ALBERT

Description: Previous studies have suggested a role for platelet CD40L in thrombosis and atherosclerosis. However, there are contradictory reports on the biologic activity of its soluble variant (sCD40L) and the involved receptor signaling pathways (CD40 vs αIIbβ3). Furthermore, CD40L mAb-associated thromboembolic complications in recent human and animal studies have raised additional questions about the pro-thrombotic properties of this molecule. This study was conducted to further investigate the function of the CD40/CD40L dyad in primary hemostasis and platelet function. CD40−/− and CD40L−/− mice and mice deficient for both genes (“double knock-out”) showed prolonged tail vein bleeding and platelet function analyzer (PFA-100) closure times as compared to their wild-type littermates, indicating an inherited defect in platelet function. Recombinant human sCD40L (rsCD40L), chemical cross-linking of which yielded a single reaction product compatible with a trimeric structure of the protein in solution, bound to CD40 on resting platelets and induced CD62P (P-selectin) expression in a concentration-dependent manner (0–5 μg/ml) from 1±1 to 23±5% positivity (means±SD, n=4–8; P

Description: Successful immunologic control of HIV infection is achieved only in rare individuals. Dendritic cells (DCs) are required for specific antigen presentation to naive T lymphocytes and for antiviral, type I interferon secretion. Two major blood DC populations are found: CD11c+ (myeloid) DCs, which secrete IL-12, and CD123+ (IL-3–receptor+) DCs (lymphoid), which secrete type I interferons in response to viral stimuli. The authors have previously found a decreased proportion of blood CD11c+ DCs in chronic HIV+ patients. In this study, 26 to 57 days after infection and before treatment, CD123+ and CD11c+ DC numbers were dramatically reduced in 13 HIV+ patients compared with 13 controls (P = .0002 and P = .001, respectively). After 6 to 12 months of highly active antiretroviral therapy, DC subpopulation average numbers remained low, but CD123+ DC numbers increased again in 5 of 13 patients. A strong correlation was found between this increase and CD4 T-cell count increase (P = .0009) and plasma viral load decrease (P = .009). Reduced DC numbers may participate in the functional impairment of HIV-specific CD4+ T cells and be responsible for the low type I interferon responsiveness already known in HIV infection. The restoration of DC numbers may be predictive of immune restoration and may be a goal for immunotherapy to enhance viral control in a larger proportion of patients.

Description: The aim was to better understand the function of von Willebrand factor (vWF) A1 domain in shear-induced platelet aggregation (SIPA), at low (200) and high shear rate (4000 seconds-1) generated by a Couette viscometer. We report on 9 fully multimerized recombinant vWFs (rvWFs) expressing type 2M or type 2B von Willebrand disease (vWD) mutations, characterized respectively by a decreased or increased binding of vWF to GPIb in the presence of ristocetin. We expressed 4 type 2M (-G561A, -E596K, -R611H, and -I662F) and 5 type 2B (rvWF-M540MM, -V551F, -V553M, -R578Q, and -L697V). SIPA was strongly impaired in all type 2M rvWFs at 200 and 4000 seconds-1. Decreased aggregation was correlated with ristocetin binding to platelets. In contrast, a distinct effect of botrocetin was observed, since type 2M rvWFs (-G561A, -E596K, and -I662F) were able to bind to platelets to the same extent as wild type rvWF (rvWF-WT). Interestingly, SIPA at 200 and 4000 seconds-1 confirmed the gain-of-function phenotype of the 5 type 2B rvWFs. Our data indicated a consistent increase of SIPA at both low and high shear rates, reaching 95% of total platelets, whereas SIPA did not exceed 40% in the presence of rvWF-WT. Aggregation was completely inhibited by monoclonal antibody 6D1 directed to GPIb, underlining the importance of vWF-GPIb interaction in type 2B rvWF. Impaired SIPA of type 2M rvWF could account for the hemorrhagic syndrome observed in type 2M vWD. Increased SIPA of type 2B rvWF could be responsible for unstable aggregates and explain the fluctuant thrombocytopenia of type 2B vWD.

Description: The aim was to better understand the function of von Willebrand factor (vWF) A1 domain in shear-induced platelet aggregation (SIPA), at low (200) and high shear rate (4000 seconds-1) generated by a Couette viscometer. We report on 9 fully multimerized recombinant vWFs (rvWFs) expressing type 2M or type 2B von Willebrand disease (vWD) mutations, characterized respectively by a decreased or increased binding of vWF to GPIb in the presence of ristocetin. We expressed 4 type 2M (-G561A, -E596K, -R611H, and -I662F) and 5 type 2B (rvWF-M540MM, -V551F, -V553M, -R578Q, and -L697V). SIPA was strongly impaired in all type 2M rvWFs at 200 and 4000 seconds-1. Decreased aggregation was correlated with ristocetin binding to platelets. In contrast, a distinct effect of botrocetin was observed, since type 2M rvWFs (-G561A, -E596K, and -I662F) were able to bind to platelets to the same extent as wild type rvWF (rvWF-WT). Interestingly, SIPA at 200 and 4000 seconds-1 confirmed the gain-of-function phenotype of the 5 type 2B rvWFs. Our data indicated a consistent increase of SIPA at both low and high shear rates, reaching 95% of total platelets, whereas SIPA did not exceed 40% in the presence of rvWF-WT. Aggregation was completely inhibited by monoclonal antibody 6D1 directed to GPIb, underlining the importance of vWF-GPIb interaction in type 2B rvWF. Impaired SIPA of type 2M rvWF could account for the hemorrhagic syndrome observed in type 2M vWD. Increased SIPA of type 2B rvWF could be responsible for unstable aggregates and explain the fluctuant thrombocytopenia of type 2B vWD.

Description: Intracoronary transfer of autologous bone marrow cells (BMCs) has been shown to promote recovery of left ventricular (LV) systolic function in patients with acute myocardial infarction. (BOOST Trial; Wollert et al. Lancet, 2004, 364 141-8). Although the mechanisms of this effect remain to be established, homing of BMCs to the infarcted LV is probably a crucial early event. We determined BMC tissue distribution after therapeutic application in nine patients with a first ST-elevation myocardial infarction, who had undergone stenting of the infarct-related artery (all male; median age 43 ys; range 36 – 66). The study was approved by the local ethics committee and all patients provided written informed consent. Time from symptom onset to percutaneous coronary intervention (PCI) was 8 h (3–27) and median maximum CK level was 1767 U/l. Cells were harvested from the posterior iliac crest during short anesthesia with etomidate and midazolam and subjected to 4% gelatine polysuccinate sedimentation to obtain a preparation of unfractionated BMCs. 2.5 ± 0,7 x 108 unfractionated BMCs (10% of the harvested cell number) were radiolabeled with 100 MBq 2′-[18F]-fluoro-deoxyglucose (FDG) and infused into the infarct-related coronary artery (i.c., n=3 patients) or injected via an antecubital vein (i.v., n=3 patients). In 3 additional patients, CD34pos cells were immunomagnetically enriched from unfractionated BMCs (Clinimacs, Miltenyi, Germany), labeled with FDG, and infused i.c. Cell transfer was performed 8±2 days after stenting. Following application of FDG labelled cells all patients received 20 ± 6 x 108 non-labeled BMCs i.c. (i.e. the cell dosage that improved cell function in the BOOST trial). More than 98% of the total radioactivity infused was cell-bound. Cell viability after FDG-labeling was 95±2%. Sixty minutes after cell transfer, all patients underwent 3D-positron emission tomography imaging. After i.c. transfer, 3.4±1.4% of FDG-labeled unfractionated BMCs were detected in the infarcted LV; the remaining activity was found primarily in the liver and spleen. After i.v. transfer, only background activity was detectable in the infarcted LV. After i.c. transfer of FDG-labeled CD34-enriched cells, 25±13% of the total activity was detectable in the infarcted LV. Unfractionated BMCs engrafted in the infarct center and border zone, CD34pos cell homing was more pronounced in the border zone. FDG-labeling can be used to monitor myocardial homing and tissue distribution of BMCs after therapeutic application. I.c. transfer is superior to i.v. application in terms of BMC homing in the the infarcted LV. CD34-enriched cells display a 7-fold higher retention in the infarcted LV as compared to unfractionated BMCs.

Description: Platelet-derived CD154 has been shown to play an important role in platelet function and arterial thrombus formation in vivo. These properties of CD154 may be mediated by signaling via the CD40 receptor and/or the α IIβ 3 integrin on the platelet surface. CD154 expressed on activated platelets can also induce an inflammatory response, including the production of tissue factor (TF) in endothelial cells and monocytes, in a CD40-dependent manner. TF-driven disseminated intravascular coagulation (DIC) with activation and subsequent sequestration of platelets are common features in murine models of hematogenous metastasis and endotoxemia. In both models, platelet- and fibrin-rich thrombi are formed in the microvasculature, and in the model of metastasis, these microthrombi are believed to enhance tumor cell lodgement, extravasation and colony formation. It is therefore reasonable to hypothesize that the CD154 pathway also plays a critical role in the processes of hematogenous tumor cell dissemination and LPS-induced consumptive coagulopathy. We have used a gene knockout model to test these hypotheses. To assess tumor cell-induced coagulopathy, wild-type (WT) mice and mice deficient for CD154 (Cd154−/−), CD40 (Cd40−/−) or both (Dbl−/−), (n=5–12 per group) were injected intravenously with B16 melanoma cells (1X106). Fifteen minutes later, blood was collected by cardiac puncture to measure platelet counts, factor X (FX) activity and plasma hemoglobin (pHb), a sensitive marker of microangiopathic hemolytic anemia following microvascular thrombosis. To assess metastasis, mice (n=10–20 per group) were injected via the tail vein with B16 tumor cells (2x105) and surface tumor nodules were counted macroscopically 18 days later. To assess endotoxin-induced coagulopathy, mice (n=10 per group) were injected intraperitoneally with LPS (50 mg/kg) and platelet count, FX and fibrinogen levels were measured after 8 hours. All groups of animals had similar baseline parameters. However, following tumor cell injection, platelet counts were reduced by 73% and 72%, in WT and Cd40−/− mice respectively, compared to 50% in Cd154−/− and 54% in Dbl−/− mice (P

Description: Recent conceptual and technical improvements have resulted in clinically meaningful levels of gene transfer into repopulating hematopoietic stem cells. At the same time, evidence is accumulating that gene therapy may induce several kinds of unexpected side effects, based on preclinical and clinical data. To assess the therapeutic potential of genetic interventions in hematopoietic cells, it will be important to derive a classification of side effects, to obtain insights into their underlying mechanisms, and to use rigorous statistical approaches in comparing data. We here review side effects related to target cell manipulation; vector production; transgene insertion and expression; selection procedures for transgenic cells; and immune surveillance. We also address some inherent differences between hematopoiesis in the most commonly used animal model, the laboratory mouse, and in humans. It is our intention to emphasize the need for a critical and hypothesis-driven analysis of “transgene toxicology,” in order to improve safety, efficiency, and prognosis for the yet small but expanding group of patients that could benefit from gene therapy.

Description: Although current allo-transplantation therapy can induce considerable graft-versus-tumor (GvT) effects in RCC patients, it is also accompanied by severe, even life-threatening side effects, mainly due to graft-versus-host disease (GvHD). Efforts aiming to improve the specificity and efficiency of allogeneic cell therapy in this disease (e.g. specific donor lymphocyte infusion or vaccination) will certainly benefit from the identification of potential anti-tumor effector mechanisms and their corresponding target structures. We recently demonstrated that RCC-reactive cytotoxic T-lymphocyte (CTL) clones can be isolated from peripheral blood of healthy donors matched with the RCC stimulators for HLA-class I. These CTL were found to recognize a broad panel of RCC antigens with restricted or ubiquitous tissue expression (Doerrschuck A, et al., Blood July 1, 2004, Epub). We now extended our analyses on peripheral blood mononuclear cells (PBMC) of further HLA-matched healthy sibling (1) and unrelated individuals (4) and compared these results with available autologous patient PBMC. While mixed lymphocyte/tumor-cell culture (MLTC) responders derived from allogeneic donors showed a robust antigen-dependent proliferation over several weeks, a weak if any proliferative response was seen with autologous MLTC populations. By analysing the fine specificity of MLTC-derived clonal CTL the majority of allogeneic effectors recognized exclusively their RCC stimulators, but not corresponding lymphoblastoid-cell lines or natural killer target K562. These CTL were restricted by various HLA-A, -B or -C molecules. We further isolated CTL clones that exhibit an extraordinary strong recognition of RCC and various epithelial tumor-cell lines. Antibody blocking experiments provided clear evidence that these CTL are restricted by a not yet defined HLA-Ib molecule and, simultaneously, by a NKG2D-dependent mechanism. Other rapidly proliferating CD3+ CD8+ CTL clones were obtained that showed a non-HLA-restricted reactivity against RCC and a minor but consistent reactivity against targets with low or absent HLA-class I expression (e.g. K562). In conclusion, our results demonstrate that a heterogeneous panel of RCC-reactive HLA-Ia/Ib-restricted CTL can be isolated from PBMC of HLA-class I-matched healthy individuals. Alternatively, CTL recognizing RCC in a non-HLA restricted manner can be obtained. Our observations might reflect the superior ability to activate and expand RCC-directed T cells from PBMC of allogeneic healthy donors compared to the autologous setting. At this point, we cannot conclude whether these various CTL populations contribute to effective anti-RCC immune responses occurring in vivo. Answering this question will certainly require to identify further CTL-defined target structures at the molecular level. This would allow us to analyse the expression of candidate antigens and the frequency of specific CTL in RCC patients after allogeneic blood stem-cell transplantation, and to correlate these findings with clinical GvT and GvH events.

Description: Background: T cells from myeloma subjects can be activated and expanded ex vivo using the Xcellerate™ Process, in which peripheral blood mononuclear cells are incubated with anti-CD3 and anti-CD28 antibody-coated magnetic beads (Xcyte™-Dynabeads®). In a previous study (Borrello et al., ASCO 2004), Xcellerated T Cells administered to myeloma subjects following high dose chemotherapy and autologous stem cell transplantation led to accelerated lymphocyte recovery and restoration of the T cell receptor repertoire. In the current study, subjects with relapsed or refractory myeloma were randomized to Xcellerated T Cells with or without one cycle of fludarabine prior to Xcellerated T Cells. Fludarabine is being used to assess the influence of lymphoablation on the anti-tumor and immune reconstitution effects of T cell therapy; it has previously been reported to have no significant activty in myeloma (Kraut et al., Invest. New Drugs, 1990). Methods: Approximately 30 subjects are planned to receive treatment. Each receives a single dose of 60–100 x 109 Xcellerated T Cells. Subjects on the fludarabine arm receive a single cycle (5 days at 25 mg/m2), completed 4 days prior to the Xcellerated T Cell infusion. Results: 17 subjects have been enrolled and 13 treated to date, with median last f/u visit of 28 days (range 0–140). Xcellerated T Cells were successfully manufactured in all subjects, with T cell expansion 136 ± 61 fold (mean ± SD), with 79.2 ± 13.8 x 109 cells infused, and final product 98.0 ± 2.0% T cells (n=13). There have been no reported serious adverse events related to Xcellerated T Cells. In the fludarabine arm, lymphocytes decreased from 1,228 ± 290/mm3 (mean ± SEM) to 402 ± 164 following fludarabine, and then increased to 1,772 ± 278 on Day 14 following T cell infusion (n=7). In the non-fludarabine arm, lymphocyte counts increased from 1,186 ± 252 to 3,204 ± 545 on Day 14 (n=4). Lymphocytes were comprised of both CD4+ and CD8+ T cells. Increases were observed in NK cells from 77 ± 26 to 121 ± 25, monocytes from 166 ± 44 to 220 ± 30 and platelets from 218 ± 16 to 235 ± 24 by Day 14 (n=11). In the non-fludarabine arm, neutrophils increased from 3.6 ± 0.9 to 4.8 ± 0.6 on Day 1. On the fludarabine arm, 3 of 6 subjects developed Grade 4 neutropenia and one developed Grade 3 thrombocytopenia. Seven subjects were evaluable for serum M-protein measurements to Day 28. One of three fludarabine treated subjects had an M-protein decrease of 38%. Conclusions: Xcellerated T Cells were well-tolerated and led to increased lymphocytes, including T cells and NK cells. Increases in other hematologic parameters, including neutrophils and platelets were also observed. In this patient population, fludarabine is lymphoablative and also can cause neutropenia and thrombocytopenia. The fludarabine schedule has been decreased from 5 to 3 days. A decrease in M-protein has been observed in one of three fludarabine-treated subjects; data on additional subjects will be presented.

Description: The receptor tyrosine kinase FLT3 is overexpressed in blasts of ~90% of acute myelogenous leukemia (AML) and the majority of B-lymphoid leukemia patients. Internal tandem duplications (ITDs) in the juxtamembrane region and point mutations in the kinase domain of FLT3 are found in ~37% of AML patients and are associated with a poor prognosis. We have recently developed a fully human monoclonal antibody (IMC-EB10) which binds with high affinity to FLT3 receptor on human leukemia cells. In the present study, a novel auristatin conjugate of the anti-FLT3 antibody (EB10-MMAF) was prepared using a dipeptide linker that allows for drug release inside the lysosomes of antigen-positive cells. The MMAF conjugates were stable in buffers and plasma. EB10-MMAF (drug/antibody raito = 8) was highly potent, and selectively inhibited the growth of FLT3-expressing leukemia cells with an IC50 of 0.19 nM and 0.08 nM for MV4;11 and BaF3-ITD cells (both positive for FLT3-ITD), 1.11 nM, 6.18 nM and 1.82 nM for REH , EOL-1, EM3 cells (all three positive for wild-type FLT3), and 135 nM for JM1 (negative for FLT3). An MMAF conjugate with a control antibody was not active in these cell lines (IC50s 〉 5.9 uM). Flow cytometric analysis with annexin V indicated that EB10-MMAF treatment induced apoptosis of leukemia cells in vitro. In vivo treatment with EB10-MMAF strongly inhibited leukemia growth and prolonged survival of mice in both EOL-1 and BaF3-ITD leukemia models. In summary, immunoconjugates composed of a fully human anti-FLT3 antibody and a potent auristatin drug may provide a valuable therapeutic approach for AML and other FLT3-positive leukemias.