Published: October 16th, 2018
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 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.
Chimeric antigen receptor (CAR) T-cell therapy has shown astonishing clinical success in the treatment of CD19+ malignancies leading to the approval of tisagenlecleucel for relapsed acute lymphoblastic leukaemia1 and axicabtagene ciloleucel for progressive large B-cell non-Hodgkin lymphoma2 in 2017.
The importance of Interactions between cancer and the immune system in both disease progression and therapeutic mechanisms is becoming increasingly recognized3,4,5. For example, it is well documented that the tumor microenvironment (TME) is awash with factors that can suppress the effector functions of immune cells6,7,8. Alternatively priming of endogenous immune cells and epitope spreading can be key in tumor eradication and long term resistance to tumor challenge9,10. Both of these phenomena cannot be evaluated in xenogeneic models that lack an immune system. Likewise, systems utilizing transgenic proteins do not accurately reflect the challenge of breaking immune tolerance which is required for epitope spreading11,12. A syngeneic model with a fully functional immune system is, therefore, paramount for modeling these important aspects of cancer disease progression and immune therapeutics.
An important caveat of CAR T-cell therapy is that lymphodepleting pre-conditioning is required for therapeutic success13,14. This is typically achieved in patients by administering chemotherapy prior to infusion of CAR T cells15,16. As a standard method, in order to mimic lymphodepletion used in the patient setting, we administer 5 Gy total body irradiation (TBI) to achieve lymphodepletion prior to administration of therapeutic CAR T cells to mice bearing systemic A20 B-cell lymphoma.
While lymphodepleting pre-conditioning is not an issue for the majority of patients, toxicity that comes with chemotherapeutic agents means that patients of low performance status are not eligible for CAR T-cell therapy. To create a test system that represents the patients ineligible for lymphodepletion, we established a lymphoreplete syngeneic mouse model in which we model CAR T-cell therapy of lymphoma. In this model, we showed that the secretion of IL-12 from within CAR T cells could lead to eradication of established lymphoma with a success rate of ~ 25%17. Moreover, we showed that endogenous immune cells were involved in cancer eradication.
Here we describe in detail the protocol for the production of mouse CAR T cells, establishing lymphoma in syngeneic mice, and treatment of lymphoma with CAR T cells with or without the use of lymphodepleting pre-conditioning. This can be used for combination studies of CAR T cells with other agents, testing CAR T cells with other transgenes or for the use of other adoptive cell therapy or immunotherapy strategies against lymphoma.
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).
2. Retroviral Transduction of T cells
3. Measurement of Transduction efficiency
4. In vitro Validation of CAR T cell Activity
5. Assess Anti-cancer Activity in Mice
For high efficiency transduction of T cells, it is necessary to obtain fresh retroviral particles. Transfection of the Plat-E cell line with pCL-Eco producer plasmid and pMP71 retrovirus plasmid gives rise to the secretion of retroviral particles into the cell supernatant. When a fluorescent marker gene, such as mCherry, is encoded in the retrovirus, successful transfection can be confirmed by fluorescence microscopy (Figure 1). Virus-containing supernatant from transfected Plat-E cells is used to transduce T cells via 2 rounds of spin-fection on fibronectin fragment-coated plates. The efficiency of transduction can be determined 4 days post transduction via flow cytometry. Successfully transduced cells express the marker gene encoded in the retrovirus (Figure 2). Transduction efficiencies range from ~ 50 - 90% efficiency with first generation receptors to ~ 10 - 40% with CAR constructs close to the retroviral packaging capacity. While marker gene expression shows successful retroviral transduction, it is paramount to show functionality of CAR T cells upon engaging with cells that express target antigen on their surface. Target cell lines modified to express luciferase can be used in luciferase assays to test the degree of cell-kill by CAR T cells directly (Figure 3A). The release of effector cytokines from CAR T cells upon co-culture with target cells, determined by ELISA, can also be used as an indirect measure of CAR T cell cytotoxicity (Figure 3B and 3C).
CAR T cells produced in this protocol can be evaluated in lymphoreplete mice by establishing systemic A20 lymphoma with a 100 mg/kg dose of cyclophosphamide (injected intravenously), 1 day prior to IV injection of 5 x 105 A20 cells (Figure 4). IP injection with luciferin and image capture using an in vivo bioluminescence imager can be used to monitor tumor burden using a constant ROI and exposure time throughout (Figure 5A-C). CAR T cells modified to express IL-12 are capable of eradicating systemic lymphoma with lymphodepleting pre-conditioning giving disease-free survival in about 25% of mice (Figure 5D). Lymphodepleting preconditioning, achieved by 5 Gy TBI 1 day prior to the IV administration of CAR T cells, significantly improves engraftment (Figure 6). In this model, first generation CAR T cells are capable of eradicating systemic A20 lymphoma, typically inducing disease-free survival in 100% of mice (Figure 7).
Figure 1. Confirmation of successful transfection of Plat E cells. Plat-E cells transfected with retroviral CAR construct and pMP71 and pcl-Eco packaging vector plasmid DNA. Successful transfection is shown by expression of the mCherry fluorescent marker gene. A) Bright field microscopy, B) fluorescence microscopy and C) merged images are shown. Magnification = 50X. Please click here to view a larger version of this figure.
Figure 2. Determining transduction efficiency by flow cytometry. Flow cytometry is used to determine the transduction efficiency of the mouse T cells on day 4 post transduction, using Zombie UV live/dead, mCherry, BV711 and BV785 for the detection of the live, CAR construct, CD4 and CD8 cells, respectively. Representative results of A) Non-transduced, B) mCherry.αmCD19.mCD3z and C) mCherry.αmCD19.mCD3z.mIL12 are shown with gating of 1) Singlets 2) Live cells 3) CD4 and CD8 4) and 5) Assessment of mCherry positive cells expressing CAR. Please click here to view a larger version of this figure.
Figure 3. Validation of CAR T-cell activity. αmCD19 CAR T cells were co-cultured with A20 lymphoma cells modified to express luciferase (1 x 104:1 x 104) for 16 h in a U-bottom 96-well plate. After co-culture, cells were pelleted, and supernatant was collected. A) Cells were re-suspended in PBS and luminometry was used to assess the viability of the target cells. Supernatant from co-culture was assessed for the presence of IFNγ (B) and IL-12 (C). The ratio of CAR T cell to target cells and length of co-culture period must be optimized for each CAR construct and target cell line. PMA and ionomycin treatment can be used as a positive control to confirm quality of T cells and their ability cells to respond. Error bars show SD. Statistical analysis was performed using one-way ANOVA. *** p < 0.001). This figure has been modified from17. Please click here to view a larger version of this figure.
Figure 4. Establishing A20 lymphoma without lymphodepletion. Cyclophosphamide can increase efficiency of lymphoma induction without causing lymphodepletion. A) Blood counts of 6-8-week-old BALB/c mice after IV delivery of 100 mg/kg of cyclophosphamide. Error bars show SD B) Lymphoma burden of 6-8-week-old BALB/c mice after IV delivery of 100 mg/kg of cyclophosphamide or saline on day -1 and IV delivery of 5 x 105 A20 cells on day 0 measured using a luminometer. C) Survival of mice in B). Error bars show SD. Statistical analysis was performed using 2-way ANOVA. ** p < 0.01, *** p < 0.001). This figure has been modified from Kueberuwa et al.17. Please click here to view a larger version of this figure.
Figure 5. Monitoring lymphoma burden and survival. Mice bearing A20 lymphoma expressing luciferase receive 100 µL intraperitoneal (IP) injections of 30 mg/mL luciferin and were imaged using an in vivo bioluminescence imaging system. A) Mice were exposed for 1 min on the ventral side and immediately flipped over to image dorsal to pick up tumor masses on both sides of the bodies (B). C) Representative results of the lymphoma burden of BALB/c mice receiving varying αmCD19 CAR T cells without lymphodepletion. Error bars show SEM. D) Survival rate of the same mice. This figure has been modified from Kueberuwa et al.17. Please click here to view a larger version of this figure.
Figure 6. Effects of lymphodepletion. A) Blood counts of 6-8-week-old BALB/c mice after receiving 5 Gy TBI at a dose rate of 0.02 Gy/min; error bars show SD. Statistical analysis by two-way ANOVA. * p < 0.05, ** p <0.01, *** p < 0.001. B) Monitoring of CD4+ and CD8+ CAR T cells in the peripheral blood of mice by flow cytometry for the mCherry marker gene 7 days post administration. Error bars show SD. This figure has been modified from Kueberuwa et al.17. Please click here to view a larger version of this figure.
Figure 7. CAR T cell activity with lymphodepleting pre-conditioning. Typical results showing the effect of 5 Gy TBI the day prior to CAR T-cell administration. A) Imaging and (B) graphical displays of imaging of mice after 100 µL intraperitoneal (IP) injections of 30 mg/mL luciferin using an in vivo bioluminescence imaging system. Error bars show SEM. C) Survival of the same mice. This figure has been modified fromKueberuwa et al.17. Please click here to view a larger version of this figure.
Syngeneic mouse models allow the testing of disease progression and therapy while maintaining an intact immune system. This is paramount when it comes to therapies that interact with the immune system and in particular for immunotherapeutic agents.
The protocol described here has two critical work streams, the first one is genetically modifying mouse T cell to express CARs. This requires 7 days from initiation to the validation of the transduction. Concomitant with the production of CAR T cells is the establishment of systemic lymphoma in mice. Should CAR T cell production fail, or quality being insufficient, there is typically not enough time to produce replacement cells before mice succumb to lymphoma. It is therefore critical that researchers using these models accurately perform tumor dosing and disease progression studies in order to successfully time the production of CAR T cells for therapeutic administration.
Typical reasons for low T-cell transduction efficiency includes poor transfection efficiency of producer cells, typically caused by poor plasmid purity or inaccurate determination of the pH of transfection media. It is recommended to check the efficiency of producer cell transfection before proceeding with the full protocol as poor transfection will limit the efficiency of T-cell transduction. Recombinant human fibronectin fragments can be collected and stored at -20 °C for re-use, however, multiple freeze-thaws result in reduced transduction efficiency. Swift processing of mouse spleens after collection is also important for obtaining high yields of viable T cells.
It should be noted that the protocol described here utilizes A20 cells expressing luciferase. This is preferred as it provides the ability to measure systemic tumor burden by bioluminescence imaging. However, in the presence of a functional immune system, responses to luciferase could skew the results. We have previously tested immune reactions of surviving mice to marker transgenes17. It is key to replicate key experiments using A20 cells free of transgenes to validate that these do not play a significant role in tumor eradication by immune cells.
While clinical agents can only be used in vivo in immune-deficient mice, the use of mouse CAR T cells against mouse cancer cells allows us to evaluate the contributions of the immune system to therapeutic efficacy or disease progression. This protocol could be utilized for the pre-clinical evaluation of CARs targeting B-cell lymphoma or other CARs with additional modifications such as secretion of IL-12 as described here. It must be noted that although the interplay between immune cells can be evaluated in syngeneic mouse models, they may not accurately recapitulate interaction in humans in vivo. Of particular note, human and mouse CARs will vary in the structure which may have downstream consequences; optimal activation and cell culture conditions for growth of T cells are different20, tissue distribution of target antigen expression may vary between humans and mice and experienced toxicities may be radically different. It is therefore essential to utilize ex vivo and xenogeneic models to corroborate results.
In summary, the syngeneic lymphodepleted and lymphoreplete model of lymphoma recapitulate patients with and without prior chemo/radiotherapy. This provides a model system in which to mimic the clinical settings to allow the testing of a range of therapeutic strategies that will be important with the coming wave of new immune therapy agents.
With the use of pre-conditioning, it will be noted that all the mice typically clear the lymphoma. With up to 90% complete response rates in humans, this is representative. However, the challenges for CD19 CAR T-cell therapy will hinge on preventing the high frequency of relapses observed that are often CD19. Relapses have not been observed in this model up to, and often beyond 100 days. Modifications to mimic the relapses seen in the clinic could help with the future challenges of CD19 CAR T-cell therapy.
David Gilham works for Celyad which is involved in the production of CAR T cells. The rest of the authors have nothing to disclose.
We would like to thank Bloodwise for funding this research (grant 13031) and the CRUK Manchester biological resource unit, imaging and cytometry and molecular biology core facilities for supporting this work.
|0.2 µm syringe filter
|0.45 µm syringe filter
|1.5ml pestle and microtube
|100X penicillin-streptomycin-glutamine (PSG)
|2-Mercaptoethanol (50 mM)
|Blasticidine S hydrochloride
|Bottle Top Filter (0.2 µm)
|Scientific Laboratory Supplies
|Brilliant Violet 711 anti-mouse CD8a Antibody
|1 in 100 staining dilution. Clone 53-6.7
|Brilliant Violet 785 anti-mouse CD4 Antibody
|1 in 100 staining dilution. Clone RM4-5
|Calcium chloride dihydrate
|Cell counting beads – CountBright absolute counting beads
|Cell Strainer 100μm
|Dulbecco’s Modified Eagle medium (DMEM) - High Glucose
|Ficoll Paque Plus
|Sold by Sigma- Aldrich
|Flow cytometer - LSR Fortessa x20
|Foetal Bovine Serum
|IL-12 p70 Mouse Uncoated ELISA Kit
|in vivo bioluminescence imaging system – in vivo xtreme II imaging system
|Ionomycin Calcium Salt
|Live/dead stain - Zombie Violet Fixable Viability Kit
|1 in 100 staining dilution
|Luminometer - Lumistart Omega
|Murine IFN-γ ELISA kit
|Phorbol 12-myristate 13-acetate (PMA)
|Platinum E cell line
|Purified NA/LE Hamster Anti-Mouse CD28
|Purified NA/LE Hamster Anti-Mouse CD3ε
|Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block)
|1 in 100 staining dilution. Clone 2.4G2
|Recombinant human fibronectin fragment - RetroNectin Reagent
|Recombinant Mouse IL-7 (carrier-free)
|Red cell lysis buffer
|RPMI 1640 Medium
|Trypsin - EDTA solution
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