Production Of Anti-drug Antibodies Research Articles

Abstract 7174: Pharmacokinetics and immunogenicity of anti-PD-1 antibody in humanized FcRn mouse models compared to immunocompetent mouse models

Abstract Introduction: The development and use of therapeutic antibodies in cancer therapy has increased significantly in recent years, led by the success of approved immune checkpoint blockade antibodies targeting PD-1 and PD-L1 axis. However, obtaining more clinically relevant pharmacokinetics (PK), safety and efficacy data for antibodies is still challenging in preclinical phase. Humanized FcRn (hFcRn) transgenic mouse model has a more faithful catabolism compared to WT mice, providing more accurate and predictable PK data of innovative antibody-based therapeutics at discovery stage[1]. In this study, we sought to investigate pharmacokinetics and immunogenicity characteristics of anti-PD-1 antibody in hFcRn transgenic mice and provide guidance for its differentiation of prediction value for drug PK and safety by comparing to WT mice. Methods: Non-tumor bearing hFcRn mice and wild type C57BL/6 mice were divided into 2 groups, 5 mice were enrolled into each group. Mice were administered anti-PD-1(Nivolumab) at 10 mg/kg single dose after grouping by intravenous (IV) injection. Blood samples at 12 timepoints (15min, 6h, 1, 2, 4, 7, 10, 14, 21, 28, 35, 42 days) post-dosing were collected for PK and Anti-Drug Antibody (ADA) analysis by MSD and ELISA. Results: Nivolumab clearance in hFcRn mice followed a typical linear profile with rapid distribution phase and slow elimination phase (t1/2 = 350 to 400 h). While the PK profile in C57BL/6 mice displayed non-linear clearance and very short elimination phase (t1/2 = 45 to 50 h). This data demonstrated that hFcRn mice have better correlated half-life with human (Nivolumab single dose by IV with 10mg/kg, t1/2 = 595 h) [2]. ADA analysis revealed that it was produced starting from Day 10 in wild type C57BL/6 mice and the frequency of ADA induced by Nivolumab in wild type mice (2/5) was higher than hFcRn mice (0/5). The variation of Nivolumab level was correlated with ADA production in wild type mice. This suggested hFcRn shows lower immunogenicity than wild type mice for human IgG drug and can potentially improve negative effect of ADA interference on PK evaluation. Conclusion: The result of this study demonstrated that the PK data in hFcRn mouse model was more predictive of human PK profile compared to wild type C57BL/6 mice. This advances innovation in antibody-based therapeutics and translational applications, with improved PK, safety and efficacy evaluation for preclinical human IgG drug development in cancer therapy.

Experimental Validation a Method for Assessing Neutralizing Antibodies of Romiplostim in Human Plasma

Introduction. Romiplostim is an analogue of the fusion protein peptide of thrombopoietin (TPO), which increases platelet count by binding and activating the human thrombopoietin receptor (TPO-R). It is used to treat thrombocytopenia associated with chronic immune thrombocytopenia. For romiplostim, one of the possible adverse reactions from the immune system is immunogenicity: the production of anti-drug antibodies to the medicinal product, including neutralising antibodies, which may affect the efficacy and safety profile of the medicinal product.Aim. Validate the procedure for determining neutralising antibodies to romiplostim in human plasma for further clinical studies of immunogenicity.Materials and methods. The study used rabbit polyclonal antibodies to romiplostim, Nplate® produced by Amgen Europe as a standard sample; a placebo produced by LLC "GEROPHARM", a cell line 32D-hTPOR clone 63 with stable expression of human TPO receptor and a chemiluminescence assay kit CellTiter-Glo® Luminescent Cell Viability Assay produced by Promega to assess specificity. The experiment was carried out on cell line 32D-hTPOR clone 63, which was seeded on the first day and the neutralizing antibody concentrations were titrated with a constant concentration of romiplostim, then the chemiluminescence was detected on the second day. Statistical processing of the results was carried out using Prism 9 software.Result and discussion. The specificity of the procedure was demonstrated; at maximum concentration, the medicinal product differs from placebo by 309 times in the residual level of cell viability. The linearity of the procedure in terms of the coefficient determination is 0.9969. The precision of the procedure was determined: the repeatability was 1–9 %, the intermediate precision was 3–18 %. The coefficient of variation in selectivity of the procedure was 22 %. For the accuracy parameter, the values for recovery/spike were determined as 90–101 %. It was proven that there was no matrix effect.Conclusion. It can be stated that the procedure is linear, specific, highly precise, correct, selective and with a proven absence of matrix effect, which allows it to be used to determine the immunogenicity of romiplostim medicinal products in clinical studies.

Treatment-emergent antidrug antibodies related to PD-1, PD-L1, or CTLA-4 inhibitors across tumor types: a systematic review

BackgroundIncreased understanding of how the immune system regulates tumor growth has innovated the use of immunotherapeutics to treat various cancers. The impact of such therapies, including programmed cell death protein...

Synthetically mannosylated antigens induce antigen-specific humoral tolerance and reduce anti-drug antibody responses to immunogenic biologics

Immunogenic biologics trigger an anti-drug antibody (ADA) response in patients that reduces efficacy and increases adverse reactions. Our laboratory has shown that targeting protein antigen to the liver microenvironment can reduce antigen-specific Tcell responses; herein, we present a strategy to increase delivery of otherwise immunogenic biologics to the liver via conjugation to a synthetic mannose polymer, p(Man). This delivery leads to reduced antigen-specific T follicular helper cell and B cell responses resulting in diminished ADA production, which is maintained throughout subsequent administrations of the native biologic. We find that p(Man)-antigen treatment impairs the ADA response against recombinant uricase, a highly immunogenic biologic, without a dependence on hapten immunodominance or control by T regulatory cells. We identify increased Tcell receptor signaling and increased apoptosis and exhaustion in Tcells as effects of p(Man)-antigen treatment via transcriptomic analyses. This modular platform may enhance tolerance to biologics, enabling long-term solutions for an ever-increasing healthcare problem.

Characterization of anti-drug antibody dynamics using a bivariate mixed hidden-markov model by nonlinear-mixed effects approach

Biological therapies may act as immunogenic triggers leading to the formation of anti-drug antibodies (ADAs). Population pharmacokinetic (PK) models can be used to characterize the relationship between ADA and drug disposition but often rely on the ADA bioassay results, which may not be sufficiently sensitive to inform on this characterization.In this work, a methodology that could help to further elucidate the underlying ADA production and impact on the drug disposition was explored. A mixed hidden-Markov model (MHMM) was developed to characterize the underlying (hidden) formation of ADA against the biologic, using certolizumab pegol (CZP), as a test drug. CZP is a PEGylated Fc free TNF-inhibitor used in the treatment of rheumatoid arthritis and other chronic inflammatory diseases.The bivariate MHMM used information from plasma drug concentrations and ADA measurements, from six clinical studies (n = 845), that were correlated through a bivariate Gaussian function to infer about two hidden states; production and no-production of ADA influencing PK. Estimation of inter-individual variability was not supported in this case. Parameters associated with the observed part of the model were reasonably well estimated while parameters associated with the hidden part were less precise. Individual state sequences obtained using a Viterbi algorithm suggested that the model was able to determine the start of ADA production for each individual, being a more assay-independent methodology than traditional population PK. The model serves as a basis for identification of covariates influencing the ADA formation, and thus has the potential to identify aspects that minimize its impact on PK and/or efficacy.

MIgM-mediated splenic marginal zone B cells targeting of folic acid for immunological evasion

Folic acid is a fully oxidized synthetic folate with high bioavailability and stability which has been extensively prescribed to prevent congenital disabilities. Here we revealed the immunosuppressive effect of folic acid by targeting splenic marginal zone B (MZB) cells. Folic acid demonstrates avid binding with the Fc domain of immunoglobulin M (IgM), targeting IgM positive MZB cells in vivo to destabilize IgM-B cell receptor (BCR) complex and block immune responses. The induced anergy of MZB cells by folic acid provides an immunological escaping window for antigens. Covalent conjugation of folic acid with therapeutic proteins and antibodies induces immunological evasion to mitigate the production of anti-drug antibodies, which is a major obstacle to the long-term treatment of biologics by reducing curative effects and/or causing adverse reactions. Folic acid acts as a safe and effective immunosuppressant via IgM-mediated MZB cells targeting to boost the clinical outcomes of biologics by inhibiting the production of anti-drug antibodies, and also holds the potential to treat other indications that adverse immune responses need to be transiently shut off.

Efficacy of Combined Initial Treatment of Methotrexate with Infliximab in Pediatric Crohn's Disease: A Pilot Study.

The combination of antitumor necrosis factor-alpha (TNF-α) agents with immunomodulators (IMMs) is a common treatment for pediatric Crohn's disease (CD). Although methotrexate (MTX) can be a first-line medication as an IMM, most clinicians in real-life practice, especially in South Korea, are more familiar with thiopurines. This study aimed to compare the efficacy and immunogenicity of MTX and azathioprine (AZA) as concurrent therapies for pediatric CD. In this pilot study, 29 newly diagnosed pediatric patients with moderate-to-severe CD were randomized to receive either MTX (n = 15) (15 mg/body surface area (BSA) per week) or oral AZA (n = 14) (0.5 mg/kg per day) in combination with Infliximab (IFX). The primary outcomes were the proportion of patients in endoscopic, biochemical, and transmural remission after 14 and 54 weeks of IFX therapy. The trough levels (TLs) of IFX and anti-drug antibody (ADA) levels were also compared. Among the 29 patients, there were no significant differences in the biochemical (p = 1.0 at week 14, p = 0.45 at week 54), endoscopic (p = 0.968 at week 14, p = 0.05 at week 54), or transmural (p = 0.103 at week 54) remission rates between the two medications during the concurrent therapy. Additionally, the trends in the IFX trough and ADA levels over time during the treatments were similar for both medications, with no significant differences (p = 0.686, p = 0.389, respectively). The MTX showed comparable efficacy to the AZA in pediatric CD patients with moderate-to-severe disease. This effectively maintained adequate IFX levels and reduced ADA production. Therefore, although additional large-scale clinical trials are needed, this study demonstrated that either MTX or AZA can be selected as IMMs in the concurrent treatment of pediatric CD, depending on individual medical institutions' circumstances.

Mitigation of Anti-Drug Antibody Production for Augmenting Anticancer Efficacy of Therapeutic Protein via Co-Injection of Nano-Rapamycin.

The induction of anti-drug antibody (ADA) is a formidable challenge for protein-based therapy. Trichosanthin (TCS) as a class of ribosome-inactivating proteins is widely studied in tumor treatment. However, the immunogenicity can induce the formation of ADA, which can cause hypersensitivity reactions and neutralize the efficacy of TCS, thus limiting its clinical application in cancer therapy. Here, a promising solution to this issueis presented by co-administration of the rapamycin nanoparticles and TCS. PEGylated rapamycin amphiphilic moleculeis designed and synthesized as a prodrug and a delivery carrier, which can self-assemble into a nanoparticle system with encapsulation of free rapamycin, a hydrophobic drug. Itis found that co-injection of the PEGylated rapamycin nanoparticles and TCS could mitigate the formation of anti-TCS antibody via inducing durable immunological tolerance. Importantly, the combination of TCS and the rapamycin nanoparticleshas an enhanced effect on inhibit the growth of breast cancer. This work provides a promising approach for protein toxin-based anticancer therapy and for promoting the clinical translation.

Quantification of anti-drug antibodies against E6011, an anti-fractalkine monoclonal antibody, in monkey and human serum, by an electrochemiluminescence assay

E6011, a humanized anti-fractalkine monoclonal antibody, is under development for the treatment of various inflammatory diseases, such as rheumatoid arthritis. Therapeutic antibodies may induce production of anti-drug antibodies (ADA) that may deteriorate efficacy and/or enhance immunogenic reaction. It is important to have an ADA assay to understand the characteristics of biotherapeutics under development. A simple and reproducible assay has thus been developed for the determination of ADA against E6011 in monkey and human serum by electrochemiluminescence (ECL) detection. An immune-complex of biotinylated E6011, ADA, and ruthenium-labeled E6011 was attached to avidin-coated wells for ECL signal detection. Screening and confirmatory cutpoints were determined to judge negative or positive ADA. Sensitivity of ADA was 1.61 and 1.34 ng/mL in monkey and human serum, respectively. Accuracy and precision of the assay were within ±20% and 20%, respectively. Drug tolerance of the assay in monkey and human sera was ensured up to 100 and 1000 μg/mL E6011 at the surrogate ADA levels of 1 and 4 μg/mL, respectively. The developed assay was successfully applied to ADA quantification in monkeys and humans in support of immunogenicity assessments.

The immunogenicity of human-origin therapeutic antibodies are associated with V gene usage.

Therapeutic antibodies can elicit unwanted immune responses in a subset of patients, which leads to the production of anti-drug antibodies (ADA). Some of these ADAs have been reported to effect the pharmacokinetics, efficacy and/or safety of the therapeutic antibodies. The sequence diversity of antibodies are generated by VDJ recombination and mutagenesis. While the antibody generation process can create a large candidate pool for identifying high-affinity antibodies, it also could produce sequences that are foreign to the human immune system. However, it is not clear how VDJ recombination and mutagenesis impact the clinical ADA rate of therapeutic antibodies. In this study, we identified a positive correlation between the clinical ADA rate and the number of introduced mutations in the antibody sequences. We also found that the use of rare V alleles in human-origin antibody therapeutics is associated with higher risk of immunogenicity. The results suggest that antibody engineering projects should start with frameworks that contain commonly used V alleles and prioritize antibody candidates with low number of mutations to reduce the risk of immunogenicity.

Reduced Graphene Oxide Electrolyte-Gated Transistor Immunosensor with Highly Selective Multiparametric Detection of Anti-Drug Antibodies.

The advent of immunotherapies with biological drugs has revolutionized the treatment of cancers and auto-immune diseases. However, in some patients the production of anti-drug antibodies (ADAs) hampers the drug efficacy. The concentration of ADAs is typically in the range of 1-10 pM, hence their immunodetection is challenging. We focus here on ADAs towards Infliximab (IFX), a drug used to treat rheumatoid arthritis and other auto-immune diseases. We report an ambipolar electrolyte-gated transistor (EGT) immunosensor based on reduced graphene oxide (rGO) channel and IFX bound to the gate electrode as the specific probe. The rGO-EGTs are easy to fabricate, exhibit low voltage operations (≤ 0.3V), a robust response within 15min, and ultra-high sensitivity (10 aM limit of detection). We propose a multiparametric analysis of the whole rGO-EGT transfer curves based on the type-I generalized extreme value distribution. We demonstrate that it allows us to selectively quantify ADAs also in the co-presence of its antagonist tumour necrosis factor alpha (TNF-α), the natural circulating target of IFX. This article is protected by copyright. All rights reserved.

Transcutaneous auricular vagus nerve stimulation may elicit anti-inflammatory actions through activation of the hypothalamic-pituitary-adrenal axis in humans

Introduction: Since the discovery of the cholinergic anti-inflammatory pathway, vagus nerve stimulation (VNS) has been suggested as a treatment option for chronic inflammatory conditions. This pathway relies on efferent vagal nerve fibers. Invasive cervical VNS activates afferent and efferent vagal nerve fibers and, thus, has the potential to directly activate the cholinergic anti-inflammatory pathway. In contrast, transcutaneous auricular VNS (taVNS) activates the auricular branch of the vagus nerve that is purely afferent and projects to the nucleus of the solitary tract. Thus, any anti-inflammatory actions of taVNS are unlikely to result from a direct activation of the cholinergic anti-inflammatory pathway. The objective of this study was to investigate alternative mechanisms by which taVNS may elicit anti-inflammatory effects. The hypothesis was tested that taVNS activates the hypothalamic-pituitary-adrenal (HPA) axis, resulting in adrenal cortisol secretion that will then modulate circulatory immune cell numbers. Methods: The study was approved by the appropriate authorities (IRB# 0054_2019 and NCT04177264). Study participants were healthy adults (n=19, 6 male, 13 female). On three consecutive days either taVNS (left ear, 10 Hz, 1-2 mA, 300 μs pulse width) or sham-taVNS (stimulator off) was applied for 10 minutes. A blood sample was drawn at the end of the third day. Plasma cortisol was determined by ELISA (K003-H1W, Arbor Assays, MI). Circulating T-helper cells (CD3+, CD4+), cytotoxic T-cells (CD3+, CD8+), B-cells (CD19+), monocytes (CD14+, CD11b+), and natural killer (NK, CD3-, CD56+) cells were counted using flow cytometry. Statistical significance was assumed at P<0.05 and trends are reported for P<0.15 based on unpaired t-tests. Results: Plasma cortisol levels tended to be higher following taVNS (72.5±9.5 ng/mL) compared to sham-taVNS (52.3±3.8 ng/mL, P=0.09). Following taVNS, the relative number of circulatory B cells was lower (0.48±0.11 % vs. 0.82±0.11 %, P<0.05), while the number of circulatory monocytes was higher (2.21±0.32 % vs 1.58±0.16 %, P=0.06) compared to sham-taVNS. T-helper cells, cytotoxic T cells, and NK cells were not significantly affected by taVNS. In conclusion, the higher plasma cortisol levels following taVNS together with the higher number of circulatory monocytes and lower number of B cells suggest that taVNS elicits anti-inflammatory actions through activation of the HPA axis, because it is known that cortisol increases circulatory monocytes and reduces B cells. The decrease in B cells following taVNS gives rise to the intriguing hypothesis that taVNS may prevent secondary treatment failure to biologic drugs in chronic inflammatory conditions by reducing production of anti-drug antibodies. Funding: This study was supported by a grant from the American Osteopathic Association (Grant No.: 19137759). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Synthetically mannosylated antigens impair the antigen-specific humoral immune response and prolong the therapeutic efficacy of immunogenic protein drugs

Abstract Immunogenic protein drugs trigger an anti-drug antibody (ADA) response in patients which reduces drug efficacy and increases adverse reactions. Our lab has previously shown that targeting antigen to the liver microenvironment can reduce antigen-specific T cell responses; herein, we present a novel strategy to further increase delivery of an antigen to the tolerogenic microenvironment of the liver via conjugation to a synthetic mannose polymer (p(Man)). This delivery leads to reduced antigen-specific T follicular helper cell and B cell responses resulting in diminished ADA production which is maintained throughout subsequent administrations of the native biologic. We found that p(Man) treatment impairs the antibody response against recombinant uricase, a highly immunogenic biologic, without a dependence on hapten-skewing or control by Tregs. Through transcriptomic analysis we identified increased TCR signaling and increased apoptosis and exhaustion in T cells as effects of p(Man)-antigen treatment, which may inform the underlying mechanism driving the reduction in ADAs. This modular platform may enhance tolerance to current and future biologics, enabling long-term therapeutic solutions for an ever-increasing healthcare problem.

The establishment of B cell-deficient Igh-J KO mouse model by gene editing and efficacy evaluation

Over the last few years, immunotherapy has made significant progress in treating various cancers with therapeutic antibodies. However, therapeutic antibodies have been validated for inducing an unintended immune response in human and animal models, which leads to the emergence of anti-drug antibodies (ADAs) and affects their effectiveness and safety. In preclinical research, ADAs production by B cells may accelerate antibody metabolism and result in missing potential candidate molecules. Thus, it is urgent to develop preclinical models that remove only B cells without affecting the function of T and NK cells. Rearrangement of immunoglobulin heavy chain J gene fragment (Igh-J) is the first link in B cell development, and immunotherapies are currently leaning toward combination treatments with PD-1/PD-L1 antibodies, here we created humanized PD-1, PD-L1 and Igh-J knockout (hPD-1/hPD-L1, Igh-J KO) mice and validated by using the reported high immunogenicity drug M7824 (a protein designed to simultaneously block PD-L1 and TGF-β pathways, poorly anti-tumor efficacy in immunocompetent mice). Phenotypic analysis revealed that human PD-1 and PD-L1 were detectable in hPD-1/hPD-L1, Igh-J KO mice, but not mouse IgM and IgD. Igh-J KO depleted B cells while increased the percentage of other immune cell types. Meanwhile, the humanization of PD-1/PD-L1 and Igh-J KO had neither effect on the overall development, differentiation, or distribution of T cell subtypes, nor on the activation of NK and T cells, indicating that mice can be used for T and NK-related immunotherapies. Furthermore, M7824 treatment of these B cell-deficient mice inhibited tumor growth significantly, with higher M7824 analog concentrations and lower ADA-positive rates. These findings demonstrate that Igh-J KO mice are an effective and stable preclinical model for testing drugs based on T and NK cells with high immunogenicity in vivo.

Validation of a Dendritic Cell and CD4+ T Cell Restimulation Assay Contributing to the Immunogenicity Risk Evaluation of Biotherapeutics.

Immunogenicity, defined as the ability to provoke an immune response, can be either wanted (i.e., vaccines) or unwanted. The latter refers to an immune response to protein or peptide therapeutics, characterized by the production of anti-drug antibodies, which may affect the efficacy and/or the safety profiles of these drugs. Consequently, evaluation of the risk of immunogenicity early in the development of biotherapeutics is of critical importance for defining their efficacy and safety profiles. Here, we describe and validate a fit-for-purpose FluoroSpot-based in vitro assay for the evaluation of drug-specific T cell responses. A panel of 24 biotherapeutics with a wide range of clinical anti-drug antibody response rates were tested in this assay. We demonstrated that using suitable cutoffs and donor cohort sizes, this assay could identify most of the compounds with high clinical immunogenicity rates (71% and 78% for sensitivity and specificity, respectively) while we characterized the main sources of assay variability. Overall, these data indicate that the dendritic cell and CD4+ T cell restimulation assay published herein could be a valuable tool to assess the risk of drug-specific T cell responses and contribute to the selection of clinical candidates in early development.

HK is the apple of FXI's eye

The pairing of coagulation factors containing a gamma‐carboxyglutamate (Gla) domain on a phosphatidylserine‐rich surface in the presence of divalent cations, such as calcium, enhances the rate of conversion of a zymogen to an active species by 1 to 2 million fold. This principle underpins the use of warfarin to prevent the vitamin K dependent posttranslational conversion of specific Glu to Gla residues in coagulation factors.1.de Lara D.V. de Melo D.O. Araujo Silva L.C. Goncalves T.S. PC J.L.S. Pharmacogenetics of clopidogrel and warfarin in the treatment of cardiovascular diseases: an overview of reviews.Pharmacogenomics. 2022; 23: 443-452Crossref PubMed Scopus (1) Google Scholar Yet, one coagulation protease, factor XI (FXI), does not contain a Gla domain and escapes regulation by warfarin. Rather, FXI comprises a trypsin‐like catalytic domain and four 90 to 91 amino acid repeats termed apple domains because of their resemblance to the forbidden fruit. The apple domain structure is shared only with its monomeric homolog prekallikrein2.Visser M. Heitmeier S. Ten Cate H. Spronk H.M.H. Role of factor XIa and plasma kallikrein in arterial and venous thrombosis.Thromb Haemost. 2020; 120: 883-993Crossref PubMed Scopus (15) Google Scholar and has formed a shroud of mystery over the mechanisms by which FXI binds to cells and partners with other proteins of the coagulation and plasma kallikrein‐kinin systems. As FXI has enjoyed a renewed interest as a druggable target for thrombosis and other inflammatory disorders,3.Srivastava P. Gailani D. The rebirth of the contact pathway: a new therapeutic target.Curr Opin Hematol. 2020; 27: 311-319Crossref PubMed Scopus (13) Google Scholar this curtain is being slowed pulled back. Thrombotic diseases present a challenge to the field of medicine as therapeutics need to maintain the hemostatic balance between clotting and anticoagulation. Proteins of the coagulation cascade have been targeted for antithrombotic therapeutics to treat deep vein thrombosis, acute myocardial infarction, and ischemic stroke. Inhibition of the activation or activity of coagulation factors, including FX and thrombin, successfully prevents thrombosis yet come with a risk for bleeding, which limits their clinical utility. FXI plays the role of bad apple in thromboinflammatory diseases including sepsis and atherosclerosis,4.Ngo A.T.P. Jordan K.R. Mueller P.A. et al.Pharmacological targeting of coagulation factor XI mitigates the development of experimental atherosclerosis in low‐density lipoprotein receptor‐deficient mice.J Thromb Haemost. 2021; 19: 1001-1017Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 5.Silasi R. Keshari R.S. Lupu C. et al.Inhibition of contact‐mediated activation of factor XI protects baboons against S aureus‐induced organ damage and death.Blood Adv. 2019; 3: 658-669Crossref PubMed Scopus (37) Google Scholar while playing a minor role in hemostasis, making it a promising therapeutic target.6.Al‐Horani R.A. Factor XI(a) inhibitors for thrombosis: an updated patent review (2016‐present).Expert Opin Ther Pat. 2020; 30: 39-55Crossref PubMed Scopus (23) Google Scholar As such, a pharmacopoeia of FXI inhibitors have been developed, including small molecule active site inhibitors,7.Pollack Jr, C.V. Kurz M.A. Hayward N.J. EP‐7041, a factor XIa inhibitor as a potential antithrombotic strategy in extracorporeal membrane oxygenation: a brief report.Crit Care Explor. 2020; 2Crossref PubMed Google Scholar antisense oligonucleotides,8.Younis H.S. Crosby J. Huh J.I. et al.Antisense inhibition of coagulation factor XI prolongs APTT without increased bleeding risk in cynomolgus monkeys.Blood. 2012; 119: 2401-2408Crossref PubMed Scopus (69) Google Scholar and monoclonal antibodies.9.Koch A.W. Schiering N. Melkko S. et al.MAA868, a novel FXI antibody with a unique binding mode, shows durable effects on markers of anticoagulation in humans.Blood. 2019; 133: 1507-1516Crossref PubMed Scopus (53) Google Scholar An additional therapeutic tool being explored here is the development of nanobodies. Nanobodies are derived from naturally occurring heavy‐chain‐only antibodies that contain a single variable domain.10.Jovcevska I. Muyldermans S. The therapeutic potential of nanobodies.BioDrugs. 2020; 34: 11-26Crossref PubMed Scopus (265) Google Scholar They are versatile tools for basic research, disease diagnosis, molecular imaging, drug delivery, and therapy.11.Wang Y. Fan Z. Shao L. et al.Nanobody‐derived nanobiotechnology tool kits for diverse biomedical and biotechnology applications.Int J Nanomedicine. 2016; 11: 3287-3303Crossref PubMed Scopus (84) Google Scholar Traditional antibody therapeutics are widely used but have many limitations because of their large size and poor penetration of tissue.12.Chames P. Van Regenmortel M. Weiss E. Baty D. Therapeutic antibodies: successes, limitations and hopes for the future.Br J Pharmacol. 2009; 157: 220-233Crossref PubMed Scopus (917) Google Scholar Repeated administration of antibody therapeutics can cause adverse immune responses through antidrug antibody production. This has the potential to diminish or totally cancel the antibody therapeutic effect.13.van Brummelen E.M. Ros W. Wolbink G. Beijnen J.H. Schellens J.H. Antidrug antibody formation in oncology: clinical relevance and challenges.Oncologist. 2016; 21: 1260-1268Crossref PubMed Scopus (62) Google Scholar The small size increased stability, and pharmacokinetic properties of nanobodies make them a promising therapeutic tool. In this issue of Journal of Thrombosis and Haemostasis, Bar Barroeta et al. identify five nanobodies that inhibit FIX activation through binding to the apple 3 domain of FXI. A new interaction between FXI and high molecular weight kininogen (HK) was discovered through characterization of a sixth nanobody that competed with FXI‐HK binding to indirectly inhibit FIX activation.14.Bar Barroeta A. Marquart J.A. Bakhtiari K. Meijer A.B. Urbanus R.T. Meijers J.C.M. Nanobodies against factor XI apple 3 domain inhibit binding of factor IX and reveal a novel binding site for high molecular weight kininogen.J Thromb Haemost. 2022; Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar Bar Barroeta et al. developed FXI nanobodies through subcutaneous injection of recombinant FXI and FXIa into two llamas. The nanobodies were isolated and screened for FXI binding. As FIX binds the apple 3 domain of FXI,15.Sun Y. Gailani D. Identification of a factor IX binding site on the third apple domain of activated factor XI.J Biol Chem. 1996; 271: 29023-29028Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar six nanobodies that bind this domain were evaluated for their potential to inhibit clotting times, FXIa activation of FIX, FXIa activity, and thrombin generation. They found that the clones 1H9 and 1D4 exhibited near‐complete inhibition of FXIa‐mediated FIX activation in a purified system. Five of the six nanobodies showed a dose‐dependent inhibition of FXIa activity, suggesting that these nanobodies compete with the FIX binding site. Consistently, these five nanobodies increased clotting time initiated by the contact pathway while reducing thrombin generation and FXIa activity, as well as FXIIa‐mediated activation of FXI. The unexpected apple fell with the observation that clone 2G3 increased clotting time initiated by the contact pathway and reduced FXIa activity, yet did not affect FXIa activation of FIX nor thrombin generation when initiated through the extrinsic pathway. Based on these puzzling observations, Bar Barroeta et al. tested whether 2G3 influenced the binding of substrates that bind to FXI, namely FXII and HK. Although 2G3 did not interfere with the activation of FXI by FXIIa, the surprising observation was that 2G3 blocked HK binding to FXI. If indeed 2G3 mapped to the apple 3 domain, this observation suggests a novel interaction site between FXI and HK. Moreover, this suggests an ill‐defined function for this interaction in thrombin generation, adding to the established role of FXI‐HK interactions in mediating FXI binding to endothelial cells and platelets, as well as negatively regulating FXI activation in the presence of DNA and polyphosphates.16.Mohammed B.M. Matafonov A. Ivanov I. et al.An update on factor XI structure and function.Thromb Res. 2018; 161: 94-105Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar The nanobodies were characterized through HDX MS to unravel how they inhibited FXI function. Perhaps not surprising at this point in the story, 2G3 did not bind the FIX binding epitope on FXI, but rather largely bound to a distinct region within the second, fourth, and fifth beta strands of the apple 3 domain (Ala193‐Ala205 and Leu239‐Phe260). Conversely, the five nanobodies that functionally inhibited FXIa‐mediated FIX activation shared a binding epitope within the apple 3 domain overlapping with the established FIX binding epitope (Figure 1).17.Bar Barroeta A. van Galen J. Stroo I. Marquart J.A. Meijer A.B. Meijers J.C.M. Hydrogen‐deuterium exchange mass spectrometry highlights conformational changes induced by factor XI activation and binding of factor IX to factor XIa.J Thromb Haemost. 2019; 17: 2047-2055Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar Yet, this still does not explain how these same apple 3 domain‐specific nanobodies inhibited FXIIa mediated activation of FXI, as FXIIa interacts with FXI via the apple 2 and apple 4 domains.18.Cheng Q. Tucker E.I. Pine M.S. et al.A role for factor XIIa‐mediated factor XI activation in thrombus formation in vivo.Blood. 2010; 116: 3981-3989Crossref PubMed Scopus (204) Google Scholar Bar Barroeta et al. speculate the nanobodies sterically interfere with FXIIa activation of FXI or that FXIIa interacts with the apple 3 domain in addition to its main binding epitope. A parallel mechanism of action has been observed for the anti‐FXI monoclonal antibody, 1A6, which binds to the apple 3 domain and inhibits FXI activation by FXIIa and FIX activation by FXIa,19.Tucker E.I. Marzec U.M. White T.C. et al.Prevention of vascular graft occlusion and thrombus‐associated thrombin generation by inhibition of factor XI.Blood. 2009; 113: 936-944Crossref PubMed Scopus (160) Google Scholar suggesting that perhaps the mechanistic apple does not fall far from the tree. In conclusion, the study by Bar Barroeta and colleagues uncovered a novel interaction site between FXI and HK within the apple 3 domain of FXI, which may represent an ideal site for targeted drug develop for the prevention or treatment of thromboinflammation. S.M., C.P., and O.M. drafted and approved the final manuscript.

Long-Term Dose Optimization of Adalimumab via Dose Spacing in Patients with Psoriasis.

Dose spacing (DS) can be useful for optimizing treatment with biologics in psoriasis patients. However, interval prolongation might increase the production of anti-drug antibodies (ADA) and, therefore, reduce the drug’s effectiveness. The long-term effects of DS with adalimumab in psoriatic patients have not been reported. The goal of our study was to evaluate the long-term follow-up of psoriatic patients after adalimumab DS regarding the clinical course and determination of circulating adalimumab, TNFα levels, and anti-adalimumab antibodies. We retrospectively included seven patients treated with adalimumab for moderate-to-severe psoriasis and benefiting from DS from 2010 to 2021. The dose interval of adalimumab was extended to three weeks for all patients and then to four weeks for three of the seven patients. Adalimumab trough levels, TNFα levels, and ADA against adalimumab were measured. For six of the seven patients, absolute PASI values remained below 3 throughout the follow-up period (median = 8.0 years; range: 1.7–11.5) after DS. All the patients were satisfied with the effectiveness of their treatment regime. Within the follow-up period, an average of 63 doses of adalimumab per patient were spared. The median adalimumab trough levels were 4.7 µg/mL (range: 1.9–12.5). TNFα levels remained under 10 pg/mL (undetectable) in all except one patient. ADA against adalimumab remained negative (<10 µg/mL) during the follow-up in all patients. Our data indicate that therapeutic drug monitoring, including the measurement of trough concentrations and ADA, together with the clinical response and patient’s preference, can be helpful for clinical decision making and treatment optimization in psoriasis.

Immunogenicity of biologics used in the treatment of inflammatory bowel disease.

Here we critically evaluate the literature on immunotherapy failure in inflammatory bowel disease patients. In particular anti-drug antibody production, and subsequently loss of response as the primary cause of immunotherapy failure in IBD patients. The benefits of shifting from the "standard" empirical dose escalation approach to therapeutic drug monitoring with anti-TNFα therapy is explored. The American Gastroenterology Association and British Society of Gastroenterology both currently recommend the use of reactive therapeutic drug monitoring to guide treatment, following loss of response in inflammatory bowel disease patients with active disease. However, further research is required to prove the efficacy of a proactive therapeutic drug monitoring approach alone in remitted IBD patients. A combination of personalised monitoring approach for anti-drug antibodies and therapeutic drug monitoring could provide beneficial treatment outcome for people with inflammatory bowel disease by predicting drug failure prior to clinical symptoms and allowing timely switching to an alternative drug.

In Silico Analysis of Therapeutic Antibody Aggregation and the Influence of Glycosylation.

The aggregation of therapeutic antibodies is a major issue for the pharmaceutical industry leading to loss of drug quality, increased dosage, and unwanted immune responses such as the production of anti-drug antibodies (ADA). As aggregation can occur at various stages of development and storage, much work has been performed to reduce or eliminate it. In this report we analyzed four antibodies available in the PDB (1IGT, 1IGY, 1HZH, and 5DK3) using the online software UCSF Chimera to study the structural features of the proteins and the associated N-linked glycans in the CH2 domains of the Fc region. To study antibody aggregation in silico we used the online software TANGO and AGGRESCAN to identify aggregation prone regions (APR) in the antibodies and the influence of the Fc glycans on hydrophobic and aromatic residues present in the APRs. In the 3D structures of 1IGT and 1IGY the glycan chains are in close enough proximity to influence and protect these hydrophobic regions. However, in the 3D structures of 1HZH and 5DK3 the glycans do not appear to influence the likely APRs of the antibodies. Therefore, in these structures we modified the Fc glycan regions by adjusting the glycosylated asparagine side chains and glycosidic bonds. We successfully adjusted the glycan chains of 1HZH and 5DK3 and reduced the distance between them and the APRs to show potential influence on aggregation. However, similar to 5DK3, the influence of glycosylation on the APRs of the antibody was limited due to the size of the glycans present in the 3D structure. This report is based on in silico studies to show how antibody glycans can influence aggregation.

Oral delivery of infliximab using nano-in-microparticles for the treatment of inflammatory bowel disease

The anti-tumor necrosis factor-α (anti-TNF-α) blocker, has shown great efficacy for the treatment of inflammatory bowel disease (IBD). However, systemic exposure to it can cause considerable safety problems due to reduced suppression of the systemic immune response and loss of response to the production of anti-drug antibodies. Thus, we try to devise a targeted vehicle system for oral administration of anti-TNF-α antibodies for the treatment of IBD. In the present study, we developed an oral Infliximab (IFX) loaded nano-in-microparticles, based on chitosan (CS)/carboxymethyl chitosan (CMC) and alginate (Alg), which could protect IFX from the harsh environment of the gastrointestinal tract and produce targeted drug delivery to the inflamed intestine. In vivo studies demonstrated that the IFX loaded nano-in-micro vehicle can alleviate colitis by ameliorating inflammation and maintaining the intestinal epithelial barrier.