Successful use of anti-CD19 CAR T cells in severe treatment-refractory stiff-person syndrome.

Treatment with autologous chimeric antigen receptor (CAR) T cells has emerged as a highly effective approach in neuroimmunological disorders such as myasthenia gravis. We report a case of successful anti-CD19 CAR T cell use in treatment-refractory stiff-person syndrome (SPS). To investigate clinical and immunological effects of anti-CD19 CAR T cell use in treatment-refractory SPS, a 69-y-old female with a 9-y history of treatment-refractory SPS with deteriorating episodes of stiffness received an infusion of autologous anti-CD19 CAR T cells (KYV-101) and was monitored clinically and immunologically for more than 6 mo. CAR T cell infusion resulted in reduced leg stiffness, drastic improvement in gait, walking speed increase over 100%, and daily walking distance improvement from less than 50 m to over 6 km within 3 mo. GABAergic medication (benzodiazepines) was reduced by 40%. KYV-101 CAR T cells were well tolerated with only low-grade cytokine release syndrome. This report of successful use of anti-CD19 CAR T cells in treatment-refractory SPS supports continued exploration of this approach in SPS and other B cell-related autoimmune disorders.

Safety, pharmacokinetics, and pharmacodynamic activity of obinutuzumab, a type 2 anti-CD20 monoclonal antibody for the desensitization of candidates for renal transplant.

The limited effectiveness of rituximab plus intravenous immunoglobulin (IVIG) in desensitization may be due to incomplete B cell depletion. Obinutuzumab is a type 2 anti-CD20 antibody that induces increased B cell depletion relative to rituximab and may therefore be more effective for desensitization. This open-label phase 1b study assessed the safety, pharmacokinetics, and pharmacodynamics of obinutuzumab in highly sensitized patients with end-stage renal disease. Patients received 1 (day 1, n = 5) or 2 (days 1 and 15; n = 20) infusions of 1000-mg obinutuzumab followed by 2 doses of IVIG on days 22 and 43. Eleven patients received additional obinutuzumab doses at the time of transplant and/or at week 24. The median follow-up duration was 9.4 months. Obinutuzumab was well tolerated, and most adverse events were grade 1-2 in severity. There were 11 serious adverse events (SAEs) in 9 patients (36%); 10 of these SAEs were infections and 4 occurred after kidney transplant. Obinutuzumab plus IVIG resulted in profound peripheral B cell depletion and appeared to reduce B cells in retroperitoneal lymph nodes. Reductions in anti-HLA antibodies, number of unacceptable antigens, and the calculated panel reactive antibody score as centrally assessed using single-antigen bead assay were limited and not clinically meaningful for most patients (NCT02586051).

Clinical pharmacology of AMG 181, a gut-specific human anti-α4ß7 monoclonal antibody, for treating inflammatory bowel diseases.

AIMS: AMG 181 pharmacokinetics/pharmacodynamics (PK/PD), safety, tolerability and effects after single subcutaneous (s.c.) or intravenous (i.v.) administration were evaluated in a randomized, double-blind, placebo-controlled study. METHODS: Healthy male subjects (n= 68) received a single dose of AMG 181 or placebo at 0.7, 2.1, 7, 21, 70 mg s.c. (or i.v.), 210 mg s.c. (or i.v.), 420 mg i.v. or placebo. Four ulcerative colitis (UC) subjects (n= 4, male : female 2:2) received 210 mg AMG 181 or placebo s.c. (3:1). AMG 181 concentration, anti-AMG 181-antibody (ADA), α4 ß7 receptor occupancy (RO), target cell counts, serum C-reactive protein, fecal biomarkers and Mayo score were measured. Subjects were followed 3-9 months after dose. RESULTS: Following s.c. dosing, AMG 181 was absorbed with a median tmax ranging between 2-10 days and a bioavailability between 82% and 99%. Cmax and AUC increased dose-proportionally and approximately dose-proportionally, respectively, within the 70-210 mg s.c. and 70-420 mg i.v. ranges. The linear ß-phase t1/2 was 31 (range 20-48) days. Target-mediated disposition occurred at serum AMG 181 concentrations of less than 1 µg ml(-1) . The PD effect on α4 ß7 RO showed an EC50 of 0.01 µg ml(-1) . Lymphocytes, eosinophils, CD4+ T cells and subset counts were unchanged. AMG 181-treated UC subjects were in remission with mucosal healing at weeks 6, 12 and/or 28. The placebo-treated UC subject experienced colitis flare at week 6. No ADA or AMG 181 treatment-related serious adverse events were observed. CONCLUSIONS: AMG 181 has PK/PD, safety, and effect profiles suitable for further testing in subjects with inflammatory bowel diseases.

Chimeric antigen receptor T cell therapy for autoimmune disease.

Infusion of T cells engineered to express chimeric antigen receptors (CARs) that target B cells has proven to be a successful treatment for B cell malignancies. This success inspired the development of CAR T cells to selectively deplete or modulate the aberrant immune responses that underlie autoimmune disease. Promising results are emerging from clinical trials of CAR T cells targeting the B cell protein CD19 in patients with B cell-driven autoimmune diseases. Further approaches are being designed to extend the application and improve safety of CAR T cell therapy in the setting of autoimmunity, including the use of chimeric autoantibody receptors to selectively deplete autoantigen-specific B cells and the use of regulatory T cells engineered to express antigen-specific CARs for targeted immune modulation. Here, we highlight important considerations, such as optimal target cell populations, CAR construct design, acceptable toxicities and potential for lasting immune reset, that will inform the eventual safe adoption of CAR T cell therapy for the treatment of autoimmune diseases.

Treatment of concomitant myasthenia gravis and Lambert-Eaton myasthenic syndrome with autologous CD19-targeted CAR T cells.

Myasthenia gravis (MG) and Lambert-Eaton myasthenic syndrome (LEMS) are autoimmune disorders affecting neuromuscular transmission. Their combined occurrence is rare, and treatment remains challenging. Two women diagnosed with concomitant MG/LEMS experienced severe, increasing disease activity despite multiple immunotherapies. Anti-CD19 chimeric antigen receptor (CAR) T cells have shown promise for treating autoimmune diseases. This report details the safe application of anti-CD19 CAR T cells for treating concomitant MG/LEMS. After CAR T cell therapy, both patients experienced rapid clinical recovery and regained full mobility. Deep B cell depletion and normalization of acetylcholine receptor and voltage-gated calcium channel N-type autoantibody levels paralleled major neurological responses. Within 2 months, both patients returned to everyday life, from wheelchair dependency to bicycling and mountain hiking, and remain stable at 6 and 4 months post-CAR T cell infusion, respectively. This report highlights the potential for anti-CD19 CAR T cells to achieve profound clinical effects in the treatment of neuroimmunological diseases.

CD19-targeted chimeric antigen receptor T cell therapy in two patients with multiple sclerosis.

BACKGROUND: Progressive multiple sclerosis (MS) is characterized by compartmentalized smoldering neuroinflammation caused by the proliferation of immune cells residing in the central nervous system (CNS), including B cells. Although inflammatory activity can be prevented by immunomodulatory therapies during early disease, such therapies typically fail to halt disease progression. CD19 chimeric antigen receptor (CAR)-T cell therapies have revolutionized the field of hematologic malignancies. Although generally considered efficacious, serious adverse events associated with CAR-T cell therapies such as immune effector cell-associated neurotoxicity syndrome (ICANS) have been observed. Successful use of CD19 CAR-T cells in rheumatic diseases like systemic lupus erythematosus and neuroimmunological diseases like myasthenia gravis have recently been observed, suggesting possible application in other autoimmune diseases. METHODS: Here, we report the first individual treatment with a fully human CD19 CAR-T cell therapy (KYV-101) in two patients with progressive MS. FINDINGS: CD19 CAR-T cell administration resulted in acceptable safety profiles for both patients. No ICANS was observed despite detection of CD19 CAR-T cells in the cerebrospinal fluid. In case 1, intrathecal antibody production in the cerebrospinal fluid decreased notably after CAR-T cell infusion and was sustained through day 64. CONCLUSIONS: CD19 CAR-T cell administration in progressive MS resulted in an acceptable safety profile. CAR-T cell presence and expansion were observed in the cerebrospinal fluid without clinical signs of neurotoxicity, which, along with intrathecal antibody reduction, indicates expansion-dependent effects of CAR-T cells on CD19+ target cells in the CNS. Larger clinical studies assessing CD19 CAR-T cells in MS are warranted. FUNDING: Both individual treatments as well the generated data were not based on external funding.

Similarities in the immunoglobulin response and VH gene usage in rhesus monkeys and humans exposed to porcine hepatocytes.

BACKGROUND: The use of porcine cells and organs as a source of xenografts for human patients would vastly increase the donor pool; however, both humans and Old World primates vigorously reject pig tissues due to xenoantibodies that react with the polysaccharide galactose alpha (1,3) galactose (alphaGal) present on the surface of many porcine cells. We previously examined the xenoantibody response in patients exposed to porcine hepatocytes via treatment(s) with bioartficial liver devices (BALs), composed of porcine cells in a support matrix. We determined that xenoantibodies in BAL-treated patients are predominantly directed at porcine alphaGal carbohydrate epitopes, and are encoded by a small number of germline heavy chain variable region (VH) immunoglobulin genes. The studies described in this manuscript were designed to identify whether the xenoantibody responses and the IgVH genes encoding antibodies to porcine hepatocytes in non-human primates used as preclinical models are similar to those in humans. Adult non-immunosuppressed rhesus monkeys (Macaca mulatta) were injected intra-portally with porcine hepatocytes or heterotopically transplanted with a porcine liver lobe. Peripheral blood leukocytes and serum were obtained prior to and at multiple time points after exposure, and the immune response was characterized, using ELISA to evaluate the levels and specificities of circulating xenoantibodies, and the production of cDNA libraries to determine the genes used by B cells to encode those antibodies. RESULTS: Xenoantibodies produced following exposure to isolated hepatocytes and solid organ liver grafts were predominantly encoded by genes in the VH3 family, with a minor contribution from the VH4 family. Immunoglobulin heavy-chain gene (VH) cDNA library screening and gene sequencing of IgM libraries identified the genes as most closely-related to the IGHV3-11 and IGHV4-59 germline progenitors. One of the genes most similar to IGHV3-11, VH3-11cyno, has not been previously identified, and encodes xenoantibodies at later time points post-transplant. Sequencing of IgG clones revealed increased usage of the monkey germline progenitor most similar to human IGHV3-11 and the onset of mutations. CONCLUSION: The small number of IGVH genes encoding xenoantibodies to porcine hepatocytes in non-human primates and humans is highly conserved. Rhesus monkeys are an appropriate preclinical model for testing novel reagents such as those developed using structure-based drug design to target and deplete antibodies to porcine xenografts.

Appraisal of the extent of chronic allograft vasculopathy in animal models: proposition of a standardized micromorphometric method.

Arterial obstruction due to intimal myproliferation is an outcome used for investigating chronic allograft vasculopathy in animal models. Since harvested tissue may be distorted while processing, discrepancy may happen in the measure of the obstruction. We propose a standardized micromorphometric method for calculating the percentage of obstruction with eliminating the variability in the shape of the vessel cross-section. The mathematical adjustment avoids the overestimation of the percentage of obstruction and improves accuracy.

Effects of JAK3 inhibition with CP-690,550 on immune cell populations and their functions in nonhuman primate recipients of kidney allografts.

BACKGROUND: Janus Kinase (JAK) 3 is a tyrosine kinase essential for proper signal transduction downstream of selected cytokine receptors and for robust T-cell and natural killer cells activation and function. JAK3 inhibition with CP-690,550 prevents acute allograft rejection. To provide further insight into the mechanisms of efficacy, we investigated the immunomodulatory effects of CP-690,550 in vitro and in vivo in nonhuman primates. METHODS: Pharmacodynamic assessments of lymphocyte activation, function, proliferation and phenotype were performed in three settings: in vitro in whole blood isolated from untransplanted cynomolgus monkeys (cynos), in vivo in blood from untransplanted cynos dosed with CP-690,550 for 8 days, and in vivo in blood from transplanted cynos immunosuppressed with CP-690,550. Cell surface activation markers expression, IL-2- enhanced IFN-gamma production, lymphocyte proliferation and immune cell phenotype analyzes were performed with multiparametric flow cytometry. RESULTS: In vitro exposure to CP-690,550 resulted in significant reduction of IL-2-enhanced IFN-gamma production by T-cells (maximum inhibition of 55-63%), T-cell surface expression of CD25 (50% inhibitory concentration (IC50); 0.18 microM) and CD71 (IC50; 1.6 microM), and T-cell proliferative capacities measured by proliferating cell nuclear antigen expression (IC50; 0.87 microM). Similar results were observed in animals dosed with CP-690,550. In addition, transplanted animals displayed significant reduction of NK cell (90% from baseline) and T-cell numbers whereas CD8 effector memory T-cell populations were unaffected. CONCLUSIONS: Potent in vitro and in vivo immunomodulatory effects of the JAK3 inhibitor CP-690,550 likely contribute to its efficacy in the prevention of organ allograft rejection.

Combined use of the JAK3 inhibitor CP-690,550 with mycophenolate mofetil to prevent kidney allograft rejection in nonhuman primates.

BACKGROUND: Immunosuppression via Janus kinase (JAK) 3 inhibition affords significant prolongation of allograft survival. We investigated the effects of an immunosuppressive regimen combining the JAK3 inhibitor CP-690,550 with mycophenolate mofetil (MMF) in nonhuman primates (NHPs). METHODS: Life-supporting kidney transplantations were performed between ABO-compatible, MLR-mismatched NHPs. Animals were treated orally twice a day with CP-690,550 and MMF (n=8) or MMF alone (n=2) and were euthanized at day 90 or earlier due to allograft rejection. RESULTS: Mean survival time (+/-SEM) in animals treated with MMF alone (23+/-1 days) was significantly extended in animals that concurrently received CP-690,550 (59.5+/-9.8 days, P=0.02). Combination animals exposed to higher levels of CP-690,550 had a significantly better survival (75.2+/-8.7 days) than animals that received less CP-690,550 (33.3+/-12.6 days, P=0.02). Three combination therapy animals were euthanized at day 90 with a subnormal renal function and early-stage acute graft rejection. Rejection, delayed by treatment, ultimately developed in other animals. Anemia and gastrointestinal intolerance was seen in combination therapy animals that otherwise did not show evidence of viral or bacterial infection besides signs consistent with subclinical pyelonephritis (n=3). One incidental lymphosarcoma was noted. CONCLUSIONS: Addition of CP-690,550 to MMF significantly improved allograft survival. The observed side effects appear amenable to improvements upon alteration of dosing strategies. Efficacy of this combination regimen suggests that it could become the backbone of calcineurin inhibitor-free regimens.

Immunosuppression by the JAK3 inhibitor CP-690,550 delays rejection and significantly prolongs kidney allograft survival in nonhuman primates.

BACKGROUND: Janus kinase 3 (JAK3) mediates signal transduction from cytokine receptors using the common chain (gammac). Because mutations in genes encoding gammac or JAK3 result in immunodeficiency, we investigated the potential of a rationally designed inhibitor of JAK3, CP-690,550, to prevent renal allograft rejection in nonhuman primates. METHODS: Life-supporting kidney transplantations were performed between mixed leukocyte reaction-mismatched, ABO blood group-matched cynomolgus monkeys. Animals were treated with CP-690,550 (n = 18) or its vehicle (controls, n = 3) and were euthanized at day 90 or earlier if there was allograft rejection. RESULTS: Mean survival time (+/- standard error of mean) in animals treated with CP-690,550 (53 +/- 7 days) was significantly longer than in control animals (7 +/- 1 days, P=0.0003) and was positively correlated with exposure to the drug (r = 0.79, P < 0.01). Four treated animals were euthanized at 90 days with a normal renal function and low-grade rejection at final pathology. Occurrence of rejection was significantly delayed in treated animals (46 +/- 7 days from transplantation vs. 7 +/- 1 days in controls, P = 0.0003). Persistent anemia, polyoma virus-like nephritis (n = 2), and urinary calcium carbonate accretions (n = 3) were seen in animals with high exposure. Natural killer cell and CD4 and CD8 T-cell numbers were significantly reduced in treated animals. Blood glucose, serum lipid levels, and arterial blood pressure were within normal range in treated animals, and no cancers were demonstrated. CONCLUSIONS: CP-690,550 is the first reported JAK3 inhibitor combining efficacy and good tolerability in a preclinical model of allotransplantation in nonhuman primates and thus has interesting potential for immunosuppression in humans.

JAK3 inhibition, a viable new modality of immunosuppression for solid organ transplants.

The field of organ transplantation has had tremendous success because of the availability of immunosuppressive drugs that efficiently prevent acute organ rejection. Numerous and severe side effects are, however, associated with all current immunosuppressive therapies and justify a search for drugs with better efficacy and safety profiles. Janus kinase (JAK) 3, a tyrosine kinase that is crucial for mediating signals from the common gamma-chain of cytokine receptors, is peculiar in that its expression, contrarily to the targets of most current immunosuppressive drugs, is limited to cells that actively participate to the immune response to allografts. The recent demonstration in stringent preclinical models that JAK3 inhibition results in efficacy for the prevention of allograft rejection with a narrow side-effect profile might lead to a new era in the field of immunosuppression.

Improved assessment of graft function by echocardiography in cynomolgus monkey recipients of hDAF-transgenic pig cardiac xenografts.

BACKGROUND: The current practice of evaluating heterotopic heart xenografts by palpation allows only detection of severe graft dysfunction, which indicates terminal graft failure. Therefore, we evaluated whether echocardiography is a better method of detecting early graft dysfunction as a marker of rejection in abdominal pig heart xenografts in cynomolgus monkeys. METHODS: Six cynomolgus monkeys received heterotopic heart transplants from pig donors transgenic for human decay-accelerating factor (hDAF). Induction therapy consisted of either cyclophosphamide or rabbit anti-thymocyte globulin. Maintenance therapy consisted of cyclosporine or tacrolimus, steroids, and sodium mycophenolate or mycophenolate mofetil, GAS914 (alphaGal oligosaccharide containing glycoconjugate), and for some animals TP10 (soluble complement receptor type 1). Echocardiography was performed immediately after transplantation and 3 times a week after surgery. We scored contractility and measured left ventricular wall thickness. Impaired contractility or increased wall thickness were considered graft dysfunction and were treated with pulse steroids. Palpation score was recorded daily. We also obtained myocardial biopsy specimens. RESULTS: Palpation score remained at 4 out of 4 in all animals until 2 to 5 days before final graft failure, whereas echocardiography detected several episodes of impaired graft function, either decreased left ventricular contractility or increased left ventricular wall thickness before graft failure. Treatment with pulse steroids improved graft function only during early episodes of graft impairment. Final graft failure was steroid resistant and caused by severe vascular rejection. CONCLUSIONS: Echocardiography is a better method of assessing graft dysfunction than is palpation. Therefore, echocardiography may detect early rejection episodes of heterotopic heart xenografts in non-human primates.

Effects of the kinase inhibitor CGP41251 (PKC 412) on lymphocyte activation and TNF-alpha production.

CGP41251 is a serine/threonine and tyrosine kinase inhibitor that is a novel anticancer agent. Because the kinases that CGP41251 inhibits play important roles in T lymphocyte activation, we hypothesized that this compound may have useful immunomodulatory properties. Here we characterized the in vitro immunomodulatory effects of CGP41251. The effects of CGP41251 on lymphocyte proliferation, expression of T cell activation surface markers, and intracellular calcium response in peripheral blood mononuclear cells (PBMC's) were measured. Intracellular IL-2, TNF-alpha, IFN-gamma expression in CGP41251-treated T cells stimulated by lectin was measured by flow cytometry. CGP41251 inhibited lectin-induced lymphocyte proliferation and upregulation of activation surface markers with a 50% inhibitory concentration (IC(50)) of 0.1 microM. CGP41251, at micromolar concentrations, blunted the intracellular calcium response during PBMC activation. CGP41251 inhibited TNF-alpha production by T cells with an IC(50) of 0.5 microM and did not significantly inhibit the production of IL-2 or IFN-gamma. In conclusion, CGP41251 potently inhibits T lymphocyte activation and function and interferes with the proximal part of the T cell activation pathway. The ability of CGP41251 to selectively block T cell TNF-alpha production warrants the evaluation of this compound on other, e.g., monocyte, immune cells and in immunological conditions that are characterized by high TNF-alpha levels such as psoriasis and inflammatory bowel diseases.

Considerations on the appropriateness of the John Cunningham virus antibody assay use in patients with rheumatoid arthritis.

OBJECTIVE: The John Cunningham virus (JCV) is a generally benign and asymptomatic polyomavirus. Due to an association of the anti-integrin agent natalizumab with progressive multifocal leukoencephalopathy (PML) in patients with multiple sclerosis (MS), a newly developed anti-JCV antibody assay has been implemented as a risk-stratification tool for natalizumab-treated patients with MS. This viewpoint offers insight and perspective regarding the potential unapproved use of the anti-JCV antibody assay in rheumatoid arthritis (RA) and examines how rheumatologists can best assist patients. METHODS: A primary literature search was conducted to identify articles on the number of cases of PML associated with natalizumab in patients with MS, the number of cases of PML associated with patients with rheumatic disease, PML incidence in the general population, serum-based assays to detect JCV exposure, and clinical PML presentation and treatment methods. RESULTS: Risk of PML in patients with RA receiving biologics appears orders of magnitude lower than that expected in natalizumab-treated patients with MS (1 in 1000). If patients with RA are risk stratified assuming an anti-JCV antibody seropositivity of 60%, theoretically 23,400 anti-JCV antibody-positive patients would have to receive rituximab before potentially observing 1 PML case. CONCLUSIONS: Data currently indicate that rheumatologists should not order the anti-JCV antibody assay for patients requiring biologics. Monitoring relevant symptoms indicative of emerging PML might provide greater value to patients, thus prompting interventional measures that could affect prognosis.

Prediction of clinical pharmacokinetics of AMG 181, a human anti-α 4 ß 7 monoclonal antibody for treating inflammatory bowel diseases.

The purpose of this study was to predict a safe starting dose of AMG 181, a human anti-α 4 ß 7 antibody for treating inflammatory bowel diseases, based on cynomolgus monkey pharmacokinetic (PK) and pharmacodynamic (PD) data. A two-compartment model with parallel linear and target-mediated drug disposition for AMG 181 PK in cynomolgus monkey was developed. The estimated parameters were allometrically scaled to predict human PK. An E max PD model was used to relate AMG 181 concentration and free α 4 ß 7 receptor data in cynomolgus monkey. AMG 181 clinical doses were selected based on observed exposures at the no adverse effect level of 80 mg·kg(-1) in monkeys, the predicted human exposures, and AMG 181 concentration expected to produce greater than 50% α 4 ß 7 receptor occupancy in humans. The predicted human AMG 181 clearance and central volume of distribution were 144 mL·day(-1) and 2900 mL, respectively. The estimated EC50 for free α 4 ß 7 receptor was 14 ng·mL(-1). At the 0.7 mg starting dose in humans, the predicted exposure margins were greater than 490,000 and AMG 181 concentrations were predicted to only briefly cover the free α 4 ß 7 receptor EC10. Predictions for both C max and AUC matched with those observed in the first-in-human study within the 7 mg subcutaneous to 420 mg intravenous dose range. The developed model aided in selection of a safe starting dose and a pharmacological relevant dose escalation strategy for testing of AMG 181 in humans. The clinically observed human AMG 181 PK data validated the modeling approach based on cynomolgus monkey data alone.

In vitro evaluation of the effects of candidate immunosuppressive drugs: flow cytometry and quantitative real-time PCR as two independent and correlated read-outs.

BACKGROUND: Immune monitoring may use flow cytometry or molecular biology techniques. Flow cytometry assays cells that are phenotypically characterized, whereas TaqMan RT-PCR starts with RNA extraction from unfractionated heterogeneous cell populations. We therefore wondered how the effects of immunosuppressive drugs on cytokine production in stimulated whole blood, as determined by flow cytometry, would correlate with those obtained with quantitative real-time PCR (TaqMan RT-PCR). METHODS: Blood drawn from naive cynomolgus monkeys was exposed to incremental amounts of cyclosporine (CsA; 300, 600, 900 and 1200 ng/ml) or tacrolimus (TRL; 8, 20, 40 and 80 ng/ml) before lectin stimulation in vitro. Blood was then either stained for CD3, IFN-gamma, IL-2, IL-4, and TNF-alpha and analyzed on a flow cytometer with various gating strategies, or submitted to RNA extraction for analysis of the above mentioned cytokines mRNA transcripts using TaqMan RT-PCR. RESULTS: Both methods revealed a parallel dose-dependent inhibition of cytokine production in stimulated blood. The 50% inhibitory concentrations (IC(50)'s) ranged from 511-771 ng/ml (CsA) and 15-29 ng/ml (TRL) with flow cytometry, and from 275-529 ng/ml (CsA) and 11-48 ng/ml (TRL) with TaqMan RT-PCR for T-helper 1 cytokines. Both assays correlated well with a Pearson product moment correlation of 0.76. Extending gating from a CD3(+) gate to a lymphocyte gate improved correlation (r = 0.85) for all cytokines investigated (except IL-2; unchanged) whereas further extending gating resulted, to the contrary, in lower correlations. Independent of gating strategy a high correlation (r = 0.97) was observed when drug IC(50)'s were considered. CONCLUSIONS: Flow cytometry and TaqMan RT-PCR may be used interchangeably to monitor the effects of candidate immunosuppressive drugs on cytokine mRNA production in lectin-stimulated whole blood.

Compared with cyclosporine, ISATX247 significantly prolongs renal-allograft survival in a nonhuman primate model.

BACKGROUND: ISATX247 is a novel calcineurin inhibitor that has shown more potency than cyclosporine in vitro. This is the first study to compare the survival times of renal allografts in nonhuman primates treated with either ISATX247 or cyclosporine. METHODS: Adult, male cynomolgus monkeys were divided into blood-group compatible and mixed-lymphocyte, stimulation-mismatched, donor-recipient pairs. Heterotopic renal transplantation and bilateral native nephrectomies were performed. The monkeys were placed into either an ISATX247 or cyclosporine treatment group. Both groups were dosed twice daily to maintain a 12-hour drug-trough level of 150 ng/mL. Whole-blood concentrations of ISATX247 and cyclosporine, complete blood counts, and serum chemistry profiles were performed three times a week. Euthanasia was performed if the serum creatinine concentration became 7 or more mg/dL or a serious complication developed. RESULTS: The group receiving ISATX247 (n=8) survived significantly (P=0.0036) longer than the group receiving cyclosporine (n=7). The mean trough blood concentration of ISATX247 was 120 +/- 32 ng/mL and cyclosporine was 189 +/- 130 ng/mL. The average area under the curve 0-12 for ISATX247 was 6045 +/- 1679 ng/mL/hr and for cyclosporine was 4919 +/- 823 ng/mL/hr. The average calcineurin inhibition at trough blood concentrations was 80 +/- 11% for ISATX247 and 48 +/- 12% for cyclosporine. CONCLUSIONS: Allografts in monkeys treated with ISATX247 survived significantly longer than those treated with cyclosporine. On the basis of survival times and degree of calcineurin inhibition, ISATX247 is a more potent immunosuppressive agent than cyclosporine in this nonhuman primate model of renal-allograft transplantation.