GD2-Targeted Immunotherapy of Neuroblastoma

Introduction

Cancer immunotherapy has been hailed as a potential “game changer” in the treatment of cancer. However, the number of antigen targets proven effective remains limited and is restricted to protein antigens, with the exception of Dinutuximab, a chimeric antibody directed against GD2, which was approved for the treatment of neuroblastoma, thereby widening the net of potential pharmaceutical targets. Altered glycosylation on glycoproteins and glycolipids is a well-known feature of cancer cells . Many tumor-associated carbohydrate antigens (TACAs), such as Tn, sialyl Tn, TF, Lewis ^x , Lewis ^y , sialyl Lewis ^x , sialyl Lewis ^a , Globo H, stage-specific embryonic antigen-3 (SSEA-3), fucosyl GM1, GM2, Neu5Gc GM3, GD2, GD3, and polysialic acids, have been identified and some of them have been associated with poor prognosis . Many clinical trials targeting various TACAs have been conducted over the past 2 decades, but only five TACA-targeted immunotherapeutics, including Theratope (sialyl-Tn-KLH vaccine), OPT-822 (Globo H-KLH) vaccine, GM2-KLH vaccine, Racotumomab (anti-Neu5GC GM3 idiotypic vaccine), and GD2-directed monoclonal antibody, have reached randomized phase III development. Unfortunately, Theratope failed to demonstrate any benefit in patients with metastatic breast cancer although subgroup analysis revealed survival benefit in patients who were on endocrine therapy . The GM2-KLH vaccine was terminated at the second interim analysis because of the detrimental outcome of overall survival . A randomized phase II/III trial of Racotumomab vaccine in 176 patients with advanced non-small-cell lung cancer (NSCLC) showed significant benefits in progression-free and overall survival and a large scale randomized phase III trial is ongoing to confirm its anticancer effect ( NCT01460472 ). The Globo H-KLH vaccine demonstrated significantly improved progression-free survival in patients who mounted anti-Globo H response in a randomized phase II/III trial . On the other hand, Dinutuximab has demonstrated an impressive improvement of event-free survival and overall survival in patients with high-risk neuroblastoma . This notable advancement led to its regulatory approval in the United States and Europe in 2015 marking the first new immunotherapeutic agent targeting a glycolipid molecule and the first novel therapeutics approved for neuroblastoma. In this chapter, we will elaborate on the past and ongoing development of GD2-targeted immunotherapy of neuroblastoma.

Structure, Biosynthesis, and Distribution of GD2

GD2 is a disialoganglioside belonging to b-series ganglioside. It comprises five monosaccharides linked to ceramide, with the carbohydrate sequence of GalNAcβ1-4(NeuAcα2-8NeuAcα2-3)Galβ1-4Glcβ1-1. The biosynthesis of GD2 starts from β-linked glucose ceramide (GlcCer) in the early Golgi apparatus followed by elongation by a series of glycosyltransferases, including galactosyltransferase I, ST3 beta-galactoside alpha-2,3-sialyltransferase 5 (ST3GAL5 or GM3 synthase), ST8 alpha-N-acetylneuraminate alpha-2,8-sialytransferase 1 (ST8SIA1 or GD3 synthase), and β1,4- N -acetylgalactosaminyltransferase (β4GalNAcT1, GM2/GD2 synthase) in the Golgi apparatus . The expression of GD2 in normal tissues is weak and restricted to the brain, peripheral pain fibers, and skin melanocytes . In contrast, it is abundantly expressed on various types of neuroectodermal cancers such as neuroblastoma (>98%) , melanoma , glioma, and small-cell lung cancer, as well as sarcomas . Notably, the expression of GD2 on primary neuroblastomas reaches ∼10 ⁷ molecules per cell . Moreover, GD2 is also found in cancer stem cells of breast adenocarcinoma and malignant phyllodes tumor of the breast . Therefore, GD2 is an ideal candidate for targeted cancer therapy.

Functions of GD2

Expression of GD2 has been found in various types of stem cells such as embryonic neural stem cells , mesenchymal stem cells (MSCs) , and neural progenitor cells . In mouse, a subpopulation of GD2-expressing bone marrow-derived mesenchymal stem cells (BM-MSCs) has been found to possess potent stemness ability. These cells displayed embryonic stem cell markers SSEA-1 and Nanog, but not hematopoietic cell markers CD45 and CD11b. The GD2 ⁺ BM-MSCs exhibited greater proliferative and clonogenic capabilities as well as better differentiation potential to adipocytes and osteoblasts, as compared to unsorted BM-MSCs . In line with these data, we have shown that malignant phyllodes tumor of the breast contained a subpopulation of GD2 ⁺ ALDH ⁺ cells, which can be induced to differentiate into neural cells of various lineages. Furthermore, the GD2 ⁺ ALDH ⁺ breast cancer cells had greater mammosphere forming ability and higher tumor-initiating frequency than GD2 ⁻ ALDH ⁻ cells, suggesting that these GD2 ⁺ ALDH ⁺ breast cancer cells possessed cancer stem cell characteristics . Similar observations have been made in H-Ras oncogene-transformed human mammary epithelial (HMLER) cells. GD2 ⁺ HMLER cells formed more mammospheres in vitro than GD2 ⁻ HMLER cells. Majority of these GD2 ⁺ HMLER cells expressed phenotype breast cancer stem cell markers and had a similar gene signature as CD44 ⁺ CD24 ⁻ breast cancer stem cells. Interestingly, induction of epithelial-mesenchymal transition by ectopic expression of either Twist or Snail in HMLER cells enhanced the percentage of GD2 ⁺ populations, along with elevated levels of GD3 synthase . Contrarily, Woo et al. found that GD2 ^high glioblastoma multiforme cancer cells had similar neurosphere formation capacity as those GD2 ^low cells . In short, GD2 ⁺ cells may be a unique subset with stemness potential in breast cancer and MSC, but not glioblastoma.

GD2 specific monoclonal antibodies have been used to examine the molecular mechanisms of GD2 in cancer cells. After removal of the Melur melanoma cells from the glass coverslips using EDTA, the GD2 was found in focal adhesion plaques by immunofluorescence staining with the GD2-specific monoclonal antibody (mAb) 126, suggesting that attachment of cultured human melanoma cells may require the direct involvement of GD2 . Incubation of GD2 positive mouse lymphoma EL4 cells with anti-GD2 mAb 14G2a resulted in changes of morphology and increase of DNA fragmentation and cell death . In human osteosarcoma cell lines Saos-2, MG-63, and SJSA-1, mAb 14G2a reduced not only cell invasion but also cell viability. The inhibition of cell invasion by mAb 14G2a was attributed to decreased expression of metalloproteinase-2 mRNA and protein, activation of phosphatidylinositide 3-kinase (PI3K), and phosphorylation of Akt . Similarly, 14G2a inhibited the activity of Akt, a mechanistic target of rapamycin (mTOR), p70S6 and 4E-BP1 , and suppressed matrix metalloproteinase-2 (MMP-2) activation by binding to CD166 in neuroblastoma cell line Neuro2a . Furthermore, mAb 14G2a induced apoptosis by activating caspase three in human neuroblastoma cell line IMR-32 . Another report showed that 14G2a decreased Aurora kinases and MYCN and induced p53 and PHLDA1 protein expression in IMR-32 cells . A microarray study of mAb 14G2a-treated IMR-32 cells revealed upregulation of JUN (jun oncogene), SVIL (supervillin) and RASSF6 (Ras association RalGDS/AF-6 domain family member 6), and downregulation of ID1 (inhibitor of DNA binding 1, dominant negative helix-loop-helix protein) and TLX2 (thyroid adenoma associated) . In line with this, the treatment of melanoma cells with another anti-GD2 mAb, 3F8, inhibited cell growth and induced apoptosis through activation of caspase 3-, 7-, and -8-dependent pathways, inhibition of the expression of anti-apoptotic molecules and cytochrome c , and augmentation of the release of caspase-9-independent apoptosis-inducing factor (AIF) from mitochondria in human melanoma HTB63 cells . In human neuroblastoma SH-SY5Y-TrkB cells, 3F8 was shown to activate Src-family kinases, phosphorylate N-methyl- d -aspartate (NMDA) receptor NR2B subunits, induce Ca ²⁺ fluxes, produce cAMP, and alter cellular morphology .

Another strategy to study the role of GD2 in cancer cells is overexpression/inhibition of GD3 synthase or GM2/GD2 synthase although this strategy may include effects not restricted to GD2. In GD3 synthase-expressing triple negative (ER ⁻ , PR ⁻ , and Her2 ⁻ ) breast cancer cell MDA-MB-231, silencing of GM2/GD2 synthase with siRNA has been shown to reduce c-Met phosphorylation and decrease proliferation. Furthermore, the proliferation of the GD3 synthase-expressing MDA-MB-231 cells decreased in the presence of anti-GD2 mAb 4G2. Moreover, anti-GD2 antibody but not anti-GD3 mAb 4F6 also diminished the phosphorylation of c-Met . Along the same line, knockout of GM2/GD2 synthase in Renca-v cells, using transcription activator-like effector nucleases technology, significantly increased cell death in the low attachment plates, suggesting that products of GM2/GD2-synthase could protect the cells from undergoing anoikis .

A few reports investigated the roles of GD2 in cells using purified GD2 ceramide. Coculture of human platelets with GD2 ceramide in vitro increased the adhesion of platelets to collagen through upregulating integrin α2β1-mediated tyrosine phosphorylation of focal adhesion kinase . As to the immune functions, Ladisch et al. showed the inhibitory activity of purified GD2 ceramide on T cell proliferation . Similarly, GD2 shed from renal cell carcinoma was shown to be incorporated by circulating T cells and induced apoptosis of the GD2-inserted T cells . In addition, purified GD2 inhibited the expression of CD83 and CD86 on CD34 ⁺ bone marrow progenitors, suggesting that GD2 impeded differentiation of CD34 ⁺ cells into dendritic cells . These findings are consistent with the notion that GD2 may act as an immune checkpoint. Interestingly, the neoexpression of GD2 on T cells has been found to cluster with T cell receptor upon stimulation of CD4 ⁺ T cells with anti-CD3/anti-CD28 antibodies. In line with this, the proliferation of CD4 T cells and the expression of IL-2/IL-2 receptor are associated with the induction of the GM2/GD2 synthase . However, CD4 ⁺ T-cell activation was normal in GM2/GD2 synthase-null mice, which express only GM3 and GD3 but no GD2 . These findings suggest that GD2 may promote tumor growth while modulating immune activation.

Cancer Immunotherapeutics

Active and passive immunotherapies are two main strategies of cancer immunotherapy. Active cancer immunotherapy aims to harness the host's immune system to attack cancer cells, whereas passive immunotherapy is to deliver tumor antigen-specific monoclonal antibodies to kill cancer cells directly or through complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). Since the initial approval of rituximab (anti-CD20 mAb) for the treatment of lymphoma in 1994, more than 50 monoclonal antibodies targeting 14 antigens have been approved for passive immunotherapy of cancer and all target protein antigens except dinutuximab (anti-GD2). Of note, the approval of ipilimumab (anti-CTLA-4) for the treatment of melanoma in 2011 as the first monoclonal antibody targeting an immune checkpoint molecule on T cells, not tumor cells , heralded a new era of game-changing cancer immunotherapy. The success of ipilimumab was closely followed by the approval of additional immune-checkpoint inhibitors, including nivolumab (Opdivo) and pembrolizumab (Keytruda) , which target PD-1 for the treatment of patients with advanced melanoma and metastatic NSCLC, and Atezolizumab (Tecentriq, Genetech) and Avelumab (Bavencio, EMD Serono), which is directed against PD-1 ligand 1 (PD-L1) for the treatment of locally advanced or metastatic urothelial carcinoma and metastatic Merkel-cell carcinoma, respectively. The promising results of these agents ensure the emergence of many more immune checkpoint blockers on the horizon.

In contrast to the growing momentum of passive immunotherapy, sipuleucel-T ( Provenge , Dendreon) is the only active immunotherapeutics approved to date . Sipuleucel-T is an autologous cellular vaccine for the treatment of metastatic prostate cancer. Autologous dendritic cells isolated from peripheral blood of patients are activated ex vivo by PA2024, which is a fusion protein combing prostatic acid phosphatase (PAP) with granulocyte macrophage colony-stimulating factor (GM-CSF). After activation, the PAP-loaded antigen-presenting cells are infused into the patient to stimulate T cells to target PAP-expressing cancer cells. It opened a new era of personalized medicine. Although hailed as a trailblazing biotechnology, it failed to gain commercial success due to the cost and requirement for freshly prepared cells with modest efficacy. Recently, a new personalized therapy, tisagenlecelucel (Kymriah, Novartis) which is autologous anti-CD19 chimeric antigen receptor (CAR) T cells, has been approved by Food and Drug Administration of the United States for the treatment of children and young adults with B-cell acute lymphoblastic leukemia. Preparation of tisagenlecleucel takes about 22 days, including isolating the T cells from the patient, genetically engineering the T cells to express anti-CD19, expanding them ex vivo before injecting these CD19-targeting T cells into the patient. In a multicenter clinical trial involving 63 patients with relapsed or resisted B-cell ALL, 83% of patients treated with tisagenlecleucel were in remission within 3 months of treatment. It was shortly followed by the approval of another CD19-directed CAR T-cell therapy, axicabtagene ciloleucel (Yescarta, Kite Pharma) for diffuse B-cell lymphoma, based on the impressive objective response rate of 82%, and the complete response rate of 54% in 110 patients with refractory B-cell lymphoma The CAR T-cell therapy will become a frequently used cancer treatment for hematological malignancies in the future.

GD2-Specific Antibodies

Various types of GD2-specific antibodies including murine monoclonal antibodies, chimeric monoclonal antibody, humanized monoclonal antibody, monoclonal antibody fused with a cytokine, and bispecific antibody, have been generated. One of them, ch14.18 has received regulatory approval for the treatment of high-risk neuroblastoma.