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1: JAG1 antibody characterization. 2: antibody inhibition of JAG1 mediated Notch signalling in tumor cell lines. 3: antibody inhibition of JAG1 signalling in vascular cells. 4: antibody inhibition of JAG1 signalling in HUVEC-HUVSMC co-culture. 5: effect of anti-Notch pathway treatment on MDA-MB231 2D growth and J1-65D titration and effect on 3D growth and gene expression. 6: J1-65D treatment of established OVCAR3 xenograft. 7: JAG1 antibodies cross-reactivity with rat Jag1. 8: anti-JAG1 treatment effect in a rat brain metastasis model.

Supplementary material and methods. Supplementary table 1: cell lines and growth conditions. Supplementary table 2: Notch ligands and control protein for plate coating. Supplementary table 3: qPCR primers. Supplementary table 4: flow cytometry antibodies. Supplementary table 5: ICC/IHC antibodies. Supplementary table 6: titration of Notch1-JAG1 binding inhibition by JAG1 antibodies. Supplementary table 7: amino acid sequence of JAG1 antibodies. Supplementary table 8: blood analysis on antibody-treated tumor-bearing rats.

The role of Notch signaling and its ligand JAGGED1 (JAG1) in tumor biology has been firmly established, making them appealing therapeutic targets for cancer treatment. Here, we report the development and characterization of human/rat-specific JAG1-neutralizing mAbs. Epitope mapping identified their binding to the Notch receptor interaction site within the JAG1 Delta/Serrate/Lag2 domain, where E228D substitution prevented effective binding to the murine Jag1 ortholog. These antibodies were able to specifically inhibit JAG1-Notch binding in vitro, downregulate Notch signaling in cancer cells, and block the heterotypic JAG1-mediated Notch signaling between endothelial and vascular smooth muscle cells. Functionally, in vitro treatment impaired three-dimensional growth of breast cancer cell spheroids, in association with a reduction in cancer stem cell number. In vivo testing showed variable effects on human xenograft growth when only tumor-expressed JAG1 was targeted (mouse models) but a more robust effect when stromal-expressed Jag1 was also targeted (rat MDA-MB-231 xenograft model). Importantly, treatment of established triple receptor-negative breast cancer brain metastasis in rats showed a significant reduction in neoplastic growth. MRI imaging demonstrated that this was associated with a substantial improvement in blood–brain barrier function and tumor perfusion. Lastly, JAG1-targeting antibody treatment did not cause any detectable toxicity, further supporting its clinical potential for cancer therapy.

The tumor suppressor p53 is widely dysregulated in cancer and represents an attractive target for immunotherapy. Because of its intracellular localization, p53 is inaccessible to classical therapeutic monoclonal antibodies, an increasingly successful class of anticancer drugs. However, peptides derived from intracellular antigens are presented on the cell surface in the context of MHC I and can be bound by T-cell receptors (TCR). Here, we report the development of a novel antibody, T1-116C, that acts as a TCR mimic to recognize an HLA-A*0201–presented wild-type p53 T-cell epitope, p5365–73(RMPEAAPPV). The antibody recognizes a wide range of cancers, does not bind normal peripheral blood mononuclear cells, and can activate immune effector functions to kill cancer cells in vitro. In vivo, the antibody targets p5365–73 peptide–expressing breast cancer xenografts, significantly inhibiting tumor growth. This represents a promising new agent for future cancer immunotherapy. Cancer Res; 77(10); 2699–711. ©2017 AACR.

Supplemental Materials and Methods from Development of a T-cell Receptor Mimic Antibody against Wild-Type p53 for Cancer Immunotherapy

CCR Translation for the Article from A New Immunostain Algorithm Classifies Diffuse Large B-Cell Lymphoma into Molecular Subtypes with High Accuracy

Supplementary Data from Immunomodulation of FOXP3+ Regulatory T Cells by the Aromatase Inhibitor Letrozole in Breast Cancer Patients

Suppl Table 1: Quantitation of T1-116C binding sites per target cell; Suppl Figure 1: Validation of the HLA-A2/p53RMP tetramer; Suppl Figure 2: ELISA screening of HLA-A2/p53RMP tetramer-reactive TCRm hybridoma supernatants; Suppl Figure 3: T1-116C staining of HL-60 cells; Suppl Figure 4: TP53 transcripts levels in cancer cell lines; Suppl Figure 5: The proteasome actively turns over p53 in NCI-H1395 lung cancer cells; Suppl Figure 6: The p53 TCRm T1-116C does not stain normal human peripheral blood mononuclear cells (PBMCs).

Purpose: We have shown previously that tumor infiltration by FOXP3+ regulatory T cells (Treg) is associated with increased relapse and shorter survival of patients with both in situ and invasive breast cancer. Because estrogen regulates Treg numbers in mice and promotes the proliferation of human Tregs, we hypothesized that blocking estrogen receptor-α signaling would abrogate Tregs and be associated with response to hormonal therapy and increased survival.Experimental Design: FOXP3+ Tregs were quantified in tumor samples collected at baseline by incisional biopsy and after 6 months at definitive surgery in 83 elderly breast cancer patients (T2-4 N0-1) enrolled in a randomized phase II trial based on 6 months of primary letrozole (2.5 mg/d) or 6 months of letrozole plus oral “metronomic” cyclophosphamide (50 mg/d).Results: Treg number ranged from 0 to 380 (median, 30) before treatment and from 0 to 300 (median, 8) after treatment. There was a significant reduction in Tregs in letrozole and letrozole-cyclophosphamide patients (P < 0.0001 and P < 0.002, respectively) after treatment. Treg number at residual histology was inversely related with response (P < 0.03 and P = 0.50, respectively) and a greater Treg reduction was observed in responding patients (P < 0.03).Conclusion: This study suggests that aromatase inhibitors may have an indirect antitumor mechanism of action through reducing Tregs in breast tumors and may be of use in estrogen receptor-α-negative tumors in combination with immunotherapy approaches.

Purpose: Hans and coworkers previously developed an immunohistochemical algorithm with ∼80% concordance with the gene expression profiling (GEP) classification of diffuse large B-cell lymphoma (DLBCL) into the germinal center B-cell–like (GCB) and activated B-cell–like (ABC) subtypes. Since then, new antibodies specific to germinal center B-cells have been developed, which might improve the performance of an immunostain algorithm.Experimental Design: We studied 84 cases of cyclophosphamide-doxorubicin-vincristine-prednisone (CHOP)–treated DLBCL (47 GCB, 37 ABC) with GCET1, CD10, BCL6, MUM1, FOXP1, BCL2, MTA3, and cyclin D2 immunostains, and compared different combinations of the immunostaining results with the GEP classification. A perturbation analysis was also applied to eliminate the possible effects of interobserver or intraobserver variations. A separate set of 63 DLBCL cases treated with rituximab plus CHOP (37 GCB, 26 ABC) was used to validate the new algorithm.Results: A new algorithm using GCET1, CD10, BCL6, MUM1, and FOXP1 was derived that closely approximated the GEP classification with 93% concordance. Perturbation analysis indicated that the algorithm was robust within the range of observer variance. The new algorithm predicted 3-year overall survival of the validation set [GCB (87%) versus ABC (44%); P < 0.001], simulating the predictive power of the GEP classification. For a group of seven primary mediastinal large B-cell lymphoma, the new algorithm is a better prognostic classifier (all “GCB”) than the Hans' algorithm (two GCB, five non-GCB).Conclusion: Our new algorithm is significantly more accurate than the Hans' algorithm and will facilitate risk stratification of DLBCL patients and future DLBCL research using archival materials. (Clin Cancer Res 2009;15(17):5494–502)

Purpose: We have shown previously that tumor infiltration by FOXP3+ regulatory T cells (Treg) is associated with increased relapse and shorter survival of patients with both in situ and invasive breast cancer. Because estrogen regulates Treg numbers in mice and promotes the proliferation of human Tregs, we hypothesized that blocking estrogen receptor-α signaling would abrogate Tregs and be associated with response to hormonal therapy and increased survival. Experimental Design: FOXP3+ Tregs were quantified in tumor samples collected at baseline by incisional biopsy and after 6 months at definitive surgery in 83 elderly breast cancer patients (T2-4 N0-1) enrolled in a randomized phase II trial based on 6 months of primary letrozole (2.5 mg/d) or 6 months of letrozole plus oral “metronomic” cyclophosphamide (50 mg/d). Results: Treg number ranged from 0 to 380 (median, 30) before treatment and from 0 to 300 (median, 8) after treatment. There was a significant reduction in Tregs in letrozole and letrozole-cyclophosphamide patients (P < 0.0001 and P < 0.002, respectively) after treatment. Treg number at residual histology was inversely related with response (P < 0.03 and P = 0.50, respectively) and a greater Treg reduction was observed in responding patients (P < 0.03). Conclusion: This study suggests that aromatase inhibitors may have an indirect antitumor mechanism of action through reducing Tregs in breast tumors and may be of use in estrogen receptor-α-negative tumors in combination with immunotherapy approaches.

Purpose: The pathogenesis and clinical heterogeneity of gastric diffuse large B-cell lymphoma (DLBCL) are poorly understood. We have comprehensively investigated the incidence and clinical significance of lymphoma-associated chromosomal translocations, particularly those involving the immunoglobulin heavy chain (IGH) gene locus, in a large series of gastric DLBCL.Experimental Design: One hundred forty-one cases of primary gastric DLBCL [58 with mucosa-associated lymphoid tissue (MALT) lymphoma and 83 without MALT lymphoma] were enrolled. Translocations involving BCL6, c-MYC, FOXP1, MALT1, and IGH were investigated using interphase fluorescence in situ hybridization. In positive cases, additional fluorescence in situ hybridization was done with appropriate probes for potential partner genes. Cases were classified into germinal center B-cell–like (GCB) or non-GCB subgroups by immunophenotyping with CD10, BCL6, and MUM1.Results: Translocations involving IGH were detected in 36 (32%) of 111 cases; their partner genes included BCL6 (n = 10), c-MYC (n = 5), and FOXP1 (n = 3) but remained unknown in the remaining 18 cases. t(14;18)/IGH-BCL2, t(14;18)/IGH-MALT1, and t(1;14)/BCL10-IGH were not detected in any case. t(11;18)/API2-MALT1 was detected in none of the cases, except for one case of DLBCL with MALT lymphoma, which showed positive signals only in MALT lymphoma cells. IGH-involved translocation was associated with younger age but not with any other clinicopathologic factors including GCB or non-GCB immunophenotypes. Cox multivariate analysis revealed that IGH-involved translocation, in addition to younger age and early stage, was an independent prognostic factor for better overall and EFSs.Conclusion: IGH-involved translocations are frequent in gastric DLBCL and seem to identify cases with favorable prognosis.

Background: The Notch pathway is an evolutionarily conserved cell-signaling system that plays an important role in both physiologic and pathologic conditions such as embryonic development and cancer. In the latter, Notch signaling has been reported to promote tumor growth by regulating different aspects of tumor biology such as cell survival, proliferation, angiogenesis, and stemness. Because of its importance, targeting of this pathway has been attempted via both small molecules (γ-secretase inhibitors; GSIs) and neutralizing antibodies (against individual Notch receptors or DLL4 ligand) but limitations still hamper the clinical use of such therapeutics, generally due to pathway complexity and toxicities caused by complete pathway inhibition (GSIs). Targeting Notch ligands such as Jagged1 (JAG1), offers the opportunity to selectively block specific elements of the pathway important in tumor biology thus avoiding normal tissue toxicities. Methods: Structural studies defined a region of JAG1 (DSL + EGF1-3) that bound Notch1, which was used as an immunogen. Using classical hybridoma technology we have generated and subsequently characterised a panel of monoclonal antibodies (mAbs) against the JAG1 ligand, both in vitro (cell signaling and cell biology assays) and in vivo (tumor xenografts in both mice and rats). Results: Four functional blocking mAbs recognised a unique JAG1 epitope within its DSL domain binding interface with Notch1, effectively blocking ligand-receptor interaction in vitro. Importantly, our mAbs were cross-reactive with rat Jag1 but not the murine orthologue. These were able to inhibit endogenous JAG1-induced signaling in tumor and stromal cells (eg. vascular smooth muscle cells). JAG1 mAb treatment reduced in vitro breast cancer 3D growth, exhibiting reduced expression of important genes such as HES1, IL6 and decreased numbers of cancer stem cells. Treatment did not affect JAG2-induced growth, or signaling mediated by other Notch ligands (eg. DLL4), confirming mAb specificity. Importantly, JAG1 mAb treatment in vivo inhibited Notch signaling and tumor growth in cancer xenograft models in two different host animals. Host body weight, blood tests and histological analysis detected no toxicity. Conclusions: We generated neutralizing mAbs able to inhibit JAG1-induced signaling both in vitro and in vivo. The ability of JAG1 blockade to impair tumor growth without toxicity indicates it has the potential to make a contribution to the current arsenal of cancer therapeutics. This mAb treatment will be subjected to further in vivo testing, both alone and in combination with other therapeutic approaches, to further expand our knowledge of its clinical potential and mechanism of action. Funding and conflicts of interest This work is supported by Cancer Research UK and the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Programme. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. We have filed a patent application, WO/2014/111704, claiming therapeutic use of the JAG1 antibodies. Citation Format: Massimo Masiero, Demin Li, Pat Whiteman, Carol Bentley, Jenny Greig, Tasneem Hassanali, Sarah Watts, Stephen Stribbling, Jenna Yates, Ji-Liang Li, Susan Mary Lea, Penny Ann Handford, Adrian Llewellyn Harris, Alison Hilary Banham. Development of therapeutic anti-Jagged1 monoclonal antibodies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C128.

Introduction: FOXP1 is frequently overexpressed in DLBCL independently to copy number variations and translocations (Goatly et. al. 2008). The prognostic significance of FOXP1 in DLBCL is controversial with some studies describing FOXP1 as a poor prognostic marker (Banham et. al. 2005) and others found no significance (Hans et. al. 2004). Truncated FOXP1 isoforms were identified in DLBCL and are associated with poor outcomes (Brown et. al. 2008). Over 10 isoforms are known, where some exhibit COOH- or NH2- truncations and absence of regulatory domains. Since truncated isoforms lack regulatory domains, we suggest that isoforms may aberrantly regulate FOXP1 target genes. We aim to determine FOXP1 function in DLBCL and specifically its target genes, and also investigate how FOXP1 isoforms may contribute to a more aggressive phenotype. Methods: Expression constructs of FOXP1 isoforms (isoforms 1, 2, 3 and 8) were kindly provided by Dr. Philip Brown and Dr. Alison Banham. A stable BJAB cell line expressing FOXP1 isoform 1 was established. Gene expression of BJAB cell lines and 4 DLBCL tumors was analysed using Illumina HT12v4 microarray. Genes with >2-fold changes in expression were investigated. VisANT analysis and GSEA was performed on these genes and candidates were validated by qRT-PCR. Promoter analysis using MatInspector identified forkhead-binding sites in candidate genes, and dual luciferase reporter gene assays were performed to ascertain promoter regulation by FOXP1 isoforms. Results: The overexpression of FOXP1 isoform 1 in BJAB cell line resulted in the differential expression of 271 genes >2-fold. A similar comparison of high/low FOXP1 expressing DLBCL patients identified 2472 genes with >2-fold level. Comparison of gene lists for both the cell line and patient samples revealed all 271 genes differentially expressed in the cell line overlapped with the patient genes, indicating these results are translatable to FOXP1 function in DLBCL tumors. The 271 differentially expressed genes were investigated by pathway analysis, though no canonical pathways were significant. Alternatively, an integrative analysis of biological network information and microarray data showed critical interactions that were not revealed by pathway analysis, and identified genes that are promising FOXP1 targets. The promoter regions of 5 genes were investigated using reporter gene assays, and FOXP1 isoforms 3 and 8 were significantly stronger repressors compared to isoforms 1 and 2. For example, protein O-fucosyltransferase 1 (POFUT1) was downregulated in the presence of isoform 1, however isoform 3 and 8 downregulated POFUT1 expression >2-fold. This gene has not previously been associated with DLBCL, though POFUT1 is implicated in notch signaling and lymphoid homeostasis (Yao et. al. 2011). We are currently investigating the other novel FOXP1 target genes, and how truncated isoforms are involved in the differential regulation of these genes. Conclusion: We have identified 271 genes that are differentially expressed by FOXP1 expression. These are novel genes for DLBCL pathogenesis, and are differentially regulated by FOXP1 isoforms, indicating truncated isoforms may have aberrant functions and may contribute to a more pathogenic phenotype. Citation Format: Emily Camilleri, Carlos Aya-Bonilla, Jamie Nourse, Philip J. Brown, Alison H. Banham, Paula Marlton, Maher K. Gandhi, Lyn R. Griffiths. FOXP1 truncated isoforms differentially regulate target genes in diffuse large B cell lymphoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B38.