Anti-CD20 Chimeric Antigen Receptor Transduced T Cells Can Recognize Very Low Antigen Expression: Determination Of The Lower Threshold Required To Activate The CAR-T Cells
Authors: Watanabe Keisuke , Terakura Seitaro , Goto Tatsunori , Hanajiri Ryo , Imahashi Nobuhiko , Shimada Kazuyuki , Nishida Tetsuya , Tomita Akihiro , Murata Makoto , Naoe Tomoki 
Published: October 2019

Background
In chimeric antigen receptor transduced T-cell (CAR-T cell) therapy, setting of the target antigen is critical in terms of both the efficacy and the possible adverse effects. However, how low expressing antigen CAR-T cells can recognize and present cytotoxicity, and how low expressing antigen would be a candidate for the target or should be avoided for the concern of off-organ effect is still unclear. Thus we developed novel anti-CD20 CAR-T cells, and investigated the threshold antigen expression level required to activate CAR-T cells.

Methods
In this study, we generated a retrovirus vector that encodes a novel CAR consisting of anti-CD20 single chain variable fragments linked to a CD3-zeta chain, a CD28 costimulatory domain, and a truncated epidermal growth factor receptor (tEGFR) as a transduction and selection marker. CD3 positive T cells from a healthy donor were activated with anti-CD3/28 beads, transduced with the CAR on day 3, enriched by selection with anti-EGFR mAb, and expanded by stimulation with gamma-irradiated B-lymphoblastoid cells. CD20 expression level of the target cells was evaluated with CD20 mean fluorescence intensity (CD20-MFI) and was quantified as CD20 specific antibody biding capacity (CD20-ABC).

Results
To determine the threshold expression level of CD20 antigen to induce cytotoxicity, we performed 51Cr release assay by anti-CD20 CAR-T cells against 30 clones of CD20 transduced CCRF-CEM (CD20-CEM) expressing variable levels of CD20 (CD20-MFI: 126-6,924/ CD20-ABC: 240-230,546 molecules) (kindly provided by Dr. A.C.M. Martens, University Medical Center Utrecht). CAR-T cells lysed CD20-CEMs equally well from low to high level of CD20 (CD20MFI: 157/ CD20ABC: 5,172 molecules or more, 40-60% of lysis at E:T ratio of 10:1), and lysed the clone expressing the lowest level of CD20 (126/ 240 molecules, 22.8±2% of lysis). However, in rituximab-induced CDC assay against the same 30 clones, a minimum CD20 MFI of 600 (ABC of 65,000 molecule) was required to induce CDC. Next, cytokine production and proliferation upon stimulation were investigated by using four representative CD20-CEM clones out of the 30 clones that cover a wide range of CD20 expression levels; Very low (VL-CEM) (MFI: 126/ ABC:240 molecules), Low (L-CEM)(252/ 26,990 molecules), Mid (M-CEM)(826/ 91,567 molecules), High (H-CEM)(6,924/ 142,722 molecules). In intracellular IFN-gamma assay, CAR-T cells produced IFN-gamma equally well (L-CEM, 45.9%, M-CEM, 35.4% and H-CEM, 38.0%, respectively), except against VL-CEM (0.2%). In CFSE division assay, CAR-T cells proliferated upon stimulation with L-CEM, M-CEM, H-CEM (31.4%, %36.1, 37.3% at 72h and, 88.9%, 90.2%, 91.1% at 96h), except with LV-CEM (0% at 72h/ 96h). In intracellular signaling assay, phosphorylation of downstream signaling molecule, CD3-zeta and ERK were evaluated. Whereas only minimal phosphorylations of CD3-zeta and ERK were observed upon VL-CEM stimulation, other 3 CEMs induced similar strength of signals in the majority of CAR-T cells, and induced phosphorylation of proximal signaling molecule, CD3-zeta according to the CD20 expression level. Finally, we evaluated cytotoxicity against low-CD20 expressing primary tumor cells and cell lines. CAR-T cells lysed RRBL1 which is a cell line established from a patient who exhibited relapse of B-cell lymphoma with very weak CD20 expression and became resistant to rituximab (47.8±3% of lysis at E:T ratio 10:1) and primary DLBCL cells isolated from pleural effusion with low CD20 expression (32.6±2.9% of lysis).

Conclusions
We observed that CAR-T cells recognize CD20 antigen with considerably low expression. A minimum number of antigen molecules required to activate CAR-T cells is very low; the threshold is a few hundreds of target antigen. For the future search of a novel target of CAR-T therapy, antigens with expression above the threshold, even if that is below the range of mAb therapy, could be also considered. Conversely, antigens with off-organ expression above the threshold may induce off-organ effect, thus should be avoided. CD20-CAR therapy may be also effective for the patients with CD20 down-regulated lymphoma and refractory to CD20 mAb therapy.

Disclosures:
No relevant conflicts of interest to declare.