MT110: A novel bispecific single-chain antibody construct with high efficacy in eradicating established tumors
Introduction
The successful treatment of late-stage cancer is one of the greatest challenges in modern medicine. This is reflected by the still unchanged average mortality rate in cancer during the past decades in industrialized countries (Antunes et al., 2003, Hoel et al., 1992, Howe et al., 2001). The invasion of organs by metastasizing tumor cells, the establishment of large tumor masses that are barely accessible to therapeutics, the genetic heterogeneity of tumor cells and the evasion of tumor cells from immune recognition by a multitude of escape mechanisms are major reasons why surgical, radiation and cytotoxic therapies in most cases are not curative but at best can retard disease progression.
Because tumor cells share all genes with normal cells, only subtle differences to normal cells will manifest which can be exploited for targeted therapies designed to very selectively eliminate or control tumor cells. Enhanced proliferation, altered metabolism, aberrant protein expression and covalent modification, mutations and altered splicing may change tumor cell surface immunogenicity, which can potentially provide useful target structures for antibody-based therapies. However, there is always the limitation that not all tumor cells show the targeted phenotype, and not all tumor cells are physically accessible to the targeted therapy. Recent years have shown that targeted tumor therapy by monoclonal antibodies of the human IgG1 isotype can lead in certain patients to significant anti-tumor responses that frequently profit from a co-therapy with standard chemotherapy or radiation (Davis et al., 2004, Hurwitz et al., 2004, Matar et al., 2004, Pegram et al., 2004). Examples are antibodies recognizing overexpressed epidermal growth factor receptors EGFR (cetuximab) (Khalil et al., 2003) and HER-2 (trastuzumab) (Vogel et al., 2001) used for treatment of solid tumours, and differentiation antigens CD20 (rituximab) (Grillo-Lopez et al., 1999, Smith, 2003), CD52 (alemtuzumab) (Faulkner et al., 2004, Hale et al., 1998, Kottaridis et al., 2000) and CD22 (epratuzumab) (Furman et al., 2004, Leonard et al., 2004) used for blood-bourne cancers. Many studies have suggested that antibody-dependent cellular cytotoxicity (ADCC) is a major contributor to the therapeutic efficacy of monoclonal antibody therapies (Clynes et al., 2000, Gennari et al., 2004, Maloney, 2001, Nagler et al., 1997).
ADCC is mediated via a bispecific function of IgG1. While the two antigen binding arms fix the antibody to the tumor-associated antigen, the Fc part of IgG1 allows to transiently tether and simultaneously activate cytotoxic immune cells bearing Fcγ receptors. Most important seem FcγR III/CD16-expressing natural killer (NK) cells, as is evident from the impact of genetic polymorphisms in CD16 on therapeutic efficacy of rituximab (Cartron et al., 2002, Dall’Ozzo et al., 2004, Weng and Levy, 2003). The continuous treatment with IgG1 for months at fairly high serum trough levels may be necessary for tumor penetration of both antibody and immune cells, a permanent decoration of tumor cells with the antibody and for the time-consuming seek-and-destroy mechanism performed by cytotoxic immune cells.
Another important class of immune cells with the potential to eradicate even large tumors are T cells (Dudley and Rosenberg, 2003, Dudley et al., 2005, Glimcher et al., 2004), which cannot be recruited by monoclonal antibodies because they lack Fcγ receptors. The increasing number of T cell escape mechanisms discovered in late-stage tumor cells impressively underscores the threat this class of immune cells is posing to tumor cells (Foss, 2002). During the past two decades, bispecific antibodies recruiting T cells have been developed (Baeuerle et al., 2003, Kufer et al., 2004), which may have the potential to circumvent some of these escape mechanisms. By binding with one arm to a common T cell signaling protein, such as CD3 and with the other arm to a tumor-associated antigen on the target cell, bispecific antibodies should theoretically elicit a polyclonal T cell response against antibody target-expressing tumor cells. Such a polyclonal response should be largely independent of regular T cell recognition molecules, such as MHC class I, a specific T cell receptor, costimulatory proteins and peptide antigens along with their processing and transport molecules. One class of bispecific antibodies with very suitable properties are bispecific CD3-specific single-chain antibody constructs, also referred to as ‘bispecific T cell engager’ (BiTE) (Baeuerle et al., 2003, Kufer et al., 2004). A CD19/CD3-bispecific BiTE has been characterized in great detail and currently is in phase I clinical trials (Dreier et al., 2003, Dreier et al., 2002, Loffler et al., 2000). Here, we report on the construction and characterization of a novel BiTE molecule called MT110 designed for the treatment of human carcinomas.
As tumor-associated antigen for recognition by MT110, we have selected the epithelial cell adhesion molecule (Ep-CAM), which is among the best characterized targets for immunotherapeutic approaches including monoclonal antibody therapies and vaccination strategies (Balzar et al., 1999, Mosolits et al., 2004, Naundorf et al., 2002, Prang et al., 2005). A particular advantage of Ep-CAM is its frequent overexpression on almost all human carcinomas (Moldenhauer et al., 1987, Momburg et al., 1987) and limited accessibility on normal epithelial tissues (Balzar et al., 1999). By targeting Ep-CAM, MT110 has the potential to be effective in a wide range of human carcinomas. A functional role of Ep-CAM overexpression in tumor proliferation, invasion and migration was recently shown for breast cancer cells (Munz et al., 2004, Osta et al., 2004), which may relate to a functional antagonism of Ep-CAM with E-cadherin (Winter et al., 2003). The finding explains why breast cancer patients with tumors overexpressing Ep-CAM have a poor overall survival (Spizzo et al., 2002, Spizzo et al., 2004).
The present study shows that MT110 is a stable and homogenous protein that shares all particular BiTE properties with another previously described BiTE molecule, bscCD19 × CD3 (Dreier et al., 2003, Dreier et al., 2002, Loffler et al., 2000). These properties include high in vitro and in vivo efficacy, and induction of lytic activity with previously unstimulated peripheral T cells at low effector-to-target ratios. Here, we also demonstrate polyclonal activation of CD8- and CD4-positive T cells by MT110, which required the presence of target-expressing cells. MT110 was highly active in two NOD/SCID mouse efficacy models. By either redirecting co-administered human T cells or tumor-resident T cells, low micrograms doses of MT110 completely prevented tumor outgrowth or led to eradication of subcutaneously growing solid tumors derived from a SW480 human cancer line and from human metastatic ovarian cancer tissue.
Section snippets
Selection of MT110 from a panel of Ep-CAM-specific BiTE constructs
EpCAM-specific single-chain antibodies used here for the generation of a panel of Ep-CAM-specific BiTE constructs were either isolated as single-chain antibodies from phage display libraries (Raum et al., 2001), or constructed from VH and VL domains cloned from various hybridoma lines. A total of 21 different Ep-CAM-specific BiTE constructs were made, expressed, purified and compared for a set of specifications deemed important for development of a highly active biological drug (see Section 3).
Generation and characterization of MT110
MT110 was selected from a panel of 21 different Ep-CAM-specific BiTE molecules based on characteristics considered to be important for a clinical development candidate. In a first round, a total of seven human Ep-CAM-specific single-chain antibodies (SCAs) were used to construct bispecific T cell engager (BiTEs) using the same human CD3ɛ-specific SCA. Ep-CAM-specific SCAs were derived either from VH and VL domains of murine monoclonal antibodies of hybridomas or isolated as SCAs from phage
Discussion
We have developed a new Ep-CAM-specific BiTE molecule with properties deemed suitable for the development of a novel therapeutic with respect to potency, in vivo efficacy, specificity, target affinity, productivity, charge homogeneity and stability. MT110 is superior in several aspects to a previously described Ep-CAM-specific BiTE molecule called bscEp-CAM × CD3 (alternative names: MT102, M79 × CD3) (Flieger et al., 2000, Kufer et al., 1997, Mack et al., 1997, Mack et al., 1995, Schlereth et al.,
Acknowledgements
This work was fully funded by Micromet AG. The authors thank Monika Becker for excellent technical assistance in the animal experiments and Dr. B. Elbe for immunohistochemistry.
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Present address: Direvo Biotech AG, 50829 Cologne, Germany.