Cell lines and Cell line Culture. 293T (cat.#CRL-3216) and Jurkat cells (cat.#TIB-152) were obtained from ATCC. 293GT cells (GripTite 293 MSR cell line cat.#R79507) and HEK293A cells (cat.#R70507) were obtained from ThermoFisher. All cell lines were grown in a humidified incubator held at 37 oC and 5% CO2. 293T cell lines were cultured in Dulbecco's Modified Eagle Medium (Invitrogen Cat.#11995-040) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Invitrogen cat.#15140-122) and Jurkat cells were cultured in RPMI 1640 medium (ThermoFisher cat.#11875-093) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. All cell lines tested negative for mycoplasma contamination.
Making 293GT CRBN knockout cell line. 293GT CRBN knockout cells were made using CRISPR-Cas9 as follows: A guide RNA targeting CRBN (CCTGTATGCAAGAACAGCAA) was cloned into a vector (pU6-aar1gRNA-nlsSpycas9nls-2apuro) that uses the U6 promoter to express the gDNA, and a CMV promoter to express both S.pyogenes Cas9 enzyme (codon optimized for expression in human cells) and a puromycin-resistant selectable marker. 293GT cells were transfected with the CRBN gDNA/CAS9 expressing plasmid and grown for three days in the presence of puromycin (2 µg/ml). Cells were replated in non-selective medium and single colonies were isolated for genomic DNA preparation and sequencing verification. A CRBN knock out clone where all copies of CRBN contain frameshifting insertion mutations was used for all future experiments.
Prolabel protein degradation assays. Cell lines stably expressing ProLabel gene of interest fusions were constructed as follows: A pLenti ProLabel gateway vector was made by inserting DNA sequence encoding the DiscoverX ProLabel tag39,40 (MSSNSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGE) downstream of the CMV promoter in the pLenti6.2/V5-DEST™ Gateway™ Vector (ThermoFisher Scientific, cat.# V36820). The IKZF1, IKZF2, IKZF3, IKZF4, IKZF2(H141Q), RIPK2, SALL4 and GSPT1 genes were cloned from Gateway entry vectors into the pLenti ProLabel vector using Gateway LR clonase (ThermoFisher cat.#11791019) and all DNA inserts were sequence verified. Viral particles were made by transfecting 293T cells with pLenti Prolabel vectors and plasmids from the Invitrogen™ ViraPower™ Lentiviral Packaging Mix (Invitrogen™ cat.#K497500). 293GT or 293GT CRBN KO cell lines were infected with viral particles carrying the ProLabel tagged genes of interest and selected with 5 µg/mL Blasticidin S HCl (ThermoFisher cat.#A1113903) for 2 weeks to make stable cell lines.
Prolabel degradation assay: ProLabel fusion expressing cell lines were trypsinized, diluted in growth medium to a concentration of 1.0 x 10^6 cells/ml, plated in solid white 384 well plates (17.5 µL per well) (Corning, cat.#3570) and incubated for 24 hours. Test compounds were serially diluted in DMSO and further diluted 25X into growth medium. Cells were treated with 2.5 µL of diluted test compounds and incubated for another 24 hours (final DMSO concentration 0.5%). Plates were removed from the incubator and equilibrated at room temperature for 30 minutes. ProLabel substrate was added according to the manufacturer’s instructions (DiscoverX PathHunter Prolabel Detection Kit, User manual: 93-0180) and the plates were incubated for three hours at room temperature. Luminescence was measured using an Envision Multilabel reader (150 ms). Readings from all DMSO wells were averaged and each compound treated well was normalized to DMSO. Results were plotted in GraphPad PRISM 9 and data were fit using the non-linear fit module (4-parameter- log dose versus response) to determine IC50 values.
Nanobit recruitment assay. CRBN was cloned into Promega SmBiT Flexi vector pFN35K and IKZF1 and IKZF2 were cloned into Promega LgBiT Flexi vector pFN33K following the Promega protocol (N2015, NanoBiT Protein:Protein Interaction System Technical Manual)41. All DNA inserts were sequence verified. 293T cells were transfected as follows: 20 µg of a mixture containing a 1 to 3 ratio of smBiT CRBN and lgBiT IKZF plasmid was added to 1 mL of OptiMEM medium (ThermoFisher cat.#31985062), mixed with 60 µL FuGene HD transfection reagent (Promega, cat.#E2311) and incubated at room temperature for 20 minutes. 293T cells were trypsinized and diluted in growth medium to a concentration of 1.0 x 10^6 cells/mL and the transfection reagents were added to 10 mL of cells. After mixing, cells (18.5 µL per well) were plated into 384 well solid white plates (Corning, cat.# 3570) and incubated for 24 hours. A 20 mM stock (in DMSO) of NAE1 inhibitor MLN4924 (AdipoGen, cat.#905579-51-3) was diluted into growth medium to a concentration of 10 µM and 2.5 µL was added to each well to a final concentration of 1.19 µM and incubated for 24 hours. Test compounds were serially diluted in DMSO and further diluted 25X into growth medium. Cells were treated with 3.0 µL of diluted test compounds and incubated for 10 minutes at room temperature (final DMSO concentration 0.5 %). 6 µL of NanoBiT substrate (Promega, Nano-Glo® Live Cell Reagent, cat.#N2011) was added to each well and the plates were incubated for 30 more minutes at room temperature. Luminescence was measured using an Envision Multilabel reader (150 ms). Readings from all DMSO wells were averaged (DMSO = 100%) and each compound treated well was normalized to DMSO. Results were plotted in GraphPad PRISM 9 and data were fit using the non-linear fit module (4-parameter- log agonist versus response). The Amax values were noted as a measure of the amplitude of the recruitment and the compound concentration at y= 400% (four times the DMSO control) was noted as a relative measure of potency.
Jurkat cell and T-cell endogenous IKZF2 degradation assay. Jurkat cells or primary CD25-enriched T cells were plated at a density of 5x104 cells per well in 96-well round bottom plates. Compounds were obtained from 10 mM stock solution in DMSO with final concentrations decreasing from 10 µM down 3-fold over 8 points, in duplicates. DMSO alone was used as a control in a similar dilution series. For assays on CD25+ primary T cells, 50 IU/mL rhIL-2 was added to the culture.
After 18-24 hours of culture, the cells were collected and stained with LIVE/DEAD® fixable viability dye (Life Technologies, cat. #L34974), washed and fixed with FOXP3 fix/perm buffer (Life Technologies, cat. #00-5523-00) followed by intracellular staining with anti-IKZF2-PECy7 (clone 22F6, BioLegend) and anti-Ikaros-BV421 (clone 16B5C71, BioLegend), and for primary cells with anti-FOXP3-APC (clone 236A/E7, eBioscience). Samples were acquired on a BD LSRFortessa flow cytometer (BD Biosciences). Analysis was performed using the by FlowJo™ Software (BD Life Sciences), and results were graphed using Prism 8 software (GraphPad Prism). IKZF2 median fluorescence intensity (MFI) and IKZF1 MFI were measured in cell lines, and %IKZF2+ FOXP3+ cells was measured in primary T cell cultures and normalized to the average of DMSO-treated samples for that culture.
Expression proteomics. Sample preparation: Cell culture, lysis, digestion, Tandem Mass Tag (TMT) labeling: Three million Jurkat cells were plated in 100 mm tissue culture dishes containing 10 mL of growth medium. Cells were exposed to DKY709 (2.5 µM final concentration) or DMSO by adding 10 µL of a 2.5 mM DKY709 stock solution (in DMSO) or 10 µL of pure DMSO as a control. After 16 hours of incubation, cells were collected into sterile 15 mL conical tubes, pelleted by centrifugation (1000 rpm for 5 minutes) and the medium was removed by aspiration. Jurkat cells were resuspended in 10 mL of ice-cold PBS (Phosphate Buffered Saline), pelleted by centrifugation (1000 rpm for 5 minutes at 4 oC) and the PBS was removed by aspiration. The pellet was resuspended in 0.5 mL of ice-cold PBS and transferred to an eppendorf tube. Cells were pelleted in a microfuge at 1000 RPM for five minutes at 4 oC. The PBS was aspirated, and the washed pellets were frozen at -80 oC for proteomic analysis.
Cell pellets were lysed in 100 µL iST-NHS lysis buffer and sonicated to shear and break the DNA aggregates. After centrifugation, the protein concentration was measured by following a BCA™ Protein Assay Kit (Thermo cat.#23227). All lysate protein concentrations for each condition were normalized to one concentration of 100 µg. An automated sample prep system called the PreON, developed by PreOmics, Inc. was then used to process the lysed material from digestion to TMT labelling and purification of peptides, in a fast and streamlined workflow42,43. The PreOn automated platform enables reproducible, high throughput sample preparation, of up to 16 samples, using reagents provided in the iST-NHS kit (PreOmics GmbH cat.#00030). Digestion of 100 µg protein was performed for a total incubation time of 2 hours at 37 oC. Each sample was then labelled with a tandem mass tag (TMTproTM, Thermo cat.#A44522) at a ratio of 5 µg tag to 1 µg protein for a total of 90 minutes at room temperature. The reaction is then quenched with 0.5% hydroxylamine TMT-labelled samples and are then combined and distributed evenly in cartridges to purify peptides. Final eluates were placed in a speed-vacuum to dry overnight.
LC-Fractionation: Following elution and concentration of peptides in a Genevac EZ 2.3 Elite vacuum concentrator, 530 µg of pooled multiplexed sample was fractionated by an offline high-pH fractionation system (Agilent HPLC 1200 series; Waters XBridge C18 3.5 µm, 150 mm x 2.1 mm column). The peptide separation was achieved with a ramp of 5-45% mobile phase B (90% acetonitrile, 5 mM ammonium formate buffer prepared from ammonium hydroxide, pH 10) in 74 min, with increase to 60% B for a total of 77 min effective gradient. Each sample was fractionated into ninety-six fractions and then manually pooled into twenty-four final fractions. Individual fractions were subsequently concentrated, and peptides reconstituted in 0.1% formic acid to 1 µg/µL.
Online LC-MS Chromatography: Tryptic peptides in each fraction were analysed using an Orbitrap Fusion™ Lumos Eclipse™ Mass Spectrometer (Thermo) equipped with an IonOptix Aurora 25 Column (1.6 µm C18, 75 µm ID x 25 cm) at an isocratic flow of 400 nL/min with a 83 min gradient of 6-35% mobile phase B (80% acetonitrile with 0.1% formic acid) using the synchronized precursor selection (SPS) mass spectrometry to the third (MS3) mode coupled with Real Time Search (RTS) function. Briefly, the first stage of mass spectrometry (MS1) was performed in the Orbitrap and scanned from 400 to 1600 mass to charge (m/z) with a resolution of 120,000. Only ions with charge state from 2+ to 6+ were selected for MS2 scans. The second stage of mass spectrometry (MS2) scan in the iontrap was set to a precursor isolation window of 0.7 m/z and normalized collision energy fixed at 32%. SPS-MS3 scans in the Orbitrap were set to 50,000 with 10 SPS precursors, an isolation window of 2, and a collision energy set to 40%.
LC-MS Data Analysis: Thermo Proteome Discoverer version 2.4 was used to process the raw mass spectrometry files. Briefly, spectra were matched against human fasta file downloaded from Uniprot (version July 2019 one gene/one protein with roughly 20,000 human reference proteins appended with common mass spec contaminants such as trypsin) using SequestHT algorithm. Peptide matches with <1% FDR as determined by Percolator were kept generating a list of identified proteins. Next, accepted peptides with TMT scores as follows SPS mass matched 65%, precursor contamination 50%, minimum average reporter ion with signal/noise greater than 10 were used for protein quantitation utilizing only non-shared peptides. In-house collection of python and R scripts were used for further data normalization, e.g. adjustment for difference in total protein loaded per TMT channel, and also for assessing protein response upon compound treatment and fold change compared to DMSO treatment (n = 3 biological replicates per treatment) using R Limma package44. Resulting p-values were corrected using commonly used Benjamini-Hochberg procedure. For clarity, the volcano plots are visualized as unadjusted p-values.
CRBN binding Assay. CRBN binding by compounds was determined by their ability to compete binding of a BodipyFL conjugated lenalidomide fluorescent probe45 as measured by fluorescence polarization. Test compounds were diluted in 90 % (v/v) DMSO/water and 100 nL was transferred to 384 well plates (Black Microtiter 384 Plate, round well; cat.#95040020 Thermo Electron Oy, Finland). 5 µL of CRBN/DDB1 protein solution (100 nM final concentration) in assay buffer (50 mM Tris/HCl at pH 7.4, 100 mM NaCl, 0.1 % (w/v) Pluronic F-127 and 1 mM TCEP) was added to each well and incubated at room temperature for 45 minutes. The binding measurement was started by addition of 5 µL of fluorescent probe in assay buffer (5 nM final concentration) and the final DMSO concentration was 0.9% (v/v). After 45 minutes at room temperature, fluorescence polarization was measured using a PHERAstar reader (BMG Labtech, Offenburg, Germany) with excitation at 485 nm and emission at 520 nm. EC50 values were calculated from the plot of percentage of protein saturation versus the test compound concentration by a logistics fit according to y = A2+(A1– A2)/(1+(x/ EC50)^ p), where y is the %-saturation value at the test compound concentration, x. A1 is the lowest saturation value, i.e. 0 %, and A2 the maximum saturation value, i.e. 100 %. The exponent, p, is the Hill coefficient.
CRBN cellular engagement assay. The ability of compounds to bind CRBN in cells was determined by measuring a compound’s ability to block the effect of a CRBN binding bifunctional degrader. 293GT ProLabel-RIPK2 cells were trypsinized, diluted to 1.0 x 10^6 cell/mL in growth medium, plated in solid white 384 well plates (17.5 µL per well) (Corning, cat.#3570) and incubated overnight. Test compounds were serially diluted in DMSO, further diluted 24X into growth medium and 2.5 µL of diluted test compounds were added to each well. Shortly afterwards, 2 µL of a lenalidomide-RIPK2 bifunctional degrader (probe 1) (Supplementary, Compounds synthesis and characterization, Scheme 6) was added to each well (final concentration 50 nM probe 1, 0.55% DMSO) and the plates were incubated for 24 hours at 37 degrees. Plates were removed from the incubator and equilibrated at room temperature for one hour. ProLabel substrate was added according to the manufacturer’s instructions (DiscoverX PathHunter Prolabel Detection Kit, User manual: 93-0180) and the plates were incubated for three hours at room temperature. Luminescence was measured using an Envision Multilabel reader (200 ms). Readings from all DMSO/probe 1 treated wells were averaged and were the negative control value (NC) and readings from 10 µM pomalidomide/ probe 1 treated wells were averaged and were the active control value (AC). Percent activity was calculated by the following equation 100[(x-NC)/(AC-NC)]. Results were plotted and IC50 values were calculated using the Novartis Helios software package.
HiBit protein degradation assays
Cell lines stably expressing HiBit-gene of interest fusions46 were constructed as follows: The lentiviral vector pLenti6.2/V5-DEST Gateway Vector (ThermoFisher cat.#V36820) was modified to express either mCherry-CHYSEL-HiBiT at the N-terminus of the protein of interest, or HiBiT-CHYSEL-mCherry at the C-terminus of the protein of interest. IKZF1 and SALL4 open reading frames were cloned to generate N-terminal HiBiT fusions, while GSPT1 was cloned to generate C-terminal HiBiT fusions. Cloning was performed using Gateway LR clonase (ThermoFisher cat.#11791019), and all DNA inserts were sequence verified. Virus was made as described above and used to infect 293GT cells (ThermoFisher Scientific cat.#R79507) and HiBit fusion stable cell lines were generated and maintained in the presence of 10 µg/ml of Blasticidine S HCl (ThermoFisher cat.#A1113903).
HiBit stable cell lines were trypsinized and diluted in growth medium to a concentration of 5 x 10^4 cells/mL for GSPT1-HiBit, 4 x 10^5 cells/mL for HiBit-IKZF1 and 2 x 10^5 cells/mL for HiBit-SALL4 expressing cells. 5 µL of cells were plated into wells of solid white 1536 well plates (Greiner cat.# 789173-A) and incubated overnight. Test compounds were serially diluted in DMSO and 10 nL of compound was dispensed directly into the wells using an Echo dispenser. After 20 hours of incubation, plates were removed from the incubator and allowed to equilibrate at room temperature for 30 minutes. 3 µL of Nano-Glo® HiBit Lytic Reagent (Promega cat.#N3050) was added to each well using a single tip bottle valve and plates were incubated for 20 minutes at room temperature. Luminescence was measured using a PHERAstar reader and readings from all DMSO wells were averaged and each compound treated well was normalized to DMSO. Results were plotted and IC50 values were calculated using the Novartis Helios software package.
High throughput solubility measurements. The high-throughput (HT) equilibrium solubility assay was performed after Zhou et al.47. Briefly, aliquots of 10 mM DMSO stock solution were plated and DMSO was removed under temperature and vacuum. pH 6.8 potassium phosphate buffer (67 mM) was added to the 96-well plate for a compound target concentration of 1 mM. The plate was sealed, incubated on a shaker at 1350 RPM and ambient temperature for 16-24 hours, and then centrifuged for 20 min at 3750 RPM to pellet the precipitate. The supernatant was transferred to another plate and centrifuged a second time. Supernatant was diluted 200-fold with 50:50 acetonitrile/water and a 4-point calibration curve was constructed using 50:50 acetonitrile/water. Analysis of the supernatant concentration was performed based on the calibration curve, using an Agilent RapidFire-MS/MS mass spectrometer system.
MDCK-LE permeability assay. Compound permeability was measured following the method described by Huth and co-workers48.
Protein production and purification. Full-length human CRBN constructs (residues 1-442) tagged with N-terminal ZZ-His was co-expressed with full-length human DDB1 (residues 1-1140) in Sf21 cells (Expression Systems) with 100 μM zinc acetate supplemented medium. Frozen cells were lysed by homogenization at pH 7.5. The soluble fraction was purified with histidine-affinity, ion-exchange and size-exclusion chromatography. Protein was concentrated to ~30 mg/mL in 20 mM HEPES, pH 7.0, 250mM NaCl, 2mM TCEP. For SPR binding assay, full-length human CRBN constructs (residues 1-442) as generated with a non-cleavable N-terminal Avi-tag with a 6xGS linker in addition to the upstream solubility tag. Avi-tagged CRBN was labeled with 100% efficiency with biotin using BirA enzyme49.
Human IKZF2 constructs (137-162 and 137-192) tagged with Twin-Strep-ZZ in a pET vector. IKZF2 constructs were expressed in E. coli BL21 (DE3) Star cells (Life Technologies cat.# C601003) in TB medium supplemented with 100 μM zinc acetate. Frozen cells were lysed by sonication at pH 8.0. The soluble fraction purified with Strepavidin-affinity, ion-exchange and size-exclusion chromatography. Protein was concentrated to 3-4 mg/mL in 20 mM HEPES, pH 6.8, 100mM NaCl.
SPR binding measurements. The binding of DKY709 and IKZF2 zinc finger proteins to CRBN or CRBN:DKY709 complexes, respectively, was measured on a Biacore 8k instrument in PBS buffer at pH 7.2 containing 5% glycerol, 150 mM NaCl, 0.01% P20 detergent, and 1 mM TCEP. For the binding of DKY709 to DDB1:N-avi-CRBN, this buffer contained 2% DMSO, and 1 mM EDTA. For the binding of IKZF2 proteins to DDB1:N-avi-CRBN:DKY709 complex, the Biacore ABA method was used to measure binding data for solutions with 5 mM DKY709 throughout the acquisition of baseline, IKZF2 association, and IKZF2 dissociation, where only the solutions for the association phase contain a 2X dilution series of IKZF2 proteins. Data analysis was performed with Biacore Insights software to normalize data relative to the baseline injections. The temperature was 15 degrees Celsius, and the flow rate was 30 µL/minute. DDB1:N-avi-CRBN, where the avi-tag is labeled with biotin, was loaded onto a streptavidin-coated Biacore sensor to ~6000-7000 RU, and the surface was exposed to 1 mM biocytin to block unoccupied streptavidin sites prior to analyses.
CRBNΔ40/DDB1ΔBPB:DKY709:IKZF2(ZF2) ternary complex crystallography.
DKY709 was first added (2 mM) to CRBNΔ40/DDB1ΔBPB in 20 mM HEPES pH7.0, 250 mM NaCl, 2 mM TCEP. After incubation on ice for 30 minutes, 3X IKZF2(ZF2) (137-162) (formulated in 25 mM MES pH6.5, 100 mM NaCl, 10 mM ZnCl2) was added and further incubated on ice for 30 minutes prior to crystallization screen. A single crystallization condition of CRBNΔ40/DDB1ΔBPB:DKY709:IKZF2(ZF2) was identified from the Qiagen Protein Complex Suite G10 (1.6 M Potassium/Sodium phosphate pH 6.5) at 4 °C using the hanging drop vapor diffusion method, after 1 week of incubation. Harvested crystals were flash cooled in liquid nitrogen following gradual equilibration into cryo protectant solution consisting of 25% (v/v) ethylene glycol supplemented to mother liquor. Diffraction data were collected at Advanced Light Source beamline 5.0.2 (λ = 1.0000 Å) using a Pilatus 6M detector and processed using process (Global Phasing Ltd.). The crystals belonged to space group P 61 2 2 with unit cell parameters a = 180.9, b = 180.9, c = 555.9 Å and α = 90°, β = 90°, γ = 120° and contained two copies of the ternary complex per asymmetric unit. The structure was solved by molecular replacement using PHASER50 with coordinates derived from the CRBNΔ40/DDB1ΔBPB:Pomalidomide:IKZF1(ZF2) complex (PDB entry 6H0F)25 as search models. Subsequent iterative model building and refinement was performed using COOT51 autoBUSTER (Global Phasing Ltd.) and PHENIX52. The structure was refined to Rwork and Rfree values of 20.8% and 24.0%, respectively, with 99.5% of the residues in Ramachandran favored and allowed regions as validated with MOLPROBITY53.
Treg procurement, isolation and expansion. Blood products obtained from healthy donors was purchased from Bioreclamation IVT (NJ), Hemacare (CA) or Stemcell Technologies (MA) in accordance with their respective IRBs. Informed consent was obtained from all participants and all ethical regulations were respected. Peripheral Blood mononuclear cells (PBMC) of were isolated from blood using density centrifugation on Ficoll-Paque (Millipore Sigma cat.#GE17-1440-03) following manufacturer instructions. CD4 enrichment by negative selection followed by CD25 enrichment by positive selection were performed using the human CD4 T cell isolation kit (cat.#130-096-533) and human CD25 microbeads (cat.#130-092-983) from Miltenyi Biotec (Cambridge, MA) according to manufacturer’s instructions. Isolated Tregs were expanded for 8-14 days in the presence of DKY709 or DMSO, using Treg expander beads (ThermoFisher, cat.#11129D) or T-cell activator beads (ThermoFisher, cat.#11161D) at a 4:1 or 3:1 ratio, respectively, in the presence of 500 U/mL rhIL-2.
Treg suppression assay. Expanded Treg cells were dispensed in co-culture with CFSE-labelled PBMCs at various Treg:PBMC ratios in the presence of T-cell activator beads or soluble anti-CD3 antibody (30 ng/mL, OKT3, Thermofisher cat.# 16-0037-81). After 3-4 days of incubation, proliferation was assessed by analysing CFSE dye dilution using flow cytometry. Analysis was performed using a BD Fortessa (BD Biosciences; BD LSRFortessa), the by FlowJo™ Software (BD Life Sciences), and results were graphed using GraphPad Prism. Teff cells that had proliferated during the co-culture were identified as having diluted CFSE and data were plotted as the proportion of CFSElow, proliferated cells in the final culture. CFSE-labelling of PBMC was performed according to manufacturer instructions (Invitrogen cat.#C34554).
Teff cell exhaustion assay. T cell functional exhaustion was modelled in vitro using a repeated stimulation protocol as previously described54. T cells were enriched from PBMCs using the Pan T cell isolation kit and an LS separation column from Miltenyi Biotec according to manufacturer’s instructions. Cells were plated in the presence of DKY709 at indicated concentrations, or DMSO, and stimulated with CD3/CD28 stimulation beads at 3:1 ratio (Thermofisher, cat.#11161D). On day 5 of culture, cells were collected, and beads removed, followed by re-plating with fresh beads in the presence of compound at the same concentration. After 5 days or restimulation, cells were treated with Cell stimulation cocktail (Life Technologies, cat.#00-4975-93) for 4 hours, then collected and stained for viability (live/dead fixable dye, Life Technologies, cat.#65-0865-14) and cell surface markers Cd3-FITC (OKZT3), CD4-BUVXX (SK3), CD8-BV510 (SK1) and PD1-BV786 (EH12.2H7), followed by fixation using the Foxp3 staining buffer kit from Ebioscience and intracellular staining for Helios-PE-Dazzle594 (22F6), IL2-BV650 (MQ1-17H12) and IFNg-AlexaFluor700 (4S.B3). Cells were acquired on a BD LSRFortessa flow cytometer (BD Biosciences). Analysis was performed by FlowJo™ Software (BD Life Sciences), and results were graphed in GraphPad Prism 8.
IL-2 production in Jurkat cells. Jurkat cells were plated at 5x104 cells/mL in 96-well round bottom plates. Compound was added at decreasing dilution from 10 µM down 5-fold, 8-points, in duplicates. After 24 hours, supernatants were collected and concentration of IL-2 was measured using MSD Human IL-2 Tissue Culture Kit (cat.#K151AHB-4, Mesoscale). Data was normalized to the DMSO-treated wells.
In vivo mouse humanization and tumor xenograft. Immunodeficient female mice of the NOD-SCID-IL2Rγ-/- (NSG) strain obtained from Jackson Laboratories and the study was carried out in accordance with the guidance from Novartis IACUC board of regulations, respecting all ethical regulations. Mice were subjected to 250 cGy of whole-body irradiation or administered bisulfan at a dose of 30 mg/kg IP and subsequently each engrafted with 50,000 hematopoietic stem cells (HSCs), from one of five donors, derived from human umbilical cord blood. Engraftment ranged from 19.3% to 98.1% hCD45+ cells at study enrolment. MDA-MB231 cell suspension in matrigel (200 µL or 10 x 106 cells per mouse) was implanted subcutaneously in NSG mice and tumor volume was monitored twice weekly. Seven days post MDA-MB231 implantation, daily oral dosing of DKY709 (100 mg/kg) and weekly intraperitoneal injection of PD-1 blocking antibody (PDR001) were initiated. After 21 days on study, blood and tumor samples were collected for terminal phenotype analysis. Blood samples were treated with ACK Lysing Buffer (Gibco, cat.#A1049201) for red blood cell lysis. Tumors were mechanically dissociated and digested using Liberase TM (Sigma-Aldrich, cat.#LIBTM-RO) / DNAse I (Roche, cat.#10104159001) with additional mechanical dissociation using the gentleMACS Dissociator (Miltenyi Biotec). Cell suspensions were then surface stained for viability (live/dead ebioscience, cat.#65-0865-14) and using antibodies against CD45-FITC (Hi30), CD8-BV510 (SK1), and CD25-BV421 (BC96) from Biolegend, and CD4-BUV737 (SK3) from BDBiosciences. Cells were fixed using the FOXP3 fix/perm kit (eBioscience, cat.#00-5523-00) and stained intracellularly with antibodies against Helios-Pe-Cy7 (22F6) and FOXP3-APC (236A/E7) from Biolegend and Invitrogen, respectively. Data was acquired using a BD LSRFortessa Cell Analyzer (BD Biosciences), analysed using the by FlowJo™ Software (BD Life Sciences) and plotted in GraphPad Prism.
In vivo monkey immunization and immunophenotyping. A non-GLP immunization study with DKY709 dosing was conducted in male cynomolgus monkeys at Covance/LabCorp. The study protocol was approved by the local IACUC board and the study was conducted in compliance with the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Office of Laboratory Animal Welfare. The study was carried out respecting all ethical regulations. Male cynomolgus monkeys (2-3 per group, aged 3-5 years) were administered 0.1 mg/kg/day DKY709; 2.5 mg mL KLH and 0.5 mL squalene adjuvant; 2.5 mg KLH, 0.5 mL squalene adjuvant and 0.1 mg/kg/day DKY709; or 2.5 mg KLH, 0.5 mL squalene adjuvant and 3 mg/kg/day DKY709. Animals were first immunized intramuscularly with KLH/Squalene or a vehicle control. On day 5 post immunization, daily oral treatment was initiated with DKY709 or a vehicle control for the indicated groups at the indicated dose, for the remainder of the study (36 days after immunization). Immunized groups received a recall immunization on day 15 after the initial immunization. Blood was collected by venipuncture in K2 EDTA tubes at the indicated timepoints. For assessment of IKZF2 degradation, samples were immediately fixed in 5:1 volume of FOXP3 fixation buffer (cat.#00-5523-00) for overnight fixation. Samples were then washed in BD FACS staining buffer (cat.#554656) and shipped to the analysis facility. Samples were subsequently stained intracellularly for CD3 (SP34-2), CD20 (2H7), CD8 (SK1) (BD bioscience), FOXP3 (236a/e7, ThermoFisher), Helios (22F6), CD16 (3G8) and Ikaros (16B5C71) (BioLegend) prior to acquisition on a flow cytometer (BD Biosciences; BD LSRFortessa). Analysis was performed by by FlowJo™ Software (BD Life Sciences), and results were graphed using GraphPad Prism 8. For assessment of T cell proliferation, blood was collected and immediately processed for surface and intracellular staining as described above with the addition of CD45, CD95, CD28 and KI67-detecting antibodies. Samples were acquired on a BD CANTO II flow cytometer. Analysis of the raw data files was performed using flow cytometry data analysis software Kaluza version 1.5 (Beckman Coulter), and results were graphed using GraphPad Prism 8.
Human clinical trial samples (PK, PD). DKY709 concentration in human plasma collected from the ongoing clinical trial (ClinicalTrials.gov NCT03891953) was measured by a Novartis-proprietary validated LC/MS/MS method to support pharmacokinetic assessment. Blood was drawn into heparin tubes, processed to PBMC and cryopreserved at Covance within 48 hours of collection. PBMC were collected from patients on the trial at C1D1, C1D2, C1D15, and C2D15 predose, and C1D1, C1D15, and C2D15 4 hours postdose. Blood samples were collected from patients by venipuncture at indicated time points and processed for PBMC cryopreservation. For characterization of Helios expression in different immune cell subsets, including T regulatory cells (Tregs), Helios expression was measured by a Novartis-proprietary validated 13-parameter flow cytometry assay. Flow cytometry was performed on cryopreserved PBMC at Navigate BioPharma Laboratory, Carlsbad, CA. PBMCs were thawed, washed with phosphate-buffered saline containing 0.1% sodium azide and 2% fetal bovine serum, and incubated with Fc Receptor Binding Inhibitor (ThermoFisher Scientific). Cells were stained with fixable viability dye (Thermo Fisher Scientific, San Diego, CA), followed by staining with fluorochrome conjugated antibodies. Cells were washed, then prepared for intracellular staining with the Foxp3 / Transcription Factor Staining Buffer Set (ThermoFisher Scientific, Carlsbad, CA), according to the manufacturer’s instructions. Intracellular staining was performed, cells were washed with 1x Permeabilization Buffer, then fixed with 0.5% formalin. Stained Samples were acquired on a BD LSRFortessa X-20 (BD Biosciences, San Jose, CA) and data were analysed using FlowJo™ Software (BD Life Sciences). Informed consent was obtained from all participants and all ethical regulations were respected.
Chemical synthesis and characterization. All chemical synthesis procedures and characterization data are provided in the Supplementary Information in the Compounds Synthesis and Characterization section.
All statistical analyses were performed using GraphPad Prism unless otherwise stated in the Methods section.
Data availability: The authors declare that the data supporting the findings of this study are available within the publication and its Supplementary Information files or have been deposited in the RCSB Protein Data Bank (PDB, http://www.rcsb.org). Further information available upon request. The PDB accession code for the human DDB1:CRBN:DKY709:IKZF2(ZF2) X-ray co-structure is 7U8F.
39. Zhao, X. et al. Homogeneous assays for cellular protein degradation using beta-galactosidase complementation: NF-kappaB/IkappaB pathway signaling. Assay and drug development technologies 1, 823–33 (2003).
40. Olson, K. R. & Eglen, R. M. Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay and drug development technologies 5, 137–44 (2007).
41. Dixon, A. S. et al. NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. ACS chemical biology 11, 400–8 (2016).
42. van Bergen, W., Heck, A. J. R. & Baggelaar, M. P. Recent advancements in mass spectrometry-based tools to investigate newly synthesized proteins. Current opinion in chemical biology 66, 102074 (2022).
43. Zaro, B. W. et al. Proteomic analysis of young and old mouse hematopoietic stem cells and their progenitors reveals post-transcriptional regulation in stem cells. eLife 9, (2020).
44. Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic acids research 43, e47 (2015).
45. Arista, L. et al. Bifunctional degraders and their methods of use. WO 2021/053495 Al, 1–513 (2021).
46. Schwinn, M. K. et al. CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide. ACS chemical biology 13, 467–474 (2018).
47. Zhou, L., Yang, L., Tilton, S. & Wang, J. Development of a high throughput equilibrium solubility assay using miniaturized shake‐flask method in early drug discovery. Journal of Pharmaceutical Sciences 96, 3052–3071 (2007).
48. Huth, F. et al. Predicting Oral Absorption for Compounds Outside the Rule of Five Property Space. Journal of pharmaceutical sciences 110, 2562–2569 (2021).
49. Fairhead, M. & Howarth, M. Site-specific biotinylation of purified proteins using BirA. Methods in molecular biology (Clifton, N.J.) 1266, 171–84 (2015).
50. McCoy, A. J. et al. Phaser crystallographic software. Journal of Applied Crystallography 40, 658–674 (2007).
51. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallographica Section D Biological Crystallography 66, 486–501 (2010).
52. Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta crystallographica. Section D, Structural biology 75, 861–877 (2019).
53. Williams, C. J. et al. MolProbity: More and better reference data for improved all-atom structure validation. Protein Science 27, 293–315 (2018).
54. Dunsford, L. S., Thoirs, R. H., Rathbone, E. & Patakas, A. A Human In Vitro T Cell Exhaustion Model for Assessing Immuno-Oncology Therapies. in 89–101 (2020). doi:10.1007/978-1-0716-0171-6_6.