National Institutes of Health Intramural funds and St Baldrick's Foundation.

Chimeric antigen receptor (CAR) T-cells targeting CD19 mediate potent effects in relapsed/refractory pre-B cell acute lymphoblastic leukemia (B-ALL) but antigen loss is a frequent cause of resistance to CD19-targeted immunotherapy. CD22 is also expressed on most B-ALL and usually retained following CD19 loss. We report results from a phase I trial testing a novel CD22-CAR in twenty-one children and adults, including 17 previously treated with CD19-directed immunotherapy. Dose dependent anti-leukemic activity was observed with complete remission in 73% (11/15) of patients receiving ≥ 1 × 106 CD22-CART cells/kg, including 5/5 patients with CD19dim/neg B-ALL. Median remission duration was 6 months. Relapses were associated with diminished CD22 site density that likely permitted escape from killing by CD22-CART cells. These results are the first to eastablish the clinical activity of a CD22-CAR in pre-B cell ALL, including in leukemia resistant to anti-CD19 immunotherapy, demonstrating comparable potency to CD19-CART at biologically active doses in B-ALL. They also highlight the critical role played by antigen density in regulating CAR function. (Funded by NCI Intramural Research Program)

Key Points A platform for the generation of clinical-grade CD19-CAR–modified TSCM. CD19-CAR–modified TSCM mediate superior antitumor responses compared with CD19-CAR T cells currently used in clinical trials.

Key Points Acute GVHD occurred in 5 of 9 patients after major histocompatibility–matched, T-cell–depleted peripheral blood stem cell transplantation plus IL-15/4-1BBL aNK-DLI. GVHD was more common in matched unrelated donor transplants and associated with higher CD3 chimerism, suggesting that aNK-DLI may augment T-cell alloreactivity.

Purpose High-dose interleukin-2 (IL-2) induces durable therapeutic responses in a small subset of patients with metastatic melanoma and renal cell carcinoma, but simple pretreatment predictors of response have not been identified. Patients and Methods To identify predictive biomarkers of clinical response, sera from patients treated with high-dose IL-2 were collected for analysis using a customized, multiplex antibody-targeted protein array platform that surveyed expression of soluble factors associated with tumor immunobiology. Soluble factors associated with clinical responses were analyzed using a multivariate permutation test, and survival outcomes were determined using Kaplan-Meier and log-rank tests. Results A training set from 10 patients identified 68 potentially relevant soluble factors that were then tested in an independent validation set of 49 patients. Class comparison revealed a cluster of 11 biomarkers that were associated with therapeutic outcome. Vascular endothelial growth factor (VEGF) and fibronectin were identified as independent predictors of response. In particular, high levels of these proteins were correlated with lack of clinical response and decreased overall survival. Conclusion Serum VEGF and fibronectin are easily measured pretreatment biomarkers that could serve to exclude patients unlikely to respond to IL-2 therapy.

The use of bone marrow derived mesenchymal stromal cells (BMSC) in the treatment of alloimmune and autoimmune conditions has generated much interest, yet an understanding of the therapeutic mechanism remains elusive. We therefore explored immune modulation by a clinical-grade BMSC product in a model of human-into-mouse xenogeneic GVHD (x-GVHD) mediated by human CD4+ Th1 cells. BMSC reversed established, lethal x-GVHD through marked inhibition of Th1 cell effector function. Gene marking studies indicated BMSC engraftment was limited to the lung; further, there was no increase in regulatory T cells, thereby suggesting a paracrine mechanism of BMSC action. BMSC recipients had increased serum CD73 expressing exosomes that promoted adenosine accumulation ex vivo. Importantly, immune modulation mediated by BMSC was fully abrogated by pharmacologic therapy with an adenosine A2A receptor antagonist. To investigate the potential clinical relevance of these mechanistic findings, patient serum samples collected pre- and post-BMSC treatment were studied for exosome content: CD73 expressing exosomes promoting adenosine accumulation were detected in post-BMSC samples. In conclusion, BMSC effectively modulate experimental GVHD through a paracrine mechanism that promotes adenosine-based immune suppression.

Background aims Autologous chimeric antigen receptor (CAR) T-cell therapies have shown promising clinical outcomes, but T-cell yields have been variable. CD19- and GD2-CAR T-cell manufacturing records were reviewed to identify sources of variability. Methods CD19-CAR T cells were used to treat 43 patients with acute lymphocytic leukemia or lymphoma and GD2-CAR T cells to treat eight patients with osteosarcoma and three with neuroblastoma. Both types of CAR T cells were manufactured using autologous peripheral blood mononuclear cells (PBMC) concentrates and anti-CD3/CD28 beads for T-cell enrichment and simulation. Results A comparison of the first 6 GD2- and the first 22 CD19-CAR T-cell products manufactured revealed that GD2-CAR T-cell products contained fewer transduced cells than CD19-CAR T-cell products (147 ± 102 × 106 vs 1502 ± 1066 × 106; P = 0.0059), and their PBMC concentrates contained more monocytes (31.4 ± 12.4% vs 18.5 ± 13.7%; P = 0.019). Among the first 28 CD19-CAR T-cell products manufactured, four had poor expansion yielding less than 1 × 106 transduced T cells per kilogram. When PBMC concentrates from these four patients were compared with the 24 others, PBMC concentrates of poorly expanding products contained greater quantities of monocytes (39.8 ± 12.9% vs. 15.3 ± 10.8%, P = 0.0014). Among the patients whose CD19-CAR T cells expanded poorly, manufacturing for two patients was repeated using cryopreserved PBMC concentrates but incorporating a monocyte depleting plastic adherence step, and an adequate dose of CAR T cells was produced for both patients. Conclusions Variability in CAR T-cell expansion is due, at least in part, to the contamination of the starting PBMC concentrates with monocytes.

Ex vivo production of highly-stimulator mature Dendritic Cells (DCs) for cellular therapy has been used to treat different pathological conditions with the aim of inducing a specific immune response. In the last decade several protocols have been developed to mature monocyte-derived DCs: each one has led to the generation of DCs showing different phenotypes and stimulatory abilities, but it is not yet known which one is the best for inducing effective immune responses. We grouped several different maturation protocols according to the downstream pathways they activated and reviewed the shared features at a transcriptomic level to reveal the potential of DCs matured by each protocol to develop Th polarized immune responses.