The great success of chimeric antigen receptor (CAR) T-cell therapy in the treatment of patients with B-cell malignancies has prompted its translation to solid tumors. In the case of glioblastoma (GBM), clinical trials have shown modest efficacy, but efforts to develop more effective anti-GBM CAR T cells are ongoing. In this study, we selected PTPRZ1 as a target for GBM treatment. We isolated six anti-human PTPRZ1 scFv from a human phage display library and produced 2nd generation CAR T cells in an RNA format. Patient-derived GBM PTPRZ1-knock-in cell lines were used to select the CAR construct that showed high cytotoxicity while consistently displaying high CAR expression (471_28z). CAR T cells incorporating 471_28z were able to release IFN-γ, IL-2, TNF-α, Granzyme B, IL-17A, IL-6, and soluble FasL, and displayed low tonic signaling. Additionally, they maintained an effector memory phenotype after in vitro killing. In addition, 471_28z CAR T cells displayed strong bystander killing against PTPRZ1-negative cell lines after pre-activation by PTPRZ1-positive tumor cells but did not kill antigen-negative non-tumor cells. In an orthotopic xenograft tumor model using NSG mice, a single dose of anti-PTPRZ1 CAR T cells significantly delayed tumor growth. Taken together, these results validate PTPRZ1 as a GBM target and prompt the clinical translation of anti-PTPRZ1 CAR T cells.

The great success of chimeric antigen receptor (CAR)-T cell therapy in B-cell malignancies has prompted its translation to solid tumors. In the case of glioblastoma (GBM), clinical trials have shown modest efficacy, but anti-GBM CAR-T cells are being intensely developed. In this study, we selected PTPRZ1 as an attractive new target for GBM treatment. We isolated six anti-human PTPRZ1 scFv from a human phage display library and produced 2 nd generation CAR-T cells in an RNA format. Patient-derived GBM PTPRZ1-knock-in cell lines were used to select the CAR construct (471_28z), which showed high cytotoxicity while consistently displaying high CAR expression. CAR-T cells incorporating 471_28z were able to release IFN-γ, IL-2, TNF-α, Granzyme B, IL-17A, IL-6, and soluble FasL, and displayed low tonic signaling. Additionally, they maintained an effector memory phenotype after in vitro killing. Importantly, 471_28z CAR-T cells displayed strong bystander killing against PTPRZ1-negative cell lines after pre-activation by PTPRZ1-positive tumor cells, but did not kill antigen-negative non-tumor cells. In an orthotopic xenograft tumor model using NSG mice, a single dose of anti-PTPRZ1 CAR-T cells significantly delayed tumor growth. Taken together, these results validate the use of PTPRZ1 as a new GBM target and prompt the use of anti-PTPRZ1 CAR-T cells for clinical translation.

The biofunctionalization of synthetic materials has extensive utility for biomedical applications, but approaches to bioconjugation typically show insufficient efficiency and controllability. We recently developed an approach by building synthetic DNA scaffolds on biomaterial surfaces that enables the precise control of cargo density and ratio, thus improving the assembly and organization of functional cargos. We used this approach to show that the modulation and phenotypic adaptation of immune cells can be regulated using our precisely functionalized biomaterials. Here, we describe the three key procedures, including the fabrication of polymeric particles engrafted with short DNA scaffolds, the attachment of functional cargos with complementary DNA strands, and the surface assembly control and quantification. We also explain the critical checkpoints needed to ensure the overall quality and expected characteristics of the biological product. We provide additional experimental design considerations for modifying the approach by varying the material composition, size or cargo types. As an example, we cover the use of the protocol for human primary T cell activation and for the identification of parameters that affect ex vivo T cell manufacturing. The protocol requires users with diverse expertise ranging from synthetic materials to bioconjugation chemistry to immunology. The fabrication procedures and validation assays to design high-fidelity DNA-scaffolded biomaterials typically require 8 d.
© 2023. Springer Nature Limited.

To determine the role of the AKT pathway in the regulating of natural Killer-induced apoptosis of acute myeloid leukemia cells and to characterize the associated molecular mechanisms.
BALB/c nude mice were injected with HL60 cells to induce a xenogenic model of subcutaneous leukemic tumors. Mice were treated with perifosine, and their spleens were analyzed using biometry, histopathology, and immunohistochemistry. Gene expression analysis in leukemia cells was performed by real-time PCR. Protein analysis of leukemia and natural Killer cells was performed by flow cytometry. AKT inhibition in HL60 cells, followed by co-culture with natural Killer cells was performed to assess cytotoxicity. Apoptosis rate was quantified using flow cytometry.
Perifosine treatment caused a reduction in leukemic infiltration in the spleens of BALB/c nude mice. In vitro , AKT inhibition reduced HL60 resistance to natural Killer-induced apoptosis. AKT inhibition suppressed the immune checkpoint proteins PD-L1, galectin-9, and CD122 in HL60 cells, but did not change the expression of their co-receptors PD1, Tim3, and CD96 on the natural Killer cell surface. In addition, the death receptors DR4, TNFR1, and FAS were overexpressed by AKT inhibition, thus increasing the susceptibility of HL60 cells to the extrinsic pathway of apoptosis.
The AKT pathway is involved in resistance to natural Killer-induced apoptosis in HL60 cells by regulating the expression of immune suppressor receptors. These findings highlight the importance of AKT in contributing to immune evasion mechanisms in acute myeloid leukemia and suggests the potential of AKT inhibition as an adjunct to immunotherapy.

Given the immune system's importance for cancer surveillance and treatment, we have investigated how it may be affected by SARS-CoV-2 infection of cancer patients. Across some heterogeneity in tumor type, stage, and treatment, virus-exposed solid cancer patients display a dominant impact of SARS-CoV-2, apparent from the resemblance of their immune signatures to those for COVID-19+ non-cancer patients. This is not the case for hematological malignancies, with virus-exposed patients collectively displaying heterogeneous humoral responses, an exhausted T cell phenotype and a high prevalence of prolonged virus shedding. Furthermore, while recovered solid cancer patients' immunophenotypes resemble those of non-virus-exposed cancer patients, recovered hematological cancer patients display distinct, lingering immunological legacies. Thus, while solid cancer patients, including those with advanced disease, seem no more at risk of SARS-CoV-2-associated immune dysregulation than the general population, hematological cancer patients show complex immunological consequences of SARS-CoV-2 exposure that might usefully inform their care.
Crown Copyright © 2021. Published by Elsevier Inc. All rights reserved.