Antibody therapy of cancer:Novel therapeutic strategies for prevention and treatment of metastases

Cancer is one of the main global health problems. Most cancer patients die from metastases rather than from the primary tumor. Therefore, it is of utmost importance to develop novel treatment options to prevent and treat metastases. A promising treatment option for cancer is immunotherapy. Immunotherapy aims to stimulate the patient’s own immune system to destroy the tumors. There are several types of immunotherapy, including antibody therapy. Antibodies have both direct effects and indirect effects. Antibodies bind tumor cells to reduce their growth (direct effect). Simultaneously, antibodies can bind immune cells, including macrophages, natural killer (NK) cells and neutrophils, creating a bridge between these cells and tumor cells. In this way, the immune cells are stimulated to eliminate the tumor cells (indirect effect). In my thesis I investigated multiple strategies to enhance the efficacy of antibody therapy. In Part I of my thesis I focused on the prevention of metastases. In Chapter 2 we discuss the process through which tumor cells detach from the primary tumor in the intestine and grow out as metastasis in the liver. As tumor cells detach from the tumor and enter the blood circulation, they are referred to as circulating tumor cells. It has been described that surgical removal of tumors in the intestine results in an increased number of circulating tumor cells. This increases the chance to develop metastases. Therefore, elimination of circulating tumor cells represents a promising treatment option to prevent development of metastases. In Chapter 3 we investigated if, for this purpose, we could use antibodies against the molecule EGFR that is highly expressed on tumor cells compared to healthy cells. We demonstrated that treatment with anti-EGFR antibodies stimulates macrophages to eat tumor cells. Therefore, we advise to treat patients with anti-EGFR antibodies prior to surgical removal of tumors in the intestine. In this way, macrophages can eat circulating tumor cells, potentially limiting development of metastases. In Chapter 4 we describe a novel method to detect circulating tumor cells in blood of cancer patients. Unfortunately, many cancer patients already present with metastases at the time of diagnosis, or develop resistance against current treatment options. Therefore, I investigated novel treatment options based on antibody therapy in Part II of my thesis. In Chapter 5, we combined antibody therapy with another type of immunotherapy: TLR stimulation. TLR are molecules expressed on immune cells that can strongly activate these cells. We demonstrated that the combination treatment enhanced the capacity of NK cells to kill tumor cells. Additionally, NK cells released certain factors that stimulate other components of the immune system, improving tumor destruction. Treatment of mice with the combination treatment resulted in reduced tumor growth and prolonged survival. In Chapter 6 we discuss the role of neutrophils in cancer, as well as potential therapeutic strategies to stimulate tumor cell killing by neutrophils. In Chapter 7 we developed a novel antibody, referred to as TrisomAb. TrisomAb simultaneously stimulates macrophages, NK cells and neutrophils, enhancing tumor cell killing. Treatment of mice with TrisomAb resulted in reduced tumor growth and prolonged survival. In Chapter 8 we investigated if TrisomAb is able to stimulate other components of the immune system. We demonstrated that treatment with TrisomAb resulted in release of certain factors that cause recruitment of other immune cells, in particular neutrophils, to the tumor. In this way, the reaction of the immune system to the tumor is strengthened, enhancing destruction of tumors

Mechanisms of colorectal liver metastasis development

Colorectal cancer (CRC) is a leading cause of cancer-related death worldwide, largely due to the development of colorectal liver metastases (CRLM). For the establishment of CRLM, CRC cells must remodel their tumor-microenvironment (TME), avoid the immune system, invade the underlying stroma, survive the hostile environment of the circulation, extravasate into the liver, reprogram the hepatic microenvironment into a permissive pre-metastatic niche, and finally, awake from a dormant state to grow out into clinically detectable CRLM. These steps form part of the invasion-metastasis cascade that relies on reciprocal interactions between the tumor and its ever-changing microenvironment. Such interplay provides a strong rational for therapeutically targeting the TME. In fact, several TME constituents, such as VEGF, TGF-β coreceptor endoglin, and CXCR4, are already targeted in clinical trials. It is, however, of utmost importance to fully understand the complex interactions in the invasion-metastasis cascade to identify novel potential therapeutic targets and prevent the establishment of CRLM, which may ultimately greatly improve patient outcome

Therapeutic exploitation of neutrophils to fight cancer

Antibody-based immunotherapy is a promising strategy in cancer treatment. Antibodies can directly inhibit tumor growth, induce complement-dependent cytotoxicity and induce Fc receptor-mediated elimination of tumor cells by macrophages and natural killer cells. Until now, however, neutrophils have been largely overlooked as potential effector cells, even though they are the most abundant type of immune cells in the circulation. Neutrophils display heterogeneity, especially in the context of cancer. Therefore, their role in cancer is debated. Nevertheless, neutrophils possess natural anti-tumor properties and appropriate stimulation, i.e. specific targeting via antibody therapy, induces potent tumor cell killing, especially via targeting of the immunoglobulin A Fc receptor (FcαRI, CD89). In this review we address the mechanisms of tumor cell killing by neutrophils and the role of neutrophils in induction of anti-tumor immunity. Moreover, possibilities for therapeutic targeting are discussed

Efficient Innate Immune Killing of Cancer Cells Triggered by Cell-Surface Anchoring of Multivalent Antibody-Recruiting Polymers

Binding of monoclonal antibodies (mAbs) onto a cell surface triggers antibody-mediated effector killing by innate immune cells through complement activation. As an alternative to mAbs, synthetic systems that can recruit endogenous antibodies from the blood stream to a cancer cell surface could be of great relevance. Herein, we explore antibody-recruiting polymers (ARPs) as a novel class of immunotherapy. ARPs consist of a cell-binding motif linked to a polymer that contains multiple small molecule antibody-binding motifs along its backbone. As a proof of concept, we employ a lipid anchor that inserts into the phospholipid cell membrane and make use of a polymeric activated ester scaffold onto which we substitute dinitrophenol as an antibody-binding motif. We demonstrate that ARPs allow for high avidity antibody binding and drive antibody recruitment to treated cells for several days. Furthermore, we show that ARP-treated cancer cells are prone to antibody-mediated killing through phagocytosis by macrophages

Detection of Circulating Tumor Cells Using the Attune NxT

Circulating tumor cells (CTCs) have been detected in many patients with different solid malignancies. It has been reported that presence of CTCs correlates with worse survival in patients with multiple types of cancer. Several techniques have been developed to detect CTCs in liquid biopsies. Currently, the only method for CTC detection that is approved by the Food and Drug Administration is CellSearch. Due to low abundance of CTCs in certain cancer types and in early stages of disease, its clinical application is currently limited to metastatic colorectal cancer, breast cancer and prostate cancer. Therefore, we aimed to develop a new method for the detection of CTCs using the Attune NxT-a flow cytometry-based application that was specifically developed to detect rare events in biological samples without the need for enrichment. When healthy donor blood samples were spiked with variable amounts of different EpCAM+EGFR+ tumor cell lines, recovery yield was on average 75%. The detection range was between 1000 and 10 cells per sample. Cell morphology was confirmed with the Attune CytPix. Analysis of blood samples from metastatic colorectal cancer patients, as well as lung cancer patients, demonstrated that increased EpCAM+EGFR+ events were detected in more than half of the patient samples. However, most of these cells showed no (tumor) cell-like morphology. Notably, CellSearch analysis of blood samples from a subset of colorectal cancer patients did not detect CTCs either, suggesting that these blood samples were negative for CTCs. Therefore, we anticipate that the Attune NxT is not superior to CellSearch in detection of low amounts of CTCs, although handling and analysis of samples is easier. Moreover, morphological confirmation is essential to distinguish between CTCs and false positive events

Epidermal Growth Factor Receptor as Target for Perioperative Elimination of Circulating Colorectal Cancer Cells

Surgical resection of the tumor is the primary treatment of colorectal cancer patients. However, we previously demonstrated that abdominal surgery promotes the adherence of circulating tumor cells (CTC) in the liver and subsequent liver metastasis development. Importantly, preoperative treatment with specific tumor-targeting monoclonal antibodies (mAb) prevented surgery-induced liver metastasis development in rats. This study investigated whether the epidermal growth factor receptor (EGFR) represents a suitable target for preoperative antibody treatment of colorectal cancer patients undergoing surgery. The majority of patients with resectable colorectal liver metastases were shown to have EGFR + CTCs. Three different anti-EGFR mAbs (cetuximab, zalutumumab, and panitumumab) were equally efficient in the opsonization of tumor cell lines. Additionally, all three mAbs induced antibody-dependent cellular phagocytosis (ADCP) of tumor cells by macrophages at low antibody concentrations in vitro, independent of mutations in EGFR signaling pathways. The plasma of cetuximab-treated patients efficiently opsonized tumor cells ex vivo and induced phagocytosis. Furthermore, neither proliferation nor migration of epithelial cells was affected in vitro, supporting that wound healing will not be hampered by treatment with low anti-EGFR mAb concentrations. These data support the use of a low dose of anti-EGFR mAbs prior to resection of the tumor to eliminate CTCs without interfering with the healing of the anastomosis. Ultimately, this may reduce the risk of metastasis development, consequently improving long-term patient outcome significantly

Augmented antibody-based anticancer therapeutics boost neutrophil cytotoxicity

Most clinically used anticancer mAbs are of the IgG isotype, which can eliminate tumor cells through NK cell-mediated antibody-dependent cellular cytotoxicity and macrophage-mediated antibody-dependent phagocytosis. IgG, however, ineffectively recruits neutrophils as effector cells. IgA mAbs induce migration and activation of neutrophils through the IgA Fc receptor (FcαRI) but are unable to activate NK cells and have poorer half-life. Here, we combined the agonistic activity of IgG mAbs and FcαRI targeting in a therapeutic bispecific antibody format. The resulting TrisomAb molecules recruited NK cells, macrophages, and neutrophils as effector cells for eradication of tumor cells in vitro and in vivo. Moreover, TrisomAb had long in vivo half-life and strongly decreased B16F10gp75 tumor outgrowth in mice. Importantly, neutrophils of colorectal cancer patients effectively eliminated tumor cells in the presence of anti-EGFR TrisomAb but were less efficient in mediating killing in the presence of IgG anti-EGFR mAb (cetuximab). The clinical application of TrisomAb may provide potential alternatives for cancer patients who do not benefit from current IgG mAb therapy

Augmented antibody-based anticancer therapeutics boost neutrophil cytotoxicity

Most clinically used anticancer mAbs are of the IgG isotype, which can eliminate tumor cells through NK cell-mediated antibody-dependent cellular cytotoxicity and macrophage-mediated antibody-dependent phagocytosis. IgG, however, ineffectively recruits neutrophils as effector cells. IgA mAbs induce migration and activation of neutrophils through the IgA Fc receptor (FcαRI) but are unable to activate NK cells and have poorer half-life. Here, we combined the agonistic activity of IgG mAbs and FcαRI targeting in a therapeutic bispecific antibody format. The resulting TrisomAb molecules recruited NK cells, macrophages, and neutrophils as effector cells for eradication of tumor cells in vitro and in vivo. Moreover, TrisomAb had long in vivo half-life and strongly decreased B16F10gp75 tumor outgrowth in mice. Importantly, neutrophils of colorectal cancer patients effectively eliminated tumor cells in the presence of anti-EGFR TrisomAb but were less efficient in mediating killing in the presence of IgG anti-EGFR mAb (cetuximab). The clinical application of TrisomAb may provide potential alternatives for cancer patients who do not benefit from current IgG mAb therapy