Project: A breast cancer toolbox: HER2-targeted immunoconjugates as therapeutic and diagnostic tools

Therapeutic recombinant antibodies mediate antineoplastic effects mostly by ‘direct’ interference with signal transduction pathways of target cells. ‘Indirect’ effects, such as the recruitment of host immune effectors, although believed to be important, reCO ill-defined. TOOLBOX candidates to exploit unprecedented synergies among antiproliferative antibody fragments, anticancer drugs, and immune effectors, hopefully obviating the pitfalls of secondary, adoptive and passive (immuno)therapy protocols, including vaccination._x000D__x000D_TOOLBOX contains recombinant therapeutic proteins, engineered drugs and cytotoxic T lymphocytes (CTL). These are combined in a multi-step approach depicted in Annex 1, Fig. 1. In the first step, a Single chain Fragment of variable antibody regions (ScFv) to the protooncogene product ErbB-2 is used for tumor targeting. This ScFv binds tumor cells and conveys a secure and universal docking site (in the form of a Strep-tag®, e.g. a small 8-amino acid epitope) that allows the specific binding of second-step reactants. These are multimers of a Streptavidin mutant called StrepTactin®, e.g. a multivalent complex of recombinant proteins capable of very high-affinity, reversible interactions with the Strep-tag. Multivalent StrepTactin® is the ideal reagent to effectively bridge ScFv-coated tumor cells to a variety of Strep-tagged anticancer agents, since it provides a high and customisable number of Strep-tag binding sites._x000D__x000D_Anticancer agents provided in the third and fourth steps are of two kinds. The first kind comprises clinically useful, established therapeutic drugs (e.g. anthracyclines and/or antiproliferative steroids) as well as newly discovered, proprietary antitumor compounds for which the Pship holds appropriate technology and freedom to operate. All these drugs bind the available valences of ScFv:StrepTactin complexes present on the tumor cell surface because they are themselves Strep-tagged. The second kind of reagents are Streptamers®, e.g. class I Human leukocyte Antigen (HLA) molecules refolded in vitro in the presence of strong viral antigens that function as docking sites for high-affinity, naturally occurring CTLs. Unlike weak, naturally occurring anti-tumor CTLs, antiviral CTLs are among the strongest known immune effectors. Their redirection would provide a quantum lap in tumor killing. The ultimate goal is to achieve an unprecedented therapeutic synergy among anticancer agents, thereby boosting the direct antitumor effect of the selected ScFv._x000D__x000D_TOOLBOX is tumor-specific, potent (4 distinct classes of drugs), multistep, modular (the independent optimization of each step is possible), versatile (many tools are available to customize each step), flexible (it contains an off-the shelf arsenal of anticancer drugs ready to be redirected in the patients), reversible (upon administration of soluble small biotin analogues), safe (all the reagents are either recombinant or synthesized by straightforward chemistry approaches), and low-cost (the ScFv may be produced in yeast). TOOLBOX permits the targeted delivery of virtually any taggable small anti-tumor molecule and/or effector cell as single agent, in combination, and in sequence. It is open to further implementation with presently unforeseen anticancer agents. TOOLBOX allows trial and error through the rapid switch among different drugs, and enables their selective concentration at the tumor site with no need to increase the total dosage._x000D__x000D_These features open the possibility of ethical applications, particularly in patients with highly aggressive breast carcinomas, like those bearing pleural neoplastic effusions. The general conditions these patients often discourage toxic and/or prolonged regimens, making TOOLBOX particularly suitable for loco-regional ex vivo administration. This project will hit phase 0 (ex vivo administration and monitorization of chemo/biological combination therapy). Phase I clinical trials (high-dosage intracavitary therapy) are foreseen to start right after project completion. _x000D__x000D_TOOLBOX is at the crossroad of several overlapping markets: recombinant therapeutics, controlled drug delivery, chemotherapy, immunotherapy, and therapeutical vaccines. It seeks to introduce substantial improvement in each of these areas. TOOLBOX widens the highly profitable market of therapeutic antibodies, and will be integrated by inexpensive, outcome-predictive ELISA/immunohistochemistry kits based on inexpensively produced ScFvs and streptactin multimers, rather than expensive antibodies produced by hybridomas._x000D__x000D_The consortium comprises one SME (IBA), a large pharmaceutical company (IBI), and two academical institutions (UNIJENA and IRE) from Germany and Italy. TOOLBOX is based on proprietary reagents (ScFv), patents (strep -tagnology), as well as unique industrial and academic expertise. The Pship comprises molecular biologists, medicinal chemists, immunologists, and medical oncologists.

Acronym	TOOLBOX (Reference Number: 5995)
Duration	01/10/2011 - 31/03/2015
Project Topic	Low-cost combination therapy will be administered by a novel, multi-step tumor targeting approach employing 3 sets of fully proprietary, patented reagents: (a) tagged antibody fragments, (b) tunable and reversible tag adaptors, and (c) tag-modified anticancer drugs and immune cells
Project Results (after finalisation)	A proof-of-concept in cell culture and first data in a mouse model that an immuntoxin using HER2/neu as a target for a desigend and refined single chain antibody fragment equipped with a Strep-tag and produced in Pichia and connected via a StrepTactin moiety to a Strep-tagged toxin or immuno active cells are able to fight tumor cells overexpression the target molecule
Network	Eurostars
Call	Eurostars Cut-Off 5

Project partner

Number	Name	Role	Country
4	IBA Gmbh	Coordinator	Germany
4	Istituto Biochimico Italiano G. Lorenzini S.p.A.	Partner	Italy
4	Italian National Cancer Institute Istituto Regina Elena IRE (IFO)	Partner	Italy
4	Universitätsklinikum Jena, University of Jena	Partner	Germany