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Biomedical subjects

R A Reisfeld

Publications and source records attributed to R A Reisfeld.

At least 91 records · Page 5Linked to original sources
PubMed1994

Effect of tissue inhibitor of the matrix metalloproteinases-2 expression on the growth and spontaneous metastasis of a human melanoma cell line.

In this study, we examined the effect of expression of tissue inhibitor of metalloproteinases-2 (TIMP-2) on the growth and dissemination of a highly metastatic human melanoma cell line (M24net). M24net melanoma cells express a number of matrix metalloproteinases (MMPs), including gelatinase A and B (MMP-2 and MMP-9) and interstitial collagenase (MMP-1) (A. M. P. Montgomery et al., Cancer Res., 53: 693-700, 1993). The activity of these proteases was effectively down-regulated by transfecting M24net cells with complementary DNA-encoding human TIMP-2. Overexpression of TIMP-2 markedly reduced melanoma growth in the skin of immunodeficient mice but did not prevent these highly malignant cells from spontaneously metastasizing to the lungs and lymph nodes of inoculated mice. We provide a mechanism to account for the growth inhibitory property of TIMP-2 based on its ability to regulate M24net cell growth in three-dimensional interstitial collagen. In the presence of this matrix, M24net cells assume a differentiated morphology and have a reduced growth rate. We present evidence that overexpression of TIMP-2 increases the susceptibility of M24net cells to growth inhibition and morphological differentiation by occluding interstitial collagen.

Animals↗
PubMed1994

A recombinant antibody-interleukin 2 fusion protein suppresses growth of hepatic human neuroblastoma metastases in severe combined immunodeficiency mice.

A genetically engineered fusion protein consisting of a human/mouse chimeric anti-ganglioside GD2 antibody (ch14.18) and recombinant human interleukin 2 (rhIL-2) was tested for its ability to target rhIL-2 to tumor sites and stimulate immune effector cells sufficiently to achieve effective tumor cell lysis in vivo. The ch14.18-IL-2 fusion protein proved more effective than equivalent doses of rhIL-2 in suppressing dissemination and growth of human neuroblastoma in an experimental hepatic metastases model of scid (severe combined immunodeficiency) mice reconstituted with human lymphokine-activated killer cells. The ch14.18-IL-2 fusion protein was also more proficient than equivalent doses of rhIL-2 in prolonging the life-span of these animals. This recombinant antibody-cytokine fusion protein may prove useful for future treatment of GD2-expressing human tumors in an adjuvant setting.

Animals↗
PubMed1994

Integrin alpha v beta 3 rescues melanoma cells from apoptosis in three-dimensional dermal collagen.

Human melanoma cells required ligation of the integrin alpha v beta 3 to sustain viability and growth in three-dimensional dermal collagen. Variant melanoma cells, lacking the alpha v subunit, progressed rapidly to apoptosis within this matrix, whereas transfection of these cells with an alpha v cDNA restored alpha v beta 3 expression and prevented apoptosis. Furthermore, inhibition of alpha v beta 3 ligation with a monoclonal antibody promoted cell death. Apoptosis of alpha v(-) cells within this matrix could be overcome by the addition of insulin or serum. However, alpha v(+) melanoma cells had a significant growth advantage in the presence of these growth factors. Initial adhesion of the melanoma cells to type I collagen depended on ligation of alpha 2 beta 1, but these cells can degrade this collagen to expose cryptic alpha v beta 3 binding sites. These findings provide evidence that the survival and growth of transformed cells may be regulated by collagen degradation and integrin-dependent anchorage to this proteolysed matrix.

Apoptosis↗
PubMed1993

Human cytotoxic T-cells suppress the growth of spontaneous melanoma metastases in SCID/hu mice.

Mice with severe combined immunodeficiency (scid) provide an excellent model for studying interactions between human tumor cells and effector cells of the immune system. Because these animals lack functional B and T lymphocytes, they can accept human tumor xenografts and transfer of human effector cells. Here, we determined the ability of a human melanoma-specific, cytotoxic T-cell line (CTL) in suppressing the growth of spontaneously metastasizing human melanoma cells M24 met (HLA-A11, A33) in scid mice. This CTL line was highly cytotoxic and restricted by HLA-A11 against M24 met melanoma cells in vitro but poorly cytotoxic when tested against a human melanoma cell line that did not express HLA-A11. In order to evaluate the efficacy of this CTL line against M24 met melanoma cells in vivo, randomized groups of animals were given injections of either RPMI culture medium, interleukin 2 (IL-2), CTLs, or CTLs + IL-2. IL-2, per se, did not significantly reduce tumor metastases; however, injection of melanoma-specific, HLA-A11 restricted CTLs into scid mice, 1 day postexcision of the previously induced primary tumor, markedly reduced the number of metastatic foci in the lung and decreased metastatic involvement in lymph nodes. The combination of these CTLs with IL-2 proved even more effective, since almost all lung metastases were eradicated and metastatic involvement in both axillary and inguinal lymph nodes was substantially reduced. Our results indicate that these human CTLs maintain their ability for specific killing of metastasizing melanoma cells in scid mice. Our data suggest that reconstitution of scid mice with a specific group of effector cells (step-wise scid/hu) may be helpful for in vivo evaluation of potentially useful cancer immunotherapy modalities.

Animals↗
PubMed1993

Production and regulation of gelatinase B by human T-cells.

Here we describe for the first time the production of gelatinase B (MMP 9; 92-kDa type IV collagenase) by human peripheral blood T-lymphocytes. Expression of this enzyme was found to be dependent on the activation status of these T-cells and to be regulated by Interleukin-2.

Cell Separation↗
PubMed1993

Melanoma-mediated dissolution of extracellular matrix: contribution of urokinase-dependent and metalloproteinase-dependent proteolytic pathways.

Constitutive overexpression of both urokinase and matrix metalloproteinase (MMP) activity is frequently observed in individual malignant tumors. In this study we describe the combined contribution of these distinct enzyme systems to the invasive phenotype of a highly metastatic human melanoma cell line (M24met). M24met cells were found to secrete a spectrum of MMPs, including interstitial collagenase, type IV collagenases (M(r) 92,000 and 72,000 progelatinases), and stromelysin. Urokinase, but not tissue-type plasminogen activator, was detected in M24met-conditioned media and on cell surfaces. The contribution of these enzymes to extracellular matrix dissolution was determined by exploiting specific inhibitors, namely tissue inhibitor of the metalloproteinases-2 and plasminogen activator inhibitor-2. Due to the coexpression of urokinase and MMP-dependent activity, M24met cells were observed to degrade multiple components of the extracellular matrix and to significantly degrade both interstitial and basement membrane matrices. Urokinase-dependent removal of matrix glycoprotein was observed to precede MMP-dependent collagenolysis as a prerequisite rate-limiting step. We present evidence which suggests that this temporal relationship is imposed by the structural architecture of the matrix such that matrix glycoprotein serves to protect associated collagen from MMP-dependent degradation. In addition to mediating significant collagenolysis, MMP activity was further implicated in the dissolution of matrix tropoelastin. Urokinase/plasmin activity was not found to be required for MMP-zymogen activation.

Amino Acid Sequence↗
PubMed1993

A mechanism for neutrophil-mediated lysis of human neuroblastoma cells.

Neutrophils mediate the lysis of human neuroblastoma cells coated with human/mouse chimeric anti-GD2 ganglioside antibody ch14.18. This study examined the mechanism(s) by which this occurs. Neutrophil degranulation was found to be a required step for lysis, since release of granular enzymes from neutrophils correlated with the lysis of antibody-coated neuroblastoma cells. In addition, agents which block degranulation specifically inhibited this process. Antibody-dependent lysis of neuroblastoma cells was enhanced by exposing neutrophils to granulocyte-macrophage colony stimulatory factor. An increased release of lytic granular molecules was found to be responsible for this lymphokine-mediated phenomenon. Among the molecules released from neutrophil granules that were shown to be involved in neuroblastoma cell lysis were defensins, M(r) 3000-4000 neutrophil granular proteins which are known to bind and permeabilize tumor cells. In addition, cathepsin-G, a neutrophil granular protease, was demonstrated for the first time to mediate the lysis of human neuroblastoma cells. The enzymatic activity of cathepsin-G was found to be required for the lysis of these tumor cells, since phenylmethylsulfonyl fluoride blocks the lytic ability of this protein.

Antibodies, Neoplasm↗
PubMed1993

Therapeutic potential of chimeric and murine anti-(epidermal growth factor receptor) antibodies in a metastasis model for human melanoma.

On many tumors, high numbers of epidermal growth factor (EGF) receptors provide a target for antibody-mediated tumor therapy. We evaluate here the therapeutic potential of a mouse/human chimeric anti-(EGF receptor) antibody and compare it to the parental murine antibody in a xenograft model for metastatic melanoma. Our model is based on the human cell line M24met, which overexpresses the EGF receptor and metastasizes spontaneously in SCID mice. Both the chimeric anti-(EGF receptor) antibody (ch225) and the mouse monoclonal antibody (m225) exhibited saturable, high-affinity binding to M24met cells and were equivalent in their ability to target M24met tumors in mice. Neither anti-(EGF receptor) antibodies nor EGF modulated the growth of M24met cells in vitro. Further analysis revealed that the EGF receptor on these cells is not phosphorylated upon EGF binding, indicating an anomalous receptor on these cells. In antibody-dependent cellular cytotoxicity experiments, ch225 and m225 were potent mediators of M24met cytolysis by effector cells. Antibody-mediated cytotoxicity revealed a marked species preference, with ch225 activating human peripheral blood mononuclear cells and m225 activating mouse splenocytes and to a lesser degree mouse macrophages. Neither antibody mediated cytolysis in the presence of human complement. In SCID mice, m225 suppressed spontaneous metastasis considerably while ch225 had only a modest effect. Our data indicate that in the M24met melanoma tumor model, anti-(EGF receptor) antibodies suppress spontaneous metastasis solely by activating immune effector cells.

Animals↗
PubMed1993

Mechanisms of cellular cytotoxicity mediated by a recombinant antibody-IL2 fusion protein against human melanoma cells.

Functional characteristics were established for a genetically engineered fusion protein between human IL2 and mouse/human chimeric mAb 225 directed against human epidermal growth factor receptor (EGFR), aberrantly expressed on human melanoma cells. The emphasis of these studies was on the mechanism(s) of action by which the ch225-IL2 fusion protein mediated cytotoxic killing of human melanoma cells by different human immune effector cells. Ch225-IL2 fusion protein bound to human EGFR with the high affinity of the parental antibody, and was as active as the equivalent amount of rhIL2. Ch225-IL2 enhanced cellular cytotoxicity mediated by freshly separated PBMC, isolated natural killer (NK) cells and activated T cells against melanoma cell lines. NK cells, which constitutively express both Fc gamma RIII and IL2R, interacted with ch225-IL2, mainly through Fc gamma RIII, while the involvement of IL2R was secondary. However, the effect of ch225-IL2 on activated T cells was most likely mediated through IL2R. These results suggest that the genetically engineered ch225-IL2 fusion protein may become a potent immunotherapeutic agent capable of stimulating various immune effector populations to effectively kill human melanoma cells.

Animals↗
PubMed1993

Colony-stimulating factor enhancement of myeloid effector cell cytotoxicity towards neuroectodermal tumour cells.

We conducted experiments to determine the optimal conditions for colony-stimulating factor-enhanced neutrophil- and mononuclear phagocyte-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) using monoclonal antibodies to disialogangliosides expressed on neuroectodermal tumour target cells. Neutrophil ADCC was most effective at effector:target ratios of 100:1, with maximal cytotoxic responses to melanoma target cells generated by 3 h. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) were the most potent stimulators of neutrophil ADCC, and enhanced ADCC activity was inhibited in the presence of antibody to Fc receptor type II (FcRII). GM-CSF and macrophage colony-stimulating factor (M-CSF) treatment of freshly isolated monocytes inhibited antibody-independent cytotoxicity but enhanced antibody-dependent responses. After 3 d in culture with CSF, 3-10-fold enhancement of ADCC against melanoma target cells was observed at effector:target cell ratios of 10:1. Greatest stimulation of macrophage ADCC was obtained when GM-CSF, M-CSF or interleukin 3 (IL-3) were used in conjunction with a secondary stimulus. Although gamma interferon (gamma-IFN) did not augment the cytotoxic capability of GM-CSF- and IL-3-stimulated macrophages, prominent cytotoxic enhancement was seen when M-CSF-stimulated macrophages were exposed to gamma-IFN. A chimaeric mouse/human monoclonal antibody was found to be equivalent in activity to the murine antibody in neutrophil ADCC; however, in macrophage ADCC assays with submaximal effector cell stimulation, the chimaeric antibody was associated with a two-fold greater response. These studies indicate that under specific conditions, CSFs capable of increasing the number and functional activity of mature myeloid effector cells enhance antibody-dependent cytotoxicity to neuroectodermal tumour target cells.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1992

Expression of tissue factor by melanoma cells promotes efficient hematogenous metastasis.

Metastasis is a multistep process which requires highly adapted interactions of tumor cells with host target organs. Compared with nonmetastatic cells, metastatic human melanoma cells express 1000-fold higher level of tissue factor (TF), the major cellular initiator of the plasma coagulation protease cascades. To explore whether TF may contribute to metastatic tumor dissemination, we analyzed the effect of specific inhibition of TF function on human melanoma metastasis in severe combined immunodeficient (SCID) mice. Using species-specific antibodies to TF, we demonstrate that initial adherence in insufficient for successful tumor cell implantation in a target organ. Rapid arrest of human tumor cells in the lungs of mice was not diminished by inhibition of TF. However, inhibition of TF receptor function and consequent reduction in local protease generation abolished prolonged adherence of tumor cells, resulting in significantly reduced numbers of tumor cells retained in the vasculature of the lungs. The growth of pulmonary metastases was also significantly inhibited by a blocking anti-TF monoclonal antibody and Fab fragments thereof, whereas a noninhibitory antibody lacked antimetastatic effects. Cell surface expression of functional TF thus contributes to melanoma progression by allowing metastatic cells to provide requisite signals for prolonged adhesive interactions and/or transmigration of tumor cells across the endothelium, resulting in successful metastatic tumor implantation.

Animals↗
PubMed1992

Long-term growth suppression of human glioma xenografts by chemoimmunoconjugates of 4-desacetylvinblastine-3-carboxyhydrazide and monoclonal antibody 9.2.27.

A conjugate of 4-desacetylvinblastine-3-carboxyhydrazide (DAVLBHY) and the glioma-reactive monoclonal antibody (mAb) 9.2.27 induced long-term suppression of tumor growth in athymic nude mice engrafted with U87MG human glioma cells. In vitro, DAVLBHY had the strongest antiproliferative activity (inhibitory concentration at which incorporation of [3H]thymidine is at 50% of untreated control is 2.0 x 10(-9) M) of seven cytotoxic drugs tested and so was chosen for conjugation to mAb 9.2.27, which reacts specifically with the core protein of chondroitin sulfate proteoglycans found in human glioblastomas. After conjugation of DAVLBHY to the carbohydrate residues of mAb 9.2.27 it retained its full binding capacity. For in vivo studies, DAVLBHY and several conjugate derivatives were evaluated by using two dosages of i.v. injections, each starting 2 days after s.c. tumor inoculation. The control tumors reached a volume of nearly 3000 mm3 within 30 days. Tumor growth was delayed by about 20 days with four i.v. injections of 0.5 mg/kg 9.2.27-DAVLBHY, which was slightly superior to the unconjugated drug. Moreover, 9.2.27-DAVLBHY produced a highly significant suppression of growth so that the average tumor volume was only 3% of that observed in untreated controls after 28 days. Four injections of this conjugate at a larger dose, 2.0 mg/kg, prevented recurrence of the tumors for 130 days in all animals tested, thus demonstrating a significant increase in the therapeutic index, since the unconjugated drug provided limited inhibition of tumor growth for only 40 days. The specificity of the antitumor effect was demonstrated in a comparison with the control conjugate, KS1/4-DAVLBHY, which despite partial tumor suppression had only a transient effect. The specific antitumor effect of 9.2.27-DAVLBHY was unexpected, since the target antigen is expressed at a relatively low density (40,000 sites/cell) on U87MG glioma cells.

Animals↗
PubMed1992

Antibody-targeted interleukin 2 stimulates T-cell killing of autologous tumor cells.

A genetically engineered fusion protein consisting of a chimeric anti-ganglioside GD2 antibody (ch14.18) and interleukin 2 (IL2) was tested for its ability to enhance the killing of autologous GD2-expressing melanoma target cells by a tumor-infiltrating lymphocyte line (660 TIL). The fusion of IL2 to the carboxyl terminus of the immunoglobulin heavy chain did not reduce IL2 activity as measured in a standard proliferation assay using either mouse or human T-cell lines. Antigen-binding activity was greater than that of the native chimeric antibody. The ability of resting 660 TIL cells to kill their autologous GD2-positive target cells was enhanced if the target cells were first coated with the fusion protein. This stimulation of killing was greater than that of uncoated cells in the presence of equivalent or higher concentrations of free IL2. Such antibody-cytokine fusion proteins may prove useful in targeting the biological effect of IL2 and other cytokines to tumor cells and in this way stimulate their immune destruction.

Antibodies, Monoclonal↗
PubMed1992

Activation of methotrexate-alpha-alanine by carboxypeptidase A-monoclonal antibody conjugate.

Carboxypeptidase A (CP-A) and monoclonal antibody KS1/4 directed against an antigen on human lung adenocarcinoma cells (UCLA-P3) were derivatized by treatment with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate and N-succinimidyl 3-(2-pyridyldithio)propionate, respectively. The derivatized proteins were reacted to produce thioether-linked enzyme-antibody conjugates. Sequential HPLC size-exclusion and DEAE chromatography separated the conjugate preparation from unreacted enzyme and antibody. On the basis of SDS-PAGE analysis and measurement of catalytic activity, the preparation contained approximately equal amounts of 1:1 and 2:1 (enzyme:antibody) conjugates; binding activity of the conjugate (1.8 x 10(5) molecules/cell) was similar to that of unreacted antibody. In vitro cytotoxicity studies with UCLA-P3 cells demonstrated the ability of cell-bound conjugate to convert the prodrug methotrexate-alpha-alanine (MTX-Ala) to methotrexate (MTX). In the absence of conjugate, ID50 values for MTX-Ala and MTX were 8.9 x 10(-6) and 5.2 x 10(-8) M, respectively. ID50 for the prodrug improved to 1.5 x 10(-6) M with cells containing bound conjugate. This potentiation of MTX-Ala cytotoxicity by conjugate-bound CP-A, which was at least 30-fold greater than that produced by a comparable amount of free enzyme, is attributed to enhanced effectiveness of MTX generated at the cell surface as opposed to the surrounding medium. Examination of the time course of cytotoxicity over a 96-h period showed that the conjugate-prodrug combination (at 2.5 x 10(-6) M) was nearly as effective as MTX in preventing cell replication. These results demonstrate the chemotherapeutic potential of carboxypeptidase-monoclonal antibody conjugates used in conjunction with MTX peptide prodrugs.

Adenocarcinoma↗
PubMed1992

A phase I study of neuroblastoma with the anti-ganglioside GD2 antibody 14.G2a.

Nine patients with neuroblastoma stage IV were treated with the murine monoclonal antibody 14.G2a, directed against disialoganglioside GD2. The antibody was injected daily for 5-10 days and the total applied dosage ranged between 100 mg/m2 and 400 mg/m2. The peak serum levels of mAb 14.G2a ranged from 28 micrograms/ml to 61 micrograms/ml. Pharmacokinetic data obtained in three patients indicated that the serum elimination of mAb 14.G2a fits a two-compartment model, with an alpha-half-time (t1/2 alpha) between 0.66 h and 1.98 h and a beta-half-time (t1/2 beta) between 30.13 h and 53.33 h. All patients presented with a human anti-(mouse IgG) antibody response either during or shortly after therapy. Eight patients showed a continuous decrease in complement component C4 during therapy, as well as an initial decrease in C3c and an initial increase in C3a, all suggesting an activation of the complement cascade. Side-effects consisted of allergic reactions like pruritus, exanthema, urticaria and of severe pain, predominantly located in the abdomen and lower extremities, which required the use of continuous intravenous morphine. Four patients additionally developed a transient hypertension and one patient experienced a transient nephrotic syndrome. Three patients were treated in an adjuvant setting and are not evaluable for tumor response. Of the remaining six patients, two had a complete remission, two showed a partial remission, and two patients did not respond to treatment.

Antibodies, Anti-Idiotypic↗