Terri McClanahan

Involvement of chemokine receptors in breast cancer metastasis

Breast cancer is characterized by a distinct metastatic pattern involving the regional lymph nodes, bone marrow, lung and liver. Tumour cell migration and metastasis share many similarities with leukocyte trafficking, which is critically regulated by chemokines and their receptors. Here we report that the chemokine receptors CXCR4 and CCR7 are highly expressed in human breast cancer cells, malignant breast tumours and metastases. Their respective ligands CXCL12/SDF-1α and CCL21/6Ckine exhibit peak levels of expression in organs representing the first destinations of breast cancer metastasis. In breast cancer cells, signalling through CXCR4 or CCR7 mediates actin polymerization and pseudopodia formation, and subsequently induces chemotactic and invasive responses. In vivo, neutralizing the interactions of CXCL12/CXCR4 significantly impairs metastasis of breast cancer cells to regional lymph nodes and lung. Malignant melanoma, which has a similar metastatic pattern as breast cancer but also a high incidence of skin metastases, shows high expression levels of CCR10 in addition to CXCR4 and CCR7. Our findings indicate that chemokines and their receptors have a critical role in determining the metastatic destination of tumour cells.

Retinoic Acid Early Inducible Genes Define a Ligand Family for the Activating NKG2D Receptor in Mice

Here we describe a family of GPI-anchored cell surface proteins that function as ligands for the mouse activating NKG2D receptor. These molecules are encoded by the retinoic acid early inducible (RAE-1) and H60 minor histocompatibility antigen genes on mouse chromosome 10 and show weak homology with MHC class I. Expression of the NKG2D ligands is low or absent on normal, adult tissues; however, they are constitutively expressed on some tumors and upregulated by retinoic acid. Ectopic expression of RAE-1 and H60 confers target susceptibility to NK cell attack. These studies identify a family of ligands for the activating NKG2D receptor on NK and T cells, which may play an important role in innate and adaptive immunity.

Characterization of the CD200 Receptor Family in Mice and Humans and Their Interactions with CD200

CD200 (OX2) is a broadly distributed cell surface glycoprotein that interacts with a structurally related receptor (CD200R) expressed on rodent myeloid cells and is involved in regulation of macrophage function. We report the first characterization of human CD200R (hCD200R) and define its binding characteristics to hCD200. We also report the identification of a closely related gene to hCD200R, designated hCD200RLa, and four mouse CD200R-related genes (termed mCD200RLa-d). CD200, CD200R, and CD200R-related genes were closely linked in humans and mice, suggesting that these genes arose by gene duplication. The distributions of the receptor genes were determined by quantitative RT-PCR, and protein expression was confirmed by a set of novel mAbs. The distribution of mouse and human CD200R was similar, with strongest labeling of macrophages and neutrophils, but also other leukocytes, including monocytes, mast cells, and T lymphocytes. Two mCD200 receptor-like family members, designated mCD200RLa and mCD200RLb, were shown to pair with the activatory adaptor protein, DAP12, suggesting that these receptors would transmit strong activating signals in contrast to the apparent inhibitory signal delivered by triggering the CD200R. Despite substantial sequence homology with mCD200R, mCD200RLa and mCD200RLb did not bind mCD200, and presently have unknown ligands. The CD200 receptor gene family resembles the signal regulatory proteins and killer Ig-related receptors in having receptor family members with potential activatory and inhibitory functions that may play important roles in immune regulation and balance. Because manipulation of the CD200-CD200R interaction affects the outcome of rodent disease models, targeting of this pathway may have therapeutic utility.

Relationship between immune gene signatures and clinical response to PD-1 blockade with pembrolizumab (MK-3475) in patients with advanced solid tumors

Meeting abstractsnnImmune checkpoint inhibition with anti–PD-1 monoclonal antibodies such as pembrolizumab has demonstrated robust, durable anti-tumor activity against many advanced malignancies. We analyzed immune-related gene expression profiles in pembrolizumab-treated patients with advanced

Patterns of PD‐1, PD‐L1, and PD‐L2 expression in pediatric solid tumors

Significant antitumor effects have been observed in a variety of malignancies via blockade of immune checkpoints. Interaction of programmed death 1 (PD‐1) with its ligands PD‐L1 and PD‐L2 suppresses T‐cell function and restricts immune‐mediated tumor killing. We examined expression of these proteins in children with solid tumors, as expression may serve as biomarkers of response to this class of drugs.

PD-1/PD-L1 and immune-related gene expression pattern in pediatric malignant brain tumors: clinical correlation with survival data in Korean population

BackgroundPD-L1 expression has been evaluated as a predictive biomarker for immunotherapy in numerous tumor types. However, very limited data are available in pediatric brain tumors. The aim of this study was to characterize PD-1 and PD-L1 expressions of four pediatric malignant brain tumors and gene expression profile.MethodsThis study included 89 pediatric patients receiving standard treatment at Seoul National University Children’s Hospital and Seoul National University Bundang Hospital between 1990 and 2014: atypical teratoid/rhabdoid tumor (AT/RT) 20; ependymoma (EPN) 20; high grade glioma (HGG) 21; and medulloblastoma (MBL) 28. We performed immunohistochemistry assays for PD-1 and PD-L1. To characterize the gene expression, a custom immune-response focused gene panel was used.ResultsPD-1 expression was positive in 7 (35%) AT/RT, 7 (35%) EPN, 4 (19%) HGG, and 3 (11%) MBL patients. PD-L1 expression was positive in 8 (40%) AT/RT, 4 (20%) EPN, and 4 (19%) HGG; negative in all MBL patients. There was no statistically significant difference in the overall survival of PD-L1 positive patients. The gene expression analysis demonstrated differences in two clustering functional categories: cell–cell signaling and antigen presentation pathway.ConclusionsAT/RT, EPN, and HGG showed a relatively higher expression rate of PD-L1 (19–40%). This suggests these tumor types might be good candidates for PD-1 checkpoint blockade. We determined that gene expression may potentially serve as a molecular tool in predicting which patients will respond to immunotherapy. Further investigation is required to better understand the predictive and prognostic role of PD-L1 in pediatric brain tumors.

Preclinical to clinical translation of anti-PD-1 blockade

Meeting abstractsnnPembrolizumab (MK-3475), a humanized monoclonal IgG4 antibody against programmed death receptor 1 (PD-1), is currently being studied in clinical trials across more than 30 types of cancers. Immunotherapy with anti–PD-1 monoclonal antibodies such as pembrolizumab shows robust,

A clinical biomarker assay to quantitate thymic stromal lymphopoietin in human plasma at sub-pg/ml level

BACKGROUNDnThymic stromal lymphopoietin (TSLP) is an attractive therapeutic target for the treatment of allergic diseases, and plasma TSLP is a potential patient selection marker in the development of therapeutic agents.nnnRESULTSnWe developed and validated an ultrasensitive electrochemiluminescence assay for measurement of TSLP in plasma with a lower limit of quantitation of 0.12 pg/ml, which allowed the quantitation of TSLP in approximately 90% of human plasma samples tested. The assay demonstrated excellent performance characteristics, including precision, accuracy, sensitivity and dilution linearity. Stability and biological variability of TSLP in plasma were also assessed for clinical sample analysis and data interpretation.nnnCONCLUSIONnThe validated TSLP assay enables assessment of circulating TSLP as a patient selection marker in the development of therapeutics to treat atopic diseases.

Abstract 4714: Blockade of LAG-3 amplifies immune activation signatures and augments curative antitumor responses to anti-PD-1 therapy in immune competent mouse models of cancer

MK-4280 is a humanized IgG4 monoclonal antibody (mAb) that binds to the immune checkpoint receptor Lymphocyte Activation Gene-3 (LAG-3) to block the interaction with its ligand, Major Histocompatibility Complex (MHC) Class II. LAG-3 is frequently co-expressed with other immune checkpoint receptors, most notably programmed cell death protein-1 (PD-1), on T cells with an exhausted phenotype. LAG-3 and PD-1 cooperate to regulate peripheral immune tolerance in healthy individuals, and, conversely, play critical roles in several diseases, including autoimmunity, graft rejection, viral infections, and cancer. Co-blockade of LAG-3 and PD-1 in immunocompetent mouse tumor models have demonstrated augmented anti-tumor activity over single agents. However, the molecular mechanisms behind these combination effects have not been fully investigated. Here, preclinical proof-of-biology studies are presented for co-targeting LAG-3 and PD-1 in cancer. c28G10-mG1-[D265A] (abbreviated 28G10-mG1) is a rat:mouse chimera that mimics MK-4280 by its ability to directly block the mouse LAG-3:MHC Class II interaction without initiating Fc-mediated effector functions. As a single agent, 28G10-mG1 demonstrated modest anti-tumor activity across several syngeneic mouse tumor models, despite evidence of systemic drug exposure and target engagement (as assessed by sLAG-3-mAb complex accumulation). The combination of 28G10-mG1 and the anti-mouse PD-1 blocking antibody mDX400 resulted in greater tumor growth inhibition and increased numbers of complete responses (CR) over mDX400 alone in the MBT-2 tumor model. Furthermore, animals that had achieved CR to combination therapy were subsequently protected from MBT-2 re-challenge, suggesting the establishment of immune memory. RT-qPCR analyses revealed up-regulation of immune-related genes, primarily at Day 4 in the blood and Day 8 in the tumor with mDX400, but not 28G10-mG1, treatment. However, when combined with mDX400, 28G10-mG1 further altered the expression of many immune-related genes that were perturbed by mDX400 single agent therapy. Genes unique to combination treatment were also observed. Significantly, immune pathway signatures associated with clinical efficacy to Keytruda were upregulated with combination therapy. Tumor transcriptome and network analysis by RNAseq revealed enrichment in several immune- and cytokine-related pathways with combination therapy compared to mDX400 single agent therapy. Taken together, these preclinical oncology studies support the concept of co-targeting LAG-3 to increase the therapeutic efficacy of PD-1 blockade. Clinical investigation of MK-4280 in combination with anti-PD-1 therapy (pembrolizumab/Keytruda) is ongoing. Citation Format: Brian B. Haines, Sarah Javaid, Long Cui, Heather Hirsch, Saso Cemerski, Terri McClanahan, Manjiri Sathe, Shuli Zhang, Michael Rosenzweig, Brian Long, Rene de Waal Malefyt. Blockade of LAG-3 amplifies immune activation signatures and augments curative antitumor responses to anti-PD-1 therapy in immune competent mouse models of cancer [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 4714. doi:10.1158/1538-7445.AM2017-4714

Abstract B114: Evaluation of the antitumor activity and molecular characterization of mouse syngeneic tumor models in response to anti-PD-1 treatment as a single agent and in combination with approved agents

Pembrolizumab (MK-3475), a humanized monoclonal IgG4 antibody against programmed death receptor 1 (PD-1), is currently being studied in clinical trials across more than 30 types of cancers. To further support the clinical development of pembrolizumab and to aid in the mechanistic understanding of anti–PD-1 immunotherapy, we generated a surrogate PD-1–blocking antibody (muDX400). We have used muDX400 to determine the antitumor activity, pharmacokinetics, and pharmacodynamics of PD-1 inhibition in multiple preclinical syngeneic tumor model systems. Response to muDX400 treatment in several syngeneic tumor models was broadly classified into 3 categories: highly responsive (ie, complete and durable tumor regressions were observed), partially responsive (ie, tumor growth inhibition was observed), and intrinsically resistant to therapy. Using a multifaceted approach, tumors from these models were extensively characterized at the molecular and cellular level by gene expression profiling, whole exome sequencing, fluorescence-activated cell sorting, and immunohistochemistry to help elucidate mechanisms of action and biology associated with response and resistance to anti-PD1 treatment. Analyses included but were not limited to the evaluation of mutational burden, immune cell activation and migration, interferon signaling, antigen presentation (major histocompatibility [MHC] class I and II), and expression of novel targets.To further evaluate mechanisms that could potentially enhance the antitumor activity of anti–PD-1 in these tumor models, muDX400 was combined with a number of different chemotherapies, targeted therapies, and other immunotherapies. In the models in which enhanced antitumor activity was evident, we evaluated the immune landscape of blood, tumors, and draining lymph nodes by molecular profiling. These data provide preclinical support to expand the clinical development of pembrolizumab into additional cancer types as both a single agent and in combination with other approved anticancer therapies. Additional studies with muDX400 are ongoing to further elucidate the mechanism of action of PD-1 blockade and to better understand the antitumor responses observed in clinical trials of pembrolizumab. Citation Format: Heather Hirsch, Ruban Mangadu, Mingmei Cai, Yanhong Ma, Uyen Phan, Yaolin Wang, Venkataraman Sriram, Joseph H. Phillips, Terri McClanahan, Brian Long, Elaine M. Pinheiro. Evaluation of the antitumor activity and molecular characterization of mouse syngeneic tumor models in response to anti-PD-1 treatment as a single agent and in combination with approved agents. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B114.