Name: A GPC3-targeting Bispecific Antibody, GPC3-S-Fab, with Potent Cytotoxicity (Video) | JoVE Journal
Uploaded: 2018-07-12T13:00:00.000+00:00
Duration: 11 min 13 s
Description: Learn to produce the bispecific antibody GPC3-S-Fab in Escherichia coli and purify it for cytotoxicity assays against liver cancer cells. This protocol enables targeted cancer immunotherapy research.

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5 minutes

This work was financially supported by the R&#38;D Plan of Guangdong Province (PR China) (2016A050503028).

All of the procedures including human blood collection were approved by the Sun Yat-Sen University Ethics Committee.
1. GPC3-S-Fab Design Strategy
<ol>
	<li>Design GPC3-S-Fab by linking a Fab of anti-GPC3 (humanized GC3313) with an anti-CD16 VHH14 (Figure 1).</li>
	<li>Synthesize and clone the VH-CH1-CD16-VHH and VL-CL into the pET26b and pET21a vectors as previously reported12.</li>
</ol>
2. Transformation and Culture
<ol>
	<li>Add 100 ng of pET26b containing VH-CH1-CD16 (Figure 1A) and 100 ng of pET21a containing VL-CL (Figure 1A) into 100 &#181;L of competent BL21 (DE3) cells. Mix well and incubate on ice for 30 min. Incubate in a 42 &#176;C water bath for 45 - 90 s, transfer, and incubate on ice for 3 - 5 min.</li>
	<li>Add 500 &#181;L of lysogeny broth (LB) medium into a tube containing BL21 (DE3) cells and then incubate at 37 &#176;C, 150 rpm for 45 - 60 min. Spread 100 &#181;L of BL21 (DE3) cells on LB-agar plates containing 50 &#181;g/mL of kanamycin and 100 &#181;g/mL of ampicillin, and then incubate at 37 &#176;C for 12 - 16 h.
	<ol>
		<li>Prepare lysogeny broth (LB) medium: 1% wt/vol tryptone, 0.5% wt/vol yeast extract, 1% wt/vol NaCl.</li>
	</ol>
	</li>
	<li>Inoculate cells from a single colony into 5 mL of super broth (SB) medium containing 50 &#181;g/mL of kanamycin and 100 &#181;g/mL of ampicillin, and culture at 37 &#176;C, 220 rpm, overnight. Then, inoculate 1 mL of culture into 100 mL of SB medium containing 50 &#181;g/mL of kanamycin and 100 &#181;g/mL of ampicillin and culture at 37 &#176;C, 220 rpm for 4 h. Then, transfer 10 mL of the culture into 1 L of SB medium containing 50 &#181;g/mL of kanamycin and 100 &#181;g/mL of ampicillin and culture at 37 &#176;C, 220 rpm.
	<ol>
		<li>Prepare super broth medium: 3.2% wt/vol tryptone, 2% wt/vol yeast extract, 0.5% wt/vol NaCl.</li>
	</ol>
	</li>
	<li>When the OD600 reaches 0.6 - 0.8, add isopropyl-b-D-thio-galactopyranoside (IPTG) to a final concentration of 0.2 mM. Culture at 16 &#176;C, 180 rpm for 36 - 48 h for periplasmic expression.</li>
</ol>
3. GPC3-S-Fab Periplasmic Purification
<ol>
	<li>Periplasmic fraction preparation
	<ol>
		<li>Collect cells by centrifuging at 4,000 x g, 4 &#176;C for 30 min, discard the medium, and weigh the cells.</li>
		<li>Resuspend the cells thoroughly in 4 mL of ice-cold sucrose solution (20 mM of Tris-HCl, pH 7.5; 25% (wt/vol) sucrose and 1 mM of EDTA) per gram of cells, and incubate on ice for 15 min.</li>
		<li>Centrifuge at 8,500 x g (table top centrifuge, fixed angle rotor), 4 &#176;C for 20 min. Remove the supernatant (the sucrose fraction) and save it on ice.</li>
		<li>Resuspend the pellets in a mix of ice-cold 5 mM of MgCl2 and 1 mM of PMSF (4 mL per gram cells). Add 40 &#181;L of the lysozyme stock (15 mg/mL) per gram, and incubate on ice for 30 min.</li>
		<li>Centrifuge at 8,500 x g (table top centrifuge, fixed angle rotor), 4 &#176;C for 20 min. Transfer the supernatant (the periplasmic fraction) and save it on ice.</li>
		<li>Combine the sucrose fraction and periplasmic fraction, and centrifuge at 30,000 x g, 4 &#176;C for 30 min. Remove the supernatant and save it in a 50 mL conical tube on ice.</li>
	</ol>
	</li>
	<li>Ni-NTA affinity purification
	<ol>
		<li>Resuspend 1 mL of Ni-NTA agarose by mixing thoroughly to achieve a homogeneous suspension, remove 1 mL of Ni-NTA agarose to a fresh 15 mL conical tube, and add 10 column volumes (CV) equilibration buffer (25 mM of Tris-HCl, pH 7.5; 1 M of NaCl) to equilibrate.</li>
		<li>Centrifuge at 400 x g for 5 min, and remove the supernatant carefully. Then, add the Ni-NTA agarose into the 50 mL tube containing the sucrose fraction and periplasmic fraction. Rock at 4 &#176;C for 2 h.</li>
		<li>Centrifuge the mixture at 400 x g, 4 &#176;C for 5 min. Carefully remove the supernatant to another fresh ice-cold tube as the unbound fraction. Transfer the Ni-NTA agarose into a gravity column.</li>
		<li>Add 10 CV of washing buffer (25 mM of Tris-HCl, pH 7.5; 1 M of NaCl; 20 mM, 30 mM or 40 mM of imidazole) to the column, and collect the elute as the washing fraction. 
		NOTE: These solutions should be added in order of increasing concentrations of imidazole.</li>
		<li>Add 3 CV of elution buffer (25 mM of Tris-HCl, pH 7.5; 300 mM of NaCl; 100 mM, 200 mM, 300 mM or 400 mM of imidazole) to the column and collect the elute as Elution fraction 1, 2, 3, and 4. 
		NOTE: These solutions should be added in order of increasing concentrations of imidazole.</li>
		<li>Dialysis the eluted fractions in 2 L of PBS (137 mM of NaCl, 2.7 mM of KCl, 10 mM of Na2HPO4, 2 mM of KH2PO4, pH 7.4) using dialysis tubing (12.4 kDa) at 4 &#176;C for 2 h. Check the presence of GPC3-S-Fab by 12% SDS-PAGE and then by Coomassie blue staining.</li>
	</ol>
	</li>
	<li>IgG-CH1 affinity purification
	<ol>
		<li>Transfer 1 mL of IgG-CH1 affinity resin into a 50 mL conical tube, and wash the beads with PBS first. Then, add the dialyzed solution containing GPC3-S-Fab after Step 3.2.6. Rock at 4 &#176;C for 2 h.</li>
		<li>Centrifuge at 400 x g for 5 min, 4 &#176;C. Carefully remove the supernatant to another fresh ice-cold tube, and transfer the resin to a gravity column.</li>
		<li>Add 10 CV of washing buffer (PBS) to the column, and collect the elution as the wash fraction.</li>
		<li>Prepare the collection tubes by adding 1 M of Tris pH 9.0 (0.1 mL per mL of each eluted fraction). Elute with 3 CV elution buffer (0.1 M of glycine-HCl, pH 2.7), and collect the eluted fraction; repeat 3 times.</li>
		<li>Dialysis the eluted fractions in 2 L of PBS using dialysis tubing (12.4 kDa) at 4 &#176;C for 2 h. Repeat twice.</li>
		<li>Determine the protein concentration by ultramicrospectrophotometer (Molar extinction coefficient: 91105. 1 A280&#160;= 0.69 mg/L).</li>
	</ol>
	</li>
</ol>
4. SDS-PAGE and Western-blotting Analysis
<ol>
	<li>Take a 10 &#181;L of the sample into 5x SDS loading buffer and mix well. Boil the protein mixture at 100 &#176;C for 5 min.
	<ol>
		<li>Prepare 5x SDS loading buffer: 250 mM of Tris-HCl, pH 6.8, 10% wt/vol SDS, 0.5% wt/vol bromophenol blue, 50% vol/vol glycerol, 5% vol/vol beta-mercaptoethanol.</li>
	</ol>
	</li>
	<li>Prepare an SDS-PAGE gel with a loading gel (5%) and separation gel (12%) as previously described26,27,28.</li>
	<li>Load 10 &#181;L of the boiled protein mixture and protein marker, and run the SDS-PAGE.</li>
	<li>Stain the gel by shaking for 20 min with Coomassie brilliant blue solution, and then perform destaining.</li>
	<li>For western blotting, after transferring to a PVDF membrane, block the membrane with TBST (20 mM of Tris-HCl, 150 mM of NaCl, 0.1% vol/vol Tween 20) + 5% wt/vol skim milk for 1 h at room temperature while shaking.</li>
	<li>Incubate the membrane with primary antibodies (anti-His or anti-Flag) diluted 1:2,000 in TBST + 5% skim milk for 1 h.</li>
	<li>Wash with TBST for 5 min at room temperature, and repeat 3 times.</li>
	<li>Incubate the membrane in secondary antibody (HRP-linked anti-mouse Fc antibody) diluted 1:2,000 in TBST + 5% skim milk for 1 h.</li>
	<li>Wash with TBST for 5 min at room temperature, and repeat 3 times.</li>
	<li>Add 1 mL of HPR substrate per film, develop and take images.</li>
</ol>
5. Gel Filtration Analysis
<ol>
	<li>Use the following column: Column volume of 24 mL; Equilibration buffer of PBS pH 7.4; Flow rate of 0.5 mL/min at room temperate; Sample volume of 200 &#181;L.</li>
	<li>Use the following protein volume and concentration: 500 &#181;L of 1.2 mg/mL GPC3-S-Fab. Centrifuge at 20,000 x g for 30 min, and take 200 &#181;L to load.</li>
	<li>For the protein marker volume and concentration, take 100 &#181;L of cytochrome C (2 mg/mL, 12.4 kDa), 140 &#181;L of anhydrase (3 mg/mL, 29 kDa), 140 &#181;L of albumin (10 mg/mL, 66 kDa) and 200 &#181;L of alcohol dehydrogenase (5 mg/mL, 150 kDa) into one tube. Centrifuge at 20,000 x g for 30 min, and take 200 &#181;L to load.</li>
	<li>Before each run, wash with at least 2 CV of distilled water at a flow rate of 0.5 mL/min.</li>
	<li>Equilibrate the column with at least 2 CV of equilibration buffer (PBS, pH 7.4) at a flow rate of 0.5 mL/min.</li>
	<li>Automatically inject the sample at 0.5 mL/min to load the sample.</li>
	<li>Elute with equilibration buffer at 0.5 mL/min and detect at 280 and 215 nm.</li>
</ol>
6. Flow Cytometry Analysis
<ol>
	<li>Culture the HepG2 (GPC3 positive), Hep3B (GPC3 positive), Huh7 (GPC3 positive), and MHCC-97H (GPC3 negative) cells in 100 mm x 20 mm plastic dishes containing DMEM medium with 10% fetal bovine serum (FBS), penicillin (100 &#181;g/mL), and streptomycin (50 &#181;g/mL) at 37 &#176;C, 5% CO2.</li>
	<li>Culture CHO (GPC3 negative) cells and CHO/GPC3 cells harboring the full-length human GPC3 cDNA (GPC3 positive) in 100 mm x 20 mm plastic dishes containing RPMI 1640 medium with 10% FBS, penicillin (100 &#181;g/mL), and streptomycin (50 &#181;g/mL) at 37 &#176;C, 5% CO2.</li>
	<li>Culture the NK92/CD16 harboring the full-length human CD16 cDNA (CD16 positive) in 25cm2 flask containing RPMI 1640 medium with 10% FBS, penicillin (100 &#181;g/mL), and streptomycin (50 &#181;g/mL) at 37 &#176;C, 5% CO2.</li>
	<li>When the cells are 70 - 90% confluent, discard the medium and wash the cells with 2 mL of PBS. Add 1 mL of 0.25% trypsin and incubate at 37 &#176;C for 2 - 3 min (or until the cells start to detach from the surface). Add 1 mL of the complete growth medium to neutralize the trypsin and re-suspend the cells by gently pipetting up and down.</li>
	<li>Count the cell numbers using a Neubauer hemocytometer and detect the cell viability by Trypan Blue.</li>
	<li>Collect 3 x 106 cells for each cell line. Add 1 mL of PBS + 0.2% BSA to re-suspend the cells. Centrifuge at 200 x g, 4 &#176;C for 5 min. Repeat twice.</li>
	<li>Add 0.3 mL of PBS + 0.2% BSA to re-suspend the cells, and transfer 100 &#181;L (approximately 1 million) to each 5 mL polystyrene round-bottom tube. 
	NOTE: Please make sure all steps from here to the tubes should be kept cold on ice. When the antibody is binding to the cell surface antigen, the complex will start to internalize. By keeping it cold, the process is slowed significantly.</li>
	<li>Add 10 &#181;L of primary antibodies (GPC3-S-Fab or human IgG1, final concentration 5 &#181;g/mL) to every tube, and incubate on ice for 1 h.</li>
	<li>Add 1 mL of PBS + 0.2% BSA to wash, and centrifuge at 200 x g, 4 &#176;C for 5 min. Repeat three times.</li>
	<li>Re-suspend in 100 &#181;L of PBS + 0.2% BSA, and then add 0.5 &#181;L of secondary antibody (anti-human IgG(H+L)-488, final concentration 10 &#181;g/mL) and incubate on ice for 1 h. NOTE: From this step, please keep the cells from the light.</li>
	<li>Add 1 mL of PBS + 0.2% BSA to wash, and centrifuge at 200 x g, 4 &#176;C for 5 min. Repeat three times.</li>
	<li>Analyze the cells by flow cytometer according to the standard protocol29. Acquire cells with a 488 nm laser for excitation.</li>
</ol>
7. Cytotoxic Assays
<ol>
	<li>Prepare tumor cells.
	<ol>
		<li>Culture HepG2, Hep3B, Huh7, MHCC-97H, and CHO cell lines as described in steps 6.1 - 6.5.</li>
		<li>Dilute cells to 0.5 &#215; 105/mL, and plate 100 &#181;L cells (5,000 cells per well) on 96-well plates. Culture 6 - 8 h to allow the cells to attach.</li>
	</ol>
	</li>
	<li>Prepare human peripheral blood mononuclear cells (PBMCs).
	<ol>
		<li>Dilute fresh prepared blood with an equal volume of PBS in a 50 mL conical tube. For example, dilute 10 mL of blood with 10 mL of PBS.</li>
		<li>Take 15 mL of lymphocyte separation medium into a fresh 50 mL conical tube. Transfer the diluted blood onto the lymphocyte separation medium along the tube side slowly. 
		NOTE: Keep the tube vertical. Do not break the surface of the lymphocyte separation medium.</li>
		<li>Centrifuge at 400 x g for 35 min at room temperature with the brake off.</li>
		<li>Slowly remove the upper plasma layer, and take the white blood layer containing PBMCs at the plasma-lymphocyte separation medium interface.</li>
		<li>Dilute the PBMCs with a double volume of PBS + 2% FBS. Centrifuge at 400 x g for 5 min.</li>
		<li>Wash the cells twice with PBS + 2% FBS. Centrifuge at 400 x g for 5 min. Count the cell numbers as described in step 6.5.</li>
	</ol>
	</li>
	<li>Prepare NK cells.
	<ol>
		<li>Prepare the PBMCs suspension at a concentration of 5 x 107 cells/mL in PBS + 2% FBS, and place them in a 5 mL polystyrene round-bottom tube.</li>
		<li>Add Human NK cell Enrichment Cocktail at 50 &#181;L/mL of cells. Mix well, and incubate at room temperature for 10 min.</li>
		<li>Resuspend the Magnetic Particles from the NK cell Enrichment kit, and mix well to ensure they are uniformly suspended by pipetting up and down vigorously more than 5 times. Transfer the resuspended Magnetic Particles at 100 &#181;L/mL to the cell suspension. Mix well, and incubate at room temperature for 5 min.</li>
		<li>Bring the cell suspension up to a total volume of 2.5 mL by adding PBS + 2% FBS. Mix the cells in the tube by gently pipetting up and down 2 - 3 times. Place the tube (without a cap) into the magnet. Let the magnetic beads settle down for 2.5 min at room temperature.</li>
		<li>Remove the magnet. In one continuous motion, invert the tube, pouring off the enriched cell suspension into a new tube.</li>
		<li>Count the cell numbers as described in step 6.5.</li>
	</ol>
	</li>
	<li>Set up the cytotoxic reaction.
	<ol>
		<li>Dilute the NK cells to 5 &#215; 105 cells/mL using the corresponding culture medium. Add 100 &#181;L of NK cell suspension to the tumor cells plated on a 96-well plate.</li>
		<li>Add 10 &#181;L of GPC3-S-Fab at different concentrations, with the highest dose at a final concentration of 30 &#181;g/mL, 3-fold dilution, 9 point (three replicates).</li>
		<li>Incubate at 37 &#176;C, 5% CO2 for 72 h.</li>
		<li>After 72 h of incubation, remove the medium, and add 100 &#181;L of DMEM medium containing 10 &#181;L of CCK8 reagent to each well in 96-well plates, and then incubate at 37 &#176;C.</li>
		<li>Read the plate at 450 nm at 1, 2, 3, and 4 h after adding the CCK8 reagent.</li>
		<li>Analyze the data. Calculate the survival rate as (OD450 GPC3-S-Fab + Effector + tumor - OD450 medium)/(OD450 tumor - OD450 medium) &#215; 100% and (OD450 GPC3-S-Fab + tumor - OD450 medium)/(OD450 tumor - OD450 medium) &#215; 100%. The effector cells are NK cells.</li>
	</ol>
	</li>
</ol>

<ol><li>Weiner, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Building+better+monoclonal+antibody-based+therapeutics%5BTitle%5D)+AND+%22Nature+Reviews+Cancer%22%5BJournal%5D)">Building better monoclonal antibody-based therapeutics.</a> Nature Reviews Cancer. 15 (6), 361-370 (2015).</li>
<li>Rathi, C., Meibohm, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clinical+pharmacology+of+bispecific+antibody+constructs%5BTitle%5D)+AND+%22The+Journal+of+Clinical+Pharmacology%22%5BJournal%5D)">Clinical pharmacology of bispecific antibody constructs.</a> The Journal of Clinical Pharmacology. 55, Suppl 3. S21-S28 (2015).</li>
<li>Weidle, U. H., Kontermann, R. E., Brinkmann, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tumor-antigen-binding+bispecific+antibodies+for+cancer+treatment%5BTitle%5D)+AND+%22Seminars+in+Oncology%22%5BJournal%5D)">Tumor-antigen-binding bispecific antibodies for cancer treatment.</a> Seminars in Oncology. 41 (5), 653-660 (2014).</li>
<li>Demarest, S. J., Glaser, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antibody+therapeutics%2C+antibody+engineering%2C+and+the+merits+of+protein+stability%5BTitle%5D)+AND+%22Current+Opinion+in+Drug+Discovery+and+Development%22%5BJournal%5D)">Antibody therapeutics, antibody engineering, and the merits of protein stability.</a> Current Opinion in Drug Discovery and Development. 11 (5), 675-687 (2008).</li>
<li>Mabry, R., Snavely, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Therapeutic+bispecific+antibodies%3A+The+selection+of+stable+single-chain+fragments+to+overcome+engineering+obstacles%5BTitle%5D)+AND+%22IDrugs%22%5BJournal%5D)">Therapeutic bispecific antibodies: The selection of stable single-chain fragments to overcome engineering obstacles.</a> IDrugs. 13 (8), 543-549 (2010).</li>
<li>Rothlisberger, D., Honegger, A., Pluckthun, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Domain+interactions+in+the+Fab+fragment%3A+a+comparative+evaluation+of+the+single-chain+Fv+and+Fab+format+engineered+with+variable+domains+of+different+stability%5BTitle%5D)+AND+%22Journal+of+Molecular+Biology%22%5BJournal%5D)">Domain interactions in the Fab fragment: a comparative evaluation of the single-chain Fv and Fab format engineered with variable domains of different stability.</a> Journal of Molecular Biology. 347 (4), 773-789 (2005).</li>
<li>Kijanka, M., Dorresteijn, B., Oliveira, S., van Bergen en Henegouwen, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanobody-based+cancer+therapy+of+solid+tumors%5BTitle%5D)+AND+%22Nanomedicine+%28London%29%22%5BJournal%5D)">Nanobody-based cancer therapy of solid tumors.</a> Nanomedicine (London). 10 (1), 161-174 (2015).</li>
<li>Muyldermans, S., Cambillau, C., Wyns, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recognition+of+antigens+by+single-domain+antibody+fragments%3A+the+superfluous+luxury+of+paired+domains%5BTitle%5D)+AND+%22Trends+in+Biochemical+Sciences%22%5BJournal%5D)">Recognition of antigens by single-domain antibody fragments: the superfluous luxury of paired domains.</a> Trends in Biochemical Sciences. 26 (4), 230-235 (2001).</li>
<li>Gonzalez-Sapienza, G., Rossotti, M. A., Tabares-da Rosa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-Domain+Antibodies+As+Versatile+Affinity+Reagents+for+Analytical+and+Diagnostic+Applications%5BTitle%5D)+AND+%22Frontiers+in+Immunology%22%5BJournal%5D)">Single-Domain Antibodies As Versatile Affinity Reagents for Analytical and Diagnostic Applications.</a> Frontiers in Immunology. 8, 977(2017).</li>
<li>Fernandes, C. F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Camelid+Single-Domain+Antibodies+As+an+Alternative+to+Overcome+Challenges+Related+to+the+Prevention%2C+Detection%2C+and+Control+of+Neglected+Tropical+Diseases%5BTitle%5D)+AND+%22Frontiers+in+Immunology%22%5BJournal%5D)">Camelid Single-Domain Antibodies As an Alternative to Overcome Challenges Related to the Prevention, Detection, and Control of Neglected Tropical Diseases.</a> Frontiers in Immunology. 8, 653(2017).</li>
<li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+bispecific+antibody%2C+S-Fab%2C+induces+potent+cancer+cell+killing%5BTitle%5D)+AND+%22Journal+of+Immunotherapy%22%5BJournal%5D)">A novel bispecific antibody, S-Fab, induces potent cancer cell killing.</a> Journal of Immunotherapy. 38 (9), 350-356 (2015).</li>
<li>Li, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+single-domain+antibody-linked+Fab+bispecific+antibody+Her2-S-Fab+has+potent+cytotoxicity+against+Her2-expressing+tumor+cells%5BTitle%5D)+AND+%22AMB+Express%22%5BJournal%5D)">A single-domain antibody-linked Fab bispecific antibody Her2-S-Fab has potent cytotoxicity against Her2-expressing tumor cells.</a> AMB Express. 6 (1), 32(2016).</li>
<li>Nakano, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anti-glypican+3+antibodies+cause+ADCC+against+human+hepatocellular+carcinoma+cells%5BTitle%5D)+AND+%22Biochemical+and+Biophysical+Research+Communications%22%5BJournal%5D)">Anti-glypican 3 antibodies cause ADCC against human hepatocellular carcinoma cells.</a> Biochemical and Biophysical Research Communications. 378 (2), 279-284 (2009).</li>
<li>Behar, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+characterization+of+anti-FcgammaRIII+%28CD16%29+llama+single-domain+antibodies+that+activate+natural+killer+cells%5BTitle%5D)+AND+%22Protein+Engineering%2C+Design%2C+and+Selection%22%5BJournal%5D)">Isolation and characterization of anti-FcgammaRIII (CD16) llama single-domain antibodies that activate natural killer cells.</a> Protein Engineering, Design, and Selection. 21 (1), 1-10 (2008).</li>
<li>Baral, T. N., Arbabi-Ghahroudi, M. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/22886257">Expression of single-domain antibodies in bacterial systems.</a> Methods in Molecular Biology. 911, 257-275 (2012).</li>
<li>Arbabi-Ghahroudi, M., Tanha, J., MacKenzie, R. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/16408159">Prokaryotic expression of antibodies.</a> Cancer and Metastasis Reviews. 24 (4), 501-519 (2005).</li>
<li>Spiess, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bispecific+antibodies+with+natural+architecture+produced+by+co-culture+of+bacteria+expressing+two+distinct+half-antibodies%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">Bispecific antibodies with natural architecture produced by co-culture of bacteria expressing two distinct half-antibodies.</a> Nature Biotechnology. 31 (8), 753-758 (2013).</li>
<li>Filmus, J., Selleck, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glypicans%3A+proteoglycans+with+a+surprise%5BTitle%5D)+AND+%22Journal+of+Clinical+Investigation%22%5BJournal%5D)">Glypicans: proteoglycans with a surprise.</a> Journal of Clinical Investigation. 108 (4), 497-501 (2001).</li>
<li>Baumhoer, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glypican+3+expression+in+human+nonneoplastic%2C+preneoplastic%2C+and+neoplastic+tissues%3A+a+tissue+microarray+analysis+of+4%2C387+tissue+samples%5BTitle%5D)+AND+%22American+Journal+of+Clinical+Pathology%22%5BJournal%5D)">Glypican 3 expression in human nonneoplastic, preneoplastic, and neoplastic tissues: a tissue microarray analysis of 4,387 tissue samples.</a> American Journal of Clinical Pathology. 129 (6), 899-906 (2008).</li>
<li>Llovet, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+molecular+signature+to+discriminate+dysplastic+nodules+from+early+hepatocellular+carcinoma+in+HCV+cirrhosis%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">A molecular signature to discriminate dysplastic nodules from early hepatocellular carcinoma in HCV cirrhosis.</a> Gastroenterology. 131 (6), 1758-1767 (2006).</li>
<li>Zhu, Z. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+glypican-3+expression+differentiates+the+majority+of+hepatocellular+carcinomas+from+benign+hepatic+disorders%5BTitle%5D)+AND+%22Gut%22%5BJournal%5D)">Enhanced glypican-3 expression differentiates the majority of hepatocellular carcinomas from benign hepatic disorders.</a> Gut. 48 (4), 558-564 (2001).</li>
<li>Hsu, H. C., Cheng, W., Lai, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cloning+and+expression+of+a+developmentally+regulated+transcript+MXR7+in+hepatocellular+carcinoma%3A+biological+significance+and+temporospatial+distribution%5BTitle%5D)+AND+%22Cancer+Research%22%5BJournal%5D)">Cloning and expression of a developmentally regulated transcript MXR7 in hepatocellular carcinoma: biological significance and temporospatial distribution.</a> Cancer Research. 57 (22), 5179-5184 (1997).</li>
<li>Flaherty, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nonclinical+evaluation+of+GMA161--an+antihuman+CD16+%28FcgammaRIII%29+monoclonal+antibody+for+treatment+of+autoimmune+disorders+in+CD16+transgenic+mice%5BTitle%5D)+AND+%22Toxicological+Sciences%22%5BJournal%5D)">Nonclinical evaluation of GMA161--an antihuman CD16 (FcgammaRIII) monoclonal antibody for treatment of autoimmune disorders in CD16 transgenic mice.</a> Toxicological Sciences. 125 (1), 299-309 (2012).</li>
<li>Sharma, R., Das, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Organ-specific+phenotypic+and+functional+features+of+NK+cells+in+humans%5BTitle%5D)+AND+%22Immunological+Research%22%5BJournal%5D)">Organ-specific phenotypic and functional features of NK cells in humans.</a> Immunological Research. 58 (1), 125-131 (2014).</li>
<li>Li, X. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+CD16a%2C+the+surface+receptor+of+Kupffer+cells%2C+on+the+growth+of+hepatocellular+carcinoma+cells%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Medicine%22%5BJournal%5D)">Effect of CD16a, the surface receptor of Kupffer cells, on the growth of hepatocellular carcinoma cells.</a> International Journal of Molecular Medicine. 37 (6), 1465-1474 (2016).</li>
<li>Fiala, G. J., Schamel, W. W., Blumenthal, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blue+native+polyacrylamide+gel+electrophoresis+%28BN-PAGE%29+for+analysis+of+multiprotein+complexes+from+cellular+lysates%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Blue native polyacrylamide gel electrophoresis (BN-PAGE) for analysis of multiprotein complexes from cellular lysates.</a> Journal of Visualized Experiments. (48), (2011).</li>
<li>Hwang, A. C., Grey, P. H., Cuddy, K., Oppenheimer, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pouring+and+running+a+protein+gel+by+reusing+commercial+cassettes%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Pouring and running a protein gel by reusing commercial cassettes.</a> Journal of Visualized Experiments. (60), (2012).</li>
<li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227 (5259), 680-685 (1970).</li>
<li>Menon, V., Thomas, R., Ghale, A. R., Reinhard, C., Pruszak, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow+cytometry+protocols+for+surface+and+intracellular+antigen+analyses+of+neural+cell+types%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Flow cytometry protocols for surface and intracellular antigen analyses of neural cell types.</a> Journal of Visualized Experiments. (94), (2014).</li>
<li>Feng, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Therapeutically+targeting+glypican-3+via+a+conformation-specific+single-domain+antibody+in+hepatocellular+carcinoma%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Science+U+S+A%22%5BJournal%5D)">Therapeutically targeting glypican-3 via a conformation-specific single-domain antibody in hepatocellular carcinoma.</a> Proceedings of the National Academy of Science U S A. 110 (12), E1083-E1091 (2013).</li>
</ol>

The authors declare no conflicts of interest.

In this study, we present a strategy to construct a new format of bsAbs, GPC3-S-Fab, which can recruit NK cells targeting GPC3 positive tumor cells. The S-Fab is based on the natural Fab format by adding an anti-CD16 VHH11,12. Compared with the bsAbs containing Fc region, GPC3-S-Fab can easily be produced in the periplasm of bacteria on a large scale.
Using the expression and purification strategy described in the protocol, we obtained...

Monoclonal antibodies are now broadly used for cancer treatment1. Due to the flexibility of antibodies, various antibody-based formats have been actively explored. Compared with monoclonal antibodies, bsAbs have two different antigen binding modules, enabling them to recognize two different targets simultaneously and efficiently trigger the recruitment of immune effector cells to target and kill tumor cells2.
Current recombinant bsAb formats can be generally assigned to two classes: Fc-containing bsAbs and bsAbs without an Fc region. Compared with Fc-containing formats that are mostly produced in mammalian cells, bsAbs without an Fc region have the advantages of smaller sizes, are more readily produced in microorganism expression systems, and can penetrate tumor tissues more efficiently3.
bsAbs without an Fc region are commonly formed by linking individual binding moieties, such as single-chain variable fragments (scFvs) or Fabs3. Without the stabilizing domains, bsAbs based on scFv fragments often have compromised thermal stability, low solubility, or an increased potential for aggregation4,5. In contrast, Fab-based bsAbs are more stable due to the heterodimerization of the CH1 and CL in the native Fab moiety4,6.
Variable domain from heavy-chain-only antibodies (VHHs, also referred to as single domain antibodies) are the active antigen-binding fragment of natural heavy chain antibodies7. VHHs have the characteristics of high affinity, specificity of conventional IgGs8, low immunogenicity, and high yields in bacterial expression9. Compared with Fv fragments, VHHs have higher thermal stability10. Compared with Fab moieties, VHHs have smaller sizes due to the lack of CH1 and CL. Thus, S-Fab, the bsAb format obtained by linking the Fab with a single domain antibody, VHH, was designed and studied for its anti-tumor effects11,12.
In this study, the construction of GPC3-S-Fab by linking the Fab of hGC3313 with an anti-CD16a VHH14 was described. The GPC3-S-Fab can be efficiently produced by periplasmic expression in Escherichia coli (E. coli). Functional studies of GPC3-S-Fab suggested that GPC3-S-Fab is a promising strategy for liver cancer therapy. Thus, the advantages of GPC3-S-Fab over alternative techniques with applicable references to previous studies include easy production and purification, and more stable bsAbs.
Mammalian expression systems and prokaryotic expression systems have been used to express various formats of BsAbs. In contrast to mammalian expression systems, E. coli-based protein expression systems have many benefits, including high yields, low cost and labor-saving, the ease of genetic manipulations, and high transformation efficiency15. For bsAbs expression in E. coli, there are two basic strategies: expression in the cytoplasm and expression in the periplasm between the cytoplasm and outer cell membranes15. Compared to the reducing environment of cytoplasm, the periplasm is a more oxidizing environment, which promotes the correct folding and co-expression of proteins16. Correct folding plays a key role in solubility, stability and function generation of bsAbs. Therefore, a signal sequence pelB was added to the N-terminus of the S-Fab to direct secretion to the periplasm of E. coli17. To ensure correct folding, solubility, thermal stability, and conformational stability, reducing the complexity and the size of an antibody is frequently employed16. The S-Fab format consists of one Fab and one VHH, which is expressed very well in bacterial systems likely due to the simple structure and small size.
GPC3 was chosen in this GPC3-S-Fab bispecific antibody format. Glypican-3 (GPC3) is a member of the heparin sulfate (HS) proteoglycan family that is anchored to the cell surface through glycosylphosphatidylinositol (GPI)18. GPC3 is overexpressed in 70% of hepatocellular carcinoma (HCC) cases, which account for the majority of liver cancers19,20,21,22. Because GPC3 is rarely expressed in normal tissues, GPC3 has been proposed as a potential target for HCC. Multiple mouse mAbs have been produced against GPC3. However, only GC33 exhibited limited anti-tumor activity 22, and it failed to exhibit clinical efficacy in patients. In this study, GPC3-S-Fab was shown to be able to recruit NK cells to kill GPC3 tumor cells14.
To recruit NK cells, anti-CD16 VHH was used. CD16a is a low affinity IgG receptor, expressed mainly on natural killer (NK) cells, macrophages, monocytes and some subtypes of T cells. It is involved in antibody-dependent cell cytotoxicity (ADCC) by NK cells23. Human NK cells can be categorized into two types, CD56-CD16+ and CD56+CD16-. In contrast to CD56+CD16&#8722; NK cells, CD56-CD16+ NK cells can release higher levels of perforin and granzyme B and thus present a strong cytotoxicity24. Kupffer cells (KCs), expressing CD16a, are the resident macrophages in liver. Kupffer cells play an important role in the suppression of liver cancer25. Thus, bsAbs targeting CD16a may be a more promising strategy than engaging T cells against liver cancer.

<table><tbody><tr><td>Shaking incubator</td><td>Thermo Fisher</td><td>MAXQ 4000</td><td></td></tr><tr><td>Shaking incubator</td><td>Zhicheng</td><td>ZWYR-D2402</td><td></td></tr><tr><td>Centrifuge</td><td>Cence</td><td>GL-10MD</td><td></td></tr><tr><td>Centrifuge</td><td>Beckman coulter</td><td>Avanti j-26S XPI</td><td></td></tr><tr><td>Centrifuge</td><td>eppendorf</td><td>5810R</td><td></td></tr><tr><td>Ultraviolet spectrophotometer</td><td>Thermo Fisher</td><td>Nanodrop</td><td></td></tr><tr><td>Analytical polyacrylamide gel electrophoresis apparatus</td><td>Mini-PROTEAN&amp;reg; Tetra</td><td>Bio-rad</td><td></td></tr><tr><td>Trans-blot apparatus</td><td>Criterion</td><td>Bio-rad</td><td></td></tr><tr><td>Imaging system</td><td>Bio-rad</td><td>chemidoc tm XRS+</td><td></td></tr><tr><td>Fast Protein Liquid chromatogram</td><td>GE Healthcare</td><td>AKTA avant</td><td></td></tr><tr><td>GF column</td><td>GE Healthcare</td><td>28-9909-44 Superdex 200 Increase 10/300 GL</td><td></td></tr><tr><td>Flow Cytometer</td><td>Beckman coulter</td><td>FC500</td><td></td></tr><tr><td>Centrifuge</td><td>eppendorf</td><td>5702R</td><td></td></tr><tr><td>Envision plate reader</td><td>TECAN</td><td>Infinite F50</td><td></td></tr><tr><td>Anti His-tag</td><td>eBioscience</td><td>14-6657-82</td><td></td></tr><tr><td>anti-Flag-tag</td><td>Sigma</td><td>F1804</td><td></td></tr><tr><td>anti-human(H&amp;amp;L)-488</td><td>A11013</td><td>Invitrogen</td><td></td></tr><tr><td>Anti-mouse IgG HRP-linked antibody</td><td>Cell Signaling</td><td>7076S</td><td></td></tr><tr><td>Ni-NTA-Agarose</td><td>Tribioscience</td><td>TBS9202-100</td><td></td></tr><tr><td>IgG-CH1 affinity resin</td><td>Thermo Fisher</td><td>194320005</td><td></td></tr><tr><td>Ficoll-Plaque Plus</td><td>GE Healthcare</td><td>17-1440-03</td><td></td></tr><tr><td>NK cell enrichment kit</td><td>Stemcell</td><td>19055</td><td></td></tr><tr><td>Magnet</td><td>Stemcell</td><td>18000</td><td></td></tr><tr><td>CCK8 kit</td><td>Dojindo</td><td>CK04</td><td></td></tr><tr><td>DMEM</td><td>Gibco</td><td>C11995500CP</td><td></td></tr><tr><td>RPMI-1640</td><td>Gibco</td><td>C11875500CP</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma</td><td>F2442</td><td></td></tr><tr><td>Trypsin</td><td>Gibco</td><td>15050-057</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140-122</td><td>Cell culture</td></tr><tr><td>Standard marker</td><td>Sigma Aldrich</td><td>MWGF200</td><td>Gel filtration</td></tr><tr><td>Isopropyl-b-D-thio-galactopyranoside (IPTG)</td><td>VWR chemicals</td><td>VWRC0487-100G</td><td></td></tr><tr><td>Dialysis tubing</td><td>Sigma Aldrich</td><td>D0655-100FT</td><td></td></tr><tr><td>Knanamycine</td><td>VWR</td><td>VWRC0408-100G</td><td></td></tr><tr><td>Ampicillin</td><td>VWR</td><td>VWRC0339-100G</td><td></td></tr><tr><td>Tryptone</td><td>Thermo Fisher</td><td>LP0042B</td><td></td></tr><tr><td>Yeast Extract</td><td>Thermo Fisher</td><td>LP0021B</td><td></td></tr><tr><td>NaCl</td><td>Sangon Biotech</td><td>A100241</td><td></td></tr><tr><td>Trizma base</td><td>Sigma Aldrich</td><td>T6791-1KG</td><td></td></tr><tr><td>EDTA</td><td>Sigma Aldrich</td><td>V900106</td><td></td></tr><tr><td>Glycine</td><td>aladdin</td><td>A110752-500g</td><td></td></tr><tr><td>KCl</td><td>aladdin</td><td>P112134-500g</td><td></td></tr><tr><td>MgCl</td><td>Sigma Aldrich</td><td>V900020</td><td></td></tr><tr><td>Agar</td><td>Sangon Biotech</td><td>A505255-0250</td><td></td></tr><tr><td>Potassium Phosphate, Monobasic Anhydrous (KH2PO4)</td><td>VWR</td><td>7778-77-0</td><td></td></tr><tr><td>Sodium Phosphate, Dibasic, Anhydrous (Na2HPO4)</td><td>VWR</td><td>7558-79-4</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>VWR</td><td>60-24-2</td><td></td></tr><tr><td>Phenylmethyl Sulfonyl Fluoride (PMSF)</td><td>VWR</td><td>329-98-6</td><td></td></tr><tr><td>Lysozyme</td><td>Sigma Aldrich</td><td>L6876-25G</td><td></td></tr><tr><td>Coomassie Brilliant Blue R250</td><td>VWR</td><td>VWRC0472-50G</td><td></td></tr><tr><td>Bromophenol blue</td><td>Sangon Biotech</td><td>A500922-25G</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>VWR</td><td>VWRC0332-100G</td><td></td></tr><tr><td>Glycerol</td><td>Sigma Aldrich</td><td>V900122</td><td></td></tr><tr><td>100mm x 20mm plastic dish</td><td>Corning</td><td>430167</td><td></td></tr><tr><td>25cm2 flask</td><td>Corning</td><td>430639</td><td></td></tr><tr><td>96 well cell culture cluster</td><td>Corning</td><td>3599</td><td></td></tr><tr><td>Sucrose</td><td>Sangon Biotech</td><td>A610498-0005</td><td></td></tr><tr><td>CHO</td><td>the Type Culture Collection of the Chinese Academy of Sciences</td><td>GNHa 3</td><td></td></tr><tr><td>MHCC-97H</td><td>the Type Culture Collection of the Chinese Academy of Sciences</td><td>SCSP-528</td><td></td></tr><tr><td>HepG2</td><td>the Type Culture Collection of the Chinese Academy of Sciences</td><td>TCHu 72</td><td></td></tr><tr><td>Huh7</td><td>the Type Culture Collection of the Chinese Academy of Sciences</td><td>TCHu182</td><td></td></tr><tr><td>Hep3B</td><td>the Type Culture Collection of the Chinese Academy of Sciences</td><td>TCHu106</td><td></td></tr><tr><td>NK92</td><td>ATCC</td><td>CRL2408</td><td></td></tr></tbody></table>

a gpc3-targeting bispecific antibody, gpc3-s-fab, with potent cytotoxicity

A GPC3-targeting Bispecific Antibody, GPC3-S-Fab, with Potent Cytotoxicity

This protocol describes the construction and functional studies of a bispecific antibody (bsAb), GPC3-S-Fab. bsAbs can recognize two different epitopes through their two different arms. bsAbs have been actively studied for their ability to directly recruit immune cells to kill tumor cells. Currently, the majority of bsAbs are produced in the form of recombinant proteins, either as Fc-containing bsAbs or as smaller bsAb derivatives without the Fc region. In this study, GPC3-S-Fab, an antibody fragment (Fab) based bispecific antibody, was designed by linking the Fab of anti-GPC3 antibody GC33 with an anti-CD16 single domain antibody. The GPC3-S-Fab can be expressed in Escherichia coli and purified by two affinity chromatographies. The purified GPC3-S-Fab can specifically bind to and kill GPC3 positive liver cancer cells by recruiting natural killer cells, suggesting a potential application of GPC3-S-Fab in liver cancer therapy.

GPC3-S-Fab purification
GPC3-S-Fab was purified from E. coli by a two-step affinity purification, first with Ni-NTA-agarose, followed by IgG-CH1 affinity purification. After the two-step affinity purification, GPC3-S-Fab was purified to homogeneity with the two chains close to 1:1 (Figure 2A). The presence of both VH-CH1-CD16 VHH and VL-CL polypeptides can be identified by ...

Here, we present a protocol to produce a bispecific antibody GPC3-S-Fab in Escherichia coli. The purified GPC3-S-Fab has potent cytotoxicity against GPC3 positive liver cancer cells.

This protocol describes the construction and functional studies of a bispecific antibody (bsAb), GPC3-S-Fab. bsAbs can recognize two different epitopes ...

a-gpc3-targeting-bispecific-antibody-gpc3-s-fab-with-potent

Research

JoVE Journal

Cancer Research

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

We describe a quantitative real-time in vitro cytolysis assay system to evaluate the potency of chimeric antigen receptor T cells targeting liquid and solid tumor cells. This protocol can be extended to assess other immune effector cells, as well as combination treatments.

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

This work describes the synthesis and characterization of a pomalidomide-based, bifunctional homo-PROTAC as a novel approach to induce ubiquitination and degradation of the E3 ubiquitin ligase cereblon (CRBN), the target of thalidomide analogs.

Chemical Inactivation of the E3 Ubiquitin Ligase Cereblon by Pomalidomide-based Homo-PROTACs

Development of a dual-functional conjugate of antigenic peptide and Fc-III mimetics (DCAF) is novel for the elimination of harmful antibodies. Here, we describe a detailed protocol for the synthesis of DCAF1 molecule, which can selectively block 4G2 antibody to eliminate antibody dependent enhancement effect during Dengue virus infection.

Targeted Antibody Blocking by a Dual-Functional Conjugate of Antigenic Peptide and Fc-III Mimetics (DCAF)

Here we present a protocol to study the in vivo localization of antibodies in mice tumor xenograft models.

Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody

Here, we describe a method for transducing human T cells with luciferase to facilitate in vivo tracking of bispecific antibody-induced T cell trafficking to tumors in studies to evaluate the anti-tumor efficacy and mechanism of T cell-engaging bispecific antibodies.

Tracking Bispecific Antibody-Induced T Cell Trafficking Using Luciferase-Transduced Human T Cells

This protocol presents an automated, image-based high-throughput technique to identify compounds modulating natural killer cell-mediated breast cancer cell killing in the presence of a therapeutic anti-HER-2 antibody.

High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds

Here, we present a method to identify compounds that modulate the ADCC mechanism, an important cancer cell-killing mechanism of antitumor antibodies. The cytotoxic effect of NK cells is measured in breast cancer cell spheroids in the presence of Trastuzumab. Image analysis identifies live and dead killer and target cells in spheroids.

Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery

Here, we present a method to differentiate and expand human iPSC-derived chimeric antigen receptor (CAR)-expressing natural killer cells with improved killing against various malignancies. This protocol demonstrates the differentiation and expansion of natural killer (NK) optimized iPSC-derived CAR-NK cells and the measurement of antitumor activity against various tumor cell lines.

Advances in Human Induced Pluripotent Stem Cell-Derived Chimeric Antigen Receptor-Expressing Natural Killer Cells

Here, we describe a method for creating double-emulsion droplets encapsulating one T cell and a cancer target cell to examine cell killing at the single-cell level. This method allows for dual quantification of both cytotoxic molecules and target-cell apoptosis within a large population of T cells.

Droplet-based Cytotoxicity Assay to Assess Chimeric Antigen Receptor T cells at the Single-cell Level

Learn to produce the bispecific antibody GPC3-S-Fab in Escherichia coli and purify it for cytotoxicity assays against liver cancer cells. This protocol enables targeted cancer immunotherapy research.

A GPC3-targeting Bispecific Antibody, GPC3-S-Fab, with Potent Cytotoxicity

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