We are thankful to University of Virginia Cancer Center Core Imaging Facility, Biomolecular Analysis Facility, Advanced Microscopy Facility and the Core Vivarium Facility for Assistance. J. T-S is an early career investigator of Ovarian Cancer Academy (OCA-DoD). This work was supported by NCI/NIH grant (R01CA233752) to J. T-S, U.S. DoD Breast Cancer Research Program (BCRP) breakthrough level-1 award to J. T-S (BC17097) and U.S. DoD Ovarian Cancer Research Program (OCRP) funding award (OC180412) to J. T-S

All the procedures involving animals handling and tumor xenografts studies were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) here at the University of Virginia and conform to the relevant regulatory standards
1. Expression and purification of antibodies
<ol>
	<li>Maintenance of CHO cells
	<ol>
		<li>Grow CHO cells in FreeStyle CHO Media supplemented with commercially available 1x glutamine supplement at 37 &#730;C shaking at 130 rpm with 5% CO2 using delong Erlenmeyer flasks either glass or disposable. 
		NOTE: It is highly recommended to use a baffled flask with vented cap for increased agitation and to improve gas transfer during shaking condition. Significantly reduced antibody yield has been obtained with regular flasks (non-baffled) due to limited agitation of the suspension culture.</li>
		<li>Maintain the cell number between 1-5 x 106 cells/mL with &#62;95% cell viability. If the cell number increases over 5 x 106 cells/mL, split the cells. Never allows CHO cells to reach below 0.2 x 106 cells/mL.</li>
	</ol>
	</li>
	<li>Transfection of CHO cells
	<ol>
		<li>Grow CHO cells in 200 mL of media (2 x 106 cells/mL) in delong Erlenmeyer baffled flasks. 
		NOTE: Suspension cultures grown in baffled flasks have always produced higher protein yields vs when grown in non-baffled flasks.</li>
		<li>In a 15 mL tube, take 5 mL of CHO FreeStyle media and add 50 &#956;g of VH clone DNA and 75 &#956;g of VL clone DNA. Vortex to mix well.</li>
		<li>Incubate the DNA mixture at room temperature for 5 min. 
		NOTE: Longer incubation reduces the protein yields.</li>
		<li>Add 750 &#956;L of 1 mg/mL polyethyleneimine (PEI) stock to the DNA solution and aggressively vortex the mixture for 30 s. Incubate at room temperature for additional 5 min. 
		NOTE: PEI must be made fresh. Multiple freeze thaw cycle of PEI reduces overall yield significantly.</li>
		<li>Add the entire mixture of DNA and PEI on the cells while manually shaking the flask. Immediately incubate the delong Erlenmeyer baffled flasks with cells at 37 &#730;C shaking at 130 rpm.</li>
	</ol>
	</li>
	<li>Expression 
	NOTE: The antibody has secretory signal peptide engineered to its N-terminal end, which helps antibodies to be secreted out into the media.
	<ol>
		<li>Grow the transfected cells at 37 &#730;C, with shaking at 130 rpm on the Day 1.</li>
		<li>On the Day 2, add 2 mL of 100x anti-clumping agent and 2 mL of 100x anti-bacterial-anti-mycotic solution. Shift the flask to lower temperature (32-34 &#730;C), with shaking at 130 rpm.</li>
		<li>On every fifth day, add 10 mL of Tryptone N1 feed, and 2 mL of 100x glutamine supplement.</li>
		<li>Keep counting the cells every third day using hemocytometer after staining an aliquot of cells with trypan blue stain. Ensure that the cell viability stay above 80%.</li>
		<li>On the Day 10 or 11, harvest the medium for antibody purification. Spin the culture at 3000 x g, 4 &#730;C for 40-60 min and then filter the clear media using 0.22 &#181;M bottle filters.</li>
	</ol>
	</li>
	<li>Purification 
	NOTE: Antibody purification is performed using commercially available Protein-A column (see Table of Materials), using a peristaltic pump.
	<ol>
		<li>Equilibrate the column with two column volume of binding buffer (20 mM sodium phosphate at pH 7.4).</li>
		<li>Pass the filtered media containing antibody (obtained in step 1.3.5) through the column at the flow rate of 1 mL/min.</li>
		<li>Wash the column with two-column volume of binding buffer.</li>
		<li>Elute the antibody into 500 &#956;L fractions using 5 mL elution buffer (30 mM sodium acetate at pH 3.4).</li>
		<li>Neutralize the pH of the eluted antibody by adding 10 &#956;L of neutralization buffer (3 M sodium acetate at pH 9) per fraction. 
		NOTE: Fraction number 3-6 contains most of the antibody. It is advisable to keep all the fractions in case the antibody is eluted in a later fraction than expected.</li>
		<li>Measure the concentration of purified antibody using a spectrophotometer by selecting the default protocol for IgG. The final concentration of the antibody is obtained in mg/mL by considering the molecular weight and absorption coefficient.</li>
	</ol>
	</li>
</ol>
2. Fluorescent labeling
NOTE: Antibodies are labeled with the infrared dye that contains an NHS ester reactive group, which couples to proteins and form a stable conjugate. This reaction is pH sensitive and works best at pH 8.5. Fluorescent conjugates labeled with the dye display an absorption maximum of 774 nm, and an emission maximum of 789 nm. pH 8.5 is key for effective conjugation.
<ol>
	<li>Dialyze 0.5 mL of the antibody using a dialysis cassette (0.1-0.5 mL) in 1 L of conjugation buffer (50 mM phosphate buffer at pH 8.5). After 4 h transfer the dialysis cassette to fresh buffer and dialyze overnight.</li>
	<li>Setup a conjugation reaction by adding 0.03 mg of IRDye 800CW per 1 mg of antibody in a reaction volume of 500 &#956;L. 
	NOTE: IRDye 800CW is dissolved in DMSO at a concentration of 10 mg/mL.</li>
	<li>Carry out labeling reactions for 2 h at 20 &#176;C. 
	NOTE: Increasing time beyond 2 h does not improve the labeling.</li>
	<li>Purify labeled conjugates by extensive dialysis against 1x PBS.</li>
	<li>Estimate the degree of labeling by measuring the absorbance of the dye at 780 nm and absorbance of the protein at 280 nm. The dye contribution to the 280 nm signal is 3%.</li>
	<li>Calculate the dye/protein ratio using this formula: 
	<img alt="Equation 1" src="/files/ftp_upload/60727/60727equ01.jpg" /> 
	where 0.03 is a correction factor for the absorbance of the dye used at 280 nm (equal to 3.0 % of its absorbance at 780 nm), &#949;Dye and &#949;Protein are molar extinction coefficients for the dye and the protein (antibody) respectively. 
	NOTE: &#949;Dye is 270,000 M-1 cm-1 and &#949;Protein is 203,000 M-1 cm-1 (for a typical IgG) in 1:1 mixture of PBS: methanol. Proteins other than IgG may have very different molar extinction coefficients. Use of correct extinction coefficient for the protein of interest is essential for accurate determination of D/P ratio.</li>
	<li>Calculate the final protein concentration using this formula: 
	<img alt="Equation 2" src="/files/ftp_upload/60727/60727equ02.jpg" /> 
	NOTE: Always confirm the antigen-binding efficacy of labeled and unlabeled antibody using ELISA (Enzyme Linked Immunosorbent Assay) or flow cytometry before proceeding to in vivo studies.</li>
</ol>
3. Mouse xenograft studies
<ol>
	<li>Preparation of tumor cells for injection
	<ol>
		<li>Grow OVCAR-3 cells in RPMI-1640 Medium, supplemented with 10% of FBS and 1x penicillin-streptomycin.</li>
		<li>The day before the injection, subculture cells into new 100 mm culture dishes with 10 mL of complete medium/dish. Use cell number of 0.5-1 x 106 cells/dish.</li>
		<li>Incubate cultures at 37 &#176;C temperature, 95% humidity and 5% CO2 for 20-24 h.</li>
		<li>On the day of injection, remove the growth medium from culture dishes. Thoroughly rinse the cell layer with Ca2+/Mg2+ free Dulbecco&#39;s phosphate-buffered saline (DPBS) to remove dead cells, cellular debris and all traces of serum, which may interfere in trypsin action.</li>
		<li>Trypsinize the cells by adding 1.0-1.5 mL of Trypsin-EDTA solution to each dish and try to spread the solution by tilting the dish all around followed by incubation at 37 &#176;C for 5-10 min. 
		NOTE: Observe cells under an inverted microscope to check the actual trypsinization status. Under trypsinization results in lower number of cell detachment from the surface of culture dish, over trypsinization induces cellular stress. So, proper trypsinization is important.</li>
		<li>Add 1.0-1.5 mL of complete growth medium to each dish to stop the trypsin action, after that re-suspend cells by pipetting gently. 
		NOTE: Gentle pipetting is important to maintain cell health.</li>
		<li>Collect the cell suspension into a 15 mL conical tube and spin at 250 x g for 5 min at room temperature.</li>
		<li>Collect the cell pellet after removing the supernatant and wash the cells by re-suspending the pellet in 1x DPBS.</li>
		<li>Spin the cell suspension at low speed 250 x g for 5 min.</li>
		<li>Add 500 &#181;L of DPBS and re-suspend cells by gentle pipetting to get single cell suspension.</li>
		<li>Count cells using hemocytometer or automated cell counter. 
		NOTE: For better accuracy, repeat the counting for three times and take an average.</li>
		<li>Adjust the volume in such a way so that the final cell density will be 1 x 108 cells/mL.</li>
	</ol>
	</li>
	<li>Subcutaneous injection of cells to develop mouse xenografts 
	NOTE: All procedures should be done in a BSL2 safety cabinet. Athymic Nude Foxn1nu/Foxn1+ mice have been used in the current study.
	<ol>
		<li>Take 50 &#181;L of the cell suspension into a 1.5 mL tube and mix with 50 &#181;L of basement membrane matrix medium. 
		NOTE: Basement membrane matrix medium tends to form a gel like state at room temperature, so carefully maintain the cells and the matrix medium mixture on ice. It is advisable to keep tubes, tips and syringes in the fridge and then transfer on ice prior to the animal injection.</li>
		<li>Agitate the mixture to avoid any cell clumping. Then, take this 100 &#181;L of the cell suspension-matrix medium mixture that contains 5 x 106 cells, into a 1 cc syringe.</li>
		<li>Gently lift the skin of the animal to separate the skin from the underlying muscle layer and slowly inject the cell suspension (100 &#181;L) under the skin (5 x 106 cells), with a 26 G needle. Wait for a few seconds before taking the needle out, so that basement matrix medium can form the semi-solid gel like structure along with cells under the skin, preventing the mixture coming out from the site of injection. 
		NOTE: Cells needs to be tested prior to injection for any contamination which may harm to immunodeficient mice. While injecting do not put the needle too deep into the skin as this may form the tumor deeper than expected.</li>
		<li>Keep the animal in a sterile cage and observe for around 20 min.</li>
		<li>Observe the mice for 2-3 weeks and allow the tumor to grow up to 500 mm3 size.</li>
	</ol>
	</li>
</ol>
4. Antibody localization using an in vivo imaging system
NOTE: In vivo imaging equipment (see Table of Materials) used in this experiment uses a set of high efficiency filters and spectral un-mixing algorithms for noninvasive visualization and tracking of cellular and genetic activity within a living organism in real time. System provides both fluorescence and bioluminescence monitoring capability.
<ol>
	<li>Inject 25 &#956;g of dye labeled antibody via tail vein.
	<ol>
		<li>Anesthetize tumor bearing mice using 2% isoflurane. Check for the lack of response to pedal reflexes.</li>
		<li>Once mice stop moving, dilate the lateral tail vein by applying worm water.</li>
		<li>Inject 25 &#956;g (in 100 &#956;L) of labeled antibody using 1 cc insulin syringe with a 26 G needle.</li>
		<li>Similarly, as a negative control, label and inject the non-specific IgG1 Isotype antibody which does not target cancer cells.</li>
	</ol>
	</li>
	<li>Perform in vivo live imaging after 8, 24, 48 h, etc. of antibody injections.
	<ol>
		<li>In the associated software, click Initialize located in the control panel and confirm that the stage temperature is 37 &#176;C.</li>
		<li>Turn on the oxygen supply, all the pumps on the anesthesia system, isoflurane gas supply to anesthetic chamber and set the isoflurane vaporizer valve to 2%.</li>
		<li>Transfer the mice to anesthetic chamber and wait till mice are completely anesthetized. Apply the eye lubricating ointment to avoid drying of their eyes.</li>
		<li>Go to the Control panel, set up the fluorescence imaging through the Imaging Wizard option and select the excitation at 773 nm and emission at 792 nm. 
		NOTE: The default auto exposure settings provide a good fluorescent image. However, the auto exposure preferences can be modified as per the needs.</li>
		<li>Transfer the anesthetized mice into the imaging chamber and assemble it on the imaging field using nose cone. The imaging stage provides the option to accommodate 5 mice at a time. 
		NOTE: Have control mice imaged along with the test mice to have a similar amount of exposure and other settings while analysis.</li>
		<li>Once everything is ready, select Acquire option on the control panel for the image acquisition.</li>
		<li>With&#160;Autoexposure settings&#160;the system generates the image within a minute. The generated image is the overlay of fluorescence on photographic image with optical fluorescence intensity displayed in units of counts or photons, or in terms of efficiency.</li>
		<li>After acquiring the image, transfer the mice from the imaging chamber back to their cage and observe for their recovery for 1 to 2 min.</li>
	</ol>
	</li>
	<li>Fine tune the image further with the tools and functions provided within the software for image analysis. Image analysis tools are in the menu bar and tool palette.
	<ol>
		<li>Under Image Adjust, adjust the brightness, contrast, or opacity and select the color scale.</li>
		<li>Use the ROI Tools to specify a region of interest (ROI) in an optical image and measure the signal intensity within the ROI. If needed, export the quantified signal data to a spreadsheet software and plot the data.</li>
		<li>For follow-up studies, analyze mice necropsies of various tissues (e.g., liver, lung, heart, kidney, spleen, brain etc.) side-by-side for a detailed non-specific distribution of fluorescently labeled antibody. 
		NOTE: Data can be further supported with tissue specific ELISA by making use of antigen (FOLR1 in this case) in 96 well assays.</li>
	</ol>
	</li>
</ol>

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Authors have no competing financial interests.

Selective and tumor tissue specific delivery of anti-cancer therapeutic agent is the key to measure efficacy and safety of a given targeted therapy13. Here we have described a quick and efficient approach to investigate the detailed tissue and tumor distribution of clinical, farletuzumab and a nonclinical BaCa antibody. The described approach is applicable to any newly generated antibody and can be used alongside of a clinically effective antibody (with desired qualities) for its tumor/organ distribution properties. Considering most antibody target receptors (such as HER2 in breast cancer) are highly overexpressed in tumor cells (tissues), in most cases their suboptimal expression and function is also critical in cell types other than tumor cells14. For example, a significant proportion of EGFR targeting clinical antibodies in colon cancer patients accumulate and cause toxicity to skin tissue15, a noncancerous tissue whose growth and differentiation requires EGFR signaling and function. Therefore, we strongly believe these sorts of preliminary tissue distribution investigations in combinations with hepatotoxicity and tissue histochemistry assays in a larger cohort of animals are key to comprehensively assess safety and therapeutic viability of the newly generated antibody. Moreover, described tissue distribution studies would also be highly applicable in immune competent mice xenograft studies, if the newly generated antibody maintains cross-reactivity to murine counterpart antigen/receptor. In syngeneic animal studies, along with tumor distribution and tissue histochemistry studies, detailed blood cytokine analysis can used to strengthen the efficacy and safety data. An attractive feature of the described approach is that it allows near accurate quantitation of antibody distribution if data is additionally supported with ELISA (against target antigen) from the tumor and other significant tissue lysates (such as liver, heart, lung, spleen, kidney etc)7. Another important feature of described method over single photon emission computed tomography (called SPECT) and position emission tomography (PET) is the cost-effectiveness16. Both SPECT and PET are very expensive and makes use of radioactive tracers for imaging, making the whole process cumbersome if testing a large cohort of animals17. In addition SPECT and PET imaging facilities are not very standard in laboratories and vivariums to study small animal models of diseases such as mice18.
One limitation with described method to achieve a near accurate quantitation of antibody tumor distribution is the dependence on high affinity target antigen binding. It is because high affinity antigen-antibody interactions may result in &#8220;target-mediated drug disposition (TMDD)&#8221; by enhanced endocytosis and shuttling to lysosomes19. Therefore, the results of the described approach will vary depending on the particular target receptor in a particular tumor type. Thus, we strongly recommend testing labeled antibody/antibodies in a large cohort of animals with tumor xenografts generated with more than one tumor cell line(s) having variable (heterogeneous) expression of target antigen receptor. It is also greatly recommended to make use of more than one fluorescent conjugate dye(s) and mice strain(s) for the proposed studies.
Considering that small size antibodies such as Fabs, scFvs, BiTes, DARTs, etc. (lacking salvage recycling by neonatal Fc receptor (FcRn) clear more rapidly from tumors (with serum half times being minutes to hours), care should be taken to compare data between different tumor types having highly variable FcRn expression. Furthermore, larger molecules (such as dual and trispecificity antibodies) that are engineered with Fc domain for salvage recycling have tissue/tumor penetration issues. In those scenarios, the described approach will not be suitable to compare tissue/tumor distribution of antibodies that differ significantly in sizes6. In terms of significance however, the described tumor and detailed tissue distribution studies along with their counterpart monospecific antibodies would serve as a key factor in an effective dual and trispecificity antibody platform design. Finally, since higher affinity and avidity-optimized antibodies generally have a significantly homogenous distribution in tumors, the target tumor epitope selection (lacking TMDD), overall antibody affinity and biological activity in a particular cancer model should always be considered before making conclusion of tumor penetration, safety and efficacy.
In summary, we have described a quick and simple method for monitoring the tumor and tissue distribution of intravenously injected antibodies. The described approach has added potential to analyze antibody-siRNA conjugates (where siRNA is labeled), antibody-drug conjugates (where drug is labeled) and antibody-nanoparticles (where nanoparticle lipids are labeled with fluorescent dye). Likewise, a uniquely engineered cysteine residue in a tumor targeting scFv (if fluorescently labeled with melamide chemistry) of chimeric antigen receptor T-cells (CAR-T) and CAR-NK will be a cost effective approach to analyze tumor/tissue distribution of these cell based therapies independent of viral transfection based GFP/RFP signals strategies.

FDA approved the first monoclonal antibody targeting CD3 (OKT3, Muromonab) in 19861,2. Since then for the next twenty years, there has been a rapid explosion in the field of antibody engineering due to the overwhelming success of antibodies against immune checkpoint inhibitors3. Beside indirect activation of immune system, antibodies are being aimed to directly flag cancer cells to precisely engage immune effector cells, trigger cytotoxicity via death receptor agonist, block tumor cell survival signaling, obstruct angiogenesis (growth of blood vessels), constrain immune checkpoint regulators, deliver radioisotopes, chemotherapy drugs and siRNA as a conjugated agents2. In addition, studying the single chain variable fragments (scFv) of various antibodies on the surface of patient derived T-cells and NK cells (CAR-T and CAR-NK) is a fast growing area of clinical investigations for cell-based therapies4.
The ultra-high affinity of antibody-based drugs that provides selectivity to antigen expressing tumor cells makes it an attractive agent. Likewise, the targeted delivery and tumor retention of a therapeutic antibody (or a chemical drug) is the key to balance efficacy over toxicity. Therefore, a large number of protein engineering based strategies that include but are not limited to bispecific5 and tri-specific antibodies6 are being exploited to significantly enhance avidity optimized tumor retention of intravenously (IV) injected therapeutics5,7. Here, we describe a simple fluorescence-based method to address the tumor and tissue distribution of potentially effective anti-cancer antibodies.
Because animal tissues possess auto-fluorescence when excited in visible spectrum, the antibodies were initially labeled with near Infrared dye (e.g., IRDye 800CW). For proofs of concept investigations, we have made use of folate receptor alpha-1 (FOLR1) targeting antibody called farletuzumab and its derivative called Bispecific anchor Cytotoxicity activator (BaCa)7 antibody that co-targets FOLR1 and death receptor-5 (DR5)8 in one recombinant antibody. FOLR1 is a well-defined overexpressed target receptor in ovarian and TNBC cancer cells, tumor xenografts and patient tumors9. Notably, there are multiple efforts to clinically exploit FOLR1 using antibody-based approaches to engage immune effector cells and antibody drug conjugates (ADC) for ovarian and breast cancers10,11.
In this methods paper, we cloned, expressed and purified clinical anti-FOLR1 (farletuzumab) along with other control antibodies using CHO expression system. IgG1 isotype and a clinical anti-idiotype mucin-16 antibody called abagovomab12 were used as negative controls. Following protein-A purification, indicated antibodies were labeled with IRDye 800CW and were administered into the tail vein of nude mice either bearing ovarian tumor xenografts or stably transfected human FOLR1 expressing murine colon cancer xenografts. The antibody localization was tracked by live imaging using in vivo imaging spectrum at multiple different time points7. This method does not require any genetic modification or injection of the substrate to enable light emission and is significantly quicker, cost effective and efficient. The general cloning, expression, purification and labeling protocol described below can be applied to any clinical and nonclinical antibody if heavy and light chain sequences are available.

<table><tbody><tr><td>FreeStyle CHO media</td><td>Gibco Life Technologies</td><td>Cat # 12651-014</td><td></td></tr><tr><td>Anti-Anti (100X)</td><td>Gibco Life Technologies</td><td>Cat # 15240-062</td><td></td></tr><tr><td>Anti-Clumping Agent</td><td>Gibco Life Technologies</td><td>Cat # 01-0057DG</td><td></td></tr><tr><td>BD Insulin Syringe</td><td>BD BioSciences</td><td>Cat #329420</td><td></td></tr><tr><td>Caliper IVIS Spectrum</td><td>PerkinElmer</td><td>Cat #124262</td><td></td></tr><tr><td>CHO CD EfficientFeed B</td><td>Gibco Life Technologies</td><td>Cat #A10240-01</td><td></td></tr><tr><td>Corning 500 mL DMEM (Dulbecco&#039;s Modified Eagle&#039;s Medium)</td><td>Corning</td><td>Cat # 10-13-CV</td><td></td></tr><tr><td>Corning 500 mL RPMI 1640</td><td>Corning</td><td>Cat # 10-040-CV</td><td></td></tr><tr><td>Cy5 conjugated Anti-Human IgG (H+L)</td><td>Jackson ImmunoResearch</td><td>Cat # 709-175-149</td><td></td></tr><tr><td>GlutaMax-I (100X)</td><td>Gibco Life Technologies</td><td>Cat # 35050-061</td><td></td></tr><tr><td>HiPure Plasmid Maxiprep kit</td><td>Invitrogen</td><td>Cat # K21007</td><td></td></tr><tr><td>HiTrap MabSelect SuRe Column</td><td>GE Healthcare</td><td>Cat # 11-0034-93</td><td></td></tr><tr><td>Infusion</td><td>Takara BioScience</td><td>STO344</td><td></td></tr><tr><td>IRDye 800CW NHS Ester</td><td>LI-COR</td><td>Cat # 929-70020</td><td></td></tr><tr><td>Isoflurane, USP</td><td>Covetrus</td><td>Cat # 11695-6777-2</td><td></td></tr><tr><td>Lubricant Eye Ointment</td><td>Refresh Lacri-Lube</td><td>Cat #4089</td><td></td></tr><tr><td>Matrigel</td><td>Corning</td><td>Cat # 354234</td><td></td></tr><tr><td>PEI transfection reagent</td><td>Thermo Fisher</td><td>Cat # BMS1003A</td><td></td></tr><tr><td>Slide-A-Lyzer Dialysis Cassettes</td><td>Thermo Scientific</td><td>Cat # 66333</td><td></td></tr><tr><td>Steritop Vacuum Filters</td><td>Millipore Express</td><td>Cat #S2GPT02RE</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Gibco Life Technologies</td><td>Cat # 15400-054</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Experimental Models: Cell lines&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Human: OVCAR-3</td><td>American Type Culture Collection</td><td>ATCC HTB-161</td><td></td></tr><tr><td>Human: CHO-K cells</td><td>Stable transformed in our lab</td><td>ATCC CCL-61</td><td></td></tr><tr><td>Mouse: 4T1</td><td>Kind gift from Dr. Chip Landen, UVA</td><td></td><td></td></tr><tr><td>Mouse: MC38</td><td>Kind gift from Dr. Suzanne Ostrand-Rosenberg, UMBC</td><td>Authenticated by STR profiling</td><td></td></tr><tr><td>Mouse: MC38 hFOLR1</td><td>Generated in our laboratory (This paper)</td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Experimental Models: Animal&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mice: athymic Nude Foxn1&lt;sup&gt;nu&lt;/sup&gt;/Foxn1&lt;sup&gt;+&lt;/sup&gt;</td><td>Envigo</td><td>Multiple Orders</td><td></td></tr><tr><td>Mice: NOD.Cg Prkdc&lt;sup&gt;scid&lt;/sup&gt; Il2rg&lt;sup&gt;tm1Wjl&lt;/sup&gt;/SzJ</td><td>Jackson Laboratory</td><td>Multiple Orders</td><td></td></tr></tbody></table>

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.

In the described methodology, first we cloned antibodies targeting folate receptor alpha-1 (FOLR1) named farletuzumab, and a bispecific antibody called BaCa consisting of farletuzumab and lexatumumab along with control antibodies such as abagovomab (sequences provided in Supplementary File 1). Details of representative variable heavy (VH) and variable light (VL) domains in DNA clones (pVH, pVL) are shown in Figure 1A. To confirm the positive clones, we carried out colony PCR using signal peptide forward (SP For) and CK Rev/CH3 Rev primers (sequences provided in Supplementary File 1). Representative results of colony PCR confirm the expected sizes of light and heavy chains (Figure 1C). Positive antibody clones were, also, confirmed using Sanger sequencing. Following confirmed pVH and pVL DNA cloning, transfections were carried out using CHO suspension cultures, followed by protein-A column affinity purifications of antibodies at 4 &#176;C (see Figure 1B and protocol for detail steps).
Representative results of purified farletuzumab along with other control IgG1 (except BaCa antibody run on non-reducing and reducing SDS PAGE are shown in Figure 2A. As evident, heavy and light chain produced 50 and 25 KDa bands after reduction. This was followed by binding confirmation of antibodies to native proteins on the cell surface. Representative farletuzumab binding to human FOLR1 on OVCAR3 cells surface is shown using flow cytometry (Figure 2B).
Next fluorescently labeled antibodies were tail vein injected into animals grafted with FOLR1 expressing tumors (Figure 3). Animals were live imaged at multiple time points using IVIS. The data confirms selective enrichment of FOLR1 and BaCa antibodies into the FOLR1+ tumors (Figure 4 and Figure 5). Importantly control antibodies (negative for tumor antigen) did not localize into tumors.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60727/60727fig01.jpg" /> 
Figure 1: Schematic of antibody cloning, expression and purification. 
(A) Shows the detail schematic of heavy (pVH) and light (pVL) chain vectors. (B) pVH vector carrying variable domain sequence of an IgG1 heavy chain and pVL vector carrying variable domain sequence of a light chain were mixed together (1:2 ratio) along with transfection reagents (such as mirus or PEI) before adding to the suspension culture of CHO or HEK cells. Cells were fed with supplemental feed next day and cultures were monitored for 10 additional days with intermittent feeding. At day 11, cells were harvested through 0.2 &#181;m PES filters followed by affinity chromatography using protein-A. Purified antibodies were next analyzed for % monomer (FPLC), binding activity (ELISA or SPR), and binding to the target antigen (FACS) on live cells. Antibodies can also be checked for in vivo assays (e.g., cell growth inhibition, cell viability assays, or inhibition of signaling intermediate phosphorylation etc). Antibodies can also be conjugated with far-red dyes e.g., IRDye 800CW for in vivo imaging. &#177; IRDye 800CW must be tested for activities prior to tumor and tissue distribution studies. (C) Agarose gels of colony PCR confirming the sizes of positive heavy (1.4 Kb) and light chain (0.8 Kb) clones. <a href="https://www.jove.com/files/ftp_upload/60727/60727fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60727/60727fig02.jpg" /> 
Figure 2: A gel-based reduction assay to confirm antibody integrity. 
(A) Four different IgG1 antibodies (schematic shown on top) were added &#177; reducing agent (such as BME or DTT) at 95&#176;C for 10 min. Antibodies were next loaded on a 10% SDS-PAGE gel followed by protein staining and imaging. Gel image in left and right clearly show the intact antibody and two separate polypeptides (~50 KDa and ~25 KDa) respectively. Please also see pVH and pVL vector maps (Figure 1) carrying cDNA corresponding to VH/VL, CH1/CK, CH2 and CH3 domains. (B) Flow cytometry confirmation of unlabeled and IRDye 800CW labeled farletuzumab binding to native FOLR1 on ovarian cancer cells. Non-reducing = Antibody run on gel with non-reducing dye, Reducing = Antibody run on gel with reducing dye, HC = Heavy chain, LC = Light chain, VL = Variable domain of light chain, VH = Variable domain of heavy chain, CK = Kappa chain <a href="https://www.jove.com/files/ftp_upload/60727/60727fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60727/60727fig03.jpg" /> 
Figure 3: Experimental schematic of tumor generation and antibody treatments. 
6-8 weeks old mice strains such as: Immunodeficient athymic nude/NSG/ Immunocompetent C57BL/6 or Balb/C mice could be easily grafted with tumor cells via subcutaneous (SQ) tumors. Similar studies could be carried out using breast fat-pad and intraperitoneal (IP) tumors. 3-4 weeks later (tumor ~200 mm3), mice were IV injected with an IRDye labeled indicated antibodies that were &#177; selective against tumor-overexpressed receptor. This was followed by live in vivo imaging. <a href="https://www.jove.com/files/ftp_upload/60727/60727fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60727/60727fig04.jpg" /> 
Figure 4: Live in-vivo imaging of human OVCAR3 tumor bearing mice. 
Randomly selected 6 to 8 weeks old (Age) and 20-25 gram (Weight) hairless athymic Nude Foxn1nu/Foxn1+ (Envigo) were grafted with FOLR1+ ovarian tumors (OVCAR-3 cells). After 3 weeks with evident tumors, mice were tail vein injected with IRDye 800CW labeled IgG1 control, abagovomab (CA-125 anti-idiotypic antibody), farletuzumab (anti-FOLR1 antibody) and BaCa (anti-FOLR1-DR5 antibody) followed by live imaging at indicated times. <a href="https://www.jove.com/files/ftp_upload/60727/60727fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60727/60727fig05v2.jpg" /> 
Figure 5: Live in-vivo imaging of human FOLR1 expressing murine MC38 cell derived tumor bearing mice. 
(A) Randomly selected 6 to 8 weeks old (Age) and 20-25 g NOD.Cg Prkdcscid Il2rgtm1Wjl/SzJ or nude mice were SQ injected with murine MC38 cells stably expressing human FOLR1. Upon tumor appearance, mice were tail vein injected with IRDye 800CW labeled IgG1 control, abagovomab (CA-125 anti-idiotypic antibody), farletuzumab (anti-FOLR1 antibody) and BaCa (anti-FOLR1-DR5 antibody) followed by live imaging at indicated times. (B) After 7 days animals were euthanized and isolated key organs (as indicated) were imaged together along with grafted tumors for relative antibody signal (IRDye 800CW) distribution. As expected, tumors remained negative with IRDye 800CW signal in IgG1 control and CA-125 anti-idiotypic antibody, abagovomab injected animals. <a href="https://www.jove.com/files/ftp_upload/60727/60727fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1: Sequences of all antibodies and primers. <a href="https://cloudflare.jove.com/files/ftp_upload/60727/Supplementary_File_1.docx" target="_blank">Please click here to download this file.</a>

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Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody

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 ...

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5.2K Views.  University of Virginia School of Medicine. Here we present a protocol to study the in vivo localization of antibodies in mice tumor xenograft models.

Video: Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody

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

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

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

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

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

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

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

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

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

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

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

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

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

Harnessing the Bioorthogonal Inverse Electron Demand Diels-Alder Cycloaddition for Pretargeted PET Imaging

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for PET imaging. However, the necessity of labeling antibodies with radionuclides with long physical half-lives often results in high background radiation dose rates to non-target tissues. In order to circumvent this issue, we have employed a pretargeted PET imaging strategy based on the inverse electron demand Diels-Alder cycloaddition reaction. The methodology decouples the antibody from the radioactivity and thus exploits the positive characteristics of antibodies, while eschewing their pharmacokinetic drawbacks. The system is composed of four steps: (1) the injection of a mAb-trans-cyclooctene (TCO) conjugate; (2) a localization time period during which the antibody accumulates in the tumor and clears from the blood; (3) the injection of the radiolabeled tetrazine; and (4) the in vivo click ligation of the components followed by the clearance of excess radioligand. In the example presented in the work at hand, a 64Cu-NOTA-labeled tetrazine radioligand and a trans-cyclooctene-conjugated humanized antibody (huA33) were successfully used to delineate SW1222 colorectal cancer tumors with high tumor-to-background contrast. Further, the pretargeting methodology produces high quality images at only a fraction of the radiation dose to non-target tissue created by radioimmunoconjugates directly labeled with 64Cu or 89Zr. Ultimately, the modularity of this protocol is one of its greatest assets, as the trans-cyclooctene moiety can be appended to any non-internalizing antibody, and the tetrazine can be attached to a wide variety of radioisotopes.

The bioorthogonal inverse electron demand Diels-Alder cycloaddition has been harnessed to create an effective and modular pretargeted PET imaging strategy for cancer. In this protocol, the steps of this methodology are described in the context of a model system employing the colorectal cancer targeted antibody huA33 and a 64Cu-labeled radioligand.

Due to their exquisite affinity and specificity, antibodies have become extremely promising vectors for the delivery of radioisotopes to cancer cells for ...

Bioengineering

Pretargeted Radioimmunotherapy Based on the Inverse Electron Demand Diels-Alder Reaction

While radioimmunotherapy (RIT) is a promising approach for the treatment of cancer, the long pharmacokinetic half-life of radiolabeled antibodies can result in high radiation doses to healthy tissues. Perhaps not surprisingly, several different strategies have been developed to circumvent this troubling limitation. One of the most promising of these approaches is pretargeted radioimmunotherapy (PRIT). PRIT is predicated on decoupling the radionuclide from the immunoglobulin, injecting them separately, and then allowing them to combine in vivo at the target tissue. This approach harnesses the exceptional tumor-targeting properties of antibodies while skirting their pharmacokinetic drawbacks, thereby lowering radiation doses to non-target tissues and facilitating the use of radionuclides with half-lives that are considered too short for use in traditional radioimmunoconjugates. Over the last five years, our laboratory and others have developed an approach to in vivo pretargeting based on the inverse electron-demand Diels-Alder (IEDDA) reaction between trans-cyclooctene (TCO) and tetrazine (Tz). This strategy has been successfully applied to pretargeted positron emission tomography (PET) and single-photon emission computed tomography (SPECT) imaging with a variety of antibody-antigen systems. In a pair of recent publications, we have demonstrated the efficacy of IEDDA-based PRIT in murine models of pancreatic ductal adenocarcinoma and colorectal carcinoma. In this protocol, we describe protocols for PRIT using a 177Lu-DOTA-labeled tetrazine radioligand ([177Lu]Lu-DOTA-PEG7-Tz) and a TCO-modified variant of the colorectal cancer targeting huA33 antibody (huA33-TCO). More specifically, we will describe the construction of huA33-TCO, the synthesis and radiolabeling of [177Lu]Lu-DOTA-PEG7-Tz, and the performance of in vivo biodistribution and longitudinal therapy studies in murine models of colorectal carcinoma.

This protocol describes the synthesis and characterization of a trans-cyclooctene (TCO)-modified antibody and a 177Lu-labeled tetrazine (Tz) radioligand for pretargeted radioimmunotherapy (PRIT). In addition, it details the use of these two constructs for in vivo biodistribution and longitudinal therapy studies in a murine model of colorectal cancer.

While radioimmunotherapy (RIT) is a promising approach for the treatment of cancer, the long pharmacokinetic half-life of radiolabeled antibodies can ...

Chemistry

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

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

Here, we describe a method for transducing human T cells with luciferase to facilitate in vivo tracking of bispecific antibody-induced T cell trafficking to tumors in studies to evaluate the anti-tumor efficacy and mechanism of T cell-engaging bispecific antibodies.

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

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.

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 ...

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 ...

In Vivo Fluorescence Imaging to Localize Antibodies in a Mouse Tumor Xenograft Model

Source: Shivange, G. et al., <a href="https://app.jove.com/v/60727">Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody.</a>&#160;J. Vis. Exp. (2020)
The video demonstrates a fluorescence imaging method for in vivo antibody localization in a mouse tumor xenograft model. It involves the injection of tumor cells in a mouse to generate solid xenografts beneath the skin. Subsequent injection of fluorescent antibodies confirms specific localization through enhanced fluorescence signals within the tumor xenograft.

1. Expression and purification of antibodies

	Maintenance of CHO cells
	
		Grow CHO cells in FreeStyle CHO Media supplemented with commercially available ...

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Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

An In Vivo Technique to Track Bispecific Antibody-Induced T Cell Trafficking to Tumors

Source: Espinosa-Cotton, M., et al. <a href="https://app.jove.com/v/64390">Tracking Bispecific Antibody-Induced T Cell Trafficking Using Luciferase-Transduced Human T Cells.</a> J. Vis. Exp. (2023).
This video demonstrates a method for tracking bispecific antibody-induced T cell trafficking to tumors in a mouse model. Luciferase-transduced effector T cells are injected into a mouse with a subcutaneous tumor cell xenograft. Bispecific antibodies are then introduced, binding to T cells and the tumor cells. The localization of the T cells is tracked using bioluminescent imaging, with the magnitude of the signal indicating the extent of T cell infiltration into the tumor.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody

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