10th European immunogenicity platform open symposium on immunogenicity of biopharmaceuticals.

Therapeutic proteins and emerging gene and cell-based therapies are attractive therapeutic tools for addressing unmet medical needs or when earlier conventional treatment approaches failed. However, the development of an immune response directed against therapeutic agents is a significant concern as it occurs in a substantial number of cases across products and indications. The specific anti-drug antibodies that develop can lead to safety adverse events as well as inhibition of drug activity or accelerated clearance, both phenomena resulting in loss of treatment efficacy. The European Immunogenicity Platform (EIP) is a meeting place for experts and newcomers to the immunogenicity field, designed to stimulate discussion amongst scientists across industry and academia, encourage interactions with regulatory agencies and share knowledge and the state-of-the-art of immunogenicity sciences with the broader scientific community. Here we report on the main topics covered during the EIP 10th Open Symposium on Immunogenicity of Biopharmaceuticals held in Lisbon, 26-27 February 2019, and the 1-d training course on practical and regulatory aspects of immunogenicity held ahead of the conference. These main topics included immunogenicity testing, clinical relevance of immunogenicity, immunogenicity prediction, regulatory aspects, tolerance induction as a mean to mitigate immunogenicity and immunogenicity in the context of gene therapy.

Real-world costing analysis for diffuse large B-cell lymphoma in British Columbia.

Introduction: Diffuse large B-cell lymphoma (dlbcl) accounts for 30%-40% of all non-Hodgkin lymphomas. Approximately 60% of patients are cured with standard treatment. Targeted treatments are being investigated and might improve disease outcomes; however, their effect on cancer drug budgets will be significant. For the present study, we conducted an analysis of real-world costs for dlbcl patients treated in British Columbia, useful for health care system planning. Methods: Patient records from a retrospective cohort of patients diagnosed with dlbcl in British Columbia during 2004-2013 were anonymously linked across multiple administrative data sources: systemic therapy, radiotherapy, hospitalizations, oncologist services, outpatient medications, and fee-for-service physician services. Using generalized linear modelling regression, time-dependent costs (in 2015 Canadian dollars) were estimated in 6-month intervals over a 5-year period. The inverse probability weighting method was applied to account for censored observations. Nonparametric bootstrapping was used to estimate standard errors for the mean cost at each time interval. Results: The cohort consisted of 678 patients (5-year overall survival: 67%). Mean age at diagnosis was 64 ± 14 years; median follow-up was 3.2 years. Mean total cost of care was highest in the first 6 months after diagnosis ($29,120; 95% confidence interval: $28,986 to $29,170) and after disease progression ($18,480; 95% confidence interval: $15,187 to $24,772). Systemic therapy and hospitalization costs were the largest cost drivers. At each time interval, costs were observed to be positively skewed. Conclusions: Our results depict real-world costs for the treatment of dlbcl patients with standard chop-r therapy. Cost-model parameters are also provided for economic modelling of dlbcl interventions.

Observation as the initial management strategy in patients with mantle cell lymphoma.

BACKGROUND: Patients with mantle cell lymphoma (MCL) follow a heterogeneous clinical course. While they generally require treatment initiation shortly after diagnosis, it is unclear whether deferring treatment in selected patients with an indolent clinical behavior affects their overall outcome. PATIENTS AND METHODS: In this population-based study, all patients diagnosed with MCL during 1998-2014 were identified in the British Columbia Cancer Agency Lymphoid Cancer Database. The associations between clinico-pathologic characteristics, including the expression of Ki67, SOX11, and TP53, and time to treatment (TtT) and OS were analyzed. RESULTS: A total of 440 patients with MCL were evaluated: 365 (83%) received early treatment and 75 (17%) were observed ≥3 months. In the observation group, 54 (72%) patients had a nodal presentation, 16 (21%) a non-nodal presentation, and 5 (7%) had only gastrointestinal involvement. Characteristics associated with deferred treatment included good performance status, no B symptoms, low LDH, non-bulky disease, non-blastoid morphology, and lower Ki67 values. The median TtT in the observation group was 35 months (range 5-79), and 60 (80%) patients were observed beyond 12 months. The median OS was significantly longer in the observation group than in the early treatment group (72 versus 52.5 months, respectively, P = 0.041). In multivariable analysis, treatment decision was not associated with OS [HR 0.804 (95% CI 0.529-1.221), P = 0.306]. CONCLUSIONS: A subgroup of patients with MCL may be safely observed from diagnosis without negatively impacting their outcomes, including patients with non-nodal presentation as well as asymptomatic patients with low burden nodal presentation and a low proliferative rate.

A time-and-motion approach to micro-costing of high-throughput genomic assays.

BACKGROUND: Genomic technologies are increasingly used to guide clinical decision-making in cancer control. Economic evidence about the cost-effectiveness of genomic technologies is limited, in part because of a lack of published comprehensive cost estimates. In the present micro-costing study, we used a time-and-motion approach to derive cost estimates for 3 genomic assays and processes-digital gene expression profiling (gep), fluorescence in situ hybridization (fish), and targeted capture sequencing, including bioinformatics analysis-in the context of lymphoma patient management. METHODS: The setting for the study was the Department of Lymphoid Cancer Research laboratory at the BC Cancer Agency in Vancouver, British Columbia. Mean per-case hands-on time and resource measurements were determined from a series of direct observations of each assay. Per-case cost estimates were calculated using a bottom-up costing approach, with labour, capital and equipment, supplies and reagents, and overhead costs included. RESULTS: The most labour-intensive assay was found to be fish at 258.2 minutes per case, followed by targeted capture sequencing (124.1 minutes per case) and digital gep (14.9 minutes per case). Based on a historical case throughput of 180 cases annually, the mean per-case cost (2014 Canadian dollars) was estimated to be $1,029.16 for targeted capture sequencing and bioinformatics analysis, $596.60 for fish, and $898.35 for digital gep with an 807-gene code set. CONCLUSIONS: With the growing emphasis on personalized approaches to cancer management, the need for economic evaluations of high-throughput genomic assays is increasing. Through economic modelling and budget-impact analyses, the cost estimates presented here can be used to inform priority-setting decisions about the implementation of such assays in clinical practice.

Electro-thermal control of aluminum-doped zinc oxide/vanadium dioxide multilayered thin films for smart-device applications.

We demonstrate the electro-thermal control of aluminum-doped zinc oxide (Al:ZnO) /vanadium dioxide (VO2) multilayered thin films, where the application of a small electric field enables precise control of the applied heat to the VO2 thin film to induce its semiconductor-metal transition (SMT). The transparent conducting oxide nature of the top Al:ZnO film can be tuned to facilitate the fine control of the SMT of the VO2 thin film and its associated properties. In addition, the Al:ZnO film provides a capping layer to the VO2 thin film, which inhibits oxidation to a more energetically favorable and stable V2O5 phase. It also decreases the SMT of the VO2 thin film by approximately 5-10 °C because of an additional stress induced on the VO2 thin film and/or an alteration of the oxygen vacancy concentration in the VO2 thin film. These results have significant impacts on technological applications for both passive and active devices by exploiting this near-room-temperature SMT.

Achievements, challenges and unmet needs for haemophilia patients with inhibitors: Report from a symposium in Paris, France on 20 November 2014.

Over the past 20 years, there have been many advances in haemophilia treatment that have allowed patients to take greater control of their disease. However, the development of factor VIII (FVIII) inhibitors is the greatest complication of the disease and a challenge in the treatment of haemophilia making management of bleeding episodes difficult and surgical procedures very challenging. A meeting to discuss the unmet needs of haemophilia patients with inhibitors was held in Paris on 20 November 2014. Topics discussed were genetic and non-genetic risk factors for the development of inhibitors, immunological aspects of inhibitor development, FVIII products and inhibitor development, generation and functional properties of engineered antigen-specific T regulatory cells, suppression of immune responses to FVIII, prophylaxis in haemophilia patients with inhibitors, epitope mapping of FVIII inhibitors, current controversies in immune tolerance induction therapy, surgery in haemophilia patients with inhibitors and future perspectives for the treatment of haemophilia patients with inhibitors. A summary of the key points discussed is presented in this paper.

IL10 receptor is a novel therapeutic target in DLBCLs.

Diffuse large B-cell lymphoma (DLBCL) is a biologically and clinically heterogeneous disease with marked genomic instability and variable response to conventional R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone) chemotherapy. More clinically aggressive cases of DLBCLs have high level of circulating interleukin 10 (IL10) cytokine and evidence of activated intracellular STAT3 (signal transducer and activator of transcription 3) signaling. We investigated the role of IL10 and its surface receptor in supporting the neoplastic phenotype of DLBCLs. We determined that IL10RA gene is amplified in 21% and IL10RB gene in 10% of primary DLBCLs. Gene expression of IL10, IL10RA and IL10RB was markedly elevated in DLBCLs. We hypothesized that DLBCLs depend for their proliferation and survival on IL10-STAT3 signaling and that blocking the IL10 receptor (IL10R) would induce cell death. We used anti-IL10R blocking antibody, which resulted in a dose-dependent cell death in all tested activated B-cell-like subtype of DLBCL cell lines and primary DLBCLs. Response of germinal center B-cell-like subtype of DLBCL cell lines to anti-IL10R antibody varied from sensitive to resistant. Cells underwent cell cycle arrest, followed by induction of apoptosis. Cell death depended on inhibition of STAT3 and, to a lesser extent, STAT1 signaling. Anti-IL10R treatment resulted in interruption of IL10-IL10R autostimulatory loop. We thus propose that IL10R is a novel therapeutic target in DLBCLs.

Inhibitors - cellular aspects and novel approaches for tolerance.

The immune response against therapeutic clotting factors VIII and IX (FVIII and FIX) is a major adverse event that can effectively thwart their effectiveness in correcting bleeding disorders. Thus, a significant number of haemophilia patients form antibodies, called inhibitors, which neutralize the procoagulant functions of therapeutic cofactors FVIII (haemophilia A) or FIX (haemophilia B). Understanding the cellular and molecular aspects of inhibitor formation is critical to designing tolerogenic therapies for clinical use. This review will focus on the basis of the immune response to FVIII, in particular, and will discuss emerging efforts to not only reduce immunogenicity but also to prevent and/or reverse inhibitor formation.

Neuroimmune semaphorin 4D is necessary for optimal lung allergic inflammation.

Neuroimmune semaphorin 4D (Sema4D) was found to be expressed and function in the nervous and immune systems. In the immune system, Sema4D is constitutively expressed on T cells and regulates T cell priming. In addition, it displays a stimulatory function on macrophages, DC, NK cells, and neutrophils. As all these cells are deeply involved in asthma pathology, we hypothesized that Sema4D plays a critical non-redundant regulatory role in allergic airway response. To test our hypothesis, we exposed Sema4D(-/-) and WT mice to OVA injections and challenges in the well-defined mouse model of OVA-induced experimental asthma. We observed a significant decrease in eosinophilic airway infiltration in allergen-treated Sema4D(-/-) mice relative to WT mice. This reduced allergic inflammatory response was associated with decreased BAL IL-5, IL-13, TGFß1, IL-6, and IL-17A levels. In addition, T cell proliferation in OVA323₋339-restimulated Sema4D(-/-) cell cultures was downregulated. We also found increased Treg numbers in spleens of Sema4D(-/-) mice. However, airway hyperreactivity (AHR) to methacholine challenges was not affected by Sema4D deficiency in either acute or chronic experimental disease setting. Surprisingly, lung DC number and activation were not affected by Sema4D deficiency. These data provide a new insight into Sema4D biology and define Sema4D as an important regulator of Th2-driven lung pathophysiology and as a potential target for a combinatory disease immunotherapy.

Endogenous expansion of regulatory T cells leads to long-term islet graft survival in diabetic NOD mice.

Donor pancreatic lymph node cells (PLNC) protect islet transplants in Non-obese diabetic (NOD) mice. We hypothesized that induced FoxP3(+) regulatory T cells (Tregs) were required for long-term islet engraftment. NOD or NOD.NON mice were treated with ALS (antilymphocyte serum) and transplanted with NOR islets +/-PLNC (5 × 10(7) ). In vivo proliferation and expansion of FoxP3(+) Tregs was monitored in spleen and PLN from ALS- and ALS/PLNC-treated recipient mice. Anti-CD25 depletion was used to determine the necessity of Tregs for tolerance. FoxP3(+) numbers significantly increased in ALS/PLNC-treated recipients compared to ALS-treated mice. In ALS/PLNC-treated mice, recipient-derived Tregs localized to the transplanted islets, and this was associated with intact, insulin-producing ß cells. Proliferation and expansion of FoxP3(+) Tregs was markedly increased in PLNC-treated mice with accepted islet grafts, but not in diabetic mice not receiving PLNC. Deletion of Tregs with anti-CD25 antibodies prevented islet graft tolerance and resulted in rejection. Adoptive transfer of Tregs to secondary NOD.scid recipients inhibited autoimmunity by cotransferred NOD effector T cells. Treg expansion induced by ALS/PLNC-treatment promoted long term islet graft survival. Strategies leading to Treg proliferation and localization to the transplant site represent a therapeutic approach to controlling recurrent autoimmunity.

BCL2 mutations in diffuse large B-cell lymphoma.

BCL2 is deregulated in diffuse large B-cell lymphoma (DLBCL) by the t(14;18) translocation, gene amplification and/or nuclear factor-κB signaling. RNA-seq data have recently shown that BCL2 is the most highly mutated gene in germinal center B-cell (GCB) DLBCL. We have sequenced BCL2 in 298 primary DLBCL biopsies, 131 additional non-Hodgkin lymphoma biopsies, 24 DLBCL cell lines and 51 germline DNAs. We found frequent BCL2 mutations in follicular lymphoma (FL) and GCB DLBCL, but low levels of BCL2 mutations in activated B-cell DLBCL, mantle cell lymphoma, small lymphocytic leukemia and peripheral T-cell lymphoma. We found no BCL2 mutations in GC centroblasts. Many mutations were non-synonymous; they were preferentially located in the flexible loop domain, with few in BCL2-homology domains. An elevated transition/transversions ratio supports that the mutations result from somatic hypermutation. BCL2 translocations correlate with, and are likely important in acquisition of, additional BCL2 mutations in GCB DLBCL and FL. DLBCL mutations were not independently associated with survival. Although previous studies of BCL2 mutations in FL have reported mutations to result in pseudo-negative BCL2 protein expression, we find this rare in de-novo DLBCL.

CD3+ and BLA.36+ cells do not occur in the epidermis and adnexal epithelia of normal equine skin.

Summary A small population of resident T lymphocytes is present in the healthy human and murine epidermis. However, resident epidermal lymphocytes have not been reported in normal skin of the horse. Skin biopsy specimens from the normal skin of 27 horses were examined histologically and immunohistochemically for the presence of lymphocytes, CD3+ cells and BLA.36+ cells in epidermis and adnexal epithelia. All examinations were negative. It appears that lymphocytes occur rarely, if at all, in the epidermis and adnexal epithelia of normal horse skin. Hence, the presence of lymphocytes in these structures should be considered abnormal.

Gene therapy for immunological tolerance: using 'transgenic' B cells to treat inhibitor formation.

B cells have been shown to function as tolerogenic antigen presenting cells (APCs) both in vivo and in vitro. We have taken advantage of this property, as well as the ability of IgG carriers to be potent 'schleppers' for tolerogenic entities, to develop a gene therapy approach to induce unresponsiveness in a number of systems, including the elimination of haemophilia inhibitors. Thus, peptide-IgG constructs have been engineered into retroviral vectors to create 'transgenic' B cells for tolerance applications. In this paper, we discuss our gene therapy approach mediated by B cells (as well as bone marrow cells) for tolerance acquisition in various mouse models for autoimmune disease and haemophilia A. The mechanisms that are the underpinning of this effort and role of regulatory T cells are discussed herein. Our results indicate that gene therapy strategies can successfully reduce the incidence and or onset of autoimmune diseases and prevent/reverse inhibitor formation in haemophilia A mice. Based on recent success with a model for tolerance with human T cell clones in vitro, plans for future application in patients are discussed.

Can we prevent immunogenicity of human protein drugs?

Monoclonal antibodies have proved to be extremely valuable additions to conventional treatment for rheumatic diseases. However, despite the general trend towards "humanisation", these drugs remain immunogenic in clinical settings, baffling drug developers. In principle, humanised and fully human monoclonal antibodies are "self" immunoglobulins and should be tolerated. In this overview, the factors that may influence this process, the nature of immunogenicity and methods to analyse and modify potential immunogenicity are discussed. Finally, novel approaches to "re-induce" immunological tolerance to these proteins, including gene therapy and the recognition of unique regulatory epitopes, are outlined.

Cutaneous melanoma with metastasis in a cynomolgus monkey (Macaca fascicularis).

BACKGROUND: A 3.3-year-old-male cynomolgus macaque (Macaca fascicularis) showed a focally extensive soft, dark, discoid dermal mass, 0.5 cm in diameter, on the dorsal surface of the right hind foot, over the fourth and fifth metatarsal bones. METHODS AND RESULTS Microscopic examination revealed a cutaneous melanoma with local lymphatic invasion, characterized by neoplastic melanocytes within the subcapsular sinus of popliteal and inguinal lymph nodes. The diagnosis was confirmed by immunohistochemistry and transmission electron microscopy. CONCLUSIONS: To our knowledge, this is the first documented case of melanoma in a cynomolgus monkey.

Prevalence, number and morphological types of multinucleated histiocytic giant cells in equine inflammatory dermatoses: a retrospective light microscopic study of skin-biopsy specimens from 362 horses.

REASONS FOR PERFORMING STUDY: Multinucleated histiocytic giant cells (MHGC) are seen frequently in skin-biopsy specimens from horses with inflammatory dermatoses. However, the prevalence, number and morphological types of these cells have not been reported. OBJECTIVE: To determine the prevalence, number and morphological types of MHGC in equine inflammatory dermatoses, and the association of these cells with specific conditions. METHODS: Skin-biopsy specimens from 335 horses with inflammatory dermatoses and from 27 horses with normal skin were evaluated for the prevalence, number and morphological types of MHGC. RESULTS: The prevalence and number of MHGC were greater in granulomatous dermatoses than in nongranulomatous dermatoses. Infectious and noninfectious dermatoses were not different in terms of prevalence or morphological types of MHGC. Foreign-body MHGC were the predominant type in almost all cases. MHGC were not seen in normal skin. CONCLUSIONS: MHGC are seen in a wide variety of equine inflammatory dermatoses, especially those that are granulomatous. Number and morphological types of MHGC are of no apparent diagnostic significance. POTENTIAL RELEVANCE: MHGC are frequently present in a wide variety of inflammatory dermatoses in the horse. Because the prevalence, number and morphological types of MHGC are of minimal diagnostic significance, special stains and tissue cultures are necessary to confirm specific diagnoses.

Prevalence of infiltrative lymphocytic mural folliculitis in equine inflammatory skin diseases.

Infiltrative lymphocytic mural folliculitis (ILMF) is a histopathological reaction pattern reported to occur in a small number of equine inflammatory dermatoses. However, the prevalence of ILMF in a variety of equine dermatoses has not been reported. Skin biopsy specimens from 250 horses with inflammatory dermatoses and from 27 horses with physically healthy skin were therefore evaluated. ILMF was present in 82% of the diseased skin specimens examined. ILMF was not seen in physically healthy skin. It appears that ILMF is frequently seen in a wide variety of equine inflammatory dermatoses and therefore is of little diagnostic significance. However, ILMF is not seen in physically healthy equine skin and the presence of lymphocytes in equine hair follicle epithelium should therefore be considered abnormal.

Malassezia otitis externa in the dog: the effect of heat-fixing otic exudate for cytological analysis.

This study was conducted on 32 dogs with Malassezia otitis externa to determine the effect of heat-fixing otic exudate on cytological analysis. Malassezia infection was confirmed by cytological examination of otic exudate. Otic discharge collected with cotton swabs was then rolled onto glass slides. One slide per dog was heat-fixed prior to staining; the other slide was not heat-fixed. The number of yeast in 10 oil-immersion fields (1000 x magnification) was counted for both slides from each dog. Heat-fixing did not systematically cause either increased or decreased numbers of Malassezia on cytology of otic exudate.

A case of multiple epitrichial sweat gland ductal carcinomas in a horse.

This case report describes a rare epitrichial sweat gland ductal carcinoma in a 14-year-old horse and is the first report of multiple carcinomas of this type in horses. Although several tumours developed, mostly on the distal extremities, over a 2-year period, the horse remained otherwise healthy. Topical treatment with imiquimod was successful for many of them.

The prevalence of apoptotic keratinocytes in equine epidermis: a retrospective light-microscopic study of skin-biopsy specimens from 253 horses with normal skin or inflammatory dermatoses.

A retrospective study was performed to assess the prevalence of apoptotic epidermal keratinocytes in biopsy specimens from 226 horses with inflammatory dermatoses and from 27 normal specimens. One or more apoptotic keratinocytes were found in specimens from 28 of 226 (12%) horses with various dermatoses, and in one of 27 (4%) specimens from normal horses. The prevalence (proportion of cases with apoptotic epidermal keratinocytes) of apoptotic keratinocytes in the group composed of discoid lupus erythematosus, erythema multiforme, photodermatitis, and systemic lupus erythematosus was significantly greater (52% prevalence in these cases) than that in a grouping of all other dermatoses (prevalence 8%). There was no correlation between the number of apoptotic keratinocytes and the extent of epidermal hyperplasia or hyperkeratosis.