Elimination of malignant clonogenic cells from human bone marrow using multiple monoclonal antibodies and complement

A clonogenic assay has been developed that utilizes Burkitt's lymphoma tumor cell lines to detect elimination of up to 5 logs of tumor cell contamination within human bone marrow. Different Burkitt's lymphoma lines bear one or more of a group of markers, including common acute lymphoblastic leukemia antigen gp26 (glycoprotein with a molecular weight of 26,000), B1, surface membrane immunoglobulin, HLA, beta 2-microglobulin, and Ia. Burkitt's tumor cells of the Namalwa line have been mixed with a 20-fold excess of irradiated human bone marrow cells. After treatment with one or more monoclonal antibodies and rabbit complement (RC), mixtures have been grown on a monolayer of irradiated human bone marrow cells and tumor cells enumerated by limiting dilution. Multiple treatments with antibody and RC were more effective than a single treatment in destroying clonogenic tumor cells which bore relevant determinants. Human serum components inhibited the lytic activity of RC in the presence of murine monoclonal antibodies. The total concentration of bone marrow cells proved critical in determining the complete elimination of tumor. Incubation of the Namalwa tumor cell line with RC and the J2 anti-gp26 eliminated more than 3 logs of malignant cells from a 20-fold excess of human bone marrow. Combinations of two monoclonal antibodies were more effective than any single antibody in eliminating Namalwa cells. A combination of three monoclonal reagents was no more effective than a combination of J2 and B1 or J2 and J5 in eliminating Namalwa cells. Treatment of human bone marrow with three antibodies and RC did not, however, produce a selective loss of nonmalignant GM-CFU-C, CFU-E, or BFU-E.     

Elimination of clonogenic malignant human T cells using monoclonal antibodies in combination with 2'-deoxycoformycin

2'Deoxycoformycin (dCF) specifically inhibits adenosine deaminase (ADA) and causes selective cytotoxicity of normal and malignant T cells. In clinical trials, dCF caused rapid lysis of malignant T lymphoblasts. Although dCF has been associated with dose-limiting nonhematopoietic toxicities, myelosuppression has not been observed. Since dCF is relatively nontoxic to hematopoietic stem cells, we tested dCF for utility in the ex vivo purging of malignant T lymphoblasts from remission leukemic bone marrow for autologous bone marrow transplantation. We found that T lymphoblast cell lines were sensitive to dCF (plus deoxyadenosine [dAdo]) under conditions that did not ablate human hematopoietic colony-forming cells. Moreover, combined pharmacologic (dCF plus dAdo) and immunologic (anti-T cell monoclonal antibodies [McAb] plus complement) purging resulted in additive reduction in clonogenic T lymphoblasts. These results provide the basis for a clinical trial of bone marrow transplantation using combined pharmacologic/immunologic purging of T lymphoblasts from patients' harvested autologous marrow.     

Elimination of clonogenic tumor cells from human bone marrow using a combination of monoclonal antibody:ricin A chain conjugates

Effective autologous bone marrow transplantation for leukemia and lymphoma is likely to depend upon the selective removal in vitro of malignant cells from normal human bone marrow precursors. Highly specific cytotoxic conjugates formed by coupling ricin A chain to monoclonal antibodies might prove useful for the selective elimination of malignant cells. Consequently, ricin A chain conjugates have been prepared with several different murine monoclonal antibodies and tested for their ability to eliminate clonogenic Burkitt's lymphoma cells from an excess of human bone marrow. The most active reagents included an antibody:A chain conjugate which bound to the nonpolymorphic chain of the la molecule and another which reacted with the mu heavy chain of cell surface immunoglobulin. Conjugates formed with anti-common acute lymphoblastic leukemia antigen, anti-Mr 26,000 glycoprotein, and anti-B1 were much less active on these Burkitt's cells, contrasting with results of complement-dependent tumor cell lysis. Tumor cell kill was partially inhibited by the addition of greater than 2 X 10(6) human bone marrow cells/ml but could be potentiated by increasing the concentration of conjugate or by the addition of 10 mM ammonium chloride. In the presence of ammonium chloride, at least 4 logs of clonogenic tumor cells could be eliminated within 24 h from a 20-fold excess of bone marrow using 10(-7) M ricin A chain linked to one or two different antibodies. Similar treatment of normal human bone marrow temporarily inhibited granulocyte-macrophage colony-forming units (cell) formation but did not compromise establishment of continuous bone marrow cultures. The degree of selective elimination of tumor cells with A chain antibody conjugates was comparable to that achieved with 4-hydroperoxycyclophosphamide or with multiple monoclonal antibodies and complement.     

Selective depletion of human myeloma clonogenic stem cells from bone marrow cell preparations by a plasma-cell reactive antibody and complement

The monoclonal antibody MM4 reacts with human myeloma cells from plasma cell dyscrasia (PCD)-derived cell lines and bone marrow (BM) biopsies from PCD patients, but not with normal BM or peripheral blood mononuclear (PBM) cells. We examined cytotoxicity of MM4 and rabbit complement (MM4:C') on mixtures of normal BM mononuclear cells and myeloma cells from three different PCD-derived cell lines, RPMI 8226, GM 1312, or ARH-77. For cell preparations containing 10% myeloma cells, treatment with MM4 (500 micrograms per 10(5) cells, 4 degrees C, 60 min) and two cycles of complement (1:8, 23 degrees C, 2 x 30 min) consistently eliminated 2 logs or more of clonogenic myeloma stem cells, as determined by colony growth assays and limiting dilution analysis (99.4%, 98.9%, and 99.96% reduction of RPMI 8226, GM 1312, and ARH-77 cells, respectively). The majority of normal marrow progenitors were spared (inhibition of CFU-C: 10-13%; BFU-E: 0%). These observations suggest that MM4 may be useful for selective depletion of human myeloma clonogenic stem cells from bone marrow ex vivo.     

Elimination of malignant clonogenic breast cancer cells from human bone marrow

Autologous bone marrow transplantation is a promising approach to the treatment of breast cancer but is at present limited to patients without bone marrow metastases. To eliminate malignant clonogenic breast cancer cells from normal human bone marrow, immunomagnetic separation has been combined with chemoseparation using 4-hydroperoxycyclophosphamide. Breast cancer cell lines have been mixed with a 10-fold excess of irradiated human bone marrow from normal donors. Mixtures have been incubated with a combination of five different monoclonal antibodies which bind to epithelial cell surface antigens of Mr 42,000, 55,000, 72,000, 200,000, and greater than 200,000. Antiglobulin coated microspheres which contained magnetite were added, and tumor cells were trapped in a magnetic field. Elimination of tumor cells from the decanted marrow was measured in a limiting dilution assay. Two treatments with antibody and microspheres permitted elimination of 2-4 logs of clonogenic breast cancer cells, depending upon the cell line studied. Similar treatment of nonirradiated normal marrow failed to affect levels of colony forming units-granulocyte-macrophage significantly. Use of immunomagnetic purging in combination with 4-hydroperoxycyclophosphamide eliminated up to 5 logs of tumor cells but reduced the recovery of colony forming units-granulocyte-macrophage. If prompt engraftment is observed following reinfusion of similarly treated marrow in phase I trials, these techniques should permit extension of autologous bone marrow transplantation to a larger population of breast cancer patients.     

Effects of monoclonal antibody and complement treatment of human marrow on hematopoiesis in continuous bone marrow culture

Long-term bone marrow cultures were established from single-cell suspensions of human bone marrow that had been treated with monoclonal antibodies and complement. Each treated cell suspension was evaluated for production of hematopoietic stem cells over 20 weeks. Treatment with antibody to HLA-DR (Ia), B1, J2, or J5 did not remove adherent cells including those differentiating to adipocytes in 17-hydroxycorticosteroid. In contrast, treatment with monoclonal antibody directed against human beta 2-microglobulin reduced adipocyte numbers by 100-fold and reduced the total adherent cell density over 70%. Cumulative total nonadherent cell and granulocyte-macrophage colony-forming units (GM-CFUc) production over 20 weeks was not significantly altered by one cycle of anti-Ia plus complement or up to three cycles of treatment with complement and anti-J2, -J5, or -B1. However, one cycle of treatment with anti-beta 2-micro-globulin depressed production of both GM-CFUc and nonadherent cells by over 100-fold compared to other treatment groups. While one cycle of treatment of anti-Ia and complement killed all detectable cells forming CFU-erythroid-granulocyte-megakaryocyte-macrophage, blast-forming units (erythroid), and GM-CFUc, GM cluster-forming cells survived. Treatment of marrow with three cycles of anti-Ia and complement removed all detectable GM colony- and GM cluster-forming cells; however, this marrow produced fewer cumulative Ia-positive GM-CFUc. Long-term bone marrow cultures may prove to be an interesting system for in vitro analysis of the effects of new immunotherapeutic agents including other monoclonal antibodies prior to clinical use.     

Elimination of clonogenic breast cancer cells from human bone marrow. A comparison of immunotoxin treatment with chemoimmunoseparation using 4-hydroperoxycyclophosphamide, monoclonal antibodies, and magnetic microspheres

Autologous bone marrow transplantation (ABMT) may aid in the management of breast cancer, but is currently limited to patients without bone marrow metastases. In earlier studies, 5 logs of malignant clonogenic breast cancer cells could be eliminated from human bone marrow using a combination of chemoseparation with 4-hydroperoxycyclophosphamide (4-HC) and immunoseparation with monoclonal antibodies and magnetic microspheres. In this report the authors compare chemoimmunoseparation to treatment with immunotoxins for elimination of tumor cells from human bone marrow and for the preservation of normal precursors. Breast cancer cells from each of five cell lines were mixed with a tenfold excess of irradiated human bone marrow cells. Treatment with a combination of five immunotoxins reduced clonogenic tumor cell growth by 1.8 to 5.5 logs depending upon the cell line. With two of the five cell lines, clonogenic tumor cells were eliminated quantitatively. Using the CAMA-1 breast cancer cell line, treatment with multiple immunotoxins was compared with chemoimmunoseparation with 4-HC, a panel of five unconjugated monoclonal antibodies and magnetic microspheres. Chemoimmunoseparation eliminated 3.5 to 5.4 logs of malignant cells, while preserving 21% of Colony-forming unit-granulocyte-macrophage (CFU-GM) and 37% of burst-forming unit-erythrocyte (BFU-E). No clonogenic breast cancer cells could be detected. Immunotoxin treatment eliminated 2.2 to 5.4 logs of clonogenic breast cancer cells, but had no effect on the bone marrow precursors. In seven of ten experiments, however, clonogenic breast cancer cells remained after immunotoxin treatment. Consequently, treatment with 4-HC, multiple murine monoclonal antibodies and magnetic microspheres provided more consistent elimination of tumor cells than separation with immunotoxins, but was significantly more toxic for marrow precursors.     

Elimination of small cell lung cancer cells in vitro from human bone marrow by a monoclonal antibody

We report here a useful method for elimination of small cell lung cancer cells in vitro from bone marrow. A monoclonal antibody, TFS-2, which mediates complement lysis and recognizes an antigen present on small cell lung cancer cells but not lymphoid cells or bone marrow cells, was used to clear infiltrated bone marrow. The antibody in the presence of complement effectively killed tumor cells, but it was not cytotoxic to bone marrow cells. When mixed populations consisting of tumor cells and bone marrow cells were treated with antibody and complement, the tumor cells were also effectively killed, except when large numbers of bone marrow cells were present, whereas TFS-2 had no significant effect on bone marrow stem cells, as judged by colony-forming unit assays.     

Selective cytotoxicity of murine monoclonal antibody LAM2 against human small-cell carcinoma in the presence of human complement: possible use for in vitro elimination of tumor cells from bone marrow

LAM2 is a murine IgM monoclonal antibody (MAb) which binds strongly to the cell membrane of human lung small-cell carcinoma (SCC) and squamous-cell carcinoma but not to normal bone-marrow cells. The cytotoxicity of this antibody in the presence of human complement was investigated in vitro by chromium release and clonogenic assays. The optimal treatment conditions included incubation with antibody for 30 min at 37 degrees C followed by 3 additions of human complement 30 min apart. Cell lysis ranged from 94 to 98% in 4 SCC cell lines at antibody dilutions of 1:100: a lower level of lysis (60%) occurred in a lung squamous-cell carcinoma cell line. The cytotoxic effect was strictly complement-dependent. No cytotoxic effect was seen with other human cell lines including lung adenocarcinoma, lung large-cell carcinoma, myeloid leukemia, and lymphoblastic leukemia. No lysis was seen with nucleated marrow cells from healthy volunteers. Normal marrow cells in excess did not inhibit SCC cell lysis. Incubation with antibody and complement resulted in a 100-fold reduction of colony formation of SCC cells, but did not reduce the number of colonies of marrow-cell precursors, including CFU-GEMM, BFU-E, and CFU-C. The selective cytotoxicity of LAM2 antibody to SCC, but not to normal bone-marrow cells, suggests that this antibody may be useful for the in vitro elimination of SCC cells from the bone marrow.     

Purging tumor cells from bone marrow by use of antibody and complement: a critical appraisal

This review highlighted several problems associated with the use of antibody and complement in the elimination of tumor cells from bone marrow that was to be used for transplantation, and it discussed some of the difficulties encountered in developing this approach in model systems. These problems should be seriously considered by any clinician contemplating this method for bone marrow purging.     

In vitro purging of clonogenic leukemic cells from human bone marrow by heat: simulation experiments for autologous bone marrow transplantation.

In order to apply a simple purging method by heat to autologous bone marrow transplantation (ABMT), we have revaluated the ability to purge clonogenic leukemic cells from the simulated marrow mixture of normal marrow cells and leukemic cell lines (HL-60, Molt-3 and HEL) in vitro by heat, using two different clonogenic assays for normal granulocyte-macrophage progenitors (CFU-GM) and leukemic cell lines. It appeared that in vitro hyperthermia (42 degrees C for 120 min) is able to selectively remove clonogenic leukemic cells from simulated tumor cell-normal marrow mixtures even when leukemic cell concentrations are increased up to 3 x 10(6) cells/ml in vitro, and results in a 4-6 log destruction of clonogenic leukemic cells/ml according to a limiting dilution assay, while leaving half of normal CFU-GM surviving. The hyperthermic purging of clonogenic leukemic cells was not affected in the presence of normal marrow cells in vitro. This high level of clonogenic leukemic cell depletion by heat correlated with that of immunologic and pharmacologic studies. These results suggest that in vitro hyperthermia could be applied effectively and safely for the elimination of residual clonogenic leukemic cells in autologous marrow grafts before ABMT.     

Detection of neuroblastoma cells in bone marrow using GD2 specific monoclonal antibodies

Three murine monoclonal antibodies (Mab) against a cell surface antigen, the disialoganglioside GD2, were investigated for detecting bone marrow metastasis in patients with neuroblastoma (NB) by indirect immunofluorescence (IF). As few as 0.01% NB cells could be detected. No Mab reactivity was found in 60 non-NB marrows. Thirty-five marrows from patients with stage III and stage IV NB at diagnosis were examined during the course of their disease. Tumor involvement was found in 74% by Mab, 55% by the two-layer soft agar clonogenic assay (CA), and 27% by conventional histochemical stains. All marrows containing NB histologically were positive for tumor by Mab; 71% were also positive by CA. Of histologically negative marrows, 63% were Mab positive, the majority (78%) of which were also positive by CA. All Mab nonreactive samples were negative histologically and by CA. We conclude that these antibodies are highly sensitive and specific in detecting NB metastasis in bone marrow.     

A novel human monoclonal antibody,TONO-1, reactive with T-lymphocytic leukemia cells

Mononuclear cells from the peripheral blood of patients with systemic lupus erythematosus (SLE) were transformed with the Epstein-Barr virus (EBV) and the resultant polyclonal B-lymphoblastoid cell lines were tested for antibody activity to membrane antigens of certain T-cell lines. B lymphoblastoid cell lines secreting specific antibodies were fused with (mouse x human) heteromyeloma SHM-D33 cells. Among the large number of hybridomas generated, one which produced a human monoclonal antibody (MAb) TONO-I (1gM, λ) was selected. MAb TONO-I proved to be reactive with 4 human T-cell lines, HPB-MLT, L-MAT, MOLT-3 and MOLT-4F, but not with B-leukemia, Burkitt's lymphoma, myelomonocytic leukemia, erythroleukemia or non-hematopoietic malignant cell lines. MAb TONO-I reacted positively with fresh leukemia cells from 2 of 7 patients with acute T-lymphocytic leukemia, but no reaction was observed in non-T-cell leukemia cases. Normal lymphocytes, monocytes, granulocytes, red blood cells and platelets in the peripheral blood did not demonstrate remarkable binding. Neither thymocytes nor bone-marrow cells from healthy volunteers were reactive. The antigens defined by MAb TONO-I were polypeptides of 57 kDa and 68 kDa. Immunohistological studies revealed no staining of thymocytes in the thymus of a 6-month-old child, but showed epithelial reticular cells and Hassall's corpuscles to stain positively. These results suggest that MAb TONO-I is directed to T-leukemic cells and some components of thymus tissue. © 1995 Wiley-Liss Inc.     

Production and characterization of monoclonal antibodies to mesenchymal stem cells derived from human bone marrow

The bone marrow (BM) serves as a reservoir for different classes of stem cells. In addition to haematopoietic stem cells, bone marrow contains a population of mesenchymal stem cells (MSCs). These cells have a multilineage differentiation capacity and are able to generate progenitors with restricted developmental potential, which include fibroblasts, osteoblasts, adipocytes, and chondrocytes. Characteristic markers have been reported for expanded MSCs, but none of these markers are specific for MSCs. Thus, the objective of this study was to produce monoclonal antibodies against MSCs. MSCs derived from human bone marrow were cultured, expanded, and immunized into mice, and spleen cells subsequently harvested were used to generate hybridoma cell lines secreting antibodies against MSCs. Hybridoma culture supernatants were screened for antibodies against MSCs by enzyme-linked immunosorbent assay (ELISA), and 33 positive clones were then screened against cell suspensions of MSCs by immunofluorescence staining and flow cytometry. Ten clones were positive in immunofluorescence staining. Among these, three hybridoma cell lines, namely, YS08, YS14, and YS18 were found to be reactive with MSC by flow cytometry, but non-reactive with human tumor cell lines and hematopoietic stem cells. YS08 and YS14 showed specific bands in Western blotting. In conclusion, we developed three monoclonal antibodies, YS08, YS14, and YS18, that recognize human MSC cell surface antigen.     

Variables affecting the killing of cultured human neuroblastoma cells with monoclonal antibody and complement

To determine incubation conditions that result in optimal in vitro killing of human tumor cells with monoclonal antibody and complement, variables affecting the killing of cultured human neuroblastoma cells with monoclonal antibody 6-19 and baby rabbit complement were studied. Neuroblastoma target cells were stained with the fluorescent dye Hoechst 33342, which enables rapid, sensitive detection of surviving cells in conjunction with trypan blue exclusion. Maximal cell lysis was obtained at an antibody concentration of 5-10 micrograms/ml. Greater than 5 logs of cell kill were obtained with appropriate treatment conditions. No antigenic modulation was detected. Complement activity was found to be the factor which limited the extent of cell kill. Specific cell lysis increased with increasing concentration of complement. As the reaction with antibody coated cells proceeded, complement activity was depleted. This resulted in the greatest cell kill occurring during the first 10-20 min of treatment. Generation of factor(s) inhibitory to complement activity was also found. However, the effect of inhibition on limiting the extent of cell kill was much less significant than the effect of complement depletion. When compared to a single incubation of the same total duration, the reduction in complement activity with duration of incubation resulted in greater killing by multiple brief incubations with fresh complement. The depletion of complement was found to increase at a greater cell concentration. This resulted in greater proportional survival as neuroblastoma cell concentration increased. Depletion of complement activity by aggregated monoclonal antibody was found to decrease cytotoxicity. This evaluation may provide a framework for optimization of target cell destruction using complement and other monoclonal antibodies and target cells.     

Inhibition by chemotherapeutic agents of human bone marrow progenitor cells and clonogenic cells of a lymphoblastic cell line

Using methylcellulose based semi-solid medium enriched with complete alpha medium and undialysed fetal calf serum, we compared the cytotoxic effects between the clonogenic cell (CFU) of a lymphoblastic leukemia cell line (MOLT-4) and normal bone marrow granulocytic progenitor cells (CFU-C) of methotrexate (MTX), vincristine (VCR), hydrocortisone (HC) and daunorubicin (DNR), as well as the reversibility by leukovorin (LV) of MTX cytotoxicity. The neoplastic cells were more markedly inhibited by MTX, VCR and HC following either brief or continuous exposure of the cell to the drugs. Inhibition by DNR was identical for both cell types. In order to reverse the inhibitory effects of MTX in MOLT-4 CFU, LV concentrations needed were at least one log order higher than MTX concentrations. At higher concentrations of MTX, LV reversal was less effective. Only one of four different human bone marrows studied were significantly inhibited by MTX under the experimental conditions. It is suggested that the high concentrations of nucleosides and deoxynucleosides present in the medium aborted the MTX cytotoxicity on CFU-C of normal human marrow but not on MOLT-4 CFU, while the differential inhibition observed with VCR and HC was due to greater sensitivity of the leukemic cells.     

Model system for removing neuroblastoma cells from bone marrow using monoclonal antibodies and magnetic immunobeads

Variables effecting removal of neuroblastoma cells from bone marrow using monoclonal antibodies and magnetic immunobeads were studied. Human neuroblastoma cell lines were labeled with the supravital DNA stain Hoechst 33342, seeded into normal bone marrow, incubated with monoclonal antibodies recognizing neuroblastoma cell surface antigens (HSAN 1.2, antibody 459, antibody 390, BA-1, and Leu-7), and then mixed with magnetic microspheres coated with goat anti-mouse immunoglobulin. Tumor cells that attached to the magnetic immunobeads were then removed from the marrow with magnets. The efficacy of tumor cell removal depended on the amount of monoclonal antibody bound to tumor cells and the immunobead/tumor cell ratio. In addition, two cycles of purging with both monoclonal antibodies and immunobeads was superior to one cycle. Using a cocktail of the five antibodies, 3 to 4 logs of tumor cells could be depleted from marrow with good recovery of viable hematopoietic cells.     

Infusion-rate independent cellular adriamycin concentrations and cytotoxicity to human bone marrow clonogenic cells (CFU-GM)

The effect of adriamycin (ADM) infusion-rate on cellular ADM concentrations and on clonogenicity of human haematopoietic cells was studied in vivo and in vitro. In patients an ADM dose of 30 mg m-2 was administered as a bolus injection, or as a 4 h or a 24 h infusion. In vitro the effect of ADM on clonogenic cell growth was determined after exposure during 5 min, 2 h and 24 h of human bone marrow cells to increasing ADM concentrations. ADM showed rapid intracellular accumulation, to levels 100-fold the plasma concentration in vivo or the incubation medium concentration in the in vitro experiments. After a bolus injection or 5 min exposure only approximately 10% of the cellular peak ADM was retained after elimination of the drug from the plasma or the incubation medium. Ninety percent of the ADM was apparently 'loosely' bound. After 4 h and 24 h constant-rate infusions and also after 2 h and 24 h incubations in vitro, the cells accumulated ADM gradually, and the subsequent washing-out of the cellular ADM was substantially less, most of the ADM being 'tightly' bound. Despite these different patterns of uptake and retention after in vivo short- and long-lasting infusion of the same total dose, the 'tightly-bound' cellular ADM concentrations were the same. Moreover, comparable cellular ADM concentrations, retained after efflux of the 'loosely-bound' cellular ADM fraction were equally cytotoxic to normal human clonogenic cells. Short-lasting cellular peak ADM concentrations which occur after a bolus injection or after short exposure to high ADM concentrations are not essential for the cytotoxic effect, in contrast to the retained, 'tightly-bound' cellular ADM levels.     

Elimination of B-lymphoma cells from human bone marrow: model experiments using monodisperse magnetic particles coated with primary monoclonal antibodies

Conditions for removing B-lymphoma cells from human bone marrow using "immunobeads" (IBs) were investigated. The IBs were prepared by coupling monoclonal antibodies directly to a new type of monodisperse magnetic polymer particles (M 450). Two monoclonal immunoglobulin M antibodies, AB-1 (CD 19), a B-cell-specific antibody, and AB-4, an HLA-DR-specific antibody, were used. The IBs were incubated with Rael Burkitt lymphoma cells admixed to fresh, mononuclear human bone marrow cells. After incubation for 30 min at 4 degrees C, the IBs were removed using cobalt samarium magnets. The number of remaining clonogenic tumor cells was assayed by the Courtenay and Mills soft agar procedure, and the clonogenic capacity of the bone marrow progenitor cells was measured by granulocyte-monocyte and granulocyte-erythroid-monocyte-megakaryocyte assays. With a ratio of tumor cells to normal bone marrow cells of 0.1 or 0.01 and a ratio of immunobeads to tumor cells in excess of 75, a tumor cell depletion of more than 3 logs was achieved with the AB-4 IBs and slightly less with the AB-1 beads. After two consecutive cycles of purification with the AB-4 beads, no colonies were found, corresponding to more than 6 logs of purification. In the case of the AB-1 beads, 4 to 5 logs of purification were achieved. The concomitant reduction in clonogenic bone marrow progenitor cells was only 30 to 40%. Flow cytometric studies showed that the tumor cell population contained appreciable proportions of cells binding only small amounts of the antibodies used. The results indicate that the IB procedure is highly efficient and capable of removing tumor cells expressing low levels of antigen. Compared to other purging methods in use the procedure described seems to offer several advantages with respect to efficacy, speed, and simplicity. By the use of a panel of suitable antibodies the new immunobead procedure may be potentially useful in autologous bone marrow transplantation of B-lymphomas and non-T-leukemias with poor prognosis.     

Selective elimination of breast cancer cells from human bone marrow using an antibody-Pseudomonas exotoxin A conjugate

A pancarcinoma monoclonal antibody (NR-LU-10), homogeneously reactive with human breast cancer cells, was conjugated to Pseudomonas exotoxin A. The immunotoxin was evaluated for its potential for purging breast cancer cells from human bone marrow. The immunotoxin NR-LU-10 antibody did not react with normal bone marrow preparations yet readily detected 1% contamination of bone marrow by MCF-7 breast cancer cells added to normal bone marrow without significantly inhibiting the colony-forming ability of bone marrow progenitor cells. NR-LU-10-Pseudomonas exotoxin A has potential for purging bone marrow of breast cancer cells without impairing the growth of bone marrow progenitor cells.