In vivo toxicity and pharmacokinetic features of B43(Anti-CD19)-Genistein immunoconjugate.

B43(anti-CD19)-Genistein immunoconjugate targets genistein, a naturally occurring protein tyrosine kinase inhibitory isoflavone to the membrane-associated anti-apoptotic CD19-LYN complexes and triggers apoptotic cell death. In this preclinical study, the toxicity profiles of B43-Genistein as well as unconjugated genistein were evaluated in mice. B43-Genistein and genistein were administered either as single bolus injections or daily injections for 10 consecutive days via the intraperitoneal route to mice. Genistein was not toxic to mice at the highest dose of 40 mg/kg and no test article-related histopathological lesions were found in any of the 64 genistein-treated mice. B43-Genistein had a significantly longer elimination half-life and slower plasma and tissue clearance than unconjugated genistein. B43-Genistein was not toxic to mice at the highest single dose of 40 mg/kg or highest cumulative dose of 100 mg/kg and no test article-related histopathological lesions were found in any of the 108 mice treated with B43-genistein. To our knowledge, this is the first preclinical toxicity and pharmacokinetic study of a tyrosine kinase inhibitor-containing immunoconjugate.

Large scale manufacturing of B43(anti-CD19)-genistein for clinical trials in leukemia and lymphoma.

We have conjugated the murine monoclonal anti-CD19 antibody B43 to the tyrosine kinase inhibitor genistein to construct an effective immunoconjugate against CD19 antigen positive hematologic malignancies. The scaled-up production and purification of B43 antibody, genistein, and B43-Genistein immunoconjugate permitted the manufacturing of a highly purified clinical-grade B43-Genistein preparation. In clonogenic assays, B43-Genistein elicited selective and potent cytotoxicity against CD19 antigen positive human leukemia cells. To our knowledge, this work represents the first effort of producing a clinical-grade genistein immunoconjugate for treatment of B-lineage leukemia and lymphoma.

In vivo toxicity and pharmacokinetic features of B43 (anti-CD19)-genistein immunoconjugate in nonhuman primates.

B43 (anti-CD19)-genistein immunoconjugate targets genistein, a naturally occurring protein tyrosine kinase-inhibitory isoflavone to the membrane-associated antiapoptotic CD19-LYN complexes and triggers apoptotic cell death. In this preclinical study, the toxicity profiles of B43-genistein as well as unconjugated genistein were evaluated in cynomolgus monkeys. B43-genistein and genistein were administered either as single bolus injections or daily injections for 5-10 consecutive days via the i.v. route to monkeys. Neither genistein nor B43-genistein was toxic to cynomolgus monkeys, and no test article-related histopathological lesions were found in any of the two genistein-treated or five B43-genistein-treated cynomolgus monkeys. B43-genistein showed a favorable pharmacokinetics in monkeys, with a plasma half-life of 10-23 h. Plasma samples from B43-genistein-treated monkeys elicited potent and CD19 antigenspecific antileukemic activity against human CD19+ leukemia cells in vitro. To our knowledge, this is the first preclinical toxicity and pharmacokinetic study of a tyrosine kinase inhibitor-containing immunoconjugate in nonhuman primates.

Granulocyte-macrophage colony-stimulating factor receptor-targeted therapy of chemotherapy- and radiation-resistant human myeloid leukemias.

Contemporary therapies for acute myeloid leukemia (AML) commonly fail to cure patients because of the emergence of drug resistance. Drug resistance in AML is multifactorial but can be associated with the overexpression of transmembrane transporter molecules, including P-glycoprotein (Pgp) or the multidrug resistance-associated protein (MRP), or associated with inactivation of the p53 tumor suppressor gene, as well as overexpression of the anti-apoptotic protein bcl-2. We are investigating if novel recombinant biotherapeutics can circumvent these resistance mechanisms to effectively treat refractory AML. To target the lethal action of diphtheria toxin (DT) to high affinity granulocyte-macrophage colony-stimulating factor (GMCSF) receptors on AML blasts, we have produced a recombinant chimeric fusion toxin, DTctGMCSF. Since DTctGMCSF enters and kills its target cells by unique mechanisms (GMCSF-receptor binding and protein synthesis inhibition) and is not similar in structure to Pgp or MRP substrates, we postulated that it would be an active agent against therapy-resistant AML. DTctGMCSF was selectively cytotoxic (IC50 1-10ng/ml) to GMCSF-receptor positive AML cells expressing the Pgp- or MRP-associated multi-drug resistant phenotypes, despite high level resistance to conventional chemotherapeutic agents. DTctGMCSF also efficiently killed AML cells deficient in p53 expression, as well as radiation-resistant AML cells and mixed lineage leukemia cells expressing high levels of bcl-2. In addition, DTctGMCSF killed > 99% of primary leukemic progenitor cells from therapy-refractory AML patients under conditions that we have previously found to not adversely affect the proliferative capacity or differentiation of pluripotent normal hematopoietic progenitor cells. DTctGMCSF may prove useful in treating myeloid leukemias that are otherwise resistant to a wide range of conventional therapies.

Induction of apoptosis in multidrug-resistant and radiation-resistant acute myeloid leukemia cells by a recombinant fusion toxin directed against the human granulocyte macrophage colony-stimulating factor receptor.

Multiagent chemotherapy regimens fail to cure more than one-half of the patients with acute myeloid leukemia (AML) because of the emergence of dominant multidrug-resistant subclones of leukemia cells. We have developed a recombinant diphtheria toxin-human granulocyte macrophage colony-stimulating factor chimeric fusion protein (DTctGMCSF) that specifically targets GMCSF receptor-positive AML cells. This novel biotherapeutic agent induced rapid apoptotic cell death of chemotherapy-resistant AML cell lines and primary leukemic cells from treatment-refractory AML patients. Our results suggest that DTctGMCSF may be useful in the treatment of AML patients whose leukemia has recurred and developed resistance to contemporary chemotherapy programs.

Differential effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on the radiation sensitivity of normal versus leukemic bone marrow progenitor cell populations.

We examined the effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF), interleukin 3 (rhIL-3) and interleukin 6 (rhIL-6) on the radiation sensitivity of normal and leukemic bone marrow progenitor cell populations. Conditioning of leukemic progenitor cells (LPC) from acute lymphoblastic leukemia (ALL) patients with rhG-CSF enhanced their radiation sensitivity, whereas conditioning with rhIL-3 or rhIL-6 had the opposite effect. In contrast to its effects on LPC derived from ALL patients, rhG-CSF reduced the radiation sensitivity of normal myeloid progenitor cells as well as LPC from acute myeloblastic leukemia (AML) patients. Differential modulation of the radiation sensitivity of LPC by rhG-CSF may provide the basis for better total body irradiation (TBI) regimens for ALL patients undergoing autologous bone marrow transplantation (BMT).

In vitro and in vivo anti-leukemic efficacy of cyclic AMP modulating agents against human leukemic B-cell precursors.

We show that the adenylate cyclase activating diterpine, forskolin, the phosphodiesterase inhibitor, aminophylline, and the permeant cAMP analog dibutyryl cAMP inhibit the in vitro clonogenic growth of leukemic B-cell precursors. We also used a SCID mouse xenograft model of refractory human B-cell precursor leukemia to evaluate the anti-leukemic effect of aminophylline in vivo. Treatment with aminophylline (6 mg/kg bolus followed by 0.1-0.5 mg/kg/hour x 7 days) significantly prolonged the event-free survival of SCID mice (median survival of control mice, 39 days, N = 79; median survival of aminophylline-treated mice, 60 days, N = 10; P < 0.0001 by log-rank test) and it was more effective than treatment with vincristine (median survival = 51 days, N = 5) or L asparaginase (median survival = 44 days, N = 5). However, aminophylline was not as effective as methylprednisolone (median survival: 103 days, N = 5). These results indicate that cAMP modulating agents may be useful in treatment of refractory human B-cell precursor leukemia.

In vitro and in vivo antileukemic activity of B43-pokeweed antiviral protein against radiation-resistant human B-cell precursor leukemia cells.

B-cell precursor (BCP) leukemia is the most common form of childhood cancer and represents one of the most radiation-resistant forms of human malignancy. In this study, we examined the antileukemic efficacy of the B43 (anti-CD19)-pokeweed antiviral protein (B43-PAP) immunotoxin against radiation-resistant BCP leukemia cells. B43-PAP caused apoptosis of radiation-resistant primary BCP leukemia cells, killed greater than 99% of radiation-resistant primary leukemic progenitor cells from BCP leukemia patients, and conferred extended survival to severe combined immunodeficiency (SCID) mice xenografted with radiation-resistant human BCP leukemia. Furthermore, the combination of B43-PAP and total body irradiation (TBI) was more effective than TBI alone in two SCID mouse bone marrow transplantation models of radiation-resistant human BCP leukemia. Thus, B43-PAP may prove useful in the treatment of radiation-resistant BCP leukemia.

In vitro and in vivo activity of topotecan against human B-lineage acute lymphoblastic leukemia cells.

Topotecan [(S)-9-dimethylaminomethyl-10-hydroxycamptothecin hydrochloride; SK&F 104864-A, NSC 609699], a water soluble semisynthetic analogue of the alkaloid camptothecin, is a potent topoisomerase I inhibitor. Here we show that topotecan stabilizes topoisomerase I/DNA cleavable complexes in radiation-resistant human B-lineage acute lymphoblastic leukemia (ALL) cells, causes rapid apoptotic cell death despite high-level expression of bcl-2 protein, and inhibits ALL cell in vitro clonogenic growth in a dose-dependent fashion. Furthermore, topotecan elicited potent antileukemic activity in three different severe combined immunodeficiency (SCID) mouse models of human poor prognosis ALL and markedly improved event-free survival of SCID mice challenged with otherwise fatal doses of human leukemia cells at systemic drug exposure levels that can be easily achieved in children with leukemia.

Sensitivity of primary clonogenic blasts from acute lymphoblastic leukemia patients to an activated cyclophosphamide, viz., mafosfamide.

Primary cyclophosphamide-naive clonogenic blasts from 32 patients with newly diagnosed acute lymphoblastic leukemia (ALL) were tested for their in vitro sensitivity to an "activated" cyclophosphamide, viz., mafosfamide, using leukemic progenitor cell (LPC) colony assays. Marked interpatient variation in the responses of LPC from newly diagnosed patients to mafosfamide prompted assessment of mafosfamide sensitivity in relation to more frequently measured parameters of newly diagnosed ALL. Only immunophenotype and sex showed a significant association with the intrinsic mafosfamide sensitivity of LPC. LPC from T-lineage ALL patients were more resistant to mafosfamide than LPC from B-lineage ALL patients, as reflected by 1.8-fold and 4.3-fold higher mean SF10 and SF20 (surviving fractions of ALL LPC of 10 and 20 microM mafosfamide, respectively) values. LPC from male patients were more resistant to mafosfamide than LPC from female patients, as reflected by 1.9-fold and 4.8-fold higher mean SF10 and SF20 values. In comparison to T-lineage ALL patients, a significantly greater fraction of B-lineage ALL patients had mafosfamide-sensitive LPC with SF10 values of < 0.25 (61% vs 11%, P = 0.01). Notably, all four cases exhibiting resistance to mafosfamide, i.e., SF20 > or = 0.5, were males with T-lineage ALL. In order to exclude the influence of sex as a confounding factor in the observed immunophenotype-mafosfamide sensitivity association, we also compared the mafosfamide sensitivities of LPC from male patients only. The means of SF10, and SF20 values of LPC from male T-lineage ALL patients were 1.5- and 3.2-fold higher than those of LPC from male B-lineage ALL patients (P < 0.1). Thus, in the male patient subgroup, the immunophenotype-mafosfamide sensitivity association remained significant.

Radiation damage repair capacity of primary clonogenic blasts in acute lymphoblastic leukemia.

The sublethal radiation damage repair capacity of primary clonogenic blasts [i.e., leukemic progenitor cells (LPC)] from 74 newly diagnosed acute lymphoblastic leukemia (ALL) patients was analyzed using LPC colony assays. We determined the (a) Dq (quasithreshold dose) and n (extrapolation number) values from single dose radiation survival curves constructed according to the single-hit multitarget model of cell survival, (b) beta values and alpha:beta ratios from single dose radiation survival curves constructed according to the linear quadratic model of cell survival, and (c) recovery factor values from survival data in split-dose experiments. Clonogenic blasts from different ALL patients varied substantially in their ability to repair sublethal radiation damage. However, in 11 of 30 (37%) T-lineage ALL cases and 13 of 44 (30%) B-lineage ALL cases, the radiation survival curves of fresh LPC were characterized by a distinct initial shoulder, providing circumstantial evidence that LPC from a significant portion of ALL patients (24 of 74 cases = 32%) are able to repair sublethal radiation damage. Leukemic progenitor cells from 23 of 34 (68%) evaluable cases evaluated had alpha:beta ratios of < 5 Gy, indicating that they may possess a substantial capacity to repair sublethal radiation damage. In order to further elucidate the repair capacity of ALL LPC, we compared the antileukemic efficacy of fractionated irradiation with 2 x 2 Gy to the antileukemic efficacy of single-dose irradiation with 1 x 4 Gy. In 20 of 28 cases (71%), a > or = 20% increase in LPC survival (recovery factor > or = 1.2) was observed when 4 Gy were delivered in two doses instead of a single dose, providing direct evidence that ALL LPC are able to repair sublethal radiation damage. Our results indicate that modifications in total body irradiation regimens, currently administered prior to bone marrow transplantation, may be necessary to diminish the probability of relapse in high-risk ALL.

Intrinsic radiation resistance of primary clonogenic blasts from children with newly diagnosed B-cell precursor acute lymphoblastic leukemia.

The radiation sensitivity of primary clonogenic blasts from 44 children with newly diagnosed B-cell precursor acute lymphoblastic leukemia (ALL) was analyzed using leukemic progenitor cell (LPC) colony assays. The derived values for SF2 (surviving fraction at 200 cGy) and alpha (initial slope of radiation survival curves constructed according to the linear quadratic model) indicated a marked interpatient heterogeneity in intrinsic radiation sensitivity of LPC populations. The SF2 values ranged from 0.01 to 1.00 (median = 0.430; mean +/- SE = 0.47 +/- 0.04), and the alpha values ranged from 0.000 to 3.272 Gy-1 (median = 0.280 Gy-1; mean +/- SE = 0.430 +/- 0.093 Gy-1). When CD19+ CD34+ versus CD19+ CD34- immunophenotypes were compared, a trend toward higher SF2 and lower alpha values were observed in LPC from CD34+ patients, consistent with greater radiation resistance. When patients were divided into three approximately equal groups based on increasing levels of CD34 expression, a clear ordering effect was observed indicating that increased CD34 expression levels are associated with significantly higher radiation resistance at the level of B-lineage LPC. The highest CD34 expression group (> or = 75% positivity) had 1.4-fold higher SF2 (P = 0.05) and twofold lower alpha values (P = 0.06) than the lowest group (< 30% positivity). Furthermore, the CD34 positivity of radiation resistant (alpha < or = 0.2 and SF2 > or = 0.5) B-cell precursor ALL cases was greater than the CD34 positivity of radiation sensitive (alpha > 0.2 and/or SF2 < 0.5) cases (56 +/- 9% versus 34 +/- 9%, P = 0.09). Whereas only 6 of 16 (38%) of radiation sensitive cases were CD34+, 11 of 15 (73%) of radiation resistant cases expressed CD34 (P = 0.04). Our results offer new insights into the inherent and/or acquired radiation resistance of primary clonogenic blasts from B-cell precursor ALL patients.

Radiation and heat sensitivity of human T-lineage acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML) clones displaying multiple drug resistance (MDR).

The hyperthermia as well as radiation responses of multidrug resistant (CEM/VLB100 with classical MDR and CEM/VM-1 with atypical MDR), methotrexate resistant (CEM/MTX) subclones of CCRF-CEM T-lineage ALL cell line were compared with those of a drug sensitive (CEM-1-3) subclone from the same parent cell line. Also analyzed were the hyperthermia as well as radiation responses of multidrug resistant (HL60/AR) and drug sensitive subclones of the HL60 AML cell line. Notably, the drug resistant subclones of CEM and HL60 were as sensitive to hyperthermia as were the drug sensitive subclones. Importantly, no thermotolerant plateau was observed in the hyperthermia survival curves of the drug resistant subclones, indicating that drug/multidrug resistance is not associated with a greater likelihood of thermal tolerance development during hyperthermia. Similarly, the drug resistant CEM and HL60 subclones were not more radiation resistant than the drug sensitive subclones. Thus, the classical or atypical forms of multidrug resistance or methotrexate resistance of the analyzed leukemic cell lines were not associated with radiation resistance. Furthermore, the radiation survival curves of the drug resistant subclones lacked a distinct initial shoulder and their n values were not greater than those of the drug sensitive subclones, suggesting that multidrug resistance is not associated with an increased ability to repair or accumulate sublethal radiation damage. Our findings provide evidence that there is no apparent association between drug/multidrug resistance and heat or radiation sensitivity of CEM T-lineage ALL or HL60 AML leukemia cells. The results of this study indicate that acquired resistance to methotrexate, vinblastine, vincristine, etoposide, actinomycin-D, adriamycin, or daunomycin, or pleiotropic multidrug resistance do not necessarily confer radiation resistance for human leukemic cells.

In vitro and in vivo radiation resistance associated with CD3 surface antigen expression in T-lineage acute lymphoblastic leukemia.

The radiobiologic features of primary clonogenic blasts (referred to also as T-lineage leukemic progenitor cells) from newly diagnosed and relapsed T-lineage acute lymphoblastic leukemia (ALL) patients were analyzed. Intrinsic radiation sensitivity differed substantially among primary clonogenic blasts from 34 newly diagnosed patients. The mean D0 (37% dose slope), SF2 (surviving fraction at 200 cGy), and alpha values (initial slope of the survival curve) were 141 +/- 15 cGy, 0.31 +/- 0.04, and 0.630 +/- 0.093 Gy-1, respectively. Among newly diagnosed cases, nine had SF2 values of greater than or equal to 0.50 and alpha values of less than or equal to 0.2 Gy-1, consistent with a marked intrinsic radiation resistance at the level of clonogenic blasts using the multitarget and linear quadratic models of cell survival. Of these nine radiation resistant cases, seven were CD3+. Furthermore, the mean D0 (162 +/- 20.8 cGy) and SF2 (0.377 +/- 0.057) values for the 20 CD3+ cases were significantly higher than the D0 (108.6 +/- 18.2 cGy) and SF2 (0.204 +/- 0.051) values for the 14 CD3- cases (P less than or equal to .05). Thus, clonogenic blasts from CD3+ newly diagnosed T-lineage ALL patients were more resistant to radiation than clonogenic blasts from CD3- newly diagnosed T-lineage ALL patients. Nineteen T-lineage ALL patients received autologous bone marrow transplants during complete remission. Pretransplant conditioning consisted of total body irradiation (TBI) combined with high-dose chemotherapy. Primary clonogenic blasts from patients who relapsed after bone marrow transplantation (BMT) displayed a particularly high degree of intrinsic radiation resistance with a mean D0 value of 333 cGy and an alpha value of 0.112 Gy-1. The expression of CD3 antigen appeared to predict the outcome of relapsed T-lineage ALL patients undergoing autologous BMT after TBI plus high-dose chemotherapy. The Kaplan-Meier estimates and standard errors of the probability of remaining in remission after BMT were 60% +/- 22% (mean relapse - free interval = 1.6 +/- 0.7 years) for CD3- patients and 0% +/- 0% (mean relapse - free interval = 0.2 +/- 0.0 years) for CD3+ patients (P = .002). Furthermore, the mean percentage of CD3-positive leukemic marrow blasts at presentation or relapse before BMT was significantly lower than the mean percentage of CD3-positive leukemic marrow blasts at relapse after BMT. Notably, in cultured leukemic bone marrow specimens from newly diagnosed as well as relapsed patients, colony blasts surviving in vitro radiation expressed CD3 more vividly than did colony blasts in unirradiated cultures.(ABSTRACT TRUNCATED AT 400 WORDS)

Stimulation of protein tyrosine phosphorylation, phosphoinositide turnover, and multiple previously unidentified serine/threonine-specific protein kinases by the Pan-B-cell receptor CD40/Bp50 at discrete developmental stages of human B-cell ontogeny.

CD40/Bp50 B-cell receptor has been implicated as having an important function for the regulation of human B-cell growth and maturation as well as antigen-driven selection of tonsillar B-cells in germinal centers. The purpose of the present study was to examine the biochemical events triggered by the engagement of the CD40 receptor in human B-lineage lymphoid cells corresponding to discrete developmental stages of human B-cell ontogeny. The engagement of the CD40 receptor on pro-B-, pre-pre-B-, pre-B-, or activated mature B-cells but not on resting mature B-cells with the agonistic anti-CD40 monoclonal antibody G28-5 resulted in enhanced tyrosine phosphorylation of four distinct phosphoproteins with molecular masses of 67, 72, 96, and 113 kDa and induced a rapid increase in the production of inositol 1,4,5-trisphosphate. Further, we have identified five electrophoretically distinct renaturable CD40-regulated serine/threonine-specific protein kinases (PK120, PK93, PK76, PK55, and PK48) that showed markedly increased in vitro activity after CD40 stimulation. Protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) abrogated the stimulation of the in vitro activity of PK120, PK93, PK55, and PK48 and attenuated the stimulation of the in vitro activity of PK76 in response to the engagement of the CD40 receptor but did not influence the enhanced tyrosine phosphorylation of cellular substrates after CD40 stimulation. Notably, genistein and herbimycin A, two potent inhibitors of tyrosine-specific protein kinases, not only abrogated the CD40-induced enhanced tyrosine phosphorylation on the 67-, 72-, 96-, and 113-kDa substrates, but they also inhibited the CD40-induced stimulation of phosphoinositide turnover as well as the CD40-induced increase of the in vitro activity of renaturable serine/threonine-specific protein kinases.(ABSTRACT TRUNCATED AT 400 WORDS)

Interleukin 7 receptor ligation stimulates tyrosine phosphorylation, inositol phospholipid turnover, and clonal proliferation of human B-cell precursors.

Functional interleukin 7 (IL-7) receptors are expressed on the surface of multiphenotypic, biphenotypic, and immature B-lineage human lymphoid precursor cells with germ-line immunoglobulin heavy-chain genes but not on more mature B-lineage lymphoid cells with rearranged and/or expressed immunoglobulin heavy-chain genes. Thus, IL-7 may have an important regulatory role during the earliest stages of human B-cell ontogeny. The engagement of the surface IL-7 receptors on immature B-cell precursor cells with recombinant human IL-7 (rhIL-7) results in enhanced tyrosine phosphorylation of multiple phosphoproteins, stimulates inositol phospholipid turnover and DNA synthesis, and promotes their clonal proliferation. These effects are (i) specific for rhIL-7, since rhIL-3, rhIL-4, rhIL-5, rhIL-6, and recombinant human granulocyte colony-stimulating factor do not elicit similar activities on IL-7 receptor-positive human pro-B cells; and (ii) mediated by IL-7 receptors, since they are not observed in IL-7 receptor-negative B-lineage lymphoid cell populations. rhIL-7-induced tyrosine phosphorylation on the 35-, 53-, 55-, 62-, 69-, 76-, 94-, 150-, 170-, and 190-kDa substrates as well as rhIL-7-induced stimulation of inositol phospholipid turnover are abrogated by the tyrosine kinase inhibitor genistein. These results demonstrate that the IL-7 receptor on immature human B-cell precursor populations is intimately linked to a functional tyrosine kinase pathway and tyrosine phosphorylation is an important and perhaps mandatory step in the generation of the IL-7 receptor-linked transmembrane signal.

Detection and characterization of human high molecular weight B cell growth factor receptors on leukemic B cells in chronic lymphocytic leukemia.

Human high molecular weight-B cell growth factor (HMW-BCGF) (60 kD) stimulates activated normal B cells, B cell precursor acute lymphoblastic leukemia (BCP-ALL) cells, hairy cell leukemia (HCL) cells, prolymphocytic leukemia (PLL) cells, and chronic lymphocytic leukemia (CLL) cells. The expression of human high molecular weight B cell growth factor (HMW-BCGF) receptors (R) on clonal populations of leukemic B cells in CLL was studied by ligand binding assays using 125I-labeled HMW-BCGF as well as by immunofluorescence/flow cytometry and Scatchard analyses using an anti-HMW-BCGF R monoclonal antibody (MAb), designated BA-5. There was a high correlation between HMW-BCGF R expression and responsiveness to HMW-BCGF. 60% of CLL cases constitutively expressed HMW-BCGF R and showed a marked proliferative response to HMW-BCGF in [3H]TdR incorporation assays as well as colony assays. Similarly, HCL cells, PLL cells, and activated normal B cells expressed functional HMW-BCGF R, as determined by ligand binding assays using 125I-HMW-BCGF, [3H]TdR incorporation assays, and reactivity with BA-5 MAb. Scatchard analyses indicated the existence of approximately 3,000 HMW-BCGF R/cell on HMW-BCGF responsive CLL cells with an apparent Ka value of 4.6 X 10(7) M-1. The concentrations of HMW-BCGF required for maximum stimulation of CLL cells were two to three orders of magnitude lower than those needed for half maximal receptor occupancy, indicating that only a small fraction of HMW-BCGF R need to be occupied to stimulate leukemic CLL B cells. Crosslinking of surface bound 125I-HMW-BCGF (60 kD) with the bivalent crosslinker DTSSP to its binding site on fresh CLL cells identified a 150-kD HMW-BCGF/HMW-BCGF R complex, suggesting an apparent molecular weight of 90 kD for the receptor protein. The growth stimulatory effects of HMW-BCGF on clonogenic CLL cells did not depend on accessory cells or costimulant factors. The anti-HMW-BCGF R monoclonal antibody BA-5 disrupted HMW-BCGF/HMW-BCGF R interactions at the level of clonogenic CLL cells and inhibited HMW-BCGF-stimulated CLL colony formation in vitro. To our knowledge, this study represents the first detailed analysis of expression, function, and structure of HMW-BCGF R on B lineage CLL cells.

Leukemic B-cell precursors constitutively express functional receptors for human interleukin-1.

This study analyzes the expression of functional interleukin-1 (IL-1) receptors on leukemic B-cell precursors (BCPs) from B-cell precursor acute lymphoblastic leukemia (BCP ALL) patients. We first investigated the specific binding of 125I-labeled recombinant IL-1 (125I-rIL-1) (4 x 10(17) cpm/mol) to fresh marrow blasts from 11 BCP ALL patients. In five of 11 cases, the binding of 125I-rIL-1 was significantly blocked by excess cold rIL-1. In these five cases, the cell-bound radioactivity ranged from 146 cpm/10(6) cells to 2,412 cpm/10(6) cells (mean +/- SE = 782 +/- 414 cpm/10(6) cells), indicating that 4 to 60 femtomols (mean +/- SE = 20 +/- 10 femtomols) of 125I-rIL-1 specifically bound per 10(7) cells. The estimated number of 125I-rIL-1 molecules bound per cell ranged from 219 to 3,618 (mean +/- SE = 1173 +/- 621). In all five cases, BCP colony formation was stimulated by 10 ng/mL (570 femtomolar) rIL-1, and the background-subtracted colony numbers ranged from 130 to 298 (mean +/- SE = 226 +/- 31). In contrast, no stimulation was observed in six cases that showed no significant 125I-rIL-1 binding. Hence, there was a high correlation between 125I-rIL-1 binding and IL-1 responsiveness, indicating that functional IL-1 receptors were detected in ligand binding assays. Scatchard plot analysis of the specific equilibrium binding data for leukemic BCPs from two IL-1-responsive BCP ALL cases yielded straight linear regression lines, indicating the existence of a single class of 132 to 154 high affinity IL-1 receptors/cell. The apparent affinity constants (Ka) values ranged from 5.2 x 10(9) mol/L-1 to 1.2 x 10(10) mol/L-1. Notably, the concentrations of IL-1 required for half-maximal receptor occupancy (kd = 83 pmol/L to 190 pmol/L) were approximately three orders of magnitude higher than those needed to elicit a half-maximal proliferative response of leukemic BCPs in colony assays (0.1 to 1.0 ng/mL = 5.7 to 57 femtomolar), indicating that only a small fraction of IL-1 receptors need to be occupied to stimulate leukemic BCPs. Notably, IL-1 unresponsive leukemic BCPs from one BCP ALL patient and two BCP ALL cell lines (REH, KM-3) did not exhibit any significant IL-1 binding (less than 10 IL-1 binding sites/cell), and two additional IL-1 unresponsive BCP ALL cell lines (NALM-6, HPB-NULL) expressed only 24 to 54 IL-1 binding sites/cell with a Ka of 7.8 to 9.8 x 10(9) mol/L-1.(ABSTRACT TRUNCATED AT 400 WORDS)