The authors would like to acknowledge Dr. Laura Johnson and Dr. Richard Morgan for providing the CAR retroviral construct. The authors also thank Giao Ngyuen for her assistance with dosimetry for whole brain irradiation. This work was supported by an NIH NCI grant 1R01CA177476-01.

This protocol is based on an experimental design where 10 mice are treated with 107 CAR T cells each. This means that 108 CAR T cells will be needed; the yield should be overestimated by 5 x 107-1 x 108 to account for loss in viability. The following protocol is scaled to generate approximately 200 x 106 cells. The cells are then administered intravenously to female C57BL/6 mice with 9 day established syngeneic EGFRvIII-positive intracranial tumors, developed from the existing KR158B astrocytoma or B16 melanoma cell lines. Concomitantly with CAR T cell generation, tumor-bearing mice are administered clinically relevant doses of lymphodepletive TMZ (60 mg/kg) and WBI (16.5 Gy).
Mice were maintained and bred under pathogen-free conditions at Duke University Medical Center (DUMC). All animal experiments were performed according to protocols approved by the Duke University Institutional Animal Care and Use Committee (IACUC).&#160;
1. Administration of Temozolomide to Tumor-bearing Mice
Days 0 through 4:
<ol>
	<li>Calculate the total number of mice to be injected and multiply this by 0.5 to determine the volume of TMZ solution needed (e.g., 30 mice x 0.5 ml = 15 ml TMZ) and add 1.5 ml to this volume to account for spillover (16.5 ml TMZ).</li>
	<li>Multiply this total volume by 3 mg/ml to determine the weight of lyophilized TMZ needed (e.g., 16.5 x 3 = 49.5 mg TMZ). Weigh this into a 50 ml conical. 
	CAUTION: TMZ is toxic by inhalation, ingestion, and contact with eyes, skin, and mucosal membranes.&#160;Consult with institution&#8217;s occupational safety and/or environmental health and wellness department for recommendations on reducing risk of exposure.&#160;</li>
	<li>Multiply the total volume by 0.15 to determine the volume of dimethyl sulfoxide (DMSO) needed (e.g., 16.5 x 0.15 = 2.475 ml DMSO).&#160;Filter sterilize this volume of DMSO (plus an additional 2 ml to account for spillover) through a 0.2 &#181;m filter into a sterile tube. Add 2.475 mL of sterile DMSO to the TMZ powder.</li>
	<li>Place the TMZ/DMSO solution in a beaker of water over a hot plate at 65 &#176;C for 10-15 min. Once TMZ is fully dissolved, the solution should become pale yellow in color; if the solution becomes pink, then the TMZ is degraded, and a new solution should be prepared with a fresh lot of TMZ.</li>
	<li>Calculate the appropriate volume of sterile saline needed by multiplying 0.85 by the total volume (0.85 x 16.5 ml = 14.025 ml saline). Add 15 ml of sterile&#160;saline to a 50 ml conical. While TMZ is dissolving in the DMSO, heat saline to 65 &#176;C.</li>
	<li>Vortex the TMZ/DMSO solution to ensure that the TMZ powder is fully dissolved and slowly add warmed saline to the TMZ/DMSO solution.</li>
	<li>Immediately after preparation, fully load 15 x 1 ml tuberculin syringes with TMZ solution for administration to 4 day established tumor bearing mice.</li>
	<li>Repeat this procedure on days 1 through 4 for a total of five TMZ administrations.</li>
</ol>
2. Whole Brain Irradiation of Tumor-bearing Mice
Days 2 through 4:
<ol>
	<li>Power on the X-ray irradiator and allow it to warm-up to full voltage. Ensure that the appropriate filter is in place and input the proper voltage and current settings (Figure 2A). 
	NOTE: The dosimetry of the irradiator should be established beforehand to determine the appropriate voltage settings and grid layout (Figure 2A,B).</li>
	<li>Calculate the length of time that is necessary to result in 5.5 Gy X-ray irradiation. For example, if X-rays are delivered at a rate of 2 Gy/min, then 2.75 min is necessary to deliver a total of 5.5 Gy. Input the appropriate time duration. 
	NOTE: Table 2 provides mouse WBI doses and their clinical equivalents in humans. In the context of high-grade gliomas, 60 Gy fractionated WBI (2 Gy fractions, 5 days/week for 6 weeks) is the clinical standard of care, and the murine equivalent used here is 16.5 Gy (5.5 Gy x 3).</li>
	<li>Prepare a fresh&#160;solution of ketamine/xylazine for systemic anesthesia by adding 2 ml ketamine and 1 ml xylazine to 17 ml saline such that the final solution is 10 mg/ml ketamine and 1 mg/ml xylazine.&#160;</li>
	<li>Weigh mice and administer 10 &#956;l of ketamine/xylazine intraperitoneally per gram of body weight such that animals receive a dose of ketamine at 100 mg/kg and 10 mg/kg xylazine. Mice should be fully sedated and visibly breathing within 2 min of ketamine/xylazine administration.</li>
	<li>To maintain ophthalmic moisture in sedated animals, gently rub a small amount of artificial tears ointment on each eye.</li>
	<li>Place sedated mice on the grid such that heads are positioned in the area that receives the highest X-ray intensity (Figure 2B,C). Shield bodies from the neck down with appropriately sized lead tubing to block systemic X-ray delivery (Figure 2D).</li>
	<li>Place positioned mice under the X-ray beam such that the laser denoting the focal point of the X-ray beam is at the coordinate (0,0) on the grid layout. Begin X-ray delivery.</li>
	<li>When X-ray delivery is complete, remove animals from the irradiator and place on a warm heating pad. Do not house sedated animals with conscious animals until animals have regained sufficient consciousness to maintain sternal recumbence. Once animals can maintain sternal recumbence, place conscious animals back in their appropriate cages.</li>
	<li>Repeat this procedure on days 3 and 4 for a total of three fractionated doses of radiation.</li>
</ol>
3. Transfection
Day -1:
<ol>
	<li>Prepare D10 media by adding 50 ml fetal bovine serum (FBS) to 500 ml of Dulbecco&#8217;s Modified Eagle Medium (DMEM).</li>
	<li>Prepare T cell media (TCM) by adding 5.5 ml L-Glutamine, 5.5 ml sodium pyruvate, 5.5 ml non-essential amino acids, and 5.5 ml pencillin/streptomycin (pen/strep) to 500 ml RPMI-1640 media. Next, add 550 &#956;l of 2-mercaptoethanol and 550 &#956;l gentamicin. Finally, add 50 ml FBS.</li>
	<li>Harvest in vitro cultured HEK293T cells and bring to a concentration of 7.5 x 106 cells/ml in D10 media.</li>
	<li>Plate 10 ml D10 media and add 1 ml of HEK293T cell suspension in each of 16 x 10 cm poly-D-lysine (PDL) coated plates.</li>
	<li>Incubate overnight at 37 &#176;C with 5% CO2.</li>
</ol>
Day 0:
<ol start="6">
	<li>Replace media with 10 ml fresh D10 at least 30 min before transfecting cells.</li>
	<li>Determine the amount of vector plasmid, pCL-Eco vector, and liposomal transfection reagent required for transfection by multiplying the total number of plates + 1 (16 + 1 = 17) by 14.1 &#956;g vector plasmid (14.1 x 17 = 239.7 &#956;g), 9.9 &#956;g pCL-Eco vector (9.9 x 17 = 168.3 &#956;g), and 60 &#956;l liposomal transfection reagent (60 x 17 = 1,020 &#956;l). All reagents should be at room temperature before preparing solutions.</li>
	<li>Label two tubes A and B. In tube A, add 239.7 &#956;g vector plasmid and 168.3 &#956;g pCL-Eco vector to (1.5 x 17) ml reduced-serum modified eagle&#8217;s medium (RS-MEM). In tube B, add 1020 &#956;l liposomal transfection reagent to (1.5 x 17) ml RS-MEM.</li>
	<li>Incubate A and B separately for 5 min at room temperature.</li>
	<li>Mix A and B together gently (vortex for 1-2 sec or invert several times) and incubate for 20 min at room temperature to form lipid/DNA complex.</li>
	<li>Add lipid/DNA complex drop wise to HEK293T cells in 10 cm dish.</li>
	<li>Incubate at 37 &#176;C for 6-8 hr or overnight (do not exceed 24 hr).</li>
	<li>After 6-8 hr incubation, replace medium with 12 ml of fresh TCM for viral production. This plated media will be used on Day 2 as viral supernatant for T cell transduction.</li>
</ol>
4. Splenectomy and T Cell Preparation
Day 0:
<ol>
	<li>Pour 10 ml of TCM into a 50 ml conical and place on ice for spleen collection.</li>
	<li>Sacrifice the appropriate number of animals by CO2 asphyxiation and secondary decapitation: Place animals in a cage receiving CO2 at a flow rate of&#160;10-30% cage volume/minute, per American Veterinary Medical Association guidelines&#160;&#160;(no more than 5 animals can be sacrificed simultaneously) until respiration terminates and for two minutes thereafter. Remove animals from the CO2 chamber and decapitate. 
	NOTE: One spleen of a 6-12 week old female C57BL/6 mouse will yield approximately 4.5-5 x 107 splenocytes. Here, 4 spleens will be harvested for approximately 200 x 106 cells.</li>
	<li>Lay the mouse such that its right side is facing up, and spray with 70% ethanol. With the forceps, grab a thin fold of skin below the left ribcage and cut a slight incision with the scissors. Peel back the skin, carefully grab a thin fold of the peritoneum with forceps, and cut a small cavity.</li>
	<li>The spleen is a small, elongated, dark red organ that resembles a flattened bean; delicately grab the spleen with the forceps and excise by cutting away the surrounding connective tissue. Place the excised spleens into the conical containing 10 ml of TCM on ice.</li>
	<li>Pour spleens over a 70 &#956;m mesh cell strainer and disaggregate by mashing with the blunt end of the inside of a 5 ml syringe to generate a single-cell suspension. A maximum of two spleens should be disaggregated per mesh strainer.</li>
	<li>Use a small volume of TCM to carefully wash the strainer following disaggregation to collect any remaining splenocytes. Pool all disaggregated spleens into a single-cell suspension. Bring final volume to 50 ml with TCM for one wash and spin at 300 x g for 10 min.</li>
	<li>Prepare a solution of 1x lysis buffer by adding 5 ml 10x lysis buffer to 45 ml sterile water. To eliminate red blood cells, resuspend pellet in 5 ml of 1x lysis buffer per spleen in a 50 ml conical, mixing well by gently pipetting up and down. If exceeding 5 spleens, use a 250 ml centrifuge tube. Place conical or centrifuge tube in a 37 &#176;C water bath for 5 min.</li>
	<li>Remove the lysis reaction from the water bath and add TCM at a 1:1 ratio with lysis buffer to neutralize the reaction. Wash by spinning at 300 x g for 10 min.</li>
	<li>Aspirate supernatant and fully resuspend pellet in TCM (2 ml/spleen, 8 ml total for 4 spleens) by pipetting up and down.</li>
	<li>Count cells by adding 10 &#956;l of cell suspension to 190 &#956;l trypan blue (1:20 dilution). Multiply the number obtained in one of the four gridded squares by 20 x 104 x total volume (8 ml) to obtain the total cell number (e.g., 125 cells x 20 x 104 x 8 ml = 200 x 106 splenocytes).</li>
	<li>Dilute cells to a concentration of 2 x 106 cells/ml in TCM, supplemented with 2 &#956;g/ml concanavalin A (ConA) and 50 IU/ml recombinant human interleukin-2 (rhIL-2). Thus, for 200 x 106 splenocytes, add 92 ml media to 8 ml cells, 200 &#956;g ConA, and 5,000 IU rhIL-2.</li>
	<li>Add 2 mL cells to each well of 24-well tissue-culture treated plates, such that 4 x 106 cells are in each well (e.g., 100 ml of cells will require approximately 4 plates).</li>
	<li>Incubate overnight at 37 &#176;C with 5% CO2.</li>
</ol>
5. Transduction
Day 1:
<ol>
	<li>Calculate the number of non-tissue culture treated 24-well plates needed for transduction by multiplying the number of spleens harvested by 2 (8 plates are needed for 4 spleens).</li>
	<li>Next, calculate the required volume of recombinant human fibronectin fragment (RHFF) solution needed to coat plates by multiplying (total number of wells + 3) x 0.5 ml (8 plates x 24 wells = 192 + 3 = 195) x 0.5 ml = 97.5 ml.</li>
	<li>Prepare 97.5 ml phosphate-buffered saline (PBS) containing RHFF at a concentration of 25 &#956;g/ml by multiplying the total volume by 25 &#956;g (97.5 x 25 = 2437.5 &#956;g). Add this amount of RHFF to 97.5 ml PBS.</li>
	<li>Coat non-tissue culture treated 24-well plates by adding 0.5 ml PBS/RHFF solution per well.</li>
	<li>Incubate overnight at 4 &#176;C.</li>
</ol>
Day 2:
<ol start="6">
	<li>Dump PBS/RHFF solution from non-tissue culture treated 24-well plates.</li>
	<li>Add 1 ml/well 2% bovine serum albumin (BSA) in PBS and incubate at room temperature for 30 min.</li>
	<li>Remove BSA by firmly upending plate. Wash by adding 2 ml PBS.</li>
	<li>Collect viral supernatant by transferring media from each HEK293T PDL plate into a 250 ml centrifuge tube and spin 10 min at 500 x g.</li>
	<li>Carefully transfer viral supernatant into a fresh 250 ml centrifuge tube, being sure not to disturb the cell pellet that may have formed.</li>
	<li>Add fresh TCM to the viral supernatant such that the final volume is 3 ml more than the amount needed for RHFF-coated wells. For example, for 192 RHFF-coated wells, bring the volume of viral supernatant to 192 + 3 ml = 195 ml.</li>
	<li>Remove cultured splenocytes from the incubator and resuspend by gently pipetting up and down 2 - 3 times in each well. Transfer to a 250 ml conical. If using a multichannel pipette, using a sterile reservoir prior to transfer will help expedite this step. Count as previously described and spin at 300 x g for 10 min.</li>
	<li>Add rhIL-2 to viral supernatant at a concentration of 50 IU/ml. For example, add 50 x 195 = 9,750 IU rhIL-2 to 195 ml of viral supernatant.</li>
	<li>Resuspend splenocytes in viral supernatant at 1 x 106 cells/ml</li>
	<li>Add 1 ml/well of splenocyte suspension to RHFF-coated 24-well plates.</li>
	<li>Spin for 90 min according to the following settings: 770 x g, acceleration = 4, brake/deceleration = 0, 32 &#176;C.</li>
	<li>Prepare a TCM solution with 50 IU/ml rhIL-2. For example, prepare a 200 TCM solution by adding 10,000 IU rhIL-2. Add 1 ml of rhIL-2/TCM to each well after centrifugation.</li>
	<li>Culture overnight at 37 &#176;C in 5% CO2.</li>
</ol>
6. CAR T cell Culture and Harvest
Days 3 and 4:
<ol>
	<li>If T cells achieve &#62;80% confluence, cells may be split (this usually occurs by day 3 or day 4). To split cells, gently pipet up and down in each well 2-3 times, and move 1 ml from each well into new wells of a fresh 24-well tissue-culture treated plate. Then, add 1 ml of fresh TCM with 50 IU/ml IL-2 to each well such that final volume is 2 ml in all wells.</li>
	<li>If cells do not reach &#62;80% confluence, perform a half media change by slowly pipetting off 1 ml of media from the top of each well. Avoid disturbing cells settled on the bottom while removing media. Add 1 ml of fresh TCM containing 50 IU/ml rhIL-2.</li>
</ol>
Day 5:
<ol start="3">
	<li>Resuspend CAR T cells by gently pipetting up and down 3 times in each well and transfer to a 250 ml centrifuge tube. Spin cells at 300 x g for 10 min.</li>
	<li>Completely aspirate supernatant without disturbing pellet. Wash once with PBS, count cells as previously described, and wash with PBS a second time. A typical CAR T cell yield is approximately 1 x 106 cells per well (8 plates x 24 wells = 192 x 106 CAR T cells).</li>
	<li>Resuspend cells in PBS at a concentration of 5 x 107/ml for an injection of 1 x 107 in a volume of 200 &#956;g (e.g., for 192 x 106 CAR T cells, resuspend washed pellet in 3.84 ml PBS).</li>
</ol>
7. CAR T cell Administration to Tumor-bearing Mice
Day 5:
<ol>
	<li>Transport cells on ice to animal facility for intravenous injection. Load 500-1,000 &#956;l into insulin syringe with 27 - 31 G needle, ensuring that all bubbles are expelled.</li>
	<li>Grab a mouse at the base of the tail and place in tube restrainer.</li>
	<li>Pull the tail taut with the vein facing upward, and glide the needle approximately 1-2 mm under the skin into the vein by inserting it parallel to the tail vein. Slowly expel 200 &#956;l into the vein. If the needle is correctly inserted into the vein, the volume will easily flow in; otherwise there will be resistance and the needle will need to be removed from the tail and re-inserted correctly.</li>
</ol>

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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+effect+of+radiation+on+the+immune+response+to+cancers.">The effect of radiation on the immune response to cancers.</a> Int J Mol Sci. 15 (1), 927-943 (2014).</li><li>Fritzell, 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=Intratumoral+temozolomide+synergizes+with+immunotherapy+in+a+T+cell-dependent+fashion.">Intratumoral temozolomide synergizes with immunotherapy in a T cell-dependent fashion.</a> Cancer Immunol Immunother. 62 (9), 1463-1474 (2013).</li><li>Park, S. 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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=An+in+vivo+immunotherapy+screen+of+costimulatory+molecules+identifies+Fc-OX40L+as+a+potent+reagent+for+the+treatment+of+established+murine+gliomas.">An in vivo immunotherapy screen of costimulatory molecules identifies Fc-OX40L as a potent reagent for the treatment of established murine gliomas.</a> Clin Cancer Res. 18 (17), 4657-4668 (2012).</li><li>Newcomb, E. W., 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=Radiotherapy+enhances+antitumor+effect+of+anti-CD137+therapy+in+a+mouse+Glioma+model.">Radiotherapy enhances antitumor effect of anti-CD137 therapy in a mouse Glioma model.</a> Radiat Res. 173 (4), 426-432 (2010).</li><li>Su, Y. B., Krown, S. E., Livingston, P. O., Wolchok, J. D., Chapman, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+lymphotoxic+is+dose-intensified+temozolomide?+The+glioblastoma+experience.">How lymphotoxic is dose-intensified temozolomide? The glioblastoma experience.</a> J Clin Oncol. 23 (18), 4235-4236 (2005).</li><li>Neyns, B., Tosoni, A., Hwu, W. J., Reardon, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dose-dense+temozolomide+regimens:+antitumor+activity,+toxicity,+and+immunomodulatory+effects.">Dose-dense temozolomide regimens: antitumor activity, toxicity, and immunomodulatory effects.</a> Cancer. 116 (12), 2868-2877 (2010).</li><li>Muranski, P., 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=Increased+intensity+lymphodepletion+and+adoptive+immunotherapy-how+far+can+we+go.">Increased intensity lymphodepletion and adoptive immunotherapy-how far can we go.</a> Nat Clin Pract Oncol. 3 (12), 668-681 (2007).</li><li>Wrzesinski, C., Restifo, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Less+is+more:+lymphodepletion+followed+by+hematopoietic+stem+cell+transplant+augments+adoptive+T-cell-based+anti-tumor+immunotherapy.">Less is more: lymphodepletion followed by hematopoietic stem cell transplant augments adoptive T-cell-based anti-tumor immunotherapy.</a> Current Opinion in Immunology. 17 (2), 195-201 (2005).</li><li>Dudley, M. E., 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=Adoptive+cell+therapy+for+patients+with+metastatic+melanoma:+evaluation+of+intensive+myeloablative+chemoradiation+preparative+regimens.">Adoptive cell therapy for patients with metastatic melanoma: evaluation of intensive myeloablative chemoradiation preparative regimens.</a> J Clin Oncol. 26 (32), 5233-5239 (2008).</li><li>Sanchez-Perez, L. 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=Myeloablative+Temozolomide+Enhances+CD8(+)+T-Cell+Responses+to+Vaccine+and+Is+Required+for+Efficacy+against+Brain+Tumors+in+Mice.">Myeloablative Temozolomide Enhances CD8(+) T-Cell Responses to Vaccine and Is Required for Efficacy against Brain Tumors in Mice.</a> Plos One. 8 (3), (2013).</li><li>Su, Y. 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=Selective+CD4++lymphopenia+in+melanoma+patients+treated+with+temozolomide:+a+toxicity+with+therapeutic+implications.">Selective CD4+ lymphopenia in melanoma patients treated with temozolomide: a toxicity with therapeutic implications.</a> J Clin Oncol. 22 (4), 610-616 (1200).</li><li>Dummer, W., 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+homeostatic+proliferation+elicits+effective+antitumor+autoimmunity.">T cell homeostatic proliferation elicits effective antitumor autoimmunity.</a> J Clin Invest. 110 (2), 185-192 (2002).</li><li>Gattinoni, L., 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=Removal+of+homeostatic+cytokine+sinks+by+lymphodepletion+enhances+the+efficacy+of+adoptively+transferred+tumor-specific+CD8++T+cells.">Removal of homeostatic cytokine sinks by lymphodepletion enhances the efficacy of adoptively transferred tumor-specific CD8+ T cells.</a> J Exp Med. 202 (7), 907-912 (2005).</li><li>Wrzesinski, C., 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=Increased+intensity+lymphodepletion+enhances+tumor+treatment+efficacy+of+adoptively+transferred+tumor-specific+T+cells.">Increased intensity lymphodepletion enhances tumor treatment efficacy of adoptively transferred tumor-specific T cells.</a> J Immunother. 33 (1), 1-7 (2010).</li><li>Hughes, 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=Transfer+of+a+TCR+gene+derived+from+a+patient+with+a+marked+antitumor+response+conveys+highly+active+T-cell+effector+functions.">Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions.</a> Hum Gene Ther. 16 (4), 457-472 (2005).</li><li>Morgan, R. 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=Recognition+of+glioma+stem+cells+by+genetically+modified+T+cells+targeting+EGFRvIII+and+development+of+adoptive+cell+therapy+for+glioma.">Recognition of glioma stem cells by genetically modified T cells targeting EGFRvIII and development of adoptive cell therapy for glioma.</a> Hum Gene Ther. 23 (10), 1043-1053 (2012).</li><li>Kerkar, S. P., 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=Genetic+engineering+of+murine+CD8++and+CD4++T+cells+for+preclinical+adoptive+immunotherapy+studies.">Genetic engineering of murine CD8+ and CD4+ T cells for preclinical adoptive immunotherapy studies.</a> J Immunother. 34 (4), 343-352 (2011).</li></ol>

The authors have no conflicts of interests to declare.

The treatment timeline described here was designed to model clinical standard of care and leverage its effects for CAR adoptive therapy. CAR T cell doses, TMZ regimens, and radiotherapy administration can be modified to enhance in vivo T cell activity, lymphodepletion, and tumor killing. TMZ regimens can be increased to yield host myeloablation and increased expansion of adoptively transferred cells30. Furthermore, the lymphodepletive effects of TMZ can be recapitulated by low-dose (4 - 6 Gy) single-f...

Glioblastoma (GBM) is the most common primary malignant brain tumor and is invariably fatal. Surgical resection coupled with non-specific standard of care chemotherapy and radiotherapy fails to completely eliminate malignant cells, resulting in a dismal prognosis of less than 15 months in patients with this disease1. In contrast, immunotherapy offers a precise approach for specifically targeting tumor cells, and thus has the potential to serve as a highly effective treatment platform with reduced risk of collateral toxicity2-4. T cells engineered ex vivo to express chimeric antigen receptors (CARs) offer a versatile strategy for tumor immunotherapy. CARs are generated by fusing the extracellular variable region of an antibody with one or more intracellular T cell signaling molecule(s), in lieu of a full-length major histocompatibility complex (MHC)-restricted T cell receptor5. This mode of antibody-like antigen recognition allows for reactive antigen-specific T cells to recognize and respond to tumor antigens in the absence of MHC and can be adapted for a virtually infinite antigen repertoire.
CAR T cells engineered against a variety of tumor antigens have shown preclinical efficacy and outstanding promise in the clinic6-9. Specifically, in the context of GBM, a CAR T cell platform targeting epidermal growth factor receptor variant III (EGFRvIII), a tumor-specific mutation expressed on the cell surface10, was shown to prolong survival in glioma-bearing mice11. Despite their versatility, however, the clinical benefit of CAR adoptive therapy has not been fully realized, due in part to tumor-associated immunosuppression and immune evasion12-16 as well as challenges in establishing and maintaining antigen-specific T cells in vivo. Leveraging standard of care (SOC) with immunotherapy can potentially overcome several of these limitations, resulting in enhanced efficacy in both the preclinical and clinical setting.
SOC for post-resection GBM consists of high-dose temozolomide (TMZ), a DNA alkylating agent17, and whole brain irradiation (WBI)1. These treatments are presumed to synergize with tumor vaccines via upregulation of tumor MHC expression18-20 and the shedding of antigens by dead tumor cells17,19,21,22. Indeed, the addition of TMZ20,23 or WBI18,24 leads to enhanced antitumor efficacy of immune-based treatments in the preclinical setting. Furthermore, like many non-specific cytotoxic chemotherapeutics, TMZ is known to cause systemic lymphopenia25,26, which can be leveraged as a means of host-conditioning for adoptive therapy platforms27-29. TMZ-mediated lymphodepletion has been shown to enhance the frequency and function of antigen-specific T cells, leading to increased efficacy of an adoptive therapy platform against intracranial tumors30. In the context of CAR therapy, lymphodepletion serves as a means of host-conditioning by both reducing the number of endogenous suppressor T cells31, and inducing homeostatic proliferation32 via reduced competition for cytokines33, thus enhancing antitumor activity11,34. Given the synergistic relationship between GBM SOC and immunotherapy platforms, evaluating novel adoptive therapies and vaccine platforms in the context of SOC is critical for drawing meaningful conclusions regarding efficacy.
In this protocol, we outline a method for the generation and intravenous administration of murine EGFRvIII-specific CAR T cells alongside TMZ and WBI in mice bearing EGFRvIII-positive intracranial tumors (see Figure 1 for treatment timeline). Briefly, CAR T cells are made ex vivo by retroviral transduction. Human embryonic kidney (HEK) 293T cells are transfected using a DNA/lipid complex (containing the CAR vector and pCL-Eco plasmids) to produce virus, which is then used to transduce activated murine splenocytes that are harvested and cultured in parallel. During the course of CAR generation, murine hosts bearing EGFRvIII-positive intracranial tumors are administered fractionated whole-brain X-ray irradiation and systemic TMZ treatment at doses comparable to clinical SOC. CAR T cells are then delivered intravenously to lymphodepleted hosts.
The following procedure is described in seven separate phases: (1) Administration of Temozolomide to Tumor-bearing Mice, (2) Whole Brain Irradiation of Tumor-bearing Mice, (3) Transfection, (4) Splenectomy and T cell Preparation, (5) Transduction, (6) CAR T cell Culture and Harvest, and (7) CAR T cell administration to Tumor-bearing Mice. These phases consist of several steps that span 6-7 days and are performed concurrently.

<table><tbody><tr><td>pCL-Eco Retrovirus Packaging Vector</td><td>Imgenex</td><td>10045P</td><td>Helper vector for generating CAR retrovirus</td></tr><tr><td>Concanavalin A</td><td>Sigma Aldrich</td><td>C2010</td><td>Non-specific mitogen to induce T cell proliferation and viral transduction</td></tr><tr><td>Retronectin</td><td>ClonTech/Takara</td><td>T100B</td><td>Facilitates retroviral transduction of T cells</td></tr><tr><td>Lipofectamine 2000</td><td>Life Technologies</td><td>11668-019</td><td>Transfection reagent</td></tr><tr><td>DMEM, high glucose, pyruvate</td><td>Life technologies</td><td>11995-065</td><td>HEK293 culture media</td></tr><tr><td>RPMI 1640</td><td>Life Technologies</td><td>11875-093</td><td>T cell culture media</td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Life technologies</td><td>11058-021</td><td>Transfection media</td></tr><tr><td>200 mM L-Glutamine</td><td>Life technologies</td><td>25030-081</td><td>T cell culture media supplement</td></tr><tr><td>100 mM Sodium Pyruvate</td><td>Life technologies</td><td>11360-070</td><td>T cell culture media supplement</td></tr><tr><td>100X MEM Non-Essential Amino Acids Solution</td><td>Life technologies</td><td>11140-050</td><td>T cell culture media supplement</td></tr><tr><td>55 mM 2-Mercaptoethanol</td><td>Life technologies</td><td>21985-023</td><td>Reducing agent to remove free radicals</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Life technologies</td><td>15140-122</td><td>T cell culture media supplement</td></tr><tr><td>Gentamicin (50 mg/ml)</td><td>Life technologies</td><td>15750-060</td><td>T cell culture media supplement</td></tr><tr><td>GemCell U.S. Origin Fetal Bovine Serum</td><td>Gemini Bio Products</td><td>100-500</td><td>Provides growth factors and nutrients for &lt;em&gt;in vitro&lt;/em&gt; cell growth</td></tr><tr><td>Bovine Serum Albumin (BSA), Fraction V&amp;ndash;Standard Grade</td><td>Gemini Bio Products</td><td>700-100P</td><td>Blocks non-specific binding of retrovirus to retronectin-coated plates</td></tr><tr><td>Pharm Lyse (10x concentrate)</td><td>BD Biosciences</td><td>555899</td><td>Lyses red blood cells during splenocyte processing</td></tr><tr><td>70 &amp;mu;m Sterile Cell Strainers</td><td>Corning</td><td>352350</td><td>Filters away large tissue particles during splenocyte processing</td></tr><tr><td>100 mm BioCoat Culture Dishes with Poly-D-Lysine</td><td>Corning</td><td>356469</td><td>Promotes HEK293 cell adhesion to maximize proliferation after transfection</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Temozolomide</td><td>Best Pharmatech</td><td>N/A</td><td>Lyophilized powder prepared on the day of administration</td></tr><tr><td>Dimethyl Sulfoxide</td><td>Sigma Life Sciences</td><td>D2650</td><td>Necessary for complete dissolution of temozolomide</td></tr><tr><td>Saline</td><td>Hospira</td><td>IM 0132 (5/04)</td><td>Solvent for temozolomide and ketamine/xylazine</td></tr><tr><td>Ketathesia HCl</td><td>Henry Schein Animal Health</td><td>11695-0701-1</td><td>Ketamine solution</td></tr><tr><td>AnaSed</td><td>Lloyd Inc</td><td>N/A</td><td>Xylazine sterile solution 100 mg/ml</td></tr></tbody></table>

generation of car t cells for adoptive therapy in the context of glioblastoma standard of care

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo to express chimeric antigen receptors specific for glioma antigens (CAR T cells). The expansion and function of adoptively transferred CAR T cells can be potentiated by the lymphodepletive and tumoricidal effects of standard of care chemotherapy and radiotherapy. We describe a method for generating CAR T cells targeting EGFRvIII, a glioma-specific antigen, and evaluating their efficacy when combined with a murine model of glioblastoma standard of care. T cells are engineered by transduction with a retroviral vector containing the anti-EGFRvIII CAR gene. Tumor-bearing animals are subjected to host conditioning by a course of temozolomide and whole brain irradiation at dose regimens designed to model clinical standard of care. CAR T cells are then delivered intravenously to primed hosts. This method can be used to evaluate the antitumor efficacy of CAR T cells in the context of standard of care.

CAR T cells are generated by transduction with the EGFRvIII CAR retroviral vector11. This vector, MSGV1, was developed from the SFGtcLuc_ITE4 vector35, which contains the murine stem cell virus (MSCV) long terminal repeats, the extended gag region and envelope splice site (splice donor, sd, and splice acceptor, sa), and viral packaging signal (&#968;). The EGFRvIII CAR containing the human anti-EGFRvIII single-chain variable fragment (scFv) 139, in tandem with murine CD8TM, CD28, 4-1BB, and CD3&#950...

Watch this Scientific Journal Video about Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care at JoVE.com

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

Adoptive T cell immunotherapy offers a promising strategy for specifically targeting and eliminating malignant gliomas. T cells can be engineered ex vivo ...

generation-car-t-cells-for-adoptive-therapy-context-glioblastoma

Research

JoVE Journal

Immunology and Infection

21.3K Views.  Duke University. The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care. 

Video: Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

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 ...

Cancer Research

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 ...

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 ...

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor (CAR) T Cell Infusions in Mice

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy and radiotherapy. As many tumors lack efficacious treatments, there is a crucial need to develop more promising therapeutic options, such as immunotherapies; the use of chimeric antigen receptor (CAR) T cell therapy&#160;directed against CNS tumors is of particular interest. Cell surface targets such as B7-H3, IL13RA2, and the disialoganglioside GD2 are highly expressed on the surface of several pediatric and adult CNS tumors,&#160;raising the opportunity to use CAR T cell therapy against these and other surface targets. To evaluate the repeated locoregional delivery of CAR T cells in preclinical murine models, an indwelling catheter system that recapitulates indwelling catheters currently being used in human clinical trials was established. Unlike stereotactic delivery, the indwelling catheter system allows for repeated dosing without the use of multiple surgeries. This protocol describes the intratumoral placement of a fixed guide cannula that has been used to successfully test serial CAR T cell infusions in orthotopic murine models of pediatric brain tumors. Following orthotopic injection and engraftment of the tumor cells in mice, intratumoral placement of a fixed guide cannula is completed on a stereotactic apparatus and secured with screws and acrylic resin. Treatment cannulas are then inserted through the fixed guide cannula for repeated CAR T cell delivery. Stereotactic placement of the guide cannula can be adjusted to deliver CAR T cells directly into the lateral ventricle or other locations in the brain. This platform offers a reliable mechanism for the preclinical testing of repeated intracranial infusions of CAR T cells and other novel therapeutics for these devastating pediatric tumors.

Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor CAR T Cell Infusions in Mice

Central nervous system (CNS) tumors are the leading cause of cancer-related death in children, and locoregional immune-based therapies are increasingly being tested for patients in clinical trials. This protocol describes methods for locoregional cannula implantation in mice for the preclinical evaluation of immunotherapeutic infusions targeting CNS tumors.

Pediatric CNS tumors are responsible for the majority of cancer-related deaths in children and have poor prognoses, despite advancements in chemotherapy ...

Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA approved in April 2011 for the treatment of patients 22 years or older with rGBM. The device delivers alternating electric fields and is programmed to ensure maximal tumor cell kill1.
Glioblastoma is the most common type of glioma and has an estimated incidence of approximately 10,000 new cases per year in the United States alone2. This tumor is particularly resistant to treatment and is uniformly fatal especially in the recurrent setting3-5. Prior to the approval of the TTF System, the only FDA approved treatment for rGBM was bevacizumab6. Bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (VEGF) protein that drives tumor angiogenesis7. By blocking the VEGF pathway, bevacizumab can result in a significant radiographic response (pseudoresponse), improve progression free survival and reduce corticosteroid requirements in rGBM patients8,9. Bevacizumab however failed to prolong overall survival in a recent phase III trial26. A pivotal phase III trial (EF-11) demonstrated comparable overall survival between physicians&#8217; choice chemotherapy and TTF Therapy but better quality of life were observed in the TTF arm10.
There is currently an unmet need to develop novel approaches designed to prolong overall survival and/or improve quality of life in this unfortunate patient population. One appealing approach would be to combine the two currently approved treatment modalities namely bevacizumab and TTF Therapy. These two treatments are currently approved as monotherapy11,12, but their combination has never been evaluated in a clinical trial. We have developed an approach for combining those two treatment modalities and treated 2 rGBM patients. Here we describe a detailed methodology outlining this novel treatment protocol and present representative data from one of the treated patients.

A novel methodology that is employed for the treatment of recurrent glioblastomas is described. This treatment approach employs the application of alternating electric tumor treating fields (TTFields), known as TTF Therapy in combination with bevacizumab, a targeted agent that is currently FDA approved as monotherapy.

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA ...

Medicine

Functional Validation of Hypoxia-Sensitive CAR-T Cells for Selective Tumors

Generation and Functional Verification of Hypoxia-Sensitive Chimeric Antigen Receptor-T Cells

Extensive studies have proven the promise of chimeric antigen receptor T (CAR-T) cell therapy in treating hematological malignancies. However, treating solid tumors remains challenging, as exemplified by the safety concerns that arise when CAR-T cells attack normal cells expressing the target antigens. Researchers have explored various approaches to enhance the tumor selectivity of CAR-T cell therapy. One representative strategy along this line is the construction of hypoxia-sensitive CAR-T cells, which are designed by fusing an oxygen-dependent degradation domain to the CAR moiety and are strategized to attain high CAR expression only in a hypoxic environment-the tumor microenvironment (TME). This paper presents a protocol for the generation of such CAR-T cells and their functional characterization, including methods to analyze the changes in CAR expression and killing capacity in response to different oxygen levels established by a mobile incubator chamber. The constructed CAR-T cells are anticipated to demonstrate CAR expression and cytotoxicity in an oxygen-sensitive manner, thus supporting their capability to distinguish between hypoxic TME and normoxic normal tissues for selective activation.

Author Spotlight: Advancements in Hypoxia-Sensitive CAR-T Therapy for Enhanced Cancer Immunotherapy

Here we present a protocol for the generation and functional verification of hypoxia-sensitive chimeric antigen receptor (CAR)-T cells. This protocol presents the lentivirus-based generation of hypoxia-sensitive CAR-T cells and their characterization, including the validation of hypoxia-dependent CAR expression and selective cytotoxicity.

Extensive studies have proven the promise of chimeric antigen receptor T (CAR-T) cell therapy in treating hematological malignancies. However, treating ...

A Nonviral Approach to Generate Transient Chimeric Antigen Receptor T Cells Using mRNA for Cancer Immunotherapy

Chimeric antigen receptor (CAR) T cell therapy has emerged as a pioneering cancer treatment, achieving unprecedented success in treating certain hematological malignancies such as lymphomas and leukemias. However, as more cancer patients receive CAR-T cell therapies, treatment-associated secondary primary malignancies are increasingly being reported partly due to unexpected CAR transgene insertion, raising serious safety concerns. To address this issue, we describe here a nonviral, non-integrating approach to generate transient CAR-T cells using mRNA. We electroporated T cells with modified mRNA encoding a human epidermal growth factor receptor 2 (HER2)-specific CAR and generated transient HER2-targeted CAR-T cells. The CAR was efficiently expressed on the T cell surface 1 day after electroporation, increased by day 2, and dramatically declined by day 5. The transient CAR-T cells exhibited potent cytotoxicity against HER2-positive SKOV-3 ovarian cancer cells and secreted high levels of IFN-&#978;. This protocol provides a step-by-step guide for developing small-scale transient CAR-T cells without permanent CAR transgene integration, describing detailed procedures for preparation of CAR mRNA, activation and transfection of T cells, assessment of CAR expression, and in vitro analysis of CAR-T cell function. This method is suitable for transient CAR-T cell generation in both preclinical and clinical studies.

This protocol outlines a detailed procedure for generating transient chimeric antigen receptor (CAR) T cells using non-integrating mRNA for cancer immunotherapy and provides reliable methods for evaluating CAR-T cells and their cytotoxic function.

Chimeric antigen receptor (CAR) T cell therapy has emerged as a pioneering cancer treatment, achieving unprecedented success in treating certain ...

Stereotactic Adoptive Transfer of Cytotoxic Immune Cells in Murine Models of Orthotopic Human Glioblastoma Multiforme Xenografts

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce efficient therapies have been proposed over the last decade. Among the promising candidates for designing novel therapeutic strategies, cellular immunotherapies have been targeted to eliminate highly invasive and chemo-radioresistant tumor cells, likely involved in a rapid and fatal relapse of this cancer. Thus, administration(s) of allogeneic GBM-reactive immune cell effectors, such as human V&#978;9V&#948;2 T lymphocytes, in the vicinity of the tumor would represents a unique opportunity to deliver efficient and highly concentrated therapeutic agents directly into the site of brain malignancies. Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a preclinical proof-of-concept for both the feasibility and the antitumor efficacy of these cellular immunotherapies that rely on stereotactic injections of allogeneic human lymphocytes within intrabrain tumor beds.

Here, we present a protocol for the preparation and the stereotaxic administration of allogeneic human lymphocytes in immunodeficient mice carrying orthotopic human primary brain tumors. This study provides a proof-of-concept for both the feasibility and the antitumor efficacy of intrabrain-delivered cellular immunotherapies.

Glioblastoma multiforme (GBM), the most frequent and aggressive primary brain cancer in adults, is generally associated with a poor prognosis, and scarce ...

Streamlined Protocol for Mouse CAR-T Cell Generation in Syngeneic Models

Efficient Generation of Murine Chimeric Antigen Receptor (CAR)-T Cells

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological malignancies and are presently undergoing development for the treatment of diverse solid tumors. So far, the preliminary evaluation of novel CAR-T cell products has predominantly taken place in xenograft tumor models using immunodeficient mice. This approach is chosen to facilitate the successful engraftment of human CAR-T cells in the experimental setting. However, syngeneic mouse models, in which tumors and CAR-T cells are derived from the same mouse strain, allow evaluation of new CAR technologies in the context of a functional immune system and comprehensive tumor microenvironment (TME). The protocol described here aims to streamline the process of mouse CAR-T cell generation by presenting standardized methods for retroviral transduction and ex vivo T cell culture. The methods described in this protocol can be applied to other CAR constructs beyond the ones used in this study to enable routine evaluation of new CAR technologies in immune-competent systems.

Author Spotlight: Enhancing CAR-T Cell Function in Syngeneic Tumor Models 

This protocol streamlines retroviral vector production and murine T cell transduction, facilitating the efficient generation of mouse CAR-T cells.

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological ...

Bioengineering

Engineering and Expansion of Human CAR Tregs for Immune Regulation Therapies

Generation of Human Chimeric Antigen Receptor Regulatory T Cells

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable hematological cancers. These successes have spurred interest in adapting the CAR platform to a small yet pivotal subset of CD4+ T cells primarily responsible for regulating and inhibiting the immune response, regulatory T cells (Tregs). The ability to redirect Tregs&#39; immunosuppressive activity to any extracellular target has enormous implications for creating cell therapies for autoimmune disease, organ transplant rejection, and graft-versus-host disease. Here, we describe in detail methodologies for bona fide Treg isolation from human peripheral blood, genetic modification of human Tregs utilizing either lentivirus or CRISPR/Cas9-aided knock-in using adeno-associated virus-mediated homologous directed repair (HDR) template delivery, and ex vivo expansion of stable human CAR Tregs. Lastly, we describe the assessment of human CAR Treg phenotypic stability and in vitro suppressive function, which provides insights into how the human CAR Tregs will behave in preclinical and clinical applications.

This protocol provides a streamlined workflow to generate and test human chimeric antigen receptor regulatory T cells (CAR Tregs).

Chimeric antigen receptor (CAR) T-cell therapy has reshaped the face of cancer treatment, leading to record remission rates in previously incurable ...

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

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