ganglioside--gd2 and Osteosarcoma

Novel therapies to treat patients with solid cancers that have developed resistance to chemotherapy represent unmet needs of considerable dimensions. In the present review, we will address the attempts to develop chimeric antigen receptor (CAR) targeted immunotherapy against osteosarcoma (OS). This aggressive cancer displays its peak incidence in children and young adults. The main cause of patient death is lung metastases with a 5-year survival as low as 5%-10% in the primary metastatic setting and 30% in the relapse situation, respectively. Effective adjuvant combination chemotherapy introduced more than 40 years ago improved the survival rates from below 20% to around 60% in patients; however, since then, no major breakthroughs have been made. The use of immune checkpoint inhibitors has been disappointing in OS, while other types of immunotherapies such as CAR T cells remain largely unexplored. Indeed, for CAR T-cell therapy to be efficacious, two main criteria need to be fulfilled: (a) CAR T cells should target an epitope selectively expressed on the cell surface of OS in order to prevent toxicities in normal tissues and (b) the target should also be widely expressed on OS metastases. These challenges have already been undertaken in OS and illustrate the difficulties in developing tomorrow's CAR-T treatment in a solid tumour. We will discuss the experiences with CAR-T therapy development and efficacy to combat the clinical challenges in OS.

Topics: Bone Neoplasms; Cancer-Associated Fibroblasts; Endopeptidases; Gangliosides; Gelatinases; Humans; Immunotherapy, Adoptive; Membrane Proteins; Osteosarcoma; Receptor, ErbB-2; Receptor, IGF Type 1; Receptors, Interleukin-11; Serine Endopeptidases; Tumor Microenvironment

To evaluate the toxicity, immunogenicity, and pharmacokinetics of a human-mouse chimeric monoclonal antibody (mAb) ch 14.18 directed against disialoganglioside (GD2) and to obtain preliminary information on its clinical efficacy, we conducted a phase I trial in 10 patients with refractory neuroblastoma and one patient with osteosarcoma.. Eleven patients were entered onto this phase I trial. They received 20 courses of mAb ch 14.18 at dose levels of 10, 20, 50, 100, and 200 mg/m2. Dose escalation was performed in cohorts of three patients; intrapatient dose escalation was also permitted.. The most prevalent toxicities were pain, tachycardia, hypertension, fever, and urticaria. Most of these toxicities were dose-dependent and rarely noted at dosages of 20 mg/m2 and less. Although the maximum-tolerated dose was not reached in this study, clinical responses were observed. These included one partial (PR) and four mixed responses (MRs) and one stable disease (SD) among 10 assessable patients. Biologic activity of ch 14.18 in vivo was shown by binding of ch 14.18 to tumor cells and complement-dependent cytotoxicity of posttreatment sera against tumor target cells. An anti-ch 14.18 immune response was detectable in seven of 10 patients studied.. In summary, with the dose schedule used, ch 14.18 appears to be clinically safe and effective, and repeated mAb administration was not associated with increased toxicities. Further clinical trials of mAb ch 14.18 in patients with neuroblastoma are warranted.

Topics: Adult; Animals; Antibodies, Monoclonal; Child; Child, Preschool; Complement C3; Complement C4; Complement Hemolytic Activity Assay; Female; Fluorescent Antibody Technique, Indirect; Gangliosides; Humans; Infusions, Intravenous; Male; Mice; Neuroblastoma; Osteosarcoma; Pain; Recombinant Fusion Proteins; Treatment Outcome

A phase I trial of a murine anti-ganglioside (GD2) monoclonal antibody (mAb) 14G2a was conducted in 14 neuroblastoma patients and 1 osteosarcoma patient to assess its safety, toxicity and pharmacokinetics in pediatric patients. The pharmacokinetics of mAb 14G2a were biphasic with a t alpha 1/2 of 2.8 +/- 2.8 h and a t beta 1/2 of 18.3 +/- 11.8 h. In general, t beta 1/2 was dose-dependent with a level of significance of P = 0.036, and it reached a plateau at doses of 250 mg/m2 or more. Overall the peak serum levels were dose-dependent at P < 0.001. However, they demonstrated an abrupt increase between doses of 100 mg/m2 and 250 mg/m2. The latter two suggest a saturable mechanism for mAb elimination. In addition, peak serum concentrations were observed earlier at higher mAb doses, which indicates the achievement of a steady state. The t beta 1/2 of mAb 14G2a in children appears to be shorter than in adults. Furthermore, 2 patients demonstrated a considerable decrease in t beta 1/2 following retreatment with 14G2a. This was paralleled by high human anti-(mouse Ig) antibody levels. This study represents the first comprehensive analysis of murine mAb pharmacokinetics in children and will be useful in the future design of mAb therapy.

Topics: Adolescent; Age Factors; Antibodies, Monoclonal; Child; Dose-Response Relationship, Immunologic; Female; Gangliosides; Humans; Male; Metabolic Clearance Rate; Neuroblastoma; Osteosarcoma

Chimeric antigen receptor (CAR) T-cell therapy of pediatric sarcomas is challenged by the paucity of targetable cell surface antigens. A candidate target in osteosarcoma (OS) is the ganglioside G. We aimed to identify mechanisms that upregulate G. G. Expression of G

Topics: Benzamides; Biphenyl Compounds; Bone Neoplasms; Brefeldin A; Cell Culture Techniques; Cytotoxicity, Immunologic; Enhancer of Zeste Homolog 2 Protein; Gangliosides; Humans; Morpholines; Osteosarcoma; Protein Synthesis Inhibitors; Pyridones; Surface Properties; T-Lymphocytes; Tumor Cells, Cultured

The cure rate for metastatic osteosarcoma has not substantially improved over the past decades. Clinical trials of anti-HER2 trastuzumab or anti-GD2 dinutuximab for metastatic or refractory osteosarcoma were not successful, and neither was immune checkpoint inhibitors (ICIs).. We tested various target antigen expressions on osteosarcoma cell lines using flow cytometry and analyzed in vitro T cell engaging BsAb (T-BsAb)-dependent T cell-mediated cytotoxicity using 4-h. GD2 and HER2 were chosen from a panel of surface markers on osteosarcoma cell lines and PDXs. Anti-GD2 BsAb or anti-HER2 BsAb exerted potent anti-tumor effect against osteosarcoma tumors in vitro and in vivo. T cells armed with anti-GD2-BsAb (GD2-EATs) or anti-HER2-BsAb (HER2-EATs) showed significant anti-tumor activities as well. Anti-PD-L1 combination treatment enhanced BsAb-armed T cell function in vivo and improved tumor control and survival of the mice, when given sequentially and continuously.. Anti-GD2 and anti-HER2 BsAbs were effective in controlling osteosarcoma. These data support the clinical investigation of GD2 and HER2 targeted T-BsAb treatment in combination with immune checkpoint inhibitors, particularly anti-PD-L1, in patients with osteosarcoma to improve their treatment outcome.

Topics: Animals; Antibodies, Bispecific; Antineoplastic Agents, Immunological; Bone Neoplasms; Cell Line, Tumor; Gangliosides; Humans; Male; Mice, Inbred BALB C; Osteosarcoma; Receptor, ErbB-2; T-Lymphocytes

The cell membrane glycolipid GD2 is expressed by multiple solid tumors, including 88% of osteosarcomas and 98% of neuroblastomas. However, osteosarcomas are highly heterogeneous, with many tumors exhibiting GD2 expression on <50% of the individual cells, while some tumors are essentially GD2-negative. Anti-GD2 immunotherapy is the current standard of care for high-risk neuroblastoma, but its application to recurrent osteosarcomas, for which no effective therapies exist, has been extremely limited. This is, in part, because the standard assays to measure GD2 expression in these heterogeneous tumors are not quantitative and are subject to tissue availability and sampling bias. To address these limitations, we evaluated a novel, sensitive radiotracer [

Topics: Animals; Antibodies, Monoclonal; Apoptosis; Bone Neoplasms; Cell Proliferation; Gangliosides; Humans; Immunotherapy; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Recurrence, Local; Osteosarcoma; Positron-Emission Tomography; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Cisplatin is an effective chemotherapeutic agent for osteosarcoma (OS) and has been shown to induce endoplasmic reticulum (ER) stress-associated apoptosis in human cancer cells. Ganglioside GD2-specific antibodies can inhibit tumor cell viability without involvement of the immune system. A recent study has shown that antiGD2 monoclonal antibody (mAb) 14G2a effectively inhibits the viability and invasiveness of human OS cells. In this study, we explored the effect of anti-GD2 mAb and cisplatin alone and in combination on ER stress-associated apoptosis in osteosarcoma cells. MG-63 and Saos-2 human OS cells were treated with cisplatin and/or an-GD2 mAb 14G2a for 48 hours. Cisplatin and 14G2a dose-dependently induced apoptosis in MG-63 and Saos-2 cells. They in combination induced 70%-77% of apoptosis in MG-63 cells and 79%-85% of apoptosis in Saos-2 cells, exhibiting a synergistic effect stronger than addition of their individual effects over the control level. Showing no significant effect on the expression of protein kinase RNA-like ER kinase (PERK), cisplatin and 14G2a exhibited a marked synergistic effect on inducing phosphorylation/activation of PERK, phosphorylation/inactivation of eukaryotic translation initiation factor 2α (eIF2α), expression of CHOP, in parallel to inducing the caspase-3 activity and apoptosis in MG-63 and Saos-2 cells. The effects were abolished by lentivirus-mediated knockdown of PERK. Particularly, PERK knockdown abolished 63% and 65% of the combined apoptotic effect of cisplatin and 14G2a on MG-63 and Saos-2 cells, respectively. In conclusion, this study provides the first evidence supporting that cisplatin and 14G2a synergize to induce ER stress-associated apoptosis in human OS cells through activating the PERK ER stress pathway by synergistically inducing phosphorylation/activation of PERK. Our findings add new insights into the pharmacologic effects of anti-GD2 mAb in anticancer treatment and suggest that cisplatin plus anti-GD2 mAb could be a new effective therapeutic strategy for OS.

Topics: Antibodies, Monoclonal; Antineoplastic Agents; Apoptosis; Bone Neoplasms; Cell Line, Tumor; Cisplatin; Drug Synergism; eIF-2 Kinase; Endoplasmic Reticulum Stress; Enzyme Activation; Gangliosides; Gene Knockdown Techniques; Humans; Osteosarcoma; Phosphorylation; Signal Transduction

Survival outcomes for patients with osteosarcoma have remained stagnant over the past 30 years. Targeting of ganglioside GD2, a glycosphingolipid on the cell surface of some tumors, with immunotherapy has resulted in improved outcomes for patients with neuroblastoma. In the current study, the expression pattern of GD2 was examined in osteosarcoma.. Immunohistochemistry was performed on osteosarcoma samples from patients at the time of initial biopsy, definitive surgery, and disease recurrence. The intensity and location of staining were scored. Cell-based enzyme-linked immunoadsorbent assay was performed on osteosarcoma cell lines to quantitate the level of GD2 expression.. Forty-four osteosarcoma samples were evaluated by immunohistochemistry, including 8 samples from the initial biopsy, 28 samples from the definitive surgery, and 8 samples from the time of disease recurrence. GD2 was expressed on all 44 osteosarcoma samples. Osteosarcoma tissue obtained at the time of disease recurrence demonstrated a higher intensity of staining compared with samples obtained at initial biopsy and definitive surgery (P = .016). The majority of osteosarcoma cell lines expressed GD2 at higher levels than the neuroblastoma cell line BE(2)-C.. Ganglioside GD2 is highly expressed on osteosarcomas. Clinical trials are needed to assess the efficacy of targeting GD2 in patients with osteosarcoma.

Topics: Antibodies, Monoclonal; Biopsy; Bone Neoplasms; Gangliosides; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Recurrence, Local; Neuroblastoma; Osteosarcoma; Primary Cell Culture; Radioimmunotherapy

Endothelin-1 (ET-1)/endothelin A receptor (ETAR) signaling is important for osteosarcoma (OS) progression. Monoclonal antibodies (mAbs) targeting ganglioside GD2 reportedly inhibit tumor cell viability independent of the immune system. A recent study suggests that ganglioside GD2 may play an important role in OS progression. In the present study, we for the first time explored the effects of anti-GD2 mAb alone or in combination with ETAR antagonist on OS cell invasiveness and viability. Human OS cell lines Saos-2, MG-63 and SJSA-1 were treated with control IgG (PK136 mAb, 50 µg/mL), anti-GD2 14G2a mAb (50 µg/mL), selective ETAR antagonist BQ123 (5 µM), or 14G2a (50 µg/mL)+BQ123 (5 µM). Cells with knockdown of ETAR (ETAR-shRNA) with or without 14G2a mAb treatment were also tested. Cells treated with selective phosphatidylinositide 3-kinase (PI3K) inhibitor BKM120 (50 µM) were used as a positive control. Our results showed that BQ123, ETAR-shRNA and 14G2a mAb individually decreased cell invasion and viability, matrix metalloproteinase-2 (MMP-2) expression and activity, PI3k activity, and phosphorylation at serine 473 (ser473) of Akt in OS cells. 14G2a mAb in combination with BQ123 or ETAR-shRNA showed significantly stronger inhibitory effects compared with each individual treatment. In all three cell lines tested, 14G2a mAb in combination with BQ123 showed the strongest inhibitory effects. In conclusion, we provide the first in vitro evidence that anti-ganglioside GD2 14G2a mAb effectively inhibits cell invasiveness, MMP-2 expression and activity, and cell viability in human OS cells. ETAR antagonist BQ123 significantly enhances the inhibitory effects of 14G2a mAb, likely mainly through inhibiting the PI3K/Akt pathway. This study adds novel insights into OS treatment, which will serve as a solid basis for future in vivo studies on the effects of combined treatment of OS with anti-ganglioside GD2 mAbs and ETAR antagonists.

Topics: Aminopyridines; Antibodies, Monoclonal; Cell Line, Tumor; Cell Survival; Endothelin A Receptor Antagonists; Gangliosides; Humans; Morpholines; Neoplasm Invasiveness; Osteosarcoma; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors

The expression and implications of gangliosides in human osteosarcomas have not been systematically analyzed. In this study, we showed that gangliosides GD3 and GD2 are highly expressed in the majority of human osteosarcoma cell lines derived from oral cavity regions. Introduction of GD3 synthase cDNA into a GD3/GD2-negative (GD3/GD2-) human osteosarcoma subline resulted in the establishment of GD3/GD2+ transfectant cells. They showed increased cell migration and invasion activities in wound healing and Boyden chamber invasion assays, respectively, compared to the control cells. When treated with serum, GD3/GD2+ cells showed stronger tyrosine phosphorylation of p130Cas, focal adhesion kinase, and paxillin than GD3/GD2- cells. In particular, paxillin underwent much stronger phosphorylation, suggesting its role in cell motility. Furthermore, we tried to dissect the roles of GD3 and GD2 in the malignant properties of the transfectant cells by establishing single ganglioside-expressing cells, that is, either GD3 or GD2. Although GD3/GD2+ cells showed the most malignant properties, GD2+ cells showed almost equivalent levels to GD3/GD2+ cells in invasion and migration activities, and in the intensities of tyrosine phosphorylation of paxillin. Among Src family kinases, Lyn was expressed predominantly, and was involved in the invasion and motility of GD3- and/or GD2-expressing transfectants. Furthermore, it was elucidated by gene silencing that Lyn was located in a different pathway from that of FAK to eventually lead paxillin activation. These results suggested that GD2/GD3 are responsible for the enhancement of the malignant features of osteosarcomas, and might be candidate targets in molecular-targeted therapy.

Topics: Bone Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Crk-Associated Substrate Protein; Focal Adhesion Kinase 1; Gangliosides; Gene Expression; Gene Silencing; Humans; Osteosarcoma; Paxillin; Phosphorylation; Sialyltransferases; Signal Transduction; src-Family Kinases; Tyrosine