We recently demonstrated that early post-transplant vaccination of patients with myeloid leukemia with irradiated autologous tumor cells, virally transduced to express the potent cytokine adjuvant GM-CSF, is a promising approach to enhance anti-leukemia immunity with minimal toxicity (Ho et al, PNAS 2009). Chronic lymphocytic leukemia (CLL) is an ideal disease to extend this treatment approach since it is incurable by conventional chemotherapy, is sensitive to graft-versus-leukemia effects, and has readily harvestable leukemia cells. However, viral transduction of lymphoid cells is inefficient, and GM-CSF-based cellular vaccines have thus far been unavailable for CLL. To address this limitation, we manufactured K562 cells engineered to secrete high and consistent levels of GM-CSF (10 micrograms GM-CSF/106 cells/24 hours), and initiated a phase I study to test the effects of a post-transplant vaccine consisting of 1 x 107 irradiated autologous CLL cells together with an equal number of irradiated bystander GM-K562 cells. CD19+CD5+CLL cells were harvested from patient peripheral blood or marrow and banked prior to salvage chemotherapy. To date, we have enrolled 13 patients with CLL, who had experienced a median of 4 prior therapies (range 2-8). Median CLL marrow involvement at transplant was 10% (range <5-50%). Patients were conditioned with a fludarabine/busulfex-based reduced-intensity regimen prior to infusion of peripheral blood stem cells from a matched related (4) or unrelated (9) donor, and received tacrolimus as GvHD prophylaxis throughout the vaccination period. Starting on day 30-45, vaccines were administered as a cycle of 6 vaccines (s.c./i.d., every week x 3, then every 2 weeks x 3). Ten of 13 patients received at least one vaccine, and 7 of 10 received 3 or more vaccines. Three of 13 developed graft-versus-host disease (GvHD) prior to day 45, precluding vaccination. Four of the 10 vaccinated subjects developed grade II GvHD. None experienced unexpected toxicities to vaccines. At a median follow-up of 1 year (range 0.5-2 years), 9 of 10 vaccinated subjects remain free of disease, and 1 patient remains in partial remission. Our studies reveal that reduced intensity transplant followed by early CLL/GM-K562 vaccination is well-tolerated and does not increase the expected rate of GvHD. Early clinical results are promising and ongoing studies are assessing the effects of the vaccination on the induction of immunity against recipient CLL cells.
<div>Abstract<p>Patients with chronic lymphocytic leukemia (CLL) who relapse after allogeneic transplant may achieve durable remission following donor lymphocyte infusion (DLI), showing the potency of donor-derived immunity in eradicating tumors. We sought to elucidate the antigenic basis of the effective graft-versus-leukemia (GvL) responses associated with DLI for the treatment of CLL by analyzing the specificity of plasma antibody responses developing in two DLI-treated patients who achieved long-term remission without graft-versus-host disease. By probing high-density protein microarrays with patient plasma, we discovered 35 predominantly intracellular antigens that elicited high-titer antibody reactivity greater in post-DLI than in pre-DLI plasma. Three antigens—C6orf130, MDS032, and ZFYVE19—were identified by both patients. Along with additional candidate antigens DAPK3, SERBP1, and OGFOD1, these proteins showed higher transcript and protein expression in B cells and CLL cells compared with normal peripheral blood mononuclear cells. DAPK3 and the shared antigens do not represent minor histocompatibility antigens, as their sequences are identical in both donor and tumor. Although ZFYVE19, DAPK3, and OGFOD1 elicited minimal antibody reactivity in 12 normal subjects and 12 chemotherapy-treated CLL patients, 5 of 12 CLL patients with clinical GvL responses were serologically reactive to these antigens. Moreover, antibody reactivity against these antigens was temporally correlated with clinical disease regression. These B-cell antigens represent promising biomarkers of effective anti-CLL immunity. Cancer Res; 70(4); 1344–55</p></div>
Summary Recent advances regarding the introduction of anti‐adhesion strategies as a novel therapeutic concept in oncology hold great promise. Here we evaluated the therapeutic potential of the new‐in‐class‐molecule selective‐adhesion‐molecule (SAM) inhibitor Natalizumab, a recombinant humanized IgG4 monoclonal antibody, which binds integrin‐α4, in multiple myeloma (MM). Natalizumab, but not a control antibody, inhibited adhesion of MM cells to non‐cellular and cellular components of the microenvironment as well as disrupted the binding of already adherent MM cells. Consequently, Natalizumab blocked both the proliferative effect of MM‐bone marrow (BM) stromal cell interaction on tumour cells, and vascular endothelial growth factor (VEGF)‐induced angiogenesis in the BM milieu. Moreover, Natalizumab also blocked VEGF‐ and insulin‐like growth factor 1 (IGF‐1)‐induced signalling sequelae triggering MM cell migration. In agreement with our in vitro results, Natalizumab inhibited tumour growth, VEGF secretion, and angiogenesis in a human severe combined immunodeficiency murine model of human MM in the human BM microenvironment. Importantly, Natalizumab not only blocked tumour cell adhesion, but also chemosensitized MM cells to bortezomib, in an in vitro therapeutically representative human MM‐stroma cell co‐culture system model. Our data therefore provide the rationale for the clinical evaluation of Natalizumab, preferably in combination with novel agents (e.g. bortezomib) to enhance MM cytotoxicity and improve patient outcome.
BCR-ABL+ K562 cells hold clinical promise as a component of cancer vaccines, either as bystander cells genetically modified to express immunostimulatory molecules, or as a source of leukemia antigens. To develop a method for detecting T-cell reactivity against K562 cell-derived antigens in patients, we exploited the dendritic cell (DC)-mediated cross-presentation of proteins generated from apoptotic cells. We used UVB irradiation to consistently induce apoptosis of K562 cells, which were then fed to autologous DCs. These DCs were used to both stimulate and detect antigen-specific CD8+ T-cell reactivity. As proof-of-concept, we used cross-presented apoptotic influenza matrix protein-expressing K562 cells to elicit reactivity from matrix protein-reactive T cells. Likewise, we used this assay to detect increased anti-CML antigen T-cell reactivity in CML patients that attained long-lasting clinical remissions following immunotherapy (donor lymphocyte infusion), as well as in 2 of 3 CML patients vaccinated with lethally irradiated K562 cells that were modified to secrete high levels of granulocyte macrophage colony-stimulating factor (GM-CSF). This methodology can be readily adapted to examine the effects of other whole tumor cell-based vaccines, a scenario in which the precise tumor antigens that stimulate immune responses are unknown.
