Name: a gpc3-targeting bispecific antibody, gpc3-s-fab, with potent cytotoxicity
Uploaded: 2018-07-12T13:00:00
Description: Watch this Scientific Journal Video about A GPC3-targeting Bispecific Antibody, GPC3-S-Fab, with Potent Cytotoxicity at JoVE.com

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>
<...

<ol>
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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 ...

This method can help answer key questions in the bispecific antibody field, such as how to produce a bispecific antibody in bacteria and determine the function of the purified antibody. The main advantage of this technique is that GPC3-S-Fab can be easily produced and purified from E.coli. To prepare the periplasmic fraction, first centrifuge the cell suspension at 4, 000 g for 30 minutes at four degrees Celsius.Then, expel the medium, and weigh the cells. After centrifugation, dissolve the cell pellet in four milliliters of ice-cold sucrose solution for each gram of cells. Incubate the cell suspension on ice for 15 minutes.After 15 minutes, centrifuge the cell suspension at 8, 500 g for 20 minutes at four degrees Celsius. Then, pipette out the supernatant containing the sucrose fraction, and store it on ice. Resuspend the cell pellet in ice-cold mixture of five-millimolar magnesium chloride and one-millimolar PMSF.Then, add 40 microliters of 15 milligrams per milliliter of the lysosome stock to the cell suspension. After adding the lysosome stock, leave the cell suspension on ice for 30 minutes. After the incubation period is over, combine the sucrose and the periplasmic fractions.Then, centrifuge the sucrose-periplasmic fraction at 30, 000 g for 30 minutes at four degrees Celsius. After centrifugation is over, transfer the supernatant in a 50-milliliter conical tube on ice. Next, mix and resuspend one milliliter of nickel-NTA agarose with the supernatant to obtain a homogeneous suspension, then one milliliter of nickel-NTA agarose to a fresh 15-milliliter conical tube.To the agarose, add 10 column volumes of the equilibration buffer. Then, centrifuge the agarose at 400 g for five minutes. Post-centrifugation, carefully remove the supernatant, and add the nickel-NTA agarose to the sucrose-periplasmic fraction.Then, rock the sucrose-periplasmic fraction-containing tube at four degrees Celsius for two hours. Again, centrifuge the mixture. Then, carefully remove the supernatant to a fresh ice-cold tube since this is the unbound fraction.After the supernatant is transferred, add the nickel-NTA agarose in the gravity column. Then, add three column volumes of elution buffer to the column. Next, collect the eluate as elution fractions one, two, three, and four, respectively.Dilute and seed 5, 000 cells in 100 microliters in each well of a 96-well plate. Incubate the plate at 37 degrees Celsius for six to eight hours for attachment. To prepare the PBMCs, first dilute the freshly prepared blood with an equal volume of phosphate-buffered saline in a 50-milliliter conical tube.Pipette 15 milliliters of lymphocyte separation medium in a fresh 50-milliliter conical tube. Next, carefully transfer the diluted blood along the walls of the tube to the lymphocyte separation medium. Centrifuge the cells at 400 g for 35 minutes at room temperature with the brakes off.Post-centrifugation, carefully draw the upper plasma layer out. Then, pipette the white blood layer containing the PBMCs. Dilute these PBMCs obtained with double the volume of phosphate-buffered saline with 2%fetal bovine serum.Then, centrifuge the PBMC suspension. Wash the cell pellet with phosphate-buffered saline containing 2%fetal bovine serum, and again centrifuge. After centrifugation, count the cell number.To prepare the natural killer cells, reconstitute the PBMCs in phosphate-buffered saline with 2%fetal bovine saline. Then, transfer the mixture in a polystyrene round-bottom tube. Add 50 microliters per milliliter of natural killer cell enrichment cocktail to the PBMC mixture.Mix the contents, and leave the tube for 10 minutes. Next, resuspend and mix the magnetic particles present in the enrichment cocktail by pipetting multiple times, such that the particles are uniformly suspended. Then, add 100 microliters per milliliter of magnetic particles to the cell suspension.Mix the cell suspension magnetic particles, and incubate at room temperature for five minutes. After five minutes, add 2.5 milliliters of phosphate-buffered saline with 2%fetal bovine serum to the cell suspension. Gently pipette two to three times to mix the cells in the tube.Place the tube on the magnet, and let the magnetic beads settle down for 2.5 minutes at room temperature. Then, in one stroke of action, invert the tube, and pour the enriched cell suspension in a new tube. Again, count the number of cells.Once the counting is over, dilute the natural killer cells in the culture medium. Then, add 100 microliters of the cell suspension to the tumor cells plated in a 96-well plate. To set up the cytotoxicity assays, add 10 microliters of varying concentrations of GPC3-S-Fab antibody to the 96-well plate.Then, incubate the plate at 37 degrees Celsius for 72 hours. After the designated incubation time, aspirate the medium. Then, add 100 microliters of DMEM medium containing 10 microliters of CCK reagent in each well of the 96-well plate.Again, incubate the plate at 37 degrees Celsius. Post-incubation, read the plate at 450 nanometers every consecutive hour until four hours after the addition of the CCK8 reagent. The GPC3-S-Fab antibody is purified using a two-step affinity purification.Here, the two panels show that the antibody is purified using the nickel-NTA agarose and the IgG-CH1 affinity purification columns, respectively. Next, the binding affinity of the GPC3-S-Fab antibody is evaluated using GPC3-positive and negative cancer cell lines. The GPC3-S-Fab binds to all the GPC3-positive cells, such as Hep3B, Huh7, Hep2G, and CHO/GPC3.Interestingly, no or minimal binding is observed when GPC3-negative cells, such as MHCC-97H and CHO, are used. The cytotoxicity of the GPC3-S-Fab is also studied in the presence of the tumor cells incubated with fresh natural killer cells. The antibody GPC3-S-Fab elicits strong cytotoxicity against HepG2, Hep3B, and Huh7 seven cells in a dose-dependent manner in the presence of the natural killer cells.However, no such cytotoxicity is observed in the absence of the natural killer cells on the MHCC-97H cells. Once mastered, this technique can be done in six hours, if it is performed properly. While attempting this procedure, it's important to remember to keep the steps on ice.Following this procedure, other methods like xenograft modeling can be performed in order to answer additional questions, like in tumor effect in vivo. After its development, this technique paved the way for researchers in the field of bispecific antibody to explore antitumor effects in vitro. After watching this video, you should have a good understanding of how to produce a functional bispecific antibody targeting the tumor cells, especially the isolation of the NK cells.

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Research

JoVE Journal

Cancer Research

Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays

Cancer patients with an aberrant regulation of the protein phosphorylation networks are often treated with the tyrosine kinase inhibitors. Response rates approaching 85% are common. Unfortunately, patients often become refractory to the treatment by altering their signal transduction pathways. An implementation of the expression profiling with microarrays can identify the overall mRNA-level changes, and proteomics can identify the overall changes in protein levels or can identify the proteins involved, but the activity of the signal transduction pathways can only be established by interrogating post-translational modifications of the proteins. As a result, the ability to identify whether a drug treatment is successful or whether resistance arose, or the ability to characterize any alterations in the signaling pathways, is an important clinical challenge. Here, we provide a detailed explanation of antibody arrays as a tool which can identify system-wide alterations in various post-translational modifications (e.g., phosphorylation). One of the advantages of using antibody arrays includes their accessibility (an array does not require either an expert in proteomics or costly equipment) and speed. The availability of arrays targeting a combination of post-translational modifications is the primary limitation. In addition, unbiased approaches (phosphoproteomics) may be more suitable for the novel discovery, whereas antibody arrays are ideal for the most widely characterized targets.

Here, we present a protocol for antibody arrays to identify alterations in signaling pathways in various cellular models. These changes, caused by drugs/hypoxia/ultra-violet light/radiation, or by overexpression/downregulation/knockouts, are important for various disease models and can indicate whether a therapy will be effective or can identify mechanisms of drugs resistance.

Cancer patients with an aberrant regulation of the protein phosphorylation networks are often treated with the tyrosine kinase inhibitors. Response rates ...

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 ...

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been used in tumor vaccines to activate innate immunity and enhance antitumor responses. This protocol demonstrates a therapeutic model using cell-based tumor vaccine consisting of Flt3L-expressing B16-F10 melanoma cells along with phenotypic and functional analysis of immune cells in the tumor microenvironment (TME). Procedures for cultured tumor cell preparation, tumor implantation, cell irradiation, tumor size measurement, intratumoral immune cell isolation, and flow cytometry analysis are described. The overall goal of this protocol is to provide a preclinical solid tumor immunotherapy model, and a research platform to study the relationship between tumor cells and infiltrating immune cells. The immunotherapy protocol described here can be combined with other therapeutic modalities, such as immune checkpoint blockade (anti-CTLA-4, anti-PD-1, anti-PD-L1 antibodies) or chemotherapy in order to improve the cancer therapeutic effect of melanoma.

The protocol presents a cancer immunotherapy model using cell-based tumor vaccination with Flt3L-expressing B16-F10 melanoma. This protocol demonstrates the procedures, including preparation of cultured tumor cells, tumor implantation, cell irradiation, measurement of tumor growth, isolation of intratumoral immune cells, and flow cytometry analysis.

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been ...

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

T cell-engaging bispecific antibodies (T-BsAbs) are in various stages of preclinical development and clinical testing for solid tumors. Factors such as valency, spatial arrangement, interdomain distance, and Fc mutations affect the anti-tumor efficacy of these therapies, commonly by influencing the homing of T cells to tumors, which remains a major challenge. Here, we describe a method to transduce activated human T cells with luciferase, allowing in vivo tracking of T cells during T-BsAb therapy studies. The ability of T-BsAbs to redirect T cells to tumors can be quantitatively evaluated at multiple time points during treatment, allowing researchers to correlate the anti-tumor efficacy of T-BsAbs and other interventions with the persistence of T cells in tumors. This method alleviates the need to sacrifice animals during treatment to histologically assess T cell infiltration and can be repeated at multiple time points to determine the kinetics of T cell trafficking during and after treatment.

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.

T cell-engaging bispecific antibodies (T-BsAbs) are in various stages of preclinical development and clinical testing for solid tumors. Factors such as ...