Engineered off-the-shelf therapeutic T cells resist host immune rejection

Abstract

Engineered T cells are effective therapies against a range of malignancies, but current approaches rely on autologous T cells, which are difficult and expensive to manufacture. Efforts to develop potent allogeneic T cells that are not rejected by the recipient's immune system require abrogating both T- and natural killer (NK)-cell responses, which eliminate foreign cells through various mechanisms. In the present study, we engineered a receptor that mediates deletion of activated host T and NK cells, preventing rejection of allogeneic T cells. Our alloimmune defense receptor (ADR) selectively recognizes 4-1BB, a cell surface receptor temporarily upregulated by activated lymphocytes. ADR-expressing T cells resist cellular rejection by targeting alloreactive lymphocytes in vitro and in vivo, while sparing resting lymphocytes. Cells co-expressing chimeric antigen receptors and ADRs persisted in mice and produced sustained tumor eradication in two mouse models of allogeneic T-cell therapy of hematopoietic and solid cancers. This approach enables generation of rejection-resistant, 'off-the-shelf', allogeneic T-cell products to produce long-term therapeutic benefit in immunocompetent recipients.

Figure 1.. 4-1BB-specific ADR T cells selectively eliminate activated T and NK cells in vitro.

a, Schematic of the ADR structure. TM, transmembrane domain. b, ADR expression on the surface of primary human T cells (ADR T, blue). Non-transduced T cells were used as control (Ctrl, grey). Data are representative of >50 independent experiments with 12 different donors. c, Counts of activated human T cells following mock transduction (Ctrl T) or gammaretroviral transduction with CD19 CAR or ADR (n=6). P value for the comparison of Ctrl T and ADR T group on day 14 was shown and was calculated by one-way ANOVA with Holm correction for multiple comparisons. d-f, Non-transduced (Ctrl) or ADR T cells were cocultured with 4-1BB⁻ cell line NALM6 (d, left), 4-1BB⁺ cell line HDLM2 (d, right), autologous resting T cells (e, left), pre-activated T cells (e, right), resting NK cells (f, left), or pre-activated NK cells (f, right) at a 1:1 effector-to-target ratio for 24 hours. Residual target cells were quantified by flow cytometry and normalized to the target-only condition. In d-f, data denote mean±SD with individual data points obtained from different donors shown on each graph. P values were determined by two-tailed paired t test.

Figure 2.. ADR protects T cells from T-cell mediated rejection in vitro.

a, Schematic of model setup for b-d. Non-transduced (Ctrl) or ADR-transduced donor TCR^KO HLA-A2⁺ T cells were mixed with mismatched recipient’s whole PBMC at a 1:10 cell ratio. b, Representative flow plots showing total frequency of donor T cells on day 0 and day 9 of coculture. c-d, Absolute counts of donor T cells (c), recipient T cells (d, left) and recipient NK cells (d, right) during coculture. e, Schematic of model setup for f-h. Donor TCR^KO T cells and recipient NK-depleted PBMC were mixed at a 1:10 cell ratio. Non-transduced TCR^KO T cells were used as control (Ctrl T). f, Representative flow plots showing percentage of CD3⁻ HLA-A2⁺ donor T cells within total alive cells on day 0 and day 9 after coculture setup. g-h, Absolute cell counts of CD3⁻ HLA-A2⁺ donor TCR^KO T cells (g) and recipient CD3⁺ HLA-A2⁻ T cells (h) at specified days after coculture setup. i, Schematic of model setup for j-l. Donor TCR^KO T cells were mixed with primed recipient alloreactive T cells at a 1:1 ratio. j, Representative flow plots showing percentage of donor T cells on day 0 and day 2 of coculture. k-l, Absolute cell counts of donor T cells (k), recipient alloreactive T cells (l) on day 2 of coculture. All data denote mean±SD. In a-h, 6 unique donor-recipient pairs were used. P values were determined by two-way ANOVA with Sidak correction for multiple comparisons, non-significant (p>0.05) values are not shown. In k-l, each dot represents a unique donor-recipient pair, and p values were determined by a two-tailed paired Student’s t test.

Figure 3.. ADR protects T cells from NK-cell mediated rejection in vitro.

a, Schematic of model setup for b-d. Donor TCR^KOβ2m^KO T cells and recipient whole PBMC were mixed at a 1:5 ratio. b, Representative flow plots showing percentage of donor T cells on day 0 and day 6 of coculture setup. c-d, Absolute cell counts of HLA-ABC⁻ donor T cells (c), recipient HLA-ABC⁺ T cells (d, left) and recipient CD56⁺ HLA-ABC⁺ NK cells (d, right) during coculture. e, Schematic of model setup for f-h. Donor TCR^KOβ2m^KO T cells were mixed with freshly isolated recipient NK cells at a 1:1 ratio. f, Representative flow plots showing total frequency of donor T cells on day 0 and day 2 of coculture. g-h, Absolute cell counts of donor T cells (g) and recipient NK cells (h) on day 3 of coculture. All data denote mean±SD. In c-d, 5 unique donor-recipient pairs were used. P values were determined by two-way ANOVA with Sidak correction for multiple comparisons and non-significant (p>0.05) values are not shown. In g-h, each dot represents a unique donor-recipient pair, and p values were determined by two-tailed paired t test.

Figure 4.. ADR T cells are protected from allogeneic rejection in vivo.

a, Schematic of model setup for b-d (Ctrl, n=5; ADR, n=7). RTC, recipient T cells. b, Representative flow plots showing frequencies of donor HLA-A2⁻ Ctrl or ADR T cells in peripheral blood of mice on days 6 and 18. c-d, Frequencies of donor HLA-A2⁻ T cells (c) and recipient HLA-A2⁺ T cells (d) at specified time points. e, Isolated recipient T cells from mice that had previously received ADR T cells were cocultured with a 1:1 mix of donor and recipient TCR^KO T cells at a 2:1 effector-to-target ratio for 72 hours. The donor:recipient cell ratio was assessed at indicated times during coculture. Recipient T cells primed in vitro against irradiated donor cells were used as a positive control (Pos Ctrl) and a mix of targets alone as a negative control (Neg Ctrl). f, Schematic of model setup for g-I, (Ctrl, n=4; ADR, n=5). g, Representative flow plots showing percentage of donor Ctrl or ADR T cells in peripheral blood on day 6 and 14. h-i, Frequencies of donor HLA-A2⁻ Ctrl or ADR T cells (h) and HLA-A2⁺ recipient PBMC (i) at specified time points. TBI, total body irradiation. In c-d and h-i, data are mean±SD. P values were determined via two-way ANOVA with Sidak correction for multiple comparisons, non-significant (p>0.05) values are not shown.

Figure 5.. T cells co-expressing ADR and CAR retain function of both receptors in vitro.

a, Left, schematic of a CAR.ADR T cell and its respective targets. Right, representative flow plot showing co-expression of a second-generation CAR and ADR on T cell surface (purple) compared to non-transduced control (Ctrl) T cells (grey). Data are representative of >20 independent experiments with 8 individual donors. b, T cells expressing either CD19 CAR or ADR alone or co-expressing both receptors were cocultured with NALM6-GFP (β2m^KO) leukemia cells (b) or autologous activated T cells (c) at a 1:2 cell ratio for 48 hours. Residual counts of live target cells were quantified by flow cytometry at the end of coculture and normalized to target-only condition. d, T cells expressing either or both CAR and ADR were cocultured with a 1:1 mix of NALM6-GFP (β2m^KO) cells and autologous activated T cells for 48 hours. Residual normalized counts of target cells are plotted. Data from 7 individual donors are shown. All data denote mean±SD. P values were determined by one-way ANOVA with Tukey (b-c) or Holm-Sidak (d) correction for multiple comparisons.

Figure 6.. CD19 CAR T cells co-expressing ADR resist alloimmune rejection in vivo and retain potent anti-tumor function.

a, Schematic of the mouse model. All cell injections were performed intravenously. b-c, Frequencies of HLA-A2⁻ donor CAR T cells (b) and HLA-A2⁺ recipient T cells (RTC) (c) in peripheral blood after tumor injection. CAR T alone, CD19 CAR T cells were given to leukemia-bearing mice without pre-engrafted recipient T cells d, Leukemia progression measured using IVIS imaging at specified time points. Individual lines denote data obtained from each animal. Open circles denote mice receiving 0.8Gy TBI while solid circles denote mice receiving 1.2Gy TBI on day −2. e, Kaplan-Meier curve showing overall animal survival in each experimental group. f, Schematic of the mouse model for g-j. g-h, Frequencies of donor HLA-A2⁻ CAR T cells (g) and HLA-A2⁺ recipient T cells (RTC) (h) in peripheral blood cells of individual animals. i, Tumor bioluminescence signals measured at specified time points. j, Kaplan-Meier curve showing survival of mice in each experimental group. P values were determined by two-sided log-rank test.