Monoclonal OKT3 Research Articles

Cytohesin Binder and Regulator Augments T Cell Receptor-induced Nuclear Factor of Activated T Cells·AP-1 Activation through Regulation of the JNK Pathway

Cytohesin binder and regulator (Cybr; also known as CYTIP, CASP, and PSCDBP) is a cytokine-induced gene preferentially expressed in hematopoietic tissues and in T helper 1 cells. Cybr protein associates with members of the cytohesin family, which are known ADP-ribosylation factors-GDP/GTP exchange factors, and its functions appear to regulate lymphocyte adhesion and cell-cell contact. Here we show that Cybr mRNA and protein levels are increased upon T cell receptor engagement. Cybr expression then influences T cell receptor-dependent signaling events, such as nuclear factor of activated T cells and AP-1 transcriptional activity. In addition, expression of Cybr results in increased T cell receptor-mediated activation of the Rho/Rac exchange factor Vav and of the JNK-p38 MAPK signaling pathway. The effects of Cybr on nuclear factor of activated T cells and AP-1 are dependent on MAPK activation, and enhanced activation of this cascade results in cooperation between the two transcription factors in the regulation of gene expression. These findings provide the first evidence that the adaptor protein Cybr not only regulates lymphocyte adhesion and cell-cell interaction but also contributes to the regulation of the signaling cascade and of the genetic program downstream of the T cell receptor.

Early post-transplant urinary IP-10 expression after kidney transplantation is predictive of short- and long-term graft function

The early identification of renal transplant recipients at enhanced risk of developing acute and subclinical rejection would allow individualized adjustment of immunosuppression before functional graft injury occurs and would exclude these patients from drug-weaning studies. Protein and reverse transcriptase-polymerase chain reaction-based analyses of candidate markers in urine open the opportunity to closely monitor kidney-transplanted patients non-invasively. The chemokine interferon-inducible protein 10 (IP-10; CXCL10) might be an interesting candidate to uncover ongoing immune processes within the graft. Urine samples from kidney-transplanted recipients were retrospectively analyzed for IP-10 mRNA and protein expression. IP-10 levels were correlated with the incidence of acute rejection episodes proven by histology and long-term graft function assessed by the glomerular filtration rate 6 months post transplantation. IP-10 expression in urine identified patients with ongoing acute rejection episodes several days before a biopsy was indicated by rising serum creatinine levels. Most importantly, elevated levels of urinary IP-10 protein within the first four postoperative weeks were predictive of graft function at 6 months even in the absence of acute rejection. These data reveal a correlation between elevated IP-10 expression in urine at early time points post-transplantation and intragraft immune activation that leads to acute rejection and compromised long-term graft function.

Targeting Gastrin-Releasing Peptide Receptors on Small Cell Lung Cancer Cells with a Bispecific Molecule that Activates Polyclonal T Lymphocytes

Gastrin-releasing peptide (GRP) is a growth factor for small cell lung cancer (SCLC). GRP belongs to the bombesin peptide family and has significant homology to bombesin. We constructed a bispecific molecule, OKT3xAntag2, by conjugating a monoclonal antibody OKT3 (anti-CD3) with a bombesin/GRP antagonist (Antag2) and evaluated cytotoxicity against SCLC cells. We tested binding of the bispecific molecule to SCLC cell lines and T cells by flow cytometry, antibody-dependent cellular cytotoxicity (ADCC) of SCLC cells in vitro and in a murine SCLC xenograft model. We studied SCLC apoptosis and necrosis during ADCC and the activity and cleavage of caspase-3, caspase-9, and poly(ADP-ribose) polymerase (PARP). The bispecific molecule functions as a cross-linker between T cells and SCLC cells, induces T cell activation, and mediates ADCC of SCLC cells; 40% to 80% growth inhibition of SCLC cells mediated by the bispecific molecule at low effector to target cell ratios was achieved. Activation of T cells by the bispecific molecule resulted in significant increases in IFNgamma production and apoptosis and necrosis of SCLC cells associated with cleavage of PARP and caspase-3. Targeted immunotherapy with the bispecific molecule-armed human T cells significantly reduced SCLC tumor burdens in a mouse model. The bispecific molecule OKT3xAntag2 mediates growth inhibition and apoptosis of SCLC cells by activated T cells through activation and cleavage of caspase-3 and PARP in vitro and in vivo. Clinical trials of this bispecific molecule through adoptive transfer of ex vivo activated T cells in GRP receptor-positive tumors, such as SCLC, are warranted.

Long-Term Trends in Allograft Survival

Kidney transplantation has changed dramatically over the past decade. Many advances have occurred that result in shorter hospital stays, decreased acute rejection, less infectious morbidity, and improved long-term allograft survival. Many factors are responsible for the improvement in kidney transplantation, and these factors are discussed in this review. These successes have led to a paradox in the care of the kidney-transplant recipient, however. As the short-term complications are prevented or successfully managed, more patients will develop chronic problems with the function of the allograft.

CD34+ Selected T-Cell Depleted Peripheral Blood Stem Cell Transplantation from HLA-Haplocompatible Donors for the Treatment of Severe Combined Immunodeficiency Disease.

We report the results of haplocompatible peripheral blood stem cell transplantation (PBSCT) utilizing CD34+ selection and T-cell depletion for 17 patients with severe combined immunodeficiency disease (SCID). Of these patients, 11 had T−B−NK+, 1 had T−B+NK+, and 5 had T−B+NK− phenotype. A total of 15 cell preparations were processed immediately after collection, in which two were shared between two twin siblings. Total viable nucleated cells (TVNC) in the original cell collections were between 5.9 and 9.13×1010 (median 7.0×1010) with 0.47–2.39% (median 0.9%) CD34+ cells. After Isolex 300i (Baxter Inc., n=14) or CliniMACS System (Miltenyi Biotec Inc., n=1) processing, a median number of 379×106 (89–970×106) TVNC were recovered, with a median viability of 98% (83–100%) and median purity of 96% (89–100%) CD34+ cells. All Isolex processed (n=14) cells were further T-cell depleted with OKT3 monoclonal antibody, yielding a median of 0.09% CD3+ cells (0.008–0.4%). One preparation that utilized the CliniMACS System yielded only 0.06% CD3+ cells, hence did not receive further T-cell depletion. Recovery of CD34+ cells after complete processing was from 13.3% to 60.2% (median 50.1%). Twelve patients (70.6%) are alive 2 months to 8.7 years post transplant. A total of five patients died from infections or transplant-related complications. Four patients suffered from autoimmune hemolytic anemia, which resulted in one death. Fourteen patients engrafted. One of three patients who did not engraft subsequently received a boost from the same donor but eventually died without engraftment, and 2 received a matched unrelated BMT with myeloablative conditioning and recovered T- and B-cell function. At last follow-up the median time for the recovery of T- and B-cell function was 8.5 months and 1 year, respectively. The dose of CD3 did not show any influence on T- or B-cell function recovery (p=0.48 and 0.09, respectively). And the dose of CD34+ cells did not influence T-cell function recovery (p=0.1), but did influence B-cell function recovery, which was statistically significant (p=0.02). The B− SCID phenotype is associated with a poorer outcome compared to the B+ SCID phenotype, with 50% and 100% survival rates, respectively. However, this result was not statistically significant (p=0.07). Of the 9 surviving patients followed for more than 2 years, most are in good general health. The body height growth curve is within the 5th and 10–25th percentiles in 3 and 4 patients, respectively. For body weight, the growth curve is within 10–25th and 50–75th percentiles in 5 and 2 patients, respectively. Five have achieved successful recovery of both T- and B-cell immunity and require no medication at last visit; however, 2 of these had graft failure following their initial haplocompatible transplant, and received a second BMT from a matched unrelated donor with conditioning. Three patients with X-linked SCID (3/5) didn't achieve B-cell reconstitution and still require IVIG replacement therapy. Based on these results, we conclude that for SCID patients who lack an HLA-matched related donor, CD34+ selected T-cell depleted haplocompatible PBSCT is an effective treatment.

OKT3 Treatment in Refractory Pediatric Heart Transplant Rejection

The anti-lymphocyte monoclonal antibody OKT3 has been shown to be effective in the management of steroid-resistant and/or fulminant heart transplant rejection in adults. In addition, some studies suggest that OKT3 may have a role in the management of transplant coronary artery disease (TxCAD). To date, there are limited data regarding the use of OKT3 treatment of refractory rejection or graft failure in children. Our study examines OKT3 treatment in steroid-resistant rejection, rejection with hemodynamic compromise, and TxCAD in children. Thirty-eight patients received 53 courses of OKT3 for treatment of rejection and/or graft dysfunction. Primary indications for OKT3 were steroid-resistant rejection (n = 27), rejection with hemodynamic compromise (n = 22), and TxCAD (n = 4). Resolution of rejection was considered absence of biopsy-proven rejection (< grade 2) or resolved TxCAD. OKT3 use in steroid-resistant rejection was associated with a lower incidence of rejection in the 3 months after OKT3 than 3 months before OKT3, median rejection episodes of 2.5 vs 0, p < 0.0001. In rejection with hemodynamic compromise, 20 subjects (91%) demonstrated improved hemodynamics after OKT3 and survived to hospital discharge. The use of OKT3 treatment for TxCAD failed to demonstrate resolution or improvement in angiographic TxCAD in any subject. Only 5 OKT3 treatment courses were stopped secondary to severe adverse side effects. OKT3 treatment in refractory pediatric heart transplant rejection is efficacious in acute rejection. OKT3 management in pediatric TxCAD is less clear, with no proven benefit identified in this study. OKT3 use in pediatric refractory heart rejection has significant side effects, but is tolerable and safe with close monitoring.

Clinical and cost-effectiveness of newer immunosuppressive regimens in renal transplantation: a systematic review and modelling study

Clinical and cost-effectiveness of newer immunosuppressive regimens in renal transplantation: a systematic review and modelling study

Unrelated donor bone marrow transplantation for children with severe aplastic anemia: minimal GVHD and durable engraftment with partial T cell depletion

Both increased graft rejection and increased graft vs host disease (GVHD) remain obstacles to success for unrelated donor (URD) BMT for patients with SAA. Partial T cell depletion (PTCD) may decrease the risk of severe GVHD, while still maintaining sufficient donor T lymphocytes to ensure engraftment. We report on 12 patients with SAA who underwent PTCD URD BMT. All patients had failed medical therapy or relapsed following initial responses, and were transfusion dependent. The median age was 6 years, and there were five males. Donors were matched for four patients, and mismatched for eight. All patients received total body irradiation with either Ara-C or thiotepa and cyclophosphamide. PTCD was accomplished using monoclonal antibody T10B9 or OKT3 and complement. All patients engrafted, with a median time of 18 days to ANC >500. Only one patient had greater than grade II acute GVHD; two patients had limited and one patient extensive chronic GVHD. Nine patients are alive and transfusion independent at a median months post BMT. Three patients died from infection or renal failure. This series suggests that an aggressive immunosuppressive conditioning regimen with PTCD results in successful engraftment and minimal GVHD in pediatric patients with SAA, even with HLA mismatched donors.

The prognostic role of CD4+ and CD8+ lymphocytes during chemoimmunotherapy in metastatic melanoma

We have observed that an early increase in the CD4+/CD8+ ratio of metastatic melanoma patients during chemoimmunotherapy is the most favourable independent prognostic factor. In this study, 87 patients with metastatic melanoma were monitored for peripheral blood lymphocyte subsets (CD4+ and CD8+) before and during chemoimmunotherapy (dacarbazine, vinblastine, lomustine and bleomycin or dacarbazine alone plus interferon-alpha) to confirm our previous observation. Blood samples were systematically obtained from patients who received either of these chemoimmunotherapies. The frequencies of peripheral blood lymphocyte subsets were monitored by flow cytometry using monoclonal antibodies OKT4 (CD4+, T-helper cells) and OKT8 (CD8+, T-suppressor cells). The overall response rate was 46.5%, and the median overall survival was 9.3 months. Patients with pre-treatment CD4+ levels above the mean level, who later responded to therapy, had a median survival of 28.1 months vs. 10.2 months for responders with low pre-treatment CD4+ levels (P=0.053, log-rank test). Responders with decreasing CD8+ levels had a median survival of 27.8 months vs. 10.8 months for responders with increasing CD8+ levels (P=0.04, log-rank test). Similarly, responding patients with an increasing CD4+/CD8+ ratio had a median survival of 28.1 months vs. 10.5 months for those with decreasing ratios (P=0.002, log-rank test). Non-responders showed no difference in median survival irrespective of low or high pre-treatment CD4+ lymphocytes, increasing or decreasing CD8+ levels, or increasing or decreasing CD4+/CD8+ ratios. This follow-up study confirms that the monitoring of CD4+ and CD8+ lymphocytes may provide important predictive and prognostic information in metastatic melanoma patients receiving chemoimmunotherapy.

Therapeutic Plasma Levels of OKT3 Muromonab Are Effective as Second-Line and Subsequent Treatment in Recipients of Stem Cell Allografts with Steroid-Resistant Acute Graft-Versus-Host Disease.

Acute graft-versus-host disease (aGvHD) remains a major complication in recipients of hematopoietic cell allografts and is associated with poor outcome. Salvage treatment is difficult after failure of steroids. OKT3 muromonab is a monoclonal murine IgG2a antibody directed against the CD3 antigen on T-lymphocytes that has proven effective in transplant settings both of solid organs and bone marrow. Here we report on the results of OKT3 treatment in 43 patients who had received stem-cell or bone marrow allografts for hematologic malignancies and subsequently been diagnosed with steroid-resistant aGvHD. Overall, 48 treatment cycles of OKT3 (5 mg intravenous injection once daily) were administered for acute GvHD of grades II (n = 12), III (n = 27) and IV (n = 9). Mean duration of OKT3 therapy was 9 (range, 1 –16) days. Twenty cycles of OKT3 were administered as second line and 28 as third-plus line (third line, n = 14; fourth line, n = 14) treatment regimens. Side effects were mild to moderate, comprising fever, dyspnea, tachycardia, and vomiting. Thirty of 42 evaluable patients responded to therapy for an overall response rate of 69 %. Complete remissions were seen in 6 (11.9 %) and partial remissions in 24 patients (57.1 %). Response to treatment was 88.8 % for skin GvHD; 53.3 % for gut aGvHD; and 8.7 % for liver involvement. Recurrent or progressive acute GvHD was observed in 23 patients (54.8 %) after a median of 9 (range, 1 – 58) days. Pharmacokinetic studies of monoclonal antibody OKT3 were performed in 17 patients, 16 of whom achieved at least a partial remission. Treatment duration was > 5 days in 16 patients. Adequate plasma levels (> 1000 ng/ml) were observed in 13 patients after a median of 6 (1 – 11) days on treatment. OKT3 concentration became undetectable at a median of merely 4 (3 – 11) days after discontinuation of therapy. Survival at 100 days from transplantation was 58.1 % and at one year, 25.5 % with the most important causes of death being infectious complications (44.7%) and GvHD (33.2%). Patients treated with OKT3 earlier in the course of aGVHD showed a trend to better survival: 35% of patients with 2nd-line therapy were alive at one year from transplantation, compared to 17.4% with 3rd- plus line treatment (p=0.21). There was also a trend to a better one-year survival for patients with grade II (33% of patients alive) when compared to grade III/IV aGvHD (16% of patients alive; p=0.24). Thus, a significant proportion of patients with corticosteroid-resistant acute GvHD showed a response to salvage treatment with OKT3. More effective antimicrobial prophylaxis and treatment are mandatory to overcome considerable treatment-related mortality in those high-risk patients.

Outcomes of transplantation with partial T-cell depletion of matched or mismatched unrelated or partially matched related donor bone marrow in children and adolescents with leukemias

Graft-versus-host disease (GVHD) remains a major barrier to successful hematopoietic stem cell transplant for patients who lack a matched related donor. Partial T-cell depletion (TCD) of the graft may decrease the risk of severe GVHD with unrelated donors (URD) and partially matched related donors (PMRD) while retaining an antileukemic effect. We analyzed our experience using URD and PMRD for pediatric patients with leukemias from 1990 to 2001. A subgroup of 'matched' URD donor pairs was retrospectively analyzed for high-resolution class I. Partial TCD was accomplished with monoclonal antibody T10B9 or OKT3 and complement. There were 76 URD (45% matched) and 28 PMRD recipients. Event-free survival (EFS) was 38.3%, and overall survival (OS) 45.1% at 3 years. On multivariate analysis, there was no difference in survival based upon marrow source, but nonrelapse mortality was higher with the use of PMRD. Relapse occurred in 6% of ALL patients, and 22.8% of AML/MDS patients. Grades III-IV GVHD was observed in only 6.7% of patients. Partial TCD allows use of matched or mismatched URD, or PMRD with little mortality from GVHD, durable engraftment, and no increase in relapse risk.

Mortalité en transplantation rénale pédiatrique : 15 ans d'expérience

The aim of the study was to analyse retrospectively the patient survival and mortality risk factors in renal transplant children at Edouard-Herriot hospital, Lyon. Patients and Methods. – One hundred and ninety four patients 0 to less than 18 years of age received a renal transplant between April 1987 and January 2002. We collected data concerning the age at transplantation, the mean duration of dialysis prior to transplantation, the type of donor (living related: LRD, or cadaver: CAD) as well as long term follow-up of all the patients. Eleven patients died during the study period and we collected mortality and morbidity factors. Results. – Mean age at transplantation was 9.5 years (range: 0.6–17.9 years); 16 children were transplanted before 2 years of age. Eighty six percent received a first graft, 10% a second graft, and 3% a third graft. One third of the patients had preemptive transplantation. The graft was carried out respectively from a LRD in 18% and from CAD in 82% of the cases. Eleven patients (5.7%) died during this period; four of them were transplanted twice. Death occurred within 2.4 years after transplantation (range: 2 days–6.3 years). Five of these deceased children developed an acute rejection episode treated with methylprednisolone pulses and OKT3 monoclonal antibodies. Four had a cytomegalovirus (CMV) infection and three required repeated surgery. The causes of death were bacterial infections (4/11), CMV infection (2/11), neurological involvement (2/11), Burkitt lymphoma (1/11) and unexplained sudden death (2/11). Conclusion. – The main causes of deaths in renal transplant children are bacterial infections. The improvement in patient survival probably will come from both better preventive approach to nosocomial infections and a less aggressive immunosuppression.

Which donor should be chosen for hematopoietic stem cell transplantation among unrelated HLA-A, -B, and -DRB1 genomically identical volunteers?

The aim of this study was to identify significant prognostic factors by using unrelated genomically HLA-A, -B and -DRB1-identical donors. Such data could help to choose the best donor. We studied 136 consecutive patients with hematologic malignancies and a median age of 32 years (range, 0–55 years) who received hematopoietic stem cell transplantation. Bone marrow grafts were given to 83 and peripheral blood stem cells to 53 patients. The cumulative incidence of grade II to IV acute graft-versus-host disease (GVHD) was 30% and of chronic GVHD was 54%. At 5 years, the overall transplant-related mortality (TRM) was 34%, and patient survival was 50%. In Cox multivariate analysis, 32 potential risk factors were analyzed. Monoclonal antibody OKT-3 during conditioning was correlated with grade II to IV acute GVHD, chronic GVHD, and TRM. HLA-DP mismatch was associated with poor TRM and poor survival. Cytomegalovirus-seropositive patients with a seronegative donor had a decreased leukemia-free survival. Five-year TRM was 14% with no risk factor, 38% with 1 risk factor, and 87% with 2 risk factors. The 5-year survival was 72%, 48%, and 30% with 0, 1, and 2 risk factors, respectively. We concluded that unrelated hematopoietic stem cell transplantation may be improved if an optimal donor and immunosuppression are chosen.

Extracellular Matrix Protein-dependent Apoptosis of T Cells in Women with a History of Recurrent Spontaneous Abortion.

The purpose of the study was to determine the role of T-cell apoptosis in extracellular matrix (ECM) environment in pregnancy maintenance in women with a history of recurrent spontaneous abortion (RSA). Thirty-nine non-pregnant women with the history of RSA (anatomic, genetic, endocrine and microbiologic causes were excluded) and 22 healthy women with the previous successful pregnancy outcome were studied. In addition, 21 women with the history of RSA were also studied at the beginning of their next pregnancy. We studied apoptosis of peripheral blood T cells after culture with monoclonal antibody (mAb) OKT-3 alone or with mAb OKT-3 following ECM proteins: collagen IV (C-IV) or fibronectin (Fn). We used Cell Death Detection ELISA for studying cell death in cell population. In addition, apoptotic peripheral blood T cells were identified by annexin V-PE staining protocol using flow cytometry. CD29+ and CD95+ T-cell surface receptors were also analyzed by flow cytometry. The significantly higher values of enrichment factor: mU of the sample (dying/dead cells) per mU of the corresponding control (viable cells) were observed after peripheral blood T-cell culture with C-IV (P = 0.0002) or Fn (P = 0.004) in samples of non-pregnant women with the history of RSA when compared with control women. The significantly higher values of enrichment factor were observed after peripheral blood T-cell culture with C-IV in samples of pregnant women with the history of RSA with successful pregnancy outcome when compared with pregnant women with the history of RSA with failed pregnancy outcome (P = 0.01). However, the percentage of apoptotic T cells stained by annexin V was significantly lower in non-pregnant RSA women compared with control (P = 0.0001). CD95 expression was significantly lower in non-pregnant RSA women compared with control (P = 0.01). Apoptosis of T cells might be an interesting possible explanation of successful pregnancy outcome in women with the history of RSA.

Pharmacologic immunosuppression.

Clinical organ transplantation only became a viable treatment option after the advent of effective pharmacologic immunosuppression. Azathioprine and steroids were among the first drugs available for pharmacologic immunosuppression allowed for the first long-term successes in kidney and liver transplantation, though survivors experienced significant adverse effects of the immunosuppression. Azathioprine is an antimetabolite which inhibits the de novo and salvage pathways of purine synthesis. This results in lymphocyte suppression but also toxicity to bone marrow, gastrointestinal tract, and liver. Mycophenolate mofetil (MMF), another antimetabolite drug, inhibits only the de novo purine synthesis pathway. Corticosteroids cause immunosuppression mainly by sequestration of CD4+ T-lymphocytes in the reticuloendothelial system and by inhibiting the transcription of cytokines. Corticosteroids have adverse effects on virtually every system in the body, producing many dose-limiting problems such as osteoporosis, obesity and glucose intolerance. The introduction of cyclosporine in 1983 allowed for further improvements in graft survival, and the incidence of acute rejection decreased. Cyclosporine and the more recently-introduced tacrolimus compose the class of immunosuppressive agents called calcineurin inhibitors. By binding calcineurin and preventing its translocation into the nucleus these drugs prevent transcription and subsequent secretion of IL-2. These drugs produce varying degrees of nephrotoxicity, neurotoxicity and glucose intolerance. Rapamycin also inhibits IL-2 expression, though by interaction with the mammalian Target of Rapamycin (mTOR) protein. The use of antibody to produce immunosuppression began with polyclonal sera developed in animals such as horses or goats. The mechanism by which polyclonal sera causes immunosuppression is not well understood, though cell-mediated cytotoxicity of lymphocytes in the circulation may be one major effect. In contrast, the monoclonal antibody OKT3 is specific for the T-cell receptor (TCR)/CD3 complex, thus preventing activation of T-lymphocutes. Most recently, human and chimeric murine monoclonal antibodies daclizumab and basiliximab have provided effective induction therapy with virtually no adverse effects. While the improved efficacy and decreased adverse effects immunosuppressive agents account for much of the progress in the field of transplantation, current immunosuppression medications not perfect. Ideally, medications would inducing graft tolerance while avoiding generalized immunosuppression and non-immunologic adverse effects. Future research will likely focus on molecular- and gene-level mechanisms to achieve this goal.

Immunosuppressive properties of anti-CD3 single-chain Fv and diabody.

The mouse anti-human CD3 monoclonal antibody OKT3 is a potent immunosuppressive agent used in clinical transplantation. However, OKT3 therapy is associated with unpleasant and often serious side effects which appear to result from cytokine release, complement activation and a human anti-mouse antibody (HAMA) response. To decrease these adverse side effects, we constructed antibody fragments comprising OKT3 variable domains without any constant domains. Single-chain Fv (scFv) monomers, dimers and trimers were generated by changing the linker length between the V(H) and V(L) domains. The linkers used were the natural extensions of the V(H) into the C(H)1 domain. The dimeric molecules (diabodies) demonstrated the best CD3-binding activity. The diabody with the six amino acid linker was produced in bacteria with a tenfold higher yield than other scFvs and possessed CD3-binding affinity approaching that of the parental mAb. In contrast to OKT3 mAb, the anti-CD3 diabody and scFv monomer did not cause any T-cell activation and cytokine release in vitro, while demonstrating CD3 modulation. In mixed lymphocyte cultures, both diabody and scFv, but not the monoclonal antibody OKT3, were able to suppress T-cell activation and secretion of IL-2 and IFN-gamma in a dose-dependent manner. The anti-CD3 diabody may provide a potent immunosuppressive drug with low toxicity and immunogenicity.

Evidence-based immunosuppression after pediatric renal transplantation—A dream?

IT IS INCREASINGLY evident that only prospective, randomized controlled clinical trials provide the highest level of evidence to compare therapeutic regimens. Clinical trials provide the framework to search for more effective and less toxic immunosuppressive agents to control solid organ transplant rejection. While systematic reviews have been presented on immunosuppression after solid organ transplantation in adults, the pediatric literature on randomized controlled clinical trials remains extremely scarce. Most trials have evaluated the safety and efficacy of recombinant human growth hormone after pediatric renal transplantation. In a relatively small trial after pediatric liver transplantation, two antihypertensive agents were compared in addition to a cyclosporine-based immunosuppressive protocol. Large-scale randomized controlled clinical trials have been few until recently. There was one trial comparing the efficacy and safety of prophylactic treatment with OKT3 monoclonal antibody versus antilymphocyte globulin induction therapy after pediatric renal transplantation. There have been several large-scale, randomized, multicenter trials in adult renal transplant recipients comparing the clinical efficacy and safety of both calcineurin inhibitors, combined with azathioprine and corticosteroids as adjunctive therapies. Acute allograft rejection was significantly reduced with tacrolimus compared with cyclosporine-based immunosuppressive therapy. There is only one similar trial in children. However, there also is a large randomized controlled trial in the United States comparing OKT3 induction therapy with IV cyclosporine. This trial has not yet been published. The aim of this manuscript is to review the existing trials to find explanations for the lack of development but obvious need for such trials in pediatric solid organ transplantation.

Enhancement of calcium signalling dynamics and stability by delayed modulation of the plasma-membrane calcium-ATPase in human T cells.

In addition to its homeostatic role of maintaining low resting levels of intracellular calcium ([Ca2+](i)), the plasma-membrane calcium-ATPase (PMCA) may actively contribute to the generation of complex Ca2+ signals. We have investigated the role of the PMCA in shaping Ca2+ signals in Jurkat human leukaemic T cells using single-cell voltage-clamp and calcium-imaging techniques. Crosslinking the T-cell receptor with the monoclonal antibody OKT3 induces a biphasic elevation in [Ca2+](i) consisting of a rapid overshoot to a level > 1 microM, followed by a slow decay to a plateau of approximately 0.5 microM. A similar overshoot was triggered by a constant level of Ca2+ influx through calcium-release-activated Ca2+ (CRAC) channels in thapsigargin-treated cells, due to a delayed increase in the rate of Ca2+ clearance by the PMCA. Following a rise in [Ca2+](i), PMCA activity increased in two phases: a rapid increase followed by a further calcium-dependent increase of up to approximately fivefold over 10-60 s, termed modulation. After the return of [Ca2+](i) to baseline levels, the PMCA recovered slowly from modulation (tau approximately 4 min), effectively retaining a 'memory' of the previous [Ca2+](i) elevation. Using a Michaelis-Menten model with appropriate corrections for cytoplasmic Ca2+ buffering, we found that modulation extended the dynamic range of PMCA activity by increasing both the maximal pump rate and Ca2+ sensitivity (reduction of K(M)). A simple flux model shows how pump modulation and its reversal produce the initial overshoot of the biphasic [Ca2+](i) response. The modulation of PMCA activity enhanced the stability of Ca2+ signalling by adjusting the efflux rate to match influx through CRAC channels, even at high [Ca2+](i) levels that saturate the transport sites and would otherwise render the cell defenceless against additional Ca2+ influx. At the same time, the delay in modulation enables small Ca2+ fluxes to transiently elevate [Ca2+](i), thus enhancing Ca2+ signalling dynamics.

Efficient gene transfer into human primary blood lymphocytes by surface-engineered lentiviral vectors that display a T cell–activating polypeptide

In contrast to oncoretroviruses, lentiviruses such as human immunodeficiency virus 1 (HIV-1) are able to integrate their genetic material into the genome of nonproliferating cells that are metabolically active. Likewise, vectors derived from HIV-1 can transduce many types of nonproliferating cells, with the exception of some particular quiescent cell types such as resting T cells. Completion of reverse transcription, nuclear import, and subsequent integration of the lentivirus genome do not occur in these cells unless they are activated via the T-cell receptor (TCR) or by cytokines or both. However, to preserve the functional properties of these important gene therapy target cells, only minimal activation with cytokines or TCR-specific antibodies should be performed during gene transfer. Here we report the characterization of HIV-1-derived lentiviral vectors whose virion surface was genetically engineered to display a T cell-activating single-chain antibody polypeptide derived from the anti-CD3 OKT3 monoclonal antibody. Interaction of OKT3 IgGs with the TCR can activate resting peripheral blood lymphocytes (PBLs) by promoting the transition from G(0) to G(1) phases of the cell cycle. Compared to unmodified HIV-1-based vectors, OKT3-displaying lentiviral vectors strongly increased gene delivery in freshly isolated PBLs by up to 100-fold. Up to 48% transduction could be obtained without addition of PBL activation stimuli during infection. Taken together, these results show that surface-engineered lentiviral vectors significantly improve transduction of primary lymphocytes by activating the target cells. Moreover these results provide a proof of concept for an approach that may have utility in various gene transfer applications, including in vivo gene delivery.

Recombinant chimeric OKT3/IgM antibodies for immune suppression: evaluation in a human CD3 transgenic mouse model

ScOKT3-γΔIgM VAEVD is a recombinant chimeric anti-CD3 antibody variant consisting of the light and heavy variable binding domains of the OKT3 monoclonal antibody and the CH3 and CH4 domains of a human IgM mutation linked by a human IgG3 hinge region. Due to the IgM Fc domains, scOKT3-γΔIgM VAEVD antibodies are able to form polymeric structures. Independent of their polymerization state, they possess in vitro CD3 modulating and immunosuppressive properties while inducing only minimal T cell activation compared to their monoclonal counterpart. To evaluate the in vivo efficacy of the antibodies, an adjuvant-induced chronic inflammation was established in human CD3 transgenic mice. Administration of four doses of 15 μg of isolated scOKT3-γΔIgM VAEVD monomers and pentamers significantly reduced diameters of inflamed ankle joints in a manner comparable to the monoclonal antibody OKT3. Additionally, the antibody treatment lead to a significant reduction of the cytokine levels (IL-2, TNF-α and INF-γ) in the mice's sera. These results suggest that scOKT3-γΔIgM VAEVD antibodies may provide a useful alternative to the OKT3 mAb for clinical immunosuppressive treatment for auto-aggressive diseases or for organ-transplantation.