Subcutaneous Administration of Monoclonal Antibodies: Pharmacology, Delivery, Immunogenicity, and Learnings From Applications to Clinical Development.

Subcutaneous (s.c.) administration of monoclonal antibodies (mAbs) can reduce treatment burden for patients and healthcare systems compared with intravenous (i.v.) infusion through shorter administration times, made possible by convenient, patient-centric devices. A deeper understanding of clinical pharmacology principles related to efficacy and safety of s.c.-administered mAbs over the past decade has streamlined s.c. product development. This review presents learnings from key constituents of the s.c. mAb development pathway, including pharmacology, administration variables, immunogenicity, and delivery devices. Restricted mAb transportation through the hypodermis explains their incomplete absorption at a relatively slow rate (pharmacokinetic (PK)) and may impact mAb-cellular interactions and/or onset and magnitude of physiological responses (pharmacodynamic). Injection volumes, formulation, rate and site of injection, and needle attributes may affect PKs and the occurrence/severity of adverse events like injection-site reactions or pain, with important consequences for treatment adherence. A review of immunogenicity data for numerous compounds reveals that incidence of anti-drug antibodies (ADAs) is generally comparable across i.v. and s.c. routes, and complementary factors including response magnitude (ADA titer), persistence over time, and neutralizing antibody presence are needed to assess clinical impact. Finally, four case studies showcase how s.c. biologics have been clinically developed: (i) by implementation of i.v./s.c. bridging strategies to streamline PD-1/PD-L1 inhibitor development, (ii) through co-development with i.v. presentations for anti-severe acute respiratory syndrome-coronavirus 2 antibodies to support rapid deployment of both formulations, (iii) as the lead route for bispecific T cell engagers (BTCEs) to mitigate BTCE-mediated cytokine release syndrome, and (iv) for pediatric patients in the case of dupilumab.

Immunogenicity of Cemiplimab: Low Incidence of Antidrug Antibodies and Cut-Point Suitability Across Tumor Types.

The immunogenicity of cemiplimab, a fully human immunoglobulin G4 monoclonal antibody directed against programmed cell death 1, was assessed in patients across multiple tumor types. The development of antidrug antibodies (ADAs) against cemiplimab was monitored using a validated bridging immunoassay. To identify ADA-positive samples in the assay, statistically determined cut points were established by analyzing baseline clinical study samples from a mixed population of different tumor types, and this validation cut point was used to assess immunogenicity in all subsequent studies. Regulatory guidance requires that ADA assay cut points be verified for appropriateness in different patient populations. Thus, for the cemiplimab ADA assay, we evaluated whether each new oncology population was comparable with the validation population used to set the cut point. Assay responses from 2393 individual serum samples from 8 different tumor types were compared with the validation population, using established statistical methods for cut-point determination and comparison, with no significant differences observed. Across tumor types, the immunogenicity of cemiplimab was low, with an overall treatment-emergent ADA incidence rate of 1.9% and 2.5% at intravenous dose regimens of 3 mg/kg every 2 weeks and 350 mg every 3 weeks, respectively. Moreover, no neutralizing antibodies to cemiplimab were detected in patients with ADA-positive samples, and there was no observed impact of cemiplimab ADAs on pharmacokinetics. Study-specific cut points may be required in some diseases, such as immune and inflammatory diseases; however, based on this analysis, in-study cut points are not required for each new oncology disease indication for cemiplimab.

Anti-drug Antibody Sample Testing and Reporting Harmonization.

A clear scientific and operational need exists for harmonized bioanalytical immunogenicity study reporting to facilitate communication of immunogenicity findings and expedient review by industry and health authorities. To address these key bioanalytical reporting gaps and provide a report structure for documenting immunogenicity results, this cross-industry group was formed to establish harmonized recommendations and a develop a submission template to facilitate agency filings. Provided here are recommendations for reporting clinical anti-drug antibody (ADA) assay results using ligand-binding assay technologies. This publication describes the essential bioanalytical report (BAR) elements such as the method, critical reagents and equipment, study samples, results, and data analysis, and provides a template for a suggested structure for the ADA BAR. This publication focuses on the content and presentation of the bioanalytical ADA sample analysis report. The interpretation of immunogenicity data, including the evaluation of the impact of ADA on safety, exposure, and efficacy, is out of scope of this publication.

Interference in a Neutralizing Antibody Assay for Odronextamab, a CD20xCD3 Bispecific mAb, from Prior Rituximab Therapy and Possible Mitigation Strategy.

A cell-based assay was developed to detect neutralizing anti-drug antibodies (NAbs) against odronextamab, a CD20xCD3 bispecific monoclonal antibody (mAb) under investigation for treatment of CD20+ B cell malignancies. In this assay, odronextamab bridges between two cell types, CD20-expressing HEK293 cells and CD3-expressing Jurkat T cells that generate a luciferase signal upon CD3 clustering. Patient samples containing NAbs directed to either arm of the bispecific drug block the odronextamab bridge formation between the cell lines thus preventing the generation of the luciferase signal. We determined that other anti-CD20 therapeutics also block bridge formation, resulting in false-positive results. In patient samples from odronextamab clinical trials, approximately 30% of baseline samples had a strong false-positive NAb signal that correlated with the presence of prior rituximab (anti-CD20) therapy. We determined that rituximab interference can be minimized by the addition of anti-rituximab antibodies in the NAb assay. Understanding and mitigating the impact of prior biologic exposure is increasingly important for implementing a successful bioanalytical strategy to support clinical drug development, especially in the immuno-oncology field. Odronextamab neutralizing antibody assay, interference, and mitigation. A Design of the odronextamab neutralizing antibody (NAb) assay where anti-CD20xCD3 drug bridges between CD20-expressing HEK293 cells and Jurkat T cells expressing an NFAT response element and luciferase reporter. True NAb prevents odronextamab from bridging between target and effector cells, thus preventing the expression of luciferase. B Interference with odronextamab from other anti-CD20 therapeutic antibodies (e.g., rituximab) from prior disease treatment generates a false-positive NAb result. Assay interference can be mitigated with an anti-idiotypic antibody against the interfering therapy.

REGEN-COV® antibody cocktail bioanalytical strategy: comparison of LC-MRM-MS and immunoassay methods for drug quantification.

Aim: In response to the COVID-19 pandemic, Regeneron developed the anti-SARS-CoV-2 monoclonal antibody cocktail, REGEN-COV® (RONAPREVE® outside the USA). Drug concentration data was important for determination of dose, so a two-part bioanalytical strategy was implemented to ensure the therapy was rapidly available for use. Results & methodology: Initially, a liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS) assay, was used to analyze early-phase study samples. Subsequently, a validated electrochemiluminescence (ECL) immunoassay was implemented for high throughput sample analysis for all samples. A comparison of drug concentration data from the methods was performed which identified strong linear correlations and for Bland-Altman, small bias. In addition, pharmacokinetic data from both methods produced similar profiles and parameters. Discussion & conclusion: This novel bioanalytical strategy successfully supported swift development of a critical targeted therapy during the COVID-19 public health emergency.

Bioanalytical Challenges due to Prior Checkpoint Inhibitor Exposure: Interference and Mitigation in Drug Concentration and Immunogenicity Assays.

Monoclonal antibodies (mAbs) are a leading class of biotherapeutics. In oncology, patients often fail on early lines of biologic therapy to a specific target. Some patients may then enroll in a new clinical trial with a mAb specific for the same target. Therefore, immunoassays designed to quantify the current mAb therapy or assess immunogenicity to the drug may be susceptible to cross-reactivity or interference with residual prior biologics. The impact of two approved anti-PD-1 mAbs, pembrolizumab and nivolumab, was tested in several immunoassays for cemiplimab, another approved anti-PD-1 mAb. The methods included a target-capture drug concentration assay, a bridging anti-drug antibody (ADA) assay and a competitive ligand-binding neutralizing antibody (NAb) assay. We also tested bioanalytical strategies to mitigate cross-reactivity or interference in these assays from other anti-PD-1 biologics. Both pembrolizumab and nivolumab cross-reacted in the cemiplimab drug concentration assay. This was mitigated by addition of antibodies specific to pembrolizumab or nivolumab. ADA specific for pembrolizumab and nivolumab did not interfere in the cemiplimab ADA assay. However, pembrolizumab and nivolumab generated a false-positive response in a target-capture NAb assay. Our results demonstrate that similar exogenous pre-existing anti-PD-1 mAbs (biotherapeutics) such as pembrolizumab and nivolumab are detected and accurately quantified in the cemiplimab drug concentration assay. However, once steady state is achieved for the new therapy, prior biologics would likely not be detected. Cross-reactivity and interference in immunoassays from previous treatment with class-specific biotherapeutic(s) pose significant bioanalytical challenges, especially in immuno-oncology.

Liquid Chromatography-Multiple Reaction Monitoring-Mass Spectrometry Assay for Quantitative Measurement of Therapeutic Antibody Cocktail REGEN-COV Concentrations in COVID-19 Patient Serum.

REGEN-COV is a cocktail of two human IgG1 monoclonal antibodies (REGN10933 + REGN10987) that targets severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and has shown great promise to reduce the SARS-CoV-2 viral load in COVID-19 patients enrolled in clinical studies. A liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS)-based method, combined with trypsin and rAspN dual enzymatic digestion, was developed for the determination of total REGN10933 and total REGN10987 concentrations in several hundreds of pharmacokinetic (PK) serum samples from COVID-19 patients participating in phase I, II, and III clinical studies. The performance characteristics of this bioanalytical assay were evaluated with respect to linearity, accuracy, precision, selectivity, specificity, and analyte stability before and after enzymatic digestion. The developed LC-MRM-MS assay has a dynamic range from 10 to 2000 µg/mL antibody drug in the human serum matrix, which was able to cover the serum drug concentration from day 0 to day 28 after drug administration in two-dose groups for the clinical PK study of REGEN-COV. The concentrations of REGEN-COV in the two-dose groups measured by the LC-MRM-MS assay were comparable to the concentrations measured by a fully validated electrochemiluminescence (ECL) immunoassay.

Molecular mechanisms linking high dose medroxyprogesterone with HIV-1 risk.

BACKGROUND: Epidemiological studies suggest that medroxyprogesterone acetate (MPA) may increase the risk of HIV-1. The current studies were designed to identify potential underlying biological mechanisms. METHODS: Human vaginal epithelial (VK2/E6E7), peripheral blood mononuclear (PBMC), and polarized endometrial (HEC-1-A) cells were treated with a range of concentrations of MPA (0.015-150 µg/ml) and the impact on gene expression, protein secretion, and HIV infection was evaluated. RESULTS: Treatment of VK2/E6E7 cells with high doses (>15 µg/ml] of MPA significantly upregulated proinflammatory cytokines, which resulted in a significant increase in HIV p24 levels secreted by latently infected U1 cells following exposure to culture supernatants harvested from MPA compared to mock-treated cells. MPA also increased syndecan expression by VK2/E6E7 cells and cells treated with 15 µg/ml of MPA bound and transferred more HIV-1 to T cells compared to mock-treated cells. Moreover, MPA treatment of epithelial cells and PBMC significantly decreased cell proliferation resulting in disruption of the epithelial barrier and decreased cytokine responses to phytohaemagglutinin, respectively. CONCLUSION: We identified several molecular mechanisms that could contribute to an association between DMPA and HIV including proinflammatory cytokine and chemokine responses that could activate the HIV promoter and recruit immune targets, increased expression of syndecans to facilitate the transfer of virus from epithelial to immune cells and decreased cell proliferation. The latter could impede the ability to maintain an effective epithelial barrier and adversely impact immune cell function. However, these responses were observed primarily following exposure to high (15-150 µg/ml) MPA concentrations. Clinical correlation is needed to determine whether the prolonged MPA exposure associated with contraception activates these mechanisms in vivo.

Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays.

The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFP enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs.

Research gaps in defining the biological link between HIV risk and hormonal contraception.

Epidemiologic data suggest an association between depot medroxyprogesterone acetate (DMPA), a progesterone-based hormonal contraceptive, and increased risk of HIV acquisition and transmission. DMPA is highly effective and is among the most commonly used form of hormonal contraception in areas of high HIV prevalence. Thus, defining the biological mechanisms that contribute to the potential negative synergy between DMPA and HIV is key and may facilitate the identification of alternative contraceptive strategies. Proposed mechanisms include thinning or disruption of the cervicovaginal epithelial barrier, induction of mucosal inflammation, interference with innate and adaptive soluble and cellular immune responses, and/or alterations in the vaginal microbiome. DMPA may also indirectly increase the risk of HIV by promoting genital herpes or other sexually transmitted infections. However, there is a paucity of rigorous in vitro, animal model and clinical data to support these potential mechanisms highlighting the need for future research.

An ITAM in a nonenveloped virus regulates activation of NF-κB, induction of beta interferon, and viral spread.

Immunoreceptor tyrosine-based activation motifs (ITAMs) are signaling domains located within the cytoplasmic tails of many transmembrane receptors and associated adaptor proteins that mediate immune cell activation. ITAMs also have been identified in the cytoplasmic tails of some enveloped virus glycoproteins. Here, we identified ITAM sequences in three mammalian reovirus proteins: µ2, σ2, and λ2. We demonstrate for the first time that µ2 is phosphorylated, contains a functional ITAM, and activates NF-κB. Specifically, µ2 and µNS recruit the ITAM-signaling intermediate Syk to cytoplasmic viral factories and this recruitment requires the µ2 ITAM. Moreover, both the µ2 ITAM and Syk are required for maximal µ2 activation of NF-κB. A mutant virus lacking the µ2 ITAM activates NF-κB less efficiently and induces lower levels of the downstream antiviral cytokine beta interferon (IFN-ß) than does wild-type virus despite similar replication. Notably, the consequences of these µ2 ITAM effects are cell type specific. In fibroblasts where NF-κB is required for reovirus-induced apoptosis, the µ2 ITAM is advantageous for viral spread and enhances viral fitness. Conversely, in cardiac myocytes where the IFN response is critical for antiviral protection and NF-κB is not required for apoptosis, the µ2 ITAM stimulates cellular defense mechanisms and diminishes viral fitness. Together, these results suggest that the cell type-specific effect of the µ2 ITAM on viral spread reflects the cell type-specific effects of NF-κB and IFN-ß. This first demonstration of a functional ITAM in a nonenveloped virus presents a new mechanism for viral ITAM-mediated signaling with likely organ-specific consequences in the host.

A single-amino-acid polymorphism in reovirus protein µ2 determines repression of interferon signaling and modulates myocarditis.

Myocarditis is indicated as the second leading cause of sudden death in young adults. Reovirus induces myocarditis in neonatal mice, providing a tractable model system for investigation of this important disease. Alpha/beta-interferon (IFN-α/ß) treatment improves cardiac function and inhibits viral replication in patients with chronic myocarditis, and the host IFN-α/ß response is a determinant of reovirus strain-specific differences in induction of myocarditis. Virus-induced IFN-ß stimulates a signaling cascade that establishes an antiviral state and further induces IFN-α/ß through an amplification loop. Reovirus strain-specific differences in induction of and sensitivity to IFN-α/ß are associated with the viral M1, L2, and S2 genes. The reovirus M1 gene-encoded µ2 protein is a strain-specific repressor of IFN-ß signaling, providing one possible mechanism for the variation in resistance to IFN and induction of myocarditis between different reovirus strains. We report here that µ2 amino acid 208 determines repression of IFN-ß signaling and modulates reovirus induction of IFN-ß in cardiac myocytes. Moreover, µ2 amino acid 208 determines reovirus replication, both in initially infected cardiac myocytes and after viral spread, by regulating the IFN-ß response. Amino acid 208 of µ2 also influences the cytopathic effect in cardiac myocytes after spread. Finally, µ2 amino acid 208 modulates myocarditis in neonatal mice. Thus, repression of IFN-ß signaling mediated by reovirus µ2 amino acid 208 is a determinant of the IFN-ß response, viral replication and damage in cardiac myocytes, and myocarditis. These results demonstrate that a single amino acid difference between viruses can dictate virus strain-specific differences in suppression of the host IFN-ß response and, consequently, damage to the heart.