A supervised machine-learning approach for the efficient development of a multi method (LC-MS) for a large number of drugs and subsets thereof: focus on oral antitumor agents.

OBJECTIVES: Accumulating evidence argues for a more widespread use of therapeutic drug monitoring (TDM) to support individualized medicine, especially for therapies where toxicity and efficacy are critical issues, such as in oncology. However, development of TDM assays struggles to keep pace with the rapid introduction of new drugs. Therefore, novel approaches for faster assay development are needed that also allow effortless inclusion of newly approved drugs as well as customization to smaller subsets if scientific or clinical situations require. METHODS: We applied and evaluated two machine-learning approaches i.e., a regression-based approach and an artificial neural network (ANN) to retention time (RT) prediction for efficient development of a liquid chromatography mass spectrometry (LC-MS) method quantifying 73 oral antitumor drugs (OADs) and five active metabolites. Individual steps included training, evaluation, comparison, and application of the superior approach to RT prediction, followed by stipulation of the optimal gradient. RESULTS: Both approaches showed excellent results for RT prediction (mean difference ± standard deviation: 2.08 % ± 9.44 % ANN; 1.78 % ± 1.93 % regression-based approach). Using the regression-based approach, the optimum gradient (4.91 % MeOH/min) was predicted with a total run time of 17.92 min. The associated method was fully validated following FDA and EMA guidelines. Exemplary modification and application of the regression-based approach to a subset of 14 uro-oncological agents resulted in a considerably shortened run time of 9.29 min. CONCLUSIONS: Using a regression-based approach, a multi drug LC-MS assay for RT prediction was efficiently developed, which can be easily expanded to newly approved OADs and customized to smaller subsets if required.

Rhodium-Catalyzed Anti-Markovnikov Transfer Hydroiodination of Terminal Alkynes.

A rhodium-catalyzed anti-Markovnikov hydroiodination of aromatic and aliphatic terminal alkynes is reported. Depending on the choice of ligand and substrate, either (E)- or (Z)-configured alkenyl iodides are obtained in high to exclusive isomeric purity. The reaction exhibits a broad substrate scope and high functional group tolerance, employing easily accessible or commercially available aliphatic iodides as HI surrogates through a shuttle process. The synthesized vinyl iodides were applied in several C-C and C-heteroatom bond-forming reactions with full retention of the stereoselectivity. The developed method could be used to significantly shorten the total synthesis of a marine cis-fatty acid. Additionally, initial deuterium-labeling experiments and stoichiometric reactions shed some light on the potential reaction mechanism.

Cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3) is localized in lysosomes and mediates resistance against kinase inhibitors.

Cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3), a splice variant of the hepatic uptake transporter OATP1B3 (liver-type; Lt-OATP1B3), is expressed in several tumor entities including colorectal carcinoma (CRC) and breast cancer. In CRC, high OATP1B3 expression has been associated with reduced progression-free and overall survival. Several kinase inhibitors used for antitumor treatment are substrates and/or inhibitors of OATP1B3 (e.g. encorafenib, vemurafenib). The functional importance of Ct-OATP1B3 has not been elucidated so far. HEK293 cells stably overexpressing Ct-OATP1B3 protein were established and compared with control cells. Confocal laser scanning microscopy, immunoblot, and proteomics-based expression analysis demonstrated that Ct-OATP1B3 protein is intracellularly localized in lysosomes of stably-transfetced cells. Cytotoxicity experiments showed that cells recombinantly expressing the Ct-OATP1B3 protein were more resistant against the kinase inhibitor encorafenib compared to control cells [e.g. encorafenib (100 µM) survival rates: 89.5% vs. 52.8%]. In line with these findings, colorectal cancer DLD1 cells endogenously expressing Ct-OATP1B3 protein had poorer survival rates when the OATP1B3 substrate bromosulfophthalein (BSP) was coincubated with encorafenib or vemurafenib compared to the incubation with the kinase inhibitor alone. This indicates a competitive inhibition of Ct-OATP1B3-mediated uptake into lysosomes by BSP. Accordingly, mass spectrometry-based drug analysis of lysosomes showed a reduced lysosomal accumulation of encorafenib in DLD1 cells additionally exposed to BSP. These results demonstrate that Ct-OATP1B3 protein is localized in the lysosomal membrane and can mediate transport of certain kinase inhibitors into lysosomes revealing a new mechanism of resistance. Significance Statement We describe the characterization of a splice variant of the liver-type uptake transporter OATP1B3 expressed in several tumor entities. This variant is localized in lysosomes mediating resistance against kinase inhibitors which are substrates of this transport protein by transporting them into lysosomes and thereby reducing the cytoplasmic concentration of these antitumor agents. Therefore, the expression of the Ct-OATP1B3 protein is associated with a better survival of cells revealing a new mechanism of drug resistance.

The immunoglobulin superfamily ligand B7H6 subjects T cell responses to NK cell surveillance.

Understanding the mechanisms that regulate T cell immunity is critical for the development of effective therapies for diseases associated with T cell dysfunction, including autoimmune diseases, chronic infections, and cancer. Co-inhibitory "checkpoint molecules," such as programmed cell death protein-1, balance excessive or prolonged immune activation by T cell-intrinsic signaling. Here, by screening for mediators of natural killer (NK) cell recognition on T cells, we identified the immunoglobulin superfamily ligand B7H6 to be highly expressed by activated T cells, including patient-infused CD19-targeting chimeric antigen receptor (CAR) T cells. Unlike other checkpoint molecules, B7H6 mediated NKp30-dependent recognition and subsequent cytolysis of activated T cells by NK cells. B7H6+ T cells were prevalent in the tissue and blood of several diseases, and their abundance in tumor tissue positively correlated with clinical response in a cohort of patients with immune checkpoint inhibitor-treated esophageal cancer. In humanized mouse models, NK cell surveillance via B7H6 limited the persistence and antitumor activity of CAR T cells, and its genetic deletion enhanced T cell proliferation and persistence. Together, we provide evidence of B7H6 protein expression by activated T cells and suggest the B7H6-NKp30 axis as a therapeutically actionable NK cell-dependent immune checkpoint that regulates human T cell function.

Dysfunctional dendritic cells limit antigen-specific T cell response in glioma.

BACKGROUND: Dendritic cells (DC), the most potent professional antigen presenting cells capable of effective cross-presentation, have been demonstrated to license T helper cells to induce antitumor immunity in solid tumors. Specific DC subtypes are recruited to the injured brain by microglial chemokines, locally adapting to distinct transcriptional profiles. In isocitrate dehydrogenase (IDH) type 1 mutant gliomas, monocyte-derived macrophages have recently been shown to display an attenuated intratumoral antigen presentation capacity as consequence of the local accumulation of the oncometabolite R-2-hydroxyglutarate. The functionality and the contribution of DC to the IDH-mutant tumor microenvironment (TME) remains unclear. METHODS: Frequencies and intratumoral phenotypes of human DC in IDH-wildtype (IDHwt) and -mutant high-grade gliomas are comparatively assessed by transcriptomic and proteomic profiling. DC functionality is investigated in experimental murine glioblastomas expressing the model antigen ovalbumin. Single-cell sequencing-based pseudotime analyses and spectral flow cytometric analyses are used to profile DC states longitudinally. RESULTS: DC are present in primary and recurrent high-grade gliomas and interact with other immune cell types within the TME. In murine glioblastomas, we find an IDH-status-associated major histocompatibility class I-restricted cross-presentation of tumor antigens by DC specifically in the tumor but not in meninges or secondary lymphoid organs of tumor-bearing animals. In single-cell sequencing-based pseudotime and longitudinal spectral flow cytometric analyses, we demonstrate an IDH-status-dependent differential, exclusively microenvironmental education of DC. CONCLUSIONS: Glioma-associated DCs are relevantly abundant in human IDHwt and mutant tumors. Glioma IDH mutations result in specifically educated, dysfunctional DCs via paracrine reprogramming of infiltrating monocytes, providing the basis for combinatorial immunotherapy concepts against IDH mutant gliomas.

The pre-existing T cell landscape determines the response to bispecific T cell engagers in multiple myeloma patients.

Bispecific T cell engagers (TCEs) have shown promise in the treatment of various cancers, but the immunological mechanism and molecular determinants of primary and acquired resistance to TCEs remain poorly understood. Here, we identify conserved behaviors of bone marrow-residing T cells in multiple myeloma patients undergoing BCMAxCD3 TCE therapy. We show that the immune repertoire reacts to TCE therapy with cell state-dependent clonal expansion and find evidence supporting the coupling of tumor recognition via major histocompatibility complex class I (MHC class I), exhaustion, and clinical response. We find the abundance of exhausted-like CD8+ T cell clones to be associated with clinical response failure, and we describe loss of target epitope and MHC class I as tumor-intrinsic adaptations to TCEs. These findings advance our understanding of the in vivo mechanism of TCE treatment in humans and provide the rationale for predictive immune-monitoring and conditioning of the immune repertoire to guide future immunotherapy in hematological malignancies.

T cell receptor dynamic and transcriptional determinants of T cell expansion in glioma-infiltrating T cells.

Background: Glioblastoma (GBM) is characterized by low numbers of glioma-infiltrating lymphocytes (GIL) with a dysfunctional phenotype. Whether this dysfunctional phenotype is fixed or can be reversed upon ex vivo culturing is poorly understood. The aim of this study was to assess T cell receptor (TCR)-dynamics and -specificities as well as determinants of in vitro GIL expansion by sequencing-based technologies and functional assays to explore the use of GIL for cell therapy. Methods: By means of flow cytometry, T cell functionality in GIL cultures was assessed from 9 GBM patients. TCR beta sequencing (TCRB-seq) was used for TCR repertoire profiling before and after in vitro expansion. Microarrays or RNA sequencing (RNA-seq) were performed from 6 micro-dissected GBM tissues and healthy brain RNA to assess the individual expression of GBM-associated antigens (GAA). GIL reactivity against in silico predicted tumor-associated antigens (TAA) and patient-individual GAA was assessed by ELISpot assay. Combined ex vivo single cell (sc)TCR-/RNA-seq and post-expansion TCRB-seq were used to evaluate transcriptional signatures that determine GIL expansion. Results: Human GIL regains cellular fitness upon in vitro expansion. Profound TCR dynamics were observed during in vitro expansion and only in one of six GIL cultures, reactivity against GAA was observed. Paired ex vivo scTCR/RNA-seq and TCRB-seq revealed predictive transcriptional signatures that determine GIL expansion. Conclusions: Profound TCR repertoire dynamics occur during GIL expansion. Ex vivo transcriptional T cell states determine expansion capacity in gliomas. Our observation has important implications for the use of GIL for cell therapy including genetic manipulation to maintain both antigen specificity and expansion capacity.

IgE type multiple myeloma exhibits hypermutated phenotype and tumor reactive T cells.

Multiple myeloma (MM) is a hematological malignancy originating from malignant and clonally expanding plasma cells. MM can be molecularly stratified, and its clonal evolution deciphered based on the Ig heavy and light chains of the respective malignant plasma cell clone. Of all MM subtypes, IgE type MM accounts for only <0.1% of cases and is associated with an aggressive clinical course and consequentially dismal prognosis. In such malignancies, adoptive transfer of autologous lymphocytes specifically targeting presented (neo)epitopes encoded by either somatically mutated or specifically overexpressed genes has resulted in substantial objective clinical regressions even in relapsed/refractory disease. However, there are no data on the genetic and immunological characteristics of this rare and aggressive entity. Here, we comprehensively profiled IgE type kappa MM on a genomic and immune repertoire level by integrating DNA- and single-cell RNA sequencing and comparative profiling against non-IgE type MM samples. We demonstrate distinct pathophysiological mechanisms as well as novel opportunities for targeting IgE type MM. Our data further provides the rationale for patient-individualized neoepitope-targeting cell therapy in high tumor mutation burden MM.

Intrathecal activation of CD8+ memory T cells in IgG4-related disease of the brain parenchyma.

IgG4-related disease (IgG4-RD) is a fibroinflammatory disorder signified by aberrant infiltration of IgG4-restricted plasma cells into a variety of organs. Clinical presentation is heterogeneous, and pathophysiological mechanisms of IgG4-RD remain elusive. There are very few cases of IgG4-RD with isolated central nervous system manifestation. By leveraging single-cell sequencing of the cerebrospinal fluid (CSF) of a patient with an inflammatory intracranial pseudotumor, we provide novel insights into the immunopathophysiology of IgG4-RD. Our data illustrate an IgG4-RD-associated polyclonal T-cell response in the CSF and an oligoclonal T-cell response in the parenchymal lesions, the latter being the result of a multifaceted cell-cell interaction between immune cell subsets and pathogenic B cells. We demonstrate that CD8+ T effector memory cells might drive and sustain autoimmunity via macrophage migration inhibitory factor (MIF)-CD74 signaling to immature B cells and CC-chemokine ligand 5 (CCL5)-mediated recruitment of cytotoxic CD4+ T cells. These findings highlight the central role of T cells in sustaining IgG4-RD and open novel avenues for targeted therapies.

An Easily Expandable Multi-Drug LC-MS Assay for the Simultaneous Quantification of 57 Oral Antitumor Drugs in Human Plasma.

Oral anticancer drugs have led to significant improvements in the treatment of multiple tumor entities. However, in patients undergoing oral antitumor therapy, plasma concentrations are highly variable, resulting in risks of reduced therapeutic effects or an increase in side effects. One important tool to reduce this variability is therapeutic drug monitoring. In this work we describe a method to simultaneously quantify the plasma concentrations of 57 oral antitumor agents. Quantification of these drugs was achieved using liquid chromatography coupled to an Orbitrap mass spectrometer. The method was fully validated according to the FDA guidelines and constitutes a simple and robust way for exposure monitoring of a wide variety of oral anticancer drugs. Applicability to clinical routine was demonstrated by the analysis of 71 plasma samples taken from 39 patients. In summary, this new multi-drug method allows simultaneous quantification of 57 oral antitumor drugs, which can be applied to exposure monitoring in clinical studies, taking into account the broad variety of oral antitumor drugs prescribed in clinical routine.