Production of anti–CTLA-4×SIRPα heterodimer

3 mL Small scale 293F Transfection Protocol (using PEI)
The small scale transfection is for initial testing of protein expression. For heterodimers, the total amount of plasmid is still the same, but can test 4:1, 2:1, 1:1, 1:2, 1:4 ratios of each arm. In other words, for example, for 1.5 ug total plasmid, can use [1.2 ug : 0.3 ug ratio, 1 ug : 0.5 ug ratio, etc.].
This small scale transfection does not use valproic acid, so the expression within the supernatant should be analyzed 48-96 hours after transfection. The timing depends on your protein expression, but 72 hours is probably a decent baseline time to check the expression via NATIVE PAGE, SDS PAGE, or ELISA.
1. Warm 293 Freestyle medium to 37°C
2. Count cells – need 4 x 10⁶ cells per mL
   a. For small scale (total volume 3 mL), need 1.5 mL (or 6 x 10⁶ cells total) cells
   b. Note – can use a minimum of 3 x 10⁶ if absolutely necessary, but 4 x 10⁶ is optimal
3. Centrifuge at 1000 rpm for 4 minutes and resuspend cells in 1.3 mL of 293 Freestyle medium (no need to wash cells before resuspension)
   a. For 3 mL transfection, use wells in a 6 well plate
4. Calculate volume of plasmid necessary for 1 ug plasmid per 1 mL, or 1.5 ug plasmid 
   a. If it is easier for calculation purposes, can also use 2 ug plasmid and then 10 ug PEI
5. Dilute plasmid in a 100 uL 293 Freestyle medium
6. Dilute 7.5 ug PEI in a separate tube, 100 uL 293 Freestyle medium
7. Add PEI to plasmid and incubate plasmid/PEI at room temperature for 5-10 minutes
8. Add the 200 uL plasmid/PEI to 1.3 mL cells for a total of 1.5 mL transfection reaction
9. Shake the transfection reaction at 85 rpm 
   a. This speed is important – cannot shake too quickly otherwise the plasmid/PEI complex won’t efficiently enter the cells
10. After shaking for 4-6 hours, add equal volume (1.5 mL) EX293 medium (warmed to 37°C)
11. Analyze 48-96 hours after initial transfection
 
400 mL Large scale 293F Transfection Protocol (using PEI)
400 mL is the baseline, but can always scale down to 100 mL or 200 mL if protein expression is high enough. Scale down plasmid and PEI accordingly.
1. Warm 293 Freestyle medium to 37°C
2. Count cells – need 4 x 10⁶ cells per mL
   a. For large scale (total volume 400 mL), need 200 mL (or 8 x 108 cells total) cells
   b. Note – can use a minimum of 3 x 10⁶ if absolutely necessary, but 4 x 10⁶ is optimal
3. Centrifuge at 1000 rpm for 5 minutes and resuspend cells in 192 mL of 293 Freestyle medium (no need to wash cells before resuspension)
   a. For 400 mL transfection, use a 1 L flask
4. Calculate volume of plasmid necessary for 1 ug plasmid per 1 mL, or 200 ug plasmid total, for anti-CTLA-4XSIRPa, plasmids are transfected with anti-CTLA-4 light chain: heavy chain: SIRPa=1:2:1, or 50 ug:100 ug:50 ug.
5. Dilute 240ug(1.2X to make sure still get 200ug after filter through 0.22 um filter) plasmid with freestyle 293 medium to 4.8ml, filter through 0.22 um filter into a 15 mL tube.
6. Dilute 600ug PEI in a separate tube (commonly use 1:3 to 1:5, test efficiency when making new batch of PEI) with freestyle 293 medium to make a final volume of 4ml.
7. Slowly add 4ml plasmid to PEI, vortex well.
8. Incubate plasmid/PEI at room temperature for 15 minutes
9. Add the 8 mL plasmid/PEI to 192 mL cells for a total of 200 mL transfection reaction
10. Shake the transfection reaction at 85 rpm 
   a. This speed is important – cannot shake too quickly otherwise the plasmid/PEI complex won’t efficiently enter the cells
11. After shaking for 4 hours, add equal volume (200 mL) EX293 medium (pre-warmed to RT), shake at 120rpm.
12. Optional: Add Penicillin/Streptomycin to help prevent contamination, stock is 10 mg/mL, add 100 ug per mL of total volume (i.e. 100x, so 4 mL)
13. 24 hours after initial transfection, add 800 uL 1.9 M valproic acid (filtered through 0.22 um filter) for a final concentration of 3.8 mM.
14. Collect supernatant and purify protein 4-6 days after initial transfection.

MATERIALS and REAGENTS
 

PEI (1mg/mL)
 

Reagents
12 M HCl 
10 M sodium hydroxide (NaOH) 
Polyethylenimine (PEI), 25 kDa, linear, powder (Polysciences Inc.) 
H2O, Milli-Q-filtered
 

Equipment
Rinse all glassware three times with high-purity H2O.
Beaker, 500-mL
Cylinder, glass, graduated, 500-mL
pH meter
Tube, conical, 50-mL
Vacuum filtration unit, 0.22-μm
 

Method
1. Pour ~450 mL of Milli-Q H2O into a 500-mL glass beaker. 
2. Add 500 mg of PEI to the beaker with stirring. 
3. Add concentrated HCl drop-wise to the solution to pH <2.0. 
4. Stir until the PEI is dissolved (~2-3 h). Maintain the pH <2.0 throughout. 
Approximately 800 μL of 12 M HCl will be required for full PEI dissolution. There may still be some small fiber particles that will not dissolve.
5. Add concentrated NaOH drop-wise to the solution to pH 7.0. 
Approximately 500 μL of 10 M NaOH will be required to neutralize the PEI solution.
6. Pour the solution into a 500-mL glass cylinder. Adjust the final volume to 500 mL with Milli-Q H2O. 
7. Filter-sterilize the solution through a 0.22-μm membrane. 
8. Store aliquots of the desired volume at -20°C.

CD OptiCHO™ Medium (293F cell growth), Invitrogen, cat#12681-011
FreeStyle™ 293 Expression Medium(transfection), GIBCO, cat#12338018
EX-CELL(TM) 293 (after transfection), Sigma, 14571c
Valproic acid sodium salt, Sigma, cat: P4543-100G

 
Protein A purification
Centrifuge supernatant containing protein to be purified for 60min at 10000rpm, 4 degree.
Pass through 0.45um filter
Wash column with dH2O, fill column with appropriate amount of protein A beads.  Typically protein-A beads will bind up to 20mg IgG per mL column.  More beads equals slower flow rate.  1-2 mL of bead volume (2-4mL 50% slurry) is usually enough per liter of supernatant for most applications binding 20-40mg IgG1. 
Wash with 10X volume of bed PBS.(2ml/min, 30min)
Load supernatant 0.5ml/min
Wash column with PBS, >10X volume of bed PBS.(2ml/min, 60min)
Setup 10 tubes each containing 0~35uL (calculate the volume needed to neutralizing to pH 7-8 when making new buffer) 1M Tris-HCl pH 9.0-9.4 to neutralize acid.
Elute protein with elution buffer (pH2.7), keep the protein on ice.
Continue to elute for 10min.
Wash with H2O for 10 min.
Wash with 20% EtOH for 10min.
Continue to concentrate protein and desalt.
 

MATERIALS and REAGENTS
 

Elution buffer
0.1M Glycine
0.15M NaCl
Adjust pH to 2.7, filter through 0.22um filter.
 

Neutralizing buffer
1M Tris-HCl. pH 9.2
Adjust pH to 7-8 (usually 80-90ul Tris-HCl per 2mL Elution buffer), filter through 0.22um filter.

CaptivA® Protein A Affinity Resin, Repligen, cat: CA-PRI-0100

Anti-mouse CTLA-4 Light chain

Signal peptide-VL-CL

METDTLLLWVLLLWVPGSTGDVMMTQSPSSLSVSAGEKATISCKSSQSLFNSNAKTNYLNWYMQKPGQSPKLLIYYASTRHTGVPDRFRGSGSGTDFTLTISSVQDEDQAFYYCQQWYDYPYTFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
 

Anti-mouse CTLA-4 Heavy chain

Signal peptide-VH-CH-Fc9

METDTLLLWVLLLWVPGSTGQVKLEESGPGLVNPSGSLSLSCSVTGTSITSGYGWNWIRQFPGQKVEWMGFIYYEGSTYYNPSIKSRISITRDTSKNQFFLQVNSVTTEDTATYYCARQTGYFDYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSALTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Mouse SIRPa

Signal peptide-SIRPa-Fc6

METDTLLLWVLLLWVPGSTGMATGKELKVTQPEKSVSVAAGDSTVLNCTLTSLLPVGPIRWYRGVGPSRLLIYSFAGEYVPRIRNVSDTTKRNNMDFSIRISNVTPADAGIYYCVKFQKGSSEPDTEIQSGGGTEVYVLAGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFKLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK