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>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonclinical+evaluation+of+GMA161--an+antihuman+CD16+(FcgammaRIII)+monoclonal+antibody+for+treatment+of+autoimmune+disorders+in+CD16+transgenic+mice.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ-specific+phenotypic+and+functional+features+of+NK+cells+in+humans.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+CD16a,+the+surface+receptor+of+Kupffer+cells,+on+the+growth+of+hepatocellular+carcinoma+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+native+polyacrylamide+gel+electrophoresis+(BN-PAGE)+for+analysis+of+multiprotein+complexes+from+cellular+lysates.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pouring+and+running+a+protein+gel+by+reusing+commercial+cassettes.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+protocols+for+surface+and+intracellular+antigen+analyses+of+neural+cell+types.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutically+targeting+glypican-3+via+a+conformation-specific+single-domain+antibody+in+hepatocellular+carcinoma.">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&#174; 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&#38;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><a name="RANGE!A50">Coomassie Brilliant Blue R250</a></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

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 ...

Watch this Scientific Journal Video about A GPC3-targeting Bispecific Antibody, GPC3-S-Fab, with Potent Cytotoxicity at JoVE.com

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

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

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Cancer Research

8.5K Views.  Sun Yat-Sen University. 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.

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

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

We describe a method for stable labeling of patient-derived xenografts (PDXs) with lentiviral particles expressing green-fluorescent protein and luciferase reporters. This method allows for tracking the growth of PDXs at the primary site, as well as detecting spontaneous and experimental metastases using in vivo imaging systems.

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy against cancer. Here we provide a protocol to identify a rational combination at the preclinical step. First, we describe a cell-based assay to assess the synergism between anticancer mAb and cytotoxic drugs, that uses the combination index equation of Chou and Talalay1. This includes the measurement of tumor cell drug- and antibody-sensitivity using an MTT assay, followed by an automated computer analysis to calculate the combination index (CI) values. CI values of &lt;1 indicate synergism between tested mAbs and cytotoxic agents1. To corroborate the in vitro findings in vivo, we further describe a method to assess the combination regimen efficacy in a xenograft tumor model. In this model, the combined regimen significantly delays tumor growth, which results in a significant extended survival in comparison to single-agent controls. Importantly, the in vivo experimentation reveals that the combination regimen is well tolerated. This protocol allows the effective evaluation of anticancer drug combinations in preclinical models and the identification of rational combination to evaluate in clinical trials.

This protocol describes how to assess synergism between an anticancer antibody and antineoplastic drugs in preclinical models by using the combination index equation of Chou and Talalay.

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy ...

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in tumorigenesis, can be directed to cancer biomarkers enabling tumor detection and characterization, and can be used for cancer therapy as mAbs or antibody-drug conjugates to activate immune effector cells, to inhibit signaling pathways, or directly kill cells carrying the specific antigen. Despite clinical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be impaired by the complexity and heterogeneity of the tumor microenvironment. Thus, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate with the living tumor microenvironment. Here, we describe In Vivo Immunofluorescence Localization (IVIL) as a new approach to study interactions of antibody-based therapeutics and diagnostics in the in vivo physiological and pathological conditions. In this technique, a therapeutic or diagnostic antigen-specific antibody is intravenously injected in&#160;vivo and localized ex vivo with a secondary antibody in isolated tumors. IVIL, therefore, reflects the in vivo biodistribution of antibody-based drugs and targeting agents. Two IVIL applications are described assessing the biodistribution and accessibility of antibody-based contrast agents for molecular imaging of breast cancer. This protocol will allow future users to adapt the IVIL method for their own antibody-based research applications.

The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining methods.

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few decades, the field of antibody-drug conjugates, bispecific antibodies, chimeric antigen receptors (CAR) and cancer immunotherapy has emerged as the most promising area of basic and therapeutic investigations. With numerous successful human trials targeting immune checkpoint receptors and CAR-T cells in leukemia and melanoma at a breakthrough pace, it is highly exciting times for oncologic therapeutics derived from variations of antibody engineering. Regrettably, a significantly large numbers of antibody and CAR based therapeutics have also proven disappointing in human trials of solid cancers because of the limited availability of immune effector cells in the tumor bed. Importantly, nonspecific distribution of therapeutic antibodies in tissues other than tumors also contribute to the lack of clinical efficacy, associated toxicity and clinical failure. As faithful translation of preclinical studies into human clinical trails are highly relied on mice tumor xenograft efficacy and safety studies, here we highlight a method to test the tumor and general tissue distribution of therapeutic antibodies. This is achieved by labeling the protein-A purified antibody with near Infrared fluorescent dye followed by live imaging of tumor bearing mice.

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

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few ...

An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes more complex, the goal of targeting mitochondria using various compounds that inflict mitochondrial damage (so-called mitocans) is becoming quite popular. Unfortunately, many existing cytotoxicity assays, such as those based on tetrazolium salts or resazurin require functional mitochondrial enzymes for their performance. The damage inflicted by compounds that target mitochondria often compromises the accuracy of these assays. Here, we describe a modified protocol based on differential staining with two fluorescent dyes, one of which is cell-permeant (Hoechst 33342) and the other of which is not (propidium iodide). The difference in staining allows living and dead cells to be discriminated. The assay is amenable to automated microscopy and image analysis, which increases throughput and reduces bias. This also allows the assay to be used in high-throughput fashion using 96-well plates, making it a viable option for drug discovery efforts, particularly when the drugs in question have some level of mitotoxicity. Importantly, results obtained by Hoechst/PI staining assay show increased consistency, both with trypan blue exclusion results and between biological replicates when the assay is compared to other methods.

The protocol describes a rapid, high-throughput, reliable, inexpensive, and unbiased assay for efficiently determining cellular viability. This assay is particularly useful when cells&#39; mitochondria have been damaged, which interferes with other assays. The assay uses automated counting of cells stained with two nuclear dyes &#8211; Hoechst 33342 and propidium iodide.

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes ...

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach in&#160;vaccine design. Antigen is delivered to DC via antibodies specific for endocytosis receptors such as DEC-205 that induce uptake, processing, and MHC class I- and II-presentation.
Efficient and reliable conjugation of the desired antigen to a suitable antibody is a critical step in DC targeting and among other factors depends on the format of the antigen. Chemical conjugation of&#160;full-length protein to purified antibodies is one possible strategy. In the past, we have successfully established cross-linking of the model antigen ovalbumin (OVA) and a DEC-205-specific IgG2a antibody (&#945;DEC-205)&#160;for in vivo DC targeting studies in mice. The first step of the protocol is the purification of the antibody from the supernatant of the NLDC (non-lymphoid dendritic cells)-145 hybridoma by affinity chromatography. The purified antibody is activated for chemical conjugation by sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate) while at the same time the sulfhydryl-groups of the OVA protein are exposed through incubation with TCEP-HCl (tris (2-carboxyethyl) phosphine hydrochloride). Excess TCEP-HCl and sulfo-SMCC are removed and the antigen is mixed with the activated antibody for overnight coupling. The resulting &#945;DEC-205/OVA conjugate is concentrated and freed from unbound OVA. Successful conjugation of OVA to &#945;DEC-205 is verified by western blot analysis and enzyme-linked immunosorbent assay (ELISA).
We have successfully used chemically crosslinked &#945;DEC-205/OVA to induce cytotoxic T cell responses in the liver and to compare different adjuvants for their potential in inducing humoral and cellular immunity following in vivo targeting of DEC-205+ DC. Beyond that, such chemically coupled antibody/antigen conjugates offer valuable tools for the efficient induction of vaccine responses to tumor antigens and have been proven to be superior to classical immunization approaches regarding the prevention and therapy of various types of tumors.

Chemical Conjugation of a Purified DEC-205-Directed Antibody with Full-Length Protein for Targeting Mouse Dendritic Cells In Vitro and In Vivo

We describe a protocol for the chemical conjugation of the model antigen ovalbumin to an endocytosis receptor-specific antibody for in vivo dendritic cell targeting. The protocol includes purification of the antibody, chemical conjugation of the antigen, as well as purification of the conjugate and the verification of efficient conjugation.

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach ...

Tumor Allotransplantation in Drosophila melanogaster with a Programmable Auto-Nanoliter Injector

This protocol describes the allotransplantation of tumors in Drosophila melanogaster using an auto-nanoliter injection apparatus. With the use of an autoinjector apparatus, trained operators can achieve more efficient and consistent transplantation results compared to those obtained using a manual injector. Here, we cover topics in a chronological fashion: from the crossing of Drosophila lines, to the induction and dissection of the primary tumor, transplantation of the primary tumor into a new adult host and continued generational transplantation of the tumor for extended studies. As a demonstration, here we use Notch intracellular domain (NICD) overexpression induced salivary gland imaginal ring tumors for generational transplantation. These tumors can first be reliably induced in a transition-zone microenvironment within larval salivary gland imaginal rings, then allografted and cultured in vivo to study continued tumor growth, evolution, and metastasis. This allotransplantation method can be useful in potential drug screening programs, as well as for studying tumor-host interactions.

Tumor Allotransplantation in Drosophila melanogaster with a Programmable Auto-Nanoliter Injector

This protocol provides detailed guidance for the initial and continued generational allotransplantation of Drosophila tumors into the abdomen of adult hosts for studying various aspects of neoplasia. Using an autoinjector apparatus, researchers can achieve improved efficiency and tumor yields compared to those achieved by traditional, manual methods.

This protocol describes the allotransplantation of tumors in Drosophila melanogaster using an auto-nanoliter injection apparatus. With the use of an ...

Robot Assisted Distal Pancreatectomy with Celiac Axis Resection (DP-CAR) for Pancreatic Cancer: Surgical Planning and Technique

Malignant pancreatic tumors involving the celiac artery can be resected with a distal pancreatectomy, splenectomy and celiac axis resection (DP-CAR), relying on collateral flow to the liver through the gastroduodenal artery (GDA). In the current manuscript, the technical conduct of robotic DP-CAR is outlined. The greater curve of the stomach is mobilized with care to avoid sacrificing the gastroepiploic vessels. The stomach and liver are retracted cephalad to facilitate dissection of the porta hepatis. The hepatic artery (HA) is dissected and encircled with a vessel loop. The gastroduodenal artery (GDA) is carefully preserved. The common HA is clamped and triphasic flow in the proper HA via the GDA is confirmed using intra-operative ultrasound. A retropancreatic tunnel is made over the superior mesenteric vein (SMV). The pancreas is divided with an endovascular stapler at the neck. The inferior mesenteric vein (IMV) and splenic vein are ligated. The HA is stapled proximal to the GDA. The entire specimen is retracted laterally with further dissection cephalad to expose the superior mesenteric artery (SMA). The SMA is then traced back to the aorta. The dissection continues cephalad along the aorta with the bipolar energy device used to divide the crural fibers and celiac nerve plexus. The specimen is mobilized from the patient's right to left until the origin of the celiac axis is identified and oriented towards the left. The trunk is circumferentially dissected and stapled. Additional dissection with hook cautery and the bipolar energy device fully mobilizes the pancreatic tail and spleen. The specimen is removed from the left lower quadrant extraction site and one drain is left in the resection bed. A final intra-operative ultrasound of the proper HA confirms pulsatile, triphasic flow in the artery and liver parenchyma. The stomach is inspected for evidence of ischemia. Robotic DP-CAR is safe, feasible and when used in conjunction with multi-modality therapy, offers potential for long-term survival in selected patients.

Robot Assisted Distal Pancreatectomy with Celiac Axis Resection DP-CAR for Pancreatic Cancer: Surgical Planning and Technique

We present our operative approach to robot assisted distal pancreatectomy, splenectomy, and celiac axis resection (DP-CAR), demonstrating that the procedure is safe and feasible with proper planning, patient selection, and surgeon experience.

Malignant pancreatic tumors involving the celiac artery can be resected with a distal pancreatectomy, splenectomy and celiac axis resection (DP-CAR), ...