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. For Procedures 1 and 2 use the mAb 7G3, which is specific for the leukemia antigen IL-3R&#945; 39.
<ol>
	<li>In a 1.7 mL microcentrifuge tube, prepare a 10 mg/mL (~1 mg total antibody) solution of 7G3 in phosphate buffered saline (PBS), pH 7.6.</li>
	<li>Dissolve sulfo-SMCC in dimethyl sulfoxide (DMSO) at a concentration of 5-10 mM. Vortexing the solution thoroughly works best in order to achieve complete dissolution.</li>
	<li>Add to the tube containing 7G3 the dissolved sulfo-SMCC solution (typically between 5-20 &#956;L depending on the molar ratio of sulfo-SMCC-to-7G3 desired). Testing sulfo-SMCC-to-7G3 ratios of 10-, 25-, and 50-to-1 is recommended. Incubate at room temperature for 1 h.</li>
	<li>Purify and concentrate the maleimide-derivatized 7G3 by using an ultrafiltration device and buffer exchange in PBS, pH 7.0. Centrifuge at 8,000 x g for approximately 5 min. Discard flow through and fill with PBS, pH 7.0 and centrifuge again.
	<ol>
		<li>Perform this 7-8 times to completely exchange the buffer. Recover the concentrated 7G3 protein by placing the filter device upside down in a clean microcentrifuge tube. Centrifuge for 2 min at 1,000 x g. Recovery volume should be approximately 0.3 mL</li>
	</ol>
	</li>
	<li>In the tube containing the maleimide-activated 7G3, add 2-fold molar excess of ChAcNLS relative to the sulfo-SMCC-to-7G3 ratio used in the previous step and incubate overnight at 4 &#176;C. For example, if a 10-to-1 sulfo-SMCC-to-7G3 ratio was used, then use a 20-to-1 ChAcNLS-to-7G3 ratio. Total volume should not be significantly changed. 
	NOTE: Although ChAcNLS does not cause precipitation during the conjugation process this is a possibility that could occur with other peptides. Observe the tube during the reaction for signs of precipitation, which is most likely caused when peptides are hydrophobic. In these cases it may be worth revisiting the peptide of interest in order to design changes to provide increased hydrophilicity.</li>
	<li>Concentrate the 7G3-ChAcNLS using an ultrafiltration device to desired concentration and buffer exchange in PBS pH 7.4 (see step 1.4). The recovery yield of total protein should be &#8805; 75% of the original starting material.</li>
	<li>Perform SDS-PAGE to evaluate the constructed 7G3-ChAcNLS candidates using a 12% polyacrylamide gel (provides sufficient separation of the ChAcNLS conjugated heavy and light chains from nonconjugated chains). Mix 10 &#956;g of 7G3-ChAcNLS in PAGE loading buffer containing 2 &#956;L &#946;-mercaptoethanol and load into well.</li>
	<li>Set the appropriate voltage (120 V for 1 h for a 12% gel) and run the electrophoresis. Stop the electrophoresis when the Coomassie dye front reaches the bottom of the gel. 
	NOTE: Do not let the Coomassie dye front run off the gel.</li>
	<li>Remove gel from electrophoresis apparatus and rinse with destaining solution containing 10% v/v of glacial acetic acid and 20% v/v methanol.</li>
	<li>Take a photo of the gel and with a ruler and measure the distance (cm) migrated for the standards and 7G3 conjugates. Calculate the retention factor (Rf) value, which is the distance migrated divided by the gel length (from where protein is loaded to the Coomassie migration front). Plot the molecular weight (MW) in log against the Rf values from each protein standard and extrapolate the size of the 7G3 conjugates.</li>
	<li>Calculate the number of peptides per antibody by dividing the difference in MW between 7G3 conjugates and unmodified 7G3 by 1768.5 g/mol (MW of ChAcNLS).</li>
	<li>Take digital images of the Coomassie-stained gel and perform densitometry analysis. At this point, selection of a lead candidate can be based on the AC with the maximum number of ChAcNLS molecules that does not contain significant aggregate species.</li>
</ol>
2. Confocal Microscopy for Intracellular Accumulation Evaluation
NOTE: It is important to test the cell selectivity of intracellular accumulation of the peptide-modified mAbs prior to developing formulations with a payload of interest. Because Tat has also been shown to have a propensity for nonspecific cell penetration, it is worth first analyzing intracellular accumulation and cell selectivity prior to undertaking costly development steps with expensive payloads. For this reason, Procedure 1 should also modify isotype specific irrelevant control mAbs. For the rest of the protocol we will work with ACs modified with 10 ChAcNLS molecules per antibody. A schematic including key steps for Procedures 2 and 3 is described in Figure 2.
CAUTION: This step of the protocol involves the handling and manipulation of paraformaldehyde. Please follow manufacturer instructions when handling.
<ol>
	<li>Treat target TF-1a cells with 200 nM of 7G3-ChAcNLS including modified control mAbs. Incubate 5 x 106 antigen-positive cells with the conjugates for 1 h at 37 &#176;C.</li>
	<li>After 1 h, remove supernatant and wash with 1 mL 3x in ice-cold PBS. Add fresh media and incubate for an additional hour at 37 &#176;C.</li>
	<li>After the additional hour, remove supernatant and wash 3x in 1 mL ice-cold PBS. Add 0.5 mL of PBS containing 0.25% trypsin and ethylenediaminetetraacetic acid (EDTA) at 37 &#176;C for 3 up to 30 min. 
	NOTE: During initial pilot testing it is recommended to not trypsinize cells in order to determine the differences in AC intracellular accumulation. This protocol promotes the use of trypsin and we demonstrate how this improves assessment of intracellular accumulation efficiency of different AC candidates.
	<ol>
		<li>Test the different times for incubation by visually checking for all cells being detached. In addition, incubate cell aliquots with viability staining solutions and perform flow cytometric analysis as previously described 40.</li>
		<li>Neutralize trypsin with 1.5 mL of cell culture RPMI 1640 media/10% fetal bovine serum. Centrifuge cells at 500 x g for 5 min, remove supernatant, and wash 3x in 0.5 mL ice-cold PBS.</li>
	</ol>
	</li>
	<li>Fix cells in 0.5 mL PBS containing 1% paraformaldehyde and 1% sucrose on ice for 30 min. Wash cells 3x in 0.5 mL ice-cold PBS and centrifuge at 250 x g for 5 min. Permeabilize cells in 0.5 mL PBS containing 0.15% Triton X-100 for 5 min on ice. Wash cells in ice-cold PBS and repeat centrifugation.</li>
	<li>Suspend cells in 0.1 mL PBS containing 2 &#956;g/mL (or the manufacturer recommended concentration) of an anti-murine (isotype specific) Fc secondary polyclonal antibody conjugated to AlexaFluor 647 for 1 h at room temperature in the dark.
	<ol>
		<li>Centrifuge at 250 x g for 5 min and wash cells 3x in 0.5 mL ice-cold PBS. Suspend cells in 0.5 mL PBS. 
		PAUSE: Cells can be stored in refrigerator. 
		NOTE: It is essential to select a secondary antibody that recognizes the correct isotype of the mAb of interest. AF647 is selected because of its far-infrared emission, which does not interfere with propidium iodide (PI) staining of the nucleus.</li>
	</ol>
	</li>
	<li>Add PI to a concentration of 10 &#956;g/mL to the container with cells. Next, mount 1 x 105 cells onto glass slides using mounting media and cover with a glass coverslip.</li>
	<li>Examine cells with a Plan Apo 60X oil immersion objective NA 1.42 on an inverted laser scanning confocal microscope. Detect PI fluorescence using the 488 nm argon laser and the spectral scanning prism set for 600 - 650 nm. For AF647 fluorescence use the 633 nm helium-neon laser and the spectral scanning prism set for 650 - 700 nm. Collect fluorescence emissions from PI and AF647 sequentially. 
	NOTE: Use the same setting throughout the evaluation and comparison of the cells. However, you will have to adjust settings for trypsinized cells compared to non-trypsinized cells.</li>
	<li>Use microscope software to acquire images. Collect images using serial horizontal optical sections of 1,024 x 1,024 pixels with 2X line averaging taken at 0.5 &#956;m intervals through the entire cell thickness. Present the images as stacked z-projections from four consecutive slices at the maximum fluorescence intensity.</li>
	<li>Analyze cells with microscope software. Record the cellular distribution pattern of the conjugates. Specifically, evaluate 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.</li>
</ol>
3. Radiolabeled AC Construction and Cellular Fractionation for the Evaluation of 64 Cu Intracellular Delivery Efficiency
CAUTION: Procedures 3 and 4 involve the handling and manipulation of radioactivity. Before performing these steps, researchers should have approved safety training and protocols approved from their home institution&#39;s radiation safety authority.
NOTE: For Procedures 3 and 4 we use the mAb A14, which is specific for the invasive bladder cancer antigen IL-5R&#945;41. Also, all experiments are time sensitive due to the short half-life of 64Cu. In general, it is best to not wait past 1 week to perform in vitro experiments and no longer than 72 h for in vivo studies.
<ol>
	<li>In a 1.7 mL tube suspend 2,2&#39;-(7-(2-((2,5-dioxopyrrolidin-1-yl)oxy)-2-oxoethyl)-1,4,7-triazonane-1,4-diyl)diacetic acid (NOTA-NHS) in DMSO to a concentration of 10 mM.</li>
	<li>React 50-fold molar excess of NOTA-NHS with A14 (2 mg starting material) in 0.1 M sodium bicarbonate at pH 8.6 for 1 h at room temperature in a volume &#8804; 0.5 mL (the final NOTA-NHS volume should not exceed 2% v/v of total volume).</li>
	<li>Purify and buffer-exchange NOTA-A14 using an ultrafiltration device in PBS, pH 7.6 (see step 1.4).</li>
	<li>For subsequent attachment of ChAcNLS, follow steps 1.2-1.6.</li>
	<li>React NOTA-A14-ChAcNLS (250 &#956;g batches) with 100 megabecquerel (MBq) of 64CuCl2 in 0.1 M sodium bicarbonate, pH 5.5 for 1 h at 37 &#176;C &#8804; 0.1 mL. 64Cu is produced on the TR-PET cyclotron at the CIMS 42.</li>
	<li>Concentrate 64Cu-A14-ChAcNLS in PBS pH 7.4 using an ultrafiltration device to the desired concentration (see step 1.4).</li>
	<li>Determine the radiolabeling efficiency of 64Cu-A14-ChAcNLS by instant thin-layer chromatography (ITLC) using chromatography strips and 0.1 M, pH 5.5 sodium citrate (ITLC eluent) as solvent. Apply 0.5 &#956;L aliquot of the reaction solution to the ITLC strip. Perform an autoradiograph of the strip and obtain a digital image.
	<ol>
		<li>Perform densitometry to obtain the proportion of bound and free 64Cu. If free 64Cu content is &#62; 5%, return to previous step and perform additional purification.</li>
		<li>In addition, perform SDS-PAGE with Coomassie staining to evaluate aggregations. Perform a second SDS-PAGE followed by an autoradiograph of the gel to determine if there are radiolabeled aggregate species.</li>
	</ol>
	</li>
	<li>Affinity binding quality check point: Incubate 64Cu-A14 conjugates at increasing concentrations (0-100 nM) in duplicate with 1 x 106 target cells per mL. To estimate nonspecific binding, mix duplicate samples of 64Cu-A14 conjugates with cells in the presence of 100-fold molar excess of unlabeled A14.
	<ol>
		<li>After 1 h incubation on ice, wash cells and count total bound antibody samples and total nonspecific bound antibody samples in a gamma counter. Graph specific binding (total bound antibody - nonspecific bound antibody) against reactive free 64Cu-A14 (nM) used in the assay. Determine the dissociation constant (Kd) by nonlinear regression using graphing software.</li>
	</ol>
	</li>
	<li>For 64Cu cellular accumulation studies: Treat cells with 100 nM of the 64Cu-labeled A14 conjugates for 1 h, 6 h, and 24 h at 37 &#176;C as previously described33. Include additional parameters such as treatment in the presence of unmodified A14 to block IL-5R&#945; sites and at 4 &#176;C to block receptor-mediated internalization.</li>
	<li>At the defined time points, as previously described in step 2.3 - 2.3.2, add 0.5 mL of PBS containing 0.25% trypsin and EDTA at 37 &#176;C followed by neutralization and washing.</li>
	<li>Incubate cells in plasma membrane lysis buffer containing 1% NP-40, 10 mM Tris pH 7.5, 10 mM NaCl, 3 mM MgCl2 buffer on ice for 10 min. Centrifuge the plasma membrane-lysed cells at 90 x g for 5 min.</li>
	<li>Remove supernatant and place in fresh tube. This represents cytoplasmic fraction. Wash nuclei 3x in ice-cold PBS and add washes to the cytoplasmic fraction.</li>
	<li>Ensure vials containing the nuclear and cytoplasmic fractions are sealed. Then place them in a gamma counter calibrated for 64Cu in order to convert raw counts to MBq.</li>
	<li>Determine the quality of nuclei isolation by performing Western blot analysis for Lamin A/C, a restricted nuclear protein, and Rab7, an abundant cytoplasmic protein on whole cell lysate, nuclear and cytoplasmic fractions.
	<ol>
		<li>Treat 5 x 106 cells with 500 &#956;L of radio-immunoprecipitation assay (RIPA) buffer and the plasma membrane lysis buffer in step 3.12 containing 1%, 2%, and 4% NP-40. Furthermore, treat the isolated nuclei in 500 &#956;L of RIPA buffer to obtain isolated nuclear proteins.</li>
		<li>Precipitate isolated nuclear, cytoplasmic and whole cell proteins using 4x the sample volume of cold acetone for 60 min at -20 &#176;C. Centrifuge at 13,000 x g for 10 min and decant supernatant being careful not to dislodge the protein pellet. Add 100 &#956;L of PBS to dissolve proteins.</li>
		<li>Load 10 &#956;g of protein into each well of a 10% SDS-PAGE gel. Perform electrophoresis as described in 1.7-1.9. Perform Western Blot transfer as previously described in refeference 43.</li>
		<li>Identify the ladder markers that best correspond to a MW of ~40 kDa. Cut the membrane in half at this marker. Probe the blot containing the higher MW proteins with Lamin A/C-specific antibodies. Probe the membrane with the lower MW proteins with the Rab 7-specific antibodies. Western blot is a well known technique and not described in this protocol.</li>
	</ol>
	</li>
</ol>
4. PET Imaging Evaluation of Tumor Targeting
NOTE: The techniques of implanting tumor cells in mice to generate heterotopic xenografts are well known and most laboratories have in-house protocols tailored for their tumor system. Thus, this is not covered in the protocol. The xenograft model will be nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice bearing IL-5R&#945;-positive invasive bladder tumors HT-1376 and HT-B9. IL-5R&#945;-positive invasive bladder tumor cells comprise &#62; 66% of total cells in the xenograft. In contrast, only ~11% of IL-5R&#945;-positive HT-B9 cells were contained in developed xenografts41. Thus this model provides an excellent example for evaluating AC tumor targeting in two tumors that represent foreseeable patient tumor heterogeneity.
<ol>
	<li>In groups containing &#8805; 4 (NOD/SCID) mice, inject 20-30 &#956;g (6-9 MBq) of 64Cu-A14-ChAcNLS, 64Cu-A14-NLS, and 64Cu-A14 into the tail vein.</li>
	<li>On the same day place a cylindrical phantom (24.8 mL) containing 5 MBq of 64Cu to convert the radioactive counts per second into injected dose per gram of tissue (%ID/g).</li>
	<li>Wait 48 h and then anesthetize mice by placing them in an induction chamber with 1.5 L/min of oxygen flow with 2% isoflurane. Apply sterile ophthalmic ointment to mouse eyes after induction and before placement into PET scanner.</li>
	<li>Rapidly transfer the mouse to a PET scanner table in the headfirst prone position with a nose cone for isoflurane. Position the respiration and rectal probes and monitor temperature and respiration rate to ensure that physiological conditions are maintained during the experiment as previously described 44.</li>
	<li>Start the PET data acquisition using a regular sampling mode setting and an energy window of 250 - 650 keV. Typically, a 45-minute static scan per mouse is sufficient to provide sufficient coincident events for reconstruction and production of high quality PET images.</li>
	<li>After the 64Cu-AC scan, remove mouse from the scanner and place it back in the cage. Allow for the mouse to sufficiently recover before returning to the animal facility.</li>
	<li>Obtain reconstructed images using 20 iterations of a three-dimensional Maximum Likelihood Expectation Maximization algorithm.</li>
	<li>Perform region of interest (ROI) analysis of the tumor and visually assessable organs using the AMIDE software.
	<ol>
		<li>Draw ROIs to obtain quantitative %ID/g data by manually using the 3D Freehand tool setting and carefully delineate the tumors or the adjacent thigh muscle. The counts per pixel in the ROI will be converted to %ID/g from previous step 4.2.</li>
	</ol>
	</li>
	<li>Compare 64Cu-A14, 64Cu-A14-ChAcNLS, 64Cu-A14-NLS images for tumor contrast, and quantitative ROI %ID/g, and tumor-to-background ratios.</li>
</ol>

The authors have nothing to disclose

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><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 &amp;mu;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...

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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 ...

initial-evaluation-antibody-conjugates-modified-with-viral-derived

Research

JoVE Journal

Bioengineering

7.6K Views.  Université de Sherbrooke. Viral afkomstige peptiden gekoppeld aan antilichaam-geconjugeerde (ACs) is een aanpak die wint dynamiek als gevolg van het potentieel van het leveren van moleculaire payloads met verhoogde tumor cel accumulatie. Gebruik makend van gemeenschappelijke methoden voor evaluatie van peptide conjugatie, AC en lading intracellulaire accumulatie en tumor gericht op, helpt dit protocol onderzoekers tijdens de cruciale eerste ontwikkelingsfasen.

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

Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this diversity, personalized medicine is developed for silencing malignancy associated genes. Nano drugs fit these needs by targeting tumor and delivering antisense oligonucleotides for silencing of genes. As drugs for the treatment are often administered repeatedly, absence of toxicity and negligible immune response are desirable. In the example presented here, a nano medicine is synthesized from the biodegradable, non-toxic and non-immunogenic platform polymalic acid by controlled chemical ligation of antisense oligonucleotides and tumor targeting molecules. The synthesis and treatment is exemplified for human Her2-positive breast cancer using an experimental mouse model. The case can be translated towards synthesis and treatment of other tumors.

An example of a nano drug based on polymalic acid is presented towards the rational design of personalized medicine that is applicable to cancer. It describes the synthesis of a nano drug to treat Her2-positive human breast cancer in a nude mouse.

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this ...

Chemistry

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Medicine

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for safe and effective therapies tailored to individual tumor immune profiles. Oncolytic viruses enable the induction of anti-tumor immune responses as well as tumor-restricted gene expression. This protocol describes the generation and ex vivo analysis of immunomodulatory oncolytic vectors. Focusing on measles vaccine viruses encoding bispecific T cell engagers as an example, the general methodology can be adapted to other virus species and transgenes. The presented workflow includes the design, cloning, rescue, and propagation of recombinant viruses. Assays to analyze replication kinetics and lytic activity of the vector as well as functionality of the isolated immunomodulator ex vivo are included, thus facilitating the generation of novel agents for further development in preclinical models and ultimately clinical translation.

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

Cancer Research

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 ...

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 ...

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 ...

Preparation of Site-Specific Cytotoxic Protein Conjugates via Maleimide-thiol Chemistry and Sortase A-Mediated Ligation

Cancer is currently the second most common cause of death worldwide. The hallmark of cancer cells is the presence of specific marker proteins such as growth factor receptors on their surface. This feature enables development of highly selective therapeutics, the protein bioconjugates, composed of targeting proteins (antibodies or receptor ligands) connected to highly cytotoxic drugs by a specific linker. Due to very high affinity and selectivity of targeting proteins the bioconjugates recognize marker proteins on the cancer cells surface and utilize receptor-mediated endocytosis to reach the cell interior. Intracellular vesicular transport system ultimately delivers the bioconjugates to the lysosomes, where proteolysis separates free cytotoxic drugs from the proteinaceous core of the bioconjugates, triggering drug-dependent cancer cell death. Currently, there are several protein bioconjugates approved for cancer treatment and large number is under development or clinical trials.
One of the main challenges in the generation of the bioconjugates is a site-specific attachment of the cytotoxic drug to the targeting protein. Recent years have brought a tremendous progress in the development of chemical and enzymatic strategies for protein modification with cytotoxic drugs. Here we present the detailed protocols for the site-specific incorporation of cytotoxic warheads into targeting proteins using a chemical method employing maleimide-thiol chemistry and an enzymatic approach that relies on sortase A-mediated ligation. We use engineered variant of fibroblast growth factor 2 and fragment crystallizable region of human immunoglobulin G as an exemplary targeting proteins and monomethyl auristatin E and methotrexate as model cytotoxic drugs. All the described strategies allow for highly efficient generation of biologically active cytotoxic conjugates of defined molecular architecture with potential for selective treatment of diverse cancers.

Here we provide detailed protocols for a site-specific labeling of proteins with cytotoxic drugs using maleimide-thiol reaction and sortase A-mediated ligation.

Cancer is currently the second most common cause of death worldwide. The hallmark of cancer cells is the presence of specific marker proteins such as ...

Biochemistry

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

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of action of the antibodies is antibody-dependent cellular cytotoxicity (ADCC), where the antibody binds to the cancer cells and engages the cellular component of the immune system, e.g., natural killer (NK) cells, to kill the tumor cells. The effectiveness of these therapies could be improved by identifying adjuvant compounds that increase the sensitivity of the cancer cells or the potency of the immune cells. In addition, undiscovered drug interactions in cancer patients co-medicated for previous conditions or cancer-associated symptoms may determine the success of the antibody therapy; therefore, such unwanted drug interactions need to be eliminated. With these goals in mind, we created a cancer ADCC model and describe here a simple protocol to find ADCC-modulating drugs. Since 3D models such as cancer cell spheroids are superior to 2D cultures in predicting in vivo responses of tumors to anticancer therapies, spheroid co-cultures of EGFP-expressing HER2+ JIMT-1 breast cancer cells and the NK92.CD16 cell lines were set up and induced with Trastuzumab, a monoclonal antibody clinically approved against HER2-positive breast cancer. JIMT-1 spheroids were allowed to form in cell-repellent U-bottom 96-well plates. On day 3, NK cells and Trastuzumab were added. The spheroids were then stained with Annexin V-Alexa 647 to measure apoptotic cell death, which was quantitated in the peripheral zone of the spheroids with an automated microscope. The applicability of our assay to identify ADCC-modulating molecules is demonstrated by showing that Sunitinib, a receptor tyrosine kinase inhibitor approved by the FDA against metastatic cancer, almost completely abolishes ADCC. The generation of the spheroids and image acquisition and analysis pipelines are compatible with high-throughput screening for ADCC-modulating compounds in cancer cell spheroids.

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

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of ...

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of various diseases. This application relies on genetic modification, self-assembly, and cysteine conjugation to fulfill the tumor-targeting application of recombinant VLPs. Compared with genetic modification alone, chemical conjugation of foreign peptides to VLPs offers a significant advantage because it allows a variety of entities, such as synthetic peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of the VLP assembly.
Here, we demonstrate how to use the hepatitis E virus nanoparticle (HEVNP), a modularized theranostic capsule, as a multifunctional delivery carrier. Functions of HEVNPs include tissue-targeting, imaging, and therapeutic delivery. Based on the well-established structural research of HEVNP, the structurally independent and surface-exposed residues were selected for cysteine replacement as conjugation sites for maleimide-linked chemical groups via thiol-selective linkages. One particular cysteine-modified HEVNP (a Cys replacement of the asparagine at 573 aa (HEVNP-573C)) was conjugated to a breast cancer cell-specific ligand, LXY30 and labeled with near-infrared (NIR) fluorescence dye (Cy5.5), rendering the tumor-targeted HEVNPs as effective diagnostic capsules (LXY30-HEVNP-Cy5.5). Similar engineering strategies can be employed with other macromolecular complexes with well-known atomic structures to explore potential applications in theranostic delivery.

We have engineered the capsid protein of hepatitis E virus as a theranostic nanoparticle (HEVNP). HEVNP self-assembles into a stable icosahedral cage in mucosal delivery. Here, we describe the modification of HEVNPs for tumor targeting by mutating surface-exposed residues to cysteines, which conjugate synthetic ligands that specifically bind tumor cells.

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...

Cellular Affinity of Particle-Stabilized Emulsion to Boost Antigen Internalization

The cellular affinity of micro-/nanoparticles is the precondition for cellular recognition, cellular uptake, and activation, which are essential for drug delivery and immune response. The present study stemmed from the observation that the effects of charge, size, and shape of solid particles on cell affinity are usually considered, but we seldom realize the essential role of softness, dynamic restructuring phenomenon, and complex interface interaction in cellular affinity. Here, we developed poly-lactic-co-glycolic acid (PLGA) nanoparticle-stabilized Pickering emulsion (PNPE) that overcame the shortcomings of rigid forms and simulated the flexibility and fluidity of pathogens. A method was set up to test the affinity of PNPE to cell surfaces and elaborate on the subsequent internalization by immune cells. The affinity of PNPE to bio-mimetic extracellular vesicles (bEVs)-the replacement for bone marrow dendritic cells (BMDCs)-was determined using a quartz crystal microbalance with dissipation monitoring (QCM-D), which allowed real-time monitoring of cell-emulsion adhesion. Subsequently, the PNPE was used to deliver the antigen (ovalbumin, OVA) and the uptake of the antigens by BMDCs was observed using confocal laser scanning microscope (CLSM). Representative results showed that the PNPE immediately decreased frequency (&#916;F) when it encountered the bEVs, indicating rapid adhesion and high affinity of the PNPE to the BMDCs. PNPE showed significantly stronger binding to the cell membrane than PLGA microparticles (PMPs) and AddaVax adjuvant (denoted as surfactant-stabilized nano-emulsion [SSE]). Furthermore, owing to the enhanced cellular affinity to the immunocytes through dynamic curvature changes and lateral diffusions, antigen uptake was subsequently boosted compared with PMPs and SSE. This protocol provides insights for designing novel formulations with high cell affinity and efficient antigen internalization, providing a platform for the development of efficient vaccines.

To rationally design efficient adjuvants, we developed poly-lactic-co-glycolic acid nanoparticle-stabilized Pickering emulsion (PNPE). The PNPE possessed unique softness and a hydrophobic interface for potent cellular contact and offered high-content antigen loading, improving the cellular affinity of the delivery system to antigen-presenting cells and inducing efficient internalization of antigens.

The cellular affinity of micro-/nanoparticles is the precondition for cellular recognition, cellular uptake, and activation, which are essential for drug ...

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

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