Immunotherapy of solid tumors: multimodal imaging strategies for chimeric antigen receptor T cell tracking in the tumor microenvironment

Abstract

Immunotherapy is an emerging building block of modern oncology, after chimeric antigen receptor (CAR) T cells demonstrated groundbreaking survival rates in hematological malignancies. However, therapy success in more common solid tumors has not been achieved yet, due to a variety of obstacles, such as a limited availability of suitable targets and decreased CAR T cells trafficking to the tumor. One of these barriers is the tumor microenvironment (TME), which is most pronounced in pancreatic ductal adenocarcinoma (PDAC). Combinatorial 2D and 3D preclinical multimodal imaging and cell tracking strategies can help to understand the mechanism that play a role in the solid tumor-specific barriers for CAR T cell migration. To this end, three non-solid CARs were characterized in vitro for killing and cytokine expression as well as for in vivo efficacy and tumor control. While in vitro results showed a potent killing and cytokine profile for all three CARs, in vivo analysis revealed a diminished killing potential of one CAR carrying an unspecifically bound IgG1-based spacer. This demonstrates the importance of imaging techniques to identify the most promising CARs for clinical transfer, to depict problematic CAR components and to unravel the underlying mechanisms. Furthermore, in vivo imaging identified the relevance of the CAR spacer domain, which is normally neglected. Favorable targeting of membrane-proximal epitopes with spacer, structural comparable to IgG1, encouraged the development of a novel spacer class, derived from sialic acid-binding immunoglobulin-type lectin (Siglec). Next, CAR T cells, incorporating the new spacers, were evaluated in vitro and ex vivo in solid and hematological malignancies. The functionality of the novel Siglec-4 derived spacer was superior to the established IgG4 and CD8α spacers in terms of the cytotoxic potential and a more potent anti-tumor marker and cytokine expression profile in comparison to IgG1-based spacers, supportive for future clinical trials. These results displayed the general functionality of CAR T cells against PDAC under the optimal conditions, in terms of target specificity, CAR composition and cell number and emphasize the implication of advanced imaging strategies for preclinical CAR T cell research. Thus, a rational multimodal imaging workflow was established and evaluated in a xenograft PDAC mouse model. First, optical 3D in vivo tracking of modified luciferase-expressing CAR T cells was applied. 3D Bioluminescence tomography (BLT) enabled the analysis of whole-body CAR T cells biodistribution and detection of pronounced CAR T cell accumulation in tumor and spleen in PDAC bearing mice. Subsequent combination with ex vivo light-sheet fluorescence microscopy (LSFM) of xenografts facilitated the generation of data visualizing whole-body and intratumoral T cell distribution of two different CARs. The addition of cyclic immunofluorescence staining (IF) provided an in-depth characterization of tumor-infiltrating CAR T cells and surrounding tumor cells, revealing strong activation and proliferation of target-specific CAR T cells. The multi-modal imaging strategy enabled the evaluation of locally applied interleukin-2 (IL-2) as a support for CAR T cells in the immunosuppressive TME of PDAC. IL-2, repeatedly injected at the tumor site was shown to negatively impact intratumoral T cell distribution and phenotype. IL-2 co-treated CAR T cells infiltrated the tumor tissue less deep and showed a more overstimulated phenotype. These cells were no longer able to perform sufficient tumor eradication and local IL-2 did not translate into an enhanced anti-tumor efficacy. Taken together, this project established optical 3D CAR T cell tracking as part of a combined in vivo and ex vivo workflow for solid tumor cell therapy, TME-redirected treatment protocols and safety-orientated research. This preclinical imaging strategy enables the in-depth characterization of combinatorial CAR T cell approaches against solid tumors and TME in a mouse model of PDAC.