Formation Of Anti-drug Antibodies Research Articles

P414 Risk factors for the development of anti-drug antibodies to infliximab and adalimumab in Crohn’s disease

Abstract Background Anti-tumor necrosis factor-α (anti-TNFa) therapy have been established as an effective maintenance treatment for complicated Crohn’s Disease (CD). However, the efficacy of Infliximab (IFX) and Adalimumab (ADM) may be affected by low serum levels and/or the presence of anti-drug antibodies (ADA). This reinforces the importance of therapeutic drug monitoring (TDM). We aim to assess the clinical benefit of proactive vs. reactive TDM. Secondly, to assess the impact of TDM on clinical management. Thirdly, to identify risk factors for low serum drug levels and the development of ADA in CD patients. Methods This was a single-centred observational cohort study performed at a tertiary hospital, comprising of total 229 CD patients: 142 received IFX and 87 received ADM, who have had a trough drug level, tested using enzyme-linked immunosorbent assay. Demographic and clinical data were retrospectively collected from electronic medical records. Fisher’s Exact Test was used to determine if there are nonrandom associations between variables. A p-value of less than 0.05 was considered statistically significant. Results One hundred and fourteen patients (49%) receiving a standard anti-TNFa regimen had subtherapeutic drug levels (67 had IFX < 3 μg/ml and 47 had ADM < 5 μg/ml). Interestingly, almost half of this cohort were asymptomatic. Reactive TDM completed among symptomatic patients have shown to have a statistically significant benefit in detecting subtherapeutic drug level (p = 0.0001). Following these results, only fifty-two patients (46%) had a change of therapy (29 IFX, 25 ADM); while the remaining sixty-two patients (54%) continued the same dosing regimen with only one documented admission within 90-days following the drug level being taken. Eight patients (4%) were found to have positive ADA, all in the presence of subtherapeutic drug levels. Two of these had a subsequent flare of their disease. They were all switched to another class of biologic therapy. Non-smoking status at diagnosis and the concomitant use of immunomodulator were found to have statistically significant associations with a therapeutic drug level (p = 0.0176 and p = 0.0001, respectively). Similarly, both of these risk factors were associated with lower risk of ADA formation (p = 0.0057 and p = 0.0165, respectively). Conclusion This study suggests that a large proportion of patients have subtherapeutic drug levels at standard dosing schedules. However, low drug levels do not correlate with a higher risk of complications if patients are in clinical remission. The results of this study also indicate that non-smoking status at diagnosis and the concomitant use of immunomodulator are associated with higher serum drug levels and lower risk of developing anti-drug antibodies.

Molecular Landscape of Anti-Drug Antibodies Reveals the Mechanism of the Immune Response Following Treatment With TNFα Antagonists.

Drugs formulated from monoclonal antibodies (mAbs) are clinically effective in various diseases. Repeated administration of mAbs, however, elicits an immune response in the form of anti-drug-antibodies (ADA), thereby reducing the drug's efficacy. Notwithstanding their importance, the molecular landscape of ADA and the mechanisms involved in their formation are not fully understood. Using a newly developed quantitative bio-immunoassay, we found that ADA concentrations specific to TNFα antagonists can exceed extreme concentrations of 1 mg/ml with a wide range of neutralization capacity. Our data further suggest a preferential use of the λ light chain in a subset of neutralizing ADA. Moreover, we show that administration of TNFα antagonists result in a vaccine-like response whereby ADA formation is governed by the extrafollicular T cell-independent immune response. Our bio-immunoassay coupled with insights on the nature of the immune response can be leveraged to improve mAb immunogenicity assessment and facilitate improvement in therapeutic intervention strategies.

Abstract PR07: Phase I/II study of mesothelin-targeted immunotoxin LMB-100 with nab-paclitaxel for patients with advanced pancreatic adenocarcinoma

Abstract Purpose: LMB-100 recombinant immunotoxin (iTox) consists of a mesothelin-binding Fab for targeting and a modified Pseudomonas exotoxin A payload. Preclinical studies showed that combining taxanes with iTox results in synergistic antitumor activity. The primary objectives of this phase I/II study were to determine the maximum tolerated dose (MTD) of LMB-100 when given with nanoalbumin bound (nab)-paclitaxel to participants with previously treated advanced pancreatic adenocarcinoma and to assess the objective response rate. Patients and Methods: Patients (n = 20) received fixed-dose nab-paclitaxel (125 mg/m2 on Days 1 and 8) with LMB-100 (65 or 100 mcg/kg on Days 1, 3, and 5) in 21-day cycles. Treatment was limited to 2-3 cycles by protocol. Safety and tolerability, pharmacokinetics, immunogenicity, and relationship of activity to tumor mesothelin expression were assessed. Results: Dose-limiting toxicity (DLT) of Grade 3 myalgia was observed in 2 of 5 patients treated with 100 mcg/kg. DLT of Grade 3 edema from capillary leak syndrome (CLS) was observed in 1 of 6 patients at MTD of 65 mcg/kg. Severe toxicities of Grade 4 left ventricular dysfunction and hypotension from CLS were observed in 1 patient in the phase II expansion, resulting in study closure. LMB-100 anti-drug antibody formation limited LMB-100 exposure in 5 of 13 patients during cycle 2. Seven of 17 patients with evaluable CA19-9 experienced >50% decrease of this tumor marker, and one patient developed a partial response. Response was associated with higher tumor mesothelin expression and Cycle 2 LMB-100 exposure. Conclusions: The combination caused serious toxicity. Despite some observed clinical activity, further testing of this combination will not be pursued. This abstract is also being presented as Poster B01. Citation Format: Christine C. Alewine, Mehwish Ahmad, Cody J. Peer, Zishuo Hu, Min-jung Lee, Akiro Yuno, Jessica Kindrick, Anish Thomas, Seth Steinberg, Jane B. Trepel, William D. Figg, Raffit Hassan, Ira Pastan. Phase I/II study of mesothelin-targeted immunotoxin LMB-100 with nab-paclitaxel for patients with advanced pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr PR07.

Abstract CN07-03: Recombinant immunotoxins for hematologic malignancies

Abstract Recombinant immunotoxins are composed of fragments of monoclonal antibodies (Mabs) and protein toxins, enabling the toxin to bind to a target cell recognized by the antibody, and the toxin to kill the cell after internalization. Recombinant immunotoxins are similar to but distinct from growth-factor fusions toxins like denileukin diftitox and Tagraxofusp, FDA-approved in 1999 and 2018, respectively. Unlike chemical conjugates, Recombinant immunotoxins have a peptide that links the cell-binding to the toxin domains. After proteolytic cleavage, the toxin separates from the binding domain, undergoes intracellular trafficking and enters the cytosol, resulting in apoptotic cell death. Recombinant immunotoxins containing Pseudomonas exotoxin A contain an Fv or Fab fragment of a Mab replacing the native cell-binding toxin domain. The first recombinant immunotoxin we made contained an anti-CD25 single-chain Fv fused to a 38 kDa fragment of Pseudomonas exotoxin called PE38. Anti-CD25 recombinant immunotoxin LMB-2 was active in several hematologic malignancies, notably hairy cell leukemia (HCL) and adult T-cell leukemia (ATL). LMB-2 achieved a high complete remission (CR) rate in ATL when combined with chemotherapy to reduce immunogenicity and progression between cycles. Improved targeting of HCL was achieved with anti-CD22 recombinant immunotoxin Moxetumomab Pasudotox (Moxe) that was stabilized with a disulfide bond in the Fv and has a high affinity for CD22. Moxe achieved investigator-assessed CR rates of 51-64% in relapsed/refractory HCL in Phase 1 and 3 trials, leading to FDA-approval in 2018. Minimal residual disease (MRD) was negative in most CRs. On the phase I trial, which had adequate follow-up, MRD eradication by the most sensitive standard assay, flow cytometry of the bone marrow aspirate (BMA), was associated with longer CR duration. Extra or ‘consolidation’ cycles past documentation of CR was also associated with longer CR duration. To detect MRD with higher sensitivity, patient specific immunoglobulin heavy chain (IgH) rearrangements were cloned and real-time quantitative PCR (RQ-PCR) performed. Using patient-specific primers and probe, 1 HCL cell could be detected in 106 normal cells. We found that phase 1-3 patients achieving MRD-free CR by blood RQ-PCR had significantly prolonged CR duration (p<0.0001) compared to those remaining blood RQ-PCR+. Both BMA flow cytometry and RQ-PCR were associated with prolonged CR duration if negative, but blood flow cytometry was not helpful since only one patient with CR remained flow-positive. Molecular remissions assessed by blood analysis may be useful to determine the number of cycles of Moxe to administer to achieve long-term CR or possibly cure. To achieve molecular remissions with fewer cycles, Moxe is being tested with rituximab to facilitate tumor reduction and decrease anti-drug antibody formation. Citation Format: Robert Kreitman, Daniel Gorelik, Maryalice Stetler-Stevenson, Constance M Yuan, Hao-Wei Wang, Hong Zhou, Katherine Potocka, Erin Fykes, Evgeny Arons, Ira Pastan. Recombinant immunotoxins for hematologic malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr CN07-03. doi:10.1158/1535-7163.TARG-19-CN07-03

Toll-like Receptor 9 Activation Accelerates Inhibitor Formation in Response to Factor VIII

Inhibitor (neutralizing anti-drug antibody) formation against factor VIII (FVIII) is currently the most serious complication of FVIII replacement therapy for hemophilia A. The role of innate immune signals in this adaptive immune response is unclear. Lipopolysaccharide (LPS, the major component of the outer membrane in Gram-negative bacteria), which activates the innate immune sensor toll-like receptor 4 (TLR4), enhances inhibitor formation in hemophilia A mice. Intriguingly, earlier data suggested that activation of the endosomal DNA sensor TLR9 may actually suppress inhibitor formation. However, we demonstrated that a TLR9 agonist induced inhibitor formation against a factor IX transgene product in gene therapy for hemophilia B, which resulted from activation of monocyte-derived dendritic cells (moDCs) and enhancement of T follicular helper (Tfh) cell responses. Tfh cells drive germinal center (GC) formation and T cell help-dependent antibody formation. The aim of this study was to elucidate the role of TLR9 signaling in FVIII inhibitor formation in hemophilia A mice. Hemophilia A (F8e16-/-) B6/129 mice were co-injected IV (n=4) with FVIII (1.5 IU) and ODN-1826 (a class B CpG oligodeoxynucleotide, 50 µg), which is a TLR9 agonist, or injected on two consecutive days (n=3), first with ODN-1826 and then with FVIII on the next day. Control mice received FVIII only (n=5). Injections were performed once weekly for 4 weeks. Blood samples, spleens and subiliac (superficial inguinal) lymph nodes were collected for ELISA and Bethesda assays, and flow cytometry analysis. In our analysis, Tfh were defined as CD4+CXCR5+PD1+Bcl6- cells, while GC B cells were defined as CD19+GL7+CD95+. Tfh cell response and GC formation in the spleen were robustly enhanced by the TLR9 agonist compared to mice injected with FVIII only (2-fold for Tfh frequencies and 6-fold for GC B cells when co-injected; P< 0.05). As a result, inhibitor titers increased >400-fold (on average from 6.4 to 2746.5 BU/ml). In contrast, anti-FVIII IgG1 levels increased only 2.5-fold. The differences between mice injected on two consecutive days and the FVIII-only group were not statistically significant. A time-course experiment was also carried out to monitor progress of immune response to FVIII (1.5 IU administered once weekly) in the absence of TLR9 agonist over time: 4 (n=4), 6 (n=4) and 8 weeks (n=5) after the initial antigen challenge. Inhibitor titers continued to rise beyond the fourth week of antigen challenges, reaching 46-fold higher values at eighth week (from 3 BU/ml in the fourth week to 138 BU/ml in the eighth week, p<0.05), while anti-FVIII IgG1 levels increased 2.5-fold (ns). These results suggest that the TLR9 agonist sped up the neutralizing response to FVIII, which otherwise progressed in the same manner (towards more neutralizing versus total anti-FVIII) but at a slower rate. Overall, TLR9 activation enhanced GC formation and accelerated neutralizing immune response to FVIII. Similar results were obtained in hemophilia A mice on a different strain background (F8e16-/- BALB/c). We propose that signaling via the innate immune receptor TLR9 leads to a more targeted immune response by reinforcing Tfh activation, likely through moDC activation, which results in enhanced germinal center formation and thus accelerated development of neutralizing antibodies. Disclosures No relevant conflicts of interest to declare.

Phase III Clinical Trial of Elizaria® and Soliris® in Adult Patients with Paroxysmal Nocturnal Hemoglobinuria: Results of Comparative Analysis of Efficacy, Safety, and Pharmacological Data

Introduction: Complement C5-inhibitor eculizumab is a current standard of care in patients with paroxysmal nocturnal hemoglobinuria (PNH). Elizaria® is the first registered biosimilar of the reference medicinal product Soliris®. Purpose: To demonstrate Elizaria® comparative efficacy and safety vs. reference product Soliris® in patients with paroxysmal nocturnal hemoglobinuria. Methods: 32 patients with PNH were stratified based on the previous eculizumab treatment status (eculizumab-naive patients/patients treated with eculizumab at a maintenance regimen before enrollment) and randomized to receive Elizaria® (n = 16) or Soliris® (n = 16). Eculizumab-naive patients with baseline LDH level ≥1.5 times the upper limit of normal started induction phase of four weekly infusion of the study medications at dose of 600 mg, with subsequent maintenance therapy at dose of 900 mg every two weeks. Previously treated patients started the study treatment at the maintenance dose. The total treatment duration was 26 weeks. Comparative evaluation of chronic hemolysis based on the area under the curve "LDH concentration-time" (LDH AUC) during the maintenance therapy period was the primary endpoint of the study. The main secondary efficacy parameters were hemoglobin dynamics, breakthrough hemolysis and transfusion dependence. PK profile as well as through eculizumab and membrane attack complex (MAC) concentration were also assessed during the treatment. Results: Thirty patients have completed the study without significant protocol deviations (16 patients in Elizaria® group and 14 patients in Soliris® group). The LDH AUC was highly variable between the patients but the means were not statistically differ between the treatment groups: 62,957.6 ± 46,066.5 U/L*day (95% CI [38,410.4; 87,504.7]) and 49,702.6 ± 26,182.1 U/L*day (95% CI [34,585.5; 64,819.7]) in Elizaria® and Soliris® groups respectively. Point estimation of the intergroup difference in LDH AUC of 13255,0 U/L*days (95% CI [-10492,9; 37002,8]) comprises 12,3% of the pre-specified non-inferiority margin of 150,635 U/L*days, that is not supposed clinically significant difference (Figure 1). The treatment groups were also comparable on all the secondary efficacy parameters (Table 1). The comparative PK parameters of eculizumab and MAC concentration have been demonstrated in the treatment groups in all studied time points. Thus, the median elimination half-life (T1/2) of eculizumab amounted to 187,7 h (IQR 165,7) in Elizaria® group and to 282,0 h (IQR 152,3) in Soliris® group (p = 0.236). The minimum concentration (Cmin) of eculizumab amounted to (64.89 ± 25.96) µg/mL in Elizaria® group and to (94.13 ± 58.76) µg/mL in Soliris® group (p = 0.065). The median residence time (MRT) of eculizumab amounted to 269,5 h (IQR 257.3) in Elizaria® group and to 393,1 h (IQR 240,9) in Soliris® group (p = 0.206). By the end of the study, the mean MAC values one hour after the study products infusion amounted to (170.41 ± 72.21) ng/mL and (214.08 ± 93.57) ng/mL in Elizaria® and Soliris® groups, respectively (Figure 2). Both Elizaria® and Soliris® demonstrated the similar safety profile. Thirteen adverse drug reactions (ADR) in 5 patients were reported in the study: 9 - in 3 Elizaria® patients, whereas 4 were reported in 2 Soliris® patients. ADRs were observed in the following system organ classes: Investigations (9.4%), Blood and lymphatic system disorders (6.3%), Infections and infestations (6.3%), Renal and urinary disorders (3.1%), General disorders and administration site conditions (3.1%), Metabolism and nutrition disorders (3.1%). No new cases of anti-drug antibody formation have been revealed during the study. Conclusion: Thus, the results of the clinical trial confirm that proposed biosimilar Elizaria® is comparable to the reference product Soliris® in term of efficacy, safety, immunogenicity and PK/PD parameters in the treatment of paroxysmal nocturnal hemoglobinuria. Disclosures Kulagin: Alexion Pharmaceuticals, Inc:: Consultancy, Honoraria; JSC GENERIUM: Consultancy, Honoraria, Research Funding. Ptushkin:Alexion Pharmaceuticals, Inc:: Consultancy, Research Funding; JSC GENERIUM: Research Funding; Janssen: Consultancy; AbbVie: Consultancy; Roche: Consultancy. Lukina:JSC GENERIUM: Research Funding; Sanofi Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Reimbursement, Research Funding; Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Reimbursement. Gapchenko:JSC GENERIUM: Employment. Markova:JSC GENERIUM: Employment. Zuev:JSC GENERIUM: Employment. Kudlay:JSC GENERIUM: Employment.

The effect of methotrexate on tumour necrosis factor concentrations in etanercept-treated rheumatoid arthritis patients.

Recently, we demonstrated that early low concentrations of circulating, adalimumab-bound TNF in RA patients treated with adalimumab was associated with future anti-drug antibody formation. Furthermore, low TNF was associated with less frequent baseline MTX use. This is remarkable, because of the anti-inflammatory effects of MTX and a potential inhibiting effect on cytokine production. We hypothesized an indirect effect of non-MTX use on low TNF concentrations via immunogenicity. To investigate the effect of MTX on TNF concentrations independent of anti-drug antibody formation, we measured TNF in RA patients treated with etanercept, a drug with low immunogenicity. TNF was quantified in 186 consecutive etanercept-treated RA patients at baseline and at weeks 4, 16 and 28. The dynamics of TNF during etanercept treatment were compared with dynamics recently published for adalimumab. We demonstrated that TNF concentrations at week 4 did not associate with baseline MTX or remission after 28 weeks. Furthermore, median (interquartile range) TNF increased from <112 (<112-<112) pg/ml at baseline to 548 (344-688) pg/ml at week 4 and remained stable at week 16 and 28 [598 (442-756) and 568 (444-755) pg/ml, respectively]. Circulating TNF did not associate with MTX usage in etanercept-treated patients. This implies that MTX does not have a direct effect on TNF concentrations in circulation and that the association between early low TNF and non-use of MTX for adalimumab is thus most likely due to anti-drug antibody formation.

Management of Anti‐drug Antibodies to Biologic Medications in Children With Inflammatory Bowel Disease

Treatment of pediatric inflammatory bowel disease (IBD) with monoclonal anti- tumor necrosis factor-alpha (TNFα) can result in immunogenicity and formation of anti-drug antibodies (ADAs). ADAs are associated with loss of clinical response and worsening disease progression. Data examining treatment interventions to overcome ADA in pediatric patients with IBD are lacking. Medical records were reviewed from 234 children and adolescents with IBD treated with infliximab or adalimumab who underwent therapeutic drug monitoring (626 tests). All patients who had detectable antibodies were further analyzed. A total 58 patients (24.8%) developed ADA while being treated with infliximab or adalimumab. The incidence of antibody development was 12.9 per 100 person-years of anti-TNF treatment. Twenty-eight patients underwent dose optimization and 54% had undetectable ADA on follow-up monitoring. The mean duration of antibody suppression was 16.8 ± 10.9 months in those who were successfully suppressed with optimization. Patients who switched to a second anti-TNF medication were not more likely to develop antibodies to the second agent. With limited therapies for IBD and the chronicity of the disease, we advocate salvage of the current anti-TNF through dose optimization in pediatric patients with antibody level <10 U/mL.

HLADQA1*05 genotype predicts anti-drug antibody formation and loss of response during infliximab therapy for inflammatory bowel disease.

Anti-drug antibodies (ADAs) are a leading contributor to infliximab loss of response and adverse drug events. It is not feasible to identify patients at risk of antibody formation before initiating infliximab. The genetic variation HLADQA1*05 (rs2097432) has been linked to infliximab antibody formation in Crohn's disease (CD). To evaluate the association between HLADQA1*05 and infliximab antibody formation, infliximab loss of response, treatment discontinuation and adverse drug events in patients with inflammatory bowel disease (IBD) METHODS: In a retrospective cohort study, infliximab-exposed patients with IBD (n=262) were screened for the genetic variation, HLADQA1*05A>G (rs2097432). Risk of infliximab ADA formation, infliximab loss of response, adverse events and discontinuation were assessed in wild-type (GG) and variant-carrying (AG or AA) individuals. Forty per cent of all participants were HLADQA1*05A>G variant carriers, with 79% of participants with infliximab antibodies carrying at least one variant allele. The risk of infliximab antibody formation was higher in HLADQA1*05A>G variant carriers (adjusted HR=7.29, 95% confidence interval (CI)=2.97-17.191, P=1.46×10-5 ) independent of age, sex, weight, dose and co-immunosuppression with an immunomodulator. Variant carrier status was associated with an increased risk of infliximab loss of response (adjusted HR=2.34, 95% CI=1.41-3.88, P=.001) and discontinuation (adjusted HR=2.27, 95% CI=1.46-3.43, P=2.53×10-4 ) although not with infliximab-associated adverse drug events. HLADQA1*05 is independently associated with a high risk of infliximab antibody formation in addition to infliximab loss of response and treatment discontinuation. There may be a role for genotype-guided application of combination therapy in IBD.

Anti-TNF combination therapy in inflammatory bowel disease: de novo or selective?

Anti-TNFs still remain the backbone of advanced therapies in inflammatory bowel diseases, but their efficacy is not universal and tends to diminish over time. As a consequence, there is the need for optimization of these treatments, and the use of combination therapy - i.e. an anti-TNF plus an immunosuppressant - is one of the main strategies. The rationale for this approach lies in the evidence that the immunosuppressant reduces the formation of antibodies directed against the anti-TNF, thus avoiding the reduction or elimination of circulating drug levels, and in the combination of the therapeutic effect of two drugs. Nowadays, two different combination therapies should be distinguished. In the "de novo" combination therapy, the anti-TNF is used in combination with an immunosuppressant from the beginning of the treatment, in order to prevent the formation of anti-drug antibodies. In the "selective" combination therapy, the immunosuppressant is added at a later time in patients who experience a loss of response during anti-TNF monotherapy due to the development of anti-drug antibodies. The purpose of this review is to summarize the available evidence on both de novo and selective combination therapy. In addition, we will express our point of view on the choice between these two different treatment modalities.

HLA-DQA1*05 Carriage Associated With Development of Anti-Drug Antibodies to Infliximab and Adalimumab in Patients With Crohn’s Disease

Anti-tumor necrosis factor (anti-TNF) therapies are the most widely used biologic drugs for treating immune-mediated diseases, but repeated administration can induce the formation of anti-drug antibodies. The ability to identify patients at increased risk for development of anti-drug antibodies would facilitate selection of therapy and use of preventative strategies. We performed a genome-wide association study to identify variants associated with time to development of anti-drug antibodies in a discovery cohort of 1240 biologic-naïve patients with Crohn's disease starting infliximab or adalimumab therapy. Immunogenicity was defined as an anti-drug antibody titer ≥10 AU/mL using a drug-tolerant enzyme-linked immunosorbent assay. Significant association signals were confirmed in a replication cohort of 178 patients with inflammatory bowel disease. The HLA-DQA1*05 allele, carried by approximately 40% of Europeans, significantly increased the rate of immunogenicity (hazard ratio [HR], 1.90; 95% confidence interval [CI], 1.60-2.25; P= 5.88× 10-13). The highest rates of immunogenicity, 92% at 1 year, were observed in patients treated with infliximab monotherapy who carried HLA-DQA1*05; conversely the lowest rates of immunogenicity, 10% at 1 year, were observed in patients treated with adalimumab combination therapy who did not carry HLA-DQA1*05. We confirmed this finding in the replication cohort (HR, 2.00; 95% CI, 1.35-2.98; P= 6.60× 10-4). This association was consistent for patients treated with adalimumab (HR, 1.89; 95% CI, 1.32-2.70) or infliximab (HR, 1.92; 95% CI, 1.57-2.33), and for patients treated with anti-TNF therapy alone (HR, 1.75; 95% CI, 1.37-2.22) or in combination with animmunomodulator (HR, 2.01; 95% CI, 1.57-2.58). In an observational study, we found a genome-wide significant association between HLA-DQA1*05 and the development of antibodies against anti-TNF agents. A randomized controlled biomarker trial is required to determine whether pretreatment testing for HLA-DQA1*05 improves patient outcomes by helpingphysicians select anti-TNF and combination therapies. ClinicalTrials.gov ID: NCT03088449.

Crohn’s Disease With Progressive Renal Impairment

Question: The patient was diagnosed with Crohn’s disease of the colon at the age of 39 years and had a chronic active disease course with rapid response to infliximab. Biopsies from the colon showed extensive inflammation with granulomas. The patient had secondary loss of response to infliximab due to formation of antidrug antibodies (ADA). She did not tolerate azathioprine or 6-mercaptopurine owing to pancreatitis and had severe hair loss when treated with methotrexate. Owing to previous good response to anti-TNF treatment, the patient was given adalimumab and subsequently certolizumab pegol, both with good initial responses, but unfortunately with subsequent loss of response caused by ADA formation. The patient also had significant extraintestinal manifestations (iridocyclitis, sacroiliitis, and peripheral arthropathies) that tended to respond to biologics. At the age of 45, the patient was switched to golimumab with good symptomatic control of the disease and extraintestinal manifestations, but fluctuating C-reactive protein levels between 0.5 and 2.5 mg/dL. At the age of 48, her estimated glomerular filtration rate decreased to <60 mL/min with more rapid decreases in periods with elevated inflammatory markers (Figure A). Urinalysis revealed proteinuria and no occult blood. Urinary excretion of protein was between 0.2 and 0.6 g/d. An initial ultrasound examination of the kidneys and urinary tracts was normal and a Tc-99-MAG3 renography showed bilateral decreased kidney function. A kidney biopsy was performed (Figure B). What is the diagnosis and treatment? Look on page 59 for the answer and see the Gastroenterology website (www.gastrojournal.org) for more information on submitting your favorite image to Clinical Challenges and images in GI. The kidney biopsy showed normal glomeruli and well-preserved tubuli without atrophy. However, granulomatous inflammation was found in the interstitial tissue. No crystal casts, amyloid deposits, or vasculitis were found. The diagnosis is granulomatous interstitial nephritis. The patient was in her childhood treated for suspected tuberculosis, but polymerase chain reaction for tuberculosis was negative, as was immunohistochemistry for Epstein–Barr virus and cytomegalovirus. There was no sign of fungal infection. Other etiologies associated with granulomatous disease, especially sarcoidosis, were also ruled out. Granulomatous interstitial nephritis is generally a rare condition and is typically secondary to exposure to drugs including antibiotics and nonsteroidal anti-inflammatory drugs.1Shah S. Carter-Monroe N. Atta M.G. Granulomatous interstitial nephritis.Clin Kidney J. 2015; 8: 516-523Crossref PubMed Scopus (51) Google Scholar The patient had been sporadically exposed to ciprofloxacin. Both adalimumab and infliximab (but not golimumab or certolizumab) have been associated with autoimmune nephritis, but not specifically to granulomatous interstitial nephritis.2Piga M. Chessa E. Ibba V. et al.Biologics-induced autoimmune renal disorders in chronic inflammatory rheumatic diseases: systematic literature review and analysis of a monocentric cohort.Autoimmun Rev. 2014; 13: 873-879Crossref PubMed Scopus (61) Google Scholar In fact, anti-tumor necrosis factor agents have been used to alleviate granulomatous interstitial nephritis in patients with Crohn’s disease.3Timmermans S.A. Christiaans M.H. Abdul-Hamid M.A. et al.Granulomatous interstitial nephritis and Crohn's disease.Clin Kidney J. 2016; 9: 556-559Crossref PubMed Scopus (9) Google Scholar First-line treatment is glucocorticoids, and the patient was kept on golimumab and had 50 mg prednisolone. The treatment was ceased after 4 weeks owing to intolerance to side effects, and lack of improvements on estimated glomerular filtration rate levels. Ileocolonoscopy and gastroscopy were without signs of active inflammation. Renal function plateaued at around 37 mL/min for some months, but a subsequent decrease to <30 mL/min required reevaluation of the condition. Owing to the patient’s tendency to form granulomas, we considered the granulomatous interstitial nephritis to be an extraintestinal manifestation-like condition to Crohn’s disease. Alternatively, the granulomatous inflammation could be a direct drug reaction to golimumab exposure. Despite the lack of activity in the intestine, a switch of treatment was considered in the face of decreasing kidney function and ongoing systemic inflammation. Because a systemic effect of the new drug was considered important and the inflammation was not driven by intestinal activity, ustekinumab was preferred over vedolizumab. The change from golimumab to ustekinumab resulted in remarkably better control of systemic inflammation with continued undetectable C-reactive protein for the first time in the disease course and was accompanied by an almost complete regain of renal function within 8 months (Figure C). Interestingly, a correlation was found between C-reactive protein levels and the speed of renal function loss (ie, the derived function of estimated glomerular filtration rate; R2 = 0.42; P < .02), suggesting that the granulomatous inflammation in the kidneys were the main driver of the systemic inflammatory response during the observed period.

Bridging the Clinical Gap for DNA-Based Antibody Therapy Through Translational Studies in Sheep.

Clinical translation of DNA-based administration of monoclonal antibodies (mAbs) is uncertain due to lack of large animal data. To bridge the clinical gap, we evaluated a panel of novel plasmid DNA (pDNA)-encoded mAbs in 40-70 kg sheep with a clinical intramuscular electroporation protocol. Injection of 4.8 mg of pDNA, encoding ovine anti-human CEA mAb (OVAC), led to peak plasma mAb titers of 300 ng/mL. OVAC remained detectable for 3 months and was boosted by a second pOVAC administration. Hyaluronidase muscle pretreatment increased OVAC concentrations up to 10-fold. These higher plasma titers, however, led to anti-drug antibodies (ADAs) toward the OVAC variable regions, resulting in loss of mAb detection and of adequate redosing. Transient immune suppression avoided ADA formation, with OVAC peaking at 3.5 μg/mL and remaining detectable for 11 months after pOVAC injection. DNA-based delivery of ovine anti-human EGFR mAb (OVAE), identical to OVAC except for the variable regions, preceded by hyaluronidase, allowed for at least three consecutive administrations in an immune-competent sheep, without ADA response. When tripling the pOVAE dose to 15 mg, transient ADAs of limited impact were observed; plasma OVAE peaked at 2.6 μg/mL and was detected up to 7 months. DNA-based anti-HER2 trastuzumab in sheep gave no detectable mAb concentrations despite previous validation in mice, highlighting the limitations of relying on small-rodent data only. In conclusion, our results highlight the potential and caveats of clinical DNA-based antibody therapy, can expedite preclinical and clinical development, and benefit the field of gene transfer as a whole.

The effect of certolizumab drug concentration and anti-drug antibodies on TNF neutralisation

Tumour necrosis factor (TNF) inhibitors like certolizumab, elicit an immunogenic response leading to the formation of anti-drug antibodies (ADAs). We sought to mechanistically investigate the relationship between certolizumab concentrations, ADAs, and the effective TNF neutralising capacity in sera of rheumatoid arthritis (RA) patients. TNF neutralising capacity of certolizumab was compared to the neutralising capacity of adalimumab. Serum samples were collected from 40 consecutive certolizumab-treated RA patients at baseline and 4, 16, 28 and 52 weeks after treatment initiation [Dutch Trial Register NTR (Nederlands Trial Register) Trial NL2824 no. 2965]. Certolizumab concentration and ADA titre were measured with a certolizumab bridging enzyme-linked immunosorbent assay (ELISA) and a drug-tolerant radioimmunoassay (RIA), respectively. TNF neutralisation by certolizumab and adalimumab, in presence or absence of ADAs, was analysed with the TNF-sensitive WEHI bioassay. Despite a high incidence of ADAs during one year of follow-up (65%; 26/40 patients), certolizumab levels of >10 μg/ml were measured in most patients. The capacity for TNF neutralisation highly correlated with certolizumab serum concentration, whereas no association with ADAs was observed. Similar results were obtained for adalimumab. The relative in vitro neutralising potency was higher for certolizumab compared to adalimumab. Anti-certolizumab antibodies were detected in a large proportion of patients, but in most cases where ADAs were detected, certolizumab was also present in high concentrations, directly correlating with in vitro neutralising capacity. These results indicate that measurement of certolizumab drug levels, rather than ADAs, have direct clinical significance.

Infliximab–Tumor Necrosis Factor Complexes Elicit Formation of Anti-Drug Antibodies

Some patients develop anti-drug antibodies (ADAs), which reduce the efficacy of infliximab, a monoclonal antibody against tumor necrosis factor (TNF), in the treatment of immune-mediated diseases, including inflammatory bowel diseases. ADAs arise inconsistently, and it is not clear what factors determine their formation. We investigated features of the immune system, the infliximab antibody, and its complex with TNF that might contribute to ADA generation. C57BL/6 mice were given injections of infliximab and recombinant human TNF or infliximab F(ab')2 fragments. Blood samples were collected every 2-3 days for 2 weeks and weekly thereafter for up to 6 weeks; infliximab-TNF complexes and ADAs were measured by enzyme-linked immunosorbent assay (ELISA). Intestinal biopsy and blood samples were obtained from patients having endoscopy who had received infliximab therapy for inflammatory bowel diseases; infliximab-TNF complexes were measured with ELISA. Infliximab-specific plasma cells were detected in patient tissue samples by using mass cytometry. We studied activation of innate immune cells in peripheral blood mononuclear cells (PBMCs) from healthy donors incubated with infliximab or infliximab-TNF complexes; toll-like receptors (TLRs) were blocked with antibodies, endocytosis was blocked with the inhibitor PitStop2, and cytokine expression was measured by real-time polymerase chain reaction and ELISAs. Uptake of infliximab and infliximab-TNF complexes by THP-1 cells was measured with confocal microscopy. Mice given increasing doses of infliximab produced increasing levels of ADAs. Blood samples from mice given injections of human TNF and infliximab contained infliximab-TNF complexes; complex formation was associated with ADA formation with an area under the curve of 0.944 (95% confidence interval, 0.851-1.000; P= .003). Intestinal tissues from patients, but not blood samples, contained infliximab-TNF complexes and infliximab-specific plasma cells. Incubation of PBMCs with infliximab-TNF complexes resulted in a 4.74-fold increase in level of interleukin (IL) 1β (IL1B) messenger RNA (P for comparison= .005), increased IL1B protein secretion, and a 2.69-fold increase in the expression of TNF messenger RNA (P for comparison= 0.013) compared with control PBMCs. Infliximab reduced only IL1B and TNF expression. Antibodies against TLR2 or TLR4 did not block the increases in IL1B or TNF expression, but endocytosis was required. THP-1 cells endocytosed higher levels of infliximab-TNF complexes than infliximab alone. In mice, we found ADA formation to increase with dose of infliximab given and concentration of infliximab-TNF complexes detected in blood. Based on studies of human intestinal tissues and blood samples, we propose that infliximab-TNF complexes formed in the intestine are endocytosed by and activate innate immune cells, which increase expression of IL1B and TNF and production of antibodies against the drug complex. It is therefore important to optimize the infliximab dose to a level that is effective but does not activate an innate immune response against the drug-TNF complex.

Innovations in Oral Therapies for Inflammatory Bowel Disease.

Prior to the biologic era, the medical management of patients with inflammatory bowel disease (IBD) was dominated by the use of aminosalicylates, corticosteroids, and immunosuppressants. In the past two decades, the advent of biologic agents that target specific components of the immune response has greatly improved the care of patients with Crohn's disease and ulcerative colitis (UC). However, not all patients respond or maintain response to biologic therapy and some patients develop adverse events that necessitate treatment discontinuation. Furthermore, sensitization with formation of anti-drug antibodies is an inherent limitation to administration of monoclonal antibodies. This circumstance has generated renewed interest in the development of novel oral small-molecule drugs (SMDs) that are effective and well tolerated. Several classes of SMDs are currently progressing through the pipeline and offer the promise of oral delivery and high potency. In this review, we summarize different mechanisms of oral drug delivery to the gastrointestinal tract, highlight key findings from phase II and III randomized trials of novel oral SMDs, and discuss how oral SMDs are likely to be integrated into future IBD treatment paradigms. The most advanced development programs currently involve evaluation of compounds blocking Janus kinase (JAK) receptors or modulating sphingosine-1-phosphate (S1P) receptors. Tofacitinib, an oral JAK inhibitor, was recently approved for the treatment of moderate-to-severe UC. Several more selective JAK-1 inhibitors, including filgotinib and upadacitinib, have also shown positive results in phase II studies and are currently enrolling in phase III development programs. Similarly, ozanimod, an S1P1 and S1P5 receptor agonist, has shown early favorable results and is enrolling in phase III trials. As these and other novel oral SMDs come to market, several questions will need to be answered. The cost effectiveness, comparative treatment efficacy, predictors of response, and relative safety of oral SMDs compared to existing therapies will need to be evaluated. Given the modest efficacy rates observed with both biologic therapies and novel SMDs to date, the potential for combination therapy based on a non-sensitizing oral option is promising and may be facilitated by development of organ-specific therapies with pharmacodynamic activity restricted to the gut to minimize systemic toxicity.

Receptor occupancy measurement of anti-PD-1 antibody drugs in support of clinical trials.

Aim: The reliable measurement of receptor occupancy (RO) provides informative data for efficacy and safety evaluation. This study aimed to assess factors affecting RO measurement of anti-PD-1 antibodies in clinical studies. Materials & methods: RO performance was assessed using different T-cell activation markers measured by flow cytometry. The validated methodology was then used in support of a clinical study. Results: The optimized active cell population was comprised of CD45RO+ or CD45RA- T cells. The bioanalytical method was validated for inter- and intra-assay precision (coefficient of variation ≤30%) and sample storage stability for 3 days. Consistent RO saturation was observed in Phase Ia clinical trial, although receptor regulation appeared to be different. The formation of anti-drug antibodies had markedly influenced pharmacokinetics and RO. Conclusion: RO measurement in combination with pharmacokinetics and anti-drug antibodies data could allow the integrated evaluation and better understanding of efficacy and safety.

Biologics in the treatment of juvenile idiopathic arthritis : A comparison of mono- and combination therapy with synthetic DMARDs

In polyarticular juvenile idiopathic arthritis, methotrexate (MTX) is still used as first-line treatment. In case of insufficient response or intolerance, anumber of biologics are now available. This faces physicians with challenging choices. Biologics are often combined with MTX, although in JIA there is little evidence and inconsistent results from various studies. In rheumatoid arthritis, combination therapy with tumor necrosis factor (TNF) inhibitors has been associated with higher efficacy. Tocilizumab appears to be highly effective as amonotherapy. MTX has aprotective effect on the formation of anti-drug antibodies, which is particularly important for the use of anti-TNF antibodies. This could also be observed in children. For golimumab, combination with MTX is mandatory according to its approval, as is the cause for abatacept. With regard to tolerability, apart from the classic side effects of MTX, there are no other significant differences concerning the combination of MTX and biologics. In case of MTX intolerance, leflunomide may be considered as an (unapproved) alternative.

BAY 1213790, a fully human IgG1 antibody targeting coagulation factor XIa: First evaluation of safety, pharmacodynamics, and pharmacokinetics

BackgroundCoagulation factor XI (FXI) contributes to the development of thrombosis but appears to play only a minor role in hemostasis and is therefore an attractive anticoagulant drug target. ObjectivesTo evaluate the safety, pharmacodynamic, and pharmacokinetic properties of BAY 1213790, a fully human immunoglobulin (Ig) G1 antibody targeting activated coagulation FXI (FXIa), in healthy men. MethodsIn this phase 1, single‐blind, parallel‐group, placebo‐controlled, dose‐escalation study, 83 healthy Caucasian men were randomized 4:1 to receive a single 60‐minute intravenous infusion of BAY 1213790 (0.015‐10 mg/kg) or placebo. Adverse events, pharmacodynamic parameters (including activated partial thromboplastin time [aPTT]) and pharmacokinetic parameters were determined. Volunteers were followed up for 150 days. ResultsBAY 1213790 demonstrated favorable safety and tolerability; there were no observed cases of bleeding or clinically relevant antidrug antibody formation. One volunteer (1.2%) experienced an infusion reaction. Following intravenous administration of BAY 1213790, dose‐dependent increases in aPTT (maximal mean increase relative to baseline: 1.85 [conventional method] and 2.17 [kaolin‐triggered method]) and rotational thromboelastometry whole blood clotting time were observed, as well as dose‐dependent reductions in FXI activity. Bleeding times did not increase following administration of BAY 1213790 and were similar for all dose cohorts, including placebo. Measurable and dose‐dependent increases in systemic exposure were detected for all doses of BAY 1213790 of 0.06 mg/kg or higher. ConclusionsBased on these safety, pharmacodynamic, and pharmacokinetic results, further evaluation of BAY 1213790 in patients with, or at risk of, thrombosis is warranted.

Recombinant human granulocyte- colony stimulating factor in women with unexplained recurrent pregnancy losses: a randomized clinical trial.

STUDY QUESTIONDoes administration of recombinant human granulocyte colony stimulating factor (rhG-CSF) in the first trimester improve pregnancy outcomes, among women with a history of unexplained recurrent pregnancy loss?SUMMARY ANSWERrhG-CSF administered in the first trimester of pregnancy did not improve outcomes among women with a history of unexplained recurrent pregnancy loss.WHAT IS KNOWN ALREADYThe only previous randomized controlled study of granulocyte colony stimulating factor in recurrent miscarriage in 68 women with unexplained primary recurrent miscarriage found a statistically significant reduction in miscarriage and improvement in live birth rates. A further four observational studies where G-CSF was used in a recurrent miscarriage population were identified in the literature, two of which confirmed statistically significant increase in clinical pregnancy and live birth rates.STUDY DESIGN, SIZE, DURATIONA randomized, double-blind, placebo controlled clinical trial involving 150 women with a history of unexplained recurrent pregnancy loss was conducted at 21 sites with established recurrent miscarriage clinics in the United Kingdom between 23 June 2014 and 05 June 2016. The study was coordinated by University of Birmingham, UK.PARTICIPANTS/MATERIALS, SETTING, METHODSOne hundred and fifty women with a history of unexplained recurrent pregnancy loss: 76 were randomized to rhG-CSF and 74 to placebo. Daily subcutaneous injections of recombinant human granulocyte – colony stimulating factor 130 μg or identical appearing placebo from as early as three to five weeks of gestation for a maximum of 9 weeks. The trial used central randomization with allocation concealment. The primary outcome was clinical pregnancy at 20 weeks of gestation, as demonstrated by an ultrasound scan. Secondary outcomes included miscarriages, livebirth, adverse events, stillbirth, neonatal birth weight, changes in clinical laboratory variables following study drug exposure, major congenital anomalies, preterm births and incidence of anti-drug antibody formation. Analysis was by intention to treat.MAIN RESULTS AND THE ROLE OF CHANCEA total of 340 participants were screened for eligibility of which 150 women were randomized. 76 women (median age, 32[IQR, 29–34] years; mean BMI, 26.3[SD, 4.2]) and 74 women (median age, 31[IQR, 26–33] years; mean BMI, 25.8[SD, 4.2]) were randomized to placebo. All women were followed-up to primary outcome, and beyond to live birth. The clinical pregnancy rate at 20 weeks, as well as the live birth rate, was 59.2% (45/76) in the rhG-CSF group, and 64.9% (48/74) in the placebo group, giving a relative risk of 0.9 (95% CI: 0.7–1.2; P = 0.48). There was no evidence of a significant difference between the groups for any of the secondary outcomes. Adverse events (AEs) occurred in 52 (68.4%) participants in rhG-CSF group and 43 (58.1%) participants in the placebo group. Neonatal congenital anomalies were observed in 1/46 (2.1%) of babies in the rhG-CSF group versus 1/49 (2.0%) in the placebo group (RR of 0.9; 95% CI: 0.1–13.4; P = 0.93).LIMITATIONS, REASONS FOR CAUTIONThis trial was conducted in women diagnosed with unexplained recurrent pregnancy loss and therefore no screening tests (commercially available) were performed for immune dysfunction related pregnancy failure/s.WIDER IMPLICATIONS OF THE FINDINGSTo our knowledge, this is the first multicentre study and largest randomized clinical trial to investigate the efficacy and safety of granulocyte human colony stimulating factor in women with recurrent miscarriages. Unlike the only available single center RCT, our trial showed no significant increase in clinical pregnancy or live births with the use of rhG-CSF in the first trimester of pregnancy.STUDY FUNDING/COMPETING INTEREST(S)This study was sponsored and supported by Nora Therapeutics, Inc., 530 Lytton Avenue, 2nd Floor, Palo Alto, CA 94301, USA. Darryl Carter was the co-founder and VP of research, Nora Therapeutics, Inc. and held shares in the company. He holds a patent for the use of recombinant human granulocyte colony stimulating factor to reduce unexplained recurrent pregnancy loss. Mark Joing, Paul Kwon and Jeff Tong were or are employees of Nora Therapeutics, Inc. No other potential conflict of interest relevant to this article was reported.TRIAL REGISTRATION NUMBEREUDRACT No: 2014-000084-40; ClinicalTrials.gov Identifier: NCT02156063TRIAL REGISTRATION DATE31 Mar 2014DATE OF FIRST PATIENT’S ENROLMENT23 Jun 2014