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WO2024097836A1 - Analyse du répertoire clonal dans l'hypersensibilité médicamenteuse - Google Patents

Analyse du répertoire clonal dans l'hypersensibilité médicamenteuse Download PDF

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WO2024097836A1
WO2024097836A1 PCT/US2023/078461 US2023078461W WO2024097836A1 WO 2024097836 A1 WO2024097836 A1 WO 2024097836A1 US 2023078461 W US2023078461 W US 2023078461W WO 2024097836 A1 WO2024097836 A1 WO 2024097836A1
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drug
sample
skin
patient
cells
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Sherrie J DIVITO
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Brigham and Womens Hospital Inc
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Brigham and Womens Hospital Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • G01N33/5044—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 involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells

Definitions

  • dtDHR delayed-type drug hypersensitivity reaction
  • dtDHR Delayed-type drug hypersensitivity reactions
  • MDE myeliform drug eruption
  • SCAR severe cutaneous adverse reactions
  • SJS/TEN Stevens-Johnson syndrome/toxic epidermal necrolysis
  • DRESS drug reaction with eosinophilia and systemic symptoms
  • a method of determining which of a plurality of drugs to which a patient has had or is having a Type 4 allergic reaction comprising: (a) obtaining a sample from the patient and culturing a portion of the sample with each of the plurality of drugs suspected of causing or having caused the allergic reaction as a test sample, and culturing another portion of the sample without any of the plurality of drugs as a control sample; (b) subjecting the test sample and the control sample to T cell receptor (TCR) sequencing; (c) detecting clonal TCR expansion to detect the presence of expanded clones in the test sample exposed to the drug as compared to non-expanded clones; and (d) identifying the drug in the test sample having clonal TCR expansion as being the drug to which the patient is having or has had an allergic reaction.
  • TCR T cell receptor
  • Also provided herein are methods comprising: (a) obtaining a sample from the patient who has had or is having a Type 4 allergic reaction, and who has been administered a plurality of drugs, one of which is suspected of causing or having caused the allergic reaction, and culturing a portion of the sample with each of the plurality of drugs suspected of causing or having caused the allergic reaction as a test sample, and culturing another portion of the sample without any of the plurality of drugs as a control sample; (b) subjecting the test sample and the control sample to T cell receptor (TCR) sequencing; (c) detecting clonal TCR expansion to detect the presence of expanded clones in the test sample exposed to the drug as compared to non-expanded clones; and (d) identifying the drug in the test sample having clonal TCR expansion as being the drug to which the patient is having or has had an allergic reaction.
  • TCR T cell receptor
  • determining whether a patient is likely to have a Type 4 drug reaction to a drug the patient has not yet taken comprising the steps of: (a) obtaining a sample from the patient and culturing a portion of the sample with the drug as a test sample, and culturing another portion of the sample without the drug as a control sample; (b) subjecting the test sample and the control sample to T cell receptor (TCR) sequencing; (c) detecting clonal TCR expansion to identify expanded clones in the test sample exposed to the drug as compared to nonexpanded clones; and (d) if the test sample has clonal TCR expansion above a first threshold, identifying the drug as being a drug that is likely to cause a Type 4 allergic reaction and should not be administered; or if the test sample does not have clonal TCR expansion, identifying the drug as being a drug that is less likely to cause a Type 4 allergic reaction and can be administered.
  • TCR T cell receptor
  • the sample from the patient is a skin sample, optionally from a reaction site.
  • the sample from the patient is a blood sample.
  • the patient has had an allergic reaction to a drug in the past.
  • the patient is having an allergic reaction when the sample is obtained, or wherein the patient has an allergic reaction that has resolved before the sample is obtained.
  • one or more concentrations of the drug to be tested are used.
  • test and control samples are incubated, optionally for 2-7 days, and thereafter restimulated with either the drug or vehicle control, and cultured, optionally for an additional 1-4 days preferably 1-2 days, before step (b).
  • the methods described herein comprise isolating migrating immune cells before step (b).
  • the methods further comprise analysing residual skin tissue for cell death and/or gross loss of integrity, and/or analysing markers in supernatant from skin culture.
  • the supernatant is analyzed for markers of T cell effector function.
  • the markers are selected from granulysin, interferon gamma (IFNg), interleukin 5 (IL5), IL 10, and IL13.
  • the residual skin tissue is analyzed for markers of keratinocyte death.
  • the marker of keratinocyte death is lactate dehydrogenase (LDH).
  • the methods further comprise enriching CD8+ T cells and/or CD14+ monocytes from PBMCs from the sample, and/or depleting CD25+ (Treg) cells from the sample, before culturing.
  • the methods further comprise identifying the drug in the test sample as being the drug to which the patient is having or has had an allergic reaction when the test sample has two or more of TCR clonal expansion, cell death and/or loss of integrity in the skin sample, and markers in supernatant from skin culture.
  • the markers are selected from granulysin, interferon gamma (IFNg), interleukin 5 (IL5), IL 10, and IL13.
  • kits for testing a patient to determine drugs to which the patient has had or is having a Type 4 allergic reaction comprising the steps of: (a) obtaining a test sample from the patient and culturing a portion of the sample with the drugs suspected of causing or having caused the patient’s allergic reaction along with culturing another portion of the sample as a vehicle control, (b) subjecting the test sample and the control sample to TCR sequencing, and (c) analyzing the results for clonal expansion to identify expanded clones in the test sample exposed to the drug as compared to vehicle control; if clonal expansion is identified, the tested drug is the drug to which the patient is having or has had an allergic reaction.
  • the test sample from the patient is a skin sample. In some embodiments, the test sample from the patient is a blood sample.
  • the patient has had an allergic reaction to a drug in the past.
  • the patient is having an allergic reaction when the test is being conducted.
  • Also provided herein are methods of determining whether a patient is likely to have a Type 4 drug reaction to a drug the patient has not yet taken comprising the steps of: (a) obtaining from the patient a test sample and culturing a portion of the sample with the drug intended for administration along with culturing another portion of the sample as a vehicle control, (b) subjecting the test sample and the control sample to TCR sequencing, and (c) analyzing the results for clonal expansion to identify expanded clones in the test sample exposed to the drug as compared to vehicle control; if clonal expansion is identified, the tested drug that is intended for administration is likely to cause a Type 4 allergic reaction and should not be administered.
  • the test sample from the patient is a skin sample. In some embodiments, the test sample from the patient is a blood sample.
  • the method is run iteratively on a series of drugs until a drug is identified to which the patient will not react.
  • one or more concentrations of the drug to be tested are used.
  • the test and control samples are incubated for 2-4 days and thereafter restimulated with either the drug or vehicle control, and cultured for an additional 1-2 days.
  • migrating cells residual skin tissue and supernatant from skin culture are employed.
  • PBMCs from the patient’s blood are collected and either CD8+ T cells and CD14+ monocytes are isolated or CD25+ (Treg) cells are depleted and then the cells cultured.
  • the sample is analyzed for markers of T cell effector function.
  • the markers are selected from granulysin, IFNg, IL5 and IL13.
  • the sample is analyzed for markers of keratinocyte death.
  • the marker of keratinocyte death is LDH.
  • HLA sequencing is additionally performed on the residual DNA and the TCR sequences of the clones and the HLA sequences are compared to known TCR and HLA sequences to identify potential public clones to determine if the same T cell clones were expanded in other patients with the same HLA allele and with the same drug reaction from the drug in question.
  • the methods further comprise treating the patient or recommending a treatment decision for the patient using a method shown in Table A.
  • FIGs. 1A-E Retrospective analysis of skin samples demonstrates variable T cell phenotypes and function across dtDHR severity.
  • C Log2counts of T cell phenotypic genes in dtDHR and healthy skin.
  • D Volcano plots highlighting significantly differentially expressed functional markers in diseased versus healthy skin.
  • E Log2counts of functional markers in dtDHR and healthy skin.
  • FIGs. 2A-C Prospective analysis by scRNAseq + CITEseq of dtDHR reveals complexity of T cell phenotypes mediating disease.
  • A 22 T cell clusters were identified from integrating scRNAseq + CITEseq across 3 SJS/TEN, 3 MDE and 3 healthy control skin and 3 healthy control blood samples. One healthy control skin sample was excluded due to low number of cells. Healthy control blood and skin were not paired. Heatmap shows clusters by phenotypic and functional markers using both genes (italicized) and protein (bolded) markers.
  • B Median percentage + range cytotoxic CD8 + T cells and Treg in skin and blood of SJS/TEN, MDE, and H.C. patients.
  • C Percentage cytotoxic T cells of total T cells in individual patient skin and blood across cytotoxic clusters identified in the heatmap (A).
  • FIG. 3 Volcano plots highlighting selected significantly differentially expressed genes between pooled SJS/TEN, MDE, and healthy controls (H.C.), in skin and in blood. Significance defined as Log2FC > ⁇ 1 and P a dj ⁇ 0.05.
  • FIGs. 4A-C TCR sequencing suggests clonal expansion and recruitment of cytotoxic CD8 + T cells in SJS/TEN but not in MDE.
  • A Clonal frequency (percentage) of the top 25 clones in skin and in blood of each dtDHR patient.
  • B Bar graph showing percent distribution across T cell phenotypic clusters of the top clone in skin (grey). If that same clone was also found in blood of that patient, it is additionally shown in open sections.
  • C Table showing fold change of clones in ex vivo culture of PBMCs from SJS/TEN patient 1 with suspected culprit drug, bupropion, at two concentrations compared to culture with vehicle alone.
  • FIGs. 5A-B TCRseq analysis of healthy control skin samples.
  • A Clonal frequency (percentage) of the top 25 clones in skin each healthy control.
  • B Bar graph showing percent distribution across T cell phenotypic clusters of the top clone in skin.
  • FIGs. 6A-D Human skin TRM can mediate MDE despite few circulating T cells.
  • C Total lymphocyte count in peripheral blood in lymphopenic vs non- lymphopenic patients with MDE. Lines show median. Significance defined as p ⁇ 0.05, two-tailed Mann-Whitney test.
  • FIGs. 7A-F TRM are present in skin after disease resolution. Mice treated with systemic drug alone did not develop skin inflammation by (A) ear thickness (mean with standard deviation of the mean, (SEM)) shown or (B) total number of CD3 + T cells and CD8 + T cells in ear skin by flow cytometry. Comparatively, mice treated with systemic and topical drug develop skin inflammation that slowly resolves by 90 days post-treatment as measured by (C) ear thickness and (D) H&E analysis. (E) Total number of CD8 + TEM (CD44 lll8ll CD62L l0 " ) in blood, TCM (CD44 high CD62L high ) in lymph node and total CD8 + T cells in ear skin quantified by flow cytometry.
  • SEM standard deviation of the mean
  • CD8 + T cells in ear skin show a TRM (CD62L low CD69 + CLA + ) phenotype by flow cytometry. Plots gated on CD3 + CD8 + T cells.
  • FIGs. 8A-F Skin TRM mediate MDE-like reaction in mice in the absence of circulating T cells.
  • A Schematic of drug challenge experiment.
  • B Ear thickness through 107 days (peak of challenge). Mean with SEM shown.
  • C Representative H&E images at day 107.
  • D Total number of CD8 + T cells in ear skin at day 107. Lines show median. Significance defined as p ⁇ 0.05, Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Only challenge without FTY720 versus primary response was significant.
  • E Percentage CD3 + T cells and CD3 + CD8 + T cell in blood, and percentage of effector CD8 + T cells (CD44 high CD62L low ) in blood at 107 days, demonstrating systemic drug challenge is associated with a systemic T cell reaction. Lines show median. Significance defined as p ⁇ 0.05, Kruskal -Wallis test followed by Dunn’s multiple comparisons test.
  • F Total number and percentage of functional CD8 + T cells in ear skin of mice treated or not with FTY720. All comparisons not significant with p > 0.05 two-tailed Mann-Whitney test.
  • A-F N > 3 mice per group. Each experiment was repeated at least twice.
  • FIGs. 9A-C T cell receptor sequencing identifies drug-reactive clones in skin affected by fixed drug eruption (FDE).
  • FDE fixed drug eruption
  • A Clinical pictures and associated clonal frequency (percentage) from two biopsy sites taken simultaneously. Two patients shown. Paired clones between each biopsy site are color coded.
  • B Clonal frequency (percentage) in an active FDE site and a non-FDE site taken simultaneously from a third patient.
  • C Clonal frequency (percentage) in an active FDE site and a non-FDE site taken simultaneously from a fourth patient. The active FDE site was re-biopsied 5 years and another 1 year after resolution.
  • FIGs. 10A-D T cell receptor sequencing identifies an expanded drug-reactive clone in skin and blood.
  • A Productive frequency (percentage) of T cell clones in skin across 4 samples: FFPE skin biopsy collected at the start of SJS/TEN, a research skin biopsy - half cultured with presumed culprit drug, palbociclib O.lng/ml, and half cultured with vehicle, and then a FFPE skin biopsy collected from an unrelated rash presenting weeks later. Each clone is color coded across samples. Biopsy - bx.
  • Immune-mediated drug reactions fall under one of six types of drug reactions per the World Health Organization classification system (5). Immune- mediated drug reactions are further classified as types 1 - 4 according to the Gell and Coombs model of hypersensitivity (6). Type 4 reactions are termed delayed-type reactions, given that they typically begin days to weeks after exposure to inciting antigen. Reactions can occur more quickly, on the order of one or more days, if history of prior antigen exposure. This timing is consistent with a T cell mediated response. Indeed, T cells and molecules typically attributed to T effector cells are consistently detected in skin biopsies or blister fluid from dtDHR (7, S).
  • Skin resident memory T cells are a unique population of memory T cells that reside long-term in skin without recirculating even during periods of immunologic quiescence (22-24). Skin TRM are increasingly implicated in the pathogenesis of several inflammatory skin diseases, most notably allergic contact dermatitis (25), which is another form of delayed-type hypersensitivity reaction, and acute graft-versus-host- disease (2d), which clinically and histologically can present identically to MDE, DRESS and SJS/TEN. Subsequently, a role for skin TRM in dtDHR has been surmised. Recent research supports that skin TRM are generated by dtDHR (27), but there are no studies to date investigating whether skin TRM mediate disease. Importantly, knowing whether TRM or other T cell subsets mediate disease is a critical step in illuminating disease pathogenesis, but moreover has significant clinical implications such as development of a reliable test to identify culprit drug, which is currently lacking in the clinic.
  • dtDHR pathobiology is historically under-researched (4) due to three main barriers: (i) the rarity and acuity of severe disease impedes prospective sample collection; (ii) skin samples obtained for clinical purposes are typically formalin fixed paraffin embedded (FFPE) which previously precluded extensive laboratory analysis; and (iii) lack of adequate mouse models.
  • FFPE formalin fixed paraffin embedded
  • the identification of clonal expansion across multiple T cell subsets implies that either a drug-reactive precursor proliferated and differentiated into multiple phenotypes during this dtDHR episode, or a prior exposure generated clones of multiple phenotypes that were reactivated upon antigen re-exposure.
  • the latter has been demonstrated in allergic contact dermatitis, another form of delayed-type hypersensitivity (25). If this is the case in dtDHR, at-risk patients could potentially be identified by testing for preexisting drug-reactive skin TRM before drug exposure.
  • pre-existing drug- reactive TRM in skin invokes a potential role for heterologous immunity in dtDHR pathogenesis whereby patients have previously generated virus-specific skin TRM that cross-react to drug (70-72). This could explain why in some cases patients react upon first exposure to drug. Further work is necessary to explore these possibilities.
  • clonal repertoire analysis T cell receptor sequencing
  • FDE active fixed drug eruption
  • FDE is another CD8 mediated form of dtDHR, supporting that clonal expansion occurs in various forms of dtDHR, and again supporting specificity.
  • drug-specific clonal expansion in test skin and blood from a patient with SJS/TEN.
  • the expanded clone matched the diseasemediating clone identified in a skin biopsy taken during active disease.
  • skin biopsy taken from an unrelated rash showed an entirely different expanded clone, again demonstrating specificity of clonal sequencing as a read-out for drug reactivity in dtDHR.
  • the methods can include an initial expert review/interpretation of history of prior disease at a clinical and/or immunologic level based on clinical history and skin biopsy, if available.
  • the methods can be practiced using samples comprising skin samples, e.g., 5- 10mm, e.g., 6mm, skin biopsies (primary test target) and/or blood samples (secondary test target) from patients.
  • skin samples e.g., 5- 10mm, e.g., 6mm
  • skin biopsies primary test target
  • blood samples secondary test target
  • the samples can be obtained after disease has resolved or during active disease.
  • the skin biopsy samples are obtained using a punch or shave biopsy method, but other methods can also be used, e.g., needle biopsies or excisional biopsies.
  • the skin sample comprises skin from a reaction site.
  • the skin sample undergoes a skin culture and preparatory method for downstream analysis.
  • the skin culture method places a portion, e.g., half, of a skin biopsy sample per well, e.g., for 2-7 days, 2-5 days, 3-5 days, or 3-4 days with a drug of interest (e.g., a potential allergic reaction trigger) and a second portion, e.g., the other half, biopsy with vehicle control.
  • a drug of interest e.g., a potential allergic reaction trigger
  • Other test compounds can be used in addition to known or approved drugs.
  • the skin is then restimulated with the same drug or vehicle control and cultured for an additional 1-2 days. The skin is reviewed grossly for changes including loss of sample integrity or signs of cell death.
  • Migratory T cells are collected, e.g., by pipetting the culture supernatant and cells non-adherent or loosely- adherent to skin biopsy into a collection tube, and then pelleting cells and removing the supernatant, prior to performing an enzymatic digestion with physical disaggregation to maximize the number of isolated viable T cells while minimizing structural cells, or alternatively keratinocyte/skin debris is collected intact (without enzymatic digestion and physical disaggregation). In addition, culture supernatant is collected.
  • PBMCs are collected from the blood sample, e.g., using a standard Ficoll gradient.
  • PBMCs potentially undergo selection based on findings in the skin samples; for example, for an SJS/TEN case collect CD8 and optionally CD14+ cells are collected, or for a spongiotic CD4+ appearing MDE reaction, the methods can include depleting Treg.
  • CD8+ cytotoxic appearing reactions CD8 T cells and CD14 positive monocytes are selected for culture if sufficient cell number.
  • CD4+ T cell / spongiotic reactions or insufficient cell number PBMCs are depleted of CD25+ (Treg) cells then cultured.
  • PBMCs are stimulated as skin in Step 3.
  • T cell receptor (TCR) repertoire analysis for clonal expansion is then performed to determine 1, 2, or all three readouts including (1) T cell receptor (TCR) repertoire analysis for clonal expansion; (2) biomarker analysis; and (3) skin integrity analysis.
  • TCR T cell receptor
  • the primary readout is based on T cell receptor (TCR) repertoire analysis on DNA extracted from the collected immune/T cells and residual skin biopsy tissue if not digested/disaggregated to identify clonal expansion.
  • TCR sequencing include next generation sequencing (NGS) and single-cell TCR sequencing methods such as TCRseq or HT-TCRP sequencing; see, e.g., Mitchell and Michels, J Life Sci (Westlake Village). 2020 Dec; 2(4): 38-58; Fu et al., Front Immunol. 2021 Nov 5: 12:777756; Fbhse et al., Eur J Immunol. 2011 Nov;41(ll):3101-13.
  • the presence of clonal expansion can be identified by comparison of frequency of each TCR clone to a threshold level that represents a control level of frequency associated with clones that have not undergone expansion, and a frequency above that threshold level indicates the presence of clonal expansion; preferably internal controls are used.
  • clonal expansion is defined as three times the frequency of nonexpanding clones within each sample.
  • the presence of specific clones identified as high-risk is indicative of a positive result independent of frequency.
  • the secondary readout is based on analyte analysis of the supernatant for particular biomarkers or a combination of biomarkers.
  • Biomarkers analyzed can include one, two, three, or all four of granulysin, interferon gamma (IFNg), interleukin 5 (IL5), IL 10, and/or IL13; optionally granulysin is analyzed alone or with one, two, or three of IFNg, IL5, IL10, and/or IL-13. Methods for analyzing these biomarkers are known in the art and can include .
  • the tertiary readout is gross analysis of the residual skin, e.g., for loss of integrity or signs of cell death. This can be performed visually, looking for signs of the skin sample breaking down, or using an assay for keratinocyte cell death such as detection of lactate dehydrogenase (LDH).
  • LDH lactate dehydrogenase
  • HLA sequencing can be performed, e.g., using residual DNA from the collected cells.
  • the HLA and TCR sequencing results from patient samples (active and/or resolved) can also be compared to a database comprising information regarding TCR/HLA/drug reactions, as added confirmation.
  • the TCR sequencing results from resolved disease can be compared to sequences from samples obtained from patients during active clinical disease, if available, as added confirmation.
  • the methods can also be used to reduce the risk of severe drug reactions.
  • skin samples and blood are obtained from a patient prior to administration of a high risk medication.
  • the skin and blood are analyzed with the drug as above.
  • a result that indicates a risk of a drug reaction would indicate that a recommendation should be made to avoid the drug, or consider giving the drug only if clinically necessary but monitoring the patient closely; a result that indicates a low or no likelihood of a drug reaction would indicate that a recommendation can be made to take the drug as normal.
  • Medications associated with a high risk of dtDHR can include abacavir; allopurinol; amoxicillin; ampicillin; azithromycin; barbiturates; carbamazepine; cenobamate; cephalosporins, fluoroquinolones, e.g., ciprofloxacin/levofloxacin/gemifloxacin;clozapine; cotrimoxazole; dapsone; diclofenac; ethosuximide; isoniazid; lamotrigine; nevirapine; non-steroidal anti-inflammatory drugs; oseltamivir; oxicams; oxcarbazepine, paracetamol/acetaminophen; penicillins; phenytoin; sulfasalazine; sulfonamides; tetracyclines, vancomycin; valproate; and zonisamide. Any drug or vaccine (either active compound or
  • FFPE skin sections 5-6 mm thick were baked, deparaffinized, and rehydrated.
  • Hematoxylin and eosin (H&E) staining was carried out by standard technique.
  • sections underwent antigen retrieval at 96°C, blocking of non-specific protein binding, and staining with primary then appropriate secondary antibodies.
  • Anti-rabbit CD3 (A0452, Dako; polyclonal), antimouse CD45RO-biotinylated (304202, Biolegend; UCHL1), anti -mouse CD45RA (158-4D3; Novus, NBP2-15193), anti-mouse CD8 (M7103, Dako; C8/144B), and antirat CLA (321302, Biolegend; HECA-452).
  • NanoStringQCPro-v. 1.18.0 82) and custom R code. Proportion of fields of view successfully counted, binding density, noise threshold, expression of positive and negative control genes, and expression of endogenous and housekeeping genes were evaluated. Samples that were found to be suboptimal were flagged and removed from the analysis. Raw counts were normalized with the geometric mean using first positive controls, then using a subset of housekeeping genes selected with an expression above a limit of detection (defined as mean expression of the negative control genes plus two times the standard deviation), and a mean value higher than 200 (value selected empirically based on average housekeeping expression). Any gene expressed below this limit was filtered out. Differential expression at the gene level was performed with the DESeq2-v.1.26.0 R package (83).
  • Adjusted p-values (p a dj) were estimated using the false discovery rate to correct for multiple comparisons.
  • Primary analysis compared each dtDHR to healthy controls. Log2FC > ⁇ 1, p a dj ⁇ 0.05 was considered significant.
  • Secondary analysis compared each form of dtDHR to each other. Log2FC > ⁇ 1, p a dj ⁇ 0.1 was considered significant.
  • Samples were processed under sterile conditions. Upon collection, skin biopsies were halved and each half frozen in 500 ml Cryostor CS10 cry opreservation media (07930; Stemcell Technologies) in liquid nitrogen (LN2). PBMCs were collected from blood by Ficoll gradient and frozen in FBS + 10% DMSO in LN2.
  • Biopsies were then rinsed in PBS, cut into small pieces and incubated with human whole skin dissociation kit without enzyme P (130-101-540; Miltenyi) for 2 hours 20 min at 37°C with agitation. Samples were washed in RPMI-1640 +10% FBS and centrifuged (1300 rpm, 4°C, 5 min). Skin was then disaggregated over a 70 pm filter, rinsed with RPMI-1640+10% FBS, and centrifuged (1300 rpm, 4°C, 5 min). PBMCs were thawed at 37°C and washed with RPMI-1640+10% FBS, then PBS. Cell pellets were resuspended in cold PBS. Cell counts and viability were determined by trypan blue exclusion.
  • RNA-seq experiments were performed by the BWH Single Cell Genomics Core. Sorted CD3 + T cells from three skin and three blood samples were pooled and resuspended in 0.4% BSA in PBS at a concentration of 1,000 cells/pl, then loaded onto a single lane (Chromium chip A, 10X Genomics) followed by encapsulation in a lipid droplet (Single Cell 5 'kit VI, 10X Genomics), then by cDNA and library generation according to the manufacturer’s protocol. Three runs were performed for a total of 18 specimens.
  • mRNA library was sequenced to an average of 50,000 reads per cell, V(D)J library and HTO (Cell Hashing antibodies) library sequenced to an average of 5,000 reads per cell, using Illumina Novaseq.
  • 10X Genomics reads were processed with Cell Ranger v3.1 for gene expression (using GRCh38 as reference genome), CITEseq and hashtag oligo counts for demultiplexing and quantification of transcript counts per putative cell. Demultiplexing of pooled samples were performed using the Seurat R package (version 4.0, Satija Lab) (85) along with R v4.0.5 (86).
  • Raw HTO counts were normalized with centered log ratio (CLR) transformation (NormalizeData func.), and then HTODemux function was used to demultiplex pooled samples and to filter out (i) negative cells with low HTO UMIs (empty droplets or failed reactions) and (ii) multiplet cells with high HTO UMIs (cells from multiple samples in a droplet) (84).
  • CLR centered log ratio
  • HTODemux function was used to demultiplex pooled samples and to filter out (i) negative cells with low HTO UMIs (empty droplets or failed reactions) and (ii) multiplet cells with high HTO UMIs (cells from multiple samples in a droplet) (84).
  • Each of the three pooled samples were repeatedly demultiplexed and 18 samples were separated into distinct Seurat objects.
  • We later pre-processed TCRseq reads with Cell Ranger v6.0.1 due to updates in the clonotype calling algorithm.
  • RNA and ADT assays from all 18 samples were log normalized and centered log ratio (CLR) normalized, respectively.
  • CD4 + CD8 + doublets and double positives were filtered out by removing cells having both more than 0.75 and 1 CLR normalized CD4 and CD8 ADT UMI counts, respectively.
  • One healthy control skin sample was removed from the analysis given Seurat integration workflow failing due to low number of cells. This filter step resulted in a scRNA dataset of 15,084 cells.
  • TCR sequences of all expanded clones from the prospective study dtDHR patients to two publicly available databases, VDJdb (Bagaev DV et al, 2019) and McPAS-TCR (89), to identify potential antigenic specificity or cross-reactivity (Glanville et al., 2017; Huang H et al., 2020; Cottrell T et al., 2021).
  • Our search included T cells with matching alpha and beta chains, or only alpha chains or only beta chains.
  • PBMCs from SJS/TEN patient 1 were thawed at 37°C and washed with complete human media containing RPMI-1640 + 4% human AB serum (Sigma), 1% Penicillin/ Streptomycin, 2mM L-glutamine, O. lmM MEM non-essential amino acids, lOmM HEPES, ImM sodium pyruvate and 50pM beta-mercaptoethanol.
  • Cell pellets were resuspended in complete human media. Cell counts and viability were determined by trypan blue exclusion.
  • PBMCs were depleted of CD25+ cells using the EasySepTM Human Pan-CD25 Positive Selection and Depletion Kit (Stemcell technologies) to remove any potential Treg.
  • CD25- T cells were recounted and plated in a 96-well flat bottom plate in triplicate in complete human media.
  • Cells were incubated with culprit drug Bupropion HCL at concentrations of 50ng/ml and lOOng/ml, and water for injection (GIBCO) as a vehicle control.
  • As a positive control wells were precoated with 500ng/ml anti-human CD3 (Biolegend) for 2 hours at 37°C, followed by 3 washes with PBS. Cells were plated with 5pg/ml anti-human CD28.
  • the 96-well plate was incubated at 37°C 5% CO2. Cells were restimulated with drug or vehicle control at day 4. On day 5, cells were collected and spun down with PBS.
  • skin biopsy was halved and cultured with Palbociclib 0.1 ng/ml or vehicle control for 4 days then re-stimulated with Palbociclib 0.1 ng/ml or vehicle for another day.
  • Non-adherent T cells were collected.
  • Skin biopsy was enzymatically digested as above with human whole skin dissociation kit (Miltenyi) and then disaggregated over a 70 pm strainer. Resultant single cells were combined with nonadherent T cells.
  • PBMC were similarly cultured with Palbociclib at 3 concentrations 0.1, 1 and 10), with Binimetinib at 0.1 and 1 or with vehicle control for the same time period and restimulated as was skin.
  • HLA-B*57:01 C57B1/6J transgenic mice were provided by D. Margulies (National Institute of Immunology, Allergy and Infectious Disease) and M. Norcross (U.S. Food and Drug Administration) 60 . Heterozygous mice were bred inhouse by crossing with C57BL/6J mice (Jackson Laboratories). Male and female mice between 6-12 weeks of age were used in experimentation. Mice were phenotyped to confirm transgene expression (60). Experiments were performed in accordance with the guidelines put forth by the Center for Animal Resources and Comparative Medicine at Harvard Medical School (HMS) (HMS IACUC approval #2016N000070).
  • HLA-B*57:01 positive (pos) mice or HLA-B*57:01 negative (neg) littermate controls were treated by intraperitoneal (i.p.) injection five days/week for 17 days with 3 mg of Abacavir (ABC) (188062-50-2; Sigma Aldrich) diluted in Water for Injection (WFI) ((60)).
  • a subset of mice underwent concurrently treatment of the left ear topically via painting three days/week with 0.2 mg ABC in 30% ethanol. Dosing was based on the animal equivalent dosage (AED) of ABC (90).
  • Vehicle control mice were treated with equal volume of WFI i.p. injection and painted topically with 30% ethanol when appropriate. All mice were depleted of CD4 + T cells by i.p.
  • mice were depleted of CD4 + T cells and treated systemically by i.p. injection with ABC or vehicle. Some mice were also treated with FTY720 (1 mg per/kg i.p. daily) (SML0700; Sigma Aldrich).
  • mice were monitored thrice weekly for clinical signs of dermatitis and ear thickness measured by electronic digital caliper for the entirety of each study. Mice were harvested at peak of disease (day 22), at disease resolution (day 90), and at peak of drug challenge (day 107).
  • PBMCs Plasma PBMCs isolated by Ficoll gradient. A portion of ear was fixed in 10% neutral buffered formalin, embedded in paraffin and sectioned at 5 microns for H&E staining by standard laboratory method. Spleen, cervical lymph nodes, and remaining ear tissue were harvested into cold PBS. Spleens and lymph nodes were disaggregated over 70 pm strainers into single cell suspensions.
  • Ear skin was cut into small pieces then incubated in 3 ml Hanks balanced salt solution (14175095; Thermofisher Scientific) supplemented with 1 mg ml -1 collagenase A (11088785103; Roche) and 40 pg ml’ 1 DNase I (10104159001; Roche) at 37°C for 3 hours in a shaking incubator.
  • RPMI+10%FBS was added to the tubes and suspension spun down at 1400 rpm for 5 min at 4°C.
  • the cell pellet was resuspended in complete RPMI media then disaggregated through a 70 pm filter. Cell counts and viability were assessed with trypan blue.
  • CLA expression was assessed by incubating cells with E-Selectin/Fc Chimera (575-ES; R&D System) in conjunction with PerCP-conjugated F(ab’)2 fragments of goat anti-human IgG F(c) antibody (109- 126-170; Jackson ImmunoResearch).
  • E-Selectin/Fc Chimera 575-ES; R&D System
  • PerCP-conjugated F(ab’)2 fragments of goat anti-human IgG F(c) antibody 109- 126-170; Jackson ImmunoResearch.
  • For intracellular cytokine staining cells were fixed and permeabilized using Cytofix/Cytoperm Fixation/Permeablization Kit (554714; BD Biosciences) according to manufacturer’s instructions and stained with FITC-IFN-y (XMG1.2), PE-TNF-a (MP6-XT22), and/or APC-Granzyme B (QA16A02) (Biolegend).
  • DGEA suggested that all three forms of disease were Thl/Tcl skewed.
  • Genes for cytolytic granule components, GZMA, GZMB, and PRF1, and IFNg signature genes, CXCL9, CXCL10 and CXCL11 were significantly upregulated in mild and severe dtDHR compared to healthy control skin (FIGs. 1D-E).
  • Analysis further demonstrated significantly increased transcription of GNLY in both SJS/TEN and DRESS, and of TNF in SJS/TEN alone compared to healthy controls (FIGs. 1D-E), similarly to prior reports (40-43).
  • the total number and percentage of T cells in each cluster in each patient is shown in Tables 4 and 5 and fold change in percentage of T cells in each cluster in each patient shown in Tables 6 and 7.
  • results were not statistically significant.
  • the percentage cytotoxic T cells of total T cells in skin correlated with more severe disease (FIGs. 2B). There was a statistically significant difference between the percentage of cytotoxic CD8 + T cells between SJS/TEN versus MDE skin. The increased percentage of cytotoxic CD8 + T cells in SJS/TEN coupled with the decrease in Treg resulted in skewing of cytotoxic T cell Treg ratio in SJS/TEN compared to MDE and healthy skin and blood (FIG. 2B).
  • Cytotoxic T cells in skin in both SJS/TEN and MDE were distributed across multiple clusters, including CD8 + T effectors, CD8 + CD56 + T cells, CD8 + TEM, CD8 + TEMRA, and CD4 + T effectors, all classically considered recruited populations, as well as CD8 + skin TRM, and gd T cells which can be recruited or skin resident (49) (FIG. 2C), suggesting that multiple T cell subsets, both resident and recruited, contributed to disease. Results were largely paralleled in blood (FIG. 2C), though there was much lower percentage of cytotoxic T cells in blood in all clusters in MDE compared to SJS/TEN, supporting that SJS/TEN had greater systemic activation and recruitment of cytotoxic T cells through circulation into skin.
  • Pseudo-bulk DGEA confirmed a prominent CD8 + Thl/Tcl signature in SJS/TEN versus healthy skin, while CD4 and CCR8, a Treg recruiting chemokine (50), were decreased.
  • the prominent cytotoxic skew was maintained when comparing SJS/TEN skin to MDE skin.
  • Functional polarization appeared more variable in MDE skin than previously suggested by bulk transcriptional profiling, with markers of Tel (GZMB), Th2 (IL5, IL10 and I 13) and Treg (CTLA4, IL2RA and IL10) observed when compared to healthy control skin, potentially reflecting variability across MDE patients.
  • MDE blood demonstrated significantly increased transcription of F0XP3, CTLA4, IL2RA and CCR8.
  • CCR8 binds CCL18, which was increased in MDE skin by bulk transcriptional profiling (Table 1), suggesting a possible mechanism by which Treg may be recruited into skin in MDE.
  • the skew toward Thl/Tcl and away from Treg was maintained in comparison of SJS/TEN to MDE blood.
  • the single expanded clone in SJS/TEN patient 1 skin and MDE patient 3 skin were each amongst the top expanded clones in blood.
  • the top expanded clone in SJS/TEN patient 3 skin that had clearly expanded by clonal frequency was also the top clone in that patient’s blood, suggesting that clonal expansion had occurred in blood, but the clonal frequency did not reach the threshold arbitrarily set by the above quantitative approach.
  • the top clone in skin of SJS/TEN patient 2 was the second highest clone in that patient’s blood, again raising the possibility of expansion in blood.
  • the top expanded clone in skin of MDE patient 1 and 2 were not detected in blood suggesting potentially skin-limited expansion (FIG. 4A).
  • top expanded clone in skin in each patient was distributed across multiple T cell phenotypic clusters including primarily activated clusters (FIG. 4B).
  • the top expanded clone in skin was a cytotoxic CD8 + T that spanned highly functional clusters in both skin and blood (FIG. 4B).
  • the top expanded clones in MDE patients 1 and 2 skin were also cytotoxic CD8 + T cells that spanned highly functional clusters but were not detected in blood.
  • the top expanded clone in MDE patient 3 skin and blood was a CD4 + T cell that spanned Treg clusters (FIG. 4B), mirroring the gene expression data from scRNAseq.
  • Healthy control skin did not have expansion by the quantitative definition (FIG. 5A), and cluster analysis revealed that the top clone in skin of each healthy control skin sample was a CD4 + T cell spanning non-functional clusters (FIG. 5B).
  • the monoclonal or oligoclonal expansion and activation observed in these patient samples reflects drug-specific activation, given that these patients were all diagnosed with active dtDHR. It is theoretically possible that the expanded T cells reacted to antigen other than drug. In rare cases, SJS/TEN can occur secondary to infection, most commonly Mycoplasma or Herpes Simplex Virus. The clinical and histologic findings in MDE can similarly occur secondary to virus alone, or to the combination of drug plus virus (the classic example being rash induced when amoxicillin is given in EBV infection). None of the 6 prospective study patients had signs or symptoms of Mycoplasma, HSV, or other viral infection, supporting drug as the antigenic source in these patients.
  • Example 4 Skin TRM can mediate MDE in the absence of circulating T cells
  • TRM may be a critical pathogenic T cell subset mediating MDE
  • TRM may be a critical pathogenic T cell subset mediating MDE
  • MDE can develop in patients that are severely lymphopenic, intimating that skin TRM alone can mediate MDE.
  • lymphopenic patients may have aberrant immune systems from their underlying disease or treatment that caused the lymphopenia, their MDE are essentially clinically and histopathologically indistinguishable to healthy patients. Lymphopenic patients also often react to the same common culprit drugs as healthy patients.
  • HLA-B*57:01 predisposes patients taking the drug abacavir to dtDHR (58, 59).
  • Cardone et al. previously generated HLA-B*57:01Tg mice that developed CD8 + T cell-mediated ear dermatitis in response to topical plus systemic abacavir exposure coupled with CD4 + T cell depletion (60).
  • mice developed a cytotoxic CD8 + T cell mediated skin-limited (MDE-like) reaction that was HLA-B*57:01 and drug dependent (FIG. 7C- D).
  • MDE-like cytotoxic CD8 + T cell mediated skin-limited
  • FIG. 7C- D drug dependent
  • CD8 + T cells were primed in secondary lymphoid organs and migrated through blood into skin to mediate disease.
  • Drug-induced dermatitis slowly resolved by day 90 (FIG. 7C).
  • Ear thickness decreased but did not return to baseline as ears were scarred; however, active inflammation resolved based on clinical and histologic evaluation (FIG. 7D).
  • mice Despite the absence of active inflammation, ear skin of HLA-B*57:01 pos , drug-treated mice demonstrated a CD8 + T cell population expressing CD62L low CD69 + CLA + consistent with skin TRM (FIGs. 7E-F). Concurrently, TEM (CD62L low CD44 high ) were observed in blood and TCM (CD62L high CD44 high ) in LN (FIG. 7E).
  • mice underwent in vivo drug challenge At day 90, HLA-B*57:01 pos mice previously treated with abacavir or vehicle were now treated systemically with abacavir or vehicle by intraperitoneal injection without topical treatment (FIG. 8 A). Mice containing drug- reactive memory T cells developed an MDE-like reaction upon drug challenge, marked by increased ear thickness and clinically and histologically evident dermatitis, faster than the primary drug-exposed mice, consistent with a memory T cell response (FIGs. 8B-C).
  • This reaction was drug specific as HLA-B*57:01 pos mice previously immunized against drug but now challenged with vehicle failed to develop a reaction.
  • This reaction included expansion and migration of CD8 + T cells in lymph node and blood, with some migration into skin, indicating that this reaction was not purely a local immune response or percutaneous reaction (FIGs. 8D-E).
  • mice A subset of drug-challenged mice was concurrently treated with FTY720, an S1PR1 agonist that prevents egress of T cells from lymphoid organs (62, 63). These mice developed dermatitis only slightly delayed to non-FTY720 treated drug- challenged mice, who had the ability to recruit T cells to skin from secondary lymphoid organs (FIGs. 8B-C). Moreover, FTY720 treated mice had a slightly reduced number of CD8 + T cells yet equivalent or higher percentage and total number of functional CD8 + T cells in ear skin compared to non-FTY720 treated mice (FIGs. 8D-F), further supporting that the main mediators of disease were skin TRM. These data directly parallel our observations in lymphopenic patients that skin TRM appear to be sufficient to mediate skin-limited reactions.
  • FDE Fixed drug eruptions
  • Example 7 Clonal repertoire analysis can identify culprit drug in skin and blood in dtDHR patient.
  • a patient with SJS/TEN was prospectively enrolled.
  • the patient provided a skin biopsy after treatment with systemic steroids and partial resolution of her rash.
  • the skin biopsy was halved.
  • One half was cultured with presumed culprit drug, palbociclib.
  • the other half was cultured with vehicle control.
  • T cells were isolated then DNA extracted and sequenced by high-throughput TCR sequencing.
  • DNA was extracted from an FFPE skin biopsy taken at initial presentation of SJS/TEN, and another taken weeks later from a clinically separate rash.
  • Clonal repertoire analysis revealed two drug-reactive clones, gray and purple, during initial presentation of SJS/TEN (FIG. 10 A) These clones expanded in culture with drug as demonstrated by increased frequency compared to vehicle control.
  • the clones were present in skin weeks later after resolution of SJS/TEN, but were unexpanded during the unrelated rash, indicated drug/reaction specificity.
  • the unrelated rash was mediated by a distinct clone (FIG. 10
  • PBMCs were collected after disease resolution and cultured with vehicle, presumed culprit drug, palbociclib, or another drug that the patient had recently taken, binimetinib (FIG. 10B).
  • the gray clone from skin was not detected in blood after disease resolution supporting that in some patients, assaying skin rather than blood may be critical.
  • culture supernatants were collected and assayed by ELISA for granulysin. In both skin and PBMC assays, granulysin was increased in culture with palbociclib compared to vehicle control. Binimetinib did not increase granulysin levels (FIGs. 10C-D).
  • Gene name is italicized if RNA was used; if protein was used, the protein name is not italicized.

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Abstract

L'invention concerne des dosages et des procédés pour identifier lequel parmi une pluralité de médicaments provoque une réaction d'hypersensibilité médicamenteuse de type retardé (dtDHR) chez un patient.
PCT/US2023/078461 2022-11-02 2023-11-02 Analyse du répertoire clonal dans l'hypersensibilité médicamenteuse Ceased WO2024097836A1 (fr)

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Citations (2)

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US20120034221A1 (en) * 2008-08-26 2012-02-09 Macrogenics Inc. T-Cell Receptor Antibodies And Methods of Use Thereof
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Publication number Priority date Publication date Assignee Title
US20120034221A1 (en) * 2008-08-26 2012-02-09 Macrogenics Inc. T-Cell Receptor Antibodies And Methods of Use Thereof
US20130136799A1 (en) * 2008-11-07 2013-05-30 Sequenta, Inc. Monitoring health and disease status using clonotype profiles

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PAN REN-YOU, CHU MU-TZU; WANG CHUANG-WEI; LEE YUN-SHIEN; LEMONNIER FRANCOIS; MICHELS AARON W.; SCHUTTE RYAN; OSTROV DAVID A.; CHEN: "Identification of drug-specific public TCR driving severe cutaneous adverse reactions", NATURE COMMUNICATIONS, NATURE PUBLISHING GROUP, UK, vol. 10, no. 1, UK, XP093171581, ISSN: 2041-1723, DOI: 10.1038/s41467-019-11396-2 *
WEN-HUNG CHUNG, REN-YOU PAN, MU-TZU CHU, SEE-WEN CHIN, YU-LIN HUANG, WEI-CHI WANG, JEN-YUN CHANG, SHUEN-IU HUNG: "Oxypurinol-Specific T Cells Possess Preferential TCR Clonotypes and Express Granulysin in Allopurinol-Induced Severe Cutaneous Adverse Reactions", JOURNAL OF INVESTIGATIVE DERMATOLOGY, ELSEVIER, NL, vol. 135, no. 9, 1 September 2015 (2015-09-01), NL , pages 2237 - 2248, XP055378732, ISSN: 0022-202X, DOI: 10.1038/jid.2015.165 *

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