Abstract
Background aims:
Intracranial (IC) locoregional delivery of chimeric antigen receptor (CAR) T cells presents in an attractive delivery method to central nervous system (CNS) tumors. While IC delivery is actively being employed in early phase clinical studies, no thaw/wash methods are published to remove the neurotoxic cryoprotectant dimethyl sulfoxide (DMSO) from CAR T cell products prior to IC administration. Thus, the aim of this study was to develop and validate a simple thaw/wash procedure.
Methods:
We developed a thaw/wash procedure that consist of product thaw at 37°C, equilibration for 5 minutes in 1 volume of preservative free normal saline (PFNS), dilution with an additional 8 volumes of PFNS, removal of DMSO through a washing step, resuspension in 2.0 mL of PFNS, and storage in a syringe at 20–25°C. Final formulated products (FPs) were assessed for quality and safety attributes and stability over 3 hours from the completion of the thaw. Stability parameters included CAR T cell viability, transgene surface expression, and cytolytic activity.
Results:
The developed procedure reduced the calculated % of DMSO to less than 0.025%. FP cell viability and recovery (vs pre-cryopreservation) were within acceptable specifications (mean viability: 85.3%, range: 83%−88%; total nucleated cell recovery mean: 76.5%, range: 65.4%−82.5%). Other prespecified Quality Assurance/Quality Control parameters including appearance/ integrity, sterility, and endotoxin level (≤1.0 EU/mL), were also met by all FPs (n=3). Three hours post thaw/wash stability was confirmed. All products maintained cell viability above 70% (mean: 80.0%, range: 79–81%), with no significant change in transgene expression or cytolytic activity of B7-H3-CAR T cells compared to thawed not diluted/washed control CAR T cells.
Conclusion:
We have developed a simple thaw/wash procedure to prepare B7-H3-CAR T cells for their locoregional delivery to the neural axis. While we focus here on CAR T cells, the methods could be readily adapted to other cryopreserved immune effector cell products.
Keywords: CAR T cells, brain tumor, intracranial, locoregional, DMSO
INTRODUCTION
Chimeric antigen receptor (CAR) T cell therapy for recurrent/refractory brain tumors holds the promise to improve outcome for these recalcitrant malignancies [1]. Preclinical studies have demonstrated that the intravenous (IV) or intracranial (IC; locoregional or intratumoral) delivery of CAR T cells results in potent antitumor activity in xenograft as well as immune competent animal models [2, 3]. Based on these results, multiple early phase clinical studies are in progress that explore the safety and efficacy of IV or locoregional delivered CAR T cells in humans [4–9]. For locoregional delivery most studies use an Ommaya or Rickham reservoir, which are a surgically placed into the resection cavity of the tumor or central nervous system (CNS) ventricles.
Compared to systemic IV infusion, IC delivery may result in a greater concentration of CAR T cells at tumor sites and abrogate the need for pre-treatment with toxic lymphodepleting chemotherapy. However, challenges exist to prepare cryopreserved cell products for IC delivery, including the need to remove the cryoprotectants dimethyl sulfoxide (DMSO), which is neurotoxic, reconstitute cells in an acceptable infusion solution, and minimize infusion volumes. Currently, there is no published methods describing cell preparation intended for IC infusion. To fill this gap, we describe here the development of a simple thaw/wash method that was validated for a clinical study that evaluates the safety and efficacy of IC-delivered B7-H3-CAR T cells for pediatric patients with brain tumors (NCT05835687).
MATERIALS AND METHODS
Generation of CAR T cells
B7-H3-CAR T cells were generated from cryopreserved apheresis mononuclear cells from healthy donors (Key Biologics; n=3) or patients (n=2). The patient-derived CAR T cell products were generated as part of a clinical protocol (NCT 04897321). The protocol was approved by the St. Jude Children’s Research Hospital Institutional Review Board (IRB); written informed consent/assent were obtained from all participants/parents in accordance with institutional guidelines and the Declaration of Helsinki. B7-H3-CAR T cell products were generated under current good manufacturing practice (cGMP) standards in the Children’s GMP at St. Jude using a standard operating procedure that was developed for St. Jude’s CD19-CAR T cell therapy study and is published [10]. Briefly, CD4+ and CD8+ selected T cells were activated with MACS®GMP T Cell TransAct™ (Miltenyi Biotec), and transduced with a cGMP-grade lentiviral vector encoding 41BBL, a P2A skip sequence, and a B7-H3-CAR with a CD28ζ endodomain.[11] Post transduction, cells were seeded and expanded in G-Rex® culture devices (Wilson Wolf) in X-VIVO™15 supplemented with 5% Human AB Serum (OTC, Heat Inactivated, Valley Biomedicals) and 10 ng/mL of IL7 and IL15 (Miltenyi Biotec). After 3 to 4 days of culture, cytokines were replenished and on day 7 or 8, CAR T cells were harvested, cryopreserved in freezing media (PlasmaLyte-A (Baxter) containing 3.75% Human Serum Albumin (HAS; Grifols), 6% pentastarch, and 5% DMSO) in a controlled rate freezer, and stored in liquid nitrogen at temperature colder than −150°C. Standard Quality Assurance/Quality Control (QA/QC) release testing was performed including sterility, viability, CAR expressing and functionality. All cryopreserved B7-H3-CAR T cell products used in this study met QA/QC specifications.
Thaw/wash Method
For process development, 5 cryopreserved cell aliquots of B7-H3-CAR T cell products from 3 healthy donors were used; for method validation, cryopreserved B7-H3-CAR T cell aliquots from 3 products (1 healthy donor and 2 patients) were used. The thaw method consists of product’s thaw, equilibration, dilution/washing and dosing/final formulation. Product aliquots in cryobags (CryoMACS, Miltenyi Biotec) with two free ports were thawed in a water bath at 37°C and transferred to an ISO-5 certified Biosafety cabinet (BSC) within the processing room. The room is an ISO-7 compliant controlled/monitored for temperature, humidity, and total/viable particle count. All spikes and open manipulations were aseptically and strictly performed in the BSCs. The cryobag was spiked/connected at one port to a 300 mL transfer bag. The second port was engaged with a needle free spike to attach 60 mL syringes prefilled with 9 product volumes of preservative free normal saline (PFNS, USP grade injectable solution, Baxter). One product volume of PFNS was slowly injected, mixed with the thawed product, and left for 5 minutes to allow equilibration. Then the reconstituted product was transferred into the 300 mL bag, further diluted with 8 product volume of PFNS, and divided into 50 mL falcon tubes for washing. Tubes were centrifuged at 600xg, 5 minutes, and combined pellets (1.0 – 2.0 mL) were resuspended in 10 mL PFNS. Post-wash cell count and viability was determined to calculate volume or aliquot required for preparation of the target CAR T cell dose. The dose volume transferred to a 15 mL tube, concentrated/volume adjusted by centrifugation (600xg, 5 minutes) and pellet (0.5 mL) resuspension to 2 mL of PFNS. The final product formulated in a 5.0 mL syringe and stored at room temperature.
Quality Assessment
In-process (post-thaw, and post-equilibration) and post-wash final product (FP) samples were assessed using approved standard operating procedures of the Human Applications Laboratory at St. Jude. Quality measures include appearance/integrity (by visual inspection), nucleated cell count (using Sysmex® XP-300: Automated Cell Counter), cell viability (Trypan blue stain), and calculated cell recovery. FPs were tested for safety attributes including Gram stain, Sterility (14-day culture for bacterial aerobic and anaerobic, and fungal growth using BD BACTEC blood culture system) and level of endotoxins using kinetic Limulus amebocyte lysate (LAL) method (Endosafe® nexgen-PTS™, Charles Rivers). To evaluate FP stability at room temperature (20–25°C), cell viability was determined at two points: 2 hours and 3 hours from the end of the thaw process. To support product stability over 3 hours, in vitro functional studies including B7-H3-CAR surface expression and B7-H3 CAR T cytotoxicity were performed in parallel at the Experimental Cellular Therapeutic Laboratory at St. Jude.
Flow Cytometry Analysis
Post-thaw/washed B7-H3-CAR T cells were cultured in X-VIVO™15 (Lonza) supplemented with 5% Human AB Serum (Vally Biomedicals, Heat Inactivated), 10 ng/mL of IL-7 and IL-15 (Miltenyi Biotec) at 2.0 × 106 cells/mL for two days, then harvested and washed with FACS buffer (PBS, Lonza; 2% FBS, Avantor). After washing, 5×105 T cells were incubated with 100 ng of recombinant human B7-H3-Fc chimeric protein (R&D Systems), washed with FACS buffer twice, and stained with mouse anti-human IgG Fc-APC antibody (Southern Biotech). The cells were then sequentially stained with PE anti-human CD137L (4–1BB ligand) antibody (Biolegend), and DAPI (a viability marker). The expression of B7-H3-CAR and 41BBL were analyzed by flow cytometry (CytoFlex, Beckman Coulter).
Cytotoxicity Assay
B7-H3-positive THP-1 cells and B7-H3-negative KG1a cells were used as target cells. Both cell lines were purchased from American Type Culture Collection (Manassas, VA, USA), and had been genetically modified to express an enhanced green fluorescent protein firefly luciferase fusion gene (ffluc-THP-1, ffluc-KG1a) as previously described [12]. B7-H3-CAR T cells were co-cultured with the respective target cells at effector to target (E:T) ratios of 1:1 and 0.1:1 in RPMI-1640 media (GE Healthcare) supplemented with 10% heat-inactivated fetal bovine serum (Avantor) (RPMI + 10% FBS). Twenty hours later, 150 μL of co-cultured cells were transferred to Costar assay 96 well plate, 50 μL of 0.6 mg/mL XenoLight D-Luciferin - K+ Salt Bioluminescent Substrate (Perkin Elmer) in RPMI (+10% FBS) media was added, and the luciferase activity was measured by multimode plate reader (Infinite M Plex, Tecan).
Statistical Analysis
FlowJo software (BD Biosciences) and Prism software (GraphPad Software, San Diego, CA) were used for flow data analysis and graph generation. Excel software (Microsoft) and Prism software were used for cytotoxicity data analysis and graph generation. For all experiments, the number of biological replicates and statistical analysis used are described in the figure legends. For comparisons between two groups, a two-tailed t-test was used.
RESULTS
Development of the thaw/wash method
The developed method includes product thaw, washing to remove DMSO and re-suspension of the target CAR T cell dose with a small volume of USP grade injectable PFNS. During the method development we found that while cell thaw without dilution had no significant impact on cell viability, direct dilution of thawed cells at 10X in PFNS resulted in a significant drop in viability to lower than 70%. Therefore, we included an equilibration step of thawed product with one volume of PFNS prior to the dilution. The developed method reduced the calculated % of DMSO in the FP from 5% in the starting material to approximately 0.0125% - 0.025% (Table 1).
Table 1:
Cell preparation method and calculated DMSO contents
| Preparation step | Calculated % of DMSO |
|---|---|
| Thaw at 37°C and equilibration for 5 minutes with equal volume of PFNS | 2.5% |
| Further dilution with 8 volumes of PFNS | 0.5% |
| Centrifugation at 600xg, 5 minutes, and combined pellets (1.0 −2.0 mL) resuspension in 10 ml PFNS | 0.05% - 0.1% |
| Calculate volume required for target CAR T cells dose and transfer to 15 mL tube | |
| Centrifugation at 600xg, 5 minutes, and pellet (0.5 mL), Suspension in 2 mL of PFNS | 0.0125% - 0.025% |
Cell viability and recovery meets product specification post wash/thaw procedure
Products were thawed, equilibrated, washed, and an aliquot containing the CAR T cell dose (30×106) was resuspended in 2.0 mL to yield the FP. Equilibration of thawed product with 1 volume of PFNS maintained cell viability above 90% (Table 2). Following dilution/washing/concentration steps, FP viability remained high (mean: 85.3%, range: 83%−88%), and the method yielded acceptable recovery of FP viable TNC vs pre-cryopreservation data (mean: 76.5%, range: 65.4%−82.5%) (Table 2). Applying aseptic techniques and the use of reagents/supplies certified for sterility and low endotoxin levels, QA/QC indicators including appearance, sterility, and endotoxin were satisfactory for all three FPs (Table 2).
Table 2:
Product analysis
| PARAMETER | SPECIFICATION | Run 1 | Run 2 | Run 3 | Mean |
|---|---|---|---|---|---|
| Apperance | Intact, no aggregates or particles seen | Intact, no aggregates or particles seen | |||
| Viability - in process post equlibration | ≥ 80% | 91% | Not determined | 92% | 91.5% |
| Viability - in process post wash | ≥ 70% | 89% | 85% | 91% | 88.3% |
| Viability - final product (FP) | ≥ 70% | 88% | 83% | 85% | 85.3% |
| Viability – 2 hours post-thaw (Stability) | ≥ 70% or stable within 5% of FP viability | 88% | 83% | 82% | 84.3% |
| Vaibility – 3 hours post thaw (Stability) | ≥ 70% or stable within 5% of FP viability | 79% | 80% | 81% | 80.0 % |
| TNC (Total) recovery | ≥ 70% recovery | 70.2% | 96.8% | 93.7% | 86.9% |
| Viable TNC recovery | ≥ 50% recovery | 65.4% | 82.5% | 81.7% | 76.5% |
| Bacterial & Fungal Stain | No Organisms Seen | No Organisms Seen | |||
| Endotoxin (EU/mL) | ≤ 1.0 EU/mL | ≤ 0.05 | ≤ 0.05 | ≤ 0.05 | |
| Sterility | Negative at 14 Days | Negative at 14 Days | |||
Final product stability
The FPs, consisting of a 2 mL suspension of 30×106 CAR-T cells in PFNS stored in a 5 mL syringe at room temperature, were assessed at 2 and 3 hours after the end of the product thaw/wash procedure for stability parameters including appearance and viability. All products remained intact with no visible aggregates or particles seen. None of the products failed the cell viability acceptable stability limit (≥70% by trypan blue). Mean viability was 84.3% and 80.0% for 2-hour and 3-hour samples, respectively (Table 2). During the method development, we have monitored cells viability for 6 hours post-thaw. Products cryopreserved with high viability (>90%), were adequately stable (>80% viability at 6 hours; data not shown). However, this was not the case for products pre-cryopreserved with lower viability where viability has obviously dropped after 3 hours post-thaw. Therefore, we have limited our stability verification to 3 hours to mitigate the risk of product’s non-conformance at the time of release.
To further assess transgene expression and functionality of CAR T cells, 2 and 3 hours after the end of the product thaw, washed cells were cultured in IL7 and IL15 for up to two days; CAR T cells that had been thawed with no washing served as controls. B7-H3-CAR and 41BBL expression were determined after 2 days of culture. Percent (mean ±1SD) of cells co-expressing B7-H3/41BBL was comparable to controls (controls: 47.0 ±3.55%, 2-hour: 43.4 ±1.64%, 3-hour: 43.7 ±1.57%) (Figure 1A,B). Of note, we have observed lower expression B7-H3-CAR of thawed cells after 2 days in culture vs % CAR expression reported initially in product release record pre-cryopreservation (mean 50.8% vs 60.1%; data not shown). We also monitored viability of cultured cells and growth kinetics over 2 days. In all cultures, there was a transient decrease in viability after 1 day of culture (Figure 1C). CAR T cells cultured from the 3-hour samples showed lower recovery after 2 days (controls: 114.8 ± 29.6%, 2-hour: 99.0 ± 19.15%, 3-hour: 74.6 ± 14.7% (Figure 1C). When cell recovery rate normalized to cell count, the low growth rate was statistically significant in 3-hour vs 2-hour sample cultures (P=0.0427) but not in the 3-hour vs control sample cultures (P=0.123). Finally, the cytolytic activity of B7-H3-CAR T cells against B7-H3-positive cells (THP-1) evaluated at an effector to target ratio of 1:1 and 0.1:1 was comparable between all three conditions (Figure 1D).
Figure 1: Transgene expression and functionality of B7-H3-CAR T-cells is not affected by thaw/wash procedure.
For method validation, three B7-H3-CAR T-cell products (1 healthy donor and 2 patient products) were thawed and washed. Two or 3 hours from end of the product thaw, washed cells were cultured in the presence of IL7/IL15 for up to 2 days. B7-H3-CAR T-cells that were not washed from the same product served as a control (n=3 donors). (A, B) CAR and 41BBL co-expression was determined after 2 days. (A) Individual FACS plots. (B) Summary data. (C) Total live cell numbers, viability by trypan blue, and cell recovery. (D) Cytotoxicity assay using firefly luciferase (ffluc)-expressing B7-H3-positive (THP-1) and B7-H3-negative (KG1a) tumor cells performed after 1 day of culture. There were no statistical differences between control and washed B7-H3-CAR T-cell products.
DISCUSSION
Immune effector cells including CAR T cell products are typically cryopreserved in 5–10% DMSO. The preparation of such products for IV infusion can be attained by direct thaw and aliquoting the target cell dose. Washing to reduce cell volume and DMSO concentration are generally not required for IV infusion. In clinical practice, it is acceptable and tolerable to infuse IV up to 1-gram DMSO/kg/day, 20 mL of product/kg, and 5.0 EU endotoxins/kg/hour [13–15]. Furthermore, directly thawed CAR T cell products are relatively stable, allowing adequate time to infusion taking in consideration that unmanipulated products would not require post-thaw quality testing. Conversely, locoregional IC infusion requires washing to reduce the DMSO content to mitigate potential toxicity, volume reduction down to a few milliliters, and additional quality testing for product release.
During method development, we have focused on cell viability, cell recovery, and FP DMSO level. Cell viability and recovery results were superior to expected values. Although the DMSO safety limit for IC infusion is not known, this method successfully reduced DMSO% as calculated to less than 0.025% below the internally set limit of 0.5%. We decided to set an upper limit of 0.5% since higher concentrations of DMSO have been reported to induce widespread apoptosis in the developing central nervous system of mice [16]. Others have reported that a single injection of 10% DMSO in the lateral ventricle of rats did not change the electrophysiological characteristics of neurons in the rat barrel cortex [17]. However, even systemic administration of DMSO can result in electrophysiological changes in preclinical models in a dose dependent manner [18]. Clearly additional studies are needed to establish an upper limit of DSMO concentration for IC administration.
Thawed product safety was assured by absence of detectable microbial contamination and very low endotoxins levels (≤ 0.05 EU/mL in all products). While there is no defined Endotoxin safety limit for IC infusion, we opted to accept up to 1.0 EU/mL which represents less than 2.0 EU in the 2-mL FP. This level is in line with the Food and Drug Administration (FDA) guidance for intrathecal administration permitting exposure to endotoxin per hour up to 0.2 EU/kg [15]. We expect that the youngest research participant enrolled on pediatric studies has a body weight of 12 kg. The endotoxin exposure for a 12 kg research participant would be less than 0.17 EU/kg (1.0 EU/mL x 2mL/12 kg). Since all prepared products applying our method revealed endotoxin level ≤ 0.05 EU/ml, it will be feasible to administer the product to a smaller patient.
Gram stain and 14-day BACTEC automated culture method validated in-house with sensitivity of 10–100 CFU/culture vial were reported negative indicating that contamination can be adequately controlled during the manual thaw/wash method applying routine cell and tissue-based (CT) lab aseptic practices. As manipulation of CT products by thaw/wash requires product testing for release, we have evaluated FP stability to allow time for testing, formulation, and transfer to the bedside. Technically two processors shall be involved in the preparation process. To avoid any delay after thaw, it is also prudent to complete many preparations related but not limited to documentation, supplies/reagents, and labeling before the start of the product thaw. The process itself takes approximately 2 hours from the start of the thaw to product release (1 hour for thaw/wash plus 1 hour for testing, formulation, and release).
Cell viability over 3 hours from the end of the product thaw, was consistently high with a mean value of 80%, indicating stability under described conditions. To further investigate CAR T cell product stability over 3 hours, we evaluated washed cells for transgene (B7-H3 CAR and 41BBL) expression and in vitro functions including growth kinetic and cytotoxicity to B7-H3-positive (THP-1) tumor cells. We observed lower CAR expression (~10%) after thaw compared to pre-cryopreservation. This difference might be related to the use of different instruments/procedures to detect CAR expression (St Jude GMP vs ECTL), actual decrease in CAR expression associated with the cryopreservation/thaw processes, and/or culturing the cells for 2 days post thaw/wash. We also observed a slight reduction in cell growth and recovery in cultures of the 3-hour post-thaw samples, however, this did not result in decreased cytolytic activity. Although additional functional studies were not part of the stability parameters, the obtained results provided additional evidence of product stability. Whether this in vitro finding would impact CAR T cell functionality in vivo needs to be determined in future studies.
We recognize that the described procedure is manual/multistep method that might not be ideal for the preparation of CAR T cells for intracranial administration for later phase clinical studies or FDA-approved CAR T cell products. Therefore, there is a need to continue to optimize the process; this may include cryopreserving cells at higher concentrations (e.g., 2×108/mL), which would significantly reduce the required wash volume, and/or automation of washing steps.
In conclusion, we have developed a simple thaw/wash procedure to prepare B7-H3-CAR T cells for their locoregional delivery to the neural axis. We will use this procedure in our clinical study entitled ‘Locoregional Delivery of B7-H3-specific Chimeric Antigen Receptor Autologous T Cells for Pediatric Patients with Primary CNS Tumors’ (Loc3CAR, NCT05835687), which just opened for patient accrual. While we focus here on CAR T cells, the methods could be readily adapted to other cryopreserved immune effector cell products.
ACKNOWLEDGEMENTS
We would like to thank Robert Ott, PhD, and Jan Lindsey for quality assurance support. The work was supported by the National Institutes of Health (NIH)/National Cancer Institute (NCI) grant P30 CA021765, and the American Lebanese Syrian Associated Charites. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
Footnotes
Conflict of interest
SG and CD are co-inventors on patent applications in the fields of cell or gene therapy for cancer. SG is a consultant of TESSA Therapeutics, a member of the Data and Safety Monitoring Board (DSMB) of Immatics, on the Scientific Advisory Board of Be Biopharma, and has received honoraria from Tidal, Catamaran Bio, and Sanofi within the last 2 years.
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