Study On The Expression Of HER2-targeted Chimeric T-cell Receptor Gene In Lymphohemopoietic Cells And Its Anti-tumor Effects

The HER2/neu oncogene is overexpressed in a variety of human malignantdisorders, including breast cancer, ovarian cancer, colon cancer, pancreatic cancer,and non-small cell lung cancer. Several lines of evidence suggest that p185HER2 isan excellent target for immune based therapy of breast cancer. HER2/neu isamplified or overexpressed in approximately 20-40% of newly diagnosed humanbreast cancers. Overexpression of HER2/neu is a predictive marker of therapeuticresponse, identifying patients at increased risk for early disease relapse and reducedoverall survival. Studies in cultured cell lines and animal models suggest a directrole for HER2 in the pathogenesis and aggressive clinical behavior of these tumors.Antibodies specific for p185HER2 inhibit the growth of cell lines and tumorsexpressing elevated levels of the receptor. Clinical trials of anti-p185HER2 mAb(Herceptin), administered to women with metastatic breast cancer, have documentedclinical responses with minimal toxicity. Chimeric T-cell receptors (chTCR) are recombinant immune receptorscomposed of two domains - an extracellular antigen binding domain and anintracellular signaling domain. The most commonly employed antigen targetingdomain is a single chain Fv (scFv), whereby the VL and VH domains of a monoclonalantibody (mAb) are tethered in a single polypeptide by a flexible linker. scFvregions exhibit similar specificities and affinities compared to normal antibodyvariable regions. Cellular activation is achieved by coupling the antigen bindingdomain to an intracellular signaling chain. The ζ-chain of the T-cell receptor andthe FcεR1γ-chain are closely related and are capable of activating T-cells and/ormyeloid cells when incorporated in chimeric receptor constructs. Thus, in a singlemolecule, the scFv-ζ/γ chTCR combines the exquisite antigen specificity of a mAband signal transduction elements necessary to activate effector cell function. Theantigenic target is an intact cell surface protein defined by a mAb. A significantpotential advantage of this design is the lack of MHC restriction and/or need forantigen processing. Such chTCR molecules have been successfully developed to target a variety oftumor antigens, including the Mov18-defined ovarian tumor antigen, TAG-72, CEA,and p185HER2. Introduction of the scFv-ζ/γ construct into T cell lines orhybridomas has resulted in a functioning chTCR with antigen specificity, as definedby cytokine release and/or cytotoxicity following stimulation of the transducedeffector cells with the appropriate antigen-bearing target cell, and antigen specificregression of tumor in animal models of T-cell adoptive immunotherapy.Objective: 1. To demonstrate if the anti-CD3/anti-CD28 activated human or murineT-cells could be efficiently transduced with a p185HER2 specific chTCR constructs. 2. To investigate if retroviral gene transfer of a chTCR could efficientlyredirects primary human T-cell responses to p185HER2 positive tumor cells. 3. To compare two different signal transduction chains in our construct side byside and find out which chain is more efficient regarding cytokine release andcytotoxicity. 4. To define the antigen specific anti-tumor effects carried by the chTCR genemodified hematopoietic stem cells in vivo in a mouse model.Methods and results: We have developed and tested chimeric T-cell receptors (TCR) specific forp185HER2, the product of the HER2/neu gene. Retroviral vectors expressing theN29γ or N29ζ receptors were constructed in pRET6, a bicistronic MPSV-basedbackbone developed in our laboratory. Amphotropic viral producer cells wereestablished in the GALV-based PG13, GP+E86 packaging cell line. Ficoll purifiedhuman peripheral blood lymphocytes (PBL) and murine spleen lymphocytes werevirally transduced using an optimized protocol incorporating activation withimmobilized anti-CD3/anti-CD28 monoclonal antibodies, followed by viral infectionin the presenc...