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.

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.

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.

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.

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.

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.

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.

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.

Short tandem repeat analysis to monitor chimerism in macaca fascicularis.

Chimerism assessment following bone marrow transplantation (BMT) in cynomolgus monkeys (cynos) has been hampered by the lack of good engraftment markers. In human BMT, such markers have been provided by short tandem repeat (STR) loci. We tested the idea that techniques effective for detecting human STR could be readily adapted to cynos. Genomic DNA was extracted from cyno unseparated blood or peripheral cell subsets. With only slight modifications, reagents for detecting human STR alleles were used to amplify and detect cyno STRs and to quantitate allelic mixtures on an automated sequencer. Of the 15 STR loci tested, only CSF1PO, D18S51, and FGA successfully amplified, with seven, seven and two alleles, respectively. CSF1PO and D18S51 heterozygosity (80% and 55%, respectively) allowed use of these two loci for chimerism quantitation after BMT. The successful adaptation of human STR reagents to monitor chimerism in transplanted cynos will facilitate the use of this species in preclinical tolerance studies.

Immunomodulatory effects of docetaxel on human lymphocytes.

Docetaxel is an antineoplastic taxoid that interferes with microtubule polymerization dynamics and is used clinically to treat advanced cancers. Because microtubules play significant roles in T lymphocyte activation and function we characterized the in vitro immunomodulatory properties of docetaxel. Effects of docetaxel on lectin-induced peripheral blood mononuclear cell (PBMC) proliferation were measured by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and proliferating cell nuclear antigen (PCNA) staining. In addition, apoptosis was measured by annexin V staining and cell activation by determination of CD25 and CD71 cell surface expression. Intracellular calcium kinetics in lectin-activated Jurkat T lymphocytes exposed to docetaxel were investigated. Th1 cytokine production was assessed in T lymphocytes by intracellular cytokine staining. Docetaxel significantly inhibited PBMC proliferation and promoted apoptosis of lectin-activated PBMCs. Docetaxel significantly decreased expression of CD71 but not that of CD25. Docetaxel altered intracellular calcium homeostasis but did not affect Th1 cytokine production in T lymphocytes. In conclusion we demonstrate that docetaxel, although exerting significant antiproliferative effects on lymphocytes and promoting activation-induced apoptosis does affect only partially lymphocyte activation and function and does not affect Th1 cytokine production. These results suggest maintenance of lymphocyte functions important for host tumor surveillance and suggest that this compound may have a role in the treatment of cancer arising organ transplant recipients.

JAK3 inhibition as a new concept for immune suppression.

Although current immunosuppressive drugs are effective, they have numerous severe side effects that mandate the search for new agents. Mutations in the gene for janus kinase (JAK)3 result in severe combined immune deficiency with severely impaired humoral and cellular immunity, an observation that has prompted the development of JAK3 inhibitors. Due to its central role in lymphocyte activation, proliferation and homeostasis, targeting the JAK/signal transducer and activator of transcription (STAT) pathway may provide the required efficacy, without the toxicities associated with current therapies. Several studies conducted in rodents have validated the proof-of-concept, with a variety of JAK3 inhibitors demonstrating efficacy for immune suppression. In addition, the selective JAK3 inhibitor CP-690550 (Pfizer Inc) significantly improved allograft survival in a stringent preclinical model in primates and exhibited a good safety profile in non-human primates. This, along with studies of protein kinase inhibitors for cancer treatment, could demonstrate that development of effective, safe and selective kinase inhibitors for immunosuppression is possible.