Addressing Domain Specificity in the Development of a Cell-Based Binding Assay for the Detection of Neutralizing Antibodies Against a CD47xPD-L1 Bispecific Antibody.

PF-07257876 is a bispecific antibody being developed for the treatment of certain advanced or metastatic solid tumors. To support clinical development of PF-07257876, neutralizing antibody (NAb) assays were developed as part of a tiered immunogenicity testing approach. Because PF-07257876 targets both CD47 and PD-L1, determination of domain specificity of a NAb response may provide additional insight relating to PK, efficacy, and safety. Due to limitations of functional cell systems, two cell-based binding assays were developed using electrochemiluminescence to detect domain-specific NAb. While both NAb assays utilized a cell-based binding approach and shared certain requirements, such as sensitivity and tolerance to potentially interfering substances, the development of each assay faced unique challenges. Among the hurdles encountered, achieving drug tolerance while preserving domain specificity for CD47 proved particularly challenging. Consequently, a sample pretreatment procedure to isolate NAb from potentially interfering substances was necessary. The sample pretreatment procedure developed was based on a bead-extraction and acid dissociation (BEAD) approach. However, the use of the standard BEAD approach with whole drug to capture NAb resulted in loss of NAb detection under certain circumstances. Specifically, mock samples containing a mixture of NAb positive controls against both binding domains of the bispecific antibody produced false-negative results in the cell-based binding assay. An adaptation made to the standard BEAD approach restored domain-specific NAb detection, while also contributing to an assay sensitivity of 1 µg/mL in the presence of a clinically relevant drug tolerance level of up to 400 µg/mL.

Blocking TNF signaling may save lives in COVID-19 infection.

Global vaccination effort and better understanding of treatment strategies provided a ray of hope for improvement in COVID-19 pandemic, however, in many countries, the disease continues to collect its death toll. The major pathogenic mechanism behind severe cases associated with high mortality is the burst of pro-inflammatory cytokines TNF, IL-6, IFNγ and others, resulting in multiple organ failure. Although the exact contribution of each cytokine is not clear, we provide an evidence that the central mediator of cytokine storm and its devastating consequences may be TNF. This cytokine is known to be involved in activated blood clotting, lung damage, insulin resistance, heart failure, and other conditions. A number of currently available pharmaceutical agents such as monoclonal antibodies and soluble TNF receptors can effectively prevent TNF from binding to its receptor(s). Other drugs are known to block NFkB, the major signal transducer molecule used in TNF signaling, or to block kinases involved in downstream activation cascades. Some of these medicines have already been selected for clinical trials, but more work is needed. A simple, rapid, and inexpensive method of directly monitoring TNF levels may be a valuable tool for a timely selection of COVID-19 patients for anti-TNF therapy.

Antitumor Necrosis Factor-like Ligand 1A Therapy Targets Tissue Inflammation and Fibrosis Pathways and Reduces Gut Pathobionts in Ulcerative Colitis.

BACKGROUND: The first-in-class treatment PF-06480605 targets the tumor necrosis factor-like ligand 1A (TL1A) molecule in humans. Results from the phase 2a TUSCANY trial highlighted the safety and efficacy of PF-06480605 in ulcerative colitis. Preclinical and in vitro models have identified a role for TL1A in both innate and adaptive immune responses, but the mechanisms underlying the efficacy of anti-TL1A treatment in inflammatory bowel disease (IBD) are not known. METHODS: Here, we provide analysis of tissue transcriptomic, peripheral blood proteomic, and fecal metagenomic data from the recently completed phase 2a TUSCANY trial and demonstrate endoscopic improvement post-treatment with PF-06480605 in participants with ulcerative colitis. RESULTS: Our results revealed robust TL1A target engagement in colonic tissue and a distinct colonic transcriptional response reflecting a reduction in inflammatory T helper 17 cell, macrophage, and fibrosis pathways in patients with endoscopic improvement. Proteomic analysis of peripheral blood revealed a corresponding decrease in inflammatory T-cell cytokines. Finally, microbiome analysis showed significant changes in IBD-associated pathobionts, Streptococcus salivarius, S. parasanguinis, and Haemophilus parainfluenzae post-therapy. CONCLUSIONS: The ability of PF-06480605 to engage and inhibit colonic TL1A, targeting inflammatory T cell and fibrosis pathways, provides the first-in-human mechanistic data to guide anti-TL1A therapy for the treatment of IBD.

Anti-TL1A Antibody PF-06480605 Safety and Efficacy for Ulcerative Colitis: A Phase 2a Single-Arm Study.

BACKGROUND & AIMS: An immune component of inflammatory bowel disease is up-regulated tumor necrosis factor-like ligand 1A (TL1A). Anti-TL1A antibodies such as PF-06480605, a fully human immunoglobulin G1 monoclonal antibody, may have therapeutic potential. METHODS: This Phase 2a, multicenter, single-arm, open-label study (TUSCANY) evaluated safety, tolerability, efficacy, pharmacokinetics, and immunogenicity in PF-06480605-treated participants with moderate to severe ulcerative colitis (UC). Participants received 500 mg intravenous PF-06480605 every 2 weeks, 7 doses total, with a 3-month follow-up period. Primary safety and efficacy endpoints were the incidence of adverse events (AEs) and week 14 endoscopic improvement (EI) (Mayo endoscopic subscore = 0 or 1), respectively. Secondary endpoints included total soluble TL1A (free/drug-bound) (sTL1A), incidence of anti-drug and neutralizing antibodies, PF-06480605 concentrations, and changes in fecal calprotectin and high-sensitivity C-reactive protein. Histology was assessed at week 14. RESULTS: The study enrolled 50 participants; 42 completed. Of 109 treatment-emergent AEs, 18 were treatment-related. The most common AEs were UC disease exacerbation and arthralgia (6 participants each). Four serious AEs, no deaths, and no malignancies were reported. Week 14 EI was observed in a statistically significant proportion of participants (38.2% [uniformly minimum-variance unbiased estimator, per protocol population]). Minimal histologic disease was observed after treatment (Robarts Histopathology Index ≤5: 33.3%; Geboes Index ≤3.2: 47.6%). sTL1A increase over time from baseline indicated sustained target engagement. Forty-one participants (82%) tested positive for anti-drug antibodies and 5 (10%) for neutralizing antibodies. CONCLUSIONS: PF-06480605 demonstrated an acceptable safety profile and statistically significant EI in participants with moderate to severe UC, warranting further study in a larger participant cohort. Tissue histopathology analyses support this conclusion. TRIAL REGISTRATION NUMBER: https://clinicaltrials.gov/NCT02840721.

First-in-human, randomized dose-escalation study of the safety, tolerability, pharmacokinetics, pharmacodynamics and immunogenicity of PF-06480605 in healthy subjects.

AIMS: Human genetic, tissue expression, proteomics, transcriptomics and nonclinical studies implicate tumour necrosis factor α-like ligand 1A (TL1A) as a novel target in inflammatory bowel disease (IBD). PF-06480605, a fully human immunoglobulin G1 monoclonal antibody, targets TL1A. This first-in-human, Phase 1, dose-escalation study assessed safety, tolerability, pharmacokinetics, pharmacodynamics and immunogenicity of intravenous (IV) and subcutaneous (SC) PF-06480605 in healthy subjects (NCT01989143). METHODS: Ninety-two subjects were randomized to single ascending doses (SAD), PF-06480605 1 mg, 3 mg, 10 mg, 30 mg, 100 mg, 300 mg, 600 mg or 800 mg IV, or multiple ascending doses (MAD), PF-06480605 3 × 500 mg IV, or 3 × 30 mg, 3 × 100 mg, or 3 × 300 mg SC every 2 weeks for three doses, or placebo. Safety, tolerability, pharmacokinetics, immunogenicity profiles and total TL1A, anti-drug antibody (ADA) and neutralizing antibody (NAb) levels were assessed at pre-determined times. RESULTS: PF-06480605 SAD up to 800 mg IV and MAD up to 300 mg ×3 SC and 500 mg ×3 IV were well tolerated. Overall, there were 45 and 44 treatment-emergent adverse events in SAD and MAD cohorts, respectively, and no deaths or serious adverse events. PF-06480605 exposure generally increased dose-dependently. ADA and NAb levels did not impact safety, pharmacokinetics, or pharmacodynamics at higher doses. Target engagement was demonstrated through dose-dependent differences in serum total soluble TL1A concentrations for PF-06480605 vs placebo cohorts. CONCLUSIONS: PF-06480605 was generally well tolerated, and binding of soluble TL1A was maintained throughout the dose interval, supporting further study of PF-06480605 in patients with IBD and other inflammatory conditions.

Mucosal Gene Expression in Pediatric and Adult Patients With Ulcerative Colitis Permits Modeling of Ideal Biopsy Collection Strategy for Transcriptomic Analysis.

Background: Transcriptional profiling has been performed on biopsies from ulcerative colitis patients. Limitations in prior studies include the variability introduced by inflammation, anatomic site of biopsy, extent of disease, and medications. We sought to more globally understand the variability of gene expression from patients with ulcerative colitis to advance our understanding of its pathogenesis and to guide clinical study design. Methods: We performed transcriptional profiling on 13 subjects, including pediatric and adult patients from 2 hospital sites. For each patient, we collected 6 biopsies from macroscopically inflamed tissue and 4 biopsies from macroscopically healthy-appearing tissue. Isolated RNA was used for microarray gene expression analysis utilizing Affymetrix Human Primeview microarrays. Ingenuity pathway analysis was used to assess over-representation of gene ontology and biological pathways. RNAseq was also performed, and differential analysis was assessed to compare affected vs unaffected samples. Finally, we modeled the minimum number of biopsies required to reliably detect gene expression across different subject numbers. Results: Transcriptional profiles co-clustered independently of the hospital collection site, patient age, sex, and colonic location, which parallels prior gene expression findings. A small set of genes not previously described was identified. Our modeling analysis reveals the number of biopsies and patients per cohort to yield reliable results in clinical studies. Conclusions: Key findings include concordance, including some expansion, of previously published gene expression studies and similarity among different age groups. We also established a reliable statistical model for biopsy collection for future clinical studies.

Development and validation of an LC-MS/MS method for quantification of cyclic guanosine 3',5'-monophosphate (cGMP) in clinical applications: a comparison with a EIA method.

An LC-MS/MS method was developed and validated to quantify endogenous cyclic guanosine 3',5'-monophosphate (cGMP) in human plasma. The LC-MS/MS and competitive enzyme immunoassay (EIA) assays were compared. cGMP concentrations of 20 human plasma samples were measured by both methods. For the MS-based assay, plasma samples were subjected to a simple protein precipitation procedure by acetonitrile prior to analysis by electrospray ionization LC-MS/MS. De-protonated analytes generated in negative ionization mode were monitored through multiple reaction monitoring (MRM). A stable isotope-labeled internal standard, (13)C(10),(15)N(5)-cGMP, which was biosynthesized in-house, was used in the LC-MS/MS method. The competitive EIA was validated using a commercially available cGMP fluorescence assay kit. The intra-assay accuracy and precision for MS-based assay for cGMP were 6-10.1% CV and -3.6% to 7.3% relative error (RE), respectively, while inter-assay precision and accuracy were 5.6-8.1% CV and -2.1% to 6.3% RE, respectively. The intra-assay accuracy and precision for EIA were 17.9-27.1% CV and -4.9% to 24.5% RE, respectively, while inter-assay precision and accuracy were 15.1-39.5% CV and -30.8% to 4.37% RE, respectively. Near the lower limits of detection, there was little correlation between the cGMP concentration values in human plasma generated by these two methods (R(2)=0.197, P=0.05). Overall, the MS-based assay offered better selectivity, recovery, precision and accuracy over a linear range of 0.5-20ng/mL. The LC-MS/MS method provides an effective tool for the quantitation of cGMP to support clinical mechanistic studies of curative pharmaceuticals.