; refs. 4, 9), and, to a lesser degree, breast carcinomas (4, 10). By contrast, the expres- sion of CAIX on normal tissues is largely restricted to the Authors' Affiliation: Bayer HealthCare AG, Berlin, Germany apical surface of cells of the stomach, bile duct mucosa Note: Supplementary data for this article are available at Molecular Cancer (4, 11), and small intestine (4, 12). The extracellular Therapeutics Online (http://mct.aacrjournals.org/). domain of this type I transmembrane protein comprises both a proteoglycan domain implicated in cell adhesion Current address for T.J. McCabe: Johnson & Johnson, PA; current address for P. Trail: Regeneron Pharmaceuticals, Inc., NY; current address for S. Ha: through homotypic interaction (13) and the carbonic Merck&Co., Inc. PA; current address for Y.S. Chang: Aileron Therapeutics, anhydrase domain that catalyzes the reversible hydration MA; current address for A. Voznesensky: Novartis Institutes for BioMedical Research, MA; current address for G. Ranges: Bayer HealthCare AG, of carbon dioxide to bicarbonate and protons (14) and is retired; and current address for P.P. Tamburini: Alexion Pharmaceuticals. involved in the regulation of the pH within the tumor Inc, CT. environment (15). Corresponding Author: Heike M. Petrul, Bayer HealthCare AG, Berlin CAIX gene expression is under the direct control 13342, Germany. Fax: 49-30-18079; E-mail: [email protected] of the transcription factor hypoxia-inducible factor-1 doi: 10.1158/1535-7163.MCT-11-0523 and is significantly upregulated by tumor hypoxia (16). Ó2011 American Association for Cancer Research. CAIX expression was found to correlate with (i) a high 340 Mol Cancer Ther; 11(2) February 2012 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst December 6, 2011; DOI: 10.1158/1535-7163.MCT-11-0523 Efficacy of Anti-CA9 Antibody–Drug Conjugate BAY 79-4620 mean vessel density, cancer stage, and degree of necrosis Cell binding by flow cytometry in head and neck carcinoma (17); (ii) poor survival in Adherent CAIX-expressing PC-3 mm2 cells, non-CAIX nasopharyngeal carcinoma (18); (iii) tumor grade, nega- expressing MiaPaCa-2 cells, and MiaPaCa-2 cells trans- tive estrogen receptor status, higher relapse rate, and poor fected with full-length CAIX were detached from their survival for invasive breast carcinoma (10). This associa- flasks with 1:10 trypsin/Versene in PBS solution for 5 to 10 tion with tumor grade and overall survival, together with minutes. Cells were spun down (1,000 rpm, 5 minutes), its cell surface distribution and restricted expression in washed once with ice-cold RPMI 10% FBS, and resus- þ þ normal tissues, implicates CAIX as an important thera- pended in ice-cold staining buffer [Ca Mg -free PBS, 2% peutic target for monoclonal antibody (mAb)-based ther- bovine serum albumin (BSA), and 0.05% sodium azide] to apy. In this article, we report the identification of a potent achieve 6 Â 106 cells/mL. Anti-CAIX IgG1 or control and selective internalizing human antibody directed human IgG1 antibodies at 25 mg/mL were incubated with against CAIX that, when conjugated to monomethylaur- 6 Â 105 cells on ice for 1 hour. Unbound antibody was istatin E (MMAE), yielded a highly efficacious antibody– removed by washing the cells with the ice-cold staining drug conjugate BAY 79-4620, with activity against a vari- buffer. The cells were fixed with 2% formaldehyde in PBS ety of solid tumor types. for 10 minutes, then washed twice with staining buffer. The cell pellet was resuspended in 100 mL ice-cold staining Materials and Methods buffer containing a 1:200 dilution of Alexa Fluor 488– labeled secondary antibody (Molecular Probes/Invitro- Reference antibodies gen) and incubated on ice for 1 hour. The unbound The hybridoma producing CAIX mAb M75 (1, 13) was antibody was washed from the cells 2 times with flow obtained from American Type Culture Collection (ATCC) buffer (PBS containing 2% BSA), and the cells were resus- and used to express and purify mAb M75 using standard pended in 1 mL flow buffer. Fluorescence-activated cell protocols. sorting (FACS) analysis of the resuspended cells was done on a Beckman FACS Calibur instrument. All cell lines Antibody discovery using the HuCAL Gold were obtained from ATCC; MiaPaCa-2-CAIX was gener- Fab-phage library ated using standard transfection methods. Cell lines are The HuCAL Gold Fab-phage library was obtained from regularly checked for authenticity by DNA fingerprinting MorphoSys AG and was prepared as described elsewhere at the DSMZ (German Collection of Microorganisms and (19), comprising a highly diverse library of 1010 different Cell Cultures), Braunschweig, Germany. monovalent phage encoded within phagemid vector pMORPH23 and allowing for monovalent CysDisplay of Assessment of cellular internalization of mAbs Fab fragments. Solid phase panning was carried out as Antibody internalization was assessed using a Cello- described in the Supplementary Data. mics Array Scan automated confocal microscope system (see Supplementary Data). Fab expression Soluble Fab fragments were produced from the isolated Generation of immunoconjugates with phage clones as described in the Supplementary Data. monomethylauristatin E mAb conjugation with MMAE was done essentially as Identification of CAIX-binding Fabs described elsewhere for conjugation with an anti-CD30 Isolated Fabs were tested for binding to the purified mAb (21). ectodomain of CAIX in an ELISA, positive Fabs were recloned in the IgG format and expressed in Chinese Cell cytotoxicity assays hamster ovary cells (see Supplementary Data). Anti- Antigen-positive and antigen-negative cells were plat- CAIX IgG antibodies were purified using protein A ed at 5,000 cells per well in 96-well plates in 100 mL media sepharose. per well overnight at 37 C, in 5% CO2 to adhere. The media was replaced with fresh media containing antibody Antibody binding kinetics using surface plasmon or antibody-vcMMAE conjugate and the plates further resonance incubated at 37 C, in 5% CO2 for 72 hours. Cytotoxicity Surface plasmon resonance (SPR) was carried out on a was assessed using the Alamar Blue cell viability assay. BIAcore 3000 instrument (BIAcore; see Supplementary Alamar Blue was added at a final concentration of 10% for Data). the last 4 hours of the incubation, and its transformation to fluorescent product by viable cells determined spectro- Immunoprecipitation fluorometrically by plate reader (544 nm excitation–590 The complexes formed between the antibodies and nm emission). biotin-labeled
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