Deciphering molecular and cellular ex vivo responses to bispecific antibodies PD1-TIM3 and PD1-LAG3 in human tumors.

BACKGROUND: Next-generation cancer immunotherapies are designed to broaden the therapeutic repertoire by targeting new immune checkpoints including lymphocyte-activation gene 3 (LAG-3) and T cell immunoglobulin and mucin-domain containing-3 (TIM-3). Yet, the molecular and cellular mechanisms by which either receptor functions to mediate its inhibitory effects are still poorly understood. Similarly, little is known on the differential effects of dual, compared with single, checkpoint inhibition. METHODS: We here performed in-depth characterization, including multicolor flow cytometry, single cell RNA sequencing and multiplex supernatant analysis, using tumor single cell suspensions from patients with cancer treated ex vivo with novel bispecific antibodies targeting programmed cell death protein 1 (PD-1) and TIM-3 (PD1-TIM3), PD-1 and LAG-3 (PD1-LAG3), or with anti-PD-1. RESULTS: We identified patient samples which were responsive to PD1-TIM3, PD1-LAG3 or anti-PD-1 using an in vitro approach, validated by the analysis of 659 soluble proteins and enrichment for an anti-PD-1 responder signature. We found increased abundance of an activated (HLA-DR+CD25+GranzymeB+) CD8+ T cell subset and of proliferating CD8+ T cells, in response to bispecific antibody or anti-PD-1 treatment. Bispecific antibodies, but not anti-PD-1, significantly increased the abundance of a proliferating natural killer cell subset, which exhibited enrichment for a tissue-residency signature. Key phenotypic and transcriptional changes occurred in a PD-1+CXCL13+CD4+ T cell subset, in response to all treatments, including increased interleukin-17 secretion and signaling toward plasma cells. Interestingly, LAG-3 protein upregulation was detected as a unique pharmacodynamic effect mediated by PD1-LAG3, but not by PD1-TIM3 or anti-PD-1. CONCLUSIONS: Our in vitro system reliably assessed responses to bispecific antibodies co-targeting PD-1 together with LAG-3 or TIM-3 using patients' tumor infiltrating immune cells and revealed transcriptional and phenotypic imprinting by bispecific antibody formats currently tested in early clinical trials.

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.

Barcoding of live peripheral blood mononuclear cells to assess immune cell phenotypes using full spectrum flow cytometry.

Barcoded flow cytometry is a multiplexing technique allowing for the simultaneous acquisition of cells from different donors or experimental conditions in a high-throughput manner. This approach allows to synchronize acquisition of samples and reduce variance introduced through the operator or technical platform. However, to date, only very few flow cytometry barcoding protocols have been developed, which often suffer from technical limitations. Here, we developed a novel barcoding protocol for a full-spectrum flow cytometry platform. We developed a 21-color immunophenotyping assay for up to 20 different samples analyzed simultaneously with comparable variance between repeated single-tube acquisition and postde-multiplexing. Barcoding offers great potential in parallelizing the analysis of complex cell populations such as peripheral blood mononuclear cells (PBMCs). Consequently, we assessed the performance of our method in situations where PBMCs were challenged with phytohaemagglutinin (PHA), a strong mitogen and broad activator of B cells and T cells, and superantigen Staphylococcus enterotoxin B (SEB) that has been reported to induce polyclonal T cell activation. PBMCs were either barcoded before pooled challenge or challenged individually pre-barcoding. Our final workflow included pooled immunophenotyping followed by machine learning aided single-cell data analysis and enabled us to identify robust PHA and SEB mode of action related phenotypic changes in PBMC immune cell lineages. Conclusively, we present a novel technique allowing the barcoded acquisition and analysis of PBMCs from up to 20 different donors and present a valid basis for the future development of complex immunophenotyping protocols.

Dendritic Cell Tumor Vaccination via Fc Gamma Receptor Targeting: Lessons Learned from Pre-Clinical and Translational Studies.

Despite significant recent improvements in the field of immunotherapy, cancer remains a heavy burden on patients and healthcare systems. In recent years, immunotherapies have led to remarkable strides in treating certain cancers. However, despite the success of checkpoint inhibitors and the advent of cellular therapies, novel strategies need to be explored to (1) improve treatment in patients where these approaches fail and (2) make such treatments widely and financially accessible. Vaccines based on tumor antigens (Ag) have emerged as an innovative strategy with the potential to address these areas. Here, we review the fundamental aspects relevant for the development of cancer vaccines and the critical role of dendritic cells (DCs) in this process. We first offer a general overview of DC biology and routes of Ag presentation eliciting effective T cell-mediated immune responses. We then present new therapeutic avenues specifically targeting Fc gamma receptors (FcγR) as a means to deliver antigen selectively to DCs and its effects on T-cell activation. We present an overview of the mechanistic aspects of FcγR-mediated DC targeting, as well as potential tumor vaccination strategies based on preclinical and translational studies. In particular, we highlight recent developments in the field of recombinant immune complex-like large molecules and their potential for DC-mediated tumor vaccination in the clinic. These findings go beyond cancer research and may be of relevance for other disease areas that could benefit from FcγR-targeted antigen delivery, such as autoimmunity and infectious diseases.

A human receptor occupancy assay to measure anti-PD-1 binding in patients with prior anti-PD-1.

Receptor occupancy (RO) assessment by flow cytometry is an important pharmacodynamic (PD) biomarker in the clinical development of large molecules such as monoclonal therapeutic antibodies (mAbs). The total-drug-bound RO assay format directly assesses mAb binding to cell surface targets using anti-drug detection antibodies. Here, we generated a flow cytometry detection antibody specifically binding to mAbs of the IgG1 P329GLALA backbone. Using this reagent, we developed a total-drug-bound RO assay format for RG7769, a bi-specific P329GLALA containing mAb targeting PD-1 and TIM3 on T cells. In its fit-for-purpose validated version, this RO assay has been used in the Phase-I dose escalation study of RG7769, informing on peripheral T cell RO and RG7769 antibody binding capacity (ABC). We assessed RG7769 RO in checkpoint-inhibitor (CPI) naïve patients and anti-PD-1 CPI experienced patients using our novel assay. Here, we show that in both groups, complete T cell RO can be achieved (~100%). However, we found that the maximum number of T cell binding sites for RG7769 pre-dosing was roughly twofold lower in patients recently having undergone anti-PD-1 treatment. We show that this is due to steric hindrance exerted by competing mAbs masking the available drug binding sites. Our findings highlight the importance of quantitative mAb assessment in addition to relative RO especially in the context of patients who have previously received anti-PD-1 treatment.

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.

Fc Gamma Receptors and Their Role in Antigen Uptake, Presentation, and T Cell Activation.

The cellular uptake, intracellular processing, and presentation of foreign antigen are crucial processes for eliciting an effective adaptive host response to the majority of pathogens. The effective recognition of antigen by T cells requires that it is first processed and then presented on MHC molecules that are expressed on other cells. A critical step leading to the presentation of antigen is delivering the foreign cargo to an intracellular compartment where the antigen can be processed and loaded onto MHC molecules. Fc-gamma receptors (FcγRs) recognize IgG-coated targets, such as opsonized pathogens or immune complexes (ICs). Cross-linking leads to internalization of the cargo with associated activation of down-stream signaling cascades. FcγRs vary in their affinity for IgG and intracellular trafficking, and therefore have an opportunity to regulate antigen presentation by controlling the shuttling and processing of their cargos. In this way, they critically influence physiological and pathophysiological adaptive immune cell functions. In this review, we will cover the contribution of FcγRs to antigen-presentation with a focus on the intracellular trafficking of IgG-ICs and the pathways that support this function. We will also discuss genetic evidence linking FcγR biology to immune cell activation and autoimmune processes as exemplified by systemic lupus erythematosus (SLE).

A simple method for measuring immune complex-mediated, Fc gamma receptor dependent antigen-specific activation of primary human T cells.

Immune complex (IC) deposition of IgG containing autologous antigens has been observed in autoimmunity. This can lead to IC-mediated antigen uptake and presentation by antigen presenting cells (APC) driving T cell dependent inflammation. IgG receptors (FcγRs) have been suggested to be involved in this process. Since ICs have been linked to autoimmune diseases, interfering with IC mediated effects on APCs and subsequent autoimmune T cell activation via FcγR blockade may be therapeutically beneficial. However, this is currently challenging due to a lack of translatable animal models and specific human in vitro assays to study IC-driven T cell responses. Here, we developed a simple cellular assay to study IC-mediated T cell activation in vitro using human peripheral blood mononuclear cells and tetanus toxoid as a model antigen. We observed that tetanus ICs led to a strong induction of T cell proliferation and release of pro-inflammatory cytokines, which are hallmarks of chronic inflammation. This process was exacerbated when compared to tetanus toxoid challenge alone. IC-mediated T cell effects were FcγR dependent and inhibited by high-dose intravenous IgG (IVIg), a drug often used for the clinical treatments of autoimmune diseases. Similar effects were also seen using a hepatitis antigen. Consequently, we propose our assay as a rapid yet robust alternative to more labour-intense and time-consuming protocols, for example involving separate maturation of dendritic cells followed by T cell co-culture to study antigen specific primary T cell activation.

Dicer1 imparts essential survival cues in Notch-driven T-ALL via miR-21-mediated tumor suppressor Pdcd4 repression.

The modulatory function of individual microRNAs (miRNAs) in Notch-driven T-cell acute lymphoblastic leukemias (T-ALLs) has recently been established. Although protumorigenic and tumor-suppressive miRNAs are implicated in disease onset in murine models of Notch-driven T-cell leukemia, whether Dicer1-processed miRNAs are essential for Notch-driven T-ALL is currently unknown. Here we used conditional and inducible genetic loss-of-function approaches to test whether the development and maintenance of Notch-driven T-ALL was dependent on Dicer1 function. Mice with specific inactivation of both Dicer1 alleles in the T-cell lineage did not develop Notch-driven T-ALL. In contrast, loss of 1 functional Dicer1 allele did not significantly perturb T-ALL onset and tumor progression. Inducible inactivation of Dicer1 in early stage polyclonal T-ALL cells was sufficient to abrogate T-ALL progression in leukemic mice, whereas late-stage monoclonal T-ALL cells were counterselected against loss of Dicer1. Lineage-tracing experiments revealed that Dicer1 deficiency led to the induction of apoptosis in T-ALL cells, whereas cell cycle progression remained unaltered. Through microarray-based miRNA profiling, we identified miR-21 as a previously unrecognized miRNA deregulated in both mouse and human T-ALL. Herein, we demonstrate that miR-21 regulates T-ALL cell survival via repression of the tumor suppressor Pdcd4.