Two cases of severe pulmonary toxicity from highly active mesothelin-directed CAR T cells.

Multiple clinical studies have treated mesothelin (MSLN)-positive solid tumors by administering MSLN-directed chimeric antigen receptor (CAR) T cells. Although these products are generally safe, efficacy is limited. Therefore, we generated and characterized a potent, fully human anti-MSLN CAR.

Novel T cells with improved in vivo anti-tumor activity generated by RNA electroporation.

The generation of T cells with maximal anti-tumor activities will significantly impact the field of T-cell-based adoptive immunotherapy. In this report, we found that OKT3/IL-2-stimulated T cells were phenotypically more heterogeneous, with enhanced anti-tumor activity in vitro and when locally administered in a solid tumor mouse model. To further improve the OKT3/IL-2-based T cell manufacturing procedure, we developed a novel T cell stimulation and expansion method in which peripheral blood mononuclear cells were electroporated with mRNA encoding a chimeric membrane protein consisting of a single-chain variable fragment against CD3 and the intracellular domains of CD28 and 4-1BB (OKT3-28BB).

A versatile system for rapid multiplex genome-edited CAR T cell generation.

The therapeutic potential of CRISPR system has already been demonstrated in many instances and begun to overlap with the rapidly expanding field of cancer immunotherapy, especially on the production of genetically modified T cell receptor or chimeric antigen receptor (CAR) T cells. Efficient genomic disruption of multiple gene loci to generate universal donor cells, as well as potent effector T cells resistant to multiple inhibitory pathways such as PD-1 and CTLA4 is an attractive strategy for cell therapy. In this study, we accomplished rapid and efficient multiplex genomic editing, and re-directing T cells with antigen specific CAR via a one-shot CRISPR protocol by incorporation of multiple gRNAs in a CAR lentiviral vector.

Multiplex Genome Editing to Generate Universal CAR T Cells Resistant to PD1 Inhibition.

Using gene-disrupted allogeneic T cells as universal effector cells provides an alternative and potentially improves current chimeric antigen receptor (CAR) T-cell therapy against cancers and infectious diseases. The CRISPR/Cas9 system has recently emerged as a simple and efficient way for multiplex genome engineering. By combining lentiviral delivery of CAR and electro-transfer of Cas9 mRNA and gRNAs targeting endogenous TCR, β-2 microglobulin (B2M) and PD1 simultaneously, to generate gene-disrupted allogeneic CAR T cells deficient of TCR, HLA class I molecule and PD1.

A Chimeric Switch-Receptor Targeting PD1 Augments the Efficacy of Second-Generation CAR T Cells in Advanced Solid Tumors.

Chimeric antigen receptor (CAR)-modified adoptive T-cell therapy has been successfully applied to the treatment of hematologic malignancies, but faces many challenges in solid tumors. One major obstacle is the immune-suppressive effects induced in both naturally occurring and genetically modified tumor-infiltrating lymphocytes (TIL) by inhibitory receptors (IR), namely PD1. We hypothesized that interfering with PD1 signaling would augment CAR T-cell activity against solid tumors.

Affinity-Tuned ErbB2 or EGFR Chimeric Antigen Receptor T Cells Exhibit an Increased Therapeutic Index against Tumors in Mice.

Target-mediated toxicity is a major limitation in the development of chimeric antigen T-cell receptors (CAR) for adoptive cell therapy of solid tumors. In this study, we developed a strategy to adjust the affinities of the scFv component of CAR to discriminate tumors overexpressing the target from normal tissues that express it at physiologic levels. A CAR-expressing T-cell panel was generated with target antigen affinities varying over three orders of magnitude.

Chimeric NKG2D CAR-expressing T cell-mediated attack of human ovarian cancer is enhanced by histone deacetylase inhibition.

NKG2D ligands (NKG2DLs) are widely expressed on ovarian cancers to various degrees, making them attractive targets for immunotherapy. Here, we applied a chimeric antigen receptor (CAR) approach for the targeting of NKG2DLs expressed on human ovarian cancer cells and evaluated the impact of pharmacological upregulation of NKG2DLs on immune recognition. Various NKG2DLs, including MICA/B and ULBP-1, -2, -3, and -4, were expressed at various levels on the surface of all established ovarian cancer cell lines and primary ovarian cancer samples tested.

Decay-accelerating factor regulates T-cell immunity in the context of inflammation by influencing costimulatory molecule expression on antigen-presenting cells.

Recent studies have indicated a role of complement in regulating T-cell immunity but the mechanism of action of complement in this process remains to be clarified. Here we studied mice deficient in decay-accelerating factor (DAF), a key membrane complement regulator whose deficiency led to increased complement-dependent T-cell immune responses in vivo. By crossing OT-II and OT-I T-cell receptor transgenic mice with DAF-knockout mice, we found that lack of DAF on T cells did not affect their responses to antigen stimulation.

Complement promotes the development of inflammatory T-helper 17 cells through synergistic interaction with Toll-like receptor signaling and interleukin-6 production.

Toll-like receptors (TLRs) and complement are 2 major components of innate immunity that provide a first-line host defense and shape the adaptive immune responses. We show here that coincidental activation of complement and several TLRs in mice led to the synergistic production of serum factors that promoted T-helper cell 17 (Th17) differentiation from anti-CD3/CD28 or antigen-stimulated T cells. Although multiple TLR-triggered cytokines were regulated by complement, Th17 cell-promoting activity in the serum was correlated with interleukin (IL)-6 induction, and antibody neutralization of IL-6 abrogated the complement effect.

Complement-dependent enhancement of CD8+ T cell immunity to lymphocytic choriomeningitis virus infection in decay-accelerating factor-deficient mice.

Decay-accelerating factor (DAF, CD55) is a GPI-anchored membrane protein that regulates complement activation on autologous cells. In addition to protecting host tissues from complement attack, DAF has been shown to inhibit CD4+ T cell immunity in the setting of model Ag immunization. However, whether DAF regulates natural T cell immune response during pathogenic infection is not known.

Regulation of Toll-like receptor-mediated inflammatory response by complement in vivo.

Toll-like receptors (TLRs) and complement are 2 components of innate immunity that are critical for first-line host defense and elicitation of adaptive immune responses. Many pathogen-associated molecular patterns activate both TLR and complement, but whether and how these 2 systems, when coactivated in vivo, interact with each other has not been well studied. We demonstrate here a widespread regulation of TLR signaling by complement in vivo.

[Establishment of eukaryotic cell line stably expressing soluble human leucocyte antigen G1 by nucleofection].

Objective: To establish a eukaryotic cell line that can express soluble human leucocyte antigen G1 (sHLA-G1) stably.

Methods: The recombinant plasmid pcDNA3-sHLA-G1 is transfected by a novel nonviral, electroporation-based gene transfer method termed nucleofection into the host cell lymphoblastoid cell line (LCL) 721.221 which does not express any HLA-classical I molecules.

[Immune tolerance inducing effects of soluble human leucocyte antigen G1 on natural killer cells and T cells].

Objective: To investigate the immune tolerance inducing effects of soluble human leucocyte antigen G1 (sHLA-G1) on natural killer (NK) cells and T cells.

Methods: A recombinant plasmid expressing sHLA-G1 was constructed and transfected into human lymphoblastoid cells LCL721.221.

Expression of local renin and angiotensinogen mRNA in cirrhotic portal hypertensive patient.

Aim: To investigate the expression of local renin and angiotensinogen mRNA in cirrhotic portal hypertensive patients.

Methods: The expression of local renin and angiotensinogen mRNA in the liver, splenic artery and vein of PH patients was detected by RT-PCR analysis.

Results: Expression of local renin mRNA in the liver of control group was (0.