Several lines of experimental evidence in in vitro and animal model systems suggest that the integrin alpha(v)beta3 plays a role in the tumorigenicity of human melanoma cells and that the blocking of alpha(v)beta3 ligand binding can inhibit tumor progression. However, there is only scanty information about the role of alpha(v)beta3 in malignant melanoma in a clinical setting. Therefore, in the present study, we have analyzed the distribution in lesions of melanocyte origin and in normal tissues of the alpha(v) integrin subunit and of the alpha(v)beta3 complex and their association with histopathological and clinical parameters of malignant melanoma. We have used as probes the monoclonal antibodies (mAbs) TP36.1 and VF27.263.15, which we have shown with a combination of serological and immunochemical assays to be specific for the alpha(v) subunit and for the alpha(v)beta3 complex, respectively. In immunohistochemical assays, mAb TP36.1 stained both benign and malignant lesions of melanocyte origin. In contrast, the reactivity of mAb VF27.263.15 was restricted to malignant lesions. Both mAbs displayed differential reactivity with primary melanoma lesions of different histotypes because they stained about 50% of acral lentiginous melanoma and superficial spreading melanoma lesions, at least 80% of nodular melanoma lesions, and none of the uveal melanoma lesions tested. Both mAbs TP36.1 and VF27.263.15 stained about 60% of lymph node metastases and 80% of cutaneous metastases. Expression of the alpha(v)beta3 complex in melanocytic lesions resembles that of intercellular adhesion molecule-1 (ICAM-1) in several respects: (a) both are expressed in a significantly (P < 0.004) larger proportion of malignant than of benign lesions; (b) expression of both molecules in primary melanoma lesions is significantly (P < 0.05) associated with lesion thickness; and (c) expression of both molecules in primary lesions from patients with stage I melanoma is significantly (P < 0.05) associated with an increased probability of disease recurrence following surgical excision. alpha(v)beta3 and ICAM-1 in primary melanoma lesions complement each other in predicting the outcome of the disease, because the association with prognosis was enhanced when primary lesions were stained by both anti-alpha(v)beta3 mAb VF27.263.15 and anti-ICAM-1 mAb CL203.4 or by neither mAb. Because alpha(v)beta3 has been suggested as a potential target of immunotherapy, its distribution in normal tissues was investigated. alpha(v)beta3 expression is restricted because it was only detected in ductal epithelium of parotid glands, thyrocytes, basal glands of the stomach, colonic and rectal epithelium glomeruli, Bowman's capsules and proximal and distal tubules of kidneys, and endometrial epithelium. These findings suggest that renal function will be a critical clinical parameter to monitor in therapies of malignant diseases relying on systemic administration of anti-alpha(v)beta3 mAb.

A correlation was recently shown between expression of the vitronectin receptor (VnR) and the tumorigenic capacity of cultured human melanoma cell lines. On the other hand, modulation of VnR expression by interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) was observed on different non-melanoma cell lines. We tested IFN-gamma, TNF-alpha and interleukin-2 (IL-2), which are presumably released by infiltrating leukocytes in the melanoma lesional environment, on three melanoma cell lines. The VnR expression was assessed using FACS analysis and radioimmunolabelling. The VnR did not show any modulation after treatment with any of the cytokines tested. By contrast, the expression of the intercellular adhesion molecule-1 (ICAM-1), tested as control, on five melanoma cell lines, was greatly enhanced by IFN-gamma and TNF-alpha. Thus, some host cytokines may preferentially induce melanoma cells to express ICAM-1 (which can increase host cytotoxic response against melanoma), other than the VnR (which instead might contribute to melanoma metastasis).

mAb LGII-612.14 derived from a BALB/c mouse immunized with interferon-gamma (IFN-gamma) treated cultured human B lymphoid cells LG-2 has been shown with serological and immunochemical assays to recognize a monomorphic determinant expressed on the beta chain of HLA-DR, -DQ and -DP antigens. The linear nature of the determinant, which is likely to be formed by residues 19-25, is indicated by the reactivity of mAb LGII-612.14 with HLA-DR, -DQ and -DP beta chains purified by electrophoresis in presence of SDS. An unusual characteristic of mAb LGII-612.14 is its reactivity with fixed tissue sections. The intensity of staining is affected by the incubation temperature, the incubation time and the fixative used. Maximal intensity of staining of formalin fixed, paraffin embedded tissue sections required an incubation time of 16 h. The intensity of staining of paraffin embedded tissues initially fixed with Bouin's solution, formalin or ethanol was similar to that of frozen tissue sections and stronger than that of tissues fixed with B5 solution. No staining was detected of paraffin embedded tissues fixed with glutaraldehyde or Zenker's solution. Comparison of the staining patterns with mAb LGII-612.14 of frozen and fixed tissue sections showed that the latter substrates provide a superior detail of tissue architecture and cellular morphology without significant loss of sensitivity. Furthermore, comparison of the characteristics of mAb LGII-612.14 with the few previously published anti-HLA class II mAb reacting with fixed tissues indicates that mAb LGII-612.14 stains formalin fixed, paraffin embedded tissues, while mAb 910D7 and TAL-1B5 stain tissues fixed with less commonly used fixatives. Furthermore, mAb LGII-612.14 is likely to yield more sensitive staining results than anti-HLA-DR, -DQ and -DP mAb KUL/05. The present results indicate that mAb LGII-612.14 represents a useful probe to apply immunohistochemical techniques to the analysis of the distribution of HLA class II antigens in fixed tissues. This will greatly facilitate the use of readily available collections of fixed tissue specimens in retrospective studies to assess the clinical significance of changes in HLA class II antigen expression which occur in various disease states.

The nonspecific accumulation of radioactivity in bone marrow, liver, and spleen in patients with melanoma injected with radiolabeled whole IgG of anti-high molecular weight-melanoma associated antigen (HMW-MAA) monoclonal antibody (mAb) hampers the application of immunoscintigraphy to visualize melanoma lesions. This nonspecific background can be reduced by utilizing fragments which do not contain the Fc portion of anti-HMW-MAA mAb. Since the in vivo targeting to melanoma lesions of radiolabeled F(ab')2 and Fab' fragments of anti-HMW-MAA mAb is critically dependent on their binding parameters, we have analyzed the effect of fragmentation on the binding characteristics of the anti-HMW-MAA mAbs 225.28, 763.74, and TP41.2. The three mAbs recognize distinct and spatially distant determinants with a heterogeneous distribution on the pool of HMW-MAA molecules synthesized by melanoma cells. The determinant recognized by mAb TP41.2 is detectable on a markedly smaller population of HMW-MAA molecules than those recognized by mAbs 225.28 and 763.74. 125I-labeled F(ab')2 fragments of the three mAbs displayed an immunoreactive fraction similar to that of the whole IgG, while Fab' fragments displayed a lower one. Fragmentation of mAbs 225.28 and TP41.2 to F(ab')2 produced a 2- and 1.5-fold reduction in their association constants but did not cause a significant change in that of mAb 763.74. Cleavage of F(ab')2 fragments to Fab' fragments produced 2-, 40-, and 7-fold reductions in the association constants of mAbs 225.28, 763.74, and TP41.2, respectively. These changes qualitatively fit the predictions of theory for univalent and bivalent mAb binding, since mAbs 763.74 and TP41.2 appear to show bivalent binding to melanoma cells and mAb 225.28 to show univalent binding. The affinity constant of IgG, F(ab')2 and Fab' fragments of mAbs 225.28, 763.74, and TP41.2 displays an inverse relationship with the extent of their time-dependent release from the membrane of melanoma cells. Since no endocytosis of mAb could be detected, the latter results suggest that radioactivity remains bound to melanoma cells in vivo for a longer time following injection of F(ab')2 fragments than following that of Fab' fragments of each of the anti-HMW-MAA mAb tested. Radiolabeled Fab' fragments of mAbs 763.74 and TP41.2 displayed a marked reduction in their reactivity with some of the antiidiotypic mAb tested. The loss of some idiotopes is likely to be caused by changes in the conformation of the molecules associated with the fragmentation of IgG and by damage during the iodination procedure.

Intravenous injections into nude mice of 5 mg/kg methotrexate (MTX) linked to the antibody to human high molecular weight-melanoma associated antigen (HMW-MAA), monoclonal antibody (mAb) 225.28, an IgG2a, on days 1, 4, 7, 10 and 14, starting 24 h after subcutaneous inoculation of 2 x 10(6) cultured human M21 melanoma cells inhibited mean tumor volume by 90% on day 14 and by 65% on day 50 after the beginning of the treatment. Injections of equimolar amounts of free MTX and MTX linked to normal mouse IgG or to an isotype-matched myeloma protein did not inhibit tumor growth significantly. MTX linked to mAb 225.28 did not inhibit the xenograft of a subline of human melanoma cell line M21 without detectable expression of HMW-MAA. In a clonogenic assay, the MTX-225.28 conjugate was three times more potent in inhibiting the growth of M21 melanoma cells than free MTX, but did not inhibit the growth of kidney carcinoma cells Caki-1, which do not express high-Mr MAA. In contrast, MTX linked to the mAb DAL K29, reacting with kidney carcinoma cells Caki-1, inhibited their growth but did not affect that of melanoma cells. M21 melanoma cells isolated from the residual tumor of a mouse treated with the MTX-225.28 conjugate did not differ in their reactivity with mAb 225.28 and in their sensitivity to MTX when compared with M21 cells from an untreated mouse.

The human high‐molecular‐weight melanoma‐associated antigen (HMW‐MAA) represents a useful marker for immunoscin‐ tigraphy in patients with melanoma. Since injection of a radiolabelled anti‐HMW‐MAA monoclonal antibody (MAb) visualizes only about 60% of melanoma lesions, approaches are being developed to increase the sensitivity of immunoscintigraphy. One of them aims at improving the immunoreactivity of radiolabelled anti‐HMW‐MAA MAbs, since this approach may improve the targeting of radiolabelled MAbs to melanoma lesions. We have previously shown that affinity chromatography on insolubilized anti‐idiotypic MAbs is a useful method for purifying immunoreactive anti‐HMW‐MAA MAb TP61.5 from 125 I‐la‐ belled MAb preparations and that not all the anti‐idiotypic MAbs are useful for this purpose. Since the increasing number of available anti‐idiotypic MAbs is likely to facilitate the application of this procedure in many antigenic systems, we have now tested criteria to select anti‐idiotypic MAbs suitable for the purification procedure. Furthermore, we have investigated the effect of the increase in immunoreactivity of 125 I‐MAb TP6 1.5 on its in vivo targeting to human melanoma lesions transplanted into nude mice. Among the 3 anti‐idiotypic MAbs tested, the most effective in purifying immunoreactive MAb TP6 1.5 mole cules following radiolabelling is MAb TK7‐110, with which 125 I‐MAb TP61.5 displays an immunoreactivity similar to that displayed with melanoma cells. This parameter may represent a useful criterion to identify anti‐idiotypic MAbs suitable for the purification procedure, if the present results are confirmed with a large number of anti‐idiotypic MAbs in different antigenic systems. We have also shown that an incubation time for up to 4 hr of 125 I‐MAb TP61.5 with insolubilized MAb TK7‐110 is the most effective in increasing immunoreactivity and in recovering immunoreactive MAb applied to the affinity matrix. The increase in the immunoreactive fraction of 125 I‐MAb TP61.5 significantly increases its specific localization in human melanoma lesions transplanted into nude mice. These results suggest that purification of radiolabelled immunoreactive anti‐HMW‐ MAA MAb TP61.5 by affinity chromatography using antiidiotypic MAb TK7‐110 represents a useful approach to increasing the sensitivity of immunoscintigraphy in patients with melanoma.

Monoclonal antibodies (MAb) to monomorphic determinants of HLA Class II antigens inhibit monocyte-dependent T cell proliferation induced by MAb OKT3 to a different extent, suggesting a differential regulatory role of the corresponding determinants in T cell proliferation. To elucidate the mechanism(s) underlying this pattern, the MAb CR10-343 and Q5/6 with high inhibitory effect and MAb CR11-462 and CR12-356 with low inhibitory effect were characterized. Cross-inhibition studies showed that the four MAb recognize distinct determinants. The determinants recognized by MAb CR10-343 and CR12-462 are spatially close. The determinants recognized by the four MAb appear to be functionally independent in MAb OKT3-induced T cell proliferation, since the inhibitory effect of the combination of MAb CR10-343 and Q5/6 and of the MAb CR11-462 and CR12-356 was additional but not synergistic. To compare the functional activity of HLA Class II determinants expressed by monocytes and by activated T cells in MAb OKT3-induced T cell proliferation, the effect of the four MAb on MAb OKT3-induced T cell proliferation in a monocyte-dependent and in a monocyte-free system was studied. Dose-response and proliferation kinetics studies showed that the four MAb display a similar inhibitory effect on MAb OKT3-induced T cell proliferation in a monocyte-free system. These results suggest fine differences in the role played by monocyte- and T cell-bound HLA Class II determinants in the regulation of MAb OKT3-induced T cell proliferation. This functional heterogeneity may enhance the flexibility of HLA Class II antigens to mediate cell-cell interactions involved in the proliferative response to a variety of mitogenic stimuli.

The intercellular adhesion molecule (ICAM‐1) has been shown to be important in interactions involying cells of the immune system and to be upregulated in a number of cell culture systems by cytokines, including immune interferon (IFN‐γ) and tumor necrosis factor‐alpha (TNF‐α). In the present study, we have determined by fluorescence‐activated cell sorter (FACS) analysis and the anti‐ICAM‐I monoclonal antibody (MAb) CL203.4 the base‐line expression of ICAM‐I and its modulation by recombinant IFN‐β, IFN‐γ and TNF‐α in early passage (< 15) human central nervous system (CNS) tumor‐derived cell cultures. These cultures were established from various malignancies, including glioblastoma multiforme (GBM), astrocytoma (AST), ganglioglioma, medulloblastoma, meningioma and a pineal tumor. ICAM‐I expression was highest in the GBM‐ and AST‐derived cell cultures and was lowest in the ganglioglioma and normal pineal cell cultures. Variable ICAM‐I expression was found, however, in tumors of the same histological group. In several cell cultures the variable expression observed by FACS was substantiated by the intensity of the molecular species immunoprecipitated by the anti‐ICAM‐I MAb CL203.4 from these cells. All the cell cultures displayed variable but consistent increases in ICAM‐I expression following treatment with IFN‐γ or TNF‐α. In general, the degree of increase in ICAM‐I expression was greatest in cultures exposed to TNF‐α. Upregulation of ICAM‐I expression in an established glioblastoma multiforme cell line was of greater magnitude and more rapid following TNF‐α treatment (within 2 to 3 hr) than exposure to IFN‐γ (by 24 hr). In several cultures, IFN‐β also increased ICAM‐I expression and enhanced the increase induced by TNF‐α. The results of the present study indicate that variable expression of ICAM‐I is a common property of early passage cultures of CNS tumors and recombinant interferons and TNF‐α can differentially upregulate ICAM‐I expression in these CNS tumor cell cultures.

Immunochemical studies have shown that the monoclonal antibody (MoAb) CL203.4, elicited with immune interferon treated cultured human melanoma cells Colo 38, recognizes intercellular adhesion molecule 1 (ICAM-1). The determinant defined by MoAb CL203.4 is distinct and spatially distant from that defined by anti-ICAM-1 MoAb RR1/1, which had been elicited with Epstein-Barr virus-transformed B-lymphocytes from a lymphocyte function associated antigen 1 deficient patient. Immunohistochemical testing with MoAb CL203.4 of surgically removed lesions of melanocyte origin has shown a markedly lower reactivity with benign than with malignant lesions. Among the latter, a higher percentage of metastatic than of primary lesions was stained by MoAb CL203.4. The higher expression of ICAM-1 in metastases than in primary lesions is unique to melanoma, since no difference was found in its distribution in primary and metastatic lesions of a variety of malignancies of different embryological origin. Reactivity with MoAb CL203.4 of primary lesions removed from patients with stage I melanoma showed a highly significant correlation with the lesion thickness and with the clinical course of the disease. The disease free interval in patients without detectable reactivity of their primary lesion with MoAb CL203.4 was significantly (P = 0.004) longer than that of patients whose primary lesion was stained with MoAb CL203.4. These results suggest that ICAM-1 may be a useful marker in the analysis of the molecular mechanism underlying the association between lesion thickness and clinical course of the disease.

A method is described to purify immunoreactive monoclonal antibodies from radiolabeled monoclonal antibody preparations. The method is based on incubation of radiolabeled monoclonal antibodies with insolubilized anti-idiotypic monoclonal antibodies to idiotopes within the antigen-combining site of monoclonal antibodies to be purified and elution of bound monoclonal antibodies with a low pH buffer. The immunoreactive fraction of the purified monoclonal antibodies was at least 82%; the yield was at least 73%. The purification procedure did not cause any detectable change in the affinity constant of the eluted monoclonal antibodies. The method is simple and rapid; the requirement for anti-idiotypic monoclonal antibodies to idiotopes within the antigen-combining site of the antibodies to be purified is not likely to represent a major limitation in the broad application of the present method, since the hybridoma technology has greatly facilitated the development of anti-idiotypic monoclonal antibodies.

Purification of murine IgG monoclonal antibodies from ascitic fluid by precipitation with caprylic acid was compared to that: 1) by sequential precipitation with caprylic acid and (NH4)2SO4; 2) by affinity chromatography on either antiidiotypic monoclonal antibodies or antimouse IgG xenoantibodies; and 3) by sequential (NH4)2SO4 precipitation, ion exchange chromatography and high pressure liquid chromatography (referred to as HPLC). In terms of yield of antibodies, precipitation with caprylic acid is comparable to sequential precipitation with caprylic acid and (NH4)2SO4, but superior to affinity chromatography on antibodies and to HPLC. In terms of purity of antibodies, precipitation with caprylic acid is less efficient than the other three methods. It should also be noted that precipitation with caprylic acid is associated with a reduction in the affinity of some antibodies and is not suitable to purify murine IgA and IgG3.

This study aimed at characterizing the mechanism(s) underlying the regulatory role of distinct determinants of HLA Class I antigens in PHA-P-induced T cell proliferation and the involvement of monocytes in this phenomenon. The anti-HLA-A2,A28 monoclonal antibodies (MoAb) CR11-351, the MoAb Q6/64 to a determinant restricted to the gene products of the I antigens HLA-B locus, and the MoAb CR10-215 and W6/32 to distinct monomorphic determinants of HLA Class I antigens were found to inhibit PHA-P-induced peripheral blood mononuclear cell (PBMC) proliferation in a dose-dependent fashion. The inhibition is specific and reflects neither inhibition of PHA-P binding to cells nor a toxic effect of the anti-HLA Class I MoAb. The latter differed in the concentration required to induce inhibition, in the influence of the concentration of PHA-P used as mitogen, in the differential effect on the donors used as a source of PBMC, and/or in the requirement of the Fc portion to induce inhibition. At variance with the information in the literature, the inhibitory effect of anti-HLA Class I MoAb on PHA-P-induced PBMC proliferation neither reflected their interaction with accessory cells nor was mediated by suppressor factors released by monocytes stimulated with PHA-P in the presence of anti-HLA Class I MoAb. Therefore, the regulatory role of HLA Class I antigens in T cell proliferation is not likely to be mediated by monocytes and/or factors released from them, but may reflect an involvement of these molecules in T cell activation pathways.

The monoclonal antibodies (MoAbs) 149.53, 225.28, and 763.74 which recognize distinct and spatially distant determinants of the human high molecular weight-melanoma associated antigen (HMW-MAA) do not influence the binding of each other to cultured human melanoma cells. In vitro incubation of melanoma cells with a combination of the three 125I-labeled anti-HMW-MAA MoAbs results in a marked additive binding only when the MoAbs are used at saturating concentrations. Injection of the combination of the three 125I-labeled MoAbs (up to 300 micrograms per mouse) into human melanoma-bearing nude mice does not increase the amount of radioactivity specifically localized in melanoma lesions above the level observed upon injection of corresponding doses of individual MoAbs. These results may reflect the low concentration of MoAbs which reaches tumor lesions in vivo. Therefore, administration of combinations of MoAbs to distinct determinants of HMW-MAA may not increase the sensitivity of immunoscintigraphy to visualize lesions in patients with melanoma.