David Schirmer

The endochondral bone protein CHM1 sustains an undifferentiated, invasive phenotype, promoting lung metastasis in Ewing sarcoma

Ewing sarcomas (ES) are highly malignant, osteolytic bone or soft tissue tumors, which are characterized by EWS–ETS translocations and early metastasis to lung and bone. In this study, we investigated the role of the BRICHOS chaperone domain‐containing endochondral bone protein chondromodulin I (CHM1) in ES pathogenesis. CHM1 is significantly overexpressed in ES, and chromosome immunoprecipitation (ChIP) data demonstrate CHM1 to be directly bound by an EWS–ETS translocation, EWS‐FLI1. Using RNA interference, we observed that CHM1 promoted chondrogenic differentiation capacity of ES cells but decreased the expression of osteolytic genes such as HIF1A, IL6, JAG1, and VEGF. This was in line with the induction of the number of tartrate‐resistant acid phosphatase (TRAP+)‐stained osteoclasts in an orthotopic model of local tumor growth after CHM1 knockdown, indicating that CHM1‐mediated inhibition of osteomimicry might play a role in homing, colonization, and invasion into bone tissues. We further demonstrate that CHM1 enhanced the invasive potential of ES cells in vitro. This invasiveness was in part mediated via CHM1‐regulated matrix metallopeptidase 9 expression and correlated with the observation that, in an xenograft mouse model, CHM1 was essential for the establishment of lung metastases. This finding is in line with the observed increase in CHM1 expression in patient specimens with ES lung metastases. Our results suggest that CHM1 seems to have pleiotropic functions in ES, which need to be further investigated, but appears to be essential for the invasive and metastatic capacities of ES.

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

ABSTRACT 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.

Human HLA-A*02:01/CHM1+ allo-restricted T cell receptor transgenic CD8+ T Cells specifically inhibit Ewing sarcoma growth in vitro and in vivo

The endochondral bone protein Chondromodulin-I (CHM1) provides oncogene addiction in Ewing sarcoma (ES). We pre-clinically tested the targetability of CHM1 by TCR transgenic, allo-restricted, peptide specific T cells to treat ES. We previously generated allo-restricted wildtype CD8+ T cells directed against the ES specific antigen CHM1319 causing specific responses against ES. However, utilization of these cells in current therapy protocols is hampered due to high complexity in production, relatively low cell numbers, and rapid T cell exhaustion. In order to provide off-the-shelf products in the future, we successfully generated HLA-A*02:01-restricted T cell receptor (TCR) transgenic T cells directed against CHM1319 by retroviral transduction. After short-term expansion a 100% purified CHM1319-TCR-transgenic T cell population expressed a CD62L+/CD45RO and CD62L+/CD45RA+ phenotype. These cells displayed specific in vitro IFNg and granzyme B release in co-culture with HLA-A*02:01+ ES cell lines expressing CHM1. When co-injected with ES cells in Rag2−/−ɣc−/− mice, CHM1-specific TCR-transgenic T cells significantly inhibited the formation of lung and liver metastases in contrast to control mice. Lungs and livers of representative mice displayed CD8+ T cell infiltration in the presence (control group treated with unspecific T cells) and in the absence (study group) of metastatic disease, respectively. Furthermore, mice receiving unspecific T cells showed signs of graft-versus-host-disease in contrast to all mice, receiving CHM1319-TCR-transgenic T cells. CHM1319 specific TCR-transgenic T cells were successfully generated causing anti-ES responses in vitro and in vivo. In the future, CHM1319-TCR-transgenic T cells may control minimal residual disease rendering donor lymphocyte infusions more efficacious and less toxic.

Abstract 3202: Transgenic antigen-specific, HLA-A*02:01-allo-restricted cytotoxic T cells recognize tumor-associated target antigen STEAP1 with high specificity

Background: Pediatric cancers, including Ewing sarcoma (ES), are only weakly immunogenic and the tumor-patients immune system often is devoid of effector T cells for tumor elimination. Based on expression profiling technology targetable tumor associated antigens (TAA) are identified and exploited for engineered T cell therapy. Here, the specific recognition and lytic potential of transgenic, allo-restricted CD8+ T cells directed against the ES-associated antigen STEAP1 was examined. Methods: Following repetitive STEAP1130 peptide-driven stimulations with HLA-A*02:01+ dendritic cells, allo-restricted HLA-A*02:01- CD8+ T cells were sorted with HLA-A*02:01/peptide multimers and expanded by limiting dilution. After functional analysis of suitable T cell clones via ELISpot, flow cytometry and xCELLigence assay, TCR α- and β-chains were identified. They were cloned into retroviral vectors, codon optimized, transfected into HLA-A*02:01- primary T cell populations and tested again for specificity and lytic capacity in vitro and in a Rag2-/-γc-/- mouse model. Results: Initially generated and transgenic T cells specifically recognized STEAP1130-pulsed or transfected cells in the context of HLA-A*02:01 with minimal cross-reactivity as determined by specific IFNγ release. They lysed cells and inhibited growth of HLA-A*02:01+ ES lines more effectively than HLA-A*02:01- ES lines. In vivo tumor growth was inhibited more effective with transgenic STEAP1130-specific T cells than with unspecific T cells. Conclusion: Our results identify TCRs capable of recognizing and inhibiting growth of STEAP1 expressing HLA-A*02:01+ ES cells in vitro and in vivo in a highly restricted manner. As STEAP1 is overexpressed in a wide variety of cancers, we anticipate these STEAP1-specific TCRs to be potentially useful for immunotherapy of other STEAP1 expressing tumors. Citation Format: David Schirmer, Richard Klar, Oxana Schmidt, Dirk Wohlleber, Wolfgang Uckert, Uwe Thiel, Felix Bohne, Dirk H. Busch, Angela M. Krackhardt, Stefan Burdach, Gunther H. Richter. Transgenic antigen-specific, HLA-A*02:01-allo-restricted cytotoxic T cells recognize tumor-associated target antigen STEAP1 with high specificity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3202.

Abstract A09: GPR64-specific CAR-transgenic T cells selectively kill Ewing sarcoma in vivo

Introduction: Pediatric cancers, including Ewing sarcoma (EwS), are only weakly immunogenic and the tumor patient’s immune system often is devoid of effector T cells for tumor elimination. Based on expression profiling technology, targetable tumor-associated antigens (TAA) are identified and exploited for engineered T-cell therapy. Here the therapeutic potential of chimeric antigen receptor (CAR) transgenic T cells directed against the G-protein coupled receptor 64 (GPR64), normally expressed only in epididymal tissue, but specifically expressed in EwS and some other sarcoma, was examined. Experimental Procedure: Two different monoclonal antibodies (mAb) directed against the extracellular region of GPR64 were generated and characterized. Subsequently, retroviral constructs containing second-generation CARs together with the scFv fragments of the respective mAbs were designed. CAR-constructs consisting of different spacers and costimulatory elements were compared (dIgG1-CD28-CD3z vs. CD8a-4-1BB-CD3z). Primary lymphocytes were transduced and tested in vitro via flow cytometry. Their activation profile and specificity was analyzed by micorarray analysis, ELISpot, and xCelligence assay as well as in immunodeficient xenograft mice. Results: Antibodies specifically stained EwS cells as determined by flow cytometry and immune histology. The signal intensity was reduced after RNAi mediated downregulation of GPR64 in EwS cell lines confirming specificity of the isolated mAbs. Further immune histology detected GPR64 expression not only in EwS but also in epithelial cancers such as renal, prostate, and pancreas carcinoma. Following sequence determination of those mAbs, two different CAR constructs were designed. Retroviruses containing such CARs transduced primary lymphocytes with high efficiency. The CAR transgenic T cells were enriched for CD8 + CAR + cells via microbead isolation and demonstrated strong proliferative capacities in vitro. Furthermore, GPR64 transmembrane cell surface target structures were specifically recognized as determined by ELISpot and xCelligence assays, but dIgG1-CD28 CAR T cells derived from one hybridoma revealed hints of early exhaustion via auto-activation. This exhaustion could be prevented by use of constructs containing CD8a-4-1BB-CD3z signaling domains for this particular hybridoma-derived scFv fragment, resulting in T-cell activation patterns more similar to TCR-transgenic T cells. Adoptive transfer of CAR-transgenic T cells into EwS tumor-bearing xenograft mice resulted in a significant suppression of tumor growth. Conclusion: CAR-transgenic T cells targeting GPR64 are a promising approach to transfer the success of CARs in hematologic malignancies to solid tumors. The cells generated in this study show strong specificity towards GPR64 and are able to control tumor cell growth. Since GPR64 expression is not restricted to EwS but also upregulated in a number of carcinomas derived from prostate, kidney, or pancreas, GPR64-specific CARs are a future treatment option for other tumor entities. Citation Format: David Schirmer, Isabel Storz, Karin Wisskirchen, Regina Feederle, Oxana Schmidt, Hinrich Abken, Ulrike Protzer, Stefan Burdach, Guenther H.S. Richter. GPR64-specific CAR-transgenic T cells selectively kill Ewing sarcoma in vivo [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A09.

Abstract B63: Therapeutic potential of GPR64 specific CAR T cells in pediatric sarcomas

Ewing Sarcoma (ES) is the second most common bone malignancy in children and young adolescents with a high potential of dissemination into lung and bones. Patients with localized disease receiving current treatment, have an approximate long-term survival of >65%. Patients with disseminated disease into the bone have an approximate long-term survival rate of only 10%, compelling the search for new therapeutic treatment modalities like engineered T cell therapy. Here the therapeutic potential of chimeric antigen receptor (CAR) transgenic T cells directed against G-protein coupled receptor 64 (GPR64), an orphan receptor with normal expression restricted to human epididymis and significant overexpression in ES, was examined. Therefore, two different monoclonal antibodies (mAb) directed against the extracellular region of GPR64 were generated and characterized. Subsequently, retroviral constructs containing second generation CARs together with the scFv fragments of the respective mAbs were designed. Primary lymphocytes were transduced and tested in vitro via flow cytometry, ELISpot and xCelligence assay. Antibodies specifically stained ES cells as determined by flow cytometry. The signal intensity was reduced after RNAi mediated down-regulation of GPR64 in ES cell lines confirming specificity of mAbs. Following sequence determination of those mAbs two different CAR constructs were designed. Retroviruses containing such CARs transduced primary lymphocytes with good efficiency. The CAR transgenic T cells could be enriched for CD8+CAR+ cells via microbead isolation and showed strong proliferative capacities in vitro. Furthermore, target structures were specifically recognized as determined by ELISpot and xCelligence assays. CAR transgenic T cells targeting GPR64 show a promising approach to transfer the success of CARs in hematological malignancies to solid tumors. The cells generated in this study show strong specificity towards GPR64 and are able to control tumor cell growth in vitro. Since GPR64 expression is not restricted to ES but also up-regulated in a number of carcinomas derived from prostate, kidney or lung, GPR64-specific CARs may also be a future treatment option for other tumor entities. Citation Format: Isabel Storz, David Schirmer, Karin Wisskirchen, Regina Feederle, Oxana Schmidt, Hinrich Abken, Ulrike Protzer, Stefan Burdach, Gunther HS Richter. Therapeutic potential of GPR64 specific CAR T cells in pediatric sarcomas. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B63.

Abstract 3764: Targeting GPR64 in pediatric sarcomas with CAR transgenic T cells

Introduction: Ewing Sarcoma (ES) is the second most common bone malignancy in children and young adolescents with a high potential of dissemination into lung and bones. Patients with disseminated disease into the bone have an approximate long-term survival rate of only 10%, compelling the search for new therapeutic treatment modalities like engineered T cell therapy. Here the therapeutic potential of chimeric antigen receptor (CAR) transgenic T cells directed against G-protein coupled receptor 64 (GPR64) was examined. Experimental procedure: Two different monoclonal antibodies (mAb) directed against the extracellular region of GPR64 were generated and characterized. Subsequently, retroviral constructs containing second generation CARs together with the scFv fragments of the respective mAbs were designed. Constructs consisting of different spacers and co-stimulatory elements were compared (dIgG1-CD28-CD3z vs. CD8a-4-1BB-CD3z). Primary lymphocytes were transduced and tested in vitro via flow cytometry, ELISpot and xCelligence assay as well as in immunodeficient xenograft mice. Results: Antibodies specifically stained ES cells as determined by flow cytometry and immune histology. The signal intensity was reduced after RNAi mediated down-regulation of GPR64 in ES cell lines confirming specificity of the isolated mAbs. Following sequence determination of those mAbs two different CAR constructs were designed. Retroviruses containing such CARs transduced primary lymphocytes with high efficiency. The CAR transgenic T cells were enriched for CD8+CAR+ cells via microbead isolation and demonstrated strong proliferative capacities in vitro. Furthermore, target structures were specifically recognized as determined by ELISpot and xCelligence assays but dIgG1-CD28 CAR T cells derived from one hybridoma revealed hints of early exhaustion via auto-activation. Conclusion: CAR transgenic T cells targeting GPR64 show a promising approach to transfer the success of CARs in hematological malignancies to solid tumors. The cells generated in this study show strong specificity towards GPR64 and are able to control tumor cell growth. Since GPR64 expression is not restricted to ES but also up-regulated in a number of carcinomas derived from prostate, kidney or lung, GPR64-specific CARs may also be a future treatment option for other tumor entities. Citation Format: David Schirmer, Isabel Storz, Karin Wisskirchen, Regina Feederle, Oxana Schmidt, Hinrich Abken, Crystall Mackall, Ulrike Protzer, Stefan Burdach, Gunther H. Richter. Targeting GPR64 in pediatric sarcomas with CAR transgenic T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3764. doi:10.1158/1538-7445.AM2017-3764

Abstract 2462: MondoA mediates in vivo aggressiveness of common ALL and may serve as a T-cell immunotherapy target

Oncogene addiction provides ideal targets for immunotherapy. We previously described MondoA (also known as MLXIP, MAX like protein X interacting protein) as a metabolic stress sensor, required for leukemogenesis. Here we report on the expression of MondoA in common acute lymphoblastic leukemia (cALL) compared to other malignancies, its role in malignancy of cALL in vivo, downstream pathways and correlation with relapse risk. Given the non-accessibility of transcription factors by drugs or chimeric antigen receptor transgenic T cells (CARs), we tested the targetability of MondoA by allo-restricted, peptide specific T cells. Our human/murine xenotransplantation model with immunodeficient RAG2-/-gc-/- mice was used (Richter et al. 2009). NALM6 and 697 cALL lines were lentivirally transduced with MondoA short hairpin RNA (shRNA). Upon successful MondoA knock down (KD), KD and control lines were injected into the mice; CD10+ blasts in blood, spleen and marrow were assessed. MondoA specific T cells were generated by priming of donor HLAA0201 negative (A2-) T-cells with A2+ dendritic cells bearing MondoA peptides, multimer-based sorting and subcloning of A2-CD8+ T-cells. For priming of T cells, five MondoA peptides were chosen by SYMPEITHI, BIMAS and NetCTL1.2. analyses. Peptide 428 stabilized best A2 expression on TAP-deficient T2 cells. Specificity and functionality of T cell clones were tested by ELISpot interferon gamma (IFg) and granzyme B assays with six MondoA+ leukemia lines (A2+, A2-). Off target effects of MondoA specific T-cell clones were assessed by IFg reactivity against the MondoA expressing A2+ NALM6 cell line vs. A2+ and A2- EBV immortalized lymphoblastoid cell lines from six donors. Peptide homology was assessed with BLAST algorithms in SWISSPROT. We found MondoA to be most strongly expressed in pediatric cALL and AML. Moreover MondoA expression was high in gastrointestinal stromal tumors and alveolar rabdomyosarcoma. MondoA KD in cALL cell lines and their subsequent analysis in xenograft mice resulted in a reduced number of leukemic blasts in blood, marrow and spleen. Spleen size and weight normalized in treated mice after MondoA KD. Further microarray analysis revealed an induction of aerobic glycolysis switch genes and hypoxia-response by MondoA. Consequently, HIF1A stabilization required MondoA expression and tied to these results, MondoA overexpression correlated with relapse risk; its expression was 63% higher in the very high-risk group as compared to the non-high-risk group of cALL. Therapeutically, MondoA-derived peptide antigens and A2+ cALL lines were successfully recognized and killed by specific, allo-restricted CD8+ T cells. In conclusion, our findings demonstrate that MondoA maintains leukemic burden and aggressiveness of cALL in vivo possibly by modulating metabolic and hypoxia stress response. Moreover, we identified MondoA as a promising target for immunotherapy of cALL. Citation Format: Alexandra Sipol, Thomas G. P. Grunewald, Juliane Schmaeh, David Schirmer, Monique L. den Boer, Rebeca Alba Rubio, Michaela Baldauf, Caroline Wernicke, Hans-Jochem Kolb, Martin Horstmann, Gunnar Cario, Guunther Richter, Stefan Burdach. MondoA mediates in vivo aggressiveness of common ALL and may serve as a T-cell immunotherapy target. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2462.