Mepolizumab, a humanized IgG1 monoclonal antibody that blocks native homodimeric interleukin-5 (IL-5) from binding to the IL-5 receptor, has recently been approved for treatment of severe eosinophilic asthma. Our initial immunogenicity assay method for phase I and II studies utilized a bridging electrochemiluminescence format with biotin and ruthenium-labelled mepolizumab linked by anti-drug antibodies (ADA). We discovered that IL-5 significantly increased in dosed subjects from a phase II study and that the increased IL-5 was in the form of a drug-bound complex. We demonstrated that the elevated drug-bound IL-5 produced false-positive response in the in vitro ADA assay, in which drug-bound IL-5 dissociated and then bridged mepolizumab conjugates to yield positive signal. To eliminate the IL-5 interference, we compared two strategies: a solid-phase immunodepletion of IL-5 and an in-solution IL-5 immunocompetition. We identified the best competitive antibody for each purpose. We found both methods demonstrated similar effectiveness in reducing the false positive signal in IL-5 spiked samples; however, the in-solution immunocompetition for IL-5 had fewer false positives in study samples. Additionally, the in-solution immunocompetition method was experimentally simpler to execute. We modified the ADA assay by adding a pre-treatment step with a mepolizumab competitive anti- IL-5 antibody. Using this new method, we retested clinical samples from two phase II studies (MEA112997 and MEA114092). The confirmed ADA positive incidence was reduced from 29% and 61% to 1% and 8% with the modified in-solution immune inhibition method. Target interference is a fairly common problem facing immunogenicity testing, and target-induced false positive cannot be distinguished from true ADA response by the commonly used drug competitive confirmation assay. The approach and method used here for resolving target interference in ADA detection will be useful for differentiating between a true ADA response and target induced false positive as well as similar challenges in other programs.

An electrochemiluminescent (ECL) bridging assay to detect anti-ofatumumab antibodies (ADA) in human serum samples was developed and validated. Using this assay format, clinical samples were first screened to identify potential ADA positive samples, which were then further tested by adding excess drug, confirming the positive signals as drug specific. However, when the method was implemented into clinical studies for ADA testing, a high positive rate was observed in the pre-dose samples collected from patients with chronic lymphocytic leukemia (CLL). Since the positive signals were not associated with ofatumumab (Ofa) treatment, and diminished after treatment, it was suspected that matrix interference might be responsible, resulting in false-positive responses. We performed a series of experimental investigations to identify, characterize, minimize or eliminate the possible false-positive responses. One possible source was identified to be CD20 (the target of Ofa) present on cell membrane fragments (CMFs). The false-positive responses caused by CD20+ CMFs could be reduced by solid-phase immunodepletion, ultracentrifugation, or inhibited by adding another anti-CD20 antibody (rituximab). As a consequence, the ADA method was modified to minimize the matrix interference caused by CD20+ CMFs and, then, validated for sample testing.

Therapeutic proteins have the potential to elicit immune responses in animals and humans (Mire-Sluis et al., 2004; Yu et al., 2006; Shankar et al., 2008). Contributors to the response could include product related factors such as chemical modifications, impurities that co-purify with product, contaminants, formulation, aggregates, and clinical factors such as dose concentration, dosing frequency, route of drug administration, rate of administration, patient underlying disease, concomitant medication, and genetic status among others (Patten and Schellekens, 2003). Further, an immune response triggered by a therapeutic enzyme may neutralize the endogenous counterpart resulting in a decrease or depletion of the therapeutic and endogenous enzymes imposing safety concerns for patients. Therefore, monitoring of anti-drug antibody (ADA) and neutralizing antibody (NAb) responses to both the recombinant therapeutic enzyme and endogenous enzyme is important during early development and subsequent clinical studies. Testing considerations for NAb detection against therapeutic enzymes have been published mostly for lysosomal storage diseases (Wang et al., 2008). NAb cross-reactivity to the endogenous counterpart has also been characterized (Sominanda et al., 2010). Here, we describe an enzymatic NAb assay which detects neutralizing antibodies to both recombinant and endogenous angiotensin-converting enzyme 2 (ACE2). NAb assay sensitivity was optimized by selecting the assay incubation time as 20 min with an enzyme concentration of 0.5 μg/mL. Four anti-ACE2 antibodies out of a commercial panel of 18 were found to have neutralizing capabilities based upon their ability to abrogate ACE2 enzymatic activity. We demonstrated assay specificity by small peptide inhibitors specific for ACE or ACE2. DX600, an ACE2 specific inhibitor did not cross-react with ACE. Conversely, captopril, an inhibitor of ACE did not inhibit ACE2. The assay specificity for ACE2 neutralizing antibodies was further demonstrated by the lack of reactivity of two species control antibodies and 14 anti-ACE2 antibodies. Moreover, we demonstrated assay specificity to human endogenous ACE2 from human epithelial cells. Three human cell lines (Calu-3, Caco-2, Huh-7) were evaluated for the cell surface expression of ACE2 by flow cytometry and Western blot. Subsequently, whole cell lysates, cell culture supernatant, and live cells were evaluated in the assay. Results demonstrated that Calu-3 had elevated levels of ACE2 compared to Caco-2 or Huh-7. Calu-3 also demonstrated elevated ACE2 enzymatic activity in all three sources and could be inhibited by the ACE2 specific inhibitor DX600 as well as the neutralizing antibodies for the recombinant ACE2. Thus, we describe here a method to detect NAb against a therapeutic enzyme and assess NAb cross-reactivity to the native endogenous enzyme. The approach of method development described here could be applied for the assessment of NAb responses to other enzymatic therapeutics.

ABSTRACT Pneumovax 23 consists of a mixture of highly purified capsular polysaccharides (Ps) from 23 of the most prevalent serotypes of Streptococcus pneumoniae . Testing of vaccine immunogenicity has been historically performed on the enzyme-linked immunosorbent assay (ELISA) platform, validated to measure immunoglobulin G (IgG) antibodies to all 23 serotypes included in Pneumovax 23. In order to significantly improve the throughput of this form of testing, we have developed and validated a direct binding electrochemiluminescence (ECL)-based multiplex assay that can measure the antibody response in human serum to eight serotypes within a single microtiter well. The pneumococcal (Pn) ECL assay is based on the Meso Scale Discovery (MSD) technology which utilizes a Sulfo-Tag-labeled anti-human IgG antibody that emits light upon electrochemical stimulation. The Pn ECL assay exhibits a wide dynamic range and provides the ability to read concentrations down to the minimum reported concentration in the Merck ELISA (0.1 μg/ml). Cross-reactivity assessment using type-specific monoclonal antibodies showed no cross talk between antigen spots within a well. By use of the WHO Pn sample reference panel, the results obtained with the Pn ECL assay were compared to the results obtained with the international Pn ELISA. The results for the Pn ECL assay satisfied the WHO-recommended acceptance criterion for concordance for all seven serotypes with published Pn ELISA values, and the overall correlation ( r value) across the seven serotypes was 0.994. The MSD methodology has great potential to be extremely useful for simultaneously quantitating IgG responses to several Pn serotypes while conserving serum volumes and laboratory testing time.

Human papillomaviruses (HPV) infect epithelial cells, including the epithelium of mucous membranes. GARDASIL™ was approved for the prevention of genital warts, vaginal, vulvar and cervical cancer caused by HPV types 6, 11, 16 and 18. A competitive Luminex immunoassay (cLIA) has been developed to measure serum levels of virus‐like particle (VLP) type‐specific antibodies. The cLIA uses neutralizing, type‐specific, R‐phycoerythrin‐labeled monoclonal antibodies to monitor the antibody response to type‐specific, neutralizing epitopes on the VLPs. Antibody responses to two distinct neutralizing epitopes on VLP‐6 were characterized using monoclonal antibodies H6.M48 and H6.1C2. Both the H6.M48 and H6.1C2 mAbs bind to HPV‐6 VLPs. Competition assays showed that the H6.M48 and H6.1C2 bind to type‐specific, overlapping, neutralizing epitopes. Avidity analysis shows that the H6.1C2 binds to the HPV 6 VLP with greater avidity than the H6.M48 mAb. Sera from individuals with confirmed HPV‐6 infection, a history of genital warts or sera from females who had been vaccinated with GARDASIL were tested in competition experiments with both of the mAbs. Results show that the H6.1C2 mAb binds to a more immunodominant epitope than the H6.M48 mAb as recognized by serum antibodies from both vaccinees and naturally infected individuals as indicated by a high prevalence in such antibodies in sera collected.

One of the most widely held hypotheses is that Alzheimer's Disease is caused by the deposition of plaques primarily composed of amyloid-β peptide aggregates in the brain tissue. Thus, a phase I, first-in-man clinical trial has been initiated in Alzheimer's patients to assess a therapeutic vaccine against the amyloid-β peptide. The safety of an amyloid-β vaccine in Alzheimer's subjects has been a concern due to cellular immune activation potentially resulting in meningoencephalitis. Although this vaccine was carefully formulated in an attempt to avoid the induction of T cells, there is still a need to monitor subjects for T cell activation. The difficulty of working with a self-antigen, such as amyloid-β, is that the levels of T cell responses tend to be lower and less prevalent than those directed against an infectious or non-self antigen. Subsequently, the sensitivity of the assay and the type of positive control needs to be considered. An exploratory interferon-gamma ELISPOT assay was developed as a biomarker to detect amyloid-β specific T cell activation in all study participants. One of the major challenges in developing a functional T cell assay is that it requires the collection of high quality peripheral blood mononuclear cells (PBMC). Once high quality PBMC were obtained, the assay development challenges included (1) working with a self-antigen and (2) lacking an amyloid- β specific positive control. PBMC from an epidemiological cohort of Alzheimer's patients were obtained for characterizing T cell responses to amyloid-β peptide in the ELISPOT assay. A contract lab was trained and qualified in using an optimized protocol for processing viable and functional PBMC from these non-vaccinated Alzheimer's subjects. This same protocol was used to train and qualify participating laboratories in the clinical trial to facilitate the availability of high quality PBMC throughout the two year study. The characterization of the interferon-gamma ELISPOT assay included identifying the appropriate cell input, peptide concentration and positive control. Although challenging, the development and characterization of an interferon-gamma ELISPOT assay capable of detecting T cells specific for amyloid-β is feasible if high quality, functional PBMC are obtained.

Immunogenicity is often a critical clinical endpoint in the assessment of vaccines prior to the submission of data to regulatory agencies. As a result, the assays used to measure immunogenicity must be highly characterized, well-controlled, and statistically supported. These goals are not easily attained, however, when the development of the assay must occur prior to the first-in-man studies. Two significant barriers exist in the development of these assays: (1) the lack of experience with the performance of a novel antigen in a clinical assay, and (2) the lack of available proper human clinical samples to create reference standards and assess sample matrices. To help to overcome these obstacles, we employed a screening experimental design to assess assay optimization. Design of experiments (DOE) is a statistical tool that allows for the evaluation of all of the key assay parameters to determine the optimal conditions for the assay, as well as determine if there are any interactions of these parameters on the response of the assay. The multivariate approach that is integral to DOE helps to overcome the lack of experience with the assay reagents by facilitating an understanding of how the variables work together in the performance of the assay. Here, we outline the use of full and fractional factorial DOE in the optimization of a clinical assay on two platforms, Luminex and ELISA, for the measurement of antibodies to the beta-amyloid peptide (Abeta) for a novel first-in-man vaccine program. Both platforms are evaluated in an attempt to determine the assay best suited to the needs of the program. We also describe the specificity experiments performed to further characterize the utility of each assay platform.

GARDASIL™ has been proven in clinical trials as a highly effective vaccine for preventing HPV 6‐, 11‐, 16‐, or 18‐related pre‐cancerous lesions and genital warts. In this study, we compared the HPV‐type specific subclass and isotype humoral immune responses following vaccination to those induced by HPV infection. Sera collected from vaccinees at month 7 and month 60 post‐vaccination, naturally infected and negative subjects were tested to determine serum levels of HPV VLP‐specific IgG, IgG1, IgG3, IgG4 and IgA using a Luminex based, direct binding, subclass and isotyping assay method. The same serum samples were also evaluated for their levels of antibodies to type‐specific, neutralizing epitopes on the VLPs using a competitive immunoassay designed to measure type‐specific, neutralizing antibodies. Vaccination with GARDASIL™ induced high serum levels of VLP‐specific IgG, IgG1, IgG3, IgG4 and IgA antibodies, and such antibodies to VLP 6, 11, 16 and 18 were also detected at Mo 60 in a high proportion of subjects. These levels were comparable to or greater than antibody levels associated with subjects naturally infected with HPV. Importantly, serum anti‐L1, HPV IgA levels induced by vaccination may provide evidence of mucosal immunity that could protect the anogenital tract from HPV infection.

ABSTRACT The Merck pneumococcal (Pn) enzyme-linked immunosorbent assays (ELISAs) for measuring antibodies to 12 serotypes (serotypes 1, 3, 4, 6B, 7F, 8, 9V, 12F, 14, 18C, 19F, and 23F) were validated in 1999. Merck Laboratories developed the Pn assays using 10 μg/ml C polysaccharide, 100 μg/ml Pn polysaccharide (PnPs) 25, and 100 μg/ml PnPs 72 for preadsorption of samples, standards, and controls in order to improve the specificity to the Pn serotypes in the vaccine. The Pn assays utilize postimmunization sera obtained from subjects immunized with PNEUMOVAX 23 as standards for measuring immunoglobulin G concentrations in sera obtained from vaccine clinical trials with adults and infants. This material was calibrated to the Pn reference standard serum, 89SF, subjected to the Merck Pn ELISA adsorbants. Comparisons were made between the Merck Pn assay and the international Pn assay, showing moderate agreement between the two assay formats. This work describes the test procedures and operating characteristics of the Merck Pn assays and the results of experiments performed to compare the Merck Pn ELISAs to the international Pn ELISAs.

ABSTRACT Weight-based assignments for immunoglobulin G1 (IgG1) and IgG2 subclass antibodies to Streptococcus pneumoniae capsular polysaccharides (PnPs) in antipneumococcal standard reference serum lot 89-S (lot 89-S), also known as lot 89-SF, have been determined for serotypes 1, 4, 5, 7F, 9V, and 18C. This extends the usefulness of lot 89-S beyond the IgG1 and IgG2 subclass assignments for serotypes 3, 6B, 14, 19F, and 23F made previously (A. Soininen, H. Kayhty, I. Seppala, and T. Wuorimaa, Clin. Diagn. Lab. Immunol. 5: 561-566, 1998) to cover 11 major serotypes associated with the highest percentage of pneumococcal disease worldwide. A method of equivalence of absorbances in enzyme immunosorbent assays was used to determine the IgG1 and IgG2 antibody concentrations for the additional serotypes in lot 89-S, based on the subclass values previously assigned for PnPs serotypes 6B, 14, and 23F. This cross-standardization method assures consistency with previous antibody assignments in that reference serum. The newly assigned subclass values for serotype 9V, and previously assigned values for serotype 14, were used to quantitate PnPs antibodies in sera from adult and pediatric subjects immunized with a pneumococcal conjugate vaccine. There was a predominance of IgG1 anti-PnPs antibodies in pediatric sera and IgG2 anti-PnPs antibodies in the adult sera. The IgG1 and IgG2 subclass assignments for the 11 PnPs serotypes in antipneumococcal standard reference serum lot 89-S are useful for quantitating and characterizing immune responses to pneumococcal infection and vaccination regimens.

ABSTRACT Human sera collected from 28 consenting adult volunteers were used to define assay conditions for meningococcal vaccine clinical trial serology. Immunoassay parameters were optimized with these test sera and the standard reference serum, CDC1992. Coating conditions for serogroup Y and W135 polysaccharide antigens were found to influence the predicted serum immunoglobulin G (IgG) antibody concentrations. Sera that displayed IgG antibody binding profiles most unlike that of CDC1992 were influenced the most by coating conditions. Our results suggest that presentation of specific epitopes is influenced by antigen-coating concentrations for serogroup Y and W135 polysaccharides.

Streptococcus pneumoniae is a major human pathogen causing pneumonia, sepsis, meningitis, and otitis media ([12][1]). It causes infections most often in young children ([12][1]) and elderly adults ([1][2]) because their immune systems are either unprepared or unable to respond effectively to

ABSTRACT A new meningococcal group C-CRM 197 conjugate vaccine (MnCC; Meningitec) has been evaluated in multiple clinical trials in the United States and most recently has been approved for routine administration in the United Kingdom. Meningococcal serogroup C (MnC)-specific immunoglobulin G (IgG) antibodies in pre- and postimmunization sera obtained from healthy U.S. adults, toddlers, and infants were quantitated by enzyme-linked immunosorbent assay (ELISA) and by an antibody-dependent, complement-mediated serum bactericidal assay (SBA). Serogroup-specific IgG antibody (micrograms per milliliter) in adults immunized either with the quadrivalent polysaccharide (A, C, Y, and W-135) vaccine or with MnCC showed a strong correlation ( r = 0.848 and 0.934, respectively) by linear regression analysis with SBA. Sera from infants immunized with the MnCC ( n = 30) and an age-matched unimmunized control group ( n = 15) were also analyzed. Linear regression analysis of serum bactericidal and IgG ELISA data from sera obtained at 2 months of age (preimmunization) showed no correlation; however, a high degree of correlation was observed at time points after two ( r = 0.877) and three ( r = 0.951) immunizations, where significant rises in anti-MnC polysaccharide antibodies occurred relative to the age-matched control group. Infants previously primed with 3 doses of MnCC were given a booster dose of conjugate vaccine at 12 to 15 months of age. The correlation coefficient of ELISA to SBA for combined pre- and postbooster data was r = 0.836 ( n = 48 pairs). In conclusion, increases in serum bactericidal activity in immunized adult, toddler, and infant populations were found to correlate very well with increases in serogroup-specific IgG concentrations, whereas the correlation between these two assays in nonimmunized 2-month-old infants was poor. Characterizing the relationship between these methods is important for understanding the significance of antigen-specific antibody concentrations relative to vaccine performance and protection from disease.