First-in-human study of oleclumab, a potent, selective anti-CD73 monoclonal antibody, alone or in combination with durvalumab in patients with advanced solid tumors.

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.

Humanised effector-null FcγRIIA antibody inhibits immune complex-mediated proinflammatory responses.

OBJECTIVE: Immune complexes (ICs) play a critical role in the pathology of autoimmune diseases. The aim of this study was to generate and characterise a first-in-class anti-FcγRIIA antibody (Ab) VIB9600 (previously known as MEDI9600) that blocks IgG immune complex-mediated cellular activation for clinical development. METHODS: VIB9600 was humanised and optimised from the IV.3 Ab. Binding affinity and specificity were determined by Biacore and ELISA. Confocal microscopy, Flow Cytometry-based assays and binding competition assays were used to assess the mode of action of the antibody. In vitro cell-based assays were used to demonstrate suppression of IC-mediated inflammatory responses. In vivo target suppression and efficacy was demonstrated in FcγRIIA-transgenic mice. Single-dose pharmacokinetic (PK)/pharmacodynamic study multiple dose Good Laboratory Practice (GLP) toxicity studies were conducted in non-human primates. RESULTS: We generated a humanised effector-deficient anti-FcγRIIA antibody (VIB9600) that potently blocks autoantibody and IC-mediated proinflammatory responses. VIB9600 suppresses FcγRIIA activation by blocking ligand engagement and by internalising FcγRIIA from the cell surface. VIB9600 inhibits IC-induced type I interferons from plasmacytoid dendritic cells (involved in SLE), antineutrophil cytoplasmic antibody (ANCA)-induced production of reactive oxygen species by neutrophils (involved in ANCA-associated vasculitis) and IC-induced tumour necrosis factor α and interleukin-6 production (involved in rheumatoid arthritis). In FcγRIIA transgenic mice, VIB9600 suppressed antiplatelet antibody-induced thrombocytopaenia, acute anti-GBM Ab-induced nephritis and anticollagen Ab-induced arthritis. VIB9600 also exhibited favourable PK and safety profiles in cynomolgus monkey studies. CONCLUSIONS: VIB9600 is a specific humanised antibody antagonist of FcγRIIA with null effector function that warrants further clinical development for the treatment of IC-mediated diseases.

A comparative study of blood cell count in four automated hematology analyzers: An evaluation of the impact of preanalytical factors.

Differential white blood cell counts are frequently used in diagnosis, patient stratification, and treatment selection to optimize therapy responses. Referral laboratories are often used but challenged with use of different hematology platforms, variable blood shipping times and storage conditions, and the different sensitivities of specific cell types. To extend the scientific literature and knowledge on the temporal commutability of blood samples between hematology analyzers, we performed a comparative ex-vivo study using four of the most utilized commercial platforms, focusing on the assessment of eosinophils given its importance in asthma management. Whole blood from healthy volunteers with and without atopy (n = 6+6) and participants with eosinophilic asthma (n = 6) were stored under different conditions (at 4, 20, 30, and 37°C, with or without agitation) and analyzed at different time points (3, 6, 24, 48 and 72h post-sampling) in parallel on the Abbott CELL-DYN Sapphire, Beckman Coulter DxH900, Siemens ADVIA 2120i and Sysmex XN-1000V. In the same blood samples, eosinophil-derived neurotoxin (EDN), eosinophil activation and death markers were analyzed. All platforms gave comparable measurements of cell differentials on fresh blood within the same day of sampling. However, by 24 hours, significant temporal and temperature-dependent differences were observed, most markedly for eosinophils. None of the platforms performed perfectly across all temperatures tested during the 72 hours, showing that handling conditions should be optimized depending on the cell type of interest and the hematology analyzer. Neither disease status (healthy vs. asthma) nor agitation of the sample affected the cell quantification result or EDN release. The eosinophil activation markers measured by flow cytometry increased with time, were influenced by temperature, and were higher in those with asthma versus healthy participants. In conclusion, hematology analyzer, time window from sampling until analysis, and temperature conditions must be considered when analyzing blood cell differentials, particularly for eosinophils, via central labs to obtain counts comparable to the values obtained in freshly sampled blood.

2023 White Paper on Recent Issues in Bioanalysis: EU IVDR 2017/746 Implementation/Impact, IVD/CDx/CLIA Approved Assays, High Dimensional Cytometry, Multiplexing Technologies, LBA Tissue Analysis, Vaccine Study Endpoints, Cell-Based Assays for Biomarkers, Cell Therapy and Vaccines (PART 2 - Recommendations on Development & Validation of Biomarkers, IVD, CDx, Cell-Based, Flow Cytometry, Ligand-Binding and Enzyme Assays; Advanced Critical Reagents Strategies).

The 17th Workshop on Recent Issues in Bioanalysis (17th WRIB) took place in Orlando, FL, USA on 19-23 June 2023. 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 17th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines.Moreover, in-depth workshops on "EU IVDR 2017/746 Implementation and impact for the Global Biomarker Community: How to Comply with these NEW Regulations" and on "US FDA/OSIS Remote Regulatory Assessments (RRAs)" were the special features of the 17th 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 2023 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 2023 edition of this comprehensive White Paper has been divided into three parts for editorial reasons.This publication (Part 2) covers the recommendations on Biomarkers, IVD/CDx, LBA and Cell-Based Assays. Part 1A (Mass Spectrometry Assays and Regulated Bioanalysis/BMV), P1B (Regulatory Inputs) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 16 of Bioanalysis, issues 9 and 7 (2024), respectively.

Flow Cytometry Method Validation Protocols.

Analytical method validation provides a means to ensure that data are credible and reproducible. This article will provide a brief introduction to analytical method validation as applied to cellular analysis by flow cytometry, along with practical procedures for four different types of validation. The first, Basic Protocol 1 (the limited validation protocol), is recommended for research and non-regulated laboratories. Next, Basic Protocol 2) presents a reasonable, fit-for-purpose validation approach appropriate for biopharma and research settings. Basic Protocol 3 addresses the type of validation performed in clinical laboratories for moderate-risk tests developed in house. Finally, Basic Protocol 4 describes the process that should be applied whenever a method is being transferred from one facility to another. All four validation plans follow the fit-for-purpose validation approach, in which the validation parameters are selected based on the intended use of the assay. These validation protocols represent the minimal requirement and may not be applicable for every intended use such as high-risk clinical assays or data to be used as a primary endpoint in a clinical trial. The recommendations presented here are consistent with the white papers published by the American Association of Pharmaceutical Scientists and the International Clinical Cytometry Society, as well as with Clinical Laboratory Standards Institute Guideline H62: Validation of Assays Performed by Flow Cytometry (CLSI, 2021). © 2023 Wiley Periodicals LLC. Basic Protocol 1: Limited validation Basic Protocol 2: Fit-for-purpose validation for biopharma and research settings Basic Protocol 3: Validation for moderate clinical risk laboratory developed tests Basic Protocol 4: Transfer validation.

2022 White Paper on Recent Issues in Bioanalysis: Enzyme Assay Validation, BAV for Primary End Points, Vaccine Functional Assays, Cytometry in Tissue, LBA in Rare Matrices, Complex NAb Assays, Spectral Cytometry, Endogenous Analytes, Extracellular Vesicles Part 2 - Recommendations on Biomarkers/CDx, Flow Cytometry, Ligand-Binding Assays Development & Validation; Emerging Technologies; Critical Reagents Deep Characterization.

The 16th Workshop on Recent Issues in Bioanalysis (16th 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 2) covers the recommendations on LBA, Biomarkers/CDx and Cytometry. Part 1 (Mass Spectrometry and ICH M10) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 15 of Bioanalysis, issues 16 and 14 (2023), respectively.

2021 White Paper on Recent Issues in Bioanalysis: ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry (Part 2 - Recommendations on Biomarkers/CDx Assays Development & Validation, Cytometry Validation & Innovation, Biotherapeutics PK LBA Regulated Bioanalysis, Critical Reagents & Positive Controls Generation).

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 three Main Workshops and seven 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 2) covers the recommendations on ISR for Biomarkers, Liquid Biopsies, Spectral Cytometry, Inhalation/Oral & Multispecific Biotherapeutics, Accuracy/LLOQ for Flow Cytometry. 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 3 (TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness) are published in volume 14 of Bioanalysis, issues 9 and 11 (2022), respectively.

High-sensitivity flow cytometric assays: Considerations for design control and analytical validation for identification of Rare events.

The current consensus recommendation papers dealing with the unique requirements for the analytical validation of assays performed by flow cytometry address the validation of sensitivity (both analytical and functional) only in general terms. In this paper, a detailed approach for designing and validating the sensitivity of rare event methods is described. The impact of panel design and optimization on the lower limit of quantification (LLOQ) and suggestions for reporting data near, or below, the LLOQ are addressed. This paper serves to provide best practices for the development, optimization, and analytical validation of flow cytometric assays designed to assess rare events. Note that this paper does not discuss clinical sensitivity validation, which addresses the positive and negative predictive value of the test result.

Best practices for the development, analytical validation and clinical implementation of flow cytometric methods for chimeric antigen receptor T cell analyses.

Chimeric Antigen Receptor (CAR) T cells are recognized as efficacious therapies with demonstrated ability to produce durable responses in blood cancer patients. Regulatory approvals and acceptance of these unique therapies by patients and reimbursement agencies have led to a significant increase in the number of next generation CAR T clinical trials. Flow cytometry is a powerful tool for comprehensive profiling of individual CAR T cells at multiple stages of clinical development, from product characterization during manufacturing to longitudinal evaluation of the infused product in patients. There are unique challenges with regard to the development and validation of flow cytometric methods for CAR T cells; moreover, the assay requirements for manufacturing and clinical monitoring differ. Based on the collective experience of the authors, this recommendation paper aims to review these challenges and present approaches to address them. The discussion focuses on describing key considerations for the design, optimization, validation and implementation of flow cytometric methods during the clinical development of CAR T cell therapies.

2020 White Paper on Recent Issues in Bioanalysis: BAV Guidance, CLSI H62, Biotherapeutics Stability, Parallelism Testing, CyTOF and Regulatory Feedback (Part 2A - Recommendations on Biotherapeutics Stability, PK LBA Regulated Bioanalysis, Biomarkers Assays, Cytometry Validation & Innovation Part 2B - Regulatory Agencies' Inputs on Bioanalysis, Biomarkers, Immunogenicity, Gene & Cell Therapy and Vaccine).

The 14th edition of the Workshop on Recent Issues in Bioanalysis (14th 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 14th 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 covers the recommendations on (Part 2A) BAV, PK LBA, Flow Cytometry Validation and Cytometry Innovation and (Part 2B) Regulatory Input. Part 1 (Innovation in Small Molecules, Hybrid LBA/LCMS & Regulated Bioanalysis), Part 3 (Vaccine, Gene/Cell Therapy, NAb Harmonization and Immunogenicity) are published in volume 13 of Bioanalysis, issues 4, and 6 (2021), respectively.

Safety and Clinical Activity of MEDI0562, a Humanized OX40 Agonist Monoclonal Antibody, in Adult Patients with Advanced Solid Tumors.

PURPOSE: Immune checkpoint blockade has demonstrated clinical benefits across multiple solid tumor types; however, resistance and relapse often occur. New immunomodulatory targets, which are highly expressed in activated immune cells, are needed. MEDI0562, an agonistic humanized mAb, specifically binds to the costimulatory molecule OX40. This first-in-human study evaluated MEDI0562 in adults with advanced solid tumors. PATIENTS AND METHODS: In this phase I, multicenter, open-label, single-arm, dose-escalation (3+3 design) study, patients received 0.03, 0.1, 0.3, 1.0, 3.0, or 10 mg/kg MEDI0562 through intravenous infusion every 2 weeks, until confirmed disease progression or unacceptable toxicity. The primary objective evaluated safety and tolerability. Secondary endpoints included antitumor activity, pharmacokinetics, immunogenicity, and pharmacodynamics. RESULTS: In total, 55 patients received ≥1 dose of MEDI0562 and were included in the analysis. The most common tumor type was squamous cell carcinoma of the head and neck (47%). Median duration of treatment was 10 weeks (range, 2-48 weeks). Treatment-related adverse events (TRAEs) occurred in 67% of patients, most commonly fatigue (31%) and infusion-related reactions (14%). Grade 3 TRAEs occurred in 14% of patients with no apparent dose relationship; no TRAEs resulted in death. Two patients had immune-related partial responses per protocol and 44% had stable disease. MEDI0562 induced increased Ki67+ CD4+ and CD8+ memory T-cell proliferation in the periphery and decreased intratumoral OX40+ FOXP3+ cells. CONCLUSIONS: MEDI0562 was safely administered at doses up to 10 mg/kg in heavily pretreated patients. On-target pharmacodynamic effects were suggested in this setting. Further evaluation with immune checkpoint inhibitors is ongoing.

Ex vivo expanded human CD4+CD25+Foxp3+ regulatory T cells prevent lethal xenogenic graft versus host disease (GVHD).

Mouse studies demonstrated that infusion of CD4+CD25+ regulatory T cells (Tregs) prevented graft versus host disease (GVHD) lethality after bone marrow transplantation (BMT). But the potential impact of human Tregs on GVHD has not been well demonstrated. In this study, we demonstrated that human Tregs enriched from peripheral blood of healthy donors could be expanded ex vivo to clinically relevant cell numbers in 2-3 weeks while maintaining Foxp3, CD25, CTLA-4, and CD62L expression as well as in vitro suppressive function. Furthermore, injection of human PBL into NOD/SCID mice induced lethal xenogenic GVHD, but co-transfer of expanded human Tregs with human PBL significantly enhanced survival, reduced GVHD symptoms, and inhibited human IgG/IgM production in the NOD/SCID mice. These results demonstrated that ex vivo expanded human Tregs retained their in vivo suppressive activity and prevented lethal xenogeneic GVHD, revealing the therapeutic potential of expanded human Tregs for GVHD.

Flow Cytometry Method Validation Protocols.

Analytical method validation provides a means to ensure that data are credible and reproducible. This unit will provide a brief introduction to analytical method validation as applied to cellular analysis by flow cytometry. In addition, the unit will provide practical procedures for three different types of validation. The first is a limited validation protocol that is applicable for research settings and non-regulated laboratories. The second is validation protocol that presents the minimum validation requirements in regulated laboratories. The third is a transfer validation protocol to be used when methods are transferred between laboratories. The recommendations presented in this unit are consistent with the white papers published by the American Association of Pharmaceutical Scientists and the International Clinical Cytometry Society, as well as with Clinical Laboratory Standards Institute Guideline H62: Validation of Assays Performed by Flow Cytometry (currently in preparation). © 2018 by John Wiley & Sons, Inc.

A CD40L-targeting protein reduces autoantibodies and improves disease activity in patients with autoimmunity.

The CD40/CD40L axis plays a central role in the generation of humoral immune responses and is an attractive target for treating autoimmune diseases in the clinic. Here, we report the generation and clinical results of a CD40L binding protein, VIB4920, which lacks an Fc domain, therefore avoiding platelet-related safety issues observed with earlier monoclonal antibody therapeutics that targeted CD40L. VIB4920 blocked downstream CD40 signaling events, resulting in inhibition of human B cell activation and plasma cell differentiation, and did not induce platelet aggregation in preclinical studies. In a phase 1 study in healthy volunteers, VIB4920 suppressed antigen-specific IgG in a dose-dependent fashion after priming and boosting with the T-dependent antigen, KLH. Furthermore, VIB4920 significantly reduced circulating Ki67+ dividing B cells, class-switched memory B cells, and a plasma cell gene signature after immunization. In a phase 1b proof-of-concept study in patients with rheumatoid arthritis, VIB4920 significantly decreased disease activity, achieving low disease activity or clinical remission in more than 50% of patients in the two higher-dose groups. Dose-dependent decreases in rheumatoid factor autoantibodies and Vectra DA biomarker score provide additional evidence that VIB4920 effectively blocked the CD40/CD40L pathway. VIB4920 demonstrated a good overall safety profile in both clinical studies. Together, these data demonstrate the potential of VIB4920 to significantly affect autoimmune disease and humoral immune activation and to support further evaluation of this molecule in inflammatory conditions.

Potent Immune Modulation by MEDI6383, an Engineered Human OX40 Ligand IgG4P Fc Fusion Protein.

Ligation of OX40 (CD134, TNFRSF4) on activated T cells by its natural ligand (OX40L, CD252, TNFSF4) enhances cellular survival, proliferation, and effector functions such as cytokine release and cellular cytotoxicity. We engineered a recombinant human OX40L IgG4P Fc fusion protein termed MEDI6383 that assembles into a hexameric structure and exerts potent agonist activity following engagement of OX40. MEDI6383 displayed solution-phase agonist activity that was enhanced when the fusion protein was clustered by Fc gamma receptors (FcγRs) on the surface of adjacent cells. The resulting costimulation of OX40 on T cells induced NFκB promoter activity in OX40-expressing T cells and induced Th1-type cytokine production, proliferation, and resistance to regulatory T cell (Treg)-mediated suppression. MEDI6383 enhanced the cytolytic activity of tumor-reactive T cells and reduced tumor growth in the context of an alloreactive human T cell:tumor cell admix model in immunocompromised mice. Consistent with the role of OX40 costimulation in the expansion of memory T cells, MEDI6383 administered to healthy nonhuman primates elicited peripheral blood CD4 and CD8 central and effector memory T-cell proliferation as well as B-cell proliferation. Together, these results suggest that OX40 agonism has the potential to enhance antitumor immunity in human malignancies. Mol Cancer Ther; 17(5); 1024-38. ©2018 AACR.

Developmental alterations in thymocyte sensitivity are actively regulated by MHC class II expression in the thymic medulla.

Developing thymocytes are positively selected if they respond to self-MHC-peptide complexes, yet mature T cells are not activated by those same self-complexes. To avoid autoimmunity, positive selection must be followed by a period of maturation when the cellular response to TCR signals is altered. The mechanisms that mediate this postselection developmental tuning remain largely unknown. Specifically, it is unknown whether developmental tuning is a preprogrammed outcome of positive selection or if it is sensitive to ongoing interactions between the thymocyte and the thymic stroma. We probed the requirement for MHC class II-TCR interactions in postselection maturation by studying single positive (SP) CD4 thymocytes from K14/A(beta)(b) mice, in which CD4 T cells cannot interact with MHC class II in the thymic medulla. We report here that SP CD4 thymocytes must receive MHC class II signals to avoid hyperactive responses to TCR signals. This hyperactivity correlates with decreased expression of CD5; however, developmental tuning can occur independently of CD5, correlating instead with differences in the distribution of Lck. Thus, the maturation of postselection SP CD4 thymocytes is an active process mediated by ongoing interactions between the T cell and MHC class II molecules. This represents a novel mechanism by which the thymic medulla prevents autoreactivity.