WO2025163315A1 - Immunoassays - Google Patents

Abstract

The present invention relates to methods of detecting one or more target molecules in a sample utilising one or more aptamer-Fc conjugates. The aptamer-Fc conjugates are compatible with immunoassay formats using secondary antibodies such as immunohistochemistry (IHC) or the like. The invention also provides aptamers, aptamer-Fc conjugates, and complexes comprising aptamer-Fc conjugates and labelled secondary agents (e.g., secondary antibodies) which may be used in such methods.

Description

IMMUNOASSAYS

Field of the Invention

The invention relates to methods of detecting one or more target molecules in a sample utilising one or more aptamer-Fc conjugates. The aptamer-Fc conjugates are compatible with immunoassay formats using secondary antibodies such as immunohistochemistry (IHC), or the like. The invention also provides aptamers, aptamer-Fc conjugates, and complexes comprising aptamer-Fc conjugates and labelled secondary agents (e.g., secondary antibodies) which may be used in such methods.

Background to the Invention

Immunohistochemistry (IHC) is a widely used laboratory technique in which an epitope specific binder (typically an antibody) recognises and binds to a biomarker of interest in a cell or tissue sample. The antigen binder may be labelled with either a fluorophore (in immunofluorescence detection) or a chromogenic reporter (in immunohistochemistry); allowing direct visualisation of the binder and hence study of the location, distribution or change of protein(s) of interest.

Immunohistochemistry is extensively used in research to study the localisation, distribution, and changes in expression of biomarkers in different parts of a tissue sample. Immunohistochemical staining has the advantage that it allows a target protein of interest to be precisely identified and located within a tissue sample. It can also be used to detect cells with specific protein expression patterns, such as altered biomarker expression in disease associated cells and tissues. This makes it a useful technique in many research applications including (but not limited to) neuroscience, oncology, and study of many pathologies.

IHC also remains the gold standard test for clinical diagnostics, and its use is increasing with the advent of personalised medicine approaches. In a clinical setting, IHC is used to identify, assess, and quantify the levels of predictive and prognostic biomarkers in many malignancies including cancers.

IHC exploits the specific binding between an antibody and an antigen to detect and localise specific antigens in cells and tissue. Typically, IHC is performed on either frozen or formalin fixed paraffin embedded (FFPE) tissue, with automated methods developed in the art for reproducible and high-volume processing. Typically, the first step in FFPE-based IHC is antigen retrieval, which involves the pretreatment of tissue to retrieve antigens masked by fixation and/or preservation of the sample, and make them more accessible to antibody binding. A primary antibody may then be added, which is capable of specifically binding to the antigen of interest. A secondary antibody may then be added, that binds to the primary antibody. To visualise the antigen-antibody interaction under light microscopy, the secondary antibody is labelled, allowing for signal amplification and use with many different primary antibodies. Examples of labels which are typically used include fluorescent molecules (used in immunofluorescence) or enzymes such as horseradish peroxidase or alkaline phosphatase which produce a coloured product after incubation with a chromogenic substrate (used in chromogenic detection).

In general, immunoassays such as IHC or the like are highly reproducible and reliable. However, owing to the presence of numerous variables that may influence an immunoassay test result and the lack of standardization among clinical laboratories, inconsistent assay results have been frequency reported in the literature. In addition, producing primary antibodies for use in immunoassays such as IHC requires a series of complicated and timeconsuming processes. Also, unlike other laboratory based immunoblotting techniques in which the staining of a protein can be checked against a molecular weight marker, in IHC it is impossible to show that the staining is truly specific to the protein of interest (rather than nonspecific staining of a non-target protein). For this reason, primary antibodies must be well- validated in a Western Blot or similar procedure before they can be used in IHC.

There are also significant challenges in development of antibodies for use in IHC etc. Many antibodies are isolated by administering a purified form of the target antigen into a suitable host animal, followed by harvesting and screening of antibodies to identify suitable candidates for further IHC development. As the administered antigen cannot undergo any fixation/preservation or retrieval processes prior to administration, it is not uncommon for the isolated antibodies to fail to recognise the antigen following IHC processing. This leads to a failure in the antibody development process.

In view of these difficulties, aptamers can offer benefits over antibodies. For example, aptamers have been demonstrated in the art as excellent probes for immunostaining of frozen or FFPE tissues. They may be isolated in vitro, without the need for animals. This means that they can be isolated using antigens that have undergone the fixation, preservation and retrieval steps required in IHC. This increases the likelihood of developing binders that are fit for purpose. Additionally, aptamers have a small size which allows increased penetration into cells and tissue, have low batch to batch variation, low cost, fast production, and stability for convenient storage. Whilst some aptamers have already been developed for use in histochemistry (“aptahistochemistry”) (Ahirwar et al. (2016) PLos ONE 11(4): e0153001 ; Aptekar et al., (2015) PLos ONE 10(8): e0134957; Zamay et al. (2017) Mol Ther Nucleic Acids 6: 150-162), these aptamers are directly conjugated with fluorescent or biotin labels and are directly detected. While these aptamers demonstrate the potential utility of aptamers in IHC; they cannot be used in existing high throughput, automated IHC workflows, as these typically use specific reagents, including specifically tailored and labelled secondary antibodies.

It is an aim of some embodiments of the present invention to at least partially mitigate some of the problems identified in the prior art by developing reagents which are more reliable, accurate, selective and/or cheaper to produce as compared to primary antibodies used in existing IHC assays yet still fit into the workflows used for high throughput, automated commercial instruments.

Summary of Certain Embodiments of the Invention

The present invention relates to the development of new reagents which may be used in existing immunoassay workflows.

In certain embodiments, the invention relates to the development of aptamers conjugated to the Fc domain of an antibody, to create a hybrid binder which combines the antigen recognition properties of the aptamer, with a secondary antibody binding Fc domain. The inventors have successfully demonstrated the functional integration of different aptamer binders into this conjugate format. This has allowed a broad target range of aptamer tools to be applied in detection assays such as IHC within existing workflows, i.e. , by simply replacing the primary, detecting, antibody with the aptamer-Fc conjugate without needing to modify existing workflows, to visual the antigen utilising a secondary antibody that specifically binds to the conjugated Fc domain.

Aptly, the “aptamer-Fc conjugate” of the invention is developed for diagnostic purposes only. As such, the aptamer-Fc conjugate is not an active therapeutic agent, nor further comprises any payload or other cargo.

Advantageously, the replacement of primary antibodies with the aptamer-Fc conjugates provides optimal staining with shorter reaction times, less restricted antigen retrieval conditions, reduced time to perform the aptamer histochemistry technique and lower background staining. Accordingly, the invention provides a method for detecting one or more target molecules in a sample.

In certain embodiments, the method comprises a step of applying to the sample an aptamer- Fc conjugate, wherein the aptamer region of the conjugate is capable of specifically binding to the target molecule. Aptly the sample comprises intact (e.g., substantially intact) tissue. For example, the method may comprise techniques of immunohistochemistry (IHC) as further described herein. Typically, the tissue may be live or unprocessed, formalin-fixed paraffin- embedded tissue (FFPE) and/or frozen. Aptly the method is an automated method.

In certain embodiments, the method further comprises a step of applying to the sample a labelled secondary agent (e.g., secondary antibody), wherein the agent is capable of specifically binding to the Fc region of the conjugate.

In certain embodiments, the method further comprises a step of detecting the presence, absence and/or level of the secondary agent.

Aptly, the sample comprises intact tissue or isolated cells. For example, the method may comprise techniques of immunohistochemistry (IHC) as further described herein. Typically, the tissues and/or cells may be live or unprocessed, FFPE and/or frozen.

Aptly, the sample is pre-treated with a nucleic acid-based blocking buffer. For example, DNA- based blockers such as salmon sperm DNA may be used. In addition, or alternatively, RNA- based blockers such as yeast tRNA may be used. In addition, or alternatively, sulphated polysaccharide-blockers such as dextran sulphate and/or heparin may be used. The techniques of the invention are therefore distinct from conventional techniques of IHC, which typically instead only use protein-based blocking buffers.

Aptly, the sample is incubated with the blocking buffer at room temperature for about an hour, overnight at about 4°C or rapidly at 37°C for up to about 30 minutes.

The invention also provides a complex comprising an aptamer-Fc conjugate and labelled secondary agent (e.g., labelled secondary antibody). As disclosed further herein, the Fc may be from a first species (e.g., rabbit) and the secondary antibody from a second species (e.g., goat, anti-rabbit). The skilled person would understand any suitable combination of Fc and labelled secondary agent may be used. In certain embodiments, the aptamer region is conjugated to the Fc region using one or more primary amines (NH2), sulfhydryl groups (SH), azide, alkyne and/or carboxyl groups (COOH).

In certain embodiments, the presence, absence and/or level of the secondary agent is visualised by light and/or fluorescent microscopy.

In certain embodiments, the presence, absence and/or level of the secondary agent is detected by immunohistochemistry (IHC) as further described herein, wherein the aptamer-Fc conjugate replaces the use of a primary antibody in a standard workflow. As used herein, the term “standard workflow” refers to known steps to detect antigens in tissue sections using antibodies, as described, for example, by Magaki et al (Methods Mol Biol. 2019; 1897;289- 298 herein incorporated by reference.

In alternative embodiments also described herein, the sample instead comprises fixed cells seeded onto a solid support. Aptly, the sample is pre-treated with a nucleic acid-based blocking buffer as described herein. Aptly the presence, absence and/or level of the secondary agent is detected by immunocytochemistry (ICC), wherein the aptamer-Fc conjugate replaces the use of a primary antibody in a standard workflow. Aptly the aptamer-Fc conjugate is not a therapeutic agent and/or does not comprise any payload or cargo.

In one non-limiting example, the invention further provides an aptamer capable of specifically binding to CD4, wherein the aptamer comprises:

(a) a nucleic acid sequence selected from any one of SEQ ID NOs: 4 to 24;

(b) a nucleic acid having at least about 85%, 90%, 95%, 99% identity or more with any one of SEQ ID NOs: 4 to 24; or

(c) a nucleic acid sequence having at least about 20 consecutive nucleotides of any one of SEQ ID NOs: 4 to 24.

In some embodiments, the invention further provides an aptamer capable of specifically binding to CD4, wherein the aptamer comprises:

(a) a nucleic acid sequence selected from SEQ ID NO: 13 or 24; or

(b) a nucleic acid having at least about 85%, 90%, 95%, 99% identity or more with SEQ ID NO: 13 or 24; or

(c) a nucleic acid sequence having at least about 20 consecutive nucleotides of any one of SEQ ID NOs 13 or 24. Aptly, the nucleic acid sequence is (or has at least about 85%, 90%, 95%, 99% identity with) SEQ ID NO: 24.

The invention also provides an aptamer-Fc conjugate capable of specifically binding to CD4, wherein the aptamer comprises:

(a) a nucleic acid sequence selected from any one of SEQ ID NOs: 4 to 24; or

(b) a nucleic acid having at least about 85%, 90%, 95%, 99% identity or more with any one of SEQ ID NOs: 4 to 24; or

(c) a nucleic acid sequence having at least about 20 consecutive nucleotides of any one of SEQ ID NOs: 4 to 24.

In some embodiments, the invention further provides an aptamer-Fc conjugate capable of specifically binding to CD4, wherein the aptamer comprises:

(a) a nucleic acid sequence selected from SEQ ID NO: 13 or 24; or

(b) a nucleic acid having at least about 85%, 90%, 95%, 99% identity or more with SEQ ID NOs: 13 or 24; or

(c) a nucleic acid sequence having at least about 20 consecutive nucleotides of SEQ ID NO: 13 or 24.

Aptly, the aptamer of the aptamer-Fc conjugate comprises SEQ ID NO:24 (or a nucleic acid having at least about 85%, 90%, 95%, 99% identity with SEQ ID NO: 24).

Detailed Description of Certain Embodiments of the Invention

Brief Description of the Figures

Certain embodiments of the present invention will be described in more detail below, with reference to the accompanying Figures in which:

Figure 1 shows the processes used to identify the best performing aptamer candidates for further evaluation. Fluorescently labelled aptamer candidates are assessed for target binding on a cell surface, by flow cytometry (A). Example data for clone CD4_7S_11 (SEQ ID NO: 13) shows the response from the aptamer (blue histograms), compared to both the autofluorescence from the cells alone (red histograms), and any non-specific binding from the unselected control (green histogram), when incubated with CD4+ cells (right) or CD4- cells (left), This data was collected for each aptamer candidate. All datasets were then averaged, and the Mean Fluorescence Intensity (MFI) plotted as a % of the autofluorescence response (B). Data is plotted for each aptamer binding to both the CD4+ cells (blue bars) and CD4- cells (orange bars) to allow comparison of their binding response and selectivity. The best performing candidates (CD4_7S_1 (SEQ ID NO: 4) and CD4_7S_11 (SEQ ID NO: 13)) are highlighted with the dotted lines. The affinity of the best performing candidates was assessed by Biolayer Interferometry (C). Aptamer candidates were immobilised on streptavidin coated BLI probes, then incubated with a concentration gradient of recombinant CD4 protein (4.7 - 300nM). The binding response at each concentration is fit globally to a 1 :1 binding model (red overlays) and the binding affinity is calculated from the association and dissociation rates for each respective phase in the assay (0-120 sec and 120-240 sec).

Figure 2 shows the data for the identification of the minimal functional fragment of the best performing candidate aptamer (CD4_7S_11 (SEQ ID NO: 13)). A panel of fragments representing different lengths and regions of the full-length aptamer were synthesised and screened by flow cytometry and the Mean Fluorescence Intensity (MFI) plotted as a % of the autofluorescence response (A). Each fragment was assessed for binding to both CD4+ cells (blue bars) and CD4- cells (orange bars). Each fragment is compared to the full-length aptamer as a positive control, and a scrambled sequence (SEQ ID NO: 45) as a negative control. The best performing fragment (CD4_7S_11_F11 (SEQ ID NO: 24)) is highlighted with the dotted lines. The affinity of the best performing fragment was assessed by Biolayer Interferometry (B). The aptamer fragment was immobilised on streptavidin coated BLI probes, then incubated with a concentration gradient of recombinant CD4 protein (4.7 - 300nM). The binding response at each concentration is fit globally to a 1 : 1 binding model (red overlays) and the binding affinity is calculated from the association and dissociation rates for each respective phase in the assay (0-120 sec and 120-240 sec).

Figure 3 shows selective binding of identified aptamer fragments by fluorescence microscopy. Aptamer fragments were incubated with CD4+ cells (upper panels) or CD4- cells (lower panels) to assess selectivity. The aptamer fragment (CD4_7S_11_F11 (SEQ ID NO: 24)) shows binding to CD4+ cells (upper left) but not CD4- cells (lower left); demonstrating selectivity. A scrambled sequence (SEQ ID NO: 45) was also assessed as a negative control and shows no binding to either cell line (right panels). Cy3 labelled aptamer fragments show as pink, Cells are also counter stained with DAPI nuclear stain (blue) to show the presence of cells in each sample.

Figure 4 shows immunofluorescence detection of CD4 positive cells in FFPE tonsil tissue, using a Cy3-labelled aptamer. Specifically, Figure 4 shows Immunohistochemistry analysis of formalin/PFA-fixed paraffin-embedded human tonsil tissue section, labelling CD4 positive cells with aptamer-Cy3 at 8pM concentration. Sections were treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 15 mins before incubation with the labelled aptamer, for 1 hour. DAPI was used as a nuclear counterstain. The immunostaining was performed manually. Image acquisition was performed with EVOS microscope. The immunofluorescence staining analysis demonstrates specific binding of Cy3 labelled CD4 aptamer to the intended CD4 cells on FFPE tonsil tissues showing as pink/red staining (a-b). Nuclei counterstaining is shown as blue. No specific aptamer staining seen on the negative tissue control (c).

Figure 5 shows IHC process using aptamer conjugated to rabbit-Fc antibody fragment. Specifically, Figure 5 shows a comparative illustration of traditional IHC with primary and secondary antibodies (left) and aptamer-Fc conjugate binding to target protein and being detected with standard secondary antibody (right).

Figure 6 shows example approach for conjugation of aptamer to rabbit-Fc antibody fragment. Specifically, Figure 6 shows an example process outline for preparation of aptamer-Fc conjugates. (A) Amine modified aptamer is treated with NHS-azide, to produce an azide functionalised aptamer. (B) Rabbit-Fc fragment is treated with NHS-DBCO, to produce a DBCO functionalised rabbit-Fc fragment. (C) Azide functionalised aptamer is incubated with DBCO functionalised rabbit-Fc fragment, to produce the aptamer-Fc conjugate.

Figure 7 shows Biolayer Interferometry (BLI)-based assessment of aptamer-Fc binding. BLI analysis shows that the aptamer-Fc retains ability to bind immobilised CD4 and simultaneously allows detection by a standard secondary antibody (red trace). No binding of secondary antibody is seen in absence of CD4 protein (violet trace), absence of aptamer-Fc conjugate (pale blue trace), absence of Fc (purple trace) or absence of secondary antibody (dark red trace). Taken together, these data shows that the aptamer-Fc conjugate can recognise immobilised CD4 and is in turn bound by secondary antibody, as required for IHC.

Figure 8 shows Immunocytochemistry (ICC) analysis of Optimer-Fc staining CD4 expressing cells: ICC using DAB stain was performed on CD4 specific cells (H9) and a CD4 counter cell line (D1.1) cultured onto coverslips. After blocking for endogenous peroxidase activity and non-specific backgrounds, Antibody/Optimer-Fc staining was performed overnight at 4 °C. Cells were stained with DAB for 2 mins and counterstained with hematoxylin for 1 minute followed by washing. Coverslips were mounted onto glass slides with aqueous mounting media. DAB staining analysis demonstrates that CD4 specific stain was obtained with Anti- CD4 antibody and CD4 Optimer-Fc conjugate as expected. No staining in the control coverslips (c) and (f) as expected. Background binding was observed with D1.1 stained with Optimer-Fc; however, more blocking steps would readily eliminate the nonspecific binding. Note: coverslip for H9 CD4 antibody was stuck to the well hence the phase contrast when imaged. Coverslip (d) could not be imaged.

Figure 9 shows chromogenic detection of CD4 positive cells in frozen human tonsil tissue using aptamer-Fc conjugates. Specifically, Figure 9 shows an immunohistochemistry analysis of frozen human tonsil tissue labelling CD4 with aptamer-Fc. Slides were treated with precooled acetone for 10 mins then washed with PBS. After blocking for endogenous peroxidase activity and non-specific background binding sites, aptamer-Fc conjugate staining was performed overnight at 4°C. Tissue were stained with DAB for 2 mins and counterstained with hematoxylin for 1 minute. Tissues were dehydrated using increasing ethanol graduate and clarified with 2 washes of xylene before cover-slipping. DAB staining analysis demonstrates comparable specific binding of CD4 aptamer-Fc (a) and anti CD4 antibody (b), to the intended CD4 cells on FFPE tonsil tissues. No CD4 staining seen on the negative tissue control (c, d).

Figure 10 shows chromogenic detection of CD4 positive cells in FFPE human tonsil tissue using aptamer-Fc staining. Specifically, Figure 10 shows IHC-FFPE staining of CD4 cells using aptamer-Fc. Chromogenic staining analysis demonstrates specific binding of CD4 aptamer-Fc to the intended CD4 cells on FFPE human tonsil tissues (a-c) as well as on gut- associated lymphoid tissue (d). Commercially available CD4 antibody was used as positive control (e) while no staining seen on the negative controls (f-h) as expected.

Sequence listing

SEQ ID NO: 1 shows a N40B Forward Primer: CCAGTGTAGACTACTCAATGC

SEQ ID NO: 2 shows a N40B Reverse Primer: GGTTGACCTGTGGATAGTAC

SEQ ID NO: 3 shows a N40B Reverse Primer binding region: GTACTATCCACAGGTCAACC

SEQ ID NO: 4 shows the full nucleic acid sequence of Aptamer CD4_7S_1 : CCAGTGTAGACTACTCAATGCGTGGGAAGGGTGGGTGGGAGCATTGATAACCCTGATA GTACTATCCACAGGTCAACC

SEQ ID NO: 5 shows the full nucleic acid sequence of Aptamer CD4_7S_1 : CCAGTGTAGACTACTCAATGCGTGGGAAGGGTGGGTGGGAGCATTGATAACCCTGATA GTACTATCCACAGGTCAACC

SEQ ID NO: 6 shows the full nucleic acid sequence of Aptamer CD4_7S_2: CCAGTGTAGACTACTCAATGCTGTGTTGACTTGATCCTGTGGTATATGGGTGGGAGGGT CGGGTACTATCCACAGGTCAACC SEQ ID NO: 7 shows the full nucleic acid sequence of Aptamer CD4_7S_3: CCAGTGTAGACTACTCAATGCTGTGTTGACTTGATCCTGTGGTATATGGGTGGGAGGGT TGGG TACTATCCACAGG TCAACC

SEQ ID NO: 8 shows the full nucleic acid sequence of Aptamer CD4_7S_4: CCAGTGTAGACTACTCAATGCTGTGTTGACTTGACCTTGGATTATGGGTTTGGGCGGGC GGGTACTATCCACAGGTCAACC

SEQ ID NO: 9 shows the full nucleic acid sequence of Aptamer CD4_7S_6: CCAGTGTAGACTACTCAATGCAGGGTGGGAGGGAGGGTATTGCATTGCCTAATCCAGG GTAGTACTATCCACAGGTCAACC

SEQ ID NO: 10 shows the full nucleic acid sequence of Aptamer CD4_7S_7: CCAGTGTAGACTACTCAATGCGTGGGAAGGGTGGGCGGGAGCATTGATAACTCGGAG GAGCGTACTATCCACAGGTCAACC

SEQ ID NO: 11 shows the full nucleic acid sequence of Aptamer CD4_7S_8: CCAGTGTAGACTACTCAATGCCGATGGGTCGGGTGGGTGGGTAGGCATTGATCGCTCC TCCGTACTATCCACAGGTCAACC

SEQ ID NO: 12 shows the full nucleic acid sequence of Aptamer CD4_7S_10: CCAGTGTAGACTACTCAATGCAGGGTGGGAGGGAGGGTATTGCATTGCCTAATCGAGG GTAGTACTATCCACAGGTCAACC

SEQ ID NO: 13 shows the full nucleic acid sequence of Aptamer CD4_7S_11 : CCAGTGTAGACTACTCAATGCGATGTGGGATGGGTGGGTTGGGTTCGCATTTTGGCCT ATAGTACTATCCACAGGTCAACC

SEQ ID NO: 14 shows the full nucleic acid sequence of Aptamer CD4_7S_19: CCAGTGTAGACTACTCAATGCGGCTGTGTGACTTGACCTCTGGATATGGGTGGGAGGG ATGGGTACTA TCCACAGGTCAACC

SEQ ID NO: 15 shows the full nucleic acid sequence of Aptamer CD4_7S_20: CCAGTGTAGACTACTCAATGCGGCTGTGTTGACTTGACCTTGGATTATGGGTTTGGGTG GGTGGGTACTATCCACAGGTCAACC

SEQ ID NO: 16 shows the full nucleic acid sequence of Aptamer CD4_T8R_11: CCAGTGTAGACTACTCAATGCCGTGGACTGGTCGGGTTTGGATTCGGCAGATGAATCA GTAGTACTATCCACAGGTCAACC

SEQ ID NO: 17 shows the full nucleic acid sequence of Aptamer CD4_T8R_17: CCAGTGTAGACTACTCAATGCTCCTATTCCGTATAGTACGTTAGGTTGGGTAGGTTGGT ACGTACTATCCACAGGTCAACC

SEQ ID NO: 18 shows the full nucleic acid sequence of Aptamer CD4_9S_1 : CCAGTGTAGACTACTCAATGCTCGACATTTCCGCCCCGACGGCCCTCCTAGTGATGGG GAGAGTACTATCCACAGGTCAACC

SEQ ID NO: 19 shows the full nucleic acid sequence of Aptamer CD4_9S_2: CCAGTGTAGACTACTCAATGCCGATGGGTCGGGGGGGTGGGTAGGCATTGATCGCTC CTTTCGTACTATCCACAGGTCAACC SEQ ID NO: 20 shows the full nucleic acid sequence of Aptamer CD4_9S_4: CCAGTGTAGACTACTCAATGCTGTGTTGACTTGATCCTGTGGTATATGGGTGGGAGGG ATGGGTACTA TCCACAGGTCAACC

SEQ ID NO: 21 shows the full nucleic acid sequence of Aptamer CD4_9S_14: CCAGTGTAGACTACTCAATGCCGATGGGTCGGGGGGTGGGTAGGCATTGATCGCTCCT TTCG TACTA TCCACAGGTCAACC

SEQ ID NO: 22 shows the full nucleic acid sequence of Aptamer CD4_9S_19: CCAGTGTAGACTACTCAATGCGGCTTCGGGAGGGGGGGCGGGTAAAAAGCCCATTGC CCTAGTACTATCCACAGGTCAACC

SEQ ID NO: 23 shows the full nucleic acid sequence of Aptamer CD4_9S_48: CCAGTGTAGACTACTCAATGCAGGGTGGGAGGGAGGGTATTGCATTGCCTAATTCAGG GTAGTACTATCCACAGGTCAACC

SEQ ID NO: 24 shows a minimal fragment (Optimer; CD4 7S_11_F11) of Aptamer 7S_11:

CCAGTGTAGACTACTCAATGCGATGTGGGATGGGTGGGTTGGGTTCGCATTTT

SEQ ID NO: 25 shows a randomised region of Aptamer CD4_7S_1 :

G TGGGAAGGGTGGGTGGGAGCA TTGA TAACCCTGA TA

SEQ ID NO: 26 shows a randomised region of Aptamer CD4_7S_1 :

G TGGGAAGGGTGGGTGGGAGCA TTGA TAACCCTGA TA

SEQ ID NO: 27 shows a randomised region of Aptamer CD4_7S_2:

TG TG TTGACTTGA TCCTG TGG TA TA TGGG TGGGAGGGTCGG

SEQ ID NO: 28 shows a randomised region of Aptamer CD4_7S_3:

TG TG TTGACTTGA TCCTG TGG TA TA TGGG TGGGAGGGTTGG

SEQ ID NO: 29 shows a randomised region of Aptamer CD4_7S_4:

TG TG TTGACTTGACCTTGGA TTA TGGG TTTGGGCGGGCGG

SEQ ID NO: 30 shows a randomised region of Aptamer CD4_7S_6:

AGGGTGGGAGGGAGGGTATTGCATTGCCTAATCCAGGGTA

SEQ ID NO: 31 shows a randomised region of Aptamer CD4_7S_7:

GTGGGAAGGGTGGGCGGGAGCATTGATAACTCGGAGGAGC

SEQ ID NO: 32 shows a randomised region of Aptamer CD4_7S_8:

CGATGGGTCGGGTGGGTGGGTAGGCATTGATCGCTCCTCC

SEQ ID NO: 33 shows a randomised region of Aptamer CD4_7S_10:

AGGGTGGGAGGGAGGGTATTGCATTGCCTAATCGAGGGTA

SEQ ID NO: 34 shows a randomised region of Aptamer CD4_7S_11:

GATGTGGGATGGGTGGGTTGGGTTCGCATTTTGGCCTATA

SEQ ID NO: 35 shows a randomised region of Aptamer CD4_7S_19:

GGCTG TG TGACTTGACCTC TGG A TA TGGG TGGGAGGGA TGG

SEQ ID NO: 36 shows a randomised region of Aptamer CD4_7S_20:

GGCTGTG TTGACTTGACCTTGGA TTA TGGG TTTGGGTGGG TGG SEQ ID NO: 37 shows a randomised region of Aptamer CD4_T8R_11 :

CGTGGACTGGTCGGGTTTGGATTCGGCAGATGAATCAGTA

SEQ ID NO: 38 shows a randomised region of Aptamer CD4_T8R_17:

TCCTATTCCGTATAGTACGTTAGGTTGGGTAGGTTGGTAC

SEQ ID NO: 39 shows a randomised region of Aptamer CD4_9S_1 :

TCGACATTTCCGCCCCGACGGCCCTCCTAGTGATGGGGAGA

SEQ ID NO: 40 shows a randomised region of Aptamer CD4_9S_2:

CGATGGGTCGGGGGGGTGGGTAGGCATTGATCGCTCCTTTC

SEQ ID NO: 41 shows a randomised region of Aptamer CD4_9S_4:

TGTGTTGACTTGA TCCTGTGG TA TATGGG TGGGAGGGA TGG

SEQ ID NO: 42 shows a randomised region of Aptamer CD4_9S_14:

CGATGGGTCGGGGGGTGGGTAGGCATTGATCGCTCCTTTC

SEQ ID NO: 43 shows a randomised region of Aptamer CD4_9S_19:

GGCTTCGGGAGGGGGGGCGGGTAAAAAGCCCATTGCCCTA

SEQ ID NO: 44 shows a randomised region of Aptamer CD4_9S_48:

AGGGTGGGAGGGAGGGTATTGCATTGCCTAATTCAGGGTA

SEQ ID NO: 45 shows a scrambled control sequence:

CGGGA TG TTTTA TCTAAACACAA TGAGAGGAGTA TTCC TOT A

SEQ ID NO: 46 shows full length sequence of CD4 (Uniprot accession number: P01730; NCBI Accession number: NP_000607.1):

MNRGVPFRHLLLVLQLALLPAATQGKKVVLGKKGDTVELTCTASQKKSIQFHWKNSNQIKIL GNQGSFLTKGPSKLNDRADSRRSLWDQGNFPLIIKNLKIEDSDTYICEVEDQKEEVQLLVFG LTANSDTHLLQGQSLTLTLESPPGSSPSVQCRSPRGKNIQGGKTLSVSQLELQDSGTWTCT VLQNQKKVEFKIDIVVLAFQKASSIVYKKEGEQVEFSFPLAFTVEKLTGSGELWWQAERASS SKSWITFDLKNKEVSVKRVTQDPKLQMGKKLPLHLTLPQALPQYAGSGNLTLALEAKTGKL HQEVNLVVMRATQLQKNLTCEVWGPTSPKLMLSLKLENKEAKVSKREKAVWVLNPEAGM WQCLLSDSGQ VLLESNIKVLPTWSTPVQPMALIVLGG VAGLLLFIGLGIFFC VRCRHRRRQA

ERMSQIKRLLSEKKTCQCPHRFQKTCSPI

Detailed Description

Further features of certain embodiments of the present invention are described below.

The practice of embodiments of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology and immunology, which are within the skill of those working in the art. Most general molecular biology, microbiology recombinant DNA technology and immunological techniques can be found in Sambrook et al, Molecular Cloning, A Laboratory Manual (2001) Cold Harbor- Laboratory Press, Cold Spring Harbor, N.Y. or Ausubel et al., Current protocols in molecular biology (1990) John Wiley and Sons, N.Y. Immunohistochemical techniques are described, for example, in Antibodies: A Laboratory Manual by Ed Harlow (Editor), David Lane (Editor) (1988, Cold Spring Harbor Laboratory Press, ISBN 0-87969-314-2), 1855; Price and Newman, “Principles and Practice of Immunoassay,” 2^nd Edition, Grove's Dictionaries (1997); and Gosling, “Immunoassays: A Practical Approach,” Oxford University Press (2000).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei- Show, 2^nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3^rd ed., Academic Press; and the Oxford University Press, provide a person skilled in the art with a general dictionary of many of the terms used in this disclosure.

Units, prefixes, and symbols are denoted in their Systeme International de Unitese (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation and nucleic acid sequences are written left to right in 5’ to 3’ orientation.

In the following, the invention will be explained in more detail by means of non-limiting examples of specific embodiments. In the example experiments, standard reagents, and buffers free from contamination are used.

Target molecule

In certain embodiments, the invention provides a method for detecting one or more target molecule(s). For example, the methods described herein may be used to detect the presence or absence and/or quantify the amount of at least one target molecule in a sample.

The term "target molecule" as used herein denotes a molecule which may be found in a tested sample, and which is capable of binding to an aptamer region of an aptamer-Fc conjugate as described herein.

In certain embodiments, the target molecule is an organic or biologically active molecule. Aptly, the target molecule is an antigen. For example, the target molecule may be a soluble antigen, a cell-surface antigen, or an antigen associated with a micelle, a liposome, or a particle. In some embodiments, the target molecule may be a protein, a polypeptide, a peptide, a ganglioside, a lipid, a hapten, an epitope, an antibody, a nucleic acid, a nucleotide, a ribonucleotide, a deoxyribonucleotide, a nanoparticle, an amino acid, a phospholipid, a carbohydrate, a steroid, a proteoglycan, a carbohydrate, or a small molecule.

In certain embodiments, a soluble antigen may be a protein, a peptide, an enzyme, a cytokine, a soluble cancer marker, an inflammation-associated marker, a hormone and/or a soluble molecule derived from a virus, bacteria or a fungus for example, a toxin or an allergen. Nonlimiting examples of a cell surface antigen in accordance with the invention are a receptor, a cell surface marker, a micro-organism associated antigen, or a receptor ligand.

In certain embodiments, the target molecule is a diagnostically relevant target. The presence, absence or level of such target molecules may provide an indication of the health or disease state of the cell, tissue, and/or individual from which the sample may be derived. The presence of the target molecule may be diagnostic, or it may serve as merely an indicator, which together with other indicators, for example a panel of indicators, points to the likelihood of one or more diseases. The quantity of the target molecule in the cell or tissue may be significant, i.e. , whether it is above or below a threshold level which is indicative of disease.

In some embodiments, the antigen is a micro-organism associated antigen. As used herein, the term “micro-organism associated antigen” is to be understood as a protein or fragment thereof encoded by the viral, bacterial, fungal, or protozoan genome (e.g., a pathogenic antigen).

In some embodiments, the antigen is a bacteria or bacterial antigen. For example, the bacteria may be C. trachomatis, B. quintana, Y. pestis, T. pallidum, Staphylococci, Streptococci or Enterococci. In some embodiments, the antigen is a virus or viral antigen. For example, the virus may be Human Herpesviruses (HHV), Human Immunodeficiency Virus (HIV), MERS- CoV, enterovirus, hepatitis C, arbovirus, hepatitis E, severe acute respiratory syndrome (SARS), SARS-CoV-2, Zika virus, West Nile virus, adenoviruses, or hantavirus. In some embodiments, the antigen is a fungus or a fungal antigen. For example, the fungus may be pythiosi, associated with fungal sinusitis, aspergillus, sporothrix, Candida albicans or Cryptococcus. In some embodiments, the antigen is a protozoa or a protozoan antigen. For example, the protozoa may be leishmania, toxoplasma, tapeworm, plasmodium or trypanosoma.

In certain embodiments, the antigen is a cancer (or tumour) marker. In general, a tumour marker may be found in the body fluids such as in blood or urine, or in body tissues such as a tissue section or biopsy. Tumour markers may be expressed or over expressed in cancer and are generally indicative of a particular disease process. Non-limiting examples of tumour antigens in accordance with the invention include tumor-specific antigens (TSAs) (e.g., neoantigens or vial antigens), tumour-associated antigens (TSAs) (e.g., carcinoembryonic antigen (CSA), prostate-specific antigen (PSA), or human-epidermal growth factor receptor 2 (HER2/neu), Cancer-Testis Antigens (CTAs) (e.g., melanoma antigen gene (MAGE) or Esophageal Squamous Cell Carcinoma-1 (ESO-1), differentiation antigens (e.g., CD19 or CD20), oncofetal antigens (e.g., alpha-fetoprotein (AFP) or human chorionic gonadotropin (hCG), glycoprotein antigens (CA-125 or CA15-3), mucin antigens (e.g., MLIC1 or MLIC16) or hormone receptors (e.g., estrogen receptor (ER) or androgen receptor (AR)). It will be appreciated that nucleic acids encoding any protein marker may also be used as a detectable marker.

In certain embodiments, CEA is associated with digestive tract cancers (e.g., colon). PSA or AR are typically associated with prostate cancer, enlarged prostate conditions (e.g., BPH) or prostasis. HER2 is typically associated with breast cancer. Metastatic patients who overexpress HER2 may benefit from treatment with anti-HER2 antibodies (e.g., Herceptin). Elevated CA125 values may be associated with ovarian cancer, endometriosis, ovarian cysts or pelvic inflammatory disease. CA15-3 may be associated with breast cancer or cirrhosis.

In certain embodiments, the tumour antigen is selected from one or more of Melan-A, S100, Chromogranin A (CgA), CDX2, Hep Par-1 , Napsin, thyroid transcription factor 1 (TTF1), cytokeratin 20 (CK20), carcinoembryonic antigen (CEA), Villin, CA125, p63, androgen receptor, cytokeratin 7(CK7), cytokeratin 19 (CK19), epithelial membrane antigen (EMA), cytokeratin 18 (CK18), cyclooxygenase-2 (COX-2), synaptophysin (SY38), CD3, CD4, CD8, CD13, CD15, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD45, CD45RO, CD56, CD99, CD133, CD235a, BerEP4, neuron specific enolase, glial fibrillary acidic protein, insulin receptor, platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptors VEGFR-1 , VEGFR-2 and/or VEGFR-3, M30, Bcl-2, p52, caspase, Fas, Kappa and Lambda light chains, Factor VIII, prostate-specific antigen (PSA), prostate specific alkaline phosphatase (PSAP), p53, c-erbB-2, matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF), B-cell immunoglobulin, B-cell lymphoma 2 (BCL2), bladder tumour antigen (BTA), c-kit/CD117, BRCA1 , BRCA2, Chromosome 17p deletion, Cyclin D1 , des-gamma-carboxy prothrombin (DCP), dihydropyrimidine dehydeogenase (DPD), epidermal growth factor receptor (EGFR), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), fms-like tyrosine kinase 3 (FLT3), Gastrin, HE4, 5-hydroxyindoleacetic acid (5-HIAA), IDH1 JDH2, IRF4, JAK2, KRAS, microsatellite instability (MSI), MYC, MYD88, myeloperoxidase (MPO), neuron-specific enolase (NSE), neurotrophic receptor kinase (NTRK), nuclear matrix protein 22, prostate cancer antigen 3 (PCA3), PML/RARa fusion, prostatic acid phosphatase (PAP), Programmed death ligand 1 (PD-L1), Programmed death 1 (PD-1), CTLA4, ROS1 , soluble mesothelin-related peptides (SMRP), somatostatin receptor, T-cell receptor, terminal transferase (TdT), Thiopurine S- methyltransferase (TPMT), thyroglobulin (TG), UGT1A1*28 variant, urine catecholamines (VMA and/or HVA), Anaplastic lymphoma kinase (ALK), alpha-fetoprotein (AFP), Beta-2- microglobulin (B2M), beta-human chorionic gonadotropin (Beta-hCG), BCR-ABL fusion, BRAF V600, CA15-3, CA27.29, CA19-9, CA-125.CA 27.29, Calcitonin, , cytokeratin fragments 21-1 , estrogen receptor (ER), progesterone receptor (PR), fibrin/fibrinogen, HE4, HER2/neu, HER3, HER4, hormone receptor (HR), KIT, lactate dehydrogenase, nuclear matrix protein 22, proliferating cell nuclear antigen (PCNA), Cadherin E, p21 , p27, Rb, protease inhibitor 6 (Pl- 6), urokinase plasminogen activator (uPA), plasminogen activator inhibitor (PAI-1), transthyretin (TTR), Apoplipoprotein A1 (ApoA1), Desmin, Actin, Vimentin (VIM), Collagen type IV, S-100, HMB45, pl.6, Ki-67 and/or Thomsen-Friedenreich (TF).

In certain embodiments, the target molecule is a small molecule. In certain embodiments, the small molecule is a therapeutic agent for example a chemotherapeutic agent which is for use in the treatment of cancer. In some non-limiting examples, the target molecule is CD4 as further described herein.

Sample

In certain embodiments, the target molecule is comprised in a sample. The sample may comprise whole blood, leukocytes, peripheral blood mononuclear cells, plasma, serum, sputum, breath, urine, semen, saliva, meningial fluid, amniotic fluid, glandular fluid, lymph fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, cells, a cellular extract, stool, tissue sample such as a biopsy or other tissue section, or cerebrospinal fluid.

The sample may comprise blood, serum, interstitial fluid, spinal fluid, cerebral fluid, tissue exudates, macerated tissue samples, cell solutions, intracellular compartments, or other biological samples. The sample may comprise cells grown in a monolayer or cells in suspension which are deposited on a slide. Samples may be unaltered or may be pretreated prior to analysis, for example being chemically fixed e.g. with formalin, and embedded in a preservative such as paraffin wax, frozen, filtered, diluted, concentrated, buffered, or otherwise treated. In certain embodiments, the sample is treated by heating, dewaxing, chemical denaturation, ultrasound, enzyme digestion, denaturant, detergent and/or oxidizing.

A sample is aptly a material provided or sampled which is believed to contain one or more target molecules of interest and which may be checked for the presence of the target.

The sample may be for example a clinical sample, a food sample, a water sample, or a sample of other environmental sampled material. In certain embodiments, the sample comprises a target molecule and a buffer solution. In certain embodiments, the sample is pre-treated, such as by mixing, addition of enzymes or markers, or purified.

In certain embodiments, the sample may be any biological material isolated from individuals, for example, biological tissues and fluids, which include, but are not limited to: Count blood, skin, plasma, serum, lymph, urine, cerebrospinal fluid, tears, swabs, tissue samples or biopsies, organs, and tumours. In certain embodiments, components of cell cultures are also included in samples.

Aptly, the sample comprises a cell and/or tissue of an individual suspected of suffering a disease or condition. Aptly, the sample comprises a cell and/or tissue of known origin and known to contain cells of tissues associated with a disease or condition, used for research applications.

Aptly the sample is a tissue and/or cell sample. For example, the sample may comprise intact (or substantially intact) tissue. Typically, the method is ex vivo. As used herein the term “ex vivo" relates to methods performed on tissues or organs extracted from a living organism. Ex vivo methods enable the study of a heterogeneous matrix, which provides a more natural, in vivo-like environment in order to study cell behaviour and function. For example, whole tissue slices retain the cytoarchitecture, as well as many of the intercellular connections and interplays.

Alternatively, the sample may comprise cultured and/or isolated cells. Typically, the method is in vitro. As used herein the term “in vitro" relates to methods performed on cells (isolated or cell lines) cultured in a highly controlled, non-living environment (e.g., glass coverslips). In certain embodiments, the sample is a tissue sample. For example, the sample comprises intact tissue. Aptly the method is ex vivo. As used herein the terms “tissue” and “intact tissue” are used interchangeably to refer to a multicellular ex vivo sample that preserves the cross- sectional spatial relationship between the cells as they existed within the subject from which the sample was obtained. An intact tissue sample can be obtained from any part of a subject or patient as described herein. For example, the four main mammalian tissues are (1) epithelium; (2) connective tissues, including blood vessels, bone and cartilage; (3) muscle tissue; and (4) nerve tissue. Aptly the source of the intact tissue sample may be solid tissue from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate.

Aptly the subject or patient from which the sample was obtained is healthy. Aptly the subject or patient from which the sample was obtained is suffering from a disease or condition or suspected of suffering from a disease or condition. Aptly the intact tissue sample may be from diseased tissue or non-diseased tissue (e.g., from a tumour). The intact tissue sample may be known to contain cells of tissues associated with a disease or condition, used for research applications.

Aptly intact tissue samples can include primary tissue samples (i.e. tissues produced by the subject) and xenografts (i.e. foreign cellular samples implanted into a subject).

Intact tissue samples can be processed by any methods known in the art compatible with methods of detecting a target molecule of interest (e.g., immunohistochemical techniques).

Aptly, the tissue sample may be a biopsy, or a section thereof, obtained from the individual. A tissue sample, such as a biopsy, can be obtained through a variety of sampling methods known to those skilled in the art, including a punch biopsy, shave biopsy, wide local excision, and other means. For example, a tumour sample may be taken from a surgical site from which the tumour is excised from an individual.

In certain embodiments, the sample is a preparation of fresh tissue and/or cells (e.g., not fixed with aldehyde-based fixative). Such samples may include, for example, biopsy materials (e.g., frozen section), cytological preparations (e.g., blood smear) and any tissue to be analysed by histochemistry. Such samples may be mounted onto a solid support (e.g., slide or cover slip), or frozen and sectioned onto slides. In certain embodiments, the sample is fixed. For example, the sample may be contacted with an alcohol or acetone-based fixative. As used herein, “mounting” a sample onto a solid support is understood to mean placing or attaching the cells or tissue to a substantially planar support. Any suitable support may be used, for example, to enable viewing the cells or tissue by a microscope. The support may be a glass slide, a membrane, a filter, a polymer slide, a chamber slide, a petri-dish, or the like.

In certain embodiments, the sample is a fixed, paraffin embedded tissue specimen. Such “FFPE” samples are fixed, usually using a formalin-based fixative, dehydrated (e.g., using xylene), embedded in a suitable embedding medium such as paraffin wax or plastic, sectioned onto a slide, deparaffinised, or otherwise treated and re-hydrated.

In certain embodiments, the sample is subjected to a variety of well-known post-collection preparative and storage techniques (e.g., nucleic acid and/or protein extraction, fixation, storage, freezing, ultrafiltration, concentration, evaporation, centrifugation, etc.).

In certain embodiments, the sample is purified or concentrated, or cells may be isolated prior to analysis. For example, the cells may be seeded onto a solid support, and fixed using techniques such as cross-linking and/or precipitation (typically using an organic solvent). The cells may be permeabilised, optionally using a detergent or organic fixative as further described herein.

Aptamers

As used herein, the terms “aptamer” or “nucleic acid molecule” are used interchangeably to refer to a non-naturally occurring nucleic acid molecule that has a desirable action on, or interaction with a target molecule. For nucleic acid aptamers, for example, a distinction is made between DNA aptamers formed from single-stranded DNA (ssDNA) and RNA aptamers formed from single-stranded RNA (ssRNA), or chemical modifications thereof, including either backbone or base modifications. Typically, the aptamer has high binding affinity to the target molecule.

In certain embodiments, the aptamer is an Optimer™. As such, the invention encompasses both aptamer-Fc conjugates (e.g., comprising full-length aptamer sequences) and Optimer-Fc conjugates (e.g., comprising Optimer sequences).

As used herein, the term “Optimer” relates to a minimal functional aptamer fragment. This is understood to mean a fragment (e.g., portion) of the full-length aptamer capable of binding to target molecule with at least the same (or improved) specificity and/or affinity as compared to the full-length aptamer. For example, the Optimer may be about 5% to about 95%, about 10% to about 90% or about 20% to about 80% of the size of the full-length aptamer. Advantageously, the small size of Optimers may provide assay benefits, including, for example, increased tissue penetration and access to epitopes. The small size of an Optimer also gives advantages in reliability and scalability of manufacture and further reductions in batch-to- batch variability. As used herein, the term “Optimer-Fc conjugate” refers to a complex formed by conjugating an Optimer with an Fc region of an antibody.

In certain embodiments, a minimal effective fragment may compete for binding to the target molecule with the full-length aptamer. By way of example, a panel of fragments representing different regions of the full-length aptamer may be produced by solid phase synthesis (incorporating a 5' biotin group or other appropriate functional group known to those skilled in the art). Each of the individual fragments may then be immobilised onto a separate streptavidin coated Biolayer Interferometry (BLI) sensor probe (or other appropriately functionalised BLI sensor probe), and the interaction with the buffered target molecule monitored using a BLI- based binding assay. A BLI screen may show which fragments retain their binding affinity and which fragments have lost their binding function. These binding and non-binding fragments may then be mapped onto the full-length aptamer sequence to identify the minimal functional fragment (Optimer™).

Aptamers are characterised by the formation of a specific three-dimensional structure that depends on the nucleic acid sequence. The three-dimensional structure of an aptamer arises due to Watson and Crick intramolecular base pairing, Hoogsteen base pairing (quadruplex), wobble pair formation or other non-canonical base interactions. This structure enables aptamers, analogous to antigen-antibody binding, to bind target structures accurately. A particular nucleic acid sequence of an aptamer may, under defined conditions, have a three-dimensional structure that is specific to a defined target structure.

The nucleic acid aptamers described herein may comprise natural or non-natural nucleotides and/or or base derivatives (or combinations thereof). In certain embodiments, the nucleic acid molecule comprises one or more modifications such that it comprises a chemical structure other than deoxyribose, ribose, phosphate, adenine (A), guanine (G), cytosine (C), thymine (T), or uracil (II). The nucleic acid molecule may be modified at the nucleobase, at the pentose or at the phosphate backbone.

In certain embodiments, the nucleic acid molecule comprises one or more modified nucleotides. Exemplary modifications include for example nucleotides comprising an alkylation, arylation or acetylation, alkoxylation, halogenation, amino group, or another functional group. Examples of modified nucleotides include 2'-fluoro ribonucleotides, 2'-NH 2 -, 2'-OCH 3 - and 2'-O-methoxyethyl ribonucleotides, which are used for RNA aptamers. The nucleic acid molecule may be wholly or partly phosphorothioate or DNA, phosphorodithioate or DNA, phosphoroselenoate or DNA, phosphorodiselenoate or DNA, locked nucleic acid (LNA), peptide nucleic acid (PNA), N3'-P5 'phosphoramidate RNA I DNAcyclohexene nucleic acid (CeNA), tricyclo DNA (tcDNA) or spiegelmer, or the phosphoramidate morpholine (PMO) components (see also Chan et al., Clinical and Experimental Pharmacology and Physiology (2006) 33, 533-540).

Some of the modifications allow nucleic acid molecules to be stabilized against nucleic acid cleaving enzymes. In the stabilization of the aptamers, a distinction can generally be made between the subsequent modification of the aptamers and the selection with already modified RNA I DNA. The stabilization does not affect the affinity of the modified RNA I DNA aptamers but prevents the rapid decomposition of the aptamers in an organism or biological solutions by RNases I DNases. An aptamer is referred to as stabilized in the context of the present invention if the half-life in biological sera is greater than one minute, preferably greater than one hour, more preferably greater than one day.

Aptamers to a target molecule may be selected using known processes. For example, an aptamer can be prepared by using the SELEX method or any related in vitro selection approach and an improved method thereof (e.g., Ellington & Szostak, (1990) Nature, 346, 818-822; Tuerk & Gold, (1990) Science, 249, 505-510). In the SELEX method, by setting strict selection conditions by increasing the number of rounds or using a competing substance, an aptamer exhibiting a stronger binding potential for the target molecule is enriched, isolated, and selected. Hence, by adjusting the number of rounds of SELEX and/or changing the competitive condition, aptamers with different binding forces, aptamers with different binding modes, and aptamers with the same binding force or binding mode but different base sequences can be obtained in some cases.

The in vitro selection method comprises a process of enriching a diverse starting library with target binding sequences through iterative rounds of target binding, recovery, and preferential amplification. The variability of a library is for example in the range of about 10¹² to 10¹⁵ different molecules. Starting from the single-stranded DNA or RNA nucleic acid molecule library, the nucleic acid molecules are interacted with the target molecule of interest. Those library members which bind best to the target, are recovered, and amplified by PCR (or RT- PCR for an RNA library). Additional diversity may be introduced into the library during the in vitro selection process by causing a mutation by using manganese ions and the like in the process. Target binding sequences are enriched cycle by cycle through various selection and amplification steps. Each aptamer selection cycle typically comprises the following sub steps: a) binding of nucleic acid molecule library to target; b) separating target-bound from unbound nucleic acid molecules; c) recovery of target-binding nucleic acid molecules; d) amplification of recovered nucleic acid molecules (e.g. PCR for DNA molecules, reverse transcription PCR for RNA molecules); and e) preparation of relevant single stranded nucleic acids from the amplified product (e.g. ssDNA purification, in vitro RNA transcription).

After each cycle, the selected and enriched nucleic acid molecule pool is used as the starting material for a next cycle. Typically, 8 to 12 cycles are run through although this number varies depending on the target type, method, and efficiency of selection.

In certain embodiments, the target used in the interaction with the nucleic acid may comprise one or more forms of the target, including, but not limited to, recombinant proteins or peptides thereof, cells expressing the protein of interest, antigen, or an unknown biomarker or tissues known to contain cells of interest. The cell or tissue sample may also be modified in a way to reflect the end application, for example through chemical fixation, paraffin wax embedding and/or an appropriate antigen retrieval method.

In certain embodiments, the method comprises analysing the nucleic acid sequence of an aptamer which has been identified as binding to the target molecule with high binding affinity. After analysing the sequence, the aptamers (including variants, mutants, fragments, and derivatives thereof) can be prepared by conventional techniques of chemical DNA and RNA synthesis, which are known to the person skilled in the art. Furthermore, the binding properties of individual aptamers to the target molecule can be investigated.

Aptamers are easily altered through chemical oligonucleotide synthesis methods. For aptamers, by predicting the secondary structure using the MFOLD program, or by predicting the steric structure by X-ray analysis or NMR analysis, it is possible to predict to some extent which nucleotide can be substituted or deleted, where to insert a new nucleotide and the like. A predicted aptamer with the new sequence can easily be chemically synthesized, and it can be determined whether the aptamer retains the activity using an existing assay system. Aptamers can be synthesized by methods known per se in the art. One of the synthesis methods is a method using an RNA polymerase. The object RNA can be obtained by chemically synthesizing a DNA having the object sequence and a promoter sequence of RNA polymerase, followed by in vitro transcription using same as a template and according to an already-known method.

Aptamers can be synthesized using DNA polymerase. DNA having an object sequence is chemically synthesized and, using same as a template, amplification is performed by a known method of polymerase chain reaction (PCR). This is converted to a single strand by an already-known method of polyacrylamide electrophoresis or enzyme treatment method. When a modified aptamer is synthesized, the efficiency of elongation reaction can be increased by using a polymerase introduced with a mutation into a specific site. The thus-obtained aptamer can be purified easily by a known method.

The DNA or RNA based aptamers can also be synthesized in a large amount by a chemical synthesis method such as amidite method, phosphoramidite method and the like. The synthesis method is a well-known method, and as described in Nucleic Acid (Vol. 2 )1] Synthesis and Analysis of Nucleic Acid (Editor: Yukio Sugiura, Hirokawa Publishing Company) and the like. A synthesizer such as Dr Oligo 96 and the like manufactured by Biolytic, or OligoPilot, OligoProcess and the like manufactured by GE Healthcare Bioscience may be used. Purification may be performed using any suitable techniques such as chromatography and the like.

In certain embodiments, the invention provides aptamers against CD4 (cluster of differentiation 4, Uniprot accession number: P01730, NCBI Accession number: NP_000607.1). Typically, such aptamers are capable of specifically binding to CD4 with high affinity and specificity. CD4 is a membrane glycoprotein (molecular weight 55 kDa) expressed on helper T lymphocytes. This integral membrane glycoprotein plays an essential role in the immune response.

In certain embodiments, the aptamers are selected from a nucleic acid molecule library such as a single-stranded DNA or RNA nucleic acid molecule library. Typically, the aptamers are selected from a “universal aptamer selection library” that is designed such that any selected aptamers need little to no adaptation to convert into any of the listed assay formats. In certain embodiments, the “universal aptamer selection library” is as defined in Example 1.

Once selected, the aptamer may be further modified before being used e.g. to remove one or both primer sequences and/or parts of the randomised region not required for target binding. Typically, aptamers of the invention comprise a first primer region (e.g. at the 5’ end), a second primer region (e.g. at the 3’ end), or both. The primer regions may serve as primer binding sites for PCR amplification of the library and selected aptamers.

The skilled person would understand different primer sequences can be selected depending, for example, on the starting library and/or aptamer selection protocol. For example, aptamers of the invention may comprise SEQ ID NOs: 1 and/or 2.

The first primer region and/or second region may comprise a detectable and/or targeting label. For example, the first and/or second primer region may be fluorescently (e.g. FAM)-labelled. In certain embodiments, the first and/or second primer region primer are phosphate (PO4) labelled.

An aptamer which binds “specifically” to CD4 (or any other specific target as described herein) is an aptamer that binds with preferential or high affinity to CD4 but does not bind or binds with only low affinity to other functionally and structurally related target molecules. For example, the aptamer may bind to CD4 without substantial cross- reactivity to other functionally and structurally related protein(s).

In certain embodiments, an aptamer binds with preferential or high affinity if it binds with a binding dissociation equilibrium constant (KD) of less than about 1 pM, less than about 500nM, less than about 400nM, less than about 300nM, less than about 200nM, less than about 100nM, less than about 90nM, less than about 80nM, less than about 70nM, less than about 60nM, less than about 50nM, less than about 40nM, less than about 30nM, less than about 20nM, less than about 10nM, less than about 1 nM or less. Binding affinity of aptamers may be measured by any method known to person skilled in the art, including, for example, surface plasmon resonance (SPR), biolayer interferometry (BLI), Isothermal Titration Colorimetry (ITC), Enzyme Linked Oligonucleotide Assay (ELONA), displacement assay and/or steady state analysis.

In certain embodiments, the aptamers of the invention bind specifically to CD4. In certain embodiments, the aptamers of the invention comprise or consist of a nucleic acid sequence selected from any one of SEQ ID NOs: 4 to 24. In certain embodiments, the aptamers of the invention comprise or consist of a nucleic acid sequence selected from any one of SEQ ID NOs: 25 to 44. In certain embodiments, the aptamers of the invention comprise or consist of a nucleic acid sequence selected from SEQ ID NOs: 13, 24 or 34 (e.g. relating to the “7S_11” aptamer). As described herein, the 7S_11 aptamer is capable of binding specifically to CD4.

In certain embodiments, the aptamers of the invention comprise or consist of the nucleic acid sequence as set forth in SEQ ID NO: 24. This sequence relates to a 7S_11_F11 fragment shown to have improved binding to CD4 as compared to full-length 7S_11. This minimal effective fragment (“Optimer”) is shown herein as the best performing aptamer against CD4.

In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 4 to 24.

In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 25 to 44.

In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the nucleotide sequence of SEQ ID NO: 13.

In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the nucleotide sequence of SEQ ID NO: 24.

In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the nucleotide sequence of SEQ ID NO: 34.

As used herein, “sequence identity” refers to the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in said sequences after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, CLUSTALW or Megalign (DNASTAR) software. For example, % nucleic acid sequence identity values can be generated using sequence comparison computer programs found on the European Bioinformatics Institute website (http://www.ebi.ac.uk).

In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence comprising at least about 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides (e.g. up to the total length) of a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity with any one of SEQ ID NOs: 4 to 24. In this context the term "about" typically means the referenced nucleotide sequence length plus or minus 10% of that referenced length.

In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence comprising at least about 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides (e.g. up to the total length) of a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity with SEQ ID NO: 13 or 24.

In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence comprising at least about 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides of a sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity with SEQ ID NO: 24.

In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence comprising at least about 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides (e.g. up to the total length) of any one of SEQ ID NOs: 4 to 24.

In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence comprising at least about 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides (e.g. up to the total length) of SEQ ID NO: 13 or 24.

In certain embodiments, aptamers of the invention comprise or consist of a nucleic acid sequence comprising at least about 20, 25, 30, 35, 40, 45, 50 or more consecutive nucleotides of SEQ ID NO: 24. Aptamers are characterised by the formation of a specific three-dimensional structure that depends on the nucleic acid sequence. The three-dimensional structure of an aptamer arises due to Watson and Crick intramolecular base pairing, Hoogsteen base pairing (quadruplex), wobble pair formation or other non-canonical base interactions. This structure enables aptamers, analogous to antigen-antibody binding, to bind target structures accurately. A nucleic acid sequence of an aptamer may, under defined conditions, have a three-dimensional structure that is specific to a defined target structure.

In certain embodiments, the aptamer comprises a secondary structure. The secondary structure analysis of the aptamers was performed by means of the free-energy minimization algorithm Mfold (M Zuker. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31(13), 3406-3415, 2003). In certain embodiments, the aptamers of the invention may contain at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide variations as compared to any one of SEQ ID NOs: 4 to 24 or 25 to 44. Positions where such variations can be introduced can be determined based on, for example, the secondary structures.

The invention also provides aptamers that compete for binding to CD4 with aptamers as described herein. In certain embodiments, the invention provides aptamers that compete for binding to CD4 with the aptamers as set forth in any one of SEQ ID NOs: 4 to 24 or 25 to 44 (e.g., SEQ ID NO: 24). In certain embodiments, competition assays or other binding assays may be used to identify an aptamer that bind to CD4. In an exemplary binding assay, immobilised CD4 is incubated in a solution comprising a first labelled aptamer that binds to CD4 and a second unlabelled aptamer that is being tested for its ability to compete with the first aptamer for binding to CD4. As a control, immobilised CD4 may be incubated in a solution comprising the first labelled aptamer but not the second unlabelled aptamer. After incubation under conditions permissive for binding of the first aptamer to CD4 excess unbound aptamer may be removed, and the amount of label associated with immobilised CD4 measured. If the amount of label associated with immobilised CD4 is substantially reduced in the test sample relative to the control sample, then that indicates that the second aptamer is competing with the first aptamer for binding to CD4.

In certain embodiments, the aptamer comprises one or more linker sequences and/or is directly labelled. As such, the aptamer may be detected directly. In addition, or alternatively, the aptamer may be conjugated to a portion of an antibody, for example the Fc region of an antibody as further described herein. In such embodiments, the aptamer may instead be detected indirectly using a labelled secondary agent (e.g., antibody) which is capable of specifically binding to the conjugated antibody portion, such as the Fc region.

In certain embodiments, the aptamer comprises one or more detectable labels. For example, the aptamer may comprise a fluorescent moiety, e.g. a fluorescent/quencher compound. Fluorescent/quencher compounds are known in the art. See, for example, Mary Katherine Johansson, Methods in Molecular Biol. 335: Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols, 2006, Didenko, ed., Humana Press, Totowa, NJ, and Marras et al., 2002, Nucl. Acids Res. 30, el22 (incorporated by reference herein).

In certain embodiments, the detectable label of the aptamer is FAM. In certain embodiments, the FAM-label is preferably situated at either end of a first or second primer region of the aptamer. The person skilled in the art would understand that the label could also be located at any suitable position within the aptamer.

In certain embodiments, the detectable label of the aptamer is selected from a fluorophore, a nanoparticle, a quantum dot, an enzyme, a radioactive isotope, a pre-defined sequence portion, a biotin, a desthiobiotin, a thiol group, an amine group, an azide, an aminoallyl group, a digoxigenin, an antibody, a catalyst, a colloidal metallic particle, a colloidal non-metallic particle, an organic polymer, a latex particle, a nanofiber, a nanotube, a dendrimer, a protein, and a liposome.

In certain embodiments, the detectable label of the aptamer is a fluorescent protein such as Green Fluorescent Protein (GFP) or any other fluorescent protein known to those skilled in the art.

In certain embodiments, the detectable label of the aptamer is an enzyme. For example, the enzyme may be selected from horseradish peroxidase, alkaline phosphatase, urease, - galactosidase, or any other enzyme known to those skilled in the art.

In certain embodiments, the nature of the detection will be dependent on the detectable label used. For example, the label may be detectable by virtue of its colour e.g. gold nanoparticles. A colour can be detected quantitatively by an optical reader or camera e.g. a camera with imaging software. In certain embodiments, the detectable label of the aptamer is a fluorescent label e.g. a quantum dot. In such embodiments, the detection means may comprise a fluorescent plate reader, strip reader or similar which is configured to record fluorescence intensity.

In embodiments in which the detectable label of the aptamer is an enzyme label, the detection means may, for example, be colorimetric, chemiluminescence and/or electrochemical (for example, using an electrochemical detector). Typically, electrochemical sensing is through conjugation of a redox reporter (e.g. methylene blue or ferrocene) to one end of the aptamer and a sensor surface to the other end. Typically, a change in aptamer conformation upon target binding changes the distance between the reporter and sensor to provide a readout.

In certain embodiments, the detectable label of the aptamer may further comprise enzymes such as horseradish peroxidase (HRP), Alkaline phosphatase (APP) or similar, to catalytically turnover a substrate to give an amplified signal.

In certain embodiments, the invention provides a complex or conjugate comprising an aptamer of the invention and a conserved antibody region such as the Fc region. Typically, the aptamers of the invention are covalently or physically conjugated to the Fc region as further described herein.

In certain embodiments, the invention provides an aptamer-Fc conjugate capable of specifically binding to CD4. Aptly the aptamer-Fc conjugate comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 4 to 24 or 25 to 44. Aptly the aptamer-Fc conjugate comprises a nucleic acid having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more with any one of SEQ ID NOs: 4 to 24 and/or comprises at least 20, 25, 30, 35, 40, 45 or 50 consecutive nucleotides of one of SEQ ID NOs: 4 to 24.

In certain embodiments, the invention provides an aptamer-Fc conjugate capable of specifically binding to CD4. Aptly the aptamer-Fc conjugate comprises a nucleic acid sequence selected from SEQ ID NOs: 13 or 24. Aptly the aptamer-Fc conjugate comprises a nucleic acid having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more with SEQ ID NOs: 13 or 24 and/or comprises at least 20, 25, 30, 35, 40, 45 or 50 consecutive nucleotides of SEQ ID NO 13 or 24.

In certain embodiments the present invention provides an aptamer-Fc conjugate capable of specifically binding to CD4, wherein the aptamer-Fc conjugate comprises a nucleic acid sequence of SEQ ID NO: 24 or a nucleic acid having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity with SEQ ID NO: 24 and/or comprises at least 20, 25, 30, 35, 40, 45 or 50 consecutive nucleotides of SEQ ID NO 24.

Aptamer-Fc conjugates

In certain embodiments, the method comprises applying to the sample an aptamer-Fc conjugate. As used herein, an “aptamer-Fc conjugate” is a complex formed by conjugating an aptamer or target binding nucleic acid molecule with an Fc (fragment crystallizable) region of an antibody. Techniques of conjugating an aptamer to the Fc region are further described herein.

The aptamer region of the conjugate is capable of specifically binding to the target molecule of interest, in the context of the sample. The Fc region of the conjugate is capable of specifically binding to the labelled secondary agent (e.g., labelled secondary antibody).

As used herein, “capable of specifically binding” refers to the ability of the aptamer to selectively attach itself to the target molecule. Selective binding means that the interaction between the molecule is highly specific, typically the result of molecular recognition where the aptamer has a binding site that matches the shape, charge, or chemical properties of the target molecule. This specific interaction allows the aptamer to recognise and attach to the target molecule while having minimal binding or affinity for other molecules in the sample.

An aptamer may be conjugated to the constant region of an antibody such as the Fc region of an antibody using any chemical modification technique which allows for the attachment of the modified aptamer to compatibly modified Fc region, while preserving the binding affinity and specificity of both components.

As used herein, the term “Fc region” is understood to mean the constant region of an antibody molecule (e.g., the tail region of the antibody that interacts with Fc receptors). The Fc region of an antibody can be obtained through various methods, including enzymatic digestion or recombinant expression. The Fc region is typically capable of specifically binding to an appropriate secondary agent as further described herein.

To facilitate conjugation, specific and compatible functional groups may be introduced to the aptamer and/or Fc region. Common function groups used for conjugation will be known to those skilled in the art. Aptly common function groups used for conjugation include, for example, primary amines (NH2), sulfhydryl groups (SH), ‘Click chemistry’ groups such as an azide or alkyne and carboxyl groups (COOH). This may be introduced during the solid phase synthesis of the aptamer using phosphoramidites carrying the functional group; or may be achieved through chemical modification of the aptamer post synthesis, or Fc fragments having existing functional groups. The functional groups may be activated using any suitable techniques known to those skilled in the art, e.g., using cross-linking reagents or chemical reactions that create reactive sites for conjugation. Common reagents include N- hydroxysuccinimide (NHS) and maleimide.

In certain embodiments, the aptamer region of the aptamer Fc conjugate comprises one or more primary amines (NH2), sulfhydryl groups (SH), azide, alkyne and/or carboxyl groups (COOH).

In certain embodiments, the Fc region of the aptamer Fc conjugate comprises one or more primary amines (NH2), sulfhydryl groups (SH), azide, alkyne and/or carboxyl groups (COOH).

In certain embodiments the aptamer-Fc conjugate is not an active agent of a pharmaceutical composition. In certain embodiments the aptamer and/or Fc region of the conjugate does not comprise any payload or cargo. Aptly, the aptamer-Fc conjugate is therefore developed for diagnostic purposes only.

The appropriately modified aptamer and Fc region may then be combined under any suitable condition which allows specific conjugation, whilst minimising any side reactions. The specific reaction conditions (e.g., pH, temperature, time) may vary depending on the conjugation chemistry and method chosen. Typically, the aptamer-Fc conjugate is then purified to remove any unreacted aptamers, Fc fragments and reaction by-products. Typical purification methods may include, for example, size exclusion chromatography, affinity chromatography, dialysis, or the like. The aptamer-Fc conjugate may then be further characterised and/or stored under any suitable conditions.

Secondary agents

In certain embodiments, one or more secondary agents are applied to the sample after the Aptamer-Fc conjugate has bound to the target molecule. As used herein, a secondary agent includes any reagent which is capable of specifically binding to the Fc region of the conjugate. The presence, absence and/or level of the target molecule may then be indirectly determined though the secondary agent. In certain embodiments, the secondary agent is a secondary antibody. As used herein, the term “secondary antibody” includes any antibody which do not directly recognise and bind to the target molecule but instead binds to the Fc region of the aptamer-Fc conjugate that has already bound to the target molecule.

In certain embodiments, the secondary antibody is raised against the species in which the Fc region of the conjugate was generated. For example, if the Fc region is derived from a rabbit antibody, a secondary antibody may be raised in a different species (for example a goat) which is then specific to rabbit IgG may be used. In this way, the secondary antibody is capable of specifically binding to the Fc region of the aptamer-Fc conjugate.

In certain embodiments, the secondary antibody is a universal anti-lg antibody that is capable of binding to the Fc region of the aptamer-Fc conjugate.

In certain embodiments, the secondary agent (e.g., secondary antibody) comprises a detectable label such as a fluorescent dye, enzyme, or gold nanoparticle. This label allows for the visualisation or quantification of the target molecule indirectly through the secondary antibody.

In certain embodiments the detectable label of the secondary agent (e.g., secondary antibody) is a fluorescent moiety, e.g., a fluorescent/quencher compound. Fluorescent/quencher compounds are known in the art, see for example Mary Katherine Johansson, Methods in Molecular Biol. 335: Fluorescent Energy Transfer Nucleic Acid Probes: Designs and Protocols, 2006, Didenko, ed., Humana Press, Totowa, NJ, and Marras et al., 2002, Nucl. Acids Res. 30, el22 (both incorporated by reference herein).

Further, moieties that result in an increase in detectable signal when in proximity of each other may be used as alternative labels in the apparatus and methods described herein, for example, as a result of fluorescence resonance energy transfer ("FRET"); suitable pairs include but are not limited to fluorescein and tetramethylrhodamine; rhodamine 6G and malachite green, and FITC and thiosemicarbazole, to name a few.

In certain embodiments, the detectable label of the secondary agent (e.g., secondary antibody) is selected from a fluorophore, a nanoparticle, a quantum dot, an enzyme, a radioactive isotope, a pre-defined sequence portion, a biotin, a desthiobiotin, a thiol group, an amine group, an azide, an aminoallyl group, a digoxigenin, an antibody, a catalyst, a colloidal metallic particle, a colloidal non-metallic particle, an organic polymer, a latex particle, a nanofiber, a nanotube, a dendrimer, a protein, and a liposome. In certain embodiments, the detectable label of the secondary agent (e.g., secondary antibody) is an enzyme. In embodiments in which the detectable label is an enzyme label, the detection means may be an electrochemical detector. The detection label may comprise enzymes such as horseradish peroxidase (HRP), Alkaline phosphatase (APP), urease, b- galactosidase or similar, to catalytically turnover a substrate to give an amplified signal.

If using an enzyme-conjugated secondary antibody (e.g., HRP or AP), a substrate solution (e.g., chromogen) may be added that will produce a visible reaction product upon enzyme action. Common substrates include diaminobenzidine (DAB), 3-Amino-9 ethylcarbazole (AEC), tetramethylbenzidine (TMB), alkaline phosphatase substrates (such as Fast Red, 5- bromo-4-chloro-3-indolyl phosphate / nitro blue tetrazolium (BCIP/NBT) or the like), NovaRED or any other suitable substrate. Factors considered in choosing a substrate may include colour produced, sensitivity, background staining, and compatibility with other detection methods.

In certain embodiments, the nature of the detection means will be dependent on the detectable label used. For example, in certain embodiments, the label may be detectable by virtue of its colour e.g., gold nanoparticles or enzymatically derived product. A colour can be detected quantitatively by an optical reader or camera e.g., a camera with imaging software. In certain embodiments, if the detectable label is a fluorescent label e.g., a quantum dot, the detection means may comprise a fluorescent reader which is configured to record fluorescence intensity for example.

Exemplary labels are visual, optical, photonic, electronic, acoustic, opto-acoustic, mass, electrochemical, electro-optical, spectrometric, enzymatic, or otherwise visually, physically, chemically, or biochemically detectable. In one embodiment of the method, the label is detected by luminescence, UV I VIS spectroscopy, enzymatically, electrochemically, or radioactively. Luminescence refers to the emission of light. In the method according to certain embodiments of the invention, for example, photoluminescence, chemiluminescence and bioluminescence are used for detection of the label. In photoluminescence or fluorescence, excitation occurs by absorption of photons. Exemplary fluorophores include, without limitation, bisbenzimidazole, fluorescein, acridine orange, Cy5, Cy3 or propidium iodide, which can be covalently coupled to secondary agents, tetramethyl-6-carboxyhodamine (TAMRA), Texas Red (TR), rhodamine, Alexa Fluor dyes.

Other tags are catalysts, colloidal metallic particles, e.g. as gold nanoparticles, colloidal non- metallic particles, quantum dots, organic polymers, latex particles, nanofibers, in particular carbon, nanotubes, in particular carbon (carbon nanotubes), dendrimers, proteins, or liposomes with signal-generating substances. Colloidal particles can be detected visually. In certain embodiments, the detectable label of the secondary agent is a radioactive isotope. The detection can also be conducted by means of radioactive isotopes with which the secondary agent is labelled, preferably 3H, 14C, 32P, 33P, 35S or 1251 , more preferably 32P, 33P or 1251. In the scintillation counting, the radioactive radiation emitted by the radioactively labelled secondary agent -target complex is measured indirectly. A scintillator substance is excited by the radioactive radiation. During the transition to the ground state, the excitation energy is released again as flashes of light, which are amplified and counted by a photomultiplier.

In certain embodiments, the detectable label of the secondary agent is selected from digoxigenin and biotin. Thus, the secondary agent may also be labelled with digoxigenin or biotin, which are bound for example by antibodies or streptavidin, which may in turn carry a label, such as e.g. an enzyme conjugate. The prior covalent linkage (conjugation) of a secondary agent with an enzyme can be accomplished in several known ways. Detection of secondary agent binding may also be radioactive in an RIA (radioactive immunoassay) with radioactive isotopes, preferably with 1251 , or by fluorescence in a FIA (fluoroimmunoassay) with fluorophores, preferably with fluorescein or FITC.

In certain embodiments the presence, absence and/or level of the secondary agent is visualised by light and/or fluorescent microscopy, as further described herein.

Immunoassays

In certain embodiments, the method of detecting a target molecule in the sample comprises an immunoassay. Immunoassay techniques as described herein are based on the reaction of the Fc region of the aptamer-Fc conjugate to its corresponding secondary agent (e.g., secondary antibody). This allows the target molecule, bound to the aptamer region of the aptamer-Fc conjugate, to be indirectly detected.

In certain embodiments, method for detecting a target molecule in a sample comprises the following steps:

(i) optionally performing one or more blocking steps, e.g., by pre-treating the sample with a nucleic acid-based and/or any other blocking buffer as described herein;

(ii) contacting the sample with the aptamer-Fc conjugate, wherein the presence of the target molecule creates a complex between the aptamer region of the aptamer-Fc conjugate and the target molecule; (iii) optionally performing one or more wash step(s);

(iv) contacting the sample with the labelled secondary agent, wherein the presence of the secondary agent creates a complex between the Fc region of the aptamer-Fc conjugate and the secondary agent;

(v) optionally performing one or more additional wash step(s); and

(vi) detecting and/or quantifying the label of the secondary agent.

The methods described herein are compatible with a wide range of immunoassay formats which may provide qualitative, semi-quantitative or quantitative results. For example, quantitative results may be generated using a standard curve created with known concentrations of the specific analyte to be detected. The response or signal from an unknown sample is plotted onto the standard curve, and a quantity or value corresponding to the target in the unknown sample is established.

In certain embodiments, the immunoassay is automated or semi-automated.

In certain embodiments, the invention comprises detecting more than one target molecule in a sample, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different target molecule. The detection of more than one target molecule may be conducted separately or simultaneously with one test sample. For separate or sequential assay of biomarkers, suitable apparatuses include clinical laboratory analyzers such as the ElecSys (Roche), the AxSym (Abbott), the Access (Beckman), the ADVIA®, the CENTAUR® (Bayer), and the NICHOLS ADVANTAGE® (Nichols Institute) immunoassay systems. Preferred apparatuses or protein chips perform simultaneous assays of a plurality of biomarkers on a single surface.

A wide variety of immunohistochemistry (I HC) techniques can be used to detect a target molecule in a sample (see ImmunoAssay: A Practical Guide, edited by Brian Law, published by Taylor & Francis, Ltd., 2005 edition). As used herein, the term “immunohistochemistry” or “IHC” encompasses techniques that utilize the visual detection of fluorescent dyes or enzymes coupled (i.e., conjugated) to a secondary agent (e.g., antibody) using fluorescent microscopy or light microscopy and includes, without limitation, direct fluorescent antibody, indirect fluorescent antibody (I FA), anticomplement immunofluorescence, avidin-biotin immunofluorescence, and immunoperoxidase assays. Exemplary assay formats include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, fluorescent, chemiluminescence, and fluorescence resonance energy transfer (FRET) or time resolved-FRET (TR-FRET) immunoassays. Examples of procedures for detecting biomarkers include biomarker immunoprecipitation followed by quantitative methods that allow size and peptide level discrimination, such as gel electrophoresis, capillary electrophoresis, planar electrochromatography, and the like.

Methods of detecting and/or quantifying a detectable label or signal generating material depend on the nature of the label. The products of reactions catalysed by appropriate enzymes can be, without limitation, fluorescent, luminescent, or radioactive or they may absorb visible or ultraviolet light. Examples of detectors suitable for detecting such detectable labels include, without limitation, x- ray film, radioactivity counters, scintillation counters, spectrophotometers, colorimeters, fluorometers, luminometers, and densitometers.

Any of the methods for detection can be performed in any format that allows for any suitable preparation, processing, and analysis of the reactions. This can be, for example, in multi-well assay plates (e.g., 96 wells or 384 wells) or using any suitable array or microarray. Stock solutions for various agents can be made manually or robotically, and all subsequent pipetting, diluting, mixing, distribution, washing, incubating, sample readout, data collection and analysis can be done robotically using commercially available analysis software, robotics, and detection instrumentation capable of detecting a detectable label.

In certain embodiments, the immunoassay is an enzyme immunoassay (EIA) such as an enzyme multiplied immunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA), IgM antibody capture ELISA (MAC ELISA), microparticle enzyme immunoassay (MEIA), capillary electrophoresis immunoassay (CEIA), radioimmunoassay (RIA), immunoradiometric assay (IRMA), fluorescence polarization immunoassay (FPIA) or chemiluminescence assay (CL).

In certain embodiments, the concentration of the target molecule can be quantitated, e.g., through endpoint titration or through measuring the visual intensity of fluorescence compared to a known reference standard. Alternatively, if using an enzyme-conjugated secondary antibody (e.g., HRP or AP), a substrate solution (e.g., DAB, alkaline phosphatase substrates or the like, as described elsewhere herein) may be added, that will produce a visible reaction product upon enzyme action.

Immunocytochemistry (ICC) In certain embodiments, the sample comprises cells (e.g., cultured and/or isolated cells). In such embodiments, the target molecule may be detected using ICC. The sequential steps in ICC can be summarized as follows:

Sample preparation, where if cultured cells are used, they are typically seeded on a solid support. If adherent cells are used, the cells may be incubated on the support, typically from about 30 minutes to about 24 or about 48 hours depending on the characteristics of the cells used.

Cell fixation, where the cells are fixed to preserve their structure and prevent protein degradation. Fixation can be done by cross-linking or by precipitating the proteins using organic solvents. Common fixatives include paraformaldehyde and methanol. Permeabilization (if required), some aptamer-Fc conjugates may require permeabilization of the cell membrane to access intracellular target proteins or epitopes. This step is not necessary for cell surface targets or epitopes. It may be achieved using solvents (e.g., alcohol, acetone, or the like) or detergents (e.g., Triton X-100, Saponins, Tween-20 or the like). Cell and tissue permeabilization kits are also commercially available for this purpose.

Blocking, where non-specific binding sites on the tissue section(s) are blocked to reduce background staining. Appropriate blocking agents may include nucleic acidbased blocking agents such as salmon sperm DNA, yeast tRNA or other non-target binding nucleic acid, known to those skilled in the art. In addition, or alternatively, common blocking agents include bovine serum albumin (BSA) or normal serum from the species in which the secondary antibody is raised.

Incubation with the aptamer-Fc conjugate, where the aptamer specifically recognises the target molecule and is applied to the sample.

Washing, where the sample is washed to remove any unbound aptamer-Fc conjugate.

Secondary agent incubation, where the secondary agent (e.g., secondary antibody) specifically binds to the Fc portion of the aptamer-Fc conjugate and is typically labelled (e.g., biotin, enzyme, fluorophore, or the like).

Washing, where the tissue sections are washed to remove any unbound secondary agents.

Detection, if using an enzyme-conjugated secondary antibody (e.g., HRP or AP), a substrate solution may be added that will produce a visible reaction product upon enzyme action. Common substrates include diaminobenzidine (DAB) or alkaline phosphatase substrates. If using a fluorophore-conjugated secondary antibody, no additional substrate may need to be added.

Counterstaining (optional), to visualise cellular structures or nuclei counterstains such as hematoxylin or eosin (H&E) or specialized nuclear stains may be added.

Mounting, the tissue section may be covered with any appropriate mounting medium to protect the sample.

Microscopy may be performed to examine the stained tissue sections under a light microscope (for enzyme-based IHC) or a fluorescence microscope (for fluorophore- based IHC) to visualise the staining patterns.

Image capture and analysis may be performed to capture images of the stained tissue sections for analysis and subsequent interpretation.

Any one or more of the above steps may be optimised depending, for example, on the aptamer-Fc conjugate being used, fixation conditions and staining conditions. Typically, appropriate controls such as negative controls (omitting the aptamer-Fc conjugate or replacing it with a scrambled nucleic acid sequence, with Fc conjugate) and positive controls (tissue with known antigen expression) are included to help validate the results.

In certain embodiments, the aptamer-Fc conjugate is titrated to optimise contrast between positively staining tissue and nonspecific background staining, with the highest dilution selected to prevent waste. For example, a range of dilutions of the aptamer-Fc conjugate may be tested on a series of tissues with the appropriate positive control. This may be combined with various combinations of dilutions of the secondary antibody in the setting of the particular antigen retrieval method and chromogen to produce optimum staining. Incubation and wash times and temperatures may also be optimised to improve the signal to ‘noise.’ For the initial titration, for example, an aptamer-Fc concentration of about 1 to about 40 pg/mL may be used.

To visualize the target molecule, the secondary antibody is typically labelled as further described herein.

Background staining may be due to non-specific aptamer or secondary antibody binding and endogenous peroxidase activity, more problematic in tissues with abundant hematopoietic elements such as bone marrow. Non-specific antibody binding can be decreased by preincubation with normal serum from the same species as the secondary antibody or with a commercially available universal blocking agent. Endogenous enzyme activity can be inhibited by pre-treating the tissue with solutions containing hydrogen peroxide prior to application of the antibody. To block non-specific binding (NSB) of DNA aptamers, the sample may be incubated with a cocktail of blocking reagents. BSA or non-albumin blockers are used for protein-based blocking; The blocking buffer could contain salmon sperm DNA, dextran sulphate, and/or heparin to block DNA-based NSBs, as well as RNA-based blockers such as yeast tRNA. Typically, the sample is incubated with the blocking buffer at room temperature for about an hour, overnight at 4 °C or rapidly at 37 °C for up to 30 minutes.

Typically, the composition of the blocking buffer is optimised to eliminate non-specific binding or nuclear staining. The key blockers and the suggested concentration ranges recommended are described below, however the exact composition can be optimised per assay.

In certain embodiments the protein-based blocker comprises Bovine Serum Albumin (BSA) optionally at a concentration of between about 2 mg/mL to about 20 mg/mL. Aptly, the blocking buffer comprises BSA at a concentration of about 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, 10 mg/mL, 11 mg/mL, 12 mg/mL, 13 mg/mL, 14 mg/mL, 15 mg/mL, 16 mg/mL, 17 mg/mL, 18 mg/mL, 19 mg/mL or 20 mg/mL.

In certain embodiments the blocking buffer comprises DNA-based blockers.

In certain embodiments the DNA-based blocker comprises salmon sperm DNA optionally at a concentration of between about 0.2 mg/mL to about 2 mg/mL. Aptly the blocking buffer comprises salmon sperm DNA at a concentration of about 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, 0.9 mg/mL, 1.0 mg/mL, 1.1 mg/mL, 1.2 mg/mL, 1.3 mg/mL, 1.4 mg/mL, 1.5 mg/mL, 1.6 mg/mL, 1.7 mg/mL, 1.8 mg/mL, 1.9 mg/mL or 2.0 mg/mL.

In certain embodiments the blocking buffer comprises sulphated polysaccharide-based blockers. As used herein a “sulphated polysaccharide” is a negatively charged biomolecule comprising monosaccharides joined together by glycosidic linkages to form a long chain, whereby at least one of the monosaccharide residues comprise at least one sulphate group.

In certain embodiments the sulphated polysaccharide-based blocker comprises dextran sulphate optionally in an amount of between about 0.1% to about 2% w/v of the composition. Aptly the blocking buffer comprises Dextran Sulphate in an amount of about 0.1 %, 0.2%, 0.3%, 0.4%, 0.5%, 0.6% 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2.0% w/v of the composition. In certain embodiments the sulphated polysaccharide-based blocker comprises heparin optionally at a concentration of between about 1 U/rnL to about 20 U/rnL. Aptly the blocking buffer comprises Heparin at a concentration of about 1 U/rnL, 2 U/rnL, 3 U/rnL, 4 U/rnL, 5 U/mL, 6 U/mL, 7 U/mL, 8 U/mL, 9 U/mL, 10 U/mL, 11 U/mL, 12 U/mL, 13 U/mL, 14 U/mL, 15 U/mL, 16 U/mL, 17 U/mL, 18 U/mL, 19 U/mL, or 20 U/mL.

In certain embodiments the blocking buffer comprises RNA-based blockers.

In certain embodiments the RNA-based blocker comprises Yeast tRNA optionally at a concentration of between about 0.2 mg/mL to about 2 mg/mL. Aptly, the blocking buffer comprises Yeast tRNA at a concentration of about 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6 mg/mL, 0.7 mg/mL, 0.8 mg/mL, 0.9 mg/mL, 1.0 mg/mL, 1.1 mg/mL, 1.2 mg/mL, 1.3 mg/mL, 1.4 mg/mL, 1.5 mg/mL, 1.6 mg/mL, 1.7 mg/mL, 1.8 mg/mL, 1.9 mg/mL or 2.0 mg/mL.

In certain embodiments the presence, absence and/or level of the secondary agent is detected by immunocytochemistry (ICC), wherein the aptamer-Fc conjugate replaces the use of a primary antibody in a standard workflow, as described herein.

Immunohistochemistry (IHC)

In certain embodiments, the sample comprises tissue (e.g., frozen, or fixed tissue sections as described herein). In such embodiments, the target molecule may be detected using IHC.

The sequential steps in IHC can be summarized as follows:

Tissue preparation, where tissue samples (e.g., FFPE or frozen sections) are sectioned to a desired thickness (typically 4-5 pm).

Deparaffinisation (if using FFPE sections), for example by immersing in xylene followed by rehydration through graded ethanol solutions.

- Antigen retrieval (optional) if target molecule is masked by formalin fixation. This may be achieved, for example, by heating the tissue sections in a buffer solution to make them more accessible to binding to the primary binding reagent e.g. the aptamer-Fc conjugate.

Blocking, where non-specific binding sites on the tissue section(s) are blocked to reduce non-specific background staining. A blocking agent may be specifically developed for the aptamer-Fc conjugate, which may include nucleic acid-based blocking agents such as salmon sperm DNA, yeast tRNA or other non-target binding nucleic acid, known to those skilled in the art. In addition, or alternatively, common blocking agents include bovine serum albumin (BSA) or normal serum from the species in which the secondary antibody is raised.

Incubation with the aptamer-Fc conjugate, where the aptamer specifically recognises the target molecule and is applied to the tissue section(s).

Washing, where the tissue sections are washed to remove any unbound aptamer-Fc conjugate,

Secondary agent incubation, where the secondary agent (e.g., secondary antibody) specifically binds to the Fc portion of the aptamer-Fc conjugate and is typically labelled (e.g., enzyme, fluorophore)

Washing, where the tissue sections are washed to remove any unbound secondary agents.

Detection, if using an enzyme-conjugated secondary antibody (e.g., HRP or AP), a substrate solution may be added that will produce a visible reaction product upon enzyme action. Common substrates include diaminobenzidine (DAB) or alkaline phosphatase substrates. If using a fluorophore-conjugated secondary antibody, no additional substrate may need to be added.

Counterstaining (optional), to visualise cellular structures or nuclei counterstains such as hematoxylin or eosin (H&E) or specialized nuclear stains may be added. Dehydration (if using aqueous mounting medium), the slides may be dehydrated through graded ethanol solutions before mounting,

Mounting, the tissue section may be covered with any appropriate mounting medium, typically a coverslip and mounting medium such as DPX, permount or an aqueous mounting medium if working with fluorescent stains,

Sealing may also be performed, to prevent drying and help preserve the slides, Microscopy may be performed to examine the stained tissue sections under a light microscope (for enzyme-based IHC) or a fluorescence microscope (for fluorophore- based IHC) to visualise the staining patterns.

Image capture, and analysis, may be performed to capture images of the stained tissue sections for analysis and subsequent interpretation.

Any one or more of the above steps may be optimised depending, for example, on the aptamer-Fc conjugate being used, antigen retrieval conditions (if required), and staining conditions. Typically, appropriate controls such as negative controls (omitting the aptamer-Fc conjugate or replacing it with a scrambled nucleic acid sequence, with Fc conjugate) and positive controls (tissue with known antigen expression) are included to help validate the results.

In certain embodiments, methods of detecting a target molecule in a sample using IHC comprising incubation with aptamer-Fc conjugates of the present invention does not require an antigen retrieval step. Without being bound by theory, aptamer-Fc conjugates of the present invention may be smaller than antibodies and capable of accessing epitopes of the target molecule without needing any antigen retrieval steps. Aptly, the aptamer region of the aptamer-Fc conjugate may be able to form a complex with the target molecule during IHC without any antigen retrieval steps.

In certain embodiments, the aptamer-Fc conjugate is titrated to optimise contrast between positively staining tissue and non-specific background staining, with the highest dilution selected to prevent waste. For example, a range of dilutions of the aptamer-Fc conjugate may be tested on a series of tissues with the appropriate positive control. This may be combined with various combinations of dilutions of the secondary antibody in the setting of the particular antigen retrieval method and chromogen to produce optimum staining. Incubation and wash times and temperatures may also be optimised to improve the signal to ‘noise.’ For the initial titration, for example, an aptamer-Fc concentration of about 1 pg/mL to about 40 pg/mL may be used.

To visualize the target molecule, the secondary antibody is typically labelled as further described herein.

Background staining may be due to non-specific aptamer or secondary antibody binding and endogenous peroxidase activity, more problematic in tissues with abundant hematopoietic elements such as bone marrow. Non-specific antibody binding can be decreased by preincubation with normal serum from the same species as the secondary antibody or with a commercially available universal blocking agent. Endogenous enzyme activity can be inhibited by pre-treating the tissue with solutions containing hydrogen peroxide prior to application of the antibody.

To block non-specific binding (NSB) of DNA aptamers, the sample may be incubated with a cocktail of blocking reagents. BSA or non-albumin blockers are used for protein-based blocking; The blocking buffer could contain salmon sperm DNA, dextran sulphate, and/or heparin to block DNA-based NSBs, as well as RNA-based blockers such as yeast tRNA. Typically, the sample is incubated with the blocking buffer at room temperature for about an hour, overnight at 4 °C or rapidly at 37 °C for up to 30 minutes.

Typically, the composition of the blocking buffer is optimised to eliminate non-specific binding or nuclear staining. The key blockers and the suggested concentration ranges are the same for IHC as already described above in the context of ICC. Again, however, the exact composition can be optimised per assay.

In certain embodiments, the presence, absence and/or level of the secondary agent is detected by immunohistochemistry (IHC), wherein the aptamer-Fc conjugate replaces the use of a primary antibody in a standard workflow as described herein.

Kits

In certain embodiments, the invention provides a kit comprising reagents to carry out the method as described herein. Typically, the kit comprises an aptamer-Fc conjugate capable of specifically binding to the target molecule. The kit may further comprise a labelled secondary agent (e.g., secondary antibody), wherein the agent is capable of specifically binding to the Fc region of the conjugate.

The aptamer-Fc conjugate and/or secondary agent may be present in an isolated or substantially purified form. They may be mixed with carriers or diluents that will not interfere with their intended use and still be regarded as substantially isolated. They may also be in a substantially purified form, in which case they will generally comprise at least 90%, e.g. at least 95%, at least 98% or at least 99% of polynucleotides of the kit.

In some embodiments, the kit further comprises instructions for using the kit to detect the target molecule. In some embodiments, the kit may further comprise one or more additional components such as reagents and/or apparatus necessary for carrying out at immunoassay, e.g., buffers, fixatives, wash solutions, blocking reagents, diluents, chromogens, enzymes, substrates, test tubes, plates, pipettes etc.

The kit may advantageously be used for carrying out any method described herein and could be employed in a variety of applications, for example in the diagnostic field or as a research tool. It will be appreciated that the parts of the kit may be packaged individually in vials or in combination in containers or multi-container units. Typically, manufacture of the kit follows standard procedures which are known to the person skilled in the art. Methods of treatment

In certain embodiments, the presence, absence, or level of the target molecule may be indicative of a disease, condition, or status of an individual from which the sample (e.g., cell or tissue) is derived. The individual may be a patient. For example, the individual may be an animal such as a dog, cat, or horse. Typically, the patient is a human.

In some embodiments, the tissue sample has previously been obtained from the subject such that the sampling itself does not form a part of the methods of the invention. The sample may have been obtained immediately prior to the method, or a number of hours, days, weeks, months, or years prior to the method. In other embodiments, a method of the invention may additionally comprise the step of obtaining the tissue sample from the subject.

In certain embodiments, the invention provides a method of determining the likelihood of a disease or condition in a patient, wherein the method comprises:

(i) detecting a target molecule in a sample according to any method as described herein, and

(ii) comparing the presence, absence and/or level of the target molecule with a reference sample or levels obtained therefrom; wherein a difference is indicative of an increased risk of the disease or condition in the patient from which the sample is taken.

In certain embodiments, reference levels may be obtained by detecting levels of the target molecule in a sample from healthy individuals or patients known not to have the disease or condition. In addition, or alternatively, reference levels may be obtained by detecting levels of the target molecule from patients known to have the disease or condition. As used herein, a “difference” is understood to mean a significant increase or decrease in the levels of the target molecule compared to the reference level(s).

In certain embodiments, the level of a target molecule is monitored in a patient over time, e.g., monitor the efficacy of one or more treatments. For example, the invention also provides a method of determining a patient’s response to a treatment, wherein the method comprises:

(i) detecting a target molecule in one or more samples from the patient over the course of a treatment, wherein the target molecule is detected according to any method as described herein, and (ii) comparing the presence, absence and/or level of the target molecule with a reference sample or levels obtained therefrom; wherein a difference in the biomarker over time (e.g., before treatment commences, at one or more points during treatment, and/or after cessation of treatment) is indicative of a response (or non-response) to the treatment.

In certain embodiments, detecting the levels of the target molecule comprises outputting, optionally on a computer, (i) an indication of the levels of the target molecule, and (ii) this indicates whether the patient is likely to have the disease or condition.

EXAMPLES

In the following, the invention will be explained in more detail by means of non-limiting examples of specific embodiments. In the example experiments, standard reagents, and buffers free from contamination are used.

EXAMPLE 1 - Aptamer selection

Recombinant His tagged CD4 protein was supplied by Sino Biological (Recombinant human CD4 Protein, HEK293 Cells, His Tag, 10400-H08H). This Recombinant human CD4 Protein comprises the extracellular domain (Met 1-Trp 390) of full length CD4 (SEQ ID NO: 46) The protein was characterised by UV spectroscopy and SDS-PAGE analysis for quality control purposes. The protein was immobilised onto His-Tag Isolation and Pulldown magnetic Dynabeads™ (ThermoFisher Scientific, UK), according to manufacturer’s protocols. The protein loading density was determined spectrophotometrical ly.

The aptamer selection process was carried out starting from synthetic ssDNA oligonucleotide sequences of an aptamer library (manufactured by IDT, Belgium). The nucleotide sequences of the aptamer library have the following structure (in a 5’ to 3’ direction):

P1 - R - P2, wherein P1 is a first primer region, R is a randomized region (40 nucleotides in length) and P2 is a further primer region wherein R or a portion thereof are involved in target molecule binding.

The following modified primers were used in the amplification of the oligomers by means of PCR: fluorescein (FAM)-labelled forward primer (P1) with the sequence: 5' - /56FAM/ CCAGTGTAGACTACTCAATGC - 3' (SEQ ID NO: 1) and P0₄-modified reverse primer (P2) with the sequence: 5' - /5Phos/ GGTTGACCTGTGGATAGTAC - 3' (SEQ ID NO: 2).

The selection process consisted of iterative selection rounds with increasingly stringent selection conditions. In Cycle 1 , 166pmol of naive aptamer library is incubated with the target immobilised beads, using binding conditions established in preliminary binding studies. The beads are washed to remove loosely bound aptamers, and the remaining aptamers are eluted in PCR mix and amplified as carried out in the preliminary binding study. The recovered amplified aptamer library is purified using AxyPrep Mag PCR Clean-up Kit (Axygen Biosciences, USA) according to manufacturers’ protocol, digested with Lambda exonuclease (EURx, Poland) at 37 °C according to manufacturers’ protocol. The nascent ssDNA is purified using AxyPrep Mag PCR Clean-up kit to produce a purified and enriched single stranded DNA library for the subsequent aptamer selection cycle. In Cycle 2 (and all subsequent rounds), the same process is followed but aptamer-target incubations are carried out with increasingly stringent conditions. Counter selection was carried out against blank His-Tag Isolation and Pulldown magnetic Dynabeads™, to remove bead binding sequences.

After aptamer selection, the refined aptamer populations were assessed for the ability to bind to CD4 using a Biolayer interferometry assay. The experiments described here were conducted using an Octet RED384 instrument (Sartorius Corporation, USA) based on manufacturers defined protocols. The aptamer population was prepared using a biotinylated primer (SEQ ID NO: 1), in the PCR reaction. Biotinylated ssDNA was then immobilised onto the surface of streptavidin coated biosensor probes (Streptavidin-SA Dip & Read Biosensors, Sartorius Corporation, USA) following manufacturer protocols. The aptamer populations were prepared at 50nM in 1x ‘high salt’ aptamer selection buffer (50mM MES pH6.2, 5mM MgCI₂, 1 mM CaCI₂, 220mM NaCI, 4.5mM KCI, 20mM Na₂SO₄, 0.01 % (v/v) Tween-20, 0.01 % (w/v) BSA). Target protein stocks were also prepared in the 1x ‘high salt’ aptamer selection buffer. All buffer I blank I baseline interactions were carried out in 1x ‘high salt’ aptamer selection buffer with no added CD4. The interaction between the immobilised aptamer population and CD4 was monitored. All data was reference corrected using a blank sensor probe (no immobilised aptamer) to allow correction of buffer effects.

Pools which have improved target binding relative to the naive library, were analysed by Next Generation Sequencing (NGS) to identify potential candidate monoclonal aptamer sequences. The population analysis was carried out using an Ion PGM Ion Torrent Next Generation Sequencing system. Libraries were prepared using Ion Torrent™ Ion Plus Fragment Library kit, according to manufacturer protocols (ThermoFisher Scientific, UK), and barcoded using Ion Xpress™ Barcode Adapters 1-96 Kit following manufacturer protocols (ThermoFisher Scientific, UK). Barcoded populations were then templated onto Ion Sphere Particles (ISP) using the Ion OneTouch™ 2 System, following manufacturer protocols (ThermoFisher Scientific, UK). After successful templating, the prepared templated ISPs were processed and loaded onto an Ion 318™ Chip Kit v2 BC following manufacturer protocols and the samples run using the Ion PGM Ion Torrent Next Generation Sequencing system. Sequence reads were then analysed in Linux to identify enriched candidate sequences within each population. In house python scripts were used to process the sequencing data and trim each sequence so that only the variable region is present in the data. Datasets were then clustered using FastAptameR2.0 (https://fastaptamer2.missouri.edu/) to group similar sequences into families or ‘clusters’. The highest read sequence from each cluster is then taken forward as the candidate sequence for synthesis and subsequent screening by Biolayer Interferometry (BLI) on the Octet RED384 instrument (Sartorius Corporation, USA). The obtained sequence data is set forth in SEQ ID NOs: 4 to 44 as described herein.

Individual aptamer candidate sequences were synthesised using standard solid phase phosphoramidite oligonucleotide synthesis methods. DNA phosphoramidites (ThermoFisher Scientific, UK) were used to prepare the candidate Optimers using a Dr Oligo 768 High throughput oligonucleotide synthesiser, following manufacturer’s instructions (Biolytic Inc. USA). Synthesised oligonucleotides were deprotected using AMA (1 :1 v/v solution of 28% ammonium hydroxide and 40% methylamine) and then purified using Glen-Pak DNA purification cartridges following the manufacturer’s instructions (Glen Research, USA). Optimers were analysed by polyacrylamide gel electrophoresis before being used in screening assays.

Each individual aptamer was then analysed for binding to their respective target; using the BLI assay described above. Candidate aptamers shown to bind to recombinant protein by BLI, were then further assessed for binding to CD4 on a cell surface, by flow cytometry. Fluorescently labelled aptamer candidates synthesised by solid phase phosphoramidite oligonucleotide synthesis as described above, incorporating a Cy3 label at the 3' terminus. Purified aptamer candidates were diluted to 1 pM in binding buffer, then added to triplicate wells pre-seeded with 0.1 million cells per well of either CD4+ H9 cells, or CD4- D1.1 cells. After incubation for 30 mins at 37°C 5% CO2; unbound aptamers were removed, and the cells were washed twice with binding buffer. Binding was quantified by flow cytometry, using an Attune flow cytometer (ThermoFisher Scientific, UK) as per manufacturer’s instructions. Autofluorescence from untreated cells was also measured, to allow background correction. The unselected library was assessed as an indication of non-specific aptamer binding to either cell type. An average fluorescence value was calculated from each triplicate dataset for each aptamer and cell type. This was then plotted as a % Mean Fluorescence Intensity (MFI) compared to the autofluorescence from the respective cell type. Aptamer candidates were chosen based on the increased binding to the CD4+ cells (blue bars in Figure 1) and minimal binding to CD4- cells (orange bars). The affinity of the best performing candidates (CD4_7S_1 (SEQ ID NO: 4) and CD4_7S_11 (SEQ ID NO: 13)) was then assessed by Biolayer Interferometry as described above. Aptamer candidates were immobilised on streptavidin coated BLI probes, then incubated with a concentration gradient of recombinant CD4 protein (4.7 - 300nM). The binding response at each concentration is fit globally to a 1 :1 binding model and the binding affinity is calculated from the association and dissociation rates for each respective phase in the assay.

After nominating a preferred aptamer candidate CD4_7S_11 (SEQ ID NO: 13), the minimal functional fragment is identified. A panel of fragments representing different lengths and regions of the full-length aptamer were synthesised and fluorescently labelled as described above. The aptamer fragments were again screened by flow cytometry as described above. The Mean Fluorescence Intensity (MFI) was plotted as a % of the autofluorescence response for the CD4+ cells (blue bars) and CD4- cells (orange bars) in Figure 2A. Each fragment is compared to the full-length aptamer as a positive control, and a scrambled sequence (SEQ ID NO: 45) as a negative control. The affinity of the best performing fragment (CD4_7S_11_F11 (SEQ ID NO: 24)) was assessed by Biolayer Interferometry as described above. The aptamer fragment was immobilised on streptavidin coated BLI probes, then incubated with a concentration gradient of recombinant CD4 protein (4.7 - 300nM). The binding response at each concentration is fit globally to a 1 : 1 binding model and the binding affinity is calculated from the association and dissociation rates for each respective phase in the assay.

The binding selectivity of the identified aptamer fragment was confirmed by fluorescence microscopy. Aptamer fragments were diluted to 1pM in binding buffer, then added to triplicate wells pre-seeded with 0.1 million cells per well of either CD4+ H9 cells, or CD4- D1.1 cells. A scrambled sequence (SEQ ID NO: 45) was also assessed as a negative control. After incubation for 30 mins at 37°C 5% CO2; unbound aptamers were removed, and the cells were washed twice with binding buffer. Cells were counter stained with DAPI nuclear stain (ThermoFisher Scientific, UK) as per manufacturer’s instructions. Binding was then assessed by fluorescence microscopy using an EVOS fluorescence microscope (ThermoFisher Scientific, UK) as per manufacturer’s instructions. Microscopy images show that aptamer fragment (CD4_7S_11_F11 (SEQ ID NO: 24)) binds to CD4+ cells (Figure 3, upper left) but not CD4- cells (lower left); demonstrating selectivity. The negative control scrambled sequence (SEQ ID NO: 45) shows no binding to either cell line (right panels). EXAMPLE 2 - Preparation of aptamer-Fc conjugates

Figure 6 illustrates a process outline for preparation of Optimer-Fc conjugates. (A) Amine modified Optimer is treated with NHS-azide, to produce an azide functionalised Optimer. (B) rabbit Fc fragment is treated with NHS-DBCO, to produce a DBCO functionalised rabbit-Fc fragment. (C) Azide functionalised Optimer is incubated with DBCO functionalised rabbit-Fc fragment, to produce the Optimer-Fc conjugate.

The target binding Optimer was synthesised and 5' amino modified, using standard solid phase phosphoramidite oligonucleotide synthesis methods. DNA phosphoramidites (ThermoFisher Scientific, UK) were used to prepare the 5' amino modified Optimer using a Dr Oligo 96 High throughput oligonucleotide synthesiser (Biolytic Inc, USA), following manufacturer’s instructions. Synthesised oligonucleotides were deprotected using AMA (1 :1 v/v solution of 28% ammonium hydroxide and 40% methylamine) and purified using Glen-Pak DNA purification cartridges (Glen Research, USA), following the manufacturer’s instructions.

The 5' amino group was converted to an azide, by treating the amino modified Optimer with a 50x excess of azidobutyric acid NHS ester (Lumiprobe GmbH, Germany) in conjugation buffer (1x PBS, pH 7.4 + 10 mM EDTA) overnight at 37 °C with shaking. Unreacted modification reagent was removed by desalting using 7 K MWCO Zeba™ Spin Desalting Columns (ThemoFisher Scientific, UK) following manufacturer’s instructions.

The rabbit IgG Fc protein (Novus Biologicals LLC, USA) was modified to carry a DBCO functional group by incubating with a 50x excess of DBCO NHS ester (Lumiprobe GmbH, Germany) in conjugation buffer (1x PBS, pH 7.4 + 10 mM EDTA) overnight at 37 °C with shaking. Unreacted modification reagent was removed by desalting using 7 K MWCO Zeba™ Spin Desalting Columns (ThemoFisher Scientific, UK) following manufacturer’s instructions.

The azide modified Optimer and DBCO modified Fc fragment were then incubated using a 2:1 molar ratio (Optimer: Fc) in conjugation buffer (1x PBS, pH 7.4 + 10 mM EDTA) overnight at 37 °C with shaking. The Optimer-Fc conjugate was purified by Size- Exclusion Chromatography using a HiLoad™ 26/600 Superdec™ 75 pg columns (Cytiva) equilibrated into purification buffer (20 mM Tris-HCI, 500 mM NaCI, pH 7.4) at a flow rate of 1 ml/min. Fractions containing the Optimer-Fc conjugate were combined and concentrated using a 5,000 MWCO Vivaspin™ 6 Centrifugal Concentrator (Sartorius Corporation, USA). Figure 5 illustrates a schematic representation of IHC/ICC imaging using a traditional antibodybased approach (left) or an Optimer-Fc based approach (right). In the antibody approach, the cell/tissue surface biomarker is recognised and bound by the primary (1°) antibody. A secondary (2°) antibody is then used which binds to the target bound primary antibody. In this example, the target binding primary antibody is from a rabbit; the secondary antibody is from a different species (in this example, from a goat). The anti-rabbit, goat secondary antibody is labelled to allow detection. This may be a fluorophore or other enzyme, but in this example, it is labelled with biotin. This allows capture of a streptavidin-Horseradish Peroxidase (HRP) conjugate, which in turn allows site specific turnover of a substrate. In this case, the substrate is DAB, which is oxidised by HRP and produces a brown precipitate at the site of action. This precipitate is then readily visualised under a light microscope.

In the Optimer-based approach; the target specific Optimer is used to recognise and bind the cell/tissue surface biomarker. The Optimer is already conjugated to the Fc domain of an antibody (in this example, a rabbit Fc domain is used). This is then bound by the same antirabbit, goat secondary antibody, and detected using strep-HRP and DAB (as for the antibodybased approach).

EXAMPLE 3 - Assessing function of aptamer-Fc conjugates

The aptamers described herein have been isolated using a recombinant protein which represents the target receptor, expressed on a cell surface. These aptamers were further characterised for binding to cells expressing the receptor, by flow cytometry and fluorescence microscopy. For use of the aptamers in commercial IHC workflows, it is desirable that the aptamers and/or minimal functional fragment Optimers are capable of binding to the target protein as it is presented in a cell and tissue sample. It is also desirable that the aptamers and/or Optimers are able to bind the target biomarker and that the Optimer is in turn bound by the secondary antibody. To demonstrate this dual binding function, the Optimer-Fc conjugate was assessed using a BLI assay. Briefly, exemplar target proteins (His tagged) were immobilised onto Ni-NTA coated BLI sensor probes (Sartorius Corporation, USA, following manufacturer protocols. The immobilised target was then interacted with the target specific Optimer-Fc conjugate. Successful binding demonstrated that the presence of the conjugated Fc domain did not interfere with the ability of the Optimer to bind its respective target. The complex between the target and Optimer-Fc conjugate was then further incubated with the ‘secondary antibody’ (31216, ThemoFisher Scientific, UK) which recognised and bound to the conjugated Fc domain. This demonstrated that the Fc domain was presented in a way which does not preclude binding of the secondary antibody. For example, Figure 7 illustrates a Biolayer Interferometry (BLI) demonstrating the concepts required to use the Optimer-Fc conjugates in IHC and ICC. In the example here, the streptavidin coated BLI biosensor probe is first incubated in aptamer binding buffer, to establish a stable ‘Baseline’ response (0-60 sec). The target protein (biotinylated CD4) was then immobilized onto the biosensor (60-360 sec). A brief wash and buffer incubation (360- 420 and 420-480 sec) are then used to show that the target loading is stable and to establish a new baseline. The CD4 loaded biosensor is then incubated with the anti-CD4 Optimer-Fc conjugate and shows a clear interaction (480-600 sec). This is followed by a dissociation phase (600-720 sec), during which the response remains stable; indicating that the Optimer- Fc conjugate has remained bound to the CD4 loaded biosensor. This is then followed by another association with the anti-rabbit goat secondary antibody (720-840 sec). A clear binding response is seen, indicating that the secondary antibody has bound. This response remains consistent during the subsequent dissociation phase (840-960 sec), indicating that the interaction is stable. Together, this data (red trace) shows that the CD4 Optimer-Fc conjugate is able to bind to immobilised target protein (CD4) and is in turn bound by the antirabbit, goat secondary antibody. This demonstrates all the interactions required for use in IHC and ICC.

Parallel control experiments show that when no CD4 is immobilised on the biosensor, there is no subsequent interaction with the Optimer-Fc conjugate or the secondary antibody (violet trace). Controls also show that if no Optimer-Fc conjugate is introduced, then no binding is seen for the secondary antibody (pale blue trace). This demonstrates that the secondary antibody does not bind to CD4 alone. Data also shows that if the Optimer alone is added (without the FC conjugate), a binding response is seen with immobilised CD4, but there is no subsequent binding to the secondary antibody (purple trace). This shows that without the conjugated Fc domain, the secondary antibody does not bind to either the Optimer alone or the CD4 protein.

Data shown in Figure 7 demonstrates a clear immobilisation of the target protein (60-360 sec) followed by an interaction between the immobilised target and the Optimer-Fc conjugate (480- 720 sec) followed by a further interaction with the secondary antibody (720-960 sec). Importantly, each interaction shows a rapid association rate (480-600 and 720-840 sec) and a slow dissociation rate (600-720 and 840-960 sec); which may be important for many diagnostic applications such as ICC and IHC.

EXAMPLE 4 - Protocol for ICC chromogenic detection Cell culture: 45,000 CD4+ target cell line (H9, HTB-176) and the CD4- counter cell line (D1.1 , ATCC-CRL-3600) were seeded onto Poly-D-lysine coated coverslips and incubated for 24 hours at 37 °C, 5% CO₂. Cells were fixed by adding 4% paraformaldehyde onto the cell culture for 10 minutes followed by centrifugation at 600 x g for 5 minutes. Fixed cells were stored in PBS + 0.1 % BSA, at 4 °C and used within 2 weeks of fixing.

Blocking non-specific background binding: Abundance of protein or DNA in complex sample matrix can cause non-specific background staining during immunocytochemistry assays. Blocking steps are performed to eliminate the background staining. This is usually performed by blocking endogenous peroxidase activity followed by blocking non-specific background interactions of the Optimers.

In this example, coverslips were blocked with hydrogen peroxide blocking solution (ab64218, Abeam, UK) following the manufacturer’s instructions; then washed three times with the wash buffer. Coverslips to be stained with Optimer-Fc conjugate were incubated for 1 hour in DNA- based 2x blocking buffer (containing 1x PBS, 50 mM glucose, 5 mM MgCI₂, 0.2 mg/mL salmon sperm DNA, 0.2 mg/mL tRNA, and 20 mg/mL BSA) at room temperature followed by three washes with 1x TBST for 5 minutes each. A commercially available anti-CD4 antibody (ab133616, Abeam, UK) was used as a positive control and slides to be stained with the antibody were incubated for 1 hour in a protein-based blocking buffer (20 % FBS) at room temperature followed by three washes with 1x TBST for 5 minutes each.

Antibody/Optimer staining: 2x blocking buffer was diluted in 1x PBS to make 1x binding buffer. Optimer-Fc conjugate was then prepared to the desired concentration of 40 pg/mL in 1x binding buffer. CD4+ expressing H9 cells were incubated with 200 pL of Optimer-Fc conjugate. Comparator H9 cells were incubated with 1/500 dilution of CD4-antibody while a separate aliquot of H9 cells were incubated with buffer only, as an untreated negative control. CD4- D1.1 cells were included as a negative tissue control to ensure Optimer-Fc conjugate stains only cells expressing CD4, they were incubated with 200 pL of Optimer-Fc. All slides were incubated for 1 hour at room temperature or overnight at 4 °C, then washed three times, with 1x TBST for 5 minutes each.

For chromogenic detection, all coverslips were treated in the same way from this point onwards. Secondary antibody staining: coverslips were incubated with 200 pL of “ready to use” HRP conjugated goat-anti- rabbit secondary antibody (ab214880, Abeam, UK) and incubated for 1 hour at room temperature. Unbound HRP conjugate was removed by washing three times with 1x TBST, for 10 minutes each.

DAB staining and haematoxylin staining: 50x DAB chromogen substrate kit (ab64238, Abeam, UK) and a 1x DAB working solution was prepared according to the manufacturer’s protocols. 200 pL was applied to cells and the chromogenic reaction was monitored for up to 10 minutes as the epitope sites turned brown. Coverslips were washed three times in deionised water for 2 minutes each. Counterstaining of the nuclei was performed by incubating with 200 pL 25 % hematoxylin for 30 seconds, followed by three washes in deionised water, for 5 minutes each.

Mounting with aqueous mounting media: coverslips were mounted with aqueous mountant. A drop of aqueous media (ab64230, Abeam, UK) was added to clear microscope slide for mounting coverslips and allowed to cure for 2 hours at room temperature before imaging. Coverslips were imaged using brightfield light microscopy.

For example, Figure 8 illustrates Immunocytochemistry (ICC) analysis of Optimer-Fc conjugate staining of CD4 expressing cells. DAB staining was performed on CD4 positive cell line (H9) and a CD4 counter cell line (D1.1) cultured onto coverslips. After blocking for endogenous peroxidase activity and non-specific backgrounds, Antibody/Optimer-Fc staining was performed overnight at 4 °C. Cells were stained with DAB for 2 mins and counterstained with hematoxylin for 1 minute followed by washing. Coverslips were mounted onto glass slides with aqueous mounting media. The immunostaining was performed manually, and Image acquisition was performed with EVOS microscope (ThermoFisher Scientific, UK).

DAB staining analysis in Figure 8 demonstrates that CD4 specific stain was obtained with Anti- CD4 antibody (ab133616, Abeam, UK) and CD4 Optimer-Fc conjugate as expected. No staining in the control coverslips (c) and (f) as expected. Background binding was observed with D1.1 stained with Optimer-Fc; however, more blocking steps would readily eliminate the nonspecific binding. Note: coverslip for H9 CD4 antibody was stuck to the well hence the phase contrast when imaged. Coverslip (d) could not be imaged.

EXAMPLE 5 - Protocol for IHC-FFPE Immunofluorescence detection Slide treatment: Prior to processing, all tissue section slides were baked in the oven at 60 °C for 30 minutes. This step is included to minimise tissue detaching from slides during process handling and is known to people skilled in the art. Deparaffinisation/dewaxing and rehydration of tissue was performed using a xylene and ethanol gradient wash (2x xylene; 2x 100 % ethanol; 1x 95 % ethanol; 1x 75 % ethanol; 50 % ethanol; 100 % distilled H₂O) for five minutes each. Slides were left in distilled water until ready for antigen retrieval step.

Antigen retrieval: Epitope on fixed tissue were retrieved by performing the antigen retrieval step, this could be performed using Heat-Induced Epitope Retrieval (HIER) or through enzymatic digestion. HIER could be achieved by exposing tissues immersed in retrieval buffer to high temperature. Suitable buffers commonly used for HIER are Tris-EDTA pH 9, Tris buffer pH 8 or Citrate buffer pH 6 or any other buffer known to the person skilled in the art. Suitable equipment for antigen retrieval are microwave, scientific pressure cooker, autoclave and incubator. Any suitable antigen retrieval method, known to those skilled in the art, may be applied.

In this example, slides were completely immersed in Citrate buffer pH 6 and heated in a microwave on high, medium, and low power for 5 minutes each. Buffers were topped up between heating steps to prevent slides from drying out. Slides were then transferred into distilled water and incubated for 10 minutes to bring them to room temperature, followed by three washes in Ix TBST.

Blocking non-specific background binding: Abundance of protein or DNA in complex sample matrix usually causes non-specific background staining during immunohistochemistry assays. Hence blocking steps are performed to eliminate the background staining.

In this example tissues slides to be stained with Optimer-Cy3 conjugate were incubated for 1 hour in DNA-based 2x blocking buffer (containing 1x PBS, 50 mM glucose, 5 mM MgCI₂, 0.2 mg/mL salmon sperm DNA, 0.2 mg/mL tRNA, and 20 mg/mL BSA) at room temperature followed by three washes in 1x TBST, for 5 minutes each. A commercially available fluorescently labelled anti-CD4 antibody (ab280849, Abeam, UK) was used as a positive control and slides to be stained with the antibody were incubated for 1 hour in a protein-based blocking buffer (20 % FBS) at room temperature followed by three washes in 1x TBST, for 5 minutes each.

Optimer folding: An equal volume of Optimer stock was diluted in 2x folding buffer (0.1M Tris pH 7.4, 5 mM MgCI₂, 1 mM CaCI₂, 20 mM NaCI, 4.5 mM KOI, 20 mM Na₂SO₄) and folded at 95 °C for 5 minutes in a thermocycler. 2x blocking buffer was diluted in 1x PBS to make 1x binding buffer and then folded optimer-Cy3 was further diluted to a working concentration of 8 pM in 1x binding buffer. Aptamers were folded no more than two hours before adding to the tissue slides.

Antibody/Optimer staining: Human tonsil tissue sections were incubated with 200 pL of Optimer-Cy3. Comparator tonsil slides were incubated with 1/100 dilution of CD4-antibody, while a separate slide was incubated with buffer only, as an untreated negative control. Colon tissue section slides were included as a negative tissue control to ensure Optimer-Cy3 conjugate stains only tissue expressing CD4. Tissues were incubated with 200 pL of Optimer- Cy3 for 1 hour at room temperature in the dark. Unbound Optimer reagents were then removed by washing three times with 1x TBST, for 10 minutes each. Counterstaining of the nuclei was performed by incubating with 200 pL of diluted NucBlue (2 drops per 1x TBS) (R37605, Invitrogen, UK) for 10 minutes, followed by three washes in cold deionised water for 5 minutes each.

For the immunofluorescence detection, all slides were treated in the same way from this point onwards.

Mounting with organic/ aqueous mounting media: Slides were mounted in Prolong Diamond mountant (Invitrogen, P36961) and cover with glass coverslips. Slides are cured for 24 hours in the dark before imaging. Tissue sections were imaged using fluorescent microscopy.

EXAMPLE 6 - Protocol for IHC chromogenic detection of CD4 on frozen tonsil tissue using Optimer-Fc conjugate

Slide treatment: Frozen tissue slides were taken from the -80 °C freezer and left to reach room temperature on the bench for over 5 minutes. The fixing agent (acetone) was precooled at -20 °C for 30 minutes. Tissues were fixed with the precooled fixing agent at room temperature for 10 minutes. Excess acetone removed with 3 washes in 1x PBS and then rinsed twice in 1x TBST. The absence of a formaldehyde-based fixative eliminates the need for an antigen retrieval step. However, if frozen tissue or cytological specimens have been fixed in formalin, antigen retrieval step(s) can be included.

Blocking non-specific background binding: Abundance of protein or DNA in complex sample matrix can cause non-specific background staining during immunohistochemistry assays. Blocking steps are performed to eliminate the background staining. This is usually performed by blocking endogenous peroxidase activity followed by blocking non-specific background interactions of the Optimers.

In this example tissues were blocked with hydrogen peroxide blocking solution (ab64218, Abeam, UK) following the manufacturer’s instructions, followed by three washes with 1x TBST. Slides to be stained with Optimer-Fc conjugate were incubated for 1 hour in DNA-based 2x blocking buffer (containing 3.28x PBS, 50 mM glucose, 5 mM MgCI₂, 2 mg/mL salmon sperm DNA, 0.2 mg/mL tRNA, and 20 mg/mL BSA) at room temperature followed by three washes in 1x TBST, for 5 minutes each. A commercially available anti-CD4 antibody (ab133616, Abeam, UK) was used as a positive control and slides to be stained with the antibody were incubated for 1 hour in a protein-based blocking buffer (20 % FBS) at room temperature followed by three washes with 1x TBST, for 5 minutes each.

Antibody/ Optimer staining: 2x blocking buffer was diluted in PBS to make 1x binding buffer. Optimer-Fc conjugate was then prepared to the desired concentration of 40 pg/mL in 1x binding buffer. Human tonsil tissue section slides were incubated with 200 pL of Optimer-Fc conjugate. Comparator tonsil slides were incubated with 1/500 dilution of CD4-antibody, while a separate slide was incubated with buffer only, as an untreated negative control. Colon tissue slides were included as a negative tissue control to ensure Optimer-Fc conjugate stains only tissue expressing CD4, they were incubated with 200 pL of Optimer-Fc. All slides were incubated for 1 hour at room temperature or overnight at 4 °C, then washed three times with 1x TBST, for 5 minutes each.

For chromogenic detection, all slides were treated in the same way from this point onwards.

Secondary antibody staining: Slides were incubated with 200 pL of “ready to use” HRP conjugated goat-anti- rabbit secondary antibody (ab214880, Abeam, UK ) and incubated for 1 hour at room temperature. Unbound HRP conjugate was removed by washing three times with 1x TBST, for 10 minutes each.

DAB staining and haematoxylin staining: 50x DAB chromogen substrate kit (ab64238, Abeam, UK) and a 1x DAB working solution was prepared according to the manufacturer’s protocols. 200 pL was applied to tissue sections and the chromogenic reaction was monitored for up to 10 minutes as the epitope sites turned brown. Tissues were washed three times in deionised water for 2 minutes each. Counterstaining of the nuclei was performed by incubating with 200 pL of 25 % hematoxylin for 30 seconds, followed by 3 washes in deionised water for 5 minutes each.

Mounting with organic/ aqueous mounting media: Slides were mounted with either organic based or aqueous mountant.

For organic based mounting, tissues were first dehydrated in an increasing gradient of ethanol washes (1x 50% ethanol; 1x 75% ethanol; 1x 95% ethanol; 2x 100% ethanol) 5 minutes each followed by two xylene washes for clearing the tissues. A drop of Histomount (008030, ThermoFisher Scientific, UK) organic mounting media was added to coverslips for mounting tissue slides. Slides are cured for 24 hours at room temperature in the fume hood before imaging.

For aqueous based mounting, a drop of aqueous media (ab64230, Abeam, UK) was added to cover slips for mounting tissue slides and allowed to cure for 2 hours at room temperature before imaging. Tissue sections were imaged using brightfield light microscopy.

For example, Figure 9 illustrates immunohistochemistry analysis of frozen human tonsil tissue labelling CD4 with Optimer-Fc. In particular, slides were treated with pre-cooled acetone for 10 mins then washed with 1x PBS. After blocking for endogenous peroxidase activity and nonspecific backgrounds, Optimer staining was performed overnight at 4 °C. Tissues were stained with DAB for 2 mins and counterstained with hematoxylin for 1 minute. Tissues were dehydrated using increasing ethanol graduate and clarified with two washes with xylene before cover slipping.

DAB staining analysis in Figure 9 demonstrates comparable specific binding of CD4 Optimer- Fc to the intended CD4 cells on FFPE tonsil tissues in (a) to CD4 antibody staining in (b). No specific CD4 staining seen on the negative tissue control (c, d).

EXAMPLE 7 - Protocol for IHC chromogenic detection of CD4 on formalin fixed paraformaldehyde fixed (FFPE) tonsil tissue using Optimer-Fc conjugate

Slide treatment: Prior to processing, all tissue slides were baked in the oven at 60 °C for 30 minutes to minimise tissue detaching from slides during processing. Deparaffinisation/dewaxing and rehydration of tissue was performed using a xylene and ethanol gradient wash (2x xylene; 2x 100 % ethanol; 1x 95 % ethanol; 1x 75 % ethanol; 50 % ethanol; 100 % distilled H₂O) for five minutes each. Slides were left in distilled water until ready for antigen retrieval step.

Antigen retrieval: Epitopes on fixed tissue were retrieved by performing the antigen retrieval step, this can be performed using Heat-Induced Epitope Retrieval (HIER) or through enzymatic digestion. In HIER, the tissues are immersed in retrieval buffer at high temperature. Commonly used buffers for HIER are Tris-EDTA pH 9, Tris buffer pH 8 or Citrate buffer pH 6 or any other buffer known to the person skilled in the art. Suitable equipment for antigen retrieval are microwave, scientific pressure cooker, autoclave and incubator. Any suitable antigen retrieval method, known to those skilled in the art, may be applied.

In this example, slides were completely immersed in Citrate buffer pH 6 and heated in a microwave on high, medium, and low power, for 5 minutes each. Slides were then transferred into distilled water and incubated for 10 minutes to bring them to room temperature, followed by three washes in wash buffer (1x TBST).

Blocking non-specific background binding: Abundance of protein or DNA in complex sample matrix can cause non-specific background staining during immunohistochemistry assays. Blocking steps are performed to eliminate the background staining. This is usually performed by blocking endogenous peroxidase activity followed by blocking non-specific background interactions of the Optimers.

In this example tissues were blocked with hydrogen peroxide blocking solution (ab64218, Abeam, UK) following the manufacturer’s protocol, followed by three washes with wash buffer. Slides to be stained with Optimer-Fc conjugate were incubated for 1 hour in DNA-based 2x blocking buffer (containing 1x PBS, 50 mM glucose, 5 mM MgCI₂, 0.2 mg/mL salmon sperm DNA, 0.2 mg/mL tRNA, and 20 mg/mL BSA) at room temperature followed by three washes in 1x TBST, for 5 minutes each. A commercially available anti-CD4 antibody (ab133616, Abeam, UK) was used as a positive control and slides to be stained with the antibody were incubated for 1 hour in a protein-based blocking buffer (20 % FBS) at room temperature followed by three washes with 1x TBST, for 5 minutes each.

Antibody/Optimer staining: 2x blocking buffer was diluted in 1x PBS to make 1x binding buffer. Optimer-Fc conjugate was then prepared to the desired concentration of 40 pg/mL in 1x binding buffer. Human tonsil tissue section slides were incubated with 200 pL of Optimer Fc. Comparator tonsil slides were incubated with 1/500 dilution of CD4-antibody, while a separate slide was incubated with buffer only, as an untreated negative control. Colon tissue slides were included as a negative tissue control to ensure Optimer-Fc conjugate stains only tissue expressing CD4, they were incubated with 200 pL of Optimer-Fc. All slides were incubated for 1 hour at room temperature or overnight at 4 °C, then washed three times with 1x TBST, for 5 minutes each.

For chromogenic detection, all slides were treated in the same way from this point onwards.

Secondary antibody staining: Slides were incubated with 200 pL of “ready to use” HRP conjugated goat-anti- rabbit secondary antibody (ab214880, Abeam, UK) and incubated for 1 hour at room temperature. Unbound HRP conjugate was removed by washing three times with 1x TBST, for 10 minutes each.

DAB staining and haematoxylin staining: 50x DAB chromogen substrate kit (ab64238, Abeam, UK) and a 1x DAB working solution was prepared according to the manufacturer’s protocols. 200 pL was applied to tissue sections and the chromogenic reaction was monitored for up to 10 minutes as the epitope sites turned brown. Tissues were washed three times in deionised water for 2 minutes each. Counterstaining of the nuclei was performed by incubating with 200 pL of 25 % hematoxylin for 30 seconds, followed by three washes in deionised water for 5 minutes each.

Mounting with organic/ aqueous mounting media: Slides were mounted with either organic based or aqueous mountant.

For organic based mounting, tissues were first dehydrated in an increasing gradient of ethanol washes (1x 50% ethanol; 1x 75% ethanol; 1x 95% ethanol; 2x 100% ethanol) 5 minutes each followed by two xylene washes for clearing the tissues. A drop of Histomount (008030, ThermoFisher Scientific, UK) organic mounting media was added to coverslips for mounting tissue slides. Slides are cured for 24 hours at room temperature in the fume hood before imaging.

For aqueous based mounting, a drop of aqueous media (ab64230, Abeam, UK) was added to cover slips for mounting tissue slides and allowed to cure for 2 hours at room temperature before imaging. Tissue sections were imaged using brightfield light microscopy.

For example, Figure 10 illustrates immunohistochemistry analysis of FFPE preserved human tonsil tissue labelling CD4 with Optimer-Fc. Chromogenic staining analysis demonstrates specific binding of CD4 aptamer-Fc to the intended CD4 cells on FFPE human tonsil tissues (a-c) as well as on gut-associated lymphoid tissue (d). Commercially available CD4 antibody was used as positive control (e) while no staining seen on the negative controls (f-h) as expected.

For example, Figure 4 illustrates analysis of formalin-fixed paraffin-embedded (FFPE) human tonsil tissue labelled CD4 with Optimer-Cy3 at 8pM. Following deparaffinisation and rehydration in ethanol gradient wash, tissue sections were treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 15 mins.

Staining of CD4+ cells was performed by incubating tissue sections with Optimer-Cy3 for 1 hour followed by nuclear counterstain with NucBlue for 10 mins. The immunostaining was performed manually, and image acquisition was performed with EVOS microscope. The immunofluorescence staining analysis of Figure 4 demonstrates specific binding of the Cy3 labelled CD4 Optimer to the intended CD4+ expressing cells on FFPE tonsil tissues showing as red stains (a-b) with the nuclei counterstaining shown as blue. No specific Optimer staining seen in colon tissue used as the negative tissue control (c). Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including, but not limited to” and they are not intended to (and do not) exclude other moieties, additives, components, integers, or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of the features and/or steps are mutually exclusive. The invention is not restricted to any details of any foregoing embodiments. The invention extends to any novel one, or novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

The reader’s attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

Claims

Claims

1. A method for detecting a target molecule in a sample, wherein the method comprises: a) applying to the sample an aptamer-Fc conjugate, wherein the sample comprises substantially intact tissue and the aptamer region of the conjugate is capable of specifically binding to the target molecule; b) applying to the sample a labelled secondary agent, wherein the agent is capable of specifically binding to the Fc region of the conjugate; and c) detecting the presence, absence and/or level of the secondary agent.

2. The method of claim 1, wherein the secondary agent is a labelled secondary antibody capable of specifically binding to the Fc region of the conjugate.

3. The method of claim 1 or 2, wherein the sample comprises frozen tissue or formalin- fixed paraffin-embedded tissue.

4. The method of any one of claims 1 to 3, wherein:

(i) the aptamer-Fc conjugate is not an active therapeutic agent; and/or

(ii) the aptamer and/or Fc region of the conjugate does not comprise any payload or cargo.

5. The method of any one of the preceding claims, wherein the aptamer region is conjugated to the Fc region using one or more primary amines (NH2), sulfhydryl groups (SH), azide, alkyne and/or carboxyl groups (COOH).

6. The method of any one of the preceding claims, wherein the presence, absence and/or level of the secondary agent is visualised by light and/or fluorescent microscopy.

7. The method of any one of the preceding claims, wherein the presence, absence and/or level of the secondary agent is detected by immunohistochemistry (IHC), wherein the aptamer-Fc conjugate replaces the use of a primary antibody in a standard workflow.

8. The method of any one of the preceding claims, wherein, the sample is pre-treated with a nucleic acid-based blocking buffer.

9. The method of claim 8, wherein the blocking buffer comprises one or more DNA- based blocker, optionally wherein the DNA-based blocker is salmon sperm DNA at a concentration of between about 0.2 mg/mL to about 2 mg/mL.

10. The method of claim 9 or 10, wherein the blocking buffer comprises one or more sulphated polysaccharide-based blocker, optionally wherein:

(i) the sulphated polysaccharide-based blocker is dextran sulphate in an amount of between about 0.1% to about 2% w/v of the composition; and/or

(ii) the sulphated polysaccharide-based blocker is heparin at a concentration of between about 1 U/rnL to about 20 U/rnL.

11. The method of any one of claims 8 to 10, wherein the blocking buffer comprises one or more RNA-based blocker, optionally wherein the RNA-based blockers are selected from yeast tRNA.

12. The method of claim 11 , wherein the RNA-based blocker is yeast tRNA at a concentration of between about 0.2 mg/mL to about 2 mg/mL.

13. The method of any one of claims 8 to 12, wherein the sample is incubated with the blocking buffer at room temperature for about 1 hour, overnight at about 4°C, or rapidly at 37°C for up to about 30 minutes.

14. The method of any one of the preceding claims, wherein:

(i) the label of the secondary agent is a fluorophore, optionally wherein the label is detected by fluorescent microscopy;

(ii) the label of the secondary agent is horseradish peroxidase and/or alkaline phosphatase, optionally wherein the label is incubated with a chromogenic substrate and detected by light microscopy; or

(iii) the label of the secondary agent is biotin, optionally wherein the label is incubated with avidin or an avidin-conjugate and detected by light microscopy.

15. The method of any one of the preceding claims, wherein the target molecule is an antigen.

16. The method of any one of the preceding claims, wherein the method comprises:

(i) pre-treating the sample with a nucleic acid-based blocking buffer; (ii) contacting the sample with the aptamer-Fc conjugate, wherein the presence of a target molecule creates a complex between the aptamer region of the aptamer-Fc conjugate and the target molecule;

(iii) optionally performing one or more wash step(s);

(iv) contacting the sample with the labelled secondary agent, wherein the presence of the secondary agent creates a complex between the Fc region of the aptamer-Fc conjugate and the secondary agent;

(v) optionally performing one or more additional wash step(s); and/or

(vi) detecting and/or quantifying the label of the secondary agent.

17. The method of claim 16, wherein the method does not comprise an antigen retrieval step prior to step (i).

18. The method of any one of the preceding claims, wherein the method is an automated method.