Alemtuzumab Levels Research Articles

Alemtuzumab and CXCL9 levels predict likelihood of sustained engraftment after reduced-intensity conditioning HCT.

Overall survival after reduced-intensity conditioning (RIC) allogeneic hematopoietic cell transplantation (HCT) using alemtuzumab, fludarabine, and melphalan is associated with high rates of mixed chimerism (MC) and secondary graft failure (GF). We hypothesized that peritransplantation alemtuzumab levels or specific patterns of inflammation would predict these risks. We assessed samples from the Bone Marrow Transplant Clinical Trials Network 1204 (NCT01998633) to study the impact of alemtuzumab levels and cytokine patterns on MC and impending or established secondary GF (defined as donor chimerism <5% after initial engraftment and/or requirement of cellular intervention). Thirty-three patients with hemophagocytic lymphohistiocytosis (n= 25) and other IEIs (n= 8) who underwent HCTs with T-cell-replete grafts were included. Patients with day 0 alemtuzumab levels ≤0.32μg/mL had a markedly lower incidence of MC, 14.3%, vs 90.9% in patients with levels >0.32μg/mL (P= .008). Impending or established secondary GF was only observed in patients with day 0 alemtuzumab levels >0.32μg/mL (P= .08). Unexpectedly, patients with impending or established secondary GF had lower CXCL9 levels. The cumulative incidence of impending or established secondary GF in patients with a day 14+ CXCL9 level ≤2394 pg/mL (day 14+ median) was 73.6% vs 0% in patients with a level >2394 pg/mL (P= .002). CXCL9 levels inversely correlated with alemtuzumab levels. These data suggest a model in which higher levels of alemtuzumab at day 0 deplete donor T cells, inhibit the graft-versus-marrow reaction (thereby suppressing CXCL9 levels), and adversely affect sustained engraftment in the nonmyeloablative HCT setting. This trial was registered at www.clinicaltrials.gov as #NCT01998633.

Development and Validation of an Efficient and Highly Sensitive Enzyme-Linked Immunosorbent Assay for Alemtuzumab Quantification in Human Serum and Plasma.

Alemtuzumab is a humanized monoclonal antibody that targets the CD52 glycoprotein expressed on most lymphocytes, subsequently inducing complement-mediated and antibody-mediated cytotoxicity. Owing to its ability to induce profound immune depletion, alemtuzumab is frequently used in patients before allogeneic hematopoietic stem cell transplantation to prevent graft rejection and acute graft-versus-host disease. In this clinical context, a stable immunoassay with high sensitivity and specificity to determine alemtuzumab levels is essential for performing pharmacokinetic and pharmacodynamic analyses; however, the available methods have several limitations. Here, we report the successful development and validation of an efficient and highly sensitive enzyme-linked immunosorbent assay technique based on commercially available reagents to quantify alemtuzumab in human serum or plasma. This enzyme-linked immunosorbent assay technique was developed and validated in accordance with the European Medicines Agency guidelines on bioanalytical method validation. The assay sensitivity (lower limit of quantification) is 0.5 ng·mL -1 , and the dynamic range is 0.78-25 ng·mL -1 . To accommodate quantification of peak concentration and concentrations below the lympholytic level (<0.1 mcg·mL -1 ), patients' serum samples were prediluted 20-400 times according to the expected alemtuzumab concentration. The overall within-run accuracy was between 96% and 105%, whereas overall within-run precision (coefficient of variation) was between 3% and 9%. The between-run assessment provided an overall accuracy between 86% and 95% and an overall coefficient of variation between 5% and 14%. The developed assay provides accurate insight into alemtuzumab exposure and its effects on the clinical response to treatment, which is key to optimizing treatment strategies.

Alemtuzumab clearance, lymphocyte count, and T‐cell chimerism after hematopoietic stem cell transplant in sickle cell disease

AbstractStudy ObjectiveAlemtuzumab is a monoclonal antibody that targets the cell surface antigen CD52 on lymphocytes. Although it is used for the treatment of hematologic malignancies, such as chronic lymphocytic leukemia, and incorporated into many hematopoietic stem cell transplant (HSCT) conditioning regimens, few studies have evaluated the pharmacology of alemtuzumab in adult patients with sickle cell disease (SCD). We therefore examined the pharmacokinetics (PK) and pharmacodynamics (PD) of alemtuzumab in adults with SCD who received a matched related donor HSCT to determine if the clearance of alemtuzumab affects transplant outcomes.DesignPK and PD analysis of patient data from a single‐center clinical trial.SettingClinical research center.PatientsTwenty‐two adult patients with SCD who received one of two nonmyeloablative allogeneic HSCT regimens: alemtuzumab and total body irradiation (Alem‐TBI) or pentostatin, cyclophosphamide, alemtuzumab, and total body irradiation (Pento‐Cy‐Alem‐TBI).Measurements and Main ResultsAlemtuzumab serum concentrations, absolute lymphocyte counts, T‐cell (CD3), and myeloid (CD14/15) chimerism were collected at distinct time points and analyzed. A semi‐mechanistic PK population model was built to understand inter‐individual differences in pharmacology. Alemtuzumab was detectable up to 28 days post‐HSCT. The mean alemtuzumab level 7 days after transplant for patients on Alem‐TBI was 818 ng/ml, significantly lower than the mean level of 1502 ng/ml for patients on Pento‐Cy‐Alem‐TBI (p &lt; 0.001), but this difference decreased as time progressed. The clearance of alemtuzumab was linear, and the half‐life was longer in the Pento‐Cy‐Alem‐TBI group (average half‐life = 61.1 h) compared to the Alem‐TBI group (average half‐life = 44.1 h) (p &lt; 0.001). The CD3 chimerism at 2 and 4 months after transplant positively correlated with alemtuzumab levels collected on day 14 after transplant (R2 = 0.40 and p = 0.004 at 2 months, R2 = 0.36 and p = 0.005 at 4 months), but this significance was lost by 6 months after HSCT. No correlation was seen between alemtuzumab levels and CD14/15 chimerism.ConclusionBetween 2 and 4 months after transplant, higher alemtuzumab levels measured 14 days after transplant correlated with patients having better engraftment, suggesting more lymphodepletion may be needed to reduce graft failure in these two non‐myeloablative matched related donor HSCT regimens.

Peri-Transplant Alemtuzumab Levels Predict Risk of Secondary Graft Failure and Inversely Impact CXCL9 Levels after RIC HCT (A Correlative Biology Study to BMT-CTN 1204 RICHI)

Peri-Transplant Alemtuzumab Levels Predict Risk of Secondary Graft Failure and Inversely Impact CXCL9 Levels after RIC HCT (A Correlative Biology Study to BMT-CTN 1204 RICHI)

Alemtuzumab Precision Dosing in Allogeneic Hematopoietic Cell Transplantation

<h3>Background</h3> Alemtuzumab is frequently used as part of reduced-intensity and reduced-toxicity conditioning regimens. High alemtuzumab levels at day 0 are associated with decreased rates of acute graft-versus-host disease (aGVHD) but increased rates of mixed chimerism, viral reactivation and delayed immune reconstitution. Prior studies performed at our center identified an ideal therapeutic range of 0.15-0.6 mcg/mL at day 0. We aimed to determine if precision dosing of alemtuzumab is feasible in the pediatric HCT setting. <h3>Methods</h3> Twelve patients with non-malignant diseases were enrolled. Alemtuzumab was dosed, and levels were checked per the schema in Figure 1. Pharmacokinetic modeling was performed on Day -5 to predict Day 0 levels. If patients were predicted to fall below 0.15 mcg/mL, simulations were performed, and a top-up dose of alemtuzumab was given on Day -3. Conditioning also included fludarabine 150 mg/m² or 5 mg/kg if <10 kg and melphalan 140 mg/m² or 4.7 mg/kg if <10 kg. GVHD prophylaxis was cyclosporine or tacrolimus plus methylprednisolone. <h3>Results</h3> Observed alemtuzumab level was within predicted range for 6 (50%) patients, and 6 (50%) patients were within the ideal therapeutic range (Figure 2). This is double the percent of patients within the ideal therapeutic range with traditional dosing (Figure 3). Four patients received a top-up dose of alemtuzumab on day -3. One patient developed acute cholecystitis, and conditioning was halted after alemtuzumab. The patient was included in alemtuzumab level analysis but excluded from clinical outcomes. Ten (91%) patients engrafted on median day +12 (range 10-15). One (9%) patient had primary graft failure with autologous neutrophil recovery and was successfully re-transplanted 8 months later. There was one (9%) case of Grade II aGVHD. Six (55%) patients had full (>95%) donor chimerism at day +100. Four (36%) patients with day 0 alemtuzumab levels ranging from 0.35-1.12 mcg/mL developed mixed chimerism (lowest chimerism 29.6-78.7% prior to Day +100). Three of these received CD34+ cell boosts, and one went on to second transplant. All 11 patients are alive and well at a median of 16 months (range 10-24) follow-up. <h3>Conclusions</h3> Our results confirm the feasibility of successful alemtuzumab precision dosing in HCT. Further dose de-escalation is needed to target the majority of patients to the goal range, and additional myelosuppression may decrease the incidence of mixed chimerism.

Impact of alemtuzumab pharmacokinetics on T-cell dynamics, graft-versus-host disease and viral reactivation in patients receiving allogeneic stem cell transplantation with an alemtuzumab-based T-cell-depleted graft

Administration of alemtuzumab (targeting the CD52 antigen) to the patient (in-vivo) or to the graft (in-vitro) before allogeneic stem cell transplantation (alloSCT) decreases the incidence of graft-versus-host disease (GvHD). Effectiveness of this treatment relies on depletion of donor T cells. Currently, no data are available on alemtuzumab pharmacokinetics and pharmacodynamics in patients who received combined in-vivo and in-vitro alemtuzumab-based T-cell depletion. In this prospective study, we analyzed alemtuzumab pharmacokinetics and its effect on the circulating T cells in 36 patients who received an allogeneic T-cell-depleted graft by addition of 20 mg alemtuzumab “to the bag” with or without prior alemtuzumab (30 mg cumulative dose intravenously) as part of the conditioning regimen. Effective T-cell depletion was shown for all patients, even though alemtuzumab plasma levels varied considerably. Peak alemtuzumab levels were observed directly after graft infusion and were not associated with the number of circulating T cells pre-infusion, but with plasma volumes of the patients. All patients engrafted, confirming feasibility of this transplantation protocol. Only three patients with low alemtuzumab levels developed acute GvHD (grade II in 2 patients and grade III in 1 patient). Persistence of circulating alemtuzumab at 3 weeks after transplantation had prevented reconstitution of CD52-positive T cells when alemtuzumab plasma levels were above 0.7 μg/mL. However, overall T-cell reconstitution did not correlate with the levels of alemtuzumab exposure, due to early reconstitution of CD52-negative alemtuzumab-resistant T cells. The protective effect of these cells likely explains the low incidence of Epstein-Barr-virus- and cytomegalovirus-related disease despite circulating alemtuzumab.

Alemtuzumab Controls Acute GVHD and Mixed Chimerism Risks in Pediatric Patients with HLH Undergoing Stem Cell Transplantation: A Correlative Biology Study from the CTN 1204 Richi Trial

A reduced intensity conditioning regimen consisting of alemtuzumab, fludarabine, and melphalan has been shown to be associated with superior survival in patients with hemophagocytic lymphohistiocytosis, but is complicated by a high risk of mixed chimerism and secondary graft failure. We hypothesized that peri-transplant alemtuzumab levels impact the risks of both acute GVHD and mixed chimerism in patients with HLH undergoing RIC HCT. We tested this hypothesis in a companion correlative biology study to the Blood and Marrow Transplant Clinical Trials Network (CTN) prospective 1204 trial, Reduced-Intensity Conditioning for Children and Adults with Hemophagocytic Syndromes or Selected Primary Immune Deficiencies. The HCT RIC regimen included alemtuzumab (mg/kg subcutaneous, maximum cumulative dose of 90mg, divided over days −14 through −10), fludarabine (150mg/m2 or 5mg/kg in patients Our cohort was 74% male with a median age of 2.1 years (range 0.4-18.9). Nine patients (39%) developed acute GVHD not associated with donor lymphocyte infusion (DLI). Acute GVHD was associated with lower Day 0 alemtuzumab levels. Sixty-seven percent of patients with Day 0 levels 0.67μg/ml (p=0.0078) (Figure 1). Seventeen patients (74%) developed mixed chimerism, defined as whole blood donor chimerism Alemtuzumab level at day 0 significantly impacts the risks of acute GVHD and mixed chimerism. Studies to test precision dosing strategies to target peri-transplant levels of alemtuzumab to appropriate therapeutic ranges are underway, and future implementation may lead to improved outcomes.

Safety and Efficacy of Donor Leukocyte Infusion for Single Unit Umbilical Cord Blood Transplantation to Accelerate Immune Reconstitution Prior to Thymopoiesis

Success of reduced intensity umbilical cord blood transplantation (UCBT) in chemotherapy naive patients is limited by an increased risk of mixed chimerism, infection, and delayed immune reconstitution (IR). In a prospective clinical trial, we utilized risk-adapted alemtuzumab dosing, tested the safety of donor leukocyte infusion (cDLI) from a fraction of the thawed graft and evaluated it's efficacy in impacting IR, viral infection, and/or donor chimerism. Patients with inborn errors of metabolism, immunity, or hematopoiesis underwent a single unit UCBT following a reduced-intensity conditioning regimen of alemtuzumab, hydroxyurea, fludarabine, melphalan, and thiotepa (ClinicalTrials.gov: NCT00744692). Up to 5% of the graft was re-cryopreserved for potential cDLI and administered in cases of declining donor Tcell chimerism, viral infection, or delayed IR. Alemtuzumab levels were obtained on the day of UCBT. Chimerism and IR were measured at standard time points. 33 patients had cDLI stored and 18 patients received cDLI at a median of 65 days post-UCBT. Indications included mixed whole blood or CD3 chimerism, viral infection, or delayed IR. All patients engrafted with neutrophils at a median of 15 days post-UCBT, however, one patient experienced secondary graft failure at day +38, despite cDLI 28 days post-UCBT. Improvement in chimerism was seen in 5 of 9 patients, viral load and/or symptoms of infection in 5 of 9 patients and absolute CD3 count in 5 of 5 patients. 15 patients developed acute grade I-III GVHD at a median of 69 days post-UCBT. In those who received cDLI, 8 developed grade I-II skin GVHD at a median of 19 days post-cDLI. 2 patients with a history of GVHD received subsequent cDLI without GVHD flare. No patients who received cDLI developed grade III or IV acute or extensive chronic GVHD, while 4 patients who did not receive cDLI developed grade III GVHD affecting skin and/or the gastrointestinal tract. There was no difference in the cumulative incidence of acute GVHD between those who received cDLI and those who did not (44% vs. 33%; p=0.81). The 3-year overall and event-free survival of all patients was 94% and 91% respectively, with treatment-related mortality (TRM) of 5% 1 year post-UCBT. We report outcomes on the largest prospective trial utilizing cord blood grafts for nonmalignant disease. cDLI is safe in patients undergoing single-unit UCBT as all engrafted. Greater than half of the patients had improvement in the primary indication for its use, without increased GVHD risk. Greatest benefit was seen in patients with delayed IR and TRM at 1 year was exceptionally low. In sum, cDLI may be a simple and effective way to address mixed chimerism, infection, and accelerate IR during the vulnerable time prior to the onset of thymopoiesis. Further characterization of the merits of cDLI should be explored in a larger clinical trial.

Pretransplant Absolute Lymphocyte Counts Impact the Pharmacokinetics of Alemtuzumab

Alemtuzumab is frequently used as part of reduced-intensity conditioning (RIC) regimens for allogeneic hematopoietic cell transplantation (HCT) in pediatric patients with nonmalignant diseases. We previously suggested an optimal day 0 targeted range of alemtuzumab, but there are no pediatric data regarding the pharmacokinetics (PK) of subcutaneous alemtuzumab to guide precision dosing trials. The goal of this study was to prospectively characterize alemtuzumab PK and to explore absolute lymphocyte count (ALC) as a predictor of interindividual variability. We prospectively enrolled 23 patients who received an alemtuzumab, fludarabine, and melphalan RIC regimen. Seventeen patients completed study and received 1 mg/kg alemtuzumab divided over 5 days subcutaneously, starting on day −14. The median age was 7 years (range, .5 to 18). Blood sampling for PK measurements and descriptive PK analyses were performed. The median maximum alemtuzumab concentration was 2.39 µg/mL (interquartile range, 1.98 to 2.92). The median terminal half-life was 5.2 days (interquartile range, 2.7 to 7.8). The median concentration at day 0 was 1.27 µg/mL (interquartile range, .35 to 1.51). Importantly, day 0 alemtuzumab levels and area under the curve negatively correlated with predose ALC and ALC area-time, respectively. In conclusion, we reported the PK of subcutaneous alemtuzumab given to pediatric allogeneic HCT patients and observed that almost all patients have persistence of lytic levels of alemtuzumab beyond day 0, at levels in excess of that needed to reduce the risk of acute graft-versus-host disease. Additionally, levels correlate with pretransplant ALC. These results will allow the development of population PK models for precision dosing trials.

Alemtuzumab Induction and Delayed Acute Rejection in Steroid-Free Simultaneous Pancreas-Kidney Transplant Recipients.

The optimal immunosuppressive regimen in simultaneous pancreas-kidney transplant (SPKT) recipients that prevents acute rejection episodes (AREs) and allows optimal outcome remains elusive. This cohort study assessed incidence and time to AREs in 73 consecutive SPKT recipients receiving alemtuzumab induction and steroid-free maintenance with tacrolimus and mycophenolate mofetil. A cohort with single high-dose antithymocyte globulin (ATG; n = 85) and triple therapy served as controls. In addition, we provided mechanistic insights in AREs after alemtuzumab depletion, including composition and alloreactivity of lymphocytes (flow cytometry and mixed lymphocyte reaction) plasma alemtuzumab levels (enzyme-linked immunosorbent assay), and maintenance drug exposure. Overall number of AREs at 3 years was significantly lower with alemtuzumab versus ATG induction (26.0% vs 43.5%; adjusted hazard ratio, 0.38; P = 0.029). Most AREs (94.6%) with ATG occurred within the first month, whereas 84.2% of AREs with alemtuzumab occurred beyond 3 months. Patients with and without an ARE in the steroid-free alemtuzumab group showed no differences in composition of lymphocytes, or in alemtuzumab levels. Of note, more than two thirds of these AREs were preceded by empiric tacrolimus and/or mycophenolate mofetil dose adjustments due to viral infections, leukopenia, or gastrointestinal symptoms. Alemtuzumab induction resulted in a significant lower incidence of AREs. Empiric dose adjustments beyond 3 months in the absence of steroids carry a significant risk for subsequent rejection in SPKT recipients.

Precision Dosing of Alemtuzumab: Population Pharmacokinetic Modeling in Pediatric Patients Undergoing Allogeneic Hematopoietic Cell Transplantation for Non-Malignant Diseases

INTRODUCTION:A commonly utilized reduced intensity conditioning (RIC) for allogeneic hematopoietic cell transplantation (HCT) of patients with non-malignant disorders consists of alemtuzumab (Campath), fludarabine, and melphalan. Our group recently showed that the risks of acute graft vs host disease (GVHD) and the development of mixed chimerism following this regimen are determined by the peri-transplant levels of alemtuzumab, and recommended a potential optimal Day 0 (D0) alemtuzumab level in the range of 0.2-0.4 µg/mL (Marsh et al, Blood 2016). Additionally, patients with Day 0 alemtuzumab levels >0.6ug/mL also had delayed immune reconstitution post-transplant.We have since conducted a prospective 'intensive sampling' pharmacokinetics (PK) study of subcutaneous alemtuzumab in pediatric patients undergoing HCT (Marsh et al. EBMT Oral Presentation 2016). PK data showed a large inter-individual variability in alemtuzumab exposure and D0 levels. Importantly, levels correlated with pre-transplant absolute lymphocyte count (ALC) and underlying diagnoses. Due to the direct effects of alemtuzumab levels on important HCT outcomes, it is desirable to optimize its dosing. Ideal dosing will be where there is adequate alemtuzumab on D0 to prevent acute GVHD but not too much so as to increase the risk of mixed chimerism and/or delay early post-HCT immune recovery. In order to support targeted precision dosing of alemtuzumab in a prospective clinical trial, we have developed a population PK (PPK) model for Bayesian adaptive control.METHODS: Pediatric patients (n=17) with non-malignant disorders receiving subcutaneous alemtuzumab as a part of their preparative regimen were prospectively enrolled in our 'intensive sampling' PK study. The median age of patients was 7.0 years (3.0-11.0) and median weight was 32.2 kg (16.2-58.9). Alemtuzumab was subcutaneously administered starting on Day -14 at a dose of 0.2 mg/kg/day for 5 days (total dose of 1 mg/kg). Alemtuzumab plasma concentrations were quantified by validated flow cytometric assay. The plasma concentrations at pre-dose, 30 minutes, and 8 hours after each dose, followed by daily levels until Day 0 were used for PPK analysis. PPK analysis was performed using nonlinear mixed effects modeling (NONMEM, version 7.2). Bayesian estimation was conducted using MW/Pharm software (version 3.6).RESULTS: A one-compartment model with first order absorption best described the PK of alemtuzumab (Figure 1). Model fit was significantly improved by including body weight with allometric scaling for clearance and the volume of distribution (p<0.01). Goodness-of-fit plots did not show systematic bias. Model parameter estimates were within the 95% CIs of median values obtained by bootstrap analysis. Bayesian estimation with the developed PPK resulted in well predicted alemtuzumab concentration-time profiles in each patient (Figure 2).CONCLUSION:This is a first study to develop a PPK model of alemtuzumab after subcutaneous administration in pediatric transplant patients. Our model can be used for Bayesian estimation of individual PK to support alemtuzumab precision dosing in pediatric patients undergoing allogeneic HCT using the alemtuzumab, fludarabine, and melphalan containing RIC. Precision dosing of alemtuzumab will in turn optimize transplant outcomes for these high risk patients, by minimizing acute GVHD and mixed chimerism, along with improving post-HCT immune reconstitution. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A Prospective Study of Alemtuzumab as a Second-Line Agent for Steroid-Refractory Acute Graft-versus-Host Disease in Pediatric and Young Adult Allogeneic Hematopoietic Stem Cell Transplantation

We describe a single-center prospective study of alemtuzumab as a second-line agent for steroid-refractory (SR) acute graft-versus-host disease (aGVHD) in pediatric and young adult allogeneic hematopoietic stem cell transplant recipients. Alemtuzumab was administered for grades II to IV aGVHD if patients did not improve within 5 days or worsened within 48 hours after corticosteroids. Interim analyses of alemtuzumab levels and response were performed after every 5 patients enrolled, resulting in 3 dosing cohorts, as follows: (1) .2 mg/kg alemtuzumab subcutaneously on days 1 to 5 (maximum of 31 mg over 5 days) and .2 mg/kg/dose (not exceeding 10 mg/dose) on days 15, 22, and 29; (2) .2 mg/kg alemtuzumab subcutaneously on days 1 to 5 (maximum of 43 mg over 5 days) and .2 mg/kg/dose on day 7, 10, 15, 22, and 29; and (3) .2 mg/kg subcutaneously on days 1 to 5 and .2 mg/kg/dose on day 7, 10, 15, and 22. Alemtuzumab levels were assessed before starting alemtuzumab and at days 1, 3, 6, 10, and 14 and weekly until day 99, where day 1 was the day of first alemtuzumab dose. Fifteen patients (median age, 10 years; range, 1.4 to 27) received alemtuzumab for grades II (6%), III (74%), and IV (20%) SR-aGVHD. The overall response rate was 67%, with complete response (CR) in 40%, partial response (PR) in 27%, and no response in 33%. The median day 6 alemtuzumab level was 2.79 µg/mL (interquartile range, 1.34 to 4.89) in patients with CR compared with .62 µg/mL (interquartile range, .25 to 1.45) in patients with PR + no response (P < .05). Ninety percent (n = 9) of patients with a CR or PR reduced corticosteroid doses within 8 weeks from first alemtuzumab dose. Side effects included fever (26%) and transient thrombocytopenia (53%). Asymptomatic viremias occurred in all patients but invasive viral disease occurred in 2 patients. One patient developed Epstein-Barr virus–post-transplantation lymphoproliferative disorder. Eighty percent (n = 12) of patients were alive at 6 months, of whom 53% (n = 8) were free of GVHD whereas 13% (n = 2) developed chronic GVHD. Alemtuzumab is an effective second-line agent for children and young adults with SR-aGVHD. Higher alemtuzumab levels are associated with CR. A real-time dose adjusted alemtuzumab study is needed to further optimize the dose of alemtuzumab in aGVHD.

Alemtuzumab levels impact acute GVHD, mixed chimerism, and lymphocyte recovery following alemtuzumab, fludarabine, and melphalan RIC HCT

AbstractReduced intensity conditioning (RIC) allogeneic hematopoietic cell transplantation (HCT) with alemtuzumab, fludarabine, and melphalan is an effective approach for patients with nonmalignant disorders. Mixed chimerism and graft-versus-host-disease (GVHD) remain limitations on success. We hypothesized that higher levels of alemtuzumab at day 0 would result in a low risk of acute GVHD, a higher risk of mixed chimerism, and delayed early lymphocyte recovery and that alemtuzumab level thresholds for increased risks of these outcomes would be definable. We collected data from 105 patients to examine the influence of peritransplant alemtuzumab levels on acute GVHD, mixed chimerism, and lymphocyte recovery. The cumulative incidences of initial grades I-IV, II-IV, and III-IV acute GVHD in patients with alemtuzumab levels ≤0.15 vs ≥0.16 μg/mL were 68% vs 18% (P < .0001), 47% vs 13% (P = .0002), and 32% vs 8%, respectively (P = .005). The cumulative incidence of mixed chimerism in patients with an alemtuzumab level ≤0.15 μg/mL was 21%, vs 42% with levels of 0.16 to 4.35 μg/mL, and 100% with levels >4.35 μg/mL (P = .003). Patients with alemtuzumab levels ≤0.15 or 0.16 to 0.56 μg/mL had higher lymphocyte counts at day +30 and higher T-cell counts at day +100 compared with patients with levels ≥0.57 μg/mL (all P < .05). We conclude that peritransplant alemtuzumab levels impact acute GVHD, mixed chimerism, and lymphocyte recovery following RIC HCT with alemtuzumab, fludarabine, and melphalan. Precision dosing trials are warranted. We recommend a day 0 therapeutic range of 0.2 to 0.4 μg/mL.

A Polyclonal Population of Piga Mutant CD52 and GPI Anchor Negative T Cells Can Give Early Immune Protection after Alemtuzumab-Based T Cell Depleted Allogeneic Stem Cell Transplantation

Alemtuzumab, a monoclonal antibody targeting the glycophosphatidylinositol (GPI) anchored CD52 protein, is used for in vivo and/or in vitro T cell depletion before allogeneic stem cell transplantation (alloSCT) to reduce the risk of graft rejection and graft versus host disease. Following profound lymphodepletion, we observed a rapid recovery of T cell numbers early after transplantation despite presence of lytic levels of residual alemtuzumab (Halkes et al. ASH 2011). In the majority of patients, a substantial portion of these T cells completely lack CD52 membrane expression, explaining why these cells escaped alemtuzumab induced cytotoxicity. The aim of the current study was to further characterize these cells and unravel the mechanism underlying the loss of CD52 membrane expression. To study the functionality of the CD52 negative T cells, tetramer staining, cytokine production analysis, and cytotoxicity assays were performed. These analyses showed that the CD52 negative T cells which were present early after alloSCT contain functional T cells specific against multiple viral targets and that their lytic capacity was comparable to that of CD52 positive counterparts, demonstrating that the function of these cells is not impaired. To investigate whether absence of CD52 expression was the result of loss of CD52 gene expression, we performed mRNA expression analysis on CD52 negative T cells purified from peripheral blood samples taken from alloSCT recipients at three months post transplantation. No loss of CD52 mRNA expression was observed. Since CD52 is tethered to the membrane via the GPI anchor, we analyzed whether loss of CD52 membrane expression resulted from loss of GPI anchor expression by flow cytometry using counterstaining with a fluorescently labeled GPI-specific aearolysin FLAER. This analysis on CD52 negative T cells from 3 alloSCT recipients revealed that loss of CD52 expression generally resulted from loss of GPI anchor expression. To study whether loss of GPI anchor expression was due to active genetic down regulation, GPI positive and GPI negative CD8+ T cell populations (n=2) were purified by fluorescent activated cell sorting followed by gene expression analysis of the 26 genes that comprise the GPI anchor biosynthesis pathway. No overall loss of expression was observed for any of the 26 genes. Since loss of GPI anchor expression in paroxysmal nocturnal hemoglobinuria and aplastic anemia has been described to be the result of mutations in PIGA, one of the 26 GPI anchor biosynthesis genes and unique for its location on the X chromosome, we performed mutation analysis on clonally isolated and expanded CD52/GPI negative (CD4+ n=53, CD8+ n=13) and CD52/GPI positive (CD4+ n=8, CD8+ n=7) populations from 3 alloSCT patients. mRNA was isolated from each clone and Sanger sequencing was performed covering the protein coding region twice, with opposing primers. Mutations were only scored when observed in both reads. Using this strategy we were able to detect mutations in 35/53 CD4+ and in 8/13 CD8+ CD52/GPI negative clones, which included point mutations (n=7), non-coding mutations (n=5), small deletions <18bp (n=13), small insertions <5bp (n=6), and exon skipping (n=12). None of the individual mutations was found more than twice within clones from the same recipient, demonstrating a highly polyclonal mutational landscape. Additionally, in CD52/GPI positive clones no mutations in the PIGA coding region where found. To investigate whether these mutations in PIGA were sufficient to induce loss of GPI anchor expression, we analyzed 35 CD52/GPI negative CD4+ T cell clones by retroviral transduction with constructs encoding wtPIGA or an empty vector. Restored GPI anchor expression and coinciding CD52 membrane expression was observed in all 35 clones upon transduction with PIGA, but not with empty vector.We conclude that loss of CD52 membrane expression in T cells isolated early after alemtuzumab-based T cell depleted alloSCT is the result of various mutations in the PIGA gene and consequential loss of GPI anchor expression. We showed that these CD52 negative populations contain functional virus-specific T cells and may therefore be essential in immune protection early after transplantation. DisclosuresOff Label Use: Alemtuzumab, conditioning of graft and/or recipient before allogeneic stem cell transplantation.

Plasma alemtuzumab levels in patients with chronic lymphocytic leukemia treated with alemtuzumab combined with chemotherapy reflect the efficacy of the treatment: a hypothesis

In the HOVON68 trial comparing subcutaneous low-dose alemtuzumab (LD-A) used together with fludarabine (F) and cyclophosphamide (C) with FC alone in high-risk chronic lymphocytic leukemia (CLL), LD-AFC resulted in significantly more clinical and molecular responses than FC, but also in more opportunistic infections. In a subgroup analysis of alemtuzumab trough levels during treatment by a sensitive enzyme-linked immunosorbent assay (ELISA) method, detectable levels were found in 4/6 complete and 0/3 partial responders. A relationship between alemtuzumab plasma levels, response and duration of lymphocytopenia was evident. We hypothesize that following combination therapy, the response may not be a function of the alemtuzumab levels, but the opposite, that plasma alemtuzumab levels are a function of the efficacy of the entire treatment, and the fewer leukemic target cells that are remaining, the higher are the levels of plasma alemtuzumab. This concept may well provide a guide for alemtuzumab dosage in future trials.

Pharmacokinetics and Clinical Activity of Very Low Dose (10mg) Alemtuzumab In Transplantation for Acute Leukemia.

AbstractAbstract 1275The impressive safety and efficacy demonstrated with the in vivo use of the rat Campath-1G led to the introduction of the commercially available humanized Campath-1H (alemtuzumab) with the same schedule, i.e. total dose (TD) 100mg and later the TD was reduced to 50mg. However, studies with such high doses of alemtuzumab revealed increased incidence of relapse and severe viral infections. The most probable explanation for the untoward effects seen turned out to be the prolonged half-life of alemtuzumab (15-21 days) as compared to rat Campath-1G (13 hours). Pharmacokinetic studies showed that the peak serum alemtuzumab levels with the 100mg and 50mg schedule were 13700 ng/ml and 2500 ng/ml, respectively. Concentrations remained at high levels >1000 ng/ml at least 4 weeks (TD 100mg) and >500 ng/ml at least 2 weeks (TD 50mg), and thus far above the lymphotoxic levels of 100ng/ml. We report here for the first time pharmacokinetic studies and clinical activity of very low dose alemtuzumab (TD 10mg) in transplantation for acute leukemia. 18 consecutive pts (median age 38y, 20–65) with acute leukemias (AML 12, ALL 6 pts) underwent sibling (39%) or unrelated (61%) allogeneic transplantation and received a TD of only 10 Campath-1H (5mg/d at d-2 and d-1) as part of the conditioning and post-transplant cyclosporine (Cya) without methotrexate. 39% of patients had advanced disease (>CR1) at tranpslant. Conditioning was BuCy=7, TBI/VP16/Cy=4, Flu/BCNU/Thiotepa +/− HD-ARA-C=10. All pts except one received peripheral blood stem cells. All pts engrafted promptly (WBC>1.000/μl on d+14, 11–21 and PLT> 20.000/μl on d+12, 7–27). 17% pts developed grade I skin acute GVHD, 11% VUD-recipients developed steroid-sensible grade II acute GvHD and further 2 (11%) VUD-recipients, who had refractory AML at transplant, developed GvHD III after early Cya discontinuation (d+43, d+95). TRM on d+100 was 0. Cya was stopped at median on d+109 (30-202). Chronic GvHD observed in only 2 patients (1 limited, 1 fulminant and fatal liver GvHD on d+143 in a HbsAg +/+ pair). 72% seropositive patients revealed CMV reactivation but no CMV disease occurred. A median absolute CD4 count higher than 200/μL was reached at 9 months. After a median follow up of 200 days (71-486), 14/18 pts are alive and in CR (Karnofsky 100%) and 4 died (relapse n=1, TRM n=3). Causes of death were liver GvHD, H1N1 infection and post-DLI GvHD. We measured serum alemtuzumab levels in 8 patients (5 AML, 3 ALL) with a sensitive (limit of detection 31.25 ng/ml) ELISA technique (performed at BioAnaLab Ltd, Oxford, UK). A total of 54 samples (median 7 samples/patient) were tested from d-2 (15 minutes after the end of the first infusion of 5mg Campath-1H) up to d+30 after transplantation. The median serum peak level was 176 ng/ml (range 135–281) and was found at day of transplantation (day 0). Alemtuzumab levels declined slowly thereafter reaching a median serum level at d+7 of 78 ng/ml (41-114) and at d+20 just above the detection limit (median 42 ng/ml). Alemtuzumab was still detected only in 1 out of 4 patients at day+30. Campath levels were not influenced by disease (AML vs ALL), body weight or body surface area (r2 test). Taken together, with our administration dose and timing (5mg/d at d-2 and d-1) we achieve the maximum peak level at day of stem cell infusion which is lymphotoxic but still 1–2 log lower as compared with the 100mg and the 50mg schedules. Levels decline fast thereafter and at the time of engraftment nearly no Campath antibody remains in the patient's serum. Such fast clearance of the low dose alemtuzumab may prove beneficial regarding safety and GvL efficacy. Though the follow-up period is still relatively short, the clinical results reported here are very encouraging. Disclosures:No relevant conflicts of interest to declare.

A Phase I Study of Escalated Dose Subcutaneous Alemtuzumab Given Weekly with Rituximab In Relapsed CLL/SLL.

Abstract Abstract 1381 Alemtuzumab is an anti-CD52 antibody originally approved for intravenous administration three times per week to CLL patients refractory to fludarabine and previously exposed to alkylators. Since that time subcutaneous administration three times per week has become widespread because of its reduced infusional toxicity and recently demonstrated equivalent efficacy. In this study we assessed the tolerability, efficacy and pharmacokinetics of administering alemtuzumab subcutaneously weekly at up to 90 mg per dose following an initial 3+3 dose escalation (see table); we further added weekly rituximab in hopes of enhancing activity in lymph nodes. Treatment was administered in up to two eight week blocks with response evaluation between; the second 8 week block continued the dose and schedule used in weeks 5–8. No more than 45 mg was given per subcutaneous injection site. 28 patients were enrolled on this study between 7/2006 and 1/2010. The median age was 62 (range 47–76), and 75% were male. The median time from diagnosis to starting study therapy was 94 mos (14-236 mos). A majority of patients (82%) had Rai stage 3–4 disease and the median number of prior therapies was 4 (1-11). 20/28 patients (71%) had high risk deletions of 17p or 11q. 13/16 (81%) had unmutated IGVH, and 14/19 (74%) were positive for ZAP70. Early study withdrawals occurred due to pre-existing and persistent thrombocytopenia requiring study therapy to be held (n=2), persistent fever attributed to alemtuzumab (n=1), PML in retrospect present prior to study entry (n=1), and a DLT (grade 3 rituximab reaction) which was observed on dose level 2 prior to dose escalation of alemtuzumab. Overall, therapy was well tolerated; injection site reactions were minimal, primarily grade 1 (n=11) with only two grade 2 events. Other toxicities were as expected with alemtuzumab in this patient population, including grade 3–4 neutropenia (54%), grade 3–4 thrombocytopenia (57%), and single cases each of grade 3 rash, AIHA, pulmonary embolism, MRSA bacteremia, diverticular abscess, pulmonary Cryptococcus, EBV lymphoma and metastatic colon cancer. The ORR by NCI-WG criteria at wk 8 was 61% (95% CI 42–76%), with CR rate 11% (95% CI 4–27%). Two of 14 patients who completed a second eight week cycle improved their response (one PR from SD, and one CR from PR). A planned endpoint of this study was to compare lymph node staging by CT to PE, and we found that using CT scans to evaluate nodal response at 8 weeks decreased the ORR rate to 14% (95% CI 6–31%), with no CRs. Bone marrow was completely cleared of disease by 8 weeks in 8 patients and by 16 weeks in an additional 4 patients. The median PFS for the entire population was 13 months with a median follow-up of 9 months in patients who have not progressed. 10 patients have died, 5 of disease, 3 of second malignancies, 1 of PML and 1 of SCT complications. The median OS from study entry is 47 months, with 10 patients having undergone subsequent SCT. Following initiation of therapy we observed a &gt;1,700X decrease in the median CD19+5+ cell count in peripheral blood by the start of week 3. Similar rapid depletion of all T and NK cell subsets was also observed, with first signs of recovery at week 28, and more definite recovery at week 40. Preliminary pharmacokinetic data demonstrated lower maximum levels of rituximab (p=0.06) and alemtuzumab (p=0.05) in patients with &gt;80% bone marrow replacement by CLL but not in those with bulky lymphadenopathy. A trend toward higher alemtuzumab levels was observed in those patients with complete bone marrow clearance (p=0.1) but not in those with objective response. In conclusion, we found that administration of alemtuzumab at 90 mg subcutaneously weekly in combination with rituximab was well-tolerated, convenient and resulted in sustained adequate blood levels of both drugs in most patients. Response rates were high although in this relapsed refractory CLL population, abdominal lymphadenopathy was common, resulting in a decreased response rate when CT scans were included in staging. PFS and OS were favorable for this novel combination regimen and many patients went on to SCT. Disclosures: Brown: Genzyme: Research Funding; Celgene: Consultancy, Research Funding; Calistoga: Consultancy; Genentech: Consultancy. Off Label Use: alternative schedule of alemtuzumab. Kipps:GlaxoSmithKline: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Genzyme: Research Funding; Memgen: Research Funding; Igenica: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi Aventis: Research Funding; Abbott Laboratories: Research Funding.

Self‐administered, subcutaneous alemtuzumab to treat residual disease in patients with chronic lymphocytic leukemia

Alemtuzumab is highly effective at treating chronic lymphocytic leukemia (CLL) in bone marrow, which is the usual site of residual disease after fludarabine-based treatment. Eliminating residual disease potentially is associated with longer remission and overall survival. The authors of this report evaluated the ability of subcutaneous alemtuzumab to treat residual disease. Patients in partial remission (PR), nodular PR (nPR), or complete remission (CR) who had disease in bone marrow established by 2-color flow cytometry analysis were enrolled and received alemtuzumab 30 mg subcutaneously 3 times weekly for 4 weeks, and patients had the option to self-administer alemtuzumab. Responders were patients in PR who converted to an nPR or a CR, patients in nPR who converted to a CR, and patients in CR who had no evidence of disease on 2-color flow cytometry analysis after treatment. There were 31 patients enrolled, of whom 29 were evaluable, and there were 23 responders (4 of 4 patients who achieved a CR, 8 of 9 patients who achieved an nPR, and 11 of 16 patients who achieved a PR. Nonresponders had significantly lower plasma alemtuzumab levels at the end of treatment. Furthermore, higher plasma alemtuzumab levels at the end of treatment were correlated with a longer response duration. Compared with the results from an historic group that received intravenous alemtuzumab for residual disease, there was a trend toward a higher response rate but a shorter response duration with subcutaneous alemtuzumab. The current results demonstrated that self-administered, subcutaneous alemtuzumab was safe and active for residual disease and that plasma alemtuzumab levels and real-time minimal residual disease evaluation are important endpoints to monitor in future alemtuzumab consolidation trials.

A Retrospective Study of Alemtuzumab Level, T Cell Chimerism and Graft Versus Host Disease Using Intermediate Dose Alemtuzumab for Matched Related and Matched Unrelated Reduced Intensity Transplantation.

Abstract 3333Poster Board III-221Alemtuzumab (CAMPATH 1H) is a well established agent for effecting in vivo T cell depletion and prevention of GVHD in reduced intensity transplants. Many studies indicate that full dose alemtuzumab (100mg in 5 daily doses of 20mg) induces profound immunodeficiency, almost completely ablating GVHD in Fludarabine and Melphalan (FM) matched related donor (MRD) and matched unrelated donor (MUD) transplants. In contrast, FM conditioning alone exposes patients to a high burden of acute and chronic GVHD. Accordingly, many transplant centres have adopted policies of intermediate alemtuzumab dosing of 50mg or less. While the pharmacokinetics, rate of T cell engraftment and incidence of GVHD are well described using full dose alemtuzumab, much less is known about the in vivo action of alemtuzumab at intermediate doses. MethodsWe report our experience of alemtuzumab at 30mg (day -2) for MRD and 60mg (30mg day -4 and day -2) for MUD transplants, which was adopted as standard GVHD prophylaxis for FM transplantation at our centre in 2006. We avoided giving alemtuzumab on day -1, since there is a steep drop in alemtuzumab level in the first 24 hours after infusion and the timing of stem cell infusion may vary considerably, especially with unrelated donor grafts. From May 2006 to May 2009, 24 patients received MRD and 27 patients received MUD transplants. Post transplant serum samples were available from 19 MRD transplants and 15 MUD transplants at day +1. In addition, day +3 samples were identified from 10 patients previously transplanted with 100mg alemtuzumab, 10 MUD receiving 60mg and 10 MRD transplants receiving 30mg. All patients gave consent for clinical follow up and post transplant serum sampling for research purposes, according to protocols approved by the local research ethics committee of Northumberland and North Tyneside. Alemtuzumab concentration was measured by a validated flow cytometry assay, as previously described. ResultsThe mean (SEM) alemtuzumab concentration (micrograms/ml) on day +1 was 2.9 (0.3) after 30mg and 4.6 (0.6) after 60mg (t test p<0.01). On day +3 the levels were 2.4 (0.2); 4.0 (0.6); 8.4 (1.9) after 30mg, 60mg and historical controls of 100mg, respectively (p<0.05 between each dosing level). There were significant inverse correlations between patient surface area and alemtuzumab concentration by linear regression for both 30mg (r2 0.51 p<0.01) and 60mg dosing (r2 0.18 p<0.05). A trend for lower alemtuzumab with increasing cell dose was also observed, although this may be related indirectly to patient weight. All patients achieved >95% myeloid engraftment by day 100. Median (range) T cell engraftment was variable and significantly higher after MUD transplants: 70% (9-99%) than MRD transplants: 21% (5-85%; Mann Witney p <0.05). T cell chimerism was inversely correlated with alemtuzumab level in MRD transplants by linear regression (r2 0.37; p <0.05) but this trend was not apparent in MUD transplants. The incidence of acute GVHD was also greater after MUD transplantation at 47% (grade I or II) compared with 11% (grade I only) for MRD recipients. There was no significant relationship between GVHD grade and alemtuzumab level in either group. There were 2/24 non-relapse deaths after MRD and 3/27 following MUD transplantation; none were due to GVHD. The incidence of chronic GVHD is currently being evaluated. ConclusionThis analysis demonstrates predictable dose and surface-area relationships with alemtuzumab level in patients receiving FM conditioning. It also reveals that significantly less than 100mg alemtuzumab confers reliable GVHD prophylaxis in both MRD and MUD recipients, although at least twice the level of alemtuzumab is required to achieve comparable GVHD control in MUD transplants. Finally, it is notable that T cell chimerism at day 100 is directly related to alemtuzumab level at day +1 in MRD transplants. We conclude that optimisation of immune reconstitution and GVHD control using alemtuzumab in vivo depends upon due consideration of both recipient and donor factors, notably the size of the recipient and the origin of the graft. DisclosuresGroves:BioAnaLab: Employment. Hale:BioAnaLab: Employment, Equity Ownership.

Prior Treatment with Alemtuzumab Interferes with T-Cell Engraftment After Allogeneic Stem Cell Transplantation in Patients with Chronic Lymphocytic Leukemia.

Abstract 3351Poster Board III-239 Objectives:The majority of patients with chronic lymphocytic leukemia (CLL) who receive allogeneic hematopoietic cell transplantation (HCT) have fludarabine-refractory disease. The most active single agent in this disease stage is alemtuzumab. Alemtuzumab has a long half-life and induces profound T-cell depletion (TCD). Since TCD may mitigate graft-versus leukemia effects we evaluated „pre-conditioning“ with alemtuzumab followed by a washout period in order to minimize in vivo T-cell depletion of the graft in a phase II study (NCT 00337519). Methods:Patients received cytoreductive treatment with 3 × 30 mg alemtuzumab weekly prior to HCT. The scheduled interval between last dose of alemtuzumab and HCT was increased from two weeks to one month during the study. The antibody level at the day of HCT was measured with an ELISA with a lower limit of detection of 31.25 ng/mL (BioAnaLab lim., Oxford, UK). The conditioning regimen contained fludarabine (150 mg/m2) and busulfan (8 mg/kg). Cyclosporine (CSA) and methotrexate (MTX) were applied as GVHD-prophylaxis. Medically fit patients with relapsed CLL were elible. Results:62 patients with a median age of 57 years were included between April, 2004 and October, 2008. A median of 3 prior regimens had been given. 55% of the patients had fludarabine-resistant disease. Two patients failed to reach HCT due to progressive disease during alemtuzumab therapy. Donors were matched siblings for 26 and matched unrelated donors for 34 patients. The median level of alemtuzumab in peripheral blood after a washout period of two weeks was 62 ng/mL (interquartile range, 45 to 196 ng/mL; minimum below the limit of detection; maximum 490 ng/mL) compared to a median level below the limit of detection after a delay of four weeks (interquartile range, between the limit of detection and 77 ng/mL, maximum 256 ng/mL) (p=0.005). Despite one month time between the last dose of alemtuzumab and HCT 4 out of 30 patients (13%) had alemtuzumab levels greater than 200 ng/mL. No primary or secondary graft failure occurred. A linear relationship between the alemtuzumab level at HCT and the time to complete CD4-T-cell chimerism (TCC) was observed (p=0.003). At day +100 a CD4 positive T-cell-chimerism (TCC) >95% had been achieved by 84% of patients with alemtuzumab levels <100 ng/mL, 83% of patients with antibody levels between 100 and 200 ng/mL and 25% of patients with antibody levels >200 ng/mL (p=0.006). All patients had a complete neutrophil-chimerism at day +100. After early taper of immunosuppression (N=2) or the application of donor lymphocyte infusions in incremental doses (N=5) mixed TCC has been converted to complete TCC in all patients. The median follow-up is 17 months (1 to 61 months). Day +100 non-relapse mortality was 2%. At two years non-relapse mortality and relapse incidence were 21% and 29%, respectively. Two-year overall survival and progression-free survival were 67% (95% CI, 51% to 83%) and 50% (95% CI, 31% to 69%). Conclusions:In patients who received alemtuzumab prior to HCT, residual drug levels may interfere with T-cell engraftment. Lineage specific T-cell chimerism should therefore be assessed prospectively in this group of patients. Persistent mixed T-cell chimerism can be converted by an early taper of immunosuppression and incremental doses of donor lymphocyte infusions. Disclosures:Schetelig:Bayer Schering: Research Funding. Platzbecker:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.