Supplementary Data
Isolation of epitope-specific TGF-β-neutralizing single-domain antibodies using next-generation DNA sequencing#
Kevin A. Henry1*, Greg Hussack1, Cathy Collins2, John C. Zwaagstra2 and C.
Roger MacKenzie1,3
1Human Health Therapeutics Portfolio, National Research Council Canada, 100
Sussex Drive, Ottawa, Ontario, Canada, K1A 0R6
2Human Health Therapeutics Portfolio, National Research Council Canada, 6100
Royalmount Avenue, Montreal, Quebec, Canada, H4P 2R2
3School of Environmental Sciences, Ontario Agricultural College, University of
Guelph, 50 Stone Road East, Guelph, Ontario, Canada, N1G 2W1
*Address correspondence to: Kevin A. Henry, Ph.D.
Human Health Therapeutics Portfolio, National Research Council Canada 100 Sussex Drive, Ottawa, Ontario, Canada, K1A 0R6
Phone: +1-613-998-3373 Fax: +1-613-952-9092
Email: kevin.henry@nrc-cnrc.gc.ca
Supplementary Table SI. Metrics for Illumina MiSeq NGS data used in this study.
No. of assembled
reads (FLASH) 972,326 378,055 106,930
No. of reads passing quality filter (FASTX)
627,441 251,473 68,757
No. of CDR3s analyzed 441,572 191,754 40,306
Supplementary Table SII. Germline IGHV gene homology and CDR lengths of sdAbs isolated in this study.
sdAb VH gene1
IMGT-CDR1 IMGT-CDR2 IMGT-CDR32 VHH1 IGHV3-23 (81.8) 8 7 14 VHH2 IGHV3-23 (85.1) 8 8 15 VHH3 IGHV3-NL1 (82.8) 8 7 5 VHH4 IGHV3-66 (81.4) 8 7 9 VHH5 IGHV3-23 (85.4) 8 8 22 VHH6 IGHV3-23 (86.1) 8 8 18 VHH7 IGHV3-23 (86.0) 8 7 13
1Nearest human IGHV gene (% homology)
1E+01. 1E+02. 1E+03. 1E+04. 1E+05. 1E+06.0 0.2 0.4 0.6 0.8 1 1.2 Immune vs. TGFb1 Immune vs. TGFb2 Immune vs. TGFb3 Preimmune vs. TGFb3
Reciprocal Serum Dilution
O D ( 4 5 0 n m )
Supplementary Figure S1. Binding of polyclonal serum antibody from the TGF-β3-immunized llama to TGF-β1, -β2 and -β3 in ELISA. Wells of NUNC
MaxiSorpTM microtiter plates were coated overnight at 4°C with 0.1 μg of each
isoform of TGF-β in 35 μL PBS. The next day, wells were blocked with 100 μL of PBS containing 5% (w/v) skim milk for 1 h at 37°C, then sera were diluted in PBS containing 1% BSA and 0.1% Tween-20 (PBS-BT), added to wells and incubated for 2 h at RT. Wells were washed 5× with PBS containing 0.1% Tween-20 (PBS-T), incubated with HRP-conjugated goat anti-llama IgG (Cedarlane) diluted 1:5,000 in PBS-BT for 1 h at RT, then washed again 5× with PBS-T and developed with 35 μL of tetramethylbenzidine (TMB) substrate (Mandel
Scientific, Guelph, Canada). After 5 min, the reaction was stopped with 35 µL of 1 M H2SO4 and the absorbance at 450 nm was measured using a MultiskanTM FC
photometer (Thermo-Fisher). Pre-immune: llama serum prepared from blood drawn before the first immunization; Immune: llama serum prepared from blood drawn 42 days after the first immunization.
0 0 0 0.01 0.1 1 10 100 0 0.2 0.4 0.6 0.8 No competitor 10% preimmune serum 10% immune serum 10% irrelevant serum [(T RII)2 trap] ( g/mL)β μ O D ( 4 5 0 n m )
Supplementary Figure S2. Binding of biotinylated (TβRII)2 trap to TGF-β3 in the
presence or absence of polyclonal serum antibody from the TGF-β3-immunized llama. Wells of NUNC MaxiSorpTM microtiter plates were coated overnight at 4°C
with 0.1 μg TGF-β3 in 35 μL PBS. The next day, wells were blocked with 200 μL of PBS containing 2% BSA for 1 h at 37°C, then (TβRII)2 trap was diluted in PBS
containing 1% BSA and 0.1% Tween-20 (PBS-BT) with or without 10% (v/v) of the indicated serum, added to wells and incubated for 1 h at RT. Wells were washed 5× with PBS containing 0.1% Tween-20 (PBS-T), incubated with 35 μL 100 ng/mL biotinylated goat anti-human TβRII antibody (R&D Systems,
Minneapolis, MN) diluted 1:10,000 in PBS-BT for 1 h at RT, then washed again 5× with PBS-T. Wells were incubated with 35 μL horseradish peroxidase (HRP)-conjugated streptavidin for 20 min at RT (R&D Systems; diluted 1:200 in PBS-BT), washed again 5× with PBS-T and developed with 35 μL of
tetramethylbenzidine (TMB) substrate (Mandel Scientific, Guelph, ON, Canada). After 5 min, the reaction was stopped with 35 µL of 1 M H2SO4 and the
absorbance at 450 nm was measured using a MultiskanTM FC photometer
(Thermo-Fisher). Pre-immune: llama serum prepared from blood drawn before the first immunization; Immune: llama serum prepared from blood drawn 42 days after the first TGF-β3 immunization; Irrelevant: llama serum prepared from blood drawn 42 days after immunization with an irrelevant antigen. EC50s: No
competitor, 0.06 μg/mL; preimmune serum, 0.16 μg/mL; immune serum, 1.0 µg/mL; irrelevant serum, 0.19 μg/mL.
0.01 0.1 1 10 100 1000 0 0.2 0.4 0.6 0.8 1 1.2 VHH3, no competitor VHH3, + 50 g/mL (T RII)2 μ β trap VHH6, no competitor VHH6, + 50 g/mL (T RII)2 μ β trap VHH7, no competitor VHH7, + 50 g/mL (T RII)2 μ β trap [VHH] (nM) O D ( 4 5 0 n m )
Supplementary Figure S3. Binding of purified sdAbs to TGF-β3 in the presence or absence of 50 μg/mL (TβRII)2 trap. Wells of NUNC MaxiSorpTM microtiter
plates were coated overnight at 4°C with 0.1 μg TGF-β3 in 35 μL PBS. The next day, wells were blocked with 200 μL of PBS containing 2% BSA for 1 h at 37°C, then sdAbs were diluted in PBS containing 1% BSA and 0.1% Tween-20 (PBS-BT) with or without 50 μg/mL of (TβRII)2 trap, added to wells and incubated for 1
h at RT. Wells were washed 5× with PBS containing 0.1% Tween-20 (PBS-T), incubated with 35 μL horseradish peroxidase (HRP)-conjugated anti-myc antibody (Santa Cruz Biotechnology, Dallas, TX; diluted 1:1,000 in PBS-BT), washed again 5× with PBS-T and developed with 35 μL of tetramethylbenzidine (TMB) substrate (Mandel Scientific, Guelph, ON, Canada). After 5 min, the reaction was stopped with 35 µL of 1 M H2SO4 and the absorbance at 450 nm
triethylamine. Twelve sdAbs are shown, unique in sequence from the sdAbs described in main text. SPR experiments were performed as described in main text.
