Anti-Endosialin Antibody–Drug Conjugate: Potential in Sarcoma and Other Malignancies Cecile Rouleau, Diego A
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Published OnlineFirst July 16, 2015; DOI: 10.1158/1535-7163.MCT-15-0312 Large Molecule Therapeutics Molecular Cancer Therapeutics Anti-Endosialin Antibody–Drug Conjugate: Potential in Sarcoma and Other Malignancies Cecile Rouleau, Diego A. Gianolio, Robert Smale, Stephanie D. Roth, Roy Krumbholz, Jay Harper, Kenneth J. Munroe, Tessa L. Green, Bruce C. Horten, Steven M. Schmid, and Beverly A. Teicher Abstract Endosialin/TEM1/CD248 is a cell surface protein expressed at were grown as xenograft tumors in nude mice. The SK-N-AS high levels by the malignant cells of about 50% of sarcomas and neuroblastoma and the A-673 Ewing sarcoma lines were select- neuroblastomas. The antibody–drug conjugate (ADC) anti-endo- ed for in vivo efficacy testing of the anti-endosialin-MC-VC- sialin-MC-VC-PABC-MMAE was selectively cytotoxic to endosia- PABC-MMAE conjugate. The treatment groups included a vehi- lin-positive cells in vitro and achieved profound and durable cle control, unconjugated anti-endosialin, an admix control antitumor efficacy in preclinical human tumor xenograft models consisting of anti-endosialinandadoseoffreeMMAEequiv- of endosialin-positive disease. MC-VC-PABC-MMAE was conju- alent to the dose administered as the ADC, and the anti- gated with anti-endosialin with 3–4 MMAE molecules per ADC. endosialin-MC-VC-PABC-MMAE conjugate. The unconjugated The anti-endosialin-MC-VC-PABC-MMAE conjugate was tested anti-endosialin had no antitumor activity and resulted in for activity in four human cell lines with varied endosialin levels. similar tumor growth as the vehicle control. The admix control The HT-1080 fibrosarcoma cells do not express endosialin, A-673 produced a modest tumor growth delay. Administration of the Ewing sarcoma cells and SK-N-AS neuroblastoma cells are mod- anti-endosialin-MC-VC-PABC-MMAE conjugate resulted in a erate expressers of endosialin, and SJSA-1 osteosarcoma cells marked prolonged tumor response of both xenograts. These express very high levels of endosialin. To determine whether proof-of-concept results break new ground and open a prom- endosialin expression was maintained in vivo, A-673 Ewing sar- ising drug discovery approach to these rare and neglected coma, SK-N-AS neuroblastoma, and SJSA-1 osteosarcoma cells tumors. Mol Cancer Ther; 14(9); 2081–9. Ó2015 AACR. Introduction types, is associated with tumor neovascularization and inflam- mation and has emerged as a molecular marker and therapeutic Endosialin/CD248/TEM1, a transmembrane glycoprotein target for sarcoma (1–12). First recognized as the antigen of an expressed on pericytes and fibroblasts during tissue develop- antibody raised in mice against human fetal fibroblasts (FB5), ment and present in the adult in several mesenchymal cell endosialin was found to be expressed by human solid tumor vasculature (13) and was detected in a subset of cells enriched Genzyme Corporation, Framingham, Massachusetts. for endothelium via selection with P1H12, an anti-CD146 Note: Supplementary data for this article are available at Molecular Cancer antibody, from a colorectal tumor specimen (14). Endosialin Therapeutics Online (http://mct.aacrjournals.org/). expression in tumor vasculature occurs mainly in pericytes and stromal fibroblasts (15–17). C. Rouleau and D.A. Gianolio contributed equally to this article. In mouse embryos, endosialin/TEM1-lacZ colocalizes with Current address for Cecile Rouleau: Tufts University, Jaharis 601, 136 Harrison most vimentin-positive cells and a large portion of CD31- or Avenue, Boston, MA; current address for Diego A. Gianolio, Sanofi Oncology, desmin-positive cells. In the mouse, endosialin is expressed 500 Kendall Street, Cambridge, MA; current address for Robert Smale, Covance, 2440 S. Sepulveda Blvd., Suite 220, Los Angeles, CA; current address for throughout embryonic and adult development in mesenchymal À/À Stephanie D. Roth, Cherokee Nation Businesses (CNT), Research Directorate, cells related to blood vessels (18). Endosialin mice have no US Army Institute of Surgical Research, 3698 Chambers Pass, Suite B, JBSA Fort defect in pericyte recruitment, suggesting a role for endosialin in Sam Houston, TX; current address for Roy Krumbholz, 15443 Grosbeak Pass, San pericyte/endothelial cell cooperation during vascular patterning À À Antonio, TX; current address for Jay Harper, MedImmune Oncology Research, (3). Endosialin / mice have higher than normal bone mass due One MedImmune Way, Gaithersburg, MD; current address for Tessa L. Green, MD to increased osteoblast-mediated bone formation (1). Growth of Anderson Cancer Center, UT MD Anderson Cancer Center, Houston, TX; current CyD/CyD address for Bruce C. Horten, Integrated Oncology-New York, 521 West 57th syngeneic tumors was reduced in CD248 mice, which lack CyD/CyD Street, New York, NY; current address for Steven Schmid, Vivo Biosciences, Inc., the endosialin cytoplasmic domain. CD248 fibroblasts 3714 Hunters Point Street, San Antonio, TX; and current address for Beverly A. have impaired PDGF-BB–induced migration, decreased matrix Teicher, National Cancer Institute, 9609 Medical Center Drive, RM 4-W602, MSC metalloproteinase secretion, and higher transcript levels of the 9735, Bethesda, MD. tumor suppressors transgelin (SM22a), Hes and Hey1 (6). þ Corresponding Author: Beverly A. Teicher, National Cancer Institute, RM Endosialin is expressed by human, but not mouse, CD8 naive þ þ 4-W062, MSC9735, 9609 Medical Center Drive, Bethesda, MD 20892. Phone: T cells, specifically CD8 CCR7 CD11a low naive T cells, and on þ þ 240-276-5972; Fax: 240-276-7895; E-mail: [email protected] CD8 T cells in the thymus. Endosialin knockdown in naive CD8 doi: 10.1158/1535-7163.MCT-15-0312 T cells increased cell proliferation; thus, endosialin has opposing þ Ó2015 American Association for Cancer Research. functions on hematopoietic (CD8 ) and stromal cells (5). www.aacrjournals.org 2081 Downloaded from mct.aacrjournals.org on October 2, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst July 16, 2015; DOI: 10.1158/1535-7163.MCT-15-0312 Rouleau et al. The function and mechanisms of regulation of endosialin are described previously (12). Synthetic procedures for the conjuga- still incompletely understood. Recently, several new anti-endo- tion of the linker-functionalized monomethylauristatin E sialin monoclonal antibodies became available, two recognize (MMAE) to anti-endosialin were performed as detailed in the C-type lectin-like domain-Sushi/SCR/CCP and four recognize the following published procedure reports (35, 36). Briefly, the sialomucin domain. In addition, a yeast-derived anti-endo- anti-endosialin-MC-VC-PABC-MMAE was prepared by partial sialin biobody-78 was developed (4, 19). reduction of the antibody interchain disulfides with 3.3 molar The earliest indication that endosialin may be expressed by equivalents of tris-(2-carboxyethyl)-phosphine hydrochloride in malignant cells was in the 1992 publication by Rettig and col- sodium borate buffer pH ¼ 8.0 for 2 hours at 37C. After cooling leagues who reported immunoreactivity of FB5 in several neuro- on ice, 4.8 molar equivalents of maleimide-linker-MMAE deriv- blastoma cell lines and mentioned FB5þ malignant cells in a ative in DMSO were added and the reaction was allowed to subset of sarcomas (13). Further evidence for endosialin expres- proceed for 30 minutes at 4C. Excess small molecule was sion by tumor cells came in 2005 with immunostaining of removed using QuadraPure DET (Sigma-Aldrich Co.) polystyrene malignant fibrous histiocytoma and liposarcoma showing tumor scavenging beads. Yields were 85% to 95% based upon protein cell immunoreactivity (20). Furthermore, endosialin expression recovery. was assessed in 86 formalin-fixed, paraffin-embedded human The drug/antibody ratio (DAR) was determined by C8 reversed- clinical sarcoma specimens. Immunoreactive tissue components phase LC/MS following published procedures (35, 36). The ADC were malignant cells, stromal cells, and vasculature. Seventy was deglycosylated with PNGase F overnight at 37C, dialyzed to (81%) were positive for endosialin, with 44 (51%) reaching at remove salt, and reduced to smaller fragments (light and heavy least 50% coverage of immunoreactive tissue components. Stain- chains) using 20 mmol/L dithiothreitol (DTT) for 30 minutes at ing intensity was scored on the scale 0, 1þ,2þ,3þ. All nine 37C. Aliquots were injected in the LC/MS instrument and elec- sarcoma subtypes tested included specimens with at least 50% trospray ionization mass spectra of light and heavy chains were immunoreactive tissue components positive with a minimum of recorded and deconvoluted, revealing conjugation profiles that 2þ staining intensity, indicating the high prevalence of endosialin included free, mono-, di-, and tri-conjugated species. The inte- in sarcomas (12). A retrospective analysis of diagnostic reports grated peak areas were then used to determine the weighted showed that endosialin can be detected in high-grade disease and average ratios for light and heavy chains. The DAR was determined metastasis. In disseminated human sarcoma xenografts, endosia- by doubling and adding the light and heavy chains ratios. The lin protein expression was maintained at different anatomic sites DAR for the ADC was in the range of 3 to 5. Aggregation was (7–9). An anti-endosialin MORAb-004, which is a humanized determined by size exclusion HPLC and the conjugate was >98% FB5 antibody, has completed phase I clinical trial and is currently monomeric. Purity was determined by C18 RP-HPLC