We have reported that immunotherapy using leukemia cell-derived heat shock proteins (HSPs) is effective against minimal residual disease (MRD) after syngeneic stem cell transplantation (SCT) in mice. However, leukemia patients after SCT are usually immunocompromised and immunologically tolerant to leukemia cells. We investigated whether the use of dendritic cells (DCs) in combination with HSP70 enhances cytotoxicity against B-cell leukemia cell line A20 in mice after syngeneic SCT. All unimmunized mice died of leukemia early after A20 cell inoculation, whereas mice immunized with HSP70 or HSP70-pulsed DCs survived significantly longer. Although only 60% of the HSP70-immunized mice survived, all mice immunized with HSP70-pulsed DCs survived without MRD. In addition, the cytotoxicities against A20 cells for splenocytes from mice immunized with HSP70-pulsed DCs were significantly higher than those of HSP70-immunized mice, and the cytotoxicities against A20 cells were significantly blocked by anti-CD8 antibody and by major histocompatibility complex class I antibody, but not by anti-CD4 antibody. Moreover, abnormalities were detected in neither the biochemical data nor the histopathologic findings. These findings indicate that the combined use of DCs and leukemia cell-derived HSP70 enhances the antileukemia effect by inducing the specific cytotoxicities of CD8+ cytotoxic T-cells, thereby eradicating MRD effectively and safely, even in an immunocompromised state after syngeneic SCT. This approach may thus be useful for further application of HSP in leukemia patients after autologous SCT.

Human peripheral blood mononuclear cells (PB-MNCs) have angiogenic properties, which make them promising cells for use in angiogenic therapy approaches in regenerative medicine. To explore an efficient method for expanding pro-angiogenic cells from PB-MNCs, we developed a novel serum-free culture system composed of X-VIVO15 medium supplemented with vascular endothelial growth factor, basic fibroblast growth factor, and thrombopoietin (TPO). Using this ex vivo culture, we obtained floating spheres composed mainly of CD11b(+) monocytes expressing c-Mpl (TPO receptor) and which exhibited acetylated low-density lipoprotein uptake and phagocytosis. Expression of IL-8, CXCR4, and vasohibin-2 mRNA was upregulated in these cells. In the presence of TPO, the number and size of the spheres were increased. In a nude mouse hind-limb ischemia model, the intramuscular injection of spheroid cells treated with TPO rescued blood perfusion more effectively than that without TPO. These results indicate that the ex vivo addition of TPO augments the pro-angiogenic activity of peripheral CD11b(+) monocytes, suggesting that this method shows promise for uses in human cell therapy aimed at the induction of vascular regeneration by activating the angiogenic properties of human peripheral blood-derived monocytes.

Survival, proliferation, and resistance to chemotherapy in CLL cells have been consistently shown to be associated with the activity of the B-cell receptor (BCR) and the associated downstream pathways activated by it. Key molecules in this pathway are Lyn and Syk (Spleen tyrosine kinase), as well as PI3K, Btk (Bruton’s tyrosine kinase), and ERK. Dasatinib, given at standard doses, allows for serum levels well above 11 nM, the IC50 for the direct suppression of Lyn kinase and Btk. We have previously shown that dasatinib used as a single agent in patients with relapsed CLL results in lymph node responses in 60% of patients and partial responses in 20% of patients as defined by NCI-WG criteria. In the current study, patients with relapsed CLL were treated with a regimen combining dasatinib at 140 mg/day, days 1-14, with fludarabine (F) 25 mg/m2/day, days 1-3, and rituximab (R) 375 mg/m2 per cycle, repeated every 28 days, for up to 6 cycles. Patients were followed closely for response with CT scans every 2 months initially. Among the first 10 patients treated, the schedule of treatment was altered to determine signal transduction effects and resultant apoptosis of the CLL cells due to the different components of the regimen. Hence, only dasatinib was given on Day 1, only fludarabine and rituximab on Day 3, and all 3 drugs on Day 4. Blood samples were obtained from the patients before dosing and at 6 hours after treatment to measure effects on the CLL cells, which were isolated by standard Ficoll separation and frozen until the assays could be performed. For these 10 patients the median time to progression (TTP) was 21 months, and the initial clinical response was as follows: Site CR CRi PR SD PD ORR 95% CI Blood 4 2 4 0 0 100% 74%, 100% Nodes 6 1 1 1 1 80% 49%, 96% CT 2 0 2 6 0 40% 15%, 70% IWCLL 2 0 2 5 1 40% 15%, 70% Key: CR=complete response in blood = < 4,000/ul lymphocytes, CR in nodes = no nodes palpable by PE, CRi = complete response with incomplete blood recovery, PR=partial response, SD=stable disease, PD=progressive disease, ORR=overall response rate, CI=confidence interval. IWCLL = International Workshop on CLL criteria. The patterns of signal transduction in response to the various drugs at 6 hours are shown in aggregate for the patients below. The numbers represent the ratio of phosphorylated protein to total protein at the times indicated with respect to their baseline levels set as 100%. The phosphorylation sites tested were p-Lyn (Y416), p-Syk (Y352), p-ERK1/2 (T202/Y204). Phosphorylation at 6 h after indicated treatment – % of baseline ± SE (N=7): Day 1 (D) Day 3 (F+R, no D) Day 4 (D+F+R) p-Lyn/Lyn: 42% ± 3% 207% ± 63% 58% ± 13% p-Syk/Syk: 34% ± 15% 122% ± 67% 36% ± 15% p-ERK/ERK : 64% ± 22% 168% ± 40% 56% ± 22% The patterns of signal transduction for the 3 patients with the most favorable outcome (TTP and OS) were compared to that for the 4 patients with the poorest clinical outcome. The baseline ratios of phospho-ERK1/2 to ERK1/2 (pre-treatment) correlated most strongly with outcome. Those with a good outcome exhibited low basal p-ERK/ERK (mean 1.0, range 0.1 to 1.8 percent), while patients with a poor outcome exhibited high basal p-ERK/ERK (mean 22.1, range 2.2 to 65.6 percent). Conclusions: For most patients, dasatinib inhibits (directly or indirectly) phosphorylation of Lyn kinase, Syk kinase, and ERK1/2 in the first 6 hours. The degree of apoptosis (to be presented, but not shown here) resulting from this is variable and is probably affected by activation of the PI3K/Akt pathway and other pathways. The long-term clinical outcome of our patients correlated strongly with the baseline phosphorylation of ERK1/2, suggesting that future treatments of patients with CLL might benefit from targeting ERK directly, or by targeting other molecules in the MAPK pathway. Disclosures Off Label Use: Dasatinib for treatment of CLL is off-label use. We present a rationale for its use in CLL patients.. Brown:Sanofi, Onyx, Vertex, Novartis, Boehringer, GSK, Roche/Genentech, Emergent, Morphosys, Celgene, Janssen, Pharmacyclics, Gilead: Consultancy. Attar:Agios: Employment. Fathi:Seattle Genetics, Inc.: Consultancy, Research Funding; Takeda pharmaceuticals International Co.: Research Funding; Exelixis: Research Funding; Ariad: Consultancy.

Human peripheral blood mononuclear cells (PB-MNCs) include some populations which have angiogenic properties. Pro-angiogenic monocytes from PB-MNCs are considered as one of candidates for angiogenic therapy in regenerative medicine. Indeed, in a recent German clinical post-infarction remodeling study (TOPCARE-AMI) for ischemic heart disease, the ex vivo culture of PB-MNCs was employed. However, in this trial, there were different therapeutic efficacies in each case, possibly due to the different expansion efficacy of the ex vivo culture of PB-MNCs using autologous serum. In order to resolve this issue, we developed a new serum-free culture system composed of X-VIVO15 medium supplemented with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Floating spheres obtained by this serum-free culture system were mainly composed of CD11b+ monocytes. Interestingly, the mRNA expression of c-Mpl (thrombopoietin receptor) was markedly elevated compared with PB-MNCs, suggesting c-Mpl agonist could increase angiogenic property of cultured CD11b+ monocytes. Therefore, we assessed the impact of c-Mpl agonists on PB-MNC cultures in our serum-free method composed of X-VIVO15 medium with VEGF and bFGF. Both recombinant human thrombopoietin (rHuTPO) and romiplostim, a clinical grade second-generation TPO-receptor agonist, successfully increased sphere formations regarding both the number and size. The expressions of angiogenic factors, IL-8, CXCR4, and vasohibin-2, mRNA of CD11b+ monocytes cultured with c-Mpl agonists were up-regulated, indicating that cultivated CD11b+ monocytes have a proangiogenic potential. Finally, we investigated the proangiogenic potential of PB-MNCs derived CD11b+ monocytes in a hindlimb ischemia model utilizing BALB/c nude mice. Mice were randomly assigned to 7 groups: control mouse group (PBS-injected), freshly isolated CD11b+ monocyte-injected mouse group, cultivated CD11b+ monocyte with 2ng/ml and 20ng/ml rHuTPO -injected mouse groups, cultivated CD11b+ monocyte with 100ng/ml and 1000ng/ml romiplostim -injected mouse groups, cultivated CD11b+ monocyte without rHuTPO and romiplostim -injected mouse group. The intramuscular injection of CD11b+ monocytes cultivated with 20 ng/ml rHuTPO into the ischemic limb completely rescued the limbs from auto-amputations or foot necrosis, while only one (10.0%) of the control mice could be rescued. In addition, the intramuscular injection of both freshly isolated CD11b+ monocytes and CD11b+ monocytes cultivated without rHuTPO and romiplostim had a weak rescue effect on the ischemic limbs (8 and 7 of 10 mice had auto-amputations or foot necrosis, respectively). The salvage rate from necrosis in cultivated CD11b+ monocyte with romiplostim-injected mouse group is also superior to that in cultivated CD11b+ monocyte without rHuTPO-injected mouse group. Analysis of blood perfusion by a laser Doppler perfusion imaging system showed a significantly higher recovery in mice receiving the CD11b+ monocytes cultivated with 2 ng/ml or 20 ng/ml rHuTPO or 100ng/ml romiplostim 1 week after surgery. The functional capillary density and surface area visualized by perfusion with BS-I lectin also significantly increased in the rHuTPO- or romiplostim-treated group. In conclusion, an ex vivo addition of c-Mpl agonists augmented the pro-angiogenic activity of peripheral CD11b+ monocytes, and this method would be promising for human cell therapy to induce vascular regeneration by activating the angiogenic property in human peripheral blood-derived monocytes. Disclosures: Mizukami: The New Energy and Industrial Technology Development Organization of Japan: Research Funding.

Background: Heat shock proteins (HSPs) are molecular chaperones binding a broad repertorie of endogenous antigenic peptides and carrying them to the MHC. Because the identification of each tumor specific antigen is not necessary, the immunotherapy using HSPs is more practical than other immunological procedures. Meanwhile, relapse due to minimal residual disease (MRD) is a big problem of autologous stem cell transplantation (SCT) against leukemia. We previously reported that immunotherapy using leukemia cell-derived HSPs is effective against MRD after syngeneic bone marrow transplantation (BMT) in mice (Sato et al. Blood, 2001). However, patients receiving SCT are usually immunocompromised due to repeated anti-cancer therapies. Accordingly, it is important to enhance the cytotoxicities (CTXs) against leukemia. Dendritic cells (DCs) are known as professional antigen-presenting cells with a specific receptor for HSPs and are expected to play a major role in immunotherapy. In this study, we evaluated whether the vaccination of DCs pulsed with HSP70 enhances the anti-leukemia effect induced by leukemia cell-derived HSP70 after syngeneic BMT and evaluated the safety of this immunotherapy. Methods: Three class-I-identical mouse tumor cell lines (A20: B-cell leukemia; T27A: myeloid leukemia; colo26: colonic carcinoma) and syngeneic balb/c mice were used in this study. HSP70 was purified from tumor cells. DCs were generated from bone marrow cells cultured with GM-CSF. DCs were pulsed with HSP70 (HSP70-pulsed-DCs) in vitro. Mice were received total body irradiation (TBI) and transplanted bone marrow cells after TBI, then inoculated 2.5 x 104 A20 cells intravenously. HSP70 or HSP70-pulsed-DCs was subcutaneously administrated. Survival days of immunized mice were compared using Kaplan and Meier methods. CTXs of splenocytes against A20 cells were determined by 51Cr release assay. Histological findings of liver and knee joint and biochemical data of serum of immunized mice were investigated. Results: All mice without immunization or immunized with DCs alone died from leukemic dissemination within 90 days after A20 inoculation, whereas mice immunized with A20-derived HSP70 (A20-HSP70) or A20-HSP70-pulsed-DCs survived long significantly. Additionally, although only 60% of the mice immunized with A20-HSP70 survived on day 120, all the mice immunized with A20-HSP70-pulsed-DCs survived with no residual leukemia cells over 120 days. Moreover, splenocytes of mice immunized with A20-HSP70-pulsed-DCs showed significantly higher CTXs against A20 cells in vitro compare to those with A20-HSP70 alone. However, no CTXs against A20 cells were induced by immunization with colo26-or T27A-HSP70-pulsed-DCs. These CTXs against A20 cells were significantly blocked by anti-CD8 and anti-MHC class-I, but not by anti-CD4. Additionally, no abnormal findings were detected either in biochemical data of serum or in histopathology of liver and joint tissue in long term immunized mice. Conclusions: Combined use of dendritic cells with leukemia cell-derived HSP70 enhances anti-leukemia effect by inducing specific cytotoxic activities against leukemia cells, and eradicates MRD effectively and safely even for immunocompromised status after syngeneic BMT. This approach would be useful for a further application of HSP in leukemia-patients after autologous SCT.