Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of Atacicept in a Randomized Trial in Healthy Caucasian and Japanese Subjects.

BACKGROUND AND OBJECTIVE: Atacicept is an inhibitor of the B lymphocyte stimulator (BLyS) and a proliferation-inducing ligand (APRIL), and is being studied in relation to immunological disease. Currently, limited data on atacicept are available in non-Caucasian subjects. Pharmacokinetic data from earlier studies of atacicept were derived using an enzyme-linked immunosorbent assay (ELISA), which was subsequently found to have inadequacies. Hence, a new bioanalytical ELISA for total atacicept was developed and validated. We conducted this randomized, double-blind, placebo-controlled phase I study to compare the safety, tolerability, pharmacokinetics, and pharmacodynamics of atacicept in healthy Japanese and Caucasian subjects while generating pharmacokinetic data using the new ELISA. METHODS: Japanese subjects aged ≥ 18 to ≤ 55 years (n = 24) were randomized (1:1:1:1) to a single subcutaneous dose of atacicept 25, 75, or 150 mg or placebo. Caucasian subjects were then enrolled to match the Japanese subjects' gender, body weight (± 20%), and height (± 15%). RESULTS: Atacicept was well tolerated and there were no clinically significant differences in treatment-emergent adverse events (TEAEs), vital signs, or laboratory parameters between the Japanese and Caucasian subjects. Most (90%) TEAEs were mild; no severe or serious TEAEs or deaths occurred. Weight-adjusted atacicept exposure was comparable between ethnicities and across doses: the Japanese/Caucasian ratio of the area under the serum concentration-time curve from time zero to the last sampling point (AUC0-t) was 107.21% (90% CI 93.42-123.02%) and the Japanese/Caucasian ratio of maximum serum concentration (Cmax) was 95.74% (90% CI 74.26-123.43%; ANCOVA). Median time to reach Cmax (tmax) was 20-60 h across all subjects. Dose-exposure relationships were comparable for the two ethnicities, with dose-normalized AUC0-t decreasing with increasing dose, indicating nonlinear pharmacokinetics for the doses examined. There were no statistically significant differences between ethnicities in the pharmacokinetics-dose relationship. Some transient dose-related decreases in mean serum immunoglobulin (Ig)A and IgM, but not IgG, were observed after atacicept administration. There were small transient increases in peripheral B cell numbers in the first 4 days after dosing that were larger with atacicept than with placebo, with no apparent dose relationship. No anti-atacicept antibodies were detected. CONCLUSION: The safety, pharmacokinetic, and pharmacodynamic profiles of atacicept in healthy Japanese subjects were comparable to those in healthy Caucasian subjects. EudraCT-ID: 2013-002703-34.

Highly immunogenic influenza virus-like particles containing B-cell-activating factor (BAFF) for multi-subtype vaccine development.

Virus-like particle (VLP) technology is an attractive platform for the development of seasonal and pandemic influenza vaccines. Influenza VLPs can be obtained by the overexpression of HA, M1, NA, and/or M2 viral proteins in insect, mammalian, or plant cells. In this study, we reported to obtain highly immunogenic influenza VLPs by molecular incorporation with B-cell-activating factor (BAFF) or proliferation-inducing ligand (APRIL). Since BAFF and APRIL act as homotrimers to interact with their receptors, we engineered the VLPs by direct fusion of BAFF or APRIL to the transmembrane anchored domain of H5HA gene. Results showed that immunizations with the HA-transmembrane anchored BAFF- or APRIL-VLPs only formulated in alum but not MPL adjuvant elicited significantly higher IgG titers in sera. However, only the BAFF-VLPs formulated in alum adjuvant elicited more broadly neutralizing antibodies against the homologous and two heterologous H5N1 clade/subclade viruses and conferred protective immunity against live virus challenges. As the multi-subtype influenza vaccines containing a variety of HA subtypes can confer broader protective immunity, we also obtained multi-subtype H5H7 BAFF-VLPs and H1H5H7 BAFF-VLPs and demonstrated that these multi-subtype BAFF-VLPs were able to induce the production of neutralizing antibodies against multiple HA subtypes. Our findings provided useful information for the development of highly immunogenic, multi-subtype influenza VLP vaccines.

Efficacy and safety of subcutaneous tabalumab in patients with systemic lupus erythematosus: results from ILLUMINATE-1, a 52-week, phase III, multicentre, randomised, double-blind, placebo-controlled study.

OBJECTIVES: Evaluate efficacy and safety of tabalumab, a human IgG4 monoclonal antibody that binds and neutralises membrane and soluble B-cell activating factor (BAFF) versus placebo plus standard of care (SoC) in patients with systemic lupus erythematosus (SLE). METHODS: This phase III, 52-week study randomised 1164 patients with moderate-to-severe SLE (Safety of Estrogens in Lupus Erythematosus National Assessment-SLE Disease Activity Index ≥6 at baseline). Patients received SoC plus subcutaneous injections of tabalumab or placebo, starting with a loading dose (240 mg) at week 0 and followed by 120 mg every two weeks (120 Q2W, n=387), 120 mg every four weeks (120 Q4W, n=389) or placebo Q2W (n=388). PRIMARY ENDPOINT: proportion of patients achieving SLE Responder Index 5 (SRI-5) response at week 52. RESULTS: Similar proportions of patients in each group achieved SRI-5 response at week 52 (120 Q2W: 31.8%; 120 Q4W: 35.2% and placebo: 29.3%). Key secondary endpoints were not met. In a sensitivity analysis not excluding patients who decreased antimalarials or immunosuppressants, SRI-5 response was achieved with 120 Q4W (37.0% vs 29.8% placebo; p=0.021), but not 120 Q2W (34.1%; p=0.171). Significant reductions in anti-dsDNA antibodies, increases in C3 and C4, and reductions in total B cells and immunoglobulins were observed with tabalumab. No differences were observed between treatment groups in percentage of deaths (120 Q2W: 0.8%; 120 Q4W: 0.5%; placebo: 0.5%), serious adverse events (AEs) (range 11.1-14.4%) or treatment-emergent AEs (range 81.1-82.3%). CONCLUSIONS: Tabalumab had biological activity-changes in anti-dsDNA, complement, B cells and immunoglobulins-consistent with BAFF pathway inhibition. Key clinical efficacy endpoints did not achieve statistical significance. Safety profiles were similar with tabalumab and placebo. TRIAL REGISTRATION NUMBER: NCT01196091.

Efficacy and safety of subcutaneous tabalumab, a monoclonal antibody to B-cell activating factor, in patients with systemic lupus erythematosus: results from ILLUMINATE-2, a 52-week, phase III, multicentre, randomised, double-blind, placebo-controlled study.

OBJECTIVES: To evaluate the efficacy and safety of tabalumab, a human IgG4 monoclonal antibody that neutralises membrane and soluble B-cell activating factor (BAFF). METHODS: This randomised, placebo-controlled study enrolled 1124 patients with moderate-to-severe systemic lupus erythematosus (SLE) (Safety of Estrogens in Lupus Erythematosus National Assessment- SLE Disease Activity Index ≥6 at baseline). Patients received standard of care plus subcutaneous study drug, starting with a loading dose (240 mg) at week 0 and followed by 120 mg every 2 weeks (120 Q2W), 120 mg every 4 weeks (120 Q4W) or placebo. Primary endpoint was proportion achieving SLE Responder Index 5 (SRI-5) improvement at week 52. RESULTS: Clinical characteristics were balanced across groups. The primary endpoint was met with 120 Q2W (38.4% vs 27.7%, placebo; p=0.002), but not with the less frequent 120 Q4W regimen (34.8%, p=0.051). Although key secondary endpoints (time to severe flare, corticosteroid sparing and fatigue) were not met, patients treated with tabalumab had greater SRI-5 response rates in a serologically active subset and improvements in more stringent SRI cut-offs, SELENA-SLEDAI, Physician's Global Assessment, anti-double-stranded DNA antibodies, complement, total B cells and immunoglobulins. The incidences of deaths, serious adverse events (AEs), and treatment-emergent AEs were similar in the 120 Q2W, 120 Q4W and placebo groups, but depression and suicidal ideation, albeit rare events, were more commonly reported with tabalumab. CONCLUSION: SRI-5 was met with 120 Q2W and although key secondary endpoints were not met, numerous other secondary endpoints significantly improved in addition to pharmacodynamic evidence of BAFF pathway blockade. The safety profile for tabalumab was similar to placebo, except for depression and suicidality, which were uncommon. TRIAL REGISTRATION NUMBER: NCT01205438.

HIV-1 adenoviral vector vaccines expressing multi-trimeric BAFF and 4-1BBL enhance T cell mediated anti-viral immunity.

Adenoviral vectored vaccines have shown considerable promise but could be improved by molecular adjuvants. Ligands in the TNF superfamily (TNFSF) are potential adjuvants for adenoviral vector (Ad5) vaccines based on their central role in adaptive immunity. Many TNFSF ligands require aggregation beyond the trimeric state (multi-trimerization) for optimal biological function. Here we describe Ad5 vaccines for HIV-1 Gag antigen (Ad5-Gag) adjuvanted with the TNFSF ligands 4-1BBL, BAFF, GITRL and CD27L constructed as soluble multi-trimeric proteins via fusion to Surfactant Protein D (SP-D) as a multimerization scaffold. Mice were vaccinated with Ad5-Gag combined with Ad5 expressing one of the SP-D-TNFSF constructs or single-chain IL-12p70 as adjuvant. To evaluate vaccine-induced protection, mice were challenged with vaccinia virus expressing Gag (vaccinia-Gag) which is known to target the female genital tract, a major route of sexually acquired HIV-1 infection. In this system, SP-D-4-1BBL or SP-D-BAFF led to significantly reduced vaccinia-Gag replication when compared to Ad5-Gag alone. In contrast, IL-12p70, SP-D-CD27L and SP-D-GITRL were not protective. Histological examination following vaccinia-Gag challenge showed a dramatic lymphocytic infiltration into the uterus and ovaries of SP-D-4-1BBL and SP-D-BAFF-treated animals. By day 5 post challenge, proinflammatory cytokines in the tissue were reduced, consistent with the enhanced control over viral replication. Splenocytes had no specific immune markers that correlated with protection induced by SP-D-4-1BBL and SP-D-BAFF versus other groups. IL-12p70, despite lack of anti-viral efficacy, increased the total numbers of splenic dextramer positive CD8+ T cells, effector memory T cells, and effector Gag-specific CD8+ T cells, suggesting that these markers are poor predictors of anti-viral immunity in this model. In conclusion, soluble multi-trimeric 4-1BBL and BAFF adjuvants led to strong protection from vaccinia-Gag challenge, but the protection was independent of standard immune markers. Soluble multi-trimeric SP-D-4-1BBL and SP-D-BAFF provide a novel technology to enhance adenoviral vector vaccines against HIV-1.

Treatment of acute lymphoblastic leukemia with an rGel/BLyS fusion toxin.

Acute lymphoblastic leukemia (ALL) is the most common malignancy affecting children and a major cause of mortality from hematopoietic malignancies in adults. A substantial number of patients become drug resistant during chemotherapy, necessitating the development of alternative modes of treatment. rGel (recombinant Gelonin)/BlyS (B-lymphocyte stimulator) is a toxin-cytokine fusion protein used for selective killing of malignant B-cells expressing receptors for B-cell-activating factor (BAFF/BLyS) by receptor-targeted delivery of the toxin, Gelonin. Here, we demonstrate that rGel/BLyS binds to ALL cells expressing BAFF receptor (BAFF-R) and upon internalization, it induces apoptosis of these cells and causes downregulation of survival genes even in the presence of stromal protection. Using an immunodeficient transplant model for human ALL, we show that rGel/BLyS prolongs survival of both Philadelphia chromosome-positive and negative ALL-bearing mice. Furthermore, we used AMD3100, a CXCR4 antagonist, to mobilize the leukemic cells protected in the bone marrow (BM) microenvironment and the combination with rGel/BLyS resulted in a significant reduction of the tumor load in the BM and complete eradication of ALL cells from the circulation. Thus, a combination treatment with the B-cell-specific fusion toxin rGel/BLyS and the mobilizing agent AMD3100 could be an effective alternative approach to chemotherapy for the treatment of primary and relapsed ALL.

Thymic stromal lymphopoietin (TSLP) acts as a potent mucosal adjuvant for HIV-1 gp140 vaccination in mice.

The development of a successful vaccine against HIV is likely to require the induction of strong and long-lasting humoral immune responses at the mucosal portal of virus entry. Hence, the design of a vaccine strategy able to induce mucosal antibodies and in particular specific IgA, may be crucial to providing immune protection. Nasal immunisation is known to induce specific IgG and IgA responses in the cervicovaginal mucosa; however, there is an urgent need for the development of safe, effective and accessible mucosal adjuvants for nasal application in humans. To reduce the potential for adverse events associated with some nasal adjuvants, we have assessed whether the B-cell-activating cytokines APRIL, BAFF and TSLP enhance humoral immune responses to HIV-1 gp140. Following intranasal immunisation, TSLP but not APRIL or BAFF induced strong humoral responses both in serum and mucosa. The adjuvant effect of TSLP on humoral responses was similar to that of cholera toxin (CT). The use of TSLP as an adjuvant skewed both the cellular and humoral immune responses towards Th2 cells. This is the first time that TSLP has been demonstrated to have a positive effect as a mucosal adjuvant, and specifically to promote mucosal and systemic responses to HIV gp140.

Biodistribution, pharmacokinetics, and nuclear imaging studies of 111In-labeled rGel/BLyS fusion toxin in SCID mice bearing B cell lymphoma.

PURPOSE: We examined the biodistribution and pharmacokinetics of (111)In-labeled rGel/BLyS, a gelonin toxin (rGel)-B lymphocyte stimulator (BLyS) fusion protein. MATERIALS AND METHODS: rGel/BLyS was labeled with In-111 through DTPA with a labeling efficiency >95%. Biodistribution/imaging studies were obtained in severe-combined immunodeficiency mice bearing diffuse large B cell lymphoma OCI-Ly10. Pharmacokinetic studies were performed in BALB/c mice. RESULTS: In vitro, DTPA-conjugated rGel/BLyS displayed selective cytotoxicity against OCI-Ly10 cells and mantle cell lymphoma JeKo cells. In vivo, rGel/BLyS exhibited a tri-exponential disposition with a rapid initial mean distribution followed by an extensive mean distribution and a long terminal elimination phase. At 48 h after injection, uptake of the radiotracer in tumors was 1.25 %ID/g, with a tumor-to-blood ratio of 13. Tumors were clearly visualized at 24-72 h post-injection. Micro-SPECT-CT images and ex vivo analyses confirmed the accumulation of rGel/BLyS in OCI-Ly10 tumors. CONCLUSIONS: (111)In-DTPA-rGel/BLyS are distributed to B cell tumors and induce apoptosis in tumors. Preclinical antitumor studies using rGel/BLyS should use a twice-per-week treatment schedule.

The rGel/BLyS fusion toxin inhibits diffuse large B-cell lymphoma growth in vitro and in vivo.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive subtype of B-cell non-Hodgkin lymphoma (NHL) and accounts for 30%to 40%of NHL. Molecules targeting nuclear factor-kappaB (NF-kappaB) are expected to be of therapeutic value in those tumors where NF-kappaB seems to play a unique survival role such as activated B-cell (ABC)-subtype DLBCL. We previously generated a rGel/BLyS fusion toxin for receptor-mediated delivery of the rGel toxin specifically to malignant B cells. In this study, we examined this fusion toxin for its ability to suppress DLBCL growth in vitro and in vivo. rGel/BLyS was specifically cytotoxic to DLBCL lines expressing all three BLyS receptors and constitutively active NF-kappaB. Treatment with rGel/BLyS induced down-regulation of the phosphorylation of inhibitory subunit of NF-kappaB (IkappaB-alpha), inhibition of NF-kappaB DNA-binding activity, and accumulation of IkappaB-alpha. In agreement with these results, we additionally found that rGel/BLyS downregulated levels of several NF-kappaB targets including Bcl-xL, Mcl-1, survivin, and x-chromosome linked inhibitor-of-apoptosis. Treatment also induced up-regulation of Bax and apoptosis through caspase-3 activation and poly ADP-ribose polymerase cleavage. Importantly, rGel/BLyS significantly inhibited tumor growth (P < .05) in a DLBCL xenograft model. Thus, our results indicate that rGel/BLyS is an excellent candidate for the treatment of aggressive NHLs that are both dependent on NF-kappaB and are resistant to conventional chemotherapeutic regimens.

Rescue of the mature B cell compartment in BAFF-deficient mice by treatment with recombinant Fc-BAFF.

BAFF deficiency in mice impairs B cell development beyond the transitional stage 1 in the spleen and thus severely reduces the size of follicular and marginal zone B cell compartments. Moreover, humoral immune responses in these mice are dramatically impaired. We now addressed the question whether the decrease in mature B cell numbers and the reduced humoral immune responses in BAFF-deficient mice could be overcome by the injection of recombinant BAFF. We therefore engineered a recombinant protein containing the human IgG1 Fc moiety fused to receptor-binding domain of human BAFF (Fc-BAFF). At 1 week after the second injection of this fusion protein a complete rescue of the marginal zone B cell compartment and a 50% rescue of the follicular B cell compartment was observed. Moreover these mice mounted a T cell-dependent humoral immune response indistinguishable from wild-type mice. By day 14 upon arrest of Fc-BAFF treatment mature B cell numbers in the blood dropped by 50%, indicating that the life span of mature B cells in the absence of BAFF is 14 days or less. Collectively these findings demonstrate that injection of Fc-BAFF in BAFF-deficient mice results in a temporary rescue of a functional mature B cell compartment.

Tumor targeting of vincristine by mBAFF-modified PEG liposomes in B lymphoma cells.

Malignant hematologic diseases are highly malignant and refractory to conventional therapies. Ligand-mediated targeting of liposomal anticancer drugs to surface receptors expressed on malignant B cells can be an effective strategy for treating B-cell malignancies. BAFF plays an important role in the maintenance of normal B-cell development and homeostasis and the expression of its receptors is significantly increased in numerous B-cell malignancies. mBAFF (a soluble BAFF mutant with amino acid 217-224 being replaced by two glycine residues) may be used as a competitive inhibitor for BAFF to treat relevant malignant hematologic diseases. It may also hold promise as a novel ligand for targeted anticancer therapy. In this study, we show that liposomes that are sterically stabilized by PEG and surface decorated with mBAFF exhibited strong affinity and specificity to cultured human Raji B lymphoma cells. Vincristine formulated in the targeted liposomes showed significantly higher levels of cytotoxicity towards Raji cells than the nontargeted liposomal drug. Therapeutic experiments in SCID mice implanted with Raji cells showed significantly prolonged survival time with targeted liposomal vincristine compared to either free VCR or vincristine formulated in nontargeted liposomes. These studies suggest the potential of the mBAFF-modified liposomal drugs in targeted therapy of B-cell malignancies.