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>

<ol>
	<li>Takahashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muromonab+CD3+(Orthoclone+OKT3).">Muromonab CD3 (Orthoclone OKT3).</a> Journal of Toxicological Sciences. 20, 483-484 (1995).</li><li>Tushir-Singh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody-siRNA+conjugates:+drugging+the+undruggable+for+anti-leukemic+therapy.">Antibody-siRNA conjugates: drugging the undruggable for anti-leukemic therapy.</a> Expert Opinion in Biological Therapy. 17, 325-338 (2017).</li><li>Gravitz, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+immunotherapy.">Cancer immunotherapy.</a> Nature. 504, 1 (2013).</li><li>Pagel, J. M., West, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimeric+Antigen+Receptor+(CAR)+T-Cell+Therapy.">Chimeric Antigen Receptor (CAR) T-Cell Therapy.</a> JAMA Oncology. 3, 1595 (2017).</li><li>Brinkmann, U., Kontermann, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+making+of+bispecific+antibodies.">The making of bispecific antibodies.</a> MAbs. 9, 182-212 (2017).</li><li>Runcie, K., Budman, D. R., John, V., Seetharamu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bi-specific+and+tri-specific+antibodies-+the+next+big+thing+in+solid+tumor+therapeutics.">Bi-specific and tri-specific antibodies- the next big thing in solid tumor therapeutics.</a> Molecular Medicine. 24, 50 (2018).</li><li>Shivange, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Single-Agent+Dual-Specificity+Targeting+of+FOLR1+and+DR5+as+an+Effective+Strategy+for+Ovarian+Cancer.">A Single-Agent Dual-Specificity Targeting of FOLR1 and DR5 as an Effective Strategy for Ovarian Cancer.</a> Cancer Cell. 34, 331-345 (2018).</li><li>Wajant, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Mode+of+Action+of+TRAIL+Receptor+Agonists-Common+Principles+and+Their+Translational+Exploitation.">Molecular Mode of Action of TRAIL Receptor Agonists-Common Principles and Their Translational Exploitation.</a> Cancers (Basel). 11 (7), 954 (2019).</li><li>Necela, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folate+receptor-alpha+(FOLR1)+expression+and+function+in+triple+negative+tumors.">Folate receptor-alpha (FOLR1) expression and function in triple negative tumors.</a> PLoS One. 10, 0122209 (2015).</li><li>Lin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antitumor+activity+of+the+human+FOLR1-specific+monoclonal+antibody,+farletuzumab,+in+an+ovarian+cancer+mouse+model+is+mediated+by+antibody-dependent+cellular+cytotoxicity.">The antitumor activity of the human FOLR1-specific monoclonal antibody, farletuzumab, in an ovarian cancer mouse model is mediated by antibody-dependent cellular cytotoxicity.</a> Cancer Biology Therapy. 14, 1032-1038 (2013).</li><li>Chen, Y., Kim, M. T., Zheng, L., Deperalta, G., Jacobson, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Characterization+of+Cross-Linked+Species+in+Trastuzumab+Emtansine+(Kadcyla).">Structural Characterization of Cross-Linked Species in Trastuzumab Emtansine (Kadcyla).</a> Bioconjugate Chemistry. 27, 2037-2047 (2016).</li><li>Bauerschlag, D. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-idiotypic+antibody+abagovomab+in+advanced+ovarian+cancer.">Anti-idiotypic antibody abagovomab in advanced ovarian cancer.</a> Future Oncology. 4, 769-773 (2008).</li><li>Narita, Y., Muro, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+in+molecular+targeted+therapy+for+gastric+cancer:+considerations+for+efficacy+and+safety.">Challenges in molecular targeted therapy for gastric cancer: considerations for efficacy and safety.</a> Expert Opinion in Drug Safety. 16, 319-327 (2017).</li><li>Jordan, N. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HER2+expression+identifies+dynamic+functional+states+within+circulating+breast+cancer+cells.">HER2 expression identifies dynamic functional states within circulating breast cancer cells.</a> Nature. 537, 102-106 (2016).</li><li>Fakih, M., Vincent, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adverse+events+associated+with+anti-EGFR+therapies+for+the+treatment+of+metastatic+colorectal+cancer.">Adverse events associated with anti-EGFR therapies for the treatment of metastatic colorectal cancer.</a> Current Oncology. 17, 18-30 (2010).</li><li>Koba, W., Jelicks, L. A., Fine, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroPET/SPECT/CT+imaging+of+small+animal+models+of+disease.">MicroPET/SPECT/CT imaging of small animal models of disease.</a> American Journal of Pathology. 182, 319-324 (2013).</li><li>van der Wall, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cost+analysis+favours+SPECT+over+PET+and+CTA+for+evaluation+of+coronary+artery+disease:+the+SPARC+study.">Cost analysis favours SPECT over PET and CTA for evaluation of coronary artery disease: the SPARC study.</a> Netherland Heart Journal. 22, 257-258 (2014).</li><li>Van Dort, M. E., Rehemtulla, A., Ross, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PET+and+SPECT+Imaging+of+Tumor+Biology:+New+Approaches+towards+Oncology+Drug+Discovery+and+Development.">PET and SPECT Imaging of Tumor Biology: New Approaches towards Oncology Drug Discovery and Development.</a> Current Computer Aided Drug Design. 4, 46-53 (2008).</li><li>Dua, P., Hawkins, E., van der Graaf, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Tutorial+on+Target-Mediated+Drug+Disposition+(TMDD)+Models.">A Tutorial on Target-Mediated Drug Disposition (TMDD) Models.</a> CPT Pharmacometrics and System Pharmacology. 4, 324-337 (2015).</li></ol>

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&#39;s Modified Eagle&#39;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>Experimental Models: Cell lines</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>Experimental Models: Animal</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mice: athymic Nude Foxn1nu/Foxn1+</td>
			<td>Envigo</td>
			<td>Multiple Orders</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice: NOD.Cg Prkdcscid Il2rgtm1Wjl/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>

Watch this Scientific Journal Video about Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody at JoVE.com

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 ...

analyzing-tumor-tissue-distribution-target-antigen-specific

Nanyang Technological University

Research

JoVE Journal

Cancer Research

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

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and manufacturing optimizations. One challenge for these development efforts, however, has been the establishment of in vitro assays that can robustly inform selection of the optimal CAR T cell products for in vivo therapeutic success. Standard in vitro tumor-lysis assays often fail to reflect the true antitumor potential of the CAR T cells due to the relatively short co-culture time and high T cell to tumor ratio. Here, we describe an in vitro co-culture method to evaluate CAR T cell recursive killing potential at high tumor cell loads. In this assay, long-term cytotoxic function and proliferative capacity of CAR T cells is examined in vitro over 7 days with additional tumor targets administered to the co-culture every other day. This assay can be coupled with profiling T cell activation, exhaustion and memory phenotypes. Using this assay, we have successfully distinguished the functional and phenotypic differences between CD4+ and CD8+ CAR T cells against glioblastoma (GBM) cells, reflecting their differential in vivo antitumor activity in orthotopic xenograft models. This method provides a facile approach to assess CAR T cell potency and to elucidate the functional variations across different CAR T cell products.

Here, we describe an in vitro co-culture method to recursively challenge tumor-targeted T cells, which allows for phenotypic and functional analysis of antitumor T cell activity.

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and ...

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 ...

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Analyzing Tumor Gene Expression Factors with the CorExplorer Web Portal

Differential gene expression analysis is an important technique for understanding disease states. The machine learning algorithm CorEx has shown utility in analyzing differential expression of groups of genes in tumor RNA-seq in a way that may be helpful for advancing precision oncology. However, CorEx produces many factors that can be challenging to analyze and connect to existing understanding. To facilitate such connections, we have built a website, CorExplorer, that allows users to interactively explore the data and answer common questions related to its analysis. We trained CorEx on RNA-seq gene expression data for four tumor types: ovarian, lung, melanoma, and colorectal. We then incorporated corresponding survival, protein-protein interactions, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichments, and heatmaps into the website for association with the factor graph visualization. Here we employ example protocols to illustrate use of the database for comprehending the significance of the learned tumor factors in the context of this external data.

We introduce the CorExplorer web portal, an resource for exploration of tumor RNA sequencing factors found by the machine learning algorithm CorEx (Correlation Explanation), and show how factors can be analyzed relative to survival, database annotations, protein-protein interactions, and one another to gain insight into tumor biology and therapeutic interventions.

Differential gene expression analysis is an important technique for understanding disease states. The machine learning algorithm CorEx has shown utility ...

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu constitutes the tumor microenvironment (TME) and can modulate response to therapy in vivo or drug response ex vivo. Conventional cancer drug discovery screens are carried out on cells cultured in a monolayer, a system critically lacking the influence of TME. Thus, experimental systems that integrate sensitive and high-throughput assays with physiological TME will strengthen the preclinical drug discovery process. Here, we introduce ex vivo tumor tissue slice culture as a platform for medium-high-throughput drug screening. Organotypic tissue slice culture constitutes precisely-cut, thin tumor sections that are maintained with the support of a porous membrane in a liquid-air interface. In this protocol, we describe the preparation and maintenance of tissue slices prepared from mouse tumors and tumors from patient-derived xenograft (PDX) models. To assess changes in tissue viability in response to drug treatment, we leveraged a biocompatible luminescence-based viability assay that enables real-time, rapid, and sensitive measurement of viable cells in the tissue. Using this platform, we evaluated dose-dependent responses of tissue slices to the multi-kinase inhibitor, staurosporine, and cytotoxic agent, doxorubicin. Further, we demonstrate the application of tissue slices for ex vivo pharmacology by screening 17 clinical and preclinical drugs on tissue slices prepared from a single PDX tumor. Our physiologically-relevant, highly-sensitive, and robust ex vivo screening platform will greatly strengthen preclinical oncology drug discovery and treatment decision making.&#160;

We introduce a protocol for measuring real-time drug response in organotypic tumor tissue slices. The experimental strategy outlined here provides a platform to carry out medium-high throughput drug screens on tissue slices derived from clinical or mouse tumors in ex vivo conditions.

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu ...

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical outcomes. Preclinical mouse models of breast cancer brain metastatic (BCBM) growth are useful but are expensive, and it is difficult to track live cells and tumor cell invasion within the brain parenchyma. Presented here is a protocol for ex vivo brain slice cultures from xenografted mice containing intracranially injected breast cancer brain-seeking clonal sublines. MDA-MB-231BR luciferase tagged cells were injected intracranially into the brains of Nu/Nu female mice, and following tumor formation, the brains were isolated, sliced, and cultured ex vivo. The tumor slices were imaged to identify tumor cells expressing luciferase and monitor their proliferation and invasion in the brain parenchyma for up to 10 days. Further, the protocol describes the use of time-lapse microscopy to image the growth and invasive behavior of the tumor cells following treatment with ionizing radiation or chemotherapy. The response of tumor cells to treatments can be visualized by live-imaging microscopy, measuring bioluminescence intensity, and performing histology on the brain slice containing BCBM cells. Thus, this ex vivo slice model may be a useful platform for rapid testing of novel therapeutic agents alone or in combination with radiation to identify drugs personalized to target an individual patient&#39;s breast cancer brain metastatic growth within the brain microenvironment.

An Ex Vivo Brain Slice Model to Study and Target Breast Cancer Brain Metastatic Tumor Growth

We introduce a protocol for measuring real-time drug and radiation response of breast cancer brain metastatic cells in an organotypic brain slice model. The methods provide a quantitative assay to investigate the therapeutic effects of various treatments on brain metastases from breast cancer in an ex vivo manner within the brain microenvironment interface.

Brain metastasis is a serious consequence of breast cancer for women as these tumors are difficult to treat and are associated with poor clinical ...

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

This protocol discusses an approach for generating epithelial organoids from primary normal and tumor mammary tissue through differential centrifugation. Furthermore, instructions are included for three-dimensional culturing as well as immunofluorescent imaging of embedded organoids.