ALBERT

Description: Peptides derived from BCR-ABL have been used to trigger immune responses in CML pts. The native peptides may have low immunogenicity because they bind with low affinity to the major histocompatibility complex (MHC) molecules. Synthetic peptides containing mutations in the sequences of these peptides can bind with higher affinity than parental peptides and may generate a cross-reactive T cell response to the native sequence (known as a heteroclitic response). We used a mixture of heteroclitic and native peptides derived from both b3a2 and b2a2 sequences in a pilot study to vaccinate pts with CML in complete cytogenetic remission on imatinib (IM) therapy with stable bcr-abl transcript levels. Montanide ISA51 and GM-CSF were used as immunological adjuvants. Pts were required to have received IM for ≥12 mo with no change in dose for at least 6 mo and were not allowed any dose increases after registration. Pts were vaccinated with the peptides corresponding to their BCR-ABL breakpoint as follows: every 2 wks x4, then once 3 wks later, followed by 10 monthly vaccinations for a total of 15 vaccinations in 12 mo. After registration, pts had 3 additional measures of bcr-abl transcripts to better define baseline values. 8 pts have been vaccinated. At enrollment, pts had a median age of 45 yrs (range 29–63) and had been receiving IM for a median of 63 mo (range 35–68 mo) on a median dose of 600mg (range 300–800mg). Pts have been followed for a median of 15 wks (range 2–20) and have received a median of 7 vaccinations (range 1 to 8). To determine the effect of vaccination on immune leukocytes, we measured peripheral blood T-cell subsets, B, NK, and dendritic cells by FACS at baseline and at 3-month intervals thereafter. The percentage of myeloid DC (DC1) and plasmacytoid DC (DC2) cells were identified by the mutually exclusive expression of CD11c and CD123; Treg cells by the surface co-expression of CD3, CD4 and CD25 together with cytoplasmic expression of CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) and FoxP3 (forkhead/winged helix transcription factor). Vaccinations have been well tolerated. The only adverse event attributable to the vaccine is local site reactions grade 1 or 2 in 5 pts. Among the 5 pts who have had an evaluation at 3 mo from the 1st vaccination, 1 has achieved a major molecular response (bcr-abl/abl

Description: Patients with sickle cell disease have an increased number of circulating activated iNKT cells while murine SCD models report increased number and activation state of iNKT cells in target organs. Furthermore, the use of a murine iNKT cell-depleting antibody in murine SCD models prevents inflammation driven end-organ damage. NKTT120 is a humanized monoclonal antibody directed to the unique invariant TCR of iNKT cells that depletes these cells by ADCC. In preclinical studies, NKTT120 has demonstrated a safe and specific dose and time dependent depletion/recovery of iNKT cells. The preclinical efficacy and safety data supported a clinical development program to show that NKTT120 demonstrates the same safety and specificity for iNKT cell depletion from the peripheral circulation in SCD patients. In this first in human phase 1 dose-escalation study, we have examined the safety of NKTT120 in adults with steady state SCD. Future studies will explore the ability of NKTT120 prevent painful vaso-occlusive crises. Objective: To determine the safety, maximum tolerated dose (MTD), pharmacokinetics, and pharmacodynamics of NKTT120 in adults with steady state SCD. The optimal dose for a phase 2 study of NKTT120 will deplete iNKT for approximately 3 months allowing for periodic dosing. Methods: A first-in-humanphase 1 study utilizing a 3+3 design to evaluate single doses escalating over a range of 7 doses from 0.001 mg/kg to 1.0 mg/kg. The primary outcome measure is safety. Secondary outcomes include blood iNKT cell depletion and recovery, pain, analgesic use, quality of life (QoL), and pulmonary function. During a screening run-in period and after dosing of NKTT120, subjects maintained a daily smartphone eDiary (eSCaPe) to report pain, respiratory symptoms and analgesic use. ASCQ-Me and PROMIS QoL questionnaires were administered at clinic visits. The screening run-in outcomes will be used as baseline comparison for values obtained post-dosing. Results: A total of 21 patients were enrolled into the 7 cohorts of the completed and closed study. The drug was delivered as a 10-minute IV push in all cohorts. No MTD was defined, as no DLTs were reported. Three subjects each were dosed at 0.001, 0.003, 0.01, 0.03, 0.1, 0.3 or 1.0 mg/kg. At leastone month of follow-up data on circulating iNKT cell numbers are available for all of the patients dosed in the study. Only iNKT cell counts were affected by NKTT120 dosing, no change in other hematologic parameters was observed in peripheral blood. No acute elevation in circulating inflammatory cytokines was seen after antibody administration. All doses of NKTT120 resulted in maximum depletion of iNKT cells at the first time point (6 hours) monitored in all patients. During the recovery period, all patients had detectable iNKT cells in their peripheral blood. In all cohorts, the time to recovery of iNKT cells correlates with the starting circulating levels, with a longer recovery time for patients with lower baseline cell numbers. T1/2 is approximately 11 days. As observed in the pre-clinical safety studies, iNKT cell depletion and recovery was dose and time dependent. At the recommended Phase 2 dose (0.3 mg/kg) no iNKT cells were detectable in the peripheral circulation for a period of several months, suggesting near complete tissue depletion at these doses requiring recovery from T cell precursors that are not targeted by NKTT120. Conclusions: In adults with SCD,NKTT120 administered up to a dose of 1.0 mg/kg specifically reduces iNKT cells without NKTT120 dose limiting toxicity. Patients at the higher dose cohorts of NKTT120 illustrate temporal pattern for iNKT cell depletion and recovery in the circulation that inform the dosing strategy for phase 2 studies. The recommended Phase 2 dose is 0.3 mg/kg administered at a 3 month interval. The Phase 2 study will highlight the reduction of iNKT cells in the suppression of the inflammatory stimuli that promote many of the pathophysiologic sequelae seen in SCD. Disclosures Eaton: NKT Therapeutics: Employment. Mazanet:NKT Therapeutics: Consultancy, Equity Ownership. Nathan:NKT Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Description: An increased number of circulating activated iNKT cells have been reported in sickle cell disease patients, and in mouse studies, iNKT cells have been reported to have increased number and activation state in target organs. Depletion of iNKT cells in a mouse model of sickle cell disease decreases inflammation and prevents end-organ damage. NKTT 120 is a humanized monoclonal antibody directed against the iNKT cell invariant TCR that depletes these cells by antibody dependent cellular cytotoxicity. Preclinical studies show that NKTT120 has high affinity and specificity for iNKT cells. NKTT120 administration as a 10 minute IV infusion produces depletion and recovery of iNKT cells in a dose and time dependent manner. Our global hypothesis is that NKTT120 will deplete iNKT cells, reduce inflammation and prevent painful vaso-occlusive crises. In this phase 1 dose-escalation study, we have examined the safety of NKTT120 in adults with steady state SCD. Objective: To determine the safety, maximum tolerated dose (MTD), pharmacokinetics, and pharmacodynamics of NKTT120 in adults with steady state SCD. The optimal dose for a phase 2 study of NKTT120 will deplete iNKT for approximately 3 months allowing for periodic dosing. Methods: Phase 1 study utilizing a 3+3 design to evaluate single doses escalating over a range of 5 doses from 0.001 mg/kg to 0.3 mg/kg thus far. The primary outcome measure is safety. Secondary outcomes include blood iNKT cell number depletion and recovery, pain, analgesic use, quality of life (QoL), and pulmonary function. During a screening run-in period and after dosing of NKTT120, subjects maintained a daily smartphone eDiary (eSCaPe) to report pain, respiratory symptoms and analgesic use. ASCQ-Me and PROMIS QoL questionnaires were administered at clinic visits. The screening run-in outcomes will be used as baseline comparison for values obtained post-dosing. Results: At leastone month of follow-up data on iNKT cell numbers are available for the first eighteen patients dosed in the study. Three subjects were dosed at each dose level of 0.001, 0.003, 0.01, 0.03, and 0.1 and 0.3 mg/kg. Only iNKT cell counts were affected by NKTT120 dosing. No change in other hematologic parameters was observed. Eleven of 15 subjects in the first 5 cohorts had recovered iNKT cells by 28 days. Of the remaining 4 patients, 3 recovered iNKT cells by 56 days and the last recovered iNKT cells within 5 months. No patient has recovered iNKT cells in cohort 6 (0.3 mg/kg) within 28 days. Time to recovery of iNKT cells correlates with the starting circulating levels, with a longer recovery time for lower cell number. NKTT120 has been well tolerated with no dose limiting toxicities reported. Conclusions: In steady state adults with SCD,NKTT120 administered up to a dose of 0.3 mg/kg specifically reduces iNKT cells without NKTT120 dose limiting toxicity. Patient cohorts at higher doses of NKTT120 are planned to further define the most effective dose and dose interval for iNKT cell depletion and recovery in the blood and target tissues. The final dose selection will support longer term studies on the reduction of iNKT cells in the suppression of the inflammatory stimuli that promote many of the pathophysiologic sequelae seen in SCD. Disclosures Vichinsky: ApoPharma: Research Funding; ARUP Research labs: Research Funding. Eaton:NKT Therapeutics: Employment. Mazanet:NKT Therapeutics: Employment.

Description: Background: Invariant NKT (iNKT) cells are a lymphocyte subset that can be rapidly activated to produce cytokines and fuel tissue inflammatory responses that are implicated in sickle cell disease (SCD) pathogenesis. In mouse models of SCD, interrupting the activation of iNKT cells decreases tissue injury. Patients with stable SCD, defined as no recent vaso-occlusive crisis (VOC), illness or transfusion, were enrolled into a phase 1 study utilizing an anti-iNKT cell monoclonal antibody, NKTT120, aimed at establishing a safe and effective dose to reduce or deplete their blood and tissue iNKT cells. Since monitoring iNKT cell response to our depleting antibody is a critical measure of both efficacy (iNKT cell depletion) and recovery (iNKT cell return to peripheral circulation) we assessed stability of the fraction of activated iNKT cells as well as their percentage as a fraction of total CD3+ lymphocytes measured over two time points. Methods: In 18 adults (12 males/6 females, 21-43 years) with stable SCD, twomeasures of blood iNKT cells were obtained at a 2 week interval during screening for entry into a Phase Ib clinical trial. In addition, we also evaluated a single sample from 7 male adult volunteers (18-31 years). Lymphocyte profiles were analyzed by FACS. A minimum of 100,000 CD3+ events were acquired to ensure a robust measure of iNKT cells (identified by TCR Va24/Vb11expression). The lower limit of quantitation of the assay was 0.01%. The iNKT cells are expressed as a percentage of CD3+ T cells. The iNKT cell activation status was defined as the percent positive for the activation marker CD69. Results: The median iNKT cell percent of CD3+ T cells was 0.04% (range 0.0-0.5) when sampled 4 weeks before enrollment and was 0.04% (0.0-0.57) when sampled 2 weeks before enrollment (A). The median percent activated iNKT cells in the Phase 1 population was 17% (range 2-54%) at week -4 and 17% (range 0-57%) at week -2. Normal volunteers had a mean of 7% (range 0-21%) (C). The iNKT measurements done at a 2 week interval within a patient at steady state were highly correlated (r=0.91, p

Description: Introduction: Major Molecular Response (MMR) is defined as a three-log reduction from a standardized baseline of BCR-ABL/control gene transcript ratio in CML patients at diagnosis. MMR has prognostic significance for progression-free survival for patients on Imatinib® therapy. Day-to-day monitoring of the MMR value in clinical laboratories is challenging due to the absence of a commercially available standardized MMR control RNA. To improve the reliability of BCR-ABL quantitation, MolecularMD has evaluated the feasibility of a single MMR control RNA valid for blood samples drawn in EDTA or PAXgene™ tubes. Material and Methods: Patient sample RNAs were interchanged between our laboratory and an International Randomized Interferon versus STI571 study (IRIS) laboratory, which had established an MMR value and international scale reporting. This exchange enabled our laboratory to establish an MMR value and reporting on an international scale using a validated conversion factor. A serial dilution of a BCR-ABL positive cell line into a human BCR-ABL negative cell line was prepared. These dilutions were tested in IRIS laboratories with established MMR value and international scale reporting and at our laboratory by QRT-PCR to determine the BCR-ABL/control gene ratio using respectively BCR and ABL control genes. We compared the BCR-ABL/ABL ratio in 104 paired PB CML patient samples drawn either in EDTA and PAXgene tubes and the BCR-ABL/BCR ratio in 32 patient samples. Stability studies were performed to evaluate the degradation of liquid and dried forms of the MMR RNA. Results: We established a conversion factor (CF) of 0.81 with an MMR value of 0.123%. Using this CF and MMR value, we created appropriate RNA dilutions that matched the MMR value using ABL as a control gene. Repeated analyzes of this MMR control RNA confirmed the accuracy of the sample with a median value of 0.124%, very close to the MMR value defined previously (0.123%). Stability studies demonstrated that the dried RNA samples could be stored several days at 37°C and freeze-thawed up-to 10 times without significant degradation. These RNA samples once reconstituted with water could also be used several times for BCR-ABL monitoring without any significant degradation. Comparison of BCR-ABL/ABL ratio between EDTA and PAXgene tubes revealed differences unlikely to have clinical impact on disease management suggesting that the MMR RNA created would be suitable under both EDTA and PAXgene extraction methodologies. Conclusions: We produced a stable MMR control RNA in large quantity for accurate monitoring of the MMR value. This MMR control RNA is now be tested in several laboratories to confirm the stability and reliability of this reagent. The MMR control RNA will be an important tool for standardizing MMR value in laboratories, and an integral part of a BCR-ABL QRT-PCR diagnostic kit.