AP1903

Generation of Suicide Gene-Modified Chimeric Antigen Receptor-Redirected T-Cells for Cancer Immunotherapy

Kentaro Minagawa, Mustafa Al-Obaidi, and Antonio Di Stasi

Abstract
Chimeric antigen receptor (CAR)-redirected T-cells are a powerful tool for the treatment of several type of cancers; however, they can cause several adverse effects including cytokine release syndrome, off-target effects resulting in potentially fatal organ damage or even death. Particularly, for CAR T-cells redirected toward acute myeloid leukemia (AML) antigens myelosuppression can be a challenge. The previously validated inducible Caspase9 (iC9) suicide gene system is one of the approaches to control the infused cells in vivo through its activation with a nontherapeutic chemical inducer of dimerizer (CID). We performed a preclinical validation using a model of CD33+ AML, and generated iC9 CAR T-cells co-ex- pressing a CAR targeting the AML-associated antigen CD33 and a selectable marker (ΔCD19). ΔCD19selected (sel.) iC9-CAR.CD33 T-cells were effective in controlling leukemia growth in vitro, and could bepartially eliminated (76%) using a chemical inducer of dimerization that activates iC9. Moreover, to completely eliminate residual cells, a second targeted agent was added. Future plans with these methods are to investigate the utility of iC9-CAR.CD33 T-cells as part of the conditioning therapy for an allogeneic hematopoietic stem cell transplant. Additional strategies that we are currently validating include (1) the modulation of the suicide gene activation, using different concentrations of the inducing agent(s), to be able to eliminate CAR T-cells modified by a regulatable gene, ideally aiming at preserving a proportion of the infused cells (and their antitumor activity) for mild to moderate toxicities, or (2) the co-expression of an inhibitory CAR aiming at sparing normal cells co-expressing an antigen not shared with the tumor.

1 Introduction
1.1 Chimeric Antigen Receptor (CAR) T-Cells for Acute Myeloid Leukemia (AML)
Chimeric antigen receptor (CAR)-redirected T-cells are a powerful tool for the treatment of several type of cancers [1]. Particularly, anti-CD19 genetically redirected CAR T-cells have shown an impressive rate of complete response in patients with relapsed/ refractory acute lymphoid leukemia (ALL). CAR T-cells have also been tested against CD123 [2–11], CD44v6 [12], CD33 [5, 8, 13–16], Lewis Y (LeY) difucosylated carbohydrate antigen [17],PR1 peptide [18], and folate receptor β [19, 20] acute myeloid leukemia (AML) associated antigens.
For clinical applications with CAR T-cells to refractory AML, only results from two small clinical trials have been reported. In one trial targeting the Le-Y antigen, 4 AML patients were enrolled, and one patient achieved cytogenetic remission [17]. The other study was a pilot trial with CAR T-cells redirected for CD33. With this trial a patient with refractory AML experienced a marked but transient decrease in the percentage of bone marrow blasts [15]. Currently several clinical trials are going on, including those with CD123 (www.clinicaltrials.gov NCT02159495, NCT02623582, NCT02937103, NCT03114670) and CD33 (NCT01864902, NCT02958397, NCT03126864) targeting

CAR T-cells.
One of the factors limiting the translation to CAR T-cell thera- pies for AML antigens is the resulting myelosuppression, since the targeted antigen is often shared between normal stem cells and/or myeloid progenitor cells [21].
Although incorporation of suicide genes is a promising strategy that could result in the elimination on demand of the infused cells [1, 11], it is not clear that it would be effective when the toxicity is already manifest. Furthermore, it could result in the loss of the therapeutic effect. Novel strategies are investigated to circumvent this “on-target off-tumor activation” such as repeated doses of transiently expressed “biodegradable” mRNA CAR [11, 21]. Addi- tionally, co-expression of an inhibitory CAR signaling with PD-1 or CTLA-4 intracellular domain could limit the cytotoxic effect toward some antigens expressed on some normal tissues [22].
Here we describe our experience and protocols for the genera- tion and characterization of anti-CD33-redirected CAR T-cells co-expressing the iC9 suicide gene and a selectable marker (ΔCD19), and express our considerations for potential additional strategies to enhance the safety of CAR T-cell therapies.

1.2 Inducible Caspase 9 Safety Switch
A suicide gene co-expressed in CAR T-cells would allow elimina- tion on demand of the introduced cells in case of toxicity. Our group has previously validated the iC9 suicide gene system, and tested the efficacy in clinical trials [23–25]. The iC9 suicide gene consists of FKBP12-F36V domain linked, via a flexible Ser-Gly- Gly-Gly-Ser linker to ΔCaspase 9, which is caspase without the physiological dimerization domain (CARD). FKBP12-F36V con- sists of a FKBP domain with a substitution at residue 36 of phenyl- alanine for valine, binding synthetic dimeric ligands, AP1903(in vivo), and closely related AP20187 (in vitro), with high selec- tivity and affinity. This construct also linked a truncated CD19 (ΔCD19) molecule as a selectable marker by 2A self-cleaving pep- tide. Previous studies showed a single 10 nM dose of the chemical inducer of dimerization (CID) induces apoptosis in up to 99% ofhigh transgene expressing T-cells. Also, this system containing CAR CD19 can eliminate these gene-modified T-cells in a dose- dependent manner in an in vivo mouse model [26]. One limitation of this approach would be the loss of the therapeutic effects. There- fore, additional strategies under investigation in our lab involve(1) a regulatable system to control the adverse event without completely eliminating the cells, or (2) the co-expression of an inhibitory CAR aiming at sparing normal cells co-expressing an antigen nonshared with the tumor.

1.3 In Vitro Preclinical Validation of iC9 CAR.CD33 T Cells
We genetically modified human activated T-cells from healthy donors or patients with acute myeloid leukemia with retroviral supernatant encoding the iC9 suicide gene, a ΔCD19 selectable marker, and a humanized third generation chimeric antigen recep- tor recognizing human CD33 (Fig. 1). We reported on the ability of these cells to effectively control leukemia growth in vitro, and the ability to conditionally and partially eliminate (76%) the gene- modified cells through activation of the suicide gene alone with the nontherapeutic dimerizing agent, or to completely eliminatethem through activation of the suicide gene in combination with a second targeted agent, especially aiming at eliminating eventual resistant cells (BCL-2 inhibitor (ABT-199), pan-BCL inhibitor (ABT-737), or Mafosfamide). All these agents are suitable for clinical application.
Albeit never performed in the clinical setting, our future plan is to investigate the utility of iC9-CAR.CD33 T-cells as part of the conditioning therapy for an allogeneic hematopoietic stem cell transplant for acute myeloid leukemia, together with other myelo- suppressive agents, while the activation of the inducible Caspase9 suicide gene would grant elimination of the infused gene-modified T-cells prior to stem cell infusion to reduce the risk of engraftment failure as the CD33 is also expressed on a proportion of the donor stem cell graft.
Here, we describe the protocol for generating gene-modified T cells targeting human CD33, co-expressing the iCaspase 9 suicide gene system and the ΔCD19 selectable marker, and describe the in vitro functional assay by us employed to characterize those cells.

2 Materials
2.1 Common Materials and Reagents
1. T cell medium: 45% Advanced RPMI 1640 (Thermo Fischer Scientific), 45% Click’s medium (Irvine Scientific), 10% FBS (GE Healthcare Life Science), 2 mM L-glutamine (Thermo Fischer Scientific).
2. Human interleukin-2 (working solution 200 IU/μL) (Miltenyi Biotec).
3. Trypsin (Thermo Fischer Scientific).
4. D-PBS (Thermo Fischer Scientific).
5. DMSO (Fischer Scientific).
6. 10 mL syringe (BD Biosciences).
7. 15 and 50 mL conical tubes (Falcon).
8. Sterile tubes (Eppendorf).
9. T25 and T75 tissue culture flasks with vented caps (Corning).
10. 24-Well plates (Corning).
11. 96-Well flat-bottom plates (Corning).
12. Cryovials (Nunc).
13. 18G and 20G needles (BD Biosciences).
14. Appropriate antibody for flow cytometry staining and acquisi- tion (see text).

2.2 Retrovirus Supernatant
1. 293T producer cells (ATCC).
2. Cationic liposome formulation (Life technologies).
3. 90% IMDM medium (Life technologies), 10% FBS (GE Healthcare Life. Science), 2 mM L-glutamine (Thermo Fischer Scientific).
4. Opti-MEM (Life technologies).
5. Peg-pam-3e plasmid containing the sequence for MoMLV gag-pol.
6. RDF plasmid containing the sequence for the RD114.
7. SFG-iC9.2A.ΔCD19.2A.CARCD33 plasmid, SFG-CARCD33 plasmid, SFG-GFP-firefly luciferase (ffLuc) plasmid.
8. 10 cm cell culture dish (Corning).
9. 0.25 μm syringe filter (Fisher Scientific).

2.3 Peripheral Mononuclear Cells (PBMCs)
1. Donor peripheral blood.
2. Lymphoprep (stem cell).

2.4 Transduction
1. No azide/low endotoxin anti-CD3 and CD28 antibodies (BD Biosciences).
2. Retronectin (Takara).
3. Non-tissue culture treated 24 well plates (Costar).

2.5 MACS Enrichment
1. MACS column (Miltenyi Biotec).
2. MACS column holder and magnet (Miltenyi Biotec).
3. MACS antihuman CD19 beads (Miltenyi Biotec).
4. MACS buffer (Milteny Biotec).

2.6 Luciferase and Flow Cytometry- Based Coculture Cytotoxicity Assays
1. X-VIVO medium (Lonza).
2. Flat-bottom 96 well assay plate with black plate, clear bottom (Costar).
3. Bright-Glo™ Luciferase Assay System (Promega).
4. PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane Labeling (Phanos Technologies).

2.7 Killing Assay
1. Chemical inducer of dimerization (CID), AP20187 (Clontech).
2. Mafosfamide (Sant Cruz Biotech).
3. ABT-199 (ApexBio Technology).
4. ABT-737 (Santa Cruz Biotech).
5. Annexin-PE assay kit (BD Biosciences).

3 Methods
3.1 Preparation of Retrovirus Supernatant
1. On Day 0, plate 1.5 106 293T cells in 10 cm culture dish in total of 12 mL IMDM medium containing 10% FBS and 2 mM L-glutamine.
2. Next day (Day 1), for each plate pipette 470 μL of serum-free media (e.g., opti-MEM) to a sterile eppendorf tube. Then, add 30 μL of a cationic liposome formulation and gently mix by pipetting up and down. Incubate for 5 min at RT.
3. After 5 min incubation, add total 12.5 μg DNA per plate to cationic liposome formulation/opti-MEM mixture of the fol- lowing plasmids.
(a) RDF 3.125 μg/plate
(b) Peg-Pam-3e 4.6875 μg/plate
(c) SFG-iC9.2A.ΔCD19.2A.CARCD33 4.6875 μg/plate Gently mix by pipetting up and down and incubate for 15 min at RT.
4. After 15 min incubation, “drop-wise” add this mixture onto the 293T cells and gently swirl to distribute evenly. Return 293T cells to the incubator.
5. At 48 h (Day 2), 293T cells should be 80–90% confluent. Harvest your supernatant and filter the supernatant through a 0.45 μm syringe filter. This may result in loss of titer.
6. Aliquot 5 mL in 15 mL conical tubes. Label the tubes with printer labels. Snap freeze retrovirus supernatant in dry ice/ethanol bath and preserve supernatant at —80 ◦C.
7. Replace the media with fresh complete medium.
8. At 72 h (Day 3), 293T cells should be 100% confluent. Harvest your supernatant and snap freeze again.

3.2 Preparation of PBMC Cells
1. Dilute heparinized peripheral blood 1:1 in D-PBS.
2. Carefully overlay 10 mL Lymphoprep with 20 mL of diluted blood in a 50 mL centrifuge tube.
3. Centrifuge at 400 g for 40 min at room temperature without break.
4. Harvest mononuclear cells with aspirating pipet and resuspend in equal volume of D-PBS (see Note 1).
5. Centrifuge at 450 × g for 10 min at room temperature.
6. Aspirate supernatant.
7. Loosen pellet by “finger-flicking” and resuspend in 20 mL of D-PBS. Remove 10 μL of cells, add 10 μL of appropriate counting solution containing 50% red cell lysis buffer, and count using a hemocytometer (see Note 2).

3.3 Cryopreservation (Optional)
1. Calculate the number of vials to be frozen, ideally at 5–10 106 cells/mL and at least 5 106 cells/vial. Prepare appropriate volume of freezing medium consisting of 40% RPMI, 50% FBS, and 10% DMSO, and place on ice.
2. Centrifuge cells for 5 min at 400 × g.
3. Resuspend cells at 5–10 106 cells/mL (20 106 cells/mL) in ice-cold freezing medium.
4. Place the pellet on ice for at least 10 min.
5. Immediately transfer to labeled cryovials.
6. Labels should record donor #, cell type, amount, concentra- tion, and date.
7. Immediately transfer the vials to a Nalgene freezing container (see Note 3).
8. Transfer immediately the Nalgene container to a 80 ◦C freezer.
9. Next day, transfer the vials to liquid nitrogen tank (see Note 4).
10. Record location and cell information in the freezer inventory form.

3.4 Generation and Transduction of Human-Activated T-cells
1. The start date for CD3/CD28 activation for peripheral blood mononuclear cells (PBMC) is designated “Day 0.” The main protocol will run from Day 0 to Day 28 (Fig. 2).
2. The average time for generation of ΔCD19 selected (sel.) iC9-CAR.CD33 T-cells takes 2–3 weeks. Expanded T cells can be frozen for later experiments.
3. Calculation of the expansion of donor PBMC is required to estimate how much number of cells from donor PBMC is needed.
3.4.1 Activation of PBMC Cells with CD3/CD28 Antibodies

1. Plate 0.5 mL of H2O containing 1 μg of antihuman CD3 antibody and 1 μg of antihuman CD28 antibody per well (see Note 5).
2. Incubate for at least 4 h in 37 ◦C incubator (see Note 6).
3. Aspirate CD3/CD28/H2O from each well of the 24 well plates. Add 1 mL of T-cell medium to each well or and incubate for 15–30 min at 37 ◦C.
4. Thaw frozen PBMCs when CD3/CD28-coated plate is ready.
5. Warm 10 mL of complete medium per 1 mL frozen cells in a 15 mL conical tube.
6. Remove the cryopreserved PBMC from liquid nitrogen (see Note 7).
7. Thaw cryopreserved PBMCs in a 37 ◦C water bath.
8. Transfer the cell suspension from all vials (see Note 8).
9. Centrifuge at 400 × g for 5 min.
10. Aspirate supernatant, loosen the pellet by “finger-flicking,” resuspend the pellet in D-PBS, and count cell number with hemocytometer (see Note 9).
11. Centrifuge at 400 × g for 5 min.
12. Aspirate supernatant, loosen the pellet by “finger-flicking,” and resuspend in T-cell medium at 1 × 106 cells/mL.
13. Aspirate media from plate, and aliquot cells into a 24 well plate at 2 mL/well and place cells in incubator.
3.4.2 IL-2 Feeding
1. On Day 2, put 1 μL of medium containing 200 units/μL of IL-2 into each well.
2. Return to incubator.
3.4.3 Retroviral Transduction
1. Prepare 1 mL of H2O containing 7 μL/mL of retronectin stock for each well to be coated (see Note 10).
2. Aliquot 1 mL retronectin solution per well.
3. Label plate, mark coated wells, and seal with parafilm. Store in 4 ◦C.
4. Prepare the required volume of retroviral supernatant (each
2.0 mL/well), and transport the retroviral supernatant on ice.
5. Transfer retronectin plates from the refrigerator. Aspirate retro- nectin from each well. Wash each well with 0.5 mL complete media (see Note 11).
6. Place 0.5 mL of thawed retroviral supernatant in each retronectin-coated well and incubate for approximately 30 min in the incubator at 37 ◦C (see Note 12).
7. Harvest the T-cells to be transduced from 24 wells plates into a sterile centrifuge tube. After cell collection, there are still cells adherent to the wells. Add 0.5 mL of cell dissociation media to each well and incubate for 5 min at room temperature. Then, add 1 mL of cold D-PBS. Harvest the rest of T-cells and transfer this suspension to the previously pooled cells.
8. Wash the cells by centrifugation at 400 × g for 5 min.
9. Aspirate the supernatant and resuspend the cell pellet in 10 mL of complete medium. Count cells and record results in worksheet.
10. Wash the cells by centrifugation at 400 × g for 5 min.
11. Adjust the cell suspension volume to 1 106 cells/mL by adding additional complete medium.
12. Aspirate half of the supernatant from retronectin plates/wells.
13. Plate 0.5 mL (0.5 106 cells) of cell suspension per well. Set aside an appropriate number of non-transduced (NT) cells as control (0.5 mL cell suspension per well, and bring up to
1.5 mL with complete medium and add IL-2 to achieve a final concentration of 100 IU/mL).
14. Add 1.5 mL of retroviral supernatant per well.
15. Add IL-2 to achieve a final concentration of 100 IU/mL.
16. Label as appropriate, and return the plates to the incubator.
17. Harvest cells by gently pipetting and pool cells suspension in to 10 mL conical centrifuge tubes 1–3 days after transduction (see Note 13).
18. Centrifuge at 400 g for 5 min. Aspirate supernatant and resuspend in a volume of complete medium estimated to give 1 × 106 cells/mL.
19. Count cells.
20. Centrifuge at 400 × g for 5 min. Adjust cell concentration to 1 × 106 mL.
21. Add IL-2 to 100 IU/mL and plate cells in 24 well tissue culture plate, at 1–2 × 106 cells/well.
22. Return to 37 ◦C incubator.
23. At 3–4 days of Intervals you can expand cells subsequently. Gently resuspend cells by pipetting and transfer 1 mL of cells to the new well of 24 well plate.
24. Add 1 mL of the new complete media containing IL-2 to each well.
25. After expansion, perform flow cytometry characterization at least for lymphocyte, and memory markers, and transgene expression (Fig. 3) (see Note 14).

3.5 ΔCD19 Selection
1. Harvest iC9.2A.ΔCD19.2A.CARCD33 transduced T cells in 50 mL tube and spin at 400g for 5 min in D-PBS (see Note15).
2. Wash again with D-PBS and centrifuge at 400 × g for 5 min.
3. Add 1 μL of anti CD19 beads with 9 μL of MACS buffer/106 cells in 50 mL tube, mix well, and incubate 30 min at 4 ◦C (see Note 16).
4. Add MACS buffer about 10 mL/107 cells and spin 300 g for 10 min (see Note 17).
5. Set MS column to the magnet system and apply 1 mL of MACS buffer and discard (see Note 18).
6. Label one 15 mL tube for negative fraction and one for positive fraction.
7. After centrifugation, resuspend cells in 500 μL of cold MACS buffer and apply cells to the column.
8. Wait until the fluid go through and collect the flow through in the negative fraction tube.
9. Apply 500 μL of cold MACS buffer three times and collect in the same negative fraction tube.
10. Remove columns, apply 1 mL cold MACS buffer and plunge cells in the positive fraction tube.
11. Count cells.
12. Add additional MACS buffer, spin, and resuspend with com- plete medium (see Note 19).

3.6 Luciferase- and Flow Cytometry- Based Coculture Cytotoxicity Assays
3.6.1 Luciferase-Based Coculture Cytotoxicity Assay [27]
1. Harvest target cells (MV4-11-eGFP-ffLuc), wash once, and resuspend 0.25 × 106/mL in X-VIVO medium.
2. While incubating targets, harvest and resuspend each effector NT, CARCD33, or ΔCD19 sel. iC9-CAR.CD33 activated T-cells at 2.5 106 cells/mL in T-cell complete medium. Add 200 μL to wells designated as Effector/Target ratio (E: T 10:1) (2.5 105/100 μL/well), in clear-flat-bottom 96 well assay plate.
3. Perform serial dilution from the wells of “E:T 10:1” by mixing, collecting 100 μL and plate in next well and repeat, until the last dilution (E:T 0.6:1) where you will discard 100 μL.
4. Add 100 μL X-VIVO media in the well designated as “maxi- mum,” and add 200 μL X-VIVO media in the well designated as “minimum.”
5. After having resuspended target cells at 0.25 106/mL, add 100 μL to each well (2.5 104/100 μL/well), and add to the well designated as “maximum.”
6. Mix each well with multichannel pipet without changing tips from the wells of “E:T ¼ 0.6:1” to the wells of “E:T ¼ 10:1.”
7. Incubate overnight.
8. Next day, carefully take the supernatant up to 150 μL from each well.
9. For measuring cell-associated luciferase activity, 50 μL of the prepared Bright-Glo™ Luciferase Assay System was added to each well.
10. Incubated for 5 min for the cells to be completely lysed.
11. The lysed mixture was directly measured for luminescence with a luminometer.
12. The percentage of specific lysis was calculated after subtraction of the minimum value of medium only with the following formula: [(maximum value of labeled targets only (max)— experimental)/max × 100] (Fig. 4).
3.6.2 Flow Cytometry- Based Coculture Cytotoxicity Assay [16, 28]
1. Harvest target cells (ex. patient samples), wash once, and resuspend 2 106/100 μL in Diluent C of the PKH26 Red Fluorescent Cell Linker Kit.
2. Dilute 1 μL of PKH26 Red Fluorescent dye to 250 μL of Diluent C in the different tube.
3. Take 100 μL of diluted PKH26 Red Fluorescent dye, and add it to the target cells.
4. Incubate at room temperature for 5 min.
5. Add 10 mL of RPMI containing 10% FBS to stop the reaction.
6. Spin and count the cells. Resuspend the cells to 0.25 106/ mL.
7. While staining the targets, harvest and resuspend each effector NT, CARCD33, or ΔCD19 sel. iC9-CAR.CD33 activated T-cells) at 1 106 cells/mL in T-cell complete medium. Add 100 μL to each well (E:T 4:1) (1 105/100 μL/well), in flat-bottom 96 well assay plate.
8. After having resuspended target cells at 0.25 106/mL, add 100 μL to each well (2.5 104/100 μL/well), and make the well of “only target cells” to add 100 μL of the medium without effector cells.
9. Mix each well with multichannel pipet.
10. Incubate overnight.
11. Next day, carefully take the supernatant up to 150 μL from each well for the cytokine assay, if necessary.
12. Directly stain with several antibodies according to the experiments.
13. Acquire by flow cytometry (Fig. 5).

3.7 Conditional Elimination of iC9.2A.Δ CD19.2A.CARCD33 Transduced T Cells [16, 23]
1. Seed 0.5 106 cells/200 μL of non, or ΔCD19 sel. iC9-CAR. CD33 transduced T cells in 96 well plate.
2. Add CID [10 nM] and/or the BCL-2 inhibitor (ABT-199 [2 or 10 μM]), and/or with the pan-BCL inhibitor (ABT- 737 [2 or 10 μM]), and/or mafosfamide [0.5 or 2 μg/mL] (see Note 20).
3. After overnight culture, take half of T cells and wash cells by centrifugation.
4. Percentage of cell killing is estimated with annexin V/7-AAD staining with the following formula: 100% (%Viability trea- ted/% Viability non-treated cells) (Fig. 6).
5. Remaining cells were washed by centrifugation, and cultured for 4~7 additional days in the presence of IL-2.
6. After 4~7 days, the cells were harvested, and assessed for per- centage of cell killing again.

3.8 Future Directions Adoptive immunotherapy strategies are attracting notable interest for the treatment of cancer, due to the potential of potent anti- tumor activity. However, novel toxicology challenges are emerging. In fact, the Foundation for the Accreditation of Cellular Therapy (FACT) has published a draft of the 1st edition FACT Standards for Immune Effector Cell Administration. These Standards are intended to promote quality in administration of immune effector cells, including CAR T-cells and therapeutic vaccines, and will be incorporated into a voluntary FACT accreditation in this field.
As the manufacture of those complex therapeutics requires considerable funding and infrastructure, until the commercializa- tion of dedicated products, one possibility would be the formation and/or the use of centralized manufacturing laboratories. For example, one interesting initiative is the Production Assistance for Cellular Therapies to facilitate the transition of laboratory research developments into clinical research products for use in regenerative medicine as well as the novel treatments for cardiac, pulmonary, and blood diseases, the diseases under the auspices of the NHLBI.
The implementation of some automated systems, eventual gene modification systems alternative to viral-derived ones can also contribute to a more stream lined, cost-effective, and safe therapeutic products [29].

4 Notes
1. During centrifugation, higher density cells pass through the barrier and cells with buoyant density of less than 1.077 g/mL (including lymphocytes, monocytes, and platelets) form an interface directly above the barrier.
2. Cells concentration is calculated as follows: the average count per square the dilution factor 104/mL. For total number of cells, cell concentrations are multiplied by the total volume.
3. Nalgene freezing container can be used up to five times with- out changing the isopropanol.
4. Cells should not be kept for longer than 5 days at 80 ◦C, ideally overnight.
5. Coat the cells with non-tissue culture treated 24 well plates. If stock concentration of antihuman CD3 and CD28 antibody is 1 mg/mL, put each 2 μL of stock antibody into 1 mL of H2O. The number of coated wells is to be determined based on the PBMC number prepared. We typically plate PBMC at 1 × 106 cells/well.
6. Anti-CD3/CD28 coated plate can alternatively be incubated overnight at 4 ◦C.
7. Keep the vial on dry ice until you are ready to thaw. ATCs can be generated from either frozen or fresh PBMCs.
8. Once the vial is thawed, clean it with alcohol wipes and place in the bio safety cabinet.
9. When thawing cells, loss of 10–20% of total cells is expected.
10. Prepare retronectin-coated wells for transduction on the previ- ous day (Day 1). Alternatively, retronectin-coated plate can be incubated for 4 h at 37 ◦C.
11. Put back retronectin in the tube and keep at 4 ◦C. We reuse it once within 30 days.
12. Alternatively, you can centrifuge plates at 1000 g for 5 min at room temperature.
13. Transduction occurs mostly within 16~24 h. When you harvest the cells, use nonenzymatic cell dissociation media to completely collect the cells.
14. After expansion, you can freeze those cells for later use. Every week perform cell count to assess expansion rate. We usually check the expression of the transgene by flow cytometry on Day 9~11 after transduction.
15. We typically use 10~20 106 transduced cells for each selection.
16. To aliquot the beads, with a syringe and sterile needle disinfect rubber part of the vial of CD19 beads and aspirate ~150 μL (need 1 μL/106 cells) in the hood. Put it in a cryovial (glass preferred) and label it appropriately. You can keep the vial at 4 ◦C.
17. Make sure centrifuge is set to “no break” to treat pellet gently.
18. Be careful to remove columns without touching the plunger. Put the plunger in a place in hood out-of-touch.
19. We usually plate cells with 1~2 × 106/mL.
20. To make final concentration of CID. First add 1 μL of CID, AP20187 stock solution (0.5 mM), in complete medium 9 μL (50 μM). Then add 3 μL of last dilution to 147 μL of complete medium (1 μM). Finally, add 2 μL of last dilution to 200 mcL of cells. For ABT-199 (1 μg/μL), add 1.74 μL in 200 μL of complete medium (10 μM). For ABT737 (2.5 μg/μL), add 0.65 μL in 200 μL of complete medium (10 μM). For mafos- famide (1 μg/μL), add 0.4 μL of stock solution into 200 μL of complete media (2 μg/mL) (Alternatively, dilute 1 μL of stock solution in 9 μL of complete medium, then use 4 μL of that dilution solution.)

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