Automated manufacture of ΔNPM1 TCR-engineered T cells for AML therapy.

Acute myeloid leukemia (AML) is a heterogeneous malignancy that requires further therapeutic improvement, especially for the elderly and for subgroups with poor prognosis. A recently discovered T cell receptor (TCR) targeting mutant nucleophosmin 1 (ΔNPM1) presents an attractive option for the development of a cancer antigen-targeted cellular therapy. Manufacturing of TCR-modified T cells, however, is still limited by a complex, time-consuming, and laborious procedure. Therefore, this study specifically addressed the requirements for a scaled manufacture of ΔNPM1-specific T cells in an automated, closed, and good manufacturing practice-compliant process. Starting from cryopreserved leukapheresis, 2E8 CD8-positive T cells were enriched, activated, lentivirally transduced, expanded, and finally formulated. By adjusting and optimizing culture conditions, we additionally reduced the manufacturing time from 12 to 8 days while still achieving a clinically relevant yield of up to 5.5E9 ΔNPM1 TCR-engineered T cells. The cellular product mainly consisted of highly viable CD8-positive T cells with an early memory phenotype. ΔNPM1 TCR CD8 T cells manufactured with the optimized process showed specific killing of AML in vitro and in vivo. The process has been implemented in an upcoming phase 1/2 clinical trial for the treatment of NPM1-mutated AML.

T cells of colorectal cancer patients' stimulated by neoantigenic and cryptic peptides better recognize autologous tumor cells.

BACKGROUND: Patients with cancers that exhibit extraordinarily high somatic mutation numbers are ideal candidates for immunotherapy and enable identifying tumor-specific peptides through stimulation of tumor-reactive T cells (Tc). METHODS: Colorectal cancers (CRC) HROC113 and HROC285 were selected based on high TMB, microsatellite instability and HLA class I expression. Their HLA ligandome was characterized using mass spectrometry, compared with the HLA ligand atlas and HLA class I-binding affinity was predicted. Cryptic peptides were identified using Peptide-PRISM. Patients' Tc were isolated from either peripheral blood (pTc) or tumor material (tumor-infiltrating Tc, TiTc) and expanded. In addition, B-lymphoblastoid cells (B-LCL) were generated and used as antigen-presenting cells. pTc and TiTc were stimulated twice for 7 days using peptide pool-loaded B-LCL. Subsequently, interferon gamma (IFNγ) release was quantified by ELISpot. Finally, cytotoxicity against autologous tumor cells was assessed in a degranulation assay. RESULTS: 100 tumor-specific candidate peptides-97 cryptic peptides and 3 classically mutated neoantigens-were selected. The neoantigens originated from single nucleotide substitutions in the genes IQGAP1, CTNNB1, and TRIT1. Cryptic and neoantigenic peptides inducing IFNγ secretion of Tc were further investigated. Stimulation of pTc and TiTc with neoantigens and selected cryptic peptides resulted in increased release of cytotoxic granules in the presence of autologous tumor cells, substantiating their improved tumor cell recognition. Tetramer staining showed an enhanced number of pTc and TiTc specific for the IQGAP1 neoantigen. Subpopulation analysis prior to peptide stimulation revealed that pTc mainly consisted of memory Tc, whereas TiTc constituted primarily of effector and effector memory Tc. This allows to infer that TiTc reacting to neoantigens and cryptic peptides must be present within the tumor microenvironment. CONCLUSION: These results prove that the analyzed CRC present both mutated neoantigenic and cryptic peptides on their HLA class I molecules. Moreover, stimulation with these peptides significantly strengthened tumor cell recognition by Tc. Since the overall number of neoantigenic peptides identifiable by HLA ligandome analysis hitherto is small, our data emphasize the relevance of increasing the target scope for cancer vaccines by the cryptic peptide category.

A Notch/STAT3-driven Blimp-1/c-Maf-dependent molecular switch induces IL-10 expression in human CD4+ T cells and is defective in Crohn´s disease patients.

Immunosuppressive Interleukin (IL)-10 production by pro-inflammatory CD4+ T cells is a central self-regulatory function to limit aberrant inflammation. Still, the molecular mediators controlling IL-10 expression in human CD4+ T cells are largely undefined. Here, we identify a Notch/STAT3 signaling-module as a universal molecular switch to induce IL-10 expression across human naïve and major effector CD4+ T cell subsets. IL-10 induction was transient, jointly controlled by the transcription factors Blimp-1/c-Maf and accompanied by upregulation of several co-inhibitory receptors, including LAG-3, CD49b, PD-1, TIM-3 and TIGIT. Consistent with a protective role of IL-10 in inflammatory bowel diseases (IBD), effector CD4+ T cells from Crohn's disease patients were defective in Notch/STAT3-induced IL-10 production and skewed towards an inflammatory Th1/17 cell phenotype. Collectively, our data identify a Notch/STAT3-Blimp-1/c-Maf axis as a common anti-inflammatory pathway in human CD4+ T cells, which is defective in IBD and thus may represent an attractive therapeutic target.

CAR T-cell Entry into Tumor Islets Is a Two-Step Process Dependent on IFNγ and ICAM-1.

Adoptive transfer of T cells expressing chimeric antigen receptors (CAR) has shown remarkable clinical efficacy against advanced B-cell malignancies but not yet against solid tumors. Here, we used fluorescent imaging microscopy and ex vivo assays to compare the early functional responses (migration, Ca2+, and cytotoxicity) of CD20 and EGFR CAR T cells upon contact with malignant B cells and carcinoma cells. Our results indicated that CD20 CAR T cells rapidly form productive ICAM-1-dependent conjugates with their targets. By comparison, EGFR CAR T cells only initially interacted with a subset of carcinoma cells located at the periphery of tumor islets. After this initial peripheral activation, EGFR CAR T cells progressively relocated to the center of tumor cell regions. The analysis of this two-step entry process showed that activated CAR T cells triggered the upregulation of ICAM-1 on tumor cells in an IFNγ-dependent pathway. The ICAM-1/LFA-1 interaction interference, through antibody or shRNA blockade, prevented CAR T-cell enrichment in tumor islets. The requirement for IFNγ and ICAM-1 to enable CAR T-cell entry into tumor islets is of significance for improving CAR T-cell therapy in solid tumors.

A Novel Siglec-4 Derived Spacer Improves the Functionality of CAR T Cells Against Membrane-Proximal Epitopes.

A domain that is often neglected in the assessment of chimeric antigen receptor (CAR) functionality is the extracellular spacer module. However, several studies have elucidated that membrane proximal epitopes are best targeted through CARs comprising long spacers, while short spacer CARs exhibit highest activity on distal epitopes. This finding can be explained by the requirement to have an optimal distance between the effector T cell and target cell. Commonly used long spacer domains are the CH2-CH3 domains of IgG molecules. However, CARs containing these spacers generally show inferior in vivo efficacy in mouse models compared to their observed in vitro activity, which is linked to unspecific Fcγ-Receptor binding and can be abolished by mutating the respective regions. Here, we first assessed a CAR therapy targeting membrane proximal CD20 using such a modified long IgG1 spacer. However, despite these mutations, this construct failed to unfold its observed in vitro cytotoxic potential in an in vivo model, while a shorter but less structured CD8α spacer CAR showed complete tumor clearance. Given the shortage of well-described long spacer domains with a favorable functionality profile, we designed a novel class of CAR spacers with similar attributes to IgG spacers but without unspecific off-target binding, derived from the Sialic acid-binding immunoglobulin-type lectins (Siglecs). Of five constructs tested, a Siglec-4 derived spacer showed highest cytotoxic potential and similar performance to a CD8α spacer in a CD20 specific CAR setting. In a pancreatic ductal adenocarcinoma model, a Siglec-4 spacer CAR targeting a membrane proximal (TSPAN8) epitope was efficiently engaged in vitro, while a membrane distal (CD66c) epitope did not activate the T cell. Transfer of the TSPAN8 specific Siglec-4 spacer CAR to an in vivo setting maintained the excellent tumor killing characteristics being indistinguishable from a TSPAN8 CD8α spacer CAR while outperforming an IgG4 long spacer CAR and, at the same time, showing an advantageous central memory CAR T cell phenotype with lower release of inflammatory cytokines. In summary, we developed a novel spacer that combines cytotoxic potential with an advantageous T cell and cytokine release phenotype, which make this an interesting candidate for future clinical applications.

Automated Manufacturing of Potent CD20-Directed Chimeric Antigen Receptor T Cells for Clinical Use.

The clinical success of gene-engineered T cells expressing a chimeric antigen receptor (CAR), as manifested in several clinical trials for the treatment of B cell malignancies, warrants the development of a simple and robust manufacturing procedure capable of reducing to a minimum the challenges associated with its complexity. Conventional protocols comprise many open handling steps, are labor intensive, and are difficult to upscale for large numbers of patients. Furthermore, extensive training of personnel is required to avoid operator variations. An automated current Good Manufacturing Practice-compliant process has therefore been developed for the generation of gene-engineered T cells. Upon installation of the closed, single-use tubing set on the CliniMACS Prodigy™, sterile welding of the starting cell product, and sterile connection of the required reagents, T cells are magnetically enriched, stimulated, transduced using lentiviral vectors, expanded, and formulated. Starting from healthy donor (HD) or lymphoma or melanoma patient material (PM), the robustness and reproducibility of the manufacturing of anti-CD20 specific CAR T cells were verified. Independent of the starting material, operator, or device, the process consistently yielded a therapeutic dose of highly viable CAR T cells. Interestingly, the formulated product obtained with PM was comparable to that of HD with respect to cell composition, phenotype, and function, even though the starting material differed significantly. Potent antitumor reactivity of the produced anti-CD20 CAR T cells was shown in vitro as well as in vivo. In summary, the automated T cell transduction process meets the requirements for clinical manufacturing that the authors intend to use in two separate clinical trials for the treatment of melanoma and B cell lymphoma.

Automated Enrichment, Transduction, and Expansion of Clinical-Scale CD62L+ T Cells for Manufacturing of Gene Therapy Medicinal Products.

Multiple clinical studies have demonstrated that adaptive immunotherapy using redirected T cells against advanced cancer has led to promising results with improved patient survival. The continuously increasing interest in those advanced gene therapy medicinal products (GTMPs) leads to a manufacturing challenge regarding automation, process robustness, and cell storage. Therefore, this study addresses the proof of principle in clinical-scale selection, stimulation, transduction, and expansion of T cells using the automated closed CliniMACS® Prodigy system. Naïve and central memory T cells from apheresis products were first immunomagnetically enriched using anti-CD62L magnetic beads and further processed freshly (n = 3) or split for cryopreservation and processed after thawing (n = 1). Starting with 0.5 × 108 purified CD3+ T cells, three mock runs and one run including transduction with green fluorescent protein (GFP)-containing vector resulted in a median final cell product of 16 × 108 T cells (32-fold expansion) up to harvesting after 2 weeks. Expression of CD62L was downregulated on T cells after thawing, which led to the decision to purify CD62L+CD3+ T cells freshly with cryopreservation thereafter. Most important in the split product, a very similar expansion curve was reached comparing the overall freshly CD62L selected cells with those after thawing, which could be demonstrated in the T cell subpopulations as well by showing a nearly identical conversion of the CD4/CD8 ratio. In the GFP run, the transduction efficacy was 83%. In-process control also demonstrated sufficient glucose levels during automated feeding and medium removal. The robustness of the process and the constant quality of the final product in a closed and automated system give rise to improve harmonized manufacturing protocols for engineered T cells in future gene therapy studies.

Role of Blimp-1 in programing Th effector cells into IL-10 producers.

Secretion of the immunosuppressive cytokine interleukin (IL) 10 by effector T cells is an essential mechanism of self-limitation during infection. However, the transcriptional regulation of IL-10 expression in proinflammatory T helper (Th) 1 cells is insufficiently understood. We report a crucial role for the transcriptional regulator Blimp-1, induced by IL-12 in a STAT4-dependent manner, in controlling IL-10 expression in Th1 cells. Blimp-1 deficiency led to excessive inflammation during Toxoplasma gondii infection with increased mortality. IL-10 production from Th1 cells was strictly dependent on Blimp-1 but was further enhanced by the synergistic function of c-Maf, a transcriptional regulator of IL-10 induced by multiple factors, such as the Notch pathway. We found Blimp-1 expression, which was also broadly induced by IL-27 in effector T cells, to be antagonized by transforming growth factor (TGF) ß. While effectively blocking IL-10 production from Th1 cells, TGF-ß shifted IL-10 regulation from a Blimp-1-dependent to a Blimp-1-independent pathway in IL-27-induced Tr1 (T regulatory 1) cells. Our findings further illustrate how IL-10 regulation in Th cells relies on several transcriptional programs that integrate various signals from the environment to fine-tune expression of this critical immunosuppressive cytokine.