We argue here that in many ways, Ewing sarcoma (EwS) is a unique tumor entity and yet, it shares many commonalities with other immunologically cold solid malignancies. From the historical ...perspective, EwS, osteosarcoma (OS) and other bone and soft-tissue sarcomas were the first types of tumors treated with the immunotherapy approach: more than 100 years ago American surgeon William B. Coley injected his patients with a mixture of heat-inactivated bacteria, achieving survival rates apparently higher than with surgery alone. In contrast to OS which exhibits recurrent somatic copy-number alterations, EwS possesses one of the lowest mutation rates among cancers, being driven by a single oncogenic fusion protein, most frequently EWS-FLI1. In spite these differences, both EwS and OS are allied with immune tolerance and low immunogenicity. We discuss here the potential mechanisms of immune escape in these tumors, including low representation of tumor-specific antigens, low expression levels of MHC-I antigen-presenting molecules, accumulation of immunosuppressive M2 macrophages and myeloid proinflammatory cells, and release of extracellular vesicles (EVs) which are capable of reprogramming host cells in the tumor microenvironment and systemic circulation. We also discuss the vulnerabilities of EwS and OS and potential novel strategies for their targeting.
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.
Here, we assess the combination of EwS tumor-associated antigen CHM1
-specific TCR-transgenic CD8
T cells and the YB-1-driven (i.e. E1A13S-deleted) oncolytic adenovirus XVir-N-31
and in a xenograft mouse model for antitumor activity and immunostimulatory properties.
both approaches specifically kill EwS cell lines in a synergistic manner over controls. This effect was confirmed
, with increased survival using the combination therapy. Further
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 CHM1
-specific TCR-transgenic CD8
T cells only in virus-tested conditions, emphasizing the superior immune-activating potential of XVir-N-31.
Our data prove synergistic antitumor effects
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.
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.
Ewing sarcoma (EwS) is a highly malignant sarcoma of bone and soft tissue with early metastatic spread and an age peak in early puberty. The prognosis in advanced stages is still dismal, and the ...long-term effects of established therapies are severe. Efficacious targeted therapies are urgently needed. Our previous work has provided preliminary safety and efficacy data utilizing T cell receptor (TCR) transgenic T cells, generated by retroviral gene transfer, targeting HLA-restricted peptides on the tumor cell derived from metastatic drivers. Here, we compared T cells engineered with either CRISPR/Cas9 or retroviral gene transfer. Firstly, we confirmed the feasibility of the orthotopic replacement of the endogenous TCR by CRISPR/Cas9 with a TCR targeting our canonical metastatic driver chondromodulin-1 (CHM1). CRISPR/Cas9-engineered T cell products specifically recognized and killed HLA-A*02:01+ EwS cell lines. The efficiency of retroviral transduction was higher compared to CRISPR/Cas9 gene editing. Both engineered T cell products specifically recognized tumor cells and elicited cytotoxicity, with CRISPR/Cas9 engineered T cells providing prolonged cytotoxic activity. In conclusion, T cells engineered with CRISPR/Cas9 could be feasible for immunotherapy of EwS and may have the advantage of more prolonged cytotoxic activity, as compared to T cells engineered with retroviral gene transfer.
Patients with stage IV alveolar rhabdomyosarcoma (RMA) have a 5-year-survival rate not exceeding 30%. Here, we assess the role of allogeneic hematopoietic stem cell transplantation (allo-HSCT) for ...these patients in comparison to standard-of-care regimens. We also compare the use of HLA-mismatched vs. HLA-matched grafts after reduced vs. myeloablative conditioning regimens, respectively.
In this retrospective analysis, we compare event-free survival (EFS), overall survival (OS), and toxicity of HLA-mismatched vs. -matched transplanted patients in uni- and multivariate analyses (total:
= 50, HLA-matched:
= 15, HLA-mismatched:
= 35). Here, the factors age at diagnosis, age at allo-HSCT, sex, Oberlin score, disease status at allo-HSCT, and HLA graft type are assessed. For 29 primarily transplanted patients, three matched non-transplanted patients per one transplanted patient were identified from the CWS registry. Outcomes were respectively compared for OS and EFS. Matching criteria included sex, age at diagnosis, favorable/unfavorable primary tumor site, and metastatic sites.
Median EFS and OS did not differ significantly between HLA-mismatched and -matched patients. In the mismatched group, incidence of acute GvHD was 0.87 (grade III-IV: 0.14) vs. 0.80 in HLA-matched patients (grade III-IV: 0.20). Transplant-related mortality (TRM) of all patients was 0.20 and did not differ significantly between HLA-mismatched and -matched groups. A proportion of 0.58 relapsed or progressed and died of disease (HLA-mismatched: 0.66, HLA-matched: 0.53) whereas 0.18 were alive in complete remission (CR) at data collection. Multivariate and competing risk analyses confirmed CR and very good partial response (VGPR) status prior to allo-HSCT as the only decisive predictor for OS (
< 0.001). Matched-pair survival analyses of primarily transplanted patients vs. matched non-transplanted patients also identified disease status prior to allo-HSCT (CR, VGPR) as the only significant predictor for EFS. Here, OS was not affected, however.
In this retrospective analysis, only a subgroup of patients with good response at allo-HSCT survived. There was no survival benefit of allo-transplanted patients compared to matched controls, suggesting the absence of a clinically relevant graft-versus-RMA effect in the current setting. The results of this analysis do not support further implementation of allo-HSCT in RMA stage IV patients.
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.
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
...(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 STEAP
-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.
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 CHM1
-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 CHM1
-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 CHM1
-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-1β and PGE
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 CHM1
/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 (EwS) is a highly malignant pediatric tumor characterized by a non-T-cell-inflamed immune-evasive phenotype. When relapsed or metastasized, survival is poor, emphasizing the need for ...novel treatment strategies. Here, we analyze the novel combination approach using the YB-1-driven oncolytic adenovirus XVir-N-31 and CDK4/6 inhibition to augment EwS immunogenicity.
In vitro, viral toxicity, replication, and immunogenicity were studied in several EwS cell lines. In vivo tumor xenograft models with transient humanization were applied to evaluate tumor control, viral replication, immunogenicity, and dynamics of innate as well as human T cells after treatment with XVir-N-31 combined with CDK4/6 inhibition. Furthermore, immunologic features of dendritic cell maturation and T-cell-stimulating capacities were assessed.
The combination approach significantly increased viral replication and oncolysis in vitro, induced HLA-I upregulation, and IFNγ-induced protein 10 expression and enhanced maturation of monocytic dendritic cells with superior capacities to stimulate tumor antigen-specific T cells. These findings were confirmed in vivo showing tumor infiltration by (i) monocytes with antigen-presenting capacities and M1 macrophage marker genes, (ii) TReg suppression in spite of adenovirus infection, (iii) superior engraftment, and (iv) tumor infiltration by human T cells. Consequently, survival was improved over controls with signs of an abscopal effect after combination treatment.
The joint forces of the YB-1-driven oncolytic adenovirus XVir-N-31 and CDK4/6 inhibition induce therapeutically relevant local and systemic antitumor effects. Innate as well as adaptive immunity against EwS is boosted in this preclinical setting, pointing toward high therapeutic potential in the clinic.
Abstract
Pediatric malignancies, in particular Ewing sarcoma (EwS), are characterized by low mutational load, low immunogenicity, and early metastasis. They recapitulate the embryonic immune ...tolerance setting and remain a challenge for established immunotherapies. These immunotherapies are not sufficient to target metastasis. Ideally, targeted therapies address gene products required for metastasis. We completed in vivo functional analyses for metastasis of 9/37 genes that we had shown to be overexpressed in EwS (Staege at al., Cancer Res 2004) and generated HLA class I restricted cytotoxic T cells against these gene products. All targets were involved in fetal development and 8/9 demonstrated functional relevance for metastasis. Allorepertoire-derived TCRs against 8/9 targets were cloned and sequenced; one target (DKK2) was nonimmunogenic. 7/8 TCRs were crossreactive, caused fratricide, or clonal TCR expansion failed (EZH2, STEAP1, PAPP-A, GPR64, ADRB3, LIPI, HOX-D1). In the tumor microenvironment we found an immunosuppressive (M0 and M2) transcriptomic signature and evidence for an immunosuppressive inflammation-associated activation of endogenous retroviral sequences. Among these most selectively expressed (n=9) and metastasis sustaining (n=8) targets, the BRICHOS chaperon domain containing antiangiogenetic bone protein chondromodulin-I (CHM1) was addressable by a non-crossreactive TCR. CHM1 is a direct downstream target of the oncogenic driver EWS-FLI1. We clinically assessed HLAA* 02:01/CHM1-specific TCR transgenic CD8+ T cells against EwS utilizing a TCR complementary determining region 3 (CDR3) recognition-sequence for the CHM1319 peptide with a Koff half-life of 113.2 ± 38.2 s. The CHM1319 motive was 130 times less homologous as compared to the 9mer ADRB3CHM1295, a crossreactive EwS target identified before. Four refractory HLA-A2+ EwS patients (pts) were treated with CHM1319-specific TCR-CDR3 transgenic T cells. Pt-derived cell lines (PDCL) were established in all cases. Pts received up to 107/kg TCR transgenic CD8+ T cells. All pts were treated with the same TCR-CDR3 recognition-sequence for CHM1. All PDCLs displayed persistent HLA-A2 expression. Transgenic T cells showed specific in vitro lysis of all PDCLs. Therapy was well tolerated and did not cause graft-versus-host disease (GvHD). Pts #1 #3 and #4 showed delayed progression, whereas pt #2, while having bone marrow (BM) involvement and accessible multifocal disease, showed partial metastatic regression associated with T-cell homing to involved lesions. In conclusion, CHM1319-TCR transgenic T cells may home to affected BM and may cause partial remission. CHM1-TCR transgenic T cells address a persistently expressed target required for metastasis, suggesting lack of immunoediting selection pressure. They proliferate in vivo without causing GvHD.
This abstract is also being presented as Poster A07.
Citation Format: Stefan Burdach, Guenther Richter, David Schirmer, Andreas Kirschner, Sebastian Schober, Valentina Evdokimova, Hendrik Gassmann, Elvira D’Ippolito, Maxim Barenboim, Dirk Busch, Poul Sorensen, Uwe Thiel. T-cell receptor (TCR)-based immunotherapy in pediatric malignancy: Addressing the challenge of early metastasis and low immunogenicity abstract. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr PR05.
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