CAR-iNKT cells targeting clonal TCRVβ chains as a precise strategy to treat T cell lymphoma

Abstract

Introduction: Most T cell receptor (TCR)Vβ chain-expressing T cell lymphomas (TCL) including those caused by Human T cell leukaemia virus type-1 (HTLV-1) have poor prognosis. We hypothesised that chimeric antigen receptor (CAR)-mediated targeting of the clonal, lymphoma-associated TCRβ chains would comprise an effective cell therapy for TCL that would minimally impact the physiological TCR repertoire.

Methods: As proof of concept, we generated CAR constructs to target four TCRVβ subunits. Efficacy of the CAR constructs was tested using conventional T cells as effectors (CAR-T). Since invariant NKT (iNKT) cell do not incite acute graft-versus-host disease and are suitable for 'off-the-shelf' immunotherapy, we generated anti-TCRVβ CAR-iNKT cells.

Results: We show that anti-TCRVβ CAR-T cells selectively kill their cognate tumour targets while leaving >90% of the physiological TCR repertoire intact. CAR-iNKT cells inhibited the growth of TCL in vivo, and were also selectively active against malignant cells from Adult T cell leukaemia/lymphoma patients without activating expression of HTLV-1.

Discussion: Thus we provide proof-of-concept for effective and selective anti-TCRVβ CAR-T and -iNKT cell-based therapy of TCL with the latter providing the option for 'off-the-shelf' immunotherapy.

Keywords: ATL; T cell lymphoma; T cell receptor; adult T cell leukaemia/lymphoma; chimeric antigen receptor (CAR) T-cells; human T cell leukaemia virus type-1; human T cell lymphotropic virus type-1 (HTLV-1); iNKT.

Figure 1

Efficacy and lack of significant off-target killing of primary T cells by Vβ-specific CAR-T cells. PBMC were transduced with anti-Vβ1, -2, -9, -11 or -CD19 CAR constructs. After three days the transduction efficiency was evaluated by protein L staining and the frequency of CD3+ T cells expressing each targeted Vβ subunit was quantified by flow cytometry. (A) Representative dot plots of CAR expression and phenotype of transduced cells. (B) Frequency of CAR-expressing cells in each culture. (C) Frequency of T cells expressing TCRVβ1, -2, -9 or -11 in transduced T cells. GS2 refers to the CAR construct containing two (G₄S₁) repeats and GS3 to the construct containing three (G₄S₁) repeats. (D) Representative dot plots showing TCRVβ1 staining in the same experiment. (E) PBMC from two normal donors were cultured alone or transduced with anti-Vβ1, 2, 9 or 11 CAR (GS2) constructs. Five days post transduction, transduction efficiency was evaluated by protein L staining, and the frequency of CD3+ T cells expressing each of 24 TCRVβ subunits was quantified. Values shown are the mean percentage of cells expressing each subunit normalised to the frequency of cells expressing that subunit the untransduced PBMC control from each individual. (F) Killing of in vitro-expanded primary T cell lines expressing TCRVβ1, 2, 9 or 11 by T cells transduced with anti-Vβ GS2/GS3 CAR constructs. Target cells were stained with CFSE co-cultured with effectors at a range of effector to target (E:T) ratios in duplicate. After 24h cells were harvested and stained with 7aad. The frequency of dead targets in each culture condition was assayed by flow cytometry. (G) Killing of a T lymphoblastic lymphoma cell line (JRT3) transduced to express TCR containing TCRVβ subunits. (H) Cytokine production and degranulation (CD107a mobilisation) by anti-Vβ2 CAR-T cells cultured alone, in the presence of TCRVβ2-transduced or untransduced JRT3-T3.5 cells or in the presence of primary T cells expressing TCRVβ2. (I) Representative data showing cytokine production and degranulation by CD4+ CAR-T and CD8+ CAR-T.

Figure 2

Cytotoxic activity of anti-TCRVβ CAR-iNKT in vitro and in vivo. (A) Killing of untransduced (JRT3) and TCR-GFP-transduced (JRT3-TCRVβ) JRT-3 T 3.5 cells by anti-Vβ CAR-iNKT cells. Target cells were stained with CFSE co-cultured with effectors at a range of effector to target (E:T) ratios in duplicate. After 24h cells were harvested and stained with 7aad. The frequency of dead targets in each culture condition was assayed by flow cytometry. Results are representative of two or more independent experiments. (B) CD107a mobilisation, IFN-γ and TNF-α production by CD3+ anti-Vβ2 and anti-CD19 CAR-iNKT when cultured alone or in the presence of untransduced JRT3-T3.5 cells (JRT), JRT3-T3.5 cells transduced with Vβ2+ TCR-GFP (Vβ+), or primary cells expressing Vβ2 (TCRVβ2+T cells). Results are from a single experiment and are representative of 2 independent experiments. (C) Representative data showing cytokine production and degranulation by CD4+, CD8+ and double negative CAR-iNKT. (D) Enhancement of killing of α-gal loaded TCRVβ2-GFP-transduced JRT3-T3.5 cells by anti-Vβ2 CAR-iNKT but not anti Vβ2 CAR-T cells. Where indicated, target cells were incubated with 200ng/ml α-gal before co-culture with effectors. Results shown are representative of two repeat experiments. (E) In vivo protocol. 5x10⁶ TCRVβ+ JRT cells were suspended in Matrigel and injected subcutaneously. On day 18, 1 x 10⁶ effector cells were injected intravenously into the tail vein. Groups consisted of untreated (n=5), Vβ2 CAR-iNKT (n=7) or CD19 CAR-iNKT (n=5). (F) Tumour volume for each group was measured periodically using calipers and (G) tumour weight was determined after the experiment was terminated. ** indicates p<0.001, * indicates P<0.05 and ns indicates no significant difference, Kruskal-Wallis test with Dunn's multiple comparisons test.

Figure 3

Killing of ex vivo CD4+CCR4+CD26- ATL cells expressing TCRVβ1 and Vβ2 by anti-TCRVβ CAR-iNKT. Cryopreserved PBMC from two ATL patients and normal donor (ND) were stained with CellTrace violet and co-incubated with anti-Vβ1 -Vβ2 or-CD19 CAR-iNKT at the indicated effector:target (PBMC) ratios in triplicate. (A) Immunophenotype of samples before co-culture. After 24h co-culture, cells were stained with a viability stain, anti-CD4, CCR4, CD26, TCRVβ1 or 2 and TCRαβ, fixed, and the frequency of dead CD4+CCR4+CD26- T cells and CCR4- and CCR4+CD26+ cells (‘rest’ of CD4+ T cells) was determined by flow cytometry. (B) Killing of cells from patient ATL01 and the normal donor by TCRVbeta1 CAR-iNKT. (C) Killing of cells from patient ATL02 and the normal donor by TCRVbeta2 CAR-iNKT.

Figure 4

Effect of CAR-iNKT on the frequency of antiviral CTL and HTLV-1 expressing CD4+ T cells. (A–C) Frequency of M1_58-66 and Tax_11-19 HLA-A*0201 pentamer+CD8+ T cells after co-culture with CAR-iNKT. CD4-depleted PBMC from three HLA-A*0201+ HTLV-1 carriers were stained with CellTraceViolet and were cultured alone or with an 1:1 ratio of anti-TCRVβ1 -Vβ2 or -CD19 CAR-iNKT. After 16-18h co-culture, cells were stained with a viability stain, and anti-CD3, -CD8 antibodies and M1_58-66 or Tax_11-19 pentamers. Cells were analysed by flow cytometry and the frequency of live Pentamer+CellTraceViolet+CD3+CD8+ cells was determined. (D, E) Frequency of HTLV-1 Tax expressing CD4+ cells after co-culture with CAR-iNKT. CellTraceViolet stained, positively selected CD4+ cells from the same donors were cultured alone or in the presence of Vβ1- Vβ2- or CD19-CAR iNKT. After 16-18h, cells were stained with a viability stain, anti-CD3,-CD4, -CD8 and -TCRVβ1 or -TCRVβ2. Cells were subsequently fixed, and stained intracellularly with anti-Tax antibody. Cells were analysed by flow cytometry and the frequency of live TCRVβ1/2+CD3+ cells and live Tax+CD4+ cells were determined.