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SUPPORTING INFORMATION for

LASIC: Light Activated Site-Specific Conjugation of Native

IgGs

James Zhe Hui^, Shereen Tamsen^, Yang Song, and Andrew Tsourkas[a]

Department of Bioengineering, School of Engineering and Applied Sciences, University

of Pennsylvania, Philadelphia PA, 19104 USA

Table of Contents Figure S1. Structure and sequence of Protein G adapter…………………………… 2 Figure S2a. Screening Protein G adapters for ability to label human IgGs………... 3 Figure S2b. Screening Protein G adapters for ability to label mice IgGs………….. 4 Figure S2c. Screening Protein G adapters for ability to label rat and other IgGs…. 5 Figure S3a. Model of Protein G binding to Fc (1FCC)………………………………. 6 Figure S3b. IgG sequence alignment………………………………………………….. 7 Figure S4. Kinetics and efficiency of K28BPA adapter protein crosslinking………. 8 Figure S5. Storage of Protein G in room temperature and under ambient lighting does not affect its ability to label IgG…………………………………………. 9 Figure S6. Modification of IgG with various functional moieties using Protein G adapters…………………………………………………………………………………… 10 Materials and methods…………………………………………………………………... 11

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SUPPLEMENTAL FIGURES:

MTFKLIINGKTLKGEITIEAVDAAEAEKIFKQYANDYGIDGEWTYDDATKTFTVTE Supplemental Figure S1. Structure and sequence of Protein G adapter. Molecular model of a Protein G Fc-binding domain showing the side-chains of the nine amino acids that were substituted by BPA in making photo-reactive Protein G adapters. The amino acid sequence of the Protein G adapter (based on the thermophilic HTB1 domain) is listed. A N37Y mutation is made to eliminate Fab binding. Residues highlighted in red indicate locations of BPA substation. Note a glycine-serine linker (GGSGGS) and the sortase motif (LPETG) would be appended to the Protein G in the finished product after STEPL. PDB file: 1FCC.

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Supplemental Figure S2a. Screening Protein G adapters for ability to label human IgGs. Human IgG1, human IgG2, human IgG3 and human IgG4 were each UV treated with one of the nine Protein G adapters (Lanes 2-10). The resulting products were analyzed using reducing SDS-PAGE gels. For human IgG2, human IgG3, and human IgG4, only the upper portions of the gels depicting heavy chains are shown.

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Supplemental Figure S2b. Screening Protein G adapters for ability to label mice IgGs. Mouse IgG1, mouse IgG2a, mouse IgG2b and mouse IgG3 were each UV treated with one of the nine Protein G adapters (Lanes 2-10). The resulting products were analyzed using reducing SDS-PAGE gels. Only the upper portions of the gels depicting heavy chains are shown.

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Supplemental Figure S2c. Screening Protein G adapters for ability to label rat and other IgGs. Upper panel: Rat IgG1, rat IgG2a, rat IgG2b and rat IgG2c were each UV treated with one of the nine Protein G adapters (Lanes 2-10). The resulting products were analyzed using reducing SDS-PAGE gels. Only the upper portions of the gels depicting heavy chains are shown. Lower panel: Rabbit polyclonal IgG and hamster IgG1 were each UV treated with one of the nine Protein G adapters (Lanes 2-10). The resulting products were analyzed using reducing SDS-PAGE gels. Only the upper portions of the gels depicting heavy chains are shown.

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Supplemental Figure S3a. Model of Protein G binding to Fc (1FCC). Molecular model showing Protein G fragments in complex with the Fc portion of the human IgG. The side-chains of IgG Met252 and Met482 are shown, as are the side-chains of A24 and K28 in Protein G, which have been substituted by BPA in making photoreactive Protein Gs. PDB: 1FCC

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235 281

hIgG1 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG

hIgG2 VAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDG

hIgG3 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDG

hIgG4 LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDG

mIgG1 PEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDD

mIgG2a LGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNN

mIgG2b EGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNN

mIgG3 LGGPSVFIFPPKPKDALMISLTPKVTCVVVDVSEDDPDVHVSWFVDN

rIgG1 --VSSVFIFPPKTKDVLTITLTPKVTCVVVDISQNDPEVRFSWFIDD

rIgG2a --VSSVFIFPPKTKDVLTITLTPKVTCVVVDISQNDPEVRFSWFIDD

rIgG2b LGGPSVFIFPPKPKDILLISQNAKVTCVVVDVSEEEPDVQFSWFVNN

rIgG2c LGRPSVFIFPPKPKDILMITLTPKVTCVVVDVSEEEPDVQFSWFVDN

rbIgG1 LGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINN

hmIgG1 LGGPSVFIFPPKPKDVLMISLTPKITCVVVDVSEEEPDVQFNWYVNN

413 447

hIgG1 DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

hIgG2 DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

hIgG3 DKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK

hIgG4 DKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

mIgG1 QKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

mIgG2a EKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

mIgG2b KTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGL

mIgG3 DTDSWLQGEIFTCSVVHEALHNHHTQKNLSRSPEL

rIgG1 KKEKWQQGNTFTCSVLHEGLHNHHTEKSLSHSPGK

rIgG2a KKETWQQGNTFTCSVLHEGLHNHHTEKSLSHSPGK

rIgG2b ERSRWDSRAPFVCSVVHEGLHNHHVEKSISRPPGK

rIgG2c DTDSWMRGDIYTCSVVHEALHNHHTQKNLSRSPGK

rbIgG1 PTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK

hmIgG1 PKSRWDQGDSFTCSVIHEALHNHHMTKTISRSLGN

Supplemental Figure S3b. IgG sequence alignment. Amino acid sequences from the Fc portions of various IgG subclasses were aligned and shown. Alignment was done using Clustal W2 program. Met252 and Met482, when present, are highlighted. The GI number of the sequences used are: 218533932 (hIgG1), P01859 (hIgG2), P01860 (hIgG3), 557804824 (hIgG4); 21304449 (mIgG1), P01863 (mIgG2a), P01863 (mIgG2b),

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P03987 (mIgG3); 623556 (rat IgG1), P20760 (rat IgG2a), P20761 (rat IgG2b), P20762 (rat IgG2c); P01870 (rabbit IgG1), 841150(hamster IgG1)

Supplemental Figure S4. Kinetics and efficiency of K28BPA adapter protein crosslinking.

(A) Non-reducing and reducing SDS-PAGE of cetuximab (Cetux, human IgG1) alone or after photocrosslinking with Protein G (PG)-based adapter possessing a K28BPA substitution. UV crosslinking was performed for varying periods of time using four equivalents of the adapter proteins. Image analysis of non-reducing gels are shown on the right. (B). Non-reducing and reducing SDS-PAGE gels of cetuximab alone or after photocrosslinking with K28BPA adapter proteins. UV crosslinking was performed for one hour and 30 minutes with various molar ratios of adapter protein-to-IgG. Image analysis of non-reducing gels are shown on the right.

2"PG 1PG 0PG

1 10385.024 10137.711 1035.619 21558.354

2 15100.409 11060.853 755.326 26916.588

3 17415.43 10369.238 338.355 28123.023

4 15705.652 10599.288 522.74 26827.68

5 16822.551 11231.823 623.861 28678.235

0"

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90"

100"

0" 2" 4" 6" 8"

%"Conj"

Equivalent"of"Protein"G"

IgG+2PG"

IgG+1PG"

Unlabeled"IgG"

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Supplemental Figure S5. Storage of Protein G in room temperature (RT) and under ambient lighting (AL) does not affect its ability to label IgG. Aliquots of Protein G adapter A24BPA were stored for 2 weeks either in room temperature under ambient fluorescent

lighting or in 4 °C and covered with aluminum foil. Samples (1M) were then UV treated

for 2 hours by themselves, or with 0.2M human IgG1 (cetuximab). The resulting products were analyzed using SDS-PAGE.

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Supplemental Figure S6. Modification of IgG with various functional moieties using Protein G adapters. Protein G adapters made with peptides containing either A) TAMRA-DBCO (lane 3), B) FAM-Azide (lane 4), or C) Bioin (lane 11), were LASIC conjugated onto human IgG1 (lane 1 and 2; 5 and 6) or mouse IgG2a (lane 9 and 10). Conjugates remained active as demonstrated by click reactions (Lane 4: Click with Peg-Azide; Lane 8: Click with PG-TAMRA-DBCO) or by Western blot with Streptavadin-IRdye800. The arrow (>) indicates Protein G-labeled heavy chains; The asterisk (*) indicates click product) Truncated image available as Figure 5.

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MATERIALS and METHODS

Materials

Clinical grade anti-EGFR antibody, cetuximab, was kindly provided by Dr. Daqing Li at the University of Pennsylvania. Human IgG2 isotype control antibody (HCA108A), human IgG3 isotype control antibody (HCA178) and human IgG4 isotype control antibody (HCA050A) were all purchased from AbD Serotec (Kidlington, UK). Mouse IgG1 anti-Dextran (10730) antibody was purchased from StemCell Technologies (Vancouver, Canada). Mouse IgG1 anti-Rat CD68 antibody (MCA341R) was purchased from AbD Serotec Inc (Kidlington, UK). Mouse IgG1 anti-Fas (DX2) antibody was purchased from R&D Systems (Minneapolis, MN). Mouse IgG2a anti-CD3 antibody (OKT3) was purchased from Bio-X-Cell (West Lebanon, NH). Mouse IgG2b isotype control antibody (14-4732) was purchased from eBioscience (San Diego, CA). Mouse IgG3 isotype control antibody (14-4742) was purchased from eBioscience (San Diego, CA). Rat IgG1 isotype control antibody (14-4301) was purchased from eBioscience (San Diego, CA). Rat IgG2a isotype control antibody (14-4321) was purchased from eBioscience (San Diego, CA). Rat IgG2b anti-ICAM1 antibody (YN-1) was kindly provided by Dr. Ann-Marie Chako at the University of Pennsylvania. Rat IgG2c isotype control antibody (400701) was purchased from Biolegend (San Diego, CA). Hamster IgG1 anti-CD3E antibody (145-2C11) was purchased from Thermo-Fisher (Waltham, MA). Polyclonal Rabbit anti-tetramethylrhodamine (A-6397) antibody was purchased from Invitrogen (Carlsbad, CA). Triglycine was purchased from Sigma-Aldrich (St Louis, MO). N-terminated triglycine peptides containing reactive groups were synthesized by Anaspec (Fremont, CA) The peptide sequences containing the reactive groups were: azide-FAM: GGGK(5(6)-FAM)GGSK(N3), MW: 1032 Da; TAMRA-Cys: GGGK(TAMRA)GGSC, MW: 1033 Da; Biotin: GGGSK(biotin), MW: 458 Da. Maleimide-PEG4-DBCO, purchased from Sigma-Aldrich, was reacted with TAMRA-Cys peptide to generate TAMRA-DBCO peptide, which was then HPLC purified. All other reagents were purchased from Thermo Fisher Scientific (Waltham, MA) unless otherwise noted.

Cloning of Protein G Fusion Protein into pSrtA Vector

A double stranded DNA fragment encoding the Protein G sequence (shown in Figure S1) fused to a C-terminal glycine-serine linker (GGSGGS) and a sortase recognition motif (LPETG) and flanked at both ends by 15 base sequences homologous to the desired NdeI and AgeI restriction sites of the destination vector pSrtA were ordered from Integrated DNA Technologies (Coralville, IA). The full amino acid sequence for the Protein G can be found in Figure S1. 50 ng of the gene fragment was then digested with NdeI and AgeI restriction enzymes (New England Biolabs, Ipswich, MA) and gel purified. The corresponding sortase pSrtA vector was also double digested with NdeI and AgeI and gel-purified. Ligation was done overnight using T4 ligase (New

of the Protein G sequence was verified by Sanger DNA sequencing using the T7 promoter as the sequencing primer. Site-directed mutagenesis of selected codons into an amber codon (TAG) was done using Quikchange Mutagenesis Kit (Agilent, Santa Clara, CA).

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Expression and Purification of Protein G Fusion Protein

The pSrtA plasmid containing the cloned Protein G sequence and the pEVOL-pBpF plasmid (Addgene.org) were cotransformed into the T7 Expression Crystal Competent Cells (New England Biolabs, Ipswich, MA). Bacterial cell cultures were

(LB) media. Cultures were scaled up to 50 mL of LB media and grown overnight under the same conditions, and then inoculated into 1 L LB media containing 50 mg/L of ampicillin and 25 mg/L of chloramphenicol.

For BPA incorporation, L-benzoylphenylalanine (Bachem, King of Prussia, PA) was added into the culture for a final concentration of 150 μM and the culture was left to grow for 30 min. Ne -D-1- thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mM and arabinose to a final concentration of 0.1% to begin the inductions of the pSrtA and pEVOL plasmids, respectively.

Cultucentrifugally pelleted at 10,000 g for 5 min, resuspended in 10 mL of B-PER lysis buffer (Pierce, Rockford, IL) containing 0.75 g/L lysozyme, 1 μg/mL DNAse, and 50 mM phenylmethylsulfonyl fluoride. Cells were lysed by incubation for 1 h in room temperature and then pulse sonicated on ice. Cell lysates were centrifuged at 15,000 g

–purification steps, all incubated for 1 h in a 10 mL Poly-Prep chromatography column (Bio-Rad, Hercules, CA) packed with 1 mL of Talon metal affinity resin (Clontech, Mountain View, CA). Supernatant was then allowed to pass through the column and resin beads were washed with 50 mL of column buffer (0.1 M Tris-HCl, pH 8.5, made with calcium free ultrapure water) at a flow rate of approximately 2 mL/min and then drained. The stopper was placed back onto the column.

Expressed Protein Ligation

Triglycine (2 μL of 250 mM solution in column buffer) or N-terminal triglycine peptides (100 μL of 2mM solution) and calcium chloride (1.5 μL of 50 mM solution in column buffer) were added into 1 mL of column buffer and then applied to the column. The resin was vortexed to ensure uniform distribution of the triglycine solution and then

buffer.

Purification and concentration of the final product can be performed using a 3 kD molecular weight cut-off (MWCO) filter (Amicon Ultra, Milipore, Temecula, CA) or size-exclusion chromatography (Zeba 7kD columns, Pierce, Rockford, IL). Alternatively, Protein G can also be purified with RP-HPLC (Varian Prostar) as was done here. A C8 300 Å 5 μm column (Agilent) was used. Protein G were eluted at 1 mL/min using a mixture of water and acetonitrile, both containing 0.1% TFA. The solvent gradient used was: 95% - 75% water over the first 10 min, then 75%-69% over the next 60 min. Absorbance was monitored at 215 nm. The collected fractions were then dried using vacuum centrifuge concentrator (Labconco, Kansas City, MO) and reconstituted in

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column buffer. Protein concentration was determined using BCA assay (Pierce, Rockford, IL).

Crosslinking

Unless otherwise stated, Protein G were crosslinked with IgGs by first mixing the IgG (final concentration 0.4 μM) and Protein G (final concentration 3.2 μM) in 0.1 M Tris-HCl buffer at the molar ratio of 1 IgG to 8 Protein G in a clear 1.5 mL centrifuge tube. Next the mixture was immediately placed on an ice bath and irradiated for 1.5 hour with 365 nm UV light using a UVP CL-1000L UV crosslinker (Upland, CA). Samples were then analyzed using SDS-PAGE gel as described below.

To assess the effect of irradiation duration on crosslinking, the samples were prepared as above, but irradiated for 15 min, 30 min, 1 h, 1.5 h and 2 h. To assess the effect of IgG to Protein G ratio on crosslinking, IgG (final concentration 0.4 μM) were mixed with Protein G at final concentrations of 0.4 μM (1 x), 0.8 μM (2 x), 1.6 μM (4 x), 3.2 μM (8 x) and 6.4 μM (16 x) and UV irradiated for 1.5 h as above. Note, since each IgG contains two heavy chains, on a per heavy chain basis, the molar ratios used would be 0.4 μM (0.5 x), 0.8 μM (1 x) , 1.6 μM (2 x), 3.2 μM (4 x) and 6.4 μM (8 x).

Analysis of crosslinking

Crosslinked products were analyzed directly using SDS-PAGE electrophoresis. For reducing SDS-PAGE, crosslinked samples were boiled for 3 min with equal volume of SDS-PAGE loading buffer (Bio-rad, Hercules, CA) containing 1:20 dilution of beta-mercaptoethanol (Bio-rad). The samples were then loaded onto AnyKd gradient PAGE gels (Bio-rad) and ran for 21 min at constant 250 Volts. For non-reducing gel, the samples were boiled as above, but using SDS-PAGE loading buffer without any beta-mercaptoethanol, and then also loaded onto an AnyKD gradient gel and ran for 1.5 h at 250 Volts. The gels were stained for protein using SimplyBlue Coomassie stain (Invitrogen).

Images of the gel were taken using a Kodak Gel Logic 100 system (Rochester, NY) with background illumination correction. The images were then analyzed using ImageJ software (http://imagej.nih.gov). Specifically, images were background corrected using the background subtraction function with a rolling ball radius of 50 pixels. Next, the lanes were selected using the rectangle tool and band intensity profiles plotted using the gel analysis functions. For reducing gels, the intensity of the crosslinked heavy chain as a percentage of total heavy chain was taken to reflect crosslinking extent. For non-reducing gels, the percentages of doubly crosslinked IgG, single crosslinked IgG and non-crosslinked IgG were all calculated as percentages of total IgG.

IgG affinity assay 4000 KB cells (ATCC, Manassas, VA) were plated in each well of a bottom-clear

96-well plate in 100 μL culture medium and kept in 37 °C cell culture incubator for three days. To fix the cells, 50 μL of 4 % paraformaldehyde was added to each well and incubated for 5 minutes. The liquid phase was then aspirated and another 50 μL 4 % paraformaldehyde was added for 15 minutes. The cells were washed with TBSTB buffer

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(25 mM Tris, 0.15 M NaCl, 0.05% Tween 20, 0.25 mg/mL BSA, pH 7.2) and blocked with TBSTB with 4% Normal Donkey Serum for 1h.

2 μL of 2 mg/mL cetuximab was mixed with 6 μL 1.3 mg/mL PG A24BPA or 6 uL H2O and UV illuminated for 30 minutes. A series of diluted product was added to the cells and incubated at room temperature for 1.5 hours. The cells were then washed with TBSTB three times and incubated in 7.5 ug/mL Donkey anti-human IgG RhodamineRedX (Jackson Laboratory) for 1.5 hours. After washing with TBST three times, the fluorescence intensity of each well is determined on a Infinity M200 (Tecan, San Jose, CA) with excitation and emission wavelengths of 565 nm and 595 nm respectively. The values were corrected by black control in which the cells were not treated with cetuximab, and fitted in Prism by one site-specific binding with Hill slope. Conjugating IgG in BSA solution Human IgG2 (0.1 mg/mL) isotype control antibody stored with 1% BSA as stabilizer (HCA108A) was purchased from AbD Serotec (Kidlington, UK). IgG2 was purified from the accompanying BSA using Protein A resin (Invitrogen) and then eluted using pH 3 0.2 M glycine buffer and buffer exchanged to 0.1 mg/mL in PBS. Human IgG2, either in purified form or with 1% BSA as stabilizer, was LASIC conjugated with three molar excesses of A24BPA TAMRA-DBCO adapter. The resulting products were then analysed using reducing SDS-PAGE gels. Labeling IgGs with reactive moieties To make LASIC adapters containing azide-FAM, TAMRA-DBCO and biotin moeities, A24BPA Protein G were ligated with the respective peptides containing these three groups during the STEPL purification process and HPLC purified as described above. Azide-FAM and TAMRA-DBCO ligated adapters were each LASIC conjugated onto human IgG1 cetuximab using two molar excesses of the adapters and 1.5 hour of UV exposure. Biotin-ligated adapter was LASIC conjugated with mouse IgG2a OKT3 using three molar excesses of the adapter and 1.5 hour of UV exposure.

One molar equivalent of PEG-azide was added to DBCO-TAMRA labeled cetuximab and the mixture was incubated for 1 hour at room temperature; the resulting click product was analyzed on a SDS-PAGE reducing gel. Two molar equivalents of DBCO-TAMRA-Protein G was added to azide-FAM labeled cetuximab and incubated for 1 hour at room temperature; the resulting click product was analyzed on a SDS-PAGE reducing gel. To detect the presence of biotin on biotin-labeled OKT3, the sample was ran on a reducing SDS-PAGE gel and transferred onto a nitrocellulose blot. The blot was then blocked using Odyssey blocking buffer (Licor, Lincoln, NE) and then treated with streptavidin-IRdye800 (Licor) per manufacturer’s instructions. After washing, the blot was imaged using an Odyssey IR imager using the 800nM channel.

Purifying mono-conjugated product Human IgG1 cetuximab (final concentration 0.4 μM) was LASIC conjugated for 30 minutes with four molar excess of A24BPA Protein G adapter. The conjugated product was then applied onto a column containing Protein A resin (Invitrogen, Carlsbad, CA). The flow-through from the column was collected. The column was then washed 3 x using PBS and then eluted using 0.2 M glycine pH 3 buffer. pH of the

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eluted product when then adjusted to 7 using 1M Tris-HCl. The LASIC product, the column flow-through and the eluted product were all analyzed using SDS-PAGE gel.