M9657 Is a Bispecific Tumor-Targeted Anti-CD137 Agonist That Induces MSLN-Dependent Antitumor Immunity without Liver Inflammation.

The costimulatory receptor CD137 (also known as TNFRSF9 or 4-1BB) sustains effective cytotoxic T-cell responses. Agonistic anti-CD137 cancer immunotherapies are being investigated in clinical trials. Development of the first-generation CD137-agonist monotherapies utomilumab and urelumab was unsuccessful due to low antitumor efficacy mediated by the epitope recognized on CD137 or hepatotoxicity mediated by Fcγ receptors (FcγR) ligand-dependent CD137 activation, respectively. M9657 was engineered as a tetravalent bispecific antibody (mAb2) in a human IgG1 backbone with LALA mutations to reduce binding to FCγRs. Here, we report that M9657 selectively binds to mesothelin (MSLN) and CD137 with similar affinity in humans and cynomolgus monkeys. In a cellular functional assay, M9657 enhanced CD8+ T cell-mediated cytotoxicity and cytokine release in the presence of tumor cells, which was dependent on both MSLN expression and T-cell receptor/CD3 activation. Both FS122m, a murine surrogate with the same protein structure as M9657, and chimeric M9657, a modified M9657 antibody with the Fab portion replaced with an anti-murine MSLN motif, demonstrated in vivo antitumor efficacy against various tumors in wild-type and human CD137 knock-in mice, and this was accompanied by activated CD8+ T-cell infiltration in the tumor microenvironment. The antitumor immunity of M9657 and FS122m depended on MSLN expression density and the mAb2 structure. Compared with 3H3, a murine surrogate of urelumab, FS122m and chimeric M9657 displayed significantly lower on-target/off-tumor toxicity. Taken together, M9657 exhibits a promising profile for development as a tumor-targeting immune agonist with potent anticancer activity without systemic immune activation and associated hepatotoxicity.

p70S6K/Akt dual inhibitor DIACC3010 is efficacious in preclinical models of gastric cancer alone and in combination with trastuzumab.

The PI3K-Akt-mTOR (PAM) pathway is implicated in tumor progression in many tumor types, including metastatic gastric cancer (GC). The initial promise of PAM inhibitors has been unrealized in the clinic, presumably due, in part, to the up-regulation of Akt signaling that occurs when the pathway is inhibited. Here we present that DIACC3010 (formerly M2698), an inhibitor of two nodes in the PAM pathway, p70S6K and Akt 1/3, blocks the pathway in in vitro and in vivo preclinical models of GC while providing a mechanism that inhibits signaling from subsequent Akt up-regulation. Utilizing GC cell lines and xenograft models, we identified potential markers of DIACC3010-sensitivity in Her2-negative tumors, i.e., PIK3CA mutations, low basal pERK, and a group of differentially expressed genes (DEGs). The combination of DIACC3010 and trastuzumab was evaluated in Her2-positive cell lines and models. Potential biomarkers for the synergistic efficacy of the combination of DIACC3010 + trastuzumab also included DEGs as well as a lack of up-regulation of pERK. Of 27 GC patient-derived xenograft (PDX) models tested in BALB/c nu/nu mice, 59% were sensitive to DIACC3010 + trastuzumab. Of the 21 HER2-negative PDX models, DIACC3010 significantly inhibited the growth of 38%. Altogether, these results provide a path forward to validate the potential biomarkers of DIACC3010 sensitivity in GC and support clinical evaluation of DIACC3010 monotherapy and combination with trastuzumab in patients with HER2- negative and positive advanced GCs, respectively.

Colocalized targeting of TGF-ß and PD-L1 by bintrafusp alfa elicits distinct antitumor responses.

BACKGROUND: Bintrafusp alfa (BA) is a bifunctional fusion protein designed for colocalized, simultaneous inhibition of two immunosuppressive pathways, transforming growth factor-ß (TGF-ß) and programmed death-ligand 1 (PD-L1), within the tumor microenvironment (TME). We hypothesized that targeting PD-L1 to the tumor by BA colocalizes the TGF-ß trap (TGF-ßRII) to the TME, enabling it to sequester TGF-ß in the tumor more effectively than systemic TGF-ß blockade, thereby enhancing antitumor activity. METHODS: Multiple technologies were used to characterize the TGF-ß trap binding avidity. BA versus combinations of anti-PD-L1 and TGF-ß trap or the pan-TGF-ß antibody fresolimumab were compared in proliferation and two-way mixed lymphocyte reaction assays. Immunophenotyping of tumor-infiltrating lymphocytes (TILs) and RNA sequencing (RNAseq) analysis assessing stromal and immune landscape following BA or the combination therapy were performed in MC38 tumors. TGF-ß and PD-L1 co-expression and their associated gene signatures in MC38 tumors and human lung carcinoma tissue were studied with single-cell RNAseq (scRNAseq) and immunostaining. BA-induced internalization, degradation, and depletion of TGF-ß were investigated in vitro. RESULTS: BA and fresolimumab had comparable intrinsic binding to TGF-ß1, but there was an ~80× avidity-based increase in binding affinity with BA. BA inhibited cell proliferation in TGF-ß-dependent and PD-L1-expressing cells more potently than TGF-ß trap or fresolimumab. Compared with the combination of anti-PD-L1 and TGF-ß trap or fresolimumab, BA enhanced T cell activation in vitro and increased TILs in MC38 tumors, which correlated with efficacy. BA induced distinct gene expression in the TME compared with the combination therapy, including upregulation of immune-related gene signatures and reduced activities in TGF-ß-regulated pathways, such as epithelial-mesenchymal transition, extracellular matrix deposition, and fibrosis. Regulatory T cells, macrophages, immune cells of myeloid lineage, and fibroblasts were key PD-L1/TGF-ß1 co-expressing cells in the TME. scRNAseq analysis suggested BA modulation of the macrophage phenotype, which was confirmed by histological assessment. PD-L1/TGF-ß1 co-expression was also seen in human tumors. Finally, BA induced TGF-ß1 internalization and degradation in the lysosomes. CONCLUSION: BA more effectively blocks TGF-ß by targeting TGF-ß trap to the tumor via PD-L1 binding. Such colocalized targeting elicits distinct and superior antitumor responses relative to single agent combination therapy.

NHS-IL12 and bintrafusp alfa combination therapy enhances antitumor activity in preclinical cancer models.

Combinatorial immunotherapy approaches are emerging as viable cancer therapeutic strategies for improving patient responses and outcomes. This study investigated whether two such immunotherapies, with complementary mechanisms of action, could enhance antitumor activity in murine tumor models. The immunocytokine NHS-IL12, and surrogate NHS-muIL12, are designed to deliver IL-12 and muIL-12, respectively, to the tumor microenvironment (TME) to activate NK cells and CD8+ T cells and increase their cytotoxic functions. Bintrafusp alfa (BA) is a bifunctional fusion protein composed of the extracellular domains of the TGF-ß receptor II to function as a TGF-ß "trap" fused to a human IgG1 antibody blocking PD-L1. With this dual-targeting strategy, BA enhances efficacy over that of monotherapies in preclinical studies. In this study, NHS-muIL12 and BA combination therapy enhanced antitumor activity, prolonged survival, and induced tumor-specific antitumor immunity. This combination therapy increased tumor-specific CD8+ T cells and induced immune profiles, consistent with the activation of both adaptive and innate immune systems. In addition, BA reduced lung metastasis in the 4T1 model. Collectively, these findings could support clinical trials designed to investigate NHS-IL12 and BA combination therapy for patients with advanced solid tumors.

Simultaneous targeting of TGF-ß/PD-L1 synergizes with radiotherapy by reprogramming the tumor microenvironment to overcome immune evasion.

Localized radiotherapy (RT) induces an immunogenic antitumor response that is in part counterbalanced by activation of immune evasive and tissue remodeling processes, e.g., via upregulation of programmed cell death-ligand 1 (PD-L1) and transforming growth factor ß (TGF-ß). We report that a bifunctional fusion protein that simultaneously inhibits TGF-ß and PD-L1, bintrafusp alfa (BA), effectively synergizes with radiotherapy, leading to superior survival in multiple therapy-resistant murine tumor models with poor immune infiltration. The BA + RT (BART) combination increases tumor-infiltrating leukocytes, reprograms the tumor microenvironment, and attenuates RT-induced fibrosis, leading to reconstitution of tumor immunity and regression of spontaneous lung metastases. Consistently, the beneficial effects of BART are in part reversed by depletion of cytotoxic CD8+ T cells. Intriguingly, targeting of the TGF-ß trap to PD-L1+ endothelium and the M2/lipofibroblast-like cell compartment by BA attenuated late-stage RT-induced lung fibrosis. Together, the results suggest that the BART combination has the potential to eradicate therapy-resistant tumors while sparing normal tissue, further supporting its clinical translation.

Identification and characterization of M6903, an antagonistic anti-TIM-3 monoclonal antibody.

T cell immunoglobulin and mucin domain-3 (TIM-3) is an immune checkpoint that regulates normal immune responses but can be exploited by tumor cells to evade immune surveillance. TIM-3 is primarily expressed on immune cells, particularly on dysfunctional and exhausted T cells, and engagement of TIM-3 with its ligands promotes TIM-3-mediated T cell inhibition. Antagonistic ligand-blocking anti-TIM-3 antibodies have the potential to abrogate T cell inhibition, activate antigen-specific T cells, and enhance anti-tumor immunity. Here we describe M6903, a fully human anti-TIM-3 antibody without effector function and with high affinity and selectivity to TIM-3. We demonstrate that M6903 blocks the binding of TIM-3 to three of its ligands, phosphatidylserine (PtdSer), carcinoembryonic antigen cell adhesion-related molecule 1 (CEACAM1), and galectin 9 (Gal-9). These results are supported by an atomic resolution crystal structure and functional assays, which demonstrate that M6903 monotherapy enhanced T cell activation. This activation was further enhanced by the combination of M6903 with bintrafusp alfa, a bifunctional fusion protein that simultaneously blocks the transforming growth factor-ß (TGF-ß) and programmed death ligand 1 (PD-L1) pathways. M6903 and bintrafusp alfa combination therapy also enhanced anti-tumor efficacy in huTIM-3 knock-in mice, relative to either monotherapy. These in vitro and in vivo data, along with favorable pharmacokinetics in marmoset monkeys, suggest that M6903 as a monotherapy warrants further pre-clinical assessment and that M6903 and bintrafusp alfa may be a promising combination therapy in the clinic.

Correction to: 33rd Annual Meeting & Pre-Conference Programs of the Society for Immunotherapy of Cancer (SITC 2018).

After publication of this supplement [1, 2], it was brought to our attention that due to an error authors were missing in the following abstracts. This has now been included in this correction.

The proportion of core species in a community varies with spatial scale and environmental heterogeneity.

Ecological communities are composed of a combination of core species that maintain local viable populations and transient species that occur infrequently due to dispersal from surrounding regions. Preliminary work indicates that while core and transient species are both commonly observed in community surveys of a wide range of taxonomic groups, their relative prevalence varies substantially from one community to another depending upon the spatial scale at which the community was characterized and its environmental context. We used a geographically extensive dataset of 968 bird community time series to quantitatively describe how the proportion of core species in a community varies with spatial scale and environmental heterogeneity. We found that the proportion of core species in an assemblage increased with spatial scale in a positive decelerating fashion with a concomitant decrease in the proportion of transient species. Variation in the shape of this scaling relationship between sites was related to regional environmental heterogeneity, with lower proportions of core species at a given scale associated with high environmental heterogeneity. Understanding this influence of scale and environmental heterogeneity on the proportion of core species may help resolve discrepancies between studies of biotic interactions, resource availability, and mass effects conducted at different scales, because the importance of these and other ecological processes are expected to differ substantially between core and transient species.

Enhanced preclinical antitumor activity of M7824, a bifunctional fusion protein simultaneously targeting PD-L1 and TGF-ß.

Antibodies targeting immune checkpoints are emerging as potent and viable cancer therapies, but not all patients respond to these as single agents. Concurrently targeting additional immunosuppressive pathways is a promising approach to enhance immune checkpoint blockade, and bifunctional molecules designed to target two pathways simultaneously may provide a strategic advantage over the combination of two single agents. M7824 (MSB0011359C) is a bifunctional fusion protein composed of a monoclonal antibody against programmed death ligand 1 (PD-L1) fused to the extracellular domain of human transforming growth factor-ß (TGF-ß) receptor II, which functions as a "trap" for all three TGF-ß isoforms. We demonstrate that M7824 efficiently, specifically, and simultaneously binds PD-L1 and TGF-ß. In syngeneic mouse models, M7824 suppressed tumor growth and metastasis more effectively than treatment with either an anti-PD-L1 antibody or TGF-ß trap alone; furthermore, M7824 extended survival and conferred long-term protective antitumor immunity. Mechanistically, the dual anti-immunosuppressive function of M7824 resulted in activation of both the innate and adaptive immune systems, which contributed to M7824's antitumor activity. Finally, M7824 was an effective combination partner for radiotherapy or chemotherapy in mouse models. Collectively, our preclinical data demonstrate that simultaneous blockade of the PD-L1 and TGF-ß pathways by M7824 elicits potent and superior antitumor activity relative to monotherapies.

Measuring the Effects of an Animal-Assisted Intervention for Pediatric Oncology Patients and Their Parents: A Multisite Randomized Controlled Trial [Formula: see text].

OBJECTIVE: This multicenter, parallel-group, randomized trial examined the effects of an animal-assisted intervention on the stress, anxiety, and health-related quality of life for children diagnosed with cancer and their parents. METHOD: Newly diagnosed patients, aged 3 to 17 years (n = 106), were randomized to receive either standard care plus regular visits from a therapy dog (intervention group), or standard care only (control group). Data were collected at set points over 4 months of the child's treatment. Measures included the State-Trait Anxiety Inventory™, Pediatric Quality of Life Inventory, Pediatric Inventory for Parents, and child blood pressure and heart rate. All instruments were completed by the child and/or his/her parent(s). RESULTS: Children in both groups experienced a significant reduction in state anxiety ( P < .001). Parents in the intervention group showed significantly decreased parenting stress ( P = .008), with no changes in stress among parents in the control group. However, no significant differences between groups over time on any measures were observed. CONCLUSIONS: Animal-assisted interventions may provide certain benefits for parents and families during the initial stages of pediatric cancer treatment.

Laminin and Matrix metalloproteinase 11 regulate Fibronectin levels in the zebrafish myotendinous junction.

BACKGROUND: Remodeling of the extracellular matrix (ECM) regulates cell adhesion as well as signaling between cells and their microenvironment. Despite the importance of tightly regulated ECM remodeling for normal muscle development and function, mechanisms underlying ECM remodeling in vivo remain elusive. One excellent paradigm in which to study ECM remodeling in vivo is morphogenesis of the myotendinous junction (MTJ) during zebrafish skeletal muscle development. During MTJ development, there are dramatic shifts in the primary components comprising the MTJ matrix. One such shift involves the replacement of Fibronectin (Fn)-rich matrix, which is essential for both somite and early muscle development, with laminin-rich matrix essential for normal function of the myotome. Here, we investigate the mechanism underlying this transition. RESULTS: We show that laminin polymerization indirectly promotes Fn downregulation at the MTJ, via a matrix metalloproteinase 11 (Mmp11)-dependent mechanism. Laminin deposition and organization is required for localization of Mmp11 to the MTJ, where Mmp11 is both necessary and sufficient for Fn downregulation in vivo. Furthermore, reduction of residual Mmp11 in laminin mutants promotes a Fn-rich MTJ that partially rescues skeletal muscle architecture. CONCLUSIONS: These results identify a mechanism for Fn downregulation at the MTJ, highlight crosstalk between laminin and Fn, and identify a new in vivo function for Mmp11. Taken together, our data demonstrate a novel signaling pathway mediating Fn downregulation. Our data revealing new regulatory mechanisms that guide ECM remodeling during morphogenesis in vivo may inform pathological conditions in which Fn is dysregulated.

The BRAF(V600E) inhibitor, PLX4032, increases type I collagen synthesis in melanoma cells.

Vertical growth phase (VGP) melanoma is frequently metastatic, a process mediated by changes in gene expression, which are directed by signal transduction pathways in the tumor cells. A prominent signaling pathway is the Ras-Raf-Mek-Erk MAPK pathway, which increases expression of genes that promote melanoma progression. Many melanomas harbor a mutation in this pathway, BRAF(V600E), which constitutively activates MAPK signaling and expression of downstream target genes that facilitate tumor progression. In BRAF(V600E) melanoma, the small molecule inhibitor, vemurafenib (PLX4032), has revolutionized therapy for melanoma by inducing rapid tumor regression. This compound down-regulates the expression of many genes. However, in this study, we document that blocking the Ras-Raf-Mek-Erk MAPK pathway, either with an ERK (PLX4032) or a MEK (U1026) signaling inhibitor, in BRAF(V600E) human and murine melanoma cell lines increases collagen synthesis in vitro and collagen deposition in vivo. Since TGFß signaling is a major mediator of collagen synthesis, we examined whether blocking TGFß signaling with a small molecule inhibitor would block this increase in collagen. However, there was minimal reduction in collagen synthesis in response to blocking TGFß signaling, suggesting additional mechanism(s), which may include activation of the p38 MAPK pathway. Presently, it is unclear whether this increased collagen synthesis and deposition in melanomas represent a therapeutic benefit or an unwanted "off target" effect of inhibiting the Ras-Raf-Erk-Mek pathway.

CXCR3 signaling in BRAFWT melanoma increases IL-8 expression and tumorigenicity.

UNLABELLED: Patients with early stage, radial growth phase (RGP) melanoma have a 97% survival rate; however, when the melanoma progresses to the invasive vertical growth phase (VGP), survival rates decrease to 15%. The targets of many clinical trials are the known genetic and molecular mechanisms involved in melanoma progression, with the most common oncogenic mutation being the BRAFV600E. However, less than half of melanomas harbor this mutation, and consequently, do not respond to the current BRAF targeted treatments. It is therefore critical to elucidate alternative mechanisms regulating melanoma progression. Increased expression of the chemokine receptor, CXCR3, on melanoma cells is correlated with increased metastasis and poor patient outcomes, suggesting a role for CXCR3 in the RGP to VGP transition. We found that endogenous CXCR3 can be induced in two RGP cell lines, BOWES (BRAFWT) and WM35 (BRAFV600E), with in vitro environmental stress and nutrient deprivation. Signaling via induced endogenous CXCR3 is linked with IL-8 expression in BOWES cells. Ectopic overexpression of CXCR3 in BOWES cells leads to increased ligand-mediated phERK, cellular migration, and IL-8 expression in vitro, and to increased tumorigenesis and lymph node metastasis in vivo. Our results demonstrate that, in BRAFWT melanomas, CXCR3 signaling mediates significant increases in IL-8 expression, suggesting that CXCR3 expression and signaling may represent a transformative event that drives the progression of BRAFWT melanomas. IMPLICATIONS: Expression of CXCR3 on BRAFWT melanoma cells may be a mediator of melanoma progression.

Epigenetic states of cells of origin and tumor evolution drive tumor-initiating cell phenotype and tumor heterogeneity.

A central confounding factor in the development of targeted therapies is tumor cell heterogeneity, particularly in tumor-initiating cells (TIC), within clinically identical tumors. Here, we show how activation of the Sonic Hedgehog (SHH) pathway in neural stem and progenitor cells creates a foundation for tumor cell evolution to heterogeneous states that are histologically indistinguishable but molecularly distinct. In spontaneous medulloblastomas that arise in Patched (Ptch)(+/-) mice, we identified three distinct tumor subtypes. Through cell type-specific activation of the SHH pathway in vivo, we determined that different cells of origin evolved in unique ways to generate these subtypes. Moreover, TICs in each subtype had distinct molecular and cellular phenotypes. At the bulk tumor level, the three tumor subtypes could be distinguished by a 465-gene signature and by differential activation levels of the ERK and AKT pathways. Notably, TICs from different subtypes were differentially sensitive to SHH or AKT pathway inhibitors, highlighting new mechanisms of resistance to targeted therapies. In summary, our results show how evolutionary processes act on distinct cells of origin to contribute to tumoral heterogeneity, at both bulk tumor and TIC levels.

Differential mechanisms of tumor progression in clones from a single heterogeneous human melanoma.

We used vertical growth phase (VGP) human VMM5 melanoma cells to ask whether the tumor microenvironment could induce matrix metalloproteinase-1 (MMP-1) in vivo, and whether this induction correlated with metastasis. We isolated two clones from parental VMM5 cells: a low MMP-1 producing clone (C4) and high producing clone (C9). When these clones were injected orthotopically (intradermally) into nude mice, both were equally tumorigenic and produced equivalent and abundant amounts of MMP-1. However, the tumors from the C4 clones displayed different growth kinetics and distinct profiles of gene expression from the C9 population. The C4 tumors, which had low MMP-1 levels in vitro, appeared to rely on growth factors and cytokines in the microenvironment to increase MMP-1 expression in vivo, while MMP-1 levels remained constant in the C9 tumors. C9 cells, but not C4 cells, grew as spheres in culture and expressed higher levels of JARID 1B, a marker associated with melanoma initiating cells. We conclude that VMM5 melanoma cells exhibit striking intra-tumor heterogeneity, and that the tumorigenicity of these clones is driven by different molecular pathways. Our data suggest that there are multiple mechanisms for melanoma progression within a tumor, which may require different therapeutic strategies.