WO2024200655A1 - Taux prédictif de pd-l1 sur des microvésicules dans l'évaluation de sa capacité à répondre à un traitement d'un cpnpc - Google Patents

Taux prédictif de pd-l1 sur des microvésicules dans l'évaluation de sa capacité à répondre à un traitement d'un cpnpc Download PDF

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WO2024200655A1
WO2024200655A1 PCT/EP2024/058488 EP2024058488W WO2024200655A1 WO 2024200655 A1 WO2024200655 A1 WO 2024200655A1 EP 2024058488 W EP2024058488 W EP 2024058488W WO 2024200655 A1 WO2024200655 A1 WO 2024200655A1
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nsclc
level
mvs
positive
tissue sample
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Annalen BLECKMANN
Kerstin MENCK
Marcel KEMPER
Nadja SCHÖNE
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Westfaelische Wilhelms Universitaet Muenster
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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
    • G01N33/57585—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites involving compounds identifiable in body fluids
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5752—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the lungs
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70532—B7 molecules, e.g. CD80, CD86
    • 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

  • the present invention relates to a novel method of evaluating whether a subject already suffering from non-small cell lung carcinoma (NSCLC) will respond to a treatment of NSCLC comprising immunotherapy, the method comprising determining a level of PD-L1 -positive MVs in a test peripheral blood (PB) sample obtained from a subject suffering from NSCLC who has not yet been treated, followed by evaluating whether said subject will be a responder to a treatment of NSCLC comprising immunotherapy, if said level of PD-L1 -positive MVs is increased relative to corresponding levels of PD-L1 -positive MVs in control PB samples obtained from subjects also suffering from NSCLC, but being non-responders to said treatment of NSCLC comprising immunotherapy. Further, the present invention relates to a data processing system comprising a processor configured to perform the method of the invention.
  • PB peripheral blood
  • Lung cancer is still the leading cause of cancer-related death worldwide (Sung et al., 2020; PMID 33538338).
  • immune checkpoint inhibitors such as antibodies against programmed cell death protein 1 (PD-1) and programmed cell death ligand 1 (PD-L1) have revolutionized state-of-the-art lung cancer treatment.
  • NSCLC patients with high PD-L1 expression on tumor cells benefit from a singleagent immunotherapy compared to standard platinum-based chemotherapy (Reck et al., 2016, PMID: 27718847; Herbst et al., 2020, PMID: 32997907; Sezer et al., 2021, PMID: 33581821).
  • dual checkpoint inhibition combined with only two cycles of chemotherapy has recently been shown to significantly improve OS in patients with advanced stage NSCLC compared to chemotherapy alone (Paz-Ares et al., 2021; PMID: 33476593).
  • tissue PD-L1 expression as determined via immunohistochemistry (IHC) is the only approved biomarker for immunotherapy.
  • IHC immunohistochemistry
  • tissue PD-L1 expression has also been shown to differ between the primary tumor and nodal metastasis (llruga et al., 2017, Journal of Thoracic Oncology 12: 458-466) which questions the reliability of just applying tissue biopsy-based PD- L1 scoring.
  • Using only tissue expression of PD-L1 as a biomarker would also require repetitive tissue biopsies for the radiological imaging to determine whether a subject does respond to the treatment given after the subject was diagnosed with NSCLC. This procedure can be very unpleasant for the patient, is also time-consuming, and is therefore associated with increased risks for the patient because it is relatively late to see whether the subject is responding to therapy or not.
  • the inventors found that the level of PD-L1 -positive microvesicles (M s) in a liquid sample, namely in a PB sample, wherein said PB sample has been obtained from a subject who already suffers from NSCLC and who has not yet been exposed to any immunotherapy, can be seen as a new biomarker and thus can be used in a method of evaluating whether said subject suffering from NSCLC and not being treated yet will respond to a treatment of NSCLC comprising immunotherapy.
  • M s microvesicles
  • Extracellular vesicles (EVs) to which the MVs as large EVs (lEVs) belong to and which are used within the method of the invention, are shed in high amounts by tumor cells, such as NSCLC cells, making their detection in PB samples in general possible.
  • tumor cells such as NSCLC cells
  • the present inventors thus discovered that the abovementioned PD-L1 is a reliable tumor-related antigen for MVs secreted by such NSCLC cells for example which was detected on MVs from all NSCLC cell lines examined by the inventors.
  • said PD-L1 positive MVs detected in PB samples in the method of the invention were derived mainly from NSCLC cells and not from platelets or leukocytes.
  • MVs are easier to isolate and analyze with common diagnostic tools, such as flow cytometry, making them an ideal cancer biomarker.
  • protocols for small EVs (sEVs, exosomes) isolation and analysis are much more time consuming and are thus not suitable for daily clinical routine such as for the method of the present invention.
  • the detected PD-L1 levels on sEVs were much lower compared to the levels on MVs.
  • PD-L1 as reliable tumor-related antigen for MVs secreted in the context of NSCLC in a test PB sample of a subject already suffering from NSCLC, but who has not yet been treated, predicts a therapy response to NSCLC.
  • This new discovery was based on the fact that the detected level of PD-L1 positive MVs in the test PB sample obtained from said NSCLC subject is increased relative to corresponding levels of PD-L1 positive MVs in control PB samples obtained from subjects who also suffer from NSCLC, but who did not respond to the treatment applied to said subjects beforehand which comprised an immunotherapy.
  • Such subjects from whom the control PB samples are then used and the detected level of PD-L1 positive MVs in said test PB sample is compared to within the method of the invention refer to the control subjects and are classified as non-responders to a treatment of NSCLC comprising immunotherapy. Evaluating whether said subject being examined with the method of the present invention will be a responder to such treatment of NSCLC, which comprises an immunotherapy, is dependent on the requirement that said level of PD-L1 positive MVs is increased relative to corresponding levels of PD-L1 positive MVs in control PB samples obtained from subjects also suffering from NSCLC, but who were not a responder to the treatment of NSCLC which comprised an immunotherapy.
  • the method of the present invention makes it possible to evaluate - before a treatment of NSCLC begins, so already at the time point of the diagnosis itself- whether the NSCLC patient will respond to an immunotherapy, meaning without evaluating any further samples during the treatment (again).
  • the present invention comprises a method of evaluating whether a subject already suffering from NSCLC will respond to a treatment of NSCLC comprising immunotherapy, the method comprising i) determining a level of PD-L1 positive MVs in a test PB sample obtained from a subject suffering from NSCLC who has not yet been treated; and ii) evaluating whether said subject will be a responder to a treatment of NSCLC comprising immunotherapy, if said level of PD-L1 positive MVs is increased relative to corresponding levels of PD-L1 positive MVs in control PB samples obtained from subjects also suffering from NSCLC, but being non-responders to said treatment of NSCLC comprising immunotherapy.
  • the present invention envisages a data processing system comprising a processor configured to perform a method comprising the steps of i) obtaining a determined level of PD-L1 positive MVs in a test PB sample obtained from a subject suffering from NSCLC who has not yet been treated; and ii) evaluating whether said subject will be a responder to a treatment of NSCLC comprising immunotherapy, if said level of PD-L1 positive MVs is increased relative to corresponding levels of PD-L1 positive MVs in control PB samples obtained from subjects also suffering from NSCLC, but being non-responders to said treatment of NSCLC comprising immunotherapy.
  • Fig. 1 Flow cytometry identifies a panel of tumor antigens expressed on lEVs from NSCLC cell lines.
  • A+B Small EVs (sEVs) and large EVs (lEVs) from H2228 and H596 cells were characterized by transmission electron microscopy (A) and NTA (B).
  • C Western blot: Expression of common positive and negative EV markers for the two distinct EV populations.
  • D Analysis of lEVs by flow cytometry. Shown is a representative FSC vs SSC plot with the IEV gate used for analysis. PBS only and size bead controls were used to confirm the specificity of the selected gate.
  • E Flow cytometry: A two-fold dilution series of H2228-IEVs was prepared and the number of events in the IEV gate was recorded during 30 sec of measurement to exclude swarm detection.
  • Fig. 2 Tumor antigens are highly expressed on lEVs.
  • A+B Expression of the selected eight tumor antigens (A) as well as PD-L1 (B) was compared on the same amount of protein from cell lysate, lEVs or sEVs from selected NSCLC cell lines with high expression of the respective antigen. Actinin-4 was included to confirm successful separation from sEVs.
  • Fig. 3 NSCLC patients do not show differences in the number or size of lEVs in plasma compared to controls.
  • A TEM of lEVs and sEVs isolated from the plasma of a NSCLC patient.
  • B Representative NTA measurements from plasma-derived lEVs and sEVs.
  • C Common IEV markers and contaminants were analyzed on plasma-derived lEVs by western blot. Cell lysates from two NSCLC cell lines as well as a plasma sample are shown as controls. Equal protein amounts were loaded for all samples.
  • D NTA: Concentration and size of lEVs in plasma of healthy controls (CTL h ), non-cancer controls (CTL nc ) and NSCLC patients (median ⁇ 95%CI).
  • Fig. 4 Tumor antigen-loaded lEVs are elevated in the blood of NSCLC patients.
  • A The percentage of lEVs positive for the indicated tumor antigen was measured by flow cytometry in healthy controls (CTL h ), non-cancer controls (CTL nc ) and NSCLC patients. Boxes mark the 25-75 percentiles (line at median) and whiskers the 5-95 percentile.
  • B Western blot of EMMPRIN and PD-L1 expression in equal protein amounts of lEVs isolated from NSCLC patients and healthy controls. Lysates from H2228 and H596 cells as well as a plasma sample were included as controls.
  • C+D ROC curves were used to determine the discriminative power of significantly elevated IEV tumor antigens alone (C) or all six combined (D).
  • Fig. 5 PD-L1 is enriched on a population of CD62P7CD45" lEVs in the plasma of NSCLC patients.
  • A+B Flow cytometry: Expression of the selected tumor antigens on lEVs from platelets (A) or HDLM2 lymphoma cells (B).
  • CD62P was included as a marker for platelet- derived lEVs and CD45 for leukocyte-derived lEVs.
  • Right curve Antigen of interest
  • CD62P + and CD45 + lEVs The percentage of CD62P + and CD45 + lEVs was measured by flow cytometry in healthy controls and NSCLC patients. Boxes mark the 25-75 percentiles (line at median) and whiskers the 5-95 percentile.
  • D+E, PD-L1 + lEVs were analyzed for their expression of CD62P and CD45. Shown is one representative scatter plot of a healthy control and NSCLC patient (D). The number of PD-L1 + /CD62P7CD45‘ lEVs was counted in both groups (E). Boxes mark the 25-75 percentiles (line at median) and whiskers the 5-95 percentile.
  • Fig. 6 lEV-associated PD-L1 predicts therapy response in patients with absent and low tissue PD-L1 expression.
  • A Kaplan-Meier survival curves of NSCLC patients according to the number of tumor antigen-positive lEVs in blood.
  • B The level of PD-L1 -positive lEVs in blood of treatment-naive patents at initial diagnosis were correlated with response to therapy at the first staging CT after 3 months (R/NR 3 ) or 6 months (R/NR 6 ). Based on the staging CT, patients were stratified as responders (R) if they showed complete or partial response or stable disease and as non-responders (NR) if they showed signs of progressive disease.
  • R responders
  • NR non-responders
  • the upper panel comprises all patients independent of the choice of therapy, the lower panel displays only patients with immunotherapy in their therapeutic regimen.
  • C+D Correlation of tPD-L1 (C) and TPS (D) with the level of PD-L1 -positive lEVs in plasma.
  • E NSCLC patients were stratified according to their tissue PD-L1 (tPD-L1) expression. Levels of PD-L1 -positive lEVs before chemo-immunotherapy (CIT) alone or in combination with mono-immunotherapy (ICI) were compared in patients classified as R or NR at the first staging CT after 3 or 6 months of treatment.
  • CIT chemo-immunotherapy
  • ICI mono-immunotherapy
  • Fig. 7 lEVs as predicitive biomarkers in NSCLC.
  • A+B Kaplan-Meier survival curves of NSCLC patients according to their expression of tissue PD-L1 (tPD-L1) (A) or response to therapy (B).
  • tPD-L1 tissue PD-L1
  • B response to therapy
  • R responders
  • NR non-responders
  • C The level of lEVs carrying the indicated tumor antigens in blood of treatment-naive patents at initial diagnosis were correlated with response to therapy at the first staging CT after 3 months (R/NR 3 ) or 6 months (R/NR 6 ).
  • the term "at least" preceding a series of elements is to be understood to refer to every element in the series.
  • the term “at least one” refers to one or more such as one, two, three, four, five, six, seven, eight, nine, ten and more.
  • the term “about” means plus or minus 20%, preferably plus or minus 10%, more preferably plus or minus 5%, most preferably plus or minus 1%.
  • the method of the present invention is applied to evaluate whether a subject already suffering from NSCLC will respond to a treatment of NSCLC comprising immunotherapy.
  • Lung carcinomas are epithelial malignancies originating primarily in the lung.
  • Therapy-oriented guidelines differentiate between small cell lung carcinomas (SCLC) and NSCLC, and in the case of NSCLC further differentiation is made according to histological, genetic and immunohistochemical parameters.
  • the NSCLC of the present invention comprises, but is not limited to, squamous cell carcinoma (SCC), large cell carcinoma (LCC), adenocarcinoma (ADC), adenosquamous carcinoma, carcinomas with pleomorphic, sarcomatoid, or sarcomatous elements, carcinoid tumor, or salivary gland carcinoma.
  • SCC squamous cell carcinoma
  • LCC large cell carcinoma
  • ADC adenocarcinoma
  • adenosquamous carcinoma carcinomas with pleomorphic, sarcomatoid, or sarcomatous elements, carcinoid tumor, or salivary gland carcinoma.
  • the NSCLC is squamous cell carcinoma.
  • the squamous cell carcinoma is papillary, clear cell, small cell, or basaloid.
  • the NSCLC is adenocarcinoma.
  • the adenocarcinoma is acinar, papillary, bronchioloalveolar carcinoma (e.g., nonmucinous, mucinous, mixed mucinous and nonmucinous or indeterminate cell type), solid adenocarcinoma with mucin, adenocarcinoma with mixed subtypes, well-differentiated fetal adenocarcinoma, mucinous (colloid) adenocarcinoma, mucinous cystadenocarcinoma, signet ring adenocarcinoma, or clear cell adenocarcinoma.
  • the NSCLC is large cell carcinoma.
  • the large cell carcinoma is large-cell neuroendocrine carcinoma, combined largecell neuroendocrine carcinoma, basaloid carcinoma, lymphoepithelioma-like carcinoma, clear cell carcinoma, or large cell carcinoma with rhabdoid phenotype.
  • the NSCLC is carcinomas with pleomorphic, sarcomatoid, or sarcomatous elements.
  • the carcinoma with pleomorphic, sarcomatoid, or sarcomatous elements is carcinomas with spindle and/or giant cells, spindle cell carcinoma, giant cell carcinoma, carcinosarcoma, or pulmonary blastoma.
  • the NSCLC is salivary gland carcinoma.
  • the carcinoma of salivary-gland type is mucoepidermoid carcinoma or adenoid cystic carcinoma.
  • the NSCLC is adenosquamous carcinoma.
  • the NSCLC is carcinoid tumor.
  • the NSCLC is adenocarcinoma (ADC).
  • NSCLC is classified in four different tumor stages l-IV according to the Union of International Cancer Control (UICC) 8 th Edition.
  • UICC International Cancer Control
  • the NSCLC is a stage I tumor (stage IA (T1 , NO, MO) or stage IB (T2a, NO, MO)), a stage II tumor (stage IIA (T2b, NO, MO) and stage IIB (T1 , N1 , MO; T2a, N1 , MO; T2b, N1 , MO; T3, NO, MO)), a stage IIIA tumor (T1 , N2, MO; T2, N2, MO; T3, N1 , MO, or T4, NO or N1 , MO), a stage IIIB tumor (T1 , N3, MO; T2, N3, MO; T3 or T4, N2, MO), a stage IIIC tumor (T3 or T4, N3, MO) or a stage IV tumor (any T, any N, M1).
  • the NSCLC is a stage IV tumor.
  • the method as defined elsewhere herein comprises said subject being examined suffering from NSCLC stage IV according to the staging classification of UICC 8 th edition. In the majority of patients in stage IV the therapeutic intent is not curative. Stage IV divides into stage IVA and IVB.
  • Stage IVA refers to the oligometastatic stage e.g., with solitary adrenal, CNS, lung, or bone metastasis, whereas stage IVB comprises multiple metastasis in different organs.
  • stage IVA there can either be separate tumor nodule in a contralateral lung lobe, pleura with nodular involvement, malignant pleural effusion, and/or malignant pericardial effusion; or an isolated distant metastasis in an extrathoracic organ.
  • multiple distant metastasis (>1) in one or more different organs can be detected.
  • step i) of the method of the present invention or after obtaining a determined level of PD-L1 positive MVs in a test PB sample obtained from a subject suffering from NSCLC who has not yet been treated, the evaluation is performed which is based on the requirement as defined in more detail below that if said level of PD-L1 positive MVs is increased relative to corresponding levels of PD-L1 positive MVs in control PB samples obtained from subjects also suffering from NSCLC, but being a non-responder to the treatment of NSCLC comprising immunotherapy. Based on this requirement it can be evaluated that said subject being examined will respond to said treatment of NSCLC as defined herein which had also been applied to the control subjects.
  • the subject being examined and to whom the method of the invention is applied to is also diagnosed with NSCLC as defined herein.
  • Such subject suffering from and being diagnosed with NSCLC has however not yet been treated in any kind of way.
  • said subject has not yet received any kind of immunotherapy as defined elsewhere herein or any targeted therapy the skilled person is familiar with or any other treatment helpful when being diagnosed with NSCLC.
  • a subject may be a mammalian species such as a rabbit, a mouse, a rat, a Guinea pig, a hamster, a dog, a cat, a pig, a cow, a goat, a sheep, a horse, a monkey, an ape or a human.
  • the subject being used in the present invention is a human. More preferably, said subject is an adult. Preferably, said adult is older than 18 years.
  • Such subject may also be considered as “patient” when referring to a human, preferably to an adult.
  • the present invention may thus also comprise the method as defined herein, wherein the subject is a human or wherein the subject is an adult, or when the subject is a human, the human is preferably an adult.
  • the subject suffering from NSCLC which is examined with the method of the invention shows specific symptoms based on the different tumor stages of NSCLC as defined herein and is diagnosed with NSCLC based on the general diagnosing test(s) the skilled person is familiar with before applying the method of the present invention.
  • a subject suffering from NSCLC (also being diagnosed with NSCLC) refers to a NSCLC subject.
  • the method of the present invention then helps to clarify whether the subject suffering from NSCLC I being diagnosed with NSCLC will then respond to a certain treatment of NSCLC which comprises an immunotherapy which then clearly helps the subject to know whether such treatment would be worth trying. This approach then tremendously reduces the time of searching for the right treatment without the subject already exposing to all different treatments.
  • the term “will respond to a treatment” means that the subject suffering from NSCLC to whom then a certain treatment comprising an immunotherapy is applied to after the method of the invention has been performed, will respond to said NSCLC treatment comprising immunotherapy.
  • a non-response which would be death or a progressive disease (PD) according to the Response Evaluation Criteria in Solid Tumors (RECIST) criteria version 1.1 can thus be excluded when there will be a response to said NSCLC treatment.
  • a non-response to such treatment thus comprises death or PD according to such RECIST as defined elsewhere herein.
  • a response to such treatment comprises a complete response (CR) or a partial response (PR) or stable disease (SD) which are also based on radiographic images evaluated with the RECIST criteria as defined below.
  • measurable lesions such as the primary tumor or metastasis
  • measurable lesions may be considered which can be accurately measured in at least one dimension with longest diameter >20 mm using conventional techniques (such as CT, MRI) or >10 mm by spiral CT scan. Lesions with longest diameter ⁇ 20 mm measured with conventional techniques or ⁇ 10 mm with spiral CT scan are considered as non-measurable lesions.
  • All measurable lesions up to a maximum of 2 lesions per organ and 5 lesions in total, representative of all involved organs should be identified as the so-called target lesions and recorded and measured at baseline (at the time of diagnosis with NSCLC before the beginning of the treatment).
  • Target lesions should be selected on the basis of their size (lesions with the longest diameter) and their suitability for accurate repeated measurements (either by imaging techniques or clinically).
  • a sum of the longest diameter (LD) for all target lesions will be calculated and reported as the baseline sum LD.
  • the baseline sum LD will be used as reference by which to characterize the objective tumor response.
  • All other lesions should be identified as non-target lesions and should also be recorded at such baseline. Measurements of these lesions are not required, but the presence or absence of each should be noted.
  • the term “responder” refers to a subject who is evaluated by the method of the invention that s/he will respond to a treatment of NSCLC which is an immunotherapy. Such subject to whom such immunotherapy treatment of NSCLC will then be applied to after the method of the invention has been performed which identified the subject as a responder, will then later on as defined above be diagnosed with a CR, a PR or a SD based on radiographic images evaluated with the RECIST as defined herein.
  • the treatment of NSCLC comprising immunotherapy when evaluating with the method of the invention whether said subject suffering from NSCLC and to whom the method of the invention is applied to, will respond to, refers to a treatment of NSCLC comprising any immunotherapy the skilled person is familiar with.
  • the treatment of NSCLC comprising an immunotherapy comprises chemo-immunotherapy (CIT) or monoimmunotherapy (ICI).
  • the invention may also comprise the method as defined herein, wherein the treatment of NSCLC comprising an immunotherapy comprises CIT and/or ICI.
  • CIT refers to a combined therapy comprising chemotherapy elements (such as platinum-based chemotherapy) as well as immunotherapy elements (such as immune checkpoint inhibitors such as antibodies against PD-1 and PD-L1) which suits the subject being examined and to whom the method of the invention has been applied to.
  • Mono-immunotherapy (ICI) only comprises immunotherapy elements such as the application of immune checkpoint inhibitors such as antibodies against PD-1 and PD-L1.
  • CIT or ICI can preferably be applied right away to said subject. This may then depend on several factors being associated with the stage of the NSCLC disease, the general condition, the course of the disease, and/or the genetic predispositions of the subject.
  • step i) of the method of the present invention when a subject as defined herein is being examined and which already suffers from NSCLC, the following parameter (also called (bio)marker) needs to be determined in said obtained test PB sample of said subject, namely a level of PD-L1 positive MVs isolated from a test PB sample.
  • the present invention also comprises the method as defined herein comprising as step i) obtaining a determined level of PD-L1 positive MVs in a test PB sample obtained from a subject suffering from NSCLC who has not yet been treated.
  • detect or “detecting” can be used interchangeably with the term “determine” or “determining” as used herein.
  • the term “detect” or “detecting” as well as the term “determine” or “determining” when used herein in combination with the words “level” or “amount”, are understood to generally refer to a quantitative or a qualitative level.
  • detect when used in the context of detecting the level of PD-L1 , “detect”, “detecting”, “determine” or “determining” are understood to generally refer to a quantitative level. Accordingly, methods according to the invention that include a quantification of PD-L1 , means i.e. the level of PD-L1 expressing MVs.
  • the invention involves detection I determination of the “level”, i.e. (relative) amount of PD-L1 positive MVs.
  • the level of specific MVs may be expressed by the (relative) amount of all MVs being detected in the PB sample.
  • the term “PD-L1 positive MVs” refers to the MVs in the PB sample (for the test or the control) expressing PD-L1 which can be detected by applying a labelled binding partner for PD-L1 such as a fluorescently-labelled antibody against PD-L1 and then comparing the difference in the signal compared to a labelled isotype control binding partner. All MVs showing a stronger signal than the isotype control are considered positive.
  • PD-L1 also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1) is a protein that in humans is encoded by the CD274 gene and refers to an immune checkpoint protein.
  • PD-L1 is a surface tumor antigen which is significantly expressed on MVs in NSCLC subjects (see Figure 4).
  • said MV marker helps to identify a subject which suffers from NSCLC from a healthy subject rendering PD-L1 on MVs a diagnostic biomarker for the detection of NSCLC in a subject (see also Figure 5).
  • MVs belong to extracellular vesicles (EVs). EVs are released by all living cells and mediate intercellular communication by carrying proteins, lipids, and nucleic acids (e.g. DNA, RNA) from the secreting to the surrounding cells (Yanez-M6 et al, 2015, J Extracell Vesicles 4). So far, two distinct EV populations have been described: small EV (sEV, exosomes) with a diameter between about 50 to about 150 nm, which were isolated from serum in e.g. Zhang et al. 2022; Tissue and Cell, vol. 79 (see pp. 2, point 2.1 , 2.2, and Figure 1 B) or in Zhang et al.
  • sEV, exosomes small EV with a diameter between about 50 to about 150 nm
  • sEV small EV
  • sEV are formed inside the cell by inward budding of endosomal membranes, while IEV bud directly from the plasma membrane.
  • NSCLC cells for example secrete MVs which carry tumor-related antigens, such as PD-L1.
  • Various techniques can be used to isolate the MVs in whole or in part from the PB sample(s).
  • the MVs can be isolated via size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, affinity capture, immunoassay, microfluidic separation, flow cytometry or combinations thereof, preferably flow cytometry as described in the Example section.
  • Such MVs e.g. secreted by NSCLC cells may carry besides PD-L1 the typical microvesicle markers such as RGAP1, ACTN4 and/or ACTIN, as well as the diagnostic marker EMMPRIN, whereas increased levels of such diagnostic markers were discovered by the inventors on MVs from NSCLC subjects (see Figure 4).
  • PD-L1 has been discovered as a biomarker expressed on MVs which have been secreted from all NSCLC cell lines being detected and further having predictive and prognostic value why the method of the invention focuses on determining the level of PD-L1 on MVs as only biomarker used.
  • step ii) of the method of the invention it has to be determined whether said level of PD-L1 positive MVs is increased relative to corresponding levels of PD-L1 positive MVs in control PB samples obtained from subjects also suffering from NSCLC, who did and do not respond to a treatment of NSCLC comprising immunotherapy. Then, if such requirement of step ii) is fulfilled it is indicative that said subject already suffering from NSCLC will respond to a NSCLC treatment comprising immunotherapy as defined herein.
  • step i) of the method of the invention If the abovementioned parameter, the level of PD-L1 positive MVs, is determined in said obtained PB sample from said subject being examined (see step i) of the method of the invention), but the requirement for said parameter in accordance with step ii) of the method is not fulfilled, meaning that said level of PD-L1 positive MVs is not increased relative to corresponding levels of PD-L1 positive MVs in control PB samples as defined herein, the subject most likely will not respond (not a CR, PR or SD) to a treatment of NSCLC comprising immunotherapy, thus being a non-responder to such treatment as defined herein. In these cases, a treatment of NSCLC comprising immunotherapy may not be performed on said subject.
  • test PB sample is being obtained as known to the person skilled in the art from said subject as defined elsewhere herein. Such sample being obtained from said subject as defined herein is thus examined for said particular parameter of step i) of the method of the present invention.
  • the PB sample as used in the method of the present invention is, essentially consists of, or includes PB from such subject suffering from NSCLC.
  • the term “essentially consists of” is understood to allow the presence of additional components in said PB sample or a composition that do not affect the properties of the sample. Examples of additional components may include, but are not limited to certain types of media, or any type of buffer.
  • the sample used in the method of the present invention is a PB sample dependent on the fact that the parameter is determined in step i) of the method of the present invention as defined elsewhere herein, since the level of PD-L1 positive MVs in PB demonstrates an increased probability of survival than it was the case in a tumor tissue (see Figure 6).
  • PB is the flowing, circulating blood of the body. It is composed of erythrocytes, leukocytes and thrombocytes as well as NSCLC cells, all secreting MVs as defined herein.
  • said PB sample may refer to a “test” PB sample. This means that said sample obtained from said subject suffering from NSCLC according to the present invention is analyzed I examined by determining said level as defined by step i) and then determining whether said level of PD-L1 positive MVs in the test PB sample is increased relative to corresponding levels of PD-L1 positive MVs in control PB samples as defined elsewhere herein.
  • the control PB samples as comparison for whether said level of PD-L1 positive MVs in the test sample as defined in step i) of the method of the present invention is increased are taken from a patient cohort who also suffer from NSCLC but when being treated with a treatment of NSCLC comprising immunotherapy (wherein the cohort was preferably treated with CIT and ICI) beforehand, said subjects in the cohort did not respond to said treatment when being evaluated as defined above which made them non-responders to said immunotherapy treatment.
  • this patient cohort may also be considered as not being healthy, since they also suffer from NSCLC and are diagnosed with NSCLC based on general diagnosing test(s) also known to a person skilled in the art.
  • non- responder(s) refer(s) to control subject(s) who received a treatment of NSCLC comprising immunotherapy (preferably CIT and ICI) and who were then diagnosed after a certain amount of time (preferably after 3 or 6 months) after having received said treatment with a progressive disease (PD) or had succumbed to their disease according to the RECIST as defined herein. If there is a PD there may be at least a 20% increase in the sum of the LD of target lesions, taking as reference the smallest sum LD recorded since the treatment started or the appearance of one or more new lesions. Additionally in PD, there may be an appearance of one or more new lesions and/or unequivocal progression of existing non-target lesions.
  • the control subjects the PB control samples have been taken from as comparison may refer to a cohort of subjects comprising PD subjects as well as deceased subjects.
  • the methods of the invention may include providing a sample from the subject or obtaining said sample from the subject.
  • the PB sample may be obtained by venous puncture following typical procedures known to a person skilled in the art and then collected in particular tubes for said further determination of said particular parameter such as comprising the isolation of one or more MVs from said PB sample as also described in the Example section for the determination of said PD-L1 marker on said one or more MVs.
  • the sample may have been taken at any desired point in time before carrying out the method of the invention. Generally, a time interval between taking the sample and carrying out the method of the invention is selected to allow analysis of the MVs.
  • the sample may have been taken on the same or on the previous day, such as about 12 hours, about 6 hours, about 4 hours, about 2 hours or less before the method of the invention is being carried out.
  • the sample is taken about 2 hours before the method of the invention is carried out.
  • said sample preferably needs to be processed within about 2 hours after having obtained said sample.
  • the PB sample may first be cleared of blood cells and cell debris by centrifugation.
  • the collected plasma can then be stored at about -20°C for several months before proceeding with the isolation and analysis of the MVs.
  • At least about 10 ml PB sample may be used from a subject being examined.
  • the term “relative to” or just “to” as used within the present invention generally means comparing “A” to “B”.
  • a level of PD-L1 positive MVs being determined in a test PB sample being obtained from a subject to be examined is then compared to the levels of PD-L1 positive MVs in control PB samples taken from a cohort of subjects which also suffer from NSCLC, so that the levels of PD-L1 positive MVs in the control PB samples can be considered as being corresponding to the level of PD-L1 positive MVs in the test PB sample obtained from said subject.
  • this comparison is carried out by comparing the parameters, whereas p-values are preferably calculated by the Mann-Whitney II test as described in the Example section.
  • the term “in comparison to” can be used interchangeably with the term “relative to” or just “to”.
  • ROC Receiver Operating Characteristic
  • a well responding subpopulation a test patient suffering from NSCLC who is a responder
  • a poorly responding subpopulation a control patient suffering from NSCLC but being a non-responder
  • From the frequency of test results level of PD-L1 positive MVs according to the method of the invention, see step i)) among such patients (control (control patient suffering NSCLC but being a non-responder) vs. test (a test patient suffering from NSCLC who is a responder)) based on gold standard, one can derive the probability of a positive test result for patients with disease plus being a responder (i.e.
  • conditional probability of correctly identifying the diseased responder subjects by testsensitivity or true positive rate (TPR)) and the probability of negative test results for patients with disease but being non-responders i.e. the conditional probability of correctly identifying diseased non-responder subjects by test -specificity or true negative rate (TNR)
  • TPR true positive rate
  • TNR true negative rate
  • the term “increased” as used herein and throughout the entire description, relates to an increased level of PD-L1 on MVs concerning the quantity of the PD-L1 positive MVs in view of their number, such term as known to the skilled person.
  • any elevation or decrease of the PD-L1 MV number can be considered as a relevant increase or decrease.
  • any recognizable alteration in form of a higher or a lower PD-L1 MV number in relation to corresponding levels of PD-L1 MVs in said control samples also concerning the quantity of the PD-L1 MVs in view of their number may be considered as an increase or decrease.
  • the increase or decrease of a PD-L1 MV number may be determined using flow cytometry as defined elsewhere herein.
  • an increase or decrease may be at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, or even 100%.
  • an increase or decrease of at least about 30% may already provide a significant statement.
  • the present invention also comprises the method as defined elsewhere herein, wherein said level of PD-L1 positive MVs is determined only if a level of PD-L1 positive cancer and/or immune cells in a cancerous tissue sample has been pre-determined, said tissue sample being obtained from said same subject suffering from NSCLC who has not yet been treated.
  • This ’’pre- determining” step may occur before step i) of the method of the invention on the same subject who comes in for being evaluated with the method of the invention. This step may be seen as refinement to the method of the invention which will be explained in more detail below.
  • the present invention may also comprise a method of evaluating whether a subject already suffering from NSCLC will respond to a treatment of NSCLC, the method comprising i) determining a level of PD-L1 positive cancer and/or immune cells in a cancerous tissue sample obtained from a subject suffering from NSCLC who has not yet been treated or obtaining a determined level of PD-L1 positive cancer and/or immune cells in a cancerous tissue sample; ii) determining a level of PD-L1 positive MVs in a test PB sample obtained from the same subject mentioned under step i) or obtaining a determined level of PD-L1 positive MVs in a test PB sample obtained from the same subject mentioned under step i); and then evaluating whether said subject will be a responder to a treatment of NSCLC comprising immunotherapy, if said level of PD-L1 positive MVs is increased relative to corresponding levels of PD-L1 positive MVs in control PB samples obtained from subjects also suffering from NSCLC,
  • not the level of PD-L1 positive MVs in PB is determined, but the level of PD-L1 positive cancer and/or immune cells in a cancerous tissue sample. It may also be comprised herein that in said particular step the level of PD-L1 on immune cells in a cancerous tissue sample is determined in addition to the level of PD-L1 on cancer cells. All definitions drawn to such level of PD-L1 positive cancer cells may also be applicable to a level of PD-L1 positive immune cells.
  • a cancerous tissue sample is a sample taken I obtained as defined herein from a primary tumor where the cellular growth is uncontrollable and it starts to spread out into the body, thus being referred to - also according to general knowledge - as a cancerous tumor (short cancer).
  • Such sample may thus also refer to a cancer tissue sample.
  • Such cancerous tissue sample may be a cancerous lung tissue sample (lung cancer tissue sample which refers to a tissue sample taken I obtained from a lung cancer) or a tissue sample of a metastasis (such as a brain metastasis) or a combination thereof.
  • lung cancer tissue sample which refers to a tissue sample taken I obtained from a lung cancer
  • tissue sample of a metastasis such as a brain metastasis
  • Such tissue sample may be obtained from the subject as defined herein by standard medically indicated biopsies or surgery known to the skilled person.
  • the present invention may thus comprise the method as defined elsewhere herein, wherein said cancerous tissue sample is a lung tissue sample or a tissue sample of a metastasis or a combination thereof.
  • the cancer cells within said sample may refer to NSCLC cells meaning with regard to a lung tissue sample they were healthy cells in the lung which then changed and grew out of control and formed such primary cancerous lung tumor (lung cancer) the sample is now taken from.
  • a tissue sample of a metastasis said cells were shed by the growing (e.g. lung) tumor and carried away via the blood or the lymph to a distant part of the body (away from the primary tumor) and formed such metastasis the sample is now taken from.
  • said immune cells of said cancerous tissue sample any immune cell is meant such as lymphocytes, neutrophils and monocytes/macrophages.
  • step i) and ii) will be performed preferably, if i) said level of PD-L1 positive cancer cells in said tissue sample is absent or low relative to all cancer cells in said tissue sample and/or if ii) said level of PD-L1 positive immune cells in said tissue sample is absent or low relative to all immune cells in said tissue sample.
  • the invention may comprise the method as defined elsewhere herein, wherein said level of PD-L1 positive MVs is only determined if such level of PD-L1 positive cancer cells in said cancerous tissue sample is absent or low relative to all cancer cells in said tissue sample and/or ii) if such level of PD-L1 positive immune cells in said cancerous tissue sample is absent or low relative to all immune cells in said tissue sample.
  • An absent level of PD-L1 on cancer cells in said tissue sample refers to the level being below 1% relative to all cancer cells in said tissue sample.
  • An absent level of PD-L1 on immune cells in said tissue sample refers to the level being below 1% relative to all immune cells in said tissue sample.
  • the present invention therefore may envisage the method as defined elsewhere herein, wherein i) an absent level of PD-L1 on cancer cells in said tissue sample is below 1% relative to all cancer cells in said tissue sample and/or ii) an absent level of PD-L1 on immune cells in said tissue sample is below 1% relative to all immune cells in said tissue sample.
  • the term “below” means not including 1%, but any percentage number independent of the decimal place which is below the number 1 and above or being 0, meaning that 0% refers to all cancer / immune cells being detected as defined herein do not express PD-L1, so no PD-L1 level.
  • a low level of PD-L1 on cancer cells in said tissue sample refers to the level being within a range of about 1% to about 49% relative to all cancer cells in said tissue sample.
  • a low level of PD-L1 on immune cells in said tissue sample refers to the level being within a range of about 1% to about 49% relative to all immune cells in said tissue sample.
  • the present invention therefore may envisage the method as defined elsewhere herein, wherein i) a low level of PD-L1 on cancer cells in said tissue sample is within a range of about 1% to about 49% relative to all cancer cells in said tissue sample and/or ii) a low level of PD-L1 on immune cells in said tissue sample is within a range of about 1% to about 49% relative to all immune cells in said tissue sample.
  • a low level of PD-L1 on cancer cells in said tissue sample is within a range of about 1% to about 49% relative to all immune cells in said tissue sample.
  • the level of A (relative) to “the level of B”, generally means comparing “A” to “B”.
  • the level of PD-L1 positive cancer I PD-L1 positive immune cells in said tissue being determined is then compared to all of the cancer / immune cells in said tissue sample, whereby preferably at least 100 cancer cells I at least 100 immune cells within said sample as defined herein may be analyzed for the determination. No control sample is needed herein as it is done so when determining the level of PD-L1 positive MVs in a test PB sample.
  • the term “above” means not including 49%, but any percentage number independent of the decimal place which is above the number 49 and below or being 100, meaning that 100% refers to all cancer cells I to all immune cells being detected as defined herein which express PD-L1.
  • a subject according to the invention may be examined first via said PD-L1 level in said tissue (tPD-L1) as defined above before performing steps i) and ii) of the method of the invention and the level of PD-L1 positive cancer cells and/or the level of PD-L1 positive immune cells in said cancerous tissue sample may be absent or low as defined above, the evaluation whether said subject is a responder to said treatment of NSCLC comprising immunotherapy according to the method of the invention is even more concrete.
  • the subject can even more significantly be evaluated as a responder to said treatment of NSCLC comprising immunotherapy such as CIT and/or ICI as defined herein so that said subject can be treated with an immunotherapy such as CIT and/or ICI.
  • the predictive power of the biomarker of the method of the invention is significantly pronounced, when the level of PD-L1 positive cancer cells and/or the level of PD-L1 positive immune cells in said cancer tissue sample is determined as being absent or low as defined herein.
  • the term “PD-L1 positive cancer or immune cells” refers to said cells in the cancerous tissue sample as defined herein expressing PD-L1 which can be detected by applying a labelled binding partner for PD-L1 such as an antibody against PD-L1 linked to an enzyme and then comparing the difference in the signal (e.g.
  • the pre-determined step as mentioned above may be included before performing steps i) and ii) of the method of the invention and the level of PD-L1 positive cancer cells and/or the level of PD-L1 positive immune cells in said cancerous tissue sample may be absent or low as defined above and the level of PD-L1 positive MVs is then not increased relative to corresponding levels of PD-L1 positive MVs in control PB samples obtained from subjects also suffering from NSCLC, but being non-responders to said treatment of NSCLC comprising immunotherapy, said subject will not be evaluated as a responder to said treatment of NSCLC comprising immunotherapy such as CIT and/or ICI (but as a nonresponder) so that said subject would not be treated with any immunotherapy such as CIT and/or ICI.
  • said level of PD-L1 positive MVs in said PB sample is detected I determined using flow cytometry.
  • Flow cytometry-based analysis is typically combined with optical detection to identify and classify cells, or in this case MVs. This allows speed, sensitivity/specificity, and a non-invasive nature of the technique.
  • labeled binding partners such as fluorescently-labeled antibodies are used, which are compounds that bind to specific structures or molecules on the surface or within MVs such as PD-L1. Such labeled binding partners are introduced into the mixture of MVs. It is envisaged that flow cytometry is combined with immunofluorescence.
  • the level of MVs defined above may be determined by detecting the specific surface marker PD-L1 on the surface of the MVs by using flow cytometry being combined with immunofluorescence. Therefore, in the present invention PD-L1 may be detected on the surface of the MVs in the PB samples where the MVs have been isolated from as defined elsewhere herein, using a flow cytometry based analysis.
  • the invention envisages that the detection of PD-L1 on MVs can be carried out in a single step or can be carried out in more than one step, e.g. two steps, three steps or four steps. In preferred embodiments, the detection is carried out in a single step.
  • a preferred method for the detection of the level of PD-L1 as defined above is flow cytometry. The skilled person in the art knows that if detection of the biomarker is to be carried out in more than one step, more than one sequences of steps can be selected. It is understood that the skilled person is able to recognize suitable sequences of steps for identifying the MVs of interest, namely the ones expressing PD-L1.
  • the steps may partly involve enriching and/or isolating subpopulations.
  • the skilled person is able to recognize suitable sequences of steps and can judge whether those steps would reasonably involve an enrichment and/or isolation step for identifying the MVs of interest.
  • Flow cytometry is a technique enabling counting, examining, and sorting microscopic particles such as biological cells, MVs suspended in a stream of fluid. It allows a simultaneous multi-parametric analysis of the physical and chemical characteristics of single events flowing through an optical detection device.
  • An illustrative example of a well-established flow cytometry based analysis in the art is FACS.
  • FACS allows sorting a heterogeneous mixture of cells I MVs into a plurality of containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. Thereby, FACS allows the sorting of subpopulations of cells I MVs of interest and their further use in in vitro and in vivo assays.
  • FACS is often used in combination with monoclonal immunoglobulins as a reagent to detect cells I MVs as having a particular antigen, indicative of an expressed protein.
  • Fluorescent signals used in flow cytometry are typically fluorescently-labeled antibody preparations or fluorescently-labeled ligands for binding to antibodies or other antigen-, epitope- or ligand-specific agent, such as with biotin/avidin binding systems or fluorescently-labeled and optionally addressable beads (e.g. LUMINEX® microspheres).
  • any desired detectable marker or combination of detectable markers can be detected by the optics of a flow cytometer.
  • Current three-laser “multidimensional”, flow cytometry devices enable up to 23 simultaneous single-cell measurements, such as two light scatter detectors and 21 fluorescence plus forward detectors allowing for example the detection of fluorescent surface/intracellular markers.
  • Current three- laser, “multidimensional”, FACS machines if used as an illustrative example of flow cytometry, enable up to 14 simultaneous single-cell measurements, such as two light scatter detectors and 12 fluorescence plus forward detectors allowing for example the detection of fluorescent surface/intracellular markers.
  • the three lasers of a flow cytometry device may be a krypton laser operating at 407 nm, an argon laser operating at 488 nm, and a dye laser operating at 595 nm.
  • a krypton laser operating at 407 nm
  • an argon laser operating at 488 nm
  • a dye laser operating at 595 nm.
  • devices with seven lasers and about 70 channels i.e. so-called spectral flow-cytometer, whereas about 40 channels may be realistic to use, which fall under the term “multidimensional” flow cytometers which may be used by the present invention.
  • the flow cytometry technique has been used extensively in relation to antigens expressed on the surface of MVs. Accordingly, the technique not only allows detecting the presence of PD-L1 on the surface of the MVs, but also detecting the presence of RNA or DNA within the MV, for example RNA encoding PD-L1. Therefore, flow cytometry according to the invention can also be used to determine the amount of nucleic acid formation from the genes, which encode PD-L1 on MVs of the sample from the subject.
  • flow cytometry technique is used for detecting the level of PD-L1 on MVs isolated from said PB sample, which demonstrates a certain strength of a signal being measured.
  • the present invention may comprise the method as defined elsewhere herein, wherein determining the level of PD-L1 positive MVs in said PB sample obtained from said subject being examined according to the present invention comprises measuring PD-L1.
  • the present invention may also comprise the method as defined herein, wherein said level of PD-L1 positive cancer cells and/or said level of PD-L1 positive immune cells in said cancerous tissue sample is detected using immunohistochemistry.
  • Immunohistochemistry is a laboratory method that also uses antibodies to check for certain markers, such as PD-L1 in a tissue sample (so a PD-L1 antibody).
  • the antibodies are usually linked to a label, such as an enzyme or a fluorescent dye. After the antibodies bind to the marker, according to the invention to PD-L1 being expressed on said cancer and/or immune cells, in the tissue sample, the enzyme or dye is activated, and the marker can then be seen under a microscope.
  • the present invention may also comprise the method as defined elsewhere herein, wherein said level of PD-L1 positive cancer cells and/or said level of PD-L1 positive immune cells in said cancerous tissue sample obtained from said subject being examined according to the present invention has been pre-determined by measuring PD-L1 , in particular using immunohistochemistry.
  • measuring e.g. PD-L1 refers to measuring the signal of detectable PD-L1 , which is attached to a binding partner as defined herein that recognizes the specific surface marker e.g. PD-L1 expressed by the specific MVs when using flow cytometry or that recognizes the specific marker e.g. PD-L1 expressed on cancer and/or immune cells in said tissue sample when using immunohistochemistry as the procedure is known to the person skilled in the art.
  • said signal may then be converted by the process called gating, which is known to a person skilled in the art, which then demonstrates the level (or relative amount) of MVs being detected.
  • the gating comprises among other factors measuring of distinct cell I MV populations using forward scatter (FSC) and side scatter (SSC).
  • FSC forward scatter
  • SSC side scatter
  • the differences between the different MV populations are based on their marker expression (e.g. PD-L1).
  • the measurement used is generally selected to be of a sensitivity of detection that allows detection of PD-L1 MVs in the range of a selected threshold value, in particular of a sensitivity of detection that allows determining whether PD-L1 MVs are above the threshold.
  • a binding partner of PD-L1 may be used, preferably linked to an excitable label such as a fluorescent dye.
  • a binding partner of PD-L1 has a detectable affinity and specificity for PD-L1.
  • binding is considered specific when the binding affinity is higher than 10' 6 M.
  • a binding partner of PD-L1 has in some embodiments an affinity of about 10' 8 M or higher, or of about 10' 9 M or higher.
  • Identification of PD-L1 either on MVs or in the tissue may be carried out using spectroscopic, photochemical, photometric, fluorometric, radiological, enzymatic or thermodynamic means.
  • Identification and enrichment or isolation of PD-L1 may likewise be carried out by using a suitable binding partner of PD-L1 (on MVs or on cells in the tissue).
  • a suitable binding partner of PD-L1 on MVs or on cells in the tissue.
  • the present invention may comprise the method as defined elsewhere herein, wherein a binding partner for PD-L1 is used.
  • Immunodetection being used in flow cytometry and/or in immunohistochemistry is generally achieved using a binding partner, which is linked to, or includes, a detectable label such as a fluorescent dye with regard to flow cytometry or such as an enzyme with regard to immunohistochemistry.
  • a binding partner of PD-L1 f.e. may be used in combination with a detectable label or the binding partner is functionally linked to a detectable label such as a fluorescent dye with regard to flow cytometry for example.
  • a respective binding partner of PD-L1 used in the method of the present invention may be an immunoglobulin or a fragment thereof, or a proteinaceous binding molecule with immunoglobulin-like functions.
  • An example of a proteinaceous binding molecule with immunoglobulin-like functions is a mutein based on a polypeptide of the lipocalin family (WO 03/029462, Beste et al., Proc Nat. Acad Sci 1999; 96:1898-1903).
  • Lipocalins such as the bilin binding protein, the human neutrophil gelatinase-associated lipocalin, human Apolipoprotein D or glycodelin, possess natural ligandbinding sites that can be modified so that they bind to selected small protein regions known as haptens.
  • Other proteinaceous binding molecules are the so-called glubodies (see e.g. international patent application WO 96/23879 or Napolitano et al., Chemistry & Biology 1996; 3(5): 359-367), proteins based on the ankyrin scaffold (Mosavi et al., Protein Science 2004; 13(6): 1435- 1448) or crystalline scaffold (e.g.
  • An immunoglobulin or a proteinaceous binding molecule with immunoglobulin-like functions as defined herein may be PEGylated or hyperglycosylated if desired.
  • a proteinaceous binding molecule with immunoglobulin-like functions is a fusion protein of one of the exemplary proteinaceous binding molecules above and an albumin-binding domain, for instance an albumin-binding domain of streptococcal protein G.
  • a proteinaceous binding molecule with immunoglobulin-like functions is a fusion protein of an immunoglobulin fragment, such as a single-chain diabody, and an immunoglobulin binding domain, for instance a bacterial immunoglobulin binding domain.
  • An immunoglobulin used in the method of the invention may be an antibody.
  • an immunoglobulin fragment thereof may be an antibody fragment having antibody activity. It generally contains an antigen binding or variable region.
  • Examples of (recombinant) antibody fragments are immunoglobulin fragments such as Fab fragments, Fab’ fragments, Fv fragments, single-chain Fv fragments (scFv), diabodies or domain antibodies (Holt, L.J., et al., Trends Biotechnol. (2003), 21, 11 , 484-490).
  • a suitable antibody may in some embodiments also be a multispecific antibody that includes several immunoglobulin fragments.
  • An immunoglobulin may be monoclonal or polyclonal.
  • polyclonal refers to immunoglobulins that are heterogenous populations of immunoglobulin molecules derived from the sera of animals immunized with an antigen or an antigenic functional derivative thereof.
  • polyclonal immunoglobulins one or more of various host animals may be immunized by injection with the antigen.
  • Various adjuvants may be used to increase the immunological response, depending on the host species.
  • “Monoclonal immunoglobulins”, also called “monoclonal antibodies”, are substantially homogenous populations of immunoglobulins to a particular antigen. They may be obtained by any technique which provides for the production of immunoglobulin molecules by continuous cell lines in culture.
  • Monoclonal immunoglobulins may be obtained by methods well known to those skilled in the art (see for example, Kohler et al., Nature (1975) 256, 495-497, and U.S. Patent No. 4,376,110).
  • An immunoglobulin or immunoglobulin fragment with specific binding affinity only for said particular biomarker as defined elsewhere herein can be isolated, enriched, or purified from a prokaryotic or eukaryotic organism. Routine methods known to those skilled in the art enable production of both immunoglobulins or immunoglobulin fragments and proteinaceous binding molecules with immunoglobulin-like functions, in both prokaryotic and eukaryotic organisms.
  • an immunoglobulin may be isolated by comparing its binding affinity to a protein of interest, e.g. PD-L1 , with its binding affinity to other polypeptides.
  • Humanized forms of the antibodies of the present invention may be generated using one of the procedures known in the art such as chimerization or CDR grafting. In general, techniques for preparing monoclonal antibodies and hybridomas are well known in the art. Any animal such as a goat, a mouse or a rabbit that is known to produce antibodies can be immunized with the selected polypeptide, e.g. PD-L1. Methods for immunization are well known in the art. Such methods include subcutaneous or intraperitoneal injection of the polypeptide.
  • the amount of polypeptide used for immunization and the immunization regimen will vary based on the animal which is immunized, including the species of mammal immunized, its immune status and the body weight of the mammal, as well as the antigenicity of the polypeptide and the site of injection.
  • a detectable label may be coupled to a binding partner of PD-L1.
  • a respective detectable label which may be coupled to a binding partner of PD-L1 as defined herein, may be an excitable fluorescent dye, a radioactive amino acid, a fluorescent protein, an enzyme, a hapten, a digoxigenin, a biotin, a metal complex, or metal and colloidal gold.
  • a suitable fluorescent dye examples include, but are not limited to, Krome Orange (KrO), fluorescein (FITC), fluorescein isothiocyanate, 5,6-carboxymethyl fluorescein, Cascade Blue®, Oregon Green®, Texas red, nitrobenz-2-oxa-1 ,3-diazol-4-yl, coumarin, dansyl chloride, rhodamine, amino-methyl coumarin, DAPI, Eosin, Erythrosin, BODIPY®, pyrene, lissamine, xanthene, acridine, a fluorescent brightener , an oxazine, phycoerythrin, a Cy dye such as Cy3, Cy3.5, Cy5, Cy5PE, Cy5.5, Cy7, Cy7PE or Cy7APC, an Alexa dye such as Alexa 647, Alexa 750 or Alexa 700, and NBD (Naphthol basic dye).
  • KrO Krome Orange
  • a suitable fluorescent protein examples include, but are not limited to, EGFP, emerald, EYFP, a phycobiliprotein such as phycoerythrin (PE) or allophycocyanin (APC), Monomeric Red Fluorescent Protein (mRFP), mOrange, mPlum and mCherry.
  • a reversibly photoswitchable fluorescent protein such as Dronpa, bsDronpa and Padron may be employed (Andresen, M., et al., Nature Biotechnology (2008) 26, 9, 1035).
  • suitable enzymes alkaline phosphatase, soybean peroxidase, or horseradish peroxidase may serve as a few illustrative examples.
  • tandem conjugates such as PE-Cy5.5 or PE-Cy7 may also be used.
  • a detectable label being coupled to a binding partner as defined herein may include a “fluorescently labelled binding partner”.
  • the subject-matter of the defined method steps of the present invention may also fully be carried out by computer program instructions running on means which, in the context of the invention, provide generic data processing functions. Such means may, for example, be embedded in a personal computer, smartphone, printer.
  • a computer-implemented invention may therefore be one which involves the use of a computer, computer network or other programmable apparatus, where one or more features are realized wholly or partly by means of a computer program.
  • the present invention comprises a data processing system comprising a processor configured to perform a method comprising the steps of obtaining a determined level of PD-L1 positive MVs in a test PB sample obtained from a subject suffering from NSCLC who has not yet been treated; followed by evaluating whether said subject will be a responder to a treatment of NSCLC comprising immunotherapy, if said level of PD-L1 positive MVs is increased relative to corresponding levels of PD-L1 positive MVs in control PB samples obtained from subjects also suffering from NSCLC, but being non-responders to said treatment of NSCLC comprising immunotherapy.
  • said specific level of PD-L1 positive MVs as defined above may be detected in a test PB sample obtained from a subject suffering from NSCLC using flow cytometry, in particular using a flow cytometry device.
  • the detected level of PD-L1 positive MVs being expressed in terms a decimal value or a percentage as described elsewhere herein may be entered into said data processing system comprising a processor which is configured to perform the abovementioned steps.
  • the step of obtaining said determined level as defined above and below may be an optional step.
  • obtaining I obtain a determined level of PD-L1 positive MVs means that the data for the determined level (in percentage) of MVs being detected in a flow cytometry device as defined elsewhere herein is entered into the data processing system by any way known to a person skilled in the art.
  • the step of comparing the detected level of PD-L1 positive MVs to the corresponding levels in the control samples for said particular evaluation is then performed as defined elsewhere herein within the processor.
  • the data processing system as defined herein may also comprise an additional step of obtaining - before the step of obtaining a determined level of PD-L1 positive MVs - i) a determined level of PD-L1 positive cancer cells and/or ii) a determined level of PD-L1 positive immune cells in a cancerous tissue sample obtained from the same subject being examined suffering from NSCLC who has not yet been treated.
  • the data for the determined level of PD-L1 positive cancer cells and/or the determined level of PD-L1 positive immune cells may be entered into the data processing system by any way known to a person skilled in the art with regard to the step of obtaining said level of PD-L1 positive cancer cells and/or said level of PD-L1 positive immune cells as defined herein. All definitions and disclosure regarding said tPD-L1 step as set forth under the method of the invention may also be applicable to the computer-implemented features defined herein and vice versa. [0086] A more meaningful characterization of modern digital data processing systems is the functional classification as either computational or input/output (“I/O”) oriented.
  • I/O input/output
  • a data processing system of the present invention may refer to a programmable or programmed device I apparatus, where one or more features are realized wholly or partly by means of a computer program.
  • a data processing system may include, but are not limited to, a computer, a smartphone, a tablet, or a chip.
  • processor may refer to one functional element being a physical unit of a data processing system, which is configured by a computer program as defined elsewhere herein to perform the specified steps mentioned above.
  • the present invention further comprises the interaction between the data processing steps and other technical means such as a flow cytometry device.
  • a flow cytometry device capable of detecting the level of PD-L1 positive MVs in a test PB sample obtained from a subject suffering from NSCLC who has not yet been treated, comprising the data processing system as defined above.
  • Said specific PD-L1 level may be detected by using a flow cytometry device, preferably using flow cytometry analysis being combined with immunofluorescence as described elsewhere herein.
  • a flow cytometry device as defined above may be a FACS.
  • said flow cytometry such as for example FACS may be connected to a computer, a tablet, a smartphone or any other technical device I apparatus being used as a further means for performing said flow cytometry analysis.
  • the present invention also envisages a computer program comprising instructions to cause the data processing system as defined above or the flow cytometry device as defined above to execute the steps of obtaining a determined level of PD-L1 positive MVs in a test PB sample obtained from a subject suffering from NSCLC who has not yet been treated and then evaluating whether said subject will be a responder to a treatment of NSCLC comprising immunotherapy, if said level of PD-L1 positive MVs is increased relative to corresponding levels of PD-L1 positive MVs in control PB samples obtained from subjects also suffering from NSCLC, but being non-responders to said treatment of NSCLC comprising immunotherapy.
  • the step of obtaining said determined level of PD-L1 positive MVs as defined above may be an optional step. Everything being defined above for said data processing system may apply mutatis mutandis to the corresponding computer program as defined herein.
  • a computer program as mentioned above may refer to a program listing written in a programming language to implement an algorithm, and to binary code loaded in a computer-based apparatus encompassing the accompanying documentation.
  • a computer program as defined above may include, but is not limited to any software or any downloadable internet link known to a person skilled in the art comprising instructions to cause the data processing system as defined elsewhere herein to execute the defined steps.
  • a computer-readable medium having stored thereon the computer program may include, but is not limited to, a USB stick, a disc, DVD, CD, CD-ROM.
  • CR complete remission/response
  • PR partial remission/response
  • SD stable disease
  • Non-responders were defined as patients who showed progressive disease (PD) or succumbed to their disease.
  • PD progressive disease
  • the expression of tPD-L1 expression was collected from routine pathological reports of tumor tissue obtained via medically indicated biopsies or surgeries and was determined by immunohistochemistry using the PD-L1 28-8 antibody clone (abeam) on the Ventana BenchMark staining platform. At least 100 tumor cells were analyzed for the determination of the TPS.
  • Table 1 Patient overview.
  • lEVs were pelleted at 17,000 g for 30 min and sEVs at 143,000 g for 90 min. EVs were washed once in PBS and used for downstream analyses. EV isolation from up to 15 ml of EDTA-anticoagulated blood (isolation from PB sample) was performed using differential ultracentrifugation as described in the prior art (Menck et al, 2017a, J Vis Exp).
  • EVs were pelleted as described above and fixed in 2.5% glutaraldehyde in Sorensen phosphate buffer. The specimens were post-fixed with 1% osmiumtetroxide, dehydrated and embedded in Epon. Sixty nanometer ultrathin sections were cut on an UltraCut E ultramicrotome (Leica) and counterstained with uranyl acetate and lead. Samples were inspected on an EM CM 10 transmission electron microscope (Phillips).
  • NTA Nanoparticle Tracking Analysis
  • EV size and concentration were measured on a ZetaView PMX-120 device equipped with a 640 nm laser and a CMOS camera (Particle Metrix). Samples were diluted in PBS to obtain a concentration of 50-400 particles/frame. For each sample, videos at 11 cell positions were recorded at 25°C with a camera sensitivity of 80-83 for sEVs and 76-79 for lEVs, respectively. Data was analyzed with the ZetaView software (v8.02.31).
  • lEVs (2.5pg) were blocked in 20 pl PBS + 1% EV-depleted FCS for 20 min and incubated with fluorescently-labeled antibodies for CA9 (#130-123-340), TROP2 (#130-115- 097, both from Miltenyi Biotec), CD147/EMMPRIN (#306207), CD227/MUC-1 (#355603), CD274/PD-L1 (#374511), CD279/PD-1 (#329905), CD326/EpCAM (#324208), EGFR (#352903), ROR1 (#357803), CD62P (#304910), CD45 (#304006, all from BioLegend), ROR2 (#FAB20641G, R&D systems), or the corresponding isotype controls (#400321, #400132, #400113 from BioLegend, #IC003G from R&D systems or #130-118-347 from Miltenyi Biotec) for 20 min at
  • Cells and EVs were lysed in RIPA buffer (50 mM Tris, 150 mM NaCI, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, pH 7.2) supplemented with protease (Sigma) and phosphatase (Roche) inhibitors. Protein concentration was measured by Lowry assay (#5000112, Bio-Rad) according to the manufacturer’s instructions. Up to 17 pg of protein were separated by SDS page (8-12% gels) and blotted onto a nitrocellulose membrane.
  • Example 2 Tumor-associated antigens are specifically enriched on IEV compared to sEV.
  • the inventors were interested to compare the expression level of the selected antigens between both EV populations, IEV and sEV. Characterizing the expression levels on the same amount of EV protein and cell lysate (Figure 2A), the inventors observed a striking enrichment of MLIC1, EMMPRIN as well as CA9 on IEV, while EGFR, ROR1 and EpCAM were expressed on both EV populations, and only ROR2 showed higher expression on sEV. Although earlier studies have described higher expression of PD-L1 on sEV from melanoma cell lines, surprisingly, the inventors detected a clear enrichment of PD-L1 on IEV of different NSCLC cell lines. In comparison, the PD-L1 levels on sEV were rather low (Figure 2B).
  • Example 3 Total IEV counts are unchanged in peripheral blood of NSCLC patients compared to controls.
  • the inventors additionally stained the IEV preparations for ApoB and ApoA1, the major components of low or high density lipoparticles, respectively ( Figure 3C). While the inventors detected small traces of ApoB in one of the investigated IEV samples, ApoA1 expression was absent, arguing against a significant contamination of lEVs with plasma lipoproteins.
  • Example 4 lEV-associated tumor antigens are diagnostic biomarkers in NSCLC.
  • R0R1 + , R0R2 + and MUC1 + lEVs were only significantly elevated compared to healthy controls, which was mainly due to increased levels of MUC1 + and R0R2 + lEVs in the non-cancer control group, pointing at an association of these two antigens with diseases other than cancer.
  • the inventors did not observe significant differences in the levels of CA9 + , PD-1 + or EpCAM + lEVs. Surprisingly, the levels of TROP2 + lEVs were decreased in NSCLC patients albeit the known overexpression of this marker in lung cancer cells.
  • Example 5 PD-L1 + lEVs in NSCLC patients do not seem to originate from platelets or leukocytes.
  • PD-L1 is known to be expressed not only on tumor cells, but also on the surface of all antigen-presenting cells as well as on platelets. Since it is known that the majority of lEVs in blood are derived from platelets and leukocytes, the inventors asked whether the striking increase of PD-L1 + lEVs indeed stemmed from the presence of tumor-IEVs or from the increased levels of lEVs secreted from platelets or leukocytes. Indeed, when comparing the blood cell counts in the samples used for IEV isolation, it was observed that the numbers of leukocytes and platelets were significantly elevated in NSCLC patients compared to healthy individuals and non-cancer controls (data not shown). There was no change in red blood cell counts. Moreover, the level of EMMPRIN- as well as PD-L1 -positive lEVs was positively and significantly correlated with platelet, but not leukocyte, counts (data not shown).
  • Example 6 PD-L1, MUC1 and EGFR on lEVs are prognostic biomarkers in NSCLC.
  • Example 7 PD-L1 + lEVs predict therapy response in patients with absent or low tissue PD-L1 levels.
  • tPD-L1 is used in routine clinical diagnostics as selection criteria to predict response to immunotherapy.
  • advanced NSCLC patients with low or absent tPD-L1 levels can benefit from immunotherapy and so far no predictive biomarker exists for this patient subgroup.
  • TPS tumor proportion score
  • the inventors grouped patients according to their tPD-L1 levels and compared the IEV PD-L1 levels in responders and non-responders to CIT or ICI. Surprisingly, the inventors detected significantly higher levels of PD-L1 -positive lEVs in responders versus non-responders in the two groups with absent or low, tPD-L1 expression, while there was no significant difference in patients with high (>49%) tPD-L1 status (Figure 6E). Comparing the predictive power of lEV-associated PD- L1 with the so far discussed scores tPD-L1 or TPS revealed a superior AUG value of 0.67 for lEVs compared to 0.55, or 0.60, respectively (Figure 6F).
  • a method of evaluating whether a subject already suffering from non-small cell lung carcinoma (NSCLC) will respond to a treatment of NSCLC comprising immunotherapy comprising i) determining a level of PD-L1 positive microvesicles (MVs) in a test peripheral blood (PB) sample obtained from a subject suffering from NSCLC who has not yet been treated; ii) evaluating whether said subject will be a responder to a treatment of NSCLC comprising immunotherapy, if said level of PD-L1 positive MVs is increased relative to corresponding levels of PD-L1 positive MVs in control PB samples obtained from subjects also suffering from NSCLC, but being non-responders to said treatment of NSCLC comprising immunotherapy.
  • PB peripheral blood
  • a low level of PD-L1 on cancer cells in said tissue sample is within a range of about 1% to about 49% relative to all cancer cells in said tissue sample, and/or wherein ii) a low level of PD-L1 on immune cells in said tissue sample is within a range of about 1% to about 49% relative to all immune cells in said tissue sample.
  • the treatment comprising immunotherapy comprises chemo-immunotherapy (CIT) or mono-immunotherapy (ICI).
  • CIT chemo-immunotherapy
  • ICI mono-immunotherapy
  • determining said level of PD-L1 positive MVs in said PB sample comprises measuring PD-L1 (CD274).
  • binding partner is an immunoglobulin or a fragment thereof, or a proteinaceous binding molecule with immunoglobulin-like functions.
  • a data processing system comprising a processor configured to perform a method comprising the steps of i) obtaining a determined level of PD-L1 positive microvesicles (MVs) in a test peripheral blood (PB) sample obtained from a subject suffering from NSCLC who has not yet been treated; ii) evaluating whether said subject will be a responder to a treatment of NSCLC comprising immunotherapy, if said level of PD-L1 positive MVs is increased relative to corresponding levels of PD-L1 positive MVs in control PB samples obtained from subjects also suffering from NSCLC, but being non-responders to said treatment of NSCLC comprising immunotherapy.
  • a flow cytometry device capable of detecting the level of PD-L1 positive MVs in a test PB sample obtained from a subject suffering from NSCLC who has not yet been treated, comprising the data processing system of item 17.
  • a computer program comprising instructions to cause the data processing system of item 17 or the flow cytometry device of item 18 to execute the steps of i) obtaining a determined level of PD-L1 positive MVs in a test PB sample obtained from a subject suffering from NSCLC who has not yet been treated; ii) evaluating whether said subject will be a responder to a treatment of NSCLC comprising immunotherapy, if said level of PD-L1 positive MVs is increased relative to corresponding levels of PD-L1 positive MVs in control PB samples obtained from subjects also suffering from NSCLC, but being non-responders to said treatment of NSCLC comprising immunotherapy.

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Abstract

La présente invention concerne une méthode permettant d'évaluer si un sujet souffrant déjà d'un carcinome pulmonaire non à petites cellules (CPNPC) répondra à un traitement du CPNPC, le traitement comprenant une immunothérapie, la méthode comprenant la détermination d'un taux de microvésicules PD-L1 positives dans un échantillon de sang périphérique obtenu auprès d'un sujet souffrant d'un CPNPC qui n'a pas encore été traité, puis à évaluer si ledit sujet répondra à un traitement du CPNPC comprenant une immunothérapie, si ledit taux de microvésicules PD-L1 positives est augmenté par rapport aux taux correspondants de microvésicules PD-L1 positives dans des échantillons de sang périphérique de contrôle obtenus auprès de sujets souffrant également d'un CPNPC, mais ne répondant pas audit traitement du CPNPC comprenant une immunothérapie. En outre, la présente invention concerne un système de traitement de données comprenant un processeur configuré pour mettre en œuvre la méthode de l'invention.
PCT/EP2024/058488 2023-03-28 2024-03-28 Taux prédictif de pd-l1 sur des microvésicules dans l'évaluation de sa capacité à répondre à un traitement d'un cpnpc Ceased WO2024200655A1 (fr)

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