Antileukemic and immunosuppressive activity of 2-chloro-2''-deoxyadenosine.

The adenosine deaminase-resistant purine deoxynucleoside 2-chloro-2'-deoxyadenosine (CdA) is markedly toxic in vitro to nondividing and proliferating normal human lymphocytes and to many leukemia cell specimens. The CdA is also effective against mouse L1210 leukemia in vivo. The present investigations have examined the pharmacology, chemotherapeutic activity, and toxicity of CdA in nine patients with advanced hematologic malignancies refractory to conventional therapy. When administered by continuous intravenous infusion, the deoxyadenosine analog was well tolerated. As monitored by radioimmunoassay, plasma CdA levels rose gradually during the infusions. The CdA was not deaminated significantly. In all patients with leukemia, the CdA lowered the blast count by at least 50%. In one patient with a T-cell leukemia-lymphoma, and in another patient with chronic myelogenous leukemia in blast crisis, the CdA infusion eliminated all detectable blasts from the blood and bone marrow. In a patient with a diffuse lymphoma complicated by severe autoimmune hemolytic anemia, CdA treatment quickly terminated the hemolytic process. Bone marrow suppression represented the dose-limiting toxicity, and was related to plasma CdA levels, cumulative drug dosage, and the rapid release of CdA that accompanied tumor cell lysis.     

Oral antilymphocyte activity and induction of apoptosis by 2-chloro-2''-arabino-fluoro-2''-deoxyadenosine.

2-Chlorodeoxyadenosine (CdA) is active in chronic lymphocytic leukemia, hairy-cell leukemia, and low-grade lymphomas. In part, this spectrum of activity may be attributable to the selective toxicity of CdA to nondividing lymphocytes and monocytes. However, CdA is unstable at acidic pH and is degraded by bacterial nucleoside phosphorylases. The present experiments demonstrate that the 2'-arabino-fluoro derivative of CdA, designated CAFdA, is also directly toxic to quiescent lymphocytes and macrophages. Unlike CdA, CAFdA was stable at pH 2 and resisted degradation by Escherichia coli nucleoside phosphorylase. Cell killing was preceded by the formation of DNA strand breaks and could be prevented by supplementation of the medium with deoxycytidine. The initial DNA damage initiated the pattern of oligonucleosomal DNA fragmentation characteristic of apoptosis. Mutant lymphoblasts, deficient in deoxycytidine kinase, with elevated cytoplasmic 5'-nucleotidase, or with expanded deoxynucleotide pools secondary to increased ribonucleotide reductase activity, were cross-resistant to both CAFdA and CdA toxicity. One-week oral treatment with CAFdA (1 mg/ml in drinking water) achieved an average plasma concentration of 0.56 microM and eliminated 90% of chronic lymphocytic leukemia cells transplanted into severe combined immunodeficiency (scid) mice. Under the same conditions, CdA was much less active. Collectively, these results suggest that CAFdA could be effective as an oral agent in indolent lymphoproliferative diseases and in autoimmune diseases where lymphocyte and monocyte depletion is desirable.     

Immucillin H, a powerful transition-state analog inhibitor of purine nucleoside phosphorylase, selectively inhibits human T lymphocytes

Transition-state theory has led to the design of Immucillin-H (Imm-H), a picomolar inhibitor of purine nucleoside phosphorylase (PNP). In humans, PNP is the only route for degradation of deoxyguanosine, and genetic deficiency of this enzyme leads to profound T cell-mediated immunosuppression. This study reports the biological effects and mechanism of action of Imm-H on malignant T cell lines and on normal activated human peripheral T cells. Imm-H inhibits the growth of malignant T cell leukemia lines with the induction of apoptosis. Imm-H also inhibits activated normal human T cells after antigenic stimulation in vitro. However, Imm-H did not inhibit malignant B cells, colon cancer cell lines, or normal human nonstimulated T cells, demonstrating the selective activity of Imm-H. The effects on leukemia cells were mediated by the cellular phosphorylation of deoxyguanosine and the accumulation of dGTP, an inhibitor of ribonucleotide diphosphate reductase. Cells were protected from the toxic effects of Imm-H when deoxyguanosine was absent or when deoxycytidine was present. Guanosine incorporation into nucleic acids was selectively blocked by Imm-H with no effect on guanine, adenine, adenosine, or deoxycytidine incorporation. Imm-H may have clinical potential for treatment of human T cell leukemia and lymphoma and for other diseases characterized by abnormal activation of T lymphocytes. The design of Imm-H from an enzymatic transition-state analysis exemplifies a powerful approach for developing high-affinity enzyme inhibitors with pharmacologic activity.     

Comparison of cytotoxicity of 2-chloro- 2'-arabino-fluoro-2'-deoxyadenosine (clofarabine) with cladribine in mononuclear cells from patients with acute myeloid and chronic lymphocytic leukemia

BACKGROUND AND OBJECTIVES: Clofarabine (CAFdA), one of the newer nucleoside drugs is undergoing a phase II clinical trial for the treatment of pediatric refractory/relapsed acute myeloid and lymphocytic leukemia. Although CAFdA is structurally similar to the clinically established analogs fludarabine and cladribine (CdA), its metabolism and mechanism of actions are significantly different. The present study investigates the in vitro cytotoxicity of CAFdA and CdA in mononuclear cells isolated from 52 patients with chronic lymphocytic (CLL) and acute myeloid leukemia (AML). DESIGN AND METHODS: We incubated the leukemic cells with drugs for 48 hours and cytotoxicity was then evaluated by the MTT dye assay. We also determined the levels of deoxycytidine and deoxyguanosine kinase with radio-chemical substrate-based assays and used a high performance liquid chromatographic method to measure cellular nucleotides in leukemia cells after 2 hours' incubation. RESULTS: Using equimolar concentrations of CAFdA and CdA, the in vitro cytotoxicity for the population was significantly higher with CAFdA than with CdA (median EC50 for CAFdA 0.12 microM and for CdA 0.15 microM, p<0.001). From the individual estimates the difference in cytotoxicity between CAFdA and CdA was more pronounced in cells from CLL patients (median EC50 for CAFdA 0.08 microM and for CdA 0.16 microM p<0.001) than in those from AML patients. We also found that CAFdA was phosphorylated more efficiently than CdA. No correlations were detected in this study between the levels of CdA and CAFdA nucleotides, enzymes levels and the in vitro responses. INTERPRETATION AND CONCLUSIONS: The greater in vitro cytotoxicity and cell metabolism of CAFdA compared to CdA confirm the high activity of CAFdA and encourage clinical trials with CAFdA in leukemic patients.     

Resistance of leukemic cells to 2-chlorodeoxyadenosine is due to a lack of calcium-dependent cytochrome c release.

The purine nucleoside 2-chlorodeoxyadenosine (CdA) is often used in leukemia therapy. Its efficacy, however, is compromised by the emergence of resistant cells. In the present study, 3 CdA-resistant cell lines were generated and characterized. Their ability to accumulate 2-chloroadenosine triphosphate (CdATP) varied, reflecting differences in activities of deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK). Nonetheless, the selected lines were uniformly resistant to CdA-induced apoptosis, as assessed by caspase activation and DNA fragmentation. In contrast, cytosols from resistant cells were capable of robust caspase activation when incubated in the presence of cytochrome c and dATP. Moreover, replacement of dATP with CdATP also resulted in caspase activation in the parental and some of the resistant cell lines. Strikingly, CdA-induced decreases in mitochondrial transmembrane potential and release of cytochrome c from mitochondria were observed in the parental cells but not in any resistant lines. The lack of cytochrome c release correlated with an increased ability of mitochondria from resistant cells to sequester free Ca2+. Consistent with this enhanced Ca2+ buffering capacity, an early increase in cytosolic Ca2+ after CdA treatment of parental cells but not resistant cells was detected. Furthermore, CdA-resistant cells were selectively cross-resistant to thapsigargin but not to staurosporine- or Fas-induced apoptosis. In addition, CdA-induced caspase-3 activation and DNA fragmentation were inhibited by the Ca2+ chelator BAPTA-AM in sensitive cells. Taken together, the data indicate that the mechanism of resistance to CdA may be dictated by changes in Ca2+-sensitive mitochondrial events.     

Possible metabolic basis for the different immunodeficient states associated with genetic deficiencies of adenosine deaminase and purine nucleoside phosphorylase.

An inherited deficiency of adenosine deaminase (Ado deaminase; adenosine aminohydrolase, EC 3.5.4.4) causes severe combined immunodeficiency disease in humans. A similar deficiency in purine nucleoside phosphorylase (Puo phosphorylase; purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) engenders a selective cellular immune deficit. To elucidate the possible metabolic basis for the contrasting immunologic phenotypes, we compared the toxicity toward mature resting human lymphocytes of the Ado deaminase substrates deoxyadenosine and adenosine and the Puo phosphorylase substrate deoxyguanosine. When Ado deaminase was inhibited, micromolar concentrations of deoxyadenosine progressively killed nondividing helper and suppressor-cytotoxic T cells, but not B cells. The toxicity required phosphorylation, with subsequent dATP formation. The deoxyadenosine analogs 2-chlorodeoxyadenosine, 2-fluorodeoxyadenosine, and adenine arabinonucleoside also killed resting T cells. Cell death was unrelated to inhibition of adenosylhomocysteinase (EC 3.3.1.1) but was preceded by a gradual decline in ATP levels. As much as 1 mM deoxyguanosine did not impair resting lymphocyte viability, despite the synthesis of dGTP. The combination of 200 microM adenosine plus 500 microM homocysteine thiolactone killed dividing lymphocytes but had no discernible toxic effect toward resting T cells, which accumulated adenosylhomocysteine over a 4-hr period but thereafter excreted the nucleoside into the culture medium. The different clinical syndromes associated with genetic deficiencies of Ado deaminase and Puo phosphorylase may be explained by the ability of dATP to kill mature resting T lymphocytes by depleting ATP levels.     

Deficiency of methylthioadenosine phosphorylase in human leukemic cells in vivo

Cells from 20 patients with leukemia and 9 with solid tumors were assayed for the enzyme methylthioadenosine phosphorylase, which function in both purine and polyamine metabolism in rapidly dividing cells. As determined by autoradiography of viable cells, and by direct enzymatic analysis, samples from one individual with pre-T-cell acute lymphoblastic leukemia and one with common acute lymphoblastic leukemia were methylthioadenosine phosphorylase deficient. In contrast, other leukemias of similar antigenic phenotype, as well as normal peripheral blood lymphocytes, thymic lymphocytes, and normal bone marrow cells, had substantial methylthioadenosine phosphorylase activity. This evidence suggests that the complete absence of methylthioadenosine phosphorylase distinguishes some leukemic cells in vivo from their nonmalignant counterparts.     

Purinogenic immunodeficiency diseases: selective toxicity of deoxyribonucleosides for T cells.

Deoxyadenosine at low concentrations and in the presence of an inhibitor of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) is markedly toxic to lymphoblast cell lines of T cell origin but does not impair growth of B cell lines. Deoxyguanosine is also more toxic for T lymphoblasts. In the presence of deoxyadenosine or deoxyguanosine, elevation of the corresponding deoxyribonucleoside triphosphate (dATP or dGTP) occurs in T cell, but not in B cell, lines. The addition of deoxycytidine or dipyridamole results in lower dATP and dGTP levels and prevents deoxyribonucleoside toxicity. These findings provide a molecular basis for the immunodeficiency observed in individuals with several inborn errors of purine metabolism.     

Deoxycytidine kinase-mediated toxicity of deoxyadenosine analogs toward malignant human lymphoblasts in vitro and toward murine L1210 leukemia in vivo.

An inherited deficiency of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) produces selective lymphopenia and immunodeficiency disease in humans. Previous experiments have suggested that lymphospecific toxicity in this condition might result from the selective accumulation of toxic deoxyadenosine nucleotides by lymphocytes with high deoxycytidine kinase, levels and low deoxynucleotide dephosphorylating activity. The present experiments were designed to determine if deoxyadenosine analogs which are not substrates for adenosine deaminase might similarly be toxic toward lymphocytes and lymphoid tumors. Two such compounds, 2-chlorodeoxyadenosine and 2-fluorodeoxyadenosine, at concentrations of 3 nM and 0.15 microM, respectively, inhibited by 50% the growth of human CCRF-CEM malignant lymphoblasts in vitro. Each was phosphorylated in intact cells by deoxycytidine kinase accumulated as the nucleoside triphosphate, and inhibited DNA synthesis more than RNA synthesis. Both deoxynucleosides had significant chemotherapeutic activity against lymphoid leukemia L1210 in mice.     

The differences in purine metabolism between T and B lymphocytes

In this study enzyme activities involved in purine metabolism were measured in T and B lymphocytes separated by E and EAC rosetting methods. Adenosine deaminase, purine nucleoside phosphorylase and HGPRTase activities were significantly elevated in T cells, compared to the activities in B cells. There were no significant differences in adenosine kinase and APRTase activities between T and B lymphocytes. In contrast, PRPPsynthetase activities were higher in B cells than in T cells. The uptake of various radiolabeled precursors by mitogen stimulated lymphocytes was studied. The uptake of 14C-formate by the mitogen stimulated lymphocytes was markedly lower, compared to that of 14C-adenosine and or 14C-purine bases. The uptake of 14C-adenosine by PHA stimulated lymphocytes was considerably higher than that of Con A or PWM stimulated lymphocytes. However, the uptake of 14C-hypoxanthine into lymphocytes stimulated with PWM was increased by comparison with unstimulated lymphocytes. From these results it seems that adenosine plays a central role in the metabolism of T cells, and that purine bases are preferentially utilized in B cells.     

Quantitation of synergism of arabinosylcytosine and cladribine against the growth of arabinosylcytosine-resistant human lymphoid cells

This report presents a quantitative analysis of the synergistic interaction of arabinosylcytosine (araC) and cladribine (CdA) in human H9-lymphoid cell lines sensitive and resistant to araC (H9-araC cells). H9-araC cells obtained by cultivation of H9 cells in the presence of 0.5 μM arabinosylcytosine (araC) had lower deoxycytidine kinase (dCK) than the parental cell line. The IC₅₀ values of araC and CdA calculated by using median-effect analysis and CalcuSyn software were: 0.55 μM and 1.16 μM for CdA and 0.0058 μM and 3.5 μM for araC in H9 and H9-araC cells, respectively. These values were reduced to 0.10 μM and 0.38 μM for CdA and to 0.004 μM and to 0.77 μM for araC when the drugs were used in combination. Computerized simulation of dose reduction index (DRI) indicated that at 50–99% growth inhibition levels, the doses of araC could be reduced by 2.0 to 11.9-fold and 2.9 to 5.3-fold and the doses of CdA by 5.9 and 183.7-fold and 3.1 to 164.8-fold in H9 and H9-araC cells, respectively, when the drugs are used in combination. Assessment by combination index (CI) analysis showed that the combination exhibited moderate to strong synergistic lympho-cytotoxic effects. CdA metabolic studies (influx and activation) in the presence of deoxyadenosine, deoxycytidine, or araC suggested that CdA enters cells by a deoxyadenosine-inhibitable transport system, which is different than that of araC and deoxycytidine transport system. Thus, in addition to the known mechanisms, other mechanisms might be involved in the metabolism of CdA. The demonstration that araC and CdA combinations exert synergistic cytotoxicity even in the resistant cells raises hope that such a combination may be useful in tumors that were found resistant to these drugs.     

Induction of an apoptotic program in cell-free extracts by 2-chloro-2′-deoxyadenosine 5′-triphosphate and cytochrome c

Adenine deoxynucleosides, such as 2-chloro-2′-deoxyadenosine (2CdA) induce apoptosis in quiescent lymphocytes, and are thus useful drugs for the treatment of indolent lymphoproliferative diseases. However, it has remained puzzling why deoxyadenosine and its analogs are toxic to a cell that is not undergoing replicative DNA synthesis. The present experiments demonstrate that the 5′-triphosphate metabolite of 2CdA (2CdA-5′-triphosphate), similar to dATP, can cooperate with cytochrome c and Apaf-1 to activate caspase-3 in a cell free system. Chronic lymphocytic leukemia cells and normal peripheral blood lymphocytes expressed both caspase-3 and apoptotic protease activating factor 1. Incubation of the lymphocytes with 2CdA induced caspase-3 activation prior to DNA degradation and cell death. Stimulation of the caspase proteolytic cascade by 2CdA-5′-triphosphate, in the context of DNA strand break formation, may provide an explanation for the potent cytotoxic effects of 2CdA toward nondividing lymphocytes.     

DNA Synthesis in Thymic-Acute Lymphoblastic Leukaemia

Deoxyribonucleoside triphosphate (dNTP) concentrations were measured in bone marrow and peripheral blood leucocytes from seven patients with acute Thy-lymphoblastic leukaemia (Thy-ALL), 12 patients with acute myeloblastic leukaemia and 15 patients with acute non-T, non-B lymphoblastic leukaemia (c-ALL), and in thymocytes from patients with myasthenia gravis. Labelled thymidine and deoxycytidine incorporation into DNA was also studied.
In Thy-ALL, dNTP concentrations were markedly increased compared with those in the other acute leukaemias. The dNTP concentrations in thymocytes were, however, similar to those in Thy-ALL. ³H-nucleoside incorporation studies showed a marked difference in labelled deoxycytidine incorporation and particularly in the deoxycytidine/thymidine DNA labelling ratio between Thy-ALL and the other cell types.
We conclude that the pathways of DNA synthesis in Thy-ALL blasts are different from those in the cells from other acute leukaemias and some but not all these differences may correspond to differences between normal cortical thymocytes and bone marrow cells.     

Differential metabolism of deoxyribonucleosides by leukaemic T cells of immature and mature phenotype

Experimental evidence has indicated that T lymphoblasts are more sensitive to deoxynucleoside toxicity than are B lymphoblasts. These data have led to the use of purine enzyme inhibitors as selective chemotherapeutic drugs in the treatment of T cell malignancies ranging from T cell acute lymphoblastic leukaemia to cutaneous T cell lymphomas. We have compared the toxicities of 2'-deoxyadenosine, 2'-deoxyguanosine, and thymidine for T cell lines derived from patients with T cell acute lymphoblastic leukaemia with those for mature T cell lines derived from patients with cutaneous T cell leukaemia/lymphoma. We have found that both deoxynucleosides are far less toxic to the mature T cell lies than to T lymphoblasts and that the mature cells accumulate much lower amounts of dATP and dGTP when exposed to deoxyadenosine and deoxyguanosine, respectively. Similar studies performed on peripheral blood cells from patients with T cell leukaemias of mature phenotype and on peripheral blood T cells demonstrate similar low amounts of deoxynucleotide accumulation. Measurements of the activities of several purine metabolizing enzymes that participate in deoxynucleoside phosphorylation or degradation do not reveal differences which would explain the toxicity of deoxynucleosides for immature, as compared to mature, T cells. We conclude that deoxynucleoside metabolism in leukaemic T cells varies with their degree of differentiation. These observations may be relevant to the design of chemotherapeutic regimes for T cell malignancies.     

Proliferation of normal and malignant human immature lymphoid cells

In this study, the proliferative activity of human B and T cell precursors in central lymphoid organs, acute lymphoblastic leukemia (ALL) cells, and permanent cell lines was investigated with double- and triple-color-labeling methods for the analysis of cell cycle-associated features such as 5-bromo-2'-deoxyuridine (BrdU) incorporation and the expression of a nuclear proliferation-associated antigen, Ki67, together with the phenotypic profile of the cells. In infant and regenerating bone marrow (BM), 41.5% +/- 4.0% of terminal deoxynucleotidyl transferase (TdT+) cells were Ki67+, and 30.0% +/- 4.0% incorporated BrdU. A similar proportion of TdT+ dividing cells was observed in adult BM. The proliferative activity of the B cell progenitors reached the peak at the pre-B stage: 80.8% +/- 7.6% and 35.3% +/- 6.1% of c mu +, RFB7- cells were Ki67+ and BrdU+, respectively. In contrast, greater than 95% of surface immunoglobulin- positive BM lymphocytes were resting cells. In infant thymus the highest dividing capacity (95% Ki67+, 60% to 90% BrdU+) was observed in large cortical thymocytes (TdT+, CD1-, cCD3+), and TdT+, CD1+ cortical thymocytes also showed a high proliferative activity (74.3% +/- 2.3% Ki67+, 22.0% +/- 1.0% BrdU+), but TdT-, mCD3+ thymic lymphocytes were mainly resting cells (less than 5% Ki67+, less than 1% BrdU+). The proliferative activity of null and common ALL blasts was significantly lower than that of normal BM TdT+ cells (15.5% +/- 4.2% Ki67+, 6.2% +/- 2.1% BrdU+; P less than .001). Dividing ALL blasts were TdT+ and expressed surface antigens detected by CD10 and/or CD19 antibodies. In T cell-ALL, the percentages of Ki67+ and BrdU+ blasts were also lower than those found in the corresponding normal immature thymocytes (13.0% +/- 3.1% and 2.4% +/- 1.3%, respectively; P less than .001). Thus, ALLs derive from actively proliferating lymphoid precursors but have a lower dividing capacity than the corresponding normal cell types. In ALL cases with heterogeneous expression of markers such as cmu and CD1, dividing blasts were distributed among both negative and positive populations, thus indicating that blasts with signs of differentiation also remain within the dividing pool.     

Production of burst-promoting activity by monoclonal antibody defined malignant T lymphocytes from patients with lymphocytic leukemia and lymphoma

We tested conditioned media from 12 patients with T lymphocyte neoplasms and four T cell lines for their ability to stimulate the in vitro growth of erythroid-burst-forming units (BFU-E) from bone marrow mononuclear cells in a methylcellulose culture system. Nine patients suffered from acute lymphocytic leukemia, two from chronic lymphocytic leukemia, and one from non-Hodgkin's lymphoma. The T lymphocytes were characterized by a series of monoclonal antibodies and their stage of development was correlated with their ability to produce burst-promoting activity (BPA). Conditioned media from cells classified as prothymocytes (three cases), common thymocytes (one case), mature thymocytes (three cases), and mature lymphocytes of the helper subtype (two cases) increased BFU-E proliferation four- to 19-fold over control values using normal bone marrow as target cells. Conditioned media from OKT8+ malignant T lymphocytes (three cases) did not enhance BFU-E proliferation. Conditioned media from cells classified as immature T cells stimulated CFU-GM proliferation in only one of seven cases even though they secreted BPA. Conditioned media from three of the four cell lines stimulated by phytohemagglutinin, enhanced BFU-E growth. Our results indicate that malignant cells that have characteristics of immature T cells are able to produce BPA. Studies using techniques to isolate homogeneous populations of normal T cell subsets are required to determine whether normal immature T lymphocytes have the same capability.     

SGX393 inhibits the CML mutant Bcr-AblT315I and preempts in vitro resistance when combined with nilotinib or dasatinib

Imatinib inhibits Bcr-Abl, the oncogenic tyrosine kinase that causes chronic myeloid leukemia. The second-line inhibitors nilotinib and dasatinib are effective in patients with imatinib resistance resulting from Bcr-Abl kinase domain mutations. Bcr-Abl(T315I), however, is resistant to all Abl kinase inhibitors in clinical use and is emerging as the most frequent cause of salvage therapy failure. SGX393 is a potent inhibitor of native and T315I-mutant Bcr-Abl kinase that blocks the growth of leukemia cell lines and primary hematopoietic cells expressing Bcr-Abl(T315I), with minimal toxicity against Bcr-Abl-negative cell lines or normal bone marrow. A screen for Bcr-Abl mutants emerging in the presence of SGX393 revealed concentration-dependent reduction in the number and range of mutations. Combining SGX393 with nilotinib or dasatinib preempted emergence of resistant subclones, including Bcr-Abl(T315I). These findings suggest that combination of a T315I inhibitor with the current clinically used inhibitors may be useful for reduction of Bcr-Abl mutants in Philadelphia chromosome-positive leukemia.     

Inhibitory effect of 2-chlorodeoxyadenosine on granulocytic, erythroid, and T-lymphocytic colony growth.

Previous studies have shown that 2-chloro-2'-deoxyadenosine (CdA) is markedly toxic to normal and malignant human lymphocytes in vitro and in vivo. Recent clinical trials have shown that CdA is a very promising drug for the treatment of lymphoid malignancies. The present investigations were designed to test the effect of CdA on the in vitro clonal growth of both myeloid progenitors and T-lymphocyte colony-forming cells (CFU-TL) obtained from normal human bone marrow and peripheral blood. Cells were exposed to CdA in doses up to 1280 nmol/L. To reduce indirect effects of CdA mediated by accessory cells, monocyte- and T-lymphocyte-depleted bone marrow cells were used for our investigations. The results show a marked inhibition of myeloid progenitor and lymphocyte colony-forming cells in a dose-dependent manner, correlating with maturation stage in that the immature progenitor cells are more sensitive to this drug. Furthermore, our studies suggest that a sequence of metabolic events previously described for lymphocytes may be operative in myeloid progenitor cells because a minimal exposure time of 48 hours is required to obtain a marked inhibition. CdA toxicity was proposed to be linked with phosphorylation by deoxycytidine-kinase (E.C. 2.7.1.74), the levels of which have been found to be high in lymphocytes, but low in granulocytes. However, the marked inhibition of myeloid progenitor cells shown in these studies suggests that other factors such as modulation of the effect of CdA by the ambient levels of other deoxynucleosides might influence the apparent sensitivity of myeloid cells.     

Preclinical studies on the use of selective antibody-ricin conjugates in autologous bone marrow transplantation

Whole-ricin immunoconjugates were synthesized with the pan-T cell antibodies T101 and 3A1 and assayed in the presence of 0.1 mol/L lactose. Their toxicity for cell lines, peripheral blood T lymphocytes, and normal bone marrow progenitors was compared with that of whole ricin. In the presence of 0.1 mol/L lactose, normal cells and cell lines exhibited the following sensitivities to ricin: 8392 (human malignant B cell line) less than E rosette-positive lymphocytes less than bone marrow progenitors less than 8402 (human T ALL) less than CEM (human T ALL). Ricin sensitivities correlated with ricin binding as determined by immunofluorescence. In the presence of lactose, peripheral blood T cells were resistant to 0.1 nmol/L ricin, but a similar concentration of T101-ricin inhibited normal and malignant T colony formation by greater than 98%. 3A1-ricin was slightly less effective. At a conjugate concentration of 0.1 nmol/L, bone marrow progenitor colony formation was inhibited by 30% or less; T101-positive cells were at least tenfold more sensitive than normal progenitors. When mixtures of 10% CEM cells and marrow cells were incubated with T101-ricin, 95% of CEM colonies were killed, and 96% of marrow granulocyte/ macrophage progenitors survived. Some free ricin was released from immunotoxin-treated cells, producing minimal inhibition of protein synthesis or cell growth. We conclude that (a) normal blood cells and malignant cell lines exhibit varying degrees of ricin sensitivity in the presence of lactose; (b) T101-ricin is at least tenfold more toxic to T lymphocytes than to bone marrow progenitor cells and is effective in mixtures of normal and malignant cells; and (c) treatment of infiltrated marrow with anti-T cell immunotoxins should safely remove target T cells without excessively damaging normal progenitors or producing excessive free ricin. Anti-T cell, whole-ricin immunotoxins merit trials for autologous transplantation.     

An antigen common to chronic lymphocytic and hairy cell leukemia cells not shared by normal lymphocytes or by other leukemic cells

Rabbit xenoantisera and mouse monoclonal antibodies prepared against human chronic lymphocytic leukemia (CLL) B cells were found to react against a single polypeptide chain with a mol wt of 69 kd found on leukemic cells of all CLL (N = 40) and B type hairy cell leukemia (HCL) patients (N = 9) examined. This common CLL-associated antigen (cCLLa) was not detectable on circulating T or B lymphocytes, thymocytes, lymph node and splenic lymphocytes, or bone marrow leukocytes from normal persons. In addition, the cCLLa was not detectable on cultured T or B lymphoblastoid cell lines or on malignant cells from other forms of lymphocytic or myelocytic leukemia. Non-Hodgkin's lymphoma cells also failed to express the antigen. Autologous cultured lymphoblastoid cell lines were established from residual normal B cells from a CLL patient whose cells were used to generate one of the antisera. Absorption of the antibody with these cultured polyclonal B cells did not affect the anti-CLL activity, which suggests that the cCLLa is not HLA related. Unlike the T cell differentiation complex gp65-71, the cCLLa was not expressed on fetal or cord blood lymphocytes or on mitogen-stimulated normal lymphocytes and was distinct from the antigen recognized by the LEU-1 antibody in spite of their similar mol wt. The cCLLa was also determined to be unrelated to the human T cell leukemia lymphoma virus (HTLV-1). One of the monoclonal antibodies generated against the cCLLa was a complement binding IgG which exhibited highly selective cytotoxic activity against 100% of cells bearing the cCLLa. Such an antibody might prove clinically useful in early diagnosis and treatment of CLL and HCL.