Engineering CAR-T cells for radiohapten capture in imaging and radioimmunotherapy applications

Abstract

Rationale: The in vivo dynamics of CAR-T cells remain incompletely understood. Novel methods are urgently needed to longitudinally monitor transferred cells non-invasively for biodistribution, functionality, proliferation, and persistence in vivo and for improving their cytotoxic potency in case of treatment failure. Methods: Here we engineered CD19 CAR-T cells ("Thor"-cells) to express a membrane-bound scFv, huC825, that binds DOTA-haptens with picomolar affinity suitable for labeling with imaging or therapeutic radionuclides. We assess its versatile utility for serial tracking studies with PET and delivery of α-radionuclides to enhance anti-tumor killing efficacy in sub-optimal adoptive cell transfer in vivo using Thor-cells in lymphoma models. Results: We show that this reporter gene/probe platform enables repeated, sensitive, and specific assessment of the infused Thor-cells in the whole-body using PET/CT imaging with exceptionally high contrast. The uptake on PET correlates with the Thor-cells on a cellular and functional level. Furthermore, we report the ability of Thor-cells to accumulate cytotoxic alpha-emitting radionuclides preferentially at tumor sites, thus increasing therapeutic potency. Conclusion: Thor-cells are a new theranostic agent that may provide crucial information for better and safer clinical protocols of adoptive T cell therapies, as well as accelerated development strategies.

Keywords: CAR-T cells; T cell tracking; alpha-particles; reporter gene; theranostic.

Figure 1

huC825-expressing CAR-T cells are not altered in their effector functions and specifically bind DOTA-haptens in vitro. (A) Schematic representation of retroviral vectors. (B) Transduction efficiency of CAR-T cells from (A) by flow cytometry is depicted for one representative donor using the 19E3 anti-CAR antibody (left) and Biotin-Pr for huC825 (right). (C) Killing of Raji tumor cells at different effector to target ratios at 24 h post co-culture as measured by total bioluminescence of the Raji tumor cells is shown. (D) The relative number of CD4⁺ and CD8⁺ cells from successfully transduced and non-transduced (NT) cells were measured by flow cytometry and compared. Mean ± S.D. is depicted for all. (E) Cytokine secretion levels as measured by Luminex are depicted using the co-culture supernatant from (C). (F) In vitro binding of [¹¹¹In]In-Pr at 1 h from a representative data set, demonstrating the specific binding of the radiolabeled DOTA-hapten to huC825-expressing T cells, whereas no significant uptake was observed in NT and 19BBz T cells (reported as mean ± se for all). Data derived from at least three different donors for (C-F).

Figure 2

[⁸⁶Y]Y-ABD PET/CT visualizes huC825-19BBz T cell trafficking to the tumor and normal tissues with exceptionally high contrast over extended periods of time. (A) Schema. Raji cells (3x10⁶) were implanted subcutaneously into NSG mice, injected with huC825-CAR or CAR-T cells (3x10⁶) intravenously seven days later, and weekly imaged by PET/CT after intravenous radiotracer ([⁸⁶Y]Y-ABD) injection. (B, C) Maximum intensity projection (MIP) and axial PET/CT images at d 7, d 14, d 21 and d 28. In mice transfused with huC825-19BBz T cells, intense uptake at the tumor site (circle, red) already at d 7, as well as uptake in normal tissues, such as lungs, and spleen. No uptake is seen in mice injected with 19BBz T cells (tumor marked by circle, blue). (D, E) Image-based biodistribution. For tumor [%ID/g]_max and [%ID/g]_mean and for normal tissues [%ID/g]_mean are provided. (mean ± SD, n = 3, *n = 2) (F) Autoradiography and immunohistochemistry of tumor tissue containing huC825-19BBz T cells confirm “homing.” Radiotracer co-localizes with CD3-positive T cell cluster (brown staining). H&E = hematoxylin and eosin.

Figure 3

Thor-cells can be characterized on a tissue level. (A) Raji cells (3x10⁶) were implanted subcutaneously into NSG mice, injected with CAR-T cells (3x10⁶) intravenously 14 days later and imaged with PET/CT 16 h post intravenous radiotracer ([⁸⁶Y]Y-ABD) injection (3.7 MBq). (B) Serial PET/CT imaging in Raji-bearing NSG mice after administration of huC825-19BBz. Maximum intensity projection (MIP) and axial images of representative mouse shown at d 7 and d 14 (n = 4). (C) IHC of CD3 (T cells) in selected tissues excised after the last imaging timepoint at d 14 post-injection of Thor-cells. Scale bar= 100 µm (D) Quantification of CD3⁺ cell population. (E) Correlation of CD3⁺ cells/section and respective radiotracer uptake on PET/CT [%ID/g]. (R²=0.23, p > 0.1 (F) Pixel by pixel overlay of CD3⁺ cell density map and PET image of tumor tissue. (G) Correlation of the CD3/PET pixel overlay, CD3 cell density in [AU], Pearson correlation, R²=0.7, p < 0.001.

Figure 4

Thor-cells' huC825 expression is dependent upon their activation and exhaustion status. (A) Raji cells and huC825-19BBz cells were co-cultured at a 1:1 ratio (1x10⁵ total cells) and huC825 levels were measured using flow cytometry with [Biotin]-Pr 16 h post co-culture. (*: p < 0.05, unpaired, two-tailed t-test). (B) Co-culture was repeated as in (A) with additional Raji cells added at d 2 and d 5. huC825 fold change MFI was normalized to the huC825 MFI from untreated huC825-19BBz cells on d 0. (C) Harvested tissues normalized huC825 MFI is depicted for mice treated as in Figure 3A. (D, E, F) Normalized CD25, LAG3 and TIM3 MFI is correlated to huC825 MFI. (Spearman's, CD25: R²=0.921, p = 0.001, LAG3: R²= -0.75623, p = 0.0228, TIM3: R²=-0.9528, p = 0.0002). Normalized MFI's for (C-F) were calculated as fold change over the FMO. 3 representative donors were utilized for (A-B).

Figure 5

Image-based dosimetry for therapeutic analogs of [⁸⁶Y]Y-ABD: [¹⁷⁷Lu]Lu-ABD, [⁹⁰Y]Y-ABD, and [²²⁵Ac]Ac-Pr. (A, B) Raji cells (3x10⁶) were implanted subcutaneously into NSG mice, injected with huC825-19BBz or 19BBz T cells (3x10⁶) intravenously seven days later and imaged by PET/CT on d 14 at 1, 3, 16 and 36 h post intravenous radiotracer ([⁸⁶Y]Y-ABD) (3.7 MBq) injection. Maximum intensity projection (MIP) and axial images of representative mice shown. n = 3 (C,D) Image-based biodistribution. For tumor [%ID/g]_max and for normal tissues [%ID/g]_mean are provided. (mean ± SD, n = 3) (E,F) MOBY mouse phantom used in Monte Carlo absorbed dose calculations. Absorbed dose maps (maximum intensity projections) for [⁹⁰Y]Y-ABD, [²²⁵Ac]Ac-Pr and [¹⁷⁷Lu]Lu-ABD estimated prospectively from [⁸⁶Y]Y-ABD biodistribution measurements (G,H,I) Organ-level mean absorbed doses from calculations in (E) for [⁹⁰Y]Y-ABD (G), [¹⁷⁷Lu]Lu-ABD (H), and [²²⁵Ac]Ac-Pr (I).