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 ...

The overall goal of these procedures is to evaluate the specificity and effectiveness of antibody conjugates for accumulating inside target cells and tumors during the initial development stages so that they can ultimately be used in the clinic. This method can help answer key question on the answer the conjugate fields, such as the efficiency of nuclear localization and the overall intercellular accumulation. An advantage of this technique is that via PET imaging researchers can assess the effectiveness and selectivity of candidate antibody conjugates.The implications of these techniques extend towards therapy and diagnosis of cancer, because ineffective tumor-cell accumulation is a major limitation with these applications for antibody conjugates. After synthesizing a colic-acid nuclear localization sequence antibody conjugate according to the text protocol, treat target TF1A cells with 200 nanomolar 7G3 colic-acid MLS antibody conjugate. Include cells treated with modified control monoclonal antibodies.Incubate five times 10 to the sixth antigen-positive cells with the conjugates at 37 degrees Celsius for one hour. Then remove the supernatant, and use one milliliter of ice-cold PBS to wash the cells three times. Add fresh medium, and incubate the cells at 37 degrees Celsius for an additional hour.Next, add 0.5 milliliters of PBS, containing 0.25%trypsin and EDTA, and incubate the cells at 37 degrees Celsius for up to 30 minutes. Then use 1.5 milliliters of RPMI1640 medium with 10%FBS to neutralize the trypsin. Centrifuge the cells at 500 times G for five minutes, remove the supernatant, and use 0.5 milliliters of ice-cold PBS to wash the cells three times again.Add 0.5 milliliters of 1%PFA and 1%sucrose and PBS to the cells, and place them on ice for 30 minutes to fix them. Then use 0.5 milliliters of ice-cold PBS to wash the cells three times, and centrifuge the samples at 250 times G for five minutes. Next, add 0.15%Triton X-100 and PBS to the cells, and permeablize them on ice for five minutes.Then, with ice-cold PBS, wash the cells, and repeat the centrifugation. Suspend the cells in 0.1 milliliters of PBS, containing two micrograms per milliliter of an antimirroring FC secondary polyclonal antibody conjugated to Alexa Fluor 647, and incubate the samples in the dark at room temperature for one hour. Centrifuge the cells at 250 times G for five minutes, and use 0.5 milliliters of ice-cold PBS to wash the cells three times.Then suspend the cells in 0.5 milliliters of PBS. Add 10 micrograms per milliliter of propidium iodide to the cells. Then use mounting medium to mount one times 10 to the fifth cells onto glass slides before covering the sample with a glass cover slip.Examine the cells with a Plan APO 60X oil-immersion objective, numerical aperture 1.42, on an inverted laser-scanning confocal microscope. Detect PI fluorescence using the 488-nanometer argon laser and the spectral-scanning prism set for 600 to 650 nanometers. For AF 647 fluorescence, use the 633-nanometer helium-neon laser and the spectral-scanning prism set for 650 to 700 nanometers.Collect images from PI and AF 647 sequentially, 1024 by 1024 pixels, with 2X line averaging, taken at 0.5-micron intervals through the entire cell thickness. Present images as Z projections. To analyze the cells, evaluate and record whether intracellular fluorescence in the cytoplasm is grouped and near the cell surface or diffuse and homogeneous.Also evaluate the relative fluorescence intensity per cell. After preparing and concentrating radiolabeled colic-acid NLS antibody conjugate according to the text protocol, determine the radiolabeling efficiency by applying 0.5 microliters of 0.1-molar PH5.5 sodium citrate, or ITLC eluent, to an ITLC strip. Form an autoradiograph of the strip, and obtain a digital image.Carry out densitometry to obtain the proportion of bound and free Copper-64. If free Copper-64 content is greater than 5%concentrate the sample further. For Copper-64 cellular accumulation studies, treat cells with 100 nanomolar of Copper-64-labeled A14 conjugates at 37 degrees Celsius for one, six, and 24 hours, as previously described.Treat the cells with unmodified A14 to block IL5R alphacytes and at four degrees Celsius to block receptor-mediated internalization. After washing the cells and incubating with fresh medium, as demonstrated earlier in this video, add 0.5 milliliters of PBS containing 0.25%trypsin and EDTA, and incubate at 37 degrees Celsius for 15 minutes. Then, after neutralizing and washing the cells as before, add plasma-membrane lysis buffer to the cells, and incubate them on ice for 10 minutes.Centrifuge the plasma-membrane-lysed cells at 90 times G for five minutes. Remove the supernatant, and transfer it to a fresh tube. This represents the cytoplasmic fraction.Then use ice-cold PBS to wash the nuclei three times, and add the washes to the cytoplasmic fraction. Ensure vials containing the nuclear and cytoplasmic fractions are sealed, then place them in a gamma counter calibrated for Copper-64 in order to convert raw counts to megabecaril. Determine the quality of nuclei isolation by performing Western-Blot analysis for lamin AC, a restricted nuclear protein;and Rab-7, an abundant cytoplasmic protein, on wholesale lysate, nuclear, and cytoplasmic fractions.Treat five times 10 to the six cells with 500 microliters of radio-amino-precipitation assay, or RIPA buffer, and plasma-membrane lysis buffer containing 1%2%and 4%NP-40. Furthermore, treat the isolated nuclei with 500 microliters of RIPA buffer to obtain isolated nuclear proteins. Add four times the sample volume of cold acetone to the isolated nuclear, cytoplasmic, and wholesale proteins, and incubate for 60 minutes at minus 20 degrees Celsius.Centrifuge the samples at 13, 000 times G for 10 minutes. Decant the supernatant, being careful not to dislodge the protein pellet. Then add 100 microliters of PBS to dissolve the proteins.After performing Western Blotting as previously described, cut the membrane in half at the 40-kilodalton molecular-weight marker. Use lamin-AC-specific antibodies to probe the blot containing the higher-molecular-weight proteins, and use Rab-7-specific antibodies to probe the lower half of the membrane. In groups containing four nodskit mice, inject into the tail vein 20 to 30 micrograms of radiolabeled A14 colic-acid NLS antibody conjugate, radiolabeled A14 NLS, and radiolabeled A14.On the same day, place a cylindrical phantom containing five megabecaril of Copper-64 into the PET scanner to convert the radioactive counts per second into injected dose per gram of tissue. 48 hours later, after anesthetizing the mice according to the text protocol, rapidly transfer a mouse to a PET scanner table in the headfirst prone position with a nosecone for isoflurane. Start the PET data acquisition using a regular sampling-mode setting and an energy window of 250 to 650 kiloelectron volts.After the scan, remove the mouse from the scanner, and place it back in the cage. As shown in this figure, the arrows show the difference when evaluating antibody conjugate accumulation with and without trypsinization. In cells that are not trypsinized, intracellular accumulation and distribution is difficult to evaluate due to the over expression of the target cell's surface receptors.The arrows show the difference in intracellular accumulation and distribution of two antibody conjugate candidates when cells are properly trypsinized. Radiolabeled A14 colic-acid NLS antibody conjugate shows nanomolar affinity for IL5R alpha. With increasing concentrations of radiolabeled A14 colic-acid NLS antibody conjugate, specific binding of A14 colic-acid NLS antibody conjugate approached saturation at concentrations of 3.5 nanomolar in both HT-1376 and HT-B9 cells.Gamma counting reveals that the increase of radioactivity in the nucleus and in the cytoplasm from radiolabeled A14 colic-acid NLS antibody conjugate is specific and increases over time. HT-1376 and HT-B9 cells were incubated with plasma-membrane lysis buffer containing 1%2%or 4%NP-40. For HT-1376 and HT-B9 cells, the lysis buffer containing one or 2%NP-40 Rap-7 was not detected in the nuclear fraction, and lamin AC was not detected in the cytoplasmic fraction.PET imaging allows for the evaluation of candidate antibody conjugates for their ability to target antigen-positive tumors, red and blue arrows, and their associated biodistribution properties in comparison to the parental non-peptide-modified antibody. One can see the signal accumulation in the tumor to help select on the potential for a developed antibody conjugate. PET imaging also allows for the evaluation of candidate antibody conjugate uptake in healthy tissues such as the liver.After mastering these techniques, researchers in the field of targeted tumor delivery of molecular payloads will be able to explore additional agents where tumor-cell accumulation is hindered due to endosome entrapment.

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7.5K vistas.  Université de Sherbrooke. The overall goal of these procedures is to evaluate the specificity and effectiveness of antibody conjugates for accumulating inside target cells and tumors during the initial development stages so that they can ultimately be used in the clinic. This method can help answer key question on the answer the conjugate fields, such as the efficiency of nuclear localization and the overall intercellular accumulation. An advantage of this technique is that via PET imaging researchers can assess the e...

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