Supplementary Figures S1-S4. Supplementary Figure S1. Development of anti-antibodies following Surek treatment; Supplementary Figure S2. Activation and proliferation of T cells after treatment with trAb Surek alone; Supplementary Figure S3. Weight loss of mice after unspecific immune activation induced by CD3 binding of Surek; Supplementary Figure S4. Cytokine milieu induced by Surek s.c.

Table S1. Statistical comparisons between neurofibroma (NF) and 3 cohorts of human neuroblastoma (NB) tissues. Table S2. Authentication of human neuroblastoma cell lines Table S3. Antibodies and dyes used for flow cytometry. Supplemental Figure Legends

Figure S1. A) Expression levels of granulocytic marker CD66b, regulatory T cell marker FoxP3 or dendritic cell marker CD83, CD11c Figure S2. A) CD34+ hematopoietic stem cells without cytokine activation lack the capacity to stimulate allogeneic T cells. Figure S3. Analysis of A) M-CSF or B) 27 other soluble factors produced by the three human neuroblastoma cell lines (SK-N-BE(2), SK-N-AS or SK-N-FI). Figure S4. Expression of PD-L1 on A) tumor-educated monocytes Figure S5. Bone marrow cells extracted from naïve mice were cultured in medium, recombinant murine M-CSF (20 ng/ml) or neuroblastoma tumor-conditioned medium (TCM). Figure S6. Mice were sacrificed 10 days after spontaneous tumors were palpable Figure S7. A) BLZ945 treatment scheme in the TH-MYCN mice bearing established spontaneous tumors was summarized. Figure S8. A) Treatment scheme combining PD-1/PD-L1 blockade and CSF-1R inhibition was illustrated.

Childhood medulloblastoma and high-risk neuroblastoma frequently present with segmental gain of chromosome 17q corresponding to aggressive tumors and poor patient prognosis. Located within the 17q-gained chromosomal segments is PPM1D at chromosome 17q23.2. PPM1D encodes a serine/threonine phosphatase, WIP1, that is a negative regulator of p53 activity as well as key proteins involved in cell cycle control, DNA repair and apoptosis. Here, we show that the level of PPM1D expression correlates with chromosome 17q gain in medulloblastoma and neuroblastoma cells, and both medulloblastoma and neuroblastoma cells are highly dependent on PPM1D expression for survival. Comparison of different inhibitors of WIP1 showed that SL-176 was the most potent compound inhibiting medulloblastoma and neuroblastoma growth and had similar or more potent effects on cell survival than the MDM2 inhibitor Nutlin-3 or the p53 activator RITA. SL-176 monotherapy significantly suppressed the growth of established medulloblastoma and neuroblastoma xenografts in nude mice. These results suggest that the development of clinically applicable compounds inhibiting the activity of WIP1 is of importance since PPM1D activating mutations, genetic gain or amplifications and/or overexpression of WIP1 are frequently detected in several different cancers.

SUMMARYMajority of cancers harbor alterations of the tumor suppressorTP53. However, childhood cancers, including unfavorable neuroblastoma, often lackTP53mutations despite frequent loss of p53 function, suggesting alternative p53 inactivating mechanisms.Here we show that p53-regulatingPPM1Dat chromosome 17q22.3 is linked to aggressive tumors and poor prognosis in neuroblastoma. We identified that WIP1-phosphatase encoded byPPM1D, is activated by frequent segmental 17q-gain further accumulated during clonal evolution, gene-amplifications, gene-fusions or gain-of-function somatic and germline mutations. Pharmacological and genetic manipulation established WIP1 as a druggable target in neuroblastoma. Genome-scale CRISPR-Cas9 screening demonstratedPPM1Dgenetic dependency inTP53wild-type neuroblastoma cell lines, and shRNAPPM1Dknockdown significantly delayed in vivo tumor formation. Establishing a transgenic mouse model overexpressingPPM1Dshowed that these mice develop cancers phenotypically and genetically similar to tumors arising in mice with dysfunctional p53 when subjected to low-dose irradiation. Tumors include T-cell lymphomas harboringNotch1-mutations,Pten-deletions and p53-accumulation, adenocarcinomas andPHOX2B-expressing neuroblastomas establishingPPM1Das abona fideoncogene in wtTP53 cancer and childhood neuroblastoma. Pharmacological inhibition of WIP1 suppressed the growth of neural tumors in nude mice proposing WIP1 as a therapeutic target in neural childhood tumors.

Despite recent progress in diagnosis and treatment, survival for children with high-risk metastatic neuroblastoma is still poor. Prostaglandin E2 (PGE2)-driven inflammation promotes tumor growth, immune suppression, angiogenesis and resistance to established cancer therapies. In neuroblastoma, cancer-associated fibroblasts (CAFs) residing in the tumor microenvironment are the primary source of PGE2. However, clinical targeting of PGE2 with current non-steroidal anti-inflammatory drugs or cyclooxygenase inhibitors has been limited due to risk of adverse side effects. By specifically targeting microsomal prostaglandin E synthase-1 (mPGES-1) activity with a small molecule inhibitor we could block CAF-derived PGE2 production leading to reduced tumor growth, impaired angiogenesis, inhibited CAF migration and infiltration, reduced tumor cell proliferation and a favorable shift in the M1/M2 macrophage ratio. In this study, we provide proof-of-principle of the benefits of targeting mPGES-1 in neuroblastoma, applicable to a wide variety of tumors. This non-toxic single drug treatment targeting infiltrating stromal cells opens up for combination treatment options with established cancer therapies.

Targeting CD38 in multiple myeloma has resulted in outstanding responses. CD38 is widely expressed on myeloma cells and other hematological malignancies. Not much is known about its expression on solid tumors and its role in the immune system. We have analysed a range of solid tumors for CD38 expression and distribution. To optimally target CD38, we have generated a novel antibody that is depleting CD38-high expressing cells, but also has immune modulatory properties. To dissect CD38 expression in solid tumors we exploited mRNA expression libraries, performed immune histochemistry (IHC) on tumor sections, and flow cytometry on patient tumor material. Bioinformatic analysis of the immune cell atlas revealed varying CD38 expression among all cancers analysed, and CD38 expression could be correlated with immune markers, e.g. Foxp3, PD-1/L1. IHC and flow cytometry confirmed CD38 expression across common cancer types, mostly confined to infiltrating lymphocyte and myeloid subsets. Expression on tumor cells was patient dependent. CD38 expression on immune cells was heterogenous and found on NK cells, T cells, suppressive myeloid cells, as well as regulatory T and B cells. Of note, high expression of CD38 was found to be correlated to a subset of exhausted T cells co-expressing PD-1 and other exhaustion markers. To target CD38 in solid tumors, we have screened a panel of CD38-binding antibodies. All antibodies have the potential to deplete CD38 positive tumor cells in vitro and in vivo. Additionally, their ability to influence effector T and NK cell activation has been evaluated. Among a panel of antibodies binding to distinct epitopes of CD38 and exerting unique functional properties, we have identified a fully human antibody, with strong capacity to deplete CD38-high cells in vitro and in vivo by varying killing mechanisms. This antibody was found to increase TCR-mediated signaling and proinflammatory cytokine secretion by human T cells, and further to enhance NK cell activation in vitro. Low dose injection to non-human primates resulted in increased expression of activation markers on both CD4 and CD8 T cells, while no T cell depletion was observed. Other selected antibodies comprise distinct modalities including strong to weak agonistic activity, differential killing properties, modulation of CD38 enzymatic activity, and offer a selection of candidates applicable for different treatment settings. In summary, we found heterogenous expression of CD38 in solid tumors, mostly confined to immune subsets. To target CD38, we present a potent anti-CD38 antibody with depleting effects on CD38-expressing cancer cells, as well as suppressive immune cells, and the capacity to increase the function of immune effector cells. This dual activity might allow to fully exploit the therapeutic potential of targeting CD38, not only in hematological malignancies but also in solid tumors. Citation Format: Nina Eissler, Simone Filosto, Jake Y. Henry, Michael F. Maguire, Kristina Witt, Andreas Lundqvist, Teresa Marafioti, Pascal Merchiers, Kevin Moulder, Beatriz Goyenechea, Haw Lu, Camilla Fairbairn, Sarah Windler, JD Aurellano, Omar Duramad, Dominic Smethurst, Sergio A. Quezada, Anne Goubier. A best in class anti-CD38 antibody with antitumor and immune-modulatory properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3812.

Abstract Background: High-risk neuroblastomas present a tumor promoting microenvironment with infiltrating cancer associated fibroblasts (CAFs) expressing the mPGES-1 enzyme, essential for prostaglandin E2 (PGE2) synthesis regulating tumor inflammation and immune suppression, angiogenesis, genetic instability, tumor progression and therapy resistance. We investigated the impact of novel therapy targeting the COX/mPGES-1/PGE2 pathway. Methods: Human neuroblastomas were investigated for immunosuppressive microenvironment and expression of the COX/mPGES-1/PGE2/EP-receptor pathway. High-risk in vivo models, human 11q-deleted xenografts and transgenic MYCN-driven tumors, were treated with a novel specific mPGES-1 inhibitor. Tumor-fibroblast co-cultures examined cell migration. Inflammatory lipid mediators were analyzed by LC-MS/MS. Tumor tissues were analyzed by immunohistochemistry, immunofluorescence and FACS. Results: Tumor microenvironment in human high-risk neuroblastomas and both 11q-deleted xenografts and MYCN-driven transgenic mice displayed mPGES-1 expression in PDGFRb+ cancer associated fibroblasts. MPGES-1 expression correlated with high-risk neuroblastoma prognosis and infiltration of tumor-promoting macrophages with M2-polarization markers CD163 and CD206. The inflammatory regulator STAT3 was active in mPGES-1 expressing CAFs. Expression of the inflammatory COX/mPGES-1/PGE2/EP-receptor pathway in experimental tumors resembled high-risk primary human neuroblastomas. Targeting mPGES-1 with a novel compound decreased PGE2, induced M1 polarization of macrophages, decreased cancer associated fibroblasts and reduced angiogenesis significantly in treated tumors. Tumor development in the xenograft model was delayed and growth of established xenografts and transgenic tumors was significantly decreased by non-toxic treatment in vivo when compared to neuroblastoma tumors in untreated animals. Tumor cell stimulated CAF migration and infiltration was inhibited by targeting mPGES-1. Conclusions: Tumor-promoting inflammation and suppression of anti-tumor immunity in neuroblastoma is mediated through prostaglandin E2 and STAT3 expression in cancer associated fibroblasts in the tumor microenvironment. Early targeting of mPGES-1 may inhibit CAF infiltration and tumor development. This novel tumor treatment targeting mPGES-1 decreases inflammatory mediators, modulates tumor-promoting microenvironment and inhibits significantly aggressive tumor growth and progression. We conclude that treatment targeting non-malignant cells in the neuroblastoma microenvironment may constitute a novel clinical therapeutic approach. Citation Format: Anna Kock, Karin Larsson, Nina Eissler, Filip Bergqvist, Joan Rauf, Marina Korotkova, John-Inge Johnsen, Per-Johan Jakobsson, Per Kogner. Targeting tumor-promoting neuroblastoma microenvironment: inhibiton of tumor development and progression by targeting mPGES-1 expressed by cancer associated fibroblasts [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 687. doi:10.1158/1538-7445.AM2017-687

Abstract Background: The most common cytogenetic lesions in the embryonal neural tumors medulloblastoma (MB) and neuroblastoma (NB) affect chromosome 17, with 17q+ or isochromosome 17q, in approximately one-third of MB with these aberrations being a significant indicator of poor clinical outcome. Similarly, in NB gain of 17q is the most powerful genetic predictor of adverse clinical outcome. 17q+ correlates with poor survival in our population-based material where we found aberrations of chromosome 17 in 85% of primary neuroblastomas, specifically, gain of PPM1D/Wip1 at 17q23. Wip1 is a serine/threonine phosphatase encoded by the gene PPM1D, described as a gatekeeper in the Mdm2-p53 regulatory loop involved in genetic stability, inflammation and a potential oncogene contributing to carcinogenesis. Methods: Comparative genomic hybridization (CGH), immunostaining, mRNA arrays, qPCR, exome- and RNA-sequencing was used to examine PPM1D/Wip1 in neuroblastoma and medulloblastoma. Genetic and pharmacological inhibition was used to analyze the function of Wip1 in preclinical neuroblastoma and medulloblastoma models. Results: CGH-array analysis detected PPM1D/Wip1 extra copies in all tumors and cell lines containing 17q-gain. Expression arrays and immunostaining showed high expression of Wip1 in neuroblastoma corresponding to poor survival. RNA sequencing confirmed PPM1D-gain and revealed truncated isoforms with oncogenic potential. Exome-sequencing detected a mutation leading to constitutive PPM1D/Wip1 activation in an aggressive metastatic infant neuroblastoma. Wip1 knockdown experiments showed significant decrease of cell viability, proliferation and colony formation as well as substantial increase of DNA-damage response in neuroblastoma and medulloblastoma cells. Tumor neuroblastoma xenograft development was significantly delayed showing median tumor development (0.10 mL) to be more than doubled (median 15 days, vs. 33 days, p<0.001) after Wip1 downregulation compared to scrambled controls. A novel Wip1 inhibitor was highly potent in cytotoxic/cytostatic effect in neuroblastoma and medulloblastoma cell lines. Furthermore, this Wip1 inhibitor significantly inhibited growth of established human neuroblastomas and medulloblastomas in nude mice after treatment (P<0.01). Conclusions: Our results show that PPM1D/Wip1 is oncogenic in neuroblastoma and medulloblastoma development. We propose three different ways on how PPM1D/Wip1 is activated: due to chromosomal gain, alternative RNA-isoforms and/or DNA-mutation. PPM1D/Wip1 provides a novel therapeutic target in neuroblastoma and medulloblastoma. Citation Format: Jelena Milosevic, Nina Eissler, Diana Treis, Malin Wicktröm, Susanne Fransson, Baldur Sveinbjornsson, Ninib Baryawno, Subazini Kosalai, Chandrasekhar Kanduri, Kazuyasu Sakaguchi, Tommy Martinsson, John Inge Johnsen, Per Kogner. PPM1D/Wip1, promising new target in childhood cancers neuroblastoma and medulloblastoma [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 1945. doi:10.1158/1538-7445.AM2017-1945

Removal of immuno-suppression has been reported to enhance antitumor immunity primed by checkpoint inhibitors. Although PD-1 blockade failed to control tumor growth in a transgenic murine neuroblastoma model, concurrent inhibition of colony stimulating factor 1 receptor (CSF-1R) by BLZ945 reprogrammed suppressive myeloid cells and significantly enhanced therapeutic effects. Microarray analysis of tumor tissues identified a significant increase of T-cell infiltration guided by myeloid cell-derived chemokines CXCL9, 10, and 11. Blocking the responsible chemokine receptor CXCR3 hampered T-cell infiltration and reduced antitumor efficacy of the combination therapy. Multivariate analysis of 59 immune-cell parameters in tumors and spleens detected the correlation between PD-L1-expressing myeloid cells and tumor burden. In vitro, anti-PD-1 antibody Nivolumab in combination with BLZ945 increased the activation of primary human T and NK cells. Importantly, we revealed a previously uncharacterized pathway, in which T cells secreted M-CSF upon PD-1 blockade, leading to enhanced suppressive capacity of monocytes by upregulation of PD-L1 and purinergic enzymes. In multiple datasets of neuroblastoma patients, gene expression of CD73 correlated strongly with myeloid cell markers CD163 and CSF-1R in neuroblastoma tumors, and associated with worse survival in high-risk patients. Altogether, our data reveal the dual role of activated T cells on myeloid cell functions and provide a rationale for the combination therapy of anti-PD-1 antibody with CSF-1R inhibitor.

Combinatorial approaches of immunotherapy hold great promise for the treatment of malignant disease. Here, we examined the potential of combining an immune checkpoint inhibitor and trifunctional bispecific antibodies (trAbs) in a preclinical melanoma mouse model using surrogate antibodies of Ipilimumab and Catumaxomab, both of which have already been approved for clinical use. The specific binding arms of trAbs redirect T cells to tumor cells and trigger direct cytotoxicity, while the Fc region activates accessory cells eventually giving rise to a long-lasting immunologic memory. We show here that T cells redirected to tumor cells by trAbs strongly upregulate CTLA-4 expression in vitro and in vivo. This suggested that blocking of CTLA-4 in combination with trAb treatment enhances T-cell activation in a tumor-selective manner. However, when mice were challenged with melanoma cells and subsequently treated with antibodies, there was only a moderate beneficial effect of the combinatorial approach in vivo with regard to direct tumor destruction in comparison to trAb therapy alone. By contrast, a significantly improved vaccination effect was obtained by CTLA-4 blocking during trAb-dependent immunization. This resulted in enhanced rejection of melanoma cells given after pre-immunization. The improved immunologic memory induced by the combinatorial approach correlated with an increased humoral antitumor response as measured in the sera and an expansion of CD4+ memory T cells found in the spleens.

Abstract Purpose: Neuroblastoma is the most common extracranial solid cancer type in childhood, and high-risk patients have poor prognosis despite aggressive multimodal treatment. Neuroblastoma-driven inflammation contributes to the induction of suppressive myeloid cells that hamper efficient antitumor immune responses. Therefore, we sought to enhance antitumor immunity by removing immunosuppression mediated by myeloid cells. Experimental Design: The prognostic values of myeloid cells are demonstrated by analyzing genomic datasets of neuroblastoma patients. The impact of tumor-derived factors on myelopoiesis and local induction of suppressive myeloid cells is dissected by in vitro culture models using freshly isolated human CD34+ hematopoietic stem cells, primary human monocytes, and murine bone marrow cells. To test the therapeutic efficacy of BLZ945 as a monotherapy or in combination with checkpoint inhibitors, we used a transgenic murine model (TH-MYCN) that develops aggressive spontaneous neuroblastoma. Results: We report that infiltrating CSF-1R+ myeloid cells predict poor clinical outcome in patients with neuroblastoma. In vitro, neuroblastoma-derived factors interfere with early development of myeloid cells and enable suppressive functions on human monocytes through M-CSF/CSF-1R interaction. In a transgenic mouse model (TH-MYCN) resembling high-risk human neuroblastoma, antagonizing CSF-1R with a selective inhibitor (BLZ945) modulates the induction of human and murine suppressive myeloid cells and efficiently limit tumor progression. While checkpoint inhibitors are insufficient in controlling tumor growth, combining BLZ945 with PD-1/PD-L1 blocking antibodies results in superior tumor control. Conclusions: Our results demonstrate the essential role of CSF-1R signaling during the induction of suppressive myeloid cells and emphasize its clinical potential as an immunotherapy for human cancers. Clin Cancer Res; 22(15); 3849–59. ©2016 AACR.

Abstract Background: The most common cytogenetic lesions in the embryonal neural tumors medulloblastoma (MB) and neuroblastoma (NB) affect chromosome 17, with 17q+ or isochromosome 17q, in approximately one-third of MB with these aberrations being a significant indicator of poor clinical outcome. Similarly, in NB gain of 17q is the most powerful genetic predictor of adverse clinical outcome. 17q+ correlates with poor survival in our population-based material where we found aberrations of chromosome 17 in 85% of primary neuroblastomas, specifically, gain of PPM1D/Wip1 at 17q23. Wip1 is a serine/threonine phosphatase encoded by the gene PPM1D, described as a gatekeeper in the Mdm2-p53 regulatory loop involved in genetic stability, inflammation and a potential oncogene contributing to carcinogenesis. Methods: Comparative genomic hybridization (CGH) was used to examine PPM1D/Wip1 in neuroblastoma and medulloblastoma tumors and cell lines. Stable Wip1 knockdown SK-N-BE(2) cells were generated by shRNA transfections and tested in vivo in tumor xenografts. Pharmacological inhibition with the p53-mdm2 modulating inhibitors RITA, Nutlin-3 and a new PPM1D/Wip1 inhibitor, was used to evaluate the function of PPM1D/Wip1 in preclinical neuroblastoma and medulloblastoma models. Results: CGH-array analysis detected PPM1D/Wip1 extra copies in all tumors and cell lines containing 17q-gain. Tumor neuroblastoma xenograft development was significantly delayed showing median tumor development (0.10 mL) to be more than doubled (median 15 days, vs. 33 days, p<0.001) after Wip1 downregulation compared to scrambled controls. A novel Wip1 inhibitor was highly potent in cytotoxic/cytostatic effect in a variety of neuroblastoma and medulloblastoma cell lines. Furthermore, this Wip1 inhibitor significantly inhibited growth of established human neuroblastoma- and medulloblastoma tumors in nude mice after treatment (P<0.01). Conclusions: Our results show that PPM1D/Wip1 is oncogenic in neuroblastoma and medulloblastoma development and provides a novel therapeutic target in these two childhood cancers of the nervous system. More studies investigating the effects of PPM1D/Wip1 inhibition are needed to evaluate its clinical significance. Citation Format: Jelena Milosevic, Diana Treis, Malin Wickstrom, Susanne Fransson, Nina Eissler, Baldur Sveinbjörnsson, Ninib Baryawno, Keiji Tanino, Galina Selivanova, Kazuyasu Sakaguchi, Tommy Martinsson, John Inge Johnsen, Per Kogner. The PPM1D encoded phosphatase Wip1 is a novel oncogene and potential therapeutic target in neuroblastoma and medulloblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 503. doi:10.1158/1538-7445.AM2015-503

AbstractTrifunctional bispecific antibodies (trAb) are novel anticancer drugs that recruit and activate different types of immune effector cells at the targeted tumor. Thus, tumor cells are effectively eliminated and a long-lasting tumor-specific T-cell memory is induced. The trAb Ektomab is directed against human CD3 on T cells and the tumor-associated ganglioside GD2, which is an attractive target for immunotherapy of melanoma in humans. To optimize clinical applicability, we studied different application routes with respect to therapeutic efficacy and tolerability by using the surrogate trAb Surek (anti-GD2 × anti-murine CD3) and a murine melanoma engineered to express GD2. We show that subcutaneous injection of the trAb is superior to the intravenous delivery pathway, which is the standard application route for therapeutic antibodies. Despite lower plasma levels after subcutaneous administration, the same tumor-protective potential was observed in vivo compared with intravenous administration of Surek. However, subcutaneously delivered Surek showed better tolerability. This could be explained by a continuous release of the antibody leading to constant plasma levels and a delayed induction of proinflammatory cytokines. Importantly, the induction of counter-regulatory mechanisms was reduced after subcutaneous application. These findings are relevant for the clinical application of trifunctional bispecific antibodies and, possibly, also other immunoglobulin constructs. Mol Cancer Ther; 14(8); 1877–83. ©2015 AACR.

Meeting abstracts Novel developments in tumor-immunotherapy show promising results in cancer therapy and might provide new opportunities for neuroblastoma treatment. To define suitable immunological targets, it is crucial to understand the role of the immune system and the mechanisms underlying

Trifunctional bispecific antibodies (trAbs) used in tumor immunotherapy have the unique ability to recruit T cells toward antigens on the tumor cell surface and, moreover, to activate accessory cells through their immunoglobulin Fc region interacting with activating Fcγ receptors. This scenario gives rise to additional costimulatory signals required for T cell-mediated tumor cell destruction and induction of an immunologic memory. Here we show in an in vitro system that most effective trAb-dependent T-cell activation and tumor cell elimination are achieved in the presence of dendritic cells (DCs). On the basis of these findings, we devise a novel approach of cancer immunotherapy that combines the specific advantages of trAbs with those of DC-based vaccination. Simultaneous delivery of trAbs and in vitro differentiated DCs resulted in a markedly improved tumor rejection in a murine melanoma model compared with monotherapy.

A major goal of tumor immunotherapy is the induction of long-lasting systemic T-cell immunity. Bispecific antibodies (bsAbs) that lack the immunoglobulin Fc region confer T-cell-mediated killing of tumor cells but do not induce long-term memory. In contrast, trifunctional bsAbs comprise an appropriate Fc region and, therefore, not only recruit T cells but also accessory cells that bear activating Fcγ receptors (FcγR), providing additional T-cell-activating signals and securing presentation of tumor-derived antigens to T cells. In this study, we show that trifunctional bsAbs induce a polyvalent T-cell response and, therefore, a vaccination effect. Mice were treated with melanoma cells and with a trifunctional bsAb directed against the melanoma target antigen ganglioside GD2 in addition to murine CD3. The trifunctional bsAb activated dendritic cells and induced a systemic immune response that was not replicated by treatment with the F(ab')2-counterpart lacking the Fc region. Restimulation of spleen and lymph node cells in vitro yielded T-cell lines that specifically produced interferon-γ in response to tumor. In addition, trifunctional bsAb-induced T cells recognized various specific peptides derived from melanoma-associated antigens. Moreover, these polyvalent responses proved to be tumor-suppressive and could not be induced by the corresponding bsF(ab')2-fragment. Taken together, our findings provide preclinical proof of concept that trifunctional bsAbs can induce tumor-specific T cells with defined antigen specificity.

A major goal of tumor immunotherapy is the induction of long-lasting systemic T-cell immunity. Bispecific antibodies (bsAbs) that lack the immunoglobulin Fc region confer T–cell-mediated killing of tumor cells but do not induce long-termmemory. In contrast, trifunctional bsAbs comprise an appropriate Fc region and, therefore, not only recruit T cells but also accessory cells that bear activating Fcg receptors (FcgR), providing additional T–cell-activating signals and securing presentation of tumor-derived antigens to T cells. In this study, we show that trifunctional bsAbs induce a polyvalent T-cell response and, therefore, a vaccination effect.Micewere treated withmelanoma cells andwith a trifunctional bsAb directed against themelanoma target antigen ganglioside GD2 in addition to murine CD3. The trifunctional bsAb activated dendritic cells and induced a systemic immune response thatwas not replicated by treatmentwith the F(ab0)2-counterpart lacking the Fc region. Restimulation of spleen and lymph node cells in vitro yielded T-cell lines that specifically produced interferon-g in response to tumor. In addition, trifunctional bsAb-induced T cells recognized various specific peptides derived from melanoma-associated antigens. Moreover, these polyvalent responses proved to be tumor-suppressive and could not be induced by the corresponding bsF(ab0)2-fragment. Taken together, our findings provide preclinical proof of concept that trifunctional bsAbs can induce tumor-specific T cells with defined antigen specificity. Cancer Res; 72(16); 1–9. 2012 AACR.

Background: Trifunctional bispecific antibodies (trAb) are a special class of bispecific molecules recruiting and activating T cells and accessory immune cells simultaneously at the targeted tumor. The new trAb Ektomab that targets the melanoma-associated ganglioside antigen GD2 and the signaling molecule human CD3 (hCD3) on T cells demonstrated potent T-cell activation and tumor cell destruction in vitro. However, the relatively low affinity for the GD2 antigen raised the question of its therapeutic capability. To further evaluate its efficacy in vivo it was necessary to establish a mouse model.Methods: We generated the surrogate trAb Surek, which possesses the identical anti-GD2 binding arm as Ektomab, but targets mouse CD3 (mCD3) instead of hCD3, and evaluated its chemical and functional quality as a therapeutic antibody homologue. The therapeutic and immunizing potential of Surek was investigated using B78-D14, a B16 melanoma transfected with GD2 and GD3 synthases and showing strong GD2 surface expression. The induction of tumor-associated and autoreactive antibodies was evaluated.Results: Despite its low affinity of approximately 10(7) M-1 for GD2, Surek exerted efficient tumor cell destruction in vitro at an EC50 of 70ng/ml [0.47nM]. Furthermore, Surek showed strong therapeutic efficacy in a dose-dependent manner and is superior to the parental GD2 mono-specific antibody, while the use of a control trAb with irrelevant target specificity had no effect. The therapeutic activity of Surek was strictly dependent on CD4(+) and CD8(+) T cells, and cured mice developed a long-term memory response against a second challenge even with GD2-negative B16 melanoma cells. Moreover, tumor protection was associated with humoral immune responses dominated by IgG2a and IgG3 tumor-reactive antibodies indicating a Th1-biased immune response. Autoreactive antibodies against the GD2 target antigen were not induced.Conclusion: Our data suggest that Surek revealed strong tumor elimination and anti-tumor immunization capabilities. The results warrant further clinical development of the human therapeutic equivalent antibody Ektomab.