Heidi H. Simmons

Isolation and characterization of an anti-CD16 single-chain Fv fragment and construction of an anti-HER2/neu/anti-CD16 bispecific scFv that triggers CD16-dependent tumor cytolysis.

Bispecific antibody (bsAb)-based clinical trials of cancer have been conducted primarily using intact murine monoclonal antibody (mAb)-derived molecules. In some of these trials, toxicity resulting from the interactions of antibody Fc domains with cellular Fc receptors has limited the doses of antibody (Ab) that can be employed. Furthermore, human anti-mouse Ab responses prohibit multiple therapy courses. These factors have decreased the efficacy of the bsAb 2B1, which targets the extracellular domains (ECD) of the HER2/neu protooncogene product and the human FcgammaRIII (CD16). To address these obstacles, we have constructed and characterized a fully human gene-fused bsAb from single-chain Fv (scFv) molecules specific for HER2/neu and CD16. The human anti-CD16 scFv component, NM3E2, was isolated from a human scFv phage display library. As binding of NM3E2 to human neutrophil-associated CD16 decreased in the presence of plasma IgG, we have concluded that NM3E2 recognizes an epitope in the vicinity of the Fc binding pocket. Furthermore, the NM3E2 scFv was found by surface plasmon resonance-based epitope mapping to share an overlapping epitope with the Leu-11c mAb. The human anti-HER2/neu scFv component, C6.5, which was previously isolated from a human scFv phage display library, was employed as fusion partner for the creation of a bispecific scFv (bs-scFv). In the presence of the C6.5 x NM3E2 bs-scFv, peripheral blood lymphocytes promoted significant lysis of human SK-OV-3 ovarian cancer cells overexpressing HER2/neu. Biodistribution studies performed in SK-OV-3 tumor-bearing scid mice revealed that 1% ID/g of 125I-labeled C6.5 x NM3E2 bs-scFv was specifically retained in tumor at 23 h following injection. These results indicated that both scFv components of the bs-scFv retained their function in the fusion protein. This bsAb should overcome some of the problems associated with the 2B1 bsAb. C6.5 x NM3E2 bs-scFv offers promise as a platform for multifunctional binding proteins with potential clinical applications as a result of its human origin, lack of an Fc domain, ease of production, high level of in vitro tumor cell cytotoxicity and highly selective tumor targeting.

Increasing the Affinity for Tumor Antigen Enhances Bispecific Antibody Cytotoxicity

We tested the hypothesis that bispecific Abs (Bsab) with increased binding affinity for tumor Ags augment retargeted antitumor cytotoxicity. We report that an increase in the affinity of Bsab for the HER2/neu Ag correlates with an increase in the ability of the Bsab to promote retargeted cytotoxicity against HER2/neu-positive cell lines. A series of anti-HER2/neu extracellular domain-directed single-chain Fv fragments (scFv), ranging in affinity for HER2/neu from 10−7 to 10−11 M, were fused to the phage display-derived NM3E2 human scFv. NM3E2 associates with the extracellular domain of human FcγRIII (CD16). The resulting series of Bsab promoted cytotoxicity of SKOV3 human ovarian carcinoma cells overexpressing HER2/neu by human PBMC preparations containing CD16-positive NK cells. The affinity for HER2/neu clearly influenced the ability of the Bsab to promote cytotoxicity of 51Cr-labeled SKOV3 cells. Lysis was 6.5% with an anti-HER2/neu KD = 1.7 × 10−7 M, 14.5% with KD = 5.7 × 10−9 M, and 21.3% with KD = 1.7 × 10−10 M at 50:1 E:T ratios. These scFv-based Bsab did not cross-link receptors and induce leukocyte calcium mobilization in the absence of tumor cell engagement. Thus, these novel Bsab structures should not induce the dose-limiting cytokine release syndromes that have been observed in clinical trials with intact IgG Bsab. Additional manipulations in Bsab structure that improve selective tumor retention or facilitate the ability of Bsab to selectively cross-link tumor and effector cells at tumor sites should further improve the utility of this therapeutic strategy.

A Single Treatment of Yttrium-90-labeled CHX-A″–C6.5 Diabody Inhibits the Growth of Established Human Tumor Xenografts in Immunodeficient Mice

Antitumor diabody molecules are noncovalent single-chain Fv dimers that recapitulate the divalent binding properties of native IgG antibodies. Diabodies are capable of substantial accumulation in tumor xenografts expressing relevant antigens in immunodeficient mouse models. With a Mr of approximately 55,000, diabodies are rapidly cleared from the circulation, resulting in tumor-to-blood ratios that significantly exceed those achieved early after the administration of monoclonal antibodies. We have evaluated the therapeutic potential of the β-emitting isotope yttrium-90 (t1/2, 64 hours) conjugated to the C6.5K-A diabody that specifically targets the HER2/neu human tumor-associated antigen. We have found that a single intravenous dose of 150 μCi (200 μg) 90Y-CHX-A″–C6.5K-A diabody substantially inhibits the growth rates of established MDA-361/DYT2 human breast tumor xenografts in athymic nude mice. In contrast, 300 μCi (300 μg) 90Y-CHX-A″–C6.5K-A diabody resulted in only a minor delay in the growth of SK-OV-3 human ovarian cancer xenografts. The maximum tolerated dose was also dependent on the tumor xenograft model used. These studies indicate that genetically engineered antitumor diabody molecules can be used as effective vehicles for radioimmunotherapy.

Isolation of anti-MISIIR scFv molecules from a phage display library by cell sorter biopanning

While cell surface antigens represent the most common targets for antibody-based cancer therapy, isolation of new antibodies specific for these targets from single-chain Fv phage display libraries has been hindered by limitations associated with traditional selection techniques. Solid phase panning is often associated with conformational changes to the target protein due to its immobilization on plastic tubes that can limit the ability of the isolated scFv to bind to conformational epitopes and solution panning methods require the use of secondary tags that often mask desired sequences and create unintended epitopes. Commonly utilized cell-based panning methods typically yield a panel of single-chain Fv (scFv) molecules that are specific for numerous cell surface antigens, often obscuring the desired clones. Here, we describe a novel cell sorter-based system to isolate single-chain Fv molecules specific for defined antigen targets expressed on stably-transformed mammalian cells. We employed these methods to isolate promising scFv clones that bind specifically to the Müllerian inhibiting substance type II receptor, a cell surface ovarian cancer antigen that has proven to be a difficult target for selection strategies.

Identification of inhibitory scFv antibodies targeting fibroblast activation protein utilizing phage display functional screens

Fibroblast activation protein (FAP) is a serine protease selectively expressed on tumor stromal fibroblasts in epithelial carcinomas and is important in cancer growth, adhesion, and metastases. As FAP enzymatic activity is a potent therapeutic target, we aimed to identify inhibitory antibodies. Using a competitive inhibition strategy, we used phage display techniques to identify 53 single‐chain variable fragments (scFvs) after three rounds of panning against FAP. These scFvs were expressed and characterized for binding to FAP by surface plasmon resonance and flow cytometry. Functional assessment of these antibodies yielded an inhibitory scFv antibody, named E3, which could attenuate 35% of FAP cleavage of the fluorescent substrate Ala‐Pro‐7‐amido‐4‐trifluoromethylcoumarin compared with nonfunctional scFv control. Furthermore, a mutant E3 scFv was identified by yeast affinity maturation. It had higher affinity (4‐fold) and enhanced inhibitory effect on FAP enzyme activity (3‐fold) than E3. The application of both inhibitory anti‐FAP scFvs significantly affected the formation of 3‐dimensional FAP‐positive cell matrix, as demonstrated by reducing the fibronectin fiber orientation from 41.18% (negative antibody control) to 34.06% (E3) and 36.15% (mutant E3), respectively. Thus, we have identified and affinity‐maturated the first scFv antibody capable of inhibiting FAP function. This scFv antibody has the potential to disrupt the role of FAP in tumor invasion and metastasis.—Zhang, J., Valianou, M., Simmons, H., Robinson, M. K., Lee, H.‐O., Mullins, S. R., Marasco, W. A., Adams, G. P., Weiner, L. M., Cheng, J. D. Identification of inhibitory ScFv antibodies targeting fibroblast activation protein utilizing phage display functional screens. FASEB J. 27, 581–589 (2013). www.fasebj.org

Abstract 2437: Engineered human monoclonal antibodies targeting the Müllerian inhibiting substance type II receptor for ovarian cancer therapy

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Currently available approaches for ovarian cancer therapy have limited success in the setting of advanced disease. Antibody-based therapeutics offer new and potentially effective strategies. Mullerian inhibiting substance (MIS, or anti-Mullerian hormone, AMH) is known for its role in embryonic development, triggering regression of the Mullerian ducts in mammalian males (in females Mullerian ducts are the source for uteri, Fallopian tubes and upper vagina). MIS signals via a heterodimeric receptor, consisting of type I (MISIR) and type II (MISIIR) receptors, where MISIIR mediates the ligand specificity and MISIR triggers the downstream signaling cascade. Approximately 64% of epithelial ovarian cancers express MISIIR with the most common expression in serous and mucinous tumors (Bakkum-Gamez et. al., Gynecol. Oncol. 2008;108(1):141). MIS has been reported to inhibit the growth of tumor xenografts in vivo (Stephen et al., Clin. Cancer Res. 2002;8(8):2640) and to trigger apoptosis of primary human ovarian cancer cells in vitro (Masaikos et al., Clin. Cancer Res. 1999;5(11):3488). This, along with the highly restricted expression pattern of MISIIR in normal tissues, suggests that MISIIR is a promising target for ovarian cancer therapy. A number of single-chain Fv molecules (scFv) and their bivalent derivatives (scFv: Fc constructs) targeting MISIIR were developed in our lab by means of screening of two large naive human scFv phage display libraries. However, to date, the potential clinical utility of these anti-MISIIR scFv molecules has been limited due to their low binding affinity. Here, we describe the screening of a third human scFv phage display library against a fusion protein representing the extracellular domain of MISIIR with the Fc domain of human IgG1 fused to its C-terminus. This screening led to selection of six unique scFv clones that specifically target MISIIR on the surface of cells with varying efficiency. One of these scFv clones, GS45, has demonstrated excellent targeting of MISIIR by flow cytometry, ELISA and surface plasmon resonance analysis. We are currently cloning GS45 into a full-length IgG molecule in order to improve its potential clinical utility. Antibodies (or antibody-based constructs, such as scFv or scFv: Fc) targeting MISIIR could be used as the basis of a variety of therapeutic strategies. They can be used on their own to induce agonistic signaling or direct antibody-dependent cell-mediated cytotoxicity (ADCC). Alternatively, MISIIR-targeting antibodies can be conjugated to cytotoxic drugs, radiolabels or imaging agents. The anti-MISIIR scFv GS45, described here, is currently being developed for ADCC and immunodrug conjugate applications. We believe it can be the basis of novel therapeutic strategies for ovarian cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2437.