Protein Expression and Purification of VHH-pHLIP and VHH
The DNA sequence coding for the VHH-pHLIP fusion (Figure S1a) and VHH alone (Figure S1b) were subcloned into a pET21b(+) vector (Biomatik). Plasmids were transformed into Rosetta (DE3) BL21 for over-expression. Colonies were selected from antibiotic-resistant plates and used to grow starter cultures in Lennox broth (LB); starter cultures were added into large volume (2 L) and grown at 37°C with shaking to an OD600 of 0.6–0.7 and induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Induced cultures were grown for an additional 4 h at 37°C with shaking and harvested by centrifugation at 10,000 rpm, 4°C for 15 min.
Cell pellets were resuspended in 50 mM NaPO4, 300 mM NaCl (pH 8.0), and lysed by sonication at 4°C (output control: 2, duty cycle: 20%, 10 min). Samples were centrifuged at 13,000 rpm, 4°C for 10 min (subsequent centrifuge steps use the same parameters). Since VHH-pHLIP and VHH alone expressed as inclusion bodies, cell pellets were washed in 50 mM NaPO4, 300 mM NaCl, 1% Triton, and 1 M urea (pH 8.0) and incubated on ice for 10 min before centrifugation. Two additional washes were done in the same buffer (no incubation) before washing twice with 50 mM NaPO4, 300 mM NaCl (pH 8.0). Inclusion body pellets were solubilized in 50 mM NaPO4, 300 mM NaCl, and 6 M urea (pH 8.0) under rotation overnight at 4°C and centrifuged at 12,000 rpm, 4°C for 25 min (removes insoluble debris) before 0.45 µm filtering (PVDF filter) and purifying by Ni-NTA affinity chromatography. HisPur™ Ni-NTA Resin (Thermo Scientific) was incubated with solubilized inclusion bodies under rotation at 4°C for 2 h. The resin was washed with 50 mM NaPO4, 300 mM NaCl, 6 M urea, and 10 mM imidazole (pH 8.0) before eluting with an increasing step gradient (100 mM, 200 mM, and so on, up to 500 mM) of imidazole. Elutions were pooled and concentrated using 10K MWCO Macrosep® Advance Centrifugal Device (Pall). Adding 50 mM NaPO4, 300 mM NaCl, 6 M urea (pH 8.0) to the top of the centrifugal device served to remove imidazole by washing it into the flow through.
Protein Refolding
Refolding conditions were based on those described by Bao, et al.64 The VHH was refolded from 6 M urea using a 1/10 dilution method. The refolding buffer contained 50 mM NaPO4, 300 mM NaCl, 352 mM L-Arg (aggregation inhibitor), 1.05% (m/v) PEG-3350 (protein stabilizer), and 1.25 mM GSH (reduced glutathione) and 1 mM GSSG (oxidized glutathione), pH 8.0. Concentrated protein had β-mercaptoethanol (BME) added to a final concentration of 1 mM to reduce any mismatched disulfide bonds. Protein was added dropwise into the refolding buffer under stirring at 4°C and allowed to stir for a minimum of 16 h. Aggregates were pelleted by centrifugation at 12 000 rpm, 4°C for 10 min, and the supernatant was concentrated using 10K MWCO Pierce™ Protein Concentrator PES (Thermo Scientific). Concentrated protein was dialyzed into 40 mM NaPO4, 200 mM NaCl (pH 8.0) in a 7K Slide-A-Lyzer® Dialysis Cassette, 0.5 mL – 3 mL capacity (Thermo Scientific). The final protein concentration was determined using the Micro BCA™ Protein Assay Kit (Thermo Scientific). Aliquots were flash-frozen in liquid nitrogen and stored at -70°C. When thawed for experiments, phenylmethylsulfonyl fluoride (PMSF; a serine protease inhibitor) was added to 1 mM final concentration.
Sample Preparation for CD and Tryptophan Fluorescence Measurements
VHH alone in 40 mM NaPO4, 200 mM NaCl (pH 8.0) was diluted to a final concentration of 10 µM. 40 mM NaPO4 (pH 8.0) was used to dilute the sample such that the final buffer composition was 40 mM NaPO4, 100 mM NaCl (pH 8.0). VHH-pHLIP in 40 mM NaPO4, 100 mM NaCl (pH 8.0) was diluted such that the final concentration in each sample was 6 µM. Any necessary sample dilution was done with 5 mM NaPO4 (pH 8.0). 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids were dried down and desiccated overnight under vacuum; lipids were rehydrated in 5 mM NaPO4 (pH 8.0) for 30 min at 37°C with occasional mixing throughout. The resulting large multilamellar vesicles went through seven freeze/thaw cycles and were then extruded through a polycarbonate membrane with 200 nm pores using a Mini-Extruder (Avanti Polar Lipids) to yield large unilamellar vesicles (LUVs). VHH-pHLIP was incubated with these LUVs at a 1:300 ratio for at least 5 min before adjusting the pH to the desired values with HCl, and samples were then incubated at RT for 30 min before analysis. Due to the inclusion of various buffers in samples, the final buffer composition was 27.3 mM NaPO4 and 63.6 mM NaCl (at experimental pH).
CD Spectroscopy
A Jasco J-815 CD spectrometer with a Peltier thermally controlled cuvette holder was used to record Far-ultraviolet (far-UV) CD spectra. Measurements were performed in a 0.1 cm quartz cuvette. CD intensities are expressed in mean residue molar ellipticity [θ] calculated from the following equation:
where θobs is the observed ellipticity in millidegrees, l is the optical path length in centimeters, c is the final molar concentration of the peptide, and n is the number of amino acid residues. CD spectra were acquired from 260 to 200 nm in 1 nm intervals at a 100 nm/min scan rate, and five scans were averaged for each sample. The spectrum of POPC liposomes was subtracted from all samples measured in the presence of liposomes.
Tryptophan Fluorescence Spectroscopy
Fluorescence emission spectra were recorded using a Fluorolog-3 spectrofluorometer (HORIBA). The excitation wavelength was set at 280 nm, and the emission spectrum was measured from 300 to 450 nm. The excitation and emission slits widths were both 5 nm.
Culture of Human Cancer Cell Lines and NK-92 Cells
Human cervical HeLa cancer and human lung cancer A549 cells were cultured in Cytiva HyClone™ Dulbecco’s Modified Eagles Medium (DMEM) high glucose, with L-glutamine and sodium pyruvate, and supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 0.1 mg/mL streptomycin. Engineered Jurkat-Lucia™ NFAT-CD16 cells with the high-affinity CD16 allotype (V158) (InvivoGen) were cultured in Gibco™ Iscove’s Modified Dulbecco’s Medium (IMDM) high glucose, with glutamine, HEPES, and sodium pyruvate, and supplemented with 10% FBS, 100 units/mL penicillin, 0.1 mg/mL streptomycin, and 0.1 mg/mL Normocin™. After the second passage, media at every other passage contains 10 µg/mL blasticidin and 0.1 mg/mL Zeocin® to maintain selective pressure. Engineered CD16+ NK-92 cells (ATCC) were cultured in Gibco™ Minimum Essential Medium Alpha (MEMα) no nucleosides, low glucose, with glutamine and sodium pyruvate, and supplemented with 0.1 mM BME, 0.2 mM inositol, 0.02 mM folic acid, 12.5% FBS, 12.5% horse serum, 200 units/mL interleukin-2 (IL-2), 100 units/mL penicillin, and 0.1 mg/mL streptomycin. Cells were cultured in a humidified atmosphere with 5% CO2 at 37°C.
Antibody Recruitment Assay
Dilutions of VHH-pHLIP were made in phosphate-buffered saline (PBS, pH 7.4), and consistency in salt concentrations was maintained by adding 40 mM NaPO4, 200 mM NaCl (pH 8.0). HeLa cells were harvested and washed once with PBS (pH 7.4). 400,000 cells were resuspended in PBS (pH 7.4) and treated in suspension with VHH-pHLIP (various dilutions) such that the final concentrations were 10 µM, 7.5 µM, 5 µΜ, or 2.5 µM. Samples were incubated for 5 min at 37°C and then treated at either pH 7.4 or 6.0 for 10 min at 37°C. Cells were washed twice with cold PBS (same pH as treatment) and blocked in PBS with 3% BSA (pH 7.4) for 10 min on ice. A 1:1000 dilution of DyLight® 488 Anti-6x His tag antibody (Abcam, ab117512) in PBS with 3% BSA (pH 7.4) was incubated with the cells for 30 min on ice. Cells were washed once with cold PBS (same pH as treatment) and fixed in 4% PFA for 10 min on ice. The cells were then resuspended in PBS (same pH as treatment) and analyzed by flow cytometry using a BD FACSCanto II flow cytometer with a 488 nm argon laser and a 530 nm/30 nm bandpass filter. Antibody recruitment with VHH alone as a negative-control experiment was done using an identical procedure, simply substituting the VHH alone for VHH-pHLIP.
Immunofluorescence Microscopy
Glass coverslips were treated with poly-L lysine for 2 h at 37°C and washed with PBS (pH 7.4) before adding cells. HeLa cells were seeded on coverslips and allowed to adhere overnight. Cells were treated with 6 µM VHH-pHLIP as described above. After treatment, coverslips were washed once with PBS with 10% FBS (same pH as treatment) before fixation with 4% PFA for 10 min at RT with rocking. Coverslips were blocked in PBS with 3% BSA (pH 7.4) for 30 min with rocking. A 1:1000 dilution of DyLight® 488 Anti-6X His tag® antibody in PBS with 3% BSA (pH 7.4) was incubated on the coverslips for 1 h at RT with rocking. Coverslips were then washed four times with PBS (pH 7.4). A 1 µg/mL solution of Hoechst 33342 (Invitrogen) in PBS (pH 7.4) was incubated on coverslips for 10 min at RT with rocking before washing twice more with PBS (pH 7.4). Coverslips were mounted on glass slides with Fluoromount (Sigma-Aldrich) and allowed to dry before sealing. Slides were imaged with a Zeiss LSM880 scanning confocal microscope with a 40x objective.
CD16 Receptor Activation Assay
VHH-pHLIP was diluted in serum-free treatment media (prepared from powdered DMEM), and consistency in salt concentrations was maintained by adding 40 mM NaPO4, 200 mM NaCl (pH 8.0). HeLa cells were seeded in a 96-well cell culture plate, grown to confluency and treated with increasing concentrations of VHH-pHLIP as described above. Cells were washed twice with cold serum-free treatment media (same pH as treatment). IMDM (without Normocin™, blasticidin, or Zeocin®) was added to each well. Jurkat-Lucia™ NFAT-CD16 cells were harvested by centrifugation and were resuspended in IMDM (without Normocin, blasticidin, or Zeocin), and 200,000 cells were added to each well for a 5:1 effector-to-target cells ratio. The plate was centrifuged at 300 rpm for 1 min before incubating for 6 h at 37°C. The plate was gently shaken before transferring the media to an opaque white 96-well plate for luminescence measurements. QUANTI-Luc reagent (InvivoGen) was added to each well, and the luminescence was measured immediately with a 0.1 s reading time on an Infinite 200 PRO Plate Reader (Tecan).
Cell Killing Assay Measured by Lactate dehydrogenase (LDH) Release
HeLa cells were seeded in a 96-well cell culture plate, grown to confluency, and treated with increasing concentrations of VHH-pHLIP as described above. Cells were washed once with DMEM (no phenol red, 10% FBS, and pen-strep). Wells used for determining the maximum LDH activity and spontaneous LDH release were washed with the same media, and fresh media was added to them for the remainder of the experiment. CD16+ NK-92 cells were harvested by centrifugation, resuspended in DMEM (no phenol red, 10% FBS, pen-strep), and 200,000 cells were added to each treated well for a 5:1 effector-to-target ratio. The plate was centrifuged at 300 rpm for 1 min before incubating for 3 h 15 min at 37°C. Following the manufacturer's recommendations, LDH activity was measured using the colorimetric CyQUANT™ LDH Cytotoxicity Assay Kit (Invitrogen). 10X Lysis Buffer was added to wells for maximum LDH activity, and MilliQ water was added to wells for spontaneous LDH release. The plate was incubated for an additional 45 min at 37°C (total 4 h incubation). The plate was centrifuged at 2,500 rpm for 5 min before transferring the supernatant to a black clear-bottom 96-well plate. The Reaction Mixture solution (containing the substrate) was added to each well and incubated in the dark at room temperature for 40 min. Absorbance at 488 nm (10 nm bandwidth) and 690 nm (50 nm bandwidth) was measured on an Infinite 200 PRO Plate Reader (Tecan). Percent Cytotoxicity was calculated using the following equation:
$$\:\%\:Cytotoxicity=\frac{\left[Treated\:LDH\:activity\right]-\left[Spontaneous\:LDH\:release\right]\:}{\left[Maximum\:LDH\:activity\right]-\left[Spontaneous\:LDH\:release\right]}\times\:100\%$$
Spontaneous LDH release and maximum LDH activity were measured after milliQ water and 10X Lysis Buffer were added, respectively.
Cell Killing Assay Measured by MTT Assay
Target cells (HeLa, A549) were seeded in a 96-well cell culture plate (10,000 cells per well) the day before each assay and allowed to adhere overnight. The following day, cells were washed twice with serum-free treatment media (pH 7.4) and treated with increasing concentrations of VHH-pHLIP as described above. Cells were washed three times with serum-free treatment media (pH 7.4) after the treatment. CD16+ NK-92 cells were harvested by centrifugation and resuspended in serum-free treatment media. Effector cells were added to each well at a 2:1 effector-to-target ratio (20,000 cells per well), and the plate was centrifuged at 300 rpm for 1 min. The plate was incubated at 37°C for a 48 h recovery period. At the end of the recovery, the wells were washed twice with serum-free treatment media to remove the haNK cells; after washing, fresh serum-free media was replaced over the cells. Cell viability in each well was determined by incubating with MTT reagent prepared fresh as a stock at 5 mg/mL in PBS. 10 µL of the stock was added to each well, and the plate was incubated for 2 h at 37°C. All media was removed from the wells, and the resulting formazan crystals were solubilized in DMSO. DMSO was also added to three unused wells to provide a background absorbance reading. The absorbance of each well was measured at 580 nm (10 nm bandwidth) on an Infinite 200 PRO Plate Reader (Tecan). The background absorbance of DMSO (from triplicate wells of DMSO only) was subtracted from each sample before calculating viability.
$$\:\%\:Cell\:Viability=\frac{\left[Treated\:{ABS}_{580}\right]}{[No\:Treatment,\:pH\:7.4\:{ABS}_{580}]}\times\:100\%$$