AKT inhibition generates potent polyfunctional clinical grade AUTO1 CAR T-cells, enhancing function and survival

BACKGROUND: AUTO1 is a fast off-rate CD19-targeting chimeric antigen receptor (CAR), which has been successfully tested in adult lymphoblastic leukemia. Tscm/Tcm-enriched CAR-T populations confer the best expansion and persistence, but Tscm/Tcm numbers are poor in heavily pretreated adult patients. To improve this, we evaluate the use of AKT inhibitor (VIII) with the aim of uncoupling T-cell expansion from differentiation, to enrich Tscm/Tcm subsets. METHODS: VIII was incorporated into the AUTO1 manufacturing process based on the semiautomated the CliniMACS Prodigy platform at both small and cGMP scale. RESULTS: AUTO1 manufactured with VIII showed Tscm/Tcm enrichment, improved expansion and cytotoxicity in vitro and superior antitumor activity in vivo. Further, VIII induced AUTO1 Th1/Th17 skewing, increased polyfunctionality, and conferred a unique metabolic profile and a novel signature for autophagy to support enhanced expansion and cytotoxicity. We show that VIII-cultured AUTO1 products from B-ALL patients on the ALLCAR19 study possess superior phenotype, metabolism, and function than parallel control products and that VIII-based manufacture is scalable to cGMP. CONCLUSION: Ultimately, AUTO1 generated with VIII may begin to overcome the product specific factors contributing to CD19+relapse

Patient characteristics.

<p>Abbreviations: ALL, acute lymphatic leukemia; AML, acute myeloid leukemia; MDS, myelodysplastic syndrome; CML, chronic myeloid leukemia; NHL, non-Hodgkin lymphoma; CLL, chronic lymphatic leukemia; Cy, <b><i>cyclophosphamide; Bus, busulphan; TBI, total body irradiation</i></b>; <b><i>Ida, idarubicin;</i></b> GVHD, graft-versus-host disease.</p

CCL20 is expressed in GVHD tissues and selectively attracts CCR6⁺ T cells.

<p>(A) CCL20 staining in skin biopsies of patients diagnosed with acute GVHD at respectively 49 (UPN 833) and 30 days (UPN 877), or chronic GVHD 127 days (UPN 722) and 321 days (UPN 741) days after allo-SCT. Squares indicate examples of single cells in the dermis in close proximity to the epidermal-dermal junction (acute GVHD) or in the epidermis which are situated in close proximity to or at the epidermal-dermal junction (chronic GVHD). Images were captured at 400X. (B) CD3 (red), CD4 (blue), and CCR6 (green) triple staining in skin biopsies of patients diagnosed with acute GVHD at respectively 49 (UPN 833) and 30 days (UPN 877), or chronic GVHD 127 days (UPN 722) and 321 days (UPN 741) days after allo-SCT. White arrows and squares indicate examples of CD3⁺CD4⁺CCR6⁺ cells, yellow arrows and squares indicate examples of CD3⁺CD4⁻CCR6⁺ cells. Single stainings of the cells in squares are depicted under the image. Images were captured at 400X.</p

Reconstitution of CD161-expressing T cells in patients after allo-SCT.

<p>(A) Percentage of circulating CD161⁺ within CD4⁺ and CD161^hi within CD8⁺ T cells in patients at 1 (<i>n</i> = 11, 20), 3 (<i>n</i> = 50, 71), 6 (<i>n</i> = 19, 24), and 12 (<i>n</i> = 19, 22) months after allo-SCT. (B) Absolute levels of circulating CD161⁺CD4⁺ and CD161^hiCD8⁺ T cells in patients at 1 (<i>n</i> = 8, 20), 3 (<i>n</i> = 50, 71), 6 (<i>n</i> = 19, 24), and 12 (<i>n</i> = 19, 22) months after allo-SCT. (C) Absolute number of circulating CD161⁻CD4⁺ and CD161^neg/lowCD8⁺ T cells in patients at 1 (<i>n</i> = 8, 20), 3 (<i>n</i> = 54, 69), 6 (<i>n</i> = 19, 24), and 12 (<i>n</i> = 19, 22) months after allo-SCT. (D) Correlation between the percentage of circulating CD161⁺CD4⁺ and CD4⁺ T cells (<i>n</i> = 58), and CD161^hiCD8⁺ T cells and CD8⁺ T cells (<i>n</i> = 70) at 3 months after allo-SCT. Lines represent median value, grey areas represent the reference range of healthy controls (mean ± SD). Statistical analysis was performed using a One-way ANOVA followed by a Bonferroni post-hoc test (CD4) or non-parametric One-way ANOVA followed by a Dunns post-hoc test (CD8). Correlations were determined by calculating the Spearman correlation coefficient (R). *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Preferential migration of CD161-expressing T cells to CCL20.

<p>(A) Comparison of CCR6 expression between CD161⁻ and CD161⁺CD4⁺ memory T cells, and CD161^neg/low and CD161^hiCD8⁺ Tem cells of healthy donors (о) and patients 3 months after allo-SCT (•). The frequency of CD161⁺CD4⁺ T cells in the measured samples was 34.6–55.7% in allo-SCT patients (<i>n</i> = 10) and 36.6–46.4% in healthy controls (<i>n</i> = 3), and the frequency of CD161^hiCD8⁺ T cells was 0.05–23.8% in allo-SCT patients (<i>n</i> = 8) and 11.5–42.4% in healthy controls (<i>n</i> = 3). Data is shown as mean fluorescent intensity (MFI). Lines represent mean value. (B) Migration of CD4⁺ and CD8⁺ T cell subsets to CCL20. CD4⁺ or CD8⁺ T cells were applied to the upper chamber and migrated to the lower chamber containing medium with or without CCL20. Migrated cells were analysed by flow cytometry. Migration is shown relative to input (mean ± SD) for CD161⁻CD4⁺ and CD161^neg/lowCD8⁺ T cells (dashed line), and CD161⁺CD4⁺ and CD161^hiCD8⁺ T cells (solid line) (<i>n</i> = 3). One representative experiment out of 2 (CD4) or 3 (CD8) independent healthy donors is shown. (C) Migration of CD4⁺ T cell subsets to CCL20. CD3⁺ T cells were applied to the upper chamber and migrated to the lower chamber containing medium with or without CCL20. Migrated cells were analysed by flow cytometry. Migration is shown relative to input (mean ± SD) for CD161⁻CD4⁺ (dashed line), and CD161⁺CD4⁺ (solid line) (n = 3). Two independent patients 3 months after allo-ST are shown. Statistical analysis was performed using Two-way ANOVA followed by a Bonferroni post-hoc test. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

PD-L1 siRNA-mediated silencing in acute myeloid leukemia enhances anti-leukemic T cell reactivity

Acute myeloid leukemia (AML) is an immune-susceptible malignancy, as demonstrated by its responsiveness to allogeneic stem cell transplantation (alloSCT). However, by employing inhibitory signaling pathways, including PD-1/PD-L1, leukemia cells suppress T cell-mediated immune attack. Notably, impressive clinical efficacy has been obtained with PD-1/PD-L1 blocking antibodies in cancer patients. Yet, these systemic treatments are often accompanied by severe toxicity, especially after alloSCT. Here, we investigated RNA interference technology as an alternative strategy to locally interfere with PD-1/PD-L1 signaling in AML. We demonstrated efficient siRNA-mediated PD-L1 silencing in HL-60 and patients' AML cells. Importantly, WT1-antigen T cell receptor(+) PD-1(+) 2D3 cells showed increased activation toward PD-L1 silenced WT1(+) AML. Moreover, PD-L1 silenced AML cells significantly enhanced the activation, degranulation, and IFN-γ production of minor histocompatibility antigen-specific CD8(+) T cells. Notably, PD-L1 silencing was equally effective as PD-1 antibody blockade. Together, our study demonstrates that PD-L1 silencing may be an effective strategy to augment AML immune-susceptibility. This provides rationale for further development of targeted approaches to locally interfere with immune escape mechanisms in AML, thereby minimizing severe toxicity. In combination with alloSCT and/or adoptive T cell transfer, this strategy could be very appealing to boost graft-versus-leukemia immunity and improve outcome in AML patients

A single infusion of UCB-NK cells inhibits growth of BM-residing human leukemia cells.

<p>The potential of UCB-NK cells to attack human leukemia <i>in vivo</i> was evaluated in NSG mice bearing K562 intra-femoral (<i>i.f</i>.) tumors. (A) Experimental study design: adult NSG mice were injected in their right femur with 10⁵ K562.LucGFP cells. The day after, mice were treated with 20×10⁶ UCB-NK cells <i>i.v.</i> in combination with IL-15 administration (0.5 µg/mouse <i>i.p.</i> every 2–3 days for 14 days), or received PBS or IL-15 alone as control (n = 6 per group). Tumor load was monitored by BLI from day 8 after K562.LucGFP cell inoculation and next every 3–4 days for 2 weeks. At later time points, only mice with undetectable tumor load were imaged. (B) Two independent anti-leukemic studies were performed with similar outcomes. Tumor load per mouse measured at day 15 following K562.LucGFP cell IF injection from experiment 1 (black circles) and 2 (open circles). (C) BLI pictures acquired at day 15 after tumor cell <i>i.f.</i> injection, and (D) tumor load in time (mean ± SD, n = 6 per group). **<i>p</i><0.01 UCB-NK cells+IL-15 vs. PBS and IL-15. Data of experiment 2 are shown. (E) Time to first tumor detection by BLI and (F) mice survival analysed according to Mantel Cox test. One mouse from the IL-15 group (experiment 1) died at day 19 after luciferine injection and was excluded from the survival analysis.</p

AKT inhibition generates potent polyfunctional clinical grade AUTO1 CAR T-cells, enhancing function and survival

Background AUTO1 is a fast off-rate CD19-targeting chimeric antigen receptor (CAR), which has been successfully tested in adult lymphoblastic leukemia. Tscm/Tcm-enriched CAR-T populations confer the best expansion and persistence, but Tscm/Tcm numbers are poor in heavily pretreated adult patients. To improve this, we evaluate the use of AKT inhibitor (VIII) with the aim of uncoupling T-cell expansion from differentiation, to enrich Tscm/Tcm subsets.Methods VIII was incorporated into the AUTO1 manufacturing process based on the semiautomated the CliniMACS Prodigy platform at both small and cGMP scale.Results AUTO1 manufactured with VIII showed Tscm/Tcm enrichment, improved expansion and cytotoxicity in vitro and superior antitumor activity in vivo. Further, VIII induced AUTO1 Th1/Th17 skewing, increased polyfunctionality, and conferred a unique metabolic profile and a novel signature for autophagy to support enhanced expansion and cytotoxicity. We show that VIII-cultured AUTO1 products from B-ALL patients on the ALLCAR19 study possess superior phenotype, metabolism, and function than parallel control products and that VIII-based manufacture is scalable to cGMP.Conclusion Ultimately, AUTO1 generated with VIII may begin to overcome the product specific factors contributing to CD19+relapse

Low-dose IL-15 mediates efficient UCB-NK cell survival and expansion <i>in vivo</i>.

<p>Adult NSG mice were injected <i>i.v.</i> with 5×10⁶ UCB-NK cells with or without supportive IL-15. Recombinant human IL-15 was administered every 2–3 days for 2 weeks at the dose of 0.5 µg/mouse/injection, starting the day of UCB-NK cell infusion. (A) Percentages of circulating human CD45⁺ cells in all CD45⁺ cells were quantified by flow cytometry at the indicated time points. Each line corresponds to one mouse. (B) Percentages of human CD45⁺ cells in all CD45⁺ cells quantified in spleen and bone marrow (leg bones) 2 weeks after UCB-NK cell infusion. (C) Expression of CD16 and CD158a/h,b,e on UCB-NK cells before and 2 weeks after infusion into NSG mice. Percentages were determined on human CD45⁺CD56⁺ NK cells isolated from spleen and bone marrow. Graphs show the mean ± SD of 5 mice.</p

Homing receptor expression profile of UCB-NK cells.

<p>(A–B) The expression level of homing receptors was analysed by flow cytometry on UCB-NK cells at the end of the culture process. (A) Dot plots gated on CD56⁺ cells from one representative donor; (B) Summary of 6 different donors analysed. (C) The capacity of UCB-NK cells to respond <i>in vitro</i> to gradients (10 to 250 ng/ml) of the chemokines CCL4 (CCR5 ligand), CCL20 (CCR6 ligand), CXCL10, CXCL11 (both CXCR3 ligand) and CXCL12 (CXCR4 ligand) was evaluated in transwell migration assays as described in materials and methods. Mean ± SEM of three independent experiments are shown, each performed with different UCB-NK cell donors. Migration towards specific chemokines was compared to non-specific migration (0 ng/ml) using a one way-ANOVA followed by Dunnett’s multiple comparison post-hoc test, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p