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

All animal experiments were conducted under the auspices of the Animals (Scientific Procedures) Act 1986 and under UK Coordinating Committee for Cancer Research guidelines. All animal studies were conducted at the CRUK-Manchester institute and approved by the local animal welfare and ethics review body (CRUK-MI AWERB).

1. Preparations

1. Maxiprep pMP71 retroviral construct plasmid and pCL-Eco retrovirus packaging plasmids¹⁸.
   NOTE: pMP71 encodes mCherry and the CAR separated by an FMDV2A sequence. This is interchangeable with other retroviral constructs. pCL-Eco encodes gag, pol and the ecotropic envelope proteins.
2. Prepare complete T cell medium (TCM) for culturing mouse T cells using RPMI 1640 medium, 10% FCS, 1% 100x penicillin-streptomycin-glutamine (PSG).
   NOTE: The solution contains 100 IU/mL penicillin, 100 µg/mL of streptomycin and 2 mM of L-glutamine), 50 μM β-mercaptoethanol and 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES).
3. Culture A20 cells in RPMI 1640, 10% FCS and 0.05 mM β-mercaptoethanol at 37 °C, 5% CO₂.
4. Culture the Platinum-E (Plat-E) cells in complete Dulbecco's modified eagle medium (DMEM) (DMEM with 10% fetal calf serum (FCS), 2 mM L-glutamine, 1 μg/mL puromycin and 10 μg/mL blasticidin) at 37 °C, 5% CO_2.
   NOTE: Plat-E cells are derived from 293T cells and express gag, pol and ecotropic envelope retroviral proteins.
5. Prepare transfection media solutions 1 and 2 immediately prior to transfection. Prepare solution 1 (pH 7.9) to contain DMEM + 10% FCS + 25 mM HEPES, solution 2 (pH 7.1) to contain DMEM + 25 mM HEPES.
6. Prepare 10 μg/mL recombinant human fibronectin fragment solution by diluting with sterile phosphate-buffered saline (PBS) and store at -20 °C until use.
7. Sterile filter all media through 0.2 μm filters prior to use (excluding recombinant human fibronectin fragment).

2. Retroviral Transduction of T cells

1. Day 1: Preparation for transfection
   1. Seed 7.5 x 10⁶ Platinum-E (Plat-E) cells in 15 cm² tissue culture dishes in 18 mL of complete DMEM and incubate overnight at 37 °C, 5% CO₂.
2. Day 2: Transfection of Plat-E retroviral packaging cell line
   1. Prepare 20.4 μg of pcl-Eco packaging vector DNA, 39.6 μg of plasmid DNA encoding retroviral CAR construct and 150 μL of 1 M CaCl₂ to final volume of 3 mL transfection solution 2 per 15 cm² dish to be transfected. Vortex for 10 s and rest for 5 min
   2. Remove DMEM media from the 15 cm² dishes and replace with 12 mL of transfection solution 1.
      CAUTION When changing the media, the 15 cm² dishes can dry at the center. This can cause substantial death of transfected Plat-E cells. Work swiftly and remove media from just 1-2 plates at a time.
   3. Add 3 mL transfection solution 2 containing DNA and CaCl₂ to each 15 cm² dish drop-wise, evenly across each plate. Gently rock plates with a side to side motion for 10 s. Incubate at 37 °C, 5% CO₂ overnight.
3. Day 3: Preparation of virus-containing supernatant for transduction
   1. Replace the media of transfected Plate-E cells with 18 mL complete TCM and return to incubator.
      CAUTION When changing the media 15 cm² dishes can dry at the center. This can cause substantial death of transfected Plat-E cells. Work swiftly and remove media from just 1-2 plates at a time.
4. Day 3: Isolation and in vitro activation of mouse splenic T cells
   1. Remove spleens from 6-8-week-old BALB/c mice as previously described by Parkinson et al.¹⁹ and immerse them in sterile, ice-cold, PBS in a 50 mL conical tube.
   2. Use tweezers to transfer a spleen to a 1.5 mL microcentrifuge tube and homogenize using a pestle with minimal force.
   3. Use a 1000 μL pipette and ~ 800 μL PBS to transfer homogenate to a 100 μm pore cell strainer affixed to a 50 mL tube containing 5 mL PBS to attain a single cell suspension. Repeat step 2.4.2 for the additional spleens. Do not exceed 3 spleens per tube.
      CAUTION Splenocytes passed through filter can form clumps if left standing. Manually swirl tubes intermittently if processing several spleens to avoid cell clumping. Remaining fragments on the cell strainer can be further mashed using a plunger from a 5 mL syringe using minimal force.
   4. Top up to 20 mL with PBS. Layer the 20 mL cell suspension gently onto 20 mL of density gradient media (Table of Materials) in a 50 mL tube. Centrifuge the resultant overlaid suspension at 800 x g for 20 min with no brake applied.
   5. Harvest cells at interface layer using a sterile Pasteur pipette and transfer to a 50 mL tube. Top up to 50 mL with PBS and centrifuge at 800 x g for 10 min to wash. Discard the supernatant and re-suspend cells in complete TCM.
   6. Count the number of cells using a hemocytometer.
   7. Culture cells at a density of 5 x 10⁶ cells/mL in complete TCM with 30 ng/mL anti-CD3ε antibody (Clone 145-2C11), 30 ng/mL anti-CD28 antibody (Clone 37.51), 100 U/mL recombinant human IL-2 and 2 ng/mL recombinant murine IL-7. Use an appropriately sized tissue culture flask for the volume of cells harvested.
      NOTE: Antigen–presenting cells are required for T-cell activation by CD3 and CD28 antibodies, if working with purified T cells it is necessary to coat plates with antibodies, or use magnetic beads (Table of Materials)
   8. Incubate mouse splenocytes at 37 °C, 5% CO₂ overnight.
5. Day 3: Preparation of plates for transduction
   1. Coat non-tissue-culture 6-well plates with 2 mL of 10 μg/mL recombinant human fibronectin fragment and incubate overnight at 4 °C.
6. Day 4: Transduction of mouse T cells
   1. Transfer recombinant human fibronectin fragment from coated plates to fresh non-tissue-culture 6-well plates. Incubate these plates overnight at 4 °C for round 2 of transduction.
   2. Add 2 mL of TCM to each well of original recombinant human fibronectin fragment-coated plates and leave for 30 min at room temperature to block non-specific binding.
   3. Harvest retrovirus-containing supernatant from transfected Plat-E cells in 15 cm tissue culture dishes and replace with 18 mL of complete TCM.
      CAUTION Work swiftly to avoid drying of Plat-E cells.
      NOTE: Success of transfection can be checked at this stage by fluorescence microscopy if utilizing a fluorescent marker gene such as mCherry (Figure 1).
   4. Filter the retrovirus-containing supernatant through 0.45 μm filter to remove cell debris. Remove TCM from recombinant human fibronectin fragment-coated 6-well plates and add 2.5 mL of filtered retrovirus-containing supernatant or to each well (use complete TCM for mock transfection). Label each well as to the addition of retrovirus or mock media.
   5. Centrifuge the plates at 1200 x g for 30 min at room temperature.
   6. Whilst plates are spinning, collect activated T cells and count using a hemocytometer.
      1. Transduction is carried out with 5 x 10⁶ activated splenocytes in a total of 5 mL/well. Pellet the required number of splenocytes for mock/transduction in separate tubes by centrifugation at 500 x g for 5 min.
      2. Re-suspend splenocytes at a density of 5 x 10⁶ cells per 2.5 mL of filtered retrovirus-containing supernatant from step 2.6.4 or TCM as a negative control. Add recombinant human IL-2 (hIL-2) and recombinant mouse IL-7 (mIL-7) to a final concentration of 200 IU/mL and 4 ng/mL respectively.
   7. Collect 6-well plates from the centrifuge at the completion of step 2.6.5 and add 2.5 mL/well re-suspended splenocytes into appropriate wells to make a final volume of 5 mL/well and a final concentration of 100 U/mL hIL-2 and 2 ng/mL mIL-7.
   8. Centrifuge the plates at 1200 x g for 90 min at room temperature. After centrifugation, incubate the plates at 37 °C, 5% CO₂ overnight.
7. Day 5: Round 2 of transduction
   1. Collect the recombinant human fibronectin fragment from the plates as this can be re-used. Repeat steps 2.6.2 – 2.6.5.
   2. Whilst plates are spinning, collect cells from the 1^st round of transduction using a Pasteur pipette. Rinse each well with 2 mL PBS, swirl and collect any remaining cells in each well.
      NOTE: Pipette up and down to re-suspend sedimented cells. Collect each control/transduction group in separate tubes.
   3. Centrifuge tubes at 500 x g for 5 min. Re-suspend cells in 2.5 mL per well of transduction with 200 IU/mL IL-2 and 4 ng/mL IL-7. Repeat steps 2.6.7 – 2.6.8.
   4. Remove cells from the centrifuge and incubate at 37 °C, 5% CO₂ for 4 h. Collect transduced cells as in steps 2.7.2-2.7.3.
   5. Count cells, centrifuge at 500 x g for 5 min and re-suspend in complete TCM at a density of 1 x 10⁶ cells/mL with 100 U/mL hIL-2 and 2ng/mL mIL-7. Transfer to a suitably sized culture flask and incubate at 37 °C, 5% CO₂.
   6. Add fresh TCM media containing 100U/mL hIL-2 and 2ng/mL mIL-7 every 2 days, maintaining a cell density of 1 x 10⁶ cells/mL.
      NOTE: Harvested splenocytes contain a variety of cell types. Under these culture conditions, non T cells die off over the course of 2-3 days. After ~ 4 days in cell culture, the number of T cell is typically equivalent to the total number of harvested splenocytes on day 0.

3. Measurement of Transduction efficiency

1. On day 4 post transduction, collect a sample of transduced or non-transduced T cells (approximately 3 x 10⁵ cells). Centrifuge the cell suspension at 500 x g for 5 min, discard the supernatant, wash the pelleted cells once with PBS and centrifuge again.
2. Discard the supernatant and add 100 μL of PBS containing a suitable amine reactive dye (e.g., live/dead stain, 1 in 100 dilution) per well. Incubate for 15 min at room temperature in the dark.
3. Wash twice with PBS and centrifuge at 500 x g for 5 min. Discard the supernatant and incubate with 50 μL of FACS buffer containing anti-mouse CD16/CD32 antibodies for Fc receptor blocking (1 in 100 dilution). Incubate for 10 min at 4 °C.
4. Directly add 50 μL of antibody staining master mix containing anti-mouse CD4-BV786 and CD8-BV711 antibodies (final concentration of 1 μL/well in FACS buffer). Incubate for 30 min at 4 °C in dark. Repeat the wash step 3.3. Re-suspend the cells in 1% PFA buffer and keep the in the dark at 4 °C until analysis by flow cytometry.
5. Analyze cells with equivalent suitable cytometer using BV711, BV785 and mCherry fluorescence as markers of CD4 and CD8 subset and CAR expression respectively gating as in (Figure 2).

4. In vitro Validation of CAR T cell Activity

1. Seed syngeneic target CD19⁺ tumor cells with or without luciferase expression at a density of 1 x 10⁴ cells in 100 μL TCM/well in a 96-well U-bottom tissue culture plate.
2. Add 1 x 10⁴ CD19 CAR T cells/well in a volume of 100 μL/well to achieve an effector to target (E:T) ratio of 1:1.
   NOTE: E:T ratios should be established for each CAR construct and target cell line.
3. Use T cells alone and tumor cells alone as negative controls and T cells stimulated by phorbol-myristate-acetate (PMA) (50 ng/mL) and ionomycin (1 μg/mL) as positive control for Interferon gamma (IFNγ) release. Co-culture cells at 37 °C, 5% CO₂ for 16-24 h.
4. Following co-culture, centrifuge the plates at 500 x g for 5 min and collect the supernatant for further IFNγ and IL-12p70 ELISA analysis.
   NOTE: This can be stored at -80 °C.
5. Re-suspend cell pellets in 100 μL of PBS containing luciferin (final concentration of 1.5 mg/mL). Incubate the plates for 10 min at 37 °C. Then measure the luminescence from each well with a suitable luminometer.
   NOTE: Exposure times must be optimized for cell lines and density. Representative results are shown in Figure 3a. Ex-vivo cytotoxicity of CAR T cells can be modified to express luciferin by co-culture with cell lines expressing target antigen. As CAR T cells kill target cells, luciferin is released, therefore a reduction in luminometry signal is correlated with cell kill. Non-transduced cells can often have an effect on target cell viability, particularly over long incubation periods. Measure the concentration of murine IFNγ and IL-12p70 in the supernatant according to the manufacturer's ELISA protocols. Representative results are shown in (Figure 3b and 3c). Ex-vivo activation of CAR T cells by co-culture with cell lines expressing target antigen can be assayed by analyzing supernatant contents using ELISA. The ratio of CAR T cell to target cells and length of co-culture period must be optimized for each CAR construct, target cell line and analyte. PMA and ionomycin treatment can be used as a positive control to confirm quality of T cells and their ability to respond.

5. Assess Anti-cancer Activity in Mice

1. Protocol 1
   1. Perform 100 mg/kg intravenous (IV) delivery of cyclophosphamide into 6 to 8-week BALB/c mice. This allows tumor engraftment without significant lymphodepletion¹⁷ (Figure 4).
      NOTE: Establishing A20 lymphoma can take over 2 months with a suboptimal take rate. This can be improved by the use of cyclophosphamide 1 day prior to the delivery of lymphoma cells. In order to study lymphoreplete mice, we identified a dose of cyclophosphamide that could increase efficiency of lymphoma without causing lymphodepletion.
   2. The next day, inject 100 µL of 5 x 10⁵ syngeneic A20 B-cell lymphoma cells modified to express luciferase and green fluorescent protein (GFP) into mice by intravenous (IV) injection.
   3. Allow the mice to develop systemic lymphoma for ~ 17 days.
   4. Confirm the presence of systemic lymphoma by intraperitoneal (IP) injection of 100 μL of 30 mg/mL luciferin and imaging using an in vivo bioluminescence imaging system.
      1. Use separators to avoid signal spillover into adjacent mice. Expose mice for 1 min on the ventral side with a constant sized region of interest.
      2. Display relative light units (RLU) as photons per second (p/s). Settings must be optimized for each tumor model; use an exposure that can pick up early detection of tumors but does not lead to saturation as tumors reach endpoints.
      3. Record total RLU for each mouse with a constant sized region of interest. (Figure 5a and b).
   5. Inject a single dose of 1 x 10⁶ CAR T cells by IV injection into lymphoreplete mice bearing established lymphoma.
      NOTE: (Important) Dosing levels must be established for each CAR construct using a dose escalation schedule to ensure that any possible toxicities arising from CAR T cells are characterized and can be addressed. Though anti-mouse CD19 CAR T cells do not display toxicities, CAR T cells can give rise to unexpected toxicities. Where multiple CAR constructs and transduction efficiencies are not identical, the total number of T cells administered should be kept equal by the addition of non-transduced T cells into cell preparations.
   6. Monitor disease progression weekly through IP injection of 100 μL of 30 mg/mL luciferin and imaging using an in vivo bioluminescence imaging system (Figure 5c).
   7. Closely monitor mice for signs of toxicity and euthanize any mice that show early signs of hind limb paralysis (HLP) or pathological tumor burden before any suffering can arise.
      NOTE: Toxicities from A20 lymphoma can include hind limb paralysis through tumor invasion of the meninges. Check regularly for early signs of altered gait. Likewise, large IP tumors can arise which can lead to discomfort shown by altered behavior.
   8. Monitor survival of mice for 60 – 100 days (Figure 5d). Perform euthanasia by a schedule-1 method upon conclusion of the experiment.
2. Protocol 2
   1. Deliver 200 mg/kg cyclophosphamide to 6 to 8-week old BALB/c mice by tail vein injection in 100 μL of PBS per mouse.
   2. On the following day, inject of 5 x 10⁵ syngeneic A20 B-cell lymphoma cells expressing luciferase and GFP in 100 μL PBS via tail vein injection.
   3. Allow the mice to develop systemic lymphomas for ~ 7-14 days
   4. Confirm systemic lymphoma by IP injection of 100 μL of 30 mg/mL luciferin and imaging using an in vivo bioluminescence imaging system.
   5. Perform 5 Gy total body irradiation (TBI) at 0.02 Gy/min for lymphodepletion.
      NOTE: Patients undergoing CAR T-cell treatments undergo a range of regimens to achieve lymphodepletion before the administration of CAR T cells which significantly increases the engraftment of adoptively transferred CAR T cells. This can be replicated in mice with total body irradiation (TBI) (Figure 6).
   6. On the next day, inject 1 x 10⁶ CAR T cells in 100 μL of PBS via tail vein injection into mice bearing established tumors.
   7. Collect blood samples via tail vein bleeds after 7 days.
   8. Add red cell lysis buffer to each blood sample, then prepare for flow cytometry as described in section 3. Analyze CAR T cell persistence in the circulation by flow cytometry (Figure 2).
      NOTE: Addition of counting beads immediately prior to cytometry allows determination of the number of CAR T cells per milliliter of blood.
   9. Monitor disease progress as described in steps 5.1.5 – 5.1.8 (Figure 7).