Enhancing CAR T function with the engineered secretion of C. perfringens neuraminidase

Abstract

Prior to adoptive transfer, CAR T cells are activated, lentivirally infected with CAR transgenes, and expanded over 9 to 11 days. An unintended consequence of this process is the progressive differentiation of CAR T cells over time in culture. Differentiated T cells engraft poorly, which limits their ability to persist and provide sustained tumor control in hematologic as well as solid tumors. Solid tumors include other barriers to CAR T cell therapies, including immune and metabolic checkpoints that suppress effector function and durability. Sialic acids are ubiquitous surface molecules with known immune checkpoint functions. The enzyme C. perfringens neuraminidase (CpNA) removes sialic acid residues from target cells, with good activity at physiologic conditions. In combination with galactose oxidase (GO), NA has been found to stimulate T cell mitogenesis and cytotoxicity in vitro. Here we determine whether CpNA alone and in combination with GO promotes CAR T cell antitumor efficacy. We show that CpNA restrains CAR T cell differentiation during ex vivo culture, giving rise to progeny with enhanced therapeutic potential. CAR T cells expressing CpNA have superior effector function and cytotoxicity in vitro. In a Nalm-6 xenograft model of leukemia, CAR T cells expressing CpNA show enhanced antitumor efficacy. Arming CAR T cells with CpNA also enhanced tumor control in xenograft models of glioblastoma as well as a syngeneic model of melanoma. Given our findings, we hypothesize that charge repulsion via surface glycans is a regulatory parameter influencing differentiation. As T cells engage target cells within tumors and undergo constitutive activation through their CARs, critical thresholds of negative charge may impede cell-cell interactions underlying synapse formation and cytolysis. Removing the dense pool of negative cell-surface charge with CpNA is an effective approach to limit CAR T cell differentiation and enhance overall persistence and efficacy.

Keywords: CAR T cells; checkpoints; differentiation; glycoproteins; immunotherapy; neuraminidase; persistence.

Graphical abstract

Figure 1

Engineering CAR T cells to secrete functional Clostridium perfringens neuraminidase (CpNA) (A) Schematic representation of the hEGFR lentiviral CAR containing an 806 scFv, which is linked via a CD8alpha hinge as well as a CD8a TM to the 4-1BB and CD3zeta intracellular signaling domains. SP, signal peptide; TM, transmembrane. A lentiviral CAR construct against human CD19 containing the FMC63 scFv is also shown. GTN is a bicistronic lentiviral construct expressing C. perfringens neuraminidase (CpNA) in tandem with a C-terminal 6× Histidine tag (6× His) as well as the transduction marker GFP. (B) After overnight stimulation with Dynabeads, T cells were co-infected with an EGFR CAR and GTN lentiviral supernatants. These cells were expanded for 3 days. Cellular lysates and supernatants were collected and immunoblotted with anti-His antibody. Relative protein loading was determined by immunoblotting for B-Actin. Representative data from two independent experiments are shown. (C) CpNA-expressing CAR T cells were generated as in (B) Surface EGFR CAR expression was measured by staining with a recombinant EGFRvIII-Fc protein (H + L) followed by anti-Fc-APC labeling. GTN levels were simultaneously detected by GFP expression. CAR + cells were defined as double-positive for CpNA (x axis) and PE (y axis). Representative flow plots from three independent experiments are shown. (D) The percentage of T cells positive for CAR expression after preparation as in (C). Representative data from one of three donors are shown. (E) CAR T cells were co-cultured with either U87-MG or Nalm6 tumor cells at a 10:1 ratio for 24 h. Cellular supernatants were collected and NA enzymatic activity was detected as described in the materials and methods. The mean ± SEM values of three independent experiments with separate donors are shown. Data were analyzed with pairwise t tests corrected for multiple comparisons using the Holm-Sidak method. ∗∗∗p < 0.001 for CAR T cells co-cultured with tumor cells expressing the CAR's target antigen versus control cells expressing an irrelevant antigen.

Figure 2

The combination of CAR T-secreted CpNA and exogenous galactose oxidase (GO) enhances T cell-mediated tumor lysis of U87 cells (A) U87-MG as well as Nalm6 tumor cells were treated with exogenous CpNA and GO for 24 h. Tumor cell lysis was measured by luciferase assay. p = 0.31 for enzyme effect on cell proliferation by a one-way ANOVA using a Tukey multiple comparison correction. (B) CD19-directed CAR T cells were co-cultured with U87 target cells. The media was conditioned with exogenous GO at various concentrations, and U87-MG tumor lysis was assessed via bioluminescence at 24 h. (C) T cells were co-infected with an EGFR-specific CAR and either GFP or CpNA lentiviral supernatants. The CAR T cells were co-cultured with luciferase-expressing U87-MG target cells, in medium conditioned with exogenous GO. After 24 h, cytotoxicity across a range of E:T ratios was measured by a luciferase-based killing assay. Values are mean ± SEM. A representative experiment from three independent replicates with separate donors is shown. ∗∗∗p = 0.0002 for 806 GTN GO versus 806 GTN at a 1:1 E:T ratio; ∗∗p = 0.0024 for 806 GTN GO versus 806 GTN at a 1:3 E:T ratio. (D) Tumor cell lysis was measured as in (C), but anti-CD19 CAR T cells were used instead of anti-EGFR CAR T cells. ∗∗∗p = 0.0002 for CD19 GTN GO versus CD19 GTN at a 1:1 E:T ratio; ∗p = 0.0108 for CD19 GTN GO versus CD19 GTN at a 1:3 E:T ratio. (E) CAR T cells, coexpressing either GFP or CpNA were treated with exogenous galactose oxidase for 24 h. Cellular supernatants were collected and IFNγ levels were detected by ELISA. (F) IL-2 levels in 24 h supernatants as detected by ELISA.

Figure 3

The combination of CpNA and GO activate T cells and promote tumor lysis in a CD2:CD58-dependent manner (A) Jurkat cells were infected with the lentiCRISPR-v2 system adapted with gRNAs for CD2, CD28, CD58, the T cell receptor alpha and beta chains (TRAC and TRBC), and a scrambled nontargeting control. The cells were selected with puromycin and knockout confirmed by flow cytometry 5 days after transduction. After an overnight incubation with NA and GO, the cells were stained with APC-conjugated anti-CD69 antibody and assessed by flow cytometry. (B) Luciferase expressing U87 and U251 cells were infected with the lentiCRISPR-v2 system encoding gRNAs against CD58 or a scrambled sequence. Knockout was confirmed after 5 days. The target cells were plated at 20 × 10³ per well and co-incubated with nontransduced or 806 CAR T cells at E:T ratios of 10:1, 3:1, 1:1, or 1:3. Wells with nontransduced T cells were treated with NA and GO (50 mU/mL and 375 mU/mL, respectively) or PBS vehicle. Tumor lysis was determined by bioluminescence assessment after 24 h. Data are means ± SEM, showing a representative experiment from three replicates with separate donors.

Figure 4

Tumor bioluminescence in NSG mice after implantation with U87 tumor cells and treatment with CpNA secreting CAR T cells (A) NSG mice were implanted by subcutaneous flank injection with 250 × 10³ luciferase-expressing U87 tumor cells. On day 7, the mice received 500 × 10³ CAR T cells in PBS injected by tail vein. The CAR T cells expressed 806 (EGFR-targeting) or anti-CD19 CARs plus co-transduced GTN (CpNA secreting) or GFP constructs. On days 8 and 21, the mice received 30 mL of GO (37,500 mU/mL in PBS) or vehicle by intra-tumoral injection. Every seventh day, the tumor bioluminescence was assessed with the IVIS Spectrum imaging system. (B) Bioluminescence measurements in mice treated with CD19 CAR T cells. Data are means ± SEM from seven replicate mice per cohort. By T test statistics, ∗p = 0.0474 for CD19 GTN GO versus CD19 GTN PBS at 21 days; ∗∗p = 0.0069 for CD19 GTN GO versus CD19 GTN PBS at 28 days; p = 0.9800 for CD19 GTN GO versus CD19 GTN PBS at 35 days. (C) Tumor volume measurements in mice treated with CD19 CAR T cells. Data are means ± SEM from seven replicate mice per cohort. By T test statistics, p = 0.1013 for CD19 GTN GO versus CD19 GTN PBS at 35 days. (D) Bioluminescence measurements in mice treated with anti-EGFR (806) CAR T cells. Data are means ± SEM from seven replicate mice per cohort. ∗∗p = 0.0040 for 806 GTN GO versus 806 GTN PBS at 21 days; p = 0.1131 for 806 GTN GO versus 806 GTN PBS at 28 days; ∗∗∗p = 0.0005 for 806 GTN GO versus 806 GTN PBS at 35 days. (E) Tumor volume measurements in mice treated with anti-EGFR (806) CAR T cells. Data are means ± SEM from seven replicate mice per cohort. ∗p = 0.0240 for 806 GTN GO versus 806 GTN PBS at 35 days.

Figure 5

Targeting sialic acids with NA enhances CAR T cell antitumor function in the Nalm-6 xenograft model of leukemia (A) NSG mice were infused by tail vein injection with 1 × 10⁶ luciferase-expressing Nalm tumor cells (day 0). On day 5, the mice received 1 × 10⁶ CAR T cells in PBS injected by tail vein. The CAR T cells expressed either tumor-targeting anti-CD19 CARs or irrelevant control 806 (EGFR-targeting) CARs, plus co-transduced GTN (CpNA secreting) or GFP constructs. Every week, the tumor bioluminescence was assessed with the IVIS Spectrum imaging system. (B) Bioluminescence measurements in Nalm6 tumor-bearing mice treated with anti-CD19 or 806 CAR T cells either secreting CpNA (GTN construct) or expressing GFP. Data are means ± SEM from starting cohorts of five mice per treatment arm. By two-way ANOVA, including all treatment arms, both time effect (∗p = 0.0485) and treatment cohort effect (∗p = 0.0226) were significant. A two-way ANOVA including only CD19 GTN and CD19 GFP arms showed significant time (∗p = 0.0446), treatment (∗p = 0.0163), and time × treatment interaction (∗∗∗p < 0.0001) effects. (C) Survival proportions of Nalm6 tumor-bearing mice in each treatment cohort. Mice were killed if found to have BLI measurements over 1 × 10¹⁰ photon flux (p/s). Analysis with log rank (Mantel-Cox) testing demonstrated that the CD19 GTN and CD19 GFP survival curves are significantly different (∗p = 0.0210). (D) Blood harvested on day 44 by retro-orbital puncture was stained with anti-human CD45 antibody in Truecount tubes (BD Biosciences) to detect adoptively transferred T cells. Data are CD45 + cells per mL of blood for individual mice plus cohort means ± SEM. By T test statistics with Welch's correction for unequal variances, the differences between CD19 GTN and CD19 GFP were not significant (p = 0.3372). (E) Images from final weekly measurement demonstrating BLI (photon flux intensity) of surviving mice, treated with either CpNA secreting (top) or control GFP-expressing (bottom) CD19-directed CAR T cells.