Enhancement of anti-sarcoma immunity by NK cells engineered with mRNA for expression of a EphA2-targeted CAR.

Paediatric sarcomas, including rhabdomyosarcoma, Ewing sarcoma and osteosarcoma, represent a group of malignancies that significantly contribute to cancer-related morbidity and mortality in children and young adults. These cancers share common challenges, including high rates of metastasis, recurrence or treatment resistance, leading to a 5-year survival rate of approximately 20% for patients with advanced disease stages. Despite the critical need, therapeutic advancements have been limited over the past three decades. The advent of chimeric antigen receptor (CAR)-based immunotherapies offers a promising avenue for novel treatments. However, CAR-T cells have faced significant challenges and limited success in treating solid tumours due to issues such as poor tumour infiltration, immunosuppressive tumour microenvironments and off-target effects. In contrast, the adaptation of CAR technology for natural killer (NK) cells has demonstrated potential in both haematological and solid tumours, suggesting a new therapeutic strategy for paediatric sarcomas. This study developed and validated a novel CAR-NK cell therapy targeting the ephrin type-A receptor-2 (EphA2) antigen, which is highly expressed in various paediatric sarcomas. CAR expression was successfully detected on the surface of NK cells post-electroporation, indicating successful transfection. Significantly, EphA2-specific CAR-NK cells demonstrated enhanced cytotoxic activity against several paediatric sarcoma cell lines in vitro, including those of rhabdomyosarcoma, Ewing sarcoma and osteosarcoma, compared to unmodified NK cells. Transient messenger RNA (mRNA) transfection of NK cells is a safe approach in genetic engineering, with further chemical modifications to mRNA enhancing stability of temporal EphA2-CAR expression in NK cells, thereby promoting prolonged protein expression. Additionally, in vivo EphA2-CAR-NK cells showed promising anti-cancer activity in rhabdomyosarcoma and osteosarcoma mouse models. The study provides a foundational basis for the clinical evaluation of EphA2-targeted CAR-NK cell therapy across a spectrum of paediatric sarcomas. The enhanced anti-tumour effects observed in vitro/vivo suggests potential for improved therapeutic outcomes in hard-to-cure paediatric sarcomas. Addressing unmet clinical needs in paediatric Sarcomas. Paediatric sarcomas, including rhabdomyosarcoma, Ewing sarcoma, and osteosarcoma, exhibit poor survival rates in advanced disease stages. The lack of significant therapeutic progress over the past three decades necessitates innovative treatment approaches. Advancing immunotherapy with CAR-NK cells. Natural killer (NK) cells modified with chimeric antigen receptors (CARs) represent a promising strategy to overcome the limitations of CAR-T cells, particularly in solid tumours. CAR-NK cells are associated with enhanced tumour targeting, reduced off-target effects, and improved safety profiles. EphA2 as a therapeutic target. EphA2, a receptor overexpressed in multiple paediatric sarcomas, is identified as a viable target for CAR-based immunotherapy due to its critical role in tumour progression and angiogenesis. Innovations in mRNA-based engineering. This study demonstrates the feasibility of transient mRNA transfection to engineer NK cells for CAR expression, offering a non-integrative and safer alternative to viral transduction. Enhancements in mRNA stability through chemical modifications, can further optimise protein expression. Preclinical efficacy of EphA2-CAR NK cells. EphA2-specific CAR-NK cells exhibit superior cytotoxicity against sarcoma cell lines in vitro and demonstrate significant anti-tumour activity in in vivo mouse models of rhabdomyosarcoma and osteosarcoma. Clinical translation potential. The findings establish a strong preclinical rationale for the clinical evaluation of EphA2-targeted CAR-NK therapy as a novel immunotherapeutic option for paediatric sarcomas. Future research directions: Combining EphA2-CAR NK cells with immune checkpoint inhibitors or other immunomodulatory agents could further enhance therapeutic outcomes and durability. Advanced preclinical models mimicking human tumour microenvironments are needed to refine and optimise this therapeutic approach.

Lam PY ，Omer N ，Wong JKM ，Tu C ，Alim L ，Rossi GR ，Victorova M ，Tompkins H ，Lin CY ，Mehdi AM ，Choo A ，Elliott MR ，Coleborn E ，Sun J ，Mercer T ，Vittorio O ，Dobson LJ ，McLellan AD ，Brooks A ，Tuong ZK ，Cheetham SW ，Nicholls W ，Souza-Fonseca-Guimaraes F ... -  《Clinical and Translational Medicine》

B7H6 is the predominant activating ligand driving natural killer cell-mediated killing in patients with liquid tumours: evidence from clinical, in silico, in vitro, and in vivo studies.

Natural killer (NK) cells are a subset of innate lymphoid cells that are inherently capable of recognizing and killing infected or tumour cells. This has positioned NK cells as a promising live drug for tumour immunotherapy, but limited success suggests incomplete knowledge of their killing mechanism. NK cell-mediated killing involves a complex decision-making process based on integrating activating and inhibitory signals from various ligand-receptor repertoires. However, the relative importance of the different activating ligand-receptor interactions in triggering NK killing remains unclear. We employed a systematic approach combining clinical, in silico, in vitro, and in vivo data analysis to quantify the impact of various activating ligands. Clinical data analysis was conducted using massive pan-cancer data (n = 10,595), where patients with high NK cell levels were stratified using CIBERSORT. Subsequently, multivariate Cox regression and Kaplan-Meier (KM) survival analysis were performed based on activating ligand expression. To examine the impact of ligand expression on NK killing at the cellular level, we assessed surface expression of five major activating ligands (B7H6, MICA/B, ULBP1, ULBP2/5/6, and ULBP3) of human tumour cell lines of diverse origins (n = 33) via flow cytometry (FACs) and their NK cell-mediated cytotoxicity on by calcein-AM assay using human primary NK cells and NK-92 cell lines. Based on this data, we quantified the contribution of each activating ligand to the NK killing activity using mathematical models and Bayesian statistics. To further validate the results, we performed calcein-AM assays upon ligand knockdown and overexpression, conjugation assays, and co-culture assays in activating ligand-downregulated/overexpressed in liquid tumour (LT) cell lines. Moreover, we established LT-xenograft mouse models to assess the efficacy of NK cell targeting toward tumours with dominant ligands. Through the clinical analysis, we discovered that among nearly all 18 activating ligands, only patients with LT who were NK cell-rich and specifically had higher B7H6 level exhibited a favorable survival outcome (p = 0.0069). This unexpected dominant role of B7H6 was further confirmed by the analysis of datasets encompassing multiple ligands and a variety of tumours, which showed that B7H6 exhibited the highest contribution to NK killing among five representative ligands. Furthermore, LT cell lines (acute myeloid leukemia (AML), B cell lymphoma, and T-acute lymphocytic leukemia (ALL)) with lowered B7H6 demonstrated decreased susceptibility to NK cell-mediated cytotoxicity compared to those with higher levels. Even within the same cell line, NK cells selectively targeted cells with higher B7H6 levels. Finally, LT-xenograft mouse models (n = 24) confirmed that higher B7H6 results in less tumour burden and longer survival in NK cell-treated LT mice (p = 0.0022). While NK cells have gained attention for their potent anti-tumour effects without causing graft-versus-host disease (GvHD), thus making them a promising off-the-shelf therapy, our limited understanding of NK killing mechanisms has hindered their clinical application. This study illuminates the crucial role of the activating ligand B7H6 in driving NK cell killing, particularly in the context of LT. Therefore, the expression level of B7H6 could serve as a prognostic marker for patients with LT. Moreover, for the development of NK cell-based immunotherapy, focusing on increasing the level of B7H6 on its cognate receptor, NKp30, could be the most effective strategy. This work was supported by the National Research Council of Science & Technology (NST) grant (CAP-18-02-KRIBB, GTL24021-000), a National Research Foundation grant (2710012258, 2710004815), and an Institute for Basic Science grant (IBS-R029-C3).

Lee S ，Chae SJ ，Jang IH ，Oh SC ，Kim SM ，Lee SY ，Kim JH ，Ko J ，Kim HJ ，Song IC ，Kim JK ，Kim TD ... -  《EBioMedicine》

Tumor-derived G-CSF induces an immunosuppressive microenvironment in an osteosarcoma model, reducing response to CAR.GD2 T-cells.

Sarcomas are rare, mesenchymal tumors, representing about 10-15% of all childhood cancers. GD2 is a suitable target for chimeric antigen receptor (CAR) T-cell therapy due to its overexpression in several solid tumors. In this preclinical study, we investigated the potential use of iCasp9.2A.GD2.CAR-CD28.4-1BBζ (CAR.GD2) T-cells as a treatment option for patients who have GD2-positive sarcomas and we sought to identify factors shaping hostile tumor microenvironment in this setting. GD2 expression was evaluated by flow-cytometry on primary tumor biopsies of pediatric sarcoma patients. GD2 expression in sarcoma cells was also evaluated in response to an enhancer of zeste homolog 2 (EZH2) inhibitor (Tazemetostat). The antitumor activity of CAR.GD2 T-cells was evaluated both in vitro and in vivo preclinical models of orthotopic and/or metastatic soft-tissue and bone sarcomas. GD2 expression was detected in 55% of the primary tumors. Notably, the Osteosarcoma and Alveolar Rhabdomyosarcomas subtypes exhibited the highest GD2 expression levels, while Ewing sarcoma showed the lowest. CAR.GD2 T-cells show a significant tumor control both in vitro and in vivo models of GD2-expressing tumors. Pretreatment with an EZH2 inhibitor (Tazemetostat) upregulating GD2 expression, sensitizes GD2dim sarcoma cells to CAR.GD2 T-cells cytotoxic activity. Moreover, in mouse models of disseminated Rhabdomyosarcomas and orthotopic Osteosarcoma, CAR.GD2 T-cells showed both a vigorous anti-tumor activity and long-term persistence as compared to un-transduced T-cells. The presence of immunosuppressive murine myeloid-derived suppressor (MDSC) cells significantly reduces long-term anti-tumour activity of infused CAR.GD2 T-cells. Tumor-derived G-CSF was found to be one of the key factors driving expansion of immunosuppressive murine and human MDSC, thus indirectly limiting the efficacy of CAR.GD2 T-cells. Our preclinical data strongly suggest that CAR.GD2 T-cells hold promise as a potential therapeutic option for the treatment of patients with GD2-positive sarcomas. Strategies to tackle hostile immunosuppressive MDSC are desirable to optimize CAR.GD2 T-cell activity.

Pezzella M ，Quintarelli C ，Quadraccia MC ，Sarcinelli A ，Manni S ，Iaffaldano L ，Ottaviani A ，Ciccone R ，Camera A ，D'Amore ML ，Di Cecca S ，Sinibaldi M ，Guercio M ，Aurigemma M ，De Falco P ，Fustaino V ，Rota R ，Pomella S ，Cassandri M ，Di Giannatale A ，Agrati C ，Bordoni V ，Guarracino F ，Massa M ，Del Baldo G ，Becilli M ，Milano GM ，Del Bufalo F ，Locatelli F ，De Angelis B ... -  《Journal of Hematology & Oncology》

Can a Liquid Biopsy Detect Circulating Tumor DNA With Low-passage Whole-genome Sequencing in Patients With a Sarcoma? A Pilot Evaluation.

A liquid biopsy is a test that evaluates the status of a disease by analyzing a sample of bodily fluid, most commonly blood. In recent years, there has been progress in the development and clinical application of liquid biopsy methods to identify blood-based, tumor-specific biomarkers for many cancer types. However, the implementation of these technologies to aid in the treatment of patients who have a sarcoma remains behind other fields of cancer medicine. For this study, we chose to evaluate a sarcoma liquid biopsy based on circulating tumor DNA (ctDNA). All human beings have normal cell-free DNA (cfDNA) circulating in the blood. In contrast with cfDNA, ctDNA is genetic material present in the blood stream that is derived from a tumor. ctDNA carries the unique genomic fingerprint of the tumor with changes that are not present in normal circulating cfDNA. A successful ctDNA liquid biopsy must be able to target these tumor-specific genetic alterations. For instance, epidermal growth factor receptor (EGFR) mutations are common in lung cancers, and ctDNA liquid biopsies are currently in clinical use to evaluate the status of disease in patients who have a lung cancer by detecting EGFR mutations in the blood. As opposed to many carcinomas, sarcomas do not have common recurrent mutations that could serve as the foundation to a ctDNA liquid biopsy. However, many sarcomas have structural changes to their chromosomes, including gains and losses of portions or entire chromosomes, known as copy number alterations (CNAs), that could serve as a target for a ctDNA liquid biopsy. Murine double minute 2 (MDM2) amplification in select lipomatous tumors or parosteal osteosarcoma is an example of a CNA due to the presence of extra copies of a segment of the long arm of chromosome 12. Since a majority of sarcomas demonstrate a complex karyotype with numerous CNAs, a blood-based liquid biopsy strategy that searches for these CNAs may be able to detect the presence of sarcoma ctDNA. Whole-genome sequencing (WGS) is a next-generation sequencing technique that evaluates the entire genome. The depth of coverage of WGS refers to how detailed the sequencing is, like higher versus lower power on a microscope. WGS can be performed with high-depth sequencing (that is, > 60×), which can detect individual point mutations, or low-depth sequencing (that is, 0.1× to 5×), referred to as low-passage whole-genome sequencing (LP-WGS), which may not detect individual mutations but can detect structural chromosomal changes including gains and losses (that is, CNAs). While similar strategies have shown favorable early results for specific sarcoma subtypes, LP-WGS has not been evaluated for applicability to the broader population of patients who have a sarcoma. Does an LP-WGS liquid biopsy evaluating for CNAs detect ctDNA in plasma samples from patients who have sarcomas representing a variety of histologic subtypes? This was a retrospective study conducted at a community-based, tertiary referral center. Nine paired (plasma and formalin-fixed paraffin-embedded [FFPE] tissue) and four unpaired (plasma) specimens from patients who had a sarcoma were obtained from a commercial biospecimen bank. Three control specimens from individuals who did not have cancer were also obtained. The paired and unpaired specimens from patients who had a sarcoma represented a variety of sarcoma histologic subtypes. cfDNA was extracted, amplified, and quantified. Libraries were prepared, and LP-WGS was performed using a NextSeq 500 next-generation sequencing machine at a low depth of sequencing coverage (∼1×). The ichorCNA bioinformatics algorithm, which was designed to detect CNAs from low-depth genomic sequencing data, was used to analyze the data. In contrast with the gold standard for diagnosis in the form of histopathologic analysis of a tissue sample, this test does not discriminate between sarcoma subtypes but detects the presence of tumor-derived CNAs within the ctDNA in the blood that should not be present in a patient who does not have cancer. The liquid biopsy was positive for the detection of cancer if the ichorCNA algorithm detected the presence of ctDNA. The algorithm was also used to quantitatively estimate the percent ctDNA within the cfDNA. The concentration of ctDNA was then calculated from the percent ctDNA relative to the total concentration of cfDNA. The CNAs of the paired FFPE tissue and plasma samples were graphically visualized using aCNViewer software. This LP-WGS liquid biopsy detected ctDNA in 9 of 13 of the plasma specimens from patients with a sarcoma. The other four samples from patients with a sarcoma and all serum specimens from patients without cancer had no detectable ctDNA. Of those 9 patients with positive liquid biopsy results, the percent ctDNA ranged from 6% to 11%, and calculated ctDNA quantities were 0.04 to 5.6 ng/mL, which are levels to be expected when ctDNA is detectable. In this small pilot study, we were able to detect sarcoma ctDNA with an LP-WGS liquid biopsy searching for CNAs in the plasma of most patients who had a sarcoma representing a variety of histologic subtypes. These results suggest that an LP-WGS liquid biopsy evaluating for CNAs to identify ctDNA may be more broadly applicable to the population of patients who have a sarcoma than previously reported in studies focusing on specific subtypes. Large prospective clinical trials that gather samples at multiple time points during the process of diagnosis, treatment, and surveillance will be needed to further assess whether this technique can be clinically useful. At our institution, we are in the process of developing a large prospective clinical trial for this purpose.

Anderson CJ ，Yang H ，Parsons J ，Ahrens WA ，Jagosky MH ，Hsu JH ，Patt JC ，Kneisl JS ，Steuerwald NM ... -  《-》

Repurposing anti-mesothelin CAR-NK immunotherapy against colorectal cancer.

Colorectal cancer (CRC) is the third most common cancer worldwide, with highly variable prognosis and response to treatment. A large subset of patients does not respond to standard treatments or develops resistance. As an alternative, adoptive immunotherapy based on chimeric antigen receptor (CAR)-transduced immune cells has been proposed, however with significant adverse events. We therefore evaluated alternative CAR targets already tested in other tumour types and employed the natural killer cell line NK-92 for CAR transduction because of its more favourable toxicity profile. As an alternative antigen, we considered mesothelin (MSLN), the most represented target in CAR-based clinical studies for solid tumours. MSLN RNA expression was analysed in large series of CRC tumours (n = 640) and cell lines (n = 150), to evaluate its distribution and to identify MSLN-overexpressing models. NK-92 cells were transduced with anti-MSLN CAR, and subsequently sorted and cloned. Activity of CAR-NK-92 cells against target-expressing ovarian and CRC cells was assessed in vitro and in vivo. Statistical significance of efficacy was evaluated by t-test and log-rank test. Large-scale expression analysis highlighted that about 10% of CRCs overexpress MSLN at levels comparable to those of ovarian cancer, a typical target of MSLN-CAR-based therapy. Intriguingly, MSLN overexpression is more frequent in poor prognosis and KRAS/BRAF-mutant CRC. Lentiviral transduction of NK-92 cells with the MSLN-CAR, followed by sorting and cloning, led to the identification of one clone, MSLN.CAR.NK-92.cl45, stably expressing the CAR and retaining the NK phenotype. As expected, the clone demonstrated significant in vitro and in vivo activity against ovarian cancer cells. When repurposed against models of CRC expressing high MSLN levels, it displayed comparable efficacy, both in vitro and in vivo. Specificity of the clone was confirmed by the absence of activity on control models with low or absent MSLN. Our results provide preclinical evidence that a subset of colorectal cancers expressing high mesothelin levels can be effectively targeted by MSLN-CAR-based immunotherapy. The potential therapeutic impact of these findings is enhanced by the fact that frequently MSLN-overexpressing CRCs display worse prognosis and resistance to standard care.

Torchiaro E ，Cortese M ，Petti C ，Basirico' M ，Invrea F ，D'Andrea A ，Franco L ，Sangiolo D ，Medico E ... -  《Journal of Translational Medicine》