TCR-transgenic T cells and YB-1-based oncolytic virotherapy improve survival in a preclinical Ewing sarcoma xenograft mouse model.

Background: Ewing sarcoma (EwS) is an aggressive and highly metastatic bone and soft tissue tumor in pediatric patients and young adults. Cure rates are low when patients present with metastatic or relapsed disease. Therefore, innovative therapy approaches are urgently needed. Cellular- and oncolytic virus-based immunotherapies are on the rise for solid cancers. Methods: Here, we assess the combination of EwS tumor-associated antigen CHM1319-specific TCR-transgenic CD8+ T cells and the YB-1-driven (i.e. E1A13S-deleted) oncolytic adenovirus XVir-N-31 in vitro and in a xenograft mouse model for antitumor activity and immunostimulatory properties. Results: In vitro both approaches specifically kill EwS cell lines in a synergistic manner over controls. This effect was confirmed in vivo, with increased survival using the combination therapy. Further in vitro analyses of immunogenic cell death and antigen presentation confirmed immunostimulatory properties of virus-infected EwS tumor cells. As dendritic cell maturation was also increased by XVir-N-31, we observed superior proliferation of CHM1319-specific TCR-transgenic CD8+ T cells only in virus-tested conditions, emphasizing the superior immune-activating potential of XVir-N-31. Conclusion: Our data prove synergistic antitumor effects in vitro and superior tumor control in a preclinical xenograft setting. Combination strategies of EwS-redirected T cells and YB-1-driven virotherapy are a highly promising immunotherapeutic approach for EwS and warrant further evaluation in a clinical setting.

YB-1-based oncolytic virotherapy in combination with CD47 blockade enhances phagocytosis of pediatric sarcoma cells.

Oncolytic viruses (OVs) selectively replicate in tumor cells resulting in lysis, spreading of new infectious units and induction of antitumor immune responses through abrogating an immunosuppressive tumor microenvironment (TME). Due to their mode of action, OVs are ideal combination partners with targeted immunotherapies. One highly attractive combination is the inhibition of the 'don't-eat-me'-signal CD47, which is known to increase the phagocytic potential of tumor-associated macrophages. In this work, we analyzed the combination approach consisting of the YB-1-based oncolytic adenovirus XVir-N-31 (XVir) and the CD47 inhibitor (CD47i) B6.H12.2 concerning its phagocytic potential. We investigate phagocytosis of XVir-, adenovirus wildtype (AdWT)-, and non-infected established pediatric sarcoma cell lines by different monocytic cells. Phagocytes (immature dendritic cells and macrophages) were derived from THP-1 cells and healthy human donors. Phagocytosis of tumor cells was assessed via FACS analysis in the presence and absence of CD47i. Additional characterization of T cell-stimulatory surface receptors as well as chemo-/cytokine analyses were performed. Furthermore, tumor cells were infected and studied for the surface expression of the 'eat-me'-signal calreticulin (CALR) and the 'don't-eat-me'-signal CD47. We herein demonstrate that (1) XVir-infected tumor cells upregulate both CALR and CD47. XVir induces higher upregulation of CD47 than AdWT. (2) XVir-infection enhances phagocytosis in general and (3) the combination of XVir and CD47i compared to controls showed by far superior enhancement of phagocytosis, tumor cell killing and innate immune activation. In conclusion, the combination of CD47i and XVir causes a significant increase in phagocytosis exceeding the monotherapies considerably accompanied by upregulation of T cell-stimulatory receptor expression and inflammatory chemo/-cytokine secretion.

Targeting the Retinoblastoma/E2F repressive complex by CDK4/6 inhibitors amplifies oncolytic potency of an oncolytic adenovirus.

CDK4/6 inhibitors (CDK4/6i) and oncolytic viruses are promising therapeutic agents for the treatment of various cancers. As single agents, CDK4/6 inhibitors that are approved for the treatment of breast cancer in combination with endocrine therapy cause G1 cell cycle arrest, whereas adenoviruses induce progression into S-phase in infected cells as an integral part of the their life cycle. Both CDK4/6 inhibitors and adenovirus replication target the Retinoblastoma protein albeit for different purposes. Here we show that in combination CDK4/6 inhibitors potentiate the anti-tumor effect of the oncolytic adenovirus XVir-N-31 in bladder cancer and murine Ewing sarcoma xenograft models. This increase in oncolytic potency correlates with an increase in virus-producing cancer cells, enhanced viral genome replication, particle formation and consequently cancer cell killing. The molecular mechanism that regulates this response is fundamentally based on the reduction of Retinoblastoma protein expression levels by CDK4/6 inhibitors.

Monocyte Maturation Mediators Upregulate CD83, ICAM-1 and MHC Class 1 Expression on Ewing's Sarcoma, Enhancing T Cell Cytotoxicity.

Ewing's sarcoma (EwS) is a pediatric solid tumor entity with low somatic mutational burden and a low rate of tumor-infiltrating T cells, indicating a low extent of immunogenicity. In EwS, immunogenicity may furthermore be significantly diminished by a predominantly M2 macrophage driven pro-tumorigenic tumor microenvironment. In the past, we demonstrated that CHM1319-specific TCR-transgenic T cells are able to control EwS growth in a preclinical mouse model as well as in a patient with metastatic disease. However, new adjuvant techniques to induce long lasting and curative CHM1319-specific TCR-transgenic T cell-mediated anti-tumor responses are needed. In this work, we sought to identify a technique to improve the cytotoxic effect of CHM1319-specific TCR-transgenic T cell by altering the immunogenic cell surface marker expression on EwS cell lines using different cytokines. We demonstrate that TNF, IL-6, IL-1ß and PGE2 cause pro-immunogenic CD83, MHC class I and II as well as ICAM-1 upregulation in EwS cell lines. This observation was associated with significantly improved recognition and killing of the tumor cells by EwS-specific CHM1319/HLA-A*02:01-restricted TCR-transgenic T cells. Conclusively, we demonstrate that the induction of an inflammatory signature renders EwS more susceptible to adoptive T cell therapy. TNF, which is upregulated during inflammatory processes, is of particular translational interest as its secretion may be induced in the patients e.g., by irradiation and hyperthermia in the clinical setting. In future clinical protocols, this finding may be important to identify appropriate conditioning regimens as well as point of time for adoptive T cell-based immunotherapy in EwS patients.

Ewing Sarcoma-Derived Extracellular Vesicles Impair Dendritic Cell Maturation and Function.

Ewing sarcoma (EwS) is an aggressive pediatric cancer of bone and soft tissues characterized by scant T cell infiltration and predominance of immunosuppressive myeloid cells. Given the important roles of extracellular vesicles (EVs) in cancer-host crosstalk, we hypothesized that EVs secreted by EwS tumors target myeloid cells and promote immunosuppressive phenotypes. Here, EVs were purified from EwS and fibroblast cell lines and exhibited characteristics of small EVs, including size (100-170 nm) and exosome markers CD63, CD81, and TSG101. Treatment of healthy donor-derived CD33+ and CD14+ myeloid cells with EwS EVs but not with fibroblast EVs induced pro-inflammatory cytokine release, including IL-6, IL-8, and TNF. Furthermore, EwS EVs impaired differentiation of these cells towards monocytic-derived dendritic cells (moDCs), as evidenced by reduced expression of co-stimulatory molecules CD80, CD86 and HLA-DR. Whole transcriptome analysis revealed activation of gene expression programs associated with immunosuppressive phenotypes and pro-inflammatory responses. Functionally, moDCs differentiated in the presence of EwS EVs inhibited CD4+ and CD8+ T cell proliferation as well as IFNγ release, while inducing secretion of IL-10 and IL-6. Therefore, EwS EVs may promote a local and systemic pro-inflammatory environment and weaken adaptive immunity by impairing the differentiation and function of antigen-presenting cells.

Sarcoma treatment in the era of molecular medicine.

Sarcomas are heterogeneous and clinically challenging soft tissue and bone cancers. Although constituting only 1% of all human malignancies, sarcomas represent the second most common type of solid tumors in children and adolescents and comprise an important group of secondary malignancies. More than 100 histological subtypes have been characterized to date, and many more are being discovered due to molecular profiling. Owing to their mostly aggressive biological behavior, relative rarity, and occurrence at virtually every anatomical site, many sarcoma subtypes are in particular difficult-to-treat categories. Current multimodal treatment concepts combine surgery, polychemotherapy (with/without local hyperthermia), irradiation, immunotherapy, and/or targeted therapeutics. Recent scientific advancements have enabled a more precise molecular characterization of sarcoma subtypes and revealed novel therapeutic targets and prognostic/predictive biomarkers. This review aims at providing a comprehensive overview of the latest advances in the molecular biology of sarcomas and their effects on clinical oncology; it is meant for a broad readership ranging from novices to experts in the field of sarcoma.

MHC Class I-Restricted TCR-Transgenic CD4+ T Cells Against STEAP1 Mediate Local Tumor Control of Ewing Sarcoma In Vivo.

In this study we report the functional comparison of T cell receptor (TCR)-engineered major histocompatibility complex (MHC) class I-restricted CD4+ versus CD8+ T cells targeting a peptide from six transmembrane epithelial antigen of the prostate 1 (STEAP1) in the context of HLA-A*02:01. STEAP1 is a tumor-associated antigen, which is overexpressed in many cancers, including Ewing sarcoma (EwS). Based on previous observations, we postulated strong antitumor potential of tumor-redirected CD4+ T cells transduced with an HLA class I-restricted TCR against a STEAP1-derived peptide. We compared CD4+ T cell populations to their CD8+ counterparts in vitro using impedance-based xCELLigence and cytokine/granzyme release assays. We further compared antitumor activity of STEAP130-TCR transgenic (tg) CD4+ versus CD8+ T cells in tumor-bearing xenografted Rag2-/-gc-/- mice. TCR tgCD4+ T cells showed increased cytotoxic features over time with similar functional avidity compared to tgCD8+ cells after 5-6 weeks of culture. In vivo, local tumor control was equal. Assessing metastatic organotropism of intraveniously (i.v.) injected tumors, only tgCD8+ cells were associated with reduced metastases. In this analysis, EwS-redirected tgCD4+ T cells contribute to local tumor control, but fail to control metastatic outgrowth in a model of xenografted EwS.

Donor lymphocyte infusions in adolescents and young adults for control of advanced pediatric sarcoma.

BACKGROUND: Allogeneic stem cell transplantation (allo-SCT) and donor lymphocyte infusions (DLI) may induce a graft-versus-tumor effect in pediatric sarcoma patients. Here, we describe general feasibility, toxicity and efficacy of DLI after allo-SCT. RESULTS: 4 of 8 patients responded. ES#4 had stable disease (SD) for 9 months after DLI and RMS#4 partial response for 8 months with combined hyperthermia/chemotherapy. In ES#4, DLI led to SD for 6 months and reverted residual disease before allo-SCT into complete remission. After DLI, ES#4 and RMS#4 developed acute GvHD (°III-°IV), ES#4 also developed chronic GvHD. 5 patients including ES#4 lived longer than expected. Median survival after allo-SCT was 2.3 years, post-relapse survival (PRS) was 13 months. Off note, HLA-mismatched DLI were associated with a trend towards increased survival after allo-SCT and increased PRS compared to HLA-matched DLI (23 versus 3 months). MATERIALS AND METHODS: We studied eight adolescents and young adults (AYAs) with advanced Ewing sarcoma (ES#1-4) and rhabdomyosarcoma (RMS#1-4) who received DLI. Escalating doses ranged from 2.5 × 104 to 1 × 108 CD3+ cells/kg body weight. AYAs were evaluated for response to DLI, graft-versus-host disease (GvHD) and survival. CONCLUSIONS: DLI after allo-SCT may control advanced pediatric sarcoma in AYAs with controllable toxicity.

Ewing sarcoma partial regression without GvHD by chondromodulin-I/HLA-A*02:01-specific allorestricted T cell receptor transgenic T cells.

Background: Chondromodulin-I (CHM1) sustains malignancy in Ewing sarcoma (ES). Refractory ES carries a dismal prognosis and patients with bone marrow (BM) metastases do not survive irrespective of therapy. We assessed HLA-A*02:01/CHM1-specific allorestricted T cell receptor (TCR) wild-type and transgenic cytotoxic (CD8+) T cells against ES. Patients and Methods: Three refractory HLA-A2+ ES patients were treated with HLA-A*02:01/peptide-specific allorepertoire-derived (i.e., allorestricted) CD8+ T cells. Patient #1 received up to 4.8 × 105/kg body weight HLA-A*02:01- allorestricted donor-derived wild-type CD8+ T cells. Patient #2 received up to 8.2 × 106/kg HLA-A*02:01- donor-derived and patient #3 up to 6 × 106/kg autologous allorestricted TCR transgenic CD8+ T cells. All patients were treated with the same TCR complementary determining region 3 allorecognition sequence for CHM1 peptide 319 (CHM1319). Results: HLA-A*02:01/CHM1319-specific allorestricted CD8+ T cells showed specific in vitro lysis of all patient-derived ES cell lines. Therapy was well tolerated and did not cause graft versus host disease (GvHD). Patients #1 and #3 showed slow progression, whereas patient #2, while having BM involvement, showed partial metastatic regression associated with T cell homing to involved lesions. CHM1319 TCR transgenic T cells could be tracked in his BM for weeks. Conclusions: CHM1319-TCR transgenic T cells home to affected BM and may cause partial disease regression. HLA-A*02:01/antigen-specific allorestricted T cells proliferate in vivo without causing GvHD.