T-cell Dependent Antibody Responses Research Articles

Preclinical immunogenicity risk assessment of biotherapeutics using CD4 T cell assays.

T-cell dependent antibody responses to biotherapeutics remain a challenge to the optimal clinical application of biotherapeutics because of their capacity to impair drug efficacy and their potential to cause safety issues. To minimize this clinical immunogenicity risk, preclinical assays measuring the capacity of biotherapeutics to elicit CD4 T cell response in vitro are commonly used. However, there is considerable variability in assay formats and a general poor understanding of their respective predictive value. In this study, we evaluated the performance of three different CD4 T cell proliferation assays in their capacity to predict clinical immunogenicity: a CD8 T cell depleted peripheral blood mononuclear cells (PBMC) assay and two co-culture-based assays between dendritic cells (DCs) and autologous CD4 T cells with or without restimulation with monocytes. A panel of 10 antibodies with a wide range of clinical immunogenicity was selected. The CD8 T cell depleted PBMC assay predicted the clinical immunogenicity in four of the eight highly immunogenic antibodies included in the panel. Similarly, five antibodies with high clinical immunogenicity triggered a response in the DC: CD4 T cell assay but the responses were of lower magnitude than the ones observed in the PBMC assay. Remarkably, three antibodies with high clinical immunogenicity did not trigger any response in either platform. The addition of a monocyte restimulation step to the DC: CD4 T cell assay did not further improve its predictive value. Overall, these results indicate that there are no CD4 T cell assay formats that can predict the clinical immunogenicity of all biotherapeutics and reinforce the need to combine results from various preclinical assays assessing antigen uptake and presentation to fully mitigate the immunogenicity risk of biotherapeutics.

Mechanisms of antibody mediated immunity – Distinct in early life

Immune responses in early life are characterized by a failure to robustly generate long-lasting protective responses against many common pathogens or upon vaccination. This is associated with a reduced ability to generate T-cell dependent high affinity antibodies. This review highlights the differences in T-cell dependent antibody responses observed between infants and adults, in particular focussing on the alterations in immune cell function that lead to reduced T follicular helper cell-B cell crosstalk within germinal centres in early life. Understanding the distinct functional characteristics of early life humoral immunity, and how these are regulated, will be critical in guiding age-appropriate immunological interventions in the very young.

Quantifying the impact of PFOA Exposure on B cell development and antibody production.

Per- and polyfluoroalkyl substances (PFAS) are synthetic chemicals; the vast majority are environmentally and biologically persistent, and some have demonstrated toxicity, including cancer, effects on metabolism, endocrine disruption, and immune dysfunction. Suppression of T-cell dependent antibody responses (TDAR) has been observed in numerous studies of PFAS but mechanisms remain elusive. Evidence from our work suggests that B cells and how they use energy are impacted by PFAS exposure. We hypothesize that a well-studied and immunotoxic PFAS, perfluorooctanoic acid (PFOA), alters B cell subclasses and markers of their metabolism. Adult male and female C57BL/6 mice were given PFOA (0 or 7.5 mg/kg) via gavage for 15 days, a duration and dose sufficient to suppress the TDAR. After dosing and immunization of subgroups, spleens were prepared to quantify B cell subsets. Flow cytometric analysis revealed decreased numbers of plasmablasts, follicular, naïve, and overall B cell subclasses in female PFOA-exposed groups. Male PFOA-exposed groups had a significant increase in follicular B cells and other subsets had decreases, including in the overall number of B cells. Twenty-four hours after naïve B cell isolation and ex vivo activation, metabolic measurements revealed a five-fold increase in metabolic markers in response to stimulation in PFOA-exposed groups compared to controls. These findings suggest that B cell development and survival may be hindered by PFOA exposure, but that activation of the remaining B cells was not. Based on these findings, PFOA mediated suppression of the primary IgM antibody response results changes to specific subsets of B cells.

Abstract 2269: Bexmarilimab induces B-cell activation and autoantibody production

Abstract Introduction: Bexmarilimab, a Clever-1 targeting humanized antibody, is a macrophage checkpoint inhibitor promoting antigen presentation and pro-inflammatory cytokine secretion. Data from the first-in-human clinical Phase I/II study (MATINS; NCT03733990) demonstrate that single agent bexmarilimab is able to ignite an interferon response with survival benefit in 30% of patients with advanced gastric cancer, cutaneous melanoma and cholangiocarcinoma (ASCO 2022). Since Clever-1 knockout mice have been reported to have enhanced T-cell antibody production and bexmarilimab increases peripheral B-cell populations, we investigated B-cell phenotype, clonality and autoantibody formation in bexmarilimab treatmented MATINS patients. Methods: We first performed comprehensive phenotyping of peripheral B-cells from a colorectal carcinoma patient showing partial response (per RECIST v1.1). Autoantibody reactivities were measured in 80 serum samples of bexmarilimab treated patients (37 pre-treatment and 43 post-treatment samples from cycles 3 and 4) and compared to serums from 53 healthy individuals using Oncimmune's SeroTag multiplex technology with an immune-oncology (IO) specific protein array comprised of 1162 antigens. Results: Single-cell sequencing together with BCR sequencing of the responder’s B-cells during cycle 4 revealed an induced and activated B-cell population consisting of four transcriptionally similar clusters expressing IGHM, IGHD, CD23 but not CD43 and one cluster expressing CD79B, PLD4 and MZB1, which was distant from the naïve CCR7 expressing B-cells and IGHA1 and IGHG1 expressing plasma blasts. The expansion of B-cells was not due to clonal expansion as no overlapping BCR clones were identified between B-cells at pre-dose and cycle 4. Autoantibody profiling revealed great inter-individual heterogeneity in the number and targets of induced antibodies among patients treated with bexmarilimab reflecting individual patterns in self- and tumor antigens. However, shared autoantibody changes against targets such as cancer testis antigens (GAGE2), typical autoimmune disease antigens (SNRPC, TPO, TOP1) and antigens related to the induction of innate immunity and interferon responses, e.g. TRIM21 were observed. Importantly, we identified a set of pre-treatment autoantibodies that were associated with longer time to disease progression and were predictive of clinical response (disease control rate [DCR], consisting of CR, PR, SD) and progression-free survival (PFS) on bexmarilimab therapy. Conclusions: Our data implies that bexmarilimab can induce activation and secondary Ig rearrangements in mature B-cells, which has been reported to occur in germinal centres during T-cell dependent antibody responses to increase B-cell diversity and affinity of antigen receptors. B cell diversity and activation was reflected in patient autoantibody production and may enhance cancer immune recognition. Citation Format: Elisa M. Vuorinen, Mari L. Björkman, Reetta Virtakoivu, Juho Jalkanen, Sofia Aakko, Akira Takeda, Petra Budde, Hans-Dieter Zucht, Manuel Brautigam, Behnaz Ahangarianabhari, Petri Bono, Maija Hollmén. Bexmarilimab induces B-cell activation and autoantibody production [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2269.

Preclinical Immunopharmacologic Assessment of KPL-404, a Novel, Humanized, Non-Depleting Antagonistic Anti-CD40 Monoclonal Antibody

The CD40/CD40L pathway plays a major role in multiple inflammatory processes involving different immune and stromal cells. Abnormal activation of this pathway has been implicated in pathogenesis of complex autoimmune diseases including systemic lupus erythematosus, rheumatoid arthritis, Graves' disease, and Sjogren's Syndrome. We completed in vitro and in vivo preclinical characterization of KPL-404, a novel humanized anti-CD40 IgG4 monoclonal antibody, to demonstrate its potency, efficacy, and pharmacokinetic profile; safety was also assessed. In vitro, KPL-404 bound recombinant human and cynomolgus monkey CD40 with comparable affinity in the nanomolar range. KPL-404 binding to cell surface CD40 did not induce antibody- or complement-mediated cytotoxicity of CD40-expressing cells. Pharmacological antagonistic activity of KPL-404 was demonstrated in vitro by inhibition of CD40-mediated downstream NF-kB activation. In the in vivo study with cynomolgus monkeys, KPL-404, administered intravenously as a single dose (10 mg/kg) or two monthly doses of 1 or 5 mg/kg, did not elicit observable safety findings, including thrombocytopenia over 8 weeks. KPL-404 engaged CD40 expressed on peripheral B cells for 2 and 4 weeks after a single administration of 5 or 10 mg/kg IV, respectively, without depletion of peripheral B cells. At 5 mg/kg IV, KPL-404 blocked both primary and secondary responses to T-cell dependent antibody responses to test antigens, KLH, and tetanus toxoid. These data illustrated the relationship between KPL-404 serum concentration and pharmacodynamic effects of CD40-targeting in circulation and in lymphoid tissues. These data support clinical development of KPL-404 in autoimmune diseases. SIGNIFICANCE STATEMENT: We aimed to develop a potent and efficacious CD40 antagonist. In vitro and in vivo findings show that KPL-404 blocks the anti-CD40 antibody that potently inhibits primary and secondary antibody responses at pharmacologically relevant concentrations, has a favorable pharmacokinetic profile, and does not deplete B cells by antibody-dependent cellular cytotoxicity or apoptosis ("nondepleting"). These findings support clinical development of KPL-404 as a potential therapeutic in autoimmune diseases.

First-in-human clinical trial to assess pharmacokinetics, pharmacodynamics, safety, and tolerability of iscalimab, an anti-CD40 monoclonal antibody.

Iscalimab is a fully human, CD40 pathway blocking, nondepleting monoclonal antibody being developed as an immunosuppressive agent. We describe a first-in-human, randomized, double-blind, placebo-controlled study investigating the safety, tolerability, pharmacokinetics, and pharmacodynamics of iscalimab in healthy subjects and rheumatoid arthritis patients. Healthy subjects (n=56) received single doses of intravenous iscalimab (0.03, 0.1, 0.3, 1, or 3mg/kg), or subcutaneous iscalimab (3mg/kg), or placebo. Rheumatoid arthritis patients (n=20) received single doses of intravenous iscalimab (10 or 30mg/kg) or placebo. Iscalimab exhibited target-mediated drug disposition resulting in dose-dependent and nonlinear pharmacokinetics. Complete (≥90%) CD40 receptor occupancy on whole blood B cells was observed at plasma concentrations >0.3-0.4µg/mL. In subjects receiving 3mg/kg iscalimab, antibody responses to keyhole limpet hemocyanin were transiently suppressed. CD40 occupancy by iscalimab prevented ex vivo human rCD154-induced expression of CD69 on B cells in whole blood. All doses were generally safe and well tolerated, with no clinically relevant changes in any safety parameters, including no evidence of thromboembolic events. Iscalimab appears to be a promising blocker of the CD40-CD154 costimulatory pathway with potential use in transplantation and other autoimmune diseases.

CD40 function in immune development and response: Cell type requirements and cytoplasmic domain function

Abstract CD40 is a TNFR family member and serves as a costimulatory molecule expressed on multiple antigen presenting cell (APC) lineages. The functional significance of CD40 expression is illustrated by human disease and mouse models of life-threatening immune deficiency. CD40-CD40L deficiency affects multiple aspects of immune development and response. However, cell type-specific requirements for CD40 and requirements for CD40 signaling domains have not been comprehensively analyzed. In this study, we use conditional CD40 knockout mice to study cell type requirements and generated CD40 intracellular domain mutants by CRISPR to determine domain-specific functions. CD40 expression on B cells but not DC is required for CD40 dependent superantigen-specific negative selection and for T-cell dependent class-switched antibody responses. TRAF2/3 binding domains of CD40 are critical for these CD40 functions, while deletion of putative TRAF6 binding domain had minimal effect. In contrast, in vivo priming of cytokine-producing T cells depends on CD40 expression on DC but not B cells and requires both TRAF2/3 and TRAF6 domains. CD40 expression on both B cells and DC is required for maximal autoimmune EAE disease induced by recombinant human MOG (rhMOG), and both TRAF2/3 and TRAF6 domains of CD40 are required for this autoimmune response. Initial results indicate that CD40 on thymic epithelial cells and DC contributes to negative selection of tissue restricted antigen-binding T cells. These results identify different CD40-dependent APC requirements and suggest distinct CD40 signaling pathways for specific immune responses. Analysis of the linkage between cell type-specific CD40 function and intracellular signaling is under study.

Adenosine deaminase-1 delineates human follicular helper T cell function and is altered with HIV

Follicular helper T cells (Tfh) play critical roles instructing, and initiating T-cell dependent antibody responses. The underlying mechanisms that enhance their function is therefore critical for vaccine development. Here we apply gene array analysis identifying adenosine deaminase (ADA) as a key molecule that delineates a human Tfh helper program in proliferating circulating Tfh (cTfh) cells and Germinal Centers Tfh (GC-Tfh). ADA-1 expression and enzymatic activity are increased in efficient cTfh2-17/GC-Tfh cells. Exogenous ADA-1 enhances less efficient cTfh1 and pro-follicular Tfh PD-1+ CXCR5+ cells to provide B cell help, while pharmacological inhibition of ADA-1 activity impedes cTfh2-17/GC-Tfh function and diminished antibody response. Mechanistically, ADA-1 controls the Tfh program by influencing IL6/IL-2 production, controlling CD26 extracellular expression and could balance signals through adenosine receptors. Interestingly, dysfunctional Tfh from HIV infected-individual fail to regulate the ADA pathway. Thus, ADA-1 regulates human Tfh and represents a potential target for development of vaccine strategy.

T Cell/B Cell Collaboration and Autoimmunity: An Intimate Relationship.

Co-ordinated interaction between distinct cell types is a hallmark of successful immune function. A striking example of this is the carefully orchestrated cooperation between helper T cells and B cells that occurs during the initiation and fine-tuning of T-cell dependent antibody responses. While these processes have evolved to permit rapid immune defense against infection, it is becoming increasingly clear that such interactions can also underpin the development of autoimmunity. Here we discuss a selection of cellular and molecular pathways that mediate T cell/B cell collaboration and highlight how in vivo models and genome wide association studies link them with autoimmune disease. In particular, we emphasize how CTLA-4-mediated regulation of CD28 signaling controls the engagement of secondary costimulatory pathways such as ICOS and OX40, and profoundly influences the capacity of T cells to provide B cell help. While our molecular understanding of the co-operation between T cells and B cells derives from analysis of secondary lymphoid tissues, emerging evidence suggests that subtly different rules may govern the interaction of T and B cells at ectopic sites during autoimmune inflammation. Accordingly, the phenotype of the T cells providing help at these sites includes notable distinctions, despite sharing core features with T cells imparting help in secondary lymphoid tissues. Finally, we highlight the interdependence of T cell and B cell responses and suggest that a significant beneficial impact of B cell depletion in autoimmune settings may be its detrimental effect on T cells engaged in molecular conversation with B cells.

Immune Responses to Animal Trypanosomosis: A Review

The aim of this document is to review the current knowledge on Trypanosome parasites of animals with emphasis on the immunological response and points to the wealth of information available for trypanosomiasis, in contrast to the numerous gaps in our understanding of immune responses to trypanosomal infections. African trypanosomes are pathogens for humans and livestock. They are single-cell, extra-cellular parasites that cause persistent infections of the blood and induce profound immune-suppression. The specific immune response to the infecting parasites is complex and involves both the humoral and cellular branches of immune systems. In trypanosomosis, parasite growth is primarily controlled through T-cell dependent antibody responses to the variable surface glycoproteins and possibly to other molecules embedded on the surface of the parasites. Cellular immune responses also occur but at the level of immune-suppression directed against B cells. Additionally, a variety of immune-modulatory cytokines like TNF-α, IFN-γ, IL-10, IL-4, IL-6, IL-12 and etc are produced during the course of infection. The center of the immune-pathology is the T-cell-independent production of antibodies to the variant surface glycoprotein of trypanosomes, the anti-VSG antibody-mediated phagocytosis of trypanosomes by macrophages, and the subsequent profound dysregulation of the macrophage system. It has, however, been demonstrated that a T-cell subset is critical in protective immunity.

Adenosine DeAminase (ADA) as an adjuvant molecule for human HIV-1 vaccine

Abstract Follicular helper T cells (Tfh) play critical role in shaping, instructing, and initiating T-cell dependent antibody responses. Understanding the underlying mechanisms that enhance their function is therefore critical for vaccine development. Using a unique gene array analysis, we have identified adenosine deaminase (ADA), as a novel key molecule that drives Tfh helper program in proliferating Germinal Centers Tfh (GC Tfh) and circulatory Tfh (cTfh) cells following their interactions with B cells. In fact, our gene array analysis showed that CD26 and ADA were exclusively up-regulated within the less efficient cTfh1 (CD4+CD45RA−CXCR5+CXCR3+) and cTfh2-17 (CD4+CD45RA−CXCR5+CXCR3+CCR6+/−) subsets, respectively. ADA enzymatic activity is significantly higher, as well, in cTfh2-17 than in the less-efficient cTfh1 cells. Exogenous ADA enhances the ability of Tfh cells to provide B cell help while inhibition of ADA activity by specific inhibitors impeded Tfh function and blunted antibody response. Our results further demonstrated that enhancement of Tfh function by ADA pathway could be due to increase in IL-6 and decrease in IL-2 production in the co-culture, and maintenance of low extracellular expression of CD26. Moreover, blocking IL-2 in cTfh2-17 co-culture from virally suppressed HIV subjects (ST) showed a significant decrease of CD26 expression associated with a rescued helper capacity. Finally, in vivo use of recombinant ADA as an adjuvant in a DNA based HIV vaccine enhanced Tfh cells differentiation and enhanced anti-Env humoral response and isotype class-switch. Thus, ADA activity fine-tunes Tfh helper program and deciphering how ADA regulate the function of Tfh subsets would benefit future vaccine adjuvant design.

Evaluation of the immunomodulatory effects of 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate in C57BL/6 mice.

2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)-propanoate was designed to replace perfluorooctanoic acid (PFOA), which has been mostly phased out of U.S. production due to environmental persistence, detectable human and wildlife serum concentrations, and reports of systemic toxicity. In rodent models, PFOA exposure suppresses T cell-dependent antibody responses (TDAR) and vaccine responses in exposed humans. To determine replacement compound effects on TDAR and related parameters, male and female C57BL/6 mice were gavaged with 0, 1, 10, or 100 mg/kg/day for 28 days. Mice immunized with antigen on day 24 were evaluated for TDAR and splenic lymphocyte subpopulations five days later. Serum and urine were collected for test compound concentrations and liver peroxisome proliferation was measured. Relative liver weight at 10 and 100 mg/kg and peroxisome proliferation at 100 mg/kg were increased in both sexes. TDAR was suppressed in females at 100 mg/kg. T lymphocyte numbers were increased in males at 100 mg/kg; B lymphocyte numbers were unchanged in both sexes. Females had less serum accumulation and higher clearance than males, and males had higher urine concentrations than females at all times and doses. While this PFOA-replacement compound appears less potent at suppressing TDAR relative to PFOA, it produces detectable changes in parameters affected by PFOA; further studies are necessary to determine its full immunomodulatory profile and potential synergism with other per- and polyfluoroalkyl substances of environmental concern.

Appearance of High-Affinity Antibodies Precedes Clinical Diagnosis of FVIII Inhibitors - Preliminary Analysis from the Hemophilia Inhibitor PUP Study (HIPS)

Background and ObjectivesThe development of neutralizing antibodies (inhibitors) to factor VIII (FVIII) represents the major complication following replacement therapy with FVIII products in patients with hemophilia A. Why some patients develop FVIII inhibitors while others do not is poorly understood. Identification of early biomarkers which predict inhibitor development in previously untreated patients (PUPs) with hemophilia A will assist in risk identification and possible early intervention strategies. Previous analysis of FVIII-specific antibodies in different cohorts of patients with severe hemophilia A and in healthy individuals indicated distinct spectra of neutralizing and non-neutralizing antibodies (Whelan SFJ et al. Blood 2013). FVIII-specific antibodies found in hemophilia A patients with inhibitors have approximately 100-fold higher apparent affinities than that of antibodies found in patients without inhibitors or in healthy individuals (Hofbauer CJ et al. Blood 2015). In one individual, high affinity FVIII-specific antibodies were detectable prior to the diagnosis of FVIII inhibitors. The Hemophilia Inhibitor PUP Study (HIPS) is a prospective multicenter observational study of PUPs with severe hemophilia A during their first 50 exposure days (EDs) to recombinant FVIII (Advate TM). The objective is to elucidate immune system changes and identify potential early biomarkers of inhibitor development in PUPs. This is the first report of FVIII-specific antibody development in the first 10 subjects who have completed the study.MethodsHIPS study procedures have been previously described (Hofbauer CJ et al. 2015; clinicaltrials.gov NCT01652027). FVIII inhibitor testing by Nijmegen methodology performed in a central laboratory (Medical College of Vienna) and FVIII-specific antibody analytics were done prior to first exposure, 7-9 days after exposure day (ED) 1 and 5-7 days after EDs 5, 10, 20, 30, 40, and 50. FVIII-specific antibody analytics, including specifications of isotypes, IgG subclasses and apparent affinities were done using methodology described in Whelan 2013 and Hofbauer 2015.Results:Currently, 24 subjects have been enrolled among 19 study sites. Data for 10 subjects were available for analysis. Among these 10 subjects, 2 developed FVIII inhibitors by study criteria (B.U. > 0.6 B.U. x 2 measurements in central laboratory) by exposure days 6 and 20 (see figure 1). A third subject is expected to meet study criteria for FVIII inhibitor due to multiple high titer measurements reported over time at a local laboratory. All three inhibitor patients revealed antibody class switching from IgG1 to IgG4 and affinity maturation resulting in the generation of high affinity FVIII-specific antibodies. High-affinity FVIII-specific IgG1 antibodies were detectable prior to inhibitor diagnosis in all 3 inhibitor subjects.One subject with a transient FVIII inhibitor showed medium affinity FVIII-specific IgG1 antibodies which persisted until ED50, but never developed high-affinity antibodies. Another subject without a detectable inhibitor developed low titer, low affinity IgG1 antibodies that persisted to ED 50 without the development of high-affinity antibodies. The remaining subjects had no inhibitor and no detectable FVIII-specific antibodies.Conclusions:Interim data from HIPS suggests that FVIII inhibitor development is preceded by the detection of high-affinity FVIII-specific antibodies which undergo class switch from IgG1 and IgG3 to IgG4.The findings confirm affinity maturation of FVIII-specific antibodies which require T-cell dependent antibody responses resulting from germinal center reactions. While low affinity IgG1 antibodies were sometimes seen in subjects without inhibitors, high-affinity antibodies were only seen in subjects who developed inhibitors. Our data indicate that high affinity FVIII-specific antibodies preceded the clinical diagnosis of an inhibitor and may serve as early biomarkers of FVIII inhibitor development facilitating early immune intervention. [Display omitted] DisclosuresReipert:Shire: Employment, Research Funding. Gangadharan:Shire: Employment, Research Funding. Hofbauer:Shire: Employment. Scheiflinger:Shire: Employment, Research Funding. Bowen:Baxalta: Research Funding. Donnachie:Baxalta: Research Funding; CSL Behring: Research Funding; Bayer Healthcare: Research Funding. Fijvandraat:CSL Behring: Research Funding; Bayer Healthcare: Research Funding; Baxalta: Membership on an entity's Board of Directors or advisory committees. Gruppo:Baxalta: Honoraria, Membership on an entity's Board of Directors or advisory committees. Male:Baxalta: Other: travel support; Novo Nordisk: Other: travel support; Biotest: Other: travel support; Bayer Healthcare: Other: travel support; CSL Behring: Other: travel support; Pfizer: Other: travel support. McGuinn:Baxalta: Research Funding; Novo Nordisk: Research Funding; Spark: Research Funding; Biogen: Research Funding. Meeks:Genentech: Membership on an entity's Board of Directors or advisory committees; Bayer Healthcare: Membership on an entity's Board of Directors or advisory committees; Grifols: Membership on an entity's Board of Directors or advisory committees; Biogen: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees. Recht:Novo Nordisk: Consultancy, Research Funding; Baxalta: Research Funding; Biogen Idec: Research Funding; Kedrion: Consultancy. Ragni:Biogen: Research Funding. Yaish:Octapharma: Consultancy; Bayer Healthcare: Consultancy; Baxalta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Santagostino:Sobi: Consultancy; Biogen Idec: Consultancy; Novo Nordisk: Consultancy; Pfizer: Consultancy; Octapharma: Consultancy; Baxalta: Consultancy; Kedrion: Consultancy; CSL Behring: Consultancy; Bayer: Consultancy; Grifols: Consultancy; Roche: Consultancy. Brown:Baxalta/Shire: Research Funding.

Regulation and Maintenance of an Adoptive T-Cell Dependent Memory B Cell Pool.

We investigated the ability of monoclonal B cells to restore primary and secondary T-cell dependent antibody responses in adoptive immune-deficient hosts. Priming induced B cell activation and expansion, AID expression, antibody production and the generation of IgM+IgG- and IgM-IgG+ antigen-experienced B-cell subsets that persisted in the lymphopenic environment by cell division. Upon secondary transfer and recall the IgM-IgG+ cells responded by the production of antigen-specific IgG while the IgM+ memory cells secreted mainly IgM and little IgG, but generated new B cells expressing germinal center markers. The recall responses were more efficient if the antigenic boost was delayed suggesting that a period of adaptation is necessary before the transferred cells are able to respond. Overall these findings indicate that reconstitution of a functional and complete memory pool requires transfer of all different antigen-experienced B cell subsets. We also found that the size of the memory B cell pool did not rely on the number of the responding naïve B cells, suggesting autonomous homeostatic controls for naïve and memory B cells. By reconstituting a stable memory B cell pool in immune-deficient hosts using a monoclonal high-affinity B cell population we demonstrate the potential value of B cell adoptive immunotherapy.

Abstract IA24: MYC in germinal center B cell physiology and pathology

Abstract Germinal centers (GC)s are sites of intense B-cell proliferation, central for T-cell dependent antibody responses. Although much is known of GC B-cell physiology, the mechanisms of cellular proliferation have remained elusive. Specifically, the role of MYC, a key cell cycle regulator, in GC B-cell proliferation has been questioned. We have identified MYC positive B-cell subpopulations in both immature and mature GCs, and shown through genetic ablation of Myc at these stages that they play indispensable roles in GC formation and maintenance, respectively. The identification of functionally critical MYC-expressing GC B-cell subsets has important implications for B-cell lymphomagenesis, which mostly originates from GC cells and frequently involves MYC deregulation through chromosomal translocations. As these translocations are generally dependent on transcription of the recombining partner loci, the MYC positive GC subpopulations may represent B-cells with a particularly high risk for malignant transformation. Citation Format: Dinis Pedro Calado. MYC in germinal center B cell physiology and pathology. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr IA24.

Fully human anti-BAFF inhibitory monoclonal antibody tabalumab does not adversely affect T-dependent antibody responses in cynomolgus monkey (Macaca fasicularis): A summary of three pre-clinical immunotoxicology evaluations

The potential immunotoxicity of tabalumab was assessed as a component of standard pre-clinical toxicology studies in cynomolgus monkeys. To evaluate potential tabalumab-associated immunosuppression after antigen challenge, cynomolgus monkeys were administered placebo control or tabalumab in three immunotoxicological safety studies. Study 1, a 4-week pilot study, evaluated biweekly intravenous (IV) control, and 0.3, 1.0, 5.0, and 15.0 mg/kg tabalumab doses. Study 2 evaluated IV control, and 0.1, 1.0, and 30.0 mg/kg tabalumab doses biweekly for 6 weeks. Study 3 evaluated IV control and 0.1, 1.0, 30.0 mg/kg, and subcutaneous (SC) 30.0 mg/kg tabalumab biweekly for 6 months, with recovery (16 weeks) to monitor standard immunotoxicity endpoints. T-cell dependent primary and secondary antibody responses to tetanus toxoid antigen challenge (4-week and 6-week studies) or keyhole limpet hemocyanin (KLH; 6-week and 6-month studies) were evaluated as a measure of immunocompetence, together with quantitation of T- and B-cell subsets. In addition, anti-tabalumab antibody formation (6-week and 6-month studies) was assessed. The results indicated that, despite expected decreases in circulating B-cell populations, no changes in follicle histopathology or organ weights, except decreases in spleen weight (after 6-months of 30 mg/kg IV/SC treatment only), were attributed to tabalumab. Non-adverse microscopic decreases in size or number of germinal centers in spleen, mesenteric, and mandibular lymph nodes occurred, but without an effect on antibody responses to KLH or tetanus. At 16-weeks recovery, microscopic compound-related changes observed after 6 months of treatment were completely reversed (0.1 mg/kg group) and partially reversed (1.0 and 30.0 mg/kg groups), while peripheral blood B cells remained 66–72% reduced from baseline. Despite reduced germinal centres in lymphoid organs, and reductions in circulating B cells, T-cell-dependent humoral immunity was maintained following tabalumab administration in three safety studies in cynomolgus monkeys.

Inhibition of the Inositol Kinase Itpkb Augments Calcium Signaling in Lymphocytes and Reveals a Novel Strategy to Treat Autoimmune Disease.

Emerging approaches to treat immune disorders target positive regulatory kinases downstream of antigen receptors with small molecule inhibitors. Here we provide evidence for an alternative approach in which inhibition of the negative regulatory inositol kinase Itpkb in mature T lymphocytes results in enhanced intracellular calcium levels following antigen receptor activation leading to T cell death. Using Itpkb conditional knockout mice and LMW Itpkb inhibitors these studies reveal that Itpkb through its product IP4 inhibits the Orai1/Stim1 calcium channel on lymphocytes. Pharmacological inhibition or genetic deletion of Itpkb results in elevated intracellular Ca2+ and induction of FasL and Bim resulting in T cell apoptosis. Deletion of Itpkb or treatment with Itpkb inhibitors blocks T-cell dependent antibody responses in vivo and prevents T cell driven arthritis in rats. These data identify Itpkb as an essential mediator of T cell activation and suggest Itpkb inhibition as a novel approach to treat autoimmune disease.

Suppression of antigen-specific antibody responses in mice exposed to perfluorooctanoic acid: Role of PPARα and T- and B-cell targeting

T-cell-dependent antibody responses (TDAR) are suppressed in female C57BL/6N mice exposed to ≥3.75 mg/kg of perfluorooctanoic acid (PFOA) for 15 days. To determine if suppression of humoral immunity by PFOA is peroxisome proliferator activated receptor alpha (PPARα)-dependent and if suppression is associated with specific targeting of T- or B-cells, three separate experiments were conducted: (1) female PPARα constitutive knockout (PPARα KO; B6.129S4-Ppartm1GonzN12) and wild-type controls (WT; C57BL/6-Tac) exposed to 0, 7.5, or 30 mg PFOA/kg for 15 days were immunized on Day 11 with a T-cell-dependent antigen and sera then collected for measures of antigen-specific IgM titers (TDAR) 5 days later; (2) female C57BL/6N WT mice exposed to 0, 0.94, 1.88, 3.75, or 7.5 mg PFOA/kg for 15 days were immunized with a T-cell-independent antigen on Day 11 and sera were then collected for analyses of antigen-specific IgM titers (TIAR) 7 days later; and (3) splenic lymphocyte phenotypes were assessed in unimmunized female C57BL/6N WT mice exposed to 0, 3.75, or 7.5 mg PFOA/kg for 10 days to investigate effects of PFOA in the absence of specific immunization. Separate groups of mice were immunized with a T-cell-dependent antigen after 11 days of exposure and splenic lymphocyte sub-populations were assessed after 13 or 15 days of exposure to assess numbers of stimulated cells. The results indicated that exposure to ≥1.88 mg PFOA/kg suppressed the TIAR; exposure to 30 mg PFOA/kg suppressed the TDAR in both PPARα KO and WT mice. The percentage of splenic B-cells was unchanged. Results obtained in the PPARα KO mice indicated that PPARα suppression of TDAR was independent of PPARα involvement. Suppression of the TIAR and the TDAR with minimal lymphocyte sub-population effects suggested that effects on humoral immunity are likely mediated by disruption of B-cell/plasma cell function.

Combined administration of RG7652, a recombinant human monoclonal antibody against PCSK9, and atorvastatin does not result in reduction of immune function.

RG7652 is a human IgG1 monoclonal antibody designed to inhibit proprotein convertase subtilisin/kexin type 9 (PCSK9) binding to hepatic low density lipoprotein receptor (LDL-r), thereby blocking PCSK9-mediated degradation of LDL-r. This therapeutic candidate is under development for the prevention of cardiovascular mortality and morbidity in dyslipidemic patients. The primary objective of this study was to evaluate the potential immunotoxicological effects of RG7652 when given to cynomolgus monkeys either alone or in combination with a daily oral dose of atorvastatin. Administration of RG7652 via subcutaneous injection every other week for 12 weeks (a total of seven doses), daily oral doses of atorvastatin (total of 85 doses), and combinations of each up to 15 and 20 mg/kg/dose, respectively, were well tolerated and there was no evidence of alteration in immune function. Administration of pharmacologically relevant doses of RG7652 in combination with atorvastatin to healthy monkeys does not result in clinically meaningful immunosuppression as measured by T-cell dependent antibody responses, natural killer cell activity, immunophenotype, or delayed type hypersensitivity. The only pharmacologically mediated changes observed during the dosing period were the anticipated changes in circulating cholesterol.

Relative sensitivities of TDAR, cytokine production, and immunophenotyping assays in immunotoxicity assessment

Additional immunotoxicity tests as part of a repeat-dose toxicity study have been recommended to improve the quality of immunotoxicity assessments. Present immunotoxicity test guidelines list many tests without concreteness of methods or principles for their use. Due to the different sensitivity and specificity of each assay, numerous different test batteries can produce diverse results and make difficult inter-laboratory comparisons. To help resolve these problems and identify some of the more sensitive tests that might be used, we established immunotoxicity in a rat model using two well-known immunosuppressants and then compared the sensitivity of three important immunotoxicity tests, i.e., measures of T-cell dependent antibody responses (TDAR), ex vivo cytokine production, and immunophenotyping. The results showed that after treatment with cyclosporin A (CsA; 20 mg kg−1 d−1) or triptolide (TP; 0.8 mg kg−1 d−1), production of interleukin (IL)-2, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and the antibody to keyhole limpet hemocyanin (KLH) were significantly inhibited; in contrast, there were only weak or sporadic changes to host histopathology and hematology. With regard to phenotyping, no significant changes were noted in the ratios of CD4+ : CD8+ cells in the treated hosts. Taken together, the results here suggest to us that cytokine production and the KLH TDAR assay are very sensitive parameters to define an agent as immunotoxic, while immunophenotyping and histopathology – while being more expensive/time-consuming – appear to be less sensitive options for an individual assessing the immunotoxic potential of a given test agent.