Deciphering the importance of culture pH on CD22 CAR T-cells characteristics

Abstract

Background

Chimeric antigen receptor (CAR) T-cells have demonstrated significant efficacy in targeting hematological malignancies, and their use continues to expand. Despite substantial efforts spent on the optimization of protocols for CAR T-cell manufacturing, critical parameters of cell culture such as pH or oxygenation are rarely actively monitored during cGMP CAR T-cell generation. A comprehensive understanding of the role that these factors play in manufacturing may help in optimizing patient-specific CAR T-cell therapy with maximum benefits and minimal toxicity.

Methods

This retrospective study examined cell culture supernatants from the manufacture of CAR T-cells for 20 patients with B-cell malignancies enrolled in a phase 1/2 clinical trial of anti-CD22 CAR T-cells. MetaFLEX was used to measure supernatant pH, oxygenation, and metabolites, and a Bio-Plex assay was used to assess protein levels. Correlations were assessed between the pH of cell culture media throughout manufacturing and cell proliferation as well as clinical outcomes. Next-generation sequencing was conducted to examine gene expression profiles of the final CAR T-cell products.

Results

A pH level at the lower range of normal at the beginning of the manufacturing process significantly correlated with measures of T-cell expansion and metabolism. Stable or rising pH during the manufacturing process was associated with clinical response, whereas a drop in pH was associated with non-response.

Conclusions

pH has potential to serve as an informative factor in predicting CAR T-cell quality and clinical outcomes. Thus, its active monitoring during manufacturing may ensure a more effective CAR T-cell product.

Background

Chimeric antigen receptor (CAR) T-cell therapy has been highly effective in patients with B-cell hematologic malignancies, including acute lymphoblastic leukemia [1,2,3,4,5] and non-Hodgins’ lymphoma [6,7,8], and ongoing research aims to expand applications to solid tumors [9, 10] and autoimmune diseases [11,12,13]. Since the initial approval of the CD19-targeted CAR T-cell, tisagenlecleucel (Kymriah), in 2017, the US Food and Drug Administration (FDA) has approved five more CAR T-cell therapies for acute lymphoblastic leukemia, non-Hodgkin lymphoma, and multiple myeloma. The rapid growth of this therapy is illustrated by the more than 700 CART trials actively recruiting patients (clinicaltrials.gov, February 2024).

As new CAR T-cell therapies become commercialized and enter into clinical use, it is essential to produce CAR T-cells that are consistently of high quality and potency. With more clinical centers aiming to establish point-of-care processes of CAR T-cell manufacturing, maintaining consistent cell culture conditions will become increasingly challenging.

Cell culture is a dynamic process, so understanding the interactions between cells, culture media, nutrients, and growth factors is critical for producing high quality and potent cell therapy products. Cell counts, viability, and expansion are routinely monitored during CAR T-cell manufacturing, but little attention is paid to variables such as metabolites, oxygen and carbon dioxide tension, or pH. These factors, however, may be associated with CAR T-cell proliferation and fitness. The pH in particular is a simple indicator that may reflect carbon dioxide tension, lactate accumulation, and rate of T-cell proliferation. Thus, monitoring these variables could be valuable in predicting properties of the final cell therapy product.

Not only could culture media characteristics serve as indicators of T-cell expansion, but they could also be a potential point of intervention. The pH of the culture media has been shown to affect T-cell proliferation, differentiation, and metabolism. T-cells cultured in acidic media (pH 6.6 and lower) have reduced activation, proliferation, and slower differentiation to antigen-specific cytotoxic T-lymphocytes [14, 15]. Moreover, in a study of tumor-infiltrating T-lymphocytes, acidic media led to suppression of T-cell function, including reduced cytokine secretion and lower expression of T-cell receptors [16]. On the other hand, while prolonged (12 days) ex vivo expansion of T-cells in an acidic environment (pH 6.6) reduced rate of proliferation, it also promoted CD8+ T-cell stemness [17]. Additionally, this extended expansion of T-cells in acidic media triggered T-cell metabolic reprogramming with restricted glycolysis and increased long chain fatty acid metabolism [17]. Studies of how changes in culture media affect T-cells in the context of CAR T-cell production specifically are limited.

Accurate monitoring and control of pH and metabolites may be an important opportunity to ensure optimal CAR T-cell performance and function. However, these factors are rarely monitored during clinical Good Manufacturing Practice (GMP) CAR T-cell generation and their effect on CAR T-cell growth and differentiation has yet to be fully investigated. In this study, we examined the pH and metabolic parameters of anti-CD22 CAR T-cell cultures and explored the relationship between culture conditions and clinical outcomes.

Methods

Patients’ cohort

The products included in this study were derived from a cohort of 20 children and young adults who were diagnosed with recurrent or refractory CD22-expressing B-cell malignancies and were enrolled on a Phase 1/2 clinical trial of anti-CD22 CAR T-cells (NCT 02315612). All patients were treated in the Pediatric Oncology Branch of the National Cancer Institute, National Institutes of Health at a dose level of 0.3 × 10⁶ cells.

CD22 CAR-T manufacturing process

All anti-CD22 CAR T-cell products were manufactured at the Center for Cellular Engineering (CCE) between September 2019 and August 2022. The CCE is compliant with good manufacturing practice (GMP) and good clinical laboratory practice (GCLP) standards and has manufactured numerous CAR T-cell products for phase 1 and 2 clinical trials at the National Institutes of Health. All the products were manufactured using autologous mononuclear cells collected by apheresis. T-cells from fresh (n = 5) or cryopreserved (n = 15) autologous mononuclear cells were selected on CliniMACS (Miltenyi Biotec) using anti-CD4 and anti-CD8 magnetic beads and stimulated with anti-CD3/CD28 Dynabeads (ThermoFisher) with a ratio of 1 cell:3 beads. Cells were seeded into culture bags at a density of 1.5–2.0 × 10⁶ viable CD3⁺ cells. Cell culture was maintained in AIM-V medium (Gibco) containing heat inactivated human AB serum (Valley Biomedical), Glutamax (Gibco) and IL2 (Aldesleukin) in a 37 °C, 5% CO₂ incubator for 9 days. On day 2 of the culturing process, cells were transduced with GMP grade CD22 lentiviral vector (Lentigen) using bag spinoculation (1000 g for a period of 2 h). On day 3 (D3), transduction was stopped by media exchange. Debeading and cell density adjustment to 0.4 × 10⁶ viable cells/mL was performed on day 4 (D4) of the culturing process. Cell density was further adjusted on Day 7 (D7) to 0.6–1 × 10⁶ viable cells/mL. All cultures were harvested on Day 9 (D9) and were either freshly infused (n = 7) or cryopreserved (n = 9). A simplified 9-day manufacturing process is illustrated in Additional file 1: Supplementary Figure 1a.

Collection of culture media supernatants

Culture media supernatants were collected at four different time points during the CD22 CAR T-cell culture (on Day 2, 3, 4 and 9). 10 mL of culture supernatant was collected before transduction on D2, when transduction was stopped on D3, and before debeading on D4. Final supernatants were collected during the cell harvest on D9. All supernatants were aliquoted (1 mL per vial), immediately frozen and stored at − 80 °C until further analysis.

Metabolite analysis

A Vi-CELL MetaFLEX bioanalyzer (Beckman Coulter) was used to measure pH, glucose, lactate, pCO₂ and pO₂ levels during the manufacturing process. At the time of the analysis, supernatants collected at various time-points during the culturing process were thawed and warmed to 24 °C. 65 uL of temperature adjusted supernatants were loaded into the MetaFLEX device for testing.

Bio-Plex assay

Culture media cytokine, chemokine, and growth factor levels were measured at 2 different time points (D2 and D9) during the culture process using the Bio-Plex 200 system (Bio-Rad Laboratories). Bio-Plex assays were performed according to the manufacturers’ instructions using commercially available Bio-Plex Pro Human Cytokine Screening 48-plex Panel Assay. All assay values are reported in pg/mL.

Bulk mRNA sequencing

We have previously described the bulk RNA sequencing procedures in detail [18]. In brief, total mRNA from CAR T-cell products was isolated using the miRNeasy Mini Kit. The concentration and quality were assessed using Nanodrop 8000 and 2100 Bioanalyzer, respectively. DNA libraries were prepared using the TruSeq Stranded Total RNA kit and sequenced on the Illumina Nextseq550 platform.

Transcriptome data analysis

Raw fastq files underwent quality control using FastQC and were further processed with Trimmomatic to remove adapter sequences and low-quality reads. After filtering, the reads were aligned to the human reference genome (GENCODE hg38) using the STAR aligner. Gene expression levels were quantified using subread (featureCounts), and TPM values were utilized for downstream analysis. For differential expression analysis, we employed the limma package in RStudio with custom scripts. Threshold for definition of differentially expressed genes (DEGs) is p value < 0.01 & |FoldChange|≥ 2. We then performed gene set enrichment analysis (GSEA), a bioinformatic method to determine whether a predefined set of genes showed statistically significant, concordant differences between two biological states. We divided the gene expression from our RNA-seq data into two sets: up-regulated and down-regulated set. Subsequently, we examined the presence of genes associated with each pathway in either the up-regulated or down-regulated set. The degree of enrichment was quantified by a normalized enrichment score (NES). A significant positive NES value suggests that members of the selected pathway gene set tend to appear in the up-regulated set, whereas a significant negative NES value indicates the opposite scenario. The analysis was conducted by the built-in function of the cluster Profiler package [19] in RStudio.

Flow cytometry

Transduction efficiency, viability and percentage of cells expressing CD3, CD4, CD8, CD14, CD15, and CD56 were measured using flow cytometry following staining with fluorescently labelled antibodies (BD Biosciences). The CD22-CAR vector transduction efficiency was detected using protein L (ThermoFisher). Untransduced cells were used to determine the positive gate for transduction efficiency. The gating strategy of protein L expression on CD22 CAR T-cells is displayed in Additional file 1: Supplementary Figure 1c. Frequencies of the above-mentioned markers were acquired on the BD FACS CantoII (BD Biosciences). Thirty thousand events in total were collected for each sample. Data were analyzed using BD FACSDiva software.

Statistical analysis

All data are presented as mean ± SEM. Graphical and statistical analysis was performed using GraphPad Prism 7 software. Statistical differences between the groups were assessed either using an unpaired t test, Mann–Whitney, or nonparametric Spearman correlation. P value less than 0.05 was considered significant, except for the next-generation sequencing data analysis.

Results

Overview of CD22 CAR-T cell expansion during the manufacturing process

The clinical characteristics of patients whose CD22 CAR T-cell products were included in the study are detailed in Table 1. In total, twenty CD22 CAR T products were analyzed. All 20 products met the 0.3 × 10⁶/kg target dose. Four products were not infused, as the patients experienced rapidly progressive disease during the manufacturing process. Seven patients received infusions of fresh CAR T-cells, and nine patients received cryopreserved CAR T-cells.

Table 1 Patients and manufacturing process characteristics

During the manufacturing process, viable total nucleated cells (TNC) were counted on days 0, 2, 4, 7 and 9. The mean TNC at the start of the manufacturing process was 470 × 10⁶ cells (range 288–565 × 10⁶). On Day 2, a mean of 343 × 10⁶ total nucleated cells underwent transduction with lentiviral vector. On Day 4, there was a drop in total nucleated cells with a mean of 52 × 10⁶ cells (range 26–76 × 10⁶). The total number of cells harvested on Day 9 was 1.1 × 10⁹ (range 0.3–1.7 × 10⁹, Fig. 1a). Fold expansion was determined between the following days of the cell culture: 0–2, 2–4, 4–7 and 7–9. The maximum fold expansion was achieved on Days 4–7 of the culturing process (mean 8.5, range 4.6–11.9) with a decrease in expansion between Days 7 and 9 (mean 2.9, range 2.0–3.9, Fig. 1b). As shown in Fig. 1c, the percentage of CD3⁺ cells increased from 47.9 in the apheresis bag to 91.2 post-CD4/CD8 selection to 99.7 at the time of the harvest of CAR T-cells. The apheresis bag and selected CD4/CD8 T-cells were analyzed for additional markers such as CD56, CD14, or CD15. Additional file 1: Supplementary Figure 1b shows the analysis of these markers. The transduction efficiency (percent of protein L⁺ cells) was assessed on Days 7 and Day 9 of the manufacturing process. On Day 7, after the fastest period of expansion, 31% transduction was observed (data not shown). By Day 9, a significantly larger proportion of cells expressed protein L, (42.8%, p = 0.0073, Fig. 1d), suggesting preferential expansion of transduced cells. The final CD4/CD8 ratio was 2.8 (Fig. 1e).

Fig. 1

Characteristics of twenty CD22 CAR T-cell products manufactured at CCE of the NIH Clinical Center. a Viable total nucleated cells (TNC) count at the start of the CD22 manufacturing process (470 × 10⁶) doubled over the time of the 9-day culturing process to 1.1 × 10⁹ of viable TNC at the harvest. b Fold expansion rate reached its maximum on D7 of the manufacturing process (8.5) with a drop at the harvest (2.9). c CD3 percentage increased over the culturing process from 48% at apheresis bag to 99.7% of CD3⁺ at the harvest. d Transduction efficiency measured via flow cytometry reached 43% of protein L⁺ cells at the culture harvest. e CD4/CD8 ratio at the harvest

The pH of the cell culture at the beginning of the manufacturing process correlated with CAR T-cell growth, expansion, and metabolic activity

Culture media supernatants were collected at 4 different time points throughout CD22 CAR T-cell manufacturing: Days 2, 3, 4, and 9. Our primary focus was on the findings on Day 2 and Day 9. Prior to transduction on Day 2, the pH of cell culture media ranged from 7.1–7.5 (Fig. 2a). pH level showed a statistically significant negative correlation with the D2 TNC count and fold expansion. On retrospective evaluation, we separated the products into 2 groups: 12 samples had relatively low pH on Day 2 (ranging from 7.093–7.289; mean 7.2), and 8 samples had high pH (ranging from 7.323–7.541; mean 7.4). The differences in pH levels between these 2 groups were statistically significant (p < 0.0001, Fig. 2a). The cell counts, fold expansion, and metabolic activity of T-cells were compared between the low pH and high pH groups. Cell products in the low pH group had significantly elevated total nucleated cell counts (414 vs 236 × 10⁶, p < 0.0061, Fig. 2b), greater fold expansion (0.85 vs 0.53, p < 0.0015, Fig. 2c), higher pCO₂ (23.2 vs 17.6 mmHg, p < 0.001, Fig. 2d) and higher lactate levels (0.73 vs 0.38 g/L, p < 0.0001, Fig. 2f). Those in the low pH group also had reduced glucose levels (1.9 vs 2.3 g/L, p < 0.0003, Fig. 2e) compared with the high pH group. These results demonstrate the relationship between lower extracellular pH and T-cell growth, expansion, and glycolysis as pH levels correlated with pCO₂, glucose, and lactate levels. On the other hand, Day 2 pH levels did not correlate with CD3% (Additional file 1: Supplementary Figure 2a) or pO₂ levels (Additional file 1: Supplementary Figure 2b).

Fig. 2

Extracellular changes in pH induced TNC, fold expansion and metabolites change at the beginning of CD22 CAR T-cells manufacturing process. a 12 samples were preserved in low pH (mean 7.2) whereas 8 samples in high pH (mean 7.4, p < 0.0001). b Higher pH negatively correlated and reduced number of total nucleated cells (236 × 10⁶) on Day 2 of the manufacturing process in comparison to 414 × 10⁶ cultured in lower pH (p < 0.0061). c pH values significantly correlated with T-cell expansion. T cells cultured in lower pH reached significantly higher expansion rate related to the ones sustained in high pH (0.85 vs 0.53, p < 0.0015). d Tension of carbon dioxide significantly correlated with pH and was decreased in high pH (23.2 vs 17.6 mmHg, p < 0.001). e Low pH induced drop in glucose levels (2.3 vs 1.9 g/L, p < 0.003) whereas opposite trend was seen for lactate levels (f) (0.38 vs 0.73, p < 0.0001). Both metabolites showed significant correlation

Cytokine and growth factor levels were also compared between low and high pH groups. On Day 2 of the manufacturing process, six analytes demonstrated significantly greater levels among samples in the low pH group compared with the high pH group (Fig. 3). Increased levels of stem cell factor (SCF, p < 0.04), macrophage colony-stimulation factor (M-CSF, p < 0.004), leukemia inhibitory factor (LIF, p < 0.0052), interleukin 15 (IL-15, p < 0.0066), interleukin receptor 2 alpha (IL2Rα, p < 0.0002), and tumor necrosis factor beta (TNF-β, p < 0.0001) were observed in products with more acidic pH. In the context of increased proliferation associated with lower pH as shown above, these findings were consistent with our expectation that more active T-cells will secrete more cytokines and growth factors into the cell culture media.

Fig. 3

Elevated levels of selected cytokines and growth factors on Day 2 of the manufacturing process were detected in cell culture supernatants collected at low pH. Lower levels of extracellular pH induced increased secretion of SCF (p < 0.04), M-CSF (p < 0.004), LIF(p < 0.0052), IL-15 (p < 0.0066), IL2Ra (p < 0.0002) and TNF-b (p < 0.0001) into cell culture supernatants compared to the ones cultured in higher pH

The pH of culture media at the end of manufacturing reflects metabolic profile, but does not correlate with expansion of final CAR T-cell products

Next, we investigated whether the pH level measured at the end of manufacturing was associated with cell or metabolic parameters. Overall, we observed a slight decline in pH values during the manufacturing process with higher values on Day 2 compared to Day 9. The Day 9 pH ranged from 7.0 to 7.4 and based on its values, the final CAR T-cell products were also separated into 2 groups representing low (9 patients, pH ranged from 7.008–7.235 with the mean pH of 7.1) and high pH (11 patients, pH ranged from 7.252 to 7.432 with mean pH of 7.3). The difference in final pH levels between these two groups was statistically significant (p < 0.0001, Fig. 4a). However, unlike the Day 2 pH groups, these final pH groups did not differ in terms of cell counts (Fig. 4b), fold expansion (Fig. 4c), CD3% (Fig. 4d), pCO₂ (Fig. 4e), or pO₂ (Fig. 4f). Higher pH at harvest correlated with higher glucose (1.91 vs 1.55 g/L; p < 0.0003, Fig. 4g) and lower lactate production (0.64 vs 0.89 g/L; p < 0.0002, Fig. 4h), suggesting reduced glycolysis and lower metabolic activity.

Fig. 4

Increased pH levels at the harvest did not affect final TNC, fold expansion or gas tension. a Significant changes (p < 0.0001) in extracellular pH were noticed in CD22 CAR T-cell products at the harvest. 9 products were collected with pH values at about 7.1 and 11 with pH 7.3. Different pH values at the harvest did not affect final TNC levels (b), fold expansion (c), CD3 percentage (d), pCO₂ (e), or pO₂ (f). High pH was correlated with significantly upregulated glucose levels (p < 0.0003) (g) and negatively with lactate levels (p < 0.0002) (h)

The Day 9 levels of 7 cytokines differed between the high and low pH groups. All 7 were elevated in the low pH group compared to the high pH group [interleukin 6 (IL6, p < 0.008), interleukin 12 subunit p70 (IL-12p70, p < 0.003), tumor necrosis factor alpha (TNF-α, p < 0.008), leukemia inhibitory factor (LIF, p < 0.007), macrophage migration inhibitory factor (MIF, p < 0.002), macrophage colony-stimulation factor (M-CSF, p < 0.003) and granulocyte macrophage colony-stimulation factor (GM-CSF, p < 0.02)] (Fig. 5). The levels of LIF and M-CSF were also greater in the low pH group on day 2 (Fig. 3), although others did not differ significantly.

Fig. 5

pH related changes in cytokines and growth factors in cell culture supernatants from CD22 CAR T-cells at the harvest. Reduced levels of IL6 (p < 0.008), IL-12p70 (p < 0.003), TNF-a (p < 0.008), LIF (p < 0.007), MIF (p < 0.002), M-CSF (p < 0.003) and GM-CSF (p < 0.02) were found in cell supernatants with higher pH

Sequencing profile of final CD22 CAR T-cells products demonstrates differences in T-cell activation pathways associated with pH

CD22 CAR T-cell final product characteristics were analyzed by RNA sequencing (Fig. 6). Sequencing analysis of 17 final products assigned 78 differentially expressed genes between pH groups at harvest: 15 downregulated and 63 upregulated under low pH. GSEA analysis revealed that genes involved in T-cell activation, lymphocyte activation, differentiation, and mediated immunity, tend to be downregulated in low pH conditions. This highlights the value of pH as an indicator during CD22 manufacturing as it can help identify more activated CAR T-cell products (Table 2 shows the list of genes involved in each pathway).

Fig. 6

Gene expression changes in CD22 CAR T-cell products separated based on final pH. Gene expression analysis of the final products cultured either in high or low pH demonstrated in total 78 differentially expressed genes: 15 downregulated and 63 upregulated in low pH condition. Blue point represents downregulated genes. Red point indicates up-regulated genes. Genes with p value < 0.01 & |Foldchange|> 2 were considered as differentially expressed genes. GSEA analysis indicates the activation of the four pathways (lymphocyte activation and differentiation, lymphocyte mediated immunity and T cells activation) in high pH group. A significant positive NES value (with FDR < 0.05) is considered indicative of the activation state of the pathway in high pH group

Table 2 List of genes involved in each pathway

Low pH at D2 is associated with clinical outcome

To determine if product pH on Day 2 correlated with clinical outcomes, we compared disease response rates between the low and high pH groups (Fig. 7). Of the 12 patients whose samples were in the low pH group on D2, 10 were infused and 2 were not due to progressive disease. Among the patients receiving the cells, 8 (80%) achieved a complete response (CR), which is defined as complete bone marrow response at 28 days. The high pH group contained 8 patients. Six patients received CAR T-cell infusions and 2 did not due to rapidly progressive disease. In contrast to the low pH group, among those with high pH, only 2 patients (33%) achieved a clinical response, while 4 (67%) did not. The relationship between low starting pH and CR was most consistent in the subsets of patients with high baseline disease burden (M3 marrow, > 25% blasts) (Additional file 1: Supplemetary Figure 3a) and prior hematopoietic stem cell transplant (HSCT) (Additional file 1: Supplemetary Figure 3b).

Fig. 7

D2 pH and its effect on clinical outcome of CD22 CAR-T cells. 10 CD22 CAR-T cells that start manufacturing process with low pH, resulted in 8 responders and 2 non-responders. On the other hand, 6 products that manufacturing process started with high pH, revealed only 2 responders and 4 non-responders

Moreover, we observed a difference in toxicity patterns between the low and high pH groups. In the low pH group, 8 patients (all those with CR) developed cytokine release syndrome (CRS), 5 (62.5%) of them with Grade 1 and 3 with (37.5%) Grade 2. Non-responders in this group did not have any toxicities. Only 25% of the patients with clinical response in the low pH group experienced neurotoxicity and 50% had IEC-HS (immune-effector cell HLH-like syndrome) (Table 3). In the high pH group, both patients with clinical response developed Grade 2 CRS, neurotoxicity, and IEC-HS. Furthermore, in the high pH group, all non-responders experienced toxicities as well: all had CRS, 2 of them had neurotoxicity, and one had IEC-HS.

Table 3 pH correlation to clinical outcome and adverse events

Analysis of pH changes over the course of manufacturing demonstrated that patients who achieved CR were more likely to have cells with stable or slightly increased pH values during the manufacturing process, whereas those who did not have CR had a drop in pH (Fig. 8a). Similar to the pattern observed with starting pH, the association between a drop in pH and lack of clinical response was most pronounced among patients with high baseline disease burden or history of prior HSCT (Fig. 8b and c).

Fig. 8

pH connection to clinical outcome. a Association between low pH at the beginning of the manufacturing process and clinical response has been proven in 80% of the patients whereas only 20% of patients demonstrated clinical response when their cells were manufactured in higher pH. b Patients with high baseline disease burden which cells were kept at lower pH displayed higher probability of clinical response. c Patients who received previous hematopoietic stem cell transplant were associated with clinical responsiveness as long as the manufacturing process of their cells started with low pH

Discussion

The aim of this study was to assess the relationship between culture conditions, specifically culture pH, and the growth kinetics and properties of CD22 CAR T-cells used for the treatment of pediatric B-cell acute lymphoblastic leukemia. Our data demonstrate that pH fluctuations during the CAR T-cell manufacturing process correlate with properties of the cellular product and may be indicative of clinical outcome. Based on our retrospective analysis, high pH at the start of manufacturing is associated with reduced number of viable nucleated cells, slowed cell expansion, and reprogrammed T-cell metabolism. Patients whose cells had a high pH at the start of the manufacturing tended to have a lower probability of clinical response. Since the data suggests that pH levels at 7.2 to 7.3 are indicative of optimal manufacturing conditions, the measure may be useful as an predictor of appropriate T-cell growth, differentiation, and clinical efficacy. We therefore propose that careful monitoring of pH during the manufacturing of cellular products is critical, and accurate pH monitoring during cGMP manufacturing should become a standard.

In our samples, lower pH generally correlated with a picture of increased T-cell proliferation. Lower pH was connected with a drop in glucose and an increase in lactate levels [20, 21]. This shows that T-cells grown in lower pH are more metabolically active compared to high pH. As the most rapid cell expansion occurs during the early days of CAR T-cell manufacturing, the Day 2 pH remains a better indicator of this process than the Day 9 pH, which is reflecting the media typically after several days of slower growth. It will be important in future studies to explore whether cell expansion patterns in vitro are recapitulated in vivo after cells are infused.

Lower pH may not only be a consequence of greater T-cell expansion but may actually be supporting further growth. Despite T-cell dysfunction in truly acidic environments, pH in the lower range of normal has been reported to be beneficial in the context of CAR T-cell expansion. In in vitro studies, reduction in extracellular pH to 6.6 significantly affected not only T cell proliferation but also reduced IL-2 and IFN-γ secretion, downregulated CD25 and CD71 expression, and upregulated CTLA4 expression leading to apoptosis [15]. Similarly, pH in the range of 6.0 to 6.5 has been shown to induce anergy in CD8+ T lymphocytes, with impaired proliferation, reduced cytokine production, decreased CD25 expression, and inhibition of STAT5 and p-ERK activation [16]. Although the pH fluctuations in our study might appear minor, the results are consistent with other studies showing that only small variations in culture pH can have a substantial effect on T-cells. Others have reported improved T-cell stimulation, expansion, and proliferation in lower pH, 7.0–7.3, compared to neutral pH [22, 23], which may be consistent with our findings. Similarly, only small variations in pH were also shown to be critical in hematopoietic cultures, mainly for progenitor cell differentiation and cloning efficiency [24].

In addition to predicting T-cell proliferation, the pH of culture medium may also help predict cytokine production. We demonstrated that cells starting out in lower pH had increased production of several cytokines including IL-6, IL12p70, TNF-a and GM-CSF, which play an important role not only in CAR T-cells’ targeted cytotoxicity, but also in development of off-target effects, such as CRS and neurotoxicity [25,26,27]. Further analysis of cytokine production during CAR T-cell manufacturing may yield important insight into which patients are at risk for developing CAR T-cell associated toxicities.

There are important limitations to this exploratory study, and further investigation will be needed to confirm the trends reported here. The sample size is limited, and since our study included only anti-CD22 CAR T-cells, the findings might not be applicable to other CAR T-cell types. Overall, the relationship between pH and T-cell characteristics likely varies with the specific manufacturing protocol, including culture media, manufacturing platform, and timing. We are, however, continuing to collect more samples as new patients are treated, including those on the ongoing CD22 trial and trials of other novel CAR T-cell products. Importantly, the data reported here were collected over the course of our standard CAR T-cell manufacturing process, and no intentional adjustments to pH were made. Therefore, we can report only correlations with other parameters rather than any causal relationships. Future work will focus on pH modification during the manufacturing process to determine whether this will have any effect on the characteristics and efficacy of the final product.

Currently there are no guidelines or standards advising monitoring of pH during cell culture for CAR T-cell manufacturing. The emerging data, however, points to pH as an important and easily accessible tool for monitoring and standardizing CAR T-cell production. Furthermore, it has potential to serve as an early indicator of clinical outcome. Since adjusting the pH of cell culture media can be a relatively straightforward and inexpensive intervention, further prospective studies are needed to determine whether actively controlling pH during manufacturing is beneficial.

Conclusions

Measurements of pH throughout CAR T-cell manufacturing correlate with cell proliferation as well as other metabolic properties of CAR T-cells. Accurate pH tracking during CAR T-cell manufacturing may serve as an important metric of the quality of the cellular product and may become a predictive marker of CAR T-cell clinical efficacy. pH should be monitored throughout CAR T-cell manufacturing, and combined with other factors, it could significantly improve the quality, compatibility, and safety of CAR T-cells.