Figure 5 ∣. mLiCAR T-cells reduce “on-target off-tumor” effects in a syngeneic mouse model of melanoma.

a, Design of constructs to recognize mouse CD19 (mCD19) antigen overexpressed in B16 murine melanoma cells. The mCD19-recognizing scFv in WT mCAR or mLiCAR was derived a mouse mAb (clone 1D3).

b, The engagement of 1D3 scFv to mCD19 antigen was quantified by NFAT-dependent luciferase (NFAT-Luc) reporter activity in Jurkat T cells. Jurkat T cells transduced with human CD19 (hCD19)-recognizing (WT hCAR) or mouse mCD19 antigen-recognizing constructs (WT mCAR, mLiCAR, or defective mLiCAR) were engaged with the corresponding tumor cells bearing noncognate (open box; B16-OVA cells) or the cognate antigens (B16-OVA-hCD19 for hCAR or B16-OVA-mCD19 for mCAR, mLiCAR, or defective mLiCAR groups) under dark (open box) or lit conditions (blue box). Blue light (40 mW/cm² at 470 nm) was applied for 20 min and then in pulsed cycles of 30 sec ON + 100 sec OFF for 8 h. n = 4 biologically independent mice (mean ± s.e.m.). P values were calculated using two-sided unpaired Student’s t-tests.

c-d, WT mCAR- (c) or mLiCAR-expressing (d) murine CD8⁺ T-cells selectively destroy mCD19-expressing melanoma cells in vivo. mLiCAR T-cells exhibit NIR-light dependent killing of B16-OVA-mCD19 tumor cells.

Left, C57BL/6J mice were intradermally inoculated with 2.5x10⁵ B16-OVA cells in the left flank (as control without mCD19 overexpression, blue circle) and 2.5x10⁵ B16-OVA-mCD19 cells (red circle) in the right flank. Mice were treated with WT mCAR T-cells + 150 μg UCNPs for 10 days (panel c), or 2x10⁶ mLiCAR T-cells + 150 μg UCNPs with subsequent exposure to NIR pulses for 10 days (panel d).

Middle, Quantification of the tumor sizes. Tumor sizes at the endpoint after UCNP removal were measured by a digital caliper with the tumor areas calculated in mm² (length x width). n = 4 biologically independent mice for panel c and n = 3 biologically independent mice for panel d (mean ± s.e.m.). P values were calculated using two-sided unpaired Student’s t-tests. Right, Representative images of isolated B16-OVA and B16-OVA-mCD19 tumors with UCNPs at day 19.

e, On-target off-tumor effects of mWT CAR and mLiCAR T-cells evaluated by the degree of B cell aplasia. Peripheral blood B cells from the WT mCAR or mLiCAR T-cell treated groups (as in panels c-d) inoculated with tumor cells were counted and compared on day 0 and day 3. B cells from peripheral blood of healthy mice were used as control. n = 4 biologically independent mice (mean ± s.e.m.). P values were calculated using two-sided unpaired Student’s t-tests.

f, Representative H&E staining images of major organs isolated from mLiCAR T-cells/UCNP/NIR treated mice bearing tumors or healthy mice subcutaneously administered with 100 μl PBS. The experiments were independently repeated four times. Scale bar, 100 μm.