Name: initial evaluation of antibody-conjugates modified with viral-derived peptides for increasing cellular accumulation and improving tumor targeting
Uploaded: 2018-03-08T12:30:00
Description: Watch this Scientific Journal Video about Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting at JoVE.com

This work was funded by the Cancer Research Society (Canada) and the CIMS. The authors thank Dr. Samia Ait-Mohand and Jean-Francois Beaudoin for assistance. Dr. Angel Lopez (University of South Australia) for mAb A14.

The in vivo animal experiments described were performed according to an approved protocol and under the ethical guidelines of the Centre Hospitalier Universitaire de Sherbrooke Ethics Committee for Animal Experiments.
1. Antibody Peptide Conjugation
NOTE: ChAcNLS can be synthesized at any commercial peptide manufacturer or university-affiliated peptide synthesis service platform. The synthesis of ChAcNLS can be found in reference 34. F...

Major goals of systemic delivery of anti-cancer agents are to increase accumulation at the tumor site, and uptake within cancer cells, and decrease unwanted side effects in healthy tissues. AC targeted delivery of molecular payloads to tumor cells is a highly promising approach to treat and detect tumors. However, the lack of efficacy caused by endosome entrapment and down stream lysosomal degradation remains an important challenge. While this protocol utilizes the ChAcNLS peptide as an example for the construction of ne...

Antibody-conjugates (ACs) are biopharmaceuticals that are maturing into a transformative class of effective drugs for improving cancer treatments and for detecting tumors. Composed of a monoclonal antibody (mAb) conjugated to molecular payloads such as radioisotopes, small molecules, and biological toxins, ACs are able to deliver these payloads to cancer cells with exquisite target antigen affinity and specificity. Thus ACs have the potential to significantly reduce nonspecific toxicity and increase payload activity at the tumor site. Therapeutically, ACs transporting cytotoxic small molecules (commonly referred to as antibody-drug conjugates) have been approved for treating patients with breast cancer and Hodgkin&#39;s lymphoma who have failed conventional treatments 1,2. In addition, ACs transporting radioisotopes (commonly referred to as radioimmunoconjugates) are also in development. An AC transporting a radioisotope for imaging is approved for identifying prostate cancer metastasis 3. With many more therapeutic ACs submitted for approval 4, optimism is high for the future of ACs to improve cancer care 5.
Nonetheless, when delivering chemotherapeutics or radioisotopes, ACs have difficulty effectively accumulating these payloads inside target cells. This aspect significantly contributes in many cases in the inability of ACs to provide long-lasting disease-free survival or high contrast tumor imaging 6,7. In general, once ACs bind their target antigen they are internalized through a process known as receptor-mediated endocytosis. The ACs are then entrapped inside endosomes and trafficked to lysosomes for degradation and payload release 8. The intracellular trafficking process poses challenges for ACs to achieve a high payload specificity and efficacy against target cancer cells. For example, many antigens such as Her2 (target for therapeutic AC Trastuzumab-emtansine) can recycle up to 85% of bound antibodies in the first 30 min 9. Furthermore, once degradation occurs, released chemotherapeutics and radioisotopes can be actively exported by increased expression and/or activity of membrane associated transport proteins 10,11. Lysosome degradation also impedes the delivery of novel biological payloads such as therapeutic enzymes and oligonucleotides that can be deactivated 12,13. In essence, the cancer cell is highly effective at abrogating the necessary intracellular accumulation of payloads delivered by ACs.
This protocol describes how to implement the concept of ACs-coupled to virus-derived peptides, specifically for escaping endosome entrapment and localizing to the cell nucleus. With such sophistication to manipulate host cell systems, it is not surprising that the development of virus-derived proteins and peptides as potential biopharmaceuticals has long been ingrained in therapeutic research 14. For millions of years viruses have evolved to acquire an exceptional collection of proteins able to exploit normal physiological mammalian cell systems in order to effectively enter host cells. For viruses that are internalized via receptor-mediated endocytosis, they are also challenged with escaping trafficking to the lysosome where the onslaught of a localized concentration of proteases can be problematic for survival. A well characterized viral-derived peptide utilized in drug delivery for escaping endosome entrapment is the human immunodeficiency virus transactivator of transcription (Tat) protein 15. Tat is able to escape endosome entrapment by sensing low-pH at which point protein conformational changes occur enabling Tat to insert itself into and disrupt the endosomal membrane 16. This results in Tat-payload conjugates able to access the cytoplasm. The second viral manipulation element related to this protocol is the approach used to deliver therapeutic genes and drugs to the nucleus 17. Viruses have evolved to successfully manipulate host cell machinery for progressing past the nuclear membrane by passing through the nuclear pore complex (NPC). Cellular macromolecules contain (or bind to proteins that contain) nuclear localization signals (NLSs) necessary for binding to nuclear transport proteins (e.g. karyopherins &#945; and &#946;), which provide the required movements through the NPC. Viruses have developed proteins to contain NLS sequences that provide them with the ability to utilize host cell transport proteins for shuttling into the nucleus 18.
Numerous ACs have previously been functionalized with Tat- and NLS-derived peptides and tested for their ability to accumulate inside cancer cells and for targeting tumors 19,20,21,22,23,24,25,26,27,28,29,30 (Table 1). Studies delivering cytotoxic payloads have demonstrated that ACs modified with virus-derived peptides are able to significantly increase cellular accumulation, cytotoxicity, and tumor killing over unmodified ACs 22,26. A common feature for this novel class of AC is their construction. Typically, peptides contain a terminal cysteine providing a free sulfhydryl group. MAbs are first reacted with a noncleavable bifunctional crosslinker containing N-hydroxysuccinimide (NHS) and maleimide groups at opposite ends. The NHS esters react with primary amines on the mAb to form amide bonds. The reacted mAb with free maleimide groups is then reacted with the sulfhydryl groups on the peptides to form a thioester bond and thus linking the peptide and mAb. Although homobifunctional crosslinkers have been used 28, heterobifunctional crosslinker are more commonly used in the construction of virus-derived peptide-ACs 22,23,26,31,32. This protocol specifically uses the crosslinker sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) for its ease of use and because it is used in the approved antibody-drug conjugate Trastuzumab-emtansine and in many virus-derived peptide-ACs 8,22,23,26,31,32. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is the primary method for initially determining conjugation efficiency and for semi-quantifying the number of peptides per mAb. Confocal microscopy using a fluorescently-labeled secondary antibody specific to the mAb is typically the method for initially evaluating intracellular distribution properties of virus-derived peptide-modified ACs. Thus far, radioisotopes are the primary payloads delivered by virus-derived peptide-modified ACs. Radioisotopes are advantageous because radioactivity in cells is easily quantified by gamma counting. In addition, ACs that are translated into mouse models of human cancers provide researchers with the ability to evaluate tumor targeting using molecular imaging modalities such as single photon emission computed tomography and positron emission tomography (PET) 23,32,33. In general, the construction and validation testing methods primarily used by researchers provide a very good assessment of ACs modified with virus-derived peptides during the initial development stage to effectively enter and deliver the payload inside target cells and to target tumors.
Tat- and NLS-modified ACs have illuminated key areas for further improving payload delivery inside cancer cells and to tumors. With respect to NLS-modified ACs, the efficiency in intracellular accumulation can be modest 23,31,34. Inefficient intracellular accumulation is caused by continued endosomal entrapment. In vivo tumor targeting can also be diminished with both Tat- and NLS-modified ACs. The active sequences of Tat and NLS contain several positive charged residues. When attached to mAbs, the overall cationic charge can be significantly increased 35. As a consequence, the Tat- and NLS-modified ACs have increased uptake in healthy tissues and increased rapid blood clearance.
Our group developed a composite compound consisting of cholic acid linked to NLS (ChAcNLS; Figure 1). ChAcNLS-modified ACs are able to increase intracellular accumulation of delivered radioisotopes and improve tumor targeting compared to NLS-modified and traditional ACs 33,34. The mechanism behind cholic acid is inspired by the ability of select nonenveloped viruses that cannot rely on membrane fusion to utilize cholic acid to trigger endosome escape through the formation of ceramide. For example, porcine enteric virus recruits cholic acid that activates sphingomyelinase, which catalyzes the hydrolysis of sphingomyelin into ceramide 36,37,38. This destabilizes endosomal membrane and allows for virus escape. Thus, cholic acid is another virus-derived component that complements NLS.
As this field moves forward and future advancements occur in payload delivery by ACs modified with virus-derived peptides, it is an opportune time to provide visual demonstrations of their biochemical and functional characteristics during initial development. Here, we describe our protocol for the initial evaluation of virus-derived peptide-modified ACs for the efficient yet simple determination of intracellular accumulation and tumor targeting during early stage development. We use the commercially available mAbs 7G3 and A14 as example model systems. Procedure 1 describes the use of SDS-PAGE as a method that allows for &#8216;go/no go&#8217; decisions for constructed ACs. Procedure 2 describes a method using trypsinization allowing for improved visualization of AC intracellular distribution and accumulation. Procedure 3 describes a method for improved intracellular fractionation to accurately determine nuclear localization. In this procedure we utilize the payload 64Cu (t1/2 = 12.7 h) because it is vulnerable to cellular efflux and is a positron emitter 10. Thus, Procedure 4 describes in vivo tumor targeting characterization by PET imaging to visualize tumor uptake relative to background (i.e. nontarget healthy tissues) and determine whether the example AC can specifically and effectively target tumors. These methods are sufficient for investigators developing ACs modified with virus-derived peptides to identify candidates for further advancement.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sulfo-SMCC</td>
			<td>Thermo Scientific</td>
			<td>22122</td>
			<td>There are many homo- and hetero-bifunctional maleimide crosslinkers to choose from.</td>
		</tr>
		<tr>
			<td>Amicon Ultra-0.5 mL Centrifugal Filters</td>
			<td>EMD Millipore</td>
			<td>UFC505096</td>
			<td>There are pack sizes of 8, 24, and 96. Choose according to your needs.</td>
		</tr>
		<tr>
			<td>Precision Plus Protein Kaleidoscope Standards</td>
			<td>BioRad</td>
			<td>1610375EDU</td>
			<td>Mulicolor recombinant proteins from 10-250 kDa.</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Thermo Scientific</td>
			<td>25200056</td>
			<td>100 or 500 mL volumes to choose from.</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 647 conjugate</td>
			<td>Thermo Scientific</td>
			<td>A-21235</td>
			<td>1-10 &#956;g/mL recommended</td>
		</tr>
		<tr>
			<td>NOTA-NHS</td>
			<td>CheMatech</td>
			<td>C100</td>
			<td></td>
		</tr>
		<tr>
			<td>Lamin A/C antibody (N-18)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-6215</td>
			<td></td>
		</tr>
		<tr>
			<td>Rab7 antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-376362</td>
			<td></td>
		</tr>
		<tr>
			<td>A14 mAb</td>
			<td>BD Biosciences</td>
			<td>555902</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE LDS Sample Buffer (4x)</td>
			<td>Thermo Scientific</td>
			<td>NP0007</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma Aldrich</td>
			<td>M3148-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>TF-1a cells</td>
			<td>ATCC</td>
			<td>ATCC CRL-2003</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>ATCC</td>
			<td>ATCC 30-2001</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA lysis and extraction buffer</td>
			<td>Thermo Scientific</td>
			<td>89900</td>
			<td></td>
		</tr>
		<tr>
			<td>AMIDE medical imaging software</td>
			<td>available at amide.sourceforge.net</td>
			<td></td>
			<td>Completely free download</td>
		</tr>
		<tr>
			<td>FluoView FV1000 Confocal Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoview Software</td>
			<td>Olympus</td>
			<td></td>
			<td>www.olympus-lifescience.com</td>
		</tr>
		<tr>
			<td>ITLC strips</td>
			<td>Biodex</td>
			<td>150-771</td>
			<td></td>
		</tr>
	</tbody>
</table>

initial evaluation of antibody-conjugates modified with viral-derived peptides for increasing cellular accumulation and improving tumor targeting

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used in cancer treatment and imaging due to increased cellular accumulation over current ACs. During early AC in vitro development, fluorescence techniques and radioimmunoassays are sufficient for determining intracellular localization, accumulation efficiency, and target cell specificity. Currently, there is no consensus on standardized methods for preparing cells for evaluating AC intracellular accumulation and localization. The initial testing of ACs modified with virus-derived peptides is critical especially if several candidates have been constructed. Determining intracellular accumulation by fluorescence can be affected by background signal from ACs at the cell surface and complicate the interpretation of accumulation. For radioimmunoassays, typically treated cells are fractionated and the radioactivity in different cell compartments measured. However, cell lysis varies from cell to cell and often nuclear and cytoplasmic compartments are not adequately isolated. This can produce misleading data on payload delivery properties. The intravenous injection of radiolabeled virus-derived peptide-modified ACs in tumor bearing mice followed by radionuclide imaging is a powerful method for determining tumor targeting and payload delivery properties at the in vivo phase of development. However, this is a relatively recent advancement and few groups have evaluated virus-derived peptide-modified ACs in this manner. We describe the processing of treated cells to more accurately evaluate virus-derived peptide-modified AC accumulation when using confocal microscopy and radioimmunoassays. Specifically, a method for trypsinizing cells to remove cell surface bound ACs. We also provide a method for improving cellular fractionation. Lastly, this protocol provides an in vivo method using positron emission tomography (PET) for evaluating initial tumor targeting properties in tumor-bearing mice. We use the radioisotope 64Cu (t1/2 = 12.7 h) as an example payload in this protocol.

For Procedure 1, the construction of 7G3 modified with ChAcNLS using sulfo-SMCC as a crosslinker is very reliable. Typically, when loaded onto a 12% gel and analyzed by SDS-PAGE, this results in distinguishable stepwise increases in MW proportional to increasing sulfo-SMCC-to-7G3 ratios used and allows for the heavy and light chains to be individually assessed for ChAcNLS conjugation (Figure 3). 7G3 reacted at 10-, 20-, 25-, and 50-to-1 sulfo-SMCC-to-7G3 ratios followed b...

Watch this Scientific Journal Video about Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting at JoVE.com

Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

Viral-derived peptides coupled to antibody-conjugates (ACs) is an approach gaining momentum due to the potential of delivering molecular payloads with increased tumor cell accumulation. Utilizing common methods to evaluate peptide conjugation, AC and payload intracellular accumulation, and tumor targeting, this protocol helps researchers during the key initial development phases.

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used ...

Research

JoVE Journal

Bioengineering

Increasing cDNA Yields from Single-cell Quantities of mRNA in Standard Laboratory Reverse Transcriptase Reactions using Acoustic Microstreaming

Correlating gene expression with cell behavior is ideally done at the single-cell level.  However, this is not easily achieved because the small amount of ...

Hydrophobic Salt-modified Nafion for Enzyme Immobilization and Stabilization

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel ...

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular ...

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...

Synthesis of Multi-walled Carbon Nanotubes Modified with Silver Nanoparticles and Evaluation of Their Antibacterial Activities and Cytotoxic Properties

In this study, multi-walled carbon nanotubes (MWCNTs) were treated with an aqueous sulfuric acid solution to form an oxygen-based functional group. Silver ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...