ALBERT

Description: Introduction For patients in need of a hematopoietic stem cell transplant (HSCT) but lacking an HLA matched donor, a haploidentical family donor is a particularly appealing alternative. However, to prevent graft-versus-host disease (GVHD), haploidentical HSCT necessitates intensive in vivo or ex vivo T-cell depletion that results in frequent and often lethal infectious complications and/or high relapse rates, thus decreasing overall survival. To overcome this limitation, we have developed a strategy that photodepletes host-reactive cells from the donor T cell graft, while preserving anti-infection and anti-leukemia reactivity. Patients and Methods In an open-label, multi-center phase 2 clinical trial (CR-AIR-007; NCT01794299), 12 of a planned 23 patients with high-risk hematologic malignancies were treated to date with this immunotherapy approach consisting of donor lymphocytes selectively allodepleted of host-reactive T-cells using photodynamic therapy (ATIR). ATIR was infused 28-32 days after haploidentical CD34-selected HSCT. No post-transplant GVHD prophylaxis was used. These patients were compared to a control group of 28 patients treated in a previous Phase 2 study with an investigational product manufactured using a process different from the Phase 1 trial and resulting mainly in dead and inactive cells instead of ATIR (CR-AIR-004). Results Twelve patients, mean age of 45 (range 21-64), 6 females/6 males with AML (n=9) and ALL (n=3) were treated with ATIR so far. ATIR consisted mainly of T-cells (〉90%), with residual B and NK cells (≤10%). Selective depletion of recipient-reactivity in each ATIR cell graft was assessed using a CFSE-based proliferation assay. Cell division numbers upon stimulation were analyzed using Modfit LT software (Fig 1A), which generated a proliferation index representing viable/reactive T-cells in donor cells (blue) and final ATIR product (green)(Fig 1B). Selective depletion of recipient-reactive T-cells with preservation of reactivity towards 3rd party antigens and anti-CD3/CD28 was observed in all ATIR cell grafts and used as a release criteria in the 007 study. Figure 1: A) CFSE-dilution pattern in Modfit LT software of ATIR stimulated with 3rd party cells. B) CFSE-based proliferation confirmed selective depletion of recipient-reactive T-cells in all grafts (representative depiction). Figure 1:. A) CFSE-dilution pattern in Modfit LT software of ATIR stimulated with 3rd party cells. B) CFSE-based proliferation confirmed selective depletion of recipient-reactive T-cells in all grafts (representative depiction). Preparative regimen consisted of A) FTBI (1200 cGy; n=5) or B) melphalan (120 mg/m2; n=7), along with thiotepa (10 mg/kg), fludarabine (30 mg/m2 x5 d) and ATG (2.5 mg/kg x4 d). Neutrophil and platelet engraftment was achieved in all patients at a median of 12 days (range: 9-35). No patient experienced graft rejection. Patients (n=28) in the 004 control group, mean age of 42 (range 18-61), 13 females/15 males had AML (n=19), ALL (n=6) or MDS (n=3). CFSE proliferation in T-cell grafts could not be assessed a posteriori due to low cell viability. These 004 patients received the same A) FTBI- (n=14), B) melphalan- (n= 10) based preparative regimen as 007 patients, except for 4 patients receiving single fraction (800 cGy) TBI. Neutrophil and platelet engraftment was achieved at a median of 16 days (range: 7-54). Three patients showed secondary graft rejection. Two patients in study CR-AIR-007 developed acute GVHD grade I (skin only) approximately 130 days post HSCT, which was of short duration, (18 and 41 days). Two patients died of infection and no patient relapsed at a mean follow-up of 8 months post HSCT (range 1-14 months). In the CR-AIR-004 control group, 2 patients developed grade I, 1 patient grade II and 3 patients grade III GvHD, none of these cases were lethal. Seventeen patients died of transplant related complications and 2 patients of relapse/disease progression. TRM is 20% in 007 group vs 63% in the 004 control group and OS is 80% in 007 group vs 35% in the 004 control group at 9 months post-transplant (Figures 2A and 2B). Figure 2A Kaplan Meier Transplant Related Mortality: 004 vs 007 (p=0.06) Figure 2B Kaplan Meier Overall Survival (OS): 004 vs 007 (p=0.03) Conclusions These data confirm that a novel immunotherapy strategy consisting of donor lymphocytes selectively photodepleted of alloreactive cells (ATIR) can be manufactured consistently and reproducibly. Results to date show that ATIR is safe and does not cause any grade III/IV GvHD. Moreover, haploidentical HSCT patients treated with ATIR demonstrate very promising TRM and OS rates when compared to the control group. Disclosures Roy: Kiadis Pharma: Consultancy, Research Funding. Foley:Hoffman-LaRoche: Advisory Board/Lectures Other, Honoraria; Lundbeck: Advisory Board/Lectures, Advisory Board/Lectures Other, Honoraria; Sanofi: Advisory Board/Lectures, Advisory Board/Lectures Other, Honoraria; Celgene: Advisory Board/Lectures, Advisory Board/Lectures Other, Honoraria; Pfizer: Advisory Board/Lectures Other, Honoraria; Novartis: Advisory Board/Lectures Other, Honoraria; Jansen: Advisory Board/Lectures Other, Honoraria; Alexion: Advisory Board/Lectures, Advisory Board/Lectures Other, Honoraria; Roche Canada: Honoraria, Research Funding, Unrestricted educational grant, Unrestricted educational grant Other. De Jong:Kiadis Pharma: Employment. Velthuis:Kiadis Pharma: Employment. Gerez:Kiadis Pharma: Employment. Reitsma:Kiadis Pharma: Employment. Wagena:Kiadis Pharma: Employment.

Description: Incidences of grade II–IV aGvhd in the range of 30–50% have been reported in sibling NMA transplant recipients despite prophylaxis with cyclosporine and methotrexate. To date, the ideal Gvhd prophylaxis regimen still remains undefined. Because tacrolimus is more potent than cyclosporine and MMF does not lead to mucositis, we hypothesized that early use of a combination of these two oral agents could offer an effective strategy to prevent Gvhd. We therefore designed an outpatient prospective phase II clinical trial with a NMA conditioning regimen consisting of daily fludarabine 30 mg/m2 and cyclophosphamide 300 mg/m2 for 5 days, followed by infusion of at least 4 × 106 CD34+ cells/kg. All donors were 6/6 matched siblings. Tacrolimus 3 mg bid orally was started on day (D)-8, adjusted to achieve levels of 10–15 nmol/L until D+50, then tapered off by D+100 or +180 according to estimated risk of relapse. MMF 1000 mg bid was given between D+1 to D+50 without taper. Enrollment criteria included age 〉55 years, an estimated increased risk of toxicity with an ablative transplant or participation in a multiple myeloma (MM) sequential therapy. Between 07/2000 and 07/2007, 131 patients (M/F: 75/56) with a median age of 54 years (range: 20–66) have received an allogeneic transplant according to our protocol. Indication for transplant included age (26%), fear of toxicity (28%) or participation in the sequential therapy (48%). Overall, 101 (77%) patients had previously received an autologous transplant. Diagnoses included MM (N=62), non Hodgkin’s lymphoma (NHL; N=46) including low grade (N=33), diffuse large cell (N=5), mantle (N=7) and Sezary (N=1), acute leukemia (N=10), and others (N=13). After infusion of a median of 6.8 × 106 CD34+ cells/kg (range 0.30 to 22.3), engraftment occurred in 95% of patients by D+180. Overall, 15 patients developed grade I–IV conventional aGvhd by D+120, with a Kaplan-Meier (KM) probability of 11.6% (95%CI: 7.1–18.5), a median of 64 (range: 31–120) days after transplant. At presentation, aGvhd grade was I in 5, II in 7, and III in 3 patients, respectively. No grade IV was observed. Organs involved included skin (13/15) or gastro-intestinal (GI; 4/15), but not liver. Additionally, 15 patients (12%; 95%CI: 7.4–19.2) developed an overlap syndrome consisting of clinical and histological features of both acute and cGvhd simultaneously, at a median of 140 (range 92–177) days post transplant. Altogether, the incidence of conventional II–IV aGvhd and overlap syndrome was 19.7% (95%CI: 13.7–27.7). In contrast, extensive cGvhd occurred in 84 patients surviving beyond D+80 (median D+148, range 83–1042), with a cumulative KM incidence at 7 years of 83.3% (95%CI: 74.3–90.6). Involved organs at diagnosis of extensive cGvhd included mouth (100%), skin (89%), liver (65%), eyes (51%), GI (20%), joints (6%) and lungs (6%), similar to other reported series. Nine of 76 (12%) patients at risk are still taking immunosuppressors after 5 years. To date, 37 patients have died; causes of death include relapse of initial malignancy (N=24), cGvhd (N=3), viral or fungal infection (N=3), or other (N=7). With a cohort median follow-up of 33 (range 2.7–86) months, KM probability of non-relapse mortality (NRM) is 15.5% (95% CI: 9.0–26.1) at 7 years. Overall survival (OS) at 1, 3 and 7 years are 88.5% (95%CI: 81.7–92.9), 71.0% (95%CI: 61.1–78.7) and 62.7% (CI: 51.4–72.1), respectively. Following NMA transplant, disease-free survival (DFS) at 3 years is highest in recipients with low-grade NHL (81.4%; 95%CI: 60.5–91.9) and lowest in intermediate grade NHL (40.0%; 95%CI: 6.6–73.4) or leukemia (35.7%; 95%CI: 13.0–59.4). We conclude that early use of tacrolimus and MMF is an effective strategy to prevent aGvhd and leads to very low NRM. Despite a high incidence of extensive cGvhd, OS, DFS and probability of discontinuing immunosuppression are excellent. Future strategies will need to focus on decreasing the incidence of extensive cGvhd without increasing the risk of relapse.

Description: Background: PBI-1402 is a novel orally active low molecular weight synthetic compound with erythropoiesis stimulating activity. Furthermore, a clinical phase I study showed that PBI-1402 induced a significant increase (100%, p 〈 0.0001, compared to placebo) of relative and absolute reticulocyte count in healthy volunteers after 21 days of oral treatment and was devoid of significant side effects. Outcomes: The objectives of this clinical phase Ib/II trial were to study the safety and tolerability of PBI-1402 and to assess its biological efficacy on hemoglobin (Hb) level and red blood cell (RBC) count in patients with Chemotherapy-Induced Anemia (CIA). Methods: An open label phase Ib/II trial, monitored by a US CRO (Pharm-Olam International), was conducted in patients developing anemia after chemotherapy treatment. Three cohorts of 6 CIA patients received 8 weeks of treatment with PBI-1402 once a day, at different doses, and were monitored every two weeks for safety, tolerability, Hb level, RBC count and blood chemistry. Patients remained on their chemotherapy during PBI-1402 treatment. Results: To date, 12 CIA patients have completed their PBI-1402 treatment. 83% of patients demonstrated a significant increase in RBC (p = 0.015) and 66% in Hb (p = 0.038). Among the responders, the mean Hb increase was 1.1 g/dL (p = 0.0007) from a baseline Hb value of 9.8 g/dL. No patient required a blood transfusion and only one patient had an Hb content below 9 g/dL (8.9 g/dL). PBI-1402 was well tolerated and no significant side effects were observed. Conclusion: PBI-1402, via a mechanism of action distinct from erythropoietin, induced sufficient erythropoiesis to raise the RBC level and Hb in CIA patients. In addition, PBI-1402 is safe and well tolerated. PBI-1402 offers the potential for a novel therapy of the anemia of CIA.

Description: Despite various strategies involving autologous Tx and use of newest drugs with anti-MM activity such as bortezomib and lenalidomide, very few patients with MM currently achieve long-term remission. Allo Tx theoretically remains an attractive option due to evidence of graft-versus-MM activity supported by small series of patients with long-term (〉10 years) complete remissions (CR) following myeloablative Tx, the association between chronic graft-versus-host disease (Gvhd) and CR, and response in 20–30% of relapsed patients following donor leucocyte infusion. However, to date, the ideal strategy using Allo Tx remains uncertain. Early studies using myeloablative regimens have been associated with high (30–50%) mortality rates and late relapses have been observed despite Gvhd. Low (10–22%) reported mortality in NMA regimens is hampered by reduced CR and higher progression rates compared to ablative regimens. Additionally, long-term outcome following NMA transplant remains unclear. In this present study, we sought to compare outcomes of 2 sequential cohorts of patients with MM who underwent Allo Tx in our institution. Between 01/01 and 10/07, patients with newly diagnosed stage II–III MM were invited to participate in a phase II prospective study consisting of vincristine, adriamycine and decadron x 4 cycles followed by autologous blood stem cell Tx with melphalan 200 mg/m2. Within 3 months post autologous Tx, enrolled patients received outpatient NMA Allo Tx from a 6/6 HLA matched sibling donor with a conditioning of fludarabine 30 mg/m2 and cyclophosphamide 300 mg/m2 for 5 days, followed by infusion of 〉4 x 106 CD34+ cells/kg. Gvhd prophylaxis was chosen to take advantage of low incidence of acute Gvhd and putative protective effect of chronic Gvhd: tacrolimus 3 mg bid was started on day (D)-8 then tapered off by D+100, with mycophenolate mofetil 1000 mg bid D+1 to D+50. Our NMA cohort was compared to MM patients who underwent full myeloablative Allo Tx between 06/90 and 01/01, mostly (N=29) with TBI; marrow was used in 54% and all received short course CSA/MTX. A total of 73 patients received NMA Tx (M/F: 43/30, median age 54 years, 73% stage III) and were compared to 39 patients with myeloablative Tx (M/F: 24/15, median age 47 years, 64% stage III). The incidence of grade II–IV acute Gvhd was significantly higher in the myeloablative group (30.7% vs 6.8%; p=0.0004); in contrast, as expected, chronic Gvhd was more frequent at 3 years following NMA Tx (90% vs 59.4%; p=0.0001). At 4 years, transplant related mortality (TRM) following myeloablative Tx was 50% compared to 17% in the NMA group (p=0.0001). Median (range) follow-up in NMA and myeloablative cohorts were 28 (3.5–82.9) and 37 (0.5–139) months, respectively. Kaplan-Meier estimates of overall survival (OS) at 2 (87% vs 59%; p=0.001) and 5 years (67% vs 44%; p=0.001) are significantly better in the NMA cohort. Similarly, disease free survival (DFS) at 2 (87% vs 48.5%; p=0.0001) and 5 years (67% vs 41%; p=0.0001) are also significantly better with the NMA Tx. In conclusion, our sequential auto-NMA Tx protocol is more effective to control MM than myeloablative Tx with significantly less TRM, better OS and DFS. These results might be explained by less advanced disease and higher incidence of chronic Gvhd in the NMA cohort. Future strategies should focus on reducing the relapse rate and incidence of extensive chronic Gvhd while preserving the graft versus MM effect.

Description: Infection and disease relapse are the two major complications occurring after haplo-mismatched stem cell transplantation (SCT). Accelerating immune reconstitution would imply broader applicability of SCT by providing a transplant opportunity to the large number of patients who cannot find an HLA-matched related or unrelated donor. We have previously reported that photodynamic therapy (PDT) using TH9402 could selectively deplete donor alloreactive cell populations while preserving lymphocytes for immune responses. We present results of an ongoing Phase I clinical trial of haplo-mismatched allogeneic stem cell transplant (SCT) supplemented with DLIs PDT depleted of host-reactive T cells. Thirteen patients with high-risk hematologic malignancies (7 AML relapsed or refractory, 1 AML in CR3, 1 refractory ALL, 2 MDS, 1 NHL relapsing after autologous SCT, 1 refractory CLL) entered the trial. Eleven pts are evaluable for acute GVHD and reconstitution. Patients (7 M, 4 F) underwent transplantation with donor cells mismatched at 3 HLA Ags: 5 patients; 2Ags: 5 pts, and DR only: 1 pt). Donor mononuclear cells (MNCs) were incubated with recipient MNCs for 4 days, exposed to ATIR™ treatment (TH9402 PDT), stored frozen, and administered on day 33±6 after transplant at 5 graded DLI dose levels: 1×104 to 8×105 CD3+ cells/kg. Anti-host cytotoxic T lymphocyte precursors (CTLp) were depleted from DLIs by approximately 1.5 logs, and flow cytometry showed greater than 90% elimination of activated T cells (CD4+CD25+ and CD8+CD25+) by ATIR. All stem cell grafts underwent in vitro immunomagnetic T cell depletion using CD34+ positive cell selection. Median age at SCT was 56 years (range: 21–60). Eight patients were in partial remission or had progressive disease, and 3 patients were in complete remission at the time of SCT. Conditioning regimen consisted of TBI (1200 cGy), thiotepa (5 mg/kg) and fludarabine (40 mg/m2/day for 5 days) followed by infusion of CD3 depleted HSC grafts. No GVHD prophylaxis was administered. Evaluable patients showed durable hematologic engraftment: median time to 〉0.5×109 granulocytes/L was 11 days (8–20), and to 〉20×109 platelets/L without transfusion, 12 days (9–137) and all achieved complete donor chimerism. No patient developed acute GVHD (grade II–IV), while 3 patients developed signs of chronic GVHD. Four of the first 6 pts developed infectious complications in the first 6 months, and all resolved rapidly with appropriate therapy, except for EBV-PTLD in the first patient (1×104 CD3). Five patients died: 1 of relapsed CLL and 4 of infections (all after day+310), and all had received DLI containing 1.3 ×105 CD3+ cells (2 pts) or less. No other patient relapsed. The first 6 pts developed 10 infectious episodes (4 lethal), while none of the 5 pts receiving the highest DLI doses of CD3+ cells/kg developed any infection (median follow-up: 318 days). The overall disease-free-survival and survival are 57% at 1 year (median follow-up: 10.5 mo). Our results indicate that the post-transplant infusion of a ATIR-PDT treated DLI is feasible, does not induce acute GVHD, and suggests a clinical benefit for patients receiving the highest DLI doses to accelerate T cell reconstitution. This PDT strategy represents an appealing alternative for older patients and those at high risk for GVHD.

Description: Prostate secretory protein 94 (PSP-94) has been shown to exert anti-tumor activity against prostate cancer cells, particularly in the form of PCK3145, a synthetic peptide corresponding to amino acids 31–45 of PSP-94. Indeed, when tested in a murine model, this peptide could reduce experimental prostate tumour growth. In addition, when evaluated in a Phase I clinical study, this peptide demonstrated a particularly interesting safety profile, with almost complete lack of toxicity. In order to determine whether PCK3145 could exert cytotoxic activity against other marrow infiltrating cancers, we tested its activity both in vitro and in vivo against non-Hodgkin’s lymphoma (NHL) and other hematologic cancers. Interestingly, PCK3145 inhibited the proliferation of human NHL (SR) and myeloma (RPMI-8226) cell lines in vitro. To explore its anti-tumor activity in vivo, the impact of PCK3145 was also measured by inoculating P815 malignant cells into syngeneic DBA mice. First, four groups of 6 DBA mice were injected subcutaneously with 2x104 P815 cells and then treated with subcutaneous injections of PCK3145, and compared to a peptide with the scrambled amino acid sequence, PCK5266 (peptide derived from amino acids 52 to 66 of PSP-94), and phosphate-buffered saline (PBS). Treatment with PCK3145 significantly decreased the growth of P815 tumours in comparison to PBS (p