T and B cell epitope analysis for the immunogenicity evaluation and mitigation of antibody-based therapeutics.

The surge in the clinical use of therapeutic antibodies has reshaped the landscape of pharmaceutical therapy for many diseases, including rare and challenging conditions. However, the administration of exogenous biologics could potentially trigger unwanted immune responses such as generation of anti-drug antibodies (ADAs). Real-world experiences have illuminated the clear correlation between the ADA occurrence and unsatisfactory therapeutic outcomes as well as immune-related adverse events. By retrospectively examining research involving immunogenicity analysis, we noticed the growing emphasis on elucidating the immunogenic epitope profiles of antibody-based therapeutics aiming for mechanistic understanding the immunogenicity generation and, ideally, mitigating the risks. As such, we have comprehensively summarized here the progress in both experimental and computational methodologies for the characterization of T and B cell epitopes of therapeutics. Furthermore, the successful practice of epitope-driven deimmunization of biotherapeutics is exceptionally highlighted in this article.

Multifaceted Approach for Quantification and Enzymatic Activity of Iduronate-2-Sulfatase to Support Developing Gene Therapy for Hunter Syndrome.

Mucopolysaccharidosis type II, commonly called Hunter syndrome, is a rare X-linked recessive disease caused by the deficiency of the lysosomal enzyme iduronate-2-sulphatase (I2S). A deficiency of I2S causes an abnormal glycosaminoglycans accumulation in the body's cells. Although enzyme replacement therapy is the standard therapy, adeno-associated viruses (AAV)-based gene therapy could provide a single-dose solution to achieve a prolonged and constant enzyme level to improve patient's quality of life. Currently, there is no integrated regulatory guidance to describe the bioanalytical assay strategy to support gene therapy products. Herein, we describe the streamlined strategy to validate/qualify the transgene protein and its enzymatic activity assays. The method validation for the I2S quantification in serum and method qualification in tissues was performed to support the mouse GLP toxicological study. Standard curves for I2S quantification ranged from 2.00 to 50.0 µg/mL in serum and 6.25 to 400 ng/mL in the surrogate matrix. Acceptable precision, accuracy, and parallelism in the tissues were demonstrated. To assess the function of the transgene protein, fit-for-purpose method qualification for the I2S enzyme activity in serum was performed. The observed data indicated that the enzymatic activity in serum increased dose-dependently in the lower I2S concentration range. The highest I2S transgene protein was observed in the liver among tissue measured, and its expression level was maintained up to 91 days after the administration of rAAV8 with a codon-optimized human I2S. In conclusion, the multifaceted bioanalytical method for I2S and its enzymatic activity were established to assess gene therapy products in Hunter syndrome.

Development of a Highly Sensitive Hybrid LC/MS Assay for the Quantitative Measurement of CTLA-4 in Human T Cells.

Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) is a check point protein expressed on the surface of T cells and plays a central role in regulating the immune response. In recent years, CTLA-4 has become a popular target for cancer immunotherapy in which blocking CTLA-4 can restore T-cell function and enhance the immune response against cancer. Currently, there are many CTLA-4 inhibitors in a variety of modalities, including cell therapies, which are being developed in both preclinical and clinical stages to further harness the potential of the target for the treatment of certain types of cancer. In drug discovery research, measuring the level of CTLA-4 in T cells is important for drug discovery and development because it provides key information for quantitative assessment of the pharmacodynamics, efficacy, and safety of the CTLA-4-based therapies. However, to our best knowledge, there is still no report of a sensitive, specific, accurate, and reliable assay for CTLA-4 measurement. In this work, an LC/MS-based method was developed to measure CTLA-4 in human T cells. The assay demonstrated high specificity with an LLOQ of 5 copies of CTLA-4 per cell when using 2.5 million T cells for analysis. As shown in the work, the assay was successfully used to measure CTLA-4 levels in subtype T-cell samples from individual healthy subjects. The assay could be applied in supporting the studies of CTLA-4-based cancer therapies.

An Automated Multicycle Immunoaffinity Enrichment Approach Developed for Sensitive Mouse IgG1 Antibody Drug Analysis in Mouse Plasma Using LC/MS/MS.

In the immuno-oncology field, surrogate mouse monoclonal antibodies are often preferred in establishing proper PK/PD/efficacy correlations as well as supporting anticipated mouse to human translation. Thus, a highly sensitive and specific bioanalytical method is needed in quantifying those surrogate mouse antibodies after dosing in mice. Unfortunately, when specific reagents, such as recombinant target antigen and anti-idiotypic antibody, are not available, measuring mouse surrogate antibody drugs in mice is very challenging for ligand binding assay (LBA) due to the severe cross reactivity potential. Different from LBA, if at least one unique surrogate peptide can be identified from the surrogate antibody sequence, the immunoaffinity enrichment based LC/MS/MS assay may be able to differentiate the analyte response from the high endogenous immunoglobulin background and provide adequate sensitivity. Herein, a new automated multicycle immunoaffinity enrichment method was recently developed to extract a surrogate mouse IgG1 (mIgG1) antibody drug from mouse plasma using a commercially available antimouse IgG1 secondary antibody. In the assay, reuse of the capture antibody up to six times mostly resolved the binding capacity issue caused by the abundant endogenous mIgG1 and made the immunoaffinity enrichment step more cost-effective. Combined with a unique surrogate peptide identified from the antibody, the LC/MS/MS assay achieved a limit of quantitation of 5 ng/mL with satisfactory assay precision, accuracy, and dynamic range. The successful implementation of this novel approach in discovery pharmacokinetic (PK) studies eliminates the dependence on specially generated immunoaffinity capturing reagents.

Insights on Droplet Digital PCR-Based Cellular Kinetics and Biodistribution Assay Support for CAR-T Cell Therapy.

Characterizing in vivo cellular kinetics and biodistribution of chimeric antigen receptor T (CAR-T) cells is critical for toxicity assessment, nonclinical and clinical efficacy studies. To date, the standardized assay to characterize CAR-T cell distribution, expansion, contraction, and persistence profiles is not readily available. To overcome this limitation and increase comparability among studies, we have established a universal protocol for analysis. We established a duplexing ddPCR protocol for the CAR-T transgene and reference gene to normalize the genomic DNA input prepared from mouse blood and tissues. The high-throughput gDNA extraction method enabled highly reproducible gDNA extraction while eliminating labor-intensive steps. The investigational CAR-T cells were intravenously injected into immunodeficient mice bearing human colorectal cancer xenografts. The blood and tissue samples were collected to measure the cellular kinetics by ddPCR and flow cytometry. The standard curves were linear throughout the calibration range with acceptable intra- and inter-day precision and accuracy. The gDNA recovery study performed by spiking in the exo-gene plasmid DNA or CAR-T cells revealed that the recovery ranged from 60 to 100% in blood and tissue homogenates. The use of both units of copy/µg gDNA and copy/µL blood met the current regulatory requirement and allowed for a systematic understanding of CAR-T cell expansion and a direct comparison with the flow cytometry data. A standardized ddPCR assay, including automated gDNA extraction procedures, has been established for evaluating cellular kinetics and biodistribution in CAR-T cell therapies.

Preclinical Antitumor Activity and Biodistribution of a Novel Anti-GCC Antibody-Drug Conjugate in Patient-derived Xenografts.

Guanylyl cyclase C (GCC) is a unique therapeutic target with expression restricted to the apical side of epithelial cell tight junctions thought to be only accessible by intravenously administered agents on malignant tissues where GCC expression is aberrant. In this study, we sought to evaluate the therapeutic potential of a second-generation investigational antibody-dug conjugate (ADC), TAK-164, comprised of a human anti-GCC mAb conjugated via a peptide linker to the highly cytotoxic DNA alkylator, DGN549. The in vitro binding, payload release, and in vitro activity of TAK-164 was characterized motivating in vivo evaluation. The efficacy of TAK-164 and the relationship to exposure, pharmacodynamic marker activation, and biodistribution was evaluated in xenograft models and primary human tumor xenograft (PHTX) models. We demonstrate TAK-164 selectively binds to, is internalized by, and has potent cytotoxic effects against GCC-expressing cells in vitro A single intravenous administration of TAK-164 (0.76 mg/kg) resulted in significant growth rate inhibition in PHTX models of metastatic colorectal cancer. Furthermore, imaging studies characterized TAK-164 uptake and activity and showed positive relationships between GCC expression and tumor uptake which correlated with antitumor activity. Collectively, our data suggest that TAK-164 is highly active in multiple GCC-positive tumors including those refractory to TAK-264, a GCC-targeted auristatin ADC. A strong relationship between uptake of 89Zr-labeled TAK-164, levels of GCC expression and, most notably, response to TAK-164 therapy in GCC-expressing xenografts and PHTX models. These data supported the clinical development of TAK-164 as part of a first-in-human clinical trial (NCT03449030).

Pharmacokinetic Characterization and Tissue Distribution of Fusion Protein Therapeutics by Orthogonal Bioanalytical Assays and Minimal PBPK Modeling.

Characterization of pharmacokinetic (PK) properties and target tissue distribution of therapeutic fusion proteins (TFPs) are critical in supporting in vivo efficacy. We evaluated the pharmacokinetic profile of an investigational TFP consisting of human immunoglobulin G4 fused to the modified interferon alpha by orthogonal bioanalytical assays and applied minimal physiologically based pharmacokinetic (PBPK) modeling to characterize the TFP pharmacokinetics in mouse. The conventional ligand binding assay (LBA), immunocapture-liquid chromatography/tandem mass spectrometry (IC-LC/MS) detecting the human IgG4 peptide or the interferon alpha peptide were developed to measure the TFP concentrations in mouse plasma and tumor. The minimal PBPK model incorporated a tumor compartment model was used for data fitting. The plasma clearance measured by LBA and IC-LC/MS was comparable in the range of 0.5-0.6 mL/h/kg. However, the tumor exposure measured by the generic human IgG4 IC-LC/MS was significantly underestimated compared with the interferon alpha specific IC-LC/MS and LBA. Furthermore, the minimal PBPK model simultaneously captured the relationship between plasma and tissue exposure. We proposed the streamlined practical strategy to characterize the plasma exposure and tumor distribution of a TFP by both LBA and IC-LC/MS. The minimal PBPK modeling was established for better understanding of pharmacokinetic profile of investigational TFPs in the biotherapeutic discovery.

An LC/MS based method to quantify DNA adduct in tumor and organ tissues.

Highly potent DNA damaging agents have become a key class of toxins for antibody-drug conjugate (ADC) based targeted therapy. However, until recently, no quantitative bioanalytical method was available to measure the toxin in the form of DNA adducts. In this work, a novel microwave assisted organic solvent extraction and LC-MS/MS based bioanalytical method was developed to extract and quantify DNA-bound toxin IGN-P1 in tissue samples. Using ADC-1 as the model ADC, the method was orthogonally checked with a radioactive method for the recovery of free toxins from DNA adducts in biological matrices. It was found that the bioanalytical method can achieve a high recovery of the IGN-P1 toxin from DNA adducts. In further assessment, tumor and organ tissue samples collected at multiple time points from in vivo studies after dosing with two other ADCs, ADC-2 and ADC-3, were measured by the method. Given the generic nature of the established bioanalytical method without the need of radiolabels, the methodology could be broadly utilized to quantitatively assess the relationship between DNA adduct levels and pharmacological/toxicological effects.

Immunocapture-LC/MS-Based Target Engagement Measurement in Tumor Plasma Membrane.

For targeted therapies, immunocapture-liquid chromatography mass spectrometry (IC-LC/MS) technology has become an important tool for determination of both drug exposures, target antigen densities, and engagement in the systemic circulation and/or in total target tissue homogenates. Although the information collected from the circulation and tissue homogenates is useful in establishing the correlations of the exposure and target engagement with the pharmacodynamic response and efficacy of a therapy, the measurement at the cell plasma membrane within the target tissue is preferred, since it is the primary action site for antigen and the target drug. However, to the best of our knowledge, IC-LC/MS-based methodologies to conduct the assays at the plasma membrane from tissue sample has been quite limited. In this paper, we reported an IC-LC/MS-based assay platform for assessing the target engagement in tumor plasma membrane prepared from the tumor tissue samples. In addition, tumor samples with guanylyl cyclase C (GCC) expression after fully human IgG1 monoclonal antibody-based antibody-drug conjugate (ADC) treatment were used as a case study. The methodology can differentiate between the total and target-drug bound fraction of GCC with minimal potential equilibrium shift between in-cell surface protein and organelle protein in tumor samples to calculate in vivo target engagement. This approach to determine in vivo target engagement in tumor plasma membrane will provide better understanding of pharmacokinetic/pharmacodynamic relationship to achieve the desired antitumor efficacy.

Perspectives on potentiating immunocapture-LC-MS for the bioanalysis of biotherapeutics and biomarkers.

The integration of ligand-binding assay and LC-MS/MS (immunocapture-LC-MS) has unleashed the combined advantages of both powerful techniques for addressing the ever increasing bioanalytical challenges for biotherapeutics and biomarker assays. The highly specific, selective and sensitive characteristics of the immunocapture-LC-MS-based assays have enabled the determination of biotherapeutics and biomarkers in biomatrices with ease of method development, less requirements on key reagents as well as structural specificity for endogenous and engineered biomolecules. In addition, the versatile immunocapture-LC-MS technology has expanded into many challenging areas to enhance mechanistic studies of drug interactions with their targets. This paper intends to summarize our perspectives on enhancing the use of immunocapture-LC-MS in drug discovery and development for emerging new modalities.

A Two-Step Immunocapture LC/MS/MS Assay for Plasma Stability and Payload Migration Assessment of Cysteine-Maleimide-Based Antibody Drug Conjugates.

Plasma stability assessment under physiological temperature is an essential step for developing and optimizing antibody drug conjugate (ADC) molecules, especially those with cleavable linkers. The assessment of plasma stability often requires monitoring multiple analytes using a combination of bioanalytical assays for free payloads, conjugated payloads (or conjugated antibodies), total antibodies, and payloads that have migrated from antibodies to plasma constituent proteins. Bioanalytical assays are needed in early drug discovery to quickly screen diverse ADC candidates of different antibody constructs, linker variants, and antibody anchor sites. To improve the sensitivity and selectivity of LC/MS/MS-based assays for the assessment, immunocapture has been widely used for extracting ADCs and unconjugated antibodies from plasma samples. In this study, a novel two-step immunocapture LC/MS/MS assay was described to allow the quantification of conjugated payloads, total antibodies, and migrated payloads forming adducts with albumin in the plasma samples for stability assessment. A target antigen immobilized on magnetic beads was used to exhaustively extract the ADC and antibody-associated species. The remaining supernatant was then extracted further with anti-albumin beads for recovering the albumin-associated adducts for quantification. The method was optimized for higher efficiency and cost-effectiveness using microwave enhanced papain-based enzymatic cleavage for measuring conjugated payloads of ADCs and lysyl endopeptidase cleavage in the total antibody assay. A maleimide linker-based ADC with a proprietary payload, TAK-001, was used to demonstrate the streamlined workflow of the ADC stability assessment. The method could provide valuable evaluation of the stability of the ADC as well as the quantitative assessment of the albumin adducts formed from the linker-payload migration in mouse and human plasma. Furthermore, the method should be readily adaptable for other ADCs using thiol-maleimide conjugation chemistry.

Concurrent Inhibition of Neurosphere and Monolayer Cells of Pediatric Glioblastoma by Aurora A Inhibitor MLN8237 Predicted Survival Extension in PDOX Models.

Purpose: Pediatric glioblastoma multiforme (pGBM) is a highly aggressive tumor in need of novel therapies. Our objective was to demonstrate the therapeutic efficacy of MLN8237 (alisertib), an orally available selective inhibitor of Aurora A kinase (AURKA), and to evaluate which in vitro model system (monolayer or neurosphere) can predict therapeutic efficacy in vivoExperimental Design: AURKA mRNA expressions were screened with qRT-PCR. In vitro antitumor effects were examined in three matching pairs of monolayer and neurosphere lines established from patient-derived orthotopic xenograft (PDOX) models of the untreated (IC-4687GBM), recurrent (IC-3752GBM), and terminal (IC-R0315GBM) tumors, and in vivo therapeutic efficacy through log rank analysis of survival times in two models (IC-4687GBM and IC-R0315GBM) following MLN8237 treatment (30 mg/kg/day, orally, 12 days). Drug concentrations in vivo and mechanism of action and resistance were also investigated.Results: AURKA mRNA overexpression was detected in 14 pGBM tumors, 10 PDOX models, and 6 cultured pGBM lines as compared with 11 low-grade gliomas and normal brains. MLN8237 penetrated into pGBM xenografts in mouse brains. Significant extension of survival times were achieved in IC-4687GBM of which both neurosphere and monolayer were inhibited in vitro, but not in IC-R0315GBM of which only neurosphere cells responded (similar to IC-3752GBM). Apoptosis-mediated MLN8237 induced cell death, and the presence of AURKA-negative and CD133+ cells appears to have contributed to in vivo therapy resistance.Conclusions: MLN8237 successfully targeted AURKA in a subset of pGBMs. Our data suggest that combination therapy should aim at AURKA-negative and/or CD133+ pGBM cells to prevent tumor recurrence. Clin Cancer Res; 24(9); 2159-70. ©2018 AACR.

Rapid quantification of a cleavable antibody-conjugated drug by liquid chromatography/tandem mass spectrometry with microwave-assisted enzymatic cleavage.

Antibody-drug conjugates (ADCs) play an increasingly important role for targeted cancer treatment. One class of ADCs has attracted particular interest in drug development. These ADCs employ a cleavable chemistry linkage for drugs and utilize the reduced interchain disulfide cysteine residues for conjugation. In this work, a novel bioanalytical method for the quantification of a cleavable antibody-conjugated drug in plasma was developed, qualified, and implemented. This novel method significantly improves throughput by combining a microwave-assisted, enzymatic cleavage of conjugated drugs from ADCs with a 96-well based sample preparation procedure to immunocapture ADCs in plasma. The released drug is subsequently quantified using a LC/MS/MS method. Our results represent a high-throughput, generic, and sensitive quantification method for antibody-conjugated microtubule inhibitors (such as MMAE) for preclinical PK/PD studies. The linear range of the standard curve for antibody conjugated drug (MMAE) was from 2.01 to 2010ng/mL with an excellent linearity (r(2)>0.997). The intra-run precision was below 8.14% and accuracy was from -7.71% to -1.08%. No matrix effect or carryover was observed for this method. This method was successfully used to measure the level of conjugated drug in a preclinical PK/PD study in mice.

Pharmacokinetics of ixazomib, an oral proteasome inhibitor, in solid tumour patients with moderate or severe hepatic impairment.

AIM: The aim of the present study was to characterize the pharmacokinetics of the oral proteasome inhibitor, ixazomib, in patients with solid tumours and moderate or severe hepatic impairment, to provide posology recommendations. METHODS: Eligible adults with advanced malignancies for which no further effective therapy was available received a single dose of ixazomib on day 1 of the pharmacokinetic cycle; patients with normal hepatic function, moderate hepatic impairment or severe hepatic impairment received 4 mg, 2.3 mg or 1.5 mg, respectively. Blood samples for single-dose pharmacokinetic characterization were collected over 336 h postdose. After sampling, patients could continue to receive ixazomib on days 1, 8 and 15 in 28-day cycles. RESULTS: Of 48 enrolled patients (13, 15 and 20 in the normal, moderate and severe groups, respectively), 43 were pharmacokinetics-evaluable. Ixazomib was rapidly absorbed (median time to reach peak concentration was 0.95-1.5 h) and highly bound to plasma proteins, with a similar mean fraction bound (~99%) across the three groups. In patients with moderate/severe hepatic impairment (combined group), the geometric least squares mean ratios (90% confidence interval) for unbound and total dose-normalized area under the plasma concentration vs. time curve from time zero to the time of the last quantifiable concentration in reference to the normal hepatic function group were 1.27 (0.75, 2.16) and 1.20 (0.79, 1.82), respectively. Seven (15%) of the 48 patients experienced a grade 3 drug-related adverse event; there were no drug-related grade 4 adverse events. CONCLUSIONS: In patients with moderate/severe hepatic impairment, unbound and total systemic exposures of ixazomib were 27% and 20% higher, respectively, vs. normal hepatic function. A reduced ixazomib starting dose of 3 mg is recommended for patients with moderate or severe hepatic impairment.

Automated non-stepwise preparation of bioanalytical calibration standards and quality controls using an ultra-low volume digitizing liquid dispenser.

RATIONALE: Stepwise preparation of calibration standards and quality controls (QCs) is one of the most routine and laborious steps in bioanalysis. An alternative non-contact dispenser using low picoliter digitized dispensing technology is evaluated for its application in non-stepwise preparation of calibration curve and QCs in bioanalysis. METHODS: Fluorescein was initially used to assess the accuracy and precision of dispense volumes with fluorescent measurement. Various concentrations of MX-1, an in-house proprietary small molecule compound, in neat solution and in dog plasma were prepared manually with calibrated pipettors and digitally by the digital dispenser. The plasma samples were extracted by protein precipitation. The resultant extracted samples and neat solutions of MX-1 were analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) using an electrospray ionization (ESI) source in positive ion mode with selected reaction monitoring (SRM) of the mass transitions. RESULTS: In the three-day precision and accuracy assessment of dispensing volumes between 13 pL to 411.2 nL, the intra-day precision and accuracy ranged from 1.4% to 10.3% and -12.7% to 12.8%, respectively. The inter-day precision and accuracy ranged from 3.5% to 7.8% and -6.6% to 10.4%, respectively. For real analysis of in vivo study samples, all 49 samples analyzed showed a less than 5% difference between calibrations with digital and manual curve preparations. The resultant pharmacokinetic (PK) parameters were physiologically comparable as well. CONCLUSIONS: Using the digitized picoliter dispensing technology, high-speed automated precise and accurate dispense of a wide range of volumes can be achieved and tests for bioanalytical standards and QC preparations passed the stringent criteria set forth for regulated bioanalysis using LC/MS/MS-based technology. The digital dispenser has been found to be a useful tool in drug discovery for automatically preparing standards and QCs in seconds with low consumption of stock solutions and blank matrices.

A novel dynamic flush method to reduce column-related carryover.

Column-related carryover affects both accuracy and precision in liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. In this work, a novel straightforward dynamic flush method to reduce the column-related carryover was developed by alternating the column flow direction of liquid chromatographic separation with a Valco switching valve and pristine instrument control software. By alternating the column flow direction, a fresh inlet is always in line to accommodate sample injection and stacking. In addition, the contaminated column inlet from the previous run is switched to the outlet position for a flush with a gradient during the next sample run. In this way, the column-related carryover can be reduced effectively without additional blank runs. It also minimizes the carryover risk between the adjacent unknown samples. The column-related carryover of the tested "sticky" compounds was reduced by 52.3-94.4% compared with the non-dynamic flush method under the same experimental conditions. The performance and reproducibility of high-performance liquid chromatography (HPLC) separation in terms of the retention time shift and peak shape are not compromised under the dynamic flush even after over 300 consecutive injections. The described novel method is simple and easy to implement for compounds with column-related carryover.

Ras-driven transcriptome analysis identifies aurora kinase A as a potential malignant peripheral nerve sheath tumor therapeutic target.

PURPOSE: Patients with neurofibromatosis type 1 (NF1) develop malignant peripheral nerve sheath tumors (MPNST), which are often inoperable and do not respond well to current chemotherapies or radiation. The goal of this study was to use comprehensive gene expression analysis to identify novel therapeutic targets. EXPERIMENTAL DESIGN: Nerve Schwann cells and/or their precursors are the tumorigenic cell types in MPNST because of the loss of the NF1 gene, which encodes the RasGAP protein neurofibromin. Therefore, we created a transgenic mouse model, CNP-HRas12V, expressing constitutively active HRas in Schwann cells and defined a Ras-induced gene expression signature to drive a Bayesian factor regression model analysis of differentially expressed genes in mouse and human neurofibromas and MPNSTs. We tested functional significance of Aurora kinase overexpression in MPNST in vitro and in vivo using Aurora kinase short hairpin RNAs (shRNA) and compounds that inhibit Aurora kinase. RESULTS: We identified 2,000 genes with probability of linkage to nerve Ras signaling of which 339 were significantly differentially expressed in mouse and human NF1-related tumor samples relative to normal nerves, including Aurora kinase A (AURKA). AURKA was dramatically overexpressed and genomically amplified in MPNSTs but not neurofibromas. Aurora kinase shRNAs and Aurora kinase inhibitors blocked MPNST cell growth in vitro. Furthermore, an AURKA selective inhibitor, MLN8237, stabilized tumor volume and significantly increased survival of mice with MPNST xenografts. CONCLUSION: Integrative cross-species transcriptome analyses combined with preclinical testing has provided an effective method for identifying candidates for molecular-targeted therapeutics. Blocking Aurora kinases may be a viable treatment platform for MPNST.

MLN0905, a small-molecule plk1 inhibitor, induces antitumor responses in human models of diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common of the non-Hodgkin lymphomas, accounting for up to 30% of all newly diagnosed lymphoma cases. Current treatment options for this disease are effective, but not always curative; therefore, experimental therapies continue to be investigated. We have discovered an experimental, potent, and selective small-molecule inhibitor of PLK1, MLN0905, which inhibits cell proliferation in a broad range of human tumor cells including DLBCL cell lines. In our report, we explored the pharmacokinetic, pharmacodynamic, and antitumor properties of MLN0905 in DLBCL xenograft models grown in mice. These studies indicate that MLN0905 modulates the pharmacodynamic biomarker phosphorylated histone H3 (pHisH3) in tumor tissue. The antitumor activity of MLN0905 was evaluated in three human subcutaneous DLBCL xenograft models, OCI LY-10, OCI LY-19, and PHTX-22L (primary lymphoma). In each model, MLN0905 yielded significant antitumor activity on both a continuous (daily) and intermittent dosing schedule, underscoring dosing flexibility. The antitumor activity of MLN0905 was also evaluated in a disseminated xenograft (OCI LY-19) model to better mimic human DLBCL disease. In the disseminated model, MLN0905 induced a highly significant survival advantage. Finally, MLN0905 was combined with a standard-of-care agent, rituximab, in the disseminated OCI LY-19 xenograft model. Combining rituximab and MLN0905 provided both a synergistic antitumor effect and a synergistic survival advantage. Our findings indicate that PLK1 inhibition leads to pharmacodynamic pHisH3 modulation and significant antitumor activity in multiple DLBCL models. These data strongly suggest evaluating PLK1 inhibitors as DLBCL anticancer agents in the clinic.