Design, Synthesis, and In Vitro and In Vivo Evaluation of Cereblon Binding Bruton's Tyrosine Kinase (BTK) Degrader CD79b Targeted Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) are an established modality that allow for targeted delivery of a potent molecule, or payload, to a desired site of action. ADCs, wherein the payload is a targeted protein degrader, are an emerging area in the field. Herein we describe our efforts of delivering a Bruton's tyrosine kinase (BTK) bifunctional degrader 1 via a CD79b mAb (monoclonal antibody) where the degrader is linked at the ligase binding portion of the payload via a cleavable linker to the mAb. The resulting CD79b ADCs, 3 and 4, exhibit in vitro degradation and cytotoxicity comparable with that of 1, and ADC 3 can achieve more sustained in vivo degradation than intravenously administered 1 with markedly reduced systemic exposure of the payload.

Structure of lipoprotein lipase in complex with GPIHBP1.

Lipoprotein lipase (LPL) plays a central role in triglyceride (TG) metabolism. By catalyzing the hydrolysis of TGs present in TG-rich lipoproteins (TRLs), LPL facilitates TG utilization and regulates circulating TG and TRL concentrations. Until very recently, structural information for LPL was limited to homology models, presumably due to the propensity of LPL to unfold and aggregate. By coexpressing LPL with a soluble variant of its accessory protein glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) and with its chaperone protein lipase maturation factor 1 (LMF1), we obtained a stable and homogenous LPL/GPIHBP1 complex that was suitable for structure determination. We report here X-ray crystal structures of human LPL in complex with human GPIHBP1 at 2.5-3.0 Å resolution, including a structure with a novel inhibitor bound to LPL. Binding of the inhibitor resulted in ordering of the LPL lid and lipid-binding regions and thus enabled determination of the first crystal structure of LPL that includes these important regions of the protein. It was assumed for many years that LPL was only active as a homodimer. The structures and additional biochemical data reported here are consistent with a new report that LPL, in complex with GPIHBP1, can be active as a monomeric 1:1 complex. The crystal structures illuminate the structural basis for LPL-mediated TRL lipolysis as well as LPL stabilization and transport by GPIHBP1.

Interaction with both ZNRF3 and LGR4 is required for the signalling activity of R-spondin.

R-spondin proteins sensitize cells to Wnt signalling and act as potent stem cell growth factors. Various membrane proteins have been proposed as potential receptors of R-spondin, including LGR4/5, membrane E3 ubiquitin ligases ZNRF3/RNF43 and several others proteins. Here, we show that R-spondin interacts with ZNRF3/RNF43 and LGR4 through distinct motifs. Both LGR4 and ZNRF3 binding motifs are required for R-spondin-induced LGR4/ZNRF3 interaction, membrane clearance of ZNRF3 and activation of Wnt signalling. Importantly, Wnt-inhibitory activity of ZNRF3, but not of a ZNRF3 mutant with reduced affinity to R-spondin, can be strongly suppressed by R-spondin, suggesting that R-spondin primarily functions by binding and inhibiting ZNRF3. Together, our results support a dual receptor model of R-spondin action, where LGR4/5 serve as the engagement receptor whereas ZNRF3/RNF43 function as the effector receptor.

The role of airway mucus and diseased pulmonary epithelium on the absorption of inhaled antibodies.

Inhaled antibody therapy for the treatment of respiratory diseases is a promising strategy to maximize pulmonary exposure and reduce side effects associated with parenteral administration. However, the development of inhaled antibodies is often challenging due to a poor understanding of key mechanisms governing antibody absorption and clearance in healthy and diseased pulmonary epithelium. Here, we utilize well established Human Bronchial Epithelial Cell (HBEC) models grown at air-liquid interface to study the absorption process of antibodies and antibody fragments. With these cellular models, we recapitulate the morphology and function of healthy and diseased pulmonary epithelium, and incorporate the mucosal barrier to enable the investigation of both cellular permeability as well as mucodiffusion. We studied the saturation of antibody transport across the HBEC barriers and estimated the impact of disease-like epithelial barriers on antibody paracellular transport. Additionally, we identified a potential role of neonatal Fc receptor (FcRn)-independent and target-mediated transcytosis in the transport of Fragment antigen-binding (Fab) and F(ab)2 antibody fragments. Lastly, our models were able to pinpoint an impaired antibody diffusion across mucus gels. These mechanistic cellular models are promising in vitro tools to inform Physiologically-based Pharmacokinetic (PBPK) computational models for dose prediction toward de-risking the development of inhaled biologics.

A CRISPR Screen Reveals Resistance Mechanisms to CD3-Bispecific Antibody Therapy.

CD3-bispecific antibodies represent an important therapeutic strategy in oncology. These molecules work by redirecting cytotoxic T cells to antigen-bearing tumor cells. Although CD3-bispecific antibodies have been developed for several clinical indications, cases of cancer-derived resistance are an emerging limitation to the more generalized application of these molecules. Here, we devised whole-genome CRISPR screens to identify cancer resistance mechanisms to CD3-bispecific antibodies across multiple targets and cancer types. By validating the screen hits, we found that deficiency in IFNγ signaling has a prominent role in cancer resistance. IFNγ functioned by stimulating the expression of T-cell killing-related molecules in a cell type-specific manner. By assessing resistance to the clinical CD3-bispecific antibody flotetuzumab, we identified core fucosylation as a critical pathway to regulate flotetuzumab binding to the CD123 antigen. Disruption of this pathway resulted in significant resistance to flotetuzumab treatment. Proper fucosylation of CD123 was required for its normal biological functions. In order to treat the resistance associated with fucosylation loss, flotetuzumab in combination with an alternative targeting CD3-bispecific antibody demonstrated superior efficacy. Together, our study reveals multiple mechanisms that can be targeted to enhance the clinical potential of current and future T-cell-engaging CD3-bispecific antibody therapies.

PCA062, a P-cadherin Targeting Antibody-Drug Conjugate, Displays Potent Antitumor Activity Against P-cadherin-expressing Malignancies.

The cell surface glycoprotein P-cadherin is highly expressed in a number of malignancies, including those arising in the epithelium of the bladder, breast, esophagus, lung, and upper aerodigestive system. PCA062 is a P-cadherin specific antibody-drug conjugate that utilizes the clinically validated SMCC-DM1 linker payload to mediate potent cytotoxicity in cell lines expressing high levels of P-cadherin in vitro, while displaying no specific activity in P-cadherin-negative cell lines. High cell surface P-cadherin is necessary, but not sufficient, to mediate PCA062 cytotoxicity. In vivo, PCA062 demonstrated high serum stability and a potent ability to induce mitotic arrest. In addition, PCA062 was efficacious in clinically relevant models of P-cadherin-expressing cancers, including breast, esophageal, and head and neck. Preclinical non-human primate toxicology studies demonstrated a favorable safety profile that supports clinical development. Genome-wide CRISPR screens reveal that expression of the multidrug-resistant gene ABCC1 and the lysosomal transporter SLC46A3 differentially impact tumor cell sensitivity to PCA062. The preclinical data presented here suggest that PCA062 may have clinical value for treating patients with multiple cancer types including basal-like breast cancer.

Identification of small-molecule inhibitors of the JIP-JNK interaction.

JNK1 (c-Jun N-terminal kinase 1) plays a crucial role in the regulation of obesity-induced insulin resistance and is implicated in the pathology of Type 2 diabetes. Its partner, JIP1 (JNK-interacting protein 1), serves a scaffolding function that facilitates JNK1 activation by MKK4 [MAPK (mitogen-activated protein kinase) kinase 4] and MKK7 (MAPK kinase 7). For example, reduced insulin resistance and JNK activation are observed in JIP1-deficient mice. On the basis of the in vivo efficacy of a cell-permeable JIP peptide, the JIP-JNK interaction appears to be a potential target for JNK inhibition. The goal of the present study was to identify small-molecule inhibitors that disrupt the JIP-JNK interaction to provide an alternative approach for JNK inhibition to ATP-competitive inhibitors. High-throughput screening was performed by utilizing a fluorescence polarization assay that measured the binding of JNK1 to the JIP peptide. Multiple chemical series were identified, revealing two categories of JIP/JNK inhibitors: 'dual inhibitors' that are ATP competitive and probably inhibit JIP-JNK binding allosterically, and 'JIP-site binders' that block binding through interaction with the JIP site. A series of polychloropyrimidines from the second category was characterized by biochemical methods and explored through medicinal-chemistry efforts. As predicted, these inhibitors also inhibited full-length JIP-JNK binding and were selective against a panel of 34 representative kinases, including ones in the MAPK family. Overall, this work demonstrates that small molecules can inhibit protein-protein interactions in vitro in the MAPK family effectively and provides strategies for similar approaches within other target families.

High-throughput biochemical kinase selectivity assays: panel development and screening applications.

Kinases represent attractive targets for drug discovery. Eight small-molecule kinase inhibitors are currently marketed in the area of oncology, and numerous others are in clinical trials. Characterization of the selectivity profiles of these compounds is important to target appropriate patient populations and to reduce the potential of toxicity due to off-target effects. The authors describe the development, validation, and utilization of a biochemical kinase assay panel for the selectivity profiling of inhibitors. The panel was developed as 29 radiometric Flashplate assays, and then an initial 13 were transitioned to a nonradiometric Caliper mobility shift assay format. Generation of high-quality data from the panel is detailed along with a comparison of the assay formats. Both assay technologies were found to be suitable for panel screening, but mobility shift assays yielded higher data quality. The selectivity data generated here should be useful in computational modeling and help facilitate, in conjunction with sequence and structural information, the rational design of inhibitors with well-defined selectivity profiles.

High yield expression of non-phosphorylated protein tyrosine kinases in insect cells.

The key role of kinases in signal transduction and cell growth regulation has been a long standing interest among academics and the pharmaceutical industry. Recombinant enzymes have been used to understand the mechanism of action as well as to screen for chemical inhibitors. The baculo-insect system has been the primary method used to obtain soluble and active kinases, usually producing a mixture of the kinase in various phosphorylation states in different conformations. To obtain a homogenous preparation of non-phosphorylated kinases is critical for biochemical, biophysical and kinetic studies aimed at understanding the mechanism of kinase activation. Taking advantage of the eukaryotic expression property of insect cells, we were able to obtain high yield expression of non-phosphorylated protein tyrosine kinases BTK, JAK3 and Eph2A through coexpression with the tyrosine phosphatase YopH, which suggests that this method can be applied to protein tyrosine kinases in general. We have demonstrated that the fully non-phosphorylated BTK obtained with this method is suitable for various biochemical and kinetic studies.

TRIAMF: A New Method for Delivery of Cas9 Ribonucleoprotein Complex to Human Hematopoietic Stem Cells.

CRISPR/Cas9 mediated gene editing of patient-derived hematopoietic stem and progenitor cells (HSPCs) ex vivo followed by autologous transplantation of the edited HSPCs back to the patient can provide a potential cure for monogenic blood disorders such as ß-hemoglobinopathies. One challenge for this strategy is efficient delivery of the ribonucleoprotein (RNP) complex, consisting of purified Cas9 protein and guide RNA, into HSPCs. Because ß-hemoglobinopathies are most prevalent in developing countries, it is desirable to have a reliable, efficient, easy-to-use and cost effective delivery method. With this goal in mind, we developed TRansmembrane Internalization Assisted by Membrane Filtration (TRIAMF), a new method to quickly and effectively deliver RNPs into HSPCs by passing a RNP and cell mixture through a filter membrane. We achieved robust gene editing in HSPCs using TRIAMF and demonstrated that the multilineage colony forming capacities and the competence for engraftment in immunocompromised mice of HSPCs were preserved post TRIAMF treatment. TRIAMF is a custom designed system using inexpensive components and has the capacity to process HSPCs at clinical scale.

Pre-clinical validation of B cell maturation antigen (BCMA) as a target for T cell immunotherapy of multiple myeloma.

Multiple myeloma has a continued need for more effective and durable therapies. B cell maturation antigen (BCMA), a plasma cell surface antigen and member of the tumor necrosis factor (TNF) receptor superfamily, is an attractive target for immunotherapy of multiple myeloma due to its high prevalence on malignant plasma cells. The current work details the pre-clinical evaluation of BCMA expression and development of a chimeric antigen receptor (CAR) targeting this antigen using a fully human single chain variable fragment (scFv). We demonstrate that BCMA is prevalently, but variably expressed by all MM with expression on 25-100% of malignant plasma cells. Extensive Immunohistochemical analysis of normal tissue expression using commercially available polyclonal antibodies demonstrated expression within B-lineage cells across a number of tissues as expected. Based upon the highly restricted expression of BCMA within normal tissues, we generated a set of novel, fully human scFv binding domains to BCMA by screening a naïve B-cell derived phage display library. Using a series of in vitro and pre-clinical in vivo studies, we identified a scFv with high specificity for BCMA and robust anti-myeloma activity when used as the binding domain of a second-generation CAR bearing a CD137 costimulatory domain. This BCMA-specific CAR is currently being evaluated in a Phase 1b clinical study in relapsed and refractory MM patients (NCT02546167).

Discovery and Optimization of HKT288, a Cadherin-6-Targeting ADC for the Treatment of Ovarian and Renal Cancers.

Despite an improving therapeutic landscape, significant challenges remain in treating the majority of patients with advanced ovarian or renal cancer. We identified the cell-cell adhesion molecule cadherin-6 (CDH6) as a lineage gene having significant differential expression in ovarian and kidney cancers. HKT288 is an optimized CDH6-targeting DM4-based antibody-drug conjugate (ADC) developed for the treatment of these diseases. Our study provides mechanistic evidence supporting the importance of linker choice for optimal antitumor activity and highlights CDH6 as an antigen for biotherapeutic development. To more robustly predict patient benefit of targeting CDH6, we incorporate a population-based patient-derived xenograft (PDX) clinical trial (PCT) to capture the heterogeneity of response across an unselected cohort of 30 models-a novel preclinical approach in ADC development. HKT288 induces durable tumor regressions of ovarian and renal cancer models in vivo, including 40% of models on the PCT, and features a preclinical safety profile supportive of progression toward clinical evaluation.Significance: We identify CDH6 as a target for biotherapeutics development and demonstrate how an integrated pharmacology strategy that incorporates mechanistic pharmacodynamics and toxicology studies provides a rich dataset for optimizing the therapeutic format. We highlight how a population-based PDX clinical trial and retrospective biomarker analysis can provide correlates of activity and response to guide initial patient selection for first-in-human trials of HKT288. Cancer Discov; 7(9); 1030-45. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 920.

Development of a multiplex UPLC-MRM MS method for quantification of human membrane transport proteins OATP1B1, OATP1B3 and OATP2B1 in in vitro systems and tissues.

OATP1B1, OATP1B3 and OATP2B1 are important members of the organic anion transporting polypeptides (OATP) family and are implicated in the hepatic disposition of endobiotics and xenobiotics. Quantitating the expression levels of human OATP1B1, OATP1B3 and OATP2B1 in in vitro systems and tissue samples could significantly improve attempts to scale up in vitro data and result in more effective in vitro-in vivo correlation of transporter-mediated effects on drug disposition, such as hepatic clearance. In the present study, a quantification method was developed, characterized, and implemented for simultaneous determination of human OATP1B1, OATP1B3 and OATP2B1 in HEK cells transfected with OATP-expressing plasmid vectors (SLCO1B1, SLCO1B3, and SLCO2B1, respectively), human hepatocytes, human brain capillary endothelial cells, and humanized mouse liver tissue using UPLC-MRM MS. Purified membrane protein standards prepared and characterized as previously reported (Protein Expr. Purif. 2008, 57, 163-71) were first used as standards for absolute quantification of the expression levels of the three human OATP membrane proteins. The specificity of the optimized MRM transitions were characterized by analyzing the tryptic digests of the membrane protein fraction of wild type HEK cells and control mouse liver tissue using the herein reported UPLC-MRM MS method. The linearity of the calibration curve spanned from 0.2 µg mL(-1) (0.040 µg mg(-1)) to 20 µg mL(-1) (4.0 µg mg(-1)), with accuracy (% RE) within 15% at all concentrations examined for all three OATPs of interest in this study. The intra- and inter-day assay accuracy (% RE) and coefficient of variations (% CV) of triplicates are all within 15% for all levels of quality control samples prepared by mixing the membrane fraction of control mouse liver tissue with the required amount of purified human OATP1B1, OATP1B3 and OATP2B1.

Endotoxin removal and prevention for pre-clinical biologics production.

The removal of endotoxin from protein solutions and its prevention are key to the success of recombinant protein production due to the possible pyogenic response in mammals caused by contaminated samples. In the pre-clinical situation, protein production is often carried out in a non-good manufacturing practice (GMP) setting, utilizing bacterial DNA for transient transfection and non-validated cleaning techniques. Here, we present our findings evaluating various options for endotoxin removal, and propose strategies for endotoxin prevention with emphasis on chromatographic separations, endotoxin-removing membranes and on-column wash strategies.

TEM8/ANTXR1 blockade inhibits pathological angiogenesis and potentiates tumoricidal responses against multiple cancer types.

Current antiangiogenic agents used to treat cancer only partially inhibit neovascularization and cause normal tissue toxicities, fueling the need to identify therapeutic agents that are more selective for pathological angiogenesis. Tumor endothelial marker 8 (TEM8), also known as anthrax toxin receptor 1 (ANTXR1), is a highly conserved cell-surface protein overexpressed on tumor-infiltrating vasculature. Here we show that genetic disruption of Tem8 results in impaired growth of human tumor xenografts of diverse origin including melanoma, breast, colon, and lung cancer. Furthermore, antibodies developed against the TEM8 extracellular domain blocked anthrax intoxication, inhibited tumor-induced angiogenesis, displayed broad antitumor activity, and augmented the activity of clinically approved anticancer agents without added toxicity. Thus, TEM8 targeting may allow selective inhibition of pathological angiogenesis.

Expression, purification and characterization of the human membrane transporter protein OATP2B1 from Sf9 insect cells.

OATP2B1 is an important member of the organic anion transporting polypeptides (OATP) family and is implicated in the intestinal and hepatic disposition of endo- and xenobiotics. The purpose of this work was to produce a highly purified protein for use as a reference standard for quantification of OATP2B1 in human tissue and in vitro assay systems. Here, we report the successful expression, purification and characterization of OATP2B1 in a heterologous expression system. Protein expressed by the Sf9-baculovirus expression system is functionally active as demonstrated by saturable uptake kinetics with a K(m) of 5.9+/-0.76 microM for estrone-3-sulfate. OATP2B1 was extracted from Sf9-membranes with ABS-14-4 detergent and purified using a one-step FLAG-tag purification method. Yield of OATP2B1 from Sf9 cells was 1.1mg per liter of culture, for a final recovery of 1.8%. SDS-PAGE resolution and Western blot of purified protein displayed multiple banding of OATP2B1-specific protein, which was thoroughly investigated to confirm homogeneity of the sample. C-terminal FLAG-tag purification and immunoblot detection, together with N-terminal sequencing, confirmed the presence of only full-length protein. Treatment with endoglycosidases had little effect on the migration pattern in SDS-PAGE, suggesting that multiple banding was not due to different glycosylation states of the protein. Amino acid analysis further confirmed the homogeneity of the protein with a calculated extinction coefficient of 80,387 cm(-1) M(-1). Physical, biochemical and functional characterization show that purified human OATP2B1 is pure, homogeneous and appropriate for use as a standard to quantitate expression of OATP2B1 in in vitro systems and tissue samples.

Prenylcysteine lyase deficiency in mice results in the accumulation of farnesylcysteine and geranylgeranylcysteine in brain and liver.

In in vitro experiments, prenylcysteine lyase (Pcly) cleaves the thioether bond of prenylcysteines to yield free cysteine and the aldehyde of the isoprenoid lipid. However, the importance of this enzyme has not yet been fully defined at the biochemical or physiologic level. In this study, we show that Pcly is expressed at high levels in mouse liver, kidney, heart, and brain. To test whether Pcly deficiency would cause prenylcysteines to accumulate in tissues and result in pathologic consequences, we produced Pcly-deficient cell lines and Pcly-deficient mice (Pcly-/-). Pcly activity levels were markedly reduced in Pcly-/- cells and tissues. Pcly-/- fibroblasts were more sensitive than wild-type fibroblasts to growth inhibition when prenylcysteines were added to the cell culture medium. To determine if the reduced Pcly enzyme activity levels led to an accumulation of prenylcysteines within cells, mass spectrometry was used to measure farnesylcysteine and geranylgeranylcysteine levels in the tissues of Pcly-/- mice and wild-type controls. These studies revealed a striking accumulation of both farnesylcysteine and geranylgeranylcysteine in the brain and liver of Pcly-/- mice. This accumulation did not appear to be accompanied by significant pathologic consequences. Pcly-/- mice were healthy and fertile, and surveys of more than 30 tissues did not uncover any abnormalities. We conclude that prenylcysteine lyase does play a physiologic role in cleaving prenylcysteines in mammals, but the absence of this activity does not lead to major pathologic consequences.