Yuri Yanishevski - Academia.edu (original) (raw)

Papers by Yuri Yanishevski

Research paper thumbnail of In Vivo., Toxicity and Pharmacokinetic Features of B43(Anti-CD19)-Genistein Immunoconjugate

Leukemia & Lymphoma, 1998

B43(anti-CD19)-Genistein immunoconjugate targets genistein, a naturally occurring protein tyrosin... more 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.

Research paper thumbnail of Treatment of human B-cell precursor leukemia in SCID mice using a combination of the investigational biotherapeutic agent B43-PAP with cytosine arabinoside

Clinical cancer research : an official journal of the American Association for Cancer Research, 1996

Combined immunochemotherapy regimens using the investigational biotherapeutic agent B43(anti-CD19... more Combined immunochemotherapy regimens using the investigational biotherapeutic agent B43(anti-CD19)-poke-weed antiviral protein (PAP) immunotoxin may offer an effective treatment for refractory B-cell precursor leukemias. The purpose of the present study was to explore and identify effective combinations of B43-PAP with standard chemotherapeutic drugs, including the anthracyclin doxorubicin, the epipodophyllotoxin etoposide, the nitrosurea carmustine, and the antimetabolite cytosine arabinoside. Here, we report that the B43-PAP plus cytosine arabinoside combination has potent antileukemic activity against human B-cell precursor leukemia in SCID mice and leads to 100% long-term event-free survival from an otherwise invariably fatal leukemia. Surprisingly, none of the other treatment protocols tested, including combinations of B43-PAP with carmustine, doxorubicin, or etoposide, proved more effective than B43-PAP alone.

Research paper thumbnail of Pharmacokinetic features, immunogenicity, and toxicity of B43(anti-CD19)-pokeweed antiviral protein immunotoxin in cynomolgus monkeys

Clinical cancer research : an official journal of the American Association for Cancer Research, 1997

We studied the pharmacokinetic features, immunogenicity, and toxicity of B43-pokeweed antiviral p... more We studied the pharmacokinetic features, immunogenicity, and toxicity of B43-pokeweed antiviral protein (PAP) immunotoxin in 13 cynomolgus monkeys. The disposition of B43-PAP in two monkeys, when administered as a single i.v. bolus dose, was characterized by a slow clearance (1-2 ml/h/kg) with a very discrete peripheral distribution. B43-PAP was retained and distributed largely in the blood as the sole compartment with no significant equilibration with the extravascular compartment. The circulating B43-PAP immunotoxin detected in monkey plasma samples by ELISA and protein immunoblotting was both immunoreactive with, and active against, human leukemic cells in vitro. In systemic immunogenicity and toxicity studies, which involved 11 cynomolgus monkeys, each monkey received a total of seven i.v. doses of B43-PAP at a specific dose level of the dose escalation schedule. B43-PAP-treated monkeys mounted a dose-dependent humoral immune response against both the mouse IgG and PAP moieties ...

Research paper thumbnail of In vivo biotherapy of HL-60 myeloid leukemia with a genetically engineered recombinant fusion toxin directed against the human granulocyte macrophage colony-stimulating factor receptor

Clinical cancer research : an official journal of the American Association for Cancer Research, 1997

Acute myeloid leukemia (AML) is the most common form of acute leukemia. Contemporary chemotherapy... more Acute myeloid leukemia (AML) is the most common form of acute leukemia. Contemporary chemotherapy regimens fail to cure most patients with AML. We have genetically engineered a recombinant diphtheria toxin human granulocyte macrophage colony-stimulating factor (GMCSF) chimeric fusion protein (DTctGMCSF) that specifically targets the GMCSF receptor on fresh human AML cells and myeloid leukemia cell lines. At a nontoxic dose level, DTctGMCSF therapy was superior to the standard chemotherapeutic agents 1-beta-D-arabinofuranosylcytosine and Adriamycin, resulting in 60% long-term event-free survival of severe combined immunodeficient mice challenged with an otherwise invariably fatal cell dose of the human HL-60 myeloid leukemia. Notably, systemic exposure levels of DTctGMCSF, which were found to be therapeutic in the severe combined immunodeficient mouse xenograft model of human HL-60 myeloid leukemia, could be achieved in cynomolgus monkeys without any significant nonhematological toxi...

Research paper thumbnail of In vivo toxicity, pharmacokinetics, and anticancer activity of Genistein linked to recombinant human epidermal growth factor

Clinical cancer research : an official journal of the American Association for Cancer Research, 1998

Epidermal growth factor receptor (EGFR)-associated protein tyrosine kinase (PTK) complexes have v... more Epidermal growth factor receptor (EGFR)-associated protein tyrosine kinase (PTK) complexes have vital anti-apoptotic functions in human breast cancer cells. We have shown previously that targeting the naturally occurring PTK inhibitor genistein to the EGFR-associated PTK complexes using the EGF-Genistein (Gen) conjugate triggers rapid apoptotic cell death in human breast cancer cells and abrogates their in vitro clonogenic growth. In the present study, we examined the in vivo toxicity profile, pharmacokinetics, and anticancer activity of EGF-Gen. No toxicities were observed in mice treated with EGF-Gen at dose levels as high as 40 mg/kg administered i.p. as a single dose or 140 mg/kg administered i.p. over 28 consecutive days. EGF-Gen significantly improved tumor-free survival in a severe combined immune deficiency (SCID) mouse xenograft model of human breast cancer, when it was administered 24 h after inoculation of tumor cells. At 100 microg/kg/day x 10 days (1 mg/kg total dose), ...

Research paper thumbnail of Methylation of mercaptopurine, thioguanine, and their nucleotide metabolites by heterologously expressed human thiopurine S-methyltransferase

Molecular pharmacology, 1995

Thiopurine S-methyltransferase (TPMT), a cytosolic enzyme that exhibits genetic polymorphism, cat... more Thiopurine S-methyltransferase (TPMT), a cytosolic enzyme that exhibits genetic polymorphism, catalyzes S-methylation of mercaptopurine (MP) and thioguanine (TG), yielding S-methylated nucleobases that are inactive, whereas S-methylated nucleotides of these thiopurines are cytotoxic. A yeast-based heterologous expression system was therefore used to characterize human TPMT-catalyzed methylation of MP, TG, and their principal nucleotide metabolites [thioinosine monophosphate (TIMP) and thioguanosine monophosphate (TGMP), respectively]. MP, TG, TIMP, and TGMP were all substrates for human TPMT, exhibiting similar Michaelis-Menten kinetic parameters (Km, 10.6-27.1 microM; Vmax, 31-59 nmol/min/mg of TPMT). Consistent with these kinetic parameters, human leukemia cells (CEM) incubated for 24 hr with 10 microM MP or TG accumulated significantly higher (2.3-fold, p = 0.01) concentrations of methyl-TIMP after MP incubation than methyl-TGMP after TG incubation, due to the 2.7-fold higher con...

Research paper thumbnail of Etoposide and antimetabolite pharmacology in patients who develop secondary acute myeloid leukemia

Leukemia, 1998

Etoposide, an effective agent for acute lymphoblastic leukemia (ALL), can cause secondary acute m... more Etoposide, an effective agent for acute lymphoblastic leukemia (ALL), can cause secondary acute myeloid leukemia (AML) in a subset of patients. Our objectives were to determine whether patients who develop secondary AML displayed altered etoposide pharmacokinetics or other pharmacologic characteristics compared to identically treated patients who did not develop AML. Children with newly diagnosed ALL were treated according to a protocol which included etoposide 300 mg/m 2 given three times over 8 days during remission induction and once every 2-4 weeks during 120 weeks of continuation therapy. Characteristic 11q23 rearrangements were documented in seven of the eight patients with AML. Etoposide clearance, time that etoposide concentrations exceeded 10 M, etoposide or etoposide catechol area-under-the-plasma-concentration vs time curve (AUC), serum albumin, and average methotrexate concentration did not differ significantly between the two groups; thiopurine methyltransferase (TPMT) activity tended to be lower in the eight children who did vs the 23 matched control children who did not develop AML (P = 0.16). Further regression analyses likewise indicated that lower TPMT activity tended to be associated with shorter onset of secondary AML (P = 0.11); it also tended to be lower among the eight index cases compared to the entire unmatched cohort of 105 identically treated children with ALL (P = 0.10). We observed no statistically significant differences in etoposide disposition and antimetabolite pharmacology between patients who did and did not develop secondary AML.

Research paper thumbnail of Reduced Folate Carrier Expression in Acute Lymphoblastic Leukemia: A Mechanism for Ploidy but not Lineage Differences in Methotrexate Accumulation

Blood

Methotrexate (MTX) is one of the most active and widely used agents for the treatment of acute ly... more Methotrexate (MTX) is one of the most active and widely used agents for the treatment of acute lymphoblastic leukemia (ALL). To elucidate the mechanism for higher accumulation of MTX polyglutamates (MTX-PG) in hyperdiploid ALL and lower accumulation in T-lineage ALL, expression of the reduced folate carrier (RFC) was assessed by reverse transcription-polymerase chain reaction in ALL blasts isolated from newly diagnosed patients. RFC expression exhibited a 60-fold range among 29 children, with significantly higher expression in hyperdiploid B-lineage ALL (median, 11.3) compared with nonhyperdiploid ALL (median, 2.1; P < .0006), but no significant difference between nonhyperdiploid B-lineage and T-lineage ALL. Furthermore, mRNA levels of RFC (mapped by FISH to chromosome 21) were significantly related to chromosome 21 copy number (P = .0013), with the highest expression in hyperdiploid ALL blasts with 4 copies of chromosome 21. To assess the functional significance of gene copy num...

Research paper thumbnail of Favorable Pharmacodynamic Features and Superior Anti-Leukemic Activity of B43 (Anti-CD 19) Immunotoxins Containing Two Pokeweed Antiviral Protein Molecules Covalently Linked to each Monoclonal Antibody Molecule

Leuk Lymphoma, 1995

Standard immunotoxin production procedures using whole IgG as the MoAb moiety yield a heterogeneo... more Standard immunotoxin production procedures using whole IgG as the MoAb moiety yield a heterogeneous mixture of 180 kDa, 210 kDa, and 240 kDa immunotoxin species with 1 to 1, 1 to 2, and 1 to 3 MoAb to toxin ratios. This heterogeneity makes it impossible to precisely deliver a predetermined immunotoxin dose to target cells and impairs the accuracy of pharmacologic studies. In this report, we describe the preparation and characterization of B43(anti-CD19)-pokeweed antiviral protein (PAP) immunotoxins containing either one or two 30 kDa PAP toxin molecules covalently linked to each 150 kDa B43 monoclonal antibody molecule. Compared to the 180 kDa immunotoxin, the 210 kDa immunotoxin displayed greater in vitro chemical stability, resulted in higher systemic exposure levels in vivo, and was a more effective anti-leukemic agent in a SCID mouse model of human B-lineage acute lymphoblastic leukemia. Taken together, the results of this study recommend the clinical evaluation of 210 kDa B43-PAP as a potentially more effective immunotoxin against relapsed B-lineage ALL.

Research paper thumbnail of Favorable Pharmacodynamic Features and Superior Anti-Leukemic Activity of B43 (Anti-CD 19) Immunotoxins Containing Two Pokeweed Antiviral Protein Molecules Covalently Linked to each Monoclonal Antibody Molecule

Standard immunotoxin production procedures using whole IgG as the MoAb moiety yield a heterogeneo... more Standard immunotoxin production procedures using whole IgG as the MoAb moiety yield a heterogeneous mixture of 180 kDa, 210 kDa, and 240 kDa immunotoxin species with 1 to 1, 1 to 2, and 1 to 3 MoAb to toxin ratios. This heterogeneity makes it impossible to precisely deliver a predetermined immunotoxin dose to target cells and impairs the accuracy of pharmacologic studies. In this report, we describe the preparation and characterization of B43(anti-CD19)-pokeweed antiviral protein (PAP) immunotoxins containing either one or two 30 kDa PAP toxin molecules covalently linked to each 150 kDa B43 monoclonal antibody molecule. Compared to the 180 kDa immunotoxin, the 210 kDa immunotoxin displayed greater in vitro chemical stability, resulted in higher systemic exposure levels in vivo, and was a more effective anti-leukemic agent in a SCID mouse model of human B-lineage acute lymphoblastic leukemia. Taken together, the results of this study recommend the clinical evaluation of 210 kDa B43-PAP as a potentially more effective immunotoxin against relapsed B-lineage ALL.

Research paper thumbnail of Enhanced Proteolysis of Thiopurine S-methyltransferase (TPMT) Encoded by Mutant Alleles in Humans (TPMT*3A, TPMT*2): Mechanisms for the Genetic Polymorphism of TPMT Activity

Proceedings of the National Academy of Sciences, Jun 10, 1997

TPMT is a cytosolic enzyme that catalyzes the S-methylation of aromatic and heterocyclic sulfhydr... more TPMT is a cytosolic enzyme that catalyzes the S-methylation of aromatic and heterocyclic sulfhydryl compounds, including medications such as mercaptopurine and thioguanine. TPMT activity exhibits autosomal codominant genetic polymorphism, and patients inheriting TPMT deficiency are at high risk of potentially fatal hematopoietic toxicity. The most prevalent mutant alleles associated with TPMT deficiency in humans have been cloned and characterized (TPMT‫2ء‬ and TPMT‫3ء‬A), but the mechanisms for loss of catalytic activity have not been elucidated. In the present study, we established that erythrocyte TPMT activity was significantly related to the amount of TPMT protein on Western blots of erythrocytes from patients with TPMT activities of 0.4-23 units͞ml pRBC (r s ‫؍‬ 0.99; P < 0.001). Similarly, heterologous expression of wild-type (TPMT‫)1ء‬ and mutant (TPMT‫2ء‬ and TPMT‫3ء‬A) human cDNAs in yeast and COS-1 cells demonstrated comparable levels of TPMT mRNA but significantly lower TPMT protein with the mutant cDNAs. Rates of protein synthesis were comparable for wild-type and mutant proteins expressed in yeast and with in vitro translation in rabbit reticulocyte lysates. In contrast, pulse-chase experiments revealed significantly shorter degradation halflives for TPMT‫2ء‬ and TPMT‫3ء‬A (ϳ0.25 hr) compared with wild-type TPMT‫1ء‬ (18 hr). The degradation of mutant proteins was impaired by ATP depletion and in yeast with mutant proteasomes (pre-1 strain) but unaffected by the lysosomal inhibitor chloroquine. These studies establish enhanced degradation of TPMT proteins encoded by TPMT‫2ء‬ and TPMT‫3ء‬A as mechanisms for lower TPMT protein and catalytic activity inherited by the predominant mutant alleles at the human TPMT locus.

Research paper thumbnail of Prevalence and growth characteristics of malignant stem cells in B- lineage acute lymphoblastic leukemia

Blood

We used a stroma-supported culture method to study the clones derived from single cells contained... more We used a stroma-supported culture method to study the clones derived from single cells contained approximately 10 3 prevalence and growth characteristics of malignant stem to 10 6 cells after 1 month of culture; other clones became cells in acute lymphoblastic leukemia (ALL). In 51 of 108 Bdetectable only after prolonged culture. Cell growth in lineage ALL samples, bone marrow-derived stroma not only stroma-coated wells correlated with the number of initially inhibited apoptosis of ALL cells but also supported their proseeded cells (1 or 10; r ! .87). However, the observed perliferation in serum-free medium. When single leukemic cells centages of positive wells seeded with 10 cells always exwere placed in the stroma-coated wells of microtiter plates, ceeded values predicted from results with single-cell-initithe percentage of wells with leukemic cell growth after 2 to ated cultures (P Ú .003 by paired t-test), suggesting 5 months of culture ranged from 6% to 20% (median, 15%; stimulation of leukemic cell growth by paracrine factors. In 5 experiments). The immunophenotypes and genetic feaconclusion, the proportion of ALL cells with clonogenic potures of cells recovered from these cultures were identical tential may be considerably higher than previously thought. to those noted before culture. All cells maintained their ᭧ 1997 by The American Society of Hematology. stroma dependency and self-renewal capacity. Leukemic MATERIALS AND METHODS C ENTRAL TO UNDERSTANDING neoplastic growth is the ability to study malignant cells with self-renewal Cells. Bone marrow samples were collected at diagnosis from capacity, the so-called cancer stem cells. This approach to 108 children with B-lineage ALL. Immunophenotyping established cancer investigation has been impeded by the lack of suitable the diagnosis in each case (ú80% of the blast cells were CD19 / , HLA-DR / and lacked surface Ig). Five samples, four collected at methods to support the long-term in vitro growth of neoplasdiagnosis and one at the time of relapse, were used for further tic cells from patients. Hence, the frequency, growth requireclonogenic studies. These cases were selected from cases with prolifments, and proliferative status of cancer stem cells remain erative activity in short-term cultures (see Results) because of the largely unknown. In acute lymphoblastic leukemia (ALL), availability of abundant cryopreserved material. Their immunophethe most common cancer of children, these issues have been notypic and karyotypic features are summarized in Table 1. Mononuaddressed almost entirely with short-term colony assays in clear cells were obtained after centrifugation on a density gradient semisolid medium. 1-15 The results suggest that leukemic (Lymphoprep; Nycomed, Oslo, Norway) and washed three times in lymphoblasts with self-renewal capacity constitute only a phosphate-buffered saline (PBS). Samples were cryopreserved and minute fraction of ALL cells (ie, one or two per thousand) used immediately after thawing. Continuously growing ALL cell lines (Nalm6, REH, 380, RS4;11, and CEM-C7) were obtained from and that the remainder of leukemic blasts are incapable of the institution's cell bank and maintained in RPMI-1640 (BioWhitgenerating self-sustaining clones. 16,17 taker, Walkersville, MD) with 10% fetal calf serum (FCS; BioWhit-We would argue that this method of assessment vastly taker), L-glutamine, and antibiotics. In all experiments, the cells' underestimates the growth potential of leukemic cell populaviability before culture exceeded 95% by trypan-blue dye exclusion. tions for two reasons. First, the majority of such cells rapidly To prepare bone marrow-derived stromal layers, we obtained die in vitro by apoptosis, which is not prevented by the mononucleated cells from bone marrow transplantation donors. 18,21-24 animal sera or exogenous cytokines commonly used in col-Preliminary studies indicated that such cells had similar capacities ony assays. 18 Thus, the pool of both resting and proliferating to maintain ALL cell viability regardless of the donor from whom leukemic cells may be drastically reduced within hours of they were collected. 22,23 Mononuclear cells were separated as described above and washed three times in RPMI-1640. They were culture. Second, it is conceivable that a proportion of leukeresuspended at 5 1 10 6 /mL in RPMI-1640, 10% FCS, and 10 06 mol/ mic stem cells are quiescent, as suggested by clinical observations of leukemic relapse years after cessation of therapy. 19,20 It seems unlikely that these cells could be driven From the Departments of Hematology-Oncology, Pharmaceutical into vigorous proliferative activity by several days of culture

Research paper thumbnail of A mathematical model of in vivo methotrexate accumulation in acute lymphoblastic leukemia

Cancer Chemotherapy and Pharmacology

Methotrexate (MTX) is one of the most widely used drugs for the treatment of childhood acute lymp... more Methotrexate (MTX) is one of the most widely used drugs for the treatment of childhood acute lymphoblastic leukemia (ALL). Interindividual differences in lymphoblast accumulation of MTX and its active metabolites, methotrexate polyglutamates (MTXPG), may contribute to the effectiveness of treatment among ALL subtypes. To better understand these differences in MTXPG accumulation, we developed a model to characterize the cellular influx and efflux of MTX, formation of MTXPG by the addition of glutamyl residues catalyzed by FPGS (folylpolyglutamate synthetase), and cleavage of glutamyl residues from MTXPG by GGH (gamma-glutamyl hydrolase). The model was fitted to in vivo intracellular MTXPG concentrations measured serially in leukemic blasts from 20 newly diagnosed patients with ALL treated with 24-h intravenous infusions of MTX. The observed median concentrations of total MTXPG at 44 h was higher in B-lineage than in T-cell ALL (1706 vs 518 pmol/10(9) cells, P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.025), consistent with the higher estimated Vmax for FPGS activity in B-lineage vs T-lineage blasts (414 vs 93 pmol/10(9) cells/h, P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.008). Simulations based on the model-estimated parameters indicated greater accumulation of MTX, MTXPGs (MTXPG(2-7)) and total MTX (MTXPG(1-7)) with longer MTX infusions and with higher MTX doses, with the highest concentrations in hyperdiploid B-lineage, intermediate in non-hyperdiploid B-lineage, and lowest in T-cell ALL. These differences provide mechanistic and treatment insights for lineage and ploidy differences in MTXPG accumulation in human leukemia cells in vivo.

Research paper thumbnail of Differences in folylpolyglutamate synthetase and dihydrofolate reductase expression in human B-lineage versus T-lineage leukemic lymphoblasts: mechanisms for …

Molecular …, 1997

Cellular accumulation of methotrexate polyglutamates (MTXPGs) is recognized as an important deter... more Cellular accumulation of methotrexate polyglutamates (MTXPGs) is recognized as an important determinant of the cytotoxicity and selectivity of methotrexate in acute lymphoblastic leukemia (ALL). We identified a significantly lower cellular accumulation of MTXPGs in T-lineage ...

Research paper thumbnail of In Vivo., Toxicity and Pharmacokinetic Features of B43(Anti-CD19)-Genistein Immunoconjugate

Leukemia & Lymphoma, 1998

B43(anti-CD19)-Genistein immunoconjugate targets genistein, a naturally occurring protein tyrosin... more 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.

Research paper thumbnail of Treatment of human B-cell precursor leukemia in SCID mice using a combination of the investigational biotherapeutic agent B43-PAP with cytosine arabinoside

Clinical cancer research : an official journal of the American Association for Cancer Research, 1996

Combined immunochemotherapy regimens using the investigational biotherapeutic agent B43(anti-CD19... more Combined immunochemotherapy regimens using the investigational biotherapeutic agent B43(anti-CD19)-poke-weed antiviral protein (PAP) immunotoxin may offer an effective treatment for refractory B-cell precursor leukemias. The purpose of the present study was to explore and identify effective combinations of B43-PAP with standard chemotherapeutic drugs, including the anthracyclin doxorubicin, the epipodophyllotoxin etoposide, the nitrosurea carmustine, and the antimetabolite cytosine arabinoside. Here, we report that the B43-PAP plus cytosine arabinoside combination has potent antileukemic activity against human B-cell precursor leukemia in SCID mice and leads to 100% long-term event-free survival from an otherwise invariably fatal leukemia. Surprisingly, none of the other treatment protocols tested, including combinations of B43-PAP with carmustine, doxorubicin, or etoposide, proved more effective than B43-PAP alone.

Research paper thumbnail of Pharmacokinetic features, immunogenicity, and toxicity of B43(anti-CD19)-pokeweed antiviral protein immunotoxin in cynomolgus monkeys

Clinical cancer research : an official journal of the American Association for Cancer Research, 1997

We studied the pharmacokinetic features, immunogenicity, and toxicity of B43-pokeweed antiviral p... more We studied the pharmacokinetic features, immunogenicity, and toxicity of B43-pokeweed antiviral protein (PAP) immunotoxin in 13 cynomolgus monkeys. The disposition of B43-PAP in two monkeys, when administered as a single i.v. bolus dose, was characterized by a slow clearance (1-2 ml/h/kg) with a very discrete peripheral distribution. B43-PAP was retained and distributed largely in the blood as the sole compartment with no significant equilibration with the extravascular compartment. The circulating B43-PAP immunotoxin detected in monkey plasma samples by ELISA and protein immunoblotting was both immunoreactive with, and active against, human leukemic cells in vitro. In systemic immunogenicity and toxicity studies, which involved 11 cynomolgus monkeys, each monkey received a total of seven i.v. doses of B43-PAP at a specific dose level of the dose escalation schedule. B43-PAP-treated monkeys mounted a dose-dependent humoral immune response against both the mouse IgG and PAP moieties ...

Research paper thumbnail of In vivo biotherapy of HL-60 myeloid leukemia with a genetically engineered recombinant fusion toxin directed against the human granulocyte macrophage colony-stimulating factor receptor

Clinical cancer research : an official journal of the American Association for Cancer Research, 1997

Acute myeloid leukemia (AML) is the most common form of acute leukemia. Contemporary chemotherapy... more Acute myeloid leukemia (AML) is the most common form of acute leukemia. Contemporary chemotherapy regimens fail to cure most patients with AML. We have genetically engineered a recombinant diphtheria toxin human granulocyte macrophage colony-stimulating factor (GMCSF) chimeric fusion protein (DTctGMCSF) that specifically targets the GMCSF receptor on fresh human AML cells and myeloid leukemia cell lines. At a nontoxic dose level, DTctGMCSF therapy was superior to the standard chemotherapeutic agents 1-beta-D-arabinofuranosylcytosine and Adriamycin, resulting in 60% long-term event-free survival of severe combined immunodeficient mice challenged with an otherwise invariably fatal cell dose of the human HL-60 myeloid leukemia. Notably, systemic exposure levels of DTctGMCSF, which were found to be therapeutic in the severe combined immunodeficient mouse xenograft model of human HL-60 myeloid leukemia, could be achieved in cynomolgus monkeys without any significant nonhematological toxi...

Research paper thumbnail of In vivo toxicity, pharmacokinetics, and anticancer activity of Genistein linked to recombinant human epidermal growth factor

Clinical cancer research : an official journal of the American Association for Cancer Research, 1998

Epidermal growth factor receptor (EGFR)-associated protein tyrosine kinase (PTK) complexes have v... more Epidermal growth factor receptor (EGFR)-associated protein tyrosine kinase (PTK) complexes have vital anti-apoptotic functions in human breast cancer cells. We have shown previously that targeting the naturally occurring PTK inhibitor genistein to the EGFR-associated PTK complexes using the EGF-Genistein (Gen) conjugate triggers rapid apoptotic cell death in human breast cancer cells and abrogates their in vitro clonogenic growth. In the present study, we examined the in vivo toxicity profile, pharmacokinetics, and anticancer activity of EGF-Gen. No toxicities were observed in mice treated with EGF-Gen at dose levels as high as 40 mg/kg administered i.p. as a single dose or 140 mg/kg administered i.p. over 28 consecutive days. EGF-Gen significantly improved tumor-free survival in a severe combined immune deficiency (SCID) mouse xenograft model of human breast cancer, when it was administered 24 h after inoculation of tumor cells. At 100 microg/kg/day x 10 days (1 mg/kg total dose), ...

Research paper thumbnail of Methylation of mercaptopurine, thioguanine, and their nucleotide metabolites by heterologously expressed human thiopurine S-methyltransferase

Molecular pharmacology, 1995

Thiopurine S-methyltransferase (TPMT), a cytosolic enzyme that exhibits genetic polymorphism, cat... more Thiopurine S-methyltransferase (TPMT), a cytosolic enzyme that exhibits genetic polymorphism, catalyzes S-methylation of mercaptopurine (MP) and thioguanine (TG), yielding S-methylated nucleobases that are inactive, whereas S-methylated nucleotides of these thiopurines are cytotoxic. A yeast-based heterologous expression system was therefore used to characterize human TPMT-catalyzed methylation of MP, TG, and their principal nucleotide metabolites [thioinosine monophosphate (TIMP) and thioguanosine monophosphate (TGMP), respectively]. MP, TG, TIMP, and TGMP were all substrates for human TPMT, exhibiting similar Michaelis-Menten kinetic parameters (Km, 10.6-27.1 microM; Vmax, 31-59 nmol/min/mg of TPMT). Consistent with these kinetic parameters, human leukemia cells (CEM) incubated for 24 hr with 10 microM MP or TG accumulated significantly higher (2.3-fold, p = 0.01) concentrations of methyl-TIMP after MP incubation than methyl-TGMP after TG incubation, due to the 2.7-fold higher con...

Research paper thumbnail of Etoposide and antimetabolite pharmacology in patients who develop secondary acute myeloid leukemia

Leukemia, 1998

Etoposide, an effective agent for acute lymphoblastic leukemia (ALL), can cause secondary acute m... more Etoposide, an effective agent for acute lymphoblastic leukemia (ALL), can cause secondary acute myeloid leukemia (AML) in a subset of patients. Our objectives were to determine whether patients who develop secondary AML displayed altered etoposide pharmacokinetics or other pharmacologic characteristics compared to identically treated patients who did not develop AML. Children with newly diagnosed ALL were treated according to a protocol which included etoposide 300 mg/m 2 given three times over 8 days during remission induction and once every 2-4 weeks during 120 weeks of continuation therapy. Characteristic 11q23 rearrangements were documented in seven of the eight patients with AML. Etoposide clearance, time that etoposide concentrations exceeded 10 M, etoposide or etoposide catechol area-under-the-plasma-concentration vs time curve (AUC), serum albumin, and average methotrexate concentration did not differ significantly between the two groups; thiopurine methyltransferase (TPMT) activity tended to be lower in the eight children who did vs the 23 matched control children who did not develop AML (P = 0.16). Further regression analyses likewise indicated that lower TPMT activity tended to be associated with shorter onset of secondary AML (P = 0.11); it also tended to be lower among the eight index cases compared to the entire unmatched cohort of 105 identically treated children with ALL (P = 0.10). We observed no statistically significant differences in etoposide disposition and antimetabolite pharmacology between patients who did and did not develop secondary AML.

Research paper thumbnail of Reduced Folate Carrier Expression in Acute Lymphoblastic Leukemia: A Mechanism for Ploidy but not Lineage Differences in Methotrexate Accumulation

Blood

Methotrexate (MTX) is one of the most active and widely used agents for the treatment of acute ly... more Methotrexate (MTX) is one of the most active and widely used agents for the treatment of acute lymphoblastic leukemia (ALL). To elucidate the mechanism for higher accumulation of MTX polyglutamates (MTX-PG) in hyperdiploid ALL and lower accumulation in T-lineage ALL, expression of the reduced folate carrier (RFC) was assessed by reverse transcription-polymerase chain reaction in ALL blasts isolated from newly diagnosed patients. RFC expression exhibited a 60-fold range among 29 children, with significantly higher expression in hyperdiploid B-lineage ALL (median, 11.3) compared with nonhyperdiploid ALL (median, 2.1; P < .0006), but no significant difference between nonhyperdiploid B-lineage and T-lineage ALL. Furthermore, mRNA levels of RFC (mapped by FISH to chromosome 21) were significantly related to chromosome 21 copy number (P = .0013), with the highest expression in hyperdiploid ALL blasts with 4 copies of chromosome 21. To assess the functional significance of gene copy num...

Research paper thumbnail of Favorable Pharmacodynamic Features and Superior Anti-Leukemic Activity of B43 (Anti-CD 19) Immunotoxins Containing Two Pokeweed Antiviral Protein Molecules Covalently Linked to each Monoclonal Antibody Molecule

Leuk Lymphoma, 1995

Standard immunotoxin production procedures using whole IgG as the MoAb moiety yield a heterogeneo... more Standard immunotoxin production procedures using whole IgG as the MoAb moiety yield a heterogeneous mixture of 180 kDa, 210 kDa, and 240 kDa immunotoxin species with 1 to 1, 1 to 2, and 1 to 3 MoAb to toxin ratios. This heterogeneity makes it impossible to precisely deliver a predetermined immunotoxin dose to target cells and impairs the accuracy of pharmacologic studies. In this report, we describe the preparation and characterization of B43(anti-CD19)-pokeweed antiviral protein (PAP) immunotoxins containing either one or two 30 kDa PAP toxin molecules covalently linked to each 150 kDa B43 monoclonal antibody molecule. Compared to the 180 kDa immunotoxin, the 210 kDa immunotoxin displayed greater in vitro chemical stability, resulted in higher systemic exposure levels in vivo, and was a more effective anti-leukemic agent in a SCID mouse model of human B-lineage acute lymphoblastic leukemia. Taken together, the results of this study recommend the clinical evaluation of 210 kDa B43-PAP as a potentially more effective immunotoxin against relapsed B-lineage ALL.

Research paper thumbnail of Favorable Pharmacodynamic Features and Superior Anti-Leukemic Activity of B43 (Anti-CD 19) Immunotoxins Containing Two Pokeweed Antiviral Protein Molecules Covalently Linked to each Monoclonal Antibody Molecule

Standard immunotoxin production procedures using whole IgG as the MoAb moiety yield a heterogeneo... more Standard immunotoxin production procedures using whole IgG as the MoAb moiety yield a heterogeneous mixture of 180 kDa, 210 kDa, and 240 kDa immunotoxin species with 1 to 1, 1 to 2, and 1 to 3 MoAb to toxin ratios. This heterogeneity makes it impossible to precisely deliver a predetermined immunotoxin dose to target cells and impairs the accuracy of pharmacologic studies. In this report, we describe the preparation and characterization of B43(anti-CD19)-pokeweed antiviral protein (PAP) immunotoxins containing either one or two 30 kDa PAP toxin molecules covalently linked to each 150 kDa B43 monoclonal antibody molecule. Compared to the 180 kDa immunotoxin, the 210 kDa immunotoxin displayed greater in vitro chemical stability, resulted in higher systemic exposure levels in vivo, and was a more effective anti-leukemic agent in a SCID mouse model of human B-lineage acute lymphoblastic leukemia. Taken together, the results of this study recommend the clinical evaluation of 210 kDa B43-PAP as a potentially more effective immunotoxin against relapsed B-lineage ALL.

Research paper thumbnail of Enhanced Proteolysis of Thiopurine S-methyltransferase (TPMT) Encoded by Mutant Alleles in Humans (TPMT*3A, TPMT*2): Mechanisms for the Genetic Polymorphism of TPMT Activity

Proceedings of the National Academy of Sciences, Jun 10, 1997

TPMT is a cytosolic enzyme that catalyzes the S-methylation of aromatic and heterocyclic sulfhydr... more TPMT is a cytosolic enzyme that catalyzes the S-methylation of aromatic and heterocyclic sulfhydryl compounds, including medications such as mercaptopurine and thioguanine. TPMT activity exhibits autosomal codominant genetic polymorphism, and patients inheriting TPMT deficiency are at high risk of potentially fatal hematopoietic toxicity. The most prevalent mutant alleles associated with TPMT deficiency in humans have been cloned and characterized (TPMT‫2ء‬ and TPMT‫3ء‬A), but the mechanisms for loss of catalytic activity have not been elucidated. In the present study, we established that erythrocyte TPMT activity was significantly related to the amount of TPMT protein on Western blots of erythrocytes from patients with TPMT activities of 0.4-23 units͞ml pRBC (r s ‫؍‬ 0.99; P < 0.001). Similarly, heterologous expression of wild-type (TPMT‫)1ء‬ and mutant (TPMT‫2ء‬ and TPMT‫3ء‬A) human cDNAs in yeast and COS-1 cells demonstrated comparable levels of TPMT mRNA but significantly lower TPMT protein with the mutant cDNAs. Rates of protein synthesis were comparable for wild-type and mutant proteins expressed in yeast and with in vitro translation in rabbit reticulocyte lysates. In contrast, pulse-chase experiments revealed significantly shorter degradation halflives for TPMT‫2ء‬ and TPMT‫3ء‬A (ϳ0.25 hr) compared with wild-type TPMT‫1ء‬ (18 hr). The degradation of mutant proteins was impaired by ATP depletion and in yeast with mutant proteasomes (pre-1 strain) but unaffected by the lysosomal inhibitor chloroquine. These studies establish enhanced degradation of TPMT proteins encoded by TPMT‫2ء‬ and TPMT‫3ء‬A as mechanisms for lower TPMT protein and catalytic activity inherited by the predominant mutant alleles at the human TPMT locus.

Research paper thumbnail of Prevalence and growth characteristics of malignant stem cells in B- lineage acute lymphoblastic leukemia

Blood

We used a stroma-supported culture method to study the clones derived from single cells contained... more We used a stroma-supported culture method to study the clones derived from single cells contained approximately 10 3 prevalence and growth characteristics of malignant stem to 10 6 cells after 1 month of culture; other clones became cells in acute lymphoblastic leukemia (ALL). In 51 of 108 Bdetectable only after prolonged culture. Cell growth in lineage ALL samples, bone marrow-derived stroma not only stroma-coated wells correlated with the number of initially inhibited apoptosis of ALL cells but also supported their proseeded cells (1 or 10; r ! .87). However, the observed perliferation in serum-free medium. When single leukemic cells centages of positive wells seeded with 10 cells always exwere placed in the stroma-coated wells of microtiter plates, ceeded values predicted from results with single-cell-initithe percentage of wells with leukemic cell growth after 2 to ated cultures (P Ú .003 by paired t-test), suggesting 5 months of culture ranged from 6% to 20% (median, 15%; stimulation of leukemic cell growth by paracrine factors. In 5 experiments). The immunophenotypes and genetic feaconclusion, the proportion of ALL cells with clonogenic potures of cells recovered from these cultures were identical tential may be considerably higher than previously thought. to those noted before culture. All cells maintained their ᭧ 1997 by The American Society of Hematology. stroma dependency and self-renewal capacity. Leukemic MATERIALS AND METHODS C ENTRAL TO UNDERSTANDING neoplastic growth is the ability to study malignant cells with self-renewal Cells. Bone marrow samples were collected at diagnosis from capacity, the so-called cancer stem cells. This approach to 108 children with B-lineage ALL. Immunophenotyping established cancer investigation has been impeded by the lack of suitable the diagnosis in each case (ú80% of the blast cells were CD19 / , HLA-DR / and lacked surface Ig). Five samples, four collected at methods to support the long-term in vitro growth of neoplasdiagnosis and one at the time of relapse, were used for further tic cells from patients. Hence, the frequency, growth requireclonogenic studies. These cases were selected from cases with prolifments, and proliferative status of cancer stem cells remain erative activity in short-term cultures (see Results) because of the largely unknown. In acute lymphoblastic leukemia (ALL), availability of abundant cryopreserved material. Their immunophethe most common cancer of children, these issues have been notypic and karyotypic features are summarized in Table 1. Mononuaddressed almost entirely with short-term colony assays in clear cells were obtained after centrifugation on a density gradient semisolid medium. 1-15 The results suggest that leukemic (Lymphoprep; Nycomed, Oslo, Norway) and washed three times in lymphoblasts with self-renewal capacity constitute only a phosphate-buffered saline (PBS). Samples were cryopreserved and minute fraction of ALL cells (ie, one or two per thousand) used immediately after thawing. Continuously growing ALL cell lines (Nalm6, REH, 380, RS4;11, and CEM-C7) were obtained from and that the remainder of leukemic blasts are incapable of the institution's cell bank and maintained in RPMI-1640 (BioWhitgenerating self-sustaining clones. 16,17 taker, Walkersville, MD) with 10% fetal calf serum (FCS; BioWhit-We would argue that this method of assessment vastly taker), L-glutamine, and antibiotics. In all experiments, the cells' underestimates the growth potential of leukemic cell populaviability before culture exceeded 95% by trypan-blue dye exclusion. tions for two reasons. First, the majority of such cells rapidly To prepare bone marrow-derived stromal layers, we obtained die in vitro by apoptosis, which is not prevented by the mononucleated cells from bone marrow transplantation donors. 18,21-24 animal sera or exogenous cytokines commonly used in col-Preliminary studies indicated that such cells had similar capacities ony assays. 18 Thus, the pool of both resting and proliferating to maintain ALL cell viability regardless of the donor from whom leukemic cells may be drastically reduced within hours of they were collected. 22,23 Mononuclear cells were separated as described above and washed three times in RPMI-1640. They were culture. Second, it is conceivable that a proportion of leukeresuspended at 5 1 10 6 /mL in RPMI-1640, 10% FCS, and 10 06 mol/ mic stem cells are quiescent, as suggested by clinical observations of leukemic relapse years after cessation of therapy. 19,20 It seems unlikely that these cells could be driven From the Departments of Hematology-Oncology, Pharmaceutical into vigorous proliferative activity by several days of culture

Research paper thumbnail of A mathematical model of in vivo methotrexate accumulation in acute lymphoblastic leukemia

Cancer Chemotherapy and Pharmacology

Methotrexate (MTX) is one of the most widely used drugs for the treatment of childhood acute lymp... more Methotrexate (MTX) is one of the most widely used drugs for the treatment of childhood acute lymphoblastic leukemia (ALL). Interindividual differences in lymphoblast accumulation of MTX and its active metabolites, methotrexate polyglutamates (MTXPG), may contribute to the effectiveness of treatment among ALL subtypes. To better understand these differences in MTXPG accumulation, we developed a model to characterize the cellular influx and efflux of MTX, formation of MTXPG by the addition of glutamyl residues catalyzed by FPGS (folylpolyglutamate synthetase), and cleavage of glutamyl residues from MTXPG by GGH (gamma-glutamyl hydrolase). The model was fitted to in vivo intracellular MTXPG concentrations measured serially in leukemic blasts from 20 newly diagnosed patients with ALL treated with 24-h intravenous infusions of MTX. The observed median concentrations of total MTXPG at 44 h was higher in B-lineage than in T-cell ALL (1706 vs 518 pmol/10(9) cells, P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.025), consistent with the higher estimated Vmax for FPGS activity in B-lineage vs T-lineage blasts (414 vs 93 pmol/10(9) cells/h, P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;0.008). Simulations based on the model-estimated parameters indicated greater accumulation of MTX, MTXPGs (MTXPG(2-7)) and total MTX (MTXPG(1-7)) with longer MTX infusions and with higher MTX doses, with the highest concentrations in hyperdiploid B-lineage, intermediate in non-hyperdiploid B-lineage, and lowest in T-cell ALL. These differences provide mechanistic and treatment insights for lineage and ploidy differences in MTXPG accumulation in human leukemia cells in vivo.

Research paper thumbnail of Differences in folylpolyglutamate synthetase and dihydrofolate reductase expression in human B-lineage versus T-lineage leukemic lymphoblasts: mechanisms for …

Molecular …, 1997

Cellular accumulation of methotrexate polyglutamates (MTXPGs) is recognized as an important deter... more Cellular accumulation of methotrexate polyglutamates (MTXPGs) is recognized as an important determinant of the cytotoxicity and selectivity of methotrexate in acute lymphoblastic leukemia (ALL). We identified a significantly lower cellular accumulation of MTXPGs in T-lineage ...