Allogeneic CD20-targeted γδ T cells exhibit innate and adaptive antitumor activities in preclinical B-cell lymphoma models

Abstract

Objectives: Autologous chimeric antigen receptor (CAR) αβ T-cell therapies have demonstrated remarkable antitumor efficacy in patients with haematological malignancies; however, not all eligible cancer patients receive clinical benefit. Emerging strategies to improve patient access and clinical responses include using premanufactured products from healthy donors and alternative cytotoxic effectors possessing intrinsic tumoricidal activity as sources of CAR cell therapies. γδ T cells, which combine innate and adaptive mechanisms to recognise and kill malignant cells, are an attractive candidate platform for allogeneic CAR T-cell therapy. Here, we evaluated the manufacturability and functionality of allogeneic peripheral blood-derived CAR+ Vδ1 γδ T cells expressing a second-generation CAR targeting the B-cell-restricted CD20 antigen.

Methods: Donor-derived Vδ1 γδ T cells from peripheral blood were ex vivo-activated, expanded and engineered to express a novel anti-CD20 CAR. In vitro and in vivo assays were used to evaluate CAR-dependent and CAR-independent antitumor activities of CD20 CAR+ Vδ1 γδ T cells against B-cell tumors.

Results: Anti-CD20 CAR+ Vδ1 γδ T cells exhibited innate and adaptive antitumor activities, such as in vitro tumor cell killing and proinflammatory cytokine production, in addition to in vivo tumor growth inhibition of B-cell lymphoma xenografts in immunodeficient mice. Furthermore, CD20 CAR+ Vδ1 γδ T cells did not induce xenogeneic graft-versus-host disease in immunodeficient mice.

Conclusion: These preclinical data support the clinical evaluation of ADI-001, an allogeneic CD20 CAR+ Vδ1 γδ T cell, and a phase 1 study has been initiated in patients with B-cell malignancies (NCT04735471).

Keywords: B‐cell lymphoma; CD20; adoptive cell therapy; chimeric antigen receptor; γδ T cells.

Conflict of interest statement

KPN, TB, AA, AM, JMR, LB, MMB, JK‐W, HS, SP, AD, CM, NTH, HT, VDR, AG‐R, M Hoopes, DS, RS, M Herrman, SEA, BTA, ZA, SP and SMH are, or were, employees of Adicet Bio, Inc., and FD, KS and KB are employees of Regeneron Pharmaceuticals, Inc.

© 2022 Adicet Bio Inc. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.

Figures

Figure 1

Binding properties of the fully human anti‐CD20 mAb clone 3H7. (a) Titration of 3H7 mAb (), therapeutic anti‐CD20 mAb rituximab () and human IgG1 control antibody () binding to parental HEK293 cells, HEK293 cells stably expressing human CD20 (HEK293‐hCD20), and Raji and Daudi human B‐cell lymphoma cell lines. Cell‐surface binding of the antibodies over a range of concentrations (1.69 pm to 100 nm) was performed at 4°C, detected by flow cytometry using APC‐labelled anti‐human IgG antibody and reported as geometric mean fluorescence intensity (geoMFI). For each dose curve, staining with the APC‐labelled anti‐human IgG antibody alone is included in the graph as a continuation of the threefold serial dilution and is represented as the lowest dose. EC50 values of the binding curves for each anti‐CD20 mAb on each CD20+ cell line are shown. Data are representative of two independent experiments. (b) Biochemical off‐rates of 3H7 mAb () and rituximab () were determined by measuring the amount of cell‐bound antibody over the course of a 3‐h time period. Antibodies (2 μg mL−1 each) were incubated with Raji cells for 2 h at room temperature. The cells were then washed, resuspended in 1% serum containing medium and incubated at 37°C. At time 0, 15, 30, 45, 60, 90, 120 and 180 min, an aliquot of cells was removed, washed, stained with PE‐labelled anti‐human Fc antibody and analysed by flow cytometry.

Figure 2

Overview of allogeneic CD20 CAR Vδ1 γδ T‐cell manufacturing process. (a) Flow chart highlighting the key steps in the manufacture of allogeneic CD20 CAR+ Vδ1 γδ T cells. Vδ1 γδ T cells present in healthy donor‐derived PBMCs are ex vivo‐activated and expanded with an agonistic anti‐Vδ1 mAb and then engineered to express the CD20 CAR by transduction with a self‐inactivating, replication‐incompetent gamma‐retroviral vector. After transduction, γδ T cells are further expanded in culture with human IL‐2 and are then enriched by αβ T‐cell depletion prior to formulation and cryopreservation. (b) Schematic diagram of the second‐generation CD20 CAR, which consists of the 3H7 scFv combined with CD8α hinge and transmembrane regions fused to the intracellular signalling domains of 4‐1BB and CD3ζ. CAR expression is regulated by the EF1a promoter. (c) The CD20 CAR+ Vδ1 γδ T‐cell manufacturing process results in a substantial fold expansion of Vδ1 γδ T cells. The mean ± standard deviation (SD) of five manufacturing runs using PBMCs from three different donors (large‐scale) and five different donors (small‐scale) are shown. (d) Average percentage of Vδ1 γδ T cells expressing the CD20 CAR from small‐scale and large‐scale manufacturing runs as measured by flow cytometry. The mean ± SD of five small‐scale manufacturing runs using PBMCs from five different donors and of seven large‐scale manufacturing runs using PBMCs from four different donors are shown. (e) Box plots of % cell composition using flow cytometric analysis of CD20 CAR+ Vδ1 T‐cell products derived from four different donors at Day 0, pre‐depletion and post‐depletion of αβ T‐cell time points (large scale). Box plot horizontal lines represent the mean, minimum and maximum values of each % cell type (Vδ1 T cells, Vδ2 T cells, αβ T cells and NK cells). (f) Cellular composition of final cell product was assessed by flow cytometry. Pie charts represent the composition of three representative donor‐derived CD20 CAR+ Vδ1 γδ T‐cell products. (g) Selective activation and expansion of Vδ1 γδ T cells with an agonistic anti‐Vδ1 mAb results in a diverse, not skewed, TCRγ repertoire. Treemaps of the TCRγ repertoire, which were created using Tableau software and are reported as TRGV (Vγ) usage in each functionally rearranged CDR3 sequence. Two donor‐derived CD20 CAR+ Vδ1 γδ T‐cell products are shown. The size of the square indicates its relative frequency in each sample. Relative frequencies of functionally rearranged TRGV (Vγ) sequences from ImMunoGeneTics (IMGT) are provided as a reference. Statistics were calculated using the paired t‐test; ns, not significant.

Figure 3

Structure and signalling capacity of the CD20 (3H7) CAR. (a) Transduction efficiency of Jurkat‐Lucia™ cells, which carry an NFAT‐inducible luciferase reporter, with the CD20 CAR gamma‐retroviral vector. Percentage of CAR‐transduced Jurkat‐Lucia cells expressing the CD20 CAR on their cell surface was measured by flow cytometry and is representative of three independent experiments. (b) The CD20 CAR, when expressed on the surface of Jurkat‐Lucia cells, does not induce antigen‐independent tonic signalling. CAR‐mediated signal transduction and activation were assessed in the presence or absence of target antigen by culturing parental (untransduced) and CAR‐transduced Jurkat‐Lucia cells either alone or at a 1:1 E:T ratio with CD20+ Raji cells. Stimulation with PMA (phorbol 12‐myristate 13‐acetate) and ionomycin was used as a positive control for NFAT activation. Duplicate samples were tested for each condition. Data are representative of three independent experiments. (c) The 3H7 CAR is expressed as dimers and higher‐order oligomers on the surface of Vδ1 γδ T cells. Untransduced (UT) or CAR‐transduced Vδ1 γδ T cells were surface‐biotinylated using EZ‐Link Sulfo‐NHS‐LC‐Biotin, lysed in Brij® O10 buffer containing a protease inhibitor cocktail and incubated with an anti‐CD20 CAR mAb coupled to Sepharose 4B beads. Immunoprecipitated proteins were resolved by non‐reducing (NR) and reducing SDS‐PAGE analysis and transferred to nitrocellulose membranes. Biotinylated CAR complexes were visualised by IRDye® 800CW Streptavidin and the Odyssey Imager. The positions of CAR dimers and oligomers (NR conditions) and monomers (R conditions) are marked. Data are representative of four independent experiments. (d) CAR‐associated phospho‐CD3ζ is only detected after CAR stimulation. CD20 CAR+ Vδ1 γδ T cells were unstimulated or stimulated on Protein L‐coated 96‐well plates for 10 min and then lysed in Milliplex® MAP Cell Signaling Universal Lysis Buffer. Lysates were resolved by reducing SDS‐PAGE analysis and then transferred to nitrocellulose membranes. Membranes were blocked and then incubated with antibodies against total CD3ζ or phospho‐CD3ζ (Tyr 142) overnight at 4°C on a shaker. CAR‐associated CD3ζ and phospho‐CD3ζ proteins were visualised using anti‐mouse IgG IR800 and the Odyssey Imager, and their positions are marked. In whole‐cell lysates, the CAR resolves as two bands: enzymatic deglycosylation of cell lysates revealed that the upper band is the glycosylated form, and that the lower band is the unglycosylated form (data not shown). Data are representative of three independent experiments.

Figure 4

Phenotypic analysis of CD20 CAR+ Vδ1 γδ T cells. (a, b) Flow cytometric analysis reveals that the majority of CAR+ Vδ1 γδ T cells in three donor‐derived CD20 CAR+ Vδ1 γδ T‐cell products exhibit a naïve‐like T‐cell memory phenotype. Expression levels of CCR7, CD62L, CD95 and CD45RO on gated naïve (CD27+ CD45RA+) CD4+ T cells from healthy donor‐derived PBMCs are shown as naïve T‐cell controls. Memory cell subsets were defined by the expression of CD27 and CD45RA: naïve‐like (CD27+ CD45RA+); central memory or TCM (CD27+ CD45RA−); effector memory or TEM (CD27− CD45RA−); and terminally differentiated effector memory or TEMRA (CD27− CD45RA+). (c) Heatmap showing percentages of CAR+ Vδ1 γδ T cells in three donor‐derived CD20 CAR+ Vδ1 γδ T‐cell products that express chemokine receptors, natural killer (NK) cell receptors and terminal differentiation markers. (d) Heatmap showing percentages of CAR+ Vδ1 γδ T cells in four donor‐derived CD20 CAR+ Vδ1 γδ T‐cell products that co‐express PD‐1 and another co‐inhibitory receptor, that is TIM‐3, LAG‐3 or TIGIT.

Figure 5

Comparison of the cytotoxic potentials of untransduced Vδ1 γδ T cells, CD20 CAR‐transduced Vδ1 γδ T cells and CD20 CAR‐transduced αβ T cells against CD20+ target cells. Cytotoxic potentials of untransduced Vδ1 γδ T cells, CD20 CAR‐transduced Vδ1 γδ T cells (71.5% CAR+) and CD20 CAR‐transduced αβ T cells (68% CAR+), all derived from the same healthy donor, were evaluated against CD20+ target cells in a 48‐h IncuCyte Immune Cell Killing Assay, in which T cells were co‐cultured with NucR‐expressing Raji or Mino target cells at E:T ratios of 10:1, 3:3:1 and 1.1:1. The viability of the NucR‐expressing targets, reported as cytotoxicity index, was monitored every 2 h over the course of 48 h with the IncuCyte Live‐Cell Analysis System. Please note that the lower the cytotoxicity index, the better the cytotoxic potential. Data are shown as mean ± SD of triplicates and are representative of three independent experiments using cell products from two different donors. The killing curves for each cell product were statistically analysed using the mixed model two‐way ANOVA. (****P < 0.0001, **P < 0.01 and *P < 0.05; NS, not significant).

Figure 6

In vitro functional analyses of preclinical large‐scale manufactured CD20 CAR+ Vδ1 γδ T cells. (a) Relative CD20 expression levels on malignant B‐cell lines [Mino, Raji, JVM‐2, WILL‐2, RR‐Raji], normal peripheral blood B cells and the CD20− Jurkat T‐ALL cell line as determined by flow cytometry. Relative CD20 expression is reported as CD20 geoMFI/isotype control geoMFI (MFI ratio). The data are representative of two independent experiments. (b) Cytotoxic potential of CD20 CAR+ Vδ1 γδ T cells against B‐lymphoma cell lines expressing varying levels of CD20 in a short‐term cytotoxicity assay. Cytotoxic potential was assessed by titrating E:T ratios in an 18‐h cytotoxicity assay in which CD20 CAR+ Vδ1 γδ T‐cell effectors were co‐cultured with RFluc‐expressing target cells. Controls included CD20 CAR+ Vδ1 γδ T cells and B‐lymphoma cell lines cultured alone. Data are shown as mean ± SD of triplicates and are representative of two independent experiments using two different donor‐derived CD20 CAR+ Vδ1 γδ T‐cell products. (c) Effect of clinically relevant concentrations of rituximab on the ability of CD20 CAR+ Vδ1 γδ T cells to recognise and kill RFluc‐expressing RR‐Raji target cells. Cytotoxic potential of CD20 CAR+ Vδ1 γδ T cells against RR‐Raji cells at a fixed 3:1 ratio was assessed after pre‐incubation of target cells with increasing concentrations of rituximab (0–400 μg mL−1) in an 18‐h luciferase cytotoxicity assay. Data are shown as mean ± SD of triplicates and are representative of two different rituximab‐resistant B‐lymphoma cell lines [**P ≤ 0.005 (t‐test)]. (d) Cytokine and chemokine production by CD20 CAR+ Vδ1 γδ T‐cell effectors after an 18‐h co‐culture with Raji cells at fixed 1.75:1 ratio. Levels of IFN‐γ, GM‐CSF, TNF‐α, IL‐2, IL‐6, IL‐8, IL‐13, MIP‐1α, MIP‐1β, RANTES, IP‐10 and IL‐17A were measured in culture medium supernatants using the Luminex platform. Data are shown as mean ± SD of triplicates and are representative of two independent experiments using two different donor‐derived CD20 CAR+ Vδ1 γδ T‐cell products. (e) Proliferative potential of CD20 CAR+ Vδ1 γδ T cells following three rounds of target antigen exposure in a 7‐day culture period. Aliquots of cells harvested on Days 2, 5 and 7 were analysed by flow cytometry to assess cellular proliferation by dye dilution. Representative data from three independent experiments using three donor‐derived CD20 CAR+ Vδ1 γδ T‐cell products are shown. (f) Cytotoxic potential of CD20 CAR+ Vδ1 γδ T cells against Raji cells in a long‐term cytotoxicity assay. Cytotoxic potential was assessed at a 1:2 E:T ratio in a 120‐h cytotoxicity assay in which CD20 CAR+ Vδ1 γδ T cells were co‐cultured with NucR‐expressing Raji cells ± 20 IU of human IL‐2. The viability of the NucR‐expressing Raji cells was monitored every 2 h over the course of 120 h with the IncuCyte Live‐Cell Analysis System. Data are shown as mean ± SD of triplicates and represent three donor‐derived CD20 CAR+ Vδ1 γδ T‐cell products.

Figure 7

In vivo and ex vivo analyses of CD20+ tumor‐bearing NSG mice treated with CD20 CAR+ Vδ1 γδ T cells. (a)In vivo efficacy of three different doses (5 × 106, 1 × 107 and 2 × 107) of viable CD20 CAR+ Vδ1 γδ T cells in combination with 13 000 IU IL‐2 in a SC Raji Burkitt lymphoma model in NSG mice (n = 5 per group). The SC xenograft model was chosen because tumor control in this model is dependent on the ability of CAR T cells to traffic to the tumor, to proliferate and to lyse tumor cells. Tumor growth, body weight and animal health were monitored two times per week after tumor implantation. Tumor volume was calculated using the formula: Volume (mm3) = (length × width2)/2. The Kruskal–Wallis test was used to assess statistical significance among the groups on Day 14 (**P = 0.0011). Data are representative of three independent experiments using one donor‐derived CD20 CAR+ Vδ1 γδ T‐cell product. (b)In vivo efficacy of three different doses (2.5 × 106, 5 × 106 and 1 × 107) of viable CD20 CAR+ Vδ1 γδ T cells in combination with 13 000 IU IL‐2 in a SC JVM‐2 mantle cell lymphoma model in NSG mice (n = 5 per group). The Kruskal–Wallis test was used to assess statistical significance among the groups on Day 30 (**P = 0.0062). Data are representative of two independent experiments using one donor‐derived CD20 CAR+ Vδ1 γδ T‐cell product. (c) Proliferative potential of untransduced and CD20 CAR+ Vδ1 γδ T cells in blood, bone marrow and tumor harvested from Raji tumor‐bearing mice on Day 6 post‐treatment. Untransduced and CD20 CAR+ Vδ1 γδ T cells were labelled with CellTrace Violet prior to adoptive transfer at a dose of 8 × 106 cells in combination with IL‐2 (n = 3 per group). Data are representative of two independent experiments using one donor‐derived CD20 CAR+ Vδ1 γδ T‐cell product. (d) Differentiation potential of untransduced and CD20 CAR+ Vδ1 γδ T cells in bone marrow and tumor harvested from Raji tumor‐bearing mice on Day 6 post‐treatment. Memory cell subsets were defined by the expression of CD27 and CD45RA: naïve‐like (CD27+ CD45RA+); TCM (CD27+ CD45RA−); TEM (CD27− CD45RA−); and TEMRA (CD27− CD45RA+). Memory phenotypes of pre‐dose untransduced and CD20 CAR+ Vδ1 γδ T cells are shown for comparison. (e) Venn diagrams of DEGs in Raji tumors treated with 1 × 107 CD20 CAR+ Vδ1 γδ T cells (red circle) from two donors and in JVM‐2 tumors treated with 1 × 107 CD20 CAR+ Vδ1 γδ T cells (blue circle) from two donors in combination with IL‐2. The overlapping area represents DEGs that are shared between the two tumor models. Gene expression levels were quantitated using the nCounter® CAR T Characterization and Human Immunology Panels on the nCounter® SPRINT Profiler according to the manufacturer’s instructions. nSolver™ software was used to normalise the data and to identify DEGs (i.e. adjusted P ≤ 0.05 and log2 fold change ≥ 1) in tumors treated with CD20 CAR+ Vδ1 γδ T cells versus tumors treated with untransduced Vδ1 γδ T cells. Gene ontology analysis was performed using Database for Annotation, Visualization and Integrated Discovery (DAVID, https://david.ncifcrf.gov/) to identify the biological pathways associated with DEGs that are unique to each tumor model and that are shared by the two tumor models.

Figure 8

Phenotypic and functional analyses of clinical‐scale CD20 CAR+ Vδ1 γδ T cells. (a) Dot plot demonstrating that 79% of clinical‐scale CD20 CAR+ Vδ1 γδ T cells exhibit a naïve‐like T‐cell memory phenotype. Data are representative of two independent experiments. (b) Cytotoxic potential of, and IFN‐γ production by, clinical‐scale CD20 CAR+ Vδ1 γδ T cells (49% CAR+) against Raji cells in an 18‐h cytotoxicity assay. Controls included CD20 CAR+ Vδ1 γδ T cells and B‐lymphoma cell lines cultured alone. Levels of IFN‐γ in culture medium supernatants were measured using the Luminex platform. Data are shown as mean ± SD of triplicates and are representative of three independent experiments. (c) Cytotoxic potential of clinical CD20 CAR+ Vδ1 γδ T cells against Raji cells in a long‐term cytotoxicity assay. Cytotoxic potential was assessed at a 1:2 E:T ratio in a 120‐h cytotoxicity assay in which CD20 CAR+ Vδ1 γδ T‐cell effectors were co‐cultured with NucR‐expressing Raji cells in the presence or absence of 20 IU of human IL‐2. The viability of the NucR‐expressing Raji cells was monitored every 2 h over the course of 120 h with the IncuCyte Live‐Cell Analysis System. Data are shown as mean ± SD of triplicates and are representative of three independent experiments. (d)In vivo efficacy of two different doses (5 × 106 and 1 × 107) of viable clinical‐scale CD20 CAR+ Vδ1 γδ T cells in combination with 13 000 IU IL‐2 in a SC Raji Burkitt lymphoma model in NSG mice (n = 7 mice per cell treatment group and n = 10 mice in the tumor‐alone group). Tumor growth, body weight and animal health were monitored two times per week after tumor implantation. The Kruskal–Wallis test was used to assess statistical significance among groups on Day 15 (***P = 0.0001). The data represent one independent experiment.