Development Of Anti-drug Antibodies Research Articles

Characterisation and application of recombinant FVIII-neutralising antibodies from haemophilia A inhibitor patients.

The development of anti-drug antibodies (ADAs) is a serious outcome of treatment strategies involving biological medicines. Coagulation factor VIII (FVIII) is used to treat haemophilia A patients, but its immunogenicity precludes a third of severe haemophiliac patients from receiving this treatment. The availability of patient-derived anti-drug antibodies can help us better understand drug immunogenicity and identify ways to overcome it. Thus, there were two aims to this work: (i) to develop and characterise a panel of recombinant, patient-derived, monoclonal antibodies covering a range of FVIII epitopes with varying potencies, kinetics and mechanism of action, and (ii) to demonstrate their applicability to assay development, evaluation of FVIII molecules and basic research. For the first objective we used recombinant antibodies to develop a rapid, sensitive, flexible and reproducible ex vivo assay that recapitulates inhibitor patient blood using blood from healthy volunteers. We also demonstrate how the panel can provide important information about the efficacy of FVIII products and reagents without the need for patient or animal material. These materials can be used as experimental exemplars or controls, as well as tools for rational, hypothesis-driven research and assay development in relation to FVIII immunogenicity and FVIII-related products.

Immunogenicity and humanization of single-domain antibodies.

Single-domain antibodies (sdAbs), the autonomous variable domains of camelid and shark heavy-chain antibodies, have many desirable properties as components of biologic drugs. However, their sequences may increase the risk of immunogenicity and antidrug antibody (ADA) development in humans, and thus, sdAbs are routinely humanized during development. Here, we review and summarize the available evidence regarding the factors governing immunogenicity of sdAbs and our current state of knowledge of strategies to mitigate immunogenicity risks by humanization. While several sdAb properties, including high homology of camelid VH Hs with human IGHV3 gene products, favor low immunogenicity in humans, epitopes absent in the human repertoire including the exposed VH :VL interface may be intrinsically immunogenic. While most clinical trials have demonstrated minimal sdAb immunogenicity, two notable exceptions (the tetrameric DR5-specific VH H TAS266 and the TNFR1-specific VH GSK1995057) illustrate that special caution must be taken in identifying preexisting ADAs against highly potent sdAbs. Nonhuman sequence alone does not adequately explain sdAb immunogenicity, as some camelid VH Hs are nonimmunogenic while some fully human VH s elicit ADAs. The presence of preexisting ADAs directed against the exposed C-termini of some sdAbs in a significant proportion of individuals awaits a molecular explanation. Whether sdAb humanization reduces or promotes immunogenicity remains unclear: reduction of nonhuman sequence content at the expense of introducing low-level aggregation in humanized variants may be counterproductive. Further work will establish thresholds for VH H and VNAR humanization to maximize human sequence content while avoiding loss of binding affinity and/or immunogenicity resulting from aggregation or decreased stability.

Anti-TROVE2 Antibody Determined by Immune-Related Array May Serve as a Predictive Marker for Adalimumab Immunogenicity and Effectiveness in RA.

Anti-drug antibody (ADAb) development is associated with secondary therapeutic failure in biologic-treated rheumatoid arthritis (RA) patients. With a treat-to-target goal, we aimed to identify biomarkers for predicting ADAb development and therapeutic response in adalimumab-treated patients. Three independent cohorts were enrolled. In Cohort-1, 24 plasma samples (6 ADAb-positive and 6 ADAb-negative patients at baseline and week 24 of adalimumab therapy, respectively) were assayed with immune-related microarray containing 1,636 correctly folded functional proteins. Next, we executed statistically powered autoantibody profiling analysis of 50 samples in Cohort-2 (24 ADAb-positive and 26 ADAb-negative patients). Subsequently, immunofluorescence assay was performed on 48 samples in Cohort-3 to correlate with ADAb titers and drug levels. The biomarkers were identified for predicting ADAb development and therapeutic response using the immune-related microarray and machine learning approach. ADAb-positive patients had lower drug levels at week 24 (median = 0.024 μg/ml) compared with ADAb-negative patients (median = 6.38 μg/ml, p < 0.001). ROC analysis based on the ADAb status revealed the top 20 autoantibodies with AUC ≥ 0.7 in differentiating both groups in Cohort-1. Analysis of Cohort-2 dataset identified a panel of 8 biomarkers (TROVE2, SSB, NDE1, ZHX2, SH3GL1, CARD9, PTPN20, and KLHL12) with 80.6% specificity, 77.4% sensitivity, and 79.0% accuracy in discriminating poor from EULAR responders. Immunofluorescence assay validated that anti-TROVE2 antibody could highly predict ADAb development and poor EULAR response (AUC 0.79 and 0.89, respectively). Multivariate regression analysis proved anti-TROVE2 antibody to be an independent predictor for developing ADAb. Immune-related protein microarray and replication analysis identified anti-TROVE2 antibody as a useful biomarker for predicting ADAb development and therapeutic response in adalimumab-treated patients.

New therapies for inflammatory bowel diseases

Currently, Inflammatory Bowel Disease (IBD) is considered an immune-mediated disease. The most widely accepted etiopathogenic hypothesis is that it is due to an inadequate interaction between the immune system and the microorganisms that form the normal intestinal flora. This abnormal interaction occurs in genetically predisposed subjects under the influence of various environmental factors (such as tobacco, diet, stress, or recurrent bacterial infections), which could act as triggers for alterations in the intestinal epithelial barrier. This would lead to an increase in the permeability of barrier, allowing the translocation of microbial products to the wall of the digestive tract, which would activate an acute cell-mediated immune response[1]. The failure of the anti-inflammatory regulatory mechanisms, together with an excess in the production of pro-inflammatory cytokines, would promote an aberrant immune response that would be self-perpetuating over time, thus giving rise to the chronic inflammation that characterizes IBD[2]. Since the first descriptions of Ulcerative Colitis (UC) and Crohn's Disease (CD), many treatments have been developed, with varying degrees of safety and efficacy. At the end of the 20th century, the first biological, called Infliximab, began to be used. A biological drug is one that has a biotechnological origin and arises from proteins derived from DNA and hybridization processes, which require living organisms as a fundamental part of the production process[3]. Undoubtedly, the appearance of biologics in the therapeutic arsenal of IBD was a very important advance in the treatment of these patients, a true revolution. Until that date, many of the patients who began to be treated with this biologic could only do with corticosteroids or by surgery. These drugs have been shown to be effective in reducing intestinal damage caused by chronic inflammation, the need for surgery and hospital admissions and, consequently, have improved the quality of life of many patients[4]. The demonstrated benefit of these drugs, especially when administered early, as well as their favorable safety profile, have led to their increasingly frequent use in the treatment of patients with IBD. They can be divided into - anti-TNF drugs (blocking the cytokine TNF-α), - Vedolizumab (blocking the integrin α4β7) and - Ustekinumab (blocking Interleukin 12 and 23). Recently, and only for use in UC, Tofacitinib (an inhibitor of the JAK-kinase pathway) has been approved and has demonstrated its efficacy in the treatment of moderate-severe active UC[5]. The management of all these drugs represents a challenge for the digestive specialist who must know their mechanisms of action and use them appropriately in patients with IBD. Anti-TNF drugs, being the oldest, are the ones with which we have the most experience and, therefore, they are usually used in the first line in those cases in which conventional drugs (corticosteroids and / or immunosuppressants) have failed. There are three currently marketed in Spain: Infliximab (for intravenous administration), Adalimumab (for subcutaneous administration) and Golimumab (for subcutaneous administration). Vedolizumab (administered intravenously), and Ustekinumab (administered subcutaneously, after a first intravenous administration) are characterized by having an excellent safety profile and a very low immunogenic potential compared to anti-TNF drugs. There are many lines of research currently underway to try to identify the clinical factors of the patient that would make one drug or another more useful in the first line. Other lines seek to identify genetic factors (it seems that mutations in the HLA DQA1 * 05 haplotype could increase immunogenicity to anti-TNF drugs[6]) and also molecular factors[7].

Ustekinumab for Ulcerative Colitis

Ustekinumab for Ulcerative Colitis

Organophosphate detoxification by membrane-engineered red blood cells

Biotherapeutics have achieved global economic success due to their high specificity towards their drug targets, providing exceptional safety and efficiency. The ongoing shift away from small molecule drugs towards biotherapeutics heightens the need to further improve the pharmacokinetics of these biological drugs. Three pervasive obstacles that limit the therapeutic capacity of biotherapeutics are proteolytic degradation, circulating half-life, and the development of anti-drug antibodies. These challenges can culminate in limited efficiency and consequently warrant the need for higher drug doses and more frequent administration. We have explored the coupling of biotherapeutics to long-lived and biocompatible red blood cells (RBCs) to address limited pharmacokinetics. Butyrylcholinesterase (BChE), for example, provides prophylactic protection against organophosphate nerve agents (OPNAs), yet the short circulation life of the drug requires extraordinary doses. Herein, we report the rapid and tunable chemical engineering of BChE to RBC membranes to create a cell-based delivery system that retains the enzyme activity and enhances stability. In a three-step process that first pre-modifies BChE with a cell-reactive polymer chain, primes the cells for engineering, and then grafts the conjugates to the cells, we attached over 2 million BChE molecules to the surface of each RBC without diminishing the bioscavenging capacity of the enzyme. Critically, this membrane-engineering approach was cell-tolerated with minimal hemolysis observed. These results provide strong evidence for the ability of engineered RBCs to serve as an enhanced biotherapeutic delivery vehicle. Statement of SignificanceOrganophosphate nerve agents (OPNAs) are one of the most lethal forms of chemical warfare. After exposure to OPNAs, a patient is given life-saving therapeutics, such as atropine and oxime. However, these drugs are limited, and the patient can still suffer from irreparable injuries. Given the toxicity of OPNAs, access to a prophylactic is vital. We have created an enhanced delivery system for prophylactic butyrylcholinesterase (BChE) by engineering this biotherapeutic to the red blood cell (RBC) surface. In three simple steps that first pre-modifies BChE with a cell-reactive polymer, primes the cells for engineering, and then grafts the conjugates to the cells, we attached over 2 million BChE molecules to a single RBC while retaining the enzyme's activity and enhancing its stability.

The optimal management of anti-drug antibodies to infliximab and identification of anti-drug antibody values for clinical outcomes in patients with inflammatory bowel disease.

Secondary loss of response (LOR) to infliximab (IFX) commonly occurs. One cause is the development of anti-drug antibodies (ADAs). Evidence regarding the optimal management of ADAs is lacking. We aim to identify the best practice of management of ADAs to IFX to avoid discontinuation of therapy and to determine specific ADA cut-off values to determine pre-specified clinical outcomes. This is a 3-year study of patients receiving IFX who developed ADAs > 8μg/ml. We reviewed the management strategies and subsequent outcomes in patients who developed ADAs. A total of 132 patients are included. Baseline characteristics include 54% male patients and mean age of 39.4years. Fifty-two percent (n = 69) of patients discontinued IFX following the development of ADAs, 33.3% (n = 44) sited as secondary to LOR. Both an increase in IFX and adjustments to combination therapy were associated with lower rates of discontinuation of IFX vs no intervention (p value < 0.001, p value < 0.001). An increase in IFX resulted in a significant difference in ADAs/IFX trough levels pre- and post-intervention (p value < 0.001, p value = 0.032). ROC curve analysis yielded significant cut-off values for ADAs and treatment failure (ADA >16μg/ml, AUC 0.642, p value 0.003), steroid use (ADA >19 μg/ml, AUC 0.61, p value 0.048) development of infusion reactions (ADA> 37 μg/ml, AUC 0.68, p value 0.045) and switch to another biologic (ADA >45 μg/ml, AUC 0.739, p value <0.001). Both escalation of IFX and combination therapy resulted in lower rates of LOR. ROC curve analysis identified significant cut-off values for ADA trough levels and important clinical outcomes.

Is There a Role for Therapeutic Drug Monitoring in Patients with Hidradenitis Suppurativa on Tumor Necrosis Factor-α Inhibitors?

Tumor necrosis factor-α inhibitors, adalimumab and infliximab, are at the forefront of biologic therapy for the management of moderate-to-severe hidradenitis suppurativa, with adalimumab as currently the only approved medication for this condition. In treating patients, primary or secondary lack of response (also termed suboptimal response) is a major burden for both patients and healthcare systems and is a challenge with biologics in part owing to the development of anti-drug antibodies following treatment. To overcome this, therapeutic drug monitoring may be conducted proactively or reactively to a patient's suboptimal response guided by measurements of trough serum drug concentrations and levels of anti-drug antibodies. While strong evidence to support the utility of therapeutic drug monitoring exists in patients with inflammatory bowel disease, current information is limited in the context of hidradenitis suppurativa. We sought to summarize the available evidence and to present the role of therapeutic drug monitoring and other dose optimization strategies in improving clinical response in patients with hidradenitis suppurativa treated with tumor necrosis factor-α inhibitors.

The efficacy of immunomodulators in the prevention and suppression of anti-drug antibodies to anti-tumor necrosis factor therapy in inflammatory bowel disease.

The development of biological agents against tumor necrosis factor (TNF) has revolutionized the management of inflammatory bowel disease (IBD), frequently achieving induction and maintenance of remission in both ulcerative colitis and Crohn’s disease. However, a loss of response due to the development of anti-drug antibodies (ADA) is seen annually in approximately 20% of IBD patients receiving anti-TNF therapy. Current evidence suggests that the use of immunomodulators (IMM), such as thiopurines (azathioprine and 6-mercaptopurine) or methotrexate, may prevent or suppress ADA formation. In this article, we present a comprehensive review of the available literature regarding the efficacy of IMM in the prevention and suppression of ADA development to anti-TNF therapy in patients with IBD.

Preclinical Safety Assessment of 2 Inhaled Single-Domain Antibodies in the Cynomolgus Monkey.

The safety of 2 single domain antibodies (dAbs) was evaluated by inhalation toxicology studies in the cynomolgus monkey. In the first case study, a 14-day repeat-dose study evaluating an anti-thymic stromal lymphopoietin (anti-TSLP) dAb resulted in minimal mononuclear inflammatory cell infiltrates in the lungs, increases in lymphocytes in bronchoalveolar lavage fluid, and development of antidrug antibodies (ADAs). In a 6-week inhalation study, there was an increase in incidence and/or severity of mononuclear cell infiltrates in the lung, increased cellularity in the tracheobronchial lymph node (TBLN), and development of ADA. The second case study evaluated a change in duration of inhalation dosing, a different route of exposure (intravenous or IV), and recovery following an off-dose period with an anti-tumor necrosis factor receptor 1 dAb. A 7-day repeat-dose inhalation study and a 14-day IV study produced no microscopic effects in the lung, whereas a 14-day inhalation study resulted in moderate increases in pulmonary perivascular/peribronchiolar/alveolar lymphocytic infiltrates and increased cellularity in the TBLN, with partial and full recovery, respectively, after 14 days. The lung and lymph node findings seen after inhalation of either dAb were considered secondary to the immunogenic response to a human protein and were considered nonadverse.

Clinicogenomic factors of biotherapy immunogenicity in autoimmune disease: A prospective multicohort study of the ABIRISK consortium.

BackgroundBiopharmaceutical products (BPs) are widely used to treat autoimmune diseases, but immunogenicity limits their efficacy for an important proportion of patients. Our knowledge of patient-related factors influencing the occurrence of antidrug antibodies (ADAs) is still limited.Methods and findingsThe European consortium ABIRISK (Anti-Biopharmaceutical Immunization: prediction and analysis of clinical relevance to minimize the RISK) conducted a clinical and genomic multicohort prospective study of 560 patients with multiple sclerosis (MS, n = 147), rheumatoid arthritis (RA, n = 229), Crohn’s disease (n = 148), or ulcerative colitis (n = 36) treated with 8 different biopharmaceuticals (etanercept, n = 84; infliximab, n = 101; adalimumab, n = 153; interferon [IFN]-beta-1a intramuscularly [IM], n = 38; IFN-beta-1a subcutaneously [SC], n = 68; IFN-beta-1b SC, n = 41; rituximab, n = 31; tocilizumab, n = 44) and followed during the first 12 months of therapy for time to ADA development. From the bioclinical data collected, we explored the relationships between patient-related factors and the occurrence of ADAs. Both baseline and time-dependent factors such as concomitant medications were analyzed using Cox proportional hazard regression models. Mean age and disease duration were 35.1 and 0.85 years, respectively, for MS; 54.2 and 3.17 years for RA; and 36.9 and 3.69 years for inflammatory bowel diseases (IBDs). In a multivariate Cox regression model including each of the clinical and genetic factors mentioned hereafter, among the clinical factors, immunosuppressants (adjusted hazard ratio [aHR] = 0.408 [95% confidence interval (CI) 0.253–0.657], p < 0.001) and antibiotics (aHR = 0.121 [0.0437–0.333], p < 0.0001) were independently negatively associated with time to ADA development, whereas infections during the study (aHR = 2.757 [1.616–4.704], p < 0.001) and tobacco smoking (aHR = 2.150 [1.319–3.503], p < 0.01) were positively associated. 351,824 Single-Nucleotide Polymorphisms (SNPs) and 38 imputed Human Leukocyte Antigen (HLA) alleles were analyzed through a genome-wide association study. We found that the HLA-DQA1*05 allele significantly increased the rate of immunogenicity (aHR = 3.9 [1.923–5.976], p < 0.0001 for the homozygotes). Among the 6 genetic variants selected at a 20% false discovery rate (FDR) threshold, the minor allele of rs10508884, which is situated in an intron of the CXCL12 gene, increased the rate of immunogenicity (aHR = 3.804 [2.139–6.764], p < 1 × 10−5 for patients homozygous for the minor allele) and was chosen for validation through a CXCL12 protein enzyme-linked immunosorbent assay (ELISA) on patient serum at baseline before therapy start. CXCL12 protein levels were higher for patients homozygous for the minor allele carrying higher ADA risk (mean: 2,693 pg/ml) than for the other genotypes (mean: 2,317 pg/ml; p = 0.014), and patients with CXCL12 levels above the median in serum were more prone to develop ADAs (aHR = 2.329 [1.106–4.90], p = 0.026). A limitation of the study is the lack of replication; therefore, other studies are required to confirm our findings.ConclusionIn our study, we found that immunosuppressants and antibiotics were associated with decreased risk of ADA development, whereas tobacco smoking and infections during the study were associated with increased risk. We found that the HLA-DQA1*05 allele was associated with an increased rate of immunogenicity. Moreover, our results suggest a relationship between CXCL12 production and ADA development independent of the disease, which is consistent with its known function in affinity maturation of antibodies and plasma cell survival. Our findings may help physicians in the management of patients receiving biotherapies.

Pretreatment and Coadministration With Methotrexate Improved Durability of Pegloticase Response: An Observational, Proof-of-Concept Case Series.

Background/ObjectivePegloticase is used for treatment of refractory gout, which has failed maximal medical management, but only 42% of patients respond completely to treatment because of the presumed development of antidrug antibodies, which rapidly clear the pegloticase molecule. Immunomodulatory medications temper antidrug antibody development in other diseases. The aim of this case series was to investigate the utility of adding methotrexate to a pegloticase regimen to increase the response durability in a real-world practice setting.MethodsIn this multicenter, proof-of-concept, observational case series, refractory tophaceous gouty arthropathy patients being started on pegloticase 8 mg every 2 weeks were identified. The patients began oral methotrexate 15 mg/wk and folic acid 1 mg/d, 1 month prior to the initial pegloticase administration, and continued throughout pegloticase treatment. Responders were defined by demonstrating ≥80% of preinfusion serum uric acid (sUA) levels <6.0 mg/dL between months 3 and 6.ResultsTen sequential patients, aged 35 to 80 years, identified between May 2017 and June 2018, from 3 separate infusion centers were followed for up to 10 months. All patients maintained methotrexate 15 mg/wk without dose adjustments. There were 143 total pegloticase infusions. All 10 patients completed a full course of pegloticase treatment with 100% response and no infusion reactions. No patients stopped pegloticase therapy because of increased sUA, loss of response, or gout flares.ConclusionsPretreatment and coadministration of methotrexate with pegloticase resulted in 100% maintenance of pegloticase sUA response with no infusion reactions. These data support the potential use of immunomodulation with methotrexate to improve durability of pegloticase response in the treatment of refractory gout.

Genetic inhibition of NFATC2 attenuates asparaginase hypersensitivity in mice

AbstractThe family of nuclear factor of activated T cells (NFAT) transcription factors plays a critical role in mediating immune responses. Our previous clinical pharmacogenetic studies suggested that NFATC2 is associated with the risk of hypersensitivity reactions to the chemotherapeutic agent L-asparaginase (ASNase) that worsen outcomes during the treatment of pediatric acute lymphoblastic leukemia. We therefore hypothesized that the genetic inhibition of NFATC2 would protect against the development of anti-ASNase antibodies and ASNase hypersensitivity. Our study demonstrates that ASNase-immunized NFATC2-deficient mice are protected against ASNase hypersensitivity and develop lower antigen-specific and total immunoglobulin E (IgE) levels compared with wild-type (WT) controls. Furthermore, ASNase-immunized NFATC2-deficient mice develop more CD4+ regulatory T cells, fewer CD4+ interleukin-4–positive (IL-4+) cells, higher IL-10/TGF-β1 levels, and lower IL-4/IL-13 levels relative to WT mice. Basophils and peritoneal mast cells from ASNase-immunized, but not naïve, NFATC2-deficient mice had lower FcεRI expression and decreased IgE-mediated mast cell activation than WT mice. Furthermore, ASNase-immunized, but not naïve, NFATC2-deficient mice developed less severe shock than WT mice after induction of passive anaphylaxis or direct histamine administration. Thus, inhibition of NFATC2 protects against ASNase hypersensitivity by impairing T helper 2 responses, which may provide a novel strategy for attenuating hypersensitivity and the development of antidrug antibodies, including to ASNase.

Assessment of clinically relevant immunogenicity for mAbs; are we over reporting ADA?

Immunogenicity is recognized as a possible clinical risk due to the development of anti drug antibodies (ADAs) that can adversely impact drug safety and efficacy. Although robust assays are currently used to assess the ADA, there is a debate on how best to generate the most appropriate immunogenicity data. There are several factors that can trigger ADA formation including the immunity status of the target population and the severity of the disease indication. Immunogenicity testing has defaulted to the most conservative approach regardless of the inherent risk of the molecule or the patient population. For low-risk biotherapeutics such as human monoclonal antibodies, ADA data that provide clinically relevant information should be prioritized when establishing immunogenicity monitoring plans.

Real-World Effectiveness of Benralizumab in Severe Eosinophilic Asthma

Benralizumab is an IL5-receptor monoclonal antibody licensed for the treatment of severe eosinophilic asthma (SEA). It has demonstrated efficacy in clinical trials in reducing asthma exacerbation rates and maintenance oral corticosteroids (mOCSs). What is the real-world effectiveness of benralizumab and what baseline characteristics are associated with response to therapy? We assessed outcomes in all SEA patients who began benralizumab treatment at our specialist center. At each dosing visit, exacerbation history, mOCS dose, spirometry, and Asthma Control Questionnaire (ACQ6) and Mini-Asthma Quality of Life Questionnaire (mAQLQ) scores were recorded. Response to treatment was defined as a reduction of≥ 50%in annualized exacerbation rate (AER) or in mOCS dose after 48weeks of treatment. Super response was defined as zero exacerbations and no mOCSs for asthma. One hundred thirty patients were included in the analysis. At 48weeks, a 72.8%reduction in AER was noted, from 4.92 ± 3.35 per year in the year preceding biologic treatment to 1.34 ± 1.71 per year (P< .001), including 57 patients (43.8%) who were exacerbation-free with benralizumab. In those receiving mOCSs (n= 74 [56.9%]), the median daily prednisolone dose fell from 10mg (interquartile range, 5-20mg) to 0mg (interquartile range, 0-5mg; P< .001), and 38 of 74 patients (51.4%) were able to discontinue mOCS therapy. Clinically and statistically significant improvements were found in ACQ6 scores, mAQLQ scores, and FEV1. Overall, 51 patients (39%) met the super responder definition and 112 patients (86%) met the responder definition. The optimal regression model of super responders vsother responders included baseline characteristics associated with a strongly eosinophilic phenotype and less severe disease. Eighteen patients (13.8%) were nonresponders to benralizumab. Evidence of chronic airway infection was observed in 6 of 18 patients, and an increase in the blood eosinophil count consistent with the development of anti-drug antibodies was observed in 5 of 18 patients. In a large real-world SEA cohort, benralizumab led to significant improvements in all clinical outcome measures. A lack of response was seen in a minority of patients and should be a focus for future investigation.

SMAC Mimetic Plus Triple-Combination Bispecific HIVxCD3 Retargeting Molecules in SHIV.C.CH505-Infected, Antiretroviral Therapy-Suppressed Rhesus Macaques.

The "shock-and-kill" human immunodeficiency virus type 1 (HIV-1) cure strategy involves latency reversal followed by immune-mediated clearance of infected cells. We have previously shown that activation of the noncanonical NF-κB pathway using an inhibitor of apoptosis (IAP), AZD5582, reverses HIV/simian immunodeficiency virus (SIV) latency. Here, we combined AZD5582 with bispecific HIVxCD3 DART molecules to determine the impact of this approach on persistence. Rhesus macaques (RMs) (n = 13) were infected with simian/human immunodeficiency virus SHIV.C.CH505.375H.dCT, and triple antiretroviral therapy (ART) was initiated after 16 weeks. After 42 weeks of ART, 8 RMs received a cocktail of 3 HIVxCD3 DART molecules having human A32, 7B2, or PGT145 anti-HIV-1 envelope (Env) specificities paired with a human anti-CD3 specificity that is rhesus cross-reactive. The remaining 5 ART-suppressed RMs served as controls. For 10 weeks, a DART molecule cocktail was administered weekly (each molecule at 1 mg/kg of body weight), followed 2 days later by AZD5582 (0.1 mg/kg). DART molecule serum concentrations were well above those considered adequate for redirected killing activity against Env-expressing target cells but began to decline after 3 to 6 weekly doses, coincident with the development of antidrug antibodies (ADAs) against each of the DART molecules. The combination of AZD5582 and the DART molecule cocktail did not increase on-ART viremia or cell-associated SHIV RNA in CD4+ T cells and did not reduce the viral reservoir size in animals on ART. The lack of latency reversal in the model used in this study may be related to low pre-ART viral loads (median, <105 copies/ml) and low preintervention reservoir sizes (median, <102 SHIV DNA copies/million blood CD4+ T cells). Future studies to assess the efficacy of Env-targeting DART molecules or other clearance agents to reduce viral reservoirs after latency reversal may be more suited to models that better minimize immunogenicity and have a greater viral burden.IMPORTANCE The most significant barrier to an HIV-1 cure is the existence of the latently infected viral reservoir that gives rise to rebound viremia upon cessation of ART. Here, we tested a novel combination approach of latency reversal with AZD5582 and clearance with bispecific HIVxCD3 DART molecules in SHIV.C.CH505-infected, ART-suppressed rhesus macaques. We demonstrate that the DART molecules were not capable of clearing infected cells in vivo, attributed to the lack of quantifiable latency reversal in this model with low levels of persistent SHIV DNA prior to intervention as well as DART molecule immunogenicity.

Predicting the Development of Anti-Drug Antibodies against Recombinant alpha-Galactosidase A in Male Patients with Classical Fabry Disease.

Fabry Disease (FD) is a rare, X-linked, lysosomal storage disease that mainly causes renal, cardiac and cerebral complications. Enzyme replacement therapy (ERT) with recombinant alpha-galactosidase A is available, but approximately 50% of male patients with classical FD develop inhibiting anti-drug antibodies (iADAs) that lead to reduced biochemical responses and an accelerated loss of renal function. Once immunization has occurred, iADAs tend to persist and tolerization is hard to achieve. Here we developed a pre-treatment prediction model for iADA development in FD using existing data from 120 classical male FD patients from three European centers, treated with ERT. We found that nonsense and frameshift mutations in the α-galactosidase A gene (p = 0.05), higher plasma lysoGb3 at baseline (p < 0.001) and agalsidase beta as first treatment (p = 0.006) were significantly associated with iADA development. Prediction performance of a Random Forest model, using multiple variables (AUC-ROC: 0.77) was compared to a logistic regression (LR) model using the three significantly associated variables (AUC-ROC: 0.77). The LR model can be used to determine iADA risk in individual FD patients prior to treatment initiation. This helps to determine in which patients adjusted treatment and/or immunomodulatory regimes may be considered to minimize iADA development risk.

Anaphylactic reactions to biological drugs.

This review summarizes the current knowledge of the pathogenic mechanisms of biologics-induced anaphylaxis, and the diagnostic and prophylactic strategies in the management of potentially reactive patients, to improve the safety profile of biologics. The recent knowledge on the topic highlights the involvement of both effector and regulatory mechanisms in the immune response to biological agents. In addition, the impact of biological's immunogenicity on hypersensitivity reactions has been confirmed in a wider number of studies, defining some details about the kinetics of antidrug antibodies development, specifically immunoglobulin G (IgG) and immunoglobulin E (IgE). Biological agents may induce anaphylaxis, mainly through the induction of antidrug antibodies. Biologics-related infusion reactions are often clinically consistent with type I hypersensitivity, but IgG antidrug antibodies may also be involved. The immune response toward biologicals is orchestrated by both effector and regulatory T cells. In addition, nonantibody-dependent mechanisms may occur. Among clinicians persists today again a low awareness, not only of the possibility to understand the immunological mechanisms behind anaphylaxis to biologicals but also the opportunity to apply potential strategies for the management of reactive patients aimed to guarantee a safe retreatment.

Increased Efficacy and Tolerability of Pegloticase in Patients With Uncontrolled Gout Co-Treated With Methotrexate: A Retrospective Study.

IntroductionGout is a painful inflammatory condition caused by chronically elevated serum uric acid levels (sUA). When standard urate-lowering therapies fail/are not tolerated, uncontrolled gout (elevated sUA, subcutaneous tophi, chronic gouty arthritis, frequent flares) can occur. Pegloticase, a recombinant uricase, converts uric acid to allantoin, a readily excreted molecule. Responder rate in trials was 42%, limited by anti-drug antibody (ADA) development. Immunomodulators attenuate ADA formation and case reports suggest immunomodulation increases pegloticase responder rates. The current study retrospectively examined responder rate in patients undergoing methotrexate/pegloticase co-therapy.MethodsPatients who underwent methotrexate/pegloticase co-treatment at a single rheumatology practice were included. Demographics, clinical, treatment, and safety parameters were collected. The primary outcome was the proportion of responders (≥ 12 biweekly pegloticase infusions, sUA < 6 mg/dl just prior to infusion 12).ResultsTen patients (nine men, 52.3 ± 13.5 years) with uncontrolled tophaceous gout (erosive damage, ulcerative tophi, frequent flares, gout-related hospitalizations) were included. Patients had failed allopurinol (100–300 mg) or febuxostat (40 mg) therapy (doses not increased because of intolerance, kidney concerns, noncompliance, or rapid tophi resolution requirement). Baseline sUA was 9.42 ± 2.05 mg/dl. Along with standard pre-infusion prophylaxis, nine patients received subcutaneous methotrexate (25 mg/week) initiated 14–35 days before pegloticase and one patient received oral methotrexate (12.5 mg/week) initiated 14 days after pegloticase. Eight patients (80%) were responders, receiving 15.5 ± 3.8 infusions (range, 12–21) over 31.8 ± 9.5 weeks. One patient had efficacy loss with mild infusion reaction during infusion 4 and one patient was lost to follow-up after infusion 5. One patient reported one gout flare. No new safety concerns emerged.ConclusionsMethotrexate/pegloticase co-therapy resulted in a higher responder rate than the established 42% with pegloticase alone. Therefore, methotrexate/pegloticase co-therapy may safely allow more patients to benefit from a full treatment course, likely through ADA attenuation.

Drug Tolerant Anti-drug Antibody Assay for Infliximab Treatment in Clinical Practice Identifies Positive Cases Earlier.

A subgroup of patients treated with infliximab lose response to the treatment and one reason for this is the development of anti-drug antibodies (ADA). If used optimally, measuring drug and ADA level could lead to a more personalized and efficient treatment regime, and enable identification of ADA-positive patients before the underlying disease flares or allergic reactions occur. With the use of a drug-tolerant ADA assay which can detect ADA irrespective of drug levels in the sample, we determined the impact of ADA on treatment failure to infliximab. The aims of this study were to estimate the real-life optimal serum infliximab (sIFX) level and set a clinical threshold value for a drug-tolerant ADA assay. Trough levels of sIFX were measured with ELISA. Free ADA was measured with two drug-sensitive methods (ELISA and a bioassay) and one drug-tolerant method (PandA). Two real-life cohorts treated with infliximab were included; a cross-sectional cohort including patients with inflammatory rheumatic diseases (n = 270) and a prospective cohort of rheumatoid arthritis (RA) patients (n = 73) followed for 1 year. Normal range of sIFX was estimated from the prospective cohort and an arbitrary optimal drug level was set to be between 1 and 6 μg/mL. Using this range, optimal sIFX was found in only 60% (163/270) of the patients in the cross-sectional cohort. These patients had significantly better treatment response than those with a drug level under 1 μg/mL, who had an ADA frequency of 34% (19/56) using the drug-tolerant method. In the prospective cohort, the drug-tolerant assay could identify 34% (53/155 samples) as ADA positive in samples with sIFX level >0.2 μg/mL. ADA were seldom detected in patients with >1 μg/mL sIFX, with three interesting exceptions. A clinically relevant ADA threshold was determined to be >3 RECL as measured with the drug-tolerant assay. In a real-life setting, there was a substantial number of patients with suboptimal drug levels and a proportion of these had ADA. Both too low and too high drug levels correlated with worse disease, but for different reasons. Adding a drug-tolerant assay enabled detection of ADA earlier and regardless of drug level at time of sampling.