A molecular glue degrader of the WIZ transcription factor for fetal hemoglobin induction.

Sickle cell disease (SCD) is a prevalent, life-threatening condition attributable to a heritable mutation in ß-hemoglobin. Therapeutic induction of fetal hemoglobin (HbF) can ameliorate disease complications and has been intently pursued. However, safe and effective small-molecule inducers of HbF remain elusive. We report the discovery of dWIZ-1 and dWIZ-2, molecular glue degraders of the WIZ transcription factor that robustly induce HbF in erythroblasts. Phenotypic screening of a cereblon (CRBN)-biased chemical library revealed WIZ as a previously unknown repressor of HbF. WIZ degradation is mediated by recruitment of WIZ(ZF7) to CRBN by dWIZ-1, as resolved by crystallography of the ternary complex. Pharmacological degradation of WIZ was well tolerated and induced HbF in humanized mice and cynomolgus monkeys. These findings establish WIZ degradation as a globally accessible therapeutic strategy for SCD.

Two cases of severe pulmonary toxicity from highly active mesothelin-directed CAR T cells.

Multiple clinical studies have treated mesothelin (MSLN)-positive solid tumors by administering MSLN-directed chimeric antigen receptor (CAR) T cells. Although these products are generally safe, efficacy is limited. Therefore, we generated and characterized a potent, fully human anti-MSLN CAR. In a phase 1 dose-escalation study of patients with solid tumors, we observed two cases of severe pulmonary toxicity following intravenous infusion of this product in the high-dose cohort (1-3 × 108 T cells per m2). Both patients demonstrated progressive hypoxemia within 48 h of infusion with clinical and laboratory findings consistent with cytokine release syndrome. One patient ultimately progressed to grade 5 respiratory failure. An autopsy revealed acute lung injury, extensive T cell infiltration, and accumulation of CAR T cells in the lungs. RNA and protein detection techniques confirmed low levels of MSLN expression by benign pulmonary epithelial cells in affected lung and lung samples obtained from other inflammatory or fibrotic conditions, indicating that pulmonary pneumocyte and not pleural expression of mesothelin may lead to dose-limiting toxicity. We suggest patient enrollment criteria and dosing regimens of MSLN-directed therapies consider the possibility of dynamic expression of mesothelin in benign lung with a special concern for patients with underlying inflammatory or fibrotic conditions.

Characterization of sabatolimab, a novel immunotherapy with immuno-myeloid activity directed against TIM-3 receptor.

Objectives: Sabatolimab is a humanized monoclonal antibody (hIgG4, S228P) directed against human T-cell immunoglobulin domain and mucin domain-3 (TIM-3). Herein, we describe the development and characterization of sabatolimab. Methods: Sabatolimab was tested for binding to its target TIM-3 and blocking properties. The functional effects of sabatolimab were tested in T-cell killing and myeloid cell cytokine assays. Antibody-mediated cell phagocytosis (ADCP) by sabatolimab was also assessed. Results: Sabatolimab was shown to (i) enhance T-cell killing and inflammatory cytokine production by dendritic cells (DCs); (ii) facilitate the phagocytic uptake of TIM-3-expressing target cells; and (iii) block the interaction between TIM-3 and its ligands PtdSer/galectin-9. Conclusion: Taken together, our results support both direct anti-leukemic effects and immune-mediated modulation by sabatolimab, reinforcing the notion that sabatolimab represents a novel immunotherapy with immuno-myeloid activity, holding promise for the treatment of myeloid cell neoplasms.

Blockade of Tim-3 binding to phosphatidylserine and CEACAM1 is a shared feature of anti-Tim-3 antibodies that have functional efficacy.

Both in vivo data in preclinical cancer models and in vitro data with T cells from patients with advanced cancer support a role for Tim-3 blockade in promoting effective anti-tumor immunity. Consequently, there is considerable interest in the clinical development of antibody-based therapeutics that target Tim-3 for cancer immunotherapy. A challenge to this clinical development is the fact that several ligands for Tim-3 have been identified: galectin-9, phosphatidylserine, HMGB1, and most recently, CEACAM1. These observations raise the important question of which of these multiple receptor:ligand relationships must be blocked by an anti-Tim-3 antibody in order to achieve therapeutic efficacy. Here, we have examined the properties of anti-murine and anti-human Tim-3 antibodies that have shown functional efficacy and find that all antibodies bind to Tim-3 in a manner that interferes with Tim-3 binding to both phosphatidylserine and CEACAM1. Our data have implications for the understanding of Tim-3 biology and for the screening of anti-Tim-3 antibody candidates that will have functional properties in vivo.

Contribution of polycomb homologues Bmi-1 and Mel-18 to medulloblastoma pathogenesis.

Bmi-1 and Mel-18 are structural homologues that belong to the Polycomb group of transcriptional regulators and are believed to stably maintain repression of gene expression by altering the state of chromatin at specific promoters. While a number of clinical and experimental observations have implicated Bmi-1 in human tumorigenesis, the role of Mel-18 in cancer cell growth has not been investigated. We report here that short hairpin RNA-mediated knockdown of either Bmi-1 or Mel-18 in human medulloblastoma DAOY cells results in the inhibition of proliferation, loss of clonogenic survival, anchorage-independent growth, and suppression of tumor formation in nude mice. Furthermore, overexpression of both Bmi-1 and Mel-18 significantly increases the clonogenic survival of Rat1 fibroblasts. In contrast, stable downregulation of Bmi-1 or Mel-18 alone does not affect the growth of normal human WI38 fibroblasts. Proteomics-based characterization of Bmi-1 and Mel-18 protein complexes isolated from cancer cells revealed substantial similarities in their respective compositions. Finally, gene expression analysis identified a number of cancer-relevant pathways that may be controlled by Bmi-1 and Mel-18 and also showed that these Polycomb proteins regulate a set of common gene targets. Taken together, these results suggest that Bmi-1 and Mel-18 may have overlapping functions in cancer cell growth.

Mapping the proteome of Drosophila melanogaster: analysis of embryos and adult heads by LC-IMS-MS methods.

Multidimensional separations combined with mass spectrometry are used to study the proteins that are present in two states of Drosophila melanogaster: the whole embryo and the adult head. The approach includes the incorporation of a gas-phase separation dimension in which ions are dispersed according to differences in their mobilities and is described as a means of providing a detailed analytical map of the proteins that are present. Overall, we find evidence for 1133 unique proteins. In total, 780 are identified in the head, and 660 are identified in the embryo. Only 307 proteins are in common to both developmental stages, indicating that there are significant differences in these proteomes. A comparison of the proteome to a database of mRNAs that are found from analysis by cDNA approaches (i.e., transcriptome) also shows little overlap. All of this information is discussed in terms of the relationship between the predicted genome, and measured transcriptomes and proteomes. Additionally, the merits and weaknesses of current technologies are assessed in some detail.

Proteome profiling for assessing diversity: analysis of individual heads of Drosophila melanogaster using LC-ion mobility-MS.

The proteomes of three heads of individual Drosophila melanogaster organisms have been analyzed and compared by a combination of liquid chromatography, ion mobility spectrometry, and mass spectrometry approaches. In total, 197 proteins are identified among all three individuals (an average of 120 +/- 20 proteins per individual), of which at least 101 proteins are present in all three individuals. Within all three datasets, more than 25 000 molecular ions (an average of 9000 +/- 2000 per individual) corresponding to protonated precursor ions of individual peptides have been observed. A comparison of peaks among the datasets reveals that peaks corresponding to protonated peptides that are found in all heads are more intense than those features that appear between pairs of or within only one of the individuals. Moreover, there is little variability in the relative intensities of the peaks common among all individuals. It appears that it is the lower abundance components of the proteome that play the most significant role in determining unique features of individuals.

Coupling capillary electrochromatography with electrospray Fourier transform mass spectrometry for characterizing complex oligosaccharide pools.

To deal with the complexity of the glycan mixtures released from glycoproteins, an efficient form of micro-column separations, capillary electrochromatography, has been combined with high-performance mass spectrometry (Fourier transform ion cyclotron resonance). Contour plots have been generated from the mixtures of O-linked oligosaccharides originated from bovine mucin and bile salt-stimulated lipase, a large glycoprotein enzyme.

Development of high-sensitivity ion trap ion mobility spectrometry time-of-flight techniques: a high-throughput nano-LC-IMS-TOF separation of peptides arising from a Drosophila protein extract.

A linear octopole trap interface for an ion mobility time-of-flight mass spectrometer has been developed for focusing and accumulating continuous beams of ions produced by electrospray ionization. The interface improves experimental efficiencies by factors of approximately 50-200 compared with an analogous configuration that utilizes a three-dimensional Paul geometry trap (Hoaglund-Hyzer, C. S.; Lee, Y. J.; Counterman, A. E.; Clemmer, D. E. Anal. Chem. 2002, 74, 992-1006). With these improvements, it is possible to record nested drift (flight) time distributions for complex mixtures in fractions of a second. We demonstrate the approach for several well-defined peptide mixtures and an assessment of the detection limits is given. Additionally, we demonstrate the utility of the approach in the field of proteomics by an on-line, three-dimensional nano-LC-ion mobility-TOF separation of tryptic peptides from the Drosophila proteome.

Quantifying peptides in isotopically labeled protease digests by ion mobility/time-of-flight mass spectrometry.

Ion mobility/time-of-flight techniques have been used to analyze mixtures of isotopically labeled peptides. The isotopic labels were generated by treatment of peptides with N-acetoxysuccinimide (or the deuterated analogue), which results in acetylation (or deuterioacetylation) of the primary amines (i.e., the N-terminus and lysine residues). The relative concentrations of a peptide in each sample are determined by comparing the peak intensities for isotopic pairs. An important consideration is that as mixtures become increasingly complex, isotopic pairs of peaks may overlap with other peaks in the mass spectrum. The influence of the acetyl and deuterioacetyl groups on the mobilities of peptides is considered. The coincidence in mobilities of isotopic pairs provides a means of distinguishing isotopic pairs from other isobaric interferences.

Conformational studies of Zn-ligand-hexose diastereomers using ion mobility measurements and density functional theory calculations.

Ion mobility studies and density functional theory calculations were used to study the structures of [Zn/diethylenetriamine/Hexose/Cl]+ complexes in an effort to probe differences in the three-dimensional conformations. This information allows us to gain insight into the structure of these complexes before collisional activation, which is the first step in understanding the stereoselective dissociations observed under collisionally activated conditions. The collision cross sections obtained from the ion mobility measurements showed that the mannose structure is more compact than the galactose and glucose complexes, respectively. Using density functional theory, candidate structures for each of the experimentally observed complexes were generated. Two criteria were used to determine the most likely structures of these complexes before activation: (1) The allowed relative energies of the molecules (between 0-90 kJ/mol) and (2) collision cross section agreement (within 2%) between the theoretically determined structures and the experimentally determined cross section. It was found that the identity of the monosaccharide made a difference in the overall conformation of the metal-ligand-monosaccharide complex. For glucose and galactose, metal coordination to O(6) was found to be favorable, with the monosaccharide occupying the 4C1 chair conformation, while for mannose, O(2) metal coordination was found with the monosaccharide in a B3,0 conformation. Coordination numbers varied between four and six for the Zn(II) metal centers. Given these results, it appears that the stereochemistry of the monosaccharide influences the conformation and metal coordination sites of the Zn(II)/monosaccharide/dien complex. These differences may influence the dissociation products observed under collisionally activated conditions.