ALBERT

Description: Autologous peripheral blood stem cell transplantation (APBSCT) is effective for the treatment of multiple myeloma (MM), however the majority of patients relapse. Recent evidence shows an improved outcome if absolute lymphocyte count is increased immediatedly following APBSCT in patients with hematologic malignancy. We designed a phase II trial evaluating a dose escalation of SQ IL-2 and standard dose GM-CSF post-transplant for myeloma patients. Patients (n=18) received melphalan 200mg/m2 with GM-CSF 250mg/m2/day beginning on day 5. IL-2 began on the day of transplant and continued 5 days per week for 4 weeks. Peripheral blood samples were obtained at baseline (pre-transplant) and every week for 4 weeks post-transplant and evaluated using flow cytometry for cell subsets using antibodies directed against CD3, CD4, CD8, CD25 and CD56. Control patients (n=11) consisted of MM patients who received melphalan 200mg/m2 with either G-CSF or GM-CSF without IL-2. IL-2 at a dose of 1 X 106IU/m2/d was not tolerated in 2 of 6 patients due to grade 4 fatique and diarrhea (n=1) and grade 4 supraventricular arrhythmia (n=1). A dose of 6 X 105 IU/m2/d was well tolerated by 12 patients. Level 3 or greater toxicities included nausea (n=5), diarrhea (n=3), anorexia (n=11), mucositis (n=9). Engraftment of neutrophils occurred on day 12 and platelets on day 18. The LOS was 21 days in IL-2 treated cohort and 18 days in control group. Absolute lymphocyte counts on days 10–15 were increased an average of 152% in the IL-2 cohort (range 25–390%) over the control group. At day 21, there was a marked increase in the number of CD3+CD8+ cytotoxic T cells (43 %, +/− 7.5%) , CD56 +NK cells ( 78.3 %, +/− 9.1), and CD8+CD56+ NKT cells (36.8 %, +/− 9.8%) compared to patients’ baseline levels. Cytotoxicity of Day #21 PBMNC in the IL-2 cohort was strikingly increased at 28.1% (18.5 – 29.2%) compared to 5.5 % (3.59 – 6.52%) at baseline when tested against a human myeloma cell line (RPMI 8226) using chromium release assays (E: T ratio = 100:1). These results demonstrate a tolerable regimen of immediate post-transplant immunotherapy with marked increase in the number and function of early cytotoxic effector cells. The enhanced immune recovery may translate into an improved outcome.

Description: We previously demonstrated a laboratory model of ex vivo expansion of mobilized peripheral blood mononuclear cells (PBMNC) that acquire the CD8+CD56+ NKT cell phenotype and aggressively destroy targets through the NKG2D receptor (Cytotherapy.2006;8:141–8). We developed a large scale culture method for clinical use that allows growth, expansion and cryopreservation of these expanded cells for infusion post-transplant. PBMNC were collected by large volume leukapheresis from healthy donors (n=3). Unselected MNC cells (2.5 × 106/ml) were cultured in Life Cell Bags (Nexell Therapeutics) with serum-free AIM V medium (Invitrogen), IL-2 (Chiron, 1000 IU/ml)and OKT3 (Ortho Biotech, 500 ng/ml) at 37° C and 5% CO2. Six bags were arranged from each collection. Fresh cytokines and medium were added on Days 3 and 5, and the cells were harvested on Day 7. The viability of the ex vivo expanded cell populations was 95% +/− 1.8%. Total MNC cell numbers expanded from 2.5 × 106/ml to 6.4 × 106/ml (2.5 +/− 0.5% fold increase). Phenotypic analyses showed CD3 cells increased from 55% (Day 0) to 94% (Day 7; +/−1.42%); the CD4 cells increased from 30% to 58% (+/− 1.48%); the CD8 cells increased from 11% to 48% (+/−3.98%); the CD56 cells increased from 17.2% to 47.8% (+/− 6.2). Cytotoxicity of Day 7 expanded cells was markedly increased at 74% (+/−7.1%) (Targets: K562 cells; E:T ratio 100:1) compared with 33% (+/− 5.4%) at baseline. We also tested if the ex vivo expanded cells could be cryopreserved for future clinical use. After 7 days in liquid nitrogen, cells were promptly thawed, washed and analyzed. The viability was 91% (+/− 2.1%) and the cytotoxicity against K562 cells was only slightly decreased (65%, +/− 5.4%), when compared to non-cryopreserved expanded cells (p = 0.18). The phenotype was not affected by cryopreservation and thawing. This large scale culture method results in growth and expansion of aggressive effector cells that are cytotoxic against tumor cells. The majority of the expanded cells are CD3+ T cells (equally distributed between CD4+ and CD8+ T cells). NK cells (CD56+) also readily expanded. After cryopreservation of the ex vivo expanded cells and a short-term storage, viability and phenotype of the cells did not change. There is minimal decrease in cytotoxicity that is not statistically significant. This large scale expansion model will be used in clinical trials at our institution to expand cells for adoptive cellular therapy following autologous PBSC transplantation in patients with a hematologic malignancy.

Description: We previously demonstrated the ability to grow and ex vivo expand mobilized peripheral blood mononuclear cells (PBMNC) from myeloma patients into aggressive cytotoxic effector cells (Blood102; 422b, 2003). These experiments were designed to test the function and mechanism of tumor cell killing of these cells. Peripheral blood stem cells (PBSC) were collected from myeloma pts after mobilizing with cyclophosphamide and rhG-CSF and cultured in Aim-V serum-free medium at 37 and 5% CO2. After 2 hrs, the non-adherent cells were removed and placed in culture with Aim-V, IL-2 (50 IU/ml) and OKT-3 (50 ng/ml) for 7 days. Cytotoxicity of the expanded cells was tested on Day 0 and Day 7 using a chromium release assay. To identify the cytotoxic potential of cell subsets, cell populations were depleted using the Auto MACS Magnetic Cell Sorter (Miltenyi Biotec Auburn, CA) and cytotoxicity assays were repeated. Since the CD8+ cell(s) were the most cytotoxic, the CD8+ cells were isolated and their mechanisms of tumor cell killing were evaluated by testing killing through MHC Class I, T cell receptor or the NKG2D receptor. The ex vivo expanded population was extremely cytotoxic and killed RPMI 8226 myeloma cells at 60% lysis (+/− 1.6%) (E:T of 100:1) when compared to 3.9 % on day 0 (+/− 0.8%). CD8+ or CD8+CD56+ cell subsets contributed to 〉 83.3 % (+/− 1.5%) of the killing. Blocking the TCR pathway (Redirected Cytotoxicity Assay) had no effect on killing and blocking the MHC Class I molecules decreased cytotoxicity by only 6%. When the NKG2D receptor was blocked, cytotoxicity by the CD8+ cells decreased by 48% (+/− 2%), demonstrating the critical role of the NKG2D in these CD8+ cell populations. The expanded cytotoxic effector cells aggressively lyse myeloma tumor cells in a MHC and non-MHC restricted fashion. These ex vivo expanded CD8+ cells likely acquire the NKG2D receptor and kill tumor cells in a non-MHC restricted manner. Since MHC expression is often low or absent on myeloma cells and the NKG2D ligands are fairly specific to tumor cells, the infusion of these ex vivo expanded cells following transplant may improve clinical outcomes.

Description: Autologous stem cell transplant is an effective modality for many patients with lymphoproliferative disorders. Still, the majority of patients with myeloma and many with lymphoma relapse after transplant. Innovative post-transplant immunotherapies are needed. We initiated a phase I immune mobilization trial utilizing dose escalation of IL-2, in combination with GM-CSF and G-CSF, in an attempt to mobilize autologous cytotoxic effector cells, along with peripheral CD34+ hematopoietic progenitor cells. IL-2 began on day 0 of mobilization and continued as a daily SQ injection for 11 days. On day 7 of mobilization, GM-CSF (7.5 mcg/kg/d) and G-CSF (5 mcg/kg/d) were initiated for 5 days (day 7–11). On day 11, leukapheresis was performed. Patients then received melphalan (200 mg/m2) followed by reinfusion of cryopreserved autologous peripheral hematopoietic progenitor cells. Phenotypic and functional analyses were performed using peripheral blood mononuclear cells (PBMNC) collected during mobilization on days 0, 7 and 11. Eleven of 12 patients treated to date completed the regimen (myeloma, n=11; NHL, n=1). One patient (NHL) was removed due to progressive disease. The first two dose levels of IL-2 have been well tolerated (dose level 1: 6x105 IU/m2/d; n=6 pts; dose level 2: 1x106 IU/m2/d; n=6 pts) with no ≥grade 3 toxicities noted. Dose escalation of IL-2 continues since the MTD has not been reached. Phenotypic analyses of PBMNC demonstrate an increase in CD3+CD8+ T cells from 17.5% (day 0 baseline) to 22.7% (day 11; p = 0.01). CD56+ NK cells increased from 18.9% (day 0) to 36.0% (day 11; p = 0.002), and CD8+CD56+ NKT cells increased from 8.2% (day 0) to 18.0% (day 11; p = 0.01). Cytotoxicity directed against a human myeloma cell line using peripheral blood lymphocytes was 8.6% at baseline and increased to 43% on day 11 of mobilization (p=0.03). All patients successfully mobilized and received an autologous transplant without delay in engraftment (ANC recovery: day 13 median; range 10–14 days; platelet recovery: day 12 median; range of 0–13 days). These results are encouraging and demonstrate effective mobilization with minimal toxicity and marked in vivo immunomodulatory effects. In addition, the enhanced cytotoxic effector cell number and killing of myeloma cells is quite promising, with additional follow up ongoing to determine the potential impact on survival.

Description: The treatment of AML in the elderly population is difficult given the inherent resistant disease, the toxic effects of therapy and the presence of co-morbid conditions. We proposed preclinical studies to investigate the potential synergy of bortezomib with melphalan against AML cells, with the anticipation of developing a clinical trial. To test the killing potential of bortezomib or melphalan, limiting dilution experiments were performed with subsequent analysis using the MTT assay. Two human AML cell lines, GDM-1 and Kasumi-1, were used as targets. Tumor cells (7.5 x 103 cells/well) were plated on 96-well tissue culture plates and incubated overnight at 37 °C with 5% CO2. At 24 hrs, varying concentrations of melphalan (doses: 100 uM to 1 uM) or bortezomib (doses: 100 nM to 1 nM) were added to each well. After 48 hrs of culture, the MTT assay was performed. Each test was run in triplicate. When using the GDM-1 cell line, the addition of melphalan alone (1 uM) resulted in 80% viability (+/− 1.7%), while bortezomib alone (1 nM) yielded 86% viability (+/− 3.6%). An increased dose of each medication decreased the viability of the GDM-1 cells. An increased dose of Melphalan (3 uM) reduced the viability to 23% (+/− 4.2 %). The viability dropped to 62% (+/− 4.9 %) with an increased dose of Bortezomib (3 nM). After combining the medications, the inhibitory activity against the GDM-1 cells required lower doses then either drug alone. For example, melphalan (1uM) with bortezomib (1 nM) reduced the viability of GDM-1 cells to 20% (+/− 2.8%); While melphalan (3 uM) and bortezomib (3 nM) further suppressed the viability to 3.7 % (+/−2.5 %). Similar results were found using the Kasumi-1 AML cell line. These results demonstrate the potential synergy of the combination of bortezomib with melphalan against AML cells. These preclinical results are currently being tested in a clinical trial using low dose melphalan with bortuzimab in elderly AML and MDS patients.

Description: We initiated an immune mobilization trial in an attempt to mobilize cytotoxic effector cells, along with CD34+ hematopoietic progenitor cells. A Prospective Phase I trial was initiated using dose escalation of IL-2, in combination with GM-CSF and G-CSF. IL-2 began on Day 0 and continued as a daily SQ injection for 11 days. On Day 7, GM-CSF (7.5 mcg/kg/d) and G-CSF (5 mcg/kg/d) were initiated for 5 days (Days 7–11). On Day 11, leukapheresis was started if the peripheral blood CD34 + cell count was 〉 5 cells/mcl. The endpoint of collection was ≥ 3 × 106 CD34+ cells/kg. After collection, patients received melphalan (200 mg/m2) followed by infusion of cryopreserved stem cells. Post-transplant GM-CSF began on Day +5 and terminated once the ANC reached 5000 cells/mcl. To date, 9 patients have been treated (myeloma, n = 8; NHL, n = 1) and 7 patients are evaluable. Six patients received IL-2 at Dose Level 1 (6 × 105 IU/m2/d). The remaining 3 patients received IL-2 at Dose Level 2 (1 × 106 IU/m2/d). The MTD of IL-2 has not been reached. One patient (NHL) was removed from the study due to progressive disease. The remaining 8 patients completed the regimen. Toxicities have been mild and have included Grade 2 fever (n=1) on Dose Level 2. All patients were successfully mobilized. The median number of CD34+ cells/kg and MNC/kg collected were 3.4 × 106 (range 2.8 – 4.4 × 106/kg) and 9.5 × 108 (range 0.4 – 1.7 × 109), respectively. Two large volume leukaphereses were required (median; range 1 – 3). Following transplant, the ANC recovered on Day 13 (median; range: 10 – 14 d) and platelets recovered on Day 12 (median; range 0 – 13 d). These preliminary results demonstrate that immune mobilization and collection of an adequate number of hematopoietic progenitor cells is feasible without suppression of hematopoiesis. Toxicities are minimal but the MTD of IL-2 has not yet been reached. Post-transplant engraftment is not delayed. As patient accrual continues, we are currently evaluating the qualitative and quantitative components of the collected cells, including Th1 vs. Th2 cells and the types of dendritic cells mobilized.