Trained Immunity-Promoting Nanobiologic Therapy Suppresses Tumor Growth and Potentiates Checkpoint Inhibition.

Trained immunity, a functional state of myeloid cells, has been proposed as a compelling immune-oncological target. Its efficient induction requires direct engagement of myeloid progenitors in the bone marrow. For this purpose, we developed a bone marrow-avid nanobiologic platform designed specifically to induce trained immunity. We established the potent anti-tumor capabilities of our lead candidate MTP10-HDL in a B16F10 mouse melanoma model. These anti-tumor effects result from trained immunity-induced myelopoiesis caused by epigenetic rewiring of multipotent progenitors in the bone marrow, which overcomes the immunosuppressive tumor microenvironment. Furthermore, MTP10-HDL nanotherapy potentiates checkpoint inhibition in this melanoma model refractory to anti-PD-1 and anti-CTLA-4 therapy. Finally, we determined MTP10-HDL's favorable biodistribution and safety profile in non-human primates. In conclusion, we show that rationally designed nanobiologics can promote trained immunity and elicit a durable anti-tumor response either as a monotherapy or in combination with checkpoint inhibitor drugs.

ELISA assay for the quantification of ipilimumab in human serum, plasma, milk, and cerebrospinal fluid.

Ipilimumab is an immune checkpoint inhibitor of the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Ipilimumab has become part of the standard of care for different types of cancer. The efficacy of these treatments is limited due to immune-related toxicity and high economic costs. Dose rationalization studies based on pharmacokinetic data may help to address these limitations. For this purpose, more sensitive analytical methods are needed. We report the development and validation of the first enzyme-linked immunosorbent assay (ELISA) for sensitive determination of ipilimumab concentrations in human serum, plasma, cerebrospinal fluid (CSF), and milk. Our assay is based on the specific capture of ipilimumab by immobilized CTLA-4. The lower limit of quantifications of ipilimumab in serum, plasma, and milk are 50 ng/mL and 10 ng/mL in CSF. The ELISA method showed long-term storage stability for at least one year at -80°C and was successfully cross-validated with ultraperformance liquid chromatography coupled with tandem mass spectrometry. The ELISA method is reliable, relatively inexpensive, and can be used in serum, plasma, CSF, and milk from patients treated with ipilimumab, as evidenced by the analysis of real clinical samples.

A multiplex UPLC-MS/MS method for the quantification of three PD-L1 checkpoint inhibitors, atezolizumab, avelumab, and durvalumab, in human serum.

BACKGROUND AND AIM: To support pharmacokinetic studies, a multiplex UPLC-MS/MS assay was developed and validated to quantify PD-L1 checkpoint inhibitors atezolizumab, avelumab, and durvalumab in serum. METHODS: A bottom-up sample pre-treatment procedure was developed to determine atezolizumab, avelumab, and durvalumab in serum. This procedure consisted of (1) precipitation of the monoclonal antibody with ammonium sulfate, (2) reduction with dithiothreitol, (3) denaturation with methanol, and (4) tryptic digestion of the protein. The unique signature peptides resulting after sample pre-treatment of the antibodies were measured using UPLC-MS/MS with a total run time of 11 minutes. The clinical application was evaluated by analyzing 114 atezolizumab patient samples. RESULTS: The developed method was found to be accurate and precise for all three analytes over a concentration range of 3.00-150 µg/mL. No endogenous interference was present in serum samples. Cross-interference experiments showed no cross-analyte interference and acceptable cross-internal standard interference. In addition, no substantial carry-over was observed. The stable isotopically labeled signature peptides were most effective in compensating for matrix effects. Recovery based on back-calculated concentrations of calibration standards and quality control samples was found to be high. The analytes were stable for at least three freeze-thaw cycles, for 42 hours at processing conditions, for at least two days at 2-8°C in the final extract, for five days before re-injection analysis at 4°C, and long-term for at least 11 months at -70°C. The assay was tested for its applicability in clinical practice. For this purpose, 114 atezolizumab patient samples were measured. CONCLUSION: A multiplex UPLC-MS/MS assay was developed and validated to quantify atezolizumab, avelumab, and durvalumab in human serum. The applicability of this method was demonstrated by the analysis of clinical atezolizumab samples. The method is suitable to support clinical pharmacokinetic studies involving atezolizumab, avelumab, or durvalumab.

Optimized sample pre-treatment procedure for the simultaneous UPLC-MS/MS quantification of ipilimumab, nivolumab, and pembrolizumab in human serum.

Ipilimumab, nivolumab, and pembrolizumab are immune checkpoint inhibiting monoclonal antibodies. Their efficacy has been proven to be correlated with clearance, and hence, bioanalytical assays to study their pharmacokinetics are of pivotal importance. We present the first kit-free sample pre-treatment procedure of only three hours for the Ultra-Performance Liquid Chromatography - tandem Mass Spectrometry (UPLC-MS/MS) simultaneous quantification of ipilimumab, nivolumab, and pembrolizumab in human serum. The conventional bottom-up sample pre-treatment steps for protein MS bioanalysis including pre-digestion purification, denaturation, reduction, alkylation, and digestion were optimized in terms of sensitivity and reproducibility. In the final, optimal sample pre-treatment procedure, samples were purified by protein precipitation with saturated ammonium sulfate solution, reduced with dithiothreitol, denatured with methanol, and digested with trypsin. The method was then validated according to European Medicines Agency (EMA) and the United States Food and Drug Administration (FDA) guidelines for bioanalytical method validation, and 4-6-20 acceptance criteria were applied. This method was selective, accurate, and precise within the range of 3-200 µg/mL for all analytes. The validated developed assay was applied to determine ipilimumab, nivolumab, and pembrolizumab concentrations in patient serum, and the results were compared to enzyme-linked immunosorbent assay (ELISA) results.

Simultaneous quantification of abemaciclib and its active metabolites in human and mouse plasma by UHPLC-MS/MS.

Abemaciclib is the third cyclin-dependent kinase 4 and 6 inhibitor approved for the treatment of advanced or metastatic breast cancer. In humans, abemaciclib is extensively metabolized by CYP3A4 with the formation of three active metabolites: N-desethylabemaciclib (M2), hydroxyabemaciclib (M20) and hydroxy-N-desethylabemaciclib (M18). These metabolites showed similar potency compared to the parent drug and were significantly abundant in plasma circulation. Thus, M2, M20, and M18 may contribute to the clinical activity of abemaciclib. For this reason, an UHPLC-MS/MS method for the simultaneous quantification of abemaciclib and its active metabolites in human and mouse plasma was developed and validated to support further clinical or preclinical investigations on this drug. Samples were processed by protein precipitation with acetonitrile, followed by supernatant dilution and filtration. Chromatographic separation was performed on a Kinetex C18 column (150 × 2.1 mm ID, 2.6 µm) using gradient elution with 10 mM ammonium bicarbonate in water (eluent A) and in methanol-water (9:1, v/v, eluent B). This method was selective, linear, accurate and precise within the range of 1-600 ng/mL for abemaciclib, 0.5-300 ng/mL for M2 and M20, and 0.2-120 ng/mL for M18. Furthermore, stability of the analytes in human and mouse plasma samples in several conditions was demonstrated. Finally, this assay was successfully used in a preclinical pharmacokinetic study, where abemaciclib and its active metabolites were identified and quantified. Inter-species differences between human and mouse samples were encountered, especially in the formation of M20, where isomers of this compound were detected in mouse plasma, but not in human plasma. This was confirmed by high resolution-mass spectrometry (HR-MS) measurements.

Quantification of anti-SARS-CoV-2 antibodies in human serum with LC-QTOF-MS.

The aim of this study was to develop the first quantitative serological test for anti-SARS-CoV-2 antibodies in human serum with liquid chromatography - quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Other assays, mostly immunoassays, are only qualitative or semi-quantitative, and hence, actual antibody concentrations after SARS-CoV-2 infection are unknown. In our assay, anti-SARS-CoV-2 antibodies were isolated with spike protein subunit 1 (S1) coupled to magnetic beads. IgG1 signature peptide GPSVFPLAPSSK was selected for quantification using ipilimumab calibration standards and SILuMAb K1 as the stable-isotope labeled internal standard. The anti-SARS-CoV-2 IgG1 calibration range was from 1.35 to 135 nM. Inter-assay accuracies were between 98.8%- 107% with inter-assay precisions between 8.37%- 13.5% measured at 3 concentration levels on three separate occasions. Anti-SARS-CoV-2 IgG1 antibodies were quantified in PCR-positive patients with mild to severe symptoms. IgM signature peptide DGFFGVPR was detected in patients that recently recovered from COVID-19. A unique and quantitative LC-QTOF-MS method to quantify anti-SARS-CoV-2 IgG1 in serum was successfully developed and its clinical applicability has been demonstrated.