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.

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).

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.

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.

Deconvoluting post-transplant immunity: cell subset-specific mapping reveals pathways for activation and expansion of memory T, monocytes and B cells.

A major challenge for the field of transplantation is the lack of understanding of genomic and molecular drivers of early post-transplant immunity. The early immune response creates a complex milieu that determines the course of ensuing immune events and the ultimate outcome of the transplant. The objective of the current study was to mechanistically deconvolute the early immune response by purifying and profiling the constituent cell subsets of the peripheral blood. We employed genome-wide profiling of whole blood and purified CD4, CD8, B cells and monocytes in tandem with high-throughput laser-scanning cytometry in 10 kidney transplants sampled serially pre-transplant, 1, 2, 4, 8 and 12 weeks. Cytometry confirmed early cell subset depletion by antibody induction and immunosuppression. Multiple markers revealed the activation and proliferative expansion of CD45RO(+)CD62L(-) effector memory CD4/CD8 T cells as well as progressive activation of monocytes and B cells. Next, we mechanistically deconvoluted early post-transplant immunity by serial monitoring of whole blood using DNA microarrays. Parallel analysis of cell subset-specific gene expression revealed a unique spectrum of time-dependent changes and functional pathways. Gene expression profiling results were validated with 157 different probesets matching all 65 antigens detected by cytometry. Thus, serial blood cell monitoring reflects the profound changes in blood cell composition and immune activation early post-transplant. Each cell subset reveals distinct pathways and functional programs. These changes illuminate a complex, early phase of immunity and inflammation that includes activation and proliferative expansion of the memory effector and regulatory cells that may determine the phenotype and outcome of the kidney transplant.

Janus kinase 3 inhibition with CP-690,550 prevents allograft vasculopathy.

Janus kinase 3 (JAK3) mediates signal transduction from cytokine receptors using the common gamma chain. The rationally designed inhibitor of JAK3, CP-690,550, prevents acute allograft rejection in rodents and in nonhuman primates. Here we investigated the ability of CP-690,550, to prevent allograft vasculopathy in a rodent model of aorta transplantation. Aortas from AxC Irish (RT1(a)) or Lewis (RT1(l)) rats were heterotopically transplanted into the infra-renal aorta of Lewis recipients and harvested at 28 or 56 days. Treated recipients received CP-690,550 by osmotic pumps (mean drug exposure of 110 +/- 38 ng/ml). Significant intimal hyperplasia was demonstrated in untreated allografts when compared with isografts at 28 days (2.08 +/- 0.85% vs. 0.43 +/- 0.2% luminal obliteration, respectively, P = 0.001) and 56 days (5.3 +/- 2.4% vs. 0.38 +/- 0.3%, P = 0.002). Treatment caused a 51% reduction in intimal hyperplasia at day 56. CP-690,550-treated animals also had a significant reduction of donor-specific IgG production and of the gene expression for suppressor of cytokine signaling-3 and with unchanged levels of expression of RANTES, IP-10 and transforming growth factor-beta1. These results are the first to show that JAK3 blockade by CP-690,550 effectively prevents allograft vasculopathy in this rat model of aorta transplantation.

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.

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.

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.

Quantitative analysis of the immunosuppressant CP-690,550 in whole blood by column-switching high-performance liquid chromatography and mass spectrometry detection.

A fast and accurate method to quantify the new immunosuppressive JAK3 inhibitor CP-690,550 in whole blood using a dual-pump liquid chromatography-liquid chromatography-mass spectrometry (LC/LC-MS) system was developed and validated in nonhuman primate blood. Before injection, blood samples were prepared by precipitation with a reagent that included methanol and acetonitrile (30:70, vol/vol) along with the internal standard (CP-istd). Column-switching LC/LC-MS analysis used online extraction followed by separation on a C8 analytic column and MS detection of the [M + H] CP-690,550 (m/z = 313.1) and CP internal standard (m/z = 288.1). Linearity was always better than r = 0.99 (n = 7) for CP-690,550 (range 2.5-750 ng/mL), with a lower limit of quantification (LLOQ) of 2.5 ng/mL. The intrarun accuracy and precision ranged from 103.0% to 105.4% and 2.7% to 4.3%, respectively (n = 5), and the interday precision ranged from 8.7% to 11.1%, and the interday accuracy ranged from 98.1% to 103.8% of nominal values (n = 14). The injection repeatability for the method was 1.3% (n = 7). Except for the LLOQ, the intraday accuracy and precision in human blood were also within 15% (n = 5). The combination of simple sample preparation and short analytic run time of this sensitive procedure makes it effective for monitoring the concentration of CP-690,550 in whole blood in organ-transplant recipients.

Immunosuppressive effects of surgery assessed by flow cytometry in nonhuman primates after nephrectomy.

Despite previous studies suggesting that surgery cause immune suppression, the underlying biologic mechanisms have not been studied using advanced immune function assays. Unilateral nephrectomy was performed in nonhuman primates. Blood was collected before surgery and at different time-points through 14 days after surgery. Lymphocyte proliferation (expression of proliferating cell nuclear antigen in cells in S/G(2)M-phase), production of intracellular cytokines [interleukin (IL)-2, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha] and expression of surface-activation antigens (CD25, CD71) on T-lymphocytes were assessed in whole blood using flow cytometry. Results were compared with nonoperated control animals. The procedure caused a decrease of 25% in absolute lymphocyte count on postoperative day 3. Inhibition of lymphocyte proliferation was maximal on postoperative day 1 (55% normalized to preoperative values) and was detectable until postoperative day 7, when it was 25%. Expression of T-cell activation antigens was decreased during the first postoperative week with a maximum on postoperative day 1 for CD71 (29%) and on postoperative day 3 for CD25 (49%). Intracellular production of cytokines by T cells was decreased only on postoperative day 1 (50% for IL-2, 29% for IFN-gamma and 22% for TNF-alpha). Immune functions returned to presurgery values by day 14. A major surgical procedure severely inhibits lymphocyte proliferation and various T-cell functions up to 1 week postoperatively.

Identification of the V genes encoding xenoantibodies in non-immunosuppressed rhesus monkeys.

The major immunological barrier that prevents the use of wild-type pig xenografts as an alternative source of organs for human xenotransplantation is antibody-mediated rejection. In this study, we identify the immunoglobulin variable region heavy (IgV(H)) chain genes encoding xenoantibodies to porcine heart and fetal porcine islet xenografts in non-immunosuppressed rhesus monkeys. We sought to compare the IgV(H) genes encoding xenoantibodies to porcine islets and solid organ xenografts. The immunoglobulin M (IgM) and IgG xenoantibody response was analysed by enzyme-linked immunosorbent assay and cDNA libraries from peripheral blood lymphocytes were prepared and sequenced. The relative frequency of IgV(H) gene usage was established by colony filter hybridization. Induced xenoantibodies were encoded by the IGHV3-11 germline progenitor, the same germline gene that encodes xenoantibodies in humans mounting active xenoantibody responses. The immune response to pig xenografts presented as solid organs or isolated cells is mediated by identical IgV(H) genes in rhesus monkeys. These animals represent a clinically relevant model to identify the immunological basis of pig-to-human xenograft rejection.

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 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.

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.

New strategies for immunosuppression: interfering with cytokines by targeting the Jak/Stat pathway.

PURPOSE OF REVIEW: Numerous immunosuppressants are available, but their adverse effects related to actions on nonlymphoid cells is problematic. Cytokines are key regulators of immune and inflammatory responses, and blocking their actions has become an important modality in treating autoimmune disorders. This review will discuss strategies to develop novel immunosuppressants that arise from advances in the understanding of cytokine signaling. RECENT FINDINGS: It is now recognized that large number of cytokines exert their effect by binding to receptors that activate the Janus kinase/signal transducer and activator of transcription pathway, so targeting intracellular signaling pathways is a logical strategy. A selective inhibitor of Janus kinase 3 has now been generated and is effective for transplant rejection in nonhuman primates and other models. Advances have also been made in understanding the functions of Stat family transcription factors, and approaches to interfering with the action of these DNA binding proteins are being devised. In addition, the identification of negative regulators of cytokine signaling offers additional therapeutic opportunities. SUMMARY: A selective inhibitor of Janus kinase 3 has now been generated and likely represents a new class of effective immunosuppressants. Strategies for targeting signal transducers and activators of transcription pathway are being intensively studied at present and hold potential promise. Multiple endogenous mechanisms exist for negatively regulating cytokine signaling; whether novel therapies can be devised that exploit these mechanisms remains to be determined.

A technique of bone marrow collection from vertebral bodies of cynomolgus macaques for transplant studies.

BACKGROUND: Strategies to induce donor-specific allograft tolerance are best tested in preclinical models developed in nonhuman primates (NHPs). Most protocols prepare the recipient by infusing hematopoietic cells from the donor. We report here a procedure to isolate and characterize large numbers of bone marrow cells (BMCs) from cynomolgus monkeys (cynos) that can then successfully be transplanted into conditioned recipients. MATERIALS AND METHODS: Vertebral columns of five cynos were excised en bloc and separated into individual vertebrae. The cancelous bone was extracted with a core puncher, fractionated, filtered, centrifuged, and resuspended in transplantation media before being analyzed by flow cytometry. In two instances, the collected BMCs were reinfused into allogeneic recipients preconditioned with a nonmyeloablative regimen. Chimerism was monitored using short-tandem repeat analysis. RESULTS: The mean total BMCs yield was 25.5 x 10(9) (range of 4.00 x 10(9) to 59 x 10(9)) with mean cell viability of 93.4% (range: 90-96%). CD34+ cells and CD3+ cells averaged 0.34 and 3.91% of total BMCs, respectively. This resulted in absolute cell number yields of 1.02 x 10(8) and 1.15 x 10(9) for CD34+ and CD3+ cells, respectively. Graft-versus-host disease was absent in both bone marrow infused animals, and a maximum level of chimerism of 18% was detected at 3 weeks after BMCs infusion. CONCLUSION: We present here the first detailed report of a procedure to retrieve and characterize large numbers of BMCs from vertebral bodies of cynos and demonstrate that cells collected with this technique have the capability of engrafting in allogenic recipients.