The authors would like to thank Dr. Vladimir Ponomarev for sharing the luciferase constructs used in the experiments described in the representative results section of this article.

The following procedures have been evaluated and approved by Memorial Sloan Kettering&#39;s Institutional Animal Care and Use Committee.
1. Transfection of 293T cells with luciferase and harvest of viral supernatant
<ol>
	<li>Culture of 293T cells
	<ol>
		<li>Prepare media by adding the following to a liter of DMEM each: 110 mL of heat-inactivated fetal bovine serum (FBS), 11 mL of penicillin-streptomycin.</li>
		<li>Thaw 5 x 106 293T cells and transfer them into a T175 flask with the media prepared as described above.</li>
		<li>Split the 293T cells 1:10 every 3 days for 6 days. Do not allow the cells to become more than 90% confluent.</li>
	</ol>
	</li>
	<li>Transfection of 293T cells 
	NOTE: The following quantities of reagents are calculated for transfecting one T175 flask of 293T cells. Adjust accordingly if more flasks are to be transduced.
	<ol>
		<li>Transfer 1.5 mL of media to each of the two 50 mL tubes using a pipette. To one tube, add 10 &#181;g of VSV-G plasmid, 20 &#181;g of Gag/pol, and 20 &#181;g of click beetle red TD tomato (CBR-TDR) luciferase plasmid and mix. To the other tube, add 100 &#181;L of DNA in vitro transfection reagent and mix. Incubate both tubes at room temperature (RT) for 5 min. 
		NOTE: All plasmids described in this manuscript were provided by Dr. Vladimir Ponomarev.</li>
		<li>Transfer the media containing the DNA in vitro transfection reagent dropwise to the tube containing the DNA plasmids (over approximately 30 s) and mix gently with a pipette. Incubate at RT for 20 min.</li>
		<li>During this 20 min incubation, detach the 293T cells by adding 5-10 mL of 0.05% trypsin. Once the cells have detached, add 10 mL of media and centrifuge at 800 x g for 5 min. Resuspend the cells in 1.5 mL of media.</li>
		<li>Add the media containing the DNA in vitro transfection reagent and the DNA plasmids to the 293T cells and incubate at 37 &#176;C for 30 min.</li>
		<li>Transfer to a T175 flask and add 18 mL of media. Incubate at 37 &#176;C overnight.</li>
		<li>The next day, carefully aspirate the media with a glass pipette without detaching the cells. Replace with 18 mL of fresh media. Incubate at 37 &#176;C overnight in an incubator.</li>
	</ol>
	</li>
	<li>Harvesting viral supernatant
	<ol>
		<li>Remove the viral supernatant using a pipette put on ice. Centrifuge at 800 x g for 5 min to pellet the cells and debris. 
		NOTE: If the cell pellet is large, the cells were likely disrupted from the flask when the supernatant was removed. These cells can be plated again in a new flask with fresh media.</li>
		<li>Filter the viral supernatant using a 0.22 &#181;m filter.</li>
		<li>Use the viral supernatant immediately. Keep it on ice or at 4 &#176;C for up to 24 h, or freeze it at -80&#176; C for long-term storage.</li>
	</ol>
	</li>
</ol>
2. Expansion and transduction of activated human T cells with luciferase
<ol>
	<li>Activation of human T cells in vitro
	<ol>
		<li>Wash the beads by adding 10 mL of phosphate buffered saline (PBS) containing 0.1% bovine serum albumin (BSA) and 2 mM ethylenediaminetetraacetic acid (EDTA) to a sterile 15 mL tube, adding beads (one bead per two T cells), placing in a magnet rack for 2 min, and suctioning out the PBS.</li>
		<li>Prepare media as described in step 1.1.1, then add IL-2 to a final concentration of 30 IU/mL and add the washed beads. Make 2.5 mL of media for every two million T cells to be activated.</li>
		<li>Count the T cells (freshly purified or previously purified, frozen, thawed, and washed) using a hemocytometer and trypan blue stain. Add T cells to the media prepared in step 2.1.2 and gently invert the tube to mix. The T cells will expand at least 20x depending on the source and general health of the cells. Determine the number of T cells to activate by calculating the number needed to treat the mice and dividing by 20. Here, 20 x 106 T cells per mouse were used based on preliminary experiments.</li>
		<li>Plate 2.5 mL of media containing two million T cells, beads, and IL-2 in each well of a 24-well tissue culture plate. Culture at 37 &#176;C in a humidified 5% CO2 incubator.</li>
		<li>Examine the T cells under a 20x microscope every day for the next 3 days to determine when to transduce with luciferase. The cells are ready when they clump together and deplete the media, turning it from red/pink to light orange.</li>
	</ol>
	</li>
	<li>Preparation of retronectin plates 
	NOTE: Retronectin is used to coat the plates used in the transduction as it enhances gene transduction15.
	<ol>
		<li>Add 2 mL of 20 &#181;g/mL retronectin in PBS to a well of an untreated 6-well plate. Incubate for 2 h at RT or 1 h at 37 &#176;C. One retronectin-coated well is required for every two million T cells to be transduced.</li>
		<li>Aspirate the retronectin without scratching the bottom of the plate.</li>
		<li>Add 3 mL of 2% BSA in PBS (sterile filtered) per well in the 6-well plate. Incubate for 30 min at RT.</li>
	</ol>
	</li>
	<li>Spinoculation
	<ol>
		<li>Aspirate the BSA without scratching the bottom of the plate.</li>
		<li>Wash the wells once with 3 mL of PBS per well. Continue or replace with fresh PBS and leave the plate covered at 4 &#176;C overnight.</li>
		<li>Add 2 mL of CBR-TDR luciferase viral supernatant (collected in step 1.3) per well.</li>
		<li>Centrifuge at 1,240 x g for 90 min at 32 &#176;C.</li>
	</ol>
	</li>
	<li>Transduction
	<ol>
		<li>Count the T cells.</li>
		<li>Aspirate the viral supernatant without scratching the bottom of the plate.</li>
		<li>Plate two million T cells per well in 6 mL of Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) containing 30 IU/mL human IL-2.</li>
		<li>Examine the T cells daily. The T cells are ready to be expanded when they are nearly confluent, usually after 2 days.</li>
		<li>When the cells are nearly confluent, gently wash them off the bottom of the plate using a pipette and transfer each well to a separate T75 flask. Add 9 mL of media for a total of 15 mL of media per flask.</li>
		<li>The next day, add an additional 15 mL of media. The cells should be ready to inject into mice the day after this addition of media. Confirm successful transduction by performing flow cytometry (the luciferase construct contains a TD tomato fluorophore that is visible in the PE channel)16.</li>
	</ol>
	</li>
</ol>
3. Engraftment of luciferase-transduced T cells in immunocompromised mice
<ol>
	<li>Prepare and count the T cells for injection.
	<ol>
		<li>Remove the T cells from the flasks and count. The cells are ready to inject when they have expanded 20x-30x, usually by day 7 post-activation.</li>
		<li>Centrifuge the T cells at 800 x g for 5 min. Resuspend in 2 mL of media and remove the beads using a magnet rack.</li>
		<li>For experiments where T cells will be injected separately from bispecific antibodies, count the T cells and resuspend so that the final concentration is 20 million T cells per 100 &#181;L of media. Skip to step 3.2. For experiments using ex vivo armed T cells (EATs), skip to step 3.1.4.</li>
		<li>For experiments using ex vivo armed T cells, count the T cells and separate them into individual 1.5 mL tubes for each group, with 20 million cells per mouse. For example, if there are three groups of five mice each, prepare three 1.5 mL tubes with 100 million T cells each.</li>
		<li>Centrifuge the 1.5 mL tubes at 800 x g for 5 min. Remove the media carefully without disturbing the T cell pellet. Resuspend in no more than 50 &#181;L of media containing the bispecific antibodies. Incubate at RT for 30 min. 
		NOTE: For the purposes of experiments conducted in this study, proprietary IgG-[L]-scFv T cell-engaging bispecific antibodies generated in the lab were used. For T cell arming, use 5 &#181;g of antibody per 20 x 106 T cells. Commercially available bispecific antibodies could also be used.</li>
		<li>Wash away excess antibodies by adding 1,450 &#181;L of media to each tube. Centrifuge at 800 x g for 5 min, carefully aspirate the media, and resuspend at a concentration of 20 million armed T cells per 100 &#181;L media.</li>
	</ol>
	</li>
	<li>Injection and engraftment into mice
	<ol>
		<li>Anesthetize the mice in a chamber supplying 3.5% (v/v) inhaled isoflurane in 1 L/min oxygen. The mice are fully anesthetized when the hind limb pedal withdrawal reflex has disappeared. 
		NOTE:&#160;Provide thermal support throughout the procedure.</li>
		<li>Using a 26 G needle, inject 20 million T cells in 100 &#181;L of media retroorbitally per mouse.</li>
		<li>Administer 1,000 U recombinant IL-2 subcutaneously to support T cell survival in vivo.</li>
		<li>Administer the bispecific antibodies retroorbitally (into the eye not used for T cell injection) or intraperitoneally. Allow the mice to recover from anesthesia and return to their cages. 
		&#8203;NOTE: Again, here, proprietary antibodies that were generated in the lab were used, but commercially available antibodies could be used instead. In the experiments here, 0.3-10 &#181;g of antibody per mouse per dose was administered. The antibodies were administered twice per week for 3-4 weeks.</li>
	</ol>
	</li>
</ol>
4. In vivo imaging of mice engrafted with luciferase-transduced T cells
NOTE: This step is to be performed on the day of imaging, which is not necessarily the same day that the T cells and/or antibodies are administered to the mice. Typically, we perform imaging 24 h after administration of the luciferase-transduced T cells.
<ol>
	<li>Preparation of D-luciferin
	<ol>
		<li>Dissolve 1 g of D-luciferin in 33.3 mL of sterile PBS (final concentration: 30 mg/mL).</li>
		<li>Aliquot the dissolved D-luciferin into 33 x 1.5 mL microcentrifuge tubes and keep the tubes at -20 &#176;C.</li>
		<li>On the day of imaging, thaw enough aliquots of 30 mg/mL D-luciferin for the number of animals to be imaged. One tube is enough for 10 animals. 
		NOTE: Refreeze the remaining D-luciferin after use. D-luciferin is stable for at least five freeze/thaw cycles.</li>
	</ol>
	</li>
	<li>Administration of D-luciferin to mice 
	NOTE: Before administering D-luciferin to the mice, open the imaging software and initialize the system. The camera may take several minutes to cool, and this should be done before the mice are injected with D-luciferin to ensure that imaging can take place 5 min after administration of the substrate.
	<ol>
		<li>Anesthetize up to five mice in a chamber supplying 3.5% (v/v) inhaled isoflurane in 1 L/min oxygen. The mice are fully anesthetized when the hind limb pedal withdrawal reflex has disappeared.</li>
		<li>Once the mice are fully anesthetized, administer 100 &#181;L of 30 mg/mL D-luciferin per mouse (3 mg per mouse) by retroorbital injection with a 26 G needle. Image this group of mice before administering D-luciferin to the next group. Place the next group of mice in the isoflurane chamber to be anesthetized while the previous group is being imaged.</li>
	</ol>
	</li>
	<li>Imaging luciferase-transduced T cells
	<ol>
		<li>Move the anesthetized mice to the light-tight chamber of the imager and continue administering 3% isoflurane at 0.5 L/min. Place the mice on their sides such that the flank bearing the xenograft is facing up toward the camera. Take another set of images with the mice in a supine position to evaluate T cell presence in the lungs.</li>
		<li>Using the Acquisition Control Panel, select Luminescent, Photograph, and Overlay. Set the exposure time to auto, the binning to medium, and F/Stop to 1. Acquire images. After the first image, set the exposure time to match that which was automatically calculated for the first image so that subsequent images may be compared directly. It may be useful to capture images with multiple exposure times to determine the optimal exposure time for achieving the brightest signal without pixel saturation.</li>
		<li>Remove the mice from isoflurane, return to their cages, and observe until they are awake and ambulatory.</li>
		<li>Repeat the steps from step 4.2.1 until all groups have been imaged.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>G&#246;kbuget, N., 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=Blinatumomab+for+minimal+residual+disease+in+adults+with+B-cell+precursor+acute+lymphoblastic+leukemia.">Blinatumomab for minimal residual disease in adults with B-cell precursor acute lymphoblastic leukemia.</a> Blood. 131 (14), 1522-1531 (2018).</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 (1), 50 (2018).</li><li>Dreier, T., 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=T+Cell+costimulus-independent+and+very+efficacious+inhibition+of+tumor+growth+in+mice+bearing+subcutaneous+or+leukemic+human+B+cell+lymphoma+xenografts+by+a+CD19-/CD3-+Bispecific+single-chain+antibody+construct.">T Cell costimulus-independent and very efficacious inhibition of tumor growth in mice bearing subcutaneous or leukemic human B cell lymphoma xenografts by a CD19-/CD3- Bispecific single-chain antibody construct.</a> The Journal of Immunology. 170 (8), 4397-4402 (2003).</li><li>Offner, S., Hofmeister, R., Romaniuk, A., Kufer, P., Baeuerle, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+regular+cytolytic+T+cell+synapses+by+bispecific+single-chain+antibody+constructs+on+MHC+class+I-negative+tumor+cells.">Induction of regular cytolytic T cell synapses by bispecific single-chain antibody constructs on MHC class I-negative tumor cells.</a> Molecular Immunology. 43 (6), 763-771 (2006).</li><li>Santich, B. H., Cheung, N. V., Klein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Editorial:+Bispecific+antibodies+for+T-cell+based+immunotherapy.">Editorial: Bispecific antibodies for T-cell based immunotherapy.</a> Frontiers in Oncology. 10, 628005 (2020).</li><li>Santich, B. H., 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=Interdomain+spacing+and+spatial+configuration+drive+the+potency+of+IgG-[L]-scFv+T+cell+bispecific+antibodies.">Interdomain spacing and spatial configuration drive the potency of IgG-[L]-scFv T cell bispecific antibodies.</a> Science Translational Medicine. 12 (534), eaax1315 (2020).</li><li>Wang, L., Hoseini, S. S., Xu, H., Ponomarev, V., Cheung, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+Fc+domains+in+T+cell-engaging+bispecific+antibodies+improves+T-cell+trafficking+and+antitumor+potency.">Silencing Fc domains in T cell-engaging bispecific antibodies improves T-cell trafficking and antitumor potency.</a> Cancer Immunology Research. 7 (12), 2013-2024 (2019).</li><li>Park, J. A., Cheung, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GD2+or+HER2+targeting+T+cell+engaging+bispecific+antibodies+to+treat+osteosarcoma.">GD2 or HER2 targeting T cell engaging bispecific antibodies to treat osteosarcoma.</a> Journal of Hematology &amp; Oncology. 13 (1), 172 (2020).</li><li>Wu, Z., Guo, H. F., Xu, H., Cheung, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+tetravalent+anti-GPA33/anti-CD3+bispecific+antibody+for+colorectal+cancers.">Development of a tetravalent anti-GPA33/anti-CD3 bispecific antibody for colorectal cancers.</a> Molecular Cancer Therapeutics. 17 (10), 2164-2175 (2018).</li><li>Lin, T. Y., Park, J. A., Long, A., Guo, H. F., Cheung, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+potent+anti-STEAP1+bispecific+antibody+to+redirect+T+cells+for+cancer+immunotherapy.">Novel potent anti-STEAP1 bispecific antibody to redirect T cells for cancer immunotherapy.</a> Journal for Immunotherapy of Cancer. 9 (9), e003114 (2021).</li><li>Hoseini, S. S., Espinosa-Cotton, M., Guo, H. F., Cheung, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overcoming+leukemia+heterogeneity+by+combining+T+cell+engaging+bispecific+antibodies.">Overcoming leukemia heterogeneity by combining T cell engaging bispecific antibodies.</a> Journal for Immunotherapy of Cancer. 8 (2), e001626 (2020).</li><li>Hoseini, S. S., Guo, H., Wu, Z., Hatano, M. N., Cheung, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+potent+tetravalent+T-cell-engaging+bispecific+antibody+against+CD33+in+acute+myeloid+leukemia.">A potent tetravalent T-cell-engaging bispecific antibody against CD33 in acute myeloid leukemia.</a> Blood Advances. 2 (11), 1250-1258 (2018).</li><li>Hoseini, S. S., 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=T+cell+engaging+bispecific+antibodies+targeting+CD33+IgV+and+IgC+domains+for+the+treatment+of+acute+myeloid+leukemia.">T cell engaging bispecific antibodies targeting CD33 IgV and IgC domains for the treatment of acute myeloid leukemia.</a> Journal for Immunotherapy of Cancer. 9 (5), e002509 (2021).</li><li>Li, H., Er Saw, P., Song, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+and+strategies+for+next-generation+bispecific+antibody-based+antitumor+therapeutics.">Challenges and strategies for next-generation bispecific antibody-based antitumor therapeutics.</a> Cellular and Molecular Immunology. 17 (5), 451-461 (2020).</li><li>Rajabzadeh, A., Hamidieh, A. A., Rahbarizadeh, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinoculation+and+retronectin+highly+enhance+the+gene+transduction+efficiency+of+Mucin-1-specific+chimeric+antigen+receptor+(CAR)+in+human+primary+T+cells.">Spinoculation and retronectin highly enhance the gene transduction efficiency of Mucin-1-specific chimeric antigen receptor (CAR) in human primary T cells.</a> BMC Molecular and Cell Biology. 22 (1), 57 (2021).</li><li>Kleeman, B., 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+guide+to+choosing+fluorescent+protein+combinations+for+flow+cytometric+analysis+based+on+spectral+overlap.">A guide to choosing fluorescent protein combinations for flow cytometric analysis based on spectral overlap.</a> Cytometry Part A. 93 (5), 556-562 (2018).</li><li>Park, J. A., Santich, B. H., Xu, H., Lum, L. G., Cheung, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+ex+vivo+armed+T+cells+using+recombinant+bispecific+antibodies+for+adoptive+immunotherapy+with+reduced+cytokine+release.">Potent ex vivo armed T cells using recombinant bispecific antibodies for adoptive immunotherapy with reduced cytokine release.</a> Journal for Immunotherapy of Cancer. 9 (5), e002222 (2021).</li><li>Park, J. A., Wang, L., Cheung, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulating+tumor+infiltrating+myeloid+cells+to+enhance+bispecific+antibody-driven+T+cell+infiltration+and+anti-tumor+response.">Modulating tumor infiltrating myeloid cells to enhance bispecific antibody-driven T cell infiltration and anti-tumor response.</a> Journal of Hematology &amp; Oncology. 14 (1), 142 (2021).</li><li>Str&#246;hlein, M. A., Heiss, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+trifunctional+antibody+catumaxomab+in+treatment+of+malignant+ascites+and+peritoneal+carcinomatosis.">The trifunctional antibody catumaxomab in treatment of malignant ascites and peritoneal carcinomatosis.</a> Future Oncology. 6 (9), 1387-1394 (2010).</li><li>Rabinovich, B. A., 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=Visualizing+fewer+than+10+mouse+T+cells+with+an+enhanced+firefly+luciferase+in+immunocompetent+mouse+models+of+cancer.">Visualizing fewer than 10 mouse T cells with an enhanced firefly luciferase in immunocompetent mouse models of cancer.</a> Proceedings of the National Academy of Sciences. 105 (38), 14342-14346 (2008).</li><li>Skovgard, M. S., 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=Imaging+CAR+T-cell+kinetics+in+solid+tumors:+Translational+implications.">Imaging CAR T-cell kinetics in solid tumors: Translational implications.</a> Molecular Therapy Oncolytics. 22, 355-367 (2021).</li></ol>

NKC reports receiving commercial research grants from Y-mAbs Therapeutics. NKC is the inventor and owner of issued patents licensed by MSK to Y-mAbs Therapeutics, Biotec Pharmacon/Lallemand, and Abpro-labs. MSK and NKC have financial interest in Y-mAbs. NKC reports receiving stock options from Eureka Therapeutics. HFG and MEC have no relevant disclosures.

While the T-BsAb blinatumomab has been approved for CD19-positive hematological malignancies, the successful implementation of T-BsAbs in solid tumors has proven much more difficult. Catumaxomab, a T-BsAb directed against epithelial cell adhesion molecule (EPCAM), was approved for the treatment of malignant ascites in ovarian cancer patients, but production of the drug was subsequently halted for commercial reasons19. No other T-BsAbs have been approved for solid tumors, underlining the challenges associated with this type of therapy. Toxicity due to cytokine release syndrome (CRS) is a common issue, although this can be managed with steroids and cytokine inhibitors. In addition to toxicity, a lack of efficacy is common in trials of T-BsAbs for solid tumors. Inducing T cell trafficking and persistence in the tumor has proven difficult, likely owing to various immunosuppressive factors in the TME14. However, even preclinically, it is often unclear why a T-BsAb that performs well in vitro fails to effectively treat tumors in vivo.
The method for tracking T cells in vivo in real time described in this article is useful to researchers for several reasons. Monitoring T cell homing provides mechanistic insight into why certain T-BsAbs succeed or fail by allowing researchers to determine when T cell trafficking has begun and for how long the T cells have persisted in the tumor during therapy. The method can be repeated at multiple time points, allowing researchers to plot the kinetics of T cell homing over the course of several weeks. (Representative results in Figure 1 show that T cells persist in the tumor for at least 28 days.) In vivo imaging of luciferase-transduced T cells also eliminates the need to sacrifice animals during treatment for histological assessment of T cell infiltration, reducing the overall cost and the number of animals needed per experiment. Because the imaging can be repeated as often as required, it also allows for a much easier and more thorough evaluation of T cell homing kinetics compared to histological analysis, which is essentially a snapshot of a single time point unless multiple animals are sacrificed at different time points. 
The most critical step of this method is the successful transduction of T cells with the luciferase construct. Unsuccessful transduction can lead to T cell death or a lack of luciferase signal in vivo. If T cells are not expanding as expected after transduction, it may be possible to reduce the viral titer with which they are transduced in order to reduce potential toxicity from the transduction process. Another approach would be to wait another day between T cell expansion steps to allow for the T cells to become more confluent before expanding them into a larger cell culture vessel. Before injecting the T cells into the mice, it is a good idea to confirm transduction success using flow cytometry (to check for expression of the TD tomato reporter) or a bioluminescent plate reader. If the transduced T cells are still not visible with bioluminescent imaging, it may be possible that the number of T cells is too low for detection using this technique. Rabinovich et al. describe a similar method using enhanced firefly luciferase to detect as few as 10 murine T cells, which may be implemented in situations requiring this level of sensitivity20. 
In summary, in vivo tracking of luciferase-transduced T cells provides a valuable tool for researchers studying T-BsAbs in immunocompromised mouse models of cancer. In addition to the applications discussed above, this method of T cell tracking has been applied to chimeric antigen receptor (CAR) T cells and could ostensibly be applied to syngeneic mouse models utilizing adoptively transferred T cells as well21. We hope that this strategy will be helpful to researchers developing translational T-BsAbs and will lead to an increased success of these therapies in patients with solid tumors.

T cell-engaging bispecific antibodies (T-BsAbs) are engineered antibodies used to provide artificial specificity to polyclonal T cells by engaging T cells through one binding arm and a tumor antigen through another binding arm. This technology has been successfully applied to hematological cancers (CD19-targeting blinatumomab1), and numerous T-BsAbs are in preclinical and clinical development for a variety of solid tumors as well2. T-BsAbs engage polyclonal T cells in a major histocompatibility complex (MHC)-independent manner, and therefore even tumors that downregulate human leukocyte antigens (HLAs) are susceptible to this type of therapy3,4. T-BsAbs have been developed in dozens of different formats, with differences in the valency and spatial arrangement of the T cell and tumor binding arms, interdomain distances, and the inclusion of an Fc domain, which affects the half-life and can induce effector functions if present5. Previous work in our lab has shown that these factors significantly affect the anti-tumor efficacy of T-BsAbs, with up to 1,000-fold differences in potency6. Through this work, we identified the IgG-[L]-scFv format as the ideal platform for T-BsAbs (see Representative Results section for more detail regarding T-BsAb formats), and have applied this platform to targets including GD2 (neuroblastoma), HER2 (breast cancer and osteosarcoma), GPA33 (colorectal cancer), STEAP1 (Ewing Sarcoma), CD19 (B cell malignancies), and CD33 (B cell malignancies)7,8,9,10,11,12,13.
One of the major challenges to successfully implementing T-BsAb therapy in solid tumors is overcoming an immunosuppressive tumor microenvironment (TME) to drive T cell trafficking to tumors14. The factors affecting T-BsAb efficacy described above have a significant impact on the ability of T-BsAbs to effectively induce T cell homing to tumors, but this effect is difficult to evaluate in an in vivo system in real time. This manuscript provides a detailed description of the use of luciferase-transduced T cells in preclinical studies of T-BsAbs to evaluate T cell trafficking to various tissues in experimental immunocompromised mouse models during treatment. The overall goal of this method is to provide a means to evaluate T cell infiltration in tumors and other tissues, as well as real-time insight into T cell homing kinetics and persistence, without the need to sacrifice animals during treatment. For the increasing number of researchers focusing on cellular immunotherapies, the ability to track T cells in vivo in preclinical animal models is crucial. We aim to provide a thorough, detailed description of the method we have employed for tracking luciferase-transduced T cells to enable other researchers to easily replicate this technique.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>293T cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma Aldrich</td>
			<td>A7030-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3/CD28 beads</td>
			<td>Gibco (ThermoFisher)</td>
			<td>11161D</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Luciferin, Potassium Salt</td>
			<td>Goldbio</td>
			<td>LUCK-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco (ThermoFisher)</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA in vitro transfection reagent (polyjet)</td>
			<td>SignaGen Laboratories</td>
			<td>SL100688</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma Aldrich</td>
			<td>E9884-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco (ThermoFisher)</td>
			<td>10437028</td>
			<td></td>
		</tr>
		<tr>
			<td>Gag/pol plasmid</td>
			<td>Addgene</td>
			<td>14887</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP plasmid</td>
			<td>Addgene</td>
			<td>11150-DNA.cg</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin-Streptomycin</td>
			<td>Gibco (ThermoFisher)</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human IL-2</td>
			<td>R&#38;D Systems</td>
			<td>202-IL-010/CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Retronectin</td>
			<td>Takara</td>
			<td>T100B</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco (ThermoFisher)</td>
			<td>25-300-120</td>
			<td></td>
		</tr>
		<tr>
			<td>VSV-G plasmid</td>
			<td>Addgene</td>
			<td>8454</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

As described in step 4.3, mice can be oriented in different positions during imaging to evaluate the presence of T cells in different tissues. Supine positioning allows for the assessment of T cells in the lungs, which is common at early time points after injection. Lateral positioning with the subcutaneous xenograft facing up is used to best assess T cell trafficking to the tumor. Female C.Cg-Rag2tm1Fwa Il2rgtm1Sug/JicTac mice were used for all experiments described in this manuscript. Figure 1 shows images from an experiment in which mice bearing GD2-positive xenografts were treated with either luciferase-transduced T cells armed ex vivo with a GD2 BsAb or luciferase-transduced T cells and GD2 BsAb injected separately17. Figure 1A shows that armed T cells trafficked to the tumor faster than unarmed T cells (Figure 1B), leaving the lungs 2 days sooner. Figure 1C shows the quantification of this phenomenon. Figure 1E-G shows that it is possible to monitor T cell trafficking for at least 28 days post-injection of the luciferase-transduced T cells, allowing researchers to track T cell homing and persistence throughout the course of treatment, unlike other techniques such as immunohistochemistry (IHC) which allow only a snapshot of T cell infiltration.
Without the ability to assess T cell trafficking in vivo, researchers testing T-BsAbs are unable to determine whether or not T cells are infiltrating and persisting in tumors during treatment. If a treatment fails, without sacrificing animals and performing IHC, it can be difficult or impossible to know whether the failure was due to a lack of T cell trafficking, persistence, T cell exhaustion, or some other cause. Figure 2A-C shows an experiment in which mice bearing breast cancer patient-derived xenografts were treated with luciferase-transduced human T cells and HER2-targeted BsAbs bearing different mutations to silence Fc functions7. Figure 2C shows that treatment with the HER2 BsAb with an unsilenced Fc had no effect on tumor growth compared to a control BsAb, whereas treatment with HER2 BsAbs containing an N297A mutation alone or in combination with a K322A mutation were able to completely halt tumor growth. Figure 2A and Figure 1B show that in groups with the silencing mutations, T cells&#39; luminescence signal reached a higher peak on day 3 post-injection and persisted at a higher level compared to control BsAb and unsilenced HER2 BsAb. Follow-up experiments showed that in mice treated with T cells and BsAbs with unsilenced Fcs, the T cells in perivascular regions of the lungs were surrounded by murine neutrophils and macrophages, suggesting an Fc-dependent sequestration of the BsAb-bound T cells which prevents migration to the tumor. The pro-tumorigenic effect of tumor-resident M2-polarized macrophages has been well described. Figure 3A,B shows that in mice bearing neuroblastoma patient-derived xenografts (PDXs), depletion of Ly6c-positive macrophages significantly increases T cell homing to tumors, resulting in decreased tumor growth and prolonged survival (Figure 3C)18. This effect is even more pronounced in osteosarcoma PDXs (Figure 3D).
In vivo T cell tracking also allows researchers to monitor how T cell trafficking kinetics are affected by other variables in antibody engineering, including structural configuration. As described in the introduction of this article, previous work in our lab underlined the importance of bivalency and cis orientation of the tumor- and T cell-binding arms for the in vivo efficacy of BsAbs. Figure 4 shows that among a panel of BsAbs targeting the tumor antigen STEAP1 (Figure 4A), the IgG-[L]-scFv format resulted in significantly more T cell infiltration on day 6 after the first dose of T cells, which persisted for the duration of treatment (Figure 4C)10. This phenomenon was not predicted by in vitro cytotoxicity assays (Figure 4B), emphasizing the importance of confirming in vitro results in in vivo models.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64390/64390fig01.jpg" /> 
Figure 1: Armed T cells exhibit faster tumor homing kinetics than BsAb-directed unarmed T cells, rapidly bypassing lung sequestration. Luciferase-transduced T cells were expanded and armed with BsAb (Luc(+) GD2-EATs). Luc(+) GD2-EATs (10 &#181;g of GD2-BsAb/2&#215;107 T cells) or Luc(+) unarmed T cells (2 x 107 cells) with or without GD2-BsAb (10 &#181;g) were administered intravenously into GD2-positive neuroblastoma PDX-bearing mice when the average tumor volume reached 100 mm3. (A) Bioluminescence images of GD2-EATs trafficking into tumors. (B) Bioluminescence images of GD2 BsAb-directed unarmed T cell trafficking into tumors over days. (C) Quantitation of T cell infiltration into tumors over time measured by bioluminescence (n = 5 mice/group) expressed as total flux or radiance (photons/s) per pixel integrated over the tumor contour (ROI). (D) Tumor growth curves of individual mice treated with GD2-EATs, GD2-BsAb plus unarmed T cells, or unarmed T cells. To test the in vivo persistence of target antigen-specific EATs, Luc(+) GD2-EATs (armed with 10 &#181;g of GD2-BsAb/2&#215;107 T cells) or Luc(+) HER2-EATs (10 &#181;g of HER2-BsAb/2&#215;107 T cells) were intravenously administered into osteosarcoma TEOS1C PDX-bearing mice. Two additional doses of non-luciferase transduced GD2-EATs or HER2-EATs were administered on day 7 and day 14. (E) Quantitation of bioluminescence of Luc(+) EATs in tumors post-treatment. (F) Tumor growth curves of individual mice treated with GD2-EATs, HER2-EATs, or unarmed T cells, or no treatment. (G) Bioluminescence images of Luc(+) GD2-EATs (upper) or Luc(+) HER2-EATs (lower) in tumors over time. Bioluminescence of Luc(+) EATs was detected over 28 days post-injection. Abbreviations: EATs = ex vivo armed T cells; ROI = region of interest. Error bars indicate the standard error of the mean. This figure has been modified from Park et al.17. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64390/64390fig01large.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/64390/64390fig02.jpg" /> 
Figure 2: T cell trafficking into HER2-positive human breast cancer PDX in DKO mice. (A) Representative bioluminescence images of T cell trafficking. (B) Quantitation of T cell trafficking into tumors over time by bioluminescence (n = 5 mice/group) expressed as total flux or radiance (photons/s) in each pixel integrated over the entire tumor contour (ROI). (C) HER2-positive breast cancer PDX (M37) growth (n = 5 mice/group) after treatment with Ctrl BsAb, HER2 BsAb, and its Fc mutants. The results shown are representative results from at least three independent experiments. The significant differences were calculated based on the area under the curve (AUC). Error bars indicate the standard error of the mean. This figure has been modified from Wang et al.7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64390/64390fig02large.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/64390/64390fig03.jpg" /> 
Figure 3: The effects of monocyte depletion on BsAb directed T cell trafficking and in vivo anti-tumor response. (A) Luciferase transduced T cells (Luc(+) T cells) or luciferase transduced GD2-BsAb armed T cells (Luc(+) GD2-EATs) were administered with anti-Ly6C antibody to the mice bearing a neuroblastoma patient-derived xenograft (PDX). (B) Bioluminescence in the lesions of the tumor was monitored. The bioluminescence images on day 7 and quantification of the bioluminescence in the lesions of the tumor. (C) In vivo anti-tumor response by GD2-EATs with anti-Ly6C antibody was tested against neuroblastoma PDXs. (D) In vivo anti-tumor effect of GD2-EATs with anti-Ly6C antibody was tested against osteosarcoma PDXs, and long-term survival was analyzed. Error bars indicate the standard error of the mean. In vivo anti-tumor effect was compared by the AUC and survival curve analyses. Tumor-infiltrating lymphocytes were quantified using the AUC of bioluminescence. Differences between samples indicated in the figure were tested for statistical significance using a two-tailed Student&#39;s t-test for two sets of data and one-way ANOVA with Tukey&#39;s post hoc test for three or more sets of data. * p &#60; 0.05; ** p &#60; 0.01; *** p &#60; 0.001; **** p &#60; 0.0001. This figure has been modified from Park et al.18. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64390/64390fig03large.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/64390/64390fig04.jpg" /> 
Figure 4: Anti-tumor activities of anti-STEAP1 T cell-engaging bispecific antibody. (A) Schematic representation of the six structural formats of STEAP1 BsAb. (B) Antibody-dependent T cell-mediated cytotoxicity (ADTC) assay against EFT cell lines (TC32 ad TC71) using different formats of STEAP1 BsAb. Effector to target (ET) cell ratio was 10:1. (C) Bioluminescence imaging (BLI) of Luc(+) T cells armed with six different formats of STEAP1 BsAb on (a) day 6 and (b) day 18 post-treatment and quantitation of bioluminescence intensity in the lesions of the tumor. Luciferase transduced T cells (Luc(+) T cells) were armed with various formats of STEAP1 BsAb (10 &#181;g of STEAP1 BsAb/2 x 107 T cells) and administered with supplementary IL-2 (1,000 IU/dose) to the mice bearing EFT PDXs (ES15a), and the BLIfollowed. Abbreviations: EFT = Ewing sarcoma family of tumors; PDXs = patient-derived tumor xenografts; STEAP = six-transmembrane epithelial antigen of prostate. Error bars indicate standard error of the mean. Tumor-infiltrating lymphocytes were quantified by calculating the area under the curve of the bioluminescence. Differences between samples indicated in the figure were tested for statistical significance using one-way ANOVA with Tukey&#39;s post hoc test. **** p &#60; 0.0001. This figure has been modified from Lin et al.10. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64390/64390fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Tracking Bispecific Antibody-Induced T Cell Trafficking Using Luciferase-Transduced Human T Cells at JoVE.com

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

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 ...

tracking-bispecific-antibody-induced-t-cell-trafficking-using

Nanyang Technological University

Research

JoVE Journal

Cancer Research

1.1K Views.  Memorial Sloan Kettering Cancer Center. 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. 

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

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the tumor microenvironment and respond adequately to tumor antigens. The recent development of improved imaging approaches, such as 2-photon microscopy, and the use of powerful mouse tumor models have shed light on some of the mechanisms that regulate anti-tumor T cell activities. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tumor samples from human patients, as well as in vitro studies. However, in vitro models lack the complex three-dimensional tumor milieu and, therefore, are incomplete approximations of in vivo T cell activities. Fresh slices made from explanted tissue represent a complex multi-cellular tumor environment that can act as an important link between co-cultured studies and animal models. Originally set up in murine lymph nodes1 and previously described in a JoVE article2, this approach has now been transposed to human tumors to examine the dynamics of both plated3&#160;as well as resident&#160;T cells4.
Here, a protocol for the preparation of human lung tumor slices, immunostaining of resident CD8 T and tumor cells, and tracking of CD8 T lymphocytes within the tumor microenvironment using confocal microscopy is described. This system is uniquely placed to screen for novel immunotherapy agents favoring T cell migration in tumors.

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen receptors (CARs) for acute lymphoblastic leukemia (ALL) andnon-Hodgkin lymphoma (NHL). The focus of the field is now on emulating these successes in other hematological malignancies where less impressive complete response rates are observed. Further engineering of CAR T cells or co-administration of other treatment modalities may successfully overcome obstacles to successful therapy in other cancer settings.
We therefore present a model in which others can conduct pre-clinical testing of CD19 CAR T cells. Results in this well tested B-cell lymphoma model are likely to be informative CAR T-cell therapy in general.
This protocol allows the reproducible production of mouse CAR T cells through calcium phosphate transfection of Plat-E producer cells with MP71 retroviral constructs and pCL-Eco packaging plasmid followed by collection of secreted retroviral particles and transduction using recombinant human fibronectin fragment and centrifugation. Validation of retroviral transduction, and confirmation of the ability of CAR T cells to kill target lymphoma cells ex vivo, through the use of flow cytometry, luminometry and enzyme-linked immunosorbent assay (ELISA), is also described.
Protocols for testing CAR T cells in vivo in lymphoreplete and lymphodepleted syngeneic mice, bearing established, systemic lymphoma are described. Anti-cancer activity is monitored by in vivo bioluminescence and disease progression. We show typical results of eradication of established B-cell lymphoma when utilizing 1st or 2nd generation CARs in combination with lymphodepleting pre-conditioning and a minority of mice achieving long term remissions when utilizing CAR T cells expressing IL-12 in lymphoreplete mice.
These protocols can be used to evaluate CD19 CAR T cells with different additional modification, combinations of CAR T cells and other therapeutic agents or adapted for the use of CAR T cells against different target antigens.

Here, we present a protocol for the production and pre-clinical testing of murine CD19 CAR T cells by retroviral transduction and utilization as a therapy against established syngeneic A20 B-cell lymphoma in BALB/c mice with or without lymphodepleting pre-conditioning.

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen ...

A Spheroid Killing Assay by CAR T Cells

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric antigen receptor (CAR) redirected T cells obtained the most spectacular results, in particular with pediatric B-acute lymphoblastic leukemia (B-ALL). Classical validation methods of CAR T cells rely on the use of specificity and functionality assays of the CAR T cells against target cells in suspension and in xenograft models. Unfortunately, observations made in vitro are often decoupled from results obtained in vivo and a lot of effort and animals could be spared by adding another step: the use of 3D culture. The production of spheroids out of potential target cells that mimic the 3D structure of the tumor cells when they are engrafted into the animal model represents an ideal alternative. Here, we report an affordable, reliable and easy method to produce spheroids from a transduced colorectal cell line as a validation tool for adoptive cell therapy (exemplified here by CD19 CAR T cells). This method is coupled with an advanced live imaging system that can follow spheroid growth, effector cells cytotoxicity and tumor cell apoptosis.

This protocol is designed to assess immunotherapeutic redirected T-cell (CAR T-cell) cytotoxicity against 3D structured cancerous cells (spheroids) in real time.

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

Preparation of Exosomes for siRNA Delivery to Cancer Cells

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, and intrinsic capability in intercellular delivery of various biomolecules. This work establishes an isolation protocol to achieve high yield and high purity of exosomes for siRNA delivery. Human Embryonic Kidney cells (HEK-293 cells) are cultured in bioreactor flasks and the culture supernatant (hereon referred to as conditioned medium) is harvested on a weekly basis to allow for enrichment of HEK-293 exosomes. The conditioned medium (CM) is pre-cleared of dead cells and cellular debris by differential centrifugation and is subjected to ultracentrifugation onto a sucrose cushion followed by a washing step, to collect the exosomes. Isolated HEK-293 exosomes are characterized for yield, morphology and exosomal marker expression by nanoparticle tracking analysis, protein quantification, electron microscopy and flow cytometry, respectively. Small interfering RNA (siRNA), fluorescently labeled with Atto655, is loaded into exosomes by electroporation and excess siRNA is removed by gel filtration. Cell uptake in PANC-1 cancer cells, after 24 h incubation at 37 &#176;C, is confirmed by flow cytometry. HEK-293 exosomes are 107.0 &#177; 8.2 nm in diameter. The exosome yield and particle-to-protein ratio (P:P) ratio are 6.99 &#177; 0.22 &#215; 1012 particle/mL and 8.3 &#177; 1.7 &#215; 1010 particle/&#181;g, respectively. The encapsulation efficiency of siRNA in exosomes is ~ 10-20%. Forty percent of the cells show positive signals for Atto655 at 24 h post-incubation. In conclusion, exosome isolation by ultracentrifugation onto sucrose cushion offers a combination of good yield and purity. siRNA could be successfully loaded into exosomes by electroporation and subsequently delivered into cancer cells in vitro. This protocol offers a standard procedure for developing siRNA-loaded exosomes for efficient delivery to cancer cells.

An exosome is a new generation of drug delivery carriers. We established an exosome isolation protocol with high yield and purity for siRNA delivery. We also encapsulated fluorescently labelled non-specific siRNA into exosomes and investigated the cellular uptake of siRNA-loaded exosomes in cancer cells.

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, ...

Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells

Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are significant limitations to its widespread use in the treatment of cancer. These limitations include the development of unique toxicities such as cytokine release syndrome (CRS) and neurotoxicity (NT) and limited expansion, effector functions, and anti-tumor activity in solid tumors. One strategy to enhance CAR-T efficacy and/or control toxicities of CAR-T cells is to edit the genome of the CAR-T cells themselves during CAR-T cell manufacturing. Here, we describe the use of CRISPR/Cas9 gene editing in CAR-T cells via transduction with a lentiviral construct containing a guide RNA to granulocyte macrophage colony-stimulating factor (GM-CSF) and Cas9. As an example, we describe CRISPR/Cas9 mediated knockout of GM-CSF. We have shown that these GM-CSFk/o CAR-T cells effectively produce less GM-CSF while maintaining critical T cell function and result in enhanced anti-tumor activity in vivo compared to wild type CAR-T cells.

Here, we present a protocol to genetically edit CAR-T cells via a CRISPR/Cas9 system.

Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are ...

Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells with chimeric antigen receptor (CAR) and expand their progeny ex vivo. We include assays to measure CAR expression as well as differentiation, proliferative capacity and cytolytic activity. We describe assays to measure effector cytokine production and inflammatory cytokine secretion in CAR T cells. Our approach provides a reliable and comprehensive method to culture CAR T cells for preclinical models of adoptive immunotherapy.

Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy

We describe an approach to reliably generate chimeric antigen receptor (CAR) T cells and test their differentiation and function in vitro and in vivo.

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells ...

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 ...

Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced killer (CIK) T cell therapy has been reported to exert significant cytotoxic effects against cancer cells and to reduce the adverse effects of surgery, radiation, and chemotherapy in cancer treatments. CIK can be derived from peripheral blood mononuclear cells (PBMCs), bone marrow, and umbilical cord blood. CIK cells are a heterogeneous subpopulation of T cells with CD3+CD56+ and natural killer (NK) phenotypic characteristics that include major histocompatibility complex (MHC)-unrestricted antitumor activity. This study describes a qualified, clinically applicable, flow cytometry-based method for the quantification of the cytolytic capability of PBMC-derived CIK cells against hematological and solid cancer cells. In the cytolytic assay, CIK cells are co-incubated at different ratios with prestained target tumor cells. After the incubation period, the number of target cells are determined by a nucleic acid-binding stain to detect dead cells. This method is applicable to both research and diagnostic applications. CIK cells possess potent cytotoxicity that could be explored as an alternative strategy for cancer treatment upon its preclinical evaluation by a cytometer setup and tracking (CS &#38; T)-based flow cytometry system.

Here, we present a protocol to perform the isolation and expansion of peripheral blood mononuclear cells-derived cytokine-induced CD3+CD56+ killer cells and illustrate their cytotoxicity effect against hematological and solid cancer cells by using an in vitro diagnosis flow cytometry system.

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced ...

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from S&#233;zary Syndrome Patients for Transcriptomic Profiling

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides (MF) and S&#233;zary syndrome (SS) represent the most common subtypes. Despite a number of studies on characterizing gene expression, genetic alterations, and epigenetic abnormalities of CTCL, the molecular pathogenesis of MF/SS remains unclear. MF refers to the more common CTCL with a skin-predominance, and is usually limited to skin, whereas&#160;SS is an aggressive leukemic variant of CTCL with widespread skin involvement and is characterized by neoplastic distribution mainly involving blood, skin, and lymph node. Focusing on clinical practice, the identification of gene expression biomarkers has&#160;enormous potential to improve diagnosis and treatment of MF/SS. Indeed, recent transcriptomic studies have identified potential diagnostic biomarkers from differences in gene expression between normal and malignant T cells, which may improve our understanding of SS biology, and reveal potential therapeutic targets. This manuscript describes a detailed reproducible protocol for the isolation of peripheral blood mononuclear cells from fresh whole blood from patients diagnosed with SS, selection of CD4+ memory T cells (CD4+CD45RO+ T cells), chemical stimulation, and preparation of RNA suitable for transcriptomic profiling to discover novel prognostic molecular markers to gain additional insight in disease etiology. The stimulation using chemical agonist to activate nuclear regulation provides more specific assessment for pathways important in the dynamic transcription regulation and gene expression and eliminates confounding defects that may arise from upstream signaling defects arising from TCR antigen loss at the cell membrane. The data obtained from comparison of transcriptome of unstimulated to stimulated SS T cells unmasks functional regulatory gene expression defects not evident from analysis of quiescent unstimulated cells. Furthermore, the method outlined from this approach can be adapted for studying T cell gene expression defects in other T cell immune diseases.

We present a simple protocol for the isolation of peripheral blood mononuclear cells from whole blood obtained from patients diagnosed with S&#233;zary Syndrome, followed by selection of CD4+ T cells, their stimulation with phorbol12-myristate13-acetate and A23187 ionophore, and preparation of RNA for transcriptomic profiling.

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides ...

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

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells

A Spheroid Killing Assay by CAR T Cells

Preparation of Exosomes for siRNA Delivery to Cancer Cells

Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells

Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy

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

Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from Sézary Syndrome Patients for Transcriptomic Profiling