Evaluation of a generic immunoassay with drug tolerance to detect immune complexes in serum samples from cynomolgus monkeys after administration of human antibodies

Current state of the art bridging ELISA technologies for detection of anti-drug antibodies (ADAs) against therapeutic antibodies bear the risk of false-negative results due to interference by circulating drug. Methods to remove the drug in the sample or sample pre-treatment techniques such as acid dissociation of the immune complexes are limited, laborious and may destroy ADAs resulting again in false-negative results. The immune complex ELISA described in this publication provides a simple solution. It is designed to analyze samples from cynomolgus monkeys dosed with human antibodies; it can be used for all human antibodies since it is independent of the specific antibody and its target. The generic applicability of the ADA assay is enabled by the use of (1) a murine anti-human Fc monoclonal antibody (MAb) as capture reagent; (2) a murine anti-cynomolgus monkey IgG MAb as detection reagent; and (3) an ADA positive control conjugate consisting of cynomolgus IgG complexed with human IgG. In its basic version, the generic ADA ELISA specifically detects only immune complexes formed in vivo. Validation of the ADA assay revealed a lower limit of quantitation of 15.6 ng/mL in serum samples. Intra-assay and inter-assay precision was characterized by a coefficient of variation of less than 10% and accuracy was within 8%. Matrix effects were low as evidenced by a mean recovery of 95%. In vitro pre-incubation of the serum samples with drug makes also the free ADA in the sample amenable to measurement by the immune complex ELISA as demonstrated by analysis of ADAs from two cynomolgus monkey studies with two different antibodies. The generic and versatile nature of this ADA assay favors its use in pilot pharmacokinetic and safety studies in cynomolgus monkeys during candidate selection of antibodies. The assay can help to explain unexpected drug clearance profiles, loss of efficacy or safety events caused by immune complexes and guide further development.     

Assessment of placental transfer and the effect on embryo-fetal development of a humanized monoclonal antibody targeting lymphotoxin-alpha in non-human primates

An enhanced embryo-fetal development study was conducted in cynomolgus monkeys using pateclizumab, a humanized IgG1 monoclonal antibody (mAb) targeting lymphotoxin-alpha. Pateclizumab administration between gestation days (GD) 20 and 132 did not induce maternal or developmental toxicities. The ratio of fetal-to-maternal serum concentration of pateclizumab was 0.73% on GD 50 and 61% by GD 139. Decreased fetal inguinal lymph node-to-body weight ratio was present in the high-dose group without microscopic abnormalities, a change attributable to inhibition of lymphocyte recruitment, which is a pharmacologic effect of pateclizumab during late lymph node development. The effect was observed in inguinal but not submandibular or mesenteric lymph nodes; this was attributed to differential susceptibility related to sequential lymph node development. Placental transfer of therapeutic IgG1 antibodies; thus, begins during the first trimester in non-human primates. Depending on the potency and dose levels administered, antibody levels in the fetus may be pharmacologically or toxicologically relevant.     

Delivery of an erythropoietin-Fc fusion protein by inhalation in humans through an immunoglobulin transport pathway.

A novel drug delivery platform has been developed that utilizes a naturally occurring receptor known as the neonatal Fc receptor (FcRn). The receptor is specific for the Fc fragment of IgG and is expressed in epithelial cells where it functions to transport immunoglobulins across these cell barriers. It has been shown that FcRn is expressed in both the upper and central airways in non-human primates as well as in humans. Pulmonary delivery of an erythropoietin- Fc fusion molecule (EpoFc) was previously demonstrated in non-human primates using this FcRn pathway. We have now conducted a phase I clinical study to test whether the FcRn pathway functioned similarly in man using human erythropoietin (Epo) fused to the Fc portion of human IgG1. The design was a three leg, non-randomized study conducted in healthy male volunteers with rising doses (3, 10, and 30 microg/kg) of the fusion protein targeted to the central lung regions. Using a target range of 10-30% vital capacity and 15 breaths per minute, approximately 70% of the lung-deposited dose of aerosolized EpoFc was delivered safely and effectively to the central lung regions. We showed dose-dependent concentrations of the fusion protein in the serum and an increase in circulating reticulocytes was evident in the highest dose group, thus demonstrating that large therapeutic molecules can be delivered to humans via the lung, with retention of biological activity, using the FcRn-mediated transport pathway.     

IL-6R distribution in normal human and cynomolgus monkey tissues

Interleukin-6 (IL-6) is a pleiotropic cytokine and a contributing factor in many diseases such as rheumatoid arthritis, Castleman’s disease, Crohn’s disease, and multiple myeloma. Since the blockade of the signaling pathway of the IL-6/interleukin-6 receptor (IL-6R)/gp130 complex is considered to have therapeutic value in such diseases, we developed an IL-6R humanized antibody (tocilizumab). In the current report, distribution of IL-6R in both normal human and cynomolgus monkey tissues was assessed as fundamental data to support preclinical and clinical studies of tocilizumab. Human and cynomolgus monkey tissue panels were stained with commercially available anti-human IL-6R and a species- and isotype-matched negative antibody, as well as assay control slides. The detection system applied used an Envision^® immunoperoxidase staining procedure with DAB reaction. Positive reactions were observed in the tissue elements of lymphatic, hematopoietic, digestive, reproductive, exocrine, endocrine, neural, muscular, epidermal, respiratory, and urinary systems of the human and cynomolgus monkey tissue panels. The current report is inclusive of a wide variety of tissues and shows the distribution of IL-6R to be similar for both human and monkey tissues. We consider this information fundamental for the support and interpretation of preclinical and clinical studies of anti-IL-6R antibody therapy.     

Highly specific,quantitative polymerase chain reaction probe for the quantification of human cells in cynomolgus monkeys

Quantification of human cells may be performed using quantitative polymerase chain reaction (qPCR). In preclinical studies, the human Alu sequence is widely used as biomarker for human DNA. However, because the Alu gene is shared by primates, its use is limited to non-primate studies. The biodistribution of human cells in primates is also necessary for translational studies. Therefore, we aimed to design a novel, human-specific primer/probe that enables the quantification of human cells in primates and other animal models. A novel primer/probe set was successfully designed based on highly repetitive LINE1 sequences. qPCR efficiency (94.95–99.21%) and linearity of calibration curves (r² = 0.996–0.999) were confirmed in tissue homogenates of cynomolgus monkey. The lower limit of detection was 10 cells per 15-mg tissue sample, a sensitivity that is equivalent to existing Alu primers/probes. The set was also effective in other animal models such as mice, rabbits, pigs, and common marmosets.To our knowledge, this is the first study describing the successful design of a human-specific qPCR primer/probe for human cell quantification in various animals, including non-human primates, using LINE1 sequence. The excellent selectivity, sensitivity, and versatility of the LINE1 primers/probes make it a promising quantification tool in preclinical biodistribution studies.     

Humanized IgG1 variants with differential binding properties to the neonatal Fc receptor: relationship to pharmacokinetics in mice and primates.

It is well established that the neonatal Fc receptor (FcRn) plays a critical role in regulating IgG homeostasis in vivo. As such, modification of the interaction of IgG with FcRn has been the focus of protein-engineering strategies designed to generate therapeutic antibodies with improved pharmacokinetic properties. In the current work, we characterized differences in interaction of IgG between mouse and primate receptors using three humanized anti-tumor necrosis factor alpha antibodies with variant IgG(1) Fc regions. The wild-type and variant IgG showed a differential combination of improved affinity, modified dissociation kinetics, and altered pH-dependent complex dissociation when evaluated on the primate and murine receptors. The observed in vitro binding differences within and between species allowed us to more completely relate these parameters to their influence on the in vivo pharmacokinetics in mice and cynomolgus monkeys. The variant antibodies have different pharmacokinetic behavior in cynomolgus monkeys and mice, which appears to be related to the unique binding characteristics observed with the murine receptor. However, we did not observe a direct relationship between increased binding affinity to the receptor and improved pharmacokinetic properties for these molecules in either species. This work provides further insights into how the FcRn/IgG interaction may be modulated to develop monoclonal antibodies with improved therapeutic properties.     

Characterization of Aggregated Antibody-Silicone Oil Complexes: From Perspectives of Morphology, 3D Image,and Fcγ Receptor Activation

Pre-filled syringes (PFS) have been in widespread use as an administration device for therapeutic antibodies in recent decades. Generally, the inner barrel and syringe of PFS are coated with silicone oil (SO) for lubrication. Multiple studies have focused on the fact that the SO adsorbs denatured antibody molecules, and induces antibody aggregation. Aggregated antibodies are recognized as a potential risk for evoking immunogenic responses in patients. The characteristics of the aggregated antibody-SO complexes, including their concentration, population, shape, three-dimensional (3D) image, and Fcγ Receptors (FcγRs) activation have been obscurely acknowledged so far. In the present work, we prepared aggregated antibody-SO complexes by agitation and analyzed using multifaceted techniques such as flow imaging, confocal fluorescence microscopy, and cell-based assays for FcγRs activation. The results emphasized that the SO accelerates the increase in sub-visible particles and antibody aggregation. The confocal fluorescence microscopy analysis revealed the high-resolution 3D images of aggregated antibody-SO complexes. The FcγRs reporter cell assay clarified that the pre-mixed and agitated Ab + SO have higher FcγRs activation capability compared to the agitated Ab. Overall, this study advances the view that SO has an effect to increase the risk of agitation-induced aggregated antibody particles.     

Reduction of matrix interferences by the combination of chaotropic salt and DMSO in a broadly applicable target-based ELISA for pharmacokinetic studies of therapeutic monoclonal antibodies

Use of a synergistic effect of DMSO together with a chaotropic salt (NaSCN or MgCl₂) allowed to drastically reduce matrix interferences in an ELISA for therapeutic monoclonal antibodies. Optimum combinations were found to be 0.4 M NaSCN together with 10.0% DMSO, and 1.0 M MgCl₂ with 15.0% DMSO. At this optimum combination, quality controls spiked with mAb at 50.0 ng/ml in eighteen individual human sera and plasmas were quantified with an overall accuracy of 102.0%. All of these QCs fulfilled the acceptance criteria of 80.0–120.0% accuracy and precision below 20.0%. The assay was also successfully applied to the quantification of two other mAbs in human serum. Furthermore, the use of the assay was extended to pre-clinical species (cynomolgus monkey and rat serum). Here, the performed validation experiments confirmed the utility of the assay and demonstrated that the assay allowed quantification of mAb from 50.0 ng/ml to 100.0 μg/ml in cynomolgus monkey serum. The method has then been applied to a pharmacokinetic study in cynomolgus monkeys. In summary, this work demonstrates the efficacy of the combination of a chaotropic salt with DMSO to minimize matrix interferences in an ELISA. The robustness thus obtained allowed the successful establishment of a cost effective, target-based ELISA format for use in pharmacokinetic studies, that is easily applicable for the quantification of mAbs in various matrices such as human, cynomolgus monkey or rat serum and plasma.     

Anti-coagulation effect of Fc fragment against anti-β2-GP1 antibodies in mouse models with APS

Anti-beta (2)-glycoprotein I (anti-β2-GP1) is one of the important pathogenesis factors responsible for thrombosis formation in patients with antiphospholipid syndrome (APS). Administration of intravenous immunoglobulin (IVIg) is a common method used to inhibit the abnormal antibody levels and decrease the mortality of APS in emergency situations. We hypothesize that the Fc fragment of IgG is the molecular structure responsible for these effects. The present study investigates the beneficial effects of both recombinant and natural human Fc fragments of heterogeneous IgG against human anti-β2-GP1 antibodies in mouse models with APS. Results showed that both recombinant and natural human Fc fragments moderately but significantly decreased the levels of serum anti-β2-GP1 antibodies and had anti-coagulation effects in human β2-GP1-immunized mice. Furthermore, both recombinant and natural human Fc fragments inhibited thrombosis formation and decreased mortality in mouse models infused intravenously with human anti-β2GP1 antibodies from patients with APS. Findings suggest that the Fc fragment might be one of the active structural units of heterogeneous IgG. Thus, recombinant human Fc fragment administration may be a useful treatment for individuals with APS.     

Qualitative and quantitative studies on human B7.1-Fc fusion protein and the application in pharmacokinetic study in rhesus monkeys

A sensitive, accurate, and precise enzyme immunoassay (EIA) for the quantification of intact human B7.1-Fc in rhesus monkey serum was validated, and the characteristics of B7.1 and Fc moiety of fusion protein were identified by surface plasmon resonance (SPR) and flow-cytometric method, respectively. B7.1-Fc bound to CD28 and CTLA-4 with K_d values of 45.1 and 9.58 nM, respectively, which were very closed to the previous reports and the function of Fc moiety of fusion protein was also confirmed by Fc receptor binding assay and IL-8 releasing assay. To monitor the intact protein, the EIA method employed a sandwich scheme in which a multiclonal anti-human IgG (Fc specific) antibody and a monoclonal anti-human B7.1 antibody were served as capture and detection antibody, respectively. This EIA has a range of reliable response of 0.5–32 ng/ml. The LLOQ was established at 0.5 ng/ml. The intra-assay precision and accuracy were 6.1–8.8% and (3.0–9.0)%, respectively with the inter-assay precision and accuracy were 5.7–11.5% and (10.7–9.1)%, respectively. Stability was established under certain conditions and no significant differences were found. This validated EIA assay was then successfully employed in the assessment of pharmacokinetic behavior of B7.1-Fc in rhesus monkeys after intravenous infusion, and a non-linear characteristics was established across the investigated dosage range (32–320 μg/kg).     

鼠疫疫苗在食蟹猴模型中的免疫学评价

目的 通过将鼠疫耶尔森菌的F1抗原和重组V抗原组成的鼠疫疫苗免疫食蟹猴,对疫苗的免疫效果进行评价.方法 将20只食蟹猴按简单随机法分成低剂量组、高剂量组和生理盐水对照组,分别于0和2周肌内免疫,并于1剂后2周和2剂后2周采血.用ELISA检测免疫动物血清中的总IgG抗体;另外,分离外周血淋巴细胞,用酶联免疫斑点试验检测分泌IFN-γ的外周血淋巴细胞.用t检验对结果进行比较.结果 免疫后,对照组均未产生抗体,而疫苗组产生了较强的抗体应答.2剂免疫后2周,低、高剂量组的抗F1抗原IgG抗体几何平均滴度分别为(4.71±0.32)1g和(5.09±0.21)lg(t=-2.76,P＜0.05),两组的抗重组V抗原IgG抗体几何平均滴度分别为(4.75±0.52) lg和(5.12±0.58) lg(t=-1.37,P＞0.05).经F1和V抗原体外刺激产生IFN-γ的外周血淋巴细胞,细胞数均无明显增加.F1抗原刺激后,低、高剂量组分泌IFN-γ的外周血淋巴细胞数分别为(1±1)/106和(1±2)/106(t=-0.16,P＞0.05).重组V抗原刺激后,两组分别为(7±15)/106和(6±7)/106(t=0.88,P＞0.05).结论 鼠疫疫苗在食蟹猴模型中能诱导较强的体液免疫应答,但不能诱导明显的细胞免疫应答.     

Validation of a de-immunization strategy for monoclonal antibodies using cynomolgus macaque as a surrogate for human

The immunogenicity of biotherapeutics presents a major challenge during the clinical development of new protein drugs including monoclonal antibodies. To address this, multiple humanization and de-immunization techniques that employ in silico algorithms and in vitro test systems have been proposed and implemented. However, the success of these approaches has been variable and to date, the ability of these techniques to predict immunogenicity has not been systematically tested in humans or other primates. This study tested whether antibody humanization and de-immunization strategies reduce the risk of anti-drug antibody (ADA) development using cynomolgus macaque as a surrogate for human. First human–cyno chimeric antibodies were constructed by grafting the variable domains of the adalimumab and golimumab monoclonal antibodies onto cynomolgus macaque IgG1 and Igκ constant domains followed by framework germlining to cyno to reduce the xenogenic content. Next, B and T cell epitopes and aggregation-prone regions were identified using common in silico methods to select domains with an ADA risk for additional modification. The resultant engineered antibodies had a comparable affinity for TNFα, demonstrated similar biophysical properties, and exhibited significantly reduced ADA levels in cynomolgus macaque compared with the parental antibodies, with a corresponding improvement in the pharmacokinetic profile. Notably, plasma concentrations of the engineered antibodies were quantifiable through 504 hours (chimeric) and 840 hours (germlined/de-immunized), compared with only 336 hours (adalimumab) or 336–672 hours (golimumab). The results point to the significant value in the investment in these engineering strategies as an important guide for monoclonal antibody optimization that can contribute to improved clinical outcomes.     

Hazard characterization of an anti-human tissue factor antibody by combining results of tissue cross-reactivity studies and distribution of hemorrhagic lesions in monkey toxicity studies

Tissue cross-reactivity (TCR) studies are conducted when developing therapeutic antibodies, but their value is sometimes questioned because the positive organs often do not match the target organs of toxicity. We conducted TCR studies in human and cynomolgus monkey tissues for the development of an anti-human tissue factor antibody (TFAb) and also for a commercially available antibody, to clarify the true distribution of the target antigen. Tissue factor (TF) was found to be distributed in a wide variety of organs and tissues, including the heart and urinary bladder, in human and monkey. Administration of the TFAb to cynomolgus monkey caused hemorrhagic lesions mainly in the heart and urinary bladder in an incidental manner. This was thought to show the physiological role of TF in regulating hemostasis in these organs. Because the distribution of antigen in human and monkey was similar, the possibility that the TFAb would have similar effects in human was judged to be high, and because of the incidental nature of the effects, that they would be difficult to avoid. Thus it was possible to prospectively characterize the hazardous potential of a therapeutic antibody by accurately evaluating the tissue distribution of the target antigen and understanding its biological nature.     

Cigarette smoking differentially affects immunoglobulin class levels in serum and saliva: An investigation and review

The aim of the present study was to compare concentrations of IgG, IgA, IgM and IgD in both serum and saliva samples from smoking and non‐smoking individuals using a protein microarray assay. The findings were also compared to previous studies. Serum and saliva were collected from 48 smoking male individuals and 48 age‐matched never‐smoker male individuals. The protein microarray assays for detection of human IgG, IgM, IgA and IgD were established and optimized using Ig class‐specific affinity‐purified goat anti‐human Ig‐Fc capture antibodies and horseradish peroxidase (HRP)‐conjugated goat anti‐human Ig‐Fc detection antibodies. The Ig class specificity of the microarray assays was verified, and the optimal dilutions of serum and saliva samples were determined for quantification of Ig levels against standard curves. We found that smoking is associated with reduced IgG concentrations and enhanced IgA concentrations in both serum and saliva. By contrast, smoking differentially affected IgM concentrations—causing increased concentrations in serum, but decreased concentrations in saliva. Smoking was associated with decreased IgD concentrations in serum and did not have a significant effect on the very low IgD concentrations in saliva. Thus, cigarette smoking differentially affects the levels of Ig classes systemically and in the oral mucosa. Although there is variation between the results of different published studies, there is a consensus that smokers have significantly reduced levels of IgG in both serum and saliva. A functional antibody deficiency associated with smoking may compromise the body's response to infection and result in a predisposition to the development of autoimmunity.     

A chimeric antibody with dual Fc regions (bisFabFc) prepared by manipulations at the IgG hinge

A new chimeric antibody for therapeutic use in human cancer is described. First the derivative FabFc was prepared by linking Fab' gamma from monoclonal antibody to Fc gamma from human normal IgG1. The bismaleimide linking agent forms a thioether bond with an SH group released by reduction of SS bonds in the hinge of each constituent. It follows that one of the original two SS bonds in the Fc hinge still has both its S atoms free, and this bond is reformed by thiol-disulphide interchange. The lone free SH in the Fc hinge can now be used to join two FabFc molecules through a similar bismaleimide linker to yield bisFabFc. As regards antibody activity against target cells, bisFabFc can be univalent, bivalent, or bispecific. Its juxtaposed dual Fc regions are designed to promote cooperative binding of effectors, and bisFabFc is indeed notably more powerful than its parent FabFc molecules in promoting complement lysis and antibody-dependent cellular cytotoxicity. However it is not possible at present to distinguish the separate contributions of Fc architecture, antibody affinity and other factors towards this improvement. In the present state of development a variety of FabFc against a given neoplasm may be prepared in high yield from mouse IgG1 and IgG2a antibodies, and when convenient dimerized to bisFabFc in any combination of specificities.     

Universal Immunoassay Applied During Early Development of Large Molecules to Understand Impact of Immunogenicity on Biotherapeutic Exposure

Immunogenicity testing during early biotherapeutic development is usually limited by resources needed for assay development, validation, and the necessity for unique product-specific controls and reagents. We describe a unique immunoassay [universal indirect species-specific assay (UNISA)] that can be applied during early phase preclinical studies to support pharmacology, pharmacokinetics (PK), and toxicology evaluation during biotherapeutic antibody candidate assessment. UNISA was evaluated across three animal species: mouse, rat, and cynomolgus monkey. For each species, a unique and specific antibody pair was generated consisting of the secondary antibody and the positive control. The secondary antibody is specific for species anti-IgG antibody while demonstrating no cross-reactivity to human antibody-based biotherapeutics. The positive control is comprised of a species-specific anti-human IgG antibody clone specific for binding to the CH2 domain of all human IgG subtypes. Applications of this platform included: (a) identifying the dose with the least immunogenicity risk; (b) characterizing the impact of immunogenicity on PK exposure profiles across multiple antibody candidates and dose regimens; and (c) characterizing the immune response specificity to the idiotype or non-idiotypic region of the biotherapeutic candidate. Due to its use of universal species-specific reagents, UNISA can overcome resource constraints and avoid extensive validation and development time to support immunogenicity testing during the early research and preclinical phase of programs. Enhanced understanding of the impact of the immunogenicity on biotherapeutic exposure and target-related immunomodulatory effects have been made possible with the use of this assay.     

磁性免疫微球在人血清白蛋白纯化中的应用

目的为了快速地从人血清中提纯人血清白蛋白，利用磁性免疫微球作为提取手段，再用间接酶联免疫法测定人血清白蛋白的回收率。方法将经过羧基修饰的聚苯乙烯微球作为载体，用EDC(碳化亚胺)活化微球表面的羧基，再将兔抗人血清白蛋白抗体包被于微球上，这种微球-抗体复合物能特异性地捕获人血清白蛋白，磁分离复合物后，通过将兔抗人血清白蛋白抗体作为捕获抗体，将酶联羊抗人血清白蛋白抗体作为检测抗体，建立起间接酶联免疫法，用于检测人血清中和磁性免疫微球上吸附人血清白蛋白的浓度，得到微球从人血清中提纯人血清白蛋白的回收率。结果第1次提纯的回收率为(86±4)%，重复利用微球2次，回收率分别为(69.0±0.6)%和(40.8±0.8)%，而提纯的人血清白蛋白的纯度为90%。结论以上结果表明，免疫磁性微球提纯人血清白蛋白的实验是有效的，为工业上大规模提纯人血清白蛋白提供了一条新的思路。     

Prevalence and Isotypic Complexity of the Anti-Chinese Hamster Ovary Host Cell Protein Antibodies in Normal Human Serum

Host cell-derived protein impurities may be present at low levels in biopharmaceutical products. Antibodies to host cell proteins are present in individuals with no known exposure to these products. In this study, antibodies to drug process-specific Chinese hamster ovary host cell-derived proteins (CHO-HCP) were measured in unexposed individuals using a validated enzyme-linked immunosorbent assay. Samples that tested positive for anti-CHO-HCP reactivity were further characterized to determine the isotypes and IgG subclasses expressed in each positive individual. The specificity of the detected anti-CHO-HCP antibody isotypes was confirmed by the competitive inhibition assay and the uncoated plate specificity testing. These antibody characterization experiments revealed that the prevalent anti-CHO-HCP antibody subclasses were predominantly IgG1 (present in 66.6% of individuals) and IgG2 (60%), with 33.3% positive for IgG3 while IgG4 was detected in low abundance. Forty percent (40%) of the individuals with IgG type reactivity were also identified as IgM-positive. Anti-CHO-HCP-specific IgE was not detected in the assays used in this study. Some individuals exhibited single isotype anti-CHO-HCP reactivity; others contained a combination of multiple antibody isotypes. Data presented in this study demonstrate the isotypic complexity and the high prevalence of anti-CHO-HCP antibodies in human serum with no known exposure to CHO cell-derived therapeutic biologics.     

Primate testing of TGN1412: right target, wrong cell

The failure of toxicity studies in non-human primates to predict the cytokine release syndrome during a first-in-man study of the CD28-specific monoclonal antibody TGN1412 has remained unexplained so far. In this issue of the BJP, work from the NIBSC first identifies the effector memory subset of human T-lymphocytes as the most likely source of the pro-inflammatory cytokines released during the study, and goes on to show that in cynomolgus monkeys, this subset lacks CD28, the target molecule of TGN1412. We discuss the implications for the TGN1412 catastrophe and for preclinical evaluation of biologicals in animal models in general.     

Development and validation of immunoassays to quantify the half-antibody exchange of an IgG4 antibody, natalizumab (Tysabri®) with endogenous IgG4

Natalizumab is a humanized IgG4 monoclonal antibody which binds human α4 integrin and is approved for treatment of multiple sclerosis and Crohn's disease. Assessment of the in vivo disposition of natalizumab presents a unique assay development challenge due to the ability of human IgG4 antibodies to undergo half-antibody exchange in vivo. Such exchange generates IgG4 molecules of mixed specificity comprising a natalizumab heavy-light chain pair coupled to an IgG4 heavy-light chain pair of unknown specificity. Since exchanged and non-exchanged species cannot be quantified independently using a single enzyme linked immunosorbent assay (ELISA), a novel quantitation strategy was developed employing two ELISAs: one measuring total natalizumab including both intact and exchanged molecules, and the second measuring only intact natalizumab. The presence and amount of exchanged natalizumab in serum is calculated by the difference in values obtained in the two assays. To evaluate assay performance, a control reagent was created from natalizumab and an irrelevant humanized monoclonal IgG4 antibody. Subsequent validation demonstrated that both assays are specific, accurate, and precise within the working ranges of the assays (1.5-10μg/mL for total and 0.5-12μg/mL for intact natalizumab assays). The mean accuracy, intra- and inter-assay precision for both assays were 82-113%, ≤9% and ≤20%, respectively. Additionally, the limits of detection of intact and exchanged natalizumab were established using statistical methods. The utility of the two-assay strategy was confirmed by analyzing samples from a pharmacokinetic study in rats using different variants of natalizumab administered along with another human IgG4 antibody as an exchange partner.