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cancer gentics project 3 excerpt 5

Submitted by jdantonio on Thu, 05/04/2017 - 18:59

We will augment the endogenous TIL lines by the creation of additional cell lines that have genetically engineered chimeric antigen receptors (CAR). These chimeric receptor will be designed to bind to one of the remaining 10 most prevalent neoantigens that we identified as previously described. We will create ten cell lines, each being given one of chimeric receptors, creating ten cell lines which each bind to a different neoantigen. We will introduce a gene which encodes for a genetically engineered T-cell receptor by a viral vector in vitro. The CTR will be  created by the fusion of the Vh and VL gene sequence segments of a monoclonal antibody that binds to our neoantigen, a hinge-Ch2-Ch3 IgG1 , a transmembrane CD4 domain, and zeta chain of the CD3 cytoplasmic domain (Park et al 2007). This creates a chimeric TCR that recognises one of our discovered neoantigen and can activate T-cell activation (Park et al 2007). The monoclonal antibodies that bind to our neoantigens will be created by using a generalized method of immunization of rodent models with our neoantigens, the rodents will create antibodies against the neoantigen and they will be harvested and screened in a manner similar to the adoptive cells for bind specificity to the neoantigen (Gerdes et al 1983). These antibody are then used to make the gene fragment that is placed in the chimeric vector. We would perform this procedure for the 10 most common neoantigens for our tumor that were not found to have endogenous TIL’s during the adoptive cell therapy. The chimeric gene would be delivered to the cells in vitro by an IVT (in vitro transcription) RNA vector which is one of the standard delivery systems for CARs (Zhao et al 2010). Once the viral vector has been incorporated into the T-Cells we would then modify them further by using CRISPR to knockout the PDL-1 and CTLA-4 receptors on the cells(Albain et al 2015).


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Park JR, DiGiusto DL, Slovak M, Wright C, Naranjo A, Wagner J, Meechoovet HB, Bautista C, Chang WC, Ostberg JR, Jensen MC. 2007. Adoptive Transfer of Chimeric Antigen Receptor Re-directed Cytolytic T Lymphocyte Clones in Patients with Neuroblastoma. Molecular Therapy 15(4):  825–833. Science Direct. <>. Accessed 2017 May 1.


Zhao Y, Moon E, Carpenito C, Paulos CM, Liu X, Brennan AL, Chew A, Carroll RG, Scholler J,

Levine BL, Albelda SM, June CH. 2010. Multiple injections of electroporated autologous

T cells expressing a chimeric antigen receptor mediate regression of human disseminated

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