BSRT Graduate School

Project 3

Building a Platform for Fast Track Development and Characterization of Engineered Antigen Receptors

Wagner, Dimitrios Laurin
Supervisor 1: 
Schmück-Henneresse, Michael
Supervisor 2: 
Volk, Hans-Dieter

Redirecting T lymphocytes using chimeric antigen receptors (CARs) is a powerful tool in the emerging field of regenerative medicine. T cells reprogrammed to recognize and kill CD19 positive cancer cells dramatically improved leukemia and lymphoma treatment, whereas the transfer of CARs specific for allogeneic antigens (Ag) into regulatory T cells was shown to induce immune tolerance in transplantation models. Recent reports suggest CARs may facilitate innovative treatments for autoimmune diseases as novel receptor designs enable CAR-T mediated depletion of pathogenic auto-Ag specific B cells and prevent autoantibody (Ab) mediated diseases (1).


CARs are fusion proteins made of a target-specific extracellular domain (usually a single chain variable fragment derived from an Ab) and an intracellular signal transduction region (varying costimulatory domains and a CD3 zeta chain) which imitate T cell receptor (TCR) signaling. Optimal signaling strength of CARs is paramount for T cell activation and the clinical success, while binding affinity/avidity and minimal tonic signaling are also of importance (2). Surprisingly, careful evaluation of CAR signaling properties is rarely performed and no standardized assays have been described. Existing assays to predict in vivo CAR-T cell function are limited to tumor-specific CARs and rely on lengthy serial co-culture experiments. Thus, they are not practical for fast iterative optimization of CAR design.


We hypothesize that signaling properties of a given CAR can predict its in vivo performance. To this end, we plan to create a reporter cell line that generates an optical output in response to Ag-receptor signaling. In Jurkat cells, which are a standard model to study TCR signaling, the expression of a variety of genes is induced upon TCR ligation, some of which correlate exclusively and gradually with Ag-mediated TCR signaling strength.


To obtain a good reporter system, Jurkat cells will be genetically modified by inserting a reporter green fluorescent protein (GFP) transgene into a TCR-inducible gene via CRISPR/Cas9 (3). Ag-receptor (TCR or CAR) signaling will lead to the expression free GFP through the self-cleaving activity of a P2A linker (Fig. 1). CAR constructs to be tested can be readily transferred to the reporter cells via the delivery system of choice (retroviral, transposon, site-specific integration). After subsequent Ag stimulation, fluorescence intensity of CAR-mediated signaling will be detected by flow cytometry over multiple time points. Results from TCR/CD19 CAR signaling will serve as performance benchmarks.


We propose to name this platform FLECS as an acronym for Flow cytometric Evaluation of CAR Signaling. It will enable standardized mass-testing of new CAR constructs and ensure the selection of the most efficient CAR for any given Ag. It will be deposited in public cell banks and shared openly to advance CAR-redirected T cell therapy in regenerative medicine.


After establishing the FLECS platform, we will focus on the development of CARs for the severe, rapidly exacerbating, demyelinating, Multiple Sclerosis (MS)-like disease Neuromyelitis Optica (NMO). In NMO, auto-Abs against immunogenic self-Ag are thought to play a causative role. Similar to a recent report (1), we designed novel antigen receptors that incorporate the immunogenic epitope derived from the self-Ag to form a chimeric auto-Ab receptor (CAAR). Thus, cytotoxic T cells will be able to recognize B cells that display surface bound immunoglobulin (sIgG) specific for the auto-Ag (Fig.2). After FLECS analysis, the CAAR with best performance will be subjected to further functional analysis in vitro and in the mouse model. Eventually, targeted elimination of auto-Ag specific B cells may offer a novel therapeutic approach to prevent chronic inflammation through auto-Ab in NMO, while FLECS might facilitate the development of similar therapeutics for other autoimmune diseases.


We are looking for a highly motivated person to take on this diverse project. Due to the interdisciplinary nature of the project, the candidate should be a team player and fluent in English. Solid knowledge in T cell immunology and a general understanding of molecular biology will be needed. Documented interest in molecular genetics and biostatistics are necessary. Medical knowledge or an understanding of disease pathology in humans is appreciated. Other Skills, that are required:

  • Advanced experience in flow cytometry (independent data acquisition and basic analysis)
  • Completed lab animal training for mice (certified according to FELASA B or equivalent)
  • Prior experience in molecular biology methods (DNA/RNA purification, PCR, plasmid isolation and transformation, ELISA, quantitative protein analysis)


Figure 1) Flow cytometric Evaluation of CAR Signaling (FLECS) platform for inexpensive, standardized in vitro assessment of CAR function.

Fig. 1: a) In reporter cells, T cell receptor (TCR) or chimeric antigen receptor (CAR) signaling activates transcription of a TCR-inducible gene which is fused to a GFP reporter transgene via a P2A linker. Upon translation, GFP is freed by self-cleavage of the P2A linker; b) anti-CD3 antibody activates TCR, leading to GFP expression (pos. control); c) CD19-specific CAR is activated in CD19 antigen-coated well leading to GFP expression (pos. control); d) Upon binding their specific antigen (Ag), test CAR constructs may vary in signaling strength and consecutive GFP expression; e) test CARs in the absence of their specific antigen ideally show no/little basal GFP expression. CD19-CARs in absence of their antigen may serve as neg. controls; f) Hypothetical results of flow cytometric analysis of GFP expression for positive controls, test CARs 1-4 (with differing signaling properties) and a negative control. (Fig. generated on



Figure 2) Chimeric auto-antibody receptor design for Neuromyelitis Optica/Multiple Sclerosis

Fig. 2: Chimeric auto-antibody receptors (CAARs) mediate recognition of pathogenic B cells by engineered CAAR-T cells through binding to surface-bound immunoglobulins (sIgG) specific for an immunogenic self-antigen. The CAAR consists of a peptide derived from the auto-antigen as its extracellular recognition domain which interacts with pathogenic auto-antibodies, a hinge domain for optimal intermembrane distance of the immunological synapse, a CD8 transmembrane (TM) domain to facilitate dimerization, a 4-1BB costimulatory domain and a CD3 zeta chain for signal transduction. (Fig. generated on




(1) Ellebrecht CT et al (2016). Reengineering chimeric antigen receptor T cells for targeted therapy of autoimmune disease. Science, Vol. 353 (6295), Pages 179-184. [PubMed]


(2) Guedan S et al (2019). Engineering and Design of Chimeric Antigen Receptors. Molecular Therapy Methods & Clinical Development., Volume 12, Pages 145-156. [PubMed]


(3) Gundry MC, ..., Wagner D, al (2016). Highly Efficient Genome Editing of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9. Cell Reports, Volume 17 (5), Pages 1453-1461. [PubMed]


(4) Pollok K et all (2017) The chronically inflamed central nervous system provides niches for long-lived plasma cells. Acta Neuropathologica Communications, Volume 5 (88) [PubMed]


(5) Relevant to new CAR designs: Gomes-Silva D, …, Wagner DL, … et al. (2017). CD7-edited T cells expressing a CD7-specific CAR for the therapy of T-cell malignancies. Blood, Volume 130 (3), Pages 285-296. [PubMed]