Advances in Brief A New Human Antitumor Immunoreagent Specific for ErbB2

Purpose: Our aim was to isolate a novel human miniantibody (scFv) that specifically targets ErbB2-positive cancer cells. ErbB2, a tyrosine kinase receptor, is overexpressed in clinically significant tumors, such as breast, ovary, and lung carcinomas. In normal tissues, it is expressed only in certain epithelial cell types. Experimental Design: A large phagemid library (Griffin.1 library) of human scFv was used for the isolation of the ErbB2-specific scFv. A very effective strategy was developed for the isolation, consisting in a double subtractive selection, the use of two different combinations of “positive,” i.e., ErbB2-bearing, and “negative” cell lines. Results: Here we report the isolation of the first human anti-ErbB2 mini-antibody endowed with antitumor action. Both in its soluble and phage format, it binds specifically to ErbB2, inhibits its autophosphorylation, is internalized by target cells, and exerts a strong and specific antiproliferative action on ErbB2-positive target cells. A correlation was found between the extent of this antiproliferative effect and the expression levels of ErbB2 on target cells, with a strong cytotoxicity for hyper-expressing cells, such as SKBR3, in which apoptosis was evidenced. Conclusions: This scFv is a potentially effective immunoreagent for diagnostics and therapeutics of certain cancers, both as a readily diffused molecule in solid tumors and as an essential asset for the construction of fully human anticancer drugs. Introduction The ErbB2 transmembrane tyrosine kinase receptor, homologous to the EGF receptor (1, 2), is highly expressed in breast, ovary, and lung carcinomas (3, 4), as well as in salivary gland and gastric tumor-derived cell lines (5, 6). Its overexpression, which occurs most commonly via gene amplification, can reach as many as 2 10 molecules/cell. In normal tissues it is expressed only in certain epithelial cell types (7). ErbB2 plays a central role in tumor progression, because it potentiates and prolongs the signal transduction cascades elicited by ligand activation of other ErbB tyrosine kinase receptors (8, 9). Overexpression of ErbB2 may also increase resistance of tumor cells to host defenses through the evasion of immune surveillance exerted by activated macrophages (10). The accessibility of ErbB2 on cell surface and its implication in the development of malignancy of these tumors make it an attractive target for immunotherapy. Several groups have isolated mouse and rat mAbs against ErbB2 extracellular domain (11–13), which display useful properties for immunotherapy. For instance, these reagents can induce endocytosis, and some of them have antiproliferative activity on tumor cells (11–15). However, as a consequence of their nonhuman origin, the use of these mAbs as immunotherapeutic drugs is limited. A clear progress in this area of research consisted of the development of the antibody humanization technology with the production of humanized versions of rodent antibodies (16) with reduced immunogenic potential. A humanized version of an anti-ErbB2 murine antibody (Herceptin) is currently in use for treatment of advanced breast cancer (17). Recently, fully human scFvs have been generated with the phage display technology through the expression of large repertoires of antibody variable regions on filamentous phages, after their fusion to a phage coat protein (18, 19). Human scFvs specific for ErbB2 have been produced, using for their selection the isolated recombinant extracellular domain (20, 21) or more recently breast tumor cells (22). Given their high affinity for the receptor, these immunoreagents may be considered precious tools as delivery vehicles for specifically directing cytotoxic agents to antigen-bearing tumor cells. However, none of these exhibit antitumor activity. Herein we describe the successful isolation from a large human scFv phagemid library (19) of a novel scFv directed against the ErbB2 receptor. We used a very effective parallel selection procedure based on intact cells either expressing high levels of the receptor antigen or virtually lacking the antigen. Recently, similar strategies have been successfully used by other Received 11/5/01; revised 2/20/02; accepted 3/8/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by grants from the Associazione Italiana per la Ricerca sul Cancro, Italy, and from the Medical Research Council, United Kingdom. D. B. P. is a recipient of an MRC Career Development Award. 2 These authors contributed equally to this work. 3 To whom requests for reprints should be addressed, at Department of Biological Chemistry, University of Naples Federico II, via Mezzocannone 16, 80134 Naples, Italy. Phone: 39-081-2534731; Fax: 39-0815521217; E-mail: [email protected]. 4 The abbreviations used are: EGF, epidermal growth factor; scFv, single-chain variable fragment; ph-A7, phages from clone A7 bearing the human anti-ErbB2 scFv; s-A7, the human anti-ErbB2 scFv in the soluble format; FACS, fluorescence-activated cell sorting; NIP, 4-hydroxy-3-nitro-5-iodophenyl-acetyl; VH, heavy chain variable region; mAb, monoclonal antibody; VL, light chain variable region; cfu, colony-forming unit. 1710 Vol. 8, 1710–1719, June 2002 Clinical Cancer Research Research. on July 17, 2017. © 2002 American Association for Cancer clincancerres.aacrjournals.org Downloaded from authors (23, 24). The human anti-ErbB2 single chain antibody was found to be endowed with biological properties not described for other anti-ErbB2 scFvs isolated thus far. This novel scFv is internalized upon specific antigen recognition by ErbB2expressing target cells; it strongly inhibits receptor autophosphorylation and displays a strong inhibitory activity on the growth of ErbB2-positive cell lines. In addition, a clear cytotoxic effect was evidenced toward ErbB2 hyper-expressing SKBR3 cells in which apoptotic death is induced. Interestingly, these features are present both in soluble scFv and in its phage format. Materials and Methods Cell Cultures. The SKBR3 human breast tumor cell line and the A431 human epidermoid carcinoma cell line (kindly provided by Menarini Research, Pomezia, Italy) were cultured in RPMI 1640 (Life Technologies, Inc., Paisley, United Kingdom). The BT-474 and MDA-MB453 human breast tumor cell lines (a kind gift of H. C. Hurst, Imperial Cancer Research Fund, London, United Kingdom), the SK-OV-3 human ovarian cell line (a kind gift of I. McNeish, Imperial Cancer Research Fund, London, United Kingdom), and the NIH-3T3 murine fibroblasts (American Type Culture Collection, Rockville, MD) were grown in DMEM (Life Technologies, Inc.). The NIH-3T3 fibroblast cell line transfected with human ErbB2, kindly provided by N. E. Hynes, Friedrich Miescher Institute, Basel, Switzerland (25), were cultured in DMEM containing 1 mg/ml G418 (Life Technologies, Inc.). Medium were supplemented with 10% FCS, 50 units/ml penicillin, and 50 g/ml streptomycin (all from Life Technologies, Inc.). Antibodies. The following antibodies were used in the current study: murine anti-M13 mAb (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom); murine mAb 9E10 directed against the myc tag protein (26); murine anti-His tag mAb (Qiagen, West Sussex, United Kingdom); murine antiErbB2 MgR6 mAb (gift from Menarini Research; Ref. 27); FITC-conjugated rabbit antimouse immunoglobulin antibody, and horseradish peroxidase-conjugated rabbit antimouse immunoglobulins (both from Dako, Cambridgeshire, United Kingdom); murine anti-phosphotyrosine mAb P-Tyr (PY99; Santa Cruz Biotechnology Inc., Santa Cruz, CA); and murine antiactin mAb (Sigma, St. Louis, MO). Anti-NIP (28) and anti-thyroglobulin scFv were kindly provided by Dr. G. Winter, Center for Protein Engineering, Cambridge, United Kingdom; gp200-MR6 scFv was isolated as described (29). Selections of scFv-Phage on Live Cells. ErbB2-positive cells were labeled as follows. The human breast tumor SKBR3 cell line, naturally expressing high levels of ErbB2, and the NIH-3T3 fibroblasts, transfected with human ErbB2, grown in 250-ml flasks (Becton Dickinson, Oxford, United Kingdom) to 70–80% confluency, were detached with the cell dissociation solution (Sigma) and washed twice with PBS. Cells were then resuspended in 1 ml of prewarmed PBS containing 15 M 5(6)-CFDA, SE (5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester mixed isomers; Molecular Probes, Eugene, OR), and incubated for 30 min at 37°C. After three washes with cold PBS, cells were resuspended (1 10 cells/ml), and the level of fluorescence analyzed by flow cytometry before each round of phage selection. Phagemid particles were rescued with M13-K07 from the Griffin library, as described previously (18). For each round of panning, phages (10 cfu) were blocked with 5% milk powder (Marvel) in PBS for 15 min. The blocked phages were incubated for 16 h at 4°C with labeled positive cells (1 10) in the presence of unlabeled negative cells (9 10) by gently rotating in a final volume of 5 ml containing 2% Marvel. Cells were then pelleted by centrifugation at 600 g for 5 min at 4°C and washed twice in 50 ml of PBS. The positive-labeled cells were sorted by FACS. To elute phages from positive cells, these were incubated with 0.5 ml PBS containing 50 mM citric acid (pH 2.5) for 5 min, and then neutralized with 0.4 ml of 1 M Tris-HCl (pH 7.4). The recovered phages were amplified by infecting Escherichia coli TG1 cells to prepare phage for the next round of selection. Phage screening was carried out by cell ELISA assays as described previously (30). Analysis of Clone Diversity. To determine the number of individual selected clones, DNA fingerprinting analysis was performed with the restriction endonuclease BstNI or BsaJI (New England Biolabs, Hertfordshire, United Kingdom) as described (18). DNA encoding the variable region of positive clones was amplified by PCR from the pHEN2 plasmid, using as primers 5 -CAGTCTATGCGGCCCCATTCA-3 (complementary to the sequence located between gene III and the c-myc peptide tag) and 5 -ATGAAATACCTATTGCCTACG-3 (pel B leader sequence). Reactions were performed with Taq DNA Polymerase (Promega, Southampton, United Kingdom) in a volume of 20 l for 30 cycles under the following conditions: 30 s denaturing at 94°C, 30 s annealing at 55°C, and 1 min extension at 72°C. The amplified products, analyzed by electrophoresis on 1% agarose gel, were used for DNA fingerprinting and sequence analyses. The nucleotide sequences encoding scFv were determined using the ABI automated sequencer (Perkin-Elmer, Warrington, United Kingdom) and were analyzed with the V-BASE sequence alignment program. Cell Lysis, Immunoprecipitation, and Western Blot Analyses. SKBR3 cell lysates were prepared by resuspending in 0.5 ml of lysis buffer [10 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.5% NP40 containing Complete proteases inhibitor; Boehringer Mannheim, Germany] 7.5 10 cells, detached previously with the cell dissociation solution (Sigma), and washed three times in PBS. After 20 min at 0°C the extracts were clarified by centrifugation at 12,000 rpm for 10 min. ErbB2 immunoprecipitation was carried out by incubating the anti-ErbB2 MgR6 mAb with the cell lysates for 3 h at 4°C. The immune complex was then collected by adsorption to protein G-Sepharose (Sigma) for 1 h at 4°C. After four washes, the proteins, released by boiling in loading buffer (31), were fractionated by 7.5% SDS-PAGE and electroblotted onto poly5 Tomlinson, I. M., Williams, S. C., Corbett, S. J., Cox, J. P. L., and Winter, G. The V BASE Directory of Human Variable Gene Sequences. MRC Centre for Protein Engineering, Hills Road, Cambridge, CB2 2QH, United Kingdom. Internet address: http://www.mrc-cpe.cam. ac.uk/imt-doc/vbase-home-page.html, 1996. 1711 Clinical Cancer Research Research. on July 17, 2017. © 2002 American Association for Cancer clincancerres.aacrjournals.org Downloaded from vinylidene difluoride membranes (Millipore Corporation, Bedford, MA). The ErbB2 protein was detected using either antiErbB2 mAbs or scFv-phage preparations, as described