Differential recognition of a BCR/ABL peptide by lymphocytes from normal donors and chronic myeloid leukemia patients.

The BCR/ABL oncogenic fusion protein transforms normal bone marrow stem cells into neoplastic cells. It has been shown that peptides derived from the junctional region of this oncogenic fusion protein can be recognized by human T-lymphocytes obtained from normal donors. In this study, we investigated the immunogenicity in patients with chronic myeloid leukemia (CML) of a 17 mer b3/a2 Bcr/abl peptide (B/A1), which was shown to induce proliferative responses in lymphocytes from normal donors. A total of 56 CML patients in chronic phase were studied. Twenty-two patients were studied at diagnosis without any treatment (group I). Fourteen patients were receiving IFN (group II), 14 patients were being treated with hydroxyurea (group III), and 6 patients were on different regimens (group IV). Patients were initially assessed for general immunological competence using both in vivo and in vitro assays. Patients were also selected for the expression of HLA-DR0401, the HLA specificity known to present peptide B/A1 to CD4 lymphocytes. With the exception of the six patients in group IV, the results of all these assays (in vitro phytohemagglutinin/tetanus toxoid responses, in vivo skin reaction to ubiquitous antigens) in CML patients did not significantly differ from those obtained in normal donors, thus excluding the presence of generalized immunosuppression. Eight patients with HLA-DR0401 and a b3/a2 type of fusion were identified and further studied. In these eight patients dendritic cells were obtained from adherent peripheral blood mononuclear cells and used to stimulate CD4 lymphocytes. No patient developed a specific response to the bcr/abl peptide, although patients' lymphocytes proliferated in response to a promiscuous tetanus toxoid peptide in all but one case. In contrast, response to the bcr/abl peptide was observed in seven of eight HLA-DR0401 healthy donors tested. These data suggest that immunocompetent, HLA-DR0401+ CML patients are unable to respond to peptide B/A1, at difference from healthy donors. The implication of these results for the immunotherapy of CML is discussed.

Mapping of HLA class I binding motifs in forty-four fusion proteins involved in human cancers.

Chromosomal translocations coding for abnormal proteins are present in several human cancers. The junctional region of fusion proteins represents a potential target for a T cell-mediated antitumor response. T lymphocytes recognize antigens in the form of short peptides that must bind to HLA molecules. Different HLA specificities can bind different peptides, thus depicting different "peptide binding motifs." It would be useful to know whether a certain fusion protein presents, within its fusion region, the binding motif(s) for a certain HLA molecule. This information would allow a more focused immunological analysis. Here we present data obtained from the screening of the fusion regions of 44 different fusion proteins for the presence of binding motifs to 34 class I HLA molecules, including all of the most frequently encountered specificities. A total of 201 independent peptides was identified (range, 0-11 peptides/fusion protein). A marked heterogeneity among the 44 different fusion proteins analyzed is evident. For example, the pml/RARalpha fusion protein present in acute promyelocytic leukemia presents no binding motif (BCR 3) at all or to a single HLA molecule (Cw*0301, BCR 1). Alternatively, the fusion proteins BCR/ABL, ALL1/AF-6, EWS/ATF-1, or NPM/ALK exhibit motifs for several common HLA specificities. Heterogeneity is also present inside a single translocation (in ALL1/ENL, for example, different subtypes match motifs with cumulative frequencies in the population from 108 to 0%). In two cases where the relative frequency of different fusion protein subtypes was available, a tendency toward an inverse relationship between frequency and the percentage of population covered by the identified binding motifs was observed. Peptides with motifs for HLA A*0201, A3, and Cw*0702 were also tested for actual binding using a stabilization assay; 13-40% showed significant HLA binding, using this assay. However, fewer fusion protein-derived peptides bound to HLA A*0201 and A3 than non-fusion protein-derived peptides. These data provide the first list of peptides derived from fusion proteins that may be assessed as potential tumor-specific antigens.

HLA binding characteristics and generation of cytotoxic lymphocytes against peptides derived from oncogenic proteins.

AIMS AND BACKGROUND: Structurally altered proteins (derived from chromosomal translocations or gene mutations) can be considered tumor specific antigens and represent an attractive target for a T-cell mediated response. T lymphocytes recognize antigens in the form of peptides bound to HLA-molecules. MATERIALS AND METHODS: Peptides derived from oncogenic proteins were screened for the presence of HLA binding motifs; actual binding were evaluated by HLA stabilization experiments using transfectants and specific anti-HLA antibodies. Specific lymphocytes were induced by in vitro peptide sensitization and screened by thymidine uptake or cellular cytotoxic assays. RESULTS: We identified peptides derived from EWS/FLI-1 fusion protein and from mutated K-RAS protein (encompassing respectively the fusion point and the mutation at position 12) that showed binding motif for HLA-Cw*0702 and HLA-A3 respectively. The actual binding of these peptides was analysed in a stabilization assay. We detected binding for the EWS/FLI-1 peptide and for 5 RAS peptides (1 wild type and 4 mutated). The effect of temperature, beta 2-microglobulin (beta 2-m) and fetal calf serum (FCS) on the binding and the stability of the HLA/peptide complex was studied. A low temperature (26 degrees C) increased the binding both in HLA-A3 and HLA-Cw*0702, while FCS reduced it. beta 2-m increased the binding to the HLA-A3 molecule but did not influence the binding to the HLA-Cw*0702. The stability of already formed complexed was somewhat different in the HLA-A3 and HLA-Cw*0702 system: both were more stable at 26 degrees C than at 37 degrees C but while the beta 2-m and FCS did not influence the stability of the HLA-A3/peptide complex, they seemed to cause opposite effects in the HLA-Cw*0702 system (beta 2-m stabilized and FCS destabilized the complex). Finally, we were able to generate a specific CD8+ CTL line against a K-RAS mutated peptide. CONCLUSIONS: Although binding motifs and actual HLA binding can be detected in several cases, the generation of a cellular response is infrequent, confirming that HLA binding is necessary but not sufficient to obtain an in vitro response. Further optimization of culture conditions, type of Antigen Presenting Cells (APC), peptides, use of stabilizers like beta 2-m are still needed.

Inhibition of the ABL kinase activity blocks the proliferation of BCR/ABL+ leukemic cells and induces apoptosis.

The BCR/ABL fusion protein transforms myeloid stem cells. Both chronic myelogenous leukemias (CML) and a subset of acute lymphoblastic leukemias (ALL) are associated with the expression of BCR/ABL proteins. This knowledge has not yet been translated into any specific tool to control ABL driven neoplastic cells growth. CGP57148B is an ATP-competitive inhibitor of the ABL protein kinase; it has been shown to inhibit the kinase activity of ABL both in vitro and in vivo and to inhibit the growth of v-abl and bcr/abl transfectants, as well as the in vitro formation of bone marrow (BM)-derived colonies in the presence of growth factors in some CML patients. These studies were performed to investigate the activity of CGP57148B on the spontaneous proliferation of both fresh and cultured, leukemic and normal, BCR/ABL positive and negative cells, and to study its mechanism of action. Six cell lines derived from BCR/ABL+ leukemias (K562, BV173, KCL22, KU812, MC3, LAMA84), thirteen BCR/ABL negative lines, both neoplastic (KG1, SU-DHL-1, U937, Daudi, NB4, NB4.306) and derived from normal cells (PHA blasts, LAK, fibroblasts, LCL, renal epithelial cells, endothelial cells, CD34(+) cells), and 14 fresh leukemic samples were tested using a tritiated thymidine uptake assay. The in vivo phosphorylation of the BCR/ABL protein was evaluated by western blot, while apoptosis was detected by the annexin V/propidium binding test. The induction of differentiation was assayed by immunofluorescence using multiple antibodies. All six BCR/ABL+ lines showed a dose dependent inhibition of their spontaneous proliferative rate, which was not accompanied by differentiation. The treatment caused, within minutes, dephosphorylation of the BCR/ABL protein, followed in 16-24 hours by a decrease in cycling cells and induction of apoptosis. No significant inhibition of DNA synthesis was observed in any BCR/ABL negative normal or neoplastic line at concentrations

In vitro transcription and translation inhibition by anti-promyelocytic leukemia (PML)/retinoic acid receptor alpha and anti-PML peptide nucleic acid.

Peptide nucleic acids (PNAs) complementary to the 15 bases around the fusion point of both genomic DNA and cDNA of the promyelocytic leukemia/retinoic acid receptor alpha (PML/ RAR alpha; P/R) hybrid gene present in acute promyelocytic leukemia cells were synthesized and shown by gel retardation experiments to specifically bind oligonucleotides corresponding to the fusion region of the P/R molecule. PNA was also able to successfully compete with anti-P/R DNA for duplex formation with P/R DNA and to displace the anti-P/R DNA from dsDNA. In vitro transcribed P/R RNA from two inserts of approximately 350 to approximately 700 bp were tested in gel acceleration experiments with fluorescein-conjugated PNA and showed stable binding (resistant to denaturing conditions) of PNA to the newly transcribed RNA. Control RNA or transcripts from the noncoding strand did not bind PNA. However, this PNA, although able to specifically clamp polymerase chain reaction, was incapable of inhibiting in vitro translation of the PML/RAR alpha mRNA, even when a bis-PNA was used. Therefore, a PNA was targeted against the start region of the P/R cDNA and against poly-purine regions of the gene. Specific inhibition of in vitro translation and transcription was shown, starting at concentrations as low as 100 nmol/L. When oligonucleotides presenting the same sequence were compared, PNA proved to be approximately 40 times more active. In conclusion, in vitro inhibition of translation and transcription of the P/R gene can be obtained with PNA; however, it is still necessary to target the ATG start region or poly-purine regions of the gene.

Lack of T-cell-mediated recognition of the fusion region of the pml/RAR-alpha hybrid protein by lymphocytes of acute promyelocytic leukemia patients.

In previous studies, it was shown that the fusion region of the pml/RAR-alpha protein, expressed by acute promyelocytic leukemia (APL) cells, can be specifically recognized in vitro by donor (D. E. ) CD4 T cells in a HLA class II DR11-restricted fashion. We present here the results on the recognition of several pml/RAR-alpha peptides by APL patients expressing HLA DR11. The in vitro immunization of peripheral blood lymphocytes from four patients in remission (S. R., F. R., M. M., P. G.) with BCR1/25, a 25-mer pml/RAR-alpha, did not elicit either a polyclonal or a clonal immune response specific to the peptide. We then generated new donor anti-pml/RAR-alpha CD4(+) T-cell clones. These clones were tested for their recognition of BCR1/25. One clone (C3/5, CD3(+), CD4(+), CD8(-)) was selected for further analysis. Clone C3/5 showed specific proliferation, cytotoxicity, and cytokine (tumor necrosis factor alpha, granulocyte-macrophage colony-stimulating factor) production when challenged with autologous lymphoblastic cell lines pulsed with peptide BCR1/25. C3/5 cells developed specific proliferation and cytotoxicity when challenged with peptide-pulsed lymphoblastic cell lines and peripheral blood lymphocytes from the four DR11(+) APL patients. APL blasts, available only from patients F. R. and P. G., were not lysed by C3/5 and were unable to present peptide BCR1/25. Incubation of APL cells with IFN-gamma failed to induce HLA class II molecules and recognition by the C3/5 clone. Since APL cells do not express HLA class II molecules, we tested in two donors (D. E. and C. H. R.) and in patients S. R. and P. G. whether the use of 9-mer peptides (BCR1/9) would generate a CD8/HLA class I-restricted response. No peptide-specific T-cell line or clone could be generated from both donors and patients. These findings are discussed in relation to possible therapeutic approaches to the immunotherapy of APL.

Experimental systemic toxicology of 4'-epidoxorubicin, a new, less cardiotoxic anthracycline antitumor agent.

The acute and chronic iv toxicity of 4'-epidoxorubicin, a new antitumor anthracycline antibiotic, was compared with doxorubicin. The LD50 of 4'-epidoxorubicin was 16.07 mg/kg in mice, 14.27 mg/kg in rats, and about 2 mg/kg in dogs; the LD50 of doxorubicin was 11.98 mg/kg in mice, 10.51 mg/kg in rats, and about 2.5 mg/kg in dogs. Rats and dogs were also dosed iv for 91 days (3 injections/week) with 4'-epidoxorubicin or doxorubicin at doses of 0.128, 0.32, and 0.8 mg/kg to rats and 0.064, 0.16, and 0.4 mg/kg to dogs. A comprehensive toxicological evaluation of the animals was carried out before, throughout, and at the end of the study. High-dose 4'-epidoxorubicin induced toxic clinical signs in dogs, and in both species caused loss of body weight, antiproliferative effects on blood-forming organs and testes, and degenerative lesions in kidneys and heart. The cardiac damage was moderate in rats and very mild in dogs; only three male rats died at this dose. The medium dose induced less pronounced changes and no heart lesions and the low dose was practically nontoxic. Doxorubicin showed similar antiproliferative activity, but more evident toxic effects, especially on the heart; many rats given the high dose died and some at the medium dose showed initial cardiac lesions. Thus 4'-epidoxorubicin appeared less toxic than doxorubicin; in particular cardiac damage was much less evident in animals chronically injected with the new drug.

Cardiomyopathy of doxorubicin in experimental animals, Factors affecting the severity, distribution and evolution of myocardial lesions.

Heart lesions induced in mice, rats, rabbits and dogs by Doxorubicin administered i.v. according to various schedules were studied by light and electron microscopy Vacuolization of myocardial cytoplasm due to distention of the sarcoplasmic reticulum, the T-tubule system and the Golgi vesicles was one of the most common findings. Myocytolysis, clumping and loss of fibrils, fragmentation of sarcomeres, swelling of mitochondria and an increase in lysosomes and residual bodies were also observed. The severity of the cardiomyopathy, quantitatively evaluated by a score system, proved to be dose-dependent. Cardiomyopathy was more severe when the treatment was given in a short period by administration of high doses than when the same cumulative dose was administered as low doses repeated for a long period. The left atrium was more severely affected than the ventricles when high doses were given, whereas it was less affected in animals given low doses. The cardiomyopathy was less severe in animals receiving the same dose in a high volume of solvent and during a long perfusion time. Threshold doses were needed both to induce the cardiomyopathy and to establish it as a progressive disease.

Quantitative experimental evaluation of adriamycin cardiotoxicity in the mouse.

Adriamycin cardiotoxicity was morphologically evaluated in mice treated iv ten times at three dose levels which were equal to fixed fractions of the LD50. Good correlation between the degenerative heart lesions, scored according to a nonparametric scoring system, and the doses was found and allowed calculation of a median cumulative cardiotoxic dose of 36.4 mg/kg of body weight (121.5 mg/m2). The method is suitable for quantitative screening of the potential cardiotoxicity of new anthracycline analogs under standardized conditions.

Mammary tumors induced in rats by adriamycin and daunomycin.

The two anthracycline antitumor antibiotics, Adriamycin and daunomycin (DM), induced a high incidence of mammary tumors, both fibroadenomas and adenocarcinomas, in female rats that received a single i.v. dose, thus confirming previous results. The incidence of DM-induced adenocarcinomas increased with the dose of the drug, whereas the incidence of Adriamycin-induced adenocarcinomas showed a plateau at 5 mg/kg and above. Adriamycin- and DM-induced fibroadenomas showed a peak at lower doses (about 5 to 6 mg/kg). With the highest DM dose (12.5 mg/kg) used, there was a slight prevalence of adenocarcinomas over fibroadenomas.

Effect of adriamycin on the activity of the succinate dehydrogenase-coenzyme Q10 reductase of the rabbit myocardium.

Rabbits were injected with adriamycin 3 days/week/7 weeks. Heart tissues of treated rabbits showed myocardial degeneration, but the tissues of controls were normal. The deficiency in the activity of the succinate dehydrogenase-coenzyme Q10 reductase in the heart tissues of the rabbits treated with adriamycin was higher (0.001 less than P less than 0.01) than that for control tissues. These data indicate a correlation of at least a part of the cardiotoxicity of adriamycin in cancer patients to inhibition of CoQ10-enzymes, since coenzyme Q10 is indispensable to bioenergetics in the myocardium of both humans and rabbit.