Minor histocompatibility antigen UTY as target for graft-versus-leukemia and graft-versus-haematopoiesis in the canine model.

Male patients with female-stem-cell donors have better prognosis compared to female-to-male combinations due to Y-encoded minor histocompatibility antigens recognized by female-alloimmune-effector lymphocytes in the context of a graft-versus-leukemia (GvL) effect. We provide data in a dog-model that the minor histocompatibility antigen UTY might be a promising target to further improve GvL-immune reactions after allogeneic-stem-cell transplantations. Female-canine-UTY-specific T cells (CTLs) were stimulated in vitro using autologous-DCs loaded with three HLA-A2-restricted-UTY-derived peptides (3-fold-expansion), and specific T cell responses were determined in 3/6 female dogs. CTLs specifically recognized/lysed autologous-female-peptide-loaded DCs, but not naïve-autologous-female DCs and monocytes. They mainly recognized bone-marrow (BM) and to a lower extent DCs, monocytes, PBMCs and B-cells from DLA-identical-male littermates and peptide-loaded T2-cells in an MHC-I-restricted manner. A UTY-/male-specific reactivity was also obtained in vivo after stimulation of a female dog with DLA-identical-male PBMCs. In summary, we demonstrated natural UTY processing and presentation in dogs. We showed that female-dog CTLs were specifically stimulated by HLA-A2-restricted-UTY peptides, thereby enabling recognition of DLA-identical-male cells, mainly BM cells. These observations suggest UTY as a promising candidate-antigen to improve GvL-reactions in the course of immunotherapy.

The potential of gene transfer into primary B-CLL cells using recombinant virus vectors.

Despite recent advances, chronic lymphocytic leukemia (CLL) as the most common leukemia remains a largely incurable disease. Modern treatment options include novel drugs like purine analogues, monoclonal antibodies and transplantation strategies. Moreover, gene transfer of immunostimulatory molecules is another, but still experimental approach that can be used to potentiate immune responses against leukemic cells. CD40 ligand (CD40L) was shown to be a promising molecule for immunotherapy of B-CLL playing a critical role in immune activation. However, CLL B cells are resistant to transduction with most currently available vector systems. Improving the efficiency and specificity of gene vectors is critical for the success of gene therapy in this area. Using replication defective adenovirus encoding CD40L (Ad-CD40L), immunologic and clinical responses were seen in CLL patients after infusion of autologous Ad-CD40L-CLL cells in a recent phase I trial. Due to the immunogenic nature of adenovirus vectors, alternative vector systems are currently explored. Recombinant adeno-associated virus (rAAV) was shown to enable efficient transduction of primary B-CLL cells. By use of a library of AAV clones with randomly modified capsids, receptor-targeting mutants with a tropism for CLL cells can be selected. Furthermore, helper-virus free Epstein-Barr virus (EBV)-based gene transfer vectors hold promise for development of CLL-targeted vaccines after remaining safety issues will be resolved. Herpes simplex virus (HSV)-based vectors, especially HSV amplicons, have favorable features for B-CLL gene transfer including high transduction efficiency, ability to infect postmitotic cells and a large packaging capacity. The challenge for the future will be to transfer these alternative vector systems into clinic and allow the detection of a CLL-specific immune response by use of defined tumor antigens. This will make it possible to establish the potential clinical role of gene therapy for CLL patients.

Immunotherapy of recurrent B-cell malignancies after allo-SCT with Bi20 (FBTA05), a trifunctional anti-CD3 x anti-CD20 antibody and donor lymphocyte infusion.

Donor lymphocyte infusions (DLIs) after allo-SCT displayed limited use in CLL and highly malignant non-Hodgkin's lymphoma (NHL). Here we studied whether Bi20 (FBTA05), a novel trifunctional bispecific antibody targeting CD20 on lymphoma cells and CD3 on T cells, could induce GVL responses in combination with DLI or mobilized PBSCT after allogeneic transplantation in these diseases. Six patients (three cases with p53-mutated CLL and three with high-grade NHL (HG-NHL)) refractory to standard therapy were treated with escalating doses of Bi20 (range 10-2000 microg) followed by DLI or SCT. Thereby, all CLL patients showed a prompt but transient clinical and hematological response. In one patient with HG-NHL, we observed a halt in progression for almost 4 months. Side effects (fever, chills and bone pain) were tolerable and appeared at antibody dose levels between 40 and 200 microg. The cytokine profile was characterized by transient increases of IL-6, IL-8 and IL-10. Neither human anti-mouse antibodies nor GVHD developed, allowing repeated treatment courses. In summary, the trifunctional antibody Bi20 induced prompt antitumor responses in extensively pretreated, p53-mutated alemtuzumab and rituximab refractory patients indicating its therapeutic potential.

Engagement of the B-cell antigen receptor (BCR) allows efficient transduction of ZAP-70-positive primary B-CLL cells by recombinant adeno-associated virus (rAAV) vectors.

Engagement of the B-cell antigen receptor (BCR) by crosslinking of the surface immunoglobulin (sIg) homodimer was studied for recombinant adeno-associated virus (rAAV)-mediated gene transfer into B-cell chronic lymphocytic leukaemia (B-CLL) cells. Leukemic cells obtained from 20 patients were stimulated with anti-sIg-directed antibodies and transduced with rAAV vectors coding for enhanced green fluorescent protein (EGFP) (AAV/EGFP) or CD40L (AAV/CD40L). Transduction of B-CLL cells was enhanced after BCR engagement compared to unstimulated controls (P=0.0356). BCR crosslinking induced a significant, dose- and time-dependent upregulation of heparan sulfate proteoglycan (HSPG), the primary receptor for AAV, on B-CLL cells (mean: 38.2 versus 1.7%; P=0.0006). A correlation of HSPG expression after BCR crosslinking with transduction efficiency by AAV/EGFP (P=0.0153) and AAV/CD40L (P=0.0347) was observed. High expression of zeta-associated protein 70 (ZAP-70) in B-CLL cells correlated with a better transduction efficiency by AAV/EGFP (P<0.0001) and AAV/CD40L (P=0.002), respectively: 48 h after transduction of ZAP-70-positive samples, transgene expression was seen in a mean of 33.8% (s.e.m. 3.7%) and 28.9% (s.e.m. 6.7%) of cells, respectively, and could be specifically blocked by heparin, a soluble competitor of HSPG (P<0.0001). In summary, engagement of the BCR on ZAP-70 positive B-CLL cells allows efficient rAAV-mediated gene delivery.