Cancer therapy with T cells expressing chimeric antigen receptors (CARs) has produced remarkable clinical responses in recent trials, but also severe side effects. Whereas most protocols use permanently reprogrammed T cells, we have developed a platform for transient CAR expression by mRNA electroporation. This approach may be useful for safe clinical testing of novel receptors, or when a temporary treatment period is desirable. Herein, we investigated therapy with transiently redirected T cells in vitro and in a xenograft mouse model. We constructed a series of CD19-specific CARs with different spacers and co-stimulatory domains (CD28, OX40 or CD28-OX40). The CAR constructs all conferred T cells with potent CD19-specific activity in vitro. Unexpectedly, the constructs incorporating a commonly used IgG1-CH2CH3 spacer showed lack of anti-leukemia activity in vivo and induced severe, partly CD19-independent toxicity. By contrast, identical CAR constructs without the CH2-domain eradicated leukemia in vivo, without notable toxicity. Follow-up studies demonstrated that the CH2CH3-spacer bound soluble mouse Fcγ-receptor I and mediated off-target T-cell activation towards murine macrophages. Our findings highlight the importance of non-signalling CAR elements and of in vivo studies. Finally, the results show that transiently redirected T cells control leukemia in mice and support the rationale for developing an mRNA-CAR platform.
BackgroundAdoptive T-cell transfer of therapeutic TCR holds great promise to specifically kill cancer cells, but relies on modifying the patient's own T cells ex vivo before injection. The manufacturing of T cells in a tailor-made setting is a long and expensive process which could be resolved by the use of universal cells. Currently, only the Natural Killer (NK) cell line NK-92 is FDA approved for universal use. In order to expand their recognition ability, they were equipped with Chimeric Antigen Receptors (CARs). However, unlike CARs, T-cell receptors (TCRs) can recognize all cellular proteins, which expand NK-92 recognition to the whole proteome.MethodsWe herein genetically engineered NK-92 to express the CD3 signaling complex, and showed that it rendered them able to express a functional TCR. Functional assays and in vivo efficacy were used to validate these cells.FindingsThis is the first demonstration that a non-T cell can exploit TCRs. This TCR-redirected cell line, termed TCR-NK-92, mimicked primary T cells phenotypically, metabolically and functionally, but retained its NK cell effector functions. Our results demonstrate a unique manner to indefinitely produce TCR-redirected lymphocytes at lower cost and with similar therapeutic efficacy as redirected T cells.InterpretationThese results suggest that an NK cell line could be the basis for an off-the-shelf TCR-based cancer immunotherapy solution.FundThis work was supported by of Norway (#254817), South-Eastern Norway Regional Health Authority (#14/00500-79), by OUS-Radiumhospitalet (Gene Therapy program) and the department of Oncology at the .
T-cell receptor (TCR) transfer is an attractive strategy to increase the number of cancer-specific T cells in adoptive cell therapy. However, recent clinical and pre-clinical findings indicate that careful consideration of the target antigen is required to limit the risk of off-target toxicity. Directing T cells against mutated proteins such as frequently occurring frameshift mutations may thus be a safer alternative to tumor-associated self-antigens. Furthermore, such frameshift mutations result in novel polypeptides allowing selection of TCRs from the non-tolerant T-cell repertoire circumventing the problem of low affinity TCRs due to central tolerance. The transforming growth factor β Receptor II frameshift mutation (TGFβRIImut) is found in Lynch syndrome cancer patients and in approximately 15% of sporadic colorectal and gastric cancers displaying microsatellite instability (MSI). The -1A mutation within a stretch of 10 adenine bases (nucleotides 709–718) of the TGFβRII gene gives rise to immunogenic peptides previously used for vaccination of MSI+ colorectal cancer patients in a Phase I clinical trial. From a clinically responding patient, we isolated a cytotoxic T lymphocyte (CTL) clone showing a restriction for HLA-A2 in complex with TGFβRIImut peptide. Its TCR was identified and shown to redirect T cells against colon carcinoma cell lines harboring the frameshift mutation. Finally, T cells transduced with the HLA-A2-restricted TGFβRIImut-specific TCR were demonstrated to significantly reduce the growth of colorectal cancer and enhance survival in a NOD/SCID xenograft mouse model.
BackgroundAlthough chemo-immunotherapy has led to an improved overall survival for most B-cell lymphoma types, relapsed and refractory disease remains a challenge. The malaria drug artesunate has previously been identified as a growth suppressor in some cancer types and was tested as a new treatment option in B-cell lymphoma.MethodsWe included artesunate in a cancer sensitivity drug screen in B lymphoma cell lines. The preclinical properties of artesunate was tested as single agent in vitro in 18 B-cell lymphoma cell lines representing different histologies and in vivo in an aggressive B-cell lymphoma xenograft model, using NSG mice. Artesunate-treated B lymphoma cell lines were analyzed by functional assays, gene expression profiling, and protein expression to identify the mechanism of action.ResultsDrug screening identified artesunate as a highly potent anti-lymphoma drug. Artesunate induced potent growth suppression in most B lymphoma cells with an IC50 comparable to concentrations measured in serum from artesunate-treated malaria patients, while leaving normal B-cells unaffected. Artesunate markedly inhibited highly aggressive tumor growth in a xenograft model. Gene expression analysis identified endoplasmic reticulum (ER) stress and the unfolded protein response as the most affected pathways and artesunate-induced expression of the ER stress markers ATF-4 and DDIT3 was specifically upregulated in malignant B-cells, but not in normal B-cells. In addition, artesunate significantly suppressed the overall cell metabolism, affecting both respiration and glycolysis.ConclusionsArtesunate demonstrated potent apoptosis-inducing effects across a broad range of B-cell lymphoma cell lines in vitro, and a prominent anti-lymphoma activity in vivo, suggesting it to be a relevant drug for treatment of B-cell lymphoma.Electronic supplementary materialThe online version of this article (10.1186/s13045-018-0561-0) contains supplementary material, which is available to authorized users.
The human polyomavirus BK (BKV) genome encodes the capsid proteins VP1 to VP3 and the three regulatory proteins, large and small tumor-antigen and the agnoprotein. Agnoprotein is a phospho-protein, but phosphorylation sites, protein kinases that mediate phosphorylation, and the biological importance of phosphorylation for the life-cycle of BK virus remain unknown. Here, we show that protein kinase C phosphorylates BKV agnoprotein at serine-11. Replacing serine-11 by either non-phosphorylable alanine or phospho-mimicking aspartic acid reduced the ability of these mutants to propagate compared to wildtype virus. Moreover, both these mutants displayed altered expression of viral proteins, which resulted from changed transrepressive property and stability of the mutated agnoprotein. Our results indicate that BKV propagation is controlled by phosphorylation of the agnoprotein and may suggest that specific inhibition of protein kinases may be used as a therapeutic strategy to hamper BK virus infection.
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