The method history report: an adaptable tool for communicating immunogenicity assay development and validation.

Over the developmental lifetime of a therapeutic protein, the immunogenicity assay validation history can become substantial, frustrating review of clinical immunogenicity within the biologics license application. In our experience, this can lead to questions by regulators, resulting in numerous information requests during the review process. To address this, we propose a new document, the method history report (MHR), which can comprehensively present the history of the immunogenicity assay for regulators, including assay development and validation. The flexibility of the MHR allows for adaptation to the specific needs of each therapeutic program, while maintaining a consistent template. Here, we detail the rationale, general outline and template for the MHR and recommend others consider adopting it for their biologics license application-related activities.

An Analysis of Patient-reported Outcomes in IXORA-S: Comparing Ixekizumab and Ustekinumab over 52 Weeks in Moderate-to-severe Psoriasis.

Patient-reported outcomes are valuable for assessing new psoriasis therapies. This study investigated patient-reported outcomes in patients with moderate-to-severe plaque psoriasis treated with ixekizumab or ustekinumab, dosed according to their respective labels, for 52 weeks (IXORA-S-NCT02561806). Patient-reported outcomes investigated included patient global assessment, pruritus, skin pain, health-related quality of life, and work productivity. Ixekizumab-treated patients reported greater improvements in patient-reported outcomes sooner after treatment compared with ustekinumab-treated patients, and maintained greater improvements in patient global assessment scores (ixekizumab 0.72, ustekinumab 1.19; p < 0.001), rates of Dermatology Life Quality Index (0, 1) (ixekizumab 71.3%, ustekinumab 56.6%, p < 0.01), and 36-item Short-form Health survey physical component summary score change from baseline (ixekizumab 5.53, ustekinumab 3.28; p < 0.05) at week 52. While clinically meaningful improvements in patient-reported outcomes resulted with either treatment, ixekizumab provided more rapid improvements in patient-reported outcomes and superior outcomes for some assessments through one year of treatment, while maintaining statistically superior improvements in skin severity, as assessed by either physicians or patients.

Ixekizumab treatment and the impact on SF-36: results from three pivotal phase III randomised controlled trials in patients with moderate-to-severe plaque psoriasis.

PURPOSE: To assess improvements in health-related quality of life (HRQoL) with ixekizumab treatment in patients with moderate-to-severe psoriasis. METHODS: Adults with plaque psoriasis were enrolled in phase III, double-blind, randomised, controlled trials (UNCOVER-1, UNCOVER-2, or UNCOVER-3). All 3 protocols included a 12-week, placebo-controlled induction period; UNCOVER-2 and UNCOVER-3 also had an active-control group (50 mg etanercept) during induction. After induction, patients in UNCOVER-1 and UNCOVER-2 entered a 48-week withdrawal (maintenance) period (Weeks 12-60), during which Week-12 sPGA (0,1) responders were rerandomized to receive placebo, or 80 mg ixekizumab every 4 weeks (Q4W) or 12 weeks. As a secondary objective, HRQoL was measured by the generic Medical Outcomes Survey Short Form-36 (SF-36) at baseline and Weeks 12 and 60. Changes in mean SF-36 Physical and Mental Component Summary (PCS and MCS) and domain scores and proportions of patients reporting improvements ≥ minimal important differences in SF-36 scores were compared between groups. RESULTS: At Week 12, ixekizumab-treated patients (both dose groups in UNCOVER-1, -2, and -3) reported statistically significantly greater improvements in mean SF-36 PCS and MCS and all 8 SF-36 domain scores versus placebo. Further, more ixekizumab-treated patients than placebo-treated patients reported at least minimal treatment responses in SF-36 PCS and MCS scores and domain scores. Overall improvements in SF-36 PCS and MCS scores were maintained through Week 60. CONCLUSIONS: Ixekizumab-treated patients reported statistically significant improvements in HRQoL at 12 weeks that persisted through 1 year.

Ixekizumab provides superior efficacy compared with ustekinumab over 52 weeks of treatment: Results from IXORA-S, a phase 3 study.

BACKGROUND: Biologics targeting interleukin 17A (IL-17A) allow for rapid clearance of psoriatic plaques, with a clinically favorable safety profile. OBJECTIVES: To compare the safety and efficacy of ixekizumab, an IL-17A antagonist, with the safety and efficacy of the IL-12/23 inhibitor ustekinumab through 52 weeks of treatment in the head-to-head trial IXORA-S. METHODS: Patients were randomized to ixekizumab (n = 136) or ustekinumab (n = 166) and dosed per the approved labels. After 1 year, efficacy was assessed via improvements in Psoriasis Area and Severity Index (PASI) score (with PASI 90 indicating a 90% or greater improvement from baseline PASI score) and a static Physician's Global Assessment (sPGA) response of either 0 or 0 or 1, with dropouts counted as nonresponders. Safety analyses included treatment-emergent adverse events (AEs). RESULTS: At week 52, significantly more ixekizumab-treated patients (P < .01) reported PASI 90 (104 [76.5%]), an sPGA response of 0 (72 [52.9%]), or an sPGA response of 0 or 1 (110 [82.1%]) responses than did ustekinumab-treated patients (PASI 90, 98 [59.0%]; sPGA response of 0, 60 [36.1%]; and sPGA response of 0 or 1, 108 [65.1%]). Treatment-emergent AEs, serious AEs, and discontinuation rates were not different between the treatment groups. Injection site reactions occurred more frequently in the ixekizumab-treated group (ixekizumab, 22 [16.3%]; ustekinumab, 2 [1.2%]) (P < .001). LIMITATIONS: This study was not designed to compare safety end points related to rare events. CONCLUSIONS: Compared with ustekinumab, ixekizumab showed superior efficacy and comparable safety outcomes through 52 weeks of treatment.

Levels of circulating follicular helper T cells, T helper 1 cells, and the prognostic significance of soluble form of CD40 ligand on survival in patients with alcoholic cirrhosis.

BACKGROUND: Excessive drinkers (ED) and patients with alcoholic liver disease (ALD) are several times more susceptible to bacterial and viral infections and have a decrease in antibody responses to vaccinations. Follicular helper T (TFH) cells are essential to select B cells in the germinal center and to produce antibodies. TFH cells express both a membrane-associated and a soluble form of CD40 ligand (sCD40L); in which the latter form is released to circulation upon T cell activation. The effect of alcohol on TFH cells has not been studied. OBJECTIVES: The goals of this study are to determine the levels of TFH and T helper 1 (Th1) cells in ED and those with alcoholic cirrhosis (AC) when compared to healthy controls and to determine the prognostic significance of sCD40L in a cohort of patients with AC. METHODS: Controls, ED, and those with AC were enrolled. Baseline demographic, laboratory tests, and peripheral blood mononuclear cells (PBMCs) were isolated and assessed via flow cytometry for TFH cells. In vitro study was performed to determine the ability of PBMCs to secrete interferon (IFN)-γ upon stimulation. Serum sCD40L were also determined and its prognostic significance was tested in a cohort of AC patients. RESULTS: The levels of circulating TFH (cTFH) cells were significantly lower in peripheral blood of subjects with ED and AC compared to controls (P<0.05). IFN-γ secretion from PBMCs upon stimulation was also lower in ED and those with cirrhosis. Serum sCD40L was significantly lower in ED and AC when compared to that in controls (P<0.0005). Its level was an independent predictor of mortality. CONCLUSIONS: Patients with AC had significantly lower level of cTFH and sCD40L. The level of sCD40L was an independent predictor of mortality in these patients.

Bcl6 promotes follicular helper T-cell differentiation and PD-1 expression in a Blimp1-independent manner in mice.

The transcription factors Bcl6 and Blimp1 have opposing roles in the development of the follicular helper T (TFH ) cells: Bcl6 promotes and Blimp1 inhibits TFH -cell differentiation. Similarly, Bcl6 activates, while Blimp1 represses, expression of the TFH -cell marker PD-1. However, Bcl6 and Blimp1 repress each other's expression, complicating the interpretation of the regulatory network. Here we sought to clarify the extent to which Bcl6 and Blimp1 independently control TFH -cell differentiation by generating mice with T-cell specific deletion of both Bcl6 and Blimp1 (double conditional KO [dcKO] mice). Our data indicate that Blimp1 does not control TFH -cell differentiation independently of Bcl6. However, a population of T follicular regulatory (TFR ) cells developed independently of Bcl6 in dcKO mice. We have also analyzed regulation of IL-10 and PD-1, two genes controlled by both Bcl6 and Blimp1, and observed that Bcl6 regulates both genes independently of Blimp1. We found that Bcl6 positively regulates PD-1 expression by inhibiting the ability of T-bet/Tbx21 to repress Pdcd1 transcription. Our data provide a novel mechanism for positive control of gene expression by Bcl6, and illuminate how Bcl6 and Blimp1 control TFH -cell differentiation.

The role of follicular helper T cells and the germinal center in HIV-1 gp120 DNA prime and gp120 protein boost vaccination.

The importance of follicular T helper (TFH) cells and the germinal center (GC) reaction in the humoral immune response has become clear in recent years, however the role of TFH cells and the GC in an HIV vaccine strategy remains unclear. In this study, we primed mice with gp120-encoding DNA and boosted with gp120 protein, a regimen previously shown to induce high titers of high affinity and cross-reactive anti-gp120 Abs. Priming with gp120 DNA caused increased TFH cell differentiation, GC B cells, and antigen-specific antibody titers, compared with priming with gp120 protein. Priming with DNA also caused more activated CD4(+) T cells to become TFH cells and more GC B cells to become memory cells. Deletion of BCL6 midway through the vaccine regimen resulted in loss of TFH cells and GCs, and, unexpectedly, increased anti-gp120 IgG titers and avidity. Our data suggests vaccination with gp120-encoding DNA elicits a stronger and more rapid TFH and GC response than gp120 protein. Furthermore, we demonstrate that the GC reaction may actually limit antigen-specific IgG secretion in the context of repeated immunizations.

A negative feedback loop mediated by the Bcl6-cullin 3 complex limits Tfh cell differentiation.

Induction of Bcl6 (B cell lymphoma 6) is essential for T follicular helper (Tfh) cell differentiation of antigen-stimulated CD4(+) T cells. Intriguingly, we found that Bcl6 was also highly and transiently expressed during the CD4(+)CD8(+) (double positive [DP]) stage of T cell development, in association with the E3 ligase cullin 3 (Cul3), a novel binding partner of Bcl6 which ubiquitinates histone proteins. DP stage-specific deletion of the E3 ligase Cul3, or of Bcl6, induced the derepression of the Bcl6 target genes Batf (basic leucine zipper transcription factor, ATF-like) and Bcl6, in part through epigenetic modifications of CD4(+) single-positive thymocytes. Although they maintained an apparently normal phenotype after emigration, they expressed increased amounts of Batf and Bcl6 at basal state and produced explosive and prolonged Tfh responses upon subsequent antigen encounter. Ablation of Cul3 in mature CD4(+) splenocytes also resulted in dramatically exaggerated Tfh responses. Thus, although previous studies have emphasized the essential role of Bcl6 in inducing Tfh responses, our findings reveal that Bcl6-Cul3 complexes also provide essential negative feedback regulation during both thymocyte development and T cell activation to restrain excessive Tfh responses.

Virus-encoded ectopic CD74 enhances poxvirus vaccine efficacy.

Vaccinia virus (VV) has been used globally as a vaccine to eradicate smallpox. Widespread use of this viral vaccine has been tempered in recent years because of its immuno-evasive properties, with restrictions prohibiting VV inoculation of individuals with immune deficiencies or atopic skin diseases. VV infection is known to perturb several pathways for immune recognition including MHC class II (MHCII) and CD1d-restricted antigen presentation. MHCII and CD1d molecules associate with a conserved intracellular chaperone, CD74, also known as invariant chain. Upon VV infection, cellular CD74 levels are significantly reduced in antigen-presenting cells, consistent with the observed destabilization of MHCII molecules. In the current study, the ability of sustained CD74 expression to overcome VV-induced suppression of antigen presentation was investigated. Viral inhibition of MHCII antigen presentation could be partially ameliorated by ectopic expression of CD74 or by infection of cells with a recombinant VV encoding murine CD74 (mCD74-VV). In contrast, virus-induced disruptions in CD1d-mediated antigen presentation persisted even with sustained CD74 expression. Mice immunized with the recombinant mCD74-VV displayed greater protection during VV challenge and more robust anti-VV antibody responses. Together, these observations suggest that recombinant VV vaccines encoding CD74 may be useful tools to improve CD4⁺ T-cell responses to viral and tumour antigens.

The transcription factor Twist1 limits T helper 17 and T follicular helper cell development by repressing the gene encoding the interleukin-6 receptor α chain.

Cytokine responsiveness is a critical component of the ability of cells to respond to the extracellular milieu. Transcription factor-mediated regulation of cytokine receptor expression is a common mode of altering responses to the external environment. We identify the transcription factor Twist1 as a component of a STAT3-induced feedback loop that controls IL-6 signals by directly repressing Il6ra. Human and mouse T cells lacking Twist1 have an increased ability to differentiate into Th17 cells. Mice with a T cell-specific deletion of Twist1 demonstrate increased Th17 and T follicular helper cell development, early onset experimental autoimmune encephalomyelitis, and increased antigen-specific antibody responses. Thus, Twist1 has a critical role in limiting both cell-mediated and humoral immunity.

Insights into the role of Bcl6 in follicular Th cells using a new conditional mutant mouse model.

The transcriptional repressor Bcl6 controls development of the follicular Th cell (T(FH)) lineage, but the precise mechanisms by which Bcl6 regulates this process are unclear. A model has been proposed whereby Bcl6 represses the differentiation of T cells into alternative effector lineages, thus favoring T(FH) cell differentiation. Analysis of T cell differentiation using Bcl6-deficient mice has been complicated by the strong proinflammatory phenotype of Bcl6-deficient myeloid cells. In this study, we report data from a novel mouse model where Bcl6 is conditionally deleted in T cells (Bcl6(fl/fl)Cre(CD4) mice). After immunization, programmed death -1 (PD-1)(high) T(FH) cells in Bcl6(fl/fl)Cre(CD4) mice are decreased >90% compared with control mice, and Ag-specific IgG is sharply reduced. Residual PD-1(high)CXCR5(+) T(FH) cells in Bcl6(fl/fl)Cre(CD4) mice show a significantly higher rate of apoptosis than do PD-1(high)CXCR5(+) T(FH) cells in control mice. Immunization of Bcl6(fl/fl)Cre(CD4) mice did not reveal enhanced differentiation into Th1, Th2, or Th17 lineages, although IL-10 expression by CD4 T cells was markedly elevated. Thus, T cell-extrinsic factors appear to promote the increased Th1, Th2, and Th17 responses in germline Bcl6-deficient mice. Furthermore, IL-10 may be a key target gene for Bcl6 in CD4 T cells, which enables Bcl6 to promote the T(FH) cell phenotype. Finally, our data reveal a novel mechanism for the role of Bcl6 in promoting T(FH) cell survival.