ganglioside--gd2 and Neoplasm-Metastasis

The ability to utilize preclinical models to predict the clinical toxicity of chimeric antigen receptor (CAR) T cells in solid tumors is tenuous, thereby necessitating the development and evaluation of gated systems. Here we found that murine GD2 CAR-T cells, specific for the tumor-associated antigen GD2, induce fatal neurotoxicity in a costimulatory domain-dependent manner. Meanwhile, human B7H3 CAR-T cells exhibit efficacy in preclinical models of neuroblastoma. Seeking a better CAR, we generated a SynNotch gated CAR-T, GD2-B7H3, recognizing GD2 as the gate and B7H3 as the target. GD2-B7H3 CAR-T cells control the growth of neuroblastoma in vitro and in metastatic xenograft mouse models, with high specificity and efficacy. These improvements come partly from the better metabolic fitness of GD2-B7H3 CAR-T cells, as evidenced by their naïve T-like post-cytotoxicity oxidative metabolism and lower exhaustion profile.

Topics: Animals; Cell Line, Tumor; Cell Survival; Cytotoxicity, Immunologic; Gangliosides; Humans; Immunotherapy, Adoptive; Mice, 129 Strain; Mice, Inbred C57BL; Neoplasm Metastasis; Neuroblastoma; Receptors, Antigen, T-Cell; Receptors, Chimeric Antigen; Tumor Burden; Xenograft Model Antitumor Assays

Breast cancer stem-like cells (BCSC) are implicated in cancer recurrence and metastasis of triple-negative breast cancer (TNBC). We have recently discovered that ganglioside GD2 expression defines BCSCs and that ST8SIA1 regulates GD2 expression and BCSC function. In this report, we show that ST8SIA1 is highly expressed in primary TNBC; its expression is positively correlated with the expression of several BCSC-associated genes such as BCL11A, FOXC1, CXCR4, PDGFRβ, SOX2, and mutations in p53. CRISPR knockout of ST8SIA1 completely inhibited BCSC functions, including

Topics: Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Gangliosides; Gene Expression Regulation, Neoplastic; Humans; Mutation; Neoplasm Metastasis; Neoplastic Stem Cells; Phenotype; Proto-Oncogene Proteins c-akt; Sialyltransferases; Signal Transduction; TOR Serine-Threonine Kinases; Triple Negative Breast Neoplasms; Tumor Suppressor Protein p53

We have identified that the ganglioside GD2 is a marker for breast cancer stem cells (BCSCs), and that targeting the enzyme GD3 synthase (GD3S, which regulates GD2 biosynthesis) reduces breast tumorigenesis. The pathways regulating GD2 expression, and their anomalous functions in BCSC, are unclear. Proteomic analysis of GD2+ and GD2- cells from breast cancer cell lines revealed the activation of NFκB signaling in GD2+ cells. Dose- and time-dependent suppression of NFκB signaling by the small molecule inhibitor BMS-345541 reduced GD2+ cells by > 90%. Likewise, BMS-345541 inhibited BCSC GD3S expression, mammosphere formation, and cell migration/invasion in vitro. Breast tumor-bearing mice treated with BMS-345541 showed a statistically significant decrease in tumor volume and exhibited prolonged survival compared to control mice, with a median survival of 78 d for the BMS-345541-treated group vs. 58 d for the controls. Moreover, in an experimental metastases model, treatment with BMS-345541 reduced the lung metastases by > 5-fold. These data suggest that GD2 expression and function,and NFκB signaling, are related, and they control BCSCs tumorigenic characteristics. Thus, the suppression of NFκB signaling by BMS-345541 is a potentially important advance in controlling breast cancer growth and metastases.

Topics: Animals; Breast Neoplasms; Carcinogenesis; Cell Line, Tumor; Gangliosides; Gene Expression Regulation, Neoplastic; Humans; I-kappa B Kinase; Imidazoles; Mice, Inbred NOD; Mice, Knockout; Mice, SCID; Neoplasm Metastasis; Neoplastic Stem Cells; Quinoxalines; RNA Interference; Sialyltransferases; Signal Transduction; Xenograft Model Antitumor Assays

Advances in the design of chimeric antigen receptors (CARs) have improved the antitumor efficacy of redirected T cells. However, functional heterogeneity of CAR T cells limits their therapeutic potential and is associated with toxicity. We proposed that CAR expression in Vα24-invariant natural killer T (NKT) cells can build on the natural antitumor properties of these cells while their restriction by monomorphic CD1d limits toxicity. Primary human NKT cells were engineered to express a CAR against the GD2 ganglioside (CAR.GD2), which is highly expressed by neuroblastoma (NB). We compared CAR.GD2 constructs that encoded the CD3ζ chain alone, with CD28, 4-1BB, or CD28 and 4-1BB costimulatory endodomains. CAR.GD2 expression rendered NKT cells highly cytotoxic against NB cells without affecting their CD1d-dependent reactivity. We observed a striking T helper 1-like polarization of NKT cells by 4-1BB-containing CARs. Importantly, expression of both CD28 and 4-1BB endodomains in the CAR.GD2 enhanced in vivo persistence of NKT cells. These CAR.GD2 NKT cells effectively localized to the tumor site had potent antitumor activity, and repeat injections significantly improved the long-term survival of mice with metastatic NB. Unlike T cells, CAR.GD2 NKT cells did not induce graft-versus-host disease. These results establish the potential of NKT cells to serve as a safe and effective platform for CAR-directed cancer immunotherapy.

Topics: 4-1BB Ligand; Animals; Antigens, CD1d; Cancer Vaccines; CD28 Antigens; Cell Line; Cell Proliferation; Cytokines; Cytotoxicity, Immunologic; Gangliosides; Graft vs Host Disease; Humans; Immunotherapy; Lymphocyte Activation; Macrophages; Mice; Natural Killer T-Cells; Neoplasm Metastasis; Neuroblastoma; Protein Structure, Tertiary; Receptors, Antigen; Retroviridae; Transduction, Genetic; Treatment Outcome

Immunocytokines (ICKs) targeting cytokines to the tumor environment using antibodies directed against a tumor-associated antigen often have a higher therapeutic index than the corresponding unconjugated cytokines. Various ICKs displaying significant antitumoral effects in several murine tumor models have already been developed, and some of them, in particular interleukin (IL)-2-based ICKs, are in Phase II clinical trials. Although sharing common biological activities with IL-2 in vitro, IL-15 is now considered as having a better potential in antitumor immunotherapeutical strategies and has been shown to be less toxic than IL-2 in preclinical studies. We previously developed the fusion protein RLI, linking a soluble form of human IL-15Rα-sushi+ domain to human IL-15. RLI showed better biological activities than IL-15 in vitro as well as higher antitumoral effects in vivo in murine and human cancer models. Here, we investigated, in the context of an ICK, the effect of associating RLI with an antibody targeting the GD2 ganglioside, a validated tumoral target expressed on many neurectodermal tumors. Anti-GD2-RLI fully retained the cytokine potential of RLI and the antibody effector functions (antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity). It displayed strong antitumor activities in two syngeneic cancer models in immunocompetent mice (subcutaneous EL4 and metastatic NXS2). Its therapeutic potency was higher than those of RLI and anti-GD2 alone or in combination. We suggest that this is related to its bifunctional (cytokine and antibody) nature.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Neoplasm; Antibody-Dependent Cell Cytotoxicity; Antigens, Neoplasm; Cell Line, Tumor; Cell Proliferation; Female; Gangliosides; Humans; Immunotherapy; Interleukin-15; Interleukin-15 Receptor alpha Subunit; Liver Neoplasms; Lymphoma, T-Cell; Male; Mice; Mice, Inbred A; Mice, Inbred C57BL; Neoplasm Metastasis; Neuroblastoma; Protein Binding; Recombinant Fusion Proteins

Genetic engineering of human T lymphocytes to express tumor-directed chimeric antigen receptors (CAR) can produce antitumor effector cells that bypass tumor immune escape mechanisms that are due to abnormalities in protein-antigen processing and presentation. Moreover, these transgenic receptors can be directed to tumor-associated antigens that are not protein-derived, such as the ganglioside GD2, which is expressed in a high proportion of melanoma cells.. We generated chimeric T cells specific for the ganglioside GD2 by joining an extracellular antigen-binding domain derived from the GD2-specific antibody sc14.G2a to cytoplasmic signaling domains derived from the T-cell receptor zeta-chain, with the endodomains of the costimulatory molecules CD28 and OX40. We expressed this CAR in human T cells and assessed the targeting of GD2-positive melanoma tumors in vitro and in a murine xenograft.. Upon coincubation with GD2-expressing melanoma cells, CAR-GD2 T lymphocytes incorporating the CD28 and OX40 endodomains secreted significant levels of cytokines in a pattern comparable with the cytokine response obtained by engagement of the native CD3 receptor. These CAR-T cells had antimelanoma activity in vitro and in our xenograft model, increasing the survival of tumor-bearing animals.. Redirecting human T lymphocytes to the tumor-associated ganglioside GD2 generates effector cells with antimelanoma activity that should be testable in subjects with disease.

Topics: Animals; Antibodies; Gangliosides; Genetic Engineering; Humans; Immunotherapy; Melanoma; Mice; Mice, SCID; Neoplasm Metastasis; Receptors, Antigen, T-Cell; T-Lymphocytes; Xenograft Model Antitumor Assays

Disialoganglioside GD2 is an established target for immunotherapy in neuroblastoma. We tested the hypothesis that active immunization against the glycolipid GD2 using DNA vaccines encoding for cyclic GD2-mimicking decapeptides (i.e., GD2 mimotopes) is effective against neuroblastoma. For this purpose, two GD2 peptide mimotopes (MA and MD) were selected based on docking experiments to anti-GD2 antibody ch14.18 (binding free energy: -41.23 kJ/mol for MA and -48.06 kJ/mol for MD) and Biacore analysis (K(d) = 12.3 x 10(-5) mol/L for MA and 5.3 x 10(-5) mol/L for MD), showing a higher affinity of MD over MA. These sequences were selected for DNA vaccine design based on pSecTag2-A (pSA) also including a T-cell helper epitope. GD2 mimicry was shown following transfection of CHO-1 cells with pSA-MA and pSA-MD DNA vaccines, with twice-higher signal intensity for cells expressing MD over MA. Finally, these DNA vaccines were tested for induction of tumor protective immunity in a syngeneic neuroblastoma model following oral DNA vaccine delivery with attenuated Salmonella typhimurium (SL 7207). Only mice receiving the DNA vaccines revealed a reduction of spontaneous liver metastases. The highest anti-GD2 humoral immune response and natural killer cell activation was observed in mice immunized with the pSA-MD, a finding consistent with superior calculated binding free energy, dissociation constant, and GD2 mimicry potential for GD2 mimotope MD over MA. In summary, we show that DNA immunization with pSA-MD may provide a useful strategy for active immunization against neuroblastoma.

Topics: Animals; Cancer Vaccines; Cell Line, Tumor; CHO Cells; Cricetinae; Gangliosides; Interferon-gamma; Mice; Neoplasm Metastasis; Neuroblastoma; Vaccination; Vaccines, DNA

Established s.c. NXS2 murine neuroblastoma tumors exhibited transient resolution after suboptimal therapy using the hu14.18-IL2 immunocytokine (IC). The hu14.18-IL2 IC is a fusion protein that has linked a molecule of interleukin 2 (IL-2) to the COOH terminus of each of the IgG heavy chains on the humanized anti-GD(2) monoclonal antibody hu14.18. To induce more potent and longer lasting in vivo antitumor effects, we tested hu14.18-IL2 IC in a regimen combining it with constant infusion IL-2 in NXS2 tumor-bearing mice. The addition of the constant infusion IL-2 augmented the antitumor response induced by treatment with the hu14.18-IL2 IC in animals with experimentally induced hepatic metastases and in animals bearing localized s.c. tumors. The combined treatment induced prolonged tumor eradication in most animals bearing s.c. tumors and involved both natural killer cells and T cells. The enhanced ability of this combined treatment to prevent tumor recurrence was not observed when a larger dose of hu14.18-IL2 IC, similar in IL-2 content to the IC plus systemic IL-2 regimen, was tested as single-agent therapy. Animals showing prolonged tumor eradication of established tumors after the combined hu14.18-IL2 plus IL-2 regimen exhibited a protective T-cell-dependent antitumor memory response against NXS2 rechallenge.

Topics: Animals; Antibodies, Monoclonal; Cell Line, Tumor; Cytotoxicity, Immunologic; Female; Gangliosides; Humans; Interleukin-2; Killer Cells, Natural; Liver Neoplasms, Experimental; Mice; Mice, Inbred Strains; Neoplasm Metastasis; Neuroblastoma; Recombinant Fusion Proteins; Spleen; Time Factors

Gangliosicle GD2 is abundant on human neuroblastoma (NB). Monoclonal antibody 3F8 targeted to GD2 may have imaging and therapeutic potential. Antigen-negative clones can escape immune-mediated attack leading to clinical resistance or recurrence.. Among 95 evaluable patients treated intravenously with 3F8 (94 Stage 4, 1 Stage 3), 66 received nonradiolabeled 3F8, 11 received 131-iodine-labeled-3F8 (8-28 mCi/kg) with autologous bone marrow rescue, and 18 received both forms of treatment. Prior to treatment, 90 patients tested positive for GD2 reactivity by bone marrow immunofluorescence (n = 68), tumor immunohistochemistry (n = 20), or diagnostic radioimmunoscintigraphy (n = 2).. Of 62 patients who had refractory or recurrent neuroblastoma following 3F8 treatment, 61 (98%) tested positive for GD2 reactivity by bone marrow immunofluorescence (n = 51) or tumor immunohistochemistry (n = 10). The sole tumor that lost GD2 expression underwent phenotypic transformation into a pheochromocytoma-like tumor.. The persistence of GD2 expression in refractory or recurrent NB suggests that complete antigen loss is an uncommon event and cannot account for treatment failure.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Antibody Specificity; Antigens, Neoplasm; Antineoplastic Combined Chemotherapy Protocols; Biomarkers, Tumor; Bone Marrow Transplantation; Cell Lineage; Child, Preschool; Combined Modality Therapy; Fatal Outcome; Female; Ganglioneuroblastoma; Gangliosides; Humans; Immunization, Passive; Immunoconjugates; Immunoglobulin G; Iodine Radioisotopes; Male; Neoplasm Metastasis; Neuroblastoma; Radioimmunodetection; Radioimmunotherapy; Remission Induction; Retrospective Studies; Transplantation, Autologous

The inability of current therapy to prevent metastases arising from uveal melanoma often results in patient mortality. With the goal of developing a treatment for metastasis, gangliosides were studied as potential tumor-associated antigens. Our report describes the production of a metastatic liver variant (MH) from a human uveal melanoma cell line (SP6.5). Cells were injected into nude mouse spleens and liver metastases collected 2 months later. After 21 days of in vitro subculture, the cells were re-injected into normal nude mice spleen; 10 cycles (MH10) were performed. Gangliosides were extracted, purified, chromatographed on HPTLC plates and sprayed with a resorcinol-HCl reagent, the sialic acid spots being quantified by densitometry. Gangliosides were analyzed in each metastatic liver variant and compared with the SP6.5 s.c. tumor. The results showed a significant increase in GM3 and a significant decrease in GD3 and GD2 in the last metastatic variants obtained (MH5, MH8, MH9 and MH1O) compared with the primary s.c. tumor, SP6.5. Such evolution in the ganglioside pattern was maintained throughout the progression of the different liver variants. Our results indicate that precursor ganglioside GM3 and gangliosides GD3 and GD2 could be associated with neoplastic evolution of malignancy of human uveal melanoma in nude mice.

Topics: Animals; G(M3) Ganglioside; Gangliosides; Humans; Liver Neoplasms; Melanoma; Mice; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Tumor Cells, Cultured; Uveal Neoplasms

Two human melanoma cell lines, derived from metastases of two patients with epithelioid malignant amelanotic melanomas, and designated IIB-MEL-LES and IIB-MEL-IAN, have been established. Both cell lines have been in continuous culture over 2 years and were propagated continuously for 85 and 75 serial passages, respectively. Morphologically, IIB-MEL-LES is composed predominantly of spindle shaped cells, whereas IIB-MEL-IAN grows as a monolayer of cuboid and stellate shaped cells with many rounded cells in suspension. Immunocytochemical studies revealed that both cell lines express S-100 protein, vimentin, and GD3 and GD2 gangliosides but are negative for keratin and collagen. Both cell lines express HLA class I and HLA-DR antigens in variable proportions. The MAGE-1 gene is expressed only by the IIB-MEL-IAN cell line, as revealed by PCR analysis. Cytogenetic analysis of both cell lines revealed abnormal karyotypes; the modal chromosome numbers of IIB-MEL-LES and IIB-MEL-IAN were 48 and 81, respectively. IIB-MEL-LES cells presented rearrangements in chromosomes 1, 14 and X, gains in chromosomes 10, 20, and 21 losses in chromosomes 15 and Y. The most frequent markers observed in IIB-MEL-IAN cells were 7q+, 10p+, 2p+, i(6p), 2q+, and 10q-. Clonal gains were observed in chromosomes 12 and 21, whereas losses were seen in chromosomes 1, 2, 3, 4, 6, 7, 11, and 17. Both cell lines were capable of forming colonies in soft agar and developed tumors when transplanted into nude mice, reproducing and maintaining the characteristics of the original tumors. These cell lines and their xenografts appear to provide useful systems for studying the biology, genetics and histogenesis of human malignant melanoma and could be utilized for the development of melanoma vaccines.

Topics: Adult; Animals; Base Sequence; DNA Restriction Enzymes; DNA, Neoplasm; Gangliosides; Histocompatibility Antigens Class I; HLA-DR Antigens; Humans; Immunohistochemistry; Immunophenotyping; Karyotyping; Male; Melanoma, Amelanotic; Mice; Mice, Nude; Molecular Sequence Data; Neoplasm Metastasis; Neoplasm Transplantation; S100 Proteins; Tumor Cells, Cultured; Vimentin

The glycosphingolipid patterns were analyzed on two clones derived from a human melanoma cell line and selected for their respectively high and low metastatic ability in immunosuppressed newborn rats. Conversely to the weakly metastatic cells which exhibited a pattern similar to that of the parental cell line, highly metastatic human melanoma cells appeared to be deficient in ganglioside biosynthesis. An accumulation of lactosylceramide was found in the latter cells, with low amounts of GM3 as the only ganglioside detected and a fourfold decreased activity of GM3 synthase (EC 2.4.99.9). After subcutaneous injection of metastatic cells in newborn rats, the cells proliferating in the tumor induced at the injection site re-expressed the four common gangliosides of melanoma: GM3, GM2, GD3 and GD2, whereas the cells growing in the lungs as metastatic nodules were deficient in ganglioside synthesis and showed an accumulation of lactosylceramide. Taken together, our results suggest that the human melanoma cells which are able to escape from the primary tumor and invade the lungs have an impaired ganglioside biosynthesis with a deficient GM3 synthase.

Topics: Animals; Animals, Newborn; Antigens, CD; G(M2) Ganglioside; G(M3) Ganglioside; Gangliosides; Glycosphingolipids; Humans; Lactosylceramides; Melanoma; Neoplasm Metastasis; Neoplasm Transplantation; Rats; Sialyltransferases; Tumor Cells, Cultured

Topics: Antibodies, Monoclonal; Gangliosides; Humans; Immunotherapy; Iodine Radioisotopes; Neoplasm Metastasis; Neuroblastoma

The cell membranes of human melanoma express a tumor-associated ganglioside, GD2. We previously established a human melanoma cell line, M14-A, that metastasizes to the lung, liver, skin, lymph nodes, and abdominal organs of nude mice in addition to forming ascites and pleural effusions. We also reported the successful in vitro production of human IgM monoclonal antibody to GD2. In the present study, we evaluated the GD2 expression of human melanoma cells at the primary and metastatic sites and their reactivity to human monoclonal anti-GD2 antibody in vivo. GD2 was expressed strongly on the melanoma cells from both primary and metastatic sites, except for cells from pleural effusions and ascites. When M14-A-bearing nude mice received systemic injections of the human monoclonal antibody, the anti-GD2 titer in the sera was reduced markedly at 2 hours, whereas the reduction was minimal in sera from tumor-free mice and mice bearing GD2-negative human M24 cells. The immune adherence test confirmed that antibody was fixed on cells of primary subcutaneous M14-A tumors and on their metastases to liver, lung, abdominal organs and skin. These results suggest that this large molecule protein can penetrate the blood-tumor barrier and bind immunologically to antigen-positive melanoma cells in vivo.

Topics: Animals; Antibodies, Monoclonal; Complement System Proteins; Female; Fluorescent Antibody Technique; Gangliosides; Guinea Pigs; Humans; Immune Adherence Reaction; Immunoglobulin M; Male; Melanoma; Mice; Mice, Inbred Strains; Mice, Nude; Neoplasm Metastasis; Neoplasm Transplantation; Rosette Formation; Transplantation, Heterologous