Design,synthesis and evaluation of anti-CD123 antibody drug conjugates

Leukemia stem cells (LSCs) account for the development of drug resistance and increased recurrence rate in acute myeloid leukemia (AML) patients. Targeted drug delivery to leukemia stem cells remains a major challenge in AML chemotherapy. Overexpressed interleukin-3 receptor alpha chain, CD123, on the surface of leukemia stem cells was reported to be a potential target in AML treatment. Here, we designed and developed an antibody drug conjugate (CD123-CPT) by integrating anti-CD123 antibody with a chemotherapeutic agent, Camptothecin (CPT), via a disulfide linker. The linker is biodegradable in the presence of Glutathione (GSH, an endogenous component in cells), which leads to release of CPT. Anti-CD123 antibody conjugates showed significant higher cellular uptake in CD123-overexpressed tumor cells. More importantly, CD123-CPT demonstrated potent inhibitory effects on CD123-overexpressed tumor cells. Consequently, these results provide a promising targeted chemotherapeutical strategy for AML treatment.     

Plasma and intracellular levels of lactate dehydrogenase, phosphohexose isomerase and lysozyme activity in acute leukemia

Plasma and intracellular levels of lactate dehydrogenase (LDH), phosphohexose isomerase (PHI) and lysozyme activities were investigated in 20 patients with acute myelocytic leukemia (AML), 18 patients with acute lymphatic leukemia (ALL) and 10 patients with chronic myelocytic leukemia in blast transformation (CML/BT). Though the plasma levels of LDH and PHI in all patients with acute leukemia were elevated as compared to control persons there was no distinctive pattern which could be of use in the classification of acute leukemia. On the other hand the intracellular levels of these enzymes could be of value in classifying acute leukemia. The leukemic lymphoblasts were characterized by low levels of PHI and lysozyme as compared to leukemic myeloblasts or to normal lymphocytes (p less than 0.01). The LDH/PHI ratio is also significantly higher in leukemic lymphoblasts than in leukemic myeloblasts or in normal lymphocytes (p always less than 0.01). These characteristics might also be made use of in identifying the blasts of CML/BT als "lymphoid" or "myeloid" in corresponding cases.     

Blood coagulation system in oncological patients treated with rubomycin, adriamycin and carminomycin]

Systems of blood coagulation in patients treated with antibiotics of the anthracycline group were studied. Rubomycin was used in the treatment of patients with acute leukemia Adriamycin and carminomycin were used in the treatment of patients with solid tumors. The antibiotics affected the process of blood coagulation mainly through their cytostatic effect on thrombocytopoesis. Thrombocytopenia induced deficit of thrombocytal factors participating in the process of blood coagulation which resulted in hypocoagulation and hemorrhagic complications. The plasmic factors did not significantly change during the antibiotic therapy. A tendency to decrease in the levels of prothrombine, fibrinase and fibrinogen was noted which was possible due to an inhibitory effect of the antibiotics on the function of the reticuloendothelial tissue cells or indirectly to suppression of the tumor process. More pronounced changes in the system of blood coagulation of patients treated with rubomycin were probably associated with inferiority of the thrombocytal apparatus of the patients with acute leukemia treated with the antibiotic.     

High expression of heat shock protein 90 alpha and its significance in human acute leukemia cells

This study investigated the expression of heat shock protein 90 alpha (Hsp90α) in acute leukemia cells. The expression of Hsp90α was investigated in leukemia cell lines and human bone marrow mononuclear cells derived from acute leukemia patients and from healthy individuals using polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay. Compared with cells from healthy individuals, the expression of Hsp90α in the untreated patients was higher. Similarly high levels were observed in remission patients. Significantly higher expression levels were observed in all the tested cell lines, and in cells from refractory and relapsed patients. No obvious relationship was observed between the occurrence of graft versus host disease and the expression of Hsp90α. The untreated patients showing higher expression levels of Hsp90α had lower complete remission rates. During remission of untreated patients, the expression of Hsp90α decreased and reached the lowest level after transplantation, but the expression increased again before relapse. Hsp90α was highly expressed in leukemia cells. The expression level of Hsp90α was associated with leukemia prognosis. However, no obvious relationship was observed between the occurrence of graft versus host disease and the expression of Hsp90α.     

Molecular diagnostics in the treatment of childhood acute lymphoblastic leukemia

Somatically acquired genetic alterations play an important role in the pathogenesis of acute lymphoblastic leukemia. The molecular analysis of these alterations has increased our understanding of the mechanisms of leukemogenesis. In addition, this information has led to improvements in our abilities to predict treatment response and to deliver the optimal intensity of treatment to individual patients. For example, the prognosis for patients with acute lymphoblastic leukemia whose leukemic cells express the TEL-AML1 fusion is favorable when they are treated on modem chemotherapy protocols, whereas patients whose leukemic lymphoblasts contain the MLL-AF4 or the BCR-ABL fusion sometimes require allogeneic hematopoietic stem cell transplantation for cure. Molecular techniques are also used to detect minimal residual disease and genetic polymorphisms that are important in optimizing drug therapy.     

Levels of selenium, zinc, copper, and antioxidant enzyme activity in patients with leukemia

Essential elements, mainly selenium and zinc, were involved in protection against oxidative stress in cells. Oxidation could lead to the formation of free radicals that have been implicated in the pathogenesis of many diseases, including leukemia. Leukemia is a neoplastic disease that is susceptible to antioxidant enzyme and essential elements alterations. This study was undertaken to examine the levels of essential elements, antioxidant enzymes activities, and their relationships with different types of leukemia. Serum selenium, zinc, and copper concentrations, red blood cell glutathione peroxidase (GPx) activities, plasma Cu−Zn superoxide dismutase (Cu−Zn SOD) activities and lipid peroxidation (LPO) levels were determined in 49 patients with different types of leukemia before initial treatment. Serum selenium and zinc concentrations were lower in leukemia patients than those of controls (p<0.01). Serum copper concentration was higher in leukemia patients than that of controls (p<0.01). The activities GPx and Cu−Zn SOD were significantly increased in leukemia patients, especially with acute leukemia (AL), acute lymphoid leukemia (ALL), and acute nonlymphoid leukemia (ANLL) (p<0.05), whereas no difference was found between those of chronic myelogeneous leukemia and the controls. The levels of LPO were normal as controls. Serum selenium concentration was not correlated with GPx, and serum levels of zinc and copper were not related to Cu−Zn SOD. Serum zinc levels had a negative correlation with the absolute peripheral blast cells, whereas serum copper had a positive correlation with the absolute peripheral blast cells. Increased GPx and Cu−Zn SOD activities and normal levels of LPO, which were a protective responses, were an indicator of mild oxidative stress; it mights indicate that the essentials elements alterations in leukemia patients were mostly dependent on tumor activity. Changes of their levels demonstrated that there are low selenium, zinc, and high copper status in leukemia patients. The decrease of plasma zinc and increase of the Cu/Zn ratio could be the index that showed an unfavorable prognosis of acute leukemia.     

Somatic deletions of genes regulating MSH2 protein stability cause DNA mismatch repair deficiency and drug resistance in human leukemia cells

DNA mismatch repair enzymes (for example, MSH2) maintain genomic integrity, and their deficiency predisposes to several human cancers and to drug resistance. We found that leukemia cells from a substantial proportion of children (～11%) with newly diagnosed acute lymphoblastic leukemia have low or undetectable MSH2 protein levels, despite abundant wild-type MSH2 mRNA. Leukemia cells with low levels of MSH2 contained partial or complete somatic deletions of one to four genes that regulate MSH2 degradation (FRAP1 (also known as MTOR), HERC1, PRKCZ and PIK3C2B); we also found these deletions in individuals with adult acute lymphoblastic leukemia (16%) and sporadic colorectal cancer (13.5%). Knockdown of these genes in human leukemia cells recapitulated the MSH2 protein deficiency by enhancing MSH2 degradation, leading to substantial reduction in DNA mismatch repair and increased resistance to thiopurines. These findings reveal a previously unrecognized mechanism whereby somatic deletions of genes regulating MSH2 degradation result in undetectable levels of MSH2 protein in leukemia cells, DNA mismatch repair deficiency and drug resistance.     

A 125I-protein A-binding assay detecting antibodies to cell surface antigens

A 125I-protein A-binding assay detecting antibodies to cell surface antigens on human blood cells was developed and evaluated using sera from multitransfused nonleukemic patients sensitized against HLA antigens. The binding assay was found to be reproducible and more sensitive than conventional HLA testing. Seven patients with acute myelogenous leukemia and two patients with acute lymphoblastic leukemia successfully treated by chemotherapy were then investigated. Sera from seven of the patients studied in partial or complete remission demonstrated significant binding to autochthonous leukemic cells obtained from bone marrow or peripheral blood. In two cases sera taken during the leukemic stage demonstrated the most pronounced binding to the patients' own leukemic cells. Sera from four patients with demonstrable significant binding to autochthonous leukemic cells failed to bind to autochthonous remission cells when both types of target cells were tested in parallel. Differences in serum concentrations of IgG, IgA, and IgM were not the cause of the demonstrated increased binding of leukemic sera to autochthonous target cells. We propose that the 125I-protein A-binding assay presented in this paper detects antibodies reacting selectively with acute leukemia cells.     

An anti-CD33 antibody-calicheamicin conjugate for treatment of acute myeloid leukemia. Choice of linker.

The anti-CD33 antibody, P67.6, has been chosen to target the potently cytotoxic calicheamicin antitumor antibiotics to acute myeloid leukemia (AML) due to the presence of CD33 on >80% of patient samples and its lack of expression outside the myeloid cell lineages, especially its lack of expression on pluripotent stem cells. Previous calicheamicin conjugates relied on the attachment of a hydrazide derivative to the oxidized carbohydrates that occur naturally on antibodies. This results in a "carbohydrate conjugate" capable of releasing active drug by hydrolysis of a hydrazone bond in the lysozomes where the pH is low. Conjugates have now been made that are formed by reacting a calicheamicin derivative containing an activated ester with the lysines of antibodies. This results in an "amide conjugate" that is stable to hydrolysis, leaving the disulfide that is present in all calicheamicin conjugates as the likely site of drug release from the conjugate. In this article, these two classes of calicheamicin-antibody conjugates are compared for potential use in AML with the anti-CD33 antibody P67.6. Conjugates of P67.6 are shown to require the site of hydrolytic release afforded by the carbohydrate conjugates in order to retain good potency and selectivity in vitro, in vivo, and ex vivo. The P67.6 carbohydrate conjugate of calicheamicin is selectively cytotoxic at <0.006 ng/mL of calicheamicin equivalents (cal equiv) toward HL-60 promyelocytic leukemia cells in tissue culture. Long-term, tumor-free survivors are seen in xenograft models when mice bearing HL-60 subcutaneous tumors are treated with the P67.6 carbohydrate conjugate at a dose of 300 microg/kg cal equiv given three times. This conjugate also selectively inhibits the formation of colonies from AML marrow samples at 2 ng/mL cal equiv. The P67.6 carbohydrate conjugate of calicheamicin therefore appears to have promise as an antibody-targeted chemotherapeutic agent for CD33-positive diseases such as AML.     

Synthesis and antiproliferative activity of novel steroidal dendrimer conjugates

We describe the synthesis of steroidal dendrimer conjugates of first and second generation with tetramethylene core and 5-hydroxy-isophtalic acid dimethyl ester as branching unit modified to incorporate ethynylestradiol or 17α-estradiol as terminal units. The steroidal dendrimer conjugates, the free drug (steroids) and dendrimer were tested against a panel of cancer cell lines (CEM, MCF7, HeLa) and normal human fibroblast (BJ). The steroidal dendrimer conjugates of first generation exhibited cytotoxic activity and induced apoptosis in chronic leukemia (CEM) as resultant activation of caspase cascade which is mainly provoked in G2/M arrested cells.     

Expression of selected genes in differentiated HL-60 cells and primary cells from human leukemias

Expression of three clones (6-1E, 7-3G and 9-5C) selected from a chronic lymphocytic leukemia cDNA library was studied by nucleic acid hybridization in human promyelocytic leukemia cells (HL-60) treated with chemical inducers of cell differentiation and in primary cells derived from 27 patients with leukemia or myelodysplastic syndrome. The differentiation of HL-60 cells into macrophage-like cells upon induction by 12-0-tetradecanoyl phorbol-13-acetate (TPA) was accompanied by rapid induction of the expression of 6-1E and 7-3G genes. The levels of expression of the 9-5C gene were not altered during macrophage-monocytic or granulocytic differentiation of HL-60 cells. The expression of the 6-1E and/or 7-3G gene was induced by TPA in four of 6 samples derived from patients who achieved complete remission, but not in any of the acute nonlymphocytic leukemia samples from patients who failed to achieve complete remission. These findings suggest that expression of the 6-1E and 7-3G genes is related to macrophage-monocytic differentiation and that alterations of these gene expressions in fresh leukemia cells after one hour of TPA treatment are of prognostic significance in predicting the response to therapy.     

In vitro culture studies of granulocyte/macrophage and erythroid progenitor cells in lymphoproliferative disorders

Granulocyte/macrophage progenitor cells (CFU-GM) and erythroid progenitor cells (BFU-E) have been assayed in peripheral blood (PB) and/or bone marrow (BM) from 12 patients with acute lymphocytic leukemia (ALL), 16 patients with chronic lymphocytic leukemia (CLL) and 31 patients with various forms of non-Hodgkin lymphoma (NHL) without BM involvement. Progenitor cell growth in PB and BM from the NHL patients did not differ statistically from controls (p greater than 0.1). CFU-GM and BFU-E per ml PB were markedly increased in ALL and CLL patients (p less than 0.001) while CFU-GM and BFU-E per plated BM cells from these patients were severely depressed (p less than 0.001). Lymphoblasts from one ALL patient failed to inhibit CFU-GM and BFU-E-derived colony growth from control PB mononuclear cells. The high levels of circulating progenitor cells in ALL and CLL patients clearly distinguish them from other cytopenic hematological malignancies, in which decreased progenitor cell levels have been demonstrated previously (acute myeloid leukemia, hairy cell leukemia). The cause of this finding and its pathophysiological implication still remains to be established.     

缓解与未缓解髓系白血病干细胞表面抗原CD34＋CD123＋表达差异研究

目的：探索缓解与未缓解急性髓系白血病干细胞表面抗原表达差异,为判定化疗疗效及其预后提供依据。方法：按照急性白血病诊断标准,根据患者入院时骨髓白血病细胞数量多少分成临床缓解与未缓解两组,以流式细胞仪分别检测骨髓中白血病干细胞表面相关抗原表达情况,比较二者之间差异。其中经标准化疗方案治疗结束后,通过复查骨髓象判定疗效并比较化疗前后白血病干细胞表面相关抗原表达变化。结果：与缓解的急性髓系白血病患者骨髓白血病干细胞相关抗原表达值相比,未缓解的患者骨髓白血病干细胞表面相关抗原表达明显升高,差异具有统计学意义（P〈0．05,0．001）;未缓解的患者经标准方案化疗后骨髓虽然已经获得完全缓解,但依然具有白血病干细胞表面抗原高表达,提示这部分患者依然有复发的可能性。结论：急性髓系白血病患者的白血病干细胞相关抗原表达值升高是急性白血病复发难治的根源之一。     

Arsenic trioxide enhances radiation response of 9L glioma in the rat brain

Arsenic trioxide (ATO) at low doses induces leukemia cells to undergo apoptosis and at higher doses causes blood flow to solid tumors to shut down. To determine whether a potential synergistic interaction exists between ATO at the non-toxic dose level in the rat and radiation, the present study was carried out with orthotopic 9L malignant gliomas growing in the brains of rats. Animals died within 50 days of treatment when 12-day-old 9L gliomas growing in the brain of Fischer rats were treated with either the drug alone (8 mg/kg) or radiation alone (25 Gy). In contrast, the overall tumor cure rate exceeded 50% at a follow-up time of 120 days after the combined treatment with radiation and ATO. Long-term surviving animals showed no clinical or disproportionately enhanced histopathological changes in the brain parenchyma. Early changes in tumor physiology showed that the vascular leakage of FITC-dextran conjugates was apparent within 8 h of drug administration. Last, the use of diffusion magnetic resonance imaging as an early surrogate marker of therapeutic efficacy corroborated the effects of drug with and without radiation on brain histology and animal survival.     

Hematopoietic growth factors in patients receiving intensive chemotherapy for malignant disorders: studies of granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3) and Flt-3 ligand (Flt3L)

The levels of hematopoietic growth factors in patients receiving intensive chemotherapy for malignant disorders were investigated using a variety of approaches. Firstly, serum levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF and Flt3-ligand (Flt3L) were examined in acute leukemia patients with treatment-induced cytopenia and complicating bacterial infections. Increased serum levels of both G-CSF and Flt3-ligand (Flt3L) were detected when these patients developed therapy-induced leukopenia, whereas GM-CSF levels were low or undetectable. Development of complicating bacterial infections then increased the serum levels of both G- and GM-CSF, and the Flt3L levels remained high during the infections. Secondly, release of growth factors was characterized for clonogenic T cells that remained in the circulation of acute leukemia patients with chemotherapy-induced cytopenia. CD4(+) and CD8(+) T cells from these patients released high levels of GM-CSF, relatively low levels of IL-3 secretion having been detected, and only a minority of the clones released detectable amounts of Flt3L. Thus, circulating T cells may contribute to the high systemic growth factor levels in cytopenic patients. Thirdly, plasma levels of GM-CSF and interleukin-3 (IL-3) were examined in patients with malignant disorders who received chemotherapy plus G-CSF for stem cell mobilization. Increased levels of GM-CSF and Flt3L were detected both in the patients' plasma and in the stem cell grafts. Despite the increased growth factor levels in neutropenic patients with complicating infections, the occurrence of febrile neutropenia did not have a major impact on normal hematopoietic reconstitution (i.e. duration of treatment-induced neutropenia) after intensive chemotherapy for acute myelogenous leukemia.     

Purification of N-terminally truncated histone H2A-monoubiquitin conjugates from leukemic cell nuclei: probable proteolytic products of ubiquitinated H2A

To gain insight into the significance of nuclear ubiquitinated proteins, two serial extracts prepared from various leukemic cells were analysed by western blotting with anti-ubiquitin antibody. Two previously unidentified ubiquitinated proteins with molecular masses of 10 and 17 kDa were found in 8 M urea-soluble extracts, obtained from Tris-buffer-insoluble materials, of acute myeloid leukemia OCI/AML 1a cells and the cells from the leukemia patients. Both proteins were successfully purified from the OCI/AML 1a cells and identified as monoubiquitin-truncated H2A conjugates, the 10 kDa ubiquitinated H2A(115-129) and the 17 kDa ubiquitinated H2A(54-129), suggesting that both proteins were produced by limited proteolysis of an intact form (23 kDa) of ubiquitinated H2A(1-129). The 17 kDa protein as well as the 23 kDa ubiquitinated histone H2A were localised in chromatin fractions of the OCI/AML cells and released by high concentrations of salt in a micrococcal nuclease-sensitive manner, suggesting their association with chromatin. In contrast, the 10 kDa protein remained insoluble even when the nuclei were treated with nuclease under high salt concentrations, presumably due to binding to the nuclear matrix. An antibody recognising H2A(70-81) also detected the 17 kDa protein in anti-ubiquitin immunoprecipitates obtained from the OCI/AML cell nuclei. In addition, the 17 kDa protein levels in THP-1 cells were transiently increased, concomitant with a decrease in the 23 kDa ubiquitinated H2A, by treatment with phorbol 12-myristate 13-acetate or all-trans-retinoic acid, both of which induce differentiation. This is the first report of probable proteolytic products of ubiquitinated H2A, which might have a role in nuclear functions.     

Monoclonal antibodies to carbohydrate antigens in autologous bone marrow transplantation

Normal and malignant myeloid cells express a highly immunogenic oligosaccharide, lacto-n-fucopentaose-III (LNF-III), that has been identified by numerous monoclonal antibodies (MoAb). We have been interested in the use of a particular monoclonal antibody to LNF-III, PM-81, in the treatment of patients with acute myelogenous leukemia using the antibody to treat bone marrow in vitro. Following in vitro treatment of bone marrow with PM-81 and another MoAb, AML-2-23, the remaining cells are used as an autograft in a patient treated with high-dose chemotherapy and radiotherapy. In order to enhance the ability of the MoAb to lyse leukemic cells in the remission bone marrow, we have explored the effect of neuraminidase treatment on leukemia cells. In this paper we describe that myeloid leukemia cells expressing low levels of LNF-III by immunofluorescence can be shown to have high levels of LNF-III after neuraminidase treatment. In addition, we show that normal bone marrow progenitor cells do not have cryptic LNF-III antigen, thus allowing the application of this finding to the clinical setting. Moreover, we have shown that leukemia colony-forming cells from one patient with acute myelogenous leukemia express cryptic LNF-III and that after exposure to neuraminidase there was an increased ability of PM-81 in the presence of complement to eliminate these colony forming cells. These data indicate that the LNF-III moiety is almost universally expressed on myeloid leukemia cells and their progenitors but not expressed on normal progenitors. Thus, it may be possible to enhance leukemia cell kill in vitro by neuraminidase treatment of bone marrow.     

Acute myeloid leukemia incidence following radiation therapy for localized or locally advanced prostate adenocarcinoma

Introduction: The effect of radiation therapy on acute myeloid leukemia incidence among prostate cancer patients has not been sufficiently elucidated despite evidence that acute myeloid leukemia is a consequence of therapeutic radiation in other primary malignancies. Therefore, we investigated the effect of definitive therapy with radiation therapy (external beam radiation therapy [EBRT] or brachytherapy) on acute myeloid leukemia incidence in a population-based cohort of patients with localized or locally advanced prostate cancer. Methods: We utilized the Surveillance, Epidemiology, and End Results database to identify a cohort of men (n = 168,612) with newly diagnosed prostate adenocarcinoma between January 1988 and December 2003. Cox proportional hazard regression was used to estimate the hazard ratios (HRs) and corresponding 95% confidence intervals (CIs) of acute myeloid leukemia incidence following definitive therapy with EBRT alone, brachytherapy alone, or surgery alone compared to no definitive therapy (i.e. no EBRT, brachytherapy, or surgery). Results: The cohort yielded 184 acute myeloid leukemia cases during 1,064,820 person-years of follow-up after prostate adenocarcinoma diagnosis. Patients treated with EBRT had a higher adjusted relative risk of developing acute myeloid leukemia than patients treated with brachytherapy or surgery when each therapy group was compared to patients who were not treated with definitive therapy (EBRT: HR = 2.05, 95% CI 1.29, 3.26; brachytherapy: HR = 1.22, 95% CI 0.46, 3.22; surgery: HR = 1.24, 95% CI 0.77, 1.98). Conclusions: Our findings suggest that acute myeloid leukemia incidence is a greater concern for patients treated with EBRT than brachytherapy for localized or locally advanced prostate adenocarcinoma.     

Type-specific separation of animal cells in aqueous two-phase systems using antibody conjugates with temperature-sensitive polymers.

A new type of aqueous two-phase system (ATPS) has been developed in which a temperature-sensitive polymer, poly-N-isopropylacrylamide [poly (NIPAM)] was used as a ligand carrier for the specific separation of animal cells. Monoclonal antibodies were modified with itaconic anhydride and copolymerized with N-isopropylacrylamide, and the ligand-conjugated carriers were added to the polyethylene glycol 8000-dextran T500 aqueous two-phase systems. The antibody-polymer conjugates were partitioned to the top phase in the absence or presence of 0.15 M NaCl. When ligand-conjugated carriers were used, more than 80% of the cells were specifically partitioned to the top phase in the presence of NaCl up to 0.1 M. The cells were partitioned almost completely to the bottom phase at 0.1 M NaCl or above, when no antibody-conjugate was added in the ATPS. As a model system, CD34-positive human acute myeloid leukemia cells (KG-1) were specifically separated from human T lymphoma cells (Jurkat) by applying anti-CD34 conjugated with poly-N-isopropylacrylamide in the aqueous two-phase system. By the temperature-induced precipitation of the polymer, about 90% of the antibody-polymer conjugates were recovered from the top phase, which gave approximately 75% cell separating efficiency in the next cycle of reuse.     

Results of randomized trials of immunotherapy for acute leukemia

Summary For more than a decade clinical trials have attempted to define the role of immunotherapy in the treatment of patients with acute leukemia. Based on animal studies which indicated that non-specific immune stimulation had an antitumor effect if the tumor burden was small, the use of immunotherapy during remission in patients with acute leukemia seemed appropriate following the initial report of the success of bacillus Calmette-Guerin (BCG) in prolonging remission duration and survival in acute lymphoblastic leukemia. Therefore a series of randomized clinical trials was initiated to confirm these original observations. In four studies comparing BCG inoculations, with or without allogeneic leukemia cells, and chemotherapy or no therapy, no advantage of immunotherapy was noted. Immunotherapy appeared to be equally as good as chemotherapy. A combination of BCG and chemotherapy showed some advantage in one study, but no advantage was noted in two other studies.In acute myeloblastic leukemia several randomized trials suggested that BCG or one of its derivatives when given alone, in combination with allogeneic cells, or with chemotherapy had a marginal effect in prolonging remission duration and survival when compared to chemotherapy or rno therapy.In conclusion, immunotherapy during remission has marginal activity in acute leukemia.