Safety, tolerability, pharmacokinetics and pharmacodynamics of the anti-CD38 cytolytic antibody TAK-079 in healthy subjects.

AIMS: This investigation characterised tolerability, pharmacokinetics and pharmacodynamics of the anti-CD38 antibody TAK-079. METHODS: A randomised, double-blind, placebo-controlled trial of a single intravenous (i.v.) infusion or subcutaneous (s.c.) injection of TAK-079 at escalating doses in healthy subjects (n = 74), who were followed for 92 days postexposure. RESULTS: TAK-079 was well tolerated. All adverse events were mild or moderate. There were no withdrawals, infusion, or injection site reactions over the tested i.v. and s.c. doses up to 0.06 and 0.6 mg kg-1 , respectively. At higher doses, transient cytokine level increases, following i.v. administration, coincided with reduction in CD38-expressing cells; clinical symptoms included mild pyrexia, headache, and postural hypotension. Following an i.v. infusion of 0.06 mg kg-1 TAK-079, maximum observed serum concentration (Cmax ) was 100.4 (%CV: 52) ng mL-1 , time to Cmax was the end of infusion and natural killer (NK_ cells were reduced 93.8 (±8.5) % from baseline levels. Following a s.c. injection of 0.6 mg kg-1 TAK-079, Cmax was 23.0 (%CV: 67) ng mL-1 with time to Cmax of 24 (range 7.98-96.02) hours, and plasmablasts were subsequently reduced 93.4 (±8.8) % from predose levels. Serum immunoglobulin (Ig)M, IgA and IgG levels were reduced by 15-60% and had not returned to baseline levels within 78 days after administration at ≥0.3 mg kg-1 s.c. Reductions in NK cells at 0.6 mg kg-1 s.c. were approximately 2-3 times more durable than at 0.06 mg kg-1 i.v. CONCLUSIONS: TAK-079 was well tolerated and s.c. administration elicited more durable reductions in plasmablasts and NK cells. This plasmacytolytic profile could be useful for treating disorders caused by plasma or NK cells, malignant counterparts, and/or pathogenic antibodies.

A Reduction in B, T, and Natural Killer Cells Expressing CD38 by TAK-079 Inhibits the Induction and Progression of Collagen-Induced Arthritis in Cynomolgus Monkeys.

Ectoenzyme CD38 is increased on lymphocytes in response to an antigenic challenge and it is hypothesized that targeting these activated lymphocytes could ameliorate pathologic activities in autoimmune diseases. The cynomolgus monkey is an appropriate model for assessing potential effects of targeting CD38 in humans because these species exhibit similar expression profiles. TAK-079 is a human monoclonal antibody (IgG1 λ ) that binds to CD38 and lyses bound cells by complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity. TAK-079 binds to monkey CD38 with an affinity at EC50 4.5 nM, and the potential activity of TAK-079 was investigated in a monkey collagen-induced arthritis model of autoimmune disease. Prophylactic administration of TAK-079 (3 mg/kg i.v. weekly) was well tolerated and prevented arthritis development compared with vehicle-treated control animals, which exhibited progressive disease with radiographic damage and worsening clinical scores over the study course. Therapeutic treatment of arthritic monkeys with TAK-079 (3 mg/kg i.v. weekly) was also well tolerated and reduced disease progression and symptoms. Arthritis scores and joint swelling were significantly lower than the vehicle control, accompanied by decreases in blood levels of C-reactive protein, alkaline phosphatase, and natural killer, B, and T cells. Histopathology, morphometry, and radiology revealed significantly less joint damage in animals exposed prophylactically to TAK-079 treatment compared with vehicle-treated animals and significantly less damage in animals treated therapeutically with TAK-079 or dexamethasone (0.1 mg/kg oral gavage daily), illustrating potential disease-modifying activity. In conclusion, these data indicate that depletion of CD38-expressing cells could be a therapeutic mechanism for treating autoimmune diseases. SIGNIFICANCE STATEMENT: This study demonstrates that targeting CD38-expressing leukocytes with a cytolytic antibody can ameliorate autoimmune disease in cynomolgus monkeys. The study gives a unique perspective into this therapeutic strategy because the three other anti-CD38 cytolytic antibodies in clinical development (daratumumab, isatuximab, and MOR202) cannot be tested in similar models because they do not crossreact with CD38 expressed by new world primates.

Antagonizing the α4ß1 integrin, but not α4ß7, inhibits leukocytic infiltration of the central nervous system in rhesus monkey experimental autoimmune encephalomyelitis.

The immune system is characterized by the preferential migration of lymphocytes through specific tissues (i.e., tissue tropism). Tissue tropism is mediated, in part, by the α(4) integrins expressed by T lymphocytes. The α(4)ß(1) integrin mediates migration of memory T lymphocytes into the CNS, whereas the α(4)ß(7) integrin mediates migration preferentially into gastrointestinal tissue. This paradigm was established primarily from investigations in rodents; thus, the objective of this investigation was to determine if blocking the α(4)ß(7) integrin exclusively would affect migration of T lymphocytes into the CNS of primates. The effects of the dual α(4)ß(1) and α(4)ß(7) antagonist natalizumab were compared with those of the α(4)ß(7) antagonist vedolizumab on experimental autoimmune encephalomyelitis in the rhesus monkey. Animals received an initial i.v. bolus of placebo, natalizumab (30 mg/kg), or vedolizumab (30 mg/kg) before intracutaneous immunization with recombinant human myelin oligodendrocyte glycoprotein and then Ab once weekly thereafter. Natalizumab prevented CNS inflammation and demyelination significantly (p < 0.05), compared with time-matched placebo control animals, whereas vedolizumab did not inhibit these effects, despite saturating the α(4)ß(7) integrin in each animal for the duration of the investigation. These results demonstrate that blocking α(4)ß(7) exclusively does not inhibit immune surveillance of the CNS in primates.

A Phase I, Randomized, Double-Blind, Placebo-Controlled, Single-Dose and Multiple-Rising-Dose Study of the BTK Inhibitor TAK-020 in Healthy Subjects.

Bruton's tyrosine kinase (BTK) is a target for treatment of hematologic malignancies and autoimmune diseases. TAK-020 is a highly selective covalent BTK inhibitor that inhibits both B cell receptor and fragment crystallizable receptor signaling. We assessed the safety/tolerability and pharmacokinetics/pharmacodynamics (PDs) of TAK-020 in healthy subjects. Each cohort of the single-rising dose (n = 72; 9 cohorts) and the multiple-rising dose (n = 48; 6 cohorts) portions of the study comprised six TAK-020-treated and two placebo-treated, subjects aged 18-55 years (inclusive). The PD effects were assessed by measuring BTK occupancy and the inhibition of fragment crystallizable epsilon receptor 1 (FcεRI)-mediated activation of basophils. Overall, treatment-emergent adverse events (TEAEs) were similar to placebo; there were no serious TEAEs or no TEAEs leading to discontinuation. TAK-020 was rapidly absorbed (median time to maximum plasma concentration (Tmax ) 45-60 minutes) with a half-life of ~ 3-9 hours at doses ≥ 2.5 mg. TAK-020 exposure was generally dose proportional for single doses ≤ 70 mg and after multiple doses of ≤ 60 mg once daily. Target occupancy was dose dependent, with doses ≥ 2.5 mg yielding maximum and sustained occupancy > 70% for > 96 hours. Single doses ≥ 4.4 mg reduced FcεRI-mediated activation of basophils by > 80% and comparable inhibition was observed with daily dosing ≥3.75 mg for 9 days. Inhibition persisted for 24-72 hours postdose and the duration generally increased with dose. TAK-020 was generally well-tolerated in healthy subjects after single and multiple doses and demonstrated target engagement and pathway modulation. The PD effects outlasted drug exposures, as expected for covalent inhibition of BTK.

The binding specificity and selective antagonism of vedolizumab, an anti-alpha4beta7 integrin therapeutic antibody in development for inflammatory bowel diseases.

Vedolizumab is a humanized monoclonal antibody that targets the alpha(4)beta(7) integrin exclusively, and modulates inflammation in the gastrointestinal tract without inducing the systemic immunosuppression that characterizes anti-alpha(4) chain monoclonal antibodies, such as natalizumab. This unique pharmacologic profile is largely attributable to four determinants. The first determinant is the restriction of the expression of the alpha(4)beta(7) integrin to subsets of leukocytes. Vedolizumab does not bind to the majority of memory CD4(+) T lymphocytes (60%), neutrophils, and most monocytes. The highest level of vedolizumab binding is to a subset (approximately 25%) of human peripheral blood memory CD4(+) T lymphocytes that include gut-homing interleukin 17 T-helper lymphocytes. Vedolizumab also binds to eosinophils at high levels, and to naive T-helper lymphocytes, naive and memory cytotoxic T lymphocytes, B lymphocytes, natural killer cells, and basophils at lower levels; vedolizumab binds to memory CD4(+) T and B lymphocytes with subnanomolar potency (EC(50) = 0.3-0.4 nM). The second determinant is binding specificity; vedolizumab binds exclusively to the alpha(4)beta(7) integrin, and not to the alpha(4)beta(1) and alpha(E)beta(7) integrins. The third determinant is selective antagonism; vedolizumab selectively inhibits adhesion of alpha(4)beta(7)-expressing cells to mucosal addressin cell adhesion molecule 1 (median inhibition concentration [IC(50)] = 0.02-0.06 microg/ml) and fibronectin (IC(50) = 0.02 microg/ml), but not vascular cell adhesion molecule 1. The fourth determinant is the gastrointestinal-specific tropism of the alpha(4)beta(7) integrin function. These pharmacologic properties of vedolizumab, in conjunction with the gastrointestinal tropism of alpha(4)beta(7) integrin function, may ultimately confer an improved risk-to-benefit profile for patients with inflammatory bowel diseases.

Effect of an experimental proteasome inhibitor on the cytoskeleton, cytosolic protein turnover, and induction in the neuronal cells in vitro.

GT-1 murine neuronal cells exposed to an experimental proteasome inhibitor (EPI) for 24h showed increased cell death via a non-apoptotic mechanism, as assessed by TUNEL and DNA fragmentation assays. Immunofluorescence staining demonstrated that EPI induced reorganization and relocation of non-ubiquinated actin microfilaments and microtubules to the perinuclear region in EPI treated cells. Immunohistochemistry analysis also demonstrated that other non-cytoskeletal proteins became ubiquitinated and/or upregulated including ubiquitin and other stress proteins. Perinuclear-centrosomal accumulation of gamma-tubulin and vimentin, key components of aggresomes, was observed in the EPI treated cells. Biochemical analysis indicated that EPI-induced accumulation of ubiquitinated protein aggregates in GT-1 cells was detergent - and mechanical - disruption resistant, a feature of aggresomes. Similar results were observed in GT-1 cells treated with lactacystin, a prototypical proteasome inhibitor, which is structurally dissimilar to EPI indicating a pharmacologic effect. In conclusion, EPI causes cytoskeletal reorganization and accumulation of diverse ubiquitinated and non-ubiquitinated proteins in the perinuclear region and potentially overloads the endoplasmic reticulum-dependent quality control mechanism. These processes acting alone, or in combination, are hypothesized to affect axonal transport or other aspects of cellular homeostasis and thus, represent events potentially relevant to the development of peripheral neuropathy associated with administration of proteasome inhibitors in nonclinical studies.

Pharmacokinetics and pharmacodynamics of the cytolytic anti-CD38 human monoclonal antibody TAK-079 in monkey - model assisted preparation for the first in human trial.

We are studying the fully human, IgG1λ cytolytic monoclonal antibody TAK-079, which binds CD38. CD38 is expressed on plasma and natural killer (NK) cells constitutively and upregulated on subsets of B and T lymphocytes upon activation. TAK-079 cross-reacts with CD38 expressed by cynomolgus monkeys and depletes subsets of NK, B, and T cells. Therefore, safety and function of TAK-079 was evaluated in this species, prior to clinical development, using bioanalytical, and flow cytometry assays. We pooled the data from eight studies in healthy monkeys (dose range 0.03-100 mg/kg) and developed mathematical models that describe the pharmacokinetics and the exposure-effect relationship for NK cells, B cells, and T cells. NK cell depletion was identified as the most sensitive pharmacodynamic effect of TAK-079. It was adequately described with a turnover model (C50  = 27.5 µg/mL on depletion rate) and complete depletion was achieved with an IV dose of 0.3 mg/kg. Intermediate effects on T-cell counts were described with a direct response model (C50  = 11.9 µg/mL) and on B-cell counts with a 4-transit-compartment model (C50  = 19.8 µg/mL on depletion rate). Our analyses substantiate the observation that NK, B and T cells are cleared by TAK-079 at different rates and required different time spans to replete the blood compartment. The models were used to simulate pharmacokinetic and cell depletion profiles in humans after applying a straightforward scaling approach for monoclonal antibodies in preparation for the first-in-human clinical trial.

Pharmacological characterization of guinea pig chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2).

Chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2), a G protein-coupled receptor activated by prostaglandin D(2) (PGD(2)), has been identified as a receptor expressed on cell types critical to the pathogenesis of asthma. The cDNA encoding guinea pig CRTH2 was cloned and mRNA expression examined in selected tissues. Transcript profiling of guinea pig CRTH2 indicated relatively high levels of expression in bone marrow, intermediate levels in brain and relatively low levels in lung, spleen, thymus, lymph node, etc. Characterization of the molecular pharmacology of guinea pig CRTH2 revealed that guinea pig CRTH2 exhibited a greater affinity for Delta(12)-PGJ(2), a stable PGD(2) metabolite relative to human CRTH2. The CRTH2 selective agonists 13,14-dihydro-15-keto PGD(2) and Delta(12)-PGJ(2) induced the recruitment of eosinophils following intradermal administration of these ligands in guinea pigs. Chemotaxis of guinea pig eosinophils was elicited by either PGD(2) or Delta(12)-PGJ(2), and was abolished by a CRTH2-specific antagonist. These results indicate that PGD(2) and the stable metabolite, Delta(12)-PGJ(2), play important roles in CRTH2 activation in the guinea pig.

Exclusive antagonism of the α4 ß7 integrin by vedolizumab confirms the gut-selectivity of this pathway in primates.

BACKGROUND: Biological therapies that antagonize specific molecules have demonstrated efficacy in inflammatory bowel diseases, but infections resulting from systemic immunosuppression underscore the need for safer therapies. The objective of this investigation was to determine if antagonism of the α(4) ß(7) integrin would exclusively yield gut-selective antiinflammatory activity in primates. METHODS: A series of experiments were conducted to investigate potential intra- and extraintestinal effects in healthy nonhuman primates dosed repeatedly with the α(4) ß(7) -exclusive antagonist vedolizumab (former versions: MLN0002, MLN02, LDP-02) for 4, 13, and 26 weeks. RESULTS: No adverse clinical effects of vedolizumab were observed in healthy cynomolgus monkeys up to the highest doses tested (100 mg/kg). Histomorphologic analyses indicated a reduction in the frequency of leukocytes in gastrointestinal tissue, but not other organs. A significant (P < 0.05) decrease in the frequency of ß 7+ lymphocytes in gastrointestinal tissues corresponded to a significant (P < 0.05) increase in α(4) ß 7+ memory helper T lymphocytes in peripheral blood. This elevation was specific to α(4) ß 7+ memory helper T lymphocytes; levels of other leukocyte subsets remained unaffected. Systemic opportunistic infections were not observed, and vedolizumab did not inhibit adaptive or innate immune responses systemically. CONCLUSIONS: These data demonstrate that blocking the α(4) ß(7) integrin exclusively yields gut-selective antiinflammatory activity in primates.

Correlation of serpin-protease expression by comparative analysis of real-time PCR profiling data.

Imbalanced protease activity has long been recognized in the progression of disease states such as cancer and inflammation. Serpins, the largest family of endogenous protease inhibitors, target a wide variety of serine and cysteine proteases and play a role in a number of physiological and pathological states. The expression profiles of 20 serpins and 105 serine and cysteine proteases were determined across a panel of normal and diseased human tissues. In general, expression of serpins was highly restricted in both normal and diseased tissues, suggesting defined physiological roles for these protease inhibitors. A high correlation in expression for a particular serpin-protease pair in healthy tissues was often predictive of a biological interaction. The most striking finding was the dramatic change observed in the regulation of expression between proteases and their cognate inhibitors in diseased tissues. The loss of regulated serpin-protease matched expression may underlie the imbalanced protease activity observed in pathological states.

Role of regulator of G protein signaling 16 in inflammation-induced T lymphocyte migration and activation.

Chemokine-induced T lymphocyte recruitment to the lung is critical for allergic inflammation, but chemokine signaling pathways are incompletely understood. Regulator of G protein signaling (RGS)16, a GTPase accelerator (GTPase-activating protein) for Galpha subunits, attenuates signaling by chemokine receptors in T lymphocytes, suggesting a role in the regulation of lymphocyte trafficking. To explore the role of RGS16 in T lymphocyte-dependent immune responses in a whole-organism model, we generated transgenic (Tg) mice expressing RGS16 in CD4(+) and CD8(+) cells. rgs16 Tg T lymphocytes migrated to CC chemokine ligand 21 or CC chemokine ligand 12 injection sites in the peritoneum, but not to CXC chemokine ligand 12. In a Th2-dependent model of allergic pulmonary inflammation, CD4(+) lymphocytes bearing CCR3, CCR5, and CXCR4 trafficked in reduced numbers to the lung after acute inhalation challenge with allergen (OVA). In contrast, spleens of sensitized and challenged Tg mice contained increased numbers of CD4(+)CCR3(+) cells producing more Th2-type cytokines (IL-4, IL-5, and IL-13), which were associated with increased airway hyperreactivity. Migration of Tg lymphocytes to the lung parenchyma after adoptive transfer was significantly reduced compared with wild-type lymphocytes. Naive lymphocytes displayed normal CCR3 and CXCR4 expression and cytokine responses, and compartmentation in secondary lymphoid organs was normal without allergen challenge. These results suggest that RGS16 may regulate T lymphocyte activation in response to inflammatory stimuli and migration induced by CXCR4, CCR3, and CCR5, but not CCR2 or CCR7.