CCR Translation for the Article from 2B4 (CD244) Signaling by Recombinant Antigen-specific Chimeric Receptors Costimulates Natural Killer Cell Activation to Leukemia and Neuroblastoma Cells

Phage display technology (PD) is a powerful technique for the generation of tumor-targeting antibodies. However, there are a number of different selection methods established in different laboratories around the world. Cell-based PD panning methods using primary tumor cells are particularly heterogeneous between laboratories, which can lead to inconsistent results. Therefore, the present study evaluated different cell-based PD selection methods regarding their potential to generate acute myeloid leukemia (AML) blast-binding antibodies. In addition to this evaluation, the present study improved the PD procedure by optimizing selection as well as depletion strategies. To the best of our knowledge, the current study demonstrated for the first time that antigen diversity during the depletion step is of importance for the enrichment of tumor-targeting phage antibodies. It is demonstrated that medium levels of depletion antigen diversity led to the most promising antibody candidates. In addition, it was determined that purification of blast cells from patients with AML by immunomagnetic separation ameliorated the selection of AML-binding phages during panning. Furthermore, suggesting a common design-related mechanism using a 'single-pot' PD library, such as the well-known Tomlinson single-chain fragment variable (scFv) library, the present study identified specific binding consensus phage particles in independent panning procedures. By means of these optimized strategies, four promising AML blast-binding phage particles were isolated and soluble scFv-Fc (scFv cloned to a fragment crystallizable of an IgG2a mouse antibody) fusion proteins were produced. These scFv-Fc antibodies bound the surface of AML blasts and were successfully internalized into their cytoplasm, indicating that they are potential immunoconjugate candidates for AML immunotherapy.

Acute myeloid leukemia (AML) is a heterogeneous disorder of the myeloid lineage. During carcinogenesis several subclones arise making treatment difficult and relapse rate high. Thus, a one-size-fits-all-treatment cannot exist. Multiple ways of targeted therapies such as immunotherapies are urgently needed. Phage display technology (PD) is a powerful method for the generation of target-specific immunotherapeutic agents. It uses the genotype-to-phenotype-linkage of bacteriophages to express different kinds of recombinant antibodies (AB) or AB fragments, e.g. single-chain-fragment-variable (scFv) at the surface of phages. So, large antibody phage libraries consisting of millions of phage-AB are generated.

Bluetongue is an infectious viral disease which can cause mortality in affected ruminants, and tremendous economic damage via impacts upon fertility, milk production and the quality of wool. The disease is caused by bluetongue virus (BTV) which is transmitted by species of Culicoides biting midge. Rapid detection of BTV is required to contain disease outbreaks and reduce economic losses. The purpose of this study was to develop a monoclonal sandwich ELISA for direct detection of BTV in infected animals. Phage display technology was used to isolate BTV specific antibody fragments by applying the human scFv Tomlinson antibody libraries directly on purified BTV-8 particles. Three unique BTV-8 specific human antibody fragments were isolated which were able to detect purified BTV particles and also BTV in serum of an infected sheep. A combination of a human/mouse scFv-Fc chimeric fusion protein and a human Fab fragment in a sandwich ELISA format was able to detect BTV specifically with a limit of detection (LOD) of 104 infectious virus particles, as determined by tissue culture titration. This approach provided pilot data towards the development of a novel diagnostic test that might be used for direct detection of BTV-8 particles.

Phage display is an effective method for the generation of target-specific human antibodies. Standard phage display panning use purified proteins, antigen-transfected cells or tumor cell lines as target structure to generate specific antibodies. However, recombinant proteins can be difficult to express and purify in their native conformation and suitable cell lines are not always available. Additionally the antigen expression profile may change during cultivation and thus differ from the malignant cells in patient. Here we describe a method for the selection of specific antibodies from phage display libraries by panning against formalin-fixed paraffin-embedded (FFPE) tissue biopsies immobilized on glass slides, using small cell lung cancer (SCLC) as a case study. The human Tomlinson single-chain variable fragment (scFv) phage libraries I and J were panned against SCLC FFPE tissue slides for positive selection and healthy lung tissue for subtraction. The specificity of the selected scFv antibodies was confirmed in vitro by ELISA on immobilized SCLC cell membranes, by flow cytometry using the SCLC cell lines NCI-H69, NCI-H82 and DMS 273, and ex vivo against tissue microarrays containing 35 different SCLC samples and 20 types of normal organs. We monitored the internalization of three selected scFv antibodies and fused them with Pseudomonas exotoxin A (ETA′) to produce immunotoxins whose cytotoxicity was confirmed by cell viability and apoptosis assays on different SCLC cell lines, achieving IC50 values of up to 23 nM. The selection of SCLC-specific scFv antibodies by panning against FFPE tissue slides circumvents the challenges of using purified antigens or cell lines for antibody selection.

Disseminated or relapsed Ewing sarcoma (EwS) has remained fatal in the majority of patients. A promising approach to preventing relapse after conventional therapy is to establish tumor antigen-specific immune control. Efficient and specific T cell memory against the tumor depends on the expansion of rare T cells with native specificity against target antigens overexpressed by the tumor. Candidate antigens in EwS include six-transmembrane epithelial antigen of the prostate-1 (STEAP1), and the human cancer/testis antigens X-antigen family member 1 (XAGE1) and preferentially expressed antigen in melanoma (PRAME). Here, we screened normal donors and EwS patients for the presence of circulating T cells reactive with overlapping peptide libraries of these antigens by IFN-γ Elispot analysis. The majority of 22 healthy donors lacked detectable memory T cell responses against STEAP1, XAGE1 and PRAME. Moreover, ex vivo detection of T cells specific for these antigens in both blood and bone marrow were limited to a minority of EwS patients and required nonspecific T cell prestimulation. Cytotoxic T cells specific for the tumor-associated antigens were efficiently and reliably generated by in vitro priming using professional antigen-presenting cells and optimized cytokine stimulation; however, these T cells failed to interact with native antigen processed by target cells and with EwS cells expressing the antigen. We conclude that EwS-associated antigens fail to induce efficient T cell receptor (TCR)-mediated antitumor immune responses even under optimized conditions. Strategies based on TCR engineering could provide a more effective means to manipulating T cell immunity toward targeted elimination of tumor cells.

Abstract The outcome of disseminated Ewing sarcoma remains poor despite intensive multimodal treatment regimens. The disease often responds well to chemotherapy, but systemic relapses occur in the majority of patients. Targeting of residual disease by cellular immunotherapy may sustain remission and improve outcome. Specifically, Ewing sarcoma cells have been shown to be exquisitely sensitive to targeting by activated NK cells. To further explore the value of cellular strategies, preclinical models are needed that mimic the anchorage-independent, multicellular growth of Ewing sarcoma micrometastases. Here, we generated Ewing sarcoma spheres from cell lines (VH-64, TC-32, TC-71, A4573) and from four low-passage cell cultures established from Ewing sarcoma biopsies at primary diagnosis or at relapse under serum-free growth conditions. Standard monolayer cultures were used for comparisons. Phenotypic analysis revealed considerable heterogeneity among individual Ewing sarcomas and between spheres and monolayers. While the Ewing sarcoma marker CD99 as well as CD133 were expressed at comparable densities, spheres had significantly higher expression of the neural crest marker CD57 (HNK-1) and of MHC class I than monolayers, whereas CD117 (c-kit) expression was lower. Side populations characterized by Hoechst dye exclusion and previously associated with cancer stem cell function were identified in one of two primary sphere cultures and in VH-64 spheres but were absent or reduced in monolayer cultures. However, cells resuspended from spheres did not form subcutaneous tumors in immunodeficient (NOD/scid) mice at higher efficiencies than monolayer cultures, arguing against higher tumorigenicity of sphere-cultured cells. VH64 spheres were significantly more resistant towards doxorubicine than monolayers, and resuspended cells from sphere cultures remained less susceptible to lysis than monolayer cultures, but among primary tumor cells, consistent differences in chemosensitivity were not observed between the two culture systems. In vitro activated allogeneic NK cells were uniformly capable to lyse single cells derived from both monolayer and sphere cultures from established cell lines and primary cell cultures. Moreover, NK cells efficiently eliminated intact Ewing sarcoma spheres. Thus, cultured Ewing sarcoma cells are highly heterogenous. Their phenotype, function and susceptibility to both chemo- and immunotherapy differs among individual cell lines and primary cultures and under variable in vitro growth conditions. Activated NK cells efficiently target Ewing sarcoma cells both as monolayers and as spheres. The sphere model may provide a useful tool to analyze the contribution of micrometastatic architecture and serum-free niches to immune evasion. Experiments with autologous NK cells and with NK cells engineered to express tumor antigen-specific chimeric receptors are ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2503. doi:1538-7445.AM2012-2503

Specific cellular immunotherapy of cancer requires efficient generation and expansion of cytotoxic T lymphocytes (CTLs) that recognize tumor-associated self-antigens. Here, we investigated the capacity of human γδ T cells to induce expansion of CD8+ T cells specific for peptides derived from the weakly immunogenic tumor-associated self-antigens PRAME and STEAP1. Coincubation of aminobisphosphonate-stimulated human peripheral blood-derived γδ T cells (Vγ9+Vδ2+), loaded with HLA-A*02-restricted epitopes of PRAME, with autologous peripheral blood CD8+ T cells stimulated the expansion of peptide-specific cytolytic effector memory T cells. Moreover, peptide-loaded γδ T cells efficiently primed antigen-naive CD45RA+ CD8+ T cells against PRAME peptides. Direct comparisons with mature DCs revealed equal potency of γδ T cells and DCs in inducing primary T-cell responses and peptide-specific T-cell activation and expansion. Antigen presentation by γδ T-APCs was not able to overcome the limited capacity of peptide-specific T cells to interact with targets expressing full-length antigen. Importantly, T cells with regulatory phenotype (CD4+CD25hiFoxP3+) were lower in cocultures with γδ T cells compared to DCs. In summary, bisphosphonate-activated γδ T cells permit generation of CTLs specific for weakly immunogenic tumor-associated epitopes. Exploiting this strategy for effective immunotherapy of cancer requires strategies that enhance the avidity of CTL responses to allow for efficient targeting of cancer.

BACKGROUND:Novel treatment strategies are needed to cure disseminated Ewing sarcoma. Primitive neuroectodermal features and a mesenchymal stem cell origin are both compatible with aberrant expression of the ganglioside antigen G(D2) and led us to explore G(D2) immune targeting in this cancer.METHODS:We investigated G(D2) expression in Ewing sarcoma by immunofluorescence staining. We then assessed the antitumour activity of T cells expressing a chimeric antigen receptor specific for G(D2) against Ewing sarcoma in vitro and in vivo.RESULTS:Surface G(D2) was detected in 10 out of 10 Ewing sarcoma cell lines and 3 out of 3 primary cell cultures. Moreover, diagnostic biopsies from 12 of 14 patients had uniform G(D2) expression. T cells specifically modified to express the G(D2)-specific chimeric receptor 14. G2a-28ζ efficiently interacted with Ewing sarcoma cells, resulting in antigen-specific secretion of cytokines. Moreover, chimeric receptor gene-modified T cells from healthy donors and from a patient exerted potent, G(D2)-specific cytolytic responses to allogeneic and autologous Ewing sarcoma, including tumour cells grown as multicellular, anchorage-independent spheres. G(D2)-specific T cells further had activity against Ewing sarcoma xenografts.CONCLUSION:G(D2) surface expression is a characteristic of Ewing sarcomas and provides a suitable target antigen for immunotherapeutic strategies to eradicate micrometastatic cells and prevent relapse in high-risk disease.

Although BCR–ABL+ stem cells in chronic myeloid leukemia (CML) resist elimination by targeted pharmacotherapy in most patients, immunological graft-versus-leukemia effects can cure the disease. Besides cytotoxic T cells, natural killer (NK) cells may have a role in immune control of CML. Here, we explored the functionality of NK cells in CML patients and in a transgenic inducible BCR–ABL mouse model. Compared with controls, NK-cell proportions among lymphocytes were decreased at diagnosis of CML and did not recover during imatinib-induced remission for 10–34 months. Functional experiments revealed limited in vitro expansion of NK cells from CML patients and a reduced degranulation response to K562 target cells both at diagnosis and during imatinib therapy. Consistent with the results in human CML, relative numbers of NK1.1+ NK cells were reduced following induction of BCR–ABL expression in mice, and the defects persisted after BCR–ABL reversion. Moreover, target-induced degranulation by expanded BCR–ABL+ NK cells was compromised. We conclude that CML is associated with quantitative and functional defects within the NK-cell compartment, which is reproduced by induced BCR–ABL expression in mice. Further work will aim at identifying the mechanisms of NK-cell deficiency in CML and at developing strategies to exploit NK cells for immunotherapy.

Primary metastatic Ewing sarcomas and alveolar rhabdomyosarcomas are often incurable by current state-of-the art treatment. Cellular immunotherapy is a promising complementary strategy to eradicate dormant and chemoresistant tumor cells and prevent sarcoma relapse. We propose that the immunostimulatory properties of activated γδ T cells can be used to induce cytolytic CD8+ T cell responses to the sarcoma-associated self antigens PRAME and STEAP-1.

Cytotoxic T cells transduced with chimeric anti-CD19 receptors prevent engraftment of primary lymphoblastic leukemia in vivo

High-risk Ewing sarcoma is still fatal in many cases. Cellular immunotherapy is a promising approach for preventing relapse by eliminating residual disease after conventional treatment. A critical prerequisite is the availability of an adequate tumor target antigen. STEAP-1 is a surface protein aberrantly expressed in various cancers. Potential obstacles to the use of STEAP-1 as a tumor target are the limited immunogenicity of STEAP-1 epitopes, clonal deletion of STEAP-1-reactive T cells, and poor presentation of STEAP-1 peptides by MHC class I on tumor cells. Here we investigated whether functional and Ewing-tumor reactive STEAP-1-specific cytotoxic T cells (CTLs) can be expanded from the repertoire of normal donors by peptide stimulation. STEAP-1 was confirmed to be expressed in 10 of 10 Ewing sarcoma cell lines by PCR. To expand STEAP-1-specific CTLs, CD8+ T cells from HLA-A2-positive healthy donors were stimulated with autologous dendritic cells pulsed with a pool of 4 STEAP-1 peptides (5 µM each), in the presence of rhIL-7, rhIL-12 and rhIL-15. Weekly restimulations were performed with peptide-pulsed K562 cells gene-modified to express human HLA-A2, CD80, CD40L and OX40L (K562aAPCs). CTLs with a predominant CD8+ effector memory CTL phenotype (CD45RA-, CCR7-) were successfully generated from 6 healthy donors. Their epitope specificity was confirmed by ELISPOT analysis after 2 to 6 rounds of restimulation. Pooled STEAP-1 peptides directly added to the CTLs induced specific secretion of IFN-γ (70 to 487 spot forming cells (SFC)/105 cells, mean 297.5±138.7 SFC/105 cells), in the absence of relevant background responses to a control peptide (0 to 50 and mean of 16.2±19.9 SFCs/105 cells). Restimulation of CTLs with individual STEAP-1 peptides demonstrated donor-dependent reactivity with one to four of the peptides, while neither one emerged as immunodominant. The CTLs specifically lysed STEAP-1 peptide pulsed target cells (52.9±4.3% at an effector-to-target ratio of 20:1), but not cells pulsed with control peptides (0.0±3.8%). In contrast, STEAP-1 specific CTLs failed to functionally interact with the HLA-A2+/STEAP-1+ Ewing sarcoma cell lines as measured by cytolysis and cytokine secretion, even after upregulation of MHC class I molecules by pretreatment with IFN-γ. Thus, while the induction of STEAP-1 peptide-specific CTLs is feasible, these CTLs do not efficiently recognize endogenously expressed antigen on Ewing sarcoma cells. Higher-avidity CTLs are likely needed for exploiting STEAP-1 as a target for adoptive immunotherapy in this disease. To define the critical requirements for recognition and lysis of STEAP-1 expressing target cells in an autologous setting, we are currently exploring activated autologous γδ T-APCs expressing full-length STEAP-1 protein by retroviral gene transfer as targets and stimulator cells for STEAP-1 specific CTLs. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1927.

Abstract Abstract 1207 Targeted pharmacologic therapy with tyrosine kinase inhibitors (TKIs) has become the first-line treatment for patients with CML. However, BCR-ABL+ stem cells resist elimination by continuous TKI treatment in most patients. By contrast, graft-versus-leukemia effects during allogeneic hematopoietic stem cell transplantation can eradicate the disease, suggesting an important role of the immune system in this disease. Besides cytotoxic T cells, natural killer (NK) cells may be involved in immune control of CML. Here, we explored numbers and functionality of NK cells in CML patients and in a transgenic inducible BCR-ABL mouse model. In 18 patients with newly diagnosed chronic-phase CML, the relative proportions of peripheral blood CD56+CD3- NK cells within the lymphocyte gate were significantly reduced compared to age-matched healthy controls (7.0 ± 5.8% versus 13.1 ± 5.1, p=0.005) and did not recover to normal levels during imatinib-induced remission (9.2 ± 5.9%, p=0.024, follow-up 10–59 months). Functional experiments showed reduced in vitro expansion of CML NK cells at diagnosis in response to stimulation with 4-1BBL/mbIL-15 transduced K562 cells (23.5 ± 14.46 fold vs 41.2 ± 7.2 fold, p=0.013) and under imatinib treatment (31.5 ± 10.5 fold, p=0.03), and a reduced degranulation response to K562 target cells by CD107 upregulation (2.8 ± 2.7% at diagnosis and 9.0 ± 13.2% under treatment, vs. 19.1 ± 8.0% in controls, p=0.003 and p=0.045, respectively). To investigate, whether the defective NK-cell compartment in CML is a consequence of the characteristic BCR-ABL-induced myeloproliferation, we addressed the quantity and functionality of NK cells in a double transgenic mouse model of human CML. Consistent with the results in human CML, the relative proportions of NK1.1+ NK cells among total splenic lymphocytes were significantly reduced in BCR-ABL induced mice (6.4 ± 3.5% vs. 14.7 ± 1.8%, p=0.005). Moreover, compared to NK cells isolated and expanded from BCR-ABL-non-induced control mice, the degranulation response of splenic NK cells from BCR-ABL+ mice to YAC-1/NIH-3T3 cells was significantly decreased (25.7 ± 1.6% vs 42.4 ± 5.6%, p=0.002), and analogous results were obtained with NK cells expanded from bone marrow of these mice (7.7 ± 4.9% vs. 25.0 ± 7.1%, p=0.033). These results suggest both quantitative and qualitative defects within the NK cell compartment in CML. Further work will aim at identifying the underlying mechanisms of the NK cell deficiency in CML, and the development of strategies to utilize NK cells for immunotherapy of CML. Disclosures: Koschmieder: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees.

Primary metastatic Ewing sarcoma involving the bone and/or bone marrow has remained largely incurable. We propose to use cytotoxic T cells (CTLs) specific for STEAP-1, a surface protein aberrantly expressed in Ewing sarcoma, as therapeutic tools to eradicate minimal residual disease and prevent tumor dissemination and relapse.