Fig 1.
Schematic representation of the technology.
A) A lentiviral expression vector was created that encodes an anti-CD19 CAR domain, a 2A cleavage site, and a secreted CD19-anti-Her2 scFv bridging protein. B) Human primary T cells were transduced and expanded. C) The T cell transduction resulted in the expression of the anti-CD19 CAR domain on the T cell surface (thus, CAR-CD19) and secretion of the bridging protein. D) The bridging protein has two domains: the anti-Her2 scFv bound to Her2 expressed on the tumor cell; the CD19 ECD bound to the anti-CD19 CAR domain on the transduced T cell. Thus the bridging protein acts as a CAR-T cell engager protein. E) The binding events created a cytotoxic immune synapse between the CAR-CD19 T cell and the Her2-positive tumor cell via recognition of CD19; this resulted in CAR T cell proliferation and tumor cell death.
Table 1.
List of major constructs used in the text.
Table 2.
Summary of bridging protein binding and cytotoxicity data.
Fig 2.
A CD19-anti-Her2 bridging protein mediates potent CAR-CD19 T cell cytotoxic activity in vitro.
A) Phenotype of transduced and expanded CAR-CD19 T cells (donor 45): CAR-CD19 T cells were 54% CAR-positive by anti-Flag-tag staining. B) The CAR-CD19 T cells were tested for cytotoxicity against Nalm6 cells at different E:T ratios (10:1, blue; 3:1, teal; 1:1 light blue) and compared to donor-matched untransduced cells (UTD) at the same E:T ratios; all comparisons to UTD controls were significant. This assay is run routinely on all CAR-CD19-based CAR T cells; the extent of cytotoxicity varies among donors. C) Bridging protein, CAR-CD19 T cells (donor 54, 58% Flag-positive) and target tumor cells were added simultaneously (red bars) or bridging proteins and tumor cells were preincubated then added to CAR-T cells (light blue bars) or bridging proteins and CAR-T cells were preincubated then added to tumor cells (black bars). The E:T ratio used was 5:1. Controls were CAR-CD19 T cells, target cells plus bridging protein only (‘BP’) or target cells alone in culture (‘none’). Nalm6 (left) and SKOV3 (right) cytotoxicity profiles are shown. The data are from duplicate wells; the experiment was performed twice with similar results. D, E) Dose response cytotoxicity curves using an E:T ratio of 10:1 CAR-CD19 T cells and target cells and varying the concentration of the CD19-anti-Her2 bridging protein or the control CD19-ECD. Target cell lines SKOV3 (D) and BT474 (E) are shown. The CAR-CD19 T cells used for the SKOV3 assay were from donor 54 (47% Flag-positive); donor 69 CAR-CD19 T cells (55% Flag-positive) were used for the BT474 assay. The data are from triplicate wells; the experiment was performed twice with similar results. All statistical tests were performed as 2-sided T tests. For Panels D and E, * indicates significance of p < 0.0001 and ** indicates significance of p < 0.02.
Table 3.
Activity of a dual antigen targeting bridging protein.
Fig 3.
Her2-bridging CAR-CD19 T cells are cytotoxic against both CD19-positive and Her2-positive cells in vitro.
(A, B) Example of the flow cytometry gating strategy (A) and the ELISA assay used (B) to determine the purity and phenotype of transduced human donor CAR-CD19 T cells that secrete a bridging protein. C) Her2-bridging CAR-CD19 T cells were added at different E:T ratios to Nalm6 cells (left) or SKOV3 cells (right) and cytotoxicity was measured. E:T ratios used were 10:1 (red bars), 3:1 (light blue bars) and 1:1 (black bars). The donor matched UTD cells are also shown at each E:T ratio.
Fig 4.
CAR T cell activity in response to restimulation.
A) Extent of CAR T cell proliferation after 2 rounds and 4 rounds of Raji cell stimulation, SKOV3 cell restimulation or no stimulation; CAR T cell types are indicated below the x-axis. B) Extent of CAR T cell-mediated cytotoxicity against target tumor cells and 1 round and 3 rounds of Raji cell stimulation, SKOV3 cell restimulation or no stimulation. The round of stimulation, cell source of stimulation is indicated, and target cell in the cytotoxicity assay are indicated. * = p, 0.001. NS = no significant differences.
Fig 5.
Her2-bridging CAR-CD19 T cells are efficacious in vivo.
A) Dose responsive elimination of pre-established leukemia in an NSG mouse model was monitored after Her2-bridging CAR-CD19 T cells (CAR-142 and CAR-374, donor 36, 99.8% CD3-positive, 67% CD8-positivem, 59% Flag-tag-positive) were administered at doses ranging from 2 x 106 to 1 x 107 cells/mouse. Luminescence values for all cohorts (A) and the treatment cohorts (B) are shown. C-E) CAR T cells (donor 45, 99.2% CD3-positive, 41% CD8-positive, 38% Flag-tag-positive) were evaluated in a SKOV3 ovarian carcinoma model in NSG mice. C) CAR-142 T cells controlled SKOV3 tumor growth while control CAR T cells (CAR-254) had no effect. D) CAR-142 T cell control of SKOV3 tumor growth as compared to CAR-Her2 (CAR-390). CAR-142 and CAR-390 fully controlled tumor growth, while control mice succumbed and had to be euthanized by day 42 (panel E). * = p < 0.001. ** = p < 0.05. NS = no significant differences.
Fig 6.
Her2-bridging CAR-CD19 T cells eliminate Her2-positive tumors and CD19-positive tumors in a serial challenge in vivo model.
A-C) CAR T cells (donor 45, 99.2% CD3-positive, 45% CD8-positive, 34% Flag-tag-positive) were evaluated in a SKOV3 ovarian carcinoma model in NSG mice. A) CAR-374 treatment eliminated preestablished SKOV3 tumors in the NSG mouse model. B) 90% of CAR-374 treated mice survived past day 36, by which point the control mice were beginning to require euthanasia. C) CAR-374 that survived the SKOV3 challenge subsequently cleared CD19-positive Nalm6 leukemia challenge; all individual mice are shown. * = p < 0.001.