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ABSTRACT: WX-G250 is a chimeric monoclonal antibody that binds to carbonic anhydrase IX(G250/MN), which is present on greater than 95% of RCCs of the clear cell subtype. The suggested working mechanism of WX-G250 is by ADCC. Because the number of activated ADCC effector cells can be increased by a low dose interleukin-2 pulsing schedule, a multicenter study was initiated to investigate whether WX-G250 combined with LD-IL-2 could lead to an improved clinical outcome in patients with progressive RCC.
A total of 35 patients with progressive clear cell RCC received weekly infusions of WX-G250 for 11 weeks combined with a daily LD-IL-2 regimen. Patients were monitored longitudinally for ADCC capacity. Radiological assessment of metastatic lesions was performed at week 16 and regularly until disease progression.
A durable clinical benefit was achieved in 8 of 35 patients (23%), including 3 with a partial response and 5 with stabilization at 24 weeks or greater. Mean survival was 22 months. In general treatment was well tolerated with little toxicity. The number of effector cells increased during treatment but lytic capacity per cell did not increase. ADCC and clinical outcome did not appear to correlate.
WX-G250 combined with LD-IL-2 in patients with metastatic RCC is safe and well tolerated. With a substantial clinical benefit and a median survival of 22 months in patients with metastatic RCC who have progressive disease at study entry combination therapy showed increased overall survival compared to WX-G250 monotherapy. Survival was at least similar to that of currently used cytokine regimens but with a favorable toxicity profile.
The Journal of Urology 02/2006; 175(1):57-62. · 3.75 Impact Factor
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M Balzar,
I H Briaire-de Bruijn,
H A Rees-Bakker,
F A Prins,
W Helfrich,
L de Leij,
G Riethmüller,
S Alberti, S O Warnaar,
G J Fleuren,
S V Litvinov
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ABSTRACT: Ep-CAM is a new type of cell adhesion molecule (CAM) which does not structurally resemble the members of the four major families (cadherins, integrins, selectins, and CAMs of the immunoglobulin superfamily) and mediates Ca(2+)-independent, homophilic adhesions. The extracellular domain of Ep-CAM consists of a cysteine-rich region, containing two type II epidermal growth factor (EGF)-like repeats, followed by a cysteine-poor region. We generated mutated Ep-CAM forms with various deletions in the extracellular domain. These deletion mutants, together with monoclonal antibodies recognizing different epitopes in the extracellular domain, were used to investigate the role of the EGF-like repeats in the formation of intercellular contacts mediated by Ep-CAM molecules. We established that both EGF-like repeats are required for the formation of Ep-CAM-mediated homophilic adhesions, including the accumulation of Ep-CAM molecules at the cell-cell boundaries, and the anchorage of the Ep-CAM adhesion complex to F-actin via alpha-actinin. Deletion of either EGF-like repeat was sufficient to inhibit the adhesion properties of the molecule. The first EGF-like repeat of Ep-CAM is required for reciprocal interactions between Ep-CAM molecules on adjacent cells, as was demonstrated with blocking antibodies. The second EGF-like repeat was mainly required for lateral interactions between Ep-CAM molecules. Lateral interactions between Ep-CAM molecules result in the formation of tetramers, which might be the first and necessary step in the formation of Ep-CAM-mediated intercellular contacts.
Molecular and Cellular Biology 05/2001; 21(7):2570-80. · 5.53 Impact Factor
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ABSTRACT: Uterine cervix represents a convenient model for the study of the gradual transformation of normal squamous epithelium via low- to high-grade squamous intraepithelial lesions (SILs). Because SIL, on the basis of the cytokeratins expressed, are thought to originate from the reserve cells, we analyzed whether SILs also show a reserve cell phenotype with respect to intercellular interactions. The changes in expression and subcellular localization of the components of the adherens junction and desmosomal complexes were investigated in normal, metaplastic, and premalignant cervical epithelium, as well as in cell cultures derived from these tissues. The results suggest that 1) during progression of SILs, E-cadherin is suppressed, with its role in cell-cell connections diminishing; 2) P-cadherin, in contrast, becomes the predominant cadherin in high-grade SILs; 3) the level of cellular alpha-catenin is dramatically decreased in high-grade SILs; 4) the level of beta-catenin is decreased during progression of SILs, with plakoglobin suggestively becoming the predominant catenin mediating connection of cadherins to the cytoskeleton; 5) the assembly of desmosomes is affected during progression of SILs and is accompanied by a dramatically decreased expression for desmogleins and desmoplakins (I, II); and 6) expression of differentiation markers (involucrin, CK13) in high-grade SILs seems to be controlled by P-cadherin as opposed to E-cadherin in the normal tissue counterpart. We conclude that during development of cervical lesions substantial (both quantitative and qualitative) changes occur in cell-cell junctions, making the interactions of cells in lesions dissimilar from those of reserve cells, basal cells, or cells of immature squamous metaplasia, despite existing morphological similarity between all of these cell types and cells of high-grade lesions.
American Journal Of Pathology 09/1999; 155(2):505-15. · 4.89 Impact Factor
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ABSTRACT: Antibody-dependent cellular cytotoxicity (ADCC) is considered to be the major mechanism through which tumour cells, upon treatment with anti-tumour MAbs, are eliminated in vivo. However, the relative importance of various parameters that influence the efficacy of ADCC is unclear. Here we present in vitro data on the impact of MAb affinity and antigen density on ADCC, as obtained by comparison of two MAbs against the tumour-associated antigen Ep-CAM. The low-affinity MAb 17-1A (Ka = 5 x 10(7)M(-1)) currently used for therapy, and the high-affinity MAb 323/A3 (Ka = 2 x 10(9) M(-1)), were compared in ADCC experiments against murine and human tumour target cells transfected with the Ep-CAM cDNA under the control of an inducible promoter to enable regulation of the target antigen expression levels. Data obtained from these studies revealed that the high-affinity MAb, in contrast to the low-affinity MAb, could mediate killing of tumour cells with low antigen expression levels. Even at comparable MAb-binding levels, ADCC mediated by the high-affinity MAb was more effective. The kinetics of ADCC was also found to be determined by the level of antigen expression, and by the affinity and the concentration of the MAb used. The efficacy of ADCC with both low- and high-affinity MAbs further depended on adhesive interactions between effector and target cells mediated by CD18. However, at every given MAb concentration these interactions were of less importance for the high-affinity MAb than for the low-affinity MAb. As heterogeneity of a target antigen expression is a common feature of all tumours, and some tumour cells express very low levels of the antigen, the use of high-affinity MAbs in immunotherapy may significantly improve the clinical results obtained to the present date in the treatment of minimal residual disease.
British Journal of Cancer 09/1998; 78(4):478-83. · 5.04 Impact Factor
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ABSTRACT: Ep-CAM, an epithelium-specific cell-cell adhesion molecule (CAM) not structurally related to the major families of CAMs, contains a cytoplasmic domain of 26 amino acids. The chemical disruption of the actin microfilaments, but not of the microtubuli or intermediate filaments, affected the localization of Ep-CAM at the cell-cell boundaries, suggesting that the molecule interacts with the actin-based cytoskeleton. Mutated forms of Ep-CAM were generated with the cytoplasmic domain truncated at various lengths. All of the mutants were transported to the cell surface in the transfectants; however, the mutant lacking the complete cytoplasmic domain was not able to localize to the cell-cell boundaries, in contrast to mutants with partial deletions. Both the disruption of the actin microfilaments and a complete truncation of the cytoplasmic tail strongly affected the ability of Ep-CAM to mediate aggregation of L cells. The capability of direct aggregation was reduced for the partially truncated mutants but remained cytochalasin D sensitive. The tail truncation did not affect the ability of the transfectants to adhere to solid-phase-adsorbed Ep-CAM, suggesting that the ability to form stable adhesions and not the ligand specificity of the molecule was affected by the truncation. The formation of intercellular adhesions mediated by Ep-CAM induced a redistribution to the cell-cell boundaries of alpha-actinin, but not of vinculin, talin, filamin, spectrin, or catenins. Coprecipitation demonstrated direct association of Ep-CAM with alpha-actinin. Binding of alpha-actinin to purified mutated and wild-type Ep-CAMs and to peptides representing different domains of the cytoplasmic tail of Ep-CAM demonstrates two binding sites for alpha-actinin at positions 289 to 296 and 304 to 314 of the amino acid sequence. The results demonstrate that the cytoplasmic domain of Ep-CAM regulates the adhesion function of the molecule through interaction with the actin cytoskeleton via alpha-actinin.
Molecular and Cellular Biology 09/1998; 18(8):4833-43. · 5.53 Impact Factor
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ABSTRACT: The contribution of noncadherin-type, Ca2+-independent cell-cell adhesion molecules to the organization of epithelial tissues is, as yet, unclear. A homophilic, epithelial Ca2+-independent adhesion molecule (Ep-CAM) is expressed in most epithelia, benign or malignant proliferative lesions, or during embryogenesis. Here we demonstrate that ectopic Ep-CAM, when expressed in cells interconnected by classic cadherins (E- or N-cadherin), induces segregation of the transfectants from the parental cell type in coaggregation assays and in cultured mixed aggregates, respectively. In the latter assay, Ep-CAM-positive transfectants behave like cells with a decreased strength of cell-cell adhesion as compared to the parental cells. Using transfectants with an inducible Ep-CAM-cDNA construct, we demonstrate that increasing expression of Ep-CAM in cadherin-positive cells leads to the gradual abrogation of adherens junctions. Overexpression of Ep-CAM has no influence on the total amount of cellular cadherin, but affects the interaction of cadherins with the cytoskeleton since a substantial decrease in the detergent-insoluble fraction of cadherin molecules was observed. Similarly, the detergent-insoluble fractions of alpha- and beta-catenins decreased in cells overexpressing Ep-CAM. While the total beta-catenin content remains unchanged, a reduction in total cellular alpha-catenin is observed as Ep-CAM expression increases. As the cadherin-mediated cell-cell adhesions diminish, Ep-CAM-mediated intercellular connections become predominant. An adhesion-defective mutant of Ep-CAM lacking the cytoplasmic domain has no effect on the cadherin-mediated cell-cell adhesions. The ability of Ep-CAM to modulate the cadherin-mediated cell-cell interactions, as demonstrated in the present study, suggests a role for this molecule in development of the proliferative, and probably malignant, phenotype of epithelial cells, since an increase of Ep-CAM expression was observed in vivo in association with hyperplastic and malignant proliferation of epithelial cells.
The Journal of Cell Biology 01/1998; 139(5):1337-48. · 10.26 Impact Factor
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ABSTRACT: The bispecific OC/TR monoclonal antibody (mAb) cross-links the CD3 molecule on T cells with the human folate-binding protein (FBP), which is highly expressed on nonmucinous ovarian carcinomas. Clinical trials of patients with ovarian carcinoma with the OC/TR mAb have shown some complete and partial responses. Most patients developed human anti-murine immunoglobulin antibodies (HAMA), which can inhibit OC/TR mAb-mediated lysis. We generated a chimeric version of the OC/TR mAb to decrease the immunogenicity of the OC/TR mAb and to allow more extended treatment schedules. Sp2/0 myeloma cells were transfected with chimeric heavy- and light-chain genes encoding the anti-CD3 mAb and the MOv18 mAb, respectively, which are reactive with FBP. The resulting cell line produced 80 micrograms/ml of total immunoglobulin G (IgG), of which 11.5% was the functionally active chimeric OC/TR mAb. Chimeric OC/TR F(ab')2 fragments mediated lysis of IGROV-1 ovarian carcinoma cells by human T cells at antibody concentrations of > or = 1 pg/ml. Specific lysis was still detectable at an effector-to-target cell ratio as low as 0.4. Two patients with ovarian carcinoma treated with F(ab')2 fragments of the murine OC/TR developed distinct HAMA titers, which were mainly anti-idiotypic and only partly directed against the murine antibody constant regions. However, of the two patients that were treated with the F(ab')2 fragments of the chimeric OC/TR mAb, only one developed a low transient HAMA response just above background level. In conclusion, the generation of chimeric OC/TR may allow more extended clinical studies of bispecific mAb-mediated immunotherapy of ovarian carcinoma.
Journla of Immunotherapy 11/1997; 20(6):496-504. · 3.27 Impact Factor
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ABSTRACT: We have reported a 27% overall anti-tumor response using i.p. immunotherapy of advanced ovarian carcinoma with autologous, ex vivo expanded, T lymphocytes re-targeted with bi-specific monoclonal antibody OC/TR, combined with soluble OC/TR and low-dose recombinant interleukin-2 (IL-2). This treatment had no effect on extraperitoneal disease. Therefore we studied in 13 patients whether this immunotherapeutic protocol resulted only in local or also in systemic immunomodulation. The phenotype of the ex vivo expanded lymphocytes was mainly CD3+, 4-, 8+, 16-, 56-. Their OC/TR-re-targeted cytolytic activity against Igrov-1 ovarian-carcinoma cells was approximately as high in responders as in non-responders. Following most therapeutic cycles, the immunophenotype of lymphocytes recovered from the peritoneal fluid was similar to that of the infused T cells (i.e., mainly CD3+, 4-, 8+) and they were coated with OC/TR. However, cytolytic activity of the recovered lymphocytes against Igrov- 1 cells was low in direct assays, and only slightly increased after additional in vitro re-targeting with OC/TR. Systemically, the i.p. immunotherapy resulted in a transient lymphopenia lasting for about 7 days, low (i.e., 5 to 13 ng/ml) serum concentrations of free, functional OC/TR, and very weak coating of circulating T lymphocytes with OC/TR. These peripheral-blood T lymphocytes did not exert OC/TR-re-targeted cytolytic activity. Thus, locoregional OC/TR-re-targeted cellular immunotherapy resulted in substantial local immunomodulation and anti-tumor effects but virtually no systemic immunomodulation.
International Journal of Cancer 11/1997; 73(2):211-9. · 5.44 Impact Factor
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ABSTRACT: Crosslinking of immunoglobulin E molecules that are bound to the Fc epsilon receptors expressed on mast cells or basophils triggers activation of these cells, resulting in the development of a type I hypersensitivity. Targeting this potent immune reaction towards tumors by using IgE that reacts with a tumor-associated antigen, may induce a local inflammation at the tumor site, and may therefore promote tumor regression. We have previously shown that murine IgE bound to tumor cells can activate murine mast cells to release TNF-alpha and histamine. To further investigate the therapeutic potential of IgE-mediated immunotherapy of carcinomas, we have developed human/murine chimeric versions, containing the murine variable regions and human constant regions, of both G250 and 323/A3 IgE. These chimeric IgEs are reactive respectively with the G250 renal cell carcinoma antigen and the Ep-CAM molecule, which is highly expressed by most carcinomas. Transfection of the respective chimeric heavy and light chain genes into recipient Sp2/0 myeloma cells yielded chimeric IgE-producing clones. Chimeric G250 and 323/A3 IgE reacted with tumor cells expressing the G250 antigen or Ep-CAM, respectively. To generate a cell line that expresses Fc receptors for human or chimeric IgE, the rat basophilic leukemia cell line RBL-7 was transfected with the human Fc epsilon RI alpha chain (RBL-7TZ) and subsequently tested for binding of chimeric IgE. Functional assays showed that both chimeric IgEs activated RBL-7TZ cells to release TNF-alpha when cultured with tumor cells that express the respective specific antigen. Furthermore, both chimeric IgEs were able to activate freshly isolated human basophils.
Human antibodies 02/1997; 8(4):169-80.
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ABSTRACT: The monoclonal antibody (MAb) G250 binds to a tumour-associated antigen, expressed in renal cell carcinoma (RCC), which has been demonstrated to be a suitable target for antibody-mediated immunotherapy. A bispecific antibody having both G250 and anti-CD3 specificity can cross-link G250 antigen-expressing RCC target cells with T cells and can mediate lysis of such targets. Therapy studies with murine antibodies are limited by immune responses to the antibodies injected (HAMA response), which can be decreased by using chimeric antibodies. We generated a chimeric bispecific G250/anti CD3 MAb by transfecting chimeric genes of heavy and light chains for both the G250 MAb and the anti-CD3 MAb into a myeloma cell line. Cytotoxicity assays revealed that the chimeric bispecific MAb was capable of mediating lysis of RCC cell lines by cloned human CD8+T cells or by IL-2-stimulated peripheral blood lymphocytes (PBLs). Lysis mediated by the MAb was specific for target cells that expressed the G250 antigen and was effective at concentrations as low as 0.01 microgram ml-1. The chimeric bispecific G250/anti-CD3 MAb produced may be an effective adjuvant to the currently used IL-2-based therapy of advanced renal cell arcinoma.
British Journal of Cancer 10/1996; 74(5):735-44. · 5.04 Impact Factor
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ABSTRACT: To evaluate the role of affinity in monoclonal antibody (mAb)-mediated treatment of carcinomas, we compared the antibodies 17-1A and 323/A3 that bind with different affinities overlapping epitopes on the epithelial adhesion molecule Ep-CAM. This comparison was performed in several models for minimal residual disease in mice grafted with Ep-CAM transfected B16 melanoma cells originating from C57BL/6 mice. These cells were either grafted subcutaneously or injected intravenously into nude BALB/c mice, or grafted subcutaneously in immunocompetent C57BL/6 mice. In the BALB/c subcutaneous model, significant therapeutic results (p < 0.05) compared with the control mAb were obtained with both mAbs 17-1A and 323/A3. However, when treating lung metastases in nude BALB/c mice that had developed after intravenous injection of the B16/Ep-CAM tumor cells, only the high-affinity 323/A3 mAb could significantly (p < 0.05) reduce the number of metastases that appeared. In syngeneic C57BL/6 mice grafted subcutaneously with B16/ Ep-CAM cells, a single 323/A3 or 17-1A mAb injection had no effect, in contrast to that observed for the nude BALB/c mouse model. However, multiple injections of the 323/A3 mAb significantly (p < 0.005) reduced the mean tumor volume, although they did not prevent tumor development. The results show that in vivo antibody-mediated effector cell activation and subsequent tumor cell elimination is determined by mAb affinity and target antigen density. Therefore, treatment of minimal residual disease with high-affinity mAb 323/ A3 is expected to improve the clinical results obtained with mAb 17-1A.
Journal of immunotherapy with emphasis on tumor immunology: official journal of the Society for Biological Therapy 08/1996; 19(4):245-56.
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ABSTRACT: Ep-CAM, an epithelial adhesion molecule, is absent in normal squamous epithelia but can be detected in some squamous carcinomas. Using a panel of monoclonal antibodies to keratinocyte differentiation and proliferation markers, we investigated the association of EP-CAM expression with differentiation-related and/or neoplastic changes in cervical epithelium. Normal endocervical glandular epithelium (Both columnar and reserve cells) appeared strongly positive for EP-CAM, whereas ectocervical squamous epithelial cells did not express this molecule. Expression of Ep-CAM (in basal cells) was sometimes observed in morphologically normal ectocervical tissue but only in areas bordering cervical intraepithelial neoplasia (CIN) lesions. At the early stages of neoplasia the expression of Ep-CAM was regularly present in squamous epithelium, in general consistent with the areas of atypical, undifferentiated cells. Thus, in CIN grades I and II, the basal/suprabasal layers of the epithelia were positive, whereas in CIN grade III lesions, up to 100% of the cells over the whole thickness of the epithelium sometimes excluding the very upper layers, expressed Ep-CAM. A clear increase, not only in number of positive cells but also in levels of Ep-CAM expression (intensity) was observed during progression from CIN I to CIN III. Expression of Ep-CAM in ectocervical lesions did not coincide with a reappearance of the simple epithelium cytokeratins (CK8 and CK18). On the other hand, expression of Ep-CAM in atypical cells of CIN lesions correlated with the disappearance of CK13, which normally marks cells undergoing squamous differentiation. As was shown with Ki-67, a marker for proliferating cell populations, the areas of Ep-CAM expression were also the areas of enhanced proliferation. Cells expressing Ep-CAM did not express involucrin, a marker for cells committed to terminal differentiation. In the majority of both squamous and adenocarcinomas of the cervix a strong expression of Ep-CAM was observed, although some decrease in the expression (both the intensity and the number of positive cells), as compared with CIN III lesions, was observed in the areas of squamous differentiation. This study demonstrates that the expression of Ep-CAM in cervical squamous epithelium is associated with abnormal proliferation of cell populations that are not committed to terminal differentiation.
American Journal Of Pathology 04/1996; 148(3):865-75. · 4.89 Impact Factor
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ABSTRACT: Immunoglobulin E (IgE) that specifically binds to antigens present on carcinoma cells may represent a useful tool to combat carcinomas. Induction of an inflammatory response at the tumor site by tumor-specific IgE may result in reduced tumor growth and tumor regression. Local mast cells may be activated to release TNF-alpha and other mediators that attract inflammatory cells, such as eosinophils and macrophages, to the tumor site. It may even be expected that eosinophils perform IgE-mediated lysis of tumor cells. The G250 IgE binds an antigen present on renal cell carcinoma. Mast cells were assayed for activation by G250 IgE in vitro in the presence of G250-positive tumor cells, by determination of the release of TNF-alpha and histamine. In parallel, G250 IgG1, IgG2a, and IgG2b, bound to tumor cells, were tested for their ability to activate mast cells. Tumor-specific IgE was capable of activating mast cells in the presence of tumor cells. This activation was specific and required the presence of the antigen on the tumor cell surface and recognition of the tumor cell by the IgE. G250 IgE mediated mast cell activation when present in the medium as well as being preloaded on either tumor cells or mast cells. Preincubation of mast cells with irrelevant IgE did not block specific G250 IgE-mediated mast cell activation. Upon activation, mast cells released histamine and TNF-alpha, as was detected in cytotoxicity assays with TNF-alpha-sensitive target cells (WEHI). G250 IgG2a also induced efficient mast cell activation, comparable to the effect of G250 IgE. Mast cells can be triggered to release mediators such as TNF-alpha by IgE in the presence of tumor cells expressing specific antigen. Whether mast cell activation contributes to antitumor effects observed during antibody-based immunotherapy of tumors deserves further investigation.
Laboratory Investigation 03/1996; 74(2):467-75. · 3.64 Impact Factor
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ABSTRACT: The therapeutic effects of the low and high affinity mAbs 17-1A and 323/A3 were investigated in nude mice xenografted with LS 180 human colorectal carcinoma cells. Treatment of mice grafted with dispersed tumor cells, before formation of a tumor nodule, was started 1 day after s.c. injection of tumor cells and consisted of a single i.p. injection of murine 17-1A or 323/A3 mAb. Tumor appearance after a single injection of either 17-1A or 323/A3 was delayed as compared to injection of an irrelevant mAb. Both 17-1A and 323/A3 reduced the tumor growth rate, and both mAbs decreased the total number of mice that eventually developed a tumor. In all experiments, 323/A3 showed consistently better treatment effects on xenografted mice than mAb 17-1A. For treatment of established tumors with mAb 17-1A or 323/A3 therapy was delayed until a tumor nodule was macroscopically detectable. One single i.p. injection of mAb 17-1A had no effect on further tumor growth and mean tumor size as compared to the control group injected with irrelevant mAb. One single i.p. injection with mAb 323/A3 reduced the tumor growth rate in some mice with established tumors and resulted in a significant difference of mean tumor size of this group as compared to the 17-1A treated mice and the control groups. Multiple injections with mAb 17-1A also had no effects on established tumors, in contrast to mAb 323/A3, where serial injections resulted in tumor growth reduction and, eventually, in some mice reduction in tumor size. In summary, we showed that in nude mice mAb 323/A3 (Ka = 2 x 10(9) M-1) is much more potent than mAb 17-1A (Ka = 5 x 10(7) M-1) in eradication of nonestablished tumor cells and treatment of small established tumors. These results suggest that high affinity mAbs like 323/A3 might dramatically improve the clinical results obtained thus far with the low affinity mAb 17-1A in the adjuvant treatment of surgically resected Dukes C colorectal cancer patients with minimal residual disease.
Cancer Research 11/1995; 55(19):4398-403. · 7.86 Impact Factor
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ABSTRACT: Tumor heterogeneity is the presence of intercellular differences, either from clonal origin or present within subpopulations of tumor cells. Recent advances in immuno-histology, flow cytometric analysis, molecular biological techniques, and tissue culture methods makes it possible to investigate tumor heterogeneity in detail. In this review data are presented to document that this hallmark of neoplastic disease results from DNA-instability and the interactions of tumor cells with their environment. The present inability to treat most patients effectively with immunotherapy may partly be due to the occurrence of tumor heterogeneity. Therefore, the heterogeneity of the tumor phenotype is discussed in conjunction with the various immunotherapeutic treatment modalities. In addition to local cytokine production by immune cells and tumor cells, and limited access of either antibodies or immune cells into the tumor, tumor heterogeneity is an important factor that determines the progress of immunotherapy of cancer. Therefore, accurate quantitative methods using antibodies and molecular probes to identify HLA-associated target peptides, tumor-associated antigens and accessory molecules, to predict which patients will have a high probability of responding to treatment, are needed.
Immunological Reviews 07/1995; 145:91-122. · 11.15 Impact Factor
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ABSTRACT: T lymphocytes of 8 patients with ovarian cancer were targeted to the tumor cells using F(ab')2 fragments of a bispecific monoclonal antibody (bsAb), specific for CD3 (a component of the T lymphocyte receptor for antigen) and for the folate receptor MOv18 (overexpressed by ovarian carcinoma cells) as part of a phase I/II study. Phase I (days 0 to 3) consisted of increasing intraperitoneal (i.p.) numbers (10(6)-10(9)) of bsAb-targeted T lymphocytes plus low-dose interleukin-2 (IL-2). Phase II (days 6 to 13, and 27 to 33) consisted of daily i.p. infusions of 10(9) targeted T lymphocytes, 2 mg soluble bsAb, and low-dose IL-2. Using enzyme-linked immunosorbent assays (ELISA), human anti-mouse antibodies (HAMA) were detected in all patients: in the serum from day 13 onwards and in the peritoneal fluid from day 20 onwards. A significant proportion of the HAMA appeared to be directed against the idiotypes of the bsAb specific for CD3 and MOv18, as suggested by (1) the clearly higher ELISA titers against OC/TR bsAb as compared to those against a monoclonal antibody (MAb) with unrelated specificity, and (2) failure to abrogate the capacity of peritoneal fluid containing HAMA to block the binding of OC/TR bsAb to MOv18+ or CD3+ cells by absorption of human anti-mouse IgG-framework antibodies in peritoneal fluid to immobilized mouse IgG. The OC/TR-targeted cytolysis of the MOv18+ ovarian carcinoma cell line Igrov-I by autologous T lymphocytes was inhibited by peritoneal fluid samples containing relatively high HAMA titers. Such inhibitory activity was never detected at the start of phase II, but coincided with the last series of i.p. infusions of targeted T lymphocytes in 2 patients.
International Journal of Cancer 03/1995; 60(4):450-7. · 5.44 Impact Factor
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ABSTRACT: Recently we have demonstrated that a 40kD human epithelium-specific glycoprotein exhibit the features of a homophilic cell-cell adhesion molecule, when expressed in transfected murine cells. We suggested the name Ep-CAM for this molecule (Litvinov et al., J. Cell Biol., 125: 437-446). Here we investigate the possible biological function of Ep-CAM in its natural environment--cells of epithelial origin. Immunolocalization of Ep-CAM in tissues and in cultures of epithelial/carcinoma cells showed that the majority of the Ep-CAM molecules are localized at cell-cell boundaries, predominantly along the whole lateral domain of polarized cells. In vitro, on single cells in suspension, the Ep-CAM molecules are present on the entire cell surface, and when the single cells grow attached, Ep-CAM is present at their pseudo-apical domain. During formation of intercellular contacts by such single cells, the majority of the Ep-CAM molecules are redistributed from the pseudoapical to the lateral domain of the cell membrane. Attachment of cells to the substrate does not cause redistribution of the molecules to the site of substrate attachment irrespective of the adhesive substrate (fibronectin, collagens, laminin, EHS-matrigel were tested). The monoclonal antibody 323/A3, reactive with the extracellular domain of the Ep-CAM molecule, has a strong negative effect on the aggregating behavior of COV362 ovarian carcinoma cells and RC-6 immortalized mammary epithelial cells. The mAb affected cell aggregation in both cell lines in the presence of Ca++, but with RC-6 cells the effect was more pronounced in low-calcium medium. The effects of the 323/A3 mAb on the already established intercellular contacts was not significant. The data presented demonstrate that the Ep-CAM molecules are functionally active in the epithelial and carcinoma cells tested, are capable of mediating Ca(++)-independent intercellular adhesions, and are not likely to be involved in cell-substrate adhesion.
Cell adhesion and communication 11/1994; 2(5):417-28.
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ABSTRACT: The epithelial glycoprotein 40 (EGP40, also known as GA733-2, ESA, KSA, and the 17-1A antigen), encoded by the GA-733-2 gene, is expressed on the baso-lateral cell surface in most human simple epithelia. The protein is also expressed in the vast majority of carcinomas and has attracted attention as a tumor marker. The function of the protein is unknown. We demonstrate here that EGP40 is an epithelium-specific intercellular adhesion molecule. The molecule mediates, in a Ca(2+)-independent manner, a homophilic cell-cell adhesion of murine cells transfected with the complete EGP40 cDNA. Two murine cell lines were tested for the effects of EGP40 expression: fibroblastic L cells and dedifferentiated mammary carcinoma L153S cells. The expression of the EGP40 protein causes morphological changes in cultures of transfected cells--increasing intercellular adhesion of the transfectants--and has a clear effect on cell aggregating behavior in suspension aggregation assays. EGP40 directs sorting in mixed cell populations, in particular, causes segregation of the transfectants from the corresponding parental cells. EGP40 expression suppresses invasive colony growth of L cells in EHS-matrigel providing tight adhesions between cells in growing colonies. EGP40 can thus be considered a new member of the intercellular adhesion molecules. In its biological behavior EGP40 resembles to some extent the molecules of the immunoglobulin superfamily of cell adhesion molecules (CAMs), although no immunoglobulin-like repeats are present in the EGP40 molecule. Certain structural similarities in general organization of the molecule exist between EGP40 and the lin-12/Notch proteins. A possible role of this adhesion molecule in formation of architecture of epithelial tissues is discussed. To reflect the function of the molecule the name Ep-CAM for EGP40 seems appropriate.
The Journal of Cell Biology 05/1994; 125(2):437-46. · 10.26 Impact Factor
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ABSTRACT: The monoclonal antibodies (MAbs) 323/A3 and 17-1A both recognize a 40-kDa carcinoma-associated epithelial glycoprotein (EGP40). MAb 17-1A has been used in many therapeutic trials as an immunotherapeutic agent to combat advanced colorectal cancer, and about 5-10% overall responses have been observed. It has been shown that MAb 323/A3 has a higher affinity than 17-1A, which might be an advantageous feature for a therapeutic agent. In our immunohistological studies different reaction patterns of these two MAbs were observed, suggesting that MAb 323/A3 reacts more intensely with carcinoma cells than MAb 17-1A. This also suggests that MAb 323/A3 might be a more effective immunotherapeutic tool. Because chimerization may reduce the immunogenicity of the murine MAb 323/A3 and increase the interaction with human effector mechanisms, we developed a chimeric form of murine MAb 323/A3. MAb 323/A3 heavy and light chain variable genes were cloned and grafted onto human C gamma 1 and C kappa domains, respectively. A chimeric antibody-producing cell line was established by transfection of the chimeric constructs into a nonproducing myeloma cell. The chimeric and murine 323/A3 MAbs were evaluated for efficacy of inducing complement-mediated cytotoxicity (CMC) and mediating antibody-dependent cellular cytotoxicity against LS 180 cells derived from human colon carcinoma. Both forms were found to mediate similar levels of CMC in the presence of human complement; however, higher levels of lysis of target cells were observed with human peripheral blood lymphocytes when the chimeric 323/A3 was used. Chimeric 323/A3 mediated higher maximal cytotoxicity than chimeric 17-1A in both CMC and antibody-dependent cellular cytotoxicity assays and was equally active as chimeric 17-1A at 100- to 1000-fold lower concentrations. The superior reactivity of chimeric 323/A3 with EGP40 on carcinoma cells and its higher cytotoxicity-mediating capacity, compared to chimeric 17-1A, are important characteristics, which support further clinical studies with chimeric MAb 323/A3 in immunotherapy of carcinomas.
Cancer Research 05/1994; 54(7):1753-9. · 7.86 Impact Factor
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ABSTRACT: The bispecific antibodies (bs-mAbs) OV-TL 3/CD3 and OC/TR (MOv18/CD3) efficiently mediate ovarian tumor cell lysis by cytotoxic T cells and activated peripheral blood lymphocytes (PBL) in vitro. OV-TL 3/CD3 and OC/TR are reactive with tumor-associated antigens on ovarian carcinoma cells (OA3 and CA-MOv18, respectively), and CD3 on activated PBL, bridging both cells and simultaneously inducing activation of the effector cells. In a comparative study we investigated the therapeutic efficacy of OV-TL 3/CD3 and OC/TR by targeting activated PBL with the bs-mAbs against intraperitoneally growing NIH:OVCAR-3 human ovarian carcinoma cells. As they have good tumor localization characteristics, HPLC-purified bispecific F(ab')2 fragments were used to target highly active PHA and IL-2-stimulated PBL effector cells. The efficacy of OV-TL 3/CD3 was compared to OC/TR with respect to tumor-associated antigen (TAA) binding on NIH:OVCAR-3 ascites cells and NIH:OVCAR-3 tumor cell lysis in vitro. In this report we show that ip ovarian cancer-bearing nude mice treated with IL-2 and activated PBL coated with bispecific F(ab')2 had a significantly longer survival than the untreated mice. No significant difference in survival was found between the OC/TR or OV-TL 3/CD3 bispecific antibody, although MOv18 expression was higher on NIH:OVCAR-3 ascites cells and PBL targeted with OC/TR induced slightly higher tumor cell lysis in vitro. Thus, the therapeutic efficacy of these bs-mAbs in vivo could not be predicted by TAA expression or bs-mAb-mediated tumor cell lysis in vitro.
Gynecologic Oncology 03/1994; 52(2):199-206. · 3.89 Impact Factor
