Anne K. Volkmer

Antibody therapy targeting the CD47 protein is effective in a model of aggressive metastatic leiomyosarcoma

Antibodies against CD47, which block tumor cell CD47 interactions with macrophage signal regulatory protein-α, have been shown to decrease tumor size in hematological and epithelial tumor models by interfering with the protection from phagocytosis by macrophages that intact CD47 bestows upon tumor cells. Leiomyosarcoma (LMS) is a tumor of smooth muscle that can express varying levels of colony-stimulating factor-1 (CSF1), the expression of which correlates with the numbers of tumor-associated macrophages (TAMs) that are found in these tumors. We have previously shown that the presence of TAMs in LMS is associated with poor clinical outcome and the overall effect of TAMs in LMS therefore appears to be protumorigenic. However, the use of inhibitory antibodies against CD47 offers an opportunity to turn TAMs against LMS cells by allowing the phagocytic behavior of resident macrophages to predominate. Here we show that interference with CD47 increases phagocytosis of two human LMS cell lines, LMS04 and LMS05, in vitro. In addition, treatment of mice bearing subcutaneous LMS04 and LMS05 tumors with a novel, humanized anti-CD47 antibody resulted in significant reductions in tumor size. Mice bearing LMS04 tumors develop large numbers of lymph node and lung metastases. In a unique model for neoadjuvant treatment, mice were treated with anti-CD47 antibody starting 1 wk before resection of established primary tumors and subsequently showed a striking decrease in the size and number of metastases. These data suggest that treatment with anti-CD47 antibodies not only reduces primary tumor size but can also be used to inhibit the development of, or to eliminate, metastatic disease.

CD47-blocking immunotherapies stimulate macrophage-mediated destruction of small-cell lung cancer.

Small-cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer with limited treatment options. CD47 is a cell-surface molecule that promotes immune evasion by engaging signal-regulatory protein alpha (SIRPα), which serves as an inhibitory receptor on macrophages. Here, we found that CD47 is highly expressed on the surface of human SCLC cells; therefore, we investigated CD47-blocking immunotherapies as a potential approach for SCLC treatment. Disruption of the interaction of CD47 with SIRPα using anti-CD47 antibodies induced macrophage-mediated phagocytosis of human SCLC patient cells in culture. In a murine model, administration of CD47-blocking antibodies or targeted inactivation of the Cd47 gene markedly inhibited SCLC tumor growth. Furthermore, using comprehensive antibody arrays, we identified several possible therapeutic targets on the surface of SCLC cells. Antibodies to these targets, including CD56/neural cell adhesion molecule (NCAM), promoted phagocytosis in human SCLC cell lines that was enhanced when combined with CD47-blocking therapies. In light of recent clinical trials for CD47-blocking therapies in cancer treatment, these findings identify disruption of the CD47/SIRPα axis as a potential immunotherapeutic strategy for SCLC. This approach could enable personalized immunotherapeutic regimens in patients with SCLC and other cancers.

Anti-KIT monoclonal antibody inhibits imatinib-resistant gastrointestinal stromal tumor growth

Gastrointestinal stromal tumor (GIST) is the most common sarcoma of the gastrointestinal tract and arises from the interstitial cells of Cajal. It is characterized by expression of the receptor tyrosine kinase CD117 (KIT). In 70–80% of GIST cases, oncogenic mutations in KIT are present, leading to constitutive activation of the receptor, which drives the proliferation of these tumors. Treatment of GIST with imatinib, a small-molecule tyrosine kinase inhibitor, inhibits KIT-mediated signaling and initially results in disease control in 70–85% of patients with KIT-positive GIST. However, the vast majority of patients eventually develop resistance to imatinib treatment, leading to disease progression and posing a significant challenge in the clinical management of these tumors. Here, we show that an anti-KIT monoclonal antibody (mAb), SR1, is able to slow the growth of three human GIST cell lines in vitro. Importantly, these reductions in cell growth were equivalent between imatinib-resistant and imatinib-sensitive GIST cell lines. Treatment of GIST cell lines with SR1 reduces cell-surface KIT expression, suggesting that mAb-induced KIT down-regulation may be a mechanism by which SR1 inhibits GIST growth. Furthermore, we also show that SR1 treatment enhances phagocytosis of GIST cells by macrophages, indicating that treatment with SR1 may enhance immune cell-mediated tumor clearance. Finally, using two xenotransplantation models of imatinib-sensitive and imatinib-resistant GIST, we demonstrate that SR1 is able to strongly inhibit tumor growth in vivo. These results suggest that treatment with mAbs targeting KIT may represent an alternative, or complementary, approach for treating GIST.

Disrupting the CD47-SIRPα anti-phagocytic axis by a humanized anti-CD47 antibody is an efficacious treatment for malignant pediatric brain tumors

Anti-CD47 antibody is effective for treating malignant pediatric brain tumors without detectable toxicity in patient-derived xenograft models. Brain tumors, meet macrophages A protein called CD47 is often expressed on the surface of tumor cells, where it serves as a “don’t eat me” signal that blocks macrophages from attacking the tumor. To overcome this signal and allow the macrophages to “eat” tumor cells, Gholamin et al. engineered a humanized antibody that blocks CD47 signaling. The researchers tested the efficacy of this antibody in patient-derived xenograft models of a variety of pediatric brain tumors. The treatment was successful at inhibiting CD47, killing tumor cells, and prolonging the animals’ survival, all without toxic effects on normal tissues. Morbidity and mortality associated with pediatric malignant primary brain tumors remain high in the absence of effective therapies. Macrophage-mediated phagocytosis of tumor cells via blockade of the anti-phagocytic CD47-SIRPα interaction using anti-CD47 antibodies has shown promise in preclinical xenografts of various human malignancies. We demonstrate the effect of a humanized anti-CD47 antibody, Hu5F9-G4, on five aggressive and etiologically distinct pediatric brain tumors: group 3 medulloblastoma (primary and metastatic), atypical teratoid rhabdoid tumor, primitive neuroectodermal tumor, pediatric glioblastoma, and diffuse intrinsic pontine glioma. Hu5F9-G4 demonstrated therapeutic efficacy in vitro and in vivo in patient-derived orthotopic xenograft models. Intraventricular administration of Hu5F9-G4 further enhanced its activity against disseminated medulloblastoma leptomeningeal disease. Notably, Hu5F9-G4 showed minimal activity against normal human neural cells in vitro and in vivo, a phenomenon reiterated in an immunocompetent allograft glioma model. Thus, Hu5F9-G4 is a potentially safe and effective therapeutic agent for managing multiple pediatric central nervous system malignancies.

CD14-expressing cancer cells establish the inflammatory and proliferative tumor microenvironment in bladder cancer

Significance Our study defines the crucial role of CD14-high bladder cancer (BC) cells in orchestrating multiple hallmarks of cancer in the early stages of BC. Inflammatory factors produced by this subpopulation of tumor cells activate angiogenesis to support establishment and maintenance of an immune-suppressive, inflammatory tumor microenvironment. Additionally, this subpopulation is able to drive tumor growth by producing factors that drive autocrine and paracrine proliferative stimulation. Here we show that a tumor-cell subpopulation establishes a tumor microenvironment orchestrating tumor-promoting inflammation and tumor-cell proliferation. Collectively, this study highlights the need to explore the broader role of CD14-expressing neoplastic cells in other solid tumors. It is noteworthy that CD14 expression is critical for IL6 secretion by these cells. Therefore, therapeutic targeting of CD14 might represent a strategy for treating cancer. Nonresolving chronic inflammation at the neoplastic site is consistently associated with promoting tumor progression and poor patient outcomes. However, many aspects behind the mechanisms that establish this tumor-promoting inflammatory microenvironment remain undefined. Using bladder cancer (BC) as a model, we found that CD14-high cancer cells express higher levels of numerous inflammation mediators and form larger tumors compared with CD14-low cells. CD14 antigen is a glycosyl-phosphatidylinositol (GPI)-linked glycoprotein and has been shown to be critically important in the signaling pathways of Toll-like receptor (TLR). CD14 expression in this BC subpopulation of cancer cells is required for increased cytokine production and increased tumor growth. Furthermore, tumors formed by CD14-high cells are more highly vascularized with higher myeloid cell infiltration. Inflammatory factors produced by CD14-high BC cells recruit and polarize monocytes and macrophages to acquire immune-suppressive characteristics. In contrast, CD14-low BC cells have a higher baseline cell division rate than CD14-high cells. Importantly, CD14-high cells produce factors that further increase the proliferation of CD14-low cells. Collectively, we demonstrate that CD14-high BC cells may orchestrate tumor-promoting inflammation and drive tumor cell proliferation to promote tumor growth.

Improving macrophage responses to therapeutic antibodies by molecular engineering of SIRPα variants.

CD47 transduces inhibitory signals through signal-regulatory protein α (SIRPα), a plasma membrane receptor expressed by macrophages. Many cancers upregulate CD47 to evade immunosurveillance. We have recently engineered SIRPα variants that potently antagonize CD47 for use as anticancer immunotherapeutics. These high-affinity SIRPα variants synergize with antineoplastic antibodies by lowering the threshold for macrophage-mediated destruction of malignant cells.

Use of a KIT-specific monoclonal antibody to bypass imatinib resistance in gastrointestinal stromal tumors

Acquired resistance to imatinib is a significant problem for the clinical management of gastrointestinal stromal tumor (GIST) patients, and second-line small molecules have shown limited efficacy in this setting. We have recently demonstrated that a monoclonal antibody targeting KIT could potentially bypass imatinib resistance in preclinical models of GIST.

MP65-15 HUMANIZED ANTI-CD47 ANTIBODY (HU-5F9-G4) FOR METASTATIC BLADDER CANCER IS SUPERIOR TO CONVENTIONAL CHEMOTHERAPY WITH CISPLATIN AND GEMCITABINE IN A MURINE BLADDER CANCER MODEL.

Simeon Springer, Baltimore, MD; Maria Del Carmen Rodriguez Pena*, Birmingham, AL; Aline Tregnago, Baltimore, MD; Diana Taheri, Tehran, Islamic Republic of Iran; Stephania Bezerra, Isabela Cunha, S~ ao Paulo, Brazil; Kazutoshi Fujita, Osaka, Japan; Dilek Baydar, Hacettepe, Turkey; Trinity Bivalacqua, Nickolas Papadopoulos, Kenneth W Kinzler, Bert Vogelstein, Baltimore, MD; George Netto, Birmingham, AL

Abstract 3629: Overcoming macrophage immunosuppression in small cell lung cancer with high-affinity SIRPa variants

CD47 allows cancer cells to evade the immune system by signaling through SIRPa, an inhibitory receptor on macrophages. Therapies that block CD47 convert tumor-promoting macrophages to a tumoricidal state within the tumor microenvironment. We recently developed next-generation CD47 antagonists by engineering the extracellular domain of SIRPa. As single-domain polypeptides, these “high-affinity SIRPa variants” have an affinity for human CD47 (KD) as low as 11.1 pM, approximately 50,000-fold improved over wild-type SIRPa. By themselves, the high-affinity SIRPa variants are inert and therefore non-toxic in mouse and primate studies. However, when combined with tumor-specific antibodies, the high-affinity SIRPa variants act as immunotherapeutic adjuvants to antibody therapies by maximizing the ability of macrophages to destroy cancer cells. In our current study, we hypothesized these novel CD47-blocking agents could be applied to the treatment of small cell lung cancer (SCLC), a cancer with poor prognosis for which no clinically-approved antibodies or immunotherapies exist. We examined a panel of human SCLC samples and found all samples tested expressed high levels of CD47 on their surface. Using purified macrophages in vitro, we found that CD47-blocking therapies were able to induce macrophage phagocytosis of SCLC cell lines and primary patient samples. As a proof-of-concept, treatment of mice bearing primary SCLC tumors with CD47-blocking antibodies was able to inhibit tumor growth and significantly prolong survival. To identify novel SCLC antigens that can be targeted in combination with the high-affinity SIRPa variants, we screened SCLC samples by high-throughput flow cytometry using LEGENDScreen comprehensive antibody arrays. We identified several new and established therapeutic targets on the surface of SCLC cells, including CD99, CD56, CD166, CD326, and CD164. We identified antibodies to these antigens that could elicit macrophage phagocytosis in vitro, validating these antigens as targets for immune-based therapies. The ability of these antibodies to induce phagocytosis was dramatically enhanced when combined with the high-affinity SIRPa variants. Future studies will test these immunotherapeutic combinations in vivo against SCLC samples to develop novel therapeutic combinations for patients. We propose this strategy as a universal method to identify new tumor antigens and overcome macrophage immunosuppression within the tumor microenvironment. Citation Format: Kipp Weiskopf, Peter J. Schnorr, Nadine Jahchan, Aaron M. Ring, Roy L. Maute, Anne K. Volkmer, Jens-Peter Volkmer, Kenan C. Garcia, Julien Sage, Irving L. Weissman. Overcoming macrophage immunosuppression in small cell lung cancer with high-affinity SIRPa variants. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3629. doi:10.1158/1538-7445.AM2014-3629