Total Antibody Concentrations Research Articles

Salivary polyreactive antibodies and Haemophilus influenzae are associated with respiratory infection severity in young children with recurrent respiratory infections.

Recurrent respiratory tract infections (rRTIs) are a common reason for immunodiagnostic testing in children, which relies on serum antibody level measurements. However, because RTIs predominantly affect the respiratory mucosa, serum antibodies may inaccurately reflect local immune defences. We investigated antibody responses in saliva and their interplay with the respiratory microbiota in relation to RTI severity and burden in young children with rRTIs. We conducted a prospective cohort study including 100 children aged <10 years with rRTIs, their family members and healthy healthcare professionals. Total and polyreactive antibody concentrations were determined in serum and saliva (ELISA); respiratory microbiota composition (16S rRNA sequencing) and respiratory viruses (quantitative PCR) were characterised in nasopharyngeal swabs. Proteomic analysis (Olink) was performed on saliva and serum samples. RTI symptoms were monitored with a daily mobile phone application and assessed using latent class analysis and negative binomial mixed models. Serum antibody levels were not associated with RTI severity. Strikingly, 28% of salivary antibodies and only 2% of serum antibodies displayed polyreactivity (p<0.001). Salivary polyreactive IgA was negatively associated with recurrent lower RTIs (adjusted OR 0.80, 95% CI 0.67-0.94) and detection of multiple respiratory viruses (adjusted OR 0.76, 95% CI 0.61-0.96). Haemophilus influenzae abundance was positively associated with RTI symptom burden (regression coefficient 0.05, 95% CI 0.02-0.08). These results highlight the importance of mucosal immunity in RTI severity and burden, and suggest that the level of salivary polyreactive IgA and H. influenzae abundance may serve as indicators of infection severity and burden in young children with rRTIs.

Intact quantitation of cysteine-conjugated antibody-drug conjugates using native mass spectrometry.

A common strategy for antibody-drug conjugate (ADC) quantitation from in vivo study samples involves measurement of total antibody, conjugated ADC, and free payload concentrations using multiple reaction monitoring (MRM) mass spectrometry. This not only provides a limited picture of biotransformation but can also involve lengthy method development. Quantitation of ADCs directly at the intact protein level in native conditions using high-resolution mass spectrometers presents the advantage of measuring exposure readout as well as monitoring the change in average drug-to-antibody ratio (DAR) and in vivo stability of new linker payloads with minimal method development. Furthermore, site-specific cysteine-conjugated ADCs often rely on non-covalent association to retain their quaternary structure, which highlights the unique capabilities of native mass spectrometry (nMS) for intact ADC quantitation. We developed an intact quantitation workflow involving three stages: automated affinity purification, nMS analysis, and data processing in batch fashion. The sample preparation method was modified to include only volatile ion-pairing reagents in the buffer systems. A capillary size-exclusion chromatography (SEC) column was coupled to a quadrupole time-of-flight high-resolution mass spectrometer for high-throughput nMS analysis. Samples from two mouse pharmacokinetic (PK) studies were analyzed using both intact quantitation workflow and the conventional MRM-based approach. A linear dynamic range of 5-100 μg/mL was achieved using 20 μL of serum sample volume. The results of mouse in vivo PK measurement using the intact quantitation workflow and the MRM-based approach were compared, revealing excellent method agreement. We demonstrated the feasibility of utilizing nMS for the quantitation of ADCs at the intact protein level in preclinical PK studies. Our results indicate that this intact quantitation workflow can serve as an alternative generic method for high-throughput analysis, enabling an in-depth understanding of ADC stability and safety in vivo.

Mucosal response of inactivated and recombinant COVID-19 vaccines in Congolese individuals.

The efficacy of immunization against an airborne pathogen depends in part on its ability to induce antibodies at the major entry site of the virus, the mucosa. Recent studies have revealed that mucosal immunity is poorly activated after vaccination with messenger RNA vaccines, thus failing in blocking virus acquisition upon its site of initial exposure. Little information is available about the induction of mucosal immunity by inactivated and recombinant coronavirus disease 2019 (COVID-19) vaccines. This study aims to investigate this topic. Saliva and plasma samples from 440 healthy Congolese were collected including (1) fully vaccinated 2 month postvaccination with either an inactivated or a recombinant COVID-19 vaccine and (2) nonvaccinated control group. Total anti-severe acute respiratory syndrome coronavirus 2 receptor-binding domain IgG and IgA antibodies were assessed using in-house enzyme-linked immunosorbent assays for both specimens. Altogether, the positivity of IgG was significantly higher in plasma than in saliva samples both in vaccinated and nonvaccinated control groups. Inversely, IgA positivity was slightly higher in saliva than in plasma of vaccinated group. The overall IgG and IgA levels were respectively over 103 and 14 times lower in saliva than in plasma samples. We found a strong positive correlation between IgG in saliva and plasma also between IgA in both specimens (r = .70 for IgG and r = .52 for IgA). Interestingly, contrary to IgG, the level of salivary IgA was not different between seropositive control group and seropositive vaccinated group. No significant difference was observed between recombinant and inactivated COVID-19 vaccines in total IgG and IgA antibody concentration release 2 months postvaccination both in plasma and saliva. Inactivated and recombinant COVID-19 vaccines in use in the Republic of Congo poorly activated mucosal IgA-mediated antibody response 2 months postvaccination.

Overcoming Soluble Target Interference in Measurement of Total Bispecific Therapeutic Antibody Concentrations.

The measurement of therapeutic drug concentrations is used to assess drug exposure and the relationship between therapeutic pharmacokinetics (PK) and pharmacodynamics (PD), which help determine the optimal dose for patients. Ligand binding assays (LBAs) are often the method of choice for evaluation of drug concentration and use either the therapeutic target protein or antibodies to the therapeutic as capture and/or detection reagents. Due to the bivalency of antibody therapeutics, heterogeneous states of the drug/target complex can exist in the presence of soluble targets which can complicate measurement of unbound drug. In the case of bispecific antibodies, measurement of drug can be even more complicated and depend upon the levels of both targets to each arm. Measuring the total drug allows for PKPD modeling prediction of human dose projections in addition to overcoming challenges associated with measuring free drug for bispecific antibodies. Here, we present a study in which a sandwich ELISA format was used to measure total anti-KLK5/KLK7 antibody concentrations. This assay utilized a non-blocking anti-idiotype (ID) antibody to one arm of the antibody for capture and an antibody to target bound to the other arm of the antibody for detection. Our qualified assay showed acceptable precision, accuracy, dilutional linearity, and reproducibility and enabled detection of a total bispecific antibody at high levels of two targets. To confirm that our assay was detecting total drug, a subset of samples was evaluated in a generic total LC-MS/MS assay.

EV-203: Phase 2 trial of enfortumab vedotin in patients with previously treated advanced urothelial carcinoma in China.

e16574 Background: Enfortumab vedotin (EV), a fully human monoclonal antibody directed against Nectin-4 delivering intracellular monomethyl auristatin E (MMAE) to tumor cells, demonstrated superior overall survival (OS) with manageable safety/tolerability over standard chemotherapy in patients (pts) with previously treated locally advanced or metastatic urothelial carcinoma (la/mUC) in the pivotal EV-301 trial. EV-301 findings led to approval in 41 countries. As EV monotherapy has not been studied in a Chinese population, EV-203 (NCT04995419) assessed efficacy, safety/tolerability, and pharmacokinetics (PK) of EV in pts with la/mUC in China. Methods: In this single-arm, open-label, phase 2 study, Chinese pts with la/mUC previously treated with platinum-based chemotherapy and PD-1/L1 inhibitor therapy received EV 1.25 mg/kg via intravenous infusion on days 1, 8, and 15 of each 28-d cycle at 6 centers. Pts had Eastern Cooperative Oncology Group performance status 0–1 and were excluded if they had preexisting grade ≥2 sensory/motor neuropathy, active central nervous system metastases, clinically significant toxicity associated with prior treatment, or history of uncontrolled diabetes mellitus. Two study centers enrolled pts for a PK cohort. Primary endpoints were confirmed objective response rate (ORR; by Response Evaluation Criteria in Solid Tumors v1.1) by independent review committee (IRC) and PK parameters of antibody–drug conjugate (ADC), total antibody (TAb), and unconjugated MMAE. Secondary endpoints included investigator-assessed confirmed ORR; investigator-/IRC-assessed duration of response (DOR), disease control rate (DCR), and progression-free survival (PFS); OS; and safety/tolerability. Results: A total of 40 Chinese pts were enrolled; 15 were in the PK cohort. Confirmed ORR by IRC was 37.5% (n/N=15/40; 95% CI: 22.7–54.2); best overall response was complete response for 1 (2.5%) pt and partial response for 14 (35.0%) pts. Investigator-assessed ORR was 42.5% (n=17). DCRs were 82.5% (n=33) by investigator and 72.5% (n=29) by IRC. Median DOR by investigator and IRC were not reached. Median PFS were 4.24 mo by investigator and 4.67 mo by IRC. Median follow-up time was 6.5 mo and median OS was not reached. Most treatment-related adverse events (TRAEs) were grade 1–2. Two pts discontinued EV due to TRAE (acute coronary syndrome and hyperglycemia/rash). Serum ADC and TAb concentrations peaked at end of EV infusion; MMAE concentrations had median peak 2–3 d post dose. Minimal ADC, TAb, and MMAE accumulation were observed with repeat EV dosing. Conclusions: EV-203 results show EV has a favorable benefit–risk profile, demonstrating meaningful clinical activity with a manageable safety profile, in pts with previously treated la/mUC in China. EV data are consistent with those from the global population in phase 2–3 EV trials of la/mUC. No new safety signals were identified. Clinical trial information: NCT04995419 .

The phenotype of Graves' orbitopathy is associated with thyrotropin receptor antibody levels.

Graves' orbitopathy (GO) is a specific inflammatory disorder of the orbit characterized by a highly heterogeneous clinical phenotype. The role of thyrotropin receptor antibodies (TSH-R-Ab) has been widely researched, however there is still no evidence that these antibodies have a direct pathogenic role in this pathology. The aim of this study was to examine their relation to the individual clinical features of GO. Ninety-one consecutive patients with GO were recruited. Total antibody concentration (TSH-R binding inhibitory immunoglobulins, TBII) and their functional activity (stimulating TSH-R-Ab, TSAb) were measured using binding immunoassay and cell-based bioassay, respectively. Both TSAb and TBII levels were significantly associated to the clinical parameters of GO activity. TSAb was a more sensitive serological marker compared to TBII pertaining to eyelid retraction and edema, proptosis, extra-orbital muscle disorders, diplopia, irritable eye symptoms, and photophobia. TSAb, but not TBII, was a significant predictive marker of conjunctival redness, chemosis, caruncle/plica inflammation, eye irritation, and orbital pain, (odds ratio: 3.096, p = 0.016; 5.833, p = 0.009; 6.443, p = 0.020; 3.167, p = 0.045; 2.893, p = 0.032; versus 2.187, p = 0.093; 2.775, p = 0.081; 3.824, p = 0.055; 0.952, p = 0.930; 2.226, p = 0.099, respectively). Neither TSAb nor TBII correlated with the level of proptosis (ρ = 0.259, p = 0.090, and ρ = 0.254, p = 0.104, respectively), however rising TSAb levels were strongly associated to the level of proptosis. TSH-R-Ab were significantly associated with GO's phenotype. Especially TSAb, as a sensitive and predictive serological biomarker, can improve diagnosis and management of GO.

Determining the Optimal SARS-CoV-2 mRNA Vaccine Dosing Interval for Maximum Immunogenicity.

Emerging evidence indicates that longer SARS-CoV-2 vaccine dosing intervals results in an enhanced immune response. However, the optimal vaccine dosing interval for achieving maximum immunogenicity is unclear. This study included samples from adult paramedics in Canada who received two doses of either BNT162b2 or mRNA-1273 vaccines and provided blood samples six months (170 to 190 days) after the first vaccine dose. The main exposure variable was vaccine dosing interval (days), categorized as "short" (first quartile), "moderate" (second quartile), "long" (third quartile), and "longest" interval (fourth quartile). The primary outcome was total spike antibody concentrations, measured using the Elecsys SARS-CoV-2 total antibody assay. Secondary outcomes included spike and receptor-binding domain (RBD) immunoglobulin G (IgG) antibody concentrations, and inhibition of angiotensin-converting enzyme 2 (ACE-2) binding to wild-type spike protein and several different Delta variant spike proteins. We fit a multiple log-linear regression model to investigate the association between vaccine dosing intervals and the antibody concentrations. A total of 564 adult paramedics (mean age 40 years, SD=10) were included. Compared to "short interval" (≤30 days), vaccine dosing intervals of the long (39-73 days) group (β= 0.31, 95% Confidence interval (CI): 0.10-0.52) and the longest (≥74 days) group (β = 0.82. 95% CI: 0.36-1.28) were associated with increased spike total antibody concentration. Compared to the short interval, the longest interval quartile was associated with higher spike IgG antibodies, while the long and longest intervals were associated with higher RBD IgG antibody concentrations. Similarly, the longest dosing intervals increased inhibition of ACE-2 binding to viral spike protein. Increased mRNA vaccine dosing intervals longer than 38 days result in higher levels of anti-spike antibodies and ACE-2 inhibition when assessed six months after the first COVID-19 vaccine.

307. Diagnostic Test Kinetics, Infectivity, and Immunological Responses Among Unvaccinated Adults During Acute SARS-CoV-2 Infection

Abstract Background Appropriate diagnostic testing can be used to inform infection control measures and reduce SARS-CoV-2 transmission, yet the test kinetics, infectivity, and immunological responses during acute, non-severe SARS-CoV-2 infection need clarity. Methods We conducted a prospective cohort study between Nov 2020-July 2021 in Seattle, Washington of 95 unvaccinated, immunocompetent adults with no prior SARS-CoV-2 infection. Nasal swabs (nasopharyngeal and anterior) and blood serum samples were serially collected at six visits over two months. Viral RNA, N and S antigen concentrations, and viral growth/infectivity were measured from nasal samples. Anti-S total antibody and IgG assays were performed on serum. We fit loess curves to quantitative data corresponding to each testing modality by days since symptom onset (DSSO) and compared qualitative test results across time points to demonstrate time-dependent agreement of PCR, N antigen, and culture results. Generalized estimating equations were used to approximate relative risk of culture positivity (a proxy for infectiousness) for positive vs. negative test results (antigen and PCR), stratified by presence/absence of symptoms and DSSO. Sampling Schema Nasal swabs and venous blood were collected at visits 1-4; venous blood only at visits 5-6. All participants were enrolled within 14 days of symptom onset (median: 6) and 7 days of a positive test (median: 4). Results Infections in this cohort (median age: 29y) were mild (no hospitalization). Median (IQR) time to negative result was 11 (4), 13 (6), and 20 (7) DSSO for culture growth, N antigen, and PCR tests, respectively. Viral RNA quantities declined more slowly than antigen and culturable virus; antibody titers rose rapidly 5-15 DSSO and plateaued 20-30 DSSO. All culture-positive samples collected 0-5 DSSO were positive by PCR, but relative risk of culture positivity (infectiousness) for positive vs. negative PCR results declined 6-10 DSSO. Relative risk of culture positivity for positive vs. negative antigen results was consistently high 0-10 DSSO, with similar results when stratified by presence of symptoms. Diagnostic test kinetics and immunological responses Diagnostic test kinetics and immunological responses measured in adults with non-severe, symptomatic SARS-CoV-2 infection: loess trendlines and 95% confidence intervals are given for SARS-CoV-2 viral load (calculated from PCR Ct value using a calibration curve), TCID50 from viral culture, mean concentrations of nucleocapsid and spike antigen proteins, and anti-S total and IgG antibody concentrations. Conclusion The results reinforce the importance of molecular PCR testing as a highly sensitive diagnostic tool but with limited utility as an indicator of viral culturability and likely infectiousness. N antigen testing may be a preferable diagnostic test within two weeks of symptom onset, especially 6-10 DSSO, because it more closely correlates with culture growth over the course of infection. Disclosures Daphne Hamilton, BA, Roche (spouse is employed by Roche): Employee Alexander L. Greninger, MD, PhD, Abbott: Contract Testing|Cepheid: Contract Testing|Gilead: Grant/Research Support|Gilead: Contract Testing|Hologic: Contract Testing|Merck: Grant/Research Support|Novavax: Contract Testing|Pfizer: Contract Testing Geoffrey S. Gottlieb, MD, PhD, Abbott Molecular Diagnostics: Grant/Research Support|Alere Technologies: Grant/Research Support|BMGF: Grant/Research Support|BMS: Grant/Research Support|Cerus Corp.: Grant/Research Support|Gilead Sciences: Grant/Research Support|Janssen Pharmaceutica: Grant/Research Support|Merck &amp; Co: Grant/Research Support|Roche Molecular Systems: Grant/Research Support|THERA Technologies/TaiMed Biologics: Grant/Research Support|ViiV Healthcare: Grant/Research Support.

SARS-CoV-2 seroprevalence and infection rate in Manila, Philippines prior to national vaccination program implementation: a repeated cross-sectional analysis

BackgroundSARS-CoV-2 seroepidemiological studies are used to guide public health decision making and to prepare for emerging infectious diseases. Disease occurrence estimates are limited in the Philippines, the country with the highest reported number of coronavirus disease-related deaths in the Western Pacific region. We aimed to estimate SARS-CoV-2 seroprevalence and infection rate among outpatient clinic attendees in Metro Manila prior to the implementation of the national coronavirus disease vaccination program.MethodsWe conducted repeated cross-sectional surveys at the animal bite clinic in San Lazaro Hospital, Manila, the Philippines across four periods, 3 months apart, between May 2020 and March 2021. Multivariable logistic regression was used to assess associations between different characteristics and infection status including seropositivity.ResultsIn total 615 participants were enrolled, ranging from 115 to 174 per period. Seroprevalence quadrupled between the first (11.3%) and second (46.8%) periods and plateaued thereafter (third—46.0%, fourth—44.6%). Among seropositive participants, total antibody concentration was comparable throughout the first to third periods but declined between the third and fourth periods. Infection prevalence was comparable across enrollment periods (range 2.9–9.5%). Post-secondary education [aOR 0.42 (95% CI 0.26, 0.67)] was protective, and frontline work [aOR 1.81 (95% CI 1.18, 2.80)] was associated with increased odds of seropositivity. Frontline work status [aOR 2.27 (95% CI 1.10, 4.75)] and large household size [aOR 2.45 (95% CI 1.18, 5.49)] were associated with increased odds of infection.ConclusionsThe quadrupling of seroprevalence over 3 months between the first and second enrollment periods coincided with the high burden of infection in Metro Manila in early 2020. Our findings suggest a limit to the rise and potential decline of population-level SARS-CoV-2 infection-induced immunity without introduction of vaccines. These results may add to our understanding of how immunity develops against emerging infectious diseases including coronaviruses.

A phase 1/2 study of REGN5093-M114, a METxMET antibody-drug conjugate, in patients with mesenchymal epithelial transition factor (MET)-overexpressing NSCLC.

TPS8593 Background: MET, also called hepatocyte growth factor receptor (HGFR), is a high-affinity transmembrane protein receptor for HGF. MET is overexpressed in various malignancies, including non-small cell lung cancer (NSCLC). MET overexpression can accompany MET exon 14 alteration or de novo/acquired MET amplification. REGN5093-M114 is an antibody drug conjugate composed of a novel linker-payload (M114, carrying the maytansine derivative M24, a potent inhibitor of microtubule assembly) covalently bound to lysine residues on a MET-targeting human IgG4p bispecific antibody, REGN5093. In preclinical models of MET overexpressing cancers, REGN5093-M114 demonstrated significant dose-dependent antitumor activity. Methods: This is an open label, phase 1/2, first-in-human, multicenter dose-escalation study with cohort expansion evaluating REGN5093-M114 in patients with MET-overexpressing NSCLC (NCT04982224). Patients must have advanced stage NSCLC for which there are no approved therapies available expected to confer clinical benefit, with tumor overexpressing MET (≥75% tumor cell staining at 2+) as centrally confirmed by immunohistochemistry. For the expansion phase, patients must have at least one lesion that is measurable by RECIST 1.1. REGN5093-M114 will be administered intravenously once every 3 weeks over 30 minutes until disease progression, intolerable adverse events, withdrawal of consent, or study withdrawal. The primary objectives in dose escalation are to evaluate safety, tolerability, PK, and maximum tolerated dose and/or recommended phase 2 dosing regimen of REGN5093-M114. PKs will include the assessment of REGN5093-M114, total antibody, and payload M24 concentrations. The primary objective in dose expansion is to assess preliminary anti-tumor activity of REGN5093-M114 in MET-overexpressing NSCLC as measured by the objective response rate. The secondary objectives of both phases of the study include an evaluation of treatment durability, and the immunogenicity of REGN5093-M114. This study is currently open to enrollment. Clinical trial information: NCT04982224.

Dynamics of specific antibodies in COVID-19 patients after recovery.

Abstract The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to an unprecedented global public health crisis. The objectives of this study were to analyse the dynamic trend in specific antibodies in the serum of patients infected with SARS-CoV-2 within 12 months after recovery and to make a preliminary assessment of the protective effect of vaccination. Eighty-seven patients with confirmed COVID-19 who were admitted to our hospital from January to February 2020 were followed after recovery. Three-millilitre blood samples were collected for specific antibody detection at four time points: 1, 6 and 12 months after recovery and 1 month after vaccination. The changes in specific immunoglobulin G (IgG) antibody and total antibody levels over 12 months were analysed. Moreover, an independent comparison of the neutralising antibody levels of patients after vaccination with those of healthy medical staff after vaccination was performed to compare the inhibition rates of the neutralising antibody to the virus. No statistically significant difference in the sex distribution between groups was observed (P &gt; 0.05). Older patients had a greater risk of developing severe and critical COVID-19 (P &lt; 0.05). The percentages of subjects positive for IgG antibodies at 1, 6 and 12 months after recovery were 88.5%, 75.9% and 50.6%, respectively. The rate of IgG antibody conversion from positive to negative was not uniform across time points: the change was slow in the first 6 months but increased significantly in the last 6 months (P &lt; 0.05). The positive rate of critically ill patients in the first 6 months was 100.0%. The trend over time in total antibody levels was similar to that of IgG antibody levels. Over 12 months, the sample/cut off value of total antibodies continued to decrease, while that of different disease severities was significantly different (P &lt; 0.05). After vaccine administration, the total antibody level exceeded the detection level in the first month, which was independent of disease severity (P &gt; 0.05). Significant differences were observed in the inhibition rate of the neutralising antibody against the virus in the disease group and the control group (P &lt; 0.05). IgG antibody produced by patients naturally infected with SARS-CoV-2 has a duration of no less than 1 year, and the change trend graph of total antibody levels was the same as that of IgG antibody levels. Under vaccine stimulation, the positive rate of IgG antibody was as high as 100%, and the total antibody concentration reached the highest level, which was independent of disease severity. Neutralising antibodies following vaccination in patients who recovered from COVID-19 had a higher inhibition rate against SARS-CoV-2 than those of vaccinated healthy controls, indicating that these COVID-19 patients had a lower risk of reinfection and were better protected.

Autoantibodies are present in the bronchoalveolar lavage but not circulation in patients with fibrotic interstitial lung disease.

BackgroundFibrotic interstitial lung disease (fILD) has previously been associated with the presence of autoantibody. While studies have focused on systemic autoimmunity, the role of local autoantibodies in the airways remains unknown. We therefore extensively characterised the airway and peripheral autoantibody profiles in patients with fILD, and assessed association with disease severity and outcome.MethodsBronchoalveolar lavage (BAL) fluid was collected from a cohort of fILD patients and total BAL antibody concentrations were quantified. An autoantigen microarray was used to measure IgG and IgA autoantibodies against 122 autoantigens in BAL from 40 idiopathic pulmonary fibrosis (IPF), 20 chronic hypersensitivity pneumonitis (CHP), 20 connective tissue disease-associated ILD (CTD-ILD) patients and 20 controls.ResultsA subset of patients with fILD but not healthy controls had a local autoimmune signature in their BAL that was not present systemically, regardless of disease. The proportion of patients with IPF with a local autoantibody signature was comparable to that of CTD-ILD, which has a known autoimmune pathology, identifying a potentially novel subset of patients. The presence of an airway autoimmune signature was not associated with reduced survival probability or changes in lung function in the cohort as a whole. Patients with IPF had increased BAL total IgA and IgG1 while subjects with CHP had increased BAL IgA, IgG1 and IgG4. In patients with CHP, increased BAL total IgA was associated with reduced survival probability.ConclusionAirway autoantibodies that are not present systemically identify a group of patients with fILD and the mechanisms by which these autoantibodies contribute to disease requires further investigation.

Multicentric evaluation of a novel point of care electrochemical ELISA platform for SARS-CoV-2 specific IgG and IgM antibody assay

New diagnostics technologies for the efficient detection and quantification of SARS-CoV-2 antibodies are very crucial to manage the COVID-19 pandemic, especially in the context of emerging vaccination paradigms. Herein, we report on a novel point-of-care Electrochemical ELISA platform with disposable screen printed electrodes functionalized with SARS-CoV-2 Spike Glycoprotein S1, to enable fast and accurate quantitative estimation of total antibody concentration (IgG and IgM) in clinical samples. The quantification is performed with a comparison of electrochemical redox current against the current produced by the spiked monoclonal antibodies with known concentration. The assay is validated through multicentric evaluation against 3 different FDA authorized Laboratory standard techniques, using both EDTA whole blood and serum samples. We demonstrate that the proposed assay has excellent sensitivity and specificity, making it a suitable candidate for epidemiological surveys and quantification of antibodies in COVID-19 vaccination programs.

Development of a rapid and sensitive quantum dot nanobead-based double-antigen sandwich lateral flow immunoassay and its clinical performance for the detection of SARS-CoV-2 total antibodies

Owing to the over-increasing demands in resisting and managing the coronavirus disease 2019 (COVID-19) pandemic, development of rapid, highly sensitive, accurate, and versatile tools for monitoring total antibody concentrations at the population level has been evolved as an urgent challenge on measuring the fatality rate, tracking the changes in incidence and prevalence, comprehending medical sequelae after recovery, as well as characterizing seroprevalence and vaccine coverage. To this end, herein we prepared highly luminescent quantum dot nanobeads (QBs) by embedding numerous quantum dots into polymer matrix, and then applied it as a signal-amplification label in lateral flow immunoassay (LFIA). After covalently linkage with the expressed recombinant SARS-CoV-2 spike protein (RSSP), the synthesized QBs were used to determine the total antibody levels in sera by virtue of a double-antigen sandwich immunoassay. Under the developed condition, the QB-LFIA can allow the rapid detection of SARS-CoV-2 total antibodies within 15 min with about one order of magnitude improvement in analytical sensitivity compared to conventional gold nanoparticle-based LFIA. In addition, the developed QB-LFIA performed well in clinical study in dynamic monitoring of serum antibody levels in the whole course of SARS-CoV-2 infection. In conclusion, we successfully developed a promising fluorescent immunological sensing tool for characterizing the host immune response to SARS-CoV-2 infection and confirming the acquired immunity to COVID-19 by evaluating the SRAS-CoV-2 total antibody level in the crowd.

Simple, rapidly electroassembled thiolated PEG‐based sensor interfaces enable rapid interrogation of antibody titer and glycosylation

Process conditions established during the development and manufacture of recombinant protein therapeutics dramatically impacts their quality and clinical efficacy. Technologies that enable rapid assessment of product quality are critically important. Here, we describe the development of sensor interfaces that directly connect to electronics and enable near real-time assessment of antibody titer and N-linked galactosylation. We make use of a spatially resolved electroassembled thiolated polyethylene glycol hydrogel that enables electroactivated disulfide linkages. For titer assessment, we constructed a cysteinylated protein G that can be linked to the thiolated hydrogel allowing for robust capture and assessment of antibody concentration. For detecting galactosylation, the hydrogel is linked with thiolated sugars and their corresponding lectins, which enables antibody capture based on glycan pattern. Importantly, we demonstrate linear assessment of total antibody concentration over an industrially relevant range and the selective capture and quantification of antibodies with terminal β-galactose glycans. We also show that the interfaces can be reused after surface regeneration using a low pH buffer. Our functionalized interfaces offer advantages in their simplicity, rapid assembly, connectivity to electronics, and reusability. As they assemble directly onto electrodes that also serve as I/O registers, we envision incorporation into diagnostic platforms including those in manufacturing settings.

Evidence that seasonal malaria chemoprevention with SPAQ influences blood and pre-erythrocytic stage antibody responses of Plasmodium falciparum infections in Niger

BackgroundIn endemic areas, children develop slowly and naturally anti-Plasmodium antibodies and become semi-immune. Seasonal Malaria Chemoprevention (SMC) with sulfadoxine-pyrimethamine + amodiaquine (SPAQ) is a new strategy to reduce malaria morbidity in West African young children. However, SMC may impact on the natural acquisition of anti-Plasmodium immunity. This paper evaluates the effect of SMC with SPAQ on antibody concentration in young children from Niger.MethodsThis research was conducted in areas benefitting from SMC since 2014 (Zinder district), without SMC (Dosso district), and with 1 year of SMC since 2016 (Gaya district). To assess the relationship between SMC and Plasmodium falciparum IgG antibody responses, the total antibody concentrations against two P. falciparum asexual stage vaccine candidate antigens, circumsporozoite protein (CSP) and glutamate-rich protein R2 (GLURP-R2), in children aged 3 to 59 months across the three areas were compared. Antibody concentrations are quantified using an enzyme-linked immunosorbent assay on the elution extracted from positive and negative malaria Rapid Diagnostic Test cassettes.ResultsThe analysis concerns two hundred and twenty-nine children aged from 3 to 59 months: 71 in Zinder, 77 in Dosso, and 81 in Gaya. In Zinder (CSP = 17.5 µg/ml and GLURP-R2 = 14.3 µg/ml) median antibody concentration observed are higher than in Gaya (CSP = 7.7 µg/ml and GLURP-R2 = 6.5 µg/ml) and Dosso (CSP = 4.5 µg/ml and GLURP-R2 = 3.6 µg/ml) (p < 0.0001).ConclusionThe research reveals some evidences which show that seasonal malaria chemoprevention with SPAQ has an effect on blood stage antibody responses and pre-erythrocytic stage of P. falciparum infections in Niger. Increased antibody titres with increased SMC/SPAQ implementation. This contradicts hypothesis that SMC/SPAQ could reduce immunity to erythrocyte and liver-stage antigens. Further studies are necessary to provide better understanding of the SMC effect on malaria immunity.

Binding affinity determination of therapeutic antibodies to membrane protein targets: Kinetic Exclusion Assay using cellular membranes for anti-CD20 antibody

Antibody-based therapeutics targeting membrane proteins have evolved as a major modality for the treatment of cancer, inflammation and autoimmune diseases. There are numerous challenges, ranging from desired epitope expression to reliable binding/functional assays which are associated with developing antibodies for this target class. Specifically, having a robust methodology for characterizing antibody interaction with a membrane protein target is essential for providing guidance on dosing, potency and thus expected efficacy. Fluorescence-activated cell sorting (FACS) has been commonly used to characterize antibodies binding to membrane protein targets. FACS provides information about the antibody-receptor complex (antibody bound to cells) and the apparent equilibrium dissociation constant (KD′) is elucidated by fitting the antibody-receptor binding isotherm as a function of total antibody concentration to a nonlinear regression model. Conversely, Kinetic Exclusion Assay (KinExA) has been used to measure solution-based equilibrium dissociation constant (KD) of antibodies. Here, KD is determined by measuring the free antibody concentration at equilibrium in a series of solutions in which the antibody is at constant concentration and the receptor (either in the membrane or the cell) is titrated. We measured the binding affinity of the anti-CD20 antibody, Rituximab, using both FACS and KinExA. There was ~25-fold difference in the binding affinity measured by these two techniques. We have explored this discrepancy through additional experiments around the mathematical framework involved in the analysis of these two different binding assays. Finally, our study concluded that KinExA enables accurate measurement of the KD for strong protein-protein interactions (sub-nanomolar values) compared to FACS.

Abstract CT125: STRO-002-GM1, a first in human, phase 1 study of STRO-002, an anti-folate receptor alpha (FRα) antibody drug conjugate (ADC), in patients with advanced platinum-resistant/refractory epithelial ovarian cancer (OC), including fallopian tube or primary peritoneal cancers

Abstract Introduction: FRα is overexpressed in 80% of OC with minimal expression in normal tissues. STRO-002 is a novel FRα-targeting ADC designed to circumvent limitations of current ADCs. STRO-002 was generated with a cell-free antibody production system (XpressCF™) and site-specific conjugation (XpressCF+™) platform. STRO-002 has a DAR of 4 and contains the tubulin-targeting 3-aminophenyl hemiasterlin warhead SC209, a potent cytotoxin that is rapidly cleared and a weak substrate for P-gp efflux pumps. STRO-002 demonstrated potent cytotoxic activity in multiple FRα-positive tumor cell lines and significant efficacy in OC xenograft models. Herein we report the results of our dose escalation study of STRO-002 in patients (pts) with OC. Methods: STRO-002-GM1 is a Phase I, open-label, multicenter, dose escalation study with dose expansion to identify the maximum tolerated dose (MTD), and recommended Phase II dose (RP2D) in pts with advanced, platinum resistant or refractory OC. There is no patient selection based on FRα expression. STRO-002 is given IV on Day 1 of each 21-day cycle (C) until disease progression. Ocular exams are performed at baseline and every other cycle. No prophylactic corticosteroid eye drops are used. Results: 20 pts have been dosed &amp; 18 pts have completed at least 1 cycle of STRO-002 at 7 dose levels: 0.5, 1.0, 1.8, 2.9, 4.3, 5.2 and 6.0 mg/kg. Median age is 60.5 (r, 48-70). Median ECOG status is 0 (r, 0-1). Median # of prior therapies is 6 (r, 2-10). 55% of pts have prior PARP inhibitors, 85% have prior bevacizumab. Median # of STRO-002 cycles is 3 (r, 1-15). One DLT of grade 3 neuropathy was reported at the 6.0 mg/kg dose level. Two patients have experienced grade 4 neutropenia, an expected and reversible side effect of STRO-002 treatment. 92% of AEs are grade 1 or 2. The most common TEAEs in ≥ 30 % of pts includes fatigue, nausea, decreased appetite and dizziness. No ocular findings have been observed. Emerging PK profile: after 1 hour infusion, total antibody concentrations declined in a log-linear manner with a 22-84 h half-life, dose-exposure relationship appeared linear without accumulation after repeat administration. Six pts have discontinued due to disease progression. 14 pts remain on treatment and dose escalation is ongoing. One patient has achieved a confirmed partial response (PR); 2 patients have confirmed stable disease (cSD) at C5, 2 pts have cSD at C7 and 1 pt has cSD at C13; 3 patients have achieved a confirmed CA-125 response. Conclusions: STRO-002 is the first hemiasterlin-containing ADC generated with cell-free protein synthesis technology to be tested in pts with solid tumors. The preliminary safety profile and evidence of efficacy and clinical benefit in the first 20 OC pts treated with STRO-002 is encouraging, particularly in this heavily pre-treated, platinum resistant/refractory patient population. No ocular toxicity signals have been observed. MTD has not been reached. Updated results and the recommended phase 2 dose will be presented. This study is registered with clinicaltrials.gov identifier NCT03748186. Citation Format: R. Wendel Naumann, Denise Uyar, John W. Moroney, Fadi S. Braiteh, Russell J. Schilder, John P. Diaz, Erika Hamilton, Sami Diab, Lainie P. Martin, David M. O'Malley, Richard T. Penson, Clifford DiLea, Michael Palumbo, Venita DeAlmeida, Shannon Matheny, Arturo Molina. STRO-002-GM1, a first in human, phase 1 study of STRO-002, an anti-folate receptor alpha (FRα) antibody drug conjugate (ADC), in patients with advanced platinum-resistant/refractory epithelial ovarian cancer (OC), including fallopian tube or primary peritoneal cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT125.

A novel TLR adjuvant increases the efficacy of vaccines against fentanyl-induced toxicity and overdose

Abstract The widespread illicit use of fentanyl and its analogs is associated with the increased incidence of fatal drug overdoses in the United States in recent years. Pre-clinical studies with vaccines targeting opioids show selectivity and efficacy but clinical data suggests vaccine efficacy against drugs of abuse may be limited by individual variability and a requirement for high and sustained antibody levels. New strategies are needed to enhance vaccine efficacy and increase the quantity and quality of the anti-drug antibody response. We investigated the ability of novel synthetic TLR adjuvants to increase anti-fentanyl antibody titers and protect from fentanyl challenge in mice. Fentanyl-based haptens were conjugated to CRM and combined with alum, novel synthetic TLR adjuvants, or combinations thereof. The most promising TLR adjuvants and combination adjuvants increased anti-fentanyl hapten total IgG, IgG1, and IgG2a antibody concentrations. Splenocyte restimulation with CRM protein showed adjuvant combinations significantly increased secretion of Th1-type cytokines compared to antigen only or single adjuvants alone. Likewise, in a fentanyl challenge model, combination adjuvanted vaccines increased protection from respiratory depression compared to mice vaccinated with antigen alone or single adjuvant controls. These data demonstrate that TLR adjuvants improve the anti-fentanyl adaptive immune response and increase protection against fentanyl challenge in mice, and may enhance vaccine-induced protection against fentanyl overdose in humans.

In vitro cytotoxicity of Auger electron-emitting [67Ga]Ga-trastuzumab

IntroductionMolecular radiotherapy exploiting short-range Auger electron-emitting radionuclides has potential for targeted cancer treatment and, in particular, is an attractive option for managing micrometastatic disease. Here, an approach using chelator-trastuzumab conjugates to target radioactivity to breast cancer cells was evaluated as a proof-of-concept to assess the suitability of 67Ga as a therapeutic radionuclide. MethodsTHP-trastuzumab and DOTA-trastuzumab were synthesised and radiolabelled with Auger electron-emitters 67Ga and 111In, respectively. Radiopharmaceuticals were tested for HER2-specific binding and internalisation, and their effects on viability (dye exclusion) and clonogenicity of HER2-positive HCC1954 and HER2–negative MDA-MB-231 cell lines was measured. Labelled cell populations were studied by microautoradiography. ResultsLabelling efficiencies for [67Ga]Ga-THP-trastuzumab and [111In]In-DOTA-trastuzumab were 90% and 98%, respectively, giving specific activities 0.52 ± 0.16 and 0.61 ± 0.11 MBq/μg (78–92 GBq/μmol). At 4 nM total antibody concentration and 200 × 103 cells/mL, [67Ga]Ga-THP-trastuzumab showed higher percentage of cell association (10.7 ± 1.3%) than [111In]In-DOTA-trastuzumab (6.2 ± 1.6%; p = 0.01). The proportion of bound activity that was internalised did not differ significantly for the two tracers (62.1 ± 1.4% and 60.8 ± 15.5%, respectively). At 100 nM, percentage cell binding of both radiopharmaceuticals was greatly reduced compared to 4 nM and did not differ significantly between the two (1.2 ± 1.0% [67Ga]Ga-THP-trastuzumab and 0.8 ± 0.9% for [111In]In-DOTA-trastuzumab). Viability and clonogenicity of HER2-positive cells decreased when each radionuclide was incorporated into cells by conjugation with trastuzumab, but not when the same level of radioactivity was confined to the medium by omitting the antibody conjugation, suggesting that 67Ga needs to be cell-bound or internalised for a therapeutic effect. Microautoradiography showed that radioactivity bound to individual cells varied considerably within the population. Conclusions[67Ga]Ga-THP-trastuzumab reduced cell viability and clonogenicity only when cell-bound, suggesting 67Ga holds promise as a therapeutic radionuclide as part of a targeted radiopharmaceutical. The causes and consequences of non-homogeneous uptake among the cell population should be explored.