Michael Hallek

Requirement of Src kinases Lyn, Hck and Fgr for BCR-ABL1-induced B-lymphoblastic leukemia but not chronic myeloid leukemia

The Abl kinase inhibitor imatinib mesylate is the preferred treatment for Philadelphia chromosome–positive (Ph+) chronic myeloid leukemia (CML) in chronic phase but is much less effective in CML blast crisis or Ph+ B-cell acute lymphoblastic leukemia (B-ALL). Here, we show that Bcr-Abl activated the Src kinases Lyn, Hck and Fgr in B-lymphoid cells. BCR-ABL1 retrovirus-transduced marrow from mice lacking all three Src kinases efficiently induced CML but not B-ALL in recipients. The kinase inhibitor CGP76030 impaired the proliferation of B-lymphoid cells expressing Bcr-Abl in vitro and prolonged survival of mice with B-ALL but not CML. The combination of CGP76030 and imatinib was superior to imatinib alone in this regard. The biochemical target of CGP76030 in leukemia cells was Src kinases, not Bcr-Abl. These results implicate Src family kinases as therapeutic targets in Ph+ B-ALL and suggest that simultaneous inhibition of Src and Bcr-Abl kinases may benefit individuals with Ph+ acute leukemia.

Genetic capsid modifications allow efficient re-targeting of adeno-associated virus type 2.

The human parvovirus adeno-associated virus type 2 (AAV2) has many features that make it attractive as a vector for gene therapy. However, the broad host range of AAV2 might represent a limitation for some applications in vivo, because recombinant AAV vector (rAAV)-mediated gene transfer would not be specific for the tissue of interest. This host range is determined by the binding of the AAV2 capsid to specific cellular receptors and/or co-receptors. The tropism of AAV2 might be changed by genetically introducing a ligand peptide into the viral capsid, thereby redirecting the binding of AAV2 to other cellular receptors. We generated six AAV2 capsid mutants by inserting a 14-amino-acid targeting peptide, L14, into six different putative loops of the AAV2 capsid protein identified by comparison with the known three-dimensional structure of canine parvovirus. All mutants were efficiently packaged. Three mutants expressed L14 on the capsid surface, and one efficiently infected wild-type AAV2-resistant cell lines that expressed the integrin receptor recognized by L14. The results demonstrate that the AAV2 capsid tolerates the insertion of a nonviral ligand sequence. This might open new perspectives for the design of targeted AAV2 vectors for human somatic gene therapy.

Consolidation with alemtuzumab in patients with chronic lymphocytic leukemia (CLL) in first remission--experience on safety and efficacy within a randomized multicenter phase III trial of the German CLL Study Group (GCLLSG).

Patients with CLL responding to initial chemotherapy with fludarabine alone (F) or in combination with cyclophosphamide (FC) were randomized for treatment with alemtuzumab (30 mg i.v. TIW, 12 weeks) or observation. Of 21 evaluable patients, 11 were randomized to alemtuzumab before the study was stopped due to severe infections in seven of 11 patients. These infections (one life-threatening pulmonary aspergillosis IV; four CMV reactivations III requiring i.v. ganciclovir; one pulmonary tuberculosis III; one herpes zoster III) were successfully treated and not associated with cumulative dose of alemtuzumab. In the observation arm, one herpes zoster infection II and one sinusitis I were documented. At 6 months after randomization, two patients in the alemtuzumab arm converted to CR, while three patients in the observation arm progressed. After alemtuzumab treatment, five of six patients achieved a molecular remission in peripheral blood while all patients in the observation arm remained MRD-positive (P=0.048). At 21.4 months median follow-up, patients receiving alemtuzumab showed a significant longer progression-free survival (no progression vs mean 24.7 months; P=0.036). In conclusion, a consolidation therapy with alemtuzumab is able to achieve molecular remissions and longer survival in CLL, but a safe treatment regimen needs to be determined.

The VP1 capsid protein of adeno-associated virus type 2 is carrying a phospholipase A2 domain required for virus infectivity.

The unique region of the VP1 protein of parvoviruses was proposed to contain a parvoviral phospholipase A2 (pvPLA2) motif. Here, PLA2 activity is shown in the unique region of adeno-associated virus type 2 (AAV-2) VP1 when expressed as an isolated domain in bacteria. Mutations in this region of the capsid protein strongly reduced the infectivity of mutant virions in comparison to wild-type AAV-2. This correlated with effects on the activity of PLA2. The mutations had no influence on capsid assembly, packaging of viral genomes into particles or binding to and entry into HeLa cells. However, a delayed onset and reduced amount of early gene expression, as measured by Rep immunofluorescence, was observed. These results suggest that pvPLA2 activity is required for a step following perinuclear accumulation of virions but prior to early gene expression.

Serum β2-Microglobulin and Serum Thymidine Kinase are Independent Predictors of Progression-Free Survival in Chronic Lymphocytic Leukemia and Immunocytoma

Chronic lymphocytic leukemia (CLL) and immunocytoma (IC) are remarkably heterogeneous with regard to their clinical course. The current staging systems can distinguish prognostic subgroups, but do not seem to predict the risk of disease progression of an individual patient with sufficient accuracy. Given the increase of treatment options for CLL and IC, additional parameters are needed to decide which patients may benefit from early or intensified treatment. It has been shown that two biochemical markers, serum beta 2-microglobulin (s-beta 2M) and serum thymidine kinase (s-TK), might identify CLL and IC patients at high risk of disease progression. Therefore, the prognostic value of these two serum parameters was compared with a panel of several established prognostic factors in a prospective clinical trial. 113 patients with CLL and 41 patients with IC (mean age +/- SD 63.9 +/- 10.7 years) were included. The following parameters were determined: histopathological diagnosis (IC vs. CLL), age, sex, performance status (Karnofsky index), B symptoms, peripheral blood lymphocyte count, platelet count, blood hemoglobin, serum lactate dehydrogenase (s-LDH), s-beta 2M, s-TK, serum creatinine, number of lymph node areas involved, prior therapy, and the time from diagnosis to inclusion in the study. Univariate analyses showed that nine parameters (Karnofsky index, peripheral blood lymphocytosis, platelet count, blood hemoglobin, lymph node areas involved, pretreatment, s-LDH, s-beta 2M, and s-TK) significantly predicted progression-free survival. In a Cox regression model, only four of these parameters provided independent prognostic information on progression-free survival: 1. s-beta 2M, 2. Karnofsky index, 3. platelet count, and 4. s-TK. The results show that s-beta 2M and s-TK independently predict progression-free survival in patients with CLL and IC, and suggest that these prognostic factors may allow an improved prediction of progression-free survival, particularly in early disease stages.

In Vitro Selection of Viral Vectors with Modified Tropism: The Adeno-associated Virus Display

Improving the efficiency and specificity of gene vectors is critical for the success of gene therapy. In an effort to generate viral mutants with controlled tropism we produced a library of adeno-associated virus (AAV) clones with randomly modified capsids and used it for the selection of receptor-targeting mutants. After several rounds of selection on different cell lines that were resistant to infection by wild-type (wt) AAV, infectious mutants were harvested at high titers. These mutants transduced target cells with an up to 100-fold increased efficiency, in a receptor-specific manner and without interacting with the primary receptor for wt AAV. The results demonstrate for the first time that a combinatorial approach based on a eukaryotic virus library allows one to generate efficient, receptor-specific targeting vectors with desired tropism.

Targeted Gene Delivery to Vascular Tissue In Vivo by Tropism-Modified Adeno-Associated Virus Vectors

Background—Gene therapy offers an unprecedented opportunity to treat diverse pathologies. Adeno-associated virus (AAV) is a promising gene delivery vector for cardiovascular disease. However, AAV transduces the liver after systemic administration, reducing its usefulness for therapies targeted at other sites. Because vascular endothelial cells (ECs) are in contact with the bloodstream and are heterogeneous between organs, they represent an ideal target for site-specific delivery of biological agents. Methods and Results—We isolated human venous EC-targeting peptides by phage display and genetically incorporated them into AAV capsids after amino acid 587. Peptide-modified AAVs transduced venous (but not arterial) ECs in vitro, whereas hepatocyte transduction was significantly lower than with native AAV. Intravenous infusion of engineered AAVs into mice produced reduced vector accumulation in liver measured 1 hour and 28 days after injection and delayed blood clearance rates compared with native AAV. Peptide-modified AAVs produced enhanced uptake of virions in the vena cava with selective transgene expression. Retargeting was dose dependent, and coinfusion of either heparin or free competing peptides indicated that uptake was principally independent of native AAV tropism and mediated via the peptide. Conclusions—AAV tropism can be genetically engineered by use of phage display–derived peptides to generate vectors that are selective for the vasculature.

Receptor targeting of adeno-associated virus vectors

Adeno-associated virus (AAV) is a promising vector for human somatic gene therapy. However, its broad host range is a disadvantage for in vivo gene therapy, because it does not allow the selective tissue- or organ-restricted transduction required to enhance the safety and efficiency of the gene transfer. Therefore, increasing efforts are being made to target AAV-2-based vectors to specific receptors. The studies summarized in this review show that it is possible to target AAV-2 to a specific cell. So far, the most promising approach is the genetic modification of the viral capsid. However, the currently available AAV-2 targeting vectors need to be improved with regard to the elimination of the wild-type AAV-2 tropism and the improvement of infectious titers. The creation of highly efficient AAV-2 targeting vectors will also require a better understanding of the transmembrane and intracellular processing of this virus.

Fludarabine plus cyclophosphamide is an efficient treatment for advanced chronic lymphocytic leukaemia (CLL): results of a phase II study of the German CLL Study Group.

The efficacy and toxicity of a combination of fludarabine and cyclophosphamide (FC) was evaluated in patients with B‐cell chronic lymphocytic leukaemia (CLL). Between April 1997 and July 1998, 36 patients with CLL (median age 59 years) received a regimen that consisted of fludarabine 30 mg/m2 in a 30‐min IV infusion, d 1–3, and cyclophosphamide 250 mg/m2 in a 30‐min IV infusion on d 1–3. Cycles were repeated every 28 d. Twenty‐one patients had received between one and three different treatment regimens prior to the study, while 15 patients had received no prior therapy. The median Eastern Cooperative Oncology Group performance score was 1. One patient was at Binet stage A, 18 were stage B and 17 patients were stage C. Objective responses, assessed according to the revised guidelines of the National Cancer Institute‐sponsored Working Group, were recorded in 29 out of 32 assessable patients (90·6%). Twenty‐four partial remissions and five complete remissions were observed. Two patients showed no change and one patient showed disease progression. At February 2000, three of the responders had relapsed. Severe neutropenia, anaemia and thrombocytopenia (Common Toxicity Criteria grade 3 and 4) were observed in 25, six and six patients (69·4%, 16·7% and 16·7%) respectively. Other side‐effects were uncommon. No treatment‐related deaths and no grade 3 or 4 infections occurred. We conclude that the combination of fludarabine and cyclophosphamide showed significant activity in patients with CLL. Myelosuppression was the major side‐effect. These results warrant further study on the FC combination in randomized trials.

The Src Family Kinase Hck Interacts with Bcr-Abl by a Kinase-independent Mechanism and Phosphorylates the Grb2-binding Site of Bcr

bcr-abl, the oncogene causing chronic myeloid leukemia, encodes a fusion protein with constitutively active tyrosine kinase and transforming capacity in hematopoietic cells. Various intracellular signaling intermediates become activated and/or associate by/with Bcr-Abl, including the Src family kinase Hck. To elucidate some of the structural requirements and functional consequences of the association of Bcr-Abl with Hck, their interaction was investigated in transiently transfected COS7 cells. Neither the complex formation of Hck kinase with Bcr-Abl nor the activation of Hck by Bcr-Abl was dependent on the Abl kinase activity. Both inactivating point mutations of Hck and dephosphorylation of Hck enhanced its complex formation with Bcr-Abl, indicating that their physical interaction was negatively regulated by Hck (auto)phosphorylation. Finally, experiments with a series of kinase negative Bcr-Abl mutants showed that Hck phosphorylated Bcr-Abl and induced the binding of Grb2 to Tyr177 of Bcr-Abl. Taken together, our results suggest that Bcr-Abl preferentially binds inactive forms of Hck by an Abl kinase-independent mechanism. This physical interaction stimulates the Hck tyrosine kinase, which may then phosphorylate the Grb2-binding site in Bcr-Abl.