Discovery and characterization of COVA322, a clinical-stage bispecific TNF/IL-17A inhibitor for the treatment of inflammatory diseases.

Biologic treatment options such as tumor necrosis factor (TNF) inhibitors have revolutionized the treatment of inflammatory diseases, including rheumatoid arthritis. Recent data suggest, however, that full and long-lasting responses to TNF inhibitors are limited because of the activation of the pro-inflammatory TH17/interleukin (IL)-17 pathway in patients. Therefore, dual TNF/IL-17A inhibition is an attractive avenue to achieve superior efficacy levels in such diseases. Based on the marketed anti-TNF antibody adalimumab, we generated the bispecific TNF/IL-17A-binding FynomAb COVA322. FynomAbs are fusion proteins of an antibody and a Fyn SH3-derived binding protein. COVA322 was characterized in detail and showed a remarkable ability to inhibit TNF and IL-17A in vitro and in vivo. Through its unique mode-of-action of inhibiting simultaneously TNF and the IL-17A homodimer, COVA322 represents a promising drug candidate for the treatment of inflammatory diseases. COVA322 is currently being tested in a Phase 1b/2a study in psoriasis ( ClinicalTrials.gov Identifier: NCT02243787).

Nonclinical safety testing of biopharmaceuticals--Addressing current challenges of these novel and emerging therapies.

Non-clinical safety testing of biopharmaceuticals can present significant challenges to human risk assessment with these often innovative and complex drugs. Hot Topics in this field were discussed recently at the 4th Annual European Biosafe General Membership meeting. In this feature article, the presentations and subsequent discussions from the main sessions are summarized. The topics covered include: (i) wanted versus unwanted immune activation, (ii) bi-specific protein scaffolds, (iii) use of Pharmacokinetic (PK)/Pharmacodynamic (PD) data to impact/optimize toxicology study design, (iv) cytokine release and challenges to human translation (v) safety testing of cell and gene therapies including chimeric antigen receptor T (CAR-T) cells and retroviral vectors and (vi) biopharmaceutical development strategies encompassing a range of diverse topics including optimizing entry of monoclonal antibodies (mAbs) into the brain, safety testing of therapeutic vaccines, non-clinical testing of biosimilars, infection in toxicology studies with immunomodulators and challenges to human risk assessment, maternal and infant anti-drug antibody (ADA) development and impact in non-human primate (NHP) developmental toxicity studies, and a summary of an NC3Rs workshop on the future vision for non-clinical safety assessment of biopharmaceuticals.

New challenges and opportunities in nonclinical safety testing of biologics.

New challenges and opportunities in nonclinical safety testing of biologics were discussed at the 3rd European BioSafe Annual General Membership meeting in November 2013 in Berlin: (i)Approaches to refine use of non-human primates in non-clinical safety testing of biologics and current experience on the use of minipigs as alternative non-rodent species.(ii)Tissue distribution studies as a useful tool to support pharmacokinetic/pharmacodynamic (PKPD) assessment of biologics, in that they provide valuable mechanistic insights at drug levels at the site of action.(iii)Mechanisms of nonspecific toxicity of antibody drug conjugates (ADC) and ways to increase the safety margins.(iv)Although biologics toxicity typically manifests as exaggerated pharmacology there are some reported case studies on unexpected toxicity.(v)Specifics of non-clinical development approaches of noncanonical monoclonal antibodies (mAbs), like bispecifics and nanobodies.

Combination of rituximab with blinatumomab (MT103/MEDI-538), a T cell-engaging CD19-/CD3-bispecific antibody, for highly efficient lysis of human B lymphoma cells.

We have compared the cytotoxic activity of rituximab with that of blinatumomab (MT103/MEDI-538), a single-chain CD19-/CD3-bispecific antibody engaging human T cells. Blinatumomab consistently led to a higher degree of lysis of human lymphoma lines than rituximab, and was active at much lower concentration. The cytotoxicity mediated by blinatumomab and rituximab both caused a potent activation of pro-caspases 3 and 7 in target cells, a key event in induction of granzyme-mediated apoptotic cell death. Combination of rituximab with blinatumomab was found to greatly enhance the activity of rituximab, in particular at low effector-to-target cell ratios and at low antibody concentration.

Strictly target cell-dependent activation of T cells by bispecific single-chain antibody constructs of the BiTE class.

Bispecific antibodies have been extensively studied in vitro and in vivo for their use in redirected tumor cell lysis. A particular challenge of bispecific antibody constructs that recognize the invariant CD3 signaling complex is a controlled polyclonal activation of T cells that, ideally, is exquisitely dependent on the presence of target cells. Otherwise, overt production of inflammatory cytokines and secondary reactions may occur as side effects, as can be observed with constitutively T-cell activating monoclonal antibodies to CD3 or CD28, and with bispecific antibodies bearing Fc gamma portions. Here we analyzed 2 distinct bispecific single-chain antibody constructs of the BiTE class, called MT110 and MT103 (or MEDI-538), for conditional T-cell activation. In the presence of target-expressing cell lines, low picomolar concentrations of the BiTE molecules were sufficient to stimulate a high percentage of peripheral human T cells to express cytokines and surface activation markers, enter into cell cycle, and induce redirected lysis of target cells. However, in the absence of target cells, the 2 BiTE molecules even at high concentrations did not detectably activate T cells. Our data show that T cell activation by monomeric forms of MT110 and MT103 is highly conditional in that it is strictly dependent on the presence of cells expressing the proper target antigen. BiTE molecules therefore qualify for a highly controlled polyclonal T-cell therapy of cancer.

A human monoclonal IgG1 potently neutralizing the pro-inflammatory cytokine GM-CSF.

The pro-inflammatory cytokine GM-CSF is aberrantly produced in many autoimmune and chronic inflammatory human diseases. GM-CSF neutralization by antibodies has been shown to have a profound therapeutic effect in animal models of rheumatoid arthritis, inflammatory lung diseases, psoriasis and multiple sclerosis. Moreover, the absence of GM-CSF in null mutant mice ameliorates or prevents certain of these diseases. Here we describe the biophysical and biological properties of a human anti-GM-CSF IgG1 antibody designated MT203, which was derived by phage display guided selection. MT203 bound with picomolar affinity to an epitope on human and macaque GM-CSF involved in high-affinity receptor interaction. As a consequence, the antibody potently prevented both GM-CSF-induced proliferation of TF-1 cells with a sub-nanomolar IC50 value and the production of the chemokine IL-8 by U937 cells. MT203 neutralized equally well recombinant (r) human (h) GM-CSF from Escherichia coli and yeast, and also normally glycosylated GM-CSF secreted by human lung epithelial cells in response to IL-1beta stimulation. Furthermore, MT203 significantly reduced both survival and activation of peripheral human eosinophils as may be required for effective treatment of inflammatory lung diseases. The antibody did not show a detectable loss of neutralizing activity after 5 days in human serum at 37 degrees C. Based on its favorable properties, MT203 has been selected for development as a novel anti-inflammatory human monoclonal antibody with therapeutic potential in a multitude of human autoimmune and inflammatory diseases.

Exchanging human Fcgamma1 with murine Fcgamma2a highly potentiates anti-tumor activity of anti-EpCAM antibody adecatumumab in a syngeneic mouse lung metastasis model.

An important mode of action shared by human IgG1 antibody therapies is antibody-dependent cellular cytotoxicity (ADCC). ADCC relies on the interaction of the antibody's Fc portion with Fc-gama receptors (FcgammaR) on immune effector cells. The anti-tumor activity of human IgG1 antibodies is frequently assessed in mouse models. Binding of human IgG1 to murine FcgammaRs is however of reduced affinity. We here show that ADCC of adecatumumab (MT201), a fully human IgG1 antibody specific for epithelial cell adhesion molecule (EpCAM/CD326), is drastically lower if human peripheral blood mononuclear cells are replaced by murine splenocytes as effector cells. When the variable domains of adecatumumab were genetically fused to a murine IgG2a backbone (yielding mu-adecatumumab), ADCC with murine effector cells was much improved, but at the same time significantly reduced with human effector cells. The serum half-lives of adecatumumab and mu-adecatumumab were determined in mice and dosing schedules established that gave similar serum trough levels during a 4-week antibody treatment. The anti-tumor activities of adecatumumab and mu-adecatumumab were then compared side-by-side in a lung metastasis mouse model established with a syngeneic B16 melanoma line expressing human EpCAM at physiologically relevant levels. Treatment of mice with mu-adecatumumab led to an almost complete prevention of lung metastases, while the human version of the antibody was much less active. This shows that adecatumumab has high anti-tumor activity when tested in a form that is better compatible with the species' immune system. Moreover, our data suggest to routinely compare in mouse models human IgG1 and murine IgG2a versions of antibodies to properly assess the contribution of ADCC to overall anti-tumor activity.

A phase I study with adecatumumab, a human antibody directed against epithelial cell adhesion molecule, in hormone refractory prostate cancer patients.

AIM OF THE STUDY: Adecatumumab (also known as MT201) is a human recombinant IgG1 monoclonal antibody binding with low affinity to epithelial cell adhesion molecule (EpCAM). To explore safety, pharmacokinetics and pharmacodynamics of adecatumumab, a phase I trial in patients with hormone refractory prostate cancer (HRPC) was performed. METHODS: Twenty patients were treated with two adecatumumab infusions on days 0 and 14 in cohorts with doses of ten up to 262 mg/m2. RESULTS: Adecatumumab was well tolerated at all doses tested, and no maximum tolerated dose reached. Most adverse events were mild or moderate with pyrexia and nausea being most frequent. The highest dose of adecatumumab induced shortly after infusion robust and transient increases of TNF-alpha serum levels. At all doses, significant transient declines of peripheral natural killer cells were observed shortly after antibody infusions. Adecatumumab had a serum half-life of 15 days, and immune responses to the antibody were not detected. CONCLUSIONS: A benign safety profile, long serum half-life and low immunogenicity do warrant further exploration of adecatumumab for treatment of EpCAM-expressing neoplasia.

N-Glucuronidation of the antiepileptic drug retigabine: results from studies with human volunteers, heterologously expressed human UGTs, human liver, kidney, and liver microsomal membranes of Crigler-Najjar type II.

Retigabine (D-23129), an N-2-amino-4-(4-fluorobenzylamino)phenylcarbamine acid ethyl ester, is a novel antiepileptic drug which is currently in phase II clinical development. This drug undergoes N-glucuronidation. We aimed to identify the principal enzymes involved in the N-glucuronidation pathway of retigabine and compared our findings with those obtained from human liver (a pool of 30 donors) and kidney microsomes (a pool of 3 donors) and with results from a human absorption, distribution, metabolism, and excretion study upon administration of 200 microCi of [(14)C]-D-23129. Essentially, microsomal assays with UGT1A1 produced only one of the 2 N-glucuronides, whereas UGT1A9 is capable of forming both N-glucuronides. The rates of metabolism for UGT1A9, human liver microsomes, and UGT1A1 were 200, 100, and 100 pmol N-glucuronide per minute per milligram of protein, respectively. At the 50 micromol/L uridine diphosphate glucoronic acid (UDPGA) concentration, UGT1A4 also catalyzed the N-glucuronidation of retigabine, the rates being approximately 5 and 6 pmol/(min.mg protein). With UGT1A9, the production of metabolites 1 and 2 proceeded at a K(m) of 38+/-25 and 45+/-15 micromol/L, whereas the K(m) for retigabine N-glucuronidation by human liver microsomal fractions was 145+/-39 micromol/L. Furthermore, a V(max) of 1.2+/-0.3 (nmol/[min.mg protein]) was estimated for human liver microsomes (4 individual donors). We investigated the potential for drug-drug interaction using the antiepileptic drugs valproic acid, lamotrigine, the tricyclic antidepressant imipramine, and the anesthetic propofol. These are commonly used medications and are extensively glucuronidated. No potential for drug-drug interactions was found at clinically relevant concentrations (when assayed with human liver microsomes or UGT1A9 enzyme preparations). Notably, the biosynthesis of retigabine-N-glucuronides was not inhibited in human liver microsomal assays in the presence of 330 micromol/L bilirubin, and glucuronidation of retigabine was also observed with microsomal preparations from human kidney and Crigler-Najjar type II liver. This suggests that lack of a particular UDP-glucuronosyltransferase (UGT) isoform (eg, UGT1A1 in kidney) or functional loss of an entire UGT1A gene does not completely abolish disposal of the drug. Finally, chromatographic separations of extracts from microsomal assays and human urine of volunteers receiving a single dose of (14)C-retigabine provided clear evidence for the presence of the 2 N-glucuronides known to be produced by UGT1A9. We therefore suggest N-glucuronidation of retigabine to be of importance in the metabolic clearance of this drug.

MT110: a novel bispecific single-chain antibody construct with high efficacy in eradicating established tumors.

We have developed a novel single-chain Ep-CAM-/CD3-bispecific single-chain antibody construct designated MT110. MT110 redirected unstimulated human peripheral T cells to induce the specific lysis of every Ep-CAM-expressing tumor cell line tested. MT110 induced a costimulation independent polyclonal activation of CD4- and CD8-positive T cells as seen by de novo expression of CD69 and CD25, and secretion of interferon gamma, tumor necrosis factor alpha, and interleukins 2, 4 and 10. CD8-positive T cells made the major contribution to redirected tumor cell lysis by MT110. With a delay, CD4-positive cells could also contribute presumably as consequence of a dramatic upregulation of granzyme B expression. MT110 was highly efficacious in a NOD/SCID mouse model with subcutaneously growing SW480 human colon cancer cells. Five daily doses of 1 microg MT110 on days 0-4 completely prevented tumor outgrowth in all mice treated. The bispecific antibody construct also led to a durable eradication of established tumors in all mice treated with 1 microg doses of MT110 on days 8-12 after tumor inoculation. Finally, MT110 could eradicate patient-derived metastatic ovarian cancer tissue growing under the skin of NOD/SCID mice. MT110 appears as an attractive bispecific antibody candidate for treatment of human Ep-CAM-overexpressing carcinomas.

High concentrations of therapeutic IgG1 antibodies are needed to compensate for inhibition of antibody-dependent cellular cytotoxicity by excess endogenous immunoglobulin G.

A common feature of human IgG1 antibodies used for cancer treatment is that their anti-tumour efficacy requires high serum trough levels and continued therapy for several months. Treatment cycles, thereby, consume several grams of IgG1 translating into significant drug needs and costs. The basis for the low in vivo efficacy, which is in contrast to high in vitro antibody-dependent cellular cytotoxicity (ADCC), is not well understood. Here, we have explored factors contributing to this discrepancy using adecatumumab (MT201), a fully human monoclonal IgG1 against epithelial cell adhesion molecule (Ep-CAM) and trastuzumab (Herceptin), a humanized IgG1 with specificity for the human epithelial growth factor receptor type 2 (HER-2) antigen. We found that physiological levels of human sera strongly inhibited ADCC of both IgG1 antibodies. Effects showed some dependence on the density of Ep-CAM and HER-2 targets, the tumour cell line tested and on effector cell and serum donors. Removal of IgG by affinity chromatography abolished the inhibitory effect of a serum pool. Inhibition of ADCC was fully restored by adding back the IgG fraction or by an equal amount of IgG from a commercial source. We further demonstrate that CD56-positive lymphocytes within human PBMC contributed >90% to ADCC and that normal serum levels of IgG effectively competed for in vitro binding of an IgG1 antibody to low-affinity Fcgamma receptor type III (CD16), as is present on natural killer (NK) cells. Competition of serum IgG for binding of therapeutic IgG1 to NK cell may be one important reason why high antibody doses are required in the clinic for treatment of cancer by an ADCC-based mechanism.

Potent inhibition of local and disseminated tumor growth in immunocompetent mouse models by a bispecific antibody construct specific for Murine CD3.

Bispecific single-chain antibody constructs specific for human CD3 have been extensively studied for antitumor activity in human xenograft models using severe combined immunodeficient mice supplemented with human T cells. High efficacy at low effector-to-target ratios, independence of T cell costimuli and a potent activation of previously unstimulated polyclonal T cells were identified as hallmarks of this class of bispecific antibodies. Here we studied a bispecific single-chain antibody construct (referred to as 'bispecific T cell engager', BiTE) in an immunocompetent mouse model. This was possible by the use of a murine CD3-specific BiTE, and a syngeneic melanoma cell line (B16F10) expressing the human Ep-CAM target. The murine CD3-specific BiTE, called 2C11x4-7 prevented in a dose-dependent fashion the outgrowth of subcutaneously growing B16/Ep-CAM tumors with daily i.v. injections of 5 or 50 microg BiTE which was most effective. Treatment with 2C11x4-7 was effective even when it was started 10 days after tumor cell inoculation but delayed treatments showed a reduction in the number of cured animals. 2C11x4-7 was also highly active in a lung tumor colony model. When treatment was started on the day of intravenous tumor cell injection, seven out of eight animals stayed free of lung tumors, and three out of eight animals when treatment was started on day 5. Our study shows that BiTEs also have a high antitumor activity in immunocompetent mice and that there is no obvious need for costimulation of T cells by secondary agents.

Intranasal loteprednol etabonate in healthy male subjects: pharmacokinetics and effects on endogenous cortisol.

Loteprednol etabonate (LE) is a glucocorticoid soft drug that is currently in development for intranasal use. The main objectives of this study were to examine the pharmacokinetics and potential effects on systemic cortisol of two intranasal suspension formulations of LE and to compare these findings with placebo and fluticasone propionate (FP, Flonase) control treatments. In this randomized, double-blind (except for FP), parallel-group study (n = 8/group), all subjects received for 14 days once daily in the morning two puffs of the following nasal spray formulations into each nostril: LE 0.1% (400 microg/day), LE 0.2% (800 microg/day), FP 0.05% (200 microg/day), and placebo. Drug trough levels were determined on days 1, 5, 12, 13, and 14, and a full pharmacokinetic profile was established on day 14, and 24-hour serum cortisol profiles were assessed prior to treatment (i.e., at baseline) and after the last dose. All subjects completed the protocol without treatment-emergent adverse findings. All formulations were rapidly absorbed (t(max) less than 1 h). The rather short mean terminal half-lives of 2.2 +/- 1.5 hours and 1.8 +/- 1.0 hours for LE 400 microg and LE 800 microg, respectively, and 4.2 +/- 1.8 hours for the 200-microg FP treatment explained the lack of any accumulation. Mean peak concentrations (C(max)) were 139 +/- 57 pg/mL with LE 400 microg and 164 +/- 54 pg/mL with LE 800 microg and thus fairly independent from dose. The 200-microg FP treatment resulted in a C(max) of only 15.5 +/- 5.9 pg/mL. Mean measured AUC(0-t) values (193 +/- 87 pg/h/mL(-1), 300 +/- 183 pg/h/mL(-1), and 40 +/- 34 pg/h/mL(-1) for LE 400 microg, LE 800 microg, and FP 200 microg, respectively) showed high variability and suggested nonlinear pharmacokinetics for the LE formulations, indicative of a less complete systemic uptake of LE from the 0.2% concentration. None of the treatments (LE 400 microg, LE 800 microg, and FP 200 microg) showed evidence for serum cortisol suppression when compared with placebo, respectively. The uptake and systemic exposure appears less complete from the 0.2% LE concentration, which principally favors this formulation for further clinical development.

Effect of R(+)alpha-lipoic acid on pyruvate metabolism and fatty acid oxidation in rat hepatocytes.

R-(+)-alpha-lipoic acid (R-LA) is the naturally occurring enantiomer of LA. It is a strong antioxidant and cofactor of key metabolic enzyme complexes catalyzing the decarboxylation of alpha-keto acids. Racemic LA (rac-LA) has shown promise in treating diabetic polyneuropathy, and some studies suggest that it improves glucose homeostasis in patients with type 2 diabetes. We examined the effects of R-LA on pyruvate metabolism and free fatty acid (FFA) oxidation in primary cultured hepatocytes isolated from 24-hour fasted rats. After overnight culture in serum-free medium, cells were pre-exposed to R-LA for 3 hours before assays. R-LA (25 to 200 micromol/L) significantly increased pyruvate oxidation ( approximately 2-fold at the highest dose tested) measured as (14)CO(2) production from [1-(14)C]pyruvate by the cells over 1 hour post-treatment. These effects correlated with proportional, significant increases in the activation state of the pyruvate dehydrogenase (PDH) complex. R-LA treatment inhibited glucose production from pyruvate by approximately 50% at 50 micromol/L R-LA and approximately 90% at 200 micromol/L. Palmitate oxidation was measured in hepatocytes cultured in the presence of albumin and physiological (0.1 mmol/L) or high (1.5 mmol/L) concentrations of FFA. The latter markedly enhanced FFA oxidation. R-LA treatment significantly inhibited FFA oxidation in both media, but was more effective in high FFA, where it reduced FFA oxidation by 48% to 82% at 25 to 200 micromol/L, respectively. Identical doses of R-LA did not affect FFA oxidation by L6 myotubes (a cell culture model for skeletal muscle) in either high or low FFA medium, but enhanced pyruvate oxidation. In conclusion, 3-hour exposure of primary cultured rat hepatocytes to R-LA at therapeutically relevant concentrations increased pyruvate oxidation, apparently by activation of the PDH complex, and decreased gluconeogenesis and FFA oxidation. These features may prove useful in the control of type 2 diabetes.

Pharmacokinetic interaction between retigabine and lamotrigine in healthy subjects.

PURPOSE: The antiepileptic drugs (AEDs) retigabine (RGB) and lamotrigine (LTG) undergo predominantly N-glucuronidation and renal excretion. This study was performed to evaluate potential pharmacokinetic interactions between both AEDs. METHODS: Twenty-nine healthy male subjects participated in the study. Group A ( n=14) received single oral 200-mg RGB doses on day 1 and day 7, and 25 mg o.i.d. LTG on days 3-8. Group B ( n=15) received single oral 200-mg LTG doses on day 1 and day 17, and was up-titrated to 300 mg RGB b.i.d. on days 6-20. Blood samples were collected to compare the pharmacokinetics of both AEDs and the N-acetyl metabolite of RGB (AWD21-360) after single and concomitant treatments. RESULTS: RGB was rapidly absorbed and eliminated with a mean half-life (t(1/2)) of 6.3+/-1.1 h and an apparent clearance (CL/F) of 0.69+/-1.4 l/h/kg. Under co-administration of LTG, mean RGB t(1/2) and area under the plasma concentration-time curve (AUC) were increased by 7.5% ( P=0.045) and 15% ( P=0.006), respectively, while CL/F was decreased by 13% ( P=0.06). Consistent results were obtained for AWD21-360. LTG was moderately rapidly absorbed, eliminated with a mean t(1/2) of 37+/-10.4 h and a CL/F of 0.028+/-0.007 l/h/kg. Under co-administration of RGB, mean LTG t(1/2) and AUC decreased by 15% and 18%, respectively, while CL/F increased by 22% (all parameters, P=0.001). CONCLUSIONS: RGB and LTG exhibit a modest pharmacokinetic interaction on each other. The slight decline in RGB clearance due to LTG is believed to result from competition for renal elimination rather than competition for glucuronidation. The induction of LTG clearance due to retigabine was unexpected since RGB did not show enzyme induction in various other drug-drug interaction studies. Further studies in patients are needed to assess the clinical relevance of these findings for concomitant treatment with both drugs in the upper recommended dose range.

Pharmacokinetic/pharmacodynamic modeling of luteinizing hormone (LH) suppression and LH surge delay by cetrorelix after single and multiple doses in healthy premenopausal women.

Cetrorelix (CET) is a potent luteinizing hormone-releasing hormone (LH-RH) antagonist and is used to prevent premature ovulation in IVF (in vitro fertilization) procedures. The objective of the present study was to develop a pharmacokinetic/pharmacodynamic (PK/PD) model for the LH suppression and LH surge delay after single doses (SD) and multiple doses (MD) of CET in healthy premenopausal women without ovarian stimulation. CET was given by subcutaneous route (SD, 0.25, 0.5, or 1 mg) on cycle day 3 and as similar multiple once-a-day doses from cycle day 3 to day 16 in two consecutive menstrual cycles. The concentration-time data of CET and LH were used for PK/PD modeling. A two-compartment model described the PK of CET with median terminal half-life estimates of 9.2 and 54.5 hours after SD and MD, respectively. An indirect-response Emax model was used to describe the LH suppression and the LH surge delay. LH suppression was linked to plasma concentrations of CET, while the delay in the LH surge was linked to the PK of CET through a hypothetical effect compartment. Since the SD regimen on day 3 did not cause significant delay, these values were used as controls in the analysis of surge delay in MD data. The IC50 (for suppression) estimate was 0.73 ng/ml for SD, and EC50 (surge delay) was 1.42 ng/ml for MD. The PK/PD model adequately described the LH suppression and the surge delay.