Transactivation of the epidermal growth factor receptor in endothelin-1-induced mitogenic signaling in human ovarian carcinoma cells.

Endothelin (ET)-1 is produced in ovarian carcinoma cells and is known to act through ET(A) receptors as an autocrine growth factor in vitro and in vivo. In OVCA 433 human ovarian carcinoma cells, ET-1 caused phosphorylation of the epidermal growth factor receptor (EGF-R) that was accompanied by phosphorylation of Shc and its recruitment complexed with Grb2. These findings suggested that an EGF-R/ras-dependent pathway may contribute to the activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (Erk) 2 and mitogenic signaling induced by ET-1 in these cells. Specific inhibition of EGF-R kinase activity by tyrphostin AG1478 prevented ET-1-induced transactivation of the EGF-R, as well as Shc phosphorylation and recruitment with Grb2. Furthermore, ET-1-induced activation of Erk 2 was partially inhibited by tyrphostin AG1478. In accord with this finding, the mitogenic action of ET-1 in OVCA 433 cells was also significantly reduced by a concentration of tyrphostin AG1478 that abolished the growth response of EGF-stimulated cells. Inhibition of protein kinase C activity, which contributes to the proliferative action of ET-1 in OVCA 433 cells, had no effect on the activation of Erk 2 by ET-1, which suggests that this effect of protein kinase C does not involve ras-independent activation of Erk 2. Inhibition by wortmannin of PI3-kinase activity, which has been implicated in ET-1 and other G protein-coupled receptor (GPCR)-mediated signaling pathways, reduced Erk 2 activation by ET-1 but had no effect on ET-1-induced EGF-R and Shc phosphorylation. These findings indicate that ET-1-induced stimulation of Erk 2 phosphorylation, and mitogenic responses in OVCA 433 ovarian cancer cells are mediated in part by signaling pathways that are initiated by transactivation of the EGF-R.

Activation of mitogenic signaling by endothelin 1 in ovarian carcinoma cells.

Endothelin 1 (ET-1) is produced in ovarian cancer cell lines and has been shown to act through ET(A) receptors as an autocrine growth factor to promote tumor cell proliferation in vitro. In OVCA 433 cells, the efficacy of ET-1 as a stimulus of [3H]thymidine incorporation was equivalent to that of epidermal growth factor. ET-1 also stimulated the rapid expression of c-fos, an action mediated by ET(A) receptors. The mitogenic action of ET-1 was not mediated by a pertussis toxin-sensitive G protein. An analysis of the effects of inhibition and depletion of protein kinase C (PKC) on mitogenic responses demonstrated that PKC was necessary but not sufficient for maximal stimulation by ET-1. In quiescent OVCA 433 cells, ET-1-induced stimulation of [3H]thymidine incorporation was prevented by two structurally distinct inhibitors of tyrosine kinase, herbimycin A and genistein. These results indicate that both PKC and protein tyrosine kinase participate in ET-1-stimulated mitogenic signaling. ET-1 rapidly stimulated tyrosine phosphorylation of several cellular proteins, among which p125FAK and p42 mitogen-activated protein kinase were identified. The additivity between the potent mitogenic actions of ET-1 and epidermal growth factor is consistent with the independence of their signal transduction pathways in ovarian cancer cells. These findings also indicate that intracellular signaling between the ET(A) receptor and a yet unidentified tyrosine kinase is involved in the mitogenic response to ET-1.

A cytoplasmic human melanoma associated antigen as a marker of activation in lymphoid cells.

A cytoplasmic glycoprotein, originally identified by the monoclonal antibody 465.12S in melanoma tumors, is significantly increased in epithelial cells of different histotype following transformation. In the present study we show that the cytoplasmic melanoma associated antigen (cyt-MAA) is drastically enhanced in lymphoid cells by polyclonal and allogeneic stimulation, as well as by transformation. Normal T-cells with helper and suppressor phenotype are far more susceptible than B-cells to this enhancement. However, among transformed lymphoid cells, the expression of the cyt-MAA does not correlate with lineage, but rather with stage of differentiation. Acute lymphoblastic leukemias represent the only exception, since in these lymphoid malignancies cyt-MAA levels are highly heterogeneous even within groups of phenotypically similar lesions. Thus, the expression of the cyt-MAA is shared by cells of distant embryological origin in early stages of their differentiation and/or during proliferation. Quantitation of the cyt-MAA may provide useful information for the classification of some lymphoid malignancies.

Effect of recombinant human leukocyte, fibroblast, and immune interferons on expression of class I and II major histocompatibility complex and invariant chain in early passage human melanoma cells.

Twenty-five early-passage (less than or equal to 8) melanoma cell lines, isolated from ten patients with metastatic melanoma, were analyzed by a combination of serological, immunochemical, and molecular methods for mRNA levels, synthesis, and surface expression of MHC class I and class II antigens prior to and following exposure to recombinant human leukocyte (IFN-alpha A), fibroblast (IFN-beta), and immune (IFN-gamma) interferon. All the cell lines expressed variable levels of HLA class I gene products that were up-regulated to different extents upon exposure to specific interferons (IFNs). HLA class II antigens were expressed in 22 of the 25 melanoma lines and IFN-gamma increased the levels of class II mRNA, protein synthesis, and surface expression in all cultures displaying baseline expression. A significant up-regulation of class II antigen expression by IFN-alpha or -beta, associated with higher levels of class II transcripts and enhanced synthesis, was found only in two early-passage human melanoma cell lines. In three lesions from the same patient which did not constitutively express class II antigens, no expression of these glycoproteins could be induced with any of the IFNs. These results indicate that IFN-gamma does not act as a de novo inducer of class II antigen expression in early-passage human melanoma cell lines. This hypothesis is further supported by analysis of class II-associated invariant chain (Ii) expression, which is expressed and induced by IFNs in a manner similar to that of class II antigens. The present study also indicates that early-passage metastatic melanoma lesions from the same patient are heterogeneous in their de novo expression of major histocompatibility antigens and in their modulation by IFNs.

Expression of endothelin 1 and endothelin A receptor in ovarian carcinoma: evidence for an autocrine role in tumor growth.

In the present study, we have investigated the expression of endothelin 1 (ET-1) and the ET(A) receptor (ET(A)R) and ET(B) receptor (ET(B)R) in primary (n = 30) and metastatic (n = 8) ovarian carcinomas and their involvement in tumor growth. By reverse transcription-PCR and Northern blot analysis, we detected ET-1 mRNA in 90% of primary and 100% of metastatic ovarian carcinomas. ET-1 mRNA expression was significantly higher in tumors than in normal ovarian tissues (n = 12; P < 0.01). ET(A)R mRNA was also detected in 84% of the carcinomas examined, whereas ET(B)R mRNA was expressed in 50% of the tumors. The in vivo presence of mature ET-1 and ET(A)R was confirmed by immunohistochemistry, demonstrating a higher expression in primary and metastatic cells. Ten primary cultures of ovarian tumors secreted ET-1 and were positive for ET-1 and ET(A)R mRNA, whereas only 40% expressed ET(B)R mRNA. Radioligand binding studies showed that ET-1-producing cells also expressed functional ET(A)R, whereas no specific ET(B)R could be demonstrated. ET-1 stimulated dose-dependent [3H]thymidine incorporation and enhanced the mitogenic effect of epidermal growth factor. The ET(A)R-selective antagonist BQ 123 strongly inhibited ET-1-stimulated growth and substantially reduced the basal growth rate of unstimulated cells, whereas the ET(B)R-selective antagonist BQ 788 had no effect. In conclusion, the present data demonstrate a novel mechanism in the growth control of ovarian carcinoma in vivo mediated by the ET-1 autocrine loop that selectively occurs via the ET(A)R.

Autocrine actions of endothelin-1 as a growth factor in human ovarian carcinoma cells.

The production of endothelin 1 (ET-1) and its receptor-mediated actions on calcium signaling and growth responses were analyzed in human ovarian carcinoma cells. Immuno-reactive ET-1 was released from three of four ovarian tumor cell lines as a function of time in amounts ranging from 56 to 74 fmol/10(6) cells. Reverse-phase HPLC and radioimmuno-assay of conditioned media from tumor cells revealed a single peak coeluting with authentic ET-1. Radioligand binding studies showed that the ET-1-producing cell lines also expressed high-affinity ETA receptors (Kd < 0.1 nM) that ranged in abundance from 2,600 to 43,600 sites/cell. In fura-2-loaded ovarian carcinoma cells, ET-1 induced dose-dependent increases in cytoplasmic Ca2+ concentration. ET-1 also stimulated thymidine incorporation in the three cell lines that expressed ET receptors. In OVCA 433 cells, BQ 123 inhibited the stimulatory actions of ET-1 on thymidine incorporation and cell proliferation, and substantially reduced the basal growth rate of unstimulated ovarian tumor cells. These results demonstrate that ET-1 is produced in ovarian cancer cells and acts as an autocrine growth factor on ETA receptors to stimulate calcium signaling and proliferative responses. Such findings suggest that ET-1 participates in the progression of neoplastic growth in certain ovarian tumors.

Gene products of the HLA-D region in normal and malignant tissues of nonlymphoid origin.

Indirect immunofluorescence staining with monoclonal antibodies has shown a differential distribution of HLA-DR, DQ, and DP antigens in normal tissues of nonlymphoid origin. The distribution of HLA-DP antigens is similar to that of HLA-DR antigens, while that of HLA-DQ antigens is more restricted. Malignant transformation of cells of nonlymphoid origin may be associated with the appearance of the gene products of the HLA-D region. HLA-DR antigens appear more frequently than the other two types of HLA class II antigens and HLA-DP antigens more frequently than HLA-DQ antigens. Differential expression of the gene products of the HLA-D region was also found in autologous metastases removed from different anatomic sites from patients with melanoma. The HLA class II phenotype of surgically removed malignant lesions did not correlate with the degree of differentiation of tumor cells and/or with the expression and/or cellular distribution of HLA class I antigens. Furthermore, in melanoma lesions, no relationship was found between the HLA class II phenotype and the expression of 3 membrane bound and 1 cytoplasmic melanoma associated antigen recognized by monoclonal antibodies. The functional significance and the practical implications of the differential expression of the gene products of the HLA-D region by tumor cells are discussed.

Regulation of the expression of class II genes of the human major histocompatibility complex in tumor cells.

The control of expression of human class II MHC genes has been studied in lymphoid and melanoma cells. Specific unmethylation of all restriction sites nearby the promoter regions has been detected in all cell lines and tissues studied, irrespective of their ability to express class II MHC products. The main functional role of DNA methylation appears, on the contrary, to be the regulation of a fraction of the nucleotide polymorphism of class II MHC genes. Constitutive expression of these genes can be modified by recombinant IFN-gamma and by the demethylating agent 5-azacytidine. Both the modifiers differentially regulate the levels of class II MHC and invariant chain products. In melanoma cells IFN-gamma derepresses transcription of a 1.2-Kb HLA-DR alpha mRNA, but does not affect the levels of a 0.8-Kb HLA-DR alpha specific mRNA. These molecular changes are triggered by IFN-gamma through a protein-synthesis-dependent pathway.

Saporin 6 conjugated to monoclonal antibody selectively kills human melanoma cells.

A novel human melanoma specific immunotoxin is described, which has been produced utilizing the murine monoclonal antibody (mAb) Ep2, IgG2a isotype, recognizing an epitope of the glycoprotein/proteoglycan high molecular weight-melanoma associated antigen. mAb Ep2 has been chemically conjugated by a disulphide bond, using the bifunctional reagent SPDP, to the plant toxin Saporin 6 (SAP) extracted from seeds of Saponaria officinalis. Cytotoxicity studies performed in vitro on melanoma cells have shown that Ep2/SAP immunotoxin efficiently kills antigen expressing cells and that its IC50 is approximately 1 x 10(-10) M, while not affecting the viability of antigen-negative melanoma cells at doses as high as 1 x 10(-7) M, therefore indicating a therapeutic index of Ep2/SAP immunotoxin higher than 1000. Kinetic studies have demonstrated that protein synthesis inhibition by Ep2/SAP is rapidly achieved, since a 90% reduction is observed within 3.1 h, and that this inhibitory activity is apparently first order with time. Furthermore, the cytotoxic activity of the immunoconjugate is not dependent, and is not influenced by, the presence in the culture medium of the lysosomotropic agent chloroquine.

Regulation of the antigenic phenotype of human melanoma cells by recombinant interferons.

The ability of interferons to modulate the antigenic phenotype of tumor cells may involve alterations in the transcription, translation, membrane expression and shedding of Major Histocompatibility Complex (MHC) and Tumor Associated Antigens (TAAs). In the present study we have investigated possible mechanisms by which recombinant human interferons, IFN-alpha, -beta and -gamma, alter the antigenic profile of long- and short-term human melanoma cultures. IFN-alpha and -beta induced similar changes in the synthesis, expression and shedding of two TAAs, a HMW-MAA and a Cyt-MAA, in the established melanoma cell line Colo 38, whereas IFN-gamma exerted a differential effect on these melanoma associated antigens. Moreover, IFN-gamma was relatively more effective than IFN-alpha or -beta in upregulating the synthesis, expression and shedding of class I MHC antigens. At the same time a dramatic differential effect of the interferons was observed with class II MHC antigens. IFN-alpha or -beta induced a modest increase in the synthesis and expression of these antigens, whereas IFN-gamma was greater than 3-fold more active in inducing the synthesis and expression of DR/DP antigens and greater than 4- and greater than 10-fold more effective in increasing the synthesis and expression, respectively, of DQ antigens. Analysis of the levels of cytoplasmic mRNA for the DR-alpha and DQ-beta genes indicated no significant difference between IFN-alpha, beta or -gamma treated cells suggesting that IFN-gamma enhancement of the synthesis of DR and DQ antigens may occur at a posttranscriptional level. In the case of a newly established human melanoma cell line (MG-3) IFN-gamma enhanced the synthesis but not the expression of DR antigens and did not alter either the synthesis or expression of DQ antigens. Our studies indicate that the effects of interferons on the antigenic phenotype of melanoma cells will vary depending on the type of interferon employed, the antigen monitored and the target cell studied. In addition, it is also apparent that some of the biosynthetic steps involved in regulating the synthesis, expression and shedding of antigens may be coordinately regulated in some melanoma cells, whereas these processes may be under independent control in other melanoma populations.

Retrovirus-derived shuttle vectors in the generation of murine bispecific MoAbs to be used in ex vivo and in vivo immunotherapy.

Mouse monoclonal antibodies specific for CD3, FcR and a melanoma associated antigen have been produced. Drug resistence of such hibrydomas has been obtained tranfecting them with plasmids containing genes conferring specific resistence. Retrovirus derived shuttle vectors with high transfection efficiency have been used for transfection. Hybrydomas were than fused and bispecific antibody producing cells selected. Purification was performed by HPLC. Such bispecific antibodies can be used in ex vivo and in vivo immunotherapy.

Recombinant human IFN-gamma, but not IFN-alpha or IFN-beta, enhances MHC- and non-MHC-encoded glycoproteins by a protein synthesis-dependent mechanism.

Despite quantitative as well as qualitative differences, all three types of IFN (IFN-alpha, IFN-beta, and IFN-gamma) modulate the synthesis as well as the expression of class I and class II histocompatibility Ag and a melanoma-associated Ag located in the plasma membrane as well as the cytoplasm of human melanoma cells. By employing inhibitors of RNA and protein synthesis it was demonstrated that IFN-alpha and -beta increase the expression of histocompatibility products and this tumor-associated Ag by a process not requiring new protein synthesis. In contrast, IFN-gamma does require de novo protein synthesis for its modulatory activity. Thus, it appears that IFN might trigger various adaptive functions in different cell lineages by inducing at least two separate sets of responses specific for either IFN-alpha and -beta or IFN-gamma. Because the induction requirements for (2'-5')-oligoadenylate synthetase as well as for the development of a cellular antiviral state by different IFN also display a similar protein synthesis dependence pattern, the present results suggest that a similar set of cellular mediators may be involved in the modulation of antigenic expression by IFN-gamma in human melanoma cells.

Accessory cell function of human melanoma cells in mitogen-induced T cell proliferation.

Six out of eight human melanoma cell lines were found to be able to function as accessory cells in PHA-induced proliferation of autologous and allogeneic T cells. The accessory cell function of the melanoma cell lines appears to be similar to that of monocytes, requires the presence of viable cells, and does not correlate with the cell surface binding sites for PHA and with the level of expression of HMW-MAA and of HLA Class I antigens. HLA Class II antigens do not appear to play a major role in these phenomena, since there is no relationship between level of expression of HLA Class II antigens and accessory cell function of melanoma cells. Furthermore, addition of anti-HLA Class II monoclonal antibodies does not affect proliferation of T cells stimulated with PHA in the presence of melanoma cells with accessory cell function. Although melanoma cells exert accessory cell function, functional and immunological assays did not detect IL-1 in the spent medium of the melanoma cell lines. Furthermore, Northern blotting analysis with IL-1 alpha and IL-1 beta probes did not detect IL-1-specific mRNA in melanoma cell lines. These results suggest that PHA-induced proliferation of T cells in the presence of melanoma cells can bypass the requirement for IL-1 or utilizes factors other than IL-1.

A third polypeptide associated with heavy and light chain subunits of class I HLA antigens in immune interferon-treated human melanoma cells.

Recombinant immune interferon (IFN-gamma) increases the synthesis, expression and shedding of class I HLA antigens by the cultured human melanoma cell line Colo 38. The magnitude of the IFN-gamma-induced changes in class I HLA antigens is dependent on both the dose and the time of exposure to IFN-gamma and is more pronounced on the heavy chain subunit than on beta 2-microglobulin (beta 2m). In addition, a third, distinct polypeptide with a molecular mass of 14 kDa is present as a major component of the class I HLA molecular pool in IFN-gamma-treated melanoma cells. As IFN-gamma preferentially increases the synthesis of the heavy chain over that of beta 2m, the newly induced 14-kDa component appears to quantitatively replace beta 2m. The stoichiometric relationship between the three molecules suggests that the IFN-gamma-induced 14-kDa component may be involved in the insertion of the heavy chain subunit into the plasma membrane.

Characterization of cytotoxic activity of saporin anti-gp185/HER-2 immunotoxins.

The oncogene HER-2/neu encodes a trans-membrane receptor of 185 kDa with tyrosine-kinase activity. Over-expression of this molecule has been reported in a significant proportion of human breast and ovarian carcinomas, characterized by a poor clinical prognosis. Two monoclonal antibodies (MAbs), recognizing distinct epitopes of the gp 185 extracellular domain, have been utilized in the present study for the production of immunotoxins (ITs) by conjugation to the type-1 RIP (ribosome-inactivating protein) plant toxin saporin 6 (SAP). These ITs have been shown to retain tumor-specificity and specifically to inhibit protein synthesis in the gp185HER-2(+) SK-BR-3 breast-carcinoma cell line with IC50 values lower then 1 nM. Kinetics of the cytotoxic activity of the ITs are characterized by a slow rate, since incubation times ranging from 24 to 60 hr, depending on the different degree of expression of the receptor, are required to determine > 90% inhibition in the incorporation of radiolabeled leucine. However, the cytotoxic activity of these ITs, as evaluated by a more sensitive clonogenic assay, appears highly potent, since we have observed that 3 to 4 logs of cells are killed upon exposure to the ITs for short times at concentrations ranging from 1 to 5 x 10(-8) M.

Production and characterization of two immunotoxins specific for M5b ANLL leukaemia.

We describe the production and functional characterization of 2 monocytic-cell-lineage-specific immunotoxins constructed with saporin emitoxin (SAP) from Saponaria officinalis. Interest in the production of these immunotoxins, of possible clinical relevance, has been raised by the availability of 2 MAbs of high specificity for circulating monocytes and M5b ANLL, thus envisaging their potential use in bone-marrow purging. SAP emitoxin was selected on the basis of the low cytotoxicity in unconjugated form, as opposed to highly specific cytotoxicity and favourable pharmacokinetical properties in the conjugated form. SPDP conjugation produced immunotoxins which retained serological specificity and protein-synthesis-inhibitory activity. The 2 immunotoxins did not interfere with bone-marrow progenitor-cell growth in a CFU-GM colony assay. On the contrary, they were capable of killing monocytic cells selectively, as demonstrated in phenotypical and functional assays. Thus these 2 novel immunotoxins appear to be promising reagents in purging autologous bone marrow prior to transplantation in patients suffering from monocytic leukaemia.

Immunotoxins to the HER-2 oncogene product: functional and ultrastructural analysis of their cytotoxic activity.

Two immunotoxins were prepared using monoclonal antibodies (mAb) directed towards two distinct epitopes of the gp185HER-2 extracellular domain, and the type I ribosome inactivating protein (RIP) plant toxin saporin 6. Cell protein synthesis inhibition assay reveals that the immunotoxins display a potent and specific cytotoxicity that is characterized by a slow rate, since the time required to inhibit incorporation of radiolabeled leucine completely ranges from 36 h to 60 h depending on the target cell line and the immunotoxin. Because this feature may hamper the immunotherapeutic use of these conjugates we analysed this further by studying the early phases of internalization of immunotoxins by immunoelectron microscopy. The results of this study have demonstrated that the distribution pattern of the immunotoxins and of the unconjugated mAb over the cell surface overlaps. Similarly the mAb and immunotoxins are internalized into the cell by two different pathways: via clathrin-coated pits or via smaller uncoated pits and vesicles. A higher degree of internalization is achieved when the two immunotoxins are used in combination. Unlike the slow kinetics of cell intoxication the process of immunotoxin endocytosis is characterized by a rapid rate of internalization (above 40% at 5 min in the SK-BR-3 cell line). Although these findings provide no clue to explain the mechanisms of the slow rate of cytotoxicity of the two immunotoxins their rapid internalization indicates that these reagents can be exploited in immunotherapeutic approaches to gp185HER-2-expressing malignancies.

A murine monoclonal antibody strictly reactive with human peripheral blood monocytes.

We have produced and characterized a novel murine monoclonal antibody (LAM7) of IgG1 isotype which appears specific for peripheral blood monocytes (PBM) on the basis of histochemical and functional studies. By indirect immunofluorescence, including FACS analysis, the antibody reacts with 90 +/- 6% of PBM and with monocytic leukemias, while it is totally unreactive with B and T lymphocytes, platelets, granulocytes, peripheral macrophages, dendritic cells, large granular lymphocytes, and nonmonocytic leukemias. The antigen-presenting capacity of peripheral blood mononuclear cells is abolished by treatment with MoAb LAM7 in an antiglobulin-complement-mediated cytotoxicity test, and restored by addition of purified PBM. The progressive disappearance of the antigen recognized by LAM7 from PBM within approximately 3 days in culture, and its absence from both bone marrow precursors and tissue macrophages, define it as a line-specific and stage-specific differentiation.

mAb KUL/05 identifies a denaturation-resistant determinant shared by class II MHC products DR, DQ and DP.

mAb KUL/05, a novel murine monoclonal antibody, reacts with molecules displaying the typical tissue distribution and molecular profile of class II MHC antigens. An extensive scrutiny employing serological and immunochemical assays on DR homozygous and DR alpha- mutant cell lines has shown that this reagent displays some additional, interesting features, namely mAb KUL/05 (a) binds in a broadly monomorphic fashion to cells of DR1 through seven specificities, (b) recognizes a determinant shared by a large proportion of DR, DQ and DP beta chains from most haplotypes, in both their monomeric and alpha chain-associated forms, and (c) reacts with frozen, acetone-fixed, as well as conventional, formalin-fixed, paraffin embedded tissues. Thus, mAb KUL/05 is likely to represent a useful adjunct for the study of the expression of class II MHC products in normal and pathological tissue specimens.

Gene transfer by retrovirus-derived shuttle vectors in the generation of murine bispecific MAbs.

This report deals with the use of gene transfer by retrovirus-derived shuttle vectors in a novel model aimed at the generation of hybrid hybridomas secreting bispecific monoclonal antibodies (biMAbs). Following this approach, two genes conferring dominant resistance trait to the neomycine analogue geneticin (G418) and to methotrexate (MTX) respectively, were infected in two established hybridoma lines, each producing a well characterized MAb. The vectors used here were replication-deficient, being dependent on the complementation of helper virus provided by packaging lines. The infection procedure involved co-cultivation of the hybridomas with irradiated packaging cell lines, previously transfected with the vectors and producing the recombinant retroviruses, not inclusive of helper virus in their genome. The packaging lines used were psi2 ecotropic cells made able to produce high titers of virus. Further, the vector pMV7 was carrying G418 resistance while the pSDHT render the cells able to survive MTX. Easy and fast transfer of the dominant selection markers yielded lines of hybridomas to be fused according to the conventional somatic fusions. The resulting double hybridomas were tested for the production of hybrid molecules retaining parental specificity and successively underwent extensive cloning. The purification system featuring the most efficiently between the true biMAbs and the parental immunoglobulins (or other combination products) proved to be HPLC on hydroxylapatite column. The method described above was successful in producing two biMAbs targeting simultaneously molecules expressed on cytotoxic cells (such as CD3 on T-lymphocytes and CD16 on NK cells) and the melanoma-associated antigen Ep2.