A Cell-Permeant Nanobody-Based Degrader That Induces Fetal Hemoglobin

Proximity-based strategies to degrade proteins have enormous therapeutic potential in medicine, but the technologies are limited to proteins for which small molecule ligands exist. The identification of such ligands for therapeutically relevant but “undruggable” proteins remains challenging. Herein, we employed yeast surface display of synthetic nanobodies to identify a protein ligand selective for BCL11A, a critical repressor of fetal globin gene transcription. Fusion of the nanobody to a cell-permeant miniature protein and an E3 adaptor creates a degrader that depletes cellular BCL11A in differentiated primary erythroid precursor cells, thereby inducing the expression of fetal hemoglobin, a modifier of clinical severity of sickle cell disease and β-thalassemia. Our strategy provides a means of fetal hemoglobin induction through reversible, temporal modulation of BCL11A. Additionally, it establishes a new paradigm for the targeted degradation of previously intractable proteins.


S3 pET-20b_2D9 Plasmid Construction
The sequence of 2D9 with a N-terminal Strep-tactin tag was codon-optimized for expression in E.
coli and cloned into a linearized pET-20b vector at the NdeI and XhoI restriction sites.

-2D9 Plasmid Construction
The sequence of ZF5.3 was codon optimized for expression in E. coli and cloned into a linearized pET20b_2D9 plasmid with a N-terminal Strep-tactin tag.
pET-20b_ZF5.3-2D9-tRNF4 Plasmid Construction: The sequence of RNF475-194 was codon-optimized for expression in E. coli and cloned into a linearized pET-20b_2D9 plasmid with a N-terminal Strep-tactin tag. Primers used are summarized in Table S1.

Expression and purification of proteins ZnF23, exZnF23 of BCL11A and exZnF23 of BCL11B.
The cDNA of ZnF23 (residues 372-430) and exZnF23 (residues 372-484) of human BCL11A, exZnF23 of human BCL11B (residues 422-528) were cloned into pET28a vector and expressed as N-terminal His6-tag fusion proteins in E. coli. Cells were resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, 1 mM DTT). After sonication and centrifugation, the supernatant was applied to the Ni 2+ -NTA resin (Qiagen) equilibrated with lysis buffer and For the first round of magnetic-activated cell sorting (MACS), 1x10 10 S. cerevisiae cells expressing a surface displayed library of synthetic nanobodies (11) were centrifuged, resuspended in selection buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.1% BSA, 5 mM maltose) and then incubated with anti-AlexaFluor647 microbeads (Miltenyi) at 4℃ for 40 min. The yeast cells were then passed through an LD column (Miltenyi) to remove any yeast expressing nanobodies that non-specifically interacted with the microbeads. Yeast cells that flowed through the column were centrifuged, resuspended in selection buffer, and incubated with 1 µM of AlexaFluor647-labeled exZnF23 of BCL11A at 4℃ for 1 hr. Yeast cells were then centrifuged, resuspended in selection buffer with anti-AlexaFluor647 microbeads, and incubated at 4℃ for 20 min before passing through an LS column (Miltenyi). The eluted yeast cells were collected and expanded to a subsequent round of MACS to further enrich for exZnF23-binding nanobodies. The second round of MACS was performed similarly to the first round but using fluorescein isothiocyanate (FITC)-labeled exZnF23 of BCL11A and anti-FITC microbeads. After MACS selections, yeast cells were costained with AlexaFluor647-and FITC-labeled exZnF23 of BCL11A and sorted by flow cytometry (Sony SH800Z). Double-positive yeast cells were selected and plated as single colonies, which were randomly picked and grown as clonal populations in 96-well plates. Yeast cells in 96-well plates were induced, stained with AlexaFluor647-or FITC-labeled exZnF23 of BCL11A and analyzed by the plate reader. Yeast DNA was extracted using standard methods and sequenced from the high activity clones.
Affinity maturation of nanobody wt2D9. Error-prone PCR was performed on nanobody wt2D9 DNA using the GeneMorph II Random Mutagenesis Kit (Agilent) and the resulting library was scaled up with a second PCR using Q5 High-Fidelity DNA Polymerase (New England Biolabs). S5 100 mL of BJ5465 S.cerevisiae cells were grown to OD600nm of 1.8 and were then made into electrocompetent yeast cells with 100 mM lithium acetate 2 . The electrocompetent cells were transformed with 56 µg of the error prone library and 17 µg of linearized pYDS649 plasmid (11) using an ECM 830 Electroporator (BTX-Harvard Apparatus) with 500 V and 15 ms single pulse.
The resulting library of nanobody 2D9 mutants has a mean mutation rate of about 1 amino acid change per nanobody clone.
1×10 6 yeast cells from the error prone library were stained with anti-HA AlexaFluor488 antibody in selection buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.1% BSA, 5mM maltose, 1mM DTT) to assess nanobody expression levels. In order to obtain high-affinity binders to exZnF23 of BCL11A, 4 rounds of MACS selections were performed, and the yeast cells were stained with 1µM of FITC-labeled exZnF23, 1µM of FITC-labeled exZnF23, 1.5µM of His6-SBP-exZnF23, and 1 µM of FITC-labeled exZnF23 to enrich for binders with higher affinities. After MACS selections, 2 rounds of FACS were performed. In the first round of FACS, the yeast cells were costained with 0.5 µM of AlexaFluro647-labeled exZnF23 and 0.75µM of FITC-labeled exZnF23.
A total of ~80,000 yeast cells from the first round of FACS were expanded and used for a second round to further enrich for high affinity nanobodies. In second round of FACS, the yeast cells were co-stained with 0.1 µM of AlexaFluro647-labeled exZnF23 and 0.15 µM of FITC-labeled exZnF23. After the second round of FACS, approximately 5,000 yeast cells were plated as single colonies using serial dilutions. 96 yeast clones were randomly selected, mini-prepped and sequenced to reveal consensus mutations contributing to affinity. The sequence of wt2D9 is found in Table S2.

Nanobody degrader production
Transformation of plasmids in BL21 cells. rpm. Cells were centrifuged for 3 min at 2500 × g and 900 µL LB media was removed. The S6 recovered cells were resuspended in 100 µL remaining media and added to ampicillin-(100 µg/mL) or kanamycin-containing (50 µg/mL) agar plates and incubated at 37 ℃ overnight.

2D9 expression and purification
The plasmid encoding Strep-2D9 was used to transform E. coli BL21 cells. Individual colonies were selected on the basis of Amp resistance and used to inoculate 50 mL of LB media supplemented with Amp (100 mg/L). The primary culture was grown overnight and then used to inoculate 6 L of ZYM-5052 autoinduction (AI) media 3 supplemented with ampicillin, which was then allowed to grow at 37 °C with shaking at 200 rpm. When the OD600nm reached 0.5, the temperature was changed to 18 °C and the cells cultured overnight. The cells were harvested by centrifugation at 6000 × g for 30 min at 4 ℃, resuspended in buffer containing 25 mM Tris-HCl pH 8.0, 300 mM NaCl, and 10% glycerol, supplemented with 1 mM PMSF and lysed via microfluidization. The lysate was clarified by centrifuging at 15,000 × g for 30 min at 4 ℃ and the cleared lysate manually added to a column with 5 mL Strep-Tactin® Sepharose® resin (IBA Lifesciences). The column was washed with 5 column volumes of 25 mM Tris-HCl pH 8.0, 300 mM NaCl, and 10% glycerol. Then protein was eluted from the resin using 25 mM Tris-HCl pH 8.0, 300 mM NaCl, and 10% glycerol supplemented with 2 mM of desthiobiotin. Elution fractions were analyzed by SDS-PAGE, and fractions containing the desired protein combined and concentrated using spin concentrators (Millipore). Following concentration, proteins were additionally purified via size exclusion chromatography (Cytiva HiLoad 26/600 Superdex-200 pg column, #GE28-9898-36). Pure proteins were concentrated, flash frozen in liquid nitrogen, and stored at −80 °C until further use.

ZF5.3-2D9 expression and purification
The plasmid encoding Strep-ZF5.3-2D9 was used to transform E. coli BL21 cells. Individual colonies were selected based on ampicillin resistance and used to inoculate 150 mL of LB media supplemented with ampicillin (100 μg/mL). The primary culture was grown overnight and then used to inoculate 6 L of LB media supplemented with ampicillin, which was then allowed to grow at 37 °C with shaking at 200 rpm. At OD600nm of 0.5, protein expression was induced by the addition of IPTG to a final concentration of 0.5 mM. After culturing for overnight at 18 °C, cells were harvested by centrifugation (6000 × g, 30 min at 4 °C), resuspended in buffer containing 25 S7 mM Tris-HCl pH 8.0, 300 mM NaCl, 0.1 mM ZnSO4, and 10% glycerol, supplemented with 1 mM PMSF, and lysed by microfluidization. The purification of this protein was identical to that of 2D9, with the exception that 0.1 mM of ZnSO4 was added to all buffers.
Individual colonies were selected based on kanamycin resistance and used to inoculate 150 mL of LB media supplemented with kanamycin (50 μg/mL). The primary culture was grown overnight and then used to inoculate 6 L of LB media supplemented with kanamycin, which was then allowed to grow at 37 °C with shaking at 200 rpm. At OD600nm of 0.5, protein expression was induced by the addition of IPTG to a final concentration of 0.5 mM. After culturing for overnight at 18 °C, cells were harvested by centrifugation at 6000 × g for 30 min at 4 °C), resuspended in buffer containing 25 mM Tris-HCl pH 8.0, 300 mM NaCl, and 10% glycerol, supplemented with 1 mM PMSF, and lysed by microfluidization. The lysate was centrifuged at 15,000 × g for 45 min at 4 o C, and to the clear lysate was added 8 M urea, pH 8.0. The lysate was manually added to a column with 3 mL Ni-NTA resin and washed with 300 mL of buffer containing 25 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole, and 8M urea. After that, protein was eluted from the resin using 25 mM Tris-HCl pH 8.0, 300 mM NaCl, 8M urea supplemented with 100 mM of imidazole. The elution fractions were analyzed by SDS page and concentrated to 1 mL. The purified proteins were refolded with 100 mL buffer containing 25mM Tris-HCl pH 8.0, 300 mM NaCl, 500 mM Larginine, 9 mM glutathione and 1 mM glutathione disulfide. The refolded protein was cleared by centrifugation at 15,000 × g for 45 min at 4 o C, passed through a 0.22 μm filter, and concentrated for future use.
Individual colonies were selected based on ampicillin resistance and used to inoculate 150 mL of LB media supplemented with kanamycin (100 μg/mL). The primary culture was grown overnight and then used to inoculate 6 L of LB media supplemented with kanamycin, which was then allowed 2D9 datasets were collected at a fixed wavelength at 0.97946 Å, using an Eiger 16M detector at beamline BL12-1. All diffraction datasets were indexed and processed by XDS 4, 5 . The structure was phased by molecular replacement in Phenix (Phaser) using model of 1NLB-H 6 . Subsequent density modification gave excellent electron-density maps, which allowed the building of the model. Iterations of refinement were carried out with Phenix Refine, and model building was performed in Coot. The statistics for data collection, processing, and refinement are listed in Table   S3. All structural figures were prepared using PyMOL.