Monoclonal antibodies against human cancer stem cells.

cancer

Review For reprint orders, please contact: [email protected]

Monoclonal antibodies against human cancer stem cells

Cancer stem cells (CSCs) are a subpopulation of tumor cells that display self-renewal and tumor initiation capacity and the ability to give rise to the heterogenous lineages of cancer cells that comprise the tumor. CSCs exhibit intrinsic mechanisms of resistance to modern cancer therapeutics, allowing them to survive current cancer therapies and to initiate tumor recurrence and metastasis. Various cell surface and transmembrane proteins expressed by CSCs, including CD44, CD47, CD123, EpCAM (CD326), CD133, IGF receptor I, and proteins of the Notch and Wnt signaling pathways have been identified. Recently, monoclonal antibodies and antibody constructs raised against these CSC proteins have shown efficacy against CSCs in human cancer xenograft mice, and some of them have demonstrated antitumor activity in clinical studies. Since current cancer therapies fail to eliminate CSCs, leading to cancer recurrence and progression, selective targeting of CSCs with monoclonal antibodies and antibody constructs may represent a novel therapeutic strategy against cancer.

Cord Naujokat Institute of Immunology, University of Heidelberg, Im Neuenheimer Feld 305, 69120 Heidelberg, Germany and Center & Network for Targeted Oncology, Muehlackerweg 8, 69239 Heidelberg, Germany Tel.: +49 6229 933977 Fax: +49 6229 933978 prof.naujokat@ gmx.de

Keywords: cancer stem cells • cancer therapy • CD123 • CD133 • CD44 • CD47 • EpCAM • IGF receptor I • monoclonal antibody • Notch • Wnt

Malignant tumor cell populations are organized as cellular hierarchies in which cancer stem cells (CSCs) constitute heterogeneous subsets of cells, which are distinguished from the bulk of the cells that they produce by their ability to indefinitely perpetuate the growth of a malignant cell population. The demonstration of distinct subsets of CSCs in most, but not all, human cancers has led to the conceptual hypothesis that malignant tumors, like physiologic proliferative tissues, are hierarchically organized and propagated by limited numbers of CSCs, which are at the apex of tumor hierarchies [1–3] . According to a consensus definition, CSCs are cells within a tumor that possess the capacity to self-renew and to give rise to the heterogeneous lineages of cancer cells that comprise the tumor [4] . CSCs can be defined experimentally by their ability to recapitulate the generation of a continuously growing tumor in serial xenotransplantation settings [4] , and specific CSC proteins and markers have been identified and used for the

10.2217/IMT.14.4 © 2014 Future Medicine Ltd

detection and characterization of CSCs [5] . Recent studies provide evidence for the clinical and therapeutic relevance of CSCs in human cancer [6–8] . CSCs possess a variety of intrinsic mechanisms of resistance to conventional chemotherapeutic drugs, radiotherapy and novel tumor-targeting drugs that permit them to survive current cancer therapies and to initiate tumor recurrence and metastasis [5,9] . Landmark studies reveal that CSCs can even be enriched by conventional anticancer drugs, as demonstrated in breast cancer patients receiving systemic cytostatic or endocrine therapy [6,10,11] . Furthermore, many novel tumor-targeted drugs, including tyrosine kinase inhibitors and monoclonal antibodies (mAbs) fail to eliminate CSCs [12–15] , so there is a pressing need for novel strategies and agents that effectively target CSCs for use in complex clinical settings, preferably in combination with conventional cytostatic drugs, tumor-targeted drugs and radiotherapy.

Immunotherapy (2014) 6(3), 291–308

part of

ISSN 1750-743X

291

Review Naujokat During the last two decades, several mAbs have emerged as effective targeted drugs for the treatment of human cancers, and various mAbs have been approved and established for the immunotherapy of cancer, most notably the mAbs rituximab (anti-CD20), cetuximab (anti-EGFR), trastuzumab (anti-HER2) and bevacizumab (anti-VEGF-A) for the treatment of lymphoma, epithelial cancer, HER2-positive breast cancer and anti-angiogenesis treatment of cancer, respectively [16,17] . Given their high target antigen specificity and generally mild toxicity, mAbs and novel antibody constructs are well positioned as potent and specific agents for the elimination of CSCs [18] . In contrast to conventional cytostatic drugs, radiatiotherapy and tumortargeted drugs, mAbs raised against CSC or cancer cell proteins exploit the host’s immune system to eliminate the targeted cells by activating humoral and cellular immune mechanisms, such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity, inhibition of receptor-mediated signal transduction, induction of apoptosis and priming of APCs and effector and memory T cells against targeted tumor antigens [19] . Therefore, targeting of CSCs and cancer cells with mAbs and antibody constructs constitutes a complex therapeutic approach that is substantially supported by the host’s immune system. Several mAbs and antibody constructs that target CSCs through binding to CSC cell surface and transmembrane proteins, which, however, can also be expressed by normal cancer cells, have been developed and validated in the last few years. Consequently, some of these novel agents exhibit not only activity against CSCs, but also against normal cancer cells, as demonstrated in human cancer xenograft mice and in Phase I–III clinical studies. Finally, recent data obtained in human cancer xenograft mice reveal that mAbs against CSCs are most effective in eliminating CSCs when combined with conventional cytostatic drugs [20,21] , envisioning the use of complex combination therapies in the future treatment of cancer. Monoclonal antibodies & antibody constructs against human CSCs Various mAbs and antibody constructs have been demonstrated to exhibit significant anti-CSC activity in human cancer xenograft mice, and some of them have been tested in clinical studies for their antitumor activity (Table 1) . Anti-CD44

The transmembrane glycoprotein CD44 is the receptor for hyaloronic acid, osteopontin, collagen, fibronection, selectin and laminin. CD44 mediates adhesive cell-to-cell and cell-to-extracellular matrix interactions

292

Immunotherapy (2014) 6(3)

through binding to hyaloronic acid and its other ligands. CD44 also participates in signal transduction processes by generating transmembrane complexes with growth factor receptors such as c-Met, EGF receptor (EGFR), HER2 and VEGF receptor [22] . Overexpression of CD44 and its splice variants is found in many cancer cells and is associated with aggressive tumor growth, invasion and metastasis [22,23] . CD44 was first described as a CSC marker in breast cancer and has subsequently been shown to be expressed on CSCs in urothelial, gastric, prostate, pancreatic, ovarian, colon and hepatocellular carcinomas and head and neck squamous cell carcinomas (HNSCC) [24] . CD44 plays a pivotal role in the regulation of normal and malignant hematopoiesis and is abundantly expressed on leukemic blasts in human acute myeloid leukemia (AML) and on AML stem cells (SCs) [25,26] . Moreover, recent evidence reveals that CD44 fulfils some of the special properties that are displayed by CSCs, including self-renewal, niche preparation, epithelial– mesenchymal transition and resistance to apoptosis [27] . Therefore, targeting of CD44 with mAbs appears to be a promising strategy to eliminate CSCs. H90 is a mouse IgG1 mAb raised against human CD44. H90 activates CD44 signaling, reverses myeloid differentiation blockage and induces myeloid differentiation in AML blasts obtained from patients with AML subtypes M1–M5 [28] . Moreover, H90 inhibits proliferation, induces terminal differentiation and mediates apoptosis in human myeloid leukemia cell lines [29,30] . Notably, H90 is the first mAb that has been shown to target CSCs. As demonstrated in a seminal study, H90 induces terminal differentiation, and inhibits engraftment, homing, proliferation and repopulation of human AMLSCs in xenograft mice engrafted with CD34 + CD38- AML SCs isolated from AML patients [25] . By contrast, H90 treatment of xenograft mice engrafted with human bone marrow CD34 + hematopoietic progenitor cells or cord blood had only a minor effect on inhibition of normal hematopoietic engraftment, indicating the specificity of H90 for AML SCs. [25] . In summary, this study revealed for the first time that CD44 is a key regulator of specific AML SC functions that are essential for homing of AML SCs to microenvironmental niches and for maintaining AML SCs in a primitive state [25] . P245 is another mouse IgG1 mAb raised against human CD44 that has been shown to reduce tumor growth and to eliminate breast CSCs in xenograft mice with human triple negative basal-like breast cancer [31] . Triple-negative (negative expression of estrogen receptor, progesterone receptor and HER2) basal-like breast cancer cells resemble many features of breast CSCs,

future science group


Review

Table 1. Monoclonal antibodies and antibody constructs against human cancer stem cells. Compound

Class

Targets

Clinical status

H90 (anti-CD44)

Mouse IgG1 mAb

AML SCs

Preclinical, xenograft mice

P245 (anti-CD44)

Mouse IgG1 mAb

Breast CSCs


[31]

H4C4 (anti-CD44)

Mouse IgG1 mAb

Pancreatic CSCs


[35]

GV5 (anti-CD44R1, variant 8–10)

Human recombinant IgG1 mAb

Uterine cervix and larynx CSCs


[36]

HNSCC, triple-negative breast cancer, CLL Metastatic and/or locally advanced, CD44-expressing malignant solid tumors AML


[38,39,41]

RO5429083/RG7356 (antiHumanized CD44; targets a glycosylated recombinant IgG1-k extracellular constant domain mAb of CD44)

B6H12.2 (anti-CD47, blocking) Mouse IgG1 mAb

Ref. [25]

Phase I clinical study

[44]

Phase I clinical study; mAb alone or in combination with cytarabine

[45]

Preclinical, xenograft mice AML SCs, urothelial CSCs, glioblastoma CSCs, colon CSCs Preclinical, xenograft mice Ovarian, breast, colon, urothelial, prostate, hepatocellular, lung, kidney and gastric cancer, HNSCC, sarcoma and glioblastoma Non-Hodgkin lymphoma, Preclinical, xenograft mice acute lymphoblastic leukemia, multiple myeloma

[48–50]

[48]

[51–53]

B6H12 (anti-CD47, blocking)

Humanized IgG1 mAb

Aggressive metastatic leiomyosarcoma, pediatric brain tumors


[54,55]

7G3 (anti-CD123)

Mouse IgG2a mAb

AML SCs


[60]

111

In-NLS-7G3 (anti-CD123 7G3 modified with 13mer peptides harboring the nuclear translocation sequence of SV-40 large T antigen, labelled with 111 Indium)

Primary AML cells Mouse IgG2a mAb, labelled with nuclear translocation sequence of SV-40 large T antigen and 111Indium


[62]

CSL360 (anti-CD123)

Humanized recombinant chimeric IgG1 mAb

Relapsed, refractory or high Phase I clinical study risk AML

[63]

CSL362 (anti-CD123)

Humanized recombinant chimeric IgG1 mAb

AML in remission

Phase I clinical study

[64]

Fusion of anti-CD123 scFv and BiTE; human anti-CD3 scFv recombinant bispecific bifunctional mAb construct

AML blasts and AML SCs

Preclinical, in vitro

[65]

Fusion of anti-CD123 scFv, anti-CD33 scFv and anti-CD16 scFv

Patient-derived AML cells

Preclinical, in vitro

[66]

Human recombinant trispecific mAb construct

AML: Acute myeloid leukemia; BiTE: Bispecific T cell engager; CLL: Chronic lymphocytic leukemia; CSC: Cancer stem cell; HNSCC: Head and neck squamous cell carcinoma; mAb: Monoclonal antibody; NSCLC: Non-small-cell lung cancer; PE38: Pseudomonas exotoxin A; SC: Stem cell; scFv: Single chain variable region fragment.


www.futuremedicine.com

293

Review Naujokat

Table 1. Monoclonal antibodies and antibody constructs against human cancer stem cells (cont.). Compound

Class

Targets

Clinical status

MT110 (solitomab) (anti-EpCAM/anti-CD3)

BiTE; human recombinant singlechain bispecific bifunctional mAb construct

Breast CSCs, liver CSCs, colon CSCs, pancreatic CSCs

Preclinical, in vitro, xenograft mice Phase I clinical study, advanced solid tumors

[72–74]

Catumaxomab (Removab™, TRION Pharma, Germany) (anti-EpCAM/anti-CD3/Fcγ)

Triomab; recombinant chimeric two half antibody, each with one light and one heavy chain from mouse IgG2a and rat IgG2b isotypes. Bispecific, trifunctional mAb construct

CSCs in malignant ascites from metastatic human ovarian, gastric, breast, pancreatic, colon, endometrial and epithelial cancer

Phase I–III clinical studies, malignant pleural effusions, malignant ascites, peritoneal carcinomatosis, ovarian, gastric, breast, pancreatic, colon and epithelial cancer, intracavitary administration

[81–85]

dCD133KDEL (Fusion of antiCD133 scFv with a truncated pseudomonas exotoxin A and with a mutated KDEL C-terminus signal)

Fusion protein; contains CSCs and tumor initiating cells in human head and an scFv targeting neck carcinomas both glycosylated and unglycosylated isoforms of human CD133, and a truncated form of PE38 with a mutated KDEL C-terminus signal providing deimmunsation and tumor cell death by preventing luminal ER protein secretion

Preclinical, in vitro, xenograft mice

[93]

CSCs in human pancreatic and hepatocellular carcinomas


[94]

BsAb-CIK (anti-CD3/ Anti-CD3/anti-CD133 anti-CD133, linked to bispecific mAb; cytokine-induced killer T cells) generated by chemical heteroconjugation of an anti-human-CD3 mouse IgG2a mAb (OKT3) and an antihuman-CD133 mouse IgG1 mAb linked via CD3 bindings to ex vivo generated and cytokine-expanded human CD3 + killer T cells

Ref.

[75,76]

AVE1642 (anti-IGF-IR)

Humanized Colon CSCs recombinant IgG1 mAb, derived from mouse anti-IGF-IR IgG1mAb EM164


[98]

Figitumumab (CP-751,871) (anti-IGF-IR)

Humanized IgG2 mAb


[99]

Colon CSCs


294




Review

Table 1. Monoclonal antibodies and antibody constructs against human cancer stem cells (cont.). Compound

Class

Targets

Humanized recombinant IgG mAb

Colon CSCs, breast CSCs, Preclinical, xenograft mice colon cancer cells with KRAS mutation, pancreatic CSCs, melanoma CSCs, ovarian CSCs, triple-negative breast cancer cells Phase I clinical studies, combination with cytostatic drugs, colon cancer, pancreatic cancer and NSCLC

REGN421 (anti-DLL4)

OMP-52M51 (anti-Notch1)

OMP-18R5 (vantictumab) (anti-Frizzled 1, 2, 5, 7, 8)

Ref.

Discontinued or terminated: Phase I–III clinical studies, patients with NSCLC, colon cancer and myeloma

Figitumumab (CP-751,871) (anti-IGF-IR) (cont.)

OMP-21M18 (demcizumab) (anti-DLL4)

Clinical status

Humanized recombinant IgG1 mAb

Humanized recombinant IgG mAb

Humanized recombinant IgG2 mAb

[97]

[20,104–109]

[110–112]

Glioblastoma


[113]

Ovarian cancer and NSCLC

Phase I clinical study, advanced solid tumors

[114]

Breast CSCs


[115]

Solid tumors

Phase I clinical study, solid tumors

[116]

Lymphoid malignancies

Phase I clinical study, lymphoid malignancies

[117]

Pancreatic CSCs, breast CSCs, colon cancer, breast cancer, lung cancer, pancreatic cancer


Solid tumors, neuroendocrine tumors

Phase I clinical study, solid tumors. antitumor activity particularly in neuroendocrine tumors

[21,119,120]

[121,122]


including expression of CD44 (high), CD24 (low) and ALDH1. This subtype of breast cancer is characterized by a high content of CSCs, aggressive proliferation, high metastatic capability and poor overall survival of the patients [32,33] . In xenograft mice with human triple negative basal-like breast cancer, P245 has been shown to significantly reduce tumor growth [31] . Treatment of the mice with doxorubicin and cyclophosphamide, a cytostatic drug combination used for the therapy of triple negative basal-like breast cancer, resulted in complete histologic tumor regression, but residual breast CSCs survived the doxorubicin/cyclophosphamide treatment and could be detected by their expression of CD44 [31] . Tumor relapse mediated by the residual CD44 + breast


CSCs occurred 4–6 weeks after complete histologic tumor regression, but the relapse could be effectively prevented when P245 was systemically injected during the tumor regression period [31] . These data provide substantial evidence that anti-CD44 mAbs such as P245 are capable of eliminating human breast CSCs and of preventing relapse of aggressive breast cancer. H4C4 is a mouse IgG1 mAb that recognizes an 85-kDa glycoprotein constant region of the human CD44 receptor. This mAb was originally developed against human peripheral blood adherent cells and displayed the unusual property of inducing in vitro homotypic aggregation of several types of human hematopoietic cells and cell lines [34] .


295

Review Naujokat Recently, it was demonstrated that H4C4 decreases tumorsphere formation of human pancreatic CSCs in vitro and inhibits human pancreatic tumor growth, metastasis and tumor recurrence in xenograft mice [35] . In particular, pretreatment of human MiaPaCa-2 pancreatic cancer cells with H4C4 dramatically decreased tumorsphere formation of CD44 + MiaPaCa-2 CSCs and totally blocked tumorigenesis after implantation of the cells into xenograft mice. In xenograft mice bearing orthotopic human pancreatic cancer established by implantation of MiaPaCa-2 cells, treatment with H4C4 resulted in significant inhibition of tumor growth and metastasis as well as an inhibition of tumor recurrence after tumor regression induced by radiotherapy. Finally, mechanistic experiments in vitro revealed that H4C4 inhibits CD44–STAT3 signaling pathways, accompanied by elimination of the pancreatic CSCs [35] . A recombinant human IgG1 mAb binding to the extracellular domain of an alternative splice variant of CD44, CD44R1(v8-v10) was recently generated by genetical reshaping and class-switching from a human CD44R1-detecting IgM to a recombinant human CD44R1-detecting IgG1, termed GV5 [36] . GV5 showed preferential binding to the CD44R1(v8-v10) splice variant, which is overexpressed in colon, urothelial, lung and larynx cancer as well as in basal-like breast cancer [37] . In xenograft mice, GV5 completely inhibited tumorigenesis and tumor formation of human ME180 uterine cervix carcinoma cells subcutaneously implanted along with orthotopic GV5 administration. Moreover, intraperitoneal injections of GV5 markedly inhibited the growth of tumors established from human HSC-3 larynx carcinoma cells implanted into xenograft mice 1 week before the first GV5 treatment [36] . Results from in vitro experiments presented in the study reveal that GV5 exerts its antitumor activity by induction of ADCC and internalization of CD44R1 [36] . RO5429083/RG7356 is a recombinant humanized IgG1-k mAb that binds to a glycosylated, extracellular constant domain of human CD44 that is present on normal CD44 as well as on all CD44 splice variants. The mAb interferes with CD44–hyaloronic acid interactions but does not influence turnover, downregulation or shedding of CD44 [38,39] . The mAb has been shown to inhibit tumor growth in xenograft mice with human HNSCC, whereas cisplatin, a cytostatic drug used for the treatment of HNSCC patients, showed only a little effect on tumor growth inhibition in HNSCC xenografts [38] . Moreover, treatment of the mice with the mAb inhibited constitutive EGFR phosphorylation in the HNSCC xenografts [38] . This finding is of considerable clinical importance because

296


constitutive EGFR phosphorylation has been shown to be associated with early relapse and poor prognosis in patients with HNSCC [40] . Finally, RO5429083/ RG7356 treatment of peripheral mononuclear cells isolated from healthy human donors resulted in the expansion of cytolytic NK cells, suggesting that the mAb is able to activate effector cells of the innate immune system [38] . A recent study demonstrates that RO5429083/ RG7356 induces significant tumor regression in xenograft mice with breast cancer established by human triple-negative MDA-MB-231 breast cancer cells [39] that contain high amounts of CSCs expressing CD44 [33] . As detected by global mass spectrometry-based phosphoproteome analysis, as well as by ELISA and immunoblot analysis, the mAb is capable of rapidly down-modulating in MDA-MB-231 xenografts the MAPK signaling pathway and its relevant downstream components operative in CSCs, including the phosphorylated (p-) p-ERK-, p-GSK3-, p-eIF4Eand p-STAT3-signaling pathways [39] . Moreover, the mAb has been shown to induce caspase-dependent apoptosis, independent of complement or cytotoxic effector cells, in patient-derived chronic lymphocytic leukemia (CLL) cells expressing high levels of CD44 and the zeta-associated 70 kDa protein ZAP70, while CLL cells lacking ZAP-70 expression were relatively resistant to the cytotoxic effects of the mAb [41] . The mAb also induces internalization of CD44 and reduces expression of ZAP-70 in CLL cells, and disrupts the CD44/ZAP-70 complex found in CLL cells but not in normal B lymphocytes [41] . Furthermore, administration of the mAb to xenograft mice engrafted with human CLL cells resulted in complete and partial clearance of engrafted ZAP-70-positive and ZAP-70-negative CLL cells, respectively [41] . As CD44 promotes disease development in CLL, and CD44 signaling has antiapoptotic effects in CLL cells [42] , targeting CD44 with RO5429083/RG7356 might be an effective therapeutic strategy against CLL, a disease known to be sustained by self-renewing stem cells [43] . These preclinical results have recently led to the initiation of two Phase I clinical studies: a study of the mAb in patients with metastatic and/or locally advanced, CD44-expressing, malignant solid tumors [44] , and a study of the mAb alone or in combination with cytarabine in patients with AML [45] . Anti-CD47

CD47 is a widely expressed transmembrane protein and a receptor for thrombospondin family members that also serves as the ligand for SIRPα [46] . SIRPα is expressed on phagocytic cells, including macrophages and dendritic cells, and when bound and activated by



CD47 SIRPα initiates a signal transduction cascade resulting in inhibition of phagocytosis [46] . Therefore, the CD47–SIRPα interaction is regarded as a mechanism that provides a ‘don’t eat me’ signal [46] . Abundant expression of CD47 is exploited by cancer cells to avoid phagocytosis by tumor-associated macrophages and is required for survival, growth and metastasis of hematopoietic and solid malignancies [46–49] . A seminal study revealed that CD47 is abundantly expressed on human AML SCs and that CD47 is much more highly expressed on AML SCs than on their normal counterparts, such as hematopoietic stem cells (HSCs) and multipotent progenitor cells [49] . As demonstrated in a large cohort of AML patients, increased CD47 expression on human AML cells is associated with poor clinical outcome and worse overall survival, providing evidence that abundant expression of CD47 on AML cells and AML SCs substantially contributes to the pathogenesis and progression of human AML [49] . It was shown in the study that the CD47-binding and CD47-blocking mouse IgG1 mAb B6H12.2 preferentially enables phagocytosis of human AML SCs by human and mouse macrophages in vitro, whereas nonblocking anti-CD47 mAbs failed to enable phagocytosis of the AML SCs by human and mouse macrophages [49] . Similar results were obtained in a study using a blocking anti-CD47 mAb and human urothelial CSCs highly expressing CD47 [50] . Furthermore, in xenograft mice, B6H12.2 prevented the engraftment of human AML SCs, and treatment of human AML xenograft mice with B6H12.2 completely eradicated AML cells by the mechanism of phagocytosis, whereas normal HSCs were not depleted [49] . Thus, human AML SCs can be targeted and eradicated with blocking anti-CD47 mAbs such as B6H12.2 capable of enabling phagocytosis of AML SCs. A comprehensive study impressively shows that CD47 is highly expressed on virtually all human solid tumor cells, as demonstrated in patient-derived ovarian, breast, colon, urothelial, prostate, hepatocellular, lung, kidney and gastric carcinomas, sarcomas and glioblastomas [48] . The study further demonstrates that anti-CD47 blocking mAb B6H12.2 enables phagocytosis of patient-derived ovarian, breast and colon carcinoma cells as well as of glioblastoma and colon CSCs by mouse and human macrophages in vitro. Administration of B6H12.2 inhibited tumor growth in xenograft mice with patient-derived glioblastoma and ovarian, breast, colon and urothelial carcinomas, and increased the survival of the xenograft mice over time. In xenograft mice with aggressive and spontaneously metastasizing patient-derived urothelial carcinoma and HNSCC, administration of B6H12.2 effectively prevented lung and lymph node metastases and also


Review

induced a significant inhibition of primary site tumor growth [48] . This study was extended to examine the effect of anti-CD47 B6H12.2 on human hematopoietic malignancies. In xenograft mice with human nonHodgkin lymphoma [51] , acute lymphoblastic leukemia [52] and multiple myeloma [53] , B6H12.2 was able to inhibit tumor growth and dissiemination by the mechanism of blocking the CD47–-SIRPα interaction and enabling phagocytosis of CD47-expressing tumor cells. Interestingly, B6H12.2 did not induce ADCC or complement-dependent cytotoxicity and exerted its antitumor activity exclusively by promoting phagocytosis of CD47-expressing cancer cells [53] . Finally, a fully humanized blocking anti-CD47 mAb (B6H12) has recently been shown to inhibit tumor growth and metastasis in xenograft mice with human aggressive metastatic leiomyosarcoma [54] and orthotopic malignant pediatric brain tumors [55] . However, B6H12 not only enables macrophage-mediated phagocytosis of CD47+ cancer cells, but also induces an antitumor cytotoxic T-cell immune response in mice with EL4 mouse lymphoma via priming of CD8 + cytotoxic T cells by the phagocytic macrophages [56] . These results collectively indicate that virtually all human solid and hematopoietic tumors cells require CD47 expression to suppress phagocytic innate immune surveillance and elimination, and that blockade of CD47 by anti-CD47 mAbs such as B6H12.2 and B6H12 represents a novel and effective strategy for cancer therapy. Anti-CD123

The mouse IgG2a mAb 7G3 recognizes the N-terminal domain of the human IL-3 receptor α chain (CD123) and functions as a specific IL-3 receptor antagonist that antagonizes IL-3 biologic activities, such as histamine release from basophil granulocytes or secretion of IL-6 and IL-8 from endothelial cells [57] . In contrast to normal HSCs, AML blasts, CD34 + leukemic progenitors and AML SCs overexpress CD123, which confers growth advantage of these cells over HSCs [58–60] . Clinically, high CD123 expression in AML is associated with higher blast counts at diagnosis and a lower complete remission rate that eventually results in poor prognosis and reduced survival [59,61] , ultimately defining CD123 as a promising target for mAb-mediated elimination of AML SCs and eradication of AML. In fact, the CD123-targeting mouse IgG2a mAb 7G3 has been shown to eliminate human AML SCs [60] . Ex vivo treatment of primary human AML cells with 7G3 selectively inhibited engraftment, repopulation ability, and bone marrow and spleen homing of the cells in xenograft mice, whereas 7G3 treatment of normal HSCs derived from human cord blood or bone marrow


297

Review Naujokat resulted in robust engraftment and hematopoietic differentiation of the human HSCs in xenograft mice. Moreover, 7G3 selectively eliminated human AML SCs in xenograft mice engrafted with primary human AML cells. It was further demonstrated that 7G3-mediated inhibition of engraftment and homing of AML SCs in xenograft mice is dependent on ADCC induced by the Fc fragment of 7G3, and that 7G3 inhibits spontaneous and IL-3-induced proliferation of AML SCs in vitro. These multiple activities of 7G3 against human AML cells and AML SCs finally led to the reduction of AML burden and to an improved long-term survival of xenograft mice engrafted with human AML [60] . Notably, the anti-CD123 mAb 7G3 has recently been used to generate an Auger electron radioimmunotherapeutic agent for targeting CD123 + AML SCs. 7G3 was modified with 13-mer peptides harboring the nuclear translocation sequence (NLS) of SV-40 large T-antigen, and with the chelating agentdiethylene triamine pentaacetic acid for labeling the mAb with the Auger electron-emitting isotope 111Indium [62] . This radioimmunotherapeutic agent, termed 111In-NLS7G3, binds effectively to primary CD123 + AML cells derived from AML patients and is rapidly imported into the nucleus where it induces DNA double-strand breaks leading to apoptosis. As demonstrated in xenograft mice with human AML, 111In-NLS-7G3 can also be used for highly sensitive imaging of CD123 + AML cells or AML SCs in single-photon emission computed tomography/computed tomography [62] . CSL360 is a recombinant chimeric IgG1 mAb derived from 7G3 that binds to the same epitope of the IL-3 receptor α chain (CD123) as 7G3. In a Phase I cinical study, 40 patients with relapsed, refractory or high-risk AML received 12 weekly intravenous infusions of CSL360 at dose levels from 0.1–10 mg/kg [63] . CSL360 treatment with a dose of ≥3 mg/kg resulted in sustained saturation and downregulation of CD123 on AML blasts as well as an inhibition of ex vivo proliferative responsiveness of AML blasts to IL-3. However, changes of the percentage of AML SCs in the patients’ bone marrow were not observed during the study. Only one patient became leukemia-free and achieved a complete remission after 12 doses of CSL360, indicating that CSL360 has no significant antileukemic activity in high-risk AML patients, and suggesting that blockade of IL-3 signaling is an ineffective therapeutic strategy for these patients [63] . Nevertheless, a Phase I clinical study of CSL362, a second generation mAb targeting CD123, in patients with CD123 + AML in remission began in 2012 [64] . Novel antibody constructs targeting CD123 and CD3 (T-cell receptor protein complex), (bispecific antibody [BsAb]) or CD123, CD33 and CD16 (trispecific

298


antibody) have been generated recently [65,66] . The BsAb construct belongs to the bispecific T-cell engager (BiTE) class of antibodies and consists of a single chain variable region fragment (scFv) targeting CD123 and a scFv targeting the T-cell receptor protein complex CD3. When expressed in CHO-S cells, CD123 scFv and CD3 scFv form a homodimer that closely resembles the structure of a natural antibody. This BsAb construct is capable of redirecting and activating resting T cells (CD3 +) via its CD3 scFv, and of simultaneously binding to CD123, thereby inducing T-cell mediated lysis and apoptosis of CD123 + AML blasts and AML SCs [65] . The trispecific antibody construct consists of scFvs binding to CD123, CD33 and CD16, and is able to target CD123/CD33 AML blasts and to mediate ADCC via binding of the Fcγ-receptor III (CD16) expressed on macrophages and NK cells. This antibody construct has been shown to induce potent ADCC of primary AML cells isolated from the peripheral blood or bone marrow of AML patients [66] . Anti-EpCAM

EpCAM (also known as CD326/ESA) is a type I transmembrane glycoprotein composed of a large extracellular domain, a single transmembrane domain and a small intracellular domain. Beyond its cell-to-cell adhesion function, EpCAM can transduce multiple oncogenic signaling and gene expression pathways to the nucleus after regulated intramembrane proteolysis of its extracellular domain and subsequent nuclear translocation of its intracellular domain [67,68] . Overexperession of EpCAM in combination with CD44 was first demonstrated for CSCs in human breast, colon and pancreatic carcinomas, and EpCAM is now regarded as a marker exclusively expressed by epithelial cancers and their CSCs [68,69] . MT110 (solitomab) is bispecific bifunctional singlechain antibody construct of the BiTE class that binds to EpCAM and to the T-cell receptor protein complex CD3 [70] . MT110 is a recombinant protein construct that consists of two single-chain antibodies linked such that a single polypeptide chain of approximately 55 kDa is created. In contrast to bispecific trifunctional antibody constructs (triomabs), MT110 and other BiTE class antibodies lack an Fcγ portion [70] . MT110 activates and redirects resting human peripheral CD4 + and CD8 + T cells to induce specific lysis and apoptosis of target cells expressing EpCAM [70,71] . First, it was demonstrated that MT110 is able to eliminate human colon cancer cells and patient-derived metastatic ovarian carcinoma in xenograft mice [70] . Next, EpCAMexpressing CSCs derived from human liver and breast cancer tissues could be eliminated in soft agar colonies by MT110 in the presence of human T cells [72] .



Moreover, CSCs isolated from patient-derived colon or pancreatic carcinomas injected together with allogeneic or autologous peripheral mononuclear cells into xenograft mice were effectively eliminated by MT110, and the CSCs failed to induce tumor outgrowth in the xenograft mice treated with MT110 [73,74] . These results suggest that EpCAM-expressing CSCs and cancer cells can effectively be eliminated by MT110. Consequently, a Phase I clinical study of MT110 in patients with locally advanced, recurrent or metastatic solid tumors expressing EpCAM has been initiated in 2008 and is ongoing and recruiting participants [75] . Interim results from this study have already been reported. Sixteen patients who received an intravenous dose of ≥24 µg/day MT110 for at least 28 days were evaluable for antitumor response: stable disease was observed in 38% of the patients, median duration of antitumor response was 155 days [76] , demonstrating an encouraging antitumor activity of MT110 in patients with advanced solid tumors. Catumaxomab (Removab®, TRION Pharma, Germany) is a chimeric antibody construct consisting of two half antibodies, each with one light and one heavy chain that originate from parental mouse IgG2a and rat IgG2b isotypes [77] . This antibody construct belongs to a novel family of bispecific, trifunctional antibodies termed triomabs, and has one FAB binding specifity directed against EpCAM and one against the T cell receptor protein complex CD3. With its Fc fragment, catumaxomab additionally binds Fcγ-receptor type I, IIa and III expressing dendritic cells, macrophages and NK cells. Therefore, the antitumor activity of catumaxomab and other triomabs results from T-cell-mediated lysis and apoptosis induction, ADCC, and phagocytosis via activation of Fcγ-receptor-positive accessory cells. Importantly, no additional activation of immune cells is necessary for tumor cell elimination by catumaxomab, which therefore represents a self-supporting system [78] . In contrast to antibody constructs of the BiTE class, triomabs exert a vaccination effect and induce a significant antitumor T-cell immune response, most probably due to their Fc-mediated interaction with Fcγ receptors expressed on dendritic cells and macrophages [79,80] . Different studies demonstrate that catumaxomab is capable of inducing regression of malignant pleural effusions, malignant ascites and peritoneal carcinomatosis in patients with advanced epithelial cancers resistant to conventional chemotherapy [81–83] . Notably, catumaxomab is able to effectively eliminate EpCAM+/CD133 + CSCs from malignant ascites of patients with advanced ovarian, gastric and pancreatic cancer [84] , indicating that catumaxomab can be therapeutically used to eliminate CSCs of epithelial cancers. Catumaxomab is currently


Review

in Phase I–III clinical studies for local and intracavitary therapy in patients with advanced epithelial cancers, and is on the market for the therapy of malignant ascites caused by epithelial cancers in the EU [85] . Other anti-EpCAM mAbs, such as edrecolomab (Panorex, mouse IgG2a mAb) and adecatumumab (MT201, recombinant human IgG1 mAb) have been tested in clinical studies, but have not yet been investigated for their activity against CSCs [86,87] . Anti-CD133

Human CD133 (prominin-1) is a transmembrane single-chain glycoprotein with two large extracellular loops containing four N-linked glycosylation sites on each extracellular loop, and two small intracellular loops [88,89] . Originally identified as a cell surface antigen present on CD34 + hematopoietic stem and progenitor cells [88] , CD133 has recently been established as a marker for CSCs in solid tumors, including brain tumors and colon, prostate, lung, ovarian pancreatic and hepatocellular carcinomas [89,90] . CD133 displays several splice variants and different glycolsylated isoforms, such as CD133–1 and CD133–2, which are bound by the mouse IgG1 mAbs AC133 and AC141, respectively [88,90,91] . Colon CSCs selectively express a distinct CD133 epitope which is bound by AC133 and which is lost upon colon CSC differentiation [92] , indicating that AC133 can be used for the selective detection and isolation of colon CSCs, whereas the specificity of AC133 for the detection of CD133 + CSCs in other solid tumors is uncertain [89,90] . dCD133KDEL is a complex fusion protein that contains an scFv targeting both glycosylated and unglycosylated isoforms of human CD133 and binding preferentially to loop 2 of the extracellular domain of CD133. dCD133KDEL also contains a truncated form of pseudomonas exotoxin A with a mutated KDEL C-terminus signal providing deimmunsation (low immunogenicity) of the fusion protein and tumor cell death by preventing luminal endoplasmatic reticulum protein secretion [93] . This antibody construct selectively binds to the CD133 receptor of HEK293 cells transfected with the human CD133 gene, and inhibited proliferation and viability of human CD133 + UMSCC-11B and CD133 + NA-SCC head and neck carcinoma cells, but did not affect CD133- UMSCC11B cells. Of note, dCD133KDEL does not inhibit viability and lineage outgrowth of human CD34 + hematopoietic progenitor cells in long-term cultures [93] . It was further demonstrated that CD133 + UMSCC-11B cells, but not CD133- UMSCC-11B cells, efficiently initiate tumors in a xenograft mice model, indicating an important role for CD133 + cells in tumor initiation of head and neck carcinoma [93] . Finally, pretreatment


299

Review Naujokat of CD133 + UMSCC-11B cells with dCD133KDEL before implantation resulted in inhibition of tumor initiation in xenograft mice, and repeated intratumoral injections of dCD133KDEL in xenograft mice with tumors initiated by CD133 + UMSCC-11B cells showed marked tumor growth inhibition and complete tumor degradation [93] . These results impressively show that novel antibody constructs like dCD133KDEL can effectively target CD133 + CSCs and tumor initiating cells and may hold therapeutic promise for CD133 + cancers. In a recent immunotherapeutic approach, a BsAb construct binding to CD3 and CD133 was generated by chemical heteroconjugation of an anti-human-CD3 mouse IgG2a mAb (OKT3) and an anti-humanCD133 mouse IgG1 mAb. This anti-CD3/anti-CD133 BsAb construct was subsequently linked to ex vivo generated, cytokine-expanded human donor-derived CD3 + cytokine-induced killer (CIK) T cells via CD3 bindings [94] . These CD3 + CIK T cells armed with the anti-CD3/anti-CD133BsAb construct (BsAb–CIK) were able to effectively and specifically kill human CD133(high) pancreatic (SW1990) and CD133(high) hepatic (Hep3B) cancer cells in coculture experiments in vitro [94] . Moreover, in xenograft mice with human pancreatic cancer established by the implantation of CD133(high) SW1990 cells, repeated intraperitoneal injection of BsAb–CIK resulted in significant tumor growth inhibition that was not observed in xenograft mice with human pancreatic cancer established by CD133(low) Capan-2 cells, demonstrating the high CD133-specificity of the BsAb–CIK [94] . This novel immunotherapeutic approach combining antibody therapy with cellular immunotherapy already yields remarkable results in immunocompromised xenograft mice and might show a pronounced effect in more immunocompetent hosts such as cancer patients. Anti-IGF receptor I

IGFs constitute polypeptide hormones with anabolic and mitogenic activities that regulate cell growth and differentiation. Deregulation of IGF signaling has been widely demonstrated in the development and progression of multiple types of human cancer, consequently leading to the development and validation of therapeutics that target IGF signaling [95] . Various humanized recombinant mAbs targeting the IGF receptor I (anti-IGF-IR), a transmembrane tyrosine kinase receptor for IGF-I and IGF-II that is overexpressed in many cancers, have been developed and tested in Phase I–III clinical studies, alone or in combination with conventional cytostatic drugs and tumor-targeted drugs [95] . Although some IGF-IR-targeting mAbs, including figitumumab (CP-751.871),

300


cixutumumab (IMC-A12), dalotuzumab (MK0646), ganitumab (AMG-479), BII022 and AVE1642 showed rather disappointing results in clinical studies [95–97] , AVE1642 and figitumumab have been demonstrated to target human colon CSCs in vitro and in xenograft mice [98,99] . AVE1642 is a humanized recombinant IgG1 mAb derived from mouse anti-IGF-IR IgG1 mAb EM164. AVE1642 inhibits growth and increases bortezomibinduced apoptosis of aggressive human myeloma cells in vitro [100] . Based on these results, a Phase I clinical study of AVE1642 in combination with the proteasome inhibitor bortezomib was conducted in patients with relapsed multiple myeloma. Despite the favorable toxicity profile of the mAb, the response rates for patients treated with AVE1642 alone or in combination with bortezomib were considered insufficient to merit further development of AVE1642 in multiple myeloma [96] . However, AVE1642 has been shown to inhibit IGF-IR signaling and proliferation of colon CSCs enriched from the human colon cancer cell line HT29. Moreover, the mAb was able to induce significant regression of tumors established by the colon CSCs in xenograft mice [98] , suggesting a therapeutic potential of AV1642 to eliminate colon CSCs. Figitumumab is a humanized IgG2 mAb binding to the IGF-IR that has been tested in Phase I–III clinical studies in patients with non-small-cell lung cancer (NSCLC) and other solid tumors with a rather disappointing outcome, leading to the discontinuation of two Phase III clinical studies due to insufficient clinical activity of the mAb [97] . Despite its weak antitumor activity in human solid tumors, figitumumab inhibited IGF-IR signaling, IGF-IR-induced Akt activation and proliferation in colon CSCs with enhanced IGF signaling that were enriched from human colon cancer cell lines [99] . Figitumumab also markedly inhibited growth of tumors established by the human colon CSCs in xenograft mice [99] . These results emphasize that targeting of IGF-IR with mAbs may be therapeutically used for the elimination of CSCs that display deregulated IGF signaling, as demonstrated so far in human colon CSCs. Anti-Delta-like ligand 4 & anti-Notch1

The Notch pathway is an evolutionary conserved signaling system that regulates cell fate specification, tissue patterning and morphogenesis by modulating differentiation, survival, proliferation and apoptosis of eukaryotic cells. In mammals, the system consists of five canonical ligands: Delta-like (DLL) 1, 3, 4, Jagged 1, 2; and four single-pass transmembrane receptors: Notch 1–4 [101] . Aberrant Notch signaling has been implicated in promoting tumorigenesis and



has been shown to critically regulate self-renewal, survival and multiresistance of CSCs [101,102] . In tumor vasculature and in tumor cells, overexpression of DLL4 is found to activate Notch signaling, and blockade of DLL4 signaling in tumors results in excessive, nonproductive tumor vascularity, paradoxically associated with poor tumor perfusion, hypoxia and decreased tumor growth [103] . A humanized recombinant IgG mAb (OMP21M18, demcizumab) that blocks DLL4 has been shown to exert antitumor activity independent of angiogenic mechanisms in xenograft mice with patient-derived colon and breast carcinomas [104] . Moreover, OMP-21M18 was able to reduce the frequency of CSCs in human colon and breast tumors in vitro and in vivo, the latter was demonstrated in explanted tumors of xenograft mice treated with OMP-21M18. The combination of OMP-21M18 with the cytostatic drugs irinotecan and paclitaxel significantly reduced the frequency of colon CSCs and breast CSCs, respectively. This was observed in vitro and in explanted human colon and breast tumors of xenograft mice treated with the mAb and the respective cytostatic drug [104] . OMP-21M18 also exhibited strong antitumor activity in xenograft mice with patient-derived colon carcinoma with oncogenic KRAS mutations that are intrinsically resistant to EGFR blockade with the mAb cetuximab [20] . In the xenograft mice, OMP-21M18 was able to eliminate colon CSCs and to significantly inhibit tumor growth, which was further enhanced when OMP21M18 was combined with irinotecan. Analysis of mutant KRAS tumors explanted from xenograft mice revealed that OMP-21M18 in combination with irinotecan produces a significant decrease in colon CSC frequency, and also induces apoptosis in colon cancer cells [20] . Similar results were obtained recently with OMP-21M18 alone and OMP-21M18 combined with the cytostatic drug gemcitabine in xenograft mice with patient-derived pancreatic carcinoma [105] . Moreover, recent studies indicate that OMP-21M18 is able to inhibit tumor growth and metastasis, and to reduce the frequency of CSCs in xenograft mice with patient-derived melanomas and ovarian carcinoma [106,107] . In xenograft mice with human B-RAF V600E melanomas resistant to B-Raf inhibitors, OMP-21M18 was able to significantly reduce tumor growth and the frequency of melanoma CSCs [108] . Finally, a recent study demonstrates that OMP21M18 delays tumor recurrence, overcomes drug resistance, inhibits epithelial–mesenchymal transition, and decreases the frequency of CSCs in xenograft mice with patient-derived breast cancer and pancreatic cancer [109] . All these promising results


Review

from preclinical studies in xenograft mice with various patient-derived cancers have recently led to the initiation of Phase I/Ib clinical studies of OMP21M18 combined with conventional cytostatic drugs in patients with colon cancer, pancreatic cancer, and NSCLC [110–112] . Another humanized IgG1 mAb raised against DLL4 is REGN421. This mAb has been developed on the basis of results from a study with polyclonal anti-mouse DLL4 antibodies that induced significant antitumor activity and nonproductive angiogenesis in mouse tumor models [113] . Interim results from a Phase I clinical study of REGN421 in patients with advanced solid tumors [114] have been reported recently and reveal a significant antitumor activity of REGN421 in patients with ovarian cancer and NSCLC [115] . OMP-52M51 is a humanized mAb that binds with high affinity and selectivity to human Notch1 receptor, thereby antagonizing Notch1 signaling [116] . A recent study shows that OMP-52M51 inhibits tumor growth and reduces the frequency of CSCs in xenograft mice with a patient-derived chemotherapy-refractory breast cancer subtype displaying an activating Notch1 mutation [116] . These results and the finding that chemotherapy-resistant breast, gastric, esophageal and hepatocellular carcinomas, cholangiocarcinomas and small cell lung cancers exhibit activating Notch1 mutations [116] have led to the initiation of Phase I dose-escalation studies of OMP-52M51 in patients with solid tumors [117] and lymphoid malignancies [118] . Anti-Frizzled

The Frizzled (FZD) family of transmembrane G protein-coupled receptors consists of ten members (FZD 1–10), which are activated by secreted lipoglycoproteins of the Wnt family. The highly conserved Wnt/FZD signaling pathway has multiple functions in animal development and cell fate determination. Deregulation of the Wnt pathway and upregulation of FZD receptors play important roles in tumorigenesis and CSC maintenance. These deregulations are observed in a variety of human cancers, including colon cancer, hepatocellular carcinoma and triple-negative breast cancer [119] . OMP-18R5 (vantictumab), a humanized recombinant IgG2 mAb binding to a conserved epitope within the extracellular domain of FZD1, FZD2, FZD5, FZD7 and FZD8, has recently been developed [21] . Through binding to FZD receptors, OMP18R5 blocks Wnt3A-induced phosphorylation of the Wnt coreceptor LPR6 and accumulation of Wnt signaling protein β-catenin, thereby inhibiting canonical Wnt signaling [21] . In contrast to the cytostatic


301

Review Naujokat drugs gemcitabine and paclitaxel, OMP-18R5 is able to reduce the frequency of CSCs in patient-derived pancreatic and breast carcinomas in xenograft mice [21,120,121] . OMP-18R5 alone, and more effectively in combination with cytostatic drugs, eliminates CSCs and inhibits tumor growth of patient-derived colon, breast, lung and pancreatic carcinomas in xenograft mice [21,120,121] , demonstrating that OMP-18R5 should be regarded as promising agent for the elimination of CSCs and the future treatment of solid tumors. Consequently, a Phase I dose-escalation study of OMP18R5 in patients with solid tumors has been initiated in 2011 and is still recruiting participants [122] . Recent interim results from this study suggest that

OMP-18R5 exerts antitumor activity particularly in patients with neuroendocrine tumors [123] . Others

There is a variety of novel mAbs and antibody constructs raised against CSC cell surface and transmembrane proteins, including CD24, CD98, CD147, GD2, leucinerich-repeat containing G-protein-coupled receptor 5, nestin, neuropilin-1, ABCG2, HDGF IL-6 receptor, sonic hedgehog, Wnt3a and MUC1. However, these mAbs and antibody constructs have not yet been demonstrated to exhibit as single agents anti-CSC activity in human cancer xenograft mice or in clinical studies. Some of these novel agents have been described elsewhere [18] .

Executive summary Cancer stem cells • Cancer stem cells (CSCs) are tumor cells with self-renewal and tumor-initiation capacity. • CSCs are at the apex of tumor hierarchies and give rise to the heterogenous lineages of cancer cells within a tumor. • Due to multiple intrinsic mechanisms of resistance, CSCs are resistant to chemotherapy, tumor-targeted drugs and radiotherapy . • CSCs are responsible for tumor recurrence after standard cancer therapies and metastasis. • CSCs can be enriched by chemo- and radio-therapy. • CSCs express distinct cell surface and transmembrane proteins that can selectively be targeted with novel mAbs and antibody constructs.

Monoclonal antibodies & antibody constructs against human CSCs • Monoclonal antibodies (mAbs) and antibody constructs against human CSCs effectively target and eliminate CSCs with high specificity and mild toxicity in human cancer xenograft mice and in humans. • They exploit the host’s immune system to eliminate the targeted cells by activating ADCC and complementdependent cytotoxicity, inhibition of receptor-mediated signaling, induction of apoptosis, and priming of APCs and effector and memory T cells. • mAbs and antibody constructs raised against CD44, CD47, CD123, EpCAM, CD133, IGF receptor I, Delta-like ligand 4 (DLL4), Notch1 and Frizzled 1, 2, 5, 7, 8 have been shown to eliminate CSCs in human cancer xenograft mice. • mAbs raised against DLL4 and Frizzled 1, 2, 5, 7, 8 (OMP-18R5) exhibit enhanced anti-CSC activity and antitumor activity when combined with conventional cytostatic drugs. • mAbs and antibody constructs raised against CD44 (RO5429083), CD123 (CSL362), EpCAM (MT110 and catumaxomab), DLL4 (OMP-21M18 and REGN421), Notch1 (OMP-52M51) and Frizzled 1,2,5,7,8 (OMP-18R5) are currently in clinical studies in patients with solid tumors or leukemias. • mAbs and antibody constructs raised against EpCAM (MT110 and catumaxomab), DLL4 (REGN421) and Frizzled 1, 2, 5, 7, 8 (OMP-18R5) have shown significant antitumor activity in clinical studies in patients with solid tumors. • Anti-CD47 mAb (B6H12) has been shown to induce an antitumor cytotoxic T-cell immune response in mice with mouse lymphoma. • Anti-EpCAM triomab (catumaxomab) has been shown to induce an antitumor T-cell immune response in patients with HNSCC and gastric cancer. • mAbs and antibody constructs raised against other CSC proteins, including CD24, CD98, CD147, GD2, leucinerich-repeat containing G-protein-coupled receptor 5, nestin, neuropilin-1, ABCG2, HDGF, IL-6 receptor, sonic hedgehog, Wnt3a and MUC1 have not yet been shown to exhibit as single agents anti-CSC activity in human cancer xenograft mice or in clinical studies.

Conclusion • mAbs and antibody constructs against CSCs constitute effective and less toxic drugs for targeting and eliminating human CSCs, thereby hopefully contributing to the eradication of cancer. • mAbs and antibody constructs against CSCs should be combined with conventional cytostatic drugs, tumor-targeted drugs or radiotherapy to achieve a maximum therapeutic effect.

302




Conclusion & future perspective Recent years have highlighted the opportunities of selectively targeting CSCs, which are regarded as one of the main driving forces in carcinogenesis and cancer. Although the rather novel CSC concept of carcinogenesis is widely accepted to date, other driving forces and mechanisms of carcinogenesis, including epigenetic modifications, genome instability, replicative immortality, immune evasion, reprogramming of energy metabolism, and most likely, a complex interplay of all these mechanisms appears to be essential for the genesis of cancer [124] . In line with the CSC concept of carcinogenesis [1–3] , CSCs constitute adequately characterized cells and represent novel and translationally relevant targets for cancer therapy [5,9] . Unfortunately, CSCs exhibit a variety of intrinsic mechanisms of resistance to conventional chemotherapeutic drugs, tumor-targeting drugs and radiotherapy, allowing them to survive standard cancer therapies and to initiate tumor recurrence and metastasis [5,9] . Fortunately, CSCs express distinct cell surface and transmembrane proteins, including CD44, CD47, EpCAM, CD133, CD123, IGF-IR, DLL4, Notch1 and FZD receptors, which can be targeted selectively and readily with mAbs and antibody constructs, without inducing a considerable extent of collateral tissue damage and even an enrichment of CSCs as observed with conventional cytostatic drugs and radiotherapy. Significant advances have been made recently in the discovery, development and validation of novel mAbs and antibody constructs that target CSCs, and the future clinical use of these novel immunotherapeutics may represent a powerful strategy for eradicating CSCs in cancer patients by exploiting the host’s immune system, thereby hopefully preventing tumor recurrence and metastasis, and contributing to the cure of cancer. There is recent evidence that some novel humanized mAbs and triomabs not only eliminate their target cells

in a spatial and temporal fashion, but are also capable of inducing a significant immunity against tumor and CSC antigens, thereby fulfilling some criteria of a cancer vaccination effect. Other mAbs targeting CSCs have been demonstrated to induce robust tumor regression in human cancer xenograft mice and in clinical studies, demonstrating their potent activity against both CSCs and cancer cells. Moreover, recent findings suggest that mAbs targeting antigens expressed on normal tumor cells are also effective in eliminating CSCs. In line with this hypothesis, a recent study shows that administration of trastuzumab (anti-HER2 mAb) inhibits tumor outgrowth and targets HER2-positive CSCs from human HER2-negative luminal breast cancer in xenograft mice [125] , ultimately demonstrating a marked plasticity and heterogeneity of CSCs in distinct tumors, and supporting the strategy of adjuvant mAb therapy (e.g., adjuvant trastuzumab therapy in HER2-negative breast cancer). Finally, the combination of CSC-targeting mAbs with conventional cytostatic drugs, novel tumor-targeted drugs and radiotherapy, as well as the establishment of appropriate biomarkers and the definition of novel clinical end points for monitoring the efficacy of combined therapeutic strategies, should improve future cancer treatment [126,127] . Acknowledgements The author dedicates this work to his patients.

Financial & competing interests disclosure The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

workshop on cancer stem cells. Cancer Res. 66, 9339–9344 (2006).

References Papers of special note have been highlighted as: • of interest •• of considerable interest

•• Consensus definition of cancer stem cells (CSCs), which is widely accepted to date.

1

Nguyen LV, Vanner R, Dirks P, Eaves CJ. Cancer stem cells: an evolving concept. Nat. Rev. Cancer 12, 133–143 (2012).

5

Yu Z, Pestell TG, Lisanti MP, Pestell RG. Cancer stem cells. Int. J. Biochem. Cell Biol. 44, 2144–2151 (2012).

2

Magee JA, Piskounova E, Morrison SJ. Cancer stem cells: impact, heterogeneity, and uncertainty. Cancer Cell 21, 283–296 (2012).

•

State-of-the-art review of CSCs.

6

Creighton CJ, Li X, Landis M et al. Residual breast cancers after conventional therapy display mesemchymal as well as tumor-initiating features. Proc. Natl Acad. Sci. USA 106, 13820–13825 (2009).

7

Tehranchi R, Woll PS, Anderson K et al. Persistant malignant stem cells in del(5q) myelodysplasia in remission. N. Engl. J. Med. 363, 1025–1037 (2010).

3

Valent P, Bonnet D, De Maria R et al. Cancer stem cell definitions and terminology: the devil is in the detail. Nat. Rev. Cancer 12, 767–775 (2012).

4

Clarke MF, Dick JE, Dirks PB et al. Cancer stem cells – perspectives on current status and future directions: AACR


Review


303

Review Naujokat 8

Gerber JM, Smith BD, Ngwang B et al. A clinically relevant population of leukemic CD34 + CD38 - cells in acute myeloid leukemia. Blood 119, 3571–3577 (2012).

23

Negi LM, Talegaonkar S, Jaggi M et al. Role of CD44 in tumour progression and strategies for targeting. J. Drug Target 20, 561–573 (2012).

9

Naujokat C, Laufer S. Targeting cancer stem cells with defined compounds and drugs. J. Cancer Res. Updates 2, 36–67 (2013).

24

Vira D, Basak SK, Veena MS et al. Cancer stem cells, microRNAs, and therapeutic strategies including natural products. Cancer Metastasis Rev. 31, 733–751 (2012).

•

Comprehensive review of the therapeutically relevant compounds and drugs against CSCs.

25

10

Yu F, Yao H, Zhu P et al. let-7 regulates self renewal and tumorigenicity of breast cancer cells. Cell 131, 1109–1123 (2007).

Jin L, Hope KJ, Zhai Q, Smadja-Joffe F, Dick JE. Targeting of CD44 eradicates human acute myeloid leukemic stem cells. Nat. Med. 2, 1167–1174 (2006).

••

First demonstration that a monolonal antibody (mAb) eliminates human CSCs in xenograft mice.

•

First demonstration that conventional chemotherapy can enrich CSCs in cancer patients.

26

Hertweck MK, Erdfelder F, Kreuzer KA. CD44 in hematological neoplasias. Ann. Hematol. 90, 493–508 (2011).

11

Li X, Lewis MT, Huang J et al. Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. J. Natl Cancer Inst. 100, 672–679 (2008).

27

Ghosh SC, Neslihan Alpay S, Klostergaard J. CD44: a validated target for improved delivery of cancer therapeutics. Expert Opin. Ther. Targets 16, 635–650 (2012).

12

Bhatia R, Holtz M, Niu N et al. Persistance of malignant hematopoietic progenitors in chronic myelogenous leukemia patients in complete cytogenetic remission following imatinib mesylate treatment. Blood 101, 4701–4707 (2003).

28

Charrad RS, Li Y, Delpech B et al. Ligation of the CD44 adhesion molecule reverses blockage of differentiation in human acute myeloid leukemia. Nat. Med. 5, 669–676 (1999).

13

Parmar A, Marz S, Rushton S et al. (2011) Stromal niche cells protect early leukemic FLT3 -ITD + progenitor cells against first-generation FLT3 tyrosine kinase inhibitors. Cancer Res. 71, 4696–4706 (2011).

29

Charrad RS, Gadhoum Z, Qi J et al. Effects of anti-CD44 monoclonal antibodies on differentiation and apoptosis of human myeloid leukemia cell lines. Blood 99, 290–299 (2002).

14

Reim F, Dombrowski Y, Ritter C et al. Immunoselection of breast and ovarian cancer cells with trastuzumab and natural killer cells: selective escape of CD44high/ CD24low/HER2low breast cancer stem cells. Cancer Res. 69, 8058–8066 (2009).

30

Gadhoum Z, Delaunay J, Maquarre E et al. The effect of anti-CD44 monoclonal antibodies on differentiation and proliferation of human acute myeloid leukemia cells. Leuk. Lymphoma 45, 1501–1510 (2004).

31

15

Cufi S, Corominas-Faja B, Vazquez-Martin A et al. Metformin-induced preferential killing of breast cancer initiating CD44 + CD24 -/low cells is sufficient to overcome primary resistance to trastuzumab in HER2 + human breast cancer xenografts. Oncotarget 3, 395–398 (2012).

Marangoni E, Lecomte N, Durant L et al. CD44 targeting reduces tumour growth and prevents post-chemotherapy relapse of human breast cancer xenografts. Br. J. Cancer 100, 918–922 (2009).

32

Idowu MO, Kmieciak M, Dumur C et al. CD44(+)/CD24(-/low) cancer stem/progenitor cells are more abundant in triple-negative invasive breast carcinoma phenotype and are associated with poor outcome. Hum. Pathol. 43, 364–373 (2012).

33

Ricardo S, Vieira AF, Gerhard R et al. Breast cancer stem cell markers CD44, CD24 and ALDH1: expression distribution within intrinsic molecular subtype. J. Clin. Pathol. 64, 937–946 (2011).

34

Cheson BD, Leonard JP. Monoclonal antibody therapy for B-cell non-Hodgkin´s lymphoma. N. Engl. J. Med. 359, 613–626 (2008).

Belitsos PC, Hildreth JE, August JT. Homotypic cell aggregation induced by anti-CD44(Pgp-1) monoclonal antibodies and related to CD44(Pgp-1) expression. J. Immunol. 144, 1661–1670 (1990).

35

Fischer M, Yen WC, Kapoun AM et al. Anti-DLL4 inhibits and reduces tumor-initiating cell frequency in colorectal tumors with oncogenic KRAS mutations. Cancer Res. 71, 1520–1525 (2011).

Tang W, Hao X, He F, Li L, Xu L. Anti-CD44 antibody treatment inhibits pancreatic cancer metastasis and postradiotherapy recurrence. Cancer Res. 71(8 Suppl. 1), Abstract 565 (2011).

36

Gurney A, Axelrod F, Bond CJ et al. Wnt pathway inhibition via the targeting of Frizzled receptors results in decreased growth and tumorigenicity of human tumors. Proc. Natl Acad. Sci. USA 109, 11717–11722 (2012).

Masuko K, Okazaki S, Satoh M et al. antitumor effect against human cancer xenografts by a fully human monoclonal anibody to a variant 8-epitope of CD44R1 expressed on cancer stem cells. PLoS ONE 7, e29728 (2012).

37

Olsson E, Honeth G, Bendahl PO et al. CD44 isoforms are heterogenously expressed in breast cancer and correlate with tumor subtypes and cancer stem cell markers. BMC Cancer 11, 418 (2011).

16

Weiner LM, Surana R, Wang S. Monoclonal antibodies: versatile platforms for cancer immunotherapy. Nat. Rev. Immunol. 10, 317–327 (2010).

17

Galluzzi L, Vacchelli E, Fridman WH et al. Trial watch: monoclonal antobodies in cancer therapy. Oncoimmunology 1, 28–37 (2012).

18

Naujokat C. Targeting human cancer stem cells with monoclonal antibodies. Clin. Cell. Immunol. S5, 007 (2012).

19

20

21

22

304

Goodison S, Urquidi V, Tarin D. CD44 cell adhesion molecules. Mod. Pathol. 52, 189–196 (1999).




38

Perez A, Neskey DM, Wen J et al. Targeting CD44 in head and neck squamous cell carcinoma (HNSCC) with a new humanized antibody RO5429083. Cancer Res. 72(8 Suppl. 1), Abstract 2521 (2012).

51

Chao MP, Tang C, Pachynski RK et al. Extranodal dissemination of non-Hodgkin lymphoma requires CD47 and is inhibited by anti-CD47 antibody therapy. Blood 118, 4890–4901 (2011).

39

Weigand S, Herting F, Maisel D et al. Global quantitative phosphoproteome analysis of human tumor xenografts treated with a CD44 antagonist. Cancer Res. 72, 4329–4339 (2012).

52

Chao MP, Alizadeh AA, Tang C et al. Therapeutic antibody targeting of CD47 eliminates human acute lymphoblastic leukemia. Cancer Res. 71, 1374–1384 (2011).

53

40

Hama T, Yuza Y, Saito Y et al. Prognostic significance of epidermal growth factor receptor phosphorylation and mutation in head and neck squamous cell carcinoma. Oncologist 14, 900–908 (2009).

Kim D, Wang J, Willingham SB et al. Anti-CD47 antibodies promote phagocytosis and inhibit the growth of human myeloma cells. Leukemia 26, 2538–2545 (2012).

54

Edris B, Weiskopf K, Volkmer AK et al. Antibody therapy targeting the CD47 protein is effective in a model of aggressive metastatic leiomyosarcoma. Proc. Natl Acad. Sci. USA 109, 6656–6661 (2012).

55

Fedorchenko O, Stiefelhagen M, Peer-Zada AA et al. CD44 regulates the apoptotic response and promotes disease development in chronic lymphocytic leukemia. Blood 121, 4126–4136 (2013).

Gholamin S, Mitra SS, Richard CE et al. Development of anti-CD47 therapy for pediatric brain tumors. Cancer Res. 73(8 Suppl. 1), Abstract 5281 (2013).

56

Kikushige Y, Ishikawa F, Miyamoto T et al. Self-renewing hematopoietic stem cell is the primary target in pathogenesis of human chronic lymphocytic leukemia. Cancer Cell 20, 246–259 (2011).

Tseng D, Volkmer JP, Willingham SB et al. Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response. Proc. Natl Acad. Sci. USA 110, 11103–11108 (2013).

57

Open-label multicenter 2-arm Phase I study of RO5429083 with dose escalation and extension cohorts, and imaging cohorts with RO5429083 and 89Zr-labeled RO5429083, in patients with metastatic and/or locally advanced, CD44expressing, malignant solid tumors (2013). http://clinicaltrials.gov/show/NCT01358903

Sun Q, Woodcock JM, Rapoport A et al. Monoclonal antibody 7G3 recognizes the N-terminal domain of the human interleukin-3 (IL-3) receptor α-chain and functions as a specific IL-3 receptor antagonist. Blood 87, 83–92 (1996).

58

Jordan CT, Upchurch D, Szilvassy SJ et al. The interleukin-3 receptor alpha chain is a unique marker for human acute myelogenous leukemia stem cells. Leukemia 14, 1777–1784 (2000).

59

Testa U, Riccioni R, Militi S et al. Elevated expression of IL-3Ralpha in acute myelogenous leukemia is associated with enhanced blast proliferation, increased cellularity, and poor prognosis. Blood 100, 2980–2988 (2002).

60

Jin L, Lee EM, Ramshaw HS et al. Monoclonal antibodymediated targeting of CD123, IL-3 receptor α chain, eliminates human acute myeloid leukemic stem cells. Cell Stem Cells 5, 31–42 (2009).

61

Vergez F, Green AS, Tamburini J et al. High levels of CD34 + CD38low/- CD123 + blasts are predictive of an adverse outcome in acute myeloid leukemias: a Groupe Ouest-Est des Leucemies Aigues et Maladies du Sang (GOELAMS) study. Haematologica 96, 1792–1798 (2011).

41

42

43

44

Zhang S, Wu CC, Fecteau JF et al. Targeting chronic lymphocytic leukemia cells with a humanized monoclonal antibody specific for CD44. Proc. Natl Acad. Sci. USA 110, 6127–6132 (2013).

45

Open-label, multicenter, dose escalation Phase 1a/b study of RO5429083, administered as intravenous infusion alone or in combination with cytarabine in patients with acute myelogenous leukemia (AML) (2013). http://clinicaltrials.gov/show/NCT01641250

46

Chao MP, Weissman IL, Majeti R. The CD47-SIRPα pathway in cancer immune evasion and potential therapeutic implications. Curr. Opin. Immunol. 24, 225–232 (2012).

47

Jaiswal S, Jamieson CH, Pang WW et al. CD47 is upregulated on circulating hematopoietic stem cells and leukemia cells to avoid phagocytosis. Cell 138, 271–285 (2009).

48

Willingham SB, Volkmer JP, Gentles AJ et al. The CD47signal regulatory protein alpha (SIRPa) interaction is a therapeutic target for human solid tumors. Proc. Natl Acad. Sci. USA 109, 6662–6667 (2012).

62

First demonstration that the CSC antigen CD47 is expressed on virtually all human solid tumor cells that can be eliminated by an anti-CD47 mAb in human xenograft mice.

Leyton JV, Hu M, Gao C et al. Auger electron radioimmunotherapeutic agent specific for the CD123 +/ CD131- phenotype of the leukemia stem cell population. J. Nucl. Med. 52, 1465–1473 (2011).

63

Majeti R, Chao MP, Alizadeh AA et al. CD47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells. Cell 138, 286–299 (2009).

Roberts AW, He S, Ritchie D et al. A Phase I study of antiCD123 monoclonal antibody (mAb) CSL360 targeting leukemia stem cells (LSC) in AML. J. Clin. Oncol. 28(15 Suppl.), Abstract e13012 (2010).

64

A Phase 1 study of CSL362 (Anti-IL3Rα/Anti-CD123 monoclonal antibody) in patients with CD123 + acute myeloid leukemia in complete remission or complete remission with incomplete platelet recovery at high risk for early relapse (2012). http://clinicaltrials.gov/show/NCT01632852

••

49

50

Chan KS, Espinosa I, Chaoa A et al. Identification, molecular characterization, clinical prognosis, and therapeutic targeting of human bladder tumor-initiating cells. Proc. Natl Acad. Sci. USA 106, 14016–14021 (2009).



Review

305

Review Naujokat

306

65

Kuo SR, Wong L, Liu JS. Engineering a CD123xCD3 bispecific scFv immunofusion for the treatment of leukemia and elimination of leukemia stem cells. Protein Eng. Des. Sel. 25, 561–570 (2012).

66

Kügler M, Stein C, Kellner C et al. A recombinant trispecific single-chain Fv derivate directed against CD123 and CD33 mediates effective elimination of acute myeloid leukaemia cells by dual targeting. Br. J. Haematol. 150, 574–586 (2010).

67

Maetzel D, Denzel S, Mack B et al. Nuclear signalling by tumour-associated antigen EpCAM. Nat. Cell Biol. 11, 162–171 (2009).

68

Imrich S, Hachmeister M, Gires O. EpCAM and its potential role in tumor-initiating cells. Cell Adh. Migr. 6, 30–38 (2012).

69

Patriarca C, Macchi RM, Marschner AK, Mellstedt H. Epithelial cell adhesion molecule (CD326) in cancer: a short review. Cancer Treat. Rev. 38, 68–75 (2012).

70

Brischwein K, Schlereth B, Guller B et al. MT110: a novel bispecific single-chain antibody construct with high efficacy in eradicating established tumors. Mol. Immunol. 43, 1129–1143 (2006).

71

Haas C, Krinner E, Brischwein K et al. Mode of cytotoxic action of T-cell engaging BiTE antibody MT110. Immunobiology 214, 441–453 (2009).

72

Münz M, Herrmann I, Friedrich M et al. Functional importance of EpCAM for the activity of tumor-initiating cancer cells and their eradication by EpCAM/CD3-bispecific antibody MT110. Cancer Res. 71(8 Suppl. 1), Abstract 1790 (2011).

73

Herrmann I, Baeuerle PA, Friedrich M et al. Highly efficient elimination of colorectal tumor-initiating cells by an EpCAM/CD3-bispecific antibody engaging human T cells. PLoS ONE 5, e13474 (2010).

cells from head and neck squamous cell carcinoma patients after radio-chemotherapy treatment. Clin. Transl. Oncol. 13, 889–898 (2011). •

First demonstration that triomabs can induce significant antitumor immunity in cancer patients.

80

Atanackovic D, Reinhard H, Meyer S et al. The trifunctional antibody catumaxomab amplifies and shapes tumor-specific immunity when applied to gastric cancer patients in the adjuvant setting. Hum. Vaccin. Immunother. doi:10.4161/ hv.26065 (2013) (Epub ahead of print).

81

Sebastian M, Kiewe P, Schuette W et al. Treatment of malignant pleural effusions with the trifunctional antibody catumaxomab (Removab) (anti-EpCAM x anti-CD3): results of a Phase 1/2 study. J. Immunother. 32, 195–202 (2009).

82

Heiss MM, Murawa P, Koralewski P et al. The trifunctional antibody catumaxomab for the treatment of malignant ascites due to epithelial cancer: results of a prospective randomized Phase II/III trial. Int. J. Cancer 127, 2209–2221 (2010).

83

Ströhlein MA, Lordick F, Rüttinger D et al. Immunotherapy of peritoneal carcinomatosis with the antibody catumaxomab in colon, gastric, or pancreatic cancer: an open-label, multicenter, Phase I/II trial. Onkologie 34, 101–108 (2011).

84

Jäger M, Schoberth A, Ruf P et al. Immunomonitoring results of a Phase II/III study of malignant ascites patients treated with the trifunctional antibody catumaxomab (antiEpCAM x anti-CD3). Cancer Res. 72, 24–32 (2012).

85

Seimetz D. Novel monoclonal antibodies for cancer treatment: the trifunctional antibody catumaxomab (removab). J. Cancer 2, 309–316 (2011).

86

Niedzwiecki D, Bertagnolli MM, Warren RS et al. Documenting the natural history of patients with resected stage II adenocarcinoma of the colon after random assignment to adjuvant treatment with edrecolomab or observation: results from the CALGB 9581. J. Clin. Oncol. 29, 3146–3152 (2011).

74

Cioffi M, Dorado J, Baeuerle PA, Heeschen C. EpCAM/ CD3-bispecific T-cell engaging antibody MT110 eliminates primary human pancreatic cancer stem cells. Clin. Cancer Res. 18, 465–474 (2012).

75

An open-label, multi-center dose escalation Phase I study to investigate the safety and tolerability of a continous infusion of the bispecific T-cell engager (BiTE) MT110 in locally advanced, recurrent or metastatic solid tumors which commonly express EpCAM and are not amenable to curative therapy (2008). http://clinicaltrials.gov/show/NCT00635596

87

Schmidt M, Scheulen ME, Dittrich C et al. An open-label, randomized Phase II study of adecatumumab, a fully human anti-EpCAM antibody, as monotherapy in patients with metastatic breast cancer. Ann. Oncol. 21, 275–282 (2010).

88

Yin AH, Miraglia S, Zanjani ED et al. AC133, a novel marker for human hematopoietic stem and progenitor cells. Blood 90, 5002–5012 (1997).

76

Fiedler WM, Wolf M, Kebenko M et al. (2012) A Phase I study of EpCAM/CD3-bispecific antibody (MT110) in patients with advanced solid tumors. J. Clin. Oncol. 30(15 Suppl.), Abstract 2504 (2012).

89

Grosse-Gehling P, Fargeas CA, Dittfeld C et al. CD133 as a biomarker for putative cancer stem cells in solid tumours: limitations, problems and challenges. J. Pathol. 229, 355–378 (2013).

77

Chelius D, Ruf P, Gruber P et al. Structural and functional characterization of the trifunctional antibody catumaxomab. Mabs 2, 309–319 (2010).

90

Bidlingmaier S, Zhu X, Liu B. The utility and limitations of glycosylated human CD133 epitopes in defining cancer stem cells. J. Mol. Med. 86, 1025–1032 (2008).

78

Seimetz D, Lindhofer H, Bokemeyer C. Development and approval of the trifunctional antibody catumaxomab (antiEpCAM x anti-CD3) as a targeted cancer immunotherapy. Cancer Treat. Rev. 36, 458–467 (2010).

91

Yu Y, Flint A, Dvorin EL, Bischoff J. AC133 -2, a novel isoform of himan AC133 stem cell antigen. J. Biol. Chem. 277, 20711–20716 (2002).

92

79

Schroeder P, Lindemann C, Dettmar K et al. Trifunctional antibodies induce efficient antitumour activity with immune

Kemper K, Sprick MR, de Bree M et al. The AC133 epitope, but not the CD133 protein, is lost upon cancer stem cell differentiation. Cancer Res. 70, 719–729 (2010).




93

Waldron NN, Kaufman DS, Oh S et al. Targeting tumorinitiating cancer cells with dCD133KDEL shows impressive tumor reductions in a xenograft model of human head and neck cancer. Mol. Cancer Ther. 10, 1829–1839 (2011).

xenografts. Cancer Res. 73(8 Suppl. 1), Abstract 3725 (2013). 108 Breviglia L, Yeung P, Fischer M et al. Anti-DLL4 reduces

tumor growth and tumorigenicity in B-RAF V600E melanomas including those with acquired resistance to B-RAF inhibitors, Cancer Res. 72(8 Suppl. 1), Abstract LB-196 (2012).

94

Huang J, Li C, Wang Y et al. Cytokine-induced killer (CIK) cells bound with antiCD3/anti-CD133 bispecific antibodies target CD133(high) cancer stem cells in vitro and in vivo. Clin. Immunol. 149, 156–168 (2013).

95

Gao J, Chang YS, Jallal B, Viner J. Targeting the insulin-like growth factor axis for the development of novel therapeutics in oncology. Cancer Res. 72, 3–12 (2012).

96

Moreau P, Cavallo F, Leleu X et al. Phase I study of the anti insulin-like growth factor 1 receptor (IGF-1R) monoclonal antibody, AVE1642, as single agent and in combination with bortezomib in patients with relapsed multiple myeloma. Leukemia 25, 872–874 (2011).

110 A Phase 1b study of FOLFIRI plus OMP-12M18 as 1st or

Scagliotti GV, Novello S. The role of the insulin-like growth factor signaling pathway in non-small cell lung cancer and other solid tumors. Cancer Treat. Rev. 38, 292–302 (2012).

111 A Phase 1b study of gemcitabine plus OMP-21M18 as 1st-line

97

98

99

Dallas NA, Xia L, Gan F et al. Chemoresistant colorectal cells, the cancer stem cell phenotype, and increased sensitivity to insulin-like growth factor-I receptor inhibition. Cancer Res. 69, 1951–1957 (2009). Hart LS, Dollof NG, Dicker DT et al. Human colon cancer stem cells are enriched by insulin-like growth factor-1 and are sensitive to figitumumab. Cell Cycle 10, 2331–2338 (2011).

100 Descamps G, Gomez-Bougie P, Venot C et al. A humanized

anti-IGF-1R monoclonal antibody (AVE1642) enhances bortezomib-induced apoptosis in myeloma lacking CD45. Br. J. Cancer 100, 366–369 (2009). 101 Shao H, Huang Q, Liu ZJ. Targeting Notch signaling

for cancer therapeutic intervention. Adv. Pharmacol. 65, 191–234 (2012). 102 Wang J, Sullenger BA, Rich JN. Notch signaling in cancer

stem cells. Adv. Exp. Med. Biol. 727, 174–185 (2012). 103 Kuhnert F, Kirshner JR, Thurston G. Dll4–Notch signaling

as a therapeutic target in tumor angiogenesis. Vasc. Cell 3, 20 (2011). 104 Hoey T, Yen WC, Axelrod F et al. DLL4 blockade inhibits

tumor growth and reduces tumor-initiating cell frequency. Cell Stem Cell 5, 168–177 (2009). ••

First demonstration of a synergistic effect of an anti-CSC mAb and conventional cytostatic drugs in the elimination of human CSCs in xenograft mice.

105 Yen WC, Fischer MM, Hynes M et al. Anti-DLL4 has

broad spectrum activity in pancreatic cancer dependent on targeting DLL4–Notch signaling in both tumor and vasculature cells. Clin. Cancer Res. 18, 5374–5386 (2012). 106 Beviglia L, Yeung P, Fischer M et al. Anti-DLL4 treatment

inhibits melanoma tumor growth, recurrence, metastases, and reduces frequency of cancer stem cells in a clinically relevant tumor model in NOD/SCID mice. Cancer Res. 71(8 Suppl. 1), Abstract 2834 (2011). 107 Yen WC, Fischer MM, Shah J et al. Anti-DLL

(demcizumab) inhibits tumor growth and reduces cancer stem cell frequency in patient-derived ovarian cancer


Review

109 Yen WC, Fischer M, Lewicki J, Gurney A, Hoey T.

Targeting cancer stem cells by an anti-DLL4 antibody inhibits epithelial-to-mesenchymal transition, delays tumor recurrence and overcomes drug resistance in breast and pancreatic cancer. Cancer Res. 72(8 Suppl. 1), Abstract 3357 (2012). 2nd-line treatment in subjects with metastatic colorectal cancer (2010). http://clinicaltrials.gov/show/NCT01189942 treatment in subjects with locally advanced or metastatic pancreatic cancer (2010). http://clinicaltrials.gov/show/NCT01189929 112 A Phase 1b study of carboplatin and pemetrexed plus OMP-

21M18 as 1st-line treatment in subjects with non-squamous non-small cell lung cancer (2010). http://clinicaltrials.gov/show/NCT01189968 113 Noguera-Troise I, Daly C, Papadopoulos NJ et al. Blockade

of Dll4 inhibits tumor growth by promoting non-productive angiogenesis. Nature 444, 1032–1037 (2006). 114 A multiple-ascending-dose study of the safety and

tolerability of REGN421(SAR153192) in patients with advanced solid malignancies. http://clinicaltrials.gov/show/NCT00871559 115 Jimeno A, LoRusso P, Strother RM et al. Phase I study

of REGN421 (R)/SAR153192, a fully-human delta-like ligand 4 (Dll4) monoclonal antibody (mAb), in patients with advanced solid tumors. J. Clin. Oncol. 31(15 Suppl.), Abstract 2502 (2013). 116 Cancilla B, Cain J, Wang M et al. Anti-Notch1 antibody

(OMP-52M51) impedes tumor growth and cancer stem cell frequency (CSC) in a chemo-refractory breast cancer xenograft model with an activating Notch1 mutation and screening for activated Notch1 across multiple solid tumor types. Cancer Res. 73(8 Suppl. 1), Abstract 3728 (2013). 117 A Phase 1 dose escalation study of OMP-52M51 in subjects

with solid tumors (2013). http://clinicaltrials.gov/show/NCT01778439 118 A Phase 1 dose escalation study of OMP-52M51 in subjects

with lymphoid malignancies (2013). http://clinicaltrials.gov/show/NCT01703572 119 Ueno K, Hirata H, Hinoda Y, Dahiya R. Frizzled homolog

proteins, microRNAs and Wnt signaling in cancer. Int. J. Cancer 132, 1731–1740 (2013). 120 Yen WC, Fischer M, Lewicki J, Gurney A, Hoey T.

(2011) Targeting cancer stem cells by a novel anti-frizzled antibody inhibits pancreatic tumor growth and induces differentiation. Cancer Res. 71(8 Suppl. 1), Abstract 973 (2011).


307

Review Naujokat 121 Lewicki J, Axelrod F, Beviglia L et al. Development of a novel

Wnt pathway antagonist antibody, OMP-18R5, that reduces tumor initiating cell frequency in breast cancer. Cancer Res. 72(8 Suppl. 1), Abstract 3356 (2012). 122 A Phase 1 dose escalation study of OMP-18R5 in subjects

with solid tumors (2011). http://clinicaltrials.gov/show/NCT01345201 123 Smith DC, Rosen LS, Chugh R et al. First-in-human

evaluation of the human monoclonal antibody vantictumab (OMP-18R5; anti-Frizzled) targeting WNT pathway in a Phase I study for patients with advanced solid tumors. J. Clin. Oncol. 31(15 Suppl.), Abstract 2540 (2013).

308


124 Hanahan D, Weinberg RA. Hallmarks of cancer: the next

generation. Cell 144, 646–674 (2011). 125 Ithimakin S, Day KC, Malik F et al. HER2 drives

luminal breast cancer stem cells in the absence of HER2 amplification: inplications for efficacy of adjuvant trastuzumab. Cancer Res. 73, 1635–1646 (2013). 126 Rasheed ZA, Kowalski J, Smith BD, Matsui W. Emerging

concepts in clinical targeting of cancer stem cells. Stem Cells 29, 883–887 (2011). 127 Ablett MP, Singh JK, Clarke RB. Stem cells in breast

tumours: are they ready for the clinic? Eur. J. Cancer 48, 2104–2116 (2012).


Monoclonal antibodies against human antithrombin III.

Monoclonal antibodies against human synovial phospholipase A2.

Development of human monoclonal antibodies against human cytomegalovirus.

Human-human hybridoma producing monoclonal antibodies against colorectal cancer-associated antigens.

Monoclonal Antibodies Against Human Cardiac Troponin I for Immunoassays II.

Establishment of monoclonal antibodies against human acidic fibroblast growth factor.

Monoclonal antibodies against mycobacterial antigens.

Human monoclonal antibodies against Plasmodium falciparum: production, stabilization and characterization.

Monoclonal antibodies against metallothioneins from the human liver.

Monoclonal antibodies against the free subunits of human chorionic gonadotrophin.

Analysis of two human monoclonal antibodies against melanoma.

Monoclonal antibodies against the human interferon-gamma receptor(s).

Two monoclonal antibodies identify antigens preferentially expressed on normal human breast cells versus breast cancer cells.

Preparation of monoclonal antibodies against bovine haptoglobin.

Monoclonal antibodies against 4-hydroxybiphenyl-UDP-glucuronosyltransferase.

Monoclonal antibodies against rat kidney antigens.

Monoclonal antibodies against purified nicotinic acetylcholine receptor.

Rat monoclonal antibodies against Aspergillus galactomannan.

Monoclonal antibodies against glycophorins and other glycoproteins.

Monoclonal antibodies against Opisthorchis viverrini antigens.

Monoclonal antibodies directed against cadherin RGD exhibit therapeutic activity against melanoma and colorectal cancer metastasis.

Murine monoclonal antibodies to human pancreatic cancer: specificity and sensitivity.

Human Cell Line-Derived Monoclonal IgA Antibodies for Cancer Immunotherapy.

Anti-idiotype monoclonal antibodies as vaccines for human cancer.