Promise and challenges of clinical non-invasive T-cell tracking in the era of cancer immunotherapy.

In the last decades, our understanding of the role of the immune system in cancer has significantly improved and led to the discovery of new immunotherapeutic targets and tools, which boosted the advances in cancer immunotherapy to fight a growing number of malignancies. Approved immunotherapeutic approaches are currently mainly based on immune checkpoint inhibitors, antibody-derived targeted therapies, or cell-based immunotherapies. In essence, these therapies induce or enhance the infiltration and function of tumor-reactive T cells within the tumors, ideally resulting in complete tumor eradication. While the clinical application of immunotherapies has shown great promise, these therapies are often accompanied either by a variety of side effects as well as partial or complete unresponsiveness of a number of patients. Since different stages of disease progression elicit different local and systemic immune responses, the ability to longitudinally interrogate the migration and expansion of immune cells, especially T cells, throughout the whole body might greatly facilitate disease characterization and understanding. Furthermore, it can serve as a tool to guide development as well as selection of appropriate treatment regiments. This review provides an overview about a variety of immune-imaging tools available to characterize and study T-cell responses induced by anti-cancer immunotherapy. Moreover, challenges are discussed that must be taken into account and overcome to use immune-imaging tools as predictive and surrogate markers to enhance assessment and successful application of immunotherapies.

Functional analysis of peripheral and intratumoral neoantigen-specific TCRs identified in a patient with melanoma.

BACKGROUND: Neoantigens derived from somatic mutations correlate with therapeutic responses mediated by treatment with immune checkpoint inhibitors. Neoantigens are therefore highly attractive targets for the development of therapeutic approaches in personalized medicine, although many aspects of their quality and associated immune responses are not yet well understood. In a case study of metastatic malignant melanoma, we aimed to perform an in-depth characterization of neoantigens and respective T-cell responses in the context of immune checkpoint modulation. METHODS: Three neoantigens, which we identified either by immunopeptidomics or in silico prediction, were investigated using binding affinity analyses and structural simulations. We isolated seven T-cell receptors (TCRs) from the patient's immune repertoire recognizing these antigens. TCRs were compared in vitro by multiparametric analyses including functional avidity, multicytokine secretion, and cross-reactivity screenings. A xenograft mouse model served to study in vivo functionality of selected TCRs. We investigated the patient's TCR repertoire in blood and different tumor-related tissues over 3 years using TCR beta deep sequencing. RESULTS: Selected mutated peptide ligands with proven immunogenicity showed similar binding affinities to the human leukocyte antigen complex and comparable disparity to their wild-type counterparts in molecular dynamic simulations. Nevertheless, isolated TCRs recognizing these antigens demonstrated distinct patterns in functionality and frequency. TCRs with lower functional avidity showed at least equal antitumor immune responses in vivo. Moreover, they occurred at high frequencies and particularly demonstrated long-term persistence within tumor tissues, lymph nodes and various blood samples associated with a reduced activation pattern on primary in vitro stimulation. CONCLUSIONS: We performed a so far unique fine characterization of neoantigen-specific T-cell responses revealing defined reactivity patterns of neoantigen-specific TCRs. Our data highlight qualitative differences of these TCRs associated with function and longevity of respective T cells. Such features need to be considered for further optimization of neoantigen targeting including adoptive T-cell therapies using TCR-transgenic T cells.

Synthesis and Preclinical Evaluation of a 68Ga-Labeled Adnectin, 68Ga-BMS-986192, as a PET Agent for Imaging PD-L1 Expression.

Blocking the interaction of the immune checkpoint molecule programmed cell death protein-1 and its ligand, PD-L1, using specific antibodies has been a major breakthrough for immune oncology. Whole-body PD-L1 expression PET imaging may potentially allow for a better prediction of response to programmed cell death protein-1-targeted therapies. Imaging of PD-L1 expression is feasible by PET with the adnectin protein 18F-BMS-986192. However, radiofluorination of proteins such as BMS-986192 remains complex and labeling yields are low. The goal of this study was therefore the development and preclinical evaluation of a 68Ga-labeled adnectin protein (68Ga-BMS-986192) to facilitate clinical trials. Methods:68Ga labeling of DOTA-conjugated adnectin (BXA-206362) was performed in NaOAc-buffer at pH 5.5 (50°C, 15 min). In vitro stability in human serum at 37°C was analyzed using radio-thin layer chromatography and radio-high-performance liquid chromatography. PD-L1 binding assays were performed using the transduced PD-L1-expressing lymphoma cell line U-698-M and wild-type U-698-M cells as a negative control. Immunohistochemical staining studies, biodistribution studies, and small-animal PET studies of 68Ga-BMS-986192 were performed using PD-L1-positive and PD-L1-negative U-698-M-bearing NSG mice. Results:68Ga-BMS-986192 was obtained with quantitative radiochemical yields of more than 97% and with high radiochemical purity. In vitro stability in human serum was at least 95% after 4 h of incubation. High and specific binding of 68Ga-BMS-986192 to human PD-L1-expressing cancer cells was confirmed, which closely correlates with the respective PD-L1 expression level determined by flow cytometry and immunohistochemistry staining. In vivo, 68Ga-BMS-986192 uptake was high at 1 h after injection in PD-L1-positive tumors (9.0 ± 2.1 percentage injected dose [%ID]/g) and kidneys (56.9 ± 9.2 %ID/g), with negligible uptake in other tissues. PD-L1-negative tumors demonstrated only background uptake of radioactivity (0.6 ± 0.1 %ID/g). Coinjection of an excess of unlabeled adnectin reduced tumor uptake of PD-L1 by more than 80%. Conclusion:68Ga-BMS-986192 enables easy radiosynthesis and shows excellent in vitro and in vivo PD-L1-targeting characteristics. The high tumor uptake combined with low background accumulation at early imaging time points demonstrates the feasibility of 68Ga-BMS-986192 for imaging of PD-L1 expression in tumors and is encouraging for further clinical applications of PD-L1 ligands.

Gene editing enables T-cell engineering to redirect antigen specificity for potent tumor rejection.

Adoptive transfer of TCR transgenic T cells holds great promise for treating various cancers. So far, mainly semi-randomly integrating vectors have been used to genetically modify T cells. These carry the risk of insertional mutagenesis, and the sole addition of an exogenous TCR potentially results in the mispairing of TCR chains with endogenous ones. Established approaches using nonviral vectors, such as transposons, already reduce the risk of insertional mutagenesis but have not accomplished site-specific integration. Here, we used CRISPR-Cas9 RNPs and adeno-associated virus 6 for gene targeting to deliver an engineered TCR gene specifically to the TCR alpha constant locus, thus placing it under endogenous transcriptional control. Our data demonstrate that this approach replaces the endogenous TCR, functionally redirects the edited T cells' specificity in vitro, and facilitates potent tumor rejection in an in vivo xenograft model.

T-cell functionality testing is highly relevant to developing novel immuno-tracers monitoring T cells in the context of immunotherapies and revealed CD7 as an attractive target.

Cancer immunotherapy has proven high efficacy in treating diverse cancer entities by immune checkpoint modulation and adoptive T-cell transfer. However, patterns of treatment response differ substantially from conventional therapies, and reliable surrogate markers are missing for early detection of responders versus non-responders. Current imaging techniques using 18F-fluorodeoxyglucose-positron-emmission-tomograpy (18F-FDG-PET) cannot discriminate, at early treatment times, between tumor progression and inflammation. Therefore, direct imaging of T cells at the tumor site represents a highly attractive tool to evaluate effective tumor rejection or evasion. Moreover, such markers may be suitable for theranostic imaging. Methods: We mainly investigated the potential of two novel pan T-cell markers, CD2 and CD7, for T-cell tracking by immuno-PET imaging. Respective antibody- and F(ab´)2 fragment-based tracers were produced and characterized, focusing on functional in vitro and in vivo T-cell analyses to exclude any impact of T-cell targeting on cell survival and antitumor efficacy. Results: T cells incubated with anti-CD2 and anti-CD7 F(ab´)2 showed no major modulation of functionality in vitro, and PET imaging provided a distinct and strong signal at the tumor site using the respective zirconium-89-labeled radiotracers. However, while T-cell tracking by anti-CD7 F(ab´)2 had no long-term impact on T-cell functionality in vivo, anti-CD2 F(ab´)2 caused severe T-cell depletion and failure of tumor rejection. Conclusion: This study stresses the importance of extended functional T-cell assays for T-cell tracer development in cancer immunotherapy imaging and proposes CD7 as a highly suitable target for T-cell immuno-PET imaging.