414. CAR Spacers Including NGFR Domains Allow Efficient T-Cell Tracking and Mediate Superior Antitumor Effects

In conclusion, we demonstrated that the incorporation of the LNGFR marker gene directly in the CAR sequence allows for a single molecule to work as a therapeutic and as a selection/tracking gene and shows an increased efficacy/safety profile compared to the IgG1-CH2CH3 spacer

637. Targeting of Myeloid Leukemia by IL-10-Engineered Human CD4+ Tr1 Cells

T regulatory type 1 (Tr1) cells, characterized by the co-expression of CD49b and LAG-3 and the ability to secrete high amounts of IL-10, control immune responses by IL-10 and TGF-beta production and by killing of myeloid cells via a Granzyme B-dependent mechanism. Tr1 cells are induced in vitro in the presence of recombinant human IL-10 or tolerogenic dendritic cells secreting high amounts of IL-10 (DC-10). Proof-of-principle clinical trials suggest that Tr1 cells can modulate Graft-versus Host Disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, their ability to mediate anti-leukemic activity or their effects of Graft versus Leukemia is largely unknown. We previously showed that enforced IL-10 expression converts human CD4+ T cells into Tr1-like (CD4IL-10) cells that suppress effector T cells in vitro and prevent xenogeneic-GvHD in humanized models. We now demonstrate that these CD4IL-10 cells selectively kill myeloid cell lines and myeloid blasts in vitro in HLA-class I-dependent but antigen-independent manner. Moreover, cytotoxic activity of CD4IL-10 cells is Granzyme B-dependent, is specific for CD13+ cells, and requires CD54 and CD112 expression on target cell lines or primary leukemic blast. Adoptive transfer of CD4IL-10 cells in humanized models mediates direct anti-leukemic activity, and does not compromise the anti-leukemic effect of allogeneic T cells while inhibits xeno-GvHD. These findings provide a strong rationale for designing personalized immunotherapy approaches using CD4IL-10 cells after allo-HSCT to cure myeloid malignancies

512 the cytokine release syndrome crucially contributes to the anti leukemic effects of cd44v6 car t cells

Background: Despite the remarkable clinical results of CD19 CAR-T cells in B-cell leukemias, their long-term efficacy is limited by the emergence of CD19-loss escape variants. Moreover, whether the cytokine release syndrome (CRS) is necessary for durable remissions is a matter of debate. Currently available xenograft models in NSG mice are not suited for studying the antitumor effects of CAR-T cells beyond 3-4 weeks, because of xenograft-versus-host disease (X-GVHD). Moreover, since NSG mice lack functional myeloid cells, the CRS does not develop. Aim: To verify whether the CRS contributes to the antileukemic effects of CAR in an innovative xenotolerant mouse model.Results: NSG mice triple transgenic for human IL-3, GM-CSF and SCF (NSG-3GS) were sub-lethally irradiated and injected intra-liver with human HSCs soon after birth, enabling an accelerated and better balanced lympho-hematopoietic reconstitution compared with NSG mice. Reconstituting human T cells were single CD4+/CD8+ T cells, representing all memory sub-populations. After ex vivo isolation and activation with CD3/CD28-beads and IL-7/IL-15, NSG-3GS T cells were transduced with a CD44v6 CAR, retaining an early-differentiated (stem-cell/central-memory) phenotype and full antitumor functionality against acute myeloid leukemia (AML). NSG-3GS-derived CD44v6 CAR T cells were subsequently infused in tumor-bearing secondary recipients previously humanized with autologous HSCs. CAR-T cells persisted in vivo for at least 6 months and mediated durable leukemia remissions (P<0.001 vs controls) in the absence of X-GVHD. Tumor clearance associated with an acute malaise syndrome, characterized by high fevers and a surge in human IL-6 levels, which was lethal in 30% of the mice. Differently from CD19 CAR-T cells, the CRS by CD44v6 CAR-T cells was significantly anticipated (3 vs 8 days), coinciding with human CD44v6+ monocyte depletion. In humanized mice, previous myeloid-cell depletion by clodronate administration completely prevented this syndrome, but associated with late leukemia relapses. Conversely, mice developing the CRS entered a state of durable and profound remission, as demonstrated by prolonged observation times and secondary transplantation. Conclusions: By using an innovative xenotolerant mouse model, we have demonstrated that the CRS is needed for sustained antileukemic effects by CD44v6 CAR-T cells

Two subsets of stem-like CD8+ memory T cell progenitors with distinct fate commitments in humans

T cell memory relies on the generation of antigen-specific progenitors with stem-like properties. However, the identity of these progenitors has remained unclear, precluding a full understanding of the differentiation trajectories that underpin the heterogeneity of antigen-experienced T cells. We used a systematic approach guided by single-cell RNA-sequencing data to map the organizational structure of the human CD8+ memory T cell pool under physiological conditions. We identified two previously unrecognized subsets of clonally, epigenetically, functionally, phenotypically and transcriptionally distinct stem-like CD8+ memory T cells. Progenitors lacking the inhibitory receptors programmed death-1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT) were committed to a functional lineage, whereas progenitors expressing PD-1 and TIGIT were committed to a dysfunctional, exhausted-like lineage. Collectively, these data reveal the existence of parallel differentiation programs in the human CD8+ memory T cell pool, with potentially broad implications for the development of immunotherapies and vaccines

Extracellular NGFR Spacers Allow Efficient Tracking and Enrichment of Fully Functional CAR-T Cells Co-Expressing a Suicide Gene

Chimeric antigen receptor (CAR)-T cell immunotherapy is at the forefront of innovative cancer therapeutics. However, lack of standardization of cellular products within the same clinical trial and lack of harmonization between different trials have hindered the clear identification of efficacy and safety determinants that should be unveiled in order to advance the field. With the aim of facilitating the isolation and in vivo tracking of CAR-T cells, we here propose the inclusion within the CAR molecule of a novel extracellular spacer based on the low-affinity nerve-growth-factor receptor (NGFR). We screened four different spacer designs using as target antigen the CD44 isoform variant 6 (CD44v6). We successfully generated NGFR-spaced CD44v6 CAR-T cells that could be efficiently enriched with clinical-grade immuno-magnetic beads without negative consequences on subsequent expansion, immuno-phenotype, in vitro antitumor reactivity, and conditional ablation when co-expressing a suicide gene. Most importantly, these cells could be tracked with anti-NGFR monoclonal antibodies in NSG mice, where they expanded, persisted, and exerted potent antitumor effects against both high leukemia and myeloma burdens. Similar results were obtained with NGFR-enriched CAR-T cells specific for CD19 or CEA, suggesting the universality of this strategy. In conclusion, we have demonstrated that the incorporation of the NGFR marker gene within the CAR sequence allows for a single molecule to simultaneously work as a therapeutic and selection/tracking gene. Looking ahead, NGFR spacer enrichment might allow good manufacturing procedures-manufacturing of standardized CAR-T cell products with high therapeutic potential, which could be harmonized in different clinical trials and used in combination with a suicide gene for future application in the allogeneic setting

MRI evaluation of sacral chordoma treated with carbon ion radiotherapy alone

Background and purpose: To compare RECIST 1.1 with volume modifications in patients with sacral chordoma not suitable for surgery treated with carbon ions radiotherapy (CIRT) alone. To evaluate patients pain before and after CIRT. To detect if baseline Apparent Diffusion Coefficient values (ADC) from Diffusion Weighted sequences could predict response to treatment. Material and methods: Patients included had one cycle of CIRT and underwent MRI before and after treatment. For each MRI, lesion maximum diameter and volume were obtained, and ADC values were analyzed within the whole lesion volume. Patients pain was evaluated with Numerical Rating Scale (NRS), considering the upper tumor level at baseline MRIs. Results: 39 patients were studied (mean follow-up 18 months). Considering RECIST 1.1 there was not a significant reduction in tumor diameters (p = 0.19), instead there was a significant reduction in tumor volume (p < 0.001), with a significant reduction in pain (p = 0.021) if the tumors were above vertebrae S2–S3 at baseline MRIs. The assessment of baseline ADC maps demonstrated higher median values and more negative skewness values in progressive disease (PD) patients versus both partial response (PR) and stable disease (SD). Conclusions: Lesion volume measurement is more accurate than maximum diameter to better stratify the response of sacral chordoma treated with CIRT. Preliminary results suggest that baseline ADC values could be predictive of response to CIRT