T cell engagers (TCEs) are becoming an integral class of biological therapeutic owing to their highly potent ability to eradicate cancer cells. Nevertheless, the widespread utility of classical CD3-targeted TCEs has been limited by narrow therapeutic index (TI) linked to systemic CD4+ T cell activation and aberrant cytokine release. One attractive approach to circumvent the systemic activation of pan CD3+ T cells and reduce the risk of cytokine release syndrome is to redirect specific subsets of T cells. A promising strategy is the use of peptide-major histocompatibility class I bispecific antibodies (pMHC-IgGs), which have emerged as an intriguing modality of TCE, based on their ability to selectively redirect highly reactive viral-specific effector memory cytotoxic CD8+ T cells to eliminate cancer cells. However, the relatively low frequency of these effector memory cells in human peripheral blood mononuclear cells (PBMCs) may hamper their redirection as effector cells for clinical applications. To mitigate this potential limitation, we report here the generation of a pMHC-IgG derivative known as guided-pMHC-staging (GPS) carrying a covalent fusion of a monovalent interleukin-2 (IL-2) mutein (H16A, F42A). Using an anti-epidermal growth factor receptor (EGFR) arm as a proof-of-concept, tumor-associated antigen paired with a single-chain HLA-A *02:01/CMVpp65 pMHC fusion moiety, we demonstrate in vitro that the IL-2-armored GPS modality robustly expands CMVpp65-specific CD8+ effector memory T cells and induces potent cytotoxic activity against target cancer cells. Similar to GPS, IL-2-armored GPS molecules induce modulated T cell activation and reduced cytokine release profile compared to an analogous CD3-targeted TCE. In vivo we show that IL-2-armored GPS, but not the corresponding GPS, effectively expands grafted CMVpp65 CD8+ T cells from unstimulated human PBMCs in an NSG mouse model. Lastly, we demonstrate that the IL-2-armored GPS modality exhibits a favorable developability profile and monoclonal antibody-like pharmacokinetic properties in human neonatal Fc receptor transgenic mice. Overall, IL-2-armored GPS represents an attractive approach for treating cancer with the potential for inducing vaccine-like antiviral T cell expansion, immune cell redirection as a TCE, and significantly widened TI due to reduced cytokine release.

AIMS:Benralizumab, a humanized, afucosylated monoclonal antibody against the interleukin 5 receptor, α subunit, causes rapid depletion of eosinophils by antibody-dependent cellular cytotoxicity. We investigated the pharmacokinetic and pharmacodynamic effects of benralizumab in patients with chronic rhinosinusitis with nasal polyps (CRSwNP) from the phase III OSTRO trial. METHODS:Patients received a placebo or 30 mg of benralizumab by subcutaneous injection every 8 weeks (first three doses every 4 weeks) to week 48; a subset of patients continued in an extended follow-up period to assess treatment durability to week 80. Serum benralizumab concentrations and blood eosinophil and basophil counts were assessed to week 80. Biomarker assessments were performed on nasal polyp tissue biopsies at week 56 and nasal lining fluid at weeks 24 and 56 to examine changes in immune cells and inflammatory mediators. RESULTS:Among 185 patients in this analysis, 93 received benralizumab. Serum benralizumab concentrations reached a steady state by week 24 (median concentration 385.52 ng mL-1); blood eosinophils were almost fully depleted and blood basophils were reduced between weeks 16 and 56. Nasal polyp tissue eosinophils decreased with benralizumab from 57.6 cells mm-2 at baseline to 0 cells mm-2 at week 56 (P < .001 vs placebo), and tissue mast cells were numerically reduced. In nasal lining fluid, eosinophil-derived neurotoxin was significantly reduced at weeks 24 and 56 (P < .001) and interleukin-17 at week 56 (P < .05) with benralizumab. CONCLUSION:Benralizumab treatment led to rapid, sustained, nearly complete depletion of eosinophils from blood and nasal polyp tissue in patients with CRSwNP.

Background MEDI1191 is an investigational therapy composed of mRNA encoding interleukin-12 (IL-12p70) in a lipid nanoparticle optimized for intratumoral injection. Previously1 2 we reported that all dose levels induce pharmacodynamic (PD) changes such as elevated serum IL-12, increased T cell infiltration and upregulation of immune gene signatures associated with anti-tumor responses. Since the actual dose and drug distribution may be affected by injected lesion size, we hypothesize that a normalized dose (ND) to lesion size will accurately reflect the dose effect and PD changes within tumor microenvironment (TME). Methods In a phase 1 dose escalation study (NCT03946800), patients received 0.1–12 µg MEDI1191 sequentially (Part 1A) or concurrently (Part 1B and Part 1D) with intravenous durvalumab (1500 mg). To determine the dose effect on PD changes within the TME, we calculated a normalized dose (µg/mm) for each patient by dividing the first actual injected dose by the longest (for non-lymph node lesions) or shortest (for lymph node lesions) diameter of injected lesions (14–130 mm). Results The first administration induced serum IL-12 levels at all dose levels and the total IL-12 exposure measured by area under the curve was induced in a dose-dependent manner. Patients dosed at 8 and 12 µg had higher normalized dose (0.08–0.75 µg/mm) than patients dosed at 0.1–3 µg (0.002–0.15 µg/mm). High normalized dose (NDhi) elevated serum IL-12 and IFN-γ levels at 24h following injection to a greater extent than low normalized dose (NDlo). To correlate the dose effect with PD changes within the TME, we looked at the immune gene signatures in 14 patients (7 patients in each dose group) with available paired biopsies at screening and day 15. The gene set enrichment analysis revealed an increased enrichment of the hallmark interferon-gamma response pathway in NDhipatients. The average scores of gene signatures such as T cell inflamed, cytotoxic activity and T cell proliferation were induced in NDhipatients, whereas NDlopatients showed decrease or no major changes. Lesions of NDhipatients exhibited an inflammatory phenotype post-treatment. The best anti-tumor PD effect was observed in a partial responder in the NDhi group, which was associated with the largest increase in tumoral CD8 T cell density (6-fold) and PD-L1 (8.5-fold) by immunohistochemistry at injected lesions. Conclusions Intratumoral injection of mRNA IL-12 can induce an immunostimulatory effect, including elevated anti-tumor gene signatures and T cell infiltration. Evidence of a dose-dependent immune modulation within the TME was observed at injected lesion sites. Trial Registration NCT03946800 References Eduardo Castañón, Dmitriy Zamarin, Benedito A Carneiro, Thomas Marron, Sandip Pravin Patel, Vivek Subbiah, Inderjit Mehmi, Honey Kumar Oberoi, Anthony El-Khoueiry, Benjamin Ridgway, Nairouz Elgeioushi, Nicholas M Durham, Emily Jennings, Michael Abadier, Paula G Fraenkel, Analia Azaro, Omid Hamid; Abstract CT004: Intratumoral (IT) MEDI1191 + durvalumab (D): Update on the first-in-human study in advanced solid tumors. Cancer Res 15 April 2023;83(8_Supplement):CT004. https://doi.org/10.1158/1538–7445.AM2023-CT004 Abadier M, Jennings E, Eyles J, et al. 708 MEDI1191 (IL-12 mRNA) induces peripheral and intratumoral immunostimulatory effect in patients with cutaneous or subcutaneous (C/SC) lesions Journal for ImmunoTherapy of Cancer 2022;10:doi: 10.1136/jitc-2022-SITC2022.0708 Ethics Approval The IRB/IEC responsible for each site reviewed and approved the final study protocol, including the final version of the ICF and any other written information and/or materials to be provided to the subjects. Institutional Review Boards, Mount Sinai Health System, New York (Board Number 19–00279).

Evolving immunogenicity assay performance expectations and a lack of harmonized neutralizing antibody validation testing and reporting tools have resulted in significant time spent by health authorities and sponsors on resolving filing queries. A team of experts within the American Association of Pharmaceutical Scientists' Therapeutic Product Immunogenicity Community across industry and the Food and Drug Administration addressed challenges unique to cell-based and non-cell-based neutralizing antibody assays. Harmonization of validation expectations and data reporting will facilitate filings to health authorities and are described in this manuscript. This team provides validation testing and reporting strategies and tools for the following assessments: (1) format selection; (2) cut point; (3) assay acceptance criteria; (4) control precision; (5) sensitivity including positive control selection and performance tracking; (6) negative control selection; (7) selectivity/specificity including matrix interference, hemolysis, lipemia, bilirubin, concomitant medications, and structurally similar analytes; (8) drug tolerance; (9) target tolerance; (10) sample stability; and (11) assay robustness.

Abstract Background EVUSHELD was developed for the prevention and treatment of mild-to-moderate COVID-19 for high-risk individuals. However, the SARS-CoV-2 virus continues to evolve in the presence of natural- and vaccine-acquired immunity, escaping previously authorized antibody therapies such as bebtelovimab and EVUSHELD. AZD3152 was selected to neutralize all known SARS-CoV-2 variants of concern and is being developed to provide immunocompromised individuals with continued protection against SARS-CoV-2. Methods Structural analyses were performed to map the AZD3152 binding site. Neutralization assays were employed to determine the potency of AZD3152 across a panel of historical and emerging SARS-CoV-2 variants. Hamsters were prophylactically administered 6.7-60 mg/kg AZD3152 or a control antibody, then challenged intranasally with 6x103 PFU WA-1 SARS-CoV-2, then monitored for weight loss; lung viral load and pathology were evaluated at days 3 and 7. A 3 week GLP repeat IM and IV dose toxicity study in nonhuman primates (NHP) was performed. Results AZD3152 binds to a conserved epitope on the spike receptor binding domain and blocks ACE2 binding. AZD3152 potently neutralizes authentic viral variants (EC50 range, 8.3 to 110.9 ng/mL) and SARS-CoV-2 pseudoviruses (EC50 range of 3.2 to 25.0 ng/mL), including XBB.1.5. Prophylactic administration of AZD3152 conferred dose-dependent protection to hamsters following challenge. Even at a suboptimal dose of 6.7 mg/kg, < 5% weight loss and 2 logs reduction in lung viral subgenomic RNA were observed. AZD3152 was not associated with any adverse findings in NHP and the no observed-adverse-effect-level was the highest dose tested (150 mg/kg). Toxicokinetics demonstrated similar systemic exposure following IM and IV dosing with bioavailability of 94.9% and half-life range of 15.2-26.5 days by IM. Conclusion These results demonstrate that AZD3152 binds a conserved epitope resulting in broad neutralization of SARS-CoV-2 variants, protects hamsters from disease, and has a favorable safety profile in NHP. Thus, AZD3152 may serve as a next-generation antibody for the prevention and treatment of COVID-19. Disclosures Joseph R Francica, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Yingyun Cai, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Seme Diallo, MS, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Kim Rosenthal, MS, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Kuishu Ren, BS, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Daniel J. Flores, MS, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Andrew Dippel, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Yuling Wu, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Xiaoru Chen, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Erin Cantu, BS, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Rakesh Choudhary, MS, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Michal Sulikowski, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Hibret Adissu, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Nydia van Dyk, MSc, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Vaheh Oganesyan, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Saravanan Rajan, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Patricia C. Ryan, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Yueh-Ming Loo, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Taylor Cohen, PhD, AstraZeneca: Employement|AstraZeneca: Stocks/Bonds Mark T. Esser, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Wade Blair, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds

The increasing complexity and diversity of antibody–drug conjugates (ADCs) have led to a need for comprehensive and informative bioanalytical methods to enhance pharmacokinetic (PK) understanding. This study aimed to evaluate the feasibility of a hybrid immunoaffinity (IA) capture microflow LC–MS/MS (μLC-MS/MS) method for ADC analysis, utilizing a minimal sample volume for PK assessments in a preclinical study. A robust workflow was established for the quantitative analysis of ADCs by the implementation of solid-phase extraction (SPE) and semi-automation in µLC-MS/MS. Utilizing the µLC-MS/MS approach in conjunction with 1 µL of ADC-dosed mouse plasma sample volume, standard curves of two representative surrogate peptides for total antibody (heavy chain, HC) and intact antibody (light chain, LC) ranged from 1.00 ng/mL (LLOQ) to 5000 ng/mL with correlation coefficients (r2) values of > 0.99. The linear range of the standard curve for payload as a surrogate for the concentration of total ADC was from 0.5 ng/mL (LLOQ) to 2000 ng/mL with high accuracy and precision (< 10

The 2022 16th Workshop on Recent Issues in Bioanalysis (WRIB) took place in Atlanta, GA, USA on September 26–30, 2022. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 16th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on ICH M10 BMV final guideline (focused on this guideline training, interpretation, adoption and transition); mass spectrometry innovation (focused on novel technologies, novel modalities, and novel challenges); and flow cytometry bioanalysis (rising of the 3rd most common/important technology in bioanalytical labs) were the special features of the 16th edition. As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues. This 2022 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2022 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity. Part 1 (Mass Spectrometry and ICH M10) and Part 2 (LBA, Biomarkers/CDx and Cytometry) are published in volume 15 of Bioanalysis, issues 16 and 15 (2023), respectively.

To combat the COVID-19 pandemic, potential therapies have been developed and moved into clinical trials at an unprecedented pace. Some of the most promising therapies are neutralizing antibodies against SARS-CoV-2. In order to maximize the therapeutic effectiveness of such neutralizing antibodies, Fc engineering to modulate effector functions and to extend half-life is desirable. However, it is critical that Fc engineering does not negatively impact the developability properties of the antibodies, as these properties play a key role in ensuring rapid development, successful manufacturing, and improved overall chances of clinical success. In this study, we describe the biophysical characterization of a panel of Fc engineered ("TM-YTE") SARS-CoV-2 neutralizing antibodies, the same Fc modifications as those found in AstraZeneca's Evusheld (AZD7442; tixagevimab and cilgavimab), in which the TM modification (L234F/L235E/P331S) reduce binding to Fc gamma R and C1q and the YTE modification (M252Y/S254T/T256E) extends serum half-life. We have previously shown that combining both the TM and YTE Fc modifications can reduce the thermal stability of the CH2 domain and possibly lead to developability challenges. Here we show, using a diverse panel of TM-YTE SARS-CoV-2 neutralizing antibodies, that despite lowering the thermal stability of the Fc CH2 domain, the TM-YTE platform does not have any inherent developability liabilities and shows an in vivo pharmacokinetic profile in human FcRn transgenic mice similar to the well-characterized YTE platform. The TM-YTE is therefore a developable, effector function reduced, half-life extended antibody platform.

Background CD73 upregulation in tumors leads to local immunosuppression. This phase I, first-in-human study evaluated oleclumab (MEDI9447), an anti-CD73 human IgG1λ monoclonal antibody, alone or with durvalumab in patients with advanced colorectal cancer (CRC), pancreatic ductal adenocarcinoma (PDAC), or epidermal growth factor receptor-mutant non-small-cell lung cancer (NSCLC). Methods Patients received oleclumab 5–40 mg/kg (dose-escalation) or 40 mg/kg (dose-expansion) intravenously every 2 weeks (Q2W), alone (escalation only) or with durvalumab 10 mg/kg intravenously Q2W. Results 192 patients were enrolled, 66 during escalation and 126 (42 CRC, 42 PDAC, 42 NSCLC) during expansion. No dose-limiting toxicities occurred during escalation. In the monotherapy and combination therapy escalation cohorts, treatment-related adverse events (TRAEs) occurred in 55 and 54%, respectively, the most common being fatigue (17 and 25%). In the CRC, PDAC, and NSCLC expansion cohorts, 60, 57, and 45% of patients had TRAEs, respectively; the most common were fatigue (15%), diarrhea (9%), and rash (7%). Free soluble CD73 and CD73 expression on peripheral T cells and tumor cells showed sustained decreases, accompanied by reduced CD73 enzymatic activity in tumor cells. Objective response rate during escalation was 0%. Response rates in the CRC, PDAC, and NSCLC expansion cohorts were 2.4% (1 complete response [CR]), 4.8% (1 CR, 1 partial response [PR]), and 9.5% (4 PRs), respectively; 6-month progression-free survival rates were 5.4, 13.2, and 16.0%. Conclusions Oleclumab ± durvalumab had a manageable safety profile, with pharmacodynamic activity reflecting oleclumab’s mechanism of action. Evidence of antitumor activity was observed in tumor types that are generally immunotherapy resistant. Clinical trial registration Clinicaltrials.gov, NCT02503774; date of registration, July 17, 2015.

Background: IL-12 is a key mediator of antitumor immune response. In preclinical models, IT IL-12 mRNA led to IFNγ release and CD8+ T cell-dependent tumor regression and potentiated PD-L1 blockade. MEDI1191, a lipid nanoparticle-formulated mRNA encoding IL-12 delivered by IT injection, drives IL-12 production and enhances antitumor immune response with improved tolerability. We hypothesized that combining MEDI1191 with PD-L1 blockade would augment antitumor immunity in vivo. Here we report updated results from the dose-escalation phase of the first-in-human study of IT MEDI1191 and IV durvalumab (D; anti-PD-L1) for advanced/metastatic solid tumors (NCT03946800). Methods: In this multicenter, open-label study, MEDI1191 was dosed sequentially (seq, Part 1A) or concurrently (conc, Part 1B) with D. In Part 1A, MEDI1191 was given IT on Days 1 and 22 followed by D 1500 mg on day 43 and then Q4W IV. In Part 1B, MEDI1191 was given IT on Days 1, 29, 57 and then Q8W, along with D on Day 1 and then Q4W. Treatment continued until progression or unacceptable toxicity for up to 2 years. Eligible adult pts had any solid tumor with cutaneous or subcutaneous lesions suitable for IT injection and progression on standard therapy for recurrent/metastatic disease. Primary objectives were safety and tolerability and determination of maximum tolerated dose (MTD); secondary objectives included preliminary antitumor activity by RECIST v1.1. Results: Starting in May 2019, 31 pts received seq MEDI1191 with D (Part 1A cohorts 0.1-12 μg; n=20) or conc MEDI1191 with D (Part 1B cohorts 1.0-3.0 μg; n=11); 23 pts had received prior anti-PD-1/PD-L1 therapy. Most common tumor types were melanoma, n=8; head and neck cancer, n=4; and breast cancer, n=4. At the data cutoff of Dec 7, 2021, there were no dose-limiting toxicities and no MTD was identified. One pt (3.2%) had a Gr ≥3 MEDI1191-related AE (Gr 3 pyrexia, resolved within 24 hr) and 1 (3.2%) had a MEDI1191-related serious AE (SAE; Gr 2 confusion). Two pts had Gr 3 D-related AEs (6.5%, pyrexia also related to MEDI1191 and pruritus; each n=1); none had a D-related SAE. There were no Gr 4 related AEs. 3 pts had partial responses (PR): 1 with head and neck cancer (unconfirmed, off study) and 2 with anti-PD-1 resistant melanoma (1 confirmed, >12 months on treatment; 1 unconfirmed, off study); 10 pts had stable disease (including the 2 unconfirmed PRs). Among pts with available biomarker data (up to 3 μg in Part 1A and 1 μg in Part 1B), MEDI1191 increased serum IL-12 in 15/17 pts, increased CD8+ T cell tumor infiltration by >2-fold in 8/14 pts and increased tumor PD-L1 expression in 6/14 pts. Conclusions: IT MEDI1191 plus systemic anti-PD-L1 was safe and feasible. Preliminary antitumor efficacy and pharmacodynamics, including tumor CD8+ T cell recruitment, were consistent with expected mechanism of action. Pts with injectable deep visceral and superficial lesions are being recruited. Citation Format: Benedito A. Carneiro, Dmitriy Zamarin, Thomas Marron, Inderjit Mehmi, Sandip P. Patel, Vivek Subbiah, Anthony El-Khoueiry, David Grand, Kirema Garcia-Reyes, Sanjay Goel, Phillip Martin, Jixin Wang, Yuling Wu, Steven Eck, Benjamin Ridgway, Nairouz Elgeioushi, Jim Eyles, Nicholas Durham, Analia Azaro, Omid Hamid. First-in-human study of MEDI1191 (mRNA encoding IL-12) plus durvalumab in patients (pts) with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT183.

The 15th edition of the Workshop on Recent Issues in Bioanalysis (15th WRIB) was held on 27 September to 1 October 2021. Even with a last-minute move from in-person to virtual, an overwhelmingly high number of nearly 900 professionals representing pharma and biotech companies, contract research organizations (CROs), and multiple regulatory agencies still eagerly convened to actively discuss the most current topics of interest in bioanalysis. The 15th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy, cell therapy and vaccines. Moreover, in-depth workshops on biomarker assay development and validation (BAV) (focused on clarifying the confusion created by the increased use of the term "Context of Use - COU"); mass spectrometry of proteins (therapeutic, biomarker and transgene); state-of-the-art cytometry innovation and validation; and, critical reagent and positive control generation were the special features of the 15th edition. This 2021 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop, and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2021 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness. Part 1A (Endogenous Compounds, Small Molecules, Complex Methods, Regulated Mass Spec of Large Molecules, Small Molecule, PoC), Part 1B (Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine) and Part 2 (ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry) are published in volume 14 of Bioanalysis, issues 9 and 10 (2022), respectively.

Background MEDI1191 is an investigational therapy composed of mRNA encoding bioactive interleukin-12 (IL-12p70) in a lipid nanoparticle optimized for intratumoral injection. We hypothesized that intratumoral injection of MEDI1191 will reprogram the immunosuppressed tumor microenvironment (TME) and increase cytotoxic T-cell recruitment, proliferation and activation. Methods In an ongoing phase 1 dose escalation study (NCT03946800), patients with C/SC lesions received 0.1-12µg MEDI1191 sequentially (Part 1A) or concurrently (Part 1B) with intravenous durvalumab (1500 mg).1 Clinical samples were collected to monitor changes in peripheral cytokines, circulating tumor (ct) DNA, peripheral and tumoral gene signatures and T cell infiltration. Results Peripheral blood cytokine analysis revealed increased serum IL-12 levels in 27/29 (93%) patients post first injection and was associated with ≥2-fold increase in serum IFN-γ in 24/29 (83%) patients. The IFN-inducible chemokines (CXCL9, CXCL10 and CXCL11) in periphery were detected at the protein and transcriptomic levels. In 11 patients whose blood samples underwent bulk RNA sequencing, markers of activated peripheral T cells were detectable over time with increased mean fold change of GZMB, IFNG and IL12RB1 gene expression in patients with partial response (PR, n=1) or stable disease (SD, n=5) patients. Pre-treatment expression of the genes encoding key mediators of cytotoxic T-cell function were potential predictors of response as observed by significantly higher expressions of peripheral IL12RB1 and IFNG genes at baseline in PR/SD patients. Using immunohistochemistry (IHC), we observed ≥ 2-fold increase in CD8 T cells (8/17, 47%) and Ki-67+CD3+ T-cells (7/17, 41%) in tumor biopsies 15 days post first injection. Patients with increased intratumoral CD8 T cells by IHC and RNA sequencing showed higher tumoral GZMB and IFNG gene expression. In 9 patients with evaluable tumor biopsies at baseline and post-treatment, we observed an increase in antitumor gene signatures, including activated CD8 (7/9, 78%), activated NK (7/9, 78%), M1 macrophages (7/9, 78%) and IFN-γ signaling (8/9, 89%). Consistent with IL-12's role as a central mediator of Th1 cell development, we observed an increase in Th1 signature and STAT4 gene (6/9, 67%). Analysis of ctDNA in plasma of 17 patients demonstrated >50% reduction in mutant allele frequency in 2/3 patients who developed PR. Conclusions Intratumoral injection of MEDI1191 induces pharmacodynamic changes within the TME such as increase in activated T cell infiltration and upregulation of anti-tumor gene signatures. There was an associated immunomodulatory effect in the periphery, including elevated IL-12, IFN-γ and activated T-cells. Trial Registration NCT03946800 Reference [1] Benedito A Carneiro, Dmitriy Zamarin, Thomas Marron, Inderjit Mehmi, Sandip P Patel, Vivek Subbiah, Anthony El-Khoueiry, David Grand, Kirema Garcia-Reyes, Sanjay Goel, Phillip Martin, Jixin Wang, Yuling Wu, Steven Eck, Benjamin Ridgway, Nairouz Elgeioushi, Jim Eyles, Nicholas Durham, Analia Azaro, Omid Hamid. First-in-human study of MEDI1191 (mRNA encoding IL-12) plus durvalumab in patients (pts) with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT183. Ethics Approval The IRB/IEC responsible for each site reviewed and approved the final study protocol, including the final version of the ICF and any other written information and/or materials to be provided to the subjects. Institutional Review Boards, Mount Sinai Health System, New York (Board Number 19-00279).

Background Ventilator-associated pneumonia caused by Pseudomonas aeruginosa (PA) in hospitalised patients is associated with high mortality. The effectiveness of the bivalent, bispecific mAb MEDI3902 (gremubamab) in preventing PA nosocomial pneumonia was assessed in PA-colonised mechanically ventilated subjects. Methods EVADE (NCT02696902) was a phase 2, randomised, parallel-group, double-blind, placebo-controlled study in Europe, Turkey, Israel, and the USA. Subjects ≥ 18 years old, mechanically ventilated, tracheally colonised with PA, and without new-onset pneumonia, were randomised (1:1:1) to MEDI3902 500, 1500 mg (single intravenous dose), or placebo. The primary efficacy endpoint was the incidence of nosocomial PA pneumonia through 21 days post-dose in MEDI3902 1500 mg versus placebo, determined by an independent adjudication committee. Results Even if the initial sample size was not reached because of low recruitment, 188 subjects were randomised (MEDI3902 500/1500 mg: n = 16/87; placebo: n = 85) between 13 April 2016 and 17 October 2019. Out of these, 184 were dosed (MEDI3902 500/1500 mg: n = 16/85; placebo: n = 83), comprising the modified intent-to-treat set. Enrolment in the 500 mg arm was discontinued due to pharmacokinetic data demonstrating low MEDI3902 serum concentrations. Subsequently, enrolled subjects were randomised (1:1) to MEDI3902 1500 mg or placebo. PA pneumonia was confirmed in 22.4% ( n = 19/85) of MEDI3902 1500 mg recipients and in 18.1% ( n = 15/83) of placebo recipients (relative risk reduction [RRR]: − 23.7%; 80% confidence interval [CI] − 83.8%, 16.8%; p = 0.49). At 21 days post-1500 mg dose, the mean (standard deviation) serum MEDI3902 concentration was 9.46 (7.91) μg/mL, with 80.6% ( n = 58/72) subjects achieving concentrations > 1.7 μg/mL, a level associated with improved outcome in animal models. Treatment-emergent adverse event incidence was similar between groups. Conclusions The bivalent, bispecific monoclonal antibody MEDI3902 (gremubamab) did not reduce PA nosocomial pneumonia incidence in PA-colonised mechanically ventilated subjects. Trial registration Registered on Clinicaltrials.gov ( NCT02696902 ) on 11th February 2016 and on EudraCT ( 2015-001706-34 ) on 7th March 2016.

Interleukin-6 (IL-6) is a pro/anti-inflammatory cytokine, the quantitative detection of which has been extensively considered for diagnosis of inflammatory associated diseases. However, there has not yet been a reliable, low-cost, and user-friendly platform developed for point-of-care (POC) detection of IL-6, which will eliminate the conventional costly, time-consuming, and complex assays. In this work, we developed a lateral flow assay for colorimetric detection of IL-6, using anti-IL-6 antibodies conjugated to gold nanoparticles (AuNPs) as the detection probes. Silver amplification technique was incorporated with the newly developed assay in order to enhance the obtained colorimetric signals, allowing sensitive detection of IL-6 in human serum in the desired physiological ranges (i.e., 5–1000 pg/mL). A limit of detection of 5 pg/mL could be achieved for IL-6 detection in serum with the amplification step which was not achievable in the standard assay. The corresponding specificity and reproducibility tests were all preformed to confirm the reliability of this assay for quantitative measurement of IL-6 in a POC manner.

treatment-emergent serious adverse event related to treatment, two (2%) patients reported an adverse event of special interest, and two (2%) reported a new-onset chronic disease. Interpretation In patients in the ICU receiving mechanical ventilation with qPCR-confirmed S aureus colonisation of the lower respiratory tract, the incidence of S aureus pneumonia at 30 days was not significantly lower following treatment with 5000 mg suvratoxumab than with placebo. Despite these negative results, monoclonal antibodies still represent one promising therapeutic option to reduce antibiotic consumption that require further exploration and studies. Funding AstraZeneca, with support from the Innovative Medicines Initiative Joint Undertaking. Copyright (c) 2021 Elsevier Ltd. All rights reserved.

The 14(th) edition of the Workshop on Recent Issues in Bioanalysis (14(th) WRIB) was held virtually on June 15-29, 2020 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations, and regulatory agencies worldwide. The 14(th) WRIB included three Main Workshops, seven Specialized Workshops that together spanned 11 days in order to allow exhaustive and thorough coverage of all major issues in bioanalysis, biomarkers, immunogenicity, gene therapy and vaccine. Moreover, a comprehensive vaccine assays track; an enhanced cytometry track and updated Industry/Regulators consensus on BMV of biotherapeutics by LCMS were special features in 2020. As in previous years, this year's WRIB continued to gather a wide diversity of international industry opinion leaders and regulatory authority experts working on both small and large molecules to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance and achieving scientific excellence on bioanalytical issues. This 2020 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the Global Bioanalytical Community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2020 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations on Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity). Part 1 (Innovation in Small Molecules, Hybrid LBA/LCMS & Regulated Bioanalysis), Part 2A (BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation) and Part 2B (Regulatory Input) are published in volume 13 of Bioanalysis, issues 4 and 5 (2020), respectively.

Abstract Multiple myeloma is a hematologic cancer that disrupts normal bone marrow function and has multiple lines of therapeutic options, but is incurable as patients ultimately relapse. We developed a novel antibody–drug conjugate (ADC) targeting CS-1, a protein that is highly expressed on multiple myeloma tumor cells. The anti–CS-1 mAb specifically bound to cells expressing CS-1 and, when conjugated to a cytotoxic pyrrolobenzodiazepine payload, reduced the viability of multiple myeloma cell lines in vitro. In mouse models of multiple myeloma, a single administration of the CS-1 ADC caused durable regressions in disseminated models and complete regression in a subcutaneous model. In an exploratory study in cynomolgus monkeys, the CS-1 ADC demonstrated a half-life of 3 to 6 days; however, no highest nonseverely toxic dose was achieved, as bone marrow toxicity was dose limiting. Bone marrow from dosed monkeys showed reductions in progenitor cells as compared with normal marrow. In vitro cell killing assays demonstrated that the CS-1 ADC substantially reduced the number of progenitor cells in healthy bone marrow, leading us to identify previously unreported CS-1 expression on a small population of progenitor cells in the myeloid–erythroid lineage. This finding suggests that bone marrow toxicity is the result of both on-target and off-target killing by the ADC.

Background MEDI9197 is an intratumorally administered toll-like receptor 7 and 8 agonist. In mice, MEDI9197 modulated antitumor immune responses, inhibited tumor growth and increased survival. This first-time-in-human, phase 1 study evaluated MEDI9197 with or without the programmed cell death ligand-1 (PD-L1) inhibitor durvalumab and/or palliative radiation therapy (RT) for advanced solid tumors. Patients and methods Eligible patients had at least one cutaneous, subcutaneous, or deep-seated lesion suitable for intratumoral (IT) injection. Dose escalation used a standard 3+3 design. Patients received IT MEDI9197 0.005–0.055 mg with or without RT (part 1), or IT MEDI9197 0.005 or 0.012 mg plus durvalumab 1500 mg intravenous with or without RT (part 3), in 4-week cycles. Primary endpoints were safety and tolerability. Secondary endpoints included pharmacokinetics, pharmacodynamics, and objective response based on Response Evaluation Criteria for Solid Tumors version 1.1. Exploratory endpoints included tumor and peripheral biomarkers that correlate with biological activity or predict response. Results From November 2015 to March 2018, part 1 enrolled 35 patients and part 3 enrolled 17 patients; five in part 1 and 2 in part 3 received RT. The maximum tolerated dose of MEDI9197 monotherapy was 0.037 mg, with dose-limiting toxicity (DLT) of cytokine release syndrome in two patients (one grade 3, one grade 4) and 0.012 mg in combination with durvalumab 1500 mg with DLT of MEDI9197-related hemorrhagic shock in one patient (grade 5) following liver metastasis rupture after two cycles of MEDI9197. Across parts 1 and 3, the most frequent MEDI9197-related adverse events (AEs) of any grade were fever (56%), fatigue (31%), and nausea (21%). The most frequent MEDI9197-related grade ≥3 events were decreased lymphocytes (15%), neutrophils (10%), and white cell counts (10%). MEDI9197 increased tumoral CD8+ and PD-L1+ cells, inducing type 1 and 2 interferons and Th1 response. There were no objective clinical responses; 10 patients in part 1 and 3 patients in part 3 had stable disease ≥8 weeks. Conclusion IT MEDI9197 was feasible for subcutaneous/cutaneous lesions but AEs precluded its use in deep-seated lesions. Although no patients responded, MEDI9197 induced systemic and intratumoral immune activation, indicating potential value in combination regimens in other patient populations. Trial registration number NCT02556463 .

Background: Tezepelumab, a human monoclonal antibody that selectively blocks thymic stromal lymphopoietin interaction (TSLP) with its receptor, improved clinical outcomes vs. placebo in patients with severe, uncontrolled asthma (Phase 2b PATHWAY study [NCT02054130]). Proteomics analysis was performed to further elucidate mechanisms of tezepelumab activity in patients with asthma. Aim: To determine which serum proteins are altered by tezepelumab. Methods: Patients were randomised to tezepelumab-treatment arms or placebo (Corren et al. NEJM 2017;377:936–46). Serum protein levels were assessed at baseline and at multiple post-treatment timepoints up to Week 52 using the Myriad Rules Based Medicine (RBM) and SomaLogic platforms (122 and 1305 analytes, respectively). Significant changes were determined by ANOVA and t-tests at each timepoint, with percent changes from baseline also being assessed. We also explored associations between baseline levels of RBM proteins and clinical biomarkers. Results: Levels of matrix remodelling (MMP-10, periostin), eosinophil-related (IL5RA, PAPP‑A), IgE and TARC proteins decreased significantly post-treatment (>10% change in tezepelumab vs. placebo at Week 52, p<0.05). At baseline, MMP-10 and periostin correlated significantly (p<0.01) with blood eosinophils (bEOS) and FENO levels. Conclusions: Tezepelumab led to a decrease in biomarkers of inflammation and matrix remodelling, which were correlated with bEOS and FENO levels at baseline. Modulation of matrix remodelling proteins may reflect a novel mechanism of action for tezepelumab in addition to the inhibition of airway inflammation. Co-sponsored by MedImmune, a member of the AstraZeneca Group, and Amgen Inc.