-
Y Wang,
J Tan,
M Sutton-Smith,
D Ditto,
M Panico,
R M Campbell, N M Varki,
J M Long,
J Jaeken,
S R Levinson,
A Wynshaw-Boris,
H R Morris,
D Le,
A Dell,
H Schachter,
J D Marth
[show abstract]
[hide abstract]
ABSTRACT: The congenital disorders of glycosylation (CDGs) are recent additions to the repertoire of inherited human genetic diseases. Frequency of CDGs is unknown since most cases are believed to be misdiagnosed or unrecognized. With few patients identified and heterogeneity in disease signs noted, studies of animal models may provide increased understanding of pathogenic mechanisms. However, features of mammalian glycan biosynthesis and species-specific variations in glycan repertoires have cast doubt on whether animal models of human genetic defects in protein glycosylation will reproduce pathogenic events and disease signs. We have introduced a mutation into the mouse germline that recapitulates the glycan biosynthetic defect responsible for human CDG type IIa (CDG-IIa). Mice lacking the Mgat2 gene were deficient in GlcNAcT-II glycosyltransferase activity and complex N-glycans, resulting in severe gastrointestinal, hematologic, and osteogenic abnormalities. With use of a lectin-based diagnostic screen for CDG-IIa, we found that all Mgat2-null mice died in early postnatal development. However, crossing the Mgat2 mutation into a distinct genetic background resulted in a low frequency of survivors. Mice deficient in complex N-glycans exhibited most CDG-IIa disease signs; however, some signs were unique to the aged mouse or are prognostic in human CDG-IIa. Unexpectedly, analyses of N-glycan structures in Mgat2-null mice revealed a novel oligosaccharide branch on the "bisecting" N-acetylglucosamine. These genetic, biochemical, and physiologic studies indicate conserved functions for N-glycan branches produced in the Golgi apparatus among two mammalian species and suggest possible therapeutic approaches to GlcNAcT-II deficiency. Our findings indicate that human genetic disease due to aberrant protein glycosylation can be modeled in the mouse to gain insights into N-glycan-dependent physiology and the pathogenesis of CDG-IIa.
Glycobiology 01/2002; 11(12):1051-70. · 3.58 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A novel mouse Siglec (mSiglec-F) belonging to the subfamily of Siglec-3-related Siglecs has been cloned and characterized. Unlike most human Siglec-3 (hSiglec-3)-related Siglecs with promiscuous linkage specificity, mSiglec-F shows a strong preference for alpha2-3-linked sialic acids. It is predominantly expressed in immature cells of the myelomonocytic lineage and in a subset of CD11b (Mac-1)-positive cells in some tissues. As with previously cloned Siglec-3-related mSiglecs, the lack of strong sequence similarity to a singular hSiglec made identification of the human ortholog difficult. We therefore conducted a comprehensive comparison of Siglecs between the human and mouse genomes. The mouse genome contains eight Siglec genes, whereas the human genome contains 11 Siglec genes and a Siglec-like gene. Although a one-to-one orthologous correspondence between human and mouse Siglecs 1, 2, and 4 is confirmed, the Siglec-3-related Siglecs showed marked differences between human and mouse. We found only four Siglec genes and two pseudogenes in the mouse chromosome 7 region syntenic to the Siglec-3-related gene cluster on human chromosome 19, which, in contrast, contains seven Siglec genes, a Siglec-like gene, and thirteen pseudogenes. Although analysis of gene maps and exon structures allows tentative assignments of mouse-human Siglec ortholog pairs, the possibility of unequal genetic recombination makes the assignments inconclusive. We therefore support a temporary lettered nomenclature for additional mouse Siglecs. Current information suggests that mSiglec-F is likely a hSiglec-5 ortholog. The previously reported mSiglec-3/CD33 and mSiglec-E/MIS are likely orthologs of hSiglec-3 and hSiglec-9, respectively. The other Siglec-3-like gene in the cluster (mSiglec-G) is probably a hSiglec-10 ortholog. Another mouse gene (mSiglec-H), without an apparent human ortholog, lies outside of the cluster. Thus, although some duplications of Siglec-3-related genes predated separation of the primate and rodent lineages (about 80-100 million years ago), this gene cluster underwent extensive duplications in the primate lineage thereafter.
Journal of Biological Chemistry 12/2001; 276(48):45128-36. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Siglecs are immunoglobulin superfamily member lectins that selectively recognize different types and linkages of sialic acids, which are major components of cell surface and secreted glycoconjugates. We report here a human Siglec-like molecule (Siglec-L1) that lacks a conserved arginine residue known to be essential for optimal sialic acid recognition by previously known Siglecs. Loss of the arginine from an ancestral molecule was caused by a single nucleotide substitution that occurred after the common ancestor of humans with the great apes but before the origin of modern humans. The chimpanzee Siglec-L1 ortholog remains fully functional and preferentially recognizes N-glycolylneuraminic acid, which is a common sialic acid in great apes and other mammals. Reintroducing the ancestral arginine into the human molecule regenerates the same properties. Thus, the single base pair mutation that replaced the arginine on human Siglec-L1 is likely to be evolutionarily related to the previously reported loss of N-glycolylneuraminic acid expression in the human lineage. Siglec-L1 and its chimpanzee Siglec ortholog also have a different expression pattern from previously reported Siglecs because they are found on the lumenal edge of epithelial cell surfaces. Notably, the human genome contains several Siglec-like pseudogenes that have independent mutations that would have replaced the arginine residue required for optimal sialic acid recognition. Thus, additional changes in the biology of sialic acids may have taken place during human evolution.
Journal of Biological Chemistry 11/2001; 276(43):40282-7. · 4.77 Impact Factor
-
Trends in Molecular Medicine 09/2001; 7(8):377. · 10.35 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Metastasis is a multistep cascade initiated when malignant cells penetrate the tissue surrounding the primary tumor and enter the bloodstream. Classic studies indicated that blood platelets form complexes around tumor cells in the circulation and facilitate metastases. In other work, the anticoagulant drug heparin diminished metastasis in murine models, as well is in preliminary human studies. However, attempts to follow up the latter observation using vitamin K antagonists failed, indicating that the primary mechanism of heparin action was unrelated to its anticoagulant properties. Other studies showed that the overexpression of sialylated fucosylated glycans in human carcinomas is associated with a poor prognosis. We have now brought all these observations together into one mechanistic explanation, which has therapeutic implications. Carcinoma cells expressing sialylated fucosylated mucins can interact with platelets, leukocytes and endothelium via the selectin family of cell adhesion molecules. The initial organ colonization of intravenously injected carcinoma cells is attenuated in P-selectin-deficient mice, in mice receiving tumor cells pretreated with O-sialoglycoprotease (to selectively remove mucins from cell surfaces), or in mice receiving a single dose of heparin prior to tumor cell injection. In each case, we found that formation of a platelet coating on cancer cells was impeded, allowing increased access of leukocytes to the tumor cells. Several weeks later, all animals showed a decrease in the extent of established metastasis, indicating a long-lasting effect of the short-term intervention. The absence of obvious synergism amongst the three treatments suggests that they all act via a common pathway. Thus, a major mechanism of heparin action in cancer may be inhibition of P-selectin-mediated platelet coating of tumor cells during the initial phase of the metastatic process. We therefore suggest that heparin use in cancer be re-explored, specifically during the time interval between initial visualization of a primary tumor until just after definitive surgical removal.
Brazilian Journal of Medical and Biological Research 07/2001; 34(6):711-7. · 1.13 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The successful induction of T cell-mediated protective immunity against poorly immunogenic malignancies remains a major challenge for cancer immunotherapy. Here, we demonstrate that the induction of tumor-protective immunity by IL-12 in a murine neuroblastoma model depends entirely on the CXC chemokine IFN-gamma-inducible protein 10 (IP-10). This was established by in vivo depletion of IP-10 with mAbs in mice vaccinated against NXS2 neuroblastoma by gene therapy with a linearized, single-chain (sc) version of the heterodimeric cytokine IL-12 (scIL-12). The efficacy of IP-10 depletion was indicated by the effective abrogation of scIL-12-mediated antiangiogenesis and T cell chemotaxis in mice receiving s.c. injections of scIL-12-producing NXS2 cells. These findings were extended by data demonstrating that IP-10 is directly involved in the generation of a tumor-protective CD8+ T cell-mediated immune response during the early immunization phase. Four lines of evidence support this contention: First, A/J mice vaccinated with NXS2 scIL-12 and depleted of IP-10 by two different anti-IP-10 mAbs revealed an abrogation of systemic-protective immunity against disseminated metastases. Second, CD8+ T cell-mediated MHC class I Ag-restricted tumor cell lysis was inhibited in such mice. Third, intracellular IFN-gamma expressed by proliferating CD8+ T cells was substantially inhibited in IP-10-depleted, scIL-12 NXS2-vaccinated mice. Fourth, systemic tumor protective immunity was completely abrogated in mice depleted of IP-10 in the early immunization phase, but not if IP-10 was depleted only in the effector phase. These findings suggest that IP-10 plays a crucial role during the early immunization phase in the induction of immunity against neuroblastoma by scIL-12 gene therapy.
The Journal of Immunology 07/2001; 166(11):6944-51. · 5.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Human intestinal epithelial cells secrete an array of chemokines known to signal the trafficking of neutrophils and monocytes important in innate mucosal immunity. We hypothesized that intestinal epithelium may also have the capacity to play a role in signaling host adaptive immunity. The CC chemokine macrophage inflammatory protein (MIP)-3alpha/CCL20 is chemotactic for immature dendritic cells and CD45RO(+) T cells that are important components of the host adaptive immune system. In these studies, we demonstrate the widespread production and regulated expression of MIP-3alpha by human intestinal epithelium. Several intestinal epithelial cell lines were shown to constitutively express MIP-3alpha mRNA. Moreover, MIP-3alpha mRNA expression and protein production were upregulated by stimulation of intestinal epithelial cells with the proinflammatory cytokines tumor necrosis factor-alpha or interleukin-1alpha or in response to infection with the enteric bacterial pathogens Salmonella or enteroinvasive Escherichia coli. In addition, MIP-3alpha was shown to function as a nuclear factor-kappaB target gene. In vitro findings were paralleled in vivo by increased expression of MIP-3alpha in the epithelium of cytokine-stimulated or bacteria-infected human intestinal xenografts and in the epithelium of inflamed human colon. Mucosal T cells, other mucosal mononuclear cells, and intestinal epithelial cells expressed CCR6, the cognate receptor for MIP-3alpha. The constitutive and regulated expression of MIP-3alpha by human intestinal epithelium is consistent with a role for epithelial cell-produced MIP-3alpha in modulating mucosal adaptive immune responses.
AJP Gastrointestinal and Liver Physiology 05/2001; 280(4):G710-9. · 3.43 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Independent studies indicate that expression of sialylated fucosylated mucins by human carcinomas portends a poor prognosis because of enhanced metastatic spread of tumor cells, that carcinoma metastasis in mice is facilitated by formation of tumor cell complexes with blood platelets, and that metastasis can be attenuated by a background of P-selectin deficiency or by treatment with heparin. The effects of heparin are not primarily due to its anticoagulant action. Other explanations have been suggested but not proven. Here, we bring together all these unexplained and seemingly disparate observations, showing that heparin treatment attenuates tumor metastasis in mice by inhibiting P-selectin-mediated interactions of platelets with carcinoma cell-surface mucin ligands. Selective removal of tumor mucin P-selectin ligands, a single heparin dose, or a background of P-selectin deficiency each reduces tumor cell-platelet interactions in vitro and in vivo. Although each of these maneuvers reduced the in vivo interactions for only a few hours, all markedly reduce long-term organ colonization by tumor cells. Three-dimensional reconstructions by using volume-rendering software show that each situation interferes with formation of the platelet "cloak" around tumor cells while permitting an increased interaction of monocytes (macrophage precursors) with the malignant cells. Finally, we show that human P-selectin is even more sensitive to heparin than mouse P-selectin, giving significant inhibition at concentrations that are in the clinically acceptable range. We suggest that heparin therapy for metastasis prevention in humans be revisited, with these mechanistic paradigms in mind.
Proceedings of the National Academy of Sciences 04/2001; 98(6):3352-7. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We previously reported an unusual carboxylated modification on N:-glycans isolated from whole bovine lung. We have now raised IgG mAbs against the modification by immunization with biotinylated aminopyridine-derivatized glycans enriched for the anionic species and screening for Abs whose reactivities were abrogated by carboxylate neutralization of bovine lung glycopeptides. One such Ab (mAb GB3.1) was inhibited by carboxylated bovine lung glycopeptides and other multicarboxylated molecules, but not by glycopeptides in which the carboxylate groups were modified. The Ab recognized an epitope constitutively expressed on bovine, human, and other mammalian endothelial cells. Stimulated, but not resting, neutrophils bound to immobilized bovine lung glycopeptides in a carboxylate-dependent manner. The binding of activated neutrophils to immobilized bovine lung glycopeptides was inhibited both by mAb GB3.1 and by soluble glycopeptides in a carboxylate-dependent manner. The Ab also inhibited extravasation of neutrophils and monocytes in a murine model of peritoneal inflammation. This inhibition of cell trafficking correlated with the increased sequestration but reduced transmigration of leukocytes that were found to be adherent to the endothelium of the mesenteric microvasculature. Taken together, these results indicate that these novel carboxylated N:-glycans are constitutively expressed on vascular endothelium and participate in acute inflammatory responses by interaction with activated neutrophils.
The Journal of Immunology 02/2001; 166(1):624-32. · 5.79 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Genetic ras mutations are infrequent in breast cancer but Ras may be pathologically activated in breast cancer by overexpression of growth factor receptors which signal through Ras. Using a highly sensitive, coupled enzymatic assay, we measured Ras activation in 20 breast cancers, two fibroadenomas, and seven normal breast samples. Ras was highly activated compared to benign tissue in 11 of the 20 cancers; 7 of these 11 cancers expressed both the epidermal growth factor (EGF) and ErbB-2/neu/HER-2 receptors with the remaining four cancers with high Ras activation expressing one of these two receptors. In the other nine cancers, Ras activation was similar to that observed in benign breast tissue with none of these cancers expressing the EGF receptor while one expressed the ErbB-2 receptor. None of the cancers tested had an activating K-ras mutation nor did any of the cancers express a truncated EGF receptor or the c-FMS receptor. The activity of mitogen-activated protein (MAP) kinase was high in the cancers, and reflected the degree of Ras activation. In cultured mammary tumor cell lines, we showed that Ras activation was ligand dependent in cells overexpressing the ErbB-2 receptor. Thus, Ras was abnormally activated in breast cancers overexpressing the EGF and/or ErbB-2 receptors indicating there are sufficient ligands in vivo to activate these receptors, and this work provides a basis for new target-based treatments of this disease.
Breast Cancer Research and Treatment 08/2000; 62(1):51-62. · 4.43 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Preclinical studies have demonstrated a relationship between DNA mismatch repair (MMR) status and sensitivity to cisplatin and carboplatin. MMR-deficient cells are resistant to both drugs, and selection for cisplatin resistance in vitro is sometimes accompanied by loss of MMR protein expression. We used immunohistochemical staining techniques to investigate hMLH1 and hMSH2 expression in paired ovarian tumor sections from 54 ovarian cancer patients before and after platinum-based therapy. We sought associations between hMLH1 and hMSH2 protein expression and clinical parameters known to be of prognostic significance as well as response to treatment and overall survival. hMLH1 and hMSH2 staining decreased significantly after platinum-based therapy. The percent of malignant cells that stained positive correlated with the intensity of nuclear staining for both proteins; staining for hMLH1 correlated well with staining for hMSH2. Unexpectedly, expression of nuclear hMLH1 correlated negatively with response to treatment. Expression of nuclear hMLH1 and hMSH2 was positively correlated with pretreatment CA125 level, and expression of nuclear hMSH2 was positively correlated with change in CA125 level after treatment. Tumor stage was associated with expression of nuclear hMSH2, and tumor histological subtype was associated with both hMLH1 and hMSH2 staining. No association was found between expression of either protein and overall survival. These results indicate that the tumor is biologically altered after chemotherapy consistent with treatment-induced selection for cells expressing lower hMLH1 and hMSH2 levels. However, immunohistochemical staining for either hMLH1 or hMSH2 was not highly predictive of drug sensitivity as measured by response or survival.
Clinical Cancer Research 05/2000; 6(4):1415-21. · 7.74 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: T lymphocyte activation evokes distinct changes in cell surface O-glycans. CD8+ T cells undergo an elimination of sialic acid on core 1 O-glycans and an induction of core 2 O-glycans until either apoptotic death or differentiation into memory cells. We find that the ST3Gal-I sialyltransferase is required for core 1 O-glycan sialylation and its deficiency induces core 2 O-glycan biosynthesis. Apoptosis ensues with the loss of peripheral CD8+ T cells in the absence of immune stimulation. Cell surface ligation of the ST3Gal-I substrate CD43 recapitulates this phenotype by a caspase 3-independent mechanism. Control of core 1 O-glycan sialylation in T lymphocytes by ST3Gal-I comprises a homeostatic mechanism that eliminates CD8+ T cells by apoptosis while facilitating the production of viable CD8+ memory T cells.
Immunity 04/2000; 12(3):273-83. · 21.64 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Selectins are adhesion molecules that mediate calcium-dependent cell-cell interactions among leukocytes, platelets, and endothelial cells. The naturally occurring vascular ligands for the selectins are mostly mucin-type glycoproteins. Increased expression and altered glycosylation of mucins are known to be prominent features of carcinoma progression. We have previously shown that all three selectins bind to colon carcinoma cell lines in a calcium-dependent fashion and that carcinoma growth and metastasis formation are attenuated in P-selectin-deficient mice. Here we show that the three recombinant soluble selectins recognize ligands within primary colon carcinoma tissue samples. Affinity chromatography showed that the ligands for all three selectins are O-sialoglycoprotease-sensitive mucins that are recognized in a calcium- and sialic acid-dependent manner. Furthermore, there are separate binding sites on the mucins for each selectin, allowing cross-binding of a single mucin molecule by more than one selectin. We also show that the selectin ligands on purified carcinoma mucins can mediate at least four different pathological interactions among platelets, leukocytes, and endothelial cells. These findings could explain some of the adhesive events of blood-borne tumor cells reported to occur with leukocytes, platelets, and endothelial cells, which are believed to play a part in modulating some early events in tumor metastases.
American Journal Of Pathology 09/1999; 155(2):461-72. · 4.89 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Intestinal epithelial cells produce an array of proinflammatory chemokines that can provide signals to mucosal immune and inflammatory cells. To determine if chemokines can also signal epithelial cells, we characterized the expression of chemokine receptors on human colon epithelial cells in vitro and in vivo.
Expression of chemokine receptor messenger RNAs (mRNAs) by the human colon epithelial cell lines HT-29, HT-29.18.C1, Caco-2, T84, HCA-7, and LS174T was assessed by reverse-transcription polymerase chain reaction. Chemokine receptors on intestinal epithelial cells in vitro were determined by flow cytometry, and expression in vivo was determined by immunostaining of human colon. Interleukin (IL)-8 and growth-related (GRO) alpha secretion were assayed by enzyme-linked immunosorbent assay.
Human colon epithelial cells constitutively expressed mRNAs for an array of CC and CXC chemokine receptors, including CCR1-8 and CXCR4, but little if any CXCR1 or CXCR2. Further studies focused on CXCR4 and CCR5 because mRNA for those chemokine receptors was abundantly expressed by each of the colon epithelial cell lines, and these receptors were present on the cell surface. Analogous to their localization on polarized cell lines, CXCR4 and CCR5 had a predominant apical and, to a lesser extent, basolateral distribution on human enterocytes, as demonstrated by immunostaining of human colon. Human colon epithelial cells stimulated with stromal cell-derived factor 1alpha and macrophage inflammatory protein (MIP)- 1alpha or MIP-1beta, which are the chemokine ligands for CXCR4 or CCR5, up-regulated production of the CXC chemokines IL-8 and GROalpha.
Human colon epithelial cells express chemokine receptors. Human colonocytes have the potential to serve as targets for chemokine signaling.
Gastroenterology 08/1999; 117(2):359-67. · 11.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The disialoganglioside GD3 is a major antigen in human melanomas that can undergo 9-O-acetylation of the outer sialic acid (giving 9-OAc-GD3). Monoclonal antibody SGR37 detects a different modification of the GD3, de-N-acetylation of the 5-N-acetyl group (giving de-N-Ac-GD3). We found that conventional immunohistochemistry of the SGR37 antigen is limited by a reduction in reactivity upon fixation with aldehydes (which presumably react with the free amino group) or with organic reagents (which can extract glycolipids). We optimized conditions for detection of this antigen in unfixed frozen tissue sections and studied its distribution in human tissues and tumors. It is expressed at low levels in a few blood vessels, infiltrating mononuclear cells in the skin and colon, and at moderate levels in skin melanocytes. In contrast, the antigen accumulates at high levels in many melanomas and in some lymphomas but not in carcinomas. In positive melanomas, expression is sometimes more intense and widespread than that of GD3. Both 9-O-acetylation and de-N-acetylation of GD3 seem to occur after its initial biosynthesis. Isotype-matched antibodies against GD3, 9-O-acetyl-GD3 and de-N-acetyl-GD3 were used to compare their subcellular localization and trafficking. 9-O-acetyl-GD3 colocalizes with GD3 predominantly on the cell surface and partly in lysosomal compartments. In contrast, de-N-acetyl-GD3 has a diffuse intracellular location. Adsorptive endocytosis of antibodies indicates that whereas GD3 remains predominantly on the cell surface, de-N-acetyl-GD3 is efficiently internalized into a compartment that is distinct from lysosomes. Rounding up of melanoma cells occurring during growth in culture is associated with relocation of the internal pool of de-N-acetyl-GD3 to the cell surface. Thus, a minor modification of the polar head group of a tumor-associated glycosphingolipid can markedly affect the subcellular localization and trafficking of the whole molecule. The high levels of the SGR37 antigen in melanomas and lymphomas, its selective endocytosis from the cell surface, and its relocation to the cell surface of rounded up cells suggest potential uses in diagnostic or therapeutic approaches to these diseases.
Cancer Research 04/1999; 59(6):1337-46. · 7.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Selectins are adhesion receptors that normally recognize certain vascular mucin-type glycoproteins bearing the carbohydrate structure sialyl-Lewisx. The clinical prognosis and metastatic progression of many epithelial carcinomas has been correlated independently with production of tumor mucins and with enhanced expression of sialyl-Lewisx. Metastasis is thought to involve the formation of tumor-platelet-leukocyte emboli and their interactions with the endothelium of distant organs. We provide a link between these observations by showing that P-selectin, which normally binds leukocyte ligands, can promote tumor growth and facilitate the metastatic seeding of a mucin-producing carcinoma. P-selectin-deficient mice showed significantly slower growth of subcutaneously implanted human colon carcinoma cells and generated fewer lung metastases from intravenously injected cells. Three potential pathophysiological mechanisms are demonstrated: first, intravenously injected tumor cells home to the lungs of P-selectin deficient mice at a lower rate; second, P-selectin-deficient mouse platelets fail to adhere to tumor cell-surface mucins; and third, tumor cells lodged in lung vasculature after intravenous injection often are decorated with platelet clumps, and these are markedly diminished in P-selectin-deficient animals.
Proceedings of the National Academy of Sciences 09/1998; 95(16):9325-30. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A major goal of tumor immunotherapy is the effective eradication of established metastases associated with the induction of a T cell-mediated protective immunity. We achieved this in a poorly immunogenic murine neuroblastoma model by gene therapy with a single chain interleukin 12 (scIL-12) fusion protein that assures equal expression of its p35 and p40 subunits. Thus, NXS2 hybrid neuroblastoma cells (C1300 x dorsal root ganglion cells), which form experimental bone marrow and liver metastases in syngeneic A/J mice, were transduced with a gene encoding murine interleukin 12, monomerized by introduction of a protein linker between the p35 and p40 protein chains of this heterodimeric cytokine. We demonstrate for the first time that subcutaneous vaccination with these transduced cells induces a protective immunity, as indicated by the complete absence of liver and bone marrow metastasis after challenge with NXS2 wild-type tumor cells. Furthermore, vaccination of animals with established liver and bone marrow metastases completely eradicated liver metastases and suppressed bone marrow metastases. The local and systemic immune response against scIL-12-transduced NXS2 cells is largely dependent on CD8(+) T cells. This was demonstrated in vivo by depletion of immunocompetent A/J mice with monoclonal anti-CD4 and anti-CD8 antibodies and in vitro by specific major histocompatibility complex, class I-restricted CD8(+) T cell-mediated killing of NXS2 and their parental C1300 neuroblastoma cells. In conclusion, we demonstrate successful anti-tumor immunotherapy with an scIL-12 fusion protein that could facilitate clinical application of interleukin 12 gene therapy.
Proceedings of the National Academy of Sciences 04/1998; 95(5):2475-80. · 9.68 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Targeted interleukin-2 (IL-2) therapy with a genetically engineered antidisialoganglioside GD2 antibody-IL-2 fusion protein induced a cell-mediated antitumor response that effectively eradicated established bone marrow and liver metastases in a syngeneic model of neuroblastoma. The mechanism involved is exclusively natural killer (NK) cell-dependent, because NK-cell deficiency abrogated the antitumor effect. In contrast, the fusion protein remained completely effective in the T-cell-deficient mice or immunocompetent mice depleted of CD8+ T cells in vivo. A strong stimulation of NK-cell activity was also shown in vitro. Immunohistology of the leukocytic infiltrate of livers from treated mice revealed a strong staining for NK cells but not for CD8+ T cells. The therapeutic effect of the fusion protein was increased when combined with NK-cell-stimulating agents, such as poly I:C or recombinant mouse interferon-gamma. In conclusion, these data show that targeted delivery of cytokines to the tumor microenvironment offers a new strategy to elicit an effective cellular immune response mediated by NK cells against metastatic neuroblastoma. This therapeutic effect may have general clinical implications for the treatment of patients with minimal residual disease who suffer from T-cell suppression after high-dose chemotherapy but are not deficient in NK cells.
Blood 04/1998; 91(5):1706-15. · 9.90 Impact Factor
-
R Xiang,
H N Lode,
C S Dolman,
T Dreier, N M Varki,
X Qian,
K M Lo,
Y Lan,
M Super,
S D Gillies,
R A Reisfeld
[show abstract]
[hide abstract]
ABSTRACT: A recombinant humanized antibody-interleukin 2 fusion protein (huKS1/4-IL-2) was used to direct IL-2 to the tumor microenvironment and elicit a T cell-mediated eradication of established pulmonary and hepatic CT26-KSA colon carcinoma metastases in syngeneic BALB/c mice. This antitumor effect was specific because a fusion protein, which was nonreactive with these tumor cells, failed to exert any such effect. The efficacy of the huKS1/4-IL-2 fusion protein in eliminating metastases was documented because mixtures of monoclonal antibody huKS1/4 with recombinant human IL-2 were ineffective and, at best, only partially reduced tumor load. Two lines of evidence indicated the eradication of metastases and the absence of minimal residual disease in animals treated with the fusion protein: first, the lack of detection of CT26-KSA cells by reverse transcription-PCR, which can detect one tumor cell in 10(6) liver cells; and second, the tripling of life span. The effector mechanism involved in this tumor eradication is dependent on T cells because the IL-2-directed therapy is ineffective in T cell-deficient SCID mice. The essential effector cells were further characterized as CD8+ T cells by in vivo depletion studies. Such T cells, isolated from tumor-bearing mice after fusion protein therapy, elicited MHC class I-restricted cytotoxicity in vitro against colon carcinoma target cells. Taken together, these data indicate that fusion protein-directed IL-2 therapy induces a T cell-dependent host immune response capable of eradicating established colon cancer metastases in an animal tumor model.
Cancer Research 12/1997; 57(21):4948-55. · 7.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Advanced (stage 4) cases of neuroblastoma, a childhood cancer of the nervous system, are associated with high relapse rates, even after intensive chemotherapy, radiotherapy, and autologous bone marrow transplantation. Therefore, the use of monoclonal antibodies directed against the neuroblastoma tumor marker disialoganglioside GD2 (GD2), in combination with recombinant human interleukin 2 (rhIL-2), is under clinical investigation. We hypothesize that targeted cytokine immunotherapy with a recombinant anti-GD2 antibody-interleukin 2 fusion protein (ch14.18-IL-2) is superior to a combination of ch14.18 and rhIL-2. Our purpose was as follows: 1) to develop a syngeneic model for murine neuroblastoma that expresses GD2 and features both experimental and spontaneous metastases to bone marrow and liver, and 2) to assess anti-GD2-targeted IL-2 therapy in this mode.
A hybrid neuroblastoma cell line was used to generate the GD2-positive NXS2 cell line. Bone marrow and liver metastases were quantified by reverse transcription-polymerase chain reaction for tyrosine hydroxylase and by organ weight or counts of macroscopic tumor foci, respectively. All P values reported are two-sided.
Injection of NXS2 cells resulted in disseminated bone marrow and liver metastases exhibiting stable, but heterogeneous expression of GD2. Treatment with fusion protein (10 microg/day for 6 days) effectively suppressed growth of both experimental and spontaneous metastases to bone marrow and liver (P<.001). In contrast, a mixture of rhIL-2 and ch14.18 at equivalent dose levels was inefficient. Only mice treated with ch14.18-IL-2 showed a twofold prolongation in life span (P<.001).
Targeted IL-2 therapy with a ch14.18-IL-2 fusion protein elicits an effective antitumor response. Our data suggest that a study of ch14.18-IL-2 as an adjuvant treatment in patients with minimal residual disease may be of value.
JNCI Journal of the National Cancer Institute 11/1997; 89(21):1586-94. · 13.76 Impact Factor
