Disialoganglioside GD2 as a therapeutic target for human diseases.

Review

Disialoganglioside GD2 as a therapeutic target for human diseases 1.

Introduction

2.

Biochemistry of GD2 and its evolution among vertebrates

3.

GD2 in human embryonic development and normal

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physiology 4.

Role of GD2 in cell signaling, tumorigenesis and metastasis

5.

GD2 as a target for antibodies for cancer diagnosis and therapy

6.

GD2 as a target for T cells

7.

GD2 as a target for drug delivery

8.

Modulating the expression of GD2 in human malignancies

9. 10.

GD2 vaccines Expert opinion

Maya Suzuki & Nai-Kong V Cheung† Memorial Sloan Kettering Cancer Center, Department of Pediatrics, New York, NY, USA

Introduction: Ganglioside GD2 is found in vertebrates and invertebrates, overexpressed among pediatric and adult solid tumors, including neuroblastoma, glioma, retinoblastoma, Ewing’s family of tumors, rhabdomyosarcoma, osteosarcoma, leiomyosarcoma, liposarcoma, fibrosarcoma, small cell lung cancer and melanoma. It is also found on stem cells, neurons, some nerve fibers and basal layer of the skin. Areas covered: GD2 provides a promising clinical target for radiolabeled antibodies, bispecific antibodies, chimeric antigen receptor (CAR)-modified T cells, drug conjugates, nanoparticles and vaccines. Here, we review its biochemistry, normal physiology, role in tumorigenesis, important characteristics as a target, as well as anti-GD2-targeted strategies. Expert opinion: Bridging the knowledge gaps in understanding the interactions of GD2 with signaling molecules within the glycosynapses, and the regulation of its cellular expression should improve therapeutic strategies targeting this ganglioside. In addition to anti-GD2 IgG mAbs, their drug conjugates, radiolabeled forms especially when genetically engineered to improve therapeutic index and novel bispecific forms or CARs to retarget T-cells are promising candidates for treating metastatic cancers. Keywords: cancer, GD2, immunotherapy, mAb Expert Opin. Ther. Targets [Early Online]

1.

Introduction

Anti-GD2 mAb is an accepted therapy for children with high-risk (HR) neuroblastoma [1,2]. A variety of GD2-targeted strategies including radiolabeled antibodies, bispecific antibodies, drug conjugates, nanoparticles and chimeric antigen receptor (CAR)-modified T cells have been tested preclinically and in clinical trials [3-6]. Besides neuroblastoma, ganglioside GD2 is expressed on many types of stem cells, as well as pediatric and adult solid tumors, including astrocytoma, retinoblastoma, Ewing’s sarcoma, rhabdomyosarcoma, osteosarcoma, leiomyosarcoma, liposarcoma, fibrosarcoma, small cell lung cancer, melanoma and breast cancer [7-14]. GD2 has a number of advantages for targeted therapy, most important being its high density on tumor cells and restricted expression on normal tissues [9,15-21]. Over the years, GD2 was ranked as the 12th most important cancer antigen out of 75 potential targets by the National Cancer Institute pilot program for the prioritization of the cancer antigens. The ranking was based on their therapeutic functions, immunogenicity, their roles in oncogenicity, specificity, expression level and percent of antigenpositive cells, stem cell expression, prevalence among human cancers, number of antigenic epitopes, and cellular location of antigen expression [22]. Here, we review its role in normal development and oncogenesis, and its important attributes as a tumor target. Of note, comprehensive updates of GD2-directed clinical trials [23] and the genetic engineering of GD2-specific antibodies [7] have recently been published. 10.1517/14728222.2014.986459 © 2015 Informa UK, Ltd. ISSN 1472-8222, e-ISSN 1744-7631 All rights reserved: reproduction in whole or in part not permitted

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M. Suzuki & N. -K. V. Cheung

Article highlights. . . . .

GD2 is expressed on the cell surface of a broad spectrum of human cancers, stem cells and neurons. By interacting with signaling proteins GD2 influences tumorigenesis, adhesion and metastasis. It can interfere with T cell immunity. It is a target for antibody-based immunotherapy and radioimmunotherapy.


This box summarizes key points contained in the article.

2. Biochemistry of GD2 and its evolution among vertebrates

In 1942, German biochemist Ernst Klenk isolated a new group of carbohydrate-rich glycolipids from ganglion cells, and named them ‘gangliosides’ [24]. Gangliosides are composed of glycosphingolipid and sialic acids (N-acetylneuraminic acid, Neu5Ac or NANA), which are nine-carbon monosaccharides. Ganglioside nomenclature is based on the number and position of the NANA residues [25]. Monosaccharides are first added to ceramide to form lactosylceramide; NANA residues are then added to form gangliosides. Each sugar is attached by specific glycosyltransferases [26]. GD2 has two NANA (a-2,8 bound sialic acid and a-2,3 bound sialic acid), and is derived from precursor GD3 by adding Gal-NAc through the enzyme GM2/GD2 synthase (b1,4-N-acetylgalactosaminyltransferase; Figure 1) [27]. The end-terminal penta-oligosaccharide constitutes the specific epitope of GD2 to which most specific antibodies are directed. This critical enzyme GM2/GD2 synthase responsible for making GD2 has been successfully exploited as a molecular marker of minimal residual neuroblastoma in the bone marrow, with major prognostic impact on patient survival [2,28]. As depicted in the synthesis pathways for gangliosides [7], the epitope neighborhood for GD2 could be clearly defined [29]. For example, GD3 and GD1b are the most common cross-reactive gangliosides recognized by anti-GD2 antibodies [29]. A GD2-derivative with a 9-O-acetyl modification on the terminal sialic acid is called O-acetyl-GD2 [30]. While most anti-GD2 antibodies cross-react with O-GD2, some do not [30]; anti-O-GD2 antibodies with no cross-reactivity with GD2 had less cross-reactivity with normal neurons [31]. Gangliosides are found on the cell surface of the nervous system in vertebrates [32]. Lower vertebrates like fish and amphibian have more polysialo-gangliosides, containing four to five NANA residues, whereas gangliosides have only one to three NANA residues in higher vertebrates, including reptiles, birds and mammals [33].

GD2 in human embryonic development and normal physiology 3.

GD2 is expressed on neural stem cells [12], mesenchymal stem cells (MSCs) [8] and peripheral sympathoadrenergic progenitors [34], and it is involved in neural differentiation and 2

proliferation [35]. While the role of polysialic acid in neuronal development has been extensively studied [36], the precise functions of gangliosides, and specifically of GD2, remain unknown. After birth, GD2 expression is restricted to the CNS, predominantly in neuronal cell bodies, and MSCs, as well as peripheral nerves and skin melanocytes at low levels [7,15]. GD2 is thought to play a role in the maintenance and repair of nervous tissues, which undergo continually progressive degenerative changes through the regulation of complement activation and subsequent inflammation, although the exact immunologic mechanism remains obscure [37]. Of note, GD2(+) MSCs have the potentials to differentiate into multiple lineages, including neurons [38].

Role of GD2 in cell signaling, tumorigenesis and metastasis

4.

GD2 is highly expressed on a variety of embryonal cancers (neuroblastoma, brain tumors, retinoblastoma, Ewing’s sarcoma, rhabdomyosarcoma), bone tumors (osteosarcoma, Ewing’s sarcoma), soft tissue sarcomas (leiomyosarcoma, liposarcoma, fibrosarcoma), neural crest derived tumors (small cell lung cancer and melanoma) and breast cancer [9,16-20]. GD2 expression in these tumor cells is regulated by the enzyme activity of GM2/GD2 synthase and/or the amount of its precursor ganglioside GD3 (Figure 1). Neuroblastoma cells and glioma cells have high levels of the GM2/GD2 synthase transcript, enzyme activity and GD2 expression, while melanoma cells have lower mRNA or enzyme activity giving rise to a higher GD3 expression [39]. GD2 is associated with proliferation, invasion and motility [7-12,40]. Gangliosides are the major components of the glycolipid-enriched microdomain (GEM)/ rafts [40], interacting with sphingomyelin, cholesterol, many glycosylphosphatidylinositol-anchored proteins and receptor tyrosine kinases (RTK). While monosialogangliosides are generally negative regulators of RTK signaling, disialogangliosides (e.g., GD2) are mostly activators [26]. GD2 induces tyrosine phosphorylation of the hepatocyte growth factor (HGF) receptor in the absence of HGF, leading to the activation of c-Met, engaging MEK/ERK and PI3K/Akt pathways, resulting in increased proliferation and cell migration in breast cancer [26,40]. GD2 also induces phosphorylation of either the focal adhesion kinase (FAK) or Lyn kinase, a member of Src family tyrosine kinase, leading to the activation of paxillin, a signal transduction protein, and it inhibits integrin-mediated cell adhesion, enhancing cell migration, invasion and motility in osteosarcoma [41-43]. Given the importance of FAK--Src signaling in migration and metastasis, and the abundant expression of GD2 in neuroblastoma, the interaction of GD2 with integrins in GEM/rafts is likely to be important for controlling the malignant potential of neuroblastoma (Figure 2) [44]. Of interest is the recent discovery of GD2 and GD3 in breast cancer stem-like cells [13,14], thought to contribute to tumor progression by their self-renewal capacity and chemoresistance. In these preclinical models, tumor growth and metastasis were

Expert Opin. Ther. Targets (2014) 19(3)

Disialoganglioside GD2 as a therapeutic target for human diseases

Ceramide

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Gal-Glc-Cer B4GALNT1 LacCer (GM2/GD2 synthase)

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Gal-Glc-Cer SA GT3 SA SA

GalNAc-Gal-Glc-Cer SA GD2 SA

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ST8SIA1 (GD3 synthase) Gal-Glc-Cer Gal-Glc-Cer SA SA GM3 GD3 SA

ST3GAL5

GalNAc-Gal-Glc-Cer SA GT2 SA SA

Gal-GalNAc-Gal-Glc-Cer SA GM1

Gal-GalNAc-Gal-Glc-Cer SA GD1b SA

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Gal-GalNAc-Gal-Glc-Cer Gal-GalNAc-Gal-Glc-Cer SA SA SA GM1b GD1a ST8SIA5

Gal-GalNAc-Gal-Glc-Cer SA GT1b SA

Gal-GalNAc-Gal-Glc-Cer SA SA SA GQ1c SA

Gal-GalNAc-Gal-Glc-Cer SA GD1 SA

Gal-GalNAc-Gal-Glc-Cer SA SA GQ 1b SA SA

Gal-GalNAc-Gal-Glc-Cer SA SA SA GP1c SA

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Figure 1. A. Biosynthesis of gangliosides by the stepwise addition of monosaccharides to ceramide. The sequential enzyme actions of ST3Gal V (GM3 synthase), ST8Sia I (GD3 synthase) and ST8Sia V (GT3 synthase) create the precursors for a-, b- and cseries gangliosides, respectively, while the 0-series gangliosides are directly derived from lactosylceramide. Nomenclature of gangliosides follows that of Svennerholm [25]. B. Structure of GD2. Adapted and modified with permission from [7]. Expert Opin. Ther. Targets (2014) 19(3)

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HGFR

RTK GD2

GD2

P P

P PI3K

MEK P


ERK

P FAK

P Akt

Proliferation migration

P Lyn P

Paxillin Migration, invasion motility

Figure 2. GD2 can induce phosphorylation of the hepatocyte growth factor receptor (HGFR) in the absence of HGF, thereby activating MEK/ERK and PI3K/Akt pathways, resulting in cell proliferation and migration. GD2 can also induce phosphorylation of FAK and Lyn kinase, and activate paxillin, resulting in increased cell migration, invasion and motility. FAK: Focal adhesion kinase; RTK: Receptor tyrosine kinases.

abrogated by GD3 synthase knockdown. GD1b is downstream of GD2 in the biosynthesis pathway (Figure 1). When the conversion enzyme, GD1b synthase, is transfected into neuroblastoma cells, the complex ganglioside derivatives from GD2 (e. g., GD1b and GT1b) are preferentially expressed; importantly, Rho/Rac1 activities are altered, and cell migration is inhibited, without affecting proliferation kinetics and cell differentiation [45]. These complex gangliosides along the ‘b’ ganglioside pathway have been noted to be more prevalent among neuroblastoma tumors with favorable clinical biology [46]. Retinoic acid, a well-established differentiating agent for neuroblastoma, is known to upregulate the GD1b/GM1a synthase as well as the complex ganglioside GD1b [47]. GD2 may also have distant effects on tumorigenesis. It has been reported to be immunosuppressive for human dendritic cells [48] and T cells [49-51], probably through myeloid-derived suppressor cells [52], and regulatory T cells [53]. Although soluble GD2 is present in the plasma of patients with clinical disease [54], its level did not correlate with patient outcome. Despite being a marker of disease that decreases with treatment, its specificity and prognostic potential when compared to other available markers are uncertain [55].

GD2 as a target for antibodies for cancer diagnosis and therapy 5.

Murine anti-GD2 mAb Murine anti-GD2 mAbs were first described in the early 1980s and notable advances have been made using these antibodies for cancer therapy. Several anti-GD2 antibodies have been described [56-58] and their crystal structures 5.1

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analyzed [59,60]. Anti-GD2 mAbs can detect GD2(+) tumor cells by immunocytology and GD2(+) tumors in patients by radioimmunoscintigraphy [8,16]. For some tumors like neuroblastoma, GD2 is an ideal target because of its abundance (107 molecules per cell [21]) on tumor cells and its stability after binding to an antibody [16]. Although GD2 is expressed in neurons, the human brain is protected from parenteral anti-GD2 mAb by the blood--brain barrier [16]. Furthermore, GD2 antigen loss from tumors is uncommon as an escape mechanism after antibody-targeted therapy [56]. This provides indirect evidence that inactivation of the specific glycosyltransferase (e.g., GD2/GM2 synthase) is incompatible with tumor survival. Anoikis is an induced apoptotic process when adherent cells detach from the extracellular matrix; tumor cells evade anoikis by overexpressing FAK or by constitutive activation of FAK by mutations [61]. As noted earlier, GD2 is involved in the activation of FAK by phosphorylation, thereby promoting cell migration; dephosphorylation of FAK should normalize anoikis and induce apoptosis. In small cell lung cancer, anti-GD2 mAb can trigger the dephosphorylation of FAK, leading to the activation of MAPK, p38 and the induction of anoikis [62], at the same time sensitizing these cells to anticancer drugs such as cisplatin [63]. Anti-GD2 mAb has also been shown to inhibit activation of PI3K/Akt pathways, thereby decreasing the viability and motility of cancer cells [64]. Besides inducing apoptosis and reducing invasiveness, anti-GD2 mAbs can hijack human leukocytes to perform Fc-dependent tumor lysis: antibody-dependent cell-mediated cytotoxicity (ADCC) and monocyte--macrophage-mediated phagocytosis [65]. Granulocytes and NK cells carry Fc-receptors, FcgRIIA (CD32) and FcRIIIA (CD16A) respectively, on their cell surface. When these Fc-receptors are engaged by the mAb bound to the tumor cells, cytotoxic granules and cytokines are released, killing tumors by ADCC [65]. These Fc-receptors can also mediate phagocytosis by activating monocytes and macrophages [65], even when they are forced into M2 macrophages by M-CSF [66,67]. When complement C1q binds to the Fc of tumorbound mAb, it initiates a complement activation cascade leading to the formation of membrane attach complex, creating pores in the cell membrane [65], and lysing tumor cells -- a process called complement-mediated cytotoxicity (CMC). Murine IgG3 antibody 3F8 was tested in the first-inhuman Phase I clinical trial in patients with neuroblastoma and malignant melanoma in late 1980s [68]. The study showed that its major side effects of pain, fever, hypertension and urticaria were severe, but controllable. Pain side effects were unexpected and did not correlate with 3F8 dose or the duration of 3F8 infusion. Furthermore, selective tumor uptake was demonstrated using Iodine-131 (131I)-labeled 3F8, with no uptake in the brain or spinal cord, and no nonspecific uptake was seen in the reticuloendothelial system (liver, spleen, lymph nodes) by radioimmunoscintigraphy [68]. Serum human anti-mouse antibody (HAMA) level was detected in some patients and those with HAMA during 3F8 infusion had



1.0

Regimen C (HR)


Proportion surviving

0.8

Regimen C (UHR) 0.6

Regimen B

0.4 Regimen A 0.2

0.0 0

5 10 15 Years from start of 3F8 immunotherapy

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Figure 3. Overall survival over 2 decades for 169 patients with high-risk (HR) GD2(+) neuroblastoma treated in first remission during consecutive eras of immunotherapy. HR referred to stage 4 metastatic disease diagnosed after 18 months of age and/ or with MYCN amplification: 3F8 alone (regimen A-HR; n = 43), 3F8 + intravenous GM-CSF + 13-cis-retinoic acid (CRA; regimen B-HR; n = 41), and 3F8 + subcutaneous GM-CSF + CRA (regimen C-HR; n = 57, and regimen C-ultra HR [UHR] because of the requirement of second-line induction; n = 28); p = 0.003 (derived from log-rank test to compare OS among these four groups). Three decades ago, curing these patients with HR neuroblastoma was unthinkable. Over the years, stem cell transplant and cis-retinoic acid have also shown no statistical benefit on survival in randomized trials [70]. Anti-GD2 3F8 antibody was likely a major contributor to the improvement in patient outcome. Adapted with permission from [2].

minimal side effects [68]. Antitumor effects were demonstrated in subsequent Phase II studies of 3F8, especially when combined with GM-CSF. 3F8 + GMCSF appeared to improve the long-term survival in patients with HR neuroblastoma; few relapses were seen after follow-up beyond 10 years. Historically, 5-year event-free survival (EFS) was 13 -- 30% in HR neuroblastoma before the era of immunotherapy; whereas 5-year progression-free survival (PFS) in HR neuroblastoma treated with 3F8 and GM-CSF in first remission was 56 -- 62% [2,69,70]. The 5-year overall survival (OS) of 61 -- 80% was highly encouraging (Figure 3) [2,69,70]. When myeloid cells were analyzed, granulocyte activation by GMCSF could be quantified by their CD11b activation epitope CBRM1/5, which correlated with the treatment outcome [71]. When NK cells were analyzed, missing ligand for inhibitory killer immunoglobulin-like receptor (KIR) was significantly associated with better tumor control [2,72,73]. ME36.1 and 14. G2a are two other murine anti-GD2 mAbs of the mouse IgG2a subclass (both were class switched from their original mouse IgG3 isotype); they had lower affinities to GD2 (KD = 19 nM and KD = 77 nM, respectively) when compared to mouse 3F8 (KD = 5 nM) [7,74]. 14.G2a was tested in clinical trials with similar side effects and antitumor activity as mouse 3F8 [23].

Chimeric and humanized anti-GD2 mAb In order to reduce HAMA, chimeric (murine human) and humanized anti-GD2 mAb were developed. Chimeric 14.18 (ch14.18) was developed by combining the heavyand light-chain variable regions of mouse 14.18, with the constant regions of the human g1 heavy chain and the human k light chain. Ch14.18 mediates ADCC significantly more efficiently than 14.G2a [75-77]. After a few Phase I/II studies, the Children’s Oncology Group (COG) started a Phase III randomized trial in 2001 of ch14.18 in combination with GM-CSF and IL-2 in patients with HR neuroblastoma, demonstrating significant improvement in OS when compared to no immunotherapy [1]. Although the impact on EFS was not significant [1,78-80], anti-GD2 immunotherapy has been accepted by oncologists world wide as a standard therapy for HR neuroblastoma. However, antibody-induced pain side effects thought to be mediated by complement activation remains a major drawback [81]. Humanized 14.18 K322A was designed to carry a point mutation K332A in the Fc region to eliminate complement activation [81]; however, in a Phase I clinical trial, pain side effects remained [82]. Murine 3F8 (m3F8) was chimerized to ch3F8, and humanized to hu3F8 carrying the human IgG1 g heavy chain [74]. 5.2


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Hu3F8 maintains a slow koff during binding to GD2 (KD = 13 nM) and mediates ADCC more efficiently than m3F8, although CMC is reduced [74]. Currently, Phase I clinical trials of hu3F8 in patients with HR neuroblastoma and GD2(+) tumors are ongoing (NCT01419834, NCT01757626, NCT01662804) [23]. Anti-GD2 antibodies have been used to target various types of payloads to tumors, including isotopes, drugs, cytokines and nanoparticles [7]. Hu14.18-IL2, an immunocytokine where IL2 is targeted to tumors using anti-GD2 mAb, was tested in Phase I/II clinical trials [83-85]. Toxicities from IL-2-related vascular permeability as well as immunogenicity were noted. CD8(+) T cells and NK cells were activated but clinical responses were seen only in patients with minimal disease [84-86]. A chimeric anti-GD2 mAb-IL15 immunocytokine (c.60C3-IL15) demonstrated higher antitumor potency than anti-GD2 mAb alone in preclinical study, but it has yet to be tested in patients [87]. 6.

GD2 as a target for T cells

Bispecific antibody Cancer immunotherapy using antigen-specific T cells has shown great therapeutic potential. Cytotoxic T lymphocytes (CTLs) carrying specific T-cell receptors scan target peptide antigens presented on the MHC class I molecules, and when there is a match, they initiate a powerful tumor lysis program. However, carbohydrate antigens like GD2 are not known to bind to MHC, unless they are attached to peptides [88]. Furthermore, tumor cells often downregulate the MHC class I expression, escaping both the afferent and the efferent arms of the T-cell response. Bispecific antibodies (BsAbs) with dual specificity (GD2 and CD3) can cross-link GD2(+) tumors, even those without MHC, to CD3(+) CTLs, thereby triggering immune synapse formation, perforin and granzyme B injection into tumor cells for cytolysis. 5F11 is another antiGD2 antibody in preclinical development where the concept of affinity maturation [29], epitope neighborhood [29] and multistep targeting (MST) [89] were extensively investigated. More recently, 5F11 was used to test the critical structural properties of tandem single-chain Fv fragment (scFv) BsAb (GD2 CD3) that impact on biologic function, highlighting the importance of VH--VL orientation, linker length and antigen affinity, as well as how they influence thermal stability, T-cell cytotoxicity and in vivo antitumor effect [90]. The importance of bivalency was tested using 3F8 in a chemically conjugated BsAb [6]. The genetic version of a bivalent (GD2 CD3) BsAb was built on the IgG-scFv platform using hu3F8 and huOKT3 showing unusually high potency in its tumoricidal properties both in vitro and in vivo [91]. Although BsAb is able to retarget even Treg to kill tumors through the granzyme--perforin pathway [92], the role of Treg in GD2GD3 BsAb therapy will need to be tested in patients. A Phase I clinical trial of the chemical conjugate BsAb (GD2 CD3) using the 3F8/OKT3 BsAb

combined with cytokines was recently initiated in patients with neuroblastoma, osteosarcoma and GD2(+) sarcoma (NCT 02173093). BsAb also can be used for MST or pretargeting strategies. When drugs, toxins or radioisotopes are carried by IgG (typical serum half life in days), therapeutic index (AUC of the isotope in tumor versus AUC in blood or normal organs) is rarely > 5:1. In MST, a BsAb targeting GD2 and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra-acetic acid (DOTA) is first injected to target to GD2(+) tumor cells (first step). After the circulating free BsAb is removed from circulation using a clearing agent (second step), radiometal (177Lu or 90Y) carried by p-aminobenzyl-DOTA (serum half-life in minutes) is administered (third step). Because of the fast clearance, a therapeutic index of 10 to 100:1 can be routinely achieved. This MST radioimmunotherapy (RIT) has shown excellent curative potential in human tumor xenografts with no obvious clinical or histologic toxicities [93]. Unlike streptavidin-based MST, which was highly immunogenic [89], these fully humanized systems should be more clinically relevant.

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Chimeric antigen receptors Anti-GD2 CAR T cell is another strategy for redirecting CTLs to tumor cells. An antigen-binding cell surface scFv is genetically engineered into CTLs to recognize GD2 on tumor cells without the need to consider their human leukocyte antigen (HLA) expression. Co-stimulatory molecules including CD28, 4-1BB, OX40 fused internally to the scFv can enhance the function and persistence of CAR T cells during the tumoricidal process. In a dual receptor platform, Epstein--Barr virus (EBV)-specific CTLs can be engineered to express anti-GD2 CAR, which can now persist through life by means of constant stimulation in vivo by latent EBV residing in most humans [94]. A Phase I study of EBV-specific anti-GD2 CAR T cells in patients with neuroblastoma showed encouraging antitumor effects and persistence of CAR T cells in blood [4]. Currently, this dual specific concept has been expanded to pools of virus-specific anti-GD2 CAR T cells (e.g., varicella zoster, adenovirus, cytomegalovirus, EBV) since EBV immunity might not be universally present, as well as to third-generation CAR constructs that incorporate two or more co-stimulatory molecules. Phase I clinical trials are actively pursued in neuroblastoma and GD2(+) sarcoma (NCT01460901, NCT01953900, NCT02107963, NCT01822652). NK cells can also be engineered using CARs. Classically, NK cells mediate ADCC by exploiting their FcgRIIIA (CD16A) receptor for antitumor mAbs. Even with CD16A activation, inhibition by inhibitory KIRs can only be partially overcome [95]. That is because the HLA ligands specific for KIRs can be re-expressed or upregulated among tumors following chemoradiotherapy or immunotherapy [72]. This may explain why among patients with neuroblastoma treated with anti-GD2 mAb, those carrying HLA class I ligand, 6.2



mismatch for their NK KIRs had better survival [72]. CARmodified NK cells have been shown to overcome inhibition by KIRs [96]; similar observation was made in preclinical models of neuroblastoma, melanoma and breast cancer [97].


7.

GD2 as a target for drug delivery

Anti-GD2 antibodies have been directly conjugated to radionuclides, toxins and drugs to enhance selectivity of cytotoxic agents. Radiolabeled anti-GD2 mAbs can be highly specific and sensitive in detecting small and occult metastases of GD2(+) tumors by PET radioimmunoscintigraphy. Besides detection, they provide quantitative information on tumor dosimetry if RIT is to be applied. 131I and technetium-99m (99mTc) have been used for labeling 3F8 and ch14.18, respectively, in studying neuroblastoma in preclinical studies and in patients [16,98,99]. Furthermore, direct injection 131I-3F8 into the cerebrospinal fluid (CSF) has been highly effective in consolidating remission among patients with recurrent neuroblastoma metastatic to the CNS, and with relapsed or HR medulloblastoma. These therapies have greatly improved the cure rate with manageable acute toxicities, which included transient headache, nausea, vomiting and fever [3,100]. Nanoparticles and liposomes can be used as carriers of drugs, enzymes, radionuclides, DNA or RNA to GD2. To deliver them to GD2(+) tumors, anti-GD2 IgG antibodies or their derivatives (e.g., scFv) have been tried. Anti-GD2 peptides have also been described, although their affinities have not been optimal [101]. Etoposide, a topoisomerase inhibitor, has been encapsulated into liposomes coupled with anti-GD2 mAbs. When tested on GD2(+) tumors, specific growth inhibition was observed, with the potential that systemic side effects of etoposide could be reduced [102]. Doxorubicin, an anthracycline, encapsulated into liposomes coupled with anti-GD2 Fab’ fragments was shown to inhibit metastatic neuroblastoma growth [103]. MicroRNA-34a, located in the chromosome 1 region commonly deleted in HR neuroblastoma, regulates multiple cancer-related genes, including MYCN, BCL2, SIRT, NOTCH1, JAG1, CCND1, CDK6 and E2F3. In vitro, its overexpression induces the activation of a caspase-mediated apoptosis pathway in neuroblastoma cell lines [104]. MicroRNA-34a-loaded silicabased nanoparticles coupled with anti-GD2 mAbs could target neuroblastoma cells and inhibit tumor growth [104]. When plasmid DNA is encapsulated into nanoparticles, anti-GD2 scFv can target them to MSCs in vitro for gene transfection [105]. Besides providing the proof of concept for selective plasmid delivery to any GD2(+) cells, it offers a novel strategy for genetic engineering of GD2(+) stem cells in tumor populations (e.g., breast cancer and mesenchymal cancers) both in vitro and in vivo. The potential of exploiting MSCs through GD2 is another exciting development in regenerative or cancer medicine [34,106-108].

Modulating the expression of GD2 in human malignancies

8.

GD2 expression on human melanoma is upregulated by IL-4 alone and in combination with IFN-g and TNF [109]. N-(4-hydroxyphenyl) retinamide (4-HPR) is a synthetic derivative of vitamin A, and 4-HPR treatment can enhance GD2 expression on neuroblastoma cell by accumulating the dihydroceramides, which are precursors of GD2, resulting in the enhancement of antitumor effects of anti-GD2 mAb [110]. 9.

GD2 vaccines

Since anti-GD2 mAb showed efficacy against neuroblastoma, using vaccines to induce anti-GD2 antibody response seemed logical. However, carbohydrate antigens are generally poorly immunogenic. By conjugating the GD2 antigen to a highly immunogenic foreign keyhole limpet hemocyanin (KLH), and administering it in the presence of a strong immune adjuvant QS-21, GD2--KLH vaccine could induce antibody response to eliminate GD2(+) micrometastatic tumor in preclinical studies [111,112]. Since anti-GD2 antibody response to the GD2--KLH conjugate vaccine was not consistent, a GD2 lactone (GD2L)--KLH vaccine was developed [113]. When tested in patients with melanoma, this conjugate vaccine induced anti-GD2 antibodies that could mediate CDC in patients, although the antibody response was transient and no long-term clinical benefit was seen [113]. However, in patients with HR neuroblastoma who re-achieved remission after one or multiple relapses, GD2L--KLH combined with GD3L--KLH and OPT-821 (QS 21 equivalent) induced anti-GD2 and anti-GD3 IgG antibody response, and the majority (> 75%) remained alive and well with long followup [114]. In addition to poor immunogenicity, gangliosides have been difficult to synthesize. To circumvent these limitations, GD2-peptide mimotopes were developed [115] to induce IgG responses in preclinical models. An anti-idiotypic mAb is another strategy to circumvent the poor immunogenicity of GD2. They can be made from rats or mice immunized with 3F8 [116] or 14G2a [83,117], respectively, and shown to induce anti-GD2 antibodies in preclinical models. These antibodies in the idiotype network were, in fact, detected in neuroblastoma patients treated with anti-GD2 mAbs [118,119]. As vaccines, these anti-idiotype antibodies mimicking GD2 have minimal toxicities, characterized by local swelling at the site of injection, low-grade fever and chills. However, measurable clinical responses in patients with melanoma treated with these vaccines were rare [120,121]. DNA vaccines encoding anti-idiotype antibodies or GD2 mimotopes have also been tested in preclinical models [122,123]. They have been shown to elicit anti-GD2 antibody response, to activate NK cells to inhibit the progression of neuroblastoma in mice, although the responses have been modest [122,124].


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10.

Expert opinion

Despite the clinical successes of GD2-specific mAbs, our knowledge of the biology of GD2 both in normal development and in tumorigenesis is generally inadequate. Of interest is the early and consistent expression of GD2 among neural crest precursors. How and what it interacts with in the lipid rafts or glycosynapses are largely unknown [125]. The structural biology of such interactions has not been carefully examined. On the other hand, how its expression is regulated, likely a result of the glycotransferases, in embryogenesis and in cancer, or in health and during sickness, is even less understood. Through the interaction of cell surface GD2 with integrins, cells such as the neural crest precursors or tumor cells most likely travel along tracks in the extracellular matrix (e.g., interacting with fibronectin) to reach distant sites. It is plausible that the embryonic machinery of the neural crest cell is now usurped by tumor cells or stem cells to travel far distances. Bridging these knowledge gaps should facilitate a rational drug design directed at GD2 and its associated signaling pathways. The tissue restriction of GD2 expression to the gray matter of the CNS and to select stem cells, when contrasted with its presence in a broad spectrum of human cancers has elevated its relevance as a target in human medicine. While exploiting this ganglioside for cancer therapeutics seems obvious, the ability to target genes via GD2 selectively to neural stem cells, sympathoadrenergic precursors, hippocampal neurons and MSCs, both ex vivo and in vivo, may offer opportunities for tissue-specific strategies in regenerative medicine. Unlike protein targets with variable homologies between their human and nonhuman counterparts, GD2 is a universal antigen identical in all known species, empowering any successful strategy with immediate relevance for other animal hosts, for example, in treating canine osteosarcoma [126] or melanoma [127], which are both GD2(+). Like many carbohydrate antibodies, GD2-specific mAb can cross-react with its epitope neighbors in its synthesis pathway, such as GD1b and GD3. Since GD1b is on the sensory neurons, this could account for some of the pain and sensory neuron toxicities from anti-GD2 antibody therapy. Unique among carbohydrate and specifically ganglioside antigens, with each additional sugar, a unique epitope is created. Hence, understanding the synthesis pathways is equivalent to mapping out the entire epitope neighborhood around the ganglioside of interest [29]. This has greatly facilitated the evolution of the next-generation anti-GD2 antibodies while preserving antigen specificity. Since each of the gangliosides neighboring GD2 can now be quantified by analytical methods [128], their differential expression and quantitation in tissues become highly relevant so as to appreciate the consequence of epitope spread. Similarly, typical for carbohydrate antibodies, GD2-specific mAbs tend to have low affinity. With the availability of high-throughput phage displays

8

and yeast displays, affinity maturation strategies, crystal structures [60], docking algorithms and in silico designs [7,129], both specificity and affinity can be greatly and rapidly improved. Although antibody--drug and antibody--radionuclides have been tested extensively, with few exceptions, their clinical relevance can be enhanced if the therapeutic index is improved. Using IgGs and scFvs directly conjugated to the toxic payloads are generally suboptimal because of their suboptimal AUC ratios. Multistep or pretargeting strategies have the tremendous promise in substantial improvement of the therapeutic ratio; they also provide a versatile platform to deliver a wide variety of payloads, including drugs, toxins, a-particle-emitting radioisotopes, nanoparticles or liposomes as long as they can be coupled with the small ligand DOTABn [93]. It provides a targeting efficiency much like the biotin--streptavidin system [89], but without its immunogenicity limitations. Perhaps the biggest challenge and implication for medicine is the ability to use recombinant antibodies to drive cells to GD2(+) sites. As professional killers, T cells hold the most promise. The advent of bispecific antibodies to redirect T cells or CAR to gene-modified T cells has already shown striking tumor responses in preclinical models and in early clinical trials. When they are combined with recombinant cytokines like IL-15 or T-cell checkpoint inhibitors, such as anti-PD1 or anti-PDL1, that potential can be further amplified. While T cells have so far provided a sound proof of principle, similar approaches can be applied to target other cell types to the tumor or to neuronal tissues. While the conventional wisdom is to inject GD2-targeted therapies intravenously, it is increasingly obvious that compartmental therapy (e.g., CSF to reach leptomeningeal disease [LM], intraperitoneal to reach malignant ascites or mesothelial seeding) can be even more effective. Partly because of the small volume of distribution in these compartments, but mostly because of the unique physiology of CSF or peritoneal fluid flows [23]; off target side effects could be substantially reduced. Intrathecal anti-GD2 antibodies have produced surprisingly few short-term or long-term side effects, while maintaining patients in long-term remissions after CNS or LM metastasis [3,130,131]. The biggest roadblock so far in the development of IgGbased GD2-targeted therapy is the pain side effect and immunogenicity of anti-GD2 antibodies [1,2,82]. Despite the removal of C1q binding and the reduction/elimination of complement activation, pain remains a major side effect [82]. It is notable that iodinated or heat-modified forms of 3F8 have decreased Fc-mediated functions and substantially less pain [132]. Furthermore, T cells redirected by CAR to GD2 have no pain side effects [94]. These observations suggest that a better understanding of the pain mechanism and appropriate engineering of these antibodies could overcome these major obstacles.



Acknowledgement We want to thank Irene Y Cheung, Mahiuddin Ahmed, and Joseph Olechnowicz for their valuable advice and suggestions.

Declaration of interest The authors were supported by Grants from the Department of Defense (PR111043), Enid A Haupt Endowed Chair, Kids


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International Symposium of Pediatric Neuro-Oncology; Singapore; 2014

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Affiliation Maya Suzuki MD & Nai-Kong V Cheung† MD PhD † Author for correspondence Memorial Sloan Kettering Cancer Center, Department of Pediatrics, 1275 York Avenue, New York, NY 10065, USA Tel: +1 646 888 2313; Fax: +1 631 422 0452; E-mail: [email protected]

Disialoganglioside GD2 in human neuroectodermal tumor cell lines and gliomas.

Hyaluronan as a therapeutic target in human diseases.

Protein S-Nitrosylation as a Therapeutic Target for Neurodegenerative Diseases.

TRPV4 as a therapeutic target for joint diseases.

Diacylglycerol kinase as a possible therapeutic target for neuronal diseases.

Angiogenesis as a therapeutic target for obesity and metabolic diseases.

A potent and specific immunotoxin for tumor cells expressing disialoganglioside GD2.

The GSK-3 family as therapeutic target for myocardial diseases.

Successful engineering of a highly potent single-chain variable-fragment (scFv) bispecific antibody to target disialoganglioside (GD2) positive tumors.

Structural design of disialoganglioside GD2 and CD3-bispecific antibodies to redirect T cells for tumor therapy.

Structure Based Refinement of a Humanized Monoclonal Antibody That Targets Tumor Antigen Disialoganglioside GD2.

Heme as a Target for Therapeutic Interventions.

LAMC2 as a therapeutic target for cancers.

Identification and Tumour-Binding Properties of a Peptide with High Affinity to the Disialoganglioside GD2.

Sestrin2: A Promising Therapeutic Target for Liver Diseases.

PERK: a novel therapeutic target for neurodegenerative diseases?

Sestrin2 as a Novel Biomarker and Therapeutic Target for Various Diseases.

Self-renewal as a therapeutic target in human colorectal cancer.

DJ-1 as a human oncogene and potential therapeutic target.

Bcl-2 as a Therapeutic Target in Human Tubulointerstitial Inflammation.

KRAS as a Therapeutic Target.

Ref-1 as an emerging therapeutic target for various human diseases: phytochemical modulation of its functions.

Bispecific antibody does not induce T-cell death mediated by chimeric antigen receptor against disialoganglioside GD2.

The Gut Microbiome as Therapeutic Target in Central Nervous System Diseases: Implications for Stroke.