A method for the phenotyping of mononuclear cells

Abstract

The present invention relates to A semi- quantitative flow cytometry method for measuring the frequency and / or the cell concentration of one or more mononuclear cell subsets in a biological sample, wherein the subset of mononuclear cells is detected by a set of markers selected from the group of: Viability, CD45, CD66b, CD19, CD3, CD4, CD8, HLA-DR, CD279, Ki67, CD45RA and CD197, wherein said frequency and / or the cell count are determined with an inter-assay coefficient of variation (CV) of less than or equal to 20%.

Description

[0001] A method for the phenotyping of mononuclear cells

[0002] Flow Cytometry (FCM) has revolutionized the study of the immune system by enabling rapid and precise multiparametric analysis at the single-cell level from millions of cells (thousands of cells or particles per second). Recent advancements in flow cytometry technology have been remarkable, with current platforms now equipped with up to nine lasers, allowing for the simultaneous detection of up to 50 fluorescent parameters. This technological leap offers an unprecedented opportunity for deep and broad immunophenotyping of various cell populations, providing richer scientific insights and enhancing the study of immune responses and biomarker exploration in clinical settings.

[0003] However, the development and implementation of high-dimensional flow cytometry (hdFCM) assays in clinical trials (CTs) present significant challenges. Traditionally, FCM assays involved multi-tube setups, sharing 4-6 consistent markers while varying phenotypic markers between tubes to measure all markers of interest. This approach often restricted each study to generating data for only a small set of biomarkers, leading to incomplete datasets where not all biomarkers were consistently measured across studies. In contrast, hdFCM offers the unique advantage of a standardized assay across multiple CTs and drug candidates, facilitating comparability between studies. This is particularly valuable in combination studies or when targeting different drug targets within the same disease area, as it can enhance our understanding of disease pathogenesis following treatment with various therapeutic molecules, thereby increasing the potential for reverse translational approaches.

[0004] Despite these benefits, the extended duration of CTs, often spanning years, necessitates the generation of comparable data over time. This requirement imposes limitations on design flexibility, leaving little room for assay customization to factor in emerging data. As a result, complex new assays need to be developed. This increased complexity and higher costs associated with developing and implementing de novo hdFCM assays have hindered the full utilization of their potential in clinical trials (Cytometry A. 2024 Nov; 105(1 l):829-837).

[0005] In conclusion, there is a need for a reliable, flexible and customizable FCM method for use in CTs.

[0006] Summary of the invention:

[0007] The present invention provides a semi- quantitative flow cytometry method for measuring the frequency and / or the cell concentration of one or more mononuclear cell subsets in a biological sample, wherein the subset of mononuclear cells is detected by a set of markers selected from the group of: Viability, CD45, CD66b, CD19, CD3, CD4, CD8, HLA-DR, CD279, Ki67, CD45RA and CD197, wherein said frequency and / or the cell count are determined with an inter-assay coefficient of variation (CV) of less than or equal to 20%.

[0008] In an embodiment of the present invention, at least one marker of a second set of markers is detected, wherein the additional set of markers comprises the following markers: CD25, CD127, FoxP3, CD95, CD28, TCF-1, TIM3, 41BB, LAG3, TIGIT, 0X40, ICOS, CD39, CD57, KLGR1, CD103, CD14, CD16, CD56, NKG2D and GranzB.In an embodiment of the present invention, the following markers of the second set of markers are detected: TCF1, TIM3, LAG3, CD95 and 41BB. (pre-set referred as “T memory stem cell Module”)

[0009] In an embodiment of the present invention, the following markers of the second set of markers are detected: CD39, CD57, KLRG1 and CD 103. (pre-set referred as “Exhaustion / senescence Module”)

[0010] In an embodiment of the present invention, the following markers of the second set of markers are detected: 0X40, TIGIT and ICOS. (pre-set referred as “Immune checkpoint Module”)

[0011] In an embodiment of the present invention, the following markers of the second set of markers are detected: CD16, CD56, CD14, NKG2D and Gran-B. (pre-set referred as “Monocytes / NK cell Module”)

[0012] In an embodiment of the present invention, the following markers of the second set of markers are detected: TCF1, TIM3, LAG3, CD95, 41BB, CD39, CD57, KLRG1, CD103, 0X40, ICOS, CD16, CD56, CD 14, NKG2D and Gran-B.

[0013] In an embodiment of the present invention, the biological sample is a patient tissue sample, preferably a whole blood sample, or peripheral blood mononuclear cells (PBMCs).

[0014] In an embodiment of the present invention, the patient is being treated with a cancer drug, an immunology drug or an inflammation drug, preferably an immunotherapy drug.

[0015] In an embodiment of the present invention, the mononuclear cells are T cells, monocytes or natural killer (NK) cells, preferably T cells.

[0016] In an embodiment of the present invention, the following marker configuration is detected: - Core markers: CD45, CD66B, CD279, CD 197, CD45RA, HLA-DR, CD8, CD3, CD 19, Ki- 67, CD4, viability;

[0017] - Regulatory T cell markers: FoxP3, CD 127 and CD25;

[0018] - NK / Monocytes markers: CD314, CD56, Gzanzyme B, CD14 and CD16;

[0019] - Senescence / exhaustion markers: CD103, CD57, KLRG1, CD39;

[0020] - Checkpoint markers: TIGIT, CD134, ICOS;

[0021] - T memory stem cell / T resident cell markers: CD95, 41BB, TCF1, CD28, TIM3 and LAG3. In an embodiment of the present invention, the following marker configuration is detected: - Core markers: CD45, CD66B, CD279, CD 197, CD45RA, HLA-DR, CD8, CD3, CD 19, Ki- 67, CD4, viability;

[0022] - Regulatory T cell markers: FoxP3, CD 127 and CD25.

[0023] - In an embodiment of the present invention, the following marker configuration is detected: - Core markers: CD45, CD66B, CD279, CD 197, CD45RA, HLA-DR, CD8, CD3, CD 19, Ki- 67, CD4, viability;

[0024] - Regulatory T cell markers: FoxP3, CD 127 and CD25.

[0025] Senescence markers: CD103, CD57, KLRG1, CD39.

[0026] In an embodiment of the present invention, the following marker configuration is detected: - Core markers: CD45, CD66B, CD279, CD 197, CD45RA, HLA-DR, CD8, CD3, CD 19, Ki- 67, CD4, viability;Regulatory T cell markers: FoxP3, CD 127 and CD25;

[0027] Senescence markers: CD 103, CD 57;

[0028] Checkpoint markers: TIGIT, ICOS.

[0029] In response to current challenges in the field, the present invention relates to the development and requested validation of a spectral FCM T-cell immunophenotyping 33-plex assay with a robust, modular backbone / drop-in modules design. This design allows for customization / modification with targeted drop-in modules, providing higher flexibility during biomarker evaluation and enabling adaptation across study phases or timepoints without compromising data robustness, enabling comparability of the data independently of the configuration applied. The modular approach enhances biomarker discovery opportunities and facilitates reverse translational approaches across studies, while also allowing for reduced panel versions for targeted hypothesis testing. All contributing to a higher efficiency, flexibility and costs / time saving during the duration of CTs.

[0030] Short description of the figures:

[0031] Fig. 1A shows a scheme of the mononuclear cells and biomarkers which are used in the assay of the present invention to classify the different populations and their functional states.

[0032] Fig. IB shows the different biomarker modules used in the assay of the present invention. The combination of biomarkers to be used in the assay of the present invention can be selected from the following marker modules: Core panel, Treg module, T stem cell / memory module, Checkpoint module, T exhaustion module and Mono-NK module. The XXX module illustrate any potential other configuration by different combination of the full listed markers considered, that would not require further validation for its implementation in a CT.

[0033] Fig. 2 shows exemplary FCM marker modules with the marker proteins, the fluorochrome and the laser. These modules can be combined into different assay configurations e.g., the Core / backbone can be combined with any of the given modules or combinations thereof.

[0034] Fig. 3 shows three exemplary FCM assay configurations: configuration 1, comprises the Core / Backbone marker module and the T regulatory cell module (Tregs); configuration 2, comprises the Core / Backbone marker module, the Treg module and the Senescence marker module; configuration 3, comprises the Core / Backbone marker module, the Treg module, a customized version of the Senescence module (including only 2 out of 4 markers of the module) and a customized version of the Checkpoint module with 12 out of 3 markers of the module. The fluorochromes and lasers used in each marker module are also shown.

[0035] Fig. 4 shows the hierarchical stepwise 2D gating strategy applied for identification of the main lineage populations defined by the core / backbone module. This comprises the following steps: before identification of any individual population, cell population autofluorescence (AF), debris and doublets are excluded to gate on total leukocytes based on CD45 expression level. Subsequently, dead cells and granulocytes are cleaned up from the targeted population. Based on CD 19 and CD3 expression, B cells and T cells are identified. T cell following analysis allows distinguishing T helper (CD4+) and T cytotoxic (CD8+) cells, classifying them into functional stages: central memory (CM), effector (EM),naive, and terminally differentiated memory CD45RA+ (TEMRA) based on CD197 and CD45RA expression. It also monitors key functional markers like HLA-DR, CD279 (PD1), and Ki67. The Treg module enables the characterization of Tregs by CD127, CD25, and FoxP3 markers. A customized combination of markers including CD 14, CD 16, and CD56, enable monitoring of different populations of Monocytes and NK cells.

[0036] Fig. 5 shows the expression patterns of markers defining the Stem memory, Exhaustion / senes-cence, Immune checkpoint and NK profiling modules.

[0037] Fig. 6: Stability and sensitivity of secondary endpoints analytes, a) Average % change from baseline (day 0) out to 4 days after ambient storage for the secondary endpoint analytes. The analytes are sorted from high to low based on the ABS count values with the dashed lines representing the 20% acceptance criteria limit, b) Samples with decreasing levels of secondary endpoint analytes were generated by mixing fully stained WB with an FMO control for either CD8 (CD8+ T Cell) or CD25 (Treg, FoxP3+ Treg) ... at different levels (CD8: Full Stain, linlO, lin 50, linlOO, lin200, lin500 and Blank; CD25 Full Stain, lin2, lin5, linlO, lin20, lin50 and Blank). Artificially generated samples were analyzed in triplicate with the full modular configuration for 3 separate AHV whole blood samples. Mean and SD of the measured absolute cell counts were plotted versus the expected values. The solid line represents the LOD and the dotted line LLOQ, where it was different to the LOD.

[0038] Fig. 7: Summary of intra-assay precision and stability of Exploratory Endpoint Analytes. a) Intra-assay precision. Whole blood from 3 AHVs was measured in triplicate and the mean for each %frequency or ABS count analyte was plotted against the %CV. The dashed line represents the 20% CV acceptance criteria limit, b) Stability. Whole blood from 3 AHVs was stained with the whole panel in singlicate, and analyte values were measured at baseline (0 days) and at 1 day intervals up 4 days. The change from baseline in % was plotted for ABS analytes from the core panel. The analytes are sorted based on ABS values. Dashed lines represent the 20% change from baseline acceptance criteria limit.

[0039] Detailed description of the invention

[0040] The T lymphocytes or T cells are key players of the adaptive immune response. They are responsible for the development of cell-mediated responses providing protection against intracellular and extracellular pathogens and contribute to the elimination of tumor or autoreactive cells. During the last decades, T cells and their subpopulations have become a central player in the development of immunotherapies to fight against diseases (e.g., cancers, autoimmune diseases, etc.).

[0041] The method of the present invention comprises a core panel of markers (first set of markers) and flexible and customizable drop-in / drop-out modules of markers (second set of markers) that enable multiple combinatorial options. The pre-defined modules, considered for validation purposes, can be modified and combined randomly, enhancing the opportunities to build up on different modules. Therefore, different final marker configurations of the assay for the immunophenotyping of T cells subpopulations can be generated, considering a pool of 21 drop-in markers that enable a comprehensive monitoring of subsets and characterization of their functionality (i.e., activation, exhaustion status, proliferation capacity).The design of the full marker configuration including all marker modules together with the core marker panel was initially drafted using established panel design recommendations (Journal of Immunological Methods Volume 475, December 2019, 112662) with fluorochrome selections driven by antigen density and predicted expression patterns, fluorochrome brightness, and reagent availability. Tools including spectral signatures, similarity matrices with complexity scores and stain index reduction predictions available within Cytek’s Full Spectrum Viewer, were leveraged during panel construction (Current Protocols in Immunology 5.4.1-5.4.38, April 2017).

[0042] The full-panel version of the assay considers all modules (i.e., Treg, Senescence -exhaustion, Checkpoints, NK / Monocytes and TSCM / TRes).

[0043] The core module (first set of markers), i.e., the backbone mandatory for all potential final combinations, defines T cells and their subsets, gathered from live leukocytes by the expression of CD3 protein. Subsequently, subsets of T helper cells (CD4+) and T cytotoxic cells (CD8+) are distinguished and further classified based on expression of CD 197 and CD45RA in their different differentiation stages, central memory (CM : CD197+CD45RA-), effector cells (EM: CD197-CD45RA-), naive cells (CD197+CD45RA+) and terminally differentiated memory CD45RA+ (TEMRA: CD197-CD45RA+). Additionally, the configuration of the core panel allows monitoring key functional markers like HLA-DR, CD279 (programmed cell death 1, PD1) and Ki67 on these subsets.(Fig.la) To monitor regulatory T cells (Tregs), the Treg module includes commonly used markers such as CD127 and CD25, along with the intracellular protein forkhead box P3 (FoxP3). Additionally, analysis of FoxP3 vs CD45RA (from core panel) allows to monitor three phenotypically and functionally distinct Treg subpopulations: resting Tregs (rTregs: CD45RA+FoxP31ow; activated Tregs (aTregs: CD45RA-FoxP3high), both possessing suppressive capacity; and a third phenotype, CD45RA-FoxP31ow, which is considered to be cytokine-secreting and non-suppressive Treg subset. The flexibility of our approach can be seen in the Treg analysis. The analysis can be potentially deeper extended by the drop-in of other markers not considered in the pre-defined module, e.g., evaluation of CD39 expression can provide evidence of activation status on Treg cells. Similarly, measurement of expression levels of PD1, TIGIT or TIM3 will enable the identification of highly suppressive Tregs.

[0044] High-dimensional flow cytometry offers unparalleled depth for immune profiling, yet its clinical research utility has been limited by logistical and technical challenges and requirements for validation to ensure robustness and comparability of the clinical data . The present invention provides a validated, innovative modular hdFCM framework that addressed these limitations, enabling flexible, cost-effective, and scalable immunophenotyping in CTs.

[0045] Through comprehensive design and optimization, from panel design and reagents selection to and sample processing and unmixing strategy, the inventors established a robust platform capable of delivering consistent pharmacodynamic data and supporting both exploratory and targeted biomarker strategies.

[0046] At its foundation, the present invention employs a 33-marker assay, combining a stable core panel with five adaptable modules validated against CLSI H62 guidelines, the assay demonstratedhigh precision (CV < 20%) and stability for up to four days at an ambient temperature. This performance was consistent across both secondary endpoints (e.g., CD8+ cytotoxic and Treg populations) and exploratory markers, with 90% of analytes showing excellent repeatability.

[0047] A key strength of the method of the present invention is its modular design, providing flexibility without compromising data integrity. Compared to earlier modular strategies — such as the 8-color assays by Riihle et al. or the 12-color Treg panel by Corselli, M et al. (BD Life Sciences - Biosciences.) — our high-dimensional spectral cytometry platform supports a more complex and comprehensive panel. While other flexible designs like the “iCoreDrop” panel by Jensen & Kim offer adaptability, the method of the present invention uniquely combines this with rigorous validation against clinical standards, minimizing the re-validation burden typically required for drop-in assays in the clinical setting.

[0048] Importantly, the inventors provide statistical validation (R2> 0.98) of data consistency across multiple complex configurations, validating an adaptable framework that represents a paradigm shift for clinical trials. This enables dynamic adaptation across trial phases — from broad exploratory profiling in early phases to targeted hypothesis testing in later stages within a single, standardized system. Consequently, the significant time and cost associated with de novo assay development and validation are drastically reduced, accelerating decision-making as trials progress.

[0049] The robust data comparability demonstrated among the core, the core + Treg, and the full panel configurations confirm the intended applicability of the method of the present invention, allowing different module combinations to be used within, or across, clinical trial phases while ensuring consistent data. By demonstrating that core panel analytes perform reliably within the full panel — the most spectrally complex and challenging combination — this invention provides strong evidence for their comparability in any less complex configuration.

[0050] The present method’s readiness for multi-center deployment is further supported by its strong performance with both fresh whole blood and cryopreserved PBMCs (R2> 0.99 for cell frequencies), offering critical logistical flexibility and enabling retrospective analyses from biobanked samples.

[0051] This modular hdFCM framework is a transformative advance for clinical trials that aligns with broader trends in high-plex technologies. Like NGS and multiplex imaging, it uses a “core and explore” model; however, while those platforms provide crucial genomic or spatial context, our framework is uniquely capable of high-throughput, single-cell protein assessment. This makes it ideal for tracking pharmacodynamic cellular changes in peripheral blood.

[0052] By ensuring data integrity, reducing validation overhead, and enabling flexible trial design, this framework maximizes the scientific and clinical value of each patient sample — ultimately accelerating the development of novel therapeutics.

[0053] Definitions:

[0054] The term “biological sample” refers to material taken from a living organism, in particular a human being, for the purpose of analysis, research, or diagnosis. This term includes blood samples taken from patients.The term “Mononuclear cell” refers to a blood cell characterized by having a single, round nucleus. This term typically refers to two main types of immune cells, namely lymphocytes and monocytes.

[0055] Lymphocytes are a subset of white blood cells that play a crucial role in the innate and adaptive immune response. Lymphocytes include:

[0056] - T cells: Involved in cell-mediated immunity and include subtypes like helper T cells (CD4+), cytotoxic T cells (CD8+), regulatory T cells and NK T cells (1% of peripheral blood T cells)

[0057] - B cells: Responsible for producing antibodies as part of the humoral immune response.

[0058] - Natural Killer (NK) cells: Part of the innate immune system, these cells can destroy virus-infected cells and tumor cells without prior sensitization.

[0059] Monocytes are a type of white blood cell (Myeloid cells) that is part of the innate immune system. They can differentiate into macrophages and dendritic cells when they migrate into tissues. These cells are involved in phagocytosis (engulfing and digesting pathogens and debris) and play a key role in inflammation and antigen presentation.

[0060] The term “Anti-inflammatory drug” refers to a medication used to reduce inflammation in the body. Anti-inflammatory drugs are commonly used to treat conditions such as arthritis, inflammatory bowel disease, and various types of pain and injury. Exemplary anti-inflammatory drugs are:

[0061] - Nonsteroidal Anti-Inflammatory Drugs (NSAIDs): These include drugs like ibuprofen, aspirin, and naproxen. They work by inhibiting enzymes (COX-1 and COX-2) involved in the production of pro-inflammatory chemicals called prostaglandins.

[0062] - Corticosteroids: These are synthetic drugs that mimic the effects of hormones like cortisol. They are powerful anti-inflammatory agents and include medications such as prednisone and dexamethasone. Corticosteroids suppress various aspects of the immune response.

[0063] - Disease-Modifying Antirheumatic Drugs (DMARDs): Used primarily in the treatment of autoimmune diseases, they include drugs like methotrexate and hydroxychloroquine, which help to slow the progression of the disease and limit joint damage.

[0064] The term “Immunological disease drug” refers to a medication designed to target and manage diseases that affect the immune system. These diseases can either involve an overactive immune response (autoimmune diseases) or an underactive immune response (immunodeficiency disorders). The goal of these drugs is to modulate the immune system to restore balance and reduce symptoms associated with the disease.

[0065] The term “cancer drug” refers to a medication designed to treat malignancies by inhibiting the proliferation or by killing of cancer cells, thereby slowing the progress of the disease or potentially curing it. These drugs can function in several ways, such as:

[0066] - Chemotherapy: Uses cytotoxic drugs to kill or damage cancer cells.

[0067] - Targeted Therapy: Involves drugs that specifically target molecular changes seen mostly in cancer cells.

[0068] - Immunotherapy: Stimulates the body’s own immune system to fight cancer.

[0069] - Hormone Therapy: Blocks or removes hormones that fuel certain cancers to stop or slow their growth.- Radiopharmaceuticals: Deliver radiation directly to cancer cells via a pharmaceutical compound.

[0070] The term “immunotherapy drug” refers to a medication designed to enhance or modify the immune system's ability to fight diseases. These drugs are primarily used in the treatment of cancer and immune-related disorders. The aim of immunotherapy is either to stimulate the immune system to attack diseased cells more efficiently or to suppress an overactive immune system that is causing harm.

[0071] The term “flow cytometry” refers to an analytical technique used to measure and analyze the physical and chemical characteristics of individual cells or particles as they flow in a fluid stream through a beam of light, typically a laser. This method allows for the rapid and simultaneous multiparameter analysis of thousands of cells or particles per second. Key components of a flow cytometry system are:

[0072] Fluidics System: Directs the flow of the sample so that cells pass single-fde through the laser beam.

[0073] Optical System: Usually consists of lasers to illuminate the cells and a series of fdters and detectors to capture light emitted or scattered by the cells as well as the fluorescent molecules used to stain these cells.

[0074] Electronics System: Converts the light signals into electronic signals that can be processed and analyzed by a computer.

[0075] Forward Scatter (FSC): A parameter that measures the amount of light scattered in the forward direction when a cell passes through the laser beam. FSC is generally related to the size of the cell or particle.

[0076] Side Scatter (SSC): A parameter that measures the light scattered at 90 degrees to the laser beam. SSC provides information about the internal complexity or granularity of the cell, such as the presence of granules or organelles.

[0077] Gating: The process of selecting a specific population of cells from a larger dataset based on their scatter and fluorescence characteristics. Gates are used to isolate specific cell types or subpopulations for further analysis.

[0078] The term “Immunophenotyping” refers to a laboratory technique used to identify and classify cells based on the expression of specific surface or intracellular proteins (antigens). These proteins are detected using antibodies that are labeled with fluorescent markers, allowing for their precise identification through flow cytometry or immunohistochemistry.

[0079] In a clinical trial, an endpoint is a specific outcome that is measured to determine if a new drug or treatment is effective and safe. There are different types of endpoints:

[0080] Primary endpoints are the main outcomes used to evaluate the primary objective of the study. For example, in a cancer trial, the primary endpoint might be patient survival.

[0081] Secondary endpoints are additional outcomes that are measured to provide more information about the treatment's effects. These might include changes in tumor size or improvements in quality of life. They can support the primary endpoint and provide further insights into the drug's mechanism of action.Exploratory endpoints are used to investigate novel or less-established aspects of a treatment. These are often used to generate new hypotheses for future research and may include measurements of biomarkers that are not yet fully understood.

[0082] The term "Semi-quantitative" describes a method or result that provides an estimate of the amount or concentration of a substance, rather than a precise, exact measurement. It indicates whether something is present in a small or large amount, but not the exact quantity. In the context of flow cytometry, "semi-quantitative" refers to measuring the relative amount of a substance (usually a protein) on or in a cell by measuring the intensity of its fluorescent signal.

[0083] Table 1: Biomarker proteins

[0084] The UniProt ID in table 1 refers to the canonical amino acid sequence of the human protein.

[0085]

[0086]

[0087] Table 2: Marker proteins for cell classification

[0088] How to interpret Table 2: The cell types are defined in a hierarchical, step-by-step manner described by marker proteins. For example, T cells are defined as follows: first, debris is excluded from the sample. Then, total leukocytes are identified by gating on CD45 expressing cells. Next, viable leukocytes are selected within the total leukocyte population by gating for those that do not show live / dead probe expression. Further, Granulocytes are excluded by gating on cells lacking the CD66B marker. Finally, T cells are identified based on the exclusive expression of CD3 and the absence of CD 19. T cells are considered those in the quadrant with CD3+ but no CD19 expression (Q3). Details can be followed in figure 4.

[0089]

[0090]

[0091] EXAMPLES:

[0092] Material & Methods:

[0093] The full method used was based on the BD Transcription Factor Buffer Set method, as described by the Supplier (BD Biosciences).

[0094] For the method development and validation under Context of use of exploratory and secondary endpoints, Whole Blood samples were drawn into 4 mL NaHep (sodium heparin) blood collection tubes from apparently healthy donors in accordance with specifics SOP (SOP-00542 Healthy Volunteer Human Biological (HBS) Sample Donation For In Vitro Experimentation). All tubes were stored ambient prior to analysis with the HDFCM assay.

[0095] The performance of the HDFCM assay requires several sample and control processing steps, including several controls to ensure high performance of the assay and to ensure robustness of the data generated. The full method implies the following steps:

[0096] 1. Sample Processing Reference Control Preparation:

[0097] Single Color Cell Reference Control

[0098] - 100 pL of normal healthy volunteer K2ETDA whole blood (no older than fresh or day 1 post blood draw) was added to appropriate polystyrene tubes.

[0099] - An extra tube was included to act as “Unstained SLW”

[0100] - 1 test worth of corresponding antibody (see table 6) was added directly to the whole blood at the bottom of the tube.

[0101] - 1 test worth of Brilliant Stain Buffer Plus was added to all tubes. Tubes were then mixed by vortexing gently.

[0102] - Samples were incubated at RT, protected from light for 30 minutes.

[0103] - 3 mL of lx BD FACS Lyse Solution was added to all tubes. Tubes were recapped, vortexed and incubated for 5 minutes at RT, protected from light. After 5 minutes, the vortex was repeated and tubes incubated for an additional 5 minutes at RT, protected from light.

[0104] - Total lysis time is 10 minutes.

[0105] - Tubes were centrifuged at 500g for 5 minutes. Supernatant was decanted and tubes blotted on a paper towel. Tubes were gently flicked to resuspend the pellet.- 2 mL of BSA Stain Buffer was added to all tubes.

[0106] - Tubes were centrifuged at 500g for 5 minutes. Supernatant was decanted and tubes blotted on a paper towel. Tubes were gently flicked to resuspend the pellet.

[0107] - Cells were resuspended in 250pL of BSA Stain Buffer and acquired on the Cytek Aurora immediately.

[0108] Single Color Bead Reference Control

[0109] - One drop of BD Compensation Beads (positive and negative control) was added to appropriate tubes.

[0110] - 1 test worth of corresponding antibody (see table 6) was added directly to the bottom of the tube.

[0111] - Samples were incubated at 2-8 °C, protected from light for 15 minutes.

[0112] - 2 mL of PBS was added to all tubes and vortexed gently.

[0113] - Tubes were centrifuged at 500g for 5 minutes. Supernatant was decanted and tubes blotted on a paper towel. Tubes were gently flicked to resuspend the pellet.

[0114] - Beads were resuspended in 250 pL of BSA Stain Buffer and acquired on the Cytek Aurora immediately.

[0115] Full Process Reference Control

[0116] - An additional tube was processed alongside samples stained only with CD57 BUV395 to act as a full process control. Sample Preparation

[0117] 2. RBC Lysis - Whole Blood Only

[0118] - 1 x 15 mL Falcon Tube was labelled per whole blood sample

[0119] - Additional 12x45mm Polystyrene FACS Tubes were labelled per sample (Unst, Tube l, Tube_2) including QC samples

[0120] - 500 pL of whole blood samples were added to 15 mL Falcon tubes.

[0121] - 1.5 mL of RT Roche RBC Lysis Buffer was added to all whole blood samples.

[0122] - Caps were secured and samples inverted 10 times to mix.

[0123] - Tubes were transferred to a rocker and incubated for 10 mins at RT.

[0124] - Tubes were centrifuged at 500 xg for 5 mins. Supernatant was carefully removed with a plastic Pasteur pipette.

[0125] - 1.5 mL of Roche RBC Lysis buffer was added to all whole blood samples.

[0126] - Caps were secured and samples inverted 10 times to mix.

[0127] - Tubes were centrifuged at 500 xg for 5 mins. Supernatant was carefully removed with a pastette.

[0128] - Cell pellet was resuspended in 450 pL of PBS and vortexed gently.

[0129] - Sample was distributed across below tubes:

[0130] i. Tube l (FMX) 200 pLii. Tube_2 (Stain) 200 pL

[0131] iii. Unstained (AF Control) 100 pL (or all of residual volume)

[0132] From this step onwards, PBMC QC samples were processed alongside Whole Blood.

[0133] 3. Surface Staining -Whole blood and QC samples

[0134] - 1 mL of PBS was added to all tubes.

[0135] - 1 pL of LiveDead Blue Viability Dye was added to all tubes

[0136] - Unstained Tubes: No LiveDead Blue Viability Dye was added

[0137] - Tubes were vortexed gently and incubated for 30 minutes at RT, protected from light.

[0138] - 2 mL of BD Stain Buffer (BSA) was added to all samples to quench Viability Dye.

[0139] - Tubes were centrifuged at 500 xg for 5 mins. Tubes were decanted and blotted dry on a paper towel.

[0140] - Tubes were vortexed gently to resuspend the cell pellet.

[0141] - 5 pL of Human TruStain FcX was added to all tubes and vortexed gently.

[0142] - Samples were incubated for 10 minutes at RT, protected from light.

[0143] - Surface cocktail was prepared by aliquoting multiples of each antibody plus a 10% overage into amber glass vials prior to dispensing to daughter tubes. See Table 8.

[0144] - 150 pL of the appropriate antibody cocktail was added to the bottom of the corresponding polystyrene tube. Tubes were vortexed gently and incubated for 30 minutes at RT, protected from light.

[0145] - Unstained Tubes: 150 pL BD Stain Buffer (BSA) was added in place of antibody cocktail. - 2 mL of BD Stain Buffer (BSA) was added to all samples.

[0146] - Tubes were centrifuged at 500 xg for 5 mins. Tubes were decanted and blotted dry on a paper towel.

[0147] - Tubes were vortexed gently to resuspend the cell pellet.

[0148] Table 3: Surface Antibody Cocktail

[0149]

[0150]

[0151] . Intracellular Stain - All Samples

[0152] 1 mL of lx BD Fix / Perm Solution was added to all tubes. Tubes were vortexed gently and incubated for 45 minutes at RT, protected from light.

[0153] 1 mL of lx BD Perm / Wash Buffer was added to all samples.

[0154] - Tubes were centrifuged at 500 xg for 5 mins. Tubes were decanted and blotted dry on a paper towel.

[0155] - Tubes were vortexed gently to resuspend the cell pellet.

[0156] - 2 mL of lx BD Perm / Wash Buffer was added to all samples.

[0157] - Tubes were centrifuged at 500 xg for 5 mins. Tubes were decanted and blotted dry on a paper towel.

[0158] - Tubes were vortexed gently to resuspend the cell pellet.

[0159] - Intracellular cocktail was prepared by aliquoting multiples of each antibody plus a 10% overage into amber glass vials prior to dispensing to daughter tubes. See Table 9.

[0160] 100 pL of the appropriate antibody cocktail was added to the bottom of the corresponding polystyrene tube. Tubes were vortexed gently and incubated for 30 minutes at RT, protected from light.

[0161] - Unstained Tubes: 100 pL Perm / Wash Buffer was added in place of antibody cocktail - 2 mL of lx BD Perm / Wash Buffer was added to all samples.- Tubes were centrifuged at 500 xg for 5 mins. Tubes were decanted and blotted dry on a paper towel.

[0162] - Tubes were vortexed gently to resuspend the cell pellet.

[0163] - 2 mL of lx BD Perm / Wash Buffer was added to all samples.

[0164] - Tubes were centrifuged at 500 xg for 5 mins. Tubes were decanted and blotted dry on a paper towel.

[0165] - Tubes were vortexed gently to resuspend the cell pellet.

[0166] - Cells were resuspended in 250 pL of IX BD Stain Buffer (BSA) and vortexed gently for a uniform suspension.

[0167] - For the validation, the full volume of sample was transferred to a 96-well U-bottomed plate, or tubes transferred to a tube rack, and acquired immediately on the Cytek Aurora Flow Cytometer.

[0168] o For the clinical sample analysis, the full volume of the sample will be transferred to a tube rack and acquired immediately on the Cytek Aurora Flow Cytometer. Table 4: Intracellular Antibody Cocktail

[0169]

[0170] Lyse-No-Wash Tubes

[0171] - During Step 32 of sample preparation, an additional FACS tube was labelled per sample (Tube_3).

[0172] - 50 pL of whole blood was added to the corresponding tubes.

[0173] - 2.5 pL of CD45 Spark Blue 550 was added to all tubes and vortexed gently.

[0174] - Samples were incubated for 15 minutes at RT, protected from light.

[0175] - 450 pL of lx FACSLyse was added to all tubes and vortexed gently.

[0176] - Samples were incubated for 15 minutes at RT, protected from light

[0177] - 500 pL of PBS was added to all tubes and vortexed gently for a uniform suspension. - 500 pL of sample was transferred to a 96-well U-bottomed plate, or tubes transferred to a tube rack, and acquired immediately on the Cytek Aurora Flow Cytometer.Flow Cytometry Analysis

[0178] Stop Gates

[0179]

[0180] - Data is acquired in SpectroFlo software integrated in the flow cytometry platform. - Data was analyzed in OMIQ. The OMIQ science platform has been assessed through the Q2 Solutions GxP validation process and approved by both Information Technology and Systems Compliance. OMIQ uses the Google Kubemetes and Q2 Solutions has a dedicated cluster that can only be accessed by approved Q2 Solutions staff through the Q2 Solutions VPN. Data files in the OMIQ cloud are pseudo-anonymized, meaning identifiers (such as accession numbers) cannot be traced back to a specific patient through OMIQ alone.

[0181] Assay validation parameters.

[0182] Assay validation in flow cytometry ensures regulatory compliance, scientific rigor, reproducibility, and cross-study comparability, all of which are critical for decision-making in CTs and eventual drug approval. Method validation for the modular High-dimensional flow cytometry (hdFCM) assay followed CLSI H62 guidelines for quasi-quantitative / semi-quantitative data. The validation of the exploratory and secondary endpoint analytes followed the fit-for-purpose (FFP) validation type 2 except for the limit of detection (LOD) and lower limit of quantitation (LLOQ) assessments which were only performed for the secondary endpoint parameters. The following validation parameters were assessed as follows:

[0183] Accuracy: As no predicate method exists for all analytes reported from the modular panel, accuracy was only assessed for T (CD3, CD4, CD8), B (CD19) and NK (CD56 / 16) cell frequencies and absolute cell count values by comparison to the same values from the BD Multitest 6 color TBNK CE / IVD Assay for 6 AHVs. Samples were analyzed in singlicate by a single operator (12 samples in total). The modular assay NK subset gating hierarchy was altered to align with the A089 BD Multitest 6 color TBNK Assay gating strategy such that a meaningful comparison could be made.Selectivity: Established through antibody selection and gating strategy developed during the assay development and optimization.

[0184] Repeatability (intra-assay): NaHep WB from 3 AHVs analyzed in triplicate by two operators across two instruments (36 samples in total). This data was also used to assess the inter-operator and inter-instrument comparability.

[0185] Reproducibly - (inter-assay): NaHep WB from 6 AHVs analyzed in singlicate across 3 independent analytical runs (instrument switched off and on, recalibrated, independent sample processing) by a single operator on a single instrument (18 samples in total).

[0186] Specimen Stability: NaHep WB from 6 AHVs analyzed within 2 hours of blood draw (baseline, day 0) and on days 1, 2, 3 and 4 after ambient storage (30 samples total).

[0187] Post-Processing Sample Stability: NaHep WB from 3 AHVs was processed and acquired within 1 horn (baseline) and after 4 horns refrigerated storage. Samples were analyzed in singlicate by a single operator on a single instrument (6 samples total).

[0188] LLOQ and LOD: For secondary endpoints only. NaHep WB from 3 AHVs were stained with the full panel or the full panel minus the CD8 or the CD25 antibody (fluorescence minus one (FMO)). Full or FMO stained samples were admixed at ratios of 1:0, 1:10, 1:50, 1:100, 1:200, 1:500 and 0:1 for CD8 and at 1:0, 1:2, 1:5, 1:10, 1:20, 1:50 and 0:1 for CD25 for Cytotoxic or Regulatory T cell subsets respectively (63 samples total).

[0189] QC Precision: Commercial PBMCs QC material from (BioIVT) was analyzed in duplicate across 5 independent analytical runs (10 samples total).

[0190] Interference: Not performed as these were not required for secondary and exploratory endpoints not related to patient care.

[0191] Precision (Repeatability & Reproducibility)

[0192] Precision refers to the closeness of agreement between a series of measurements obtained from the same sample. It is a measure of random error and is usually expressed as a Coefficient of Variation (CV).

[0193] Intra-assay Precision (Repeatability): This measures the variability of results when the same sample is tested multiple times within the same experiment (same day, same operator, same instrument). It shows how consistent the immediate process is.

[0194] Inter-assay Precision (Reproducibility): This measures the variability of results when the same sample is tested across different conditions — such as on different days, with different operators, or on different instruments. This is crucial for ensuring that results from a long-term study are comparable.

[0195] Specificity

[0196] Specificity is the ability of the assay to detect and measure only the target of interest without interference from other substances.

[0197] In a Flow Cytometry Context: This means ensuring your antibody panel only stains the intended cell population. Specificity is confirmed using Fluorescence Minus One (FMO) controls, where you stain with all antibodies except one. This reveals the background spread from the other dyes into theempty channel, helping you set an accurate gate and prove that the signal in that channel is specific to your target antibody.

[0198] Limit of Detection (LOD)

[0199] The LOD is the smallest number or frequency of a specific cell type that can be reliably distinguished from the background noise of a sample that does not contain the cells of interest. It is the point where you can confidently say that a signal is real and not just random instrument noise.

[0200] Lower Limit of Quantitation (LLOQ)

[0201] The LLOQ is the smallest number or frequency of a specific cell type that can be measured with a defined level of accuracy and precision. Below this limit, you might be able to detect the cells (you are above the LOD), but you cannot report an exact number with confidence.

[0202] Assay Validation Results

[0203] The definition of the performance characteristics of the assay ensures that any pharmacodynamic changes identified by the panel can be attributed to biology and not technical variability in the assay. The validation scope was driven by the intended use of the data as defined in a clinical protocol. The majority of the analytes were validated as exploratory endpoints with CD8+ cytotoxic and regulatory T cells analytes validated as secondary endpoints.

[0204] Secondary endpoint analytes

[0205] For the 6 secondary endpoint analytes, the acceptance criteria were met for repeatability, reproducibility, specimen stability out to day 4 from blood draw stored ambient and processed sample stability out to 4 hours stored refrigerated and QC Precision (Figure 4 and Supp. Info). The assay LLOQ was defined as the highest LLOQ value of the three donors. The LLOQ was determined to be 6.4 cells / pL for CD8+ ABS values, 1.6 cells / pL for CD4+CD1271oCD25+ ABS and 1.2 cells / pL for CD4+CD1271oCD25+FoxP3+ ABS values (Figure 5).

[0206] Exploratory endpoint analytes

[0207] For the 208 exploratory endpoint analytes defined for the full panel, 188 (90%) met the acceptance criteria of <20%CV for repeatability (Figure 6a) and 115 (55%) were considered stable out to 4 days from blood draw with a <20% change from day 0 values. For the core panel, 47 out of 62 (76%) of the analytes showed a stability of 4 days from blood draw (Figure 6b). Most of the analytes that did not meet the acceptance criteria for repeatability and stability were low frequency tertiary analytes where minor absolute differences in values resulted in larger relative %CV or change from baseline in percentage.

[0208] Validation in accordance with CLSI H62 guidelines confirmed the assay's reliability across exploratory and secondary endpoints, demonstrating high accuracy, selectivity, repeatability, and reproducibility. Key secondary endpoints, including CD8+ cytotoxic and regulatory T cells, achieved precision, with repeatability and inter-operator variability under 20% coefficient of variation (CV), while specimen stability was maintained over four days of ambient storage.

Claims

Claims1. A semi- quantitative flow cytometry method for measuring the frequency and / or the cell concentration of one or more mononuclear cell subsets in a biological sample, wherein the subset of mononuclear cells is detected by a set of markers selected from the group of: Viability, CD45, CD66b, CD 19, CD3, CD4, CD8, HLA-DR, CD279, Ki67, CD45RA and CD197, wherein said frequency and / or the cell count are determined with an inter-assay coefficient of variation (CV) of less than or equal to 20%.

2. The flow cytometry method of claim 1, wherein at least one marker of a second set of markers is detected, wherein the additional set of markers comprises the following markers: CD25, CD 127, FoxP3, CD95, CD28, TCF-1, TIM3, 41BB, LAG3, TIGIT, 0X40, ICOS, CD39, CD57, KLRG1, CD103, CD14, CD16, CD56, NKG2D and Granz-B.

3. The flow cytometry method of claims 1 or 2, wherein the following markers of the second set of markers are detected: TCF1, TIM3, LAG3, CD95 and 41BB.

4. The flow cytometry method of claim 1 or 2, wherein the following markers of the second set of markers are detected: CD39, CD57, KLRG1 and CD103.

5. The flow cytometry method of claim 1 or 2, wherein the following markers of the second set of markers are detected: 0X40 and ICOS.

6. The flow cytometry method of claim 1 or 2, wherein the following markers of the second set of markers are detected: CD16, CD56, CD14, NKG2D and Granz-B.

7. The flow cytometry method of claims 1 - 6, wherein the following markers of the second set of markers are detected: TCF1, TIM3, LAG3, CD95, 41BB, CD39, CD57, KLRG1, CD103, 0X40, ICOS, CD16, CD56, CD14, NKG2D and GranzB.

8. The flow cytometry method of claims 1 - 7, wherein the biological sample is a patient tissue sample, preferably a blood sample.

9. The flow cytometry method of claims 1 - 8, wherein the patient is being treated with a cancer drug, an immunology drug or an inflammation drug, preferably an immunotherapy drug.

10. The flow cytometry method of claims 1 - 9, wherein the mononuclear cells are T-cells, monocytes or NK cells, preferably T-cells.

11. The flow cytometry method of claims 1 - 10, wherein the following marker configuration ted:- Core markers: Viability, CD45, CD66B, CD279, CD197, CD45RA, HLA-DR, CD8, CD3, CD 19, Ki-67, CD4;- T Cell regulatory markers: FoxP3, CD127 and CD25;- NK / Monocytes markers: NKG2D, CD56, Granz-B, CD 14 and CD 16;Senescence markers: CD103, CD57, KLRG1, CD39;- Checkpoint markers: TIGIT, 0X40, ICOS;- T memory stem cell / T resource cell markers: CD95, 4 IBB, TCF1, CD28, TIM3 and LAG3.

12. The method of claims 1 - 10, wherein the following marker configuration is detected:- Core markers: Viability, CD45, CD66B, CD279, CD197, CD45RA, HLA-DR, CD8, CD3, CD19, Ki-67, CD4;- T Cell regulatory markers: Fox P3, CD127 and CD25.

13. The method of claims 1 - 10, wherein the following marker configuration is detected:- Core markers: Viability, CD45, CD66B, CD279, CD197, CD45RA, HLA-DR, CD8, CD3, CD19, Ki-67, CD4;- T Cell regulatory markers: Fox P3, CD127 and CD25:Senescence markers: CD103, CD57, KLRG1, CD39.

14. The method of claims 1 - 10, wherein the following marker configuration is detected:- Core markers: Viability, CD45, CD66B, CD279, CD197, CD45RA, HLA-DR, CD8, CD3, CD19, Ki-67, CD4;- T Cell regulatory markers: Fox P3, CD127 and CD25;- Senescence markers: CD103, CD57;- Checkpoint markers: TIGIT, ICOS.

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