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WO2025046128A1 - Détection d'analytes cibles à l'aide d'une amplification de signal à médiation enzymatique - Google Patents

Détection d'analytes cibles à l'aide d'une amplification de signal à médiation enzymatique Download PDF

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Publication number
WO2025046128A1
WO2025046128A1 PCT/EP2024/074376 EP2024074376W WO2025046128A1 WO 2025046128 A1 WO2025046128 A1 WO 2025046128A1 EP 2024074376 W EP2024074376 W EP 2024074376W WO 2025046128 A1 WO2025046128 A1 WO 2025046128A1
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nucleic acid
enzyme
binding
product
tsa
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Patrick MICKE
Carina STRELL
Agata ZIEBA-WICHER
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Navinci Diagnostics AB
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Navinci Diagnostics AB
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens

Definitions

  • the present disclosure and invention lie in the field of nucleic acid detection, and in particular in the detection of nucleic acid or non-nucleic acid target analytes using proximity assays which generate a nucleic acid product, particularly a rolling circle amplification product (RCP), as the means by which the target analyte is detected and distinguished from other analytes, in other words as the signal, or reporter, for the target analyte.
  • a nucleic acid product e.g. RCP
  • TSA Tyramide Signal Amplification kits for use in the methods.
  • analyte detection assays involve the detection of a target nucleic acid molecule which has been generated either from a target nucleic acid analyte or a copy or amplicon thereof, or from assay reagents, e.g. probes or oligonucleotides used therewith, as a detection assay reaction product which acts as a reporter (i.e. a signal, or proxy) for the target analyte, and which is detected in order to detect the target analyte.
  • assay reagents e.g. probes or oligonucleotides used therewith
  • the target nucleic acid molecule actually detected is typically an amplification product (amplicon), and especially an amplification product which comprises multiple copies of a monomer, or repeating unit.
  • the target nucleic acid molecule, which is detected as the assay product is an RCP.
  • Rolling circle amplification is an isothermal amplification technigue reguiring a circular amplification template.
  • Amplification of the circular template using a strand-displacing polymerase provides a concatenated RCA product (RCP), comprising multiple copies (i.e. monomer repeats) of a seguence complementary to that of the RCA template.
  • RCP concatenated RCA product
  • Such a concatemer typically forms a ball or “blob”, which may readily be visualised and detected, and thus RCA-based assays have been adopted for the detection of nucleic acids, and indeed, more generally, as reporter systems for the detection of any target analyte.
  • Both target nucleic acids which may themselves be circularised directly, or probes, e.g.
  • RNAscopeTM was developed for in situ hybridisation for detection of RNA, it exemplifies the principle of using sandwich-type, or intermediate, hybridisation probes each providing multiple binding sites for labelled detection probes, to generate a detectable nucleic acid product (a so-called hybridisation assembly) comprising multiple labels.
  • TSA has been used to provide amplified signals in the context of analyte detection, it has not previously been combined with a signal amplification method such as RCA, HCR, or hybridisation assembly methods.
  • the present inventors propose a new method to increase the sensitivity of proximity-based detection methods which rely on generating nucleic acid products as amplified signals, by combining the signal amplification power of such methods (e.g. RCA or HCR etc.) with that of TSA, further to amplify the signal provided by the proximity-based nucleic acid product generation reaction (e.g. the RCA, HCR, or nucleic acid hybridisation assembly reaction).
  • the nucleic acid product e.g. the RCA product (RCP)
  • RCP RCA product
  • the nucleic acid product provides an amplified signal to detect the analyte, which advantageously can be localised to the analyte, and this is further amplified by performing a TSA reaction which is targeted, or localised, to the nucleic acid product.
  • the specific binding partner may be coupled to a nucleic acid, which may be detected using an RCA strategy, e.g. in an assay which uses or generates a circular nucleic acid molecule which can be the RCA template, or a similar strategy which generates an alternative nucleic acid product.
  • Analytes of particular interest may thus include nucleic acid molecules, such as DNA (e.g. genomic DNA, mitochondrial DNA, plastid DNA, viral DNA, etc.) and RNA (e.g. mRNA, microRNA, rRNA, snRNA, viral RNA, etc.), and synthetic and/or modified nucleic acid molecules, (e.g.
  • DNA e.g. genomic DNA, mitochondrial DNA, plastid DNA, viral DNA, etc.
  • RNA e.g. mRNA, microRNA, rRNA, snRNA, viral RNA, etc.
  • synthetic and/or modified nucleic acid molecules e.g.
  • nucleic acid domains comprising or consisting of synthetic or modified nucleotides such as LNA, PNA, morpholino, etc.
  • proteinaceous molecules such as peptides, polypeptides, proteins or prions or any molecule which includes a protein or polypeptide component, etc., or fragments thereof, or a lipid or carbohydrate molecule, or any molecule which comprise a lipid or carbohydrate component.
  • the analyte may be a single molecule or a complex that contains two or more molecular subunits, e.g. including but not limited to protein- nucleic acid, e.g. protein-DNA, complexes, which may or may not be covalently bound to one another, and which may be the same or different.
  • analyte may also be a protein complex or protein interaction, or more generally a complex of interaction comprising or involving a protein.
  • a complex or interaction may thus be a homo- or hetero-multimer.
  • Aggregates of molecules, e.g. proteins may also be target analytes, for example aggregates of the same protein or different proteins.
  • the analyte may also be a complex between proteins or peptides and nucleic acid molecules such as DNA or RNA, e.g. interactions between proteins and nucleic acids, e.g. regulatory factors, such as transcription factors, and DNA or RNA.
  • the individual members of an interaction may be detected using proximity probes, each specific for a member of the interaction.
  • An exemplary interaction is for example the PD-1/PD-L1 interaction. Analogously interactions with other immune checkpoints or regulatory proteins may be detected.
  • a proximity assay detects proximity between two target molecules
  • the analyte may simply be the proximal localisation of two or more target molecules or entities together, without necessarily requiring binding, or a physical association between them. Such a proximity may indicate that the molecules/entities are interacting, but this is not a requirement.
  • Proximity in this context means that the two target molecules/entities are sufficiently close to one another to enable them to be detected by a proximity assay. For example, this may in practice meant that they lie, or are located, within a distance of no more than 100, 90, 80 or more particularly 70 nm of each other, e.g. 10-80, 20-80, 20-70, 30-70, 40-70nm of each other etc.
  • a single molecule target such as a protein may be detected by detecting two separate epitopes on the molecule.
  • the target analyte may be a protein or component of a proteinaceous molecule which is detected on the surface of a cell, or vesicle or other cellular or sub-cellular compartment.
  • the target analyte may be a variant of a target sequence.
  • Target sequences may commonly occur in variant forms, for example allelic variants, or mutant and wild-type sequences and it may be desirable which variant is present.
  • the target nucleotide sequence may be one of a number of different variants of the nucleic acid sequence which may occur in a target nucleic acid molecule.
  • the term “nucleotide sequence” is used herein synonymously and interchangeably with “nucleic acid sequence”.
  • detecting is used broadly herein to include any means of determining the presence of the analyte (i.e. if it is present or not) or any form of measurement of the analyte. Thus “detecting” may include determining, measuring, assessing or assaying the presence or absence or amount or location of analyte in any way. Quantitative and qualitative determinations, measurements or assessments are included, including semi-quantitative. Such determinations, measurements or assessments may be relative, for example when two or more different analytes in a sample are being detected, or absolute. As such, the term “quantifying" when used in the context of quantifying a target analyte(s) in a sample can refer to absolute or to relative quantification.
  • Absolute quantification may be accomplished by inclusion of known concentration(s) of one or more control analytes and/or referencing the detected level of the target analyte with known control analytes (e.g. through generation of a standard curve).
  • relative quantification can be accomplished by comparison of detected levels or amounts between two or more different target analytes to provide a relative quantification of each of the two or more different analytes, i.e., relative to each other.
  • the method may be for the localised detection of target analyte.
  • Localised detection means that the signal giving rise to the detection of the analyte is localised to the analyte, in this case the nucleic acid product and the TSA product are localised to the target analyte.
  • the analyte may therefore be detected in or at its location in the sample.
  • the spatial position (or localization) of the analyte within the sample may be determined (or “detected”). This means that the analyte may be localised to, or within, the cell in which it is expressed, or to a position within a cell or tissue sample.
  • “localised detection” may include determining, measuring, assessing or assaying the presence or amount and location, or absence, of the analyte in any way.
  • the method may be used for the in situ detection of an analyte.
  • the method may be used for the localised, particularly in situ, detection of nucleic acids, e.g. mRNA, or proteins or protein interactions or modifications, including more particularly the localised, particularly in situ, detection of proteins or protein interactions or modifications in a sample of cells.
  • the term "in situ" refers to the detection of a target analyte in its native context, i.e. in the cell or tissue in which it normally occurs. Thus, this may refer to the natural or native localization of a target analyte. In other words, the analyte may be detected where, or as, it occurs in its native environment or situation. Thus, the analyte is not moved from its normal location, i.e. it is not isolated or purified in any way, or transferred to another location or medium etc. Typically, this term refers to the analyte as it occurs within a cell or within a cell or tissue sample, e.g. its native localization within the cell or tissue and/or within its normal or native cellular environment.
  • in situ detection includes detecting the target analyte within a tissue sample, and particularly a tissue section. In other embodiments the method can be carried out on a sample of isolated cells, such that the cells themselves are not in situ.
  • the detection is not localized, or not in situ.
  • the method includes embodiments in which the target analyte is not present (e.g. is not fixed) in its native context. This may include embodiments in which a target analyte is immobilized, e.g. on a solid support, for example in or on an array.
  • the method can be carried out in solution or in suspension.
  • the analyte can be in solution.
  • the method can be performed on a sample comprising an isolated analyte.
  • the method can be performed where the analyte is suspended in a sample, for example where the analyte is a cell, or an aggregate etc.
  • the analyte may be present in or on a cell which is in suspension in the sample, or which is immobilized in the sample etc.
  • the analyte is present within a sample.
  • the sample may be any sample which contains any amount of target analyte which is to be detected, from any source or of any origin.
  • a sample may thus be any clinical or non-clinical sample, and may be any biological, clinical or environmental sample in which the target analyte may occur. All biological and clinical samples are included, e.g. any cell or tissue sample of an organism, or any body fluid or preparation derived therefrom, as well as samples such as cell cultures, cell preparations, cell lysates etc.
  • Environmental samples e.g. soil and water samples or food samples, are also included.
  • the samples may be freshly prepared for use in the method of the present invention, or they may be prior-treated in any convenient way e.g. for storage.
  • the target analyte may be detected in situ, as it naturally occurs in the sample.
  • the target analyte may be present in a sample at a fixed, detectable or visualisable position in the sample.
  • the sample will thus be any sample which reflects the normal or native ("/n situ") localisation of the target analyte, i.e. any sample in which it normally or natively occurs.
  • Such a sample will advantageously be a cell or tissue sample.
  • samples such as cultured or harvested or biopsied cell or tissue samples in which the target analyte may be detected to reveal the localisation of the target analyte relative to other features of the sample.
  • the sample may be a cell or tissue sample possessing a high autofluorescence, in particular a human tissue sample.
  • the sample may be a cancer tissue sample.
  • samples may also include, for example, dehydrated or fixed biological fluids, and nuclear material such as chromosome/chromatin preparations, e.g. on microscope slides.
  • the samples may be freshly prepared or they may be prior-treated in any convenient way such as by fixation or freezing. Accordingly, fresh, frozen or fixed cells or tissues may be used, e.g. FFPE tissue (Formalin Fixed Paraffin Embedded).
  • Analytes, including cells, or cells which carry or contain an analyte may be immobilised on a solid support or surface, e.g. a slide, well or beads or other particles etc., using techniques and reagents well known the art, e.g. capture probes and such like, or by chemical bonding or cross-linking etc.
  • representative samples may include any material which may contain a target analyte, including for example foods and allied products, clinical and environmental samples, etc.
  • the sample may be a biological sample, which may contain any viral or cellular material, including all prokaryotic or eukaryotic cells, viruses, bacteriophages, mycoplasmas, protoplasts and organelles.
  • Such biological material may thus comprise all types of mammalian and non-mammalian animal cells, plant cells, algae including blue-green algae, fungi, bacteria, protozoa etc.
  • Representative samples thus include clinical samples, e.g. whole blood and blood- derived products such as plasma, serum and buffy coat, blood cells, other circulating cells (e.g.
  • tumour cells circulating tumour cells
  • urine faeces, cerebrospinal fluid or any other body fluids (e.g. respiratory secretions, saliva, milk, etc.), tissues, biopsies, as well as other samples such as cell cultures, cell suspensions, conditioned media or other samples of cell culture constituents, etc.
  • samples such as cell cultures, cell suspensions, conditioned media or other samples of cell culture constituents, etc.
  • the sample may be pre-treated in any convenient or desired way to prepare for use in the present methods, for example by cell lysis or purification, fixing of cells, isolation of the analyte, immobilisation etc.
  • the present methods may be used to select a target analyte in an in situ (i.e. a native) setting, it is also contemplated that the method may be employed to select a target analyte in any detection system, including where a target analyte has been isolated or purified from its native setting.
  • the sample may thus be a direct product of a target analyte isolation procedure, or of a cell lysis procedure, or it may further be fractionated or purified in some way.
  • the analyte may be a synthetic molecule such as a cDNA or an amplicon etc.
  • the sample may be any material or medium containing such a molecule, e.g. a reaction mixture.
  • the sample can be a preparation of cells, e.g. a cell suspension.
  • an RCP may be a product of an any type of proximity-based detection reaction which comprises an RCA step, for example a proximity probe assay in which a circular nucleic acid molecule is generated - see the DuolinkTM PLA of SigmaAldrich for example, and the modified PLA which uses so-called Unfold proximity probes, which comprise hairpins which are opened, or unfolded, by cleavage to release nucleic acid domains which may be circularised to form an RCA template (see Klaesson et al, 2018, Scientific Reports 8, 5400).
  • a typical PLA generates a template circle upon interaction of the nucleic acid domains of proximity probes, when bound in proximity to their target.
  • the detection sequence in the RCP may be equivalent to a sequence present in the target nucleic acid sequence itself (e.g. the nucleic acid domain of a proximity probe).
  • a detection sequence e.g. tag or barcode sequence
  • the detection sequence may be present in the gap oligonucleotide which is hybridised between the respective hybridised ends of the padlock probe, where they are hybridised to non-adjacent sequences in the target molecule.
  • a new nucleic acid molecule may be generated in a sample (i.e. a nucleic acid molecule that was not present in the original sample and was not one of the components added to the sample) by one or more molecules that interact with, e.g. bind to, the target analyte.
  • the detection of the generated nucleic acid molecule is indicative of the target analyte in a sample.
  • the generated molecule may be a circular molecule, or it may template the circularisation of another molecule, such as a padlock probe for the generated molecule.
  • hybridisation probes may be used to detect nucleic acid analytes, which are provided with, or designed to hybridise to, circular RCA templates or circularisable RCA template molecules, e.g. proximity variants of FISH and smFISH assays in which the hybridisation probes comprise, in addition to a target-specific binding domain for hybridisation to a target molecule, a second domain, which does not hybridise to the target nucleic acid but which functions analogously to the nucleic acid domains described above, e.g. which contains a binding site for a circular or circularisable probe, which may, upon circularisation if necessary, be subjected to RCA.
  • Such hybridisation proximity probes may be provided as hairpins which open upon binding of the probe to its target, releasing the second domains for interaction.
  • Proximity extension assays may generate an extended nucleic acid molecule wherein the nucleic acid domain of one proximity probe is extended using the nucleic acid domain of another proximity probe as extension template.
  • the extended molecule may be detected by hybridisation of a circular or circularisable oligonucleotide which acts as an RCA template for an RCA reaction.
  • nucleic acid product may be generated. Such products may be the result of nucleic acid amplification, nucleic acid polymerisation or nucleic acid hybridisation reactions.
  • nucleic acid products are generated in detection assays as a means of signal amplification.
  • a nucleic acid product generated in the methods herein may be characterized by comprising a detection sequence, at high concentration or high copy number, including in certain embodiments, at a spatially defined site or position.
  • a product may originate from localized amplification or polymerization reactions (such as RCA or HCR), or from the hybridization of multiple hybridization probes at proximal locations on the same nucleic acid molecule.
  • Such a hybridization system may allow a branched nucleic acid structure to be built up, or assembled, for example akin to a RNAscopeTM product as mentioned above.
  • RCA-based methods where an RCP is generated, where necessary, the binding of proximity probes may be followed by a reaction to circularise a circularisable probe or added circularisable oligonucleotide(s), again according to well-known procedures.
  • Ligation reactions for circularisation of such probes or oligonucleotides are also well known and described in the art, and a variety of different template-directed ligases may be used, including temperature sensitive and thermostable ligases, such as bacteriophage T4 DNA ligase, bacteriophage T7 ligase, E. coli ligase, Taq ligase, Tth ligase, Ampligase® and Pfu ligase.
  • a suitable ligase and any reagents that are necessary and/or desirable may be combined with the sample/reaction mixture and maintained under conditions sufficient for ligation to occur. Ligation reaction conditions are well known in the art and may depend on the ligase enzyme used.
  • reaction mixtures are prepared.
  • the various reagents and constituent components of a reaction mixture may be combined in any convenient order.
  • all of the various constituent components may be combined at the same time to produce the reaction mixture, and/or they may be added sequentially, according to the needs or steps of the method.
  • the method may comprise various steps of contacting reagents with the sample, or with a reaction mixture.
  • contacting is used broadly herein to include bringing the reagents in question into contact. Thus, one may be added to the other and vice versa, or they may each be introduced to each other etc. This time or order of addition, or contact with the sample etc., may depend on the precise nature of the method, or method step, which is performed.
  • the RCP or other nucleic acid product that is provided or generated in the method herein comprises multiple copies of a detection sequence which is indicative of the target analyte.
  • a detection sequence which is indicative of the target analyte.
  • the detection sequence is used in the initiation of the TSA reaction.
  • the detection sequence provides a binding site for localisation or capture of the enzyme that performs the TSA reaction. Since the RCP is a concatemer of monomer repeats, multiple binding sites for the enzyme are provided. Analogously, other nucleic acid products also comprise multiple binding sites for the enzyme.
  • the method comprises hybridising a multiplicity of oligonucleotides to the nucleic acid product, or more particularly to the detection sequences present in the repeats/copies (monomer units) thereof, which allow an enzyme to be bound to the nucleic acid product.
  • the term “multiple” or “multiplicity” means two or more, e.g. at least 2, 3, 4, 5, 6, 10, 20, 30, 50, 70 or 100 or more. It will be understood that whilst each of the repeat units/copies or monomers of the nucleic acid product comprise the binding site for the enzyme, in practice not all of these binding sites may (or will) be occupied by an enzyme. It suffices that a number, or multiplicity, of such binding sites are bound by an enzyme.
  • the enzyme is bound to a detection sequence in at least one monomer of the nucleic acid product, but preferably to multiple detection sequences.
  • an enzyme capture oligonucleotide may be used, which targets the enzyme to the detection sequences in the nucleic acid product.
  • the enzyme-capture oligonucleotide thus comprises a sequence, or binding site, which is complementary to the detection sequence, or a part thereof, i.e. it is capable of hybridising to the detection sequence.
  • the capture oligonucleotide may be attached, directly or indirectly, to the enzyme. This attachment may be reversible, or in other words, the enzyme may be removably attached to the capture oligonucleotide.
  • the enzyme-capture oligonucleotide comprises biotin.
  • the enzyme-capture oligonucleotide is allowed to hybridise to its complementary binding sites in the nucleic acid product (i.e. to the detection sequences). Subsequently, the nucleic acid product with the hybridised enzymecapture oligonucleotide is contacted with an enzyme agent comprising the enzyme linked to streptavidin, thereby attaching the enzyme to the nucleic acid product.
  • the captured enzyme i.e. the nucleic acid product with attached enzyme
  • the enzyme catalyses a reaction which deposits a substrate reaction product in the vicinity of the enzyme. Since the enzyme is attached to the nucleic acid product, the substrate reaction product is deposited in the vicinity of the nucleic acid product, allowing the localised detection of the nucleic acid product. Conveniently, the substrate is labelled to allow its detection.
  • the labelling may be direct or indirect.
  • the substrate may be directly conjugated with, or attached to, a label (as is depicted in Figure 1 for example), or it may be attached to a moiety which allows it to be attached to a label, e.g.
  • the hybridised nucleic acid product-capture oligonucleotide-enzyme is contacted with a signal localisation agent comprising (i) a substrate for the enzyme and (ii) either a label or a first binding moiety for binding to a labelling agent.
  • a signal localisation agent comprising a label
  • the label is deposited along with the substrate reaction product.
  • the signal localisation agent may alternatively be referred to as a TSA reagent.
  • the reaction mixture comprising the deposited substrate reaction product is contacted with a labelling agent comprising a label linked to a second binding moiety cognate to the first binding moiety, and the first and second binding moieties are allowed to bind.
  • the labelling agent, and hence the label is attached to the substrate reaction product.
  • the label is then detected, to detect the nucleic acid product, and hence the target analyte.
  • the first and second binding moieties may be members of an affinity binding pair as described above for the first and second capture moieties.
  • TSA reaction and various detection methods using TSA are known in the art and, generally speaking, known methods and reagents may be used to perform the various steps of the TSA reaction as set out above.
  • TSA protocols and reagents have been commercialised. Reference may be made in this regard to the ThermoFisher website pages relating to TSA. Further, Akoya Biosciences, Inc. have developed the TSA method for use in connection with spatial biology, and the localised detection of analytes in cells.
  • a readout molecule (such as an enzyme, more specifically horseradish peroxidase) is conjugated to an oligonucleotide which has a complementary sequence to a barcode oligonucleotide.
  • the readout moiety and detection moiety hybridise the readout of which can be detected (for example, by utilising TSA).
  • the barcode oligonucleotide may thus be seen as a capture oligonucleotide, which operates in a manner akin to the capture oligonucleotides, enzyme agents, and capture moieties etc, as described herein.
  • the TSA reaction may be performed using reagents, known and available in the art, notably known TSA enzymes and substrates, which can be used or adapted for use in accordance with the methods herein, e.g. used to prepare enzyme-capture oligonucleotides, enzyme agents, TSA reagents or signal localisation agents etc.
  • This incudes for example reagents available commercially from, amongst others, Akoya Biosciences, Inc. or ThermoFisher.
  • the enzyme is typically a peroxidase, which term refers broadly to any enzyme having peroxidase activity, e.g. soybean peroxidase or, more commonly, horseradish peroxidase (HRP).
  • the peroxidase acts in the presence of hydrogen peroxide to catalyse the deposition of the substrate, i.e. to convert the substrate into a reactive species, or reactive reaction product, which is able to react with molecules in its vicinity, and thereby bind to, or to become localised to, those molecules, and hence to the vicinity of enzyme and/or proteins in the vicinity of the enzyme.
  • reactive species i.e. to convert the substrate into a reactive species, or reactive reaction product, which is able to react with molecules in its vicinity, and thereby bind to, or to become localised to, those molecules, and hence to the vicinity of enzyme and/or proteins in the vicinity of the enzyme.
  • reactive species i.e. to convert the substrate into a reactive species, or reactive reaction product, which is able to react with molecules in its vicinity, and thereby bind to, or to become localised to, those molecules, and hence to the vicinity of enzyme and/or proteins in the vicinity of the enzyme.
  • reactive reaction product which may include the analyte, or an
  • the substrate can be any substrate that may be converted by a peroxidase or analogous enzyme to a reactive product capable of being deposited.
  • the reactive product is capable of reacting with molecules in its vicinity, most notably with proteins, and in particular with tyrosine residues in proteins.
  • the substrate is a tyramide compound, that is a derivative of tyramine, a compound that contains a tyramine or tyramide derivative, p-hydroxy-cinnamic acid, or a derivative of p-hydroxy cinnamic acid.
  • the label may be any label known or used in the art in detection assays. In an embodiment it is an optical label. This includes any optical label that may be detected directly or indirectly. In other words, the label may directly or indirectly signal-giving. By indirectly signal-giving it is typically meant that the label undergoes a process or conversion before it can be detected, for example it is enzymatically converted, or converted by a chemical reaction to a product that may be detected.
  • the label may be a fluorescent label, a coloured label, a chromogenic label, a quantum dot, a mass-tag label, or a particle.
  • the label is dye or stain. It is convenient to use a label that may be visualised, and particularly which may be detected microscopically, and more particularly by imaging.
  • fluorescent or coloured labels are known in the art and are commercially available. This includes traditional well-known labels such as C3, Cy5, Cy7, FITC, FAM and TAMRA etc. Further, the Opal dyes available from Akoya Biosciences may be used. This includes Opal fluorophores developed or commercialised for in TSA detection protocols. Suitable labels are also available from ThermoFisher, e.g. Alexa Fluor dyes.
  • the detection step will depend on the label which is used and also on the sample, and the format off the detection method, but generally any convenient or desired detection modality may be used (and the label may be selected in accordance with that).
  • the method may be carried out in heterogenous or homogenous formats. That is, it may be performed on a solid phase (or support), or in solution or suspension (i.e. without a solid phase or support), or indeed both, since a solid phase may be introduced at a later stage.
  • the label is detected microscopically, and particularly by imaging.
  • Techniques and instruments for such detection e.g. fluorescent microscopes and image analysis software etc are well known in the art.
  • techniques for detecting coloured labels, e.g. by bright field microscopy are also well known in the art.
  • detection methods may also be used, for example flow cytometry.
  • flow cytometry to detect fluorescently labelled products in detection assays is well known, for example.
  • Protocols for performing multiplexed TSA detection assay in such a manner are known and described in the art, for example in US 2021/0222234 (Akoya Biosciences, Inc.) or WO 2021/226516 (Leland Stanford Junior University), and such protocols may be followed, or adapted for use in the methods herein.
  • Such methods typically involve removing enzymes, and/or probes, between cycles. Labelling of the TSA product may take place in individual cycles, or the labelling steps may be performed together. Detection may take place in each cycle, or it may take place in one step, after all the cycles have been performed, and all TSA products have been labelled.
  • images may be taken in each cycle, and may then be combined or overlaid, or otherwise merged or consolidated.
  • a single imaging step may be performed at the end of all the cycles.
  • Antigen retrieval processes which are known in the art may be used for this purpose, which may remove both the proximity probes, and the enzyme. These may use heat and/or enzymatic or chemical processes for the removal, for example HIER (heat-induced antigen (epitope) retrieval). HIER methods involve heating to high temperatures, e.g. to at least 60, 70, 80, 85, 90 or 95 °C for a period of time, e.g. for at least 10, 15 or 20 minutes, or longer. Generally, lower temperatures require longer incubations, for example, 60 °C overnight. HIER may include the use of steamers, microwave ovens, pressure cookers and hot water baths etc. Suitable solutions, e.g. buffers for use in antigen retrieval, are known in the art. Enzymatic methods include protease-induced epitope retrieval (PIER), using for example, pepsin, trypsin, or proteinase K.
  • PIER protease-induced epitope retrieval
  • the steps are performed as described above, with a first pair, or a first set of two or more pairs, of proximity probes to detect a selected (first) target analyte or first sub-set of target analytes, and the TSA product(s) is/are labelled with a first label or first set of labels.
  • the enzyme whether attached directly to the capture oligonucleotide or via a separate enzyme agent which binds to the capture oligonucleotide, is removed, for example by HIER. If HIER is used, the proximity probes are also removed from the sample.
  • hybridisation or “hybridises” as used herein refers to the formation of a duplex between nucleotide sequences which are sufficiently complementary to form duplexes via Watson-Crick base pairing, or any analogous base-pair interactions.
  • Two nucleotide sequences are "complementary" to one another when those molecules share base pair organization homology.
  • a region of complementarity in a molecule or probe or sequence refers to a portion of that molecule or probe or sequence that is capable of forming a duplex.
  • Hybridisation does not require 100% complementarity between the sequences, and hence regions of complementarity to one another do not require the sequences to be fully complementary, although this is not excluded.
  • the methods may further comprise counterstaining the sample to aid visualisation, before the step of detecting the TSA product, or detecting the label.
  • the nuclear stain DAPI may be used.
  • Other suitable stains are known in the art, and may be used according to choice.
  • the methods herein may be used in combination with other detection assays, including commercially available assays, and may be integrated in a workflow with one or more other detection assays.
  • Such other assays may be assays to detect other analytes in the sample.
  • This may allow a particular target analyte, for example an interaction (e.g. a protein-protein interaction (PPI), such as PD-1/PD-L1), to be assessed in the context of its local environment, for example the tissue environment, or tumour microenvironment (TME).
  • PPI protein-protein interaction
  • TME tumour microenvironment
  • Both cellular and functional data can be integrated in this way, for example, the proximity-TSA method revealing functional information, e.g.
  • the present method can provide a powerful tool for characterising a particular tissue or environment in relation to a particular interaction etc.
  • the methods herein may be used for an in situ analysis of the PD-1/PD-L1 interaction, or other checkpoint interactions, in the TME. In this way the understanding of immune responses may be deepened, and identification of spatial signatures improved, e.g. for improved patient stratification.
  • Such other detection assays thus include any assays for detecting an analyte in a sample, for example immunoassays, including immunohistochemical (IHC) procedures, or any other detection assay, e.g. immunofluorescence staining assays to detect one or more protein analytes present in the sample.
  • the one or more other detection assays may be performed before or after the present method.
  • the same sample preparation steps may prepare the sample for both the present method and the other assay(s), e.g. antigen retrieval, dewaxing, fixing etc.
  • further steps may be performed.
  • directly or indirectly labelled antibodies or other binding partners for one or more target analytes may be added to the sample, together in combination or sequentially.
  • the subsequent detection assay may be a multiplex assay, which may be performed simultaneously or sequentially.
  • the other detection assay may employ TSA-labelling technology to detect the antibody.
  • Different labels e.g. different Opal dyes, may be used to detect different analytes.
  • the method provided herein may further comprise the following steps:
  • step (vi) detecting said detection antibody of step (v) by:
  • the amplification process for the Alexa 647 fluorophore was observed to have comparable efficiency to the previously used Alexa 594 fluorophore in the PLA-TSA experiment.
  • the detected signals showcased a marked difference between the conventional PLA and the enhanced PLA-TSA techniques.
  • the intensity of the PLA-TSA method was augmented several-fold for both the PDGFR and pantyrosine antibody pairs, irrespective of the Alexa dyes used.
  • This enhancement in the PLA-TSA approach highlights its heightened sensitivity and precision, making it especially valuable for probing interactions that have historically been elusive due to limitations in antibody efficacy and inherently weak signals.
  • a multiplex TSA-based immunofluorescent staining of a non-small cell lung cancer (NSCLC) tissue core The primary objective of this staining was to simultaneously visualize multiple markers - DAPI, PD1-PDL1 (PLA), panCK, pSTAT3, pSMAD2, and CD8.
  • PDA PD1-PDL1
  • panCK panCK
  • pSTAT3, pSMAD2 panCK
  • CD8 CD8
  • IF-TSA Tyramide Signal Amplification
  • the Proximity Ligation Assay was integrated with TSA.
  • FFPE paraffin-embedded non-small cell lung cancer
  • the NSCLC tissue samples were first subjected to Proximity Ligation Assay (PLA) to detect the interactions between PD1 and PDL1. Following the primary antibody binding specific for these interactions, tissues underwent PLA and TSA. Post washing, secondary antibodies conjugated to unique oligonucleotides were introduced. After the probes were bound, the ligation and amplification steps were conducted. This RCA product was detected using complementary oligonucleotides labeled with biotin. Streptavidin conjugated with horseradish peroxidase (HRP) was added next, binding the HRP enzyme to the amplified DNA. The tissue was treated with a tyramide substrate conjugated to a fluorophore.
  • PHA Proximity Ligation Assay
  • Example 3 PDGFRB-GRB2 PLA combined with several markers including CD31, DAPI, Active YAP, FAP and panCK
  • IF-TSA multiplex immunofluorescent panel was employed.
  • Bond RXm Autostainer for the multiplex process, we initiated deparaffinization and antigen retrieval using a pH9 buffer. Subsequent cycles only required antigen retrieval with a pH6 buffer. Post-retrieval, slides were blocked and washed. Primary antibodies, differing in diluents, were applied and incubated at RT. After washing, secondary antibodies were added based on the primary antibody's origin and incubated. Fluorophores were then added after another wash. Following staining cycles, another antigen retrieval was done, succeeded by multiple washes.
  • Elevated stromal platelet-derived growth factor receptor beta (PDGFRP) expression is a harbinger of poor prognosis in various solid tumors. Its role in NSCLC, however, remains ambiguous. Recognizing that mere PDGFRp expression does not equate to its active signaling, understanding its activation state might offer deeper insights. In this study, we employed the advanced PLA-TSA in situ technique to pinpoint activated PDGFRp, deciphering its clinical relevance and prognostic nuances in NSCLC.
  • Exemplary protocol for in situ proximity ligation assay (PLA)-TSA assay using Naveni® PLA reagents including Naveni® PLA PD-1/PD-L1
  • Reaction 2 repeat the process with Buffer 2 and Enzyme 2, following the same dilution and application guidelines. Incubate for 90 minutes.

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Abstract

La présente invention concerne un procédé de détection d'un analyte cible dans un échantillon comprenant la génération d'un produit d'acide nucléique par un dosage de proximité, en tant que signal pour l'analyte, la réalisation d'une amplification d'amplification de signal de tyramine à médiation enzymatique (TSA) du produit d'acide nucléique pour générer un produit TSA localisé sur le produit d'acide nucléique, et la détection du produit TSA. Le procédé peut être utilisé dans le contexte d'un quelconque dosage de proximité qui génère un produit d'acide nucléique, mais trouve une utilité particulière dans des dosages de ligature de proximité (PLA) qui génèrent un produit d'amplification par cercle roulant (RCP). En combinant la puissance d'amplification de signal de procédés de détection basés sur la proximité avec celle de TSA, la sensibilité de tels procédés est augmentée. Le signal fourni par la réaction de génération de produit d'acide nucléique à base de proximité est en outre amplifié, le produit de réaction TSA agissant en tant qu'étiquette ou signal pour le produit d'acide nucléique.
PCT/EP2024/074376 2023-08-31 2024-08-30 Détection d'analytes cibles à l'aide d'une amplification de signal à médiation enzymatique Pending WO2025046128A1 (fr)

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