OKT3 Treatment Research Articles

Myeloid and dendritic cells enhance therapeutics-induced cytokine release syndrome features in humanized BRGSF-HIS preclinical model.

Despite their efficacy, some immunotherapies have been shown to induce immune-related adverse events, including the potentially life-threatening cytokine release syndrome (CRS), calling for reliable and translational preclinical models to predict potential safety issues and investigate their rescue. Here, we tested the reliability of humanized BRGSF mice for the assessment of therapeutics-induced CRS features in preclinical settings. BRGSF mice reconstituted with human umbilical cord blood CD34+ cells (BRGSF-CBC) were injected with anti-CD3 antibody (OKT3), anti-CD3/CD19 bispecific T-cell engager Blinatumomab, or VISTA-targeting antibody. Human myeloid and dendritic cells' contribution was investigated in hFlt3L-boosted BRGSF-CBC mice. OKT3 treatment was also tested in human PBMC-reconstituted BRGSF mice (BRGSF-PBMC). Cytokine release, immune cell distribution, and clinical signs were followed. OKT3 injection in BRGSF-CBC mice induced hallmark features of CRS, specifically inflammatory cytokines release, modifications of immune cell distribution and activation, body weight loss, and temperature drop. hFlt3L-boosted BRGSF-CBC mice displayed enhanced CRS features, revealing a significant role of myeloid and dendritic cells in this process. Clinical CRS-managing treatment Infliximab efficiently attenuated OKT3-induced toxicity. Comparison of OKT3 treatment's effect on BRGSF-CBC and BRGSF-PBMC mice showed broadened CRS features in BRGSF-CBC mice. CRS-associated features were also observed in hFlt3L-boosted BRGSF-CBC mice upon treatment with other T-cell or myeloid-targeting compounds. These data show that BRGSF-CBC mice represent a relevant model for the preclinical assessment of CRS and CRS-managing therapies. They also confirm a significant role of myeloid and dendritic cells in CRS development and exhibit the versatility of this model for therapeutics-induced safety assessment.

PBMC-Humanized Mouse Model to Assess Individual Variability in Efficacy and Toxicity of Universal CD19 Chimeric Antigen Receptor T Cell Immunotherapy

Chimeric antigen receptor T cell (CART) immunotherapy has been accumulating extraordinary breakthroughs in fighting against hematological malignancies. Particularly, universal, “off-the-shelf”, CARTs (uCARTs) are emerging as an attractive alternative to patient-derived CARTs, offering cost-effectiveness, immediate availability for treatment, and independence from patients' lymphopenic conditions. However, uCARTs share the same safety concerns with autologous CARTs while the efficacy and persistence of uCARTs vary by each product and remain uncertain. Further, T cells sourced from healthy donors for uCARTs may manifest individual-specific immune characteristics upon interaction with cancer cells and the patient's immune system. These characteristics often remain latent when assessed in vitro and complicate the prediction of uCART therapy's toxicity and efficacy on an individual basis. We previously demonstrated that the PBMC-humanized mouse model can capture efficacy and toxicity of autologous and allogeneic CD19 CARTs. We showed that the CART-induced toxicity varied by the combination of PBMC donors and CART cell donors. Here, we extend the use of PBMC-humanized mice to assess the potential toxicity, efficacy, and persistence of uCARTs. Female NSG™-DKO mice (n = 19-20 mice per donor) were irradiated and reconstituted with human PBMCs sourced from four selected donors (Donor A, B, C, and D) out of 58 available in our PBMC bank. The selection criteria were based on their pre-characterized in vivo human cytokine induction profiles, specifically IFN-gamma response levels to OKT3 treatment (Donor A: 268 pg/ml; B: 1936 pg/ml; C: 4862 pg/ml; D: 13538 pg/ml). PBMC-humanized mice were grouped into three different CD19 CART treatments; a) Allogeneic CARTs were manufactured from a healthy donor's T cells containing CD28 costimulatory domain (19-28z) and no genetic modification was made for “off-the-shelf” availability; b) uCART 1 cells (19-28z) were knocked out of TCRab via CRISPR-cas9 (19-28z); c) uCART 2 cells were knocked out of CD3 and TCRab using TALENs and magnetically depleted for remaining CD3/TCRab, and contain 4-1BB costimulatory domain. Body weight change and clinical scores were measured throughout the experiments. We measured the levels of human CD45, human CD19, and CART expansion from blood collected 8 days post-treatment using flow cytometry. We measured 54 human cytokine levels from serum collected 48 hours post-treatment using a multiplex assay. CART-induced toxicity, measured by body weight loss, dramatically varied by PBMC donors and the treatments (Figure 1). uCART 2 induced significant body weight loss in Donor B and D reaching the lowest 6 days post-treatment. Notably, all mice humanized with Donor C and treated with uCART 2 reached a humane endpoint within 12 days post-treatment. We only observed noticeable body weight loss from uCART 1 in Donor C. Allogeneic CART did not induce body weight loss across the four donors. Efficacy, measured by the % of elimination of human CD19 cells in peripheral blood compared to the PBS-treated group, also varied by PBMC donor and the treatments (Figure 2). Overall, Allogeneic CART showed the best elimination of CD19 cells. Mice humanized with Donor D responded with the poorest efficacy of the three CART treatments. CART expansion was observed only in uCART 2 treated mice that reached a humane endpoint within 6 days post-treatment. CART persistence measured 16 days post-treatment was not observed across three CARTs and this corresponds to the clinical data of uCARTs. Principal component analysis of human cytokine data revealed that uCART 2 induced the most distinct cytokine responses compared to the other CARTs, driven by IFN-gamma, IL-10, IL-5, IL-3, and GM-CSF. In Donor-A humanized mice, the same set of human cytokines (IFN-gamma, IL-10, IL-5, IL-3, GM-CSF, IL-13) was induced in response to the three CARTs, while the degree of induction varied by treatment. In contrast, Donor D-humanized mice showed vastly different responses to each treatment; uCART 2 induced significant levels of IL-6, TNF-beta, MIG/CXCL9, MDC, MCP-3, IL-13, and IL-2. In sum, we show that PBMC-humanized mice capture individual variations in the toxicity and efficacy of uCARTs. This platform can be used to preclinically assess efficacy and persistence of uCARTs as well as used to predict toxicity from the interaction between the host and healthy T cell donor of uCARTs.

Abstract 4240: In vivo assessment of AICD and cytokine release upon treatment of OKT3 and CD3 bispecific antibody in homozygous CD3EDG HuGEMM™

Abstract Introduction: CD3 bispecific antibodies have transformed the therapeutic landscape of immunotherapy for some hematological malignancies, however, the clinical therapeutic effects of CD3 bispecific therapies is still under investigation for solid tumors. Moreover, CD3 bispecific antibody-mediated tumor targeting as well as T cell engagement have resulted in cytokine release syndrome (CRS), increased on-target/off-tumor toxicities and poor T cell infiltration, which may affect safety and limit therapeutic efficacy. To better understand the relationship between T cell activation and systemic cytokine release in the context of CD3 bispecific antibody treatment, a homozygous human CD3EDG HuGEMM࣪ preclinical model was developed. Methods: The homozygous CD3EDG HuGEMM mice, which express human CD3ε/CD3δ/CD3γ on the surface of T cells, were subcutaneously inoculated with MC38 or MC38-hEpCAM HuCELL™ syngeneic tumors and treated with vehicle, anti-mPD-1 (RMP1-14), OKT3, and anti-hCD3×hEpCAM. For efficacy studies, mouse body weight and tumor volume were measured twice weekly and tumor growth inhibition (TGI%) was determined. To investigate OKT3 or CD3 bispecifics-induced cytokine release, mouse body weight was monitored daily and several inflammatory and proinflammatory cytokines in the serum were detected by MSD. Results: Firstly, anti-mPD-1 treatment (5 mg/kg and 15 mg/kg i.p.) of homozygous CD3EDG mice engrafted with MC38 syngeneic tumors led to 42% and 62% TGI, respectively. OKT3 treatment of MC38-hEpCAM bearing homozygous CD3EDG HuGEMM promoted tumor growth (TGI= -80%), likely due to activation-induced cell death (AICD) of T cells. In addition, CRS was induced within 24 hours of OKT3 dosing, with IL-2, IL-6, and TNF-α peaking 2 hr post the 1st dose, and IFN-γ, IL-10 and IL-5 peaking 6 hr post the 1st dose. In contrast, no significant cytokine release was observed upon anti-hCD3×hEpCAM treatment despite remarkable TGI, indicating that the delicate balance of in vivo T cell activation and T cell cytotoxicity is crucial for effective tumor killing by CD3 bispecific antibodies or engagers. Conclusions: Our CD3EDG HuGEMM and HuCELL models provide a predictive platform for in vivo efficacy and safety assessment of CD3 bispecific or trispecific antibodies. The uncoupling of systemic cytokine release and T cell cytotoxicity via CD3 bispecific or trispecific antibodies provides a biological rationale to clinically explore preventative treatment approaches to avoid or reduce CRS-related toxicity, as well as combination approaches to increase selectivity and efficacy. Citation Format: Daniel XF He. In vivo assessment of AICD and cytokine release upon treatment of OKT3 and CD3 bispecific antibody in homozygous CD3EDG HuGEMM™ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4240.

A donor-dependent in vivo model for single agent and drug combination cytokine release syndrome safety evaluation.

2612 Background: Although antibodies and CART cells therapies have been successfully used for cancer therapy, they can have lethal adverse effects such as cytokine release syndrome (CRS). The animal models and in vitro human PBMC assays presently in use can’t reliably predict the CRS in patients. A predictive marker for identifying patients at risk for developing CRS upfront would improve the safety of immune-oncology drug development. Methods: We have developed a rapid, sensitive and reproducible in vivo humanized mouse model for quantitating CRS. The NSG mouse and its derivatives are engrafted with human PBMCs. On day 6 we induced cytokines release with pembrolizumab, avelumab, atezolizumab, ipilimumab, anti-CD28, ATG and OKT3 in single dose; as well as combination treatments involving pembrolizumab, lenalidomide, ATG and anti-CD28. Furthermore, we compared our method versus the in vitro PBMC assay. The cytokine levels were also compared to the dose response. Results: There are about 10-15% CD45+ human cells on day 5 of engraftment; and among of them, there were approximately 70% CD3 T cells and 25% CD56 NK cells. All tested cytokines, human IFN-γ, IL-2, IL-4, IL-6, IL-10 and TNF were upregulated after 2 and 6 hours of OKT3, ATG, anti-CD28, pembrolizumab, avelumab and atezolizumab drug treatment. Mouse’s rectal temperatures dropped from 37-38 °C to about 36 °C at 6 hours’ time point in the treated groups. There is various cytokines release levels, low to high response in different donors with anti-CD28 treatment. All donors showed high response to OKT3. The cytokine release levels were consistent with a dose response or variable PBMC engraftment. The cytokine levels were also higher in some drug combination studies such as pembrolizumab combined with lenalidomide or ATG; anti-CD28 combined with ATG. Our in vivo method was able to determine CRS missed in the in vitro testing method. Conclusions: We have developed a rapid, sensitive and reproducible novel in vivo PBMC humanized mouse model that is able to differentiate human PBMC donors based on individual safety response to single agent and combination therapeutics of immune checkpoint inhibitors and possibly CAR-T therapy. This assay could be employed in future drug development.

LONG-TERM SURVIVAL AFTER CARDIAC TRANSPLANTATION: THE BRITISH COLUMBIA EXPERIENCE

Orthotopic heart transplantation (OHT) continues to be the gold standard for the management of patients with end-stage heart failure. However, there are regional variations in perioperative care and outcomes. Long-term survival data published by the International Society of Heart and Lung Transplantation may not be reflective of center specific outcomes. We undertook this analysis to review our single center outcomes in patients receiving OHT. This is a retrospective cohort analysis of all patients who underwent OHT between December 1, 1988 and October 21, 2014 through the BC Heart Transplant Program. Overall long-term survival at 25 years was determined. Survival at 10-years was stratified by induction strategy, sex and treatment era (early: 1998-2001, late: 2002-2014), and compared using the log-rank test. Over the study period, a total of 406 patients were transplanted, 6 of whom were re-transplanted and excluded from our analysis. Of the remaining 400 patients, 307 (76.8%) were males and the average age was 52.1 years. The median follow-up time was 6.6 years. Overall long-term survival was 34.5% at 25 years with a median survival of 15.5 years (Figure 1). Conditional survival beyond 1-year was 41.3% at 25 years with a median survival of 18.5 years. Survival at 10-years in the early era (59.8%) was significantly lower versus late era (76.9%; p=0.04). Survival at 10-years amongst male (68.4%) versus female (72.0%) recipients was similar (p=0.67). Basiliximab, rabbit anti-thymocyte globulin (RATG), and OKT3 were used in 184, 99, and 87 patients, respectively. There was a significant difference in 10-year survival amongst the basiliximab (78.8%) RATG (68.7%) and OKT3 (55.2%) treatment groups (p=0.02), but no difference between the basiliximab and RATG groups (p=0.70). Our long-term survival for OHT patients is acceptable at 34.5 % at 25 years. There are no differences in outcomes based on the commonly used induction agents or sex. The difference in survival based on treatment era may be driven by choice of induction and immunosuppression strategies as well as improvements in pre- and post-transplant care.

Clinical and pathological analysis of acute rejection following orthotopic liver transplantation.

Acute rejection is one of the most important factors for prognosis following liver transplantation. With the use of potent immunosuppressants, acute rejection does not always present typical manifestations. Moreover, other complications often occur concomitantly after liver transplantation, which makes early diagnosis of acute rejection more difficult. Acute rejection is best diagnosed by liver biopsy. Differentiation of clinical manifestations and pathological features plays an important role in achieving individualized immunosuppressive treatment and prolonging long term survival of patients given orthotopic liver transplants. From January 2004 to December 2006, 516 orthotopic liver transplantations were performed at the First Affiliated Hospital, Sun Yat-sen University. For patients who suffered acute rejection, clinical manifestations, histopathological features, diagnosis and anti-rejection treatment were summarized and analyzed. In 86 cases (16.7%), of the 516 recipients, 106 episodes of acute rejection occurred, which included 9 with histopathological borderline changes, 36 Banff I rejections, 48 Banff II and 13 Banff III. Among these, 36 were cured by adjusting the dose of immunosuppressant and 65 were reversed by methylprednisolone pulse treatment. Five were methylprednisolone resistant, 3 of whom were given OKT3 treatment and 2 underwent liver retransplantation. Due to potent immunosuppressive agents, acute rejection following an orthotopic liver transplantation lacks typical clinical manifestations and pathological features. Acute rejection is best diagnosed by liver biopsy. Designing rational individualized immunosuppressive regimen based on clinical and pathological features of acute rejection plays an important role in prolonging long term survival of patients.

OKT3 prevents xenogeneic GVHD and allows reliable xenograft initiation from unfractionated human hematopoietic tissues

AbstractImmunodeficient mice are now readily engrafted with human hematopoietic cells. However, these mice are susceptible to graft-versus-host disease (GVHD) induced by the engraftment and rapid expansion of coinjected human T cells. Therefore, highly purified sample populations must be used, adding significant time, expense, and effort. Here, we have explored in vivo and in vitro methods utilizing anti-T–cell antibodies to circumvent this problem. Intraperitoneal injection of the antibody within 48 hours prevented GVHD. Alternatively, short-term in vitro incubation of cells with antibody immediately before transplant was equally effective. Although in vitro antithymocyte globulin treatment resulted in a dramatic loss of SCID-repopulating cells (SRCs), treatment with OKT3 or UCHT1 abrogated GVHD risk and preserved engraftment potential. Leukemia samples that presented with substantial human T-cell contamination were effectively rescued from GVHD. In addition, OKT3 treatment of unfractionated cord blood resulted in robust engraftment of primary and secondary mice that was indistinguishable from grafts obtained using purified CD34+cells. Limiting dilution analysis of unfractionated blood demonstrated a SRC frequency of 1 in 300 to 500 CD34+cells, similar to that of purified hematopoietic stem and progenitor cells. This protocol streamlines xenograft studies while significantly reducing the cost and time of the procedure.

Steroid-resistant acute rejection after cadaveric liver transplantation: Experience from one single center

Steroid-resistant acute rejection (SRAR) is an infrequent event under current immunosuppressant but still a risk factor leading to graft loss and patients' death after liver transplantation. There are several strategies for managing this complication according to current literatures, but none of the treatment seems convincing and widely accepted. Here we retrospectively analyzed the clinical data of a cohort of patients to gain an insight into this complication. A total of 962 adult patients receiving whole liver grafts at a single center between January 2004 and December 2012 were studied. One hundred and forty-two recipients experienced 158 episodes of acute rejection after the operation, 14 recipients had no response to steroid bolus treatment. The clinical data was analyzed retrospectively. Incidence rate of acute rejection after liver transplant in our single center was 14.7% (142/962), among them 8.8% (14/158) were steroid-resistant. These episodes occurred on 19days (6-72days) after the operation, 3 were controlled by anti-T3-receptor antibody (OKT3) treatment, 4 were reversed by IL-2 receptor inhibitors combining with MMF treatment, 2 were reversed by antithymocyte globulin (ATG) treatment. Five did not recover and 2 received retransplantation. Mortality associated with SRAR was 28.6% (4/14, 1 died from acute liver failure, 1 from chronic liver failure, 1 from renal failure after retransplantation and 1 from pulmonary infection after OKT3 treatment). SRAR is a severe complication with high mortality after liver transplantation; ATG might serve as a potential treatment.

Mortality after Steroid Resistant Acute Cellular Rejection and Chronic Rejection Episodes in Adult Intestinal Transplant: Report from a Single Center in Induction/Preconditioning Era

Aim: Steroid resistant ACR and chronic rejection (CR) are still a major concern after intestinal transplantation, especially using grafts without liver: over last 20 years the presence of liver has been shown to have a protective effect on rejection episodes. We report our experience from a single center on adult recipients. Subjects and Methods: We transplanted 48 adult recipients using 49 grafts, from 2001 up to 2011: 85.7% of them were represented by grafts without liver (isolated or modified multivisceral allografts). Induction protocol was used initially on 12 transplants, accomplished by Daclizumab plus Tacrolimus and Steroids, while later we developed a preconditioning protocol, based on Alemtuzumab plus Tacrolimus, on 35 allografts; in 2 cases we preconditioned the recipients by Thymoglobulin followed by Tacrolimus. Results: Overall patient survival was 41.5 % at 10 years while graft survival was 39%. Steroid resistant ACR population: 13 recipients experienced episodes of steroid resistant ACR. Three of them were successfully treated by OKT3, 1 by Alemtuzumab and 1 by Thymoglobuline, while 1 case underwent re-transplantation after OKT3 treatment. Seven recipients died after ACR therapy mainly for sepsis: 2 after Thymoglobulin, 2 with OKT3, 1 after Infliximab, and 2 after combined treatment (Alemtuzumab plus Thymoglobulin and Thymoglobulin plus Infliximab). Twelve allografts out of 13 were grafts without liver. Chronic rejection population: 5 recipients were affected by CR (1 of them primarly transplanted in another center): 4 of them experienced indeterminate to mild episodes of ACR (only one as early as 30 days after transplant) before developing CR while only 1 had a severe episode of rejection. Two of them are still alive: 1 underwent graftectomy without increasing immunosuppressive treatment and was relisted on TPN while 1 recovered oral feeding (supported by TPN) after partial bowel resection plus stoma. The remnant 3 patients died for sepsis, 1 after retransplantation. Majority of them had mesenteric fibrosis as main pathologic feature of CR and diagnosis was always performed on resected specimen. Overall mortality: Steroid resistant ACR mortality was 50% while CR mortality was 60%: overall, 10 patients out of 19 (52.6%) died after steroid resistant ACR or CR. Notably, mortality after Daclizumab was 17.7% in steroid resistant ACR and CR population while after Alemtuzumab the percentage achieved 28.8%. The remnant transplant population (no ACR or steroid sensitive ACR - 27 patients) was affected by mortality percentage of 40.7% (p= NS). Conclusions: In our series steroid resistant ACR affected 29.1 % of our intestinal allografts (all but one without liver) while CR was shown on 8% of our resected bowels, all isolated grafts: CR was often preceded by ACR episodes. Mortality related to steroid resistant ACR and CR still affects intestinal transplant population in induction/preconditioning era.

Mortality after Steroid Resistant Acute Cellular Rejection and Chronic Rejection Episodes in Adult Intestinal Transplant: Report from a Single Center in Induction/Preconditioning Era

Aim: Steroid resistant ACR and chronic rejection (CR) are still a major concern after intestinal transplantation, especially using grafts without liver: over last 20 years the presence of liver has been shown to have a protective effect on rejection episodes. We report our experience from a single center on adult recipients. Subjects and Methods: We transplanted 48 adult recipients using 49 grafts, from 2001 up to 2011: 85.7% of them were represented by grafts without liver (isolated or modified multivisceral allografts). Induction protocol was used initially on 12 transplants, accomplished by Daclizumab plus Tacrolimus and Steroids, while later we developed a preconditioning protocol, based on Alemtuzumab plus Tacrolimus, on 35 allografts; in 2 cases we preconditioned the recipients by Thymoglobulin followed by Tacrolimus. Results: Overall patient survival was 41.5 % at 10 years while graft survival was 39%. Steroid resistant ACR population: 13 recipients experienced episodes of steroid resistant ACR. Three of them were successfully treated by OKT3, 1 by Alemtuzumab and 1 by Thymoglobuline, while 1 case underwent re-transplantation after OKT3 treatment. Seven recipients died after ACR therapy mainly for sepsis: 2 after Thymoglobulin, 2 with OKT3, 1 after Infliximab, and 2 after combined treatment (Alemtuzumab plus Thymoglobulin and Thymoglobulin plus Infliximab). Twelve allografts out of 13 were grafts without liver. Chronic rejection population: 5 recipients were affected by CR (1 of them primarly transplanted in another center): 4 of them experienced indeterminate to mild episodes of ACR (only one as early as 30 days after transplant) before developing CR while only 1 had a severe episode of rejection. Two of them are still alive: 1 underwent graftectomy without increasing immunosuppressive treatment and was relisted on TPN while 1 recovered oral feeding (supported by TPN) after partial bowel resection plus stoma. The remnant 3 patients died for sepsis, 1 after retransplantation. Majority of them had mesenteric fibrosis as main pathologic feature of CR and diagnosis was always performed on resected specimen. Overall mortality: Steroid resistant ACR mortality was 50% while CR mortality was 60%: overall, 10 patients out of 19 (52.6%) died after steroid resistant ACR or CR. Notably, mortality after Daclizumab was 17.7% in steroid resistant ACR and CR population while after Alemtuzumab the percentage achieved 28.8%. The remnant transplant population (no ACR or steroid sensitive ACR - 27 patients) was affected by mortality percentage of 40.7% (p= NS). Conclusions: In our series steroid resistant ACR affected 29.1 % of our intestinal allografts (all but one without liver) while CR was shown on 8% of our resected bowels, all isolated grafts: CR was often preceded by ACR episodes. Mortality related to steroid resistant ACR and CR still affects intestinal transplant population in induction/preconditioning era.

De Novo Crohn’s Disease in a Renal Transplant Recipient

The prevalence of inflammatory bowel disease (IBD) after renal transplantation is affected by the immune tolerance and the modality of immunosuppression. Mycophenolate mofetil (MMF) may have a promoting effect on the development of posttransplantation erosive enterocolitis and a Crohn’s disease-like pattern of colitis. We have presented a 40-year-old man with end-stage renal disease due to chronic glomerulonephritis who commenced hemodialysis for 2 months before receipt of a live unrelated renal transplant. He developed early posttransplantation diabetes mellitus and an anti graft rejection episode, which responded to a methylprednisolone pulse and OKT3 treatment. His immunosuppressive regimen included prednisolone, MMF, and tacrolimus. Three years after transplantation, he developed mild constitutional symptoms, mouth ulcerations, and chronic intermittent bloody diarrhea. Colonoscopy showed active segmental colitis with aphthous ulcers, involving the proximal descending colon and the splenic flexure. Colonic biopsies showed distended and branched crypts in the ascending colon, moderate active chronic colitis with regenerative atypia, skipping appearance, and ulceration in the splenic flexure and descending colon. The edematous crypts were associated with ulcerations in the sigmoid colon and rectum. The features were highly suggestive of Crohn’s disease. He was successfully treated with high-dose steroids and 5-aminosalicylic acid. Subsequently, he developed chronic transplant glomerulopathy and restarted hemodialysis. We concluded that de novo Crohn’s disease may develop in renal transplant recipients despite immunosuppressive therapy especially with MMF immunosuppression.

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.

OKT3 Therapy in Addition to Tacrolimus Is Associated with Improved Long-Term Function in Patients with Steroid Refractory Renal Allograft Rejection

Background/Aims: The aim of this study was to evaluate long-term allograft salvage rates of patients with steroid refractory allograft rejection after kidney transplantation and to identify factors indicating a successful outcome. Patients and Methods: Fifty patients with continuing rejection after high-dose steroids were included in the study. Baseline immunosuppression was switched from cyclosporine to tacrolimus in all patients. Twenty patients additionally received OKT3 as antirejection therapy. Patients having received a cadaveric renal transplant in 1995, excluding patients with steroid resistant rejection, were chosen as a control cohort. Results: Patient survival rates were 96% (n = 48) and 90% (n = 45) and allograft survival rates were 66% (n = 33) and 62% (n = 31) after 5 and 7 years following steroid refractory renal allograft rejection. Graft survival within the control cohort was 73% after 5 years and 69% after 7 years. Creatinine clearance increased from 20 ± 15 ml/min/1.73 m² at the start of tacrolimus therapy to 37 ± 29 ml/min/1.73 m² and to 32 ± 26 ml/min/1.73 m² after 5 and 7 years. OKT3 treatment predicted successful rescue therapy (p = 0.005 and p = 0.04 after 5 and 7 years). Conclusion: Our data indicate a reasonable graft survival in steroid refractory renal allograft rejection using tacrolimus. OKT3 treatment in addition to tacrolimus therapy may be beneficial for long-term allograft survival.

OKT3 Treatment for Steroid-Resistant Acute Rejection in Kidney Transplantation

Orthoclone (OKT3, Ortho Biotech Inc, USA) monoclonal antilymphocyte antibody is a powerful T-cell–specific immunosuppressive agent. OKT3 has been used for induction therapy in kidney and liver transplantation, as well as to treat acute or steroid-resistant acute rejection episodes (ARE). This study was a retrospective analysis of 43 renal transplant recipients who developed steroid-resistant ARE and were treated with OKT3 between September 1994 and June 2004. The recipients were 36 men and 7 women of mean age 32.7 ± 11.6 years (range, 19 to 48 years). The mean time from transplantation to OKT3 treatment was 7.2 ± 6.7 months. Thirty-four episodes (79.1%) responded to OKT3 therapy with improved graft function, but the remaining 9 (20.9%) grafts did not respond. Among the 34 OKT3 responders, the mean serum creatinine decreased from 3.96 ± 2.5 mg/dL to 2.45 ± 1.77 mg/dL after treatment. Eleven (25.6%) of the 43 patients experienced minor side effects: fever, dyspnea, tachycardia, bradycardia. One patient (2.3%) developed acute pulmonary edema; one (2.3%), cytomegalovirus infection; and eight (18.6%), bacterial infections. The 1-, 3-, and 5-year graft survival rates for the 34 patients who responded to OKT3 therapy were 96%, 93%, and 85%, respectively. All patients are currently alive. The results indicate that OKT3 is a safe, effective treatment choice for steroid-resistant ARE in kidney transplantation.

Changing impact of cytomegalovirus in liver transplantation - a single centre experience of more than 1000 transplantations without ganciclovir prophylaxis

As cytomegalovirus (CMV) disease was a leading cause of death following liver transplantation in earlier reports, general CMV prophylaxis is widely used. We re-evaluated the impact of CMV in a recent time period under balanced immunosuppression and effective CMV diagnostics and therapy. A retrospective analysis of 1200 liver transplantations between 1988 and 2000 was performed comparing the incidence of CMV infection and disease and patient survival rates in two different time periods (before and after availability of the pp65-antigenaemia assay). In addition, risk factors for CMV in the recent time period were analysed. No ganciclovir prophylaxis was administered during the whole study period. The incidence of CMV tissue invasive disease decreased from 9.4% in period I to 2.7% in period II, whereas the incidence of viral syndrome was about 6% in both periods. Especially CMV pneumonia and generalized disease were almost abandoned in period II. Patients with tissue invasive disease, but not with infection or viral syndrome had reduced survival rates in both periods. However, the disease-specific mortality was 10% and 0% respectively. The overall rate of CMV infection in period II was low (25.9%). Risk factors for CMV infection in the univariate analysis were: Initial nonfunction, D+R- seroconstellation, acute liver failure, triple or quadruple immunosuppression, OKT3 or ATG treatment, transfusion of >10 packed red cells, steroid boluses, postoperative mechanical ventilation and retransplantation. In the multivariate analysis only quadruple or triple immunosuppression, OKT3-treatment, transplantation for acute liver failure and initial nonfunction. The incidence of CMV tissue invasive disease as well as the disease-specific mortality has markedly decreased during the last years. Using routine surveillance with the pp65-antigenaemia assay, CMV infection and disease rates compare well to data with long-term ganciclovir prophylaxis. As D+R- patients still more often develop symptomatic disease, pre-emptive therapy could be useful in this patient group.

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.

Repeated Steroid Pulse Therapies in HCV-Positive Liver Recipients: Significant Risk Factor for HCV-Related Graft Loss

Background The optimal immunosuppressive regimen for HCV-positive liver transplant recipients has not been established. Treatment for acute cellular rejection (ACR) with steroids is associated with increased viral replication and graft hepatitis. We retrospectively analyzed 232 patients after orthotopic liver transplantation (OLT) for HCV cirrhosis to determine the influence of methylprednisolone pulse therapy on long-term outcome after OLT. Methods Two hundred thirty-two liver transplants were performed in HCV-positive recipients between 1989 and 2001. Median follow-up was 4.4 years and median age of patients was 53 years (range 15 to 72 years). Immunosuppression consisted of tacrolimus (Tac) or cyclosporine (CyA) in different protocols. All rejection episodes were histologically proven. Results Twenty-eight of 232 (12.06%) graft losses were due to severe hepatitis C reinfection. Of 232 patients, 105 showed a minimum of one episode of ACR (45.25%). Of 232 patients, 71 (30.6%) received methylprednisolone pulse therapy once and 15 of 232 required OKT3 treatment for steroid-resistant rejection (6.4%). Of 232 patients, 19 (8.1%) required repeated steroid pulse therapy due to more than one episode of ACR. In patients with more than one episode of ACR, the risk of HCV-related graft loss was significantly enhanced (6/19, P < .05). The primary immunosuppression had no influence on the outcome in our data. Conclusion Our data show that outcome of HCV-positive patients who require repeated steroid pulse therapy (RSPT) for ACR is significant worse than that in patients with a single pulse therapy. Therefore RSPT should be avoided in HCV-positive transplant recipients. New strategies to manage acute rejection are required for these patients.

Anti-TNF-Alpha Therapy for Acute Rejection in Intestinal Transplantation

Introduction We present our experience with infliximab rescue therapy for steroid- and OKT3-resistant rejection after intestinal transplantation (ITx). Methods Twelve ITx and one multivisceral transplant recipients were immunosuppressed with tacrolimus, rapamycin, daclizumab, steroids ( n = 10) or tacrolimus, campath, and steroids ( n = 3). Results In two patients, severe acute rejection did not resolve despite steroid bolus therapy plus 5 to 10 days of OKT3 treatment. Signs of moderate rejection persisted in the distal portions of the grafts. Treatment with infliximab, a chimeric anti-TNF-alpha antibody (four infusions of 3 mg/kg body weight), induced a complete remission of histological and clinical signs of rejection. Two further patients with steroid-resistant rejection received two courses of infliximab (3 mg/kg body weight) as antirejection therapy. All rejection episodes resolved completely. Conclusions Infliximab effectively treats steroid and OKT3 resistant acute rejection episodes of intestinal transplantations.

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.

Anti-CD3 antibodies OKT3 and hOKT3γ1(Ala-Ala) induce proliferation of T cells but impair expansion of alloreactive T cells; aspecifc T cell proliferation induced by Anti-CD3 antibodies correlates with impaired expansion of alloreactive T cells

OKT3, a mouse anti-human CD3 monoclonal antibody (mAb), has been used for decades to reverse acute transplant rejection. A humanized OKT3 with two alanine replacements on the CH2 domain, hOKT3γ1(Ala-Ala), was generated to reduce side effects of OKT3. This study reports the effects of OKT3 and hOKT3γ1(Ala-Ala) on responder T cells in mixed leukocyte cultures (MLC). T cells were purified from peripheral blood mononuclear cells (PBMC) and labeled with CFSE before culture with irradiated allogeneic PBMC in a MLC. Multiparameter flow cytometric analysis of MLC demonstrated logarithmic expansion of T cells with a memory phenotype. Treatment with either OKT3 or OKT3γ1(Ala-Ala) prevented the logarithmic expansion of these cells. Instead, both OKT3 and hOKT3γ1(Ala-Ala) induced nonspecific expansion of T cells with both naïve and memory phenotype. The proliferating T cells in OKT3 or hOKT3γ1(Ala-Ala) treated cultures expressed low levels of CD25, and showed a diminished Granzyme B expression compared to that of the untreated MLC, suggesting that these cells may not be fully functional. An array of cytokines in the MLC supernatant was analyzed, and IL-5 and IL-13 were significantly reduced by OKT3 or hOKT3γ1(Ala-Ala) treatment. These results demonstrated that OKT3 and hOKT3γ1(Ala-Ala) have similar effects on T cells in MLC. Both antibodies inhibited the logarithmic expansion of allo-reactive T cells, probably through inducing suboptimal proliferation of specific and non-specific T cells. This in vitro study provided one possible mechanism of the therapeutic effects of OKT3 and hOKT3γ1(Ala-Ala) antibodies.