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WO2025172529A1 - Nouveaux procédés d'évaluation d'une réponse à un médicament spécifique à une cellule - Google Patents

Nouveaux procédés d'évaluation d'une réponse à un médicament spécifique à une cellule

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Publication number
WO2025172529A1
WO2025172529A1 PCT/EP2025/054024 EP2025054024W WO2025172529A1 WO 2025172529 A1 WO2025172529 A1 WO 2025172529A1 EP 2025054024 W EP2025054024 W EP 2025054024W WO 2025172529 A1 WO2025172529 A1 WO 2025172529A1
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Prior art keywords
cells
cell
specifically
test compound
well
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English (en)
Inventor
Tea PEMOVSKA
Philipp Bernhard STABER
Carmen Marita SCHWEICKER
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Medizinische Universitaet Wien
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Medizinische Universitaet Wien
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • markers selected from one or more fluorescence-labeled antibodies binding to cellular antigens, specifically selected from antibodies binding to antigens of a cell population, and/or one or more cellular dyes, wherein at least one cellular dye binds to dead cells;
  • the multi-well microtiter plate contains 1 to 1000, specifically 10-1000, more specifically 50 to 500, or more different test compounds, specifically at different concentrations, specifically the test and control compounds are chosen based on the type of cells present in the sample.
  • the cellular dye is selected from the group consisting of 4',6-Diamidin-2-phenylindol (DAPI), live-death cell staining fluorescent dyes, cell-surface marker, specifically a disease specific or patient specific cell-surface marker.
  • DAPI 4',6-Diamidin-2-phenylindol
  • live-death cell staining fluorescent dyes cell-surface marker, specifically a disease specific or patient specific cell-surface marker.
  • the fluorescence-labeled antibodies are selectively targeting diseased cells.
  • Fig. 2. Comparison of detection of distinct immune cell populations with modified iQue3® protocol as described herein and a standard flow cytometry instrument (CytoFLEX®) which required significantly more sample input and volume.
  • Fig. 5 Case study of a CD20 negative T-cell rich B-NHL case patient. Selective AUC of DAPI negative, CD22 and CD79a positive relative to CD3 positive immune background cells are sorted. Note, among other compounds, vandetanib and copanlisib demonstrate a highly selective AUC score relative to CD3 cells of >0.2.
  • Fig. 6 Case study of a CD20 negative T-cell rich B-NHL case of a 37 year old patient. After failing 4 previous treatment lines, a real-time biopsy was undertaken and the method described herein was performed on the biopsy specimen.
  • healthy cells are defined as expressing antigens, such as surface antigens, or surface or cell markers characteristic for healthy cells or not expressing cell markers characteristic for diseased cells, i.e. disease cell markers.
  • antigens such as surface antigens, or surface or cell markers characteristic for healthy cells or not expressing cell markers characteristic for diseased cells, i.e. disease cell markers.
  • the disease may be cancer.
  • PBMCs for use in the methods described herein can be isolated from whole blood using any suitable method known in the art or described herein.
  • the protocol described by Panda and Ravindran (2016) may be used.
  • density gradient centrifugation is used for isolation, which separates whole blood into components separated by layers, e.g., a top layer of plasma, followed by a layer of PBMCs and a bottom fraction of polymorphonuclear cells (such as neutrophils and eosinophils) and erythrocytes.
  • the polymorphonuclear cells can be further isolated by lysing the red blood cells, i.e. non-nucleated cells.
  • Common density gradients useful for such centrifugation include, but are not limited to, Ficoll® gradient.
  • the method described herein requires significantly less cells compared to known methods for the determination of the selectivity of test compounds, specifically only 8-50 %, 20-50 %, or 25-50 % of the cells used by known methods, more specifically 8 %, 10 %, 12 %, 14 %, 16 %, 18 %, 20 %, 21 %, 22 %, 23 %, 24 %, 25 %, 26 %, 27 %, 28 %, 29 %, 30 %, 31 %, 32 %, 33 %, 34 %, 35 %, 36 %, 37 %, 38 %, 39 %, 40 %, 41 %, 42 %, 43 %, 44 %, 45 %, 46 %, 47 %, 48 %, 49 %, or 50 % cells compared to flow cytometry methods for determining drug sensitivity and/or selectivity described in prior art, specifically Majumder M. M. (2016).
  • the number of seeded cells can be further reduced to a minimum of 1 .000 cells, more specifically 1 x 1 o 3 , 2 x 10 3 , 3 x 10 3 , 4 x 10 3 cells.
  • Incubation is carried out in a culture medium and is performed under conditions to allow uniform cell growth of all cell populations of the sample.
  • the culture medium to be used in the methods of the invention can be any medium applicable for sample cell culture.
  • the culture medium is a liquid medium with nutrients and substances necessary for cultivation of the cells.
  • concentrations of each of the test compounds are contained in the wells.
  • the test components can be present in the wells at concentrations that correspond to the concentrations of these components as they are present in the subject's body upon administration.
  • the concentration of the test compound is in the range of 0.01 to 100.000 nM, specifically in the range of 0.01 nM to 100 pM, more specifically 1 nM, 10nM, 50nM, 100nM, 500nM, 1000 nM, 2000 nM, 3000 nM, 4000 nM/ 5000 nM, 6000 nM, 7000 nM, 8000 nM, 9000 nM, 10.000 nM, 20.000nM, 30.000nM, 40.000nM, 50.000nM, 60.000nM, 70.000nM, 80.000nM, 90.000nM, 100.000nM, 10 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80
  • solvents used in pharmaceutical industry are: cyclopentyl methyl ether solvents, water, octanoic acid-based supramolecular solvent, n-butanol and acetone, bio-based green solvent disulfides, and eutectic solvents.
  • SA comprehensive list of solvents is given by the US FDA (Q3C, Tables and List; August 2018, Revision 4, https://www.fda.gov/media/133650/download, accessed on Dec 13, 2023).
  • the solvents are solvents or excipients of a test compound, e.g. dimethyl sulfoxide (DMSO).
  • 1 , 2, 3, 4, 5, or more cellular dyes bind to dead cells.
  • Screening is performed on a high-throughput flow cytometer. In contrast to automated microscopy, it allows screening of viable cells that are not fixed with formaldehyde or any other similar agent. Specifically, it is excluded to fix the cells with formaldehyde or any other similar agent.
  • the gating is continued by a second gating which is performed on viable single cells and selection of population of interest selected from the group consisting of single marker positive, single marker negative, double marker positive, double marker negative, multiple marker positive, multiple marker negative cell population, and any combinations thereof, based on the staining performed of the cells with the fluorescent- labeled antibodies and cellular dyes and the screening of the so labeled cells by high throughput flow cytometry.
  • the cell counts are determined in each of the gates obtained by the first and second gating in each well of the multi-well microtiter plate.
  • the method can be specified by the following steps:
  • the minimum and maximum response, slope, and IC50 or EC50 are calculated for the dose-response curve described above and an area under the curve (AUC) is calculated thereby obtaining a range between 0 and 1 , wherein a value higher or equal to 0.12 indicates sensitivity to the test compounds.
  • the cutoff for evaluating the sensitivity of the test compounds based on the AUC depends on the indication, specifically the cutoff for evaluating the sensitivity of the test compounds based on the AUC is between 0.10 and 0.30, more specifically the cutoff for evaluating the sensitivity of the test compounds based on the AUC is 0.10, 0.11 , 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.20, 0.21 , 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, or 0.30.
  • the cutoff is 0.12.
  • the 0.12 cutoff for a significant effect is demonstrated.
  • test compounds’ action obtained above is ranked for the cell population of interest.
  • the methods of the present invention quantify the selective ability of a test compound such to kill diseased over non-diseased cells in order to determine whether a subject suffering from a disease will respond or is responsive to treatment with the test compound.
  • the methods of the present invention give a highly accurate information about whether a subject suffering from a disease will respond or is responsive to treatment with a test compound. Specifically, the AUC value as a measure of the quality by which a method can distinguish two classes using the method of the present invention.
  • the sensitivity to the test compounds may be determined based on dose response relationship and accompanying AUC such that: i. if the percentage of survival per concentration is lower than 80% for 2, 3, 4, 5, 6, 7,8, 9, 10, ... , 1000 or more test compound concentrations and the AUC is higher or equal to 0.12, the drug is determined to have activity in the cell population, and ii. if the percentage of survival per concentration is higher than 80% for majority of the tested compound concentrations and the AUC is below 0.12, the test compound is determined to have no activity in the particular cell population.
  • AUC is 0 in cases where the maximum response is 10% or lower or IC50/EC50 equals or is beyond the maximum drug concentration tested.
  • reagent refers to any substance necessary to execute the method described herein, specifically the term reagent may include but is not limited to one or more of culture media, antibodies, dyes, buffers, compounds, beads or DMSO.
  • the term “reagent” refers to any substance necessary to execute the method described herein, specifically the term reagent may include but is not limited to antibodies against CD1a, antibodies against CD1c, antibodies against CD2, antibodies against CD3, antibodies against CD4, antibodies against CD5, antibodies against CD7, antibodies against CD8, antibodies against CD8a, antibodies against CD10, antibodies against CD11 b, antibodies against CD13, antibodies against CD14, antibodies against CD16, antibodies against CD19, antibodies against CD20, antibodies against 22, antibodies against CD24, antibodies against CD27, antibodies against CD30, antibodies against CD33, antibodies against CD34, antibodies against CD38, antibodies against CD45, antibodies against CD45RA, antibodies against CD56, antibodies against CD64, antibodies against CD66b, antibodies against CD68, antibodies against CD79a, antibodies against CD79b, antibodies against CD114, antibodies against CD115, antibodies against CD116, antibodies against CD117, antibodies against CD123, antibodies against CD138, antibodies against CD184, antibodies against CD235a, antibodies against CD274, antibodies against CD279, antibodies against CD319, antibodies against antibodies against CD1
  • markers selected from one or more fluorescence-labeled antibodies binding to cellular antigens, specifically selected from antibodies binding to antigens of a cell population, and/or one or more cellular dyes, wherein at least one cellular dye binds to dead cells;
  • sample is any sample containing viable cells, specifically it is selected from peripheral blood, urine, bone marrow, skin, or any other organ of interest, fresh biopsies, more specifically the sample was frozen.
  • diseased cells are cancer cells, specifically selected from the group consisting of hematological cancer cells, leukemia cells, lymphoma cells, and solid cancer cells.
  • control compound is DMSO, or a drug solvent, specifically a solvent of the test compound. 6.
  • each well contains a single test compound at a single dose, combinations of two or more test compounds, or a control compound.
  • test and control compounds are chosen based on the type of cells present in the sample.
  • centrifugal force of the centrifugation is determined to spin down the cells to the well bottom and to allow subsequent resuspension of the cells, specifically it is at 100 x g.
  • cellular dye is selected from the group consisting of 4',6-Diamidin-2-phenylindol (DAPI), live-death cell staining fluorescent dyes, cell-surface marker, specifically a disease specific or patient specific cell-surface marker.
  • DAPI 4',6-Diamidin-2-phenylindol
  • test compound action refers to activation or inhibition of cell proliferation, cell viability, cell differentiation, mean fluorescent intensity of a cellular marker, induction of cell death, clonal/sub-clonal drug response tracking, potentiating, or diminishing the effect of cellular therapies such as CAR T cells or other immunotherapies.
  • sensitivity to the test compounds is determined based on dose response relationship and accompanying AUC such that: i. if the percentage of survival per concentration is lower than 80% for at least two test compound concentrations and the AUC is equal to or higher than 0.12, the drug is determined to have activity in the cell population, and ii. if the percentage of survival per concentration is higher than 80% for majority of the tested compound concentrations and the AUC is below 0.12 the test compound is determined to have no activity in the particular cell population.
  • selectivity to the test compound is determined based on differences in the AUC between cell populations such that: i. if the difference in the AUC value is equal to or higher than 0.12, the test compound is considered to exhibit selective effects in a cell population, specifically in a disease cell population, and ii. if the difference in the AUC value is below 0.12, the test compound is deemed to exhibit non-selective or no effect in a population, specifically in a disease-cell population.
  • the report comprises one or more of: i. general information of sample and assay characteristics including staining information, quality control (QC) metrics of the instrument and screen, and gating strategy; ii. summary treatment recommendation; iii. overall test compound sensitivity results displayed as bar graphs providing a ranking of the effectiveness of the test compound on each of the cell populations with the test compounds, specifically annotated per drug class; iv. disease-specific drug action results displayed as bar graphs providing a ranking of the selective hits in each of the cell populations with the test compound, specifically annotated per drug class; v. the most relevant result graph highlighted for each subject in concordance to a pathology or laboratory medicine report from the same subject; and vi. dose response curves of the test compound per cell population.
  • Test kit comprising i. a multi-well microtiter plate comprising 1-1000, specifically 10-1000, or more immobilized test compounds, wherein said test compounds are present in at least 2-5 different concentrations, and wherein at least one of each test compound concentration corresponds to the concentration in which the test compound is present in a subject's body cells after administration at a prescribed dosage; ii. reagents for performing the method of any one of embodiments 1 to 18; iii. cellular dyes, wherein at least one cellular dye binds to dead cells; iv. a list of candidate antibodies binding to two or more cellular antigens; and a leaflet or a QR code providing information for performing the method of any one of embodiments 1 to 18.
  • FIG. 1 A schematic representation of a general workflow in the case of a patient diagnosed with cancer is shown in Fig. 1.
  • a cancer biopsy which contains cancer and normal microenvironmental cells is taken from the patient diagnosed with cancer.
  • the single cells from the biopsy are incubated with drugs.
  • the plate is centrifuged and the supernatant is aspirated.
  • the goal of the latter step is to miniaturize the assay and increase the sample concentration. This reduces the sample volume, the antibody staining volume and, hence, the costs.
  • the cells are stained with patient-specific markers and High-throughput - flow cytometry is performed.
  • the drugs are ranked based on their cancer selective effect. Finally, the results lead to an informed patient treatment decision.
  • Example 2 Protocol - High-throughput flow cytometry drug screening (single-cell flow cytometry fPM (scfcfPM))
  • Prewarm cell culture media of choice for example RPMI + 10% FCS + 1 % penicillin/streptomycin
  • Prewarm cell culture media of choice for example RPMI + 10% FCS + 1 % penicillin/streptomycin
  • BM bone marrow
  • lymph node to generate a single cell suspension by either Ficoll® gradient centrifugation or mechanical dissociation.
  • Ficoll® gradient centrifugation For other starting materials use appropriate cell dissociation procedure.
  • DAPI can be used for assessing the viability of the sample cells
  • Install a low-volume peristaltic pump e.g., Random Access Dispensing (RAD) module for the Multiflo FX
  • RAD Random Access Dispensing
  • Multiflo FX Multiflo FX
  • the second plate can be stained while the first is running on the iQue 3® screener
  • the second plate can be run after the first plate is finished on the iQue 3® screener
  • Gating strategy (Fig. 3) a. FSC/SSC make a first all cells gate b. Eliminate duplets by plotting the height or width against the area for forward scatter or side scatter. Doublets have increased area whilst similar height to single cells. c. Eliminate dead cells by gating for DAPI-negative cells from the singlets gate d. Continue gating on viable singlet cells to select single marker positive, double marker positive, and/or triple marker positive cells and corresponding negative cell populations. The double and triple-marker positive cell populations are selected with overlay graphs of the respective single-marker positive populations. e. Determine the cell counts and percentages in each of the gates in each well from the 384-well assay plate. f. Export cell counts from all populations of interest and match with the drug well annotation sheet
  • AUC values range between 0 and 1 (the higher the value the more activity the drug has in a given cell population).
  • Fig. 2 shows a comparison of the detection of distinct immune cell populations in peripheral blood mononuclear cells (PBMCs) with the protocol of Example 2 and a standard flow cytometry method for measuring different cell populations, which requires significantly more sample input and volume.
  • PBMCs peripheral blood mononuclear cells
  • a standard flow cytometry method for measuring different cell populations which requires significantly more sample input and volume.
  • cells were seeded at a density of 12000 cells/well in 50pl in a 384 well plate and incubated overnight at 37°C. The next day, the plate was centrifuged at 100 x g for 5 min, the supernatant was aspirated, and cells were stained with antibodies against CD4, CD8a, CD19, CD14, DAPI, or a combination thereof.
  • Example 2 After 30 min incubation, the plate was run on the iQue 3® instrument as described in Example 2 (“inventive method”). For the CytoFLEX comparison (“comparative method”), approximately 50000 cells per condition were washed with PBS and were either left unstained or stained (1 :500 antibody dilution) with DAPI or antibodies against CD4, CD8a, CD19, CD14, or multistained. The samples were incubated for 30min, washed with PBS, resuspended in 300pl FACS buffer, and measured on a benchtop flow cytometry instrument.
  • Fig. 3-6 show examples of a workflow of a CD20 negative T-cell rich B-NHL case patient who has failed multiple prior treatment lines and whose biopsy sample has undergone the procedure described in detail in Example 2.
  • Fig. 3 shows the gating strategy of the flow-cytometry plots of the indicated patient case.
  • the tumor cells were positive for CD79A, CD22, and negative for CD3. Background immune T-cells were highly CD3 positive.
  • Fig. 3A shows the forward versus side scatter to identify cells of interest.
  • Fig. 3B shows the differentiation between live and dead cells with DAPI staining, whereas DAPI negative cell population signifies live cells.
  • Fig. 3C-J show the density and histogram plots for CD19, CD79a, CD22, and CD3 staining.
  • Fig. 3K-L show the overlay dot plots depicting the cancer cell populations in this sample (double positive CD22+CD79a+; Fig. 3K and CD3-CD79a+; Fig. 3L).
  • Fig. 4 shows dose response curves of copanlisib (PI3K inhibitor) and vandetanib (VEGFR/Ret inhibitor) in DAPI negative, CD3 positive, CD19 positive, CD22 positive, CD79a positive and CD22-CD79a double positive cells expressed as percentage survival following normalization to DMSO controls.
  • Fig. 5 shows sorted selective AUC of DAPI negative, CD22and CD79a positive relative to CD3 positive immune background cells. Note, among other compounds, vandetanib and copanlisib demonstrate a highly selective AUC score relative to CD3 cells of >0.2.
  • Fig. 6 shows a case study of a CD20 negative T cell rich B-NHL case of a 37 years old patient. Based on the marker profile, the selective drug report, and the toxicity profile of the compounds, a molecular tumor board recommended the regimen: Anti- CD19 antibody tafasitamab plus copanlisib or vandetanib. After a remission was achieved, an allogeneic stem cell transplantation with an available donor was performed. The treating physician and the patient decided to start the treatment consisting of tafasitamab and vandetanib. The patient was treated accordingly for 6 months, achieved a complete metabolic remission and thus underwent allogenic stem cell transplantation as consolidation. One year after allogenic stem cell transplantation, the patient remained in complete remission and leads a normal life.
  • Example 5 Functional and genomic based precision medicine in blood cancer patients: Feasibility results of a multicentric, prospective, randomized controlled trial
  • PM Precision medicine
  • oncology genomics has been the dominant tool in performing PM. Since many cancer patients lack actionable alterations to accurately match patients to effective therapies, there is a need to extend the advantages of PM to a larger proportion of cancer patients. Additional methods need to be explored, and one important alternative approach is functional PM, a strategy by which living patient cancer cells are exposed to therapies and measured to predict clinical response.
  • Prognosis is dismal for aggressive hematological cancer patients relapsing or refractory upon standard treatments. If tumor-containing biopsies can be obtained timely and safely, these patients are candidates for PM programs or studies.
  • the inventive method referred to as drug-screening-based single-cell (sc) high-throughput (HT) flow cytometry (fc) functional PM (scfcfPM) (see Example 3 for details), provides clinical benefit to advanced hematological cancer patients.
  • scfcfPM drug-screening-based single-cell
  • HT high-throughput
  • fc flow cytometry
  • scfcfPM functional PM
  • PC - physicians’ choice
  • a “real-time biopsy” solid tissue biopsy, bone marrow aspirate, or peripheral blood draws) containing viable tumor cells was collected from each patient. Samples are subjected to image-based (mbfPM) and/or high-throughput (HT) flow cytometry-based fPM (scfcfPM), as well as gPM testing.
  • the compounds used in the screening drug collection were as follows: Venetoclax, Selinexor, Azathioprine, Capecitabine, Cladribine, Clofarabine, Cytarabine, Decitabine, Fludarabine, 5-fluorouracil, Gemcitabine, 6-mercaptopurine, Methotrexate, Nelarabine, Leflunomide, Pemetrexed, 5-azacitidine, Bendamustine, Busulfan, Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cyclophosphamide, Ifosfamide, Melphalan, Mitomycin C, Temozolomide, Docetaxel, Paclitaxel, Vinblastine, Vincristine, Vindesine, Hydroxyurea, Arsenic trioxide, Bortezomib, Carfilzomib, Ixazomib, ATRA, Bexarotene, Etoposide, Mitoxantrone, Topotecan, Pixantron
  • the tumor board consisted of at least two hemoncologists, one pathologist, one molecular biologist, and one pharmacist. If a PM assay failed or did not identify a treatment rationale, the study protocol allowed switching to the other experimental arm.
  • Flow cytometry-based scfcfPM was feasible in 86% of tests; microscopy-based mbfPM was feasible in 64% of tests; gPM was feasible in 86% of tests.
  • gPM identified a median of 5 (range: 1-13) genetic aberrations per patient, of which a median of 1 (range: 0-5) aberration was conceived as an actionable genetic target (Table 2).
  • Table 2 A detailed summary of genetic aberrations in B-NHL, T-NHL and leukemia subgroups is shown in Fig. 8 A-C.
  • the inventive scfcfPM method described herein surprisingly provided the best results in that feasibility of therapy and availability of report were significantly better and the results were also received in a shorter time period which is very important to allow early onset of targeted cancer therapy.
  • the scfcfPM method could deliver technically valid test results with 5 * 10 6 cells. This low cell number has the potential to maximize clinical benefit since it is oftentimes difficult or even impossible to obtain enough viable cells suitable for other fPM methods, such as mbfPM.
  • the data of example 4 were compared to the state of the art, specifically Majumder (2016).
  • the following was compared; the outcome of ranking selective drug responses with the inventive method (using the non-cancer cell population present in the same biopsy), in this case, the CD19+ cell population, versus using the mean drug response profile of CD19+ cell populations from two different healthy donors as used in the method of Majumder (2016).
  • vandetanib the therapy the patient of example 4 was treated with and achieved complete remission, came up as one of the 3 top hits (Fig. 9A).
  • vandetanib did not score in the top 5 selective hits with the method according to Majumder (2016) (Fig. 9B).
  • the top 3 hits are considered for a therapy recommendation, and even if extended to the top 5 selective hits, only one drug is an overlapping hit with both analysis methods.
  • Doramapimod the top selective hit with the inventive method, is an investigational compound that could not be considered a treatment choice.
  • Fig. 9 shows a comparison of selective drug sensitivity analysis methods with a case study of a CD20 negative T-cell rich B-NHL case patient.
  • the results of the inventive method and the state of the art method are shown in Fig.9A and Fig. 9B, respectively.
  • Selective AUC of CD22 and CD79a double-positive cells relative to CD19 positive immune background cells from the same patient A) and from two different healthy donors B) are sorted and visualized with a bar graph.
  • vandetanib is top scoring when the patient’s own CD19 cell population is used as a comparator, whereas it does not score in the top 5 selective hits when compared to averaged drug response profiles from CD19 cell populations from two different healthy donors as in the method according to Majumder (2016) making it highly unlikely to be considered as a therapy recommendation.

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Abstract

L'invention concerne un nouveau procédé rapide pour déterminer la sensibilité et la sélectivité spécifiques à une maladie de composés de test dans des échantillons de faible volume sur la base d'une reconnaissance de cellule basée sur la cytométrie en flux haut débit. L'invention concerne en outre un kit de test pour mettre en oeuvre le procédé.
PCT/EP2025/054024 2024-02-16 2025-02-14 Nouveaux procédés d'évaluation d'une réponse à un médicament spécifique à une cellule Pending WO2025172529A1 (fr)

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Citations (3)

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