Development Of Anti-drug Antibodies Research Articles

Comparative pharmacokinetics of porcine and human anti-influenza hemagglutinin monoclonal antibodies in outbred pigs and minipigs.

Assessing the pharmacokinetics of monoclonal antibodies (mAbs) in relevant animal models is essential for designing improved formulations and developing mAb delivery platforms. We have established the pig, a large natural host animal for influenza with many similarities to humans, as a robust model for testing the therapeutic efficacy of anti-influenza mAbs and evaluating mAb delivery platforms. Here, we compared the pharmacokinetic characteristics of two anti-influenza hemagglutinin mAbs, human 2-12C and porcine pb27, in Göttingen minipigs and Landrace × Large White outbred pigs. Minipigs offer the advantage of a more stable weight, whereas outbred pigs are more readily available but exhibit rapid growth. Outbred pigs and minipigs showed similar pharmacokinetics and a similar porcine pb27 half-life (half-life of 15.7 days for outbred pigs and 16.6 days for minipigs). In contrast, the half-life of human 2-12C was more rapid in two of the minipigs but not in the outbred pigs, correlating with the development of antidrug antibodies in the two minipigs. Our results demonstrate that both outbred pigs and minipigs are appropriate models for pharmacokinetic studies and the evaluation of mAb delivery platforms, potentially bridging the gap between small animals and human trials.

Factors Associated with the Development of Anti-drug Antibodies to TNFi and the Consequences for Axial Spondyloarthritis: A Two-year Follow-up Study.

To evaluate the development of anti-drug antibodies (ADAb) against tumor necrosis factor inhibitors (TNFi) therapy during a 2-year period and search the factors linked to patients with axial spondyloarthritis (axSpA). Biologic-naive patients with axSpA were included in this observational study. Serum drug levels and ADAb were measured at weeks 12, 24, 52, and 104 of treatment by enzyme-linked immunosorbent assay (ELISA). The development of ADAb and factors related to ADAb over time were investigated using generalized estimating equations (GEE). A total of 180 patients with axSpA (116 male, mean (±SD) 45.6 (±11.9) years) who started TNFi treatment (etanercept (32.2%), adalimumab (27.2%), golimumab (20.6%), infliximab (20%)) were included. In the etanercept treatment group, only 1 patient had ADAb at 12 weeks and 24 weeks. Anti-drug antibodies against TNFi drugs were present in the adalimumab group in 32.7% of patients and in the infliximab group in 21.2% of patients at 12 weeks, and the proportion of ADAb-positive patients were found to be stable throughout the follow-up for adalimumab- and infliximab-treated patients. In the golimumab group, one patient had ADAb against golimumab at 12 weeks and the proportion of ADAb-positive patients increased throughout follow-up. In longitudinal analysis, baseline age, TNFi type, longitudinal Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and ASDAS-CRP scores, serum C-eeactive protein (CRP) levels, presence of adverse events and treatment discontinuation were associated with the presence of ADAb. The development of ADAb against TNFi therapy is associated with younger age, high disease activity, the development of adverse events and more common treatment discontinuation in patients with axSpA during 2-year follow-up.

HLA DQA1*05 and risk of anti-TNF treatment failure and anti-drug antibody development in children with Crohn's Disease: HLA DQA1*05 and Pediatric Crohn's Disease.

HLA DQA1*05 has been associated with the development of anti-drug antibodies (ADA) to tumor necrosis factor antagonists (anti-TNF) and treatment failure among adults with Crohn's disease (CD). However, findings from other studies have been inconsistent with limited pediatric data. We analyzed banked serum from patients with CD < 21 years of age enrolled in COMBINE, a multi-center, prospective randomized trial of anti-TNF monotherapy vs. combination with methotrexate. The primary outcome was a composite of factors indicative of treatment failure. The secondary outcome was ADA development. A trend towards increased treatment failure among HLA DQA1*05 positive participants was not significant (HR 1.58, 95% CI 0.95-2.62; p=0.08). After stratification by HLA DQA1*05 and by methotrexate vs. placebo, patients who were HLA DQA1*05 negative and assigned to methotrexate experienced less treatment failures than HLA DQA1*05 positive patients on placebo (HR 0.31, 95% CI 0.13-0.70; p=0.005).A trend toward increased ADA development among HLA DQA1*05 positive participants was not significant (odds ratio [OR] 1.96, 95% CI 0.90-4.31, p=0.09). After further stratification, HLA DQA1*05 negative participants assigned to methotrexate were less likely to develop ADA relative to HLA DQA1*05 positive patients on placebo (OR 0.12, 95% CI 0.03-0.55; p=0.008). In a randomized trial of children with CD initiating anti-TNF, 40% were HLA DQ-A1*05 positive, which was associated with a trend toward increased risk of both treatment failure and ADA. These risks were mitigated, but not eliminated, by adding oral methotrexate. HLA DQ-A1*05 is an important biomarker for prognosis and risk stratification.

Immunogenicity dynamics and covariate effects after satralizumab administration predicted with a hidden Markov model.

Immunogenicity is the propensity of a therapeutic protein to generate an immune response to itself. While reporting of antidrug antibodies (ADAs) is increasing, model-based analysis of such data is seldom performed. Model-based characterization of factors affecting the emergence and dissipation of ADAs may inform drug development and/or improve understanding in clinical practice. This analysis aimed to predict ADA dynamics, including the potential influence of individual covariates, following subcutaneous satralizumab administration. Satralizumab is a humanized IgG2 monoclonal recycling IL-6 receptor antagonist antibody approved for treating neuromyelitis optica spectrum disorder (NMOSD). Longitudinal pharmacokinetic (PK) and ADA data from 154 NMOSD patients in two pivotal Phase 3 studies (NCT02028884, NCT02073279) and PK data from one Phase 1 study (SA-001JP) in 72 healthy volunteers were available for this analysis. An existing population PK model was adapted to derive steady-state concentration without ADA for each patient. A mixed hidden Markov model (mHMM) was developed whereby three different states were identified: one absorbing Markov state for non-ADA developer, and two dynamic and inter-connected Markov states-transient ADA negative and positive. Satralizumab exposure and body mass index impacted transition probabilities and, therefore, the likelihood of developing ADAs. In conclusion, the mHMM model was able to describe the time course of ADA development and identify patterns of ADA development in NMOSD patients following treatment with satralizumab, which may allow for the formulation of strategies to reduce the emergence or limit the impact of ADA in the clinical setting.

Comprehensive evaluation of anti-emicizumab antibodies in acquired hemophilia A: a detailed case study and methodological evaluation

BackgroundAcquired hemophilia A (AHA) is a rare and severe bleeding disorder characterized by autoantibodies inhibiting coagulation factor (F)VIII. Current treatment of AHA involves bypassing agents or FVIII replacement therapy, yet their efficacy is limited in cases of high inhibitor titers. Emicizumab, a humanized bispecific monoclonal antibody, has shown promising hemostatic effectiveness in persons with congenital hemophilia A (HA) and AHA, but a minority of patients developed anti-drug antibodies (ADAs), compromising its efficacy. MethodsWe present a comprehensive characterization of anti-emicizumab antibodies in a patient with AHA experiencing diminished emicizumab efficacy from week 10 of treatment. We developed a method for analysis of anti-emicizumab antibodies, distinguishing immunoglobulin (Ig) subclasses and IgG subtype profiling. ADAs’ neutralizing activity was evaluated using a modified clotting assay. ResultsThe Luminex-based assay demonstrated the presence of anti-emicizumab antibodies, confirmed through competitive analysis. We detected anti-emicizumab IgG antibodies in the patient’s plasma between week 16 and 29. IgG1 and IgG2 subclasses were identified. Longitudinal analysis showed IgG isotype antibodies against both emicizumab and FVIII. Anti-emicizumab antibodies exhibited noninhibitory activity, confirmed by a modified Bethesda assay. Moreover, no accelerated clearance of emicizumab was observed in plasma samples from the patient. ConclusionThis study presents the first documented case of anti-emicizumab antibodies in AHA. Our study provides insights into ADA development against emicizumab, emphasizing the need for monitoring tools. The developed method enables comprehensive evaluation of ADAs, aiding in personalized treatment strategies. These findings contribute to understanding emicizumab's immunogenicity profile in AHA, facilitating its optimized clinical use.

Reactive Immunomodulator Addition to Infliximab Monotherapy Restores Clinical Response in Inflammatory Bowel Disease: A Meta-Analysis.

Patients with inflammatory bowel disease (IBD) receiving infliximab (IFX) commonly experience immunogenic loss of response (LOR) by formation of anti-drug antibodies (ADAs). An immunomodulator (IMM) used in combination with initial IFX induction is known to reduce ADA development and improve clinical outcomes. We aimed to assess the impact of reactively adding an IMM to patients on IFX monotherapy. We conducted a retrospective cohort study and systematic review with meta-analysis of patients with IBD demonstrating immunologic LOR, with or without clinical LOR, that had an IMM (azathioprine, 6-mercaptopurine, or methotrexate) reactively added (reactive combination therapy; rCT) to combat elevated ADAs and raise IFX level. Data were extracted for pooled effect size estimation using random-effects models, and ADA and IFX trough levels were compared pre- and post-IMM initiation. We identified 6 patients who received rCT due to rising ADA titers and low IFX levels. Median ADA titer decreased from 506ng/mL (interquartile range (IQR) [416-750]) to 76.5ng/mL (IQR [25.8-232]), an 85% decrease (p = 0.031). Median IFX trough increased from 0.4µg/mL (IQR [0.4-0.48]) to 8.25µg/mL (IQR [3.7-9.6]), a 20.6-fold increase (p = 0.038). Meta-analysis pooled effect size of 7 studies with 89 patients showed an 87% ADA titer reduction [95% confidence interval (CI) = 72-94%], 6.7-fold increased IFX trough (95% CI = 2.4-18.7), and 76% clinical remission rescue rate (95% CI = 59-93%). These results suggest rCT is a valid rescue strategy in patients with immunogenic LOR to IFX to reduce ADA titers, restore therapeutic IFX levels, and recapture clinical remission of IBD.

Development and characterization of dendritic cell internalization and activation assays contributing to the immunogenicity risk evaluation of biotherapeutics.

Immunogenicity refers to the ability of a substance, such as a therapeutic drug, to elicit an immune response. While beneficial in vaccine development, undesirable immunogenicity can compromise the safety and efficacy of therapeutic proteins by inducing anti-drug antibodies (ADAs). These ADAs can reduce drug bioavailability and alter pharmacokinetics, necessitating comprehensive immunogenicity risk assessments starting at early stages of drug development. Given the complexity of immunogenicity, an integrated approach is essential, as no single assay can universally recapitulate the immune response leading to the formation of anti-drug antibodies. To better understand the Dendritic Cell (DC) contribution to immunogenicity, we developed two flow cytometry-based assays: the DC internalization assay and the DC activation assay. Monocyte-derived dendritic cells (moDCs) were generated from peripheral blood mononuclear cells (PBMCs) and differentiated over a five-day period. The internalization assay measured the accumulation rate of therapeutic antibodies within moDCs, while the activation assay assessed the expression of DC activation markers such as CD40, CD80, CD86, CD83, and DC-SIGN (CD209). To characterize these two assays further, we used a set of marketed therapeutic antibodies. The study highlights that moDCs differentiated for 5 days from freshly isolated monocytes were more prone to respond to external stimuli. The internalization assay has been shown to be highly sensitive to the molecule tested, allowing the use of only 4 donors to detect small but significant differences. We also demonstrated that therapeutic antibodies were efficiently taken up by moDCs, with a strong correlation with their peptide presentation on MHC-II. On the other hand, by monitoring DC activation through a limited set of activation markers including CD40, CD83, and DC-SIGN, the DC activation assay has the potential to compare a series of compounds. These two assays provide a more comprehensive understanding of DC function in the context of immunogenicity, highlighting the importance of both internalization and activation processes in ADA development. The DC internalization and activation assays described here address key gaps in existing immunogenicity assessment methods by providing specific and reliable measures of DC function. The assays enhance our ability to pre-clinically evaluate the immunogenic potential of biotherapeutics, thereby improving their safety and efficacy. Future work should focus on further validating these assays and integrating them into a holistic immunogenicity risk assessment framework.

Immune-Mediated Liver Effects Associated With Administration of a Human Anti-IL-21 Receptor Antibody (ATR-107) in Rats.

The toxicity of ATR-107, a human anti-interleukin-21 receptor (IL-21R) monoclonal antibody (mAb), was evaluated in CD-1 mice and cynomolgus monkeys after single-dose intravenous (IV) administration, and in Sprague-Dawley (SD) rats and cynomolgus monkeys after weekly IV and subcutaneous (SC) administration in 13-week toxicity studies that included recovery. Adverse liver necrosis, diffuse bridging fibrosis, and higher liver enzymes occurred in rats in the low-dose IV group (10 mg/kg), but not at 50 or 250 mg/kg IV, and not following SC administration despite overlapping systemic ATR-107 exposures. Similar findings were not seen in mice or cynomolgus monkeys. A series of investigative rat toxicity studies showed liver findings only occurred after administration of at least 3 weekly doses, only occurred in rats that developed anti-drug antibodies (ADAs), and the incidence was associated with higher ADAs titers. However, the presence of ADAs did not always result in liver injury. Liver findings did not occur in nude rats, which had high ATR-107 exposures and no ADAs. These findings suggest an adaptive immune response with formation of ADAs was necessary for development of ATR-107-related liver findings, and that liver injury can occur in rats secondary to development of ADAs following repeated administration of a human therapeutic mAb.

HLA-DQA1*05 Allele Carriage and Anti-TNF Therapy Persistence in Inflammatory Bowel Disease

Abstract Introduction Carriage of the HLA-DQA1*05 allele is associated with development of antidrug antibodies (ADAs) to antitumor necrosis factor (anti-TNF) therapy in patients with Crohn’s disease. However, ADA is not uniformly associated with treatment failure. We aimed to determine the impact of carriage of HLA-DQA1*05 allele on outcome of biologic therapy evaluated by drug persistence. Methods A multicenter, retrospective study of 877 patients with inflammatory bowel disease (IBD) treated with anti-TNF therapy with HLA-DQA1*05 genotypes were generated by imputation from whole genome sequence using the HIBAG package, in R. Primary end point was anti-TNF therapy persistence, (time to therapy failure), segregated by HLA-DQA1*05 allele genotype and development of a risk score to predict anti-TNF therapy failure, incorporating HLA-DQA1*05 allele genotype status (LORisk score). Results In all, 877 patients receiving anti-TNF therapy were included in our study; 543 (62%) had no copy, 281 (32%) one copy, and 53 (6%) 2 copies of HLA-DQA1*05 allele. Mean time to anti-TNF therapy failure in patients with 2 copies of HLA-DQA1*05 allele was significantly shorter compared with patients with 0 or 1 copy at 700 days’ follow-up: 418 vs 541 vs 513 days, respectively (P = .012). Factors independently associated with time to anti-TNF therapy failure included carriage of HLA-DQA1*05 allele (hazard ratio [HR], 1.2, P = .02; female gender HR, 1.6, P &amp;lt; .001; UC phenotype HR, 1.4, P = .009; and anti-TNF therapy type [infliximab], HR, 1.5, P = .002). The LORisk score was significantly associated with shorter time to anti-TNF therapy failure (P &amp;lt; .001). Conclusions Carriage of 2 HLA-DQA1*05 alleles is associated with less favorable outcomes for patients receiving anti-TNF therapy with shorter time to therapy failure. HLA-DQA1*05 genotype status in conjunction with clinical factors may aid in therapy selection in patients with IBD.

A first-in-human, phase I, dose-escalation study to evaluate pharmacokinetics, safety, and tolerability of KD6001, a novel anti-CTLA-4 monoclonal antibody (mAb) in patients with advanced solid tumors.

e14501 Background: KD6001, a novel fully human anti-CTLA4 IgG1 monoclonal antibody, exhibits immunomodulatory effects, enhances T-cell-mediated antitumor immune response and shows promising activity in preclinical models of solid tumors. Here we present the pharmacokinetics, safety and tolerability data from the phase 1 study of KD6001 (NCT05230290) in patients with advanced solid tumors. Methods: patients were enrolled in a 3+3 dose escalation phase 1 study with the following pre-defined KD6001 cohorts: 0.2mg/kg, 1mg/kg, 3mg/kg or 6mg/kg, every 2 weeks via intravenous infusion for 4 cycles. The primary objective was to evaluate the safety and tolerability of KD6001 and determine the MTD/RP2D. The secondary objectives were to measure antitumor activity, immunogenicity and pharmacokinetics (PK). All AEs are graded using NCI CTCAE v5.0. Efficacy assessments are per RECIST 1.1. Blood samples are obtained for pharmacokinetics (PK) and for immunogenicity assessments, measured by the development of anti-drug antibodies. Results: The study had been completed. Thirteen pts with melanoma had been enrolled and treated at 0.2 mg/kg (n = 1), 1mg/kg (n = 3), 3 mg/kg (n = 6) and 6mg/kg (n = 3) at Beijing Cancer Hospital. The average age was 57.2 years (range 34-68). Baseline ECOG scores were 0 (n = 5), 1(n = 8) with all pts. Seven pts (53.85%) received ≥ 2 lines of previous systemic therapies and 12 pts (92.31%) received previous PD-1/PD-L1 treatment. KD6001 was well-tolerated with no dose-limiting toxicities (DLTs) observed, nor was the MTD reached at the highest doses administered. All the thirteen pts had at least one adverse event during treatment, and eleven of them (84.62%) had Treatment Related Adverse Events (TRAEs), most of which were Grades 1-2. There were no permanent drug discontinuation or deaths due to adverse events. Pharmacokinetic analysis indicated a linear correlate between the drug exposure (Cmax, AUC0-t and AUC0-inf) and study drug dosages evaluated. The geometric mean T1/2 was 10 days (CV%: 31%). The peak concentration and exposure level increased proportionally to dose from 0.2mg/kg to 6 mg/kg. No anti-drug antibodies were detected in any of the patient’s serum samples. Clinical efficacy was observed in 12 evaluable pts: 3 patients at 3mg/kg had a stable disease (SD) and 1 patient at 3mg/kg had a confirmed partial response (PR). The ORR of the 3mg/kg dose group was 16.7% (1/6), the DCR was 66.67% (4/6). The median PFS was 109 days and the median OS was not reached. Conclusions: KD6001 showed promising antitumor activity and a tolerable safety profile in patients with advanced solid tumors. Hence, 3mg/kg was selected as the RP2D. Clinical trial information: NCT05230290 .

MULTIDRUG RESISTANT DYSLIPIDEMIA: A CASE REPORT

Abstract Introduction Recently introduced lipid–lowering agents (PCSK9–i, bempedoic acid) can efficiently treat statin–intolerant patients, who couldn‘t reach LDL target before. Nonetheless, there are still cases where the pharmacological management may have strong limitations. Case Report A 71 year–old woman, with multiple cardiovascular risk factors (diabetes, hypertension and familial dylipidemia, LDL–C 276) underwent heart transplantation for a hypertrophic cardiomyopathy with an evolution towards a dilated phenotype in 2005. Before the transplant, she was on low–dose statin treatment (pravastatin 10 mg/die) that was suspended years later owing to hyper–CKemia (600 U/L), despite her being totally asymptomatic. A second attempt with simvastatin/ezetimibe 40/10 mg resulted again in elevation of CK levels, associated with significant muscular pain. Thus, just ezetimibe was continued. In 2020, 15 years after the transplant, the patient underwent a coronary angiography showing a moderate coronary allograft vasculopathy (ISHLT CAV2). Specifically, a sub occlusive ostial left main coronary artery lesion was treated by percutaneous angioplasty and stent implantation. Since a lower LDL target was needed (LDL–C 55 mg/dL), Alirocumab was introduced. However, 6 months later, no significant LDL levels reduction had been observed. The shift to evolocumab led to the same result, although the patient reported full therapeutic compliance. In the suspicion of anti–drug antibodies development, we opted for Inclisiran without achieving a significant decrease in LDL–C levels. In parallel with the search for genetic mutations in the PCSK9 gene and other genes involved in cholesterol metabolism (including LDLR, APOB, and LDLRAP1 and resulting positive only for common variants in LDLR receptor), therapy with bempedoic acid was initiated, again without substantial benefit (LDL–C 118 mg/dL). Conclusion Despite cholesterol management has been simplified by the introduction of new drugs, it may still be complex in patients requiring very low LDL–C levels in some specific cases. Drugs with a LDL receptor–independent mechanism of action (e.g., evinacumab) may allow the achievement of therapeutic goals suggested by clinical guidelines.

MULTIDRUG RESISTANT DYSLIPIDEMIA: A CASE REPORT

Objective: Recently introduced lipid-lowering agents (PCSK9-i, bempedoic acid) can efficiently treat statin-intolerant patients, who couldn’t reach LDL target before. Nonetheless, there are still cases where the pharmacological management may have strong limitations. Design and method: A 71 year-old woman, with multiple cardiovascular risk factors (diabetes, hypertension and familial dylipidemia, LDL-C 276) underwent heart transplantation for a hypertrophic cardiomyopathy with an evolution towards a dilated phenotype in 2005. Before the transplant, she was on low-dose statin treatment (pravastatin 10 mg/die) that was suspended years later owing to hyper-CKemia (600 U/L), despite her being totally asymptomatic. A second attempt with simvastatin/ezetimibe 40/10 mg resulted again in elevation of CK levels, associated with significant muscular pain. Thus, just ezetimibe was continued. In 2020, 15 years after the transplant, the patient underwent a coronary angiography showing a moderate coronary allograft vasculopathy (ISHLT CAV2). Specifically, a sub occlusive ostial left main coronary artery lesion was treated by percutaneous angioplasty and stent implantation. Since a lower LDL target was needed (LDL-C 55 mg/dL), Results: Alirocumab was introduced. However, 6 months later, no significant LDL levels reduction had been observed. The shift to evolocumab led to the same result, although the patient reported full therapeutic compliance. In the suspicion of anti-drug antibodies development, we opted for Inclisiran without achieving a significant decrease in LDL-C levels. In parallel with the search for genetic mutations in the PCSK9 gene and other genes involved in cholesterol metabolism (including LDLR, APOB, and LDLRAP1 and resulting positive only for common variants in LDLR receptor), therapy with bempedoic acid was initiated, again without substantial benefit (LDL-C 118 mg/dL). Conclusions: Despite cholesterol management has been simplified by the introduction of new drugs, it may still be complex in patients requiring very low LDL-C levels in some specific cases. Drugs with a LDL receptor-independent mechanism of action (e.g., evinacumab) may allow the achievement of therapeutic goals suggested by clinical guidelines.

Adalimumab (ADA) in Pediatric Non-infectious Uveitis: An Observational Study.

Pediatric uveitis is a rare but sight-threatening condition. Prompt and adequate treatment is crucial to preserve vision and avoid long-term complications. In cases that are resistant to corticosteroids and disease-modifying anti-rheumatic drugs (DMARDs), anti-tumor necrosis (anti-TNF) biologic agents are usually added. In this study, we report our experience with adalimumab (ADA) anti-TNF use in this group of patients. This is a retrospective observational study conducted in a tertiary pediatric uveitis clinic, in Manchester Royal Eye Hospital. All patients were pediatric patients (aged 2-18 years old) under follow-up during the period of six months. The patients' data were analyzed according to the diagnosis, age of onset of uveitis, systemic medications used before and concomitantly withADA, duration of uveitis before starting ADA, its effect, and time to notice the therapeutic effect in controlling inflammation. Finally, cases were reviewed for the development of anti-drug antibodies. Forty-two patients were included in the study. Idiopathic uveitis was diagnosed in 47.6% of patients and 40.5% of patients were associated with juvenile idiopathic arthritis (JIA). Most (97.6%) of patients were using topical steroids before starting ADA and 95.2% continued using steroids after established ADA use, but systemic steroid use was reduced from 33.3% to 14.3%. The most common non-biologic DMARD used before ADA was methotrexate (MTX) (90.5%). One-third of the patients started ADA between 6 and 12 months after the diagnosis of uveitis, while this percentage dropped to 9.5% the year after diagnosis. Seventy-eight percent of patients acquired complete clinical control of inflammation on ADA use. Almost 78.6% of patients showed a full response in less than six months. In eight patients who were not controlled or were transiently controlled on ADA, three patients had positive anti-drug antibodies. In one patient, antidrug antibodies were identified after 12 years of ADA use, and in another, after 4 years. Adalimumab is an effective, well-tolerated drug in children with uveitis refractory to non-biologic DMARD therapy. DMARDs were usually used alongside ADA in this cohort and few patients had confirmed ADA antibodies.

Implementation of a Three-Way Comparability Assessment for a Bioanalytical Anti-Drug Antibody Method

Immunogenicity evaluation is a critical part of drug development. Regulatory guidelines from multiple health agencies provide recommendations for the development and validation of anti-drug antibody (ADA) assays to assess immunogenicity in clinical trials. These recommendations primarily describe an ADA method run in one bioanalytical laboratory supporting a biotherapeutic molecule; however, there are increasing instances that may necessitate the support of the ADA method being run in more than one laboratory. A program can rapidly expand into multiple clinical studies within one or multiple countries, where the most appropriate way to support the program is by having multiple laboratories perform ADA sample analysis. In addition, there may be certain country-specific challenges that may make it infeasible to transport samples outside of the country for analysis. China for example has a lengthy sample exportation process that has potential to negatively impact study timelines. If multiple laboratories analyze samples using the same ADA method, comparable method performance should be established. Here, we describe a three-way assessment of ADA assay comparability between two US-based bioanalytical laboratories and one based in China.Graphical

Mechanisms and management of loss of response to anti-TNF therapy for patients with Crohn's disease: 3-year data from the prospective, multicentre PANTS cohort study

We sought to report the effectiveness of infliximab and adalimumab over the first 3 years of treatment and to define the factors that predict anti-TNF treatment failure and the strategies that prevent or mitigate loss of response. Personalised Anti-TNF therapy in Crohn's disease (PANTS) is a UK-wide, multicentre, prospective observational cohort study reporting the rates of effectiveness of infliximab and adalimumab in anti-TNF-naive patients with active luminal Crohn's disease aged 6 years and older. At the end of the first year, sites were invited to enrol participants still receiving study drug into the 2-year PANTS-extension study. We estimated rates of remission across the whole cohort at the end of years 1, 2, and 3 of the study using a modified survival technique with permutation testing. Multivariable regression and survival analyses were used to identify factors associated with loss of response in patients who had initially responded to anti-TNF therapy and with immunogenicity. Loss of response was defined in patients who initially responded to anti-TNF therapy at the end of induction and who subsequently developed symptomatic activity that warranted an escalation of steroid, immunomodulatory, or anti-TNF therapy, resectional surgery, or exit from study due to treatment failure. This study was registered with ClinicalTrials.gov, NCT03088449, and is now complete. Between March 19, 2014, and Sept 21, 2017, 389 (41%) of 955 patients treated with infliximab and 209 (32%) of 655 treated with adalimumab in the PANTS study entered the PANTS-extension study (median age 32·5 years [IQR 22·1-46·8], 307 [51%] of 598 were female, and 291 [49%] were male). The estimated proportion of patients in remission at the end of years 1, 2, and 3 were, for infliximab 40·2% (95% CI 36·7-43·7), 34·4% (29·9-39·0), and 34·7% (29·8-39·5), and for adalimumab 35·9% (95% CI 31·2-40·5), 32·9% (26·8-39·2), and 28·9% (21·9-36·3), respectively. Optimal drug concentrations at week 14 to predict remission at any later timepoints were 6·1-10·0 mg/L for infliximab and 10·1-12·0 mg/L for adalimumab. After excluding patients who had primary non-response, the estimated proportions of patients who had loss of response by years 1, 2, and 3 were, for infliximab 34·4% (95% CI 30·4-38·2), 54·5% (49·4-59·0), and 60·0% (54·1-65·2), and for adalimumab 32·1% (26·7-37·1), 47·2% (40·2-53·4), and 68·4% (50·9-79·7), respectively. In multivariable analysis, loss of response at year 2 and 3 for patients treated with infliximab and adalimumab was predicted by low anti-TNF drug concentrations at week 14 (infliximab: hazard ratio [HR] for each ten-fold increase in drug concentration 0·45 [95% CI 0·30-0·67], adalimumab: 0·39 [0·22-0·70]). For patients treated with infliximab, loss of response was also associated with female sex (vs male sex; HR 1·47 [95% CI 1·11-1·95]), obesity (vs not obese 1·62 [1·08-2·42]), baseline white cell count (1·06 [1·02-1·11) per 1 × 109 increase in cells per L), and thiopurine dose quartile. Among patients treated with adalimumab, carriage of the HLA-DQA1*05 risk variant was associated with loss of response (HR 1·95 [95% CI 1·17-3·25]). By the end of year 3, the estimated proportion of patients who developed anti-drug antibodies associated with undetectable drug concentrations was 44·0% (95% CI 38·1-49·4) among patients treated with infliximab and 20·3% (13·8-26·2) among those treated with adalimumab. The development of anti-drug antibodies associated with undetectable drug concentrations was significantly associated with treatment without concomitant immunomodulator use for both groups (HR for immunomodulator use: infliximab 0·40 [95% CI 0·31-0·52], adalimumab 0·42 [95% CI 0·24-0·75]), and with carriage of HLA-DQA1*05 risk variant for infliximab (HR for carriage of risk variant: infliximab 1·46 [1·13-1·88]) but not for adalimumab (HR 1·60 [0·92-2·77]). Concomitant use of an immunomodulator before or on the day of starting infliximab was associated with increased time without the development of anti-drug antibodies associated with undetectable drug concentrations compared with use of infliximab alone (HR 2·87 [95% CI 2·20-3·74]) or introduction of an immunomodulator after anti-TNF initiation (1·70 [1·11-2·59]). In years 2 and 3, 16 (4%) of 389 patients treated with infliximab and 11 (5%) of 209 treated with adalimumab had adverse events leading to treatment withdrawal. Nine (2%) patients treated with infliximab and two (1%) of those treated with adalimumab had serious infections in years 2 and 3. Only around a third of patients with active luminal Crohn's disease treated with an anti-TNF drug were in remission at the end of 3 years of treatment. Low drug concentrations at the end of the induction period predict loss of response by year 3 of treatment, suggesting higher drug concentrations during the first year of treatment, particularly during induction, might lead to better long-term outcomes. Anti-drug antibodies associated with undetectable drug concentrations of infliximab, but not adalimumab, can be predicted by carriage of HLA-DQA1*05 and mitigated by concomitant immunomodulator use for both drugs. Guts UK, Crohn's and Colitis UK, Cure Crohn's Colitis, AbbVie, Merck Sharp and Dohme, Napp Pharmaceuticals, Pfizer, and Celltrion Healthcare.

Antibiotics influence the risk of anti-drug antibody formation during anti-TNF therapy in Chinese inflammatory bowel disease patients.

Aims: The formation of anti-drug antibodies (ADAs) during anti-tumor necrosis factor (anti-TNF) therapy is reported to lead to reducing serum drug levels, which may bring about a loss of response to treatment. Previous research has suggested an association between specific antibiotic classes and ADA formation during anti-TNF therapy. However, there are few studies specifically examining this association in Chinese inflammatory bowel disease (IBD) patients. Therefore, our study aimed to evaluate the possible effect of antibiotic use on ADA formation to anti-TNF therapy in Chinese patients with IBD. Methods: A total of 166 patients with IBD, including 149 with Crohn's disease (CD) and 17 with ulcerative colitis (UC), were included in this retrospective analysis. These patients were initially treated with anti-TNF therapy (infliximab or adalimumab) after January 2018 and reviewed with available ADA levels before October 2023. After univariable analysis of all the variables, a multivariate Cox proportional hazards model was used to assess the association between antibiotic use and ADA development. Results: Among 166 IBD patients treated with infliximab (108/166, 65.1%) or adalimumab (58/166, 34.9%), 31 patients (18.7%) were measured as positive ADA levels. Cox proportional hazard model demonstrated an increased risk of ADA formation in IBD patients who used β-lactam-β-lactamase inhibitor combinations (BL-BLIs) (HR = 5.143, 95%CI 1.136-23.270, p = 0.033), or nitroimidazoles (HR = 4.635, 95%CI 1.641-13.089, p = 0.004) during 12months before the ADA test. On the contrary, a reduced risk was noted in patients treated with fluoroquinolones (HR = 0.258, 95% CI 0.072-0.924, p = 0.037). Moreover, the median serum infliximab or adalimumab concentration in patients with positive ADA levels was significantly lower than that in patients with negative ADA levels (infliximab: 0.30 vs. 1.85μg/mL, p < 0.0001; adalimumab: 0.45 vs. 7.55μg/mL, p = 0.0121). Conclusion: ADA development is associated with various antibiotic classes. BL-BLIs and nitroimidazoles might increase the risk of ADA formation during anti-TNF therapy in Chinese IBD patients, while the treatment with fluoroquinolones could probably reduce such risk. There were certain limitations in the retrospective analysis of the study, therefore, the results are just for reference, and other studies are needed to further confirm our findings.

Phase 1, first-in-human study of TYRP1-TCB (RO7293583), a novel TYRP1-targeting CD3 T-cell engager, in metastatic melanoma: active drug monitoring to assess the impact of immune response on drug exposure.

Although checkpoint inhibitors (CPIs) have improved outcomes for patients with metastatic melanoma, those progressing on CPIs have limited therapeutic options. To address this unmet need and overcome CPI resistance mechanisms, novel immunotherapies, such as T-cell engaging agents, are being developed. The use of these agents has sometimes been limited by the immune response mounted against them in the form of anti-drug antibodies (ADAs), which is challenging to predict preclinically and can lead to neutralization of the drug and loss of efficacy. TYRP1-TCB (RO7293583; RG6232) is a T-cell engaging bispecific (TCB) antibody that targets tyrosinase-related protein 1 (TYRP1), which is expressed in many melanomas, thereby directing T cells to kill TYRP1-expressing tumor cells. Preclinical studies show TYRP1-TCB to have potent anti-tumor activity. This first-in-human (FIH) phase 1 dose-escalation study characterized the safety, tolerability, maximum tolerated dose/optimal biological dose, and pharmacokinetics (PK) of TYRP1-TCB in patients with metastatic melanoma (NCT04551352). Twenty participants with cutaneous, uveal, or mucosal TYRP1-positive melanoma received TYRP1-TCB in escalating doses (0.045 to 0.4 mg). All participants experienced ≥1 treatment-related adverse event (TRAE); two participants experienced grade 3 TRAEs. The most common toxicities were grade 1-2 cytokine release syndrome (CRS) and rash. Fractionated dosing mitigated CRS and was associated with lower levels of interleukin-6 and tumor necrosis factor-alpha. Measurement of active drug (dual TYPR1- and CD3-binding) PK rapidly identified loss of active drug exposure in all participants treated with 0.4 mg in a flat dosing schedule for ≥3 cycles. Loss of exposure was associated with development of ADAs towards both the TYRP1 and CD3 domains. A total drug PK assay, measuring free and ADA-bound forms, demonstrated that TYRP1-TCB-ADA immune complexes were present in participant samples, but showed no drug activity in vitro. This study provides important insights into how the use of active drug PK assays, coupled with mechanistic follow-up, can inform and enable ongoing benefit/risk assessment for individuals participating in FIH dose-escalation trials. Translational studies that lead to a better understanding of the underlying biology of cognate T- and B-cell interactions, ultimately resulting in ADA development to novel biotherapeutics, are needed.

Sensitive B-cell receptor repertoire analysis shows repopulation correlates with clinical response to rituximab in rheumatoid arthritis

BackgroundAlthough B-cell depleting therapy in rheumatoid arthritis (RA) is clearly effective, response is variable and does not correlate with B cell depletion itself.MethodsThe B-cell receptor (BCR) repertoire was prospectively analyzed in peripheral blood samples of twenty-eight RA patients undergoing rituximab therapy. Timepoints of achieved BCR-depletion and -repopulation were defined based on the percentage of unmutated BCRs in the repertoire. The predictive value of early BCR-depletion (within one-month post-treatment) and early BCR-repopulation (within 6 months post-treatment) on clinical response was assessed.ResultsWe observed changes in the peripheral blood BCR repertoire after rituximab treatment, i.e., increased clonal expansion, decreased clonal diversification and increased mutation load which persisted up to 12 months after treatment, but started to revert at month 6. Early BCR depletion was not associated with early clinical response but late depleters did show early response. Patients with early repopulation with unmutated BCRs showed a significant decrease in disease activity in the interval 6 to 12 months. Development of anti-drug antibodies non-significantly correlated with more BCR repopulation.ConclusionOur findings indicate that rather than BCR-depletion it is repopulation with unmutated BCRs, possibly from naïve B cells, which induces remission. This suggests that (pre-existing) differences in B-cell turnover between patients explain the interindividual differences in early clinical effect.

Immunogenicity of avelumab in patients with metastatic Merkel cell carcinoma or advanced urothelial carcinoma.

Like other monoclonal antibodies, immune checkpoint inhibitors may be immunogenic in some patients, potentially affecting pharmacokinetics (PKs) and clinical outcomes. In post hoc analyses, we characterized antidrug antibody (ADA) development with avelumab monotherapy in patients with metastatic Merkel cell carcinoma (mMCC) from the JAVELIN Merkel 200 trial (first-line [1L; N = 116] and second-line or later [≥2L; N = 88] cohorts) or with advanced urothelial carcinoma (aUC) from the JAVELIN Bladder 100 (1L maintenance [N = 350]) and JAVELIN Solid Tumor (≥2L [N = 249]) trials. Treatment-emergent ADAs developed in a numerically higher proportion of patients with aUC (1L maintenance, 19.1%; ≥2L, 18.1%) versus mMCC (1L, 8.2%; ≥2L, 8.9%); incidences within tumor types were similar by line of therapy. In PK analyses, numerically lower avelumab trough concentration and higher baseline clearance were observed in treatment-emergent ADA+ versus ADA- subgroups; however, differences were not clinically relevant. Numerical differences in overall survival, progression-free survival, or objective response rate by ADA status were observed; however, no clinically meaningful trends were identified. Proportions of patients with treatment-emergent adverse events (TEAEs; any grade or grade 3/4), serious TEAEs, TEAEs leading to treatment discontinuation, or infusion-related reactions were similar, with overlapping 80% confidence intervals between ADA subgroups. Efficacy and safety observations were similar in subgroups defined by early development of ADA+ status during treatment. In conclusion, no meaningful differences in PKs, efficacy, and safety were observed between subgroups of avelumab-treated patients with different ADA status. Overall, these data suggest that ADAs are not relevant for treatment decisions with avelumab.

Immunogenicity and Loss of Effectiveness of Biologic Therapy for Inflammatory Bowel Disease Patients Due to Anti-Drug Antibody Development.

Many patients with inflammatory bowel disease (IBD) experience a loss of effectiveness to biologic therapy (i.e., anti-TNF therapy, etc.). Therefore, in addition to the adverse effects of the treatment, these patients also face failure to achieve and maintain remission. Immunogenicity, the process of production of antibodies to biological agents, is fundamental to the evolution of loss of response to treatment in IBD patients. The presence of these antibodies in patients is linked to decreased serum drug levels and inhibited biological activity. However, immunogenicity rates exhibit significant variability across inflammatory disease states, immunoassay formats, and time periods. In this review, we aimed to elucidate the immunogenicity and immune mechanisms of antibody formation to biologics, the loss of therapy response, clinical results of biological treatment for IBD from systematic reviews and meta-analyses, as well as to summarize the most recent strategies for overcoming immunogenicity and approaches for managing treatment failure in IBD.