Nucleic acid aptamers

Abstract

Disclosed herein are aptamers specific for PD1, PDL1 and complexes of PD1-PDL1, and probes specific for such aptamers. Also disclosed are methods of quantifying the fraction of PD1 occupied by PDL1 and the fraction of PDL1 occupied by PD1 in a biospecimen including, but not limited to, fresh, frozen or formalin-fixed, paraffin embedded tissue, cells in suspension and soluble protein complexes. Also disclosed are methods for selecting and using aptamers to quantify the fractional occupancy of one molecule by another, including receptors, ligands and other members of a molecular complex, in mammalian tissue.

Description

[0001] NUCLEIC ACID APTAMERS

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims priority to United States Provisional Application Number 63 / 764,295, which was filed on February 27, 2025. The entire content of the application referenced above is hereby incorporated by reference herein.

[0004] STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under CA97274 and CA86862 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] BACKGROUND

[0007] Molecular complexes, including ligand-receptor complexes, have significant therapeutic and diagnostic potential in clinical settings as well as in research. While the significance of ligand-receptor complexes in health and disease pathogenesis is well known, ligand-receptor complexes are understudied as biomarkers mainly due to the limitations in detection technology. Commercially available diagnostic methodologies, largely based on monoclonal antibody (mAb) technology, are well suited for detecting receptors and ligands that exist separately, but the ability to determine whether receptors are occupied by ligand is more difficult with mAb technologies. This has hindered the evolution of diagnostic technologies that can accurately measure changes in the occupancy of receptors by ligands, or ligands by receptors, in the body during health and diseases, thus missing a whole class of potentially valuable clinical biomarkers. In addition, the inability to generate mAbs against molecular complexes also narrows the range of antigens that could be used diagnostically in diseases, including cancers. There is an on-going need to directly quantify the fraction of receptors occupied by a ligand in a given biospecimen, which would provide an additional and valuable clinical and research tool in diagnosis, determining likelihood of response to a given therapy or assessing whether an ongoing therapy is targeting a ligand-receptor target as intended.

[0008] SUMMARYIn certain aspects, provided herein is a PD1 -binding aptamer comprising a polynucleotide 45-55 nucleotides in length comprising SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0009] In certain aspects, provided herein is a PDL1 -binding aptamer comprising a polynucleotide 45-55 nucleotides in length comprising SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0010] In certain aspects, provided herein is a PD1-PDL1 complex-binding aptamer comprising a polynucleotide 45-55 nucleotides in length comprising SEQ ID NO:7, SEQ ID NO:8, or SEQ ID N0:9.

[0011] In certain aspects, provided herein is a composition comprising an aptamer as described above operably linked to a tag.

[0012] In certain aspects, provided herein is a labelled probe comprising

[0013] (a) a polynucleotide selected from the group consisting of a probe for P17 aptamer (SEQ ID NO: 10), a probe for P150 aptamer (SEQ ID NO: 11), a probe for Cl 13 aptamer (SEQ ID NO: 12), a probe for C18 aptamer (SEQ ID NO: 13), a probe for L42 aptamer (SEQ ID NO: 14) or a probe for L50 aptamer (SEQ ID NO: 15), and (b) a label.

[0014] In certain aspects, provided herein is a method of quantifying a fractional occupancy (FO) of PD1 by PDL1 or of PDL1 by PD1 in a biospecimen, the method comprising: contacting the sample with a first aptamer that specifically binds to PD1, with a second aptamer that specifically binds to PDL1, and with a third aptamer that specifically binds to PD1 / PDL1 complex, and concurrently determining the concentration of bound first aptamer, second aptamer and third aptamer.

[0015] In certain aspects, provided herein is a method of quantifying in a biospecimen fractional occupancy (FO) of one member of a molecular complex by another member of the molecular complex, the method comprising: contacting the biospecimen with a first aptamer that specifically binds to the first member of the molecular complex, with a second aptamer that specifically binds to the second member of the molecular complex, and with a third aptamer that specifically binds to the complex of the first member and the second member, and concurrently determining the concentration of bound first aptamer, second aptamer and third aptamer in the biospecimen.

[0016] BRIEF DESCRIPTION OF THE DRAWINGSFigures 1A-1C. General Scheme of LIRECAP Assay. Scheme of LIRECAP assay including the two-aptamer LIRECAP assay (Figure 1 A) and the three-aptamer LIRECAP assay (Figure IB). Expected two-aptamer and three-aptamer PD1-PDL1 LIRECAP clinical assay results related to T cell infiltration and PDL1 expression in tumors (Figure 1C).

[0017] Figures 2A-2C. SELEX to enrich aptamer pools and identify key candidate sequences that are specific for PD1, PDL1 or the PD1-PDL1 complex. Scheme of LIRECAP SELEX (Figure 2A). Enrichment of aptamer pools observed with sequential rounds of SELEX using PD1, PDL1 and the PD1-PDL1 complex as targets (Figure 2B). Preferential binding of aptamer pools enriched for binding to PD1, PDL1 or Complex with sequential rounds of SELEX (Figure 2C).

[0018] Figures 3A-3D. Lead PD1, PDL1 and PDl-PDLl-complex aptamer candidates identified by SELEX. Fold enrichment of select aptamer sequences following multiple rounds of SELEX using PD1, PDL1 and PD1-PDL1 complex as targets (Figure 3A). Predicted secondary structures of lead candidate aptamers demonstrating significant structural heterogeneity including PDl-binding aptamers (Figure 3B), PDLl-binding aptamers (Figure 3C) and Complex-binding aptamers (Figure 3D).

[0019] Figures 4A-4C. Specific binding of P aptamer candidates to beads coated with PD1, PDL1 and PD1-PDL1 complex proteins. Binding of P aptamer candidate P10 (Figure 4A), P17 (Figure 4B) and Pl 50 (Figure 4C).

[0020] Figures 5A-5D. Specific binding of P aptamer candidates to beads coated with PD1 protein with various levels of FO by PDL1. Scheme illustrating production of beads coated by PD1 with various levels of FO by PDL1 (Figure 5A). Binding of specific aptamers to PD1 beads based on FO by PDL1 for P10 (Figure 5B), P17 (Figure 5C) and Pl 50 (Figure 5D).

[0021] Figures 6A-6B. Specific binding of P aptamer candidates to cells expressing PD1 with various levels of FO by PDL1. Scheme illustrating production of cells that express PD1 and are coated with PDL1 to achieve various levels of FO (Figure 6A). Binding of P aptamers to cells that express PD1 with various levels of FO by PDL1 (Figure 6B).

[0022] Figures 7A-7C. Specific binding of C aptamer candidates to beads coated with PD1, PDL1 and PD1-PDL1 complex proteins. Binding of C aptamer candidate C18 (Figure 7 A), C52 (Figure 7B) and Cl 13 (Figure 7C).

[0023] Figures 8A-8D. Binding of C aptamer candidates to beads coated with PD1 protein with various levels of FO by PDL1. Scheme illustrating production of beads coated by PD1 with various levels of FO by PDL1 (Figure 8A). Binding of specific aptamers to PD1 beads based on FO by PDL1 for C18 (Figure 8B), C52 (Figure 8C) and Cl 13 (Figure 8D).Figures 9A-9B. Binding of complex (C) aptamer candidates to cells expressing PD1 with various levels of FO by PDL1. Scheme illustrating production of cells that express PD1 and are coated with PDL1 to achieve various levels of FO (Figure 9A). Binding of C aptamers to cells that express PD1 with various levels of FO by PDL1 (Figure 9B).

[0024] Figures 10A-10C. Specific binding of L aptamer candidates to beads coated with PD1, PDL1 and PD1-PDL1 protein. Binding of L aptamer candidate L42 (Figure 10A), L45 (Figure 10B) andL50 (Figure 10C).

[0025] Figures 11A-11D. Binding of L aptamer candidates to beads coated with PDL1 protein with various levels of FO by PD1. Scheme illustrating production of beads coated by PDL1 with various levels of FO by PD1 (Figure 11 A). Binding of specific aptamers to PDL1 beads based on FO by PD1 for L42 (Figure 1 IB), L45 (Figure 1 IB) and L50 (Figure 1 ID).

[0026] Figures 12A-12B. Binding of L aptamer candidates to cells expressing PDL1 with various levels of FO by PD1. Scheme illustrating production of cells that express PDL1 coated with various levels of FO by PD1 (Figure 12A). Binding of L aptamers to cells that express PDL1 with various levels of FO by PD1 (Figure 12B).

[0027] Figure 13. Probes and primers for PD1-PDL1 LIRECAP assay. The probes are the following: SEQ ID NO: 10 (Pl 7 probe), SEQ ID NO:11 (P150 probe), SEQ ID NO: 12 (Cl 13 probe), SEQ ID NO: 13 (C18 probe), SEQ ID NO: 14 (L42 probe), and SEQ ID NO: 15 (L50 probe). The common PCR primer pairs are SEQ ID NO: 16 and SEQ ID NO: 17.

[0028] Figures 14A-14B. Two-aptamer LIRECAP assay PD1 beads with various levels of PD1 FO by PDL1 Aptamers P17 and C18. Scheme illustrating production of PD1 coated beads with various levels of FO by PDL1 for pilot evaluation of two-aptamer PD1-PDL1 LIRECAP assay (Figure 14A). Results of pilot LIRECAP assay showing strong correlation between C:P aptamer ratio and FO of PD1 by PDL1 (Figure 14B).

[0029] Figures 15A-15C. Use of P17 and C18 as aptamers in two-aptamer PD1-PDL1 LIRECAP assay using biospecimen produced through co-culture of cells expressing PD1 (Jurkat) and PDL1 (Raji). Scheme illustrating co-culture conditions involving cells that express PD1 (Jurkat) mixed with Raji cells including various ratios of PDL1(+) and PDLl(-) Raji cells to produce conditions with various levels of PD1 FO by PDL1 based on cell-cell interaction (Figure 15A). Demonstration that the level of expression of PDL1 by Raji cells in the co-culture impacts on signaling via PD1 on Jurkat cells as indicated by decreased Jurkat cell luminescence as the percent of Raji cells expressing PDL1 increases (Figure 15B). Results of pilot LIRECAP assay on cell co-culture showing strong correlation between C:P aptamer ratio and FO of PD1 by PDL1 (Figure 15C).Figures 16A-16D. Use of P17 and C18 as aptamers in two-aptamer PD1-PDL1 LIRECAP assay of FFPE scrolls. Scheme illustrating process for generating FFPE scrolls of PD1 positive cell pellets with various numbers of PDL1(+) and PDLl(-) cells to produce various levels of FO by PDL1 (Figure 16A). Proximity ligation assay (PLA) on FFPE scrolls providing semi-quantitative evidence for increased proximity of PD1 and PDL1 in the FFPE samples (Figure 16B). PD1-PDL1 LIRECAP assay showing higher C:P aptamer ratios in samples with high PD1 FO by PDL1 compared to samples with low PD1 FO by PDL1 (Figure 16C). PD1-PDL1 LIRECAP assay showing a correlation between the C:P ratio and FO of PD1 by PDL1 (Figure 16D).

[0030] Figures 17A-17D. Technical, intratumoral and interpatient variability of two-aptamer LIRECAP assay in clinical samples. High PLA score on samples containing tumor that expresses high PDL1 (Figure 17A). Technical variability of LIRECAP assay (Figure 17B). Intratumoral variability of LIRECAP assay (Figure 17C). Interpatient variability of LIRECAP assay (Figure 17D).

[0031] Figure 18. Illustration of a 3-color LIRECAP assay using FFPE scrolls from Jurkat-Raji co-culture system.

[0032] Figure 19. Results of three-aptamer LIRECAP assay using scrolls of FFPE tissue obtained from co-culture of Jurkat and Raji cells with varying expression of PD1 and PDL1 respectively.

[0033] DETAILED DESCRIPTION

[0034] The ability to directly quantify the fraction of receptors occupied by a ligand in a given biospecimen could provide an additional and valuable clinical and research tool in diagnosis, determining likelihood of response to a given therapy or assessing whether an ongoing therapy is targeting a ligand-receptor target as intended. To address this need, a biomarker platform was developed to quantify the fraction of receptors occupied by a ligand using pairs of RNA aptamers, where one aptamer binds preferentially to the unoccupied receptor and the other to the ligand-receptor complex. Bound aptamer is quantified using techniques such as RT-qPCR with colorimetric probes specific for each aptamer. The aptamer ratio quantified in this manner correlates with the fraction of receptors occupied by the ligand. This assay, termed the LIRECAP (Ligand— REceptor Complex-binding APtamer) assay, was initially developed to determine the fraction of soluble CD25 occupied by IL2 in the serum. This proof of concept led to an expanded approach, described herein, to assess molecular complexes, including ligand-receptor complexes on cells, in tumors or in tissue. Among receptors and ligands where quantification of such complexes is likely to be highly impactful is the PD1 / PDL1 system.

[0035] Therapy directed at interfering with the interaction of Programmed cell death protein 1 (PD1) with its ligand PDL1, known as anti-PDl checkpoint blockade, has generated great clinical and commercial excitement in the field of cancer therapeutics. PD1 is expressed by activated T lymphocytes including many of those found in the tumor microenvironment. When PDL1, a prominent ligand for PD1, binds to PD1, it sends an “off’ signal to the T cell. This reduces the ability of the T cell to kill the cell expressing PDL1. If the cell expressing PDL1 is a cancer cell, that cancer cell is less likely to be killed by the T cell. Treatment with antibodies that interfere with the PD1-PDL1 interaction removes this off signal and allows the T cells to remain activated. This can result in an enhanced anti-cancer T cell immune response with significant benefit to the patient. Unfortunately, only a minority of patients benefit from anti-PDl therapy. One of the main reasons many patients treated with anti-PDl do not benefit from therapy is the inability to identify which patients would benefit from the treatment ahead of time. At present, biomarkers for predicting the success of anti-PDl therapy are suboptimal. Measurement of total PDL1 expression by cancer cells in a tumor is often used as predictive biomarker for anti-PDl or anti-PDLl therapy, but is far from ideal. Better biomarker assays capable of directly analyzing PD1-PDL1 complexes could be superior in predicting clinical response to PD1 checkpoint blockade.

[0036] The LIRECAP assay platform was redesigned, applied and expanded upon to develop such an assay that would allow for quantification of PD1, PDL1 and the PD1-PDL1 complex in tissue biospecimens. This assay is the focus of this invention which includes specific components of the PD1-PDL1 LIRECAP assay. This invention also includes technological improvements and novel processes to identify and utilize aptamers in the LIRECAP assay that could be applied to research and clinical care of other molecular pathways were understanding fractional occupancy of a receptor by a ligand, or one molecule by another, in a biospecimen would be of value.

[0037] The invention described herein involves a composition of matter that include aptamers that bind to PD1, PDL1 and the PD1-PDL1 complex, and probes that identify such aptamers. It also includes the novel process by which similar aptamers can be identified used to assess the fractional occupancy (FO) of one molecule by another in tissue. Complexes as used herein refers to groups of molecules having at least 2, at least 3, at least 4, at least 5, at least 6 or greater distinct molecules that are co-located and interact with each other. The molecules can be described as members of a complex. The molecules can be selected from combinations oforganic, inorganic molecules or partially organic molecules. Examples of molecules include proteins, antibodies, active pharmaceutical ingredients including chemotherapeutics, other drugs and markers used in diagnostics. Complexes are formed when more than one molecule is located in a close enough proximity to another molecule that an aptamer can specifically bind to the combination of the more than one molecule. In some instances, the complexes are formed by a receptor-ligand, an antibody-epitope binding, a first domain and a second domain of a multi-domain protein, or an active pharmaceutic ingredient and its associated active site.

[0038] Some processes described herein allow for the making of aptamers that preferentially bind to a particular complex as compared to the aptamers’ ability to bind to the individual members of the complex, which other aptamers bind to the individual members of the complex more extensively than to the complex itself. One of ordinary skill in the art will appreciate that specific binding is a relative term meaning that an aptamer binds more specifically to one molecule than it does to another molecule. This preferential binding is referred to as specific binding and it can be assessed using any method known in the art. For example, a particular aptamer can be separately contacted with an immobilized complex, an individual immobilized member of the complex, and a second immobilized individual member from the complex. The three samples can then be subjected to increasingly stringent physical conditions, for example changes in ionic strength, surfactant concentrations, temperatures, and the like. The amount of aptamer that remains bound can then be determined, and, if there is a higher concentration or quantity of aptamer bound to the complex as compared to the individual molecules from that complex, the aptamer can be characterized as specifically binding to the complex. One of ordinary skill in the art will appreciate that specific binding can be additionally characterized by the strength of the binding. One example of characterizing the strength of the specific binding of an aptamer to a complex is by the amount of aptamer that remains bound to the complex as compared to the amount of aptamer that remains bound to one or more of the individual molecules that make up the complex. Expressed in this way an aptamer can be characterized as having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% more binding to the complex.

[0039] One of ordinary skill in the art will appreciate that by using a set of two or three aptamers, a first aptamer that preferentially binds to a complex, a second aptamer that preferentially binds to an unbound member of the complex, and, in some embodiments, a third aptamer that preferentially binds to a different unbound member of the complex, one can quantify the amount of complexes in a sample as compared to the amount of unbound individual molecules that, when together, make up the complex. Accordingly, methods of detecting therelative amount of bound and unbound complex members are described herein. This includes using either two aptamers (one that binds to an unbound complex member and one that binds to the complex) or three aptamers (one that binds to an unbound complex member, one that binds to the other unbound complex member and one that binds to the complex). These methods can be used as research tools and diagnostics.

[0040] One of ordinary skill in the art will appreciate that molecular complexes are dynamic, and that there is ongoing formation and dissolution of such complexes in a living organism and during the testing of biologic specimens even after those specimens have been procured.

[0041] Furthermore, agents designed to assess such complexes, such as antibodies and aptamers, can impact on such “on” and “off’ rates, and so influence the measurement of such complexes. Accordingly, methods for stabilizing such complexes, and for selection of aptamers using stabilized complexes, are described herein. These methods allow for evaluation of molecular complexes in biological samples that have been stabilized and so are not influenced by formation or dissolution of complexes that takes place after the biological specimen has been procured. Furthermore, this allows for analysis of a broader range of biospecimens by this method, including clinical biospecimens that are formalin fixed and paraffin embedded (FFPE) to be evaluated using these methods.

[0042] Much of the prior art related to complexes involves the study of soluble complexes that are found in the circulation or other bodily fluids. The aptamers described herein are useful, among other things, for binding to complexes that are located on or near the extracellular region of a cell or in tissues. For example, a molecule in the complex can have an extracellular domain that is then associated with a second molecule to form a complex. The extracellular domain does not have to be from a molecule that is completely extracellular, one of ordinary skill in the art will appreciate that many molecules are partially extracellular and that a complex can be formed with the exposed portion of the molecule. One of ordinary skill in the art will also appreciate that a complex can be formed by molecules expressed by a single cell, by molecules on two cells with each contributing a molecule to the complex that is bound by the aptamer, by molecules where one or both are not on a cell but are in the extracellular matrix, by molecules on non-cellular structures such as extracellular vesicles and exosomes, or by molecules where one or both are in soluble form.

[0043] The aptamers described herein can be 45-55 nucleotides in length. For example, the aptamer can be 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55 nucleotides in length.

[0044] The aptamers described herein can be additionally modified to include at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 modifications that increase the stability or specificity of the aptamer. Suchmodifications are characterized as differences to the structure of the nucleotide sequence of the aptamer as compared to a natural nucleotide sequence. These modifications are described in detail herein.

[0045] While the aptamers described herein are RNA aptamers, a similar approach could be developed using DNA aptamers.

[0046] One of ordinary skill in the art will appreciate that aptamers that specifically bind to a complex can be identified and sequenced and that once the sequence of the aptamer is known one of ordinary skill in the art can make alterations, substitutions and / or deletions to the aptamer sequence and test the modified sequence for its ability to selectively bind to a complex. One of ordinary skill in the art will appreciate that when a specific aptamer is referred to such reference includes sequences that share at least 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, or 50% sequence identity to the identified sequence.

[0047] The aptamers described herein can also be linked to additional moieties. These moieties can be useful for detecting the presence of the aptamer, isolating molecules bound to the aptamer, delivering a therapeutic molecule to the location of the complex or stabilizing the complex. One of ordinary skill in the art will appreciate that there are a variety of moieties that can be used to fulfill these functions and any moiety known in the art to be useful can be used. Exemplary moieties are described further herein.

[0048] In certain embodiments, the present invention provides for evaluation of aptamer binding of cells after those cells have been isolated or isolating cells after the aptamers have been added.

[0049] In certain embodiments, the present invention provides a set of two aptamers comprising: (a) a first aptamer that specifically binds to a complex, wherein the complex comprises at least two members, wherein each member is a polypeptide, and (b) a second aptamer that specifically binds to an unbound member of the complex.

[0050] In certain embodiments, the present invention provides a set of three aptamers comprising: (a) a first aptamer that specifically binds to a complex, wherein the complex comprises at least two members, wherein each member is a molecule, (b) a second aptamer that specifically binds to a first unbound member of the complex, and (c) a third aptamer that specifically binds to a second unbound member of the complex.

[0051] In certain embodiments, the present invention provides a method of diagnosing a disease in a mammal, predicting likelihood of response of that disease to therapy, or monitoring the response of that disease to therapy, wherein the disease is associated with an increased or decreased amount of complex relative to unbound molecules in a sample from the mammal. This method is applicable when the complex comprises at least a first molecule and a secondmolecule, the method comprising: (a) quantifying an amount of a first aptamer that is bound to the complex, a second aptamer that is bound to a first unbound member of the complex, and a third aptamer that is bound to a second unbound member of the complex. Together, analysis of these bound aptamers reveals the amount of complexes in a sample as compared to the amount of unbound members of the complex to determine a relative amount of bound and unbound members of a complex.

[0052] Methods of making and using the aptamers that bind selectively to complexes are also described. The aptamers can be selected and produced using any method known in the art. For example, methods based upon the SELEX method can be used to identify aptamers.

[0053] The PD1-PDL1 interaction is particularly attractive for such a diagnostic innovation because of the importance of this interaction in controlling the immune response to cancer, the clinical benefit of treatments geared towards interfering with the interaction between PD1 and PDL1 for many patients with cancer, and the lack of a good prognostic marker that demonstrates which cancer patients will, and will not, respond to such therapy.

[0054] In certain embodiments, the present invention provides a two-aptamer method based on the LIRECAP assay of quantifying a fraction of PD1 occupied by PDL1 in a sample, the method comprising: contacting the sample with a first aptamer that specifically binds to a PD1 / PDL1 complex, contacting the sample with a second aptamer that specifically binds to the unoccupied PD1 and determining the amount of bound first aptamer and second aptamer.

[0055] In certain embodiments, the present invention provides a three-aptamer method based on the LIRECAP assay of quantifying a fraction of PD1 occupied by PDL1 or a fraction of PDL1 occupied by PD1 in a sample, the method comprising: contacting the sample with a first aptamer that specifically binds to a PD1-PDL1 complex, contacting the sample with a second aptamer that specifically binds to the unoccupied PDL1, contacting the sample with a third aptamer that specifically binds to the unoccupied PDL1, and determining the amount of bound first aptamer, second aptamer and third aptamer.

[0056] Aptamers, Compositions and Methods

[0057] In certain aspects, provided herein is a PD1 -binding aptamer comprising a polynucleotide 45-55 nucleotides in length comprising SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0058] In certain aspects, provided herein is a PDL1 -binding aptamer comprising a polynucleotide 45-55 nucleotides in length comprising SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.In certain aspects, provided herein is aPDl-PDLl complex-binding aptamer comprising a polynucleotide 45-55 nucleotides in length comprising SEQ ID NO:7, SEQ ID NO:8, or SEQ ID N0:9.

[0059] In certain aspects, the polynucleotide is 51 or 52 nucleotides in length.

[0060] In certain aspects, provided herein is a composition comprising an aptamer as described above operably linked to a tag.

[0061] In certain aspects, the tag is selected from a molecule that can be detected using optical sensors, molecular size sensors, or isotopic sensors.

[0062] In certain aspects, the aptamer is bound to a substrate.

[0063] In certain aspects, the aptamer is operably linked to a therapeutic moiety.

[0064] In certain aspects, the therapeutic moiety is selected from nucleic acid-based therapeutics, chemotherapeutics, immunotherapeutics, and small molecule therapeutics.

[0065] In certain aspects, provided herein is a composition comprising an aptamer bound to a complex, wherein the complex comprises at least two polypeptides, wherein the aptamer is the PD1 / PDL1 complex-binding aptamer comprising a polynucleotide 45-55 nucleotides in length comprising SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9.

[0066] In certain aspects, the at least two peptides are selected from the group consisting of PD1 and PDL1.

[0067] In certain aspects, the aptamer specifically binds to the complex.

[0068] In certain aspects, the aptamer bound to the complex additionally comprises a tag.

[0069] In certain aspects, provided herein is a composition comprising an aptamer bound to a PD1, wherein the aptamer is the PD1 aptamer comprising a polynucleotide 45-55 nucleotides in length comprising SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.

[0070] In certain aspects, the aptamer bound to the complex additionally comprises a tag.

[0071] In certain aspects, provided herein is a composition comprising an aptamer bound to a PDL1, wherein the aptamer is PDL1 aptamer comprising a polynucleotide 45-55 nucleotides in length comprising SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0072] In certain aspects, the aptamer bound to the complex additionally comprises a tag.

[0073] In certain aspects, provided herein is a labelled probe comprising: (a) a polynucleotide selected from the group consisting of a probe for P17 aptamer (SEQ ID NO: 10), a probe forP150 aptamer (SEQ ID NO: 11), a probe for Cl 13 aptamer (SEQ ID NO: 12), a probe for C18 aptamer (SEQ ID NO: 13), a probe for L42 aptamer (SEQ ID NO: 14) or a probe for L50 aptamer (SEQ ID NO: 15), and (b) a label.

[0074] In certain aspects, provided herein is a method of quantifying a fractional occupancy (FO) of PD1 by PDL1 or of PDL1 by PD1 in a biospecimen, the method comprising: contacting the sample with a first aptamer that specifically binds to PD1, with a second aptamer that specifically binds to PDL1, and with a third aptamer that specifically binds to PD1 / PDL1 complex, and concurrently determining the concentration of bound first aptamer, second aptamer and third aptamer.

[0075] In certain aspects, the biospecimen is a fluid, a cell sample, or tissue.

[0076] In certain aspects, the biospecimen is a fluid, and the fluid is blood, saliva, cerebrospinal fluid, or ascites.

[0077] In certain aspects, the biospecimen is a cell sample, and the cell sample is a fine needle aspirate.

[0078] In certain aspects, the biospecimen is fresh, frozen or fixed in preservative.

[0079] In certain aspects, the biospecimen is evaluated without additional manipulation, placed on a slide, minced, macerated, sonicated or enzymatically digested prior to analysis.

[0080] In certain aspects, the quantification of FO of PD1 by PDL1 or PDL1 by PD1 is performed using a two-aptamer assay.

[0081] In certain aspects, the tag on the first aptamer is distinct from the tag on the second aptamer.

[0082] In certain aspects, the quantification of FO of PD1 by PDL1 or PDL1 by PD1 is performed using of a three-aptamer assay.

[0083] In certain aspects, the tag on the first aptamer is distinct from the tag on the second and third aptamers, the tag on the second aptamer is distinct from the tag on the first aptamer and third aptamer, and the tag on the third aptamer is distinct from the tag on the first aptamer and the second aptamer.

[0084] In certain aspects, the concentration of bound aptamers is determined by PCR or using a single set of primers.

[0085] In certain aspects, each aptamer is individually linked to a tag, and the concentration of bound aptamers is determined by detecting the tags.

[0086] In certain aspects, the detection is by using optical, molecular or isotopic sensors.In certain aspects, assessment of FO of PD1 by PDL1 or PDL1 by PD1 is done in the biospecimen as a whole.

[0087] In certain aspects, assessment of FO of PD1 by PDL1 or PDL1 or PD1 is done using spatial techniques to assess FO in multiple areas of a single or multiple biospecimens.

[0088] In certain aspects, provided herein is a method of quantifying in a biospecimen fractional occupancy (FO) of one member of a molecular complex by another member of the molecular complex, the method comprising: contacting the biospecimen with a first aptamer that specifically binds to the first member of the molecular complex, with a second aptamer that specifically binds to the second member of the molecular complex, and with a third aptamer that specifically binds to the complex of the first member and the second member, and acconcurrently determining the concentration of bound first aptamer, second aptamer and third aptamer in the biospecimen.

[0089] In certain aspects, the first member is a ligand, the second member is a receptor, and the complex comprises a ligand / receptor complex.

[0090] In certain aspects, the biospecimen is a fluid, a cell sample, or tissue.

[0091] In certain aspects, the biospecimen is a fluid, and the fluid is blood, saliva, cerebrospinal fluid, or ascites.

[0092] In certain aspects, the biospecimen is a cell sample, and the cell sample is a fine needle aspirate.

[0093] In certain aspects, the biospecimen is fresh, frozen or fixed in preservative.

[0094] In certain aspects, the biospecimen is evaluated without additional manipulation, placed on a slide, minced, macerated, sonicated or enzymatically digested prior to analysis.

[0095] In certain aspects, the quantification of FO of the receptor by PDL1 or the ligand by the receptor is performed using a two-aptamer assay.

[0096] In certain aspects, the quantification of FO of the receptor by PDL1 or the ligand by the receptor is performed using of a three-aptamer assay.

[0097] In certain aspects, the concentration of bound aptamers is determined by PCR or using a single set of primers.

[0098] In certain aspects, each aptamer is individually linked to a tag, and the concentration of bound aptamers is determined by detecting the tags.

[0099] In certain aspects, the detection is by using optical, molecular or isotopic sensors.

[0100] In certain aspects, assessment of FO of the receptor by the ligand or the ligand by the receptor is done in the biospecimen as a whole.In certain aspects, assessment of FO of the receptor by the ligand or the ligand by the receptor is done using spatial techniques to assess FO in multiple areas of a single or multiple biospecimens.

[0101] Molecular complexes

[0102] Molecular complexes, including those that form between ligands and receptors, heterodimeric and other multimeric molecules, play a central role in mediating a broad range of biological processes. Many therapies for a broad range of diseases are designed to impact on such molecular complexes. One example is regulation of immune cells as mediated by the interaction of PD1 and PDL1. Agents that block such interactions have been established as effective therapeutics for a number of cancers. Numerous studies have illustrated the importance of PD1-PDL1 complexes on the immune response to cancer. Measurement of the individual molecules involved in these interactions, e.g. PD1 and PDL1, has been used to predict likelihood of clinical response in a variety of settings. Most such studies probe with either monoclonal antibodies or labeled ligand that recognize both the unoccupied receptors as well as receptors occupied by ligand. The ability to directly identify and quantify complexes such as the PD1-PDL1 complex, as opposed to assessing its individual components, and comparing the prevalence of such complexes relative to that of its molecular components in their uncomplexed state, could provide an additional and valuable tool for assessing the presence or absence of such complexes in tissues or fluids. Doing so using nucleic acid aptamers in a novel fashion is the focus of this invention.

[0103] Aptamers

[0104] Nucleic acid aptamers are short oligonucleotides that recognize target antigens in a manner analogous to antibodies. The specificity of aptamers, including RNA aptamers, is, in part, based on their nucleotide sequence, which determines their secondary and tertiary structures. RNA aptamers bind to targets via structural complementarity and through forces, including van der Waals forces, hydrogen bonding and electrostatic interaction, and can have affinities similar to those of antibodies.

[0105] Generally, RNA aptamers are generated by a process called SELEX (Systematic Evolution of Ligand by Exponential enrichment) that involves sequential enrichment of a diverse RNA library against known or unknown protein or cellular targets until high-affinity binders are selected. SELEX is generally done using the native primary target, i.e., it does notrequire antigen processing and presentation. Thus, RNA aptamers can be developed against target antigens that are not easily targeted by antibodies, such as self-antigens, and even subtle structural changes in the targets, such as in molecular complexes or antigens that are altered with processing and presentation. The nucleic acid nature of RNA aptamers allows them to be sequenced, synthesized, multiplied and modified easily. RNA aptamers, such as VEGF-binding aptamers, can be effective therapeutics; although their therapeutic utility has been limited by their short half-life in vivo, which is not a huge issue when using RNA aptamers as in vitro diagnostic agents.

[0106] Aptamers are single stranded oligonucleotides that can naturally fold into different 3-dimensional structures, which have the capability of binding specifically to biosurfaces, a target compound or a moiety. The term “conformational change” refers to the process by which a nucleic acid, such as an aptamer, adopts a different secondary or tertiary structure. The term “fold” may be substituted for conformational change.

[0107] Aptamers have advantages over more traditional affinity molecules such as antibodies in that they are very stable, can be easily synthesized, and can be chemically manipulated with relative ease. Aptamer synthesis is potentially far cheaper and more reproducible than antibody production. Aptamers are produced by solid phase chemical synthesis, an accurate and reproducible process with consistency among production batches. An aptamer can be produced in large quantities by polymerase chain reaction (PCR) and once the sequence is known, can be assembled from individual naturally occurring nucleotides and / or synthetic nucleotides.

[0108] Aptamers can be stored stably at room temperature, and, if denatured, aptamers can easily be renatured, a feature not shared by antibodies. Furthermore, aptamers have the potential to measure concentrations of ligand in orders of magnitude lower (parts per trillion or even quadrillion) than antibody-based diagnostic tests. These characteristics of aptamers make them attractive for diagnostic applications.

[0109] Aptamers are typically oligonucleotides that may be single stranded oligodeoxynucleotides, oligoribonucleotides, or modified oligodeoxynucleotide or oligoribonucleotides. The term “modified” encompasses nucleotides with a covalently modified base and / or sugar. For example, modified nucleotides include nucleotides having sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position. Thus, modified nucleotides may also include 2' substituted sugars such as 2'-O-methyl-, 2'-O-amino, 2'-O-alkyl, 2'-O-allyl, 2'-S-alkyl, 2'-S-allyl, 2'-fluoro-, 2'-halo or 2-azido-ribose; carbocyclic sugar analogues; a-anomeric sugars; epimeric sugars such as arabinose, xyloses or lyxoses; pyranose sugars;furanose sugars; and sedoheptulose.

[0110] Modified nucleotides are known in the art and include, by example and not by way of limitation, alkylated purines and / or pyrimidines; acylated purines and / or pyrimidines; or other heterocycles. These classes of pyrimidines and purines are known in the art and include, pseudoisocytosine; N4, N4-ethanocytosine; 8-hydroxy-N6-methyladenine; 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil; 5 -fluorouracil; 5 -bromouracil; 5-carboxymethylaminomethyl-2 -thiouracil; 5 -carboxymethylaminomethyl uracil; dihydrouracil; inosine; N6-isopentyl-adenine; 1 -methyladenine; 1 -methylpseudouracil; 1-methylguanine; 2,2-dimethylguanine; 2-methyladenine; 2-methylguanine; 3 -methylcytosine; 5-methylcytosine; N6-methyladenine; 7-methylguanine; 5-methylaminomethyl uracil; 5-methoxy amino methyl-2 -thiouracil; P-D-mannosylqueosine; 5-methoxycarbonylmethyluracil; 5 -methoxyuracil; 2-methylthio-N6-isopentenyladenine; uracil-5-oxyacetic acid methyl ester; pseudouracil; 2-thiocytosine; 5-methyl-2 thiouracil, 2-thiouracil; 4-thiouracil; 5 -methyluracil; N-uracil-5-oxyacetic acid methylester; uracil 5-oxyacetic acid; queosine; 2-thiocytosine; 5 -propyluracil; 5-propylcytosine; 5 -ethyluracil; 5-ethylcytosine; 5-butyluracil; 5 -pentyluracil; 5 -pentylcytosine; 2,6,-diaminopurine; methylpsuedouracil; 1-methylguanine; and 1 -methylcytosine.

[0111] The aptamers can be synthesized using conventional phosphodiester linked nucleotides and using standard solid or solution phase synthesis techniques which are known in the art. Linkages between nucleotides may use alternative linking molecules. For example, linking groups of the formula P(O)S, (thioate); P(S)S, (dithioate); P(O)NR'2; P(O)R'; P(O)OR6; CO; or CONR'2 wherein R is H (or a salt) or alkyl (1-12C) and R6 is alkyl (1-9C) is joined to adjacent nucleotides through -O- or -S-.

[0112] Additional modifications to the aptamer include 2'O-fluoro modification of the pyrimidines. In other embodiments, all of the nucleotides in the aptamer are 2'-O-fluoro modified. Alternatively, the pyrimidines, or all the nucleotides, may be modified with 2'-methyl (both pyrimidines and purines).

[0113] Generation of Aptamers

[0114] Aptamers can be isolated from combinatorial libraries through an iterative process of in vitro selection known as SELEX (Systemic Evolution of Ligands by Exponential enrichment). Aptamers exhibit specificity and affinity comparable to or exceeding that of antibodies, and can be generated against most targets. Unlike antibodies, aptamers can be synthesized in a chemical process and hence offer significant advantages in terms of reduced production cost and muchsimpler regulatory approval process. Also, aptamers are not expected to exhibit significant immunogenicity in vivo.

[0115] Aptamers specific for a given biomolecule can be identified using techniques known in the art. Briefly, these techniques typically involve the binding of the molecular target with a random mixture of oligonucleotides. The aptamer-molecular target complex is separated from the unbound oligonucleotides. The aptamer is recovered from the separated complex and amplified. This cycle is repeated to identify those aptamer sequences with the highest affinity for the molecular target.

[0116] The SELEX process is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules. Each SELEX-identified nucleic acid ligand is a specific ligand of a given target compound or molecule. The SELEX process is based on the unique insight that nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets.

[0117] SELEX relies as a starting point upon a large library of single stranded oligonucleotides comprising randomized sequences derived from chemical synthesis on a standard DNA synthesizer. The oligonucleotides can be modified or unmodified DNA, RNA or DNA / RNA hybrids. In some embodiments, the pool comprises 100% random or partially random oligonucleotides. In other embodiments, the pool comprises random or partially random oligonucleotides containing at least one fixed sequence and / or conserved sequence incorporated within randomized sequence. In other embodiments, the pool comprises random or partially random oligonucleotides containing at least one fixed sequence and / or conserved sequence at its 5' and / or 3' end, which may comprise a sequence shared by all the molecules of the oligonucleotide pool. Fixed sequences are sequences common to oligonucleotides in the pool which are incorporated for a pre-selected purpose such as, CpG motifs, hybridization sites for PCR primers, promoter sequences for RNA polymerases (e.g., T3, T4, T7, and SP6), restriction sites, or homopolymeric sequences, such as poly A or poly T tracts, catalytic cores, sites for selective binding to affinity columns, and other sequences to facilitate cloning and / or sequencing of an oligonucleotide of interest. Conserved sequences are sequences, other than the previously described fixed sequences, shared by a number of aptamers that bind to the same target.

[0118] The oligonucleotides of the pool can include a randomized sequence portion as well as fixed sequences necessary for efficient amplification. Typically, the oligonucleotides of the starting pool contain fixed 5' and 3' terminal sequences which flank an internal region of randomnucleotides. The randomized nucleotides can be produced in a number of ways including chemical synthesis and size selection from randomly cleaved cellular nucleic acids. Sequence variation in test nucleic acids can also be introduced or increased by mutagenesis before or during the selection / amplification iterations.

[0119] The random sequence portion of the oligonucleotide can be of any length, can comprise ribonucleotides and / or deoxyribonucleotides, and can include modified or non-natural nucleotides or nucleotide analogs. Random oligonucleotides can be synthesized from phosphodiester-linked nucleotides using solid phase oligonucleotide synthesis techniques well known in the art. Random oligonucleotides can also be synthesized using solution phase methods, such as triester synthesis methods. Sufficiently large regions of random sequence in the sequence design increases the likelihood that each synthesized molecule is likely to represent a unique sequence.

[0120] The starting library of oligonucleotides may be generated by automated chemical synthesis on a DNA synthesizer. To synthesize randomized sequences, mixtures of all four nucleotides are added at each nucleotide addition step during the synthesis process, allowing for random incorporation of nucleotides. As stated above, in one embodiment, random oligonucleotides comprise entirely random sequences; however, in other embodiments, random oligonucleotides can comprise stretches of nonrandom or partially random sequences. Partially random sequences can be created by adding the four nucleotides in different molar ratios at each addition step.

[0121] The starting library of oligonucleotides may be either RNA or DNA. In those instances where an RNA library is to be used as the starting library, it is typically generated by transcribing a DNA library in vitro using T7 RNA polymerase or modified T7 RNA polymerases and then purifying the transcribed products. The RNA or DNA library is then mixed with the target under conditions favorable for binding and subjected to stepwise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. The target can be molecules of a certain type or types, cells of a certain type or types or any other target or targets of a certain type or types. The identity of the type or types of the molecules, cells, or other targets can be known, suspected, or unknown. More specifically, starting with a mixture containing the starting pool of nucleic acids, the SELEX method includes steps of: (a) contacting the mixture with the target under conditions favorable for binding; (b) partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules; (c) dissociating the nucleic acid-target complexes; (d) amplifying the nucleic acids dissociated from the nucleicacid-target complexes to yield a ligand-enriched mixture of nucleic acids; and (e) reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific, high affinity nucleic acid ligands to the target molecule. In those instances where RNA aptamers are being selected, the SELEX method further comprises the steps of: (i) reverse transcribing the nucleic acids dissociated from the nucleic acid-target complexes before amplification in step (d); and (ii) transcribing the amplified nucleic acids from step (d) before restarting the process.

[0122] Within a nucleic acid mixture containing a large number of possible sequences and structures, there is a wide range of binding affinities for a given target. Those which have the higher affinity constants for the target are most likely to bind to the target. After partitioning, dissociation and amplification, these aptamers are used to generate a second nucleic acid pool enriched for the higher binding affinity candidates. Additional rounds of selection progressively favor the best ligands until the resulting nucleic acid mixture is predominantly composed of only one or a few sequences. These can then be cloned, sequenced and individually tested for binding affinity as pure ligands or aptamers.

[0123] Cycles of selection and amplification are repeated until a desired goal is achieved. In the most general case, selection / amplification is continued until no significant improvement in binding strength is achieved on repetition of the cycle. Generally, nucleic acid aptamer molecules are selected in a 5 to 20 cycle procedure. In one embodiment, heterogeneity is introduced only in the initial selection stages and does not occur throughout the replicating process. In many cases, it is not necessarily desirable to perform the iterative steps of SELEX until a single nucleic acid ligand is identified. The target-specific nucleic acid ligand solution may include a family of nucleic acid structures or motifs that have a number of conserved sequences and a number of sequences which can be substituted or added without significantly affecting the affinity of the nucleic acid ligands to the target. By terminating the SELEX process prior to completion, it is possible to determine the sequence of a number of members of the nucleic acid ligand solution family.

[0124] Counter-SELEX is a method for improving the specificity of nucleic acid ligands to a target molecule by eliminating nucleic acid ligand sequences with cross-reactivity to one or more non-target molecules. Counter-SELEX is comprised of the steps of: (a) preparing a candidate mixture of nucleic acids; (b) contacting the increased affinity nucleic acids with the one or more non-target molecules such that nucleic acid ligands that bind the non-target molecule(s) are removed; (c) discarding the nucleic acids that bind the non-targets from the candidate mixture; (c) contacting the remaining candidate mixture with the target, whereinnucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; (d) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; (e) dissociating the increased affinity nucleic acids from the target; and (f) amplifying the nucleic acids with specific affinity only to the target molecule to yield a mixture of nucleic acids enriched for nucleic acid sequences with a relatively higher affinity and specificity for binding to the target molecule. As described above for SELEX, cycles of selection and amplification are repeated as necessary until a desired goal is achieved.

[0125] The SELEX method may encompass the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand. Examples of such modifications include chemical substitutions at the ribose and / or phosphate and / or base positions. One of ordinary skill in the art will appreciate that one or more of the modifications described herein can be included in the desired aptamer and that the modified aptamer can be tested for binding using any method known in the art. For example, oligonucleotides can contain nucleotide derivatives chemically modified at the 2' position of ribose, 5' position of pyrimidines, and 8' position of purines, 2'-modified pyrimidines, and nucleotides modified with 2'-amino (2'-NH2), 2'- fluoro (2'-F), and / or 2'-O-methyl (2'-0Me) substituents.

[0126] In some embodiments, one or more modifications of the aptamers contemplated in this invention include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrophobicity, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole. Modifications to generate oligonucleotide populations which are resistant to nucleases can also include one or more substitute internucleotide linkages, altered sugars, altered bases, or combinations thereof. Such modifications include, but are not limited to, 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil; backbone modifications, phosphorothioate or alkyl phosphate modifications, methylations, and unusual base-pairing combinations such as the isobases - isocytidine and isoguanosine. Modifications can also include 3' and 5' modifications such as capping.

[0127] Pre-SELEX process modifications or those made by incorporation into the SELEX process can, for example, yield nucleic acid ligands with both specificity for their SELEX target and improved stability, e.g., in vivo stability. Post-SELEX process modifications made to nucleic acid ligands can, for example, result in improved stability, e.g., in vivo stability withoutadversely affecting the binding capacity of the nucleic acid ligand. The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and nonoligonucleotide functional units as described in U.S. Pat. No. 5,637,459 and U.S. Pat. No.

[0128] 5,683,867 (which are incorporated by reference). The SELEX method further encompasses combining selected nucleic acid ligands with lipophilic or non-immunogenic high molecular weight compounds in a diagnostic or therapeutic complex. These patents and applications teach the combination of a broad array of shapes and other properties, with the efficient amplification and replication properties of oligonucleotides, and with the desirable properties of other molecules.

[0129] The aptamers with specificity and binding affinity to the target(s) of the present invention are typically selected by the SELEX process as described herein. As part of the SELEX process, the sequences selected to bind to the target can then optionally be minimized to determine the minimal sequence having the desired binding affinity. The selected sequences and / or the minimized sequences are optionally optimized by performing random or directed mutagenesis of the sequence to increase binding affinity or alternatively to determine which positions in the sequence are essential for binding activity. Additionally, selections can be performed with sequences incorporating modified nucleotides to stabilize the aptamer molecules against degradation in vivo.

[0130] Modification of aptamers

[0131] Once they have been identified by SELEX or a related technology, the aptamers of the present invention can be operably linked to one or more entities. In certain embodiments, the entity is a fluorescent tag, affinity tag, a protein, a solid substrate, a cell surface, or a cellular component.

[0132] In certain embodiments, the aptamer is linked to the entity by means of a linker. In certain embodiments, the linker is a binding pair. In certain embodiments, the “binding pair” refers to two molecules which interact with each other through any of a variety of molecular forces including, for example, ionic, covalent, hydrophobic, van der Waals, and hydrogen bonding, so that the pair have the property of binding specifically to each other. “Specific” binding means that the binding pair members exhibit binding to each other under conditions where they do not bind to one of any number of other molecules. Examples of binding pairs are biotin-avidin, hormone-receptor, receptor-ligand, enzyme-substrate, IgG-protein A, antigenantibody, and the like. In certain embodiments, a first member of the binding pair comprisesavidin or streptavidin and a second member of the binding pair comprises biotin. In certain embodiments, the aptamer is linked to the entity by means of a covalent bond.

[0133] A number of “molecular beacons” (such as fluorescence compounds) can be attached to aptamers to provide a means for signaling the presence of and / or quantifying a target chemical or biological agent. Other exemplary detection labels that could be attached to the aptamers include biotin, any fluorescent dye or tracer, amine modification, horseradish peroxidase, alkaline phosphatase, etc.

[0134] In certain embodiments, the aptamer is operably linked to a detection means and to a solid substrate. For example, the aptamer may be linked to a fluorescent dye and to a magnetic bead.

[0135] Small molecules can be linked to the aptamer. These include but are not limited to siRNA sequences, miRNAs, small molecule inhibitors, cytotoxic chemicals, chelators for housing radionuclides (for diagnostic / imaging applications, as well as development of targeted radiotherapies, see, e.g., BioorgMed Chem. 2011 Jul l;19(13):4080-90), nanoparticles containing all of the above plus DNA vectors and / or mRNA sequences or other types of small molecules, depending on the use of the ligand as a diagnostic agent or as a therapeutic agent. In certain embodiments, the small molecule is a molecule capable of modulating cell activity, including but not limited to biologic and pharmacologic inhibitors / agonists, siRNA, or miRNA. In certain embodiments, the small molecules are biologic or pharmacologic agents that can influence T cell activity.

[0136] Chemistries that can be used to link molecules to the aptamer are known in the art, such as disulfide linkages, amino linkages, covalent linkages, etc.

[0137] Stabilization of ligand-receptor complexes prior to selection of aptamers Molecular complexes are dynamic, and there is ongoing formation and dissolution of such complexes in a living organism and during the testing of biologic specimens after those specimens have been procured. Furthermore, agents designed to assess such complexes, such as antibodies and aptamers, can impact on such “on” and “off’ rates, and so influence the measurement of such complexes. To avoid this problem, the targets including molecules, cells and tissues, used for selection of aptamers via SELEX, can be fixed in a way that cross-links and stabilizes such complexes. This can be done using formalin and other agents that cross-link proteins. Fixing biospecimens before using such specimens to select aptamers allows for selection of aptamers that recognize epitopes that are not altered by such fixation. Suchaptamers can then be used to analyze molecular complexes in fixed tissue such as FFPE tissue or cells that have been fixed in formalin using the method described here.

[0138] Therefore, in certain embodiments, the complexes or cells are stabilized either by cold temperature incubation or formalin fixing. For example, in cold temperature incubation, each step of LIRECAP SELEX is performed at 4°C in order to reduce the dissociation of the complex. For example, for formalin fixing, the ligand-receptor complex was fully stabilized by fixing the target-bound beads with 2% formaldehyde before applying the aptamer pool to the bead-bound targets.

[0139] Generation of aptamers binding to PD1 and PDL1 and PD1-PDL1 complex for the PD1-PDL1 LIRECAP assay

[0140] In the current invention, a modification of SELEX was used to select aptamers for the PD1-PDL1 LIRECAP assay to quantify unbound PD1, unbound PDL1 and the PD1-PDL1 complex in a biospecimen. This includes both a 2-aptmer LIRECAP assay (based on one aptamer that binds to an unbound complex member such as PD1 and one aptamer that binds to the complex such as PD1-PDL1) or three-aptamer LIRECAP assay (one aptamer that binds to an unbound complex member such as PD1, one aptamer that binds to the other unbound complex member such as PDL1 and one aptamer that binds to the complex). The two-aptamer LIRECAP focuses on PD1 fractional occupancy (FO) by PDL1 which, based on current understanding of checkpoint blockade, provides prognostic information on whether anti-PDl -based therapy will reverse the immune inhibition resulting from the interaction between PD1 and PDL1. The three-aptamer LIRECAP provides information on PD1 FO by PDL1, while also providing information on FO of PDL1 by PD1, could add further prognostic information.

[0141] Two-aptamer and three-aptamer LIRECAP assays

[0142] The current embodiment includes two, three or more aptamers - each with predominant binding towards unoccupied receptor (PD1), unoccupied ligand (PDL1) or ligand-receptor complex (PD1-PDL1). A modification of the traditional SELEX approach (8 rounds of SELEX against native targets) was developed to improve selection of aptamers for the LIRECAP assay. Unoccupied PD1, unoccupied PDL1 and the PD1-PDL1 complex, were immobilized on Dynabeads and used as targets for SELEX selection of aptamers. Selection of aptamers following formalin fixation of targets minimized complex dissociation and identified aptamers that bound to target and can be used in FFPE biospecimens. Additional modifications included use of RNase to enzymatically eliminate aptamers that are not bound to target. Negativeselection using the counter-SELEX approach was used to remove sequences that are not desired as outlined in Table 1 below.

[0143] Table 1

[0144]

[0145] Bioinformatics were used to choose the most highly enriched aptamers from each aptamer pool after multiple rounds of SELEX, with 3-6 aptamers identified per target (PD1, PDL1, PD1-PDL1 complex). Aptamers based on the identified sequences were synthesized and screened to determine the best candidate aptamers for developing a two-aptamer and a three-aptamer PD1-PDL1 LIRECAP assay. Those aptamers specific for PD1 were designated as “P” aptamers, those specific for PDL1 were designated as “L” aptamers, and those specific for the PD1-PDL1 complex were designated as “C” aptamers.

[0146] Concurrent measurement of aptamer concentrations

[0147] A key aspect of the present invention that applies to both the two-aptamer and the three-aptamer LIRECAP assays is that the aptamers have the same 5' and 3' ends and are of similar lengths. This allows multiple aptamers that recognize different targets to be amplified using the same set of primers, in a single sample and in the same assay simultaneously which provides for a robust internal control. In one embodiment of the present invention, the method involves the amplification of selected RNAs which can include 2 aptamers, 3 aptamers or more.

[0148] “Amplifying” utilizes methods such as the polymerase chain reaction (PCR), ligation amplification (or ligase chain reaction, LCR), strand displacement amplification, nucleic acid sequence-based amplification, and amplification methods based on the use of Q-beta replicase. These methods are well known and widely practiced in the art. Reagents and hardware for conducting PCR are commercially available. One of ordinary skill in the art will appreciate that in some methods of amplification at least one type of aptamer can be immobilized on a solid surface.

[0149] According to the methods of the present invention, the amplification may be carried out by any means known to the art. Examples of suitable amplification techniques include, but are not limited to, polymerase chain reaction (including, for RNA amplification, reverse-transcriptase polymerase chain reaction), ligase chain reaction, strand displacement amplification, transcription-based amplification, self-sustained sequence replication (or “3 SR”), the QP replicase system, nucleic acid sequence-based amplification (or “NASBA”), the repair chain reaction (or “RCR”), and boomerang DNA amplification (or “BDA”).

[0150] The nucleotides incorporated into the amplification product may be natural or modified nucleotides (modified before or after amplification), and the nucleotides may be selected to optimize subsequent electrochemical detection steps.

[0151] Polymerase chain reaction (PCR) may be carried out in accordance with known techniques. See, e.g., U.S. Patent Numbers 4,683,195; 4,683,202; 4,800,159; and 4,965,188 (which are incorporated by reference). In general, PCR involves, first, treating a nucleic acid sample (e.g, in the presence of a heat stable DNA polymerase) with one oligonucleotide primer for each strand of the specific sequence to be detected under hybridizing conditions so that an extension product of each primer is synthesized that is complementary to each nucleic acid strand, with the primers sufficiently complementary to each strand of the specific sequence to hybridize therewith so that the extension product synthesized from each primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer, and then treating the sample under denaturing conditions to separate the primer extension products from their templates if the sequence or sequences to be detected are present. These steps are cyclically repeated until the desired degree of amplification is obtained.

[0152] Detection of the amplified sequence may be carried out by adding to the reaction product an oligonucleotide probe capable of hybridizing to the reaction product (e.g, an oligonucleotide probe of the present invention), the probe carrying a detectable label, and then detecting the label in accordance with known techniques. Where the nucleic acid to be amplified is RNA, amplification may be carried out by initial conversion to DNA by reverse transcriptase in accordance with known techniques.

[0153] Strand displacement amplification (SDA) may be carried out in accordance with known techniques. For example, SDA may be carried out with a single amplification primer or a pair of amplification primers, with exponential amplification being achieved with the latter. In general, SDA amplification primers comprise, in the 5' to 3' direction, a flanking sequence (the DNA sequence of which is noncritical), a restriction site for the restriction enzyme employed in the reaction, and an oligonucleotide sequence (e.g., an oligonucleotide probe of the present invention) that hybridizes to the target sequence to be amplified and / or detected. The flanking sequence, which serves to facilitate binding of the restriction enzyme to the recognition site and provides a DNA polymerase priming site after the restriction site has been nicked, is about 15 to20 nucleotides in length in one embodiment. The restriction site is functional in the SDA reaction. The oligonucleotide probe portion is about 13 to 15 nucleotides in length in one embodiment of the invention.

[0154] Ligase chain reaction (LCR) is also carried out in accordance with known techniques. In general, the reaction is carried out with two pairs of oligonucleotide probes: one pair binds to one strand of the sequence to be detected; the other pair binds to the other strand of the sequence to be detected. Each pair together completely overlaps the strand to which it corresponds. The reaction is carried out by, first, denaturing (e.g., separating) the strands of the sequence to be detected, then reacting the strands with the two pairs of oligonucleotide probes in the presence of a heat stable ligase so that each pair of oligonucleotide probes is ligated together, then separating the reaction product, and then cyclically repeating the process until the sequence has been amplified to the desired degree. Detection may then be carried out in like manner as described above with respect to PCR.

[0155] The sample may be processed in any suitable manner known to those ordinarily skilled in the art. For example, the sample may be solubilized in solution, macerated, enzymatically digested or sonicated to allow aptamers access to their molecular targets. Suitable conditions are well known to those ordinarily skilled in the art. Alternatively, the sample may be immobilized on a solid support, whereby the sample may be contacted with the aptamer by immersing the solid support having the aptamer immobilized thereon in the solution containing the sample.

[0156] Aptamers specific to the PD1-PDL1 LIRECAP assay

[0157] In certain embodiments of the present invention, aptamers identified using the above novel approach are used to assess FO of PD1 by PDL1, and FO of PDL1 by PD1 in a biospecimen.

[0158] In certain embodiments, the present invention provides a PD1 -binding aptamer comprising a polynucleotide 45-55 nucleotides in length comprising SEQ ID NO:1 (P10), SEQ ID NO:2 (P17), SEQ ID NO:3 (P150).

[0159] In certain embodiments, the present invention provides a PDL1 -binding aptamer comprising a polynucleotide 45-55 nucleotides in length comprising SEQ ID NO:4 (L42), SEQ ID NO:5 (L45), SEQ ID NO:6 (L50).

[0160] In certain embodiments, the present invention provides a PD1 / PDL1 complex-binding aptamer comprising a polynucleotide 45-55 nucleotides in length comprising SEQ ID NO:7 (C18), SEQ IDNO:8 (C52), SEQ ID NO:9 (Cl 13).Diseases and Conditions Amendable to the Methods of the Invention The novel processes described herein to identify aptamers that are specific for complexes as well as for the individual molecules that make us such complexes is useful in additional embodiments of the present invention where a mammal has a condition where quantification of FO of a receptor by a ligand, or one molecule by another, would be of research or clinical value. Such aptamers selected using the novel process described here could be used to develop a LIRECAP or related assay that quantifies the FO of a receptor by a ligand, or of one molecule by another molecule, in order to diagnose the disease, determine the likelihood of response to treatment of that disease, or assess whether a given therapy of that disease is having the desired outcome.

[0161] Such an embodiment of the present invention includes LIRECAP assays designed around other immune checkpoint inhibitors used to treat cancer including but not limited to PDL2, LAG3, TIM3, TIGIT and CTLA4.

[0162] Such an embodiment of the present invention includes application of this technology to diagnosis in a mammal that has or is suspected of having autoimmune disease. Such autoimmune diseases include but are not limited to lupus, rheumatoid arthritis, multiple sclerosis, psoriasis, Sjogren’s syndrome, celiac disease, type I diabetes, Graves disease, scleroderma, ulcerative colitis, Crohn’s disease, alopecia areata and autoimmune vasculitis.

[0163] Such an embodiment of the present invention includes application of this technology to diagnosis in a mammal that is at elevated risk or is suspected of being at elevated risk of having rejection of a transplant. Types of transplants include, but are not limited to, organ, such as kidney, liver, heart, or lung; and tissue, such as skin, bone, or heart valve. In certain embodiments, the transplant rejection is suspected to be amenable to treatment by modulation of the activity of effector and regulatory T cells. These diseases include, but are not necessarily limited to, islet transplantation for Type I diabetes or pancreatic transplantation for Type I diabetes.

[0164] Such an embodiment of the present invention includes application of this technology to diagnosis in a mammal where there would be benefit from understanding the FO of molecule by a therapeutic molecule. In such a case, determining FO of that molecule by a drug could impact on selection of a therapeutic molecule or dosing of the therapeutic molecule. As used herein, the term “therapeutic molecule” refers to any small molecule that has a beneficial effect on the recipient. Thus, “therapeutic molecule” embraces both therapeutic and prophylactic small molecules.Spatial analysis

[0165] In one embodiment of the present invention, the aptamers are used to determine and quantify the relative amount of bound to unbound members of a complex in biospecimen as a whole. As described above, these methods involve using a set of two or of three aptamers with different specificity. The amount of bound aptamers can be determined by expanding the aptamers simultaneously using the same set of 5' and 3' primers in a single RT qPCR or similar assay, thereby providing a robust internal control. The amplification is consistent for all the aptamers involved, and thus the amount of amplified nucleic acid quantity is directly proportional to the initial quantity of bound aptamers. The concentration of each aptamer individually can be determined simultaneously in the same assay by using a probe specific for the variable region of each aptamer. By measuring the amount of each aptamer that binds in a sample, a determination of the relative amount of complexes versus unbound members can be made in a variety of tissues and biospecimens including saliva, blood, tissue samples, etc.

[0166] In additional embodiments, the aptamers described herein can be used to mark different areas of a biospecimen to assess FO of one molecule by another in different areas of a biospecimen. The aptamers described herein can be substituted for antibodies in commonly used assays including flow cytometry, fluorescence microscopy, immunohistochemistry, in situ hybridization, and other approaches to spatial analysis of molecular expression known to one with ordinary skill in the art. In this embodiment, the assay could be used to determine the spatial location of complexes and uncomplexed molecules within a biospecimen.

[0167] For example, in embodiments of the present invention, molecular beacons are attached to aptamers to provide a means for signaling and detecting target complexes and unbound members of complexes at the regional, cellular or subcellular level in a biospecimen. Molecular beacons, for example, can employ fluorescence resonance energy transfer-based methods to provide fluorescence signals in the presence of a particular analyte / biomarker of interest. The aptamer acts as a sensor to detect the presence of a specific target analyte / biomarker. Upon detection of the analyte / biomarker, the aptamer communicates with a molecular beacon to generate a detectable signal.

[0168] Similarly, amplifying fluorescent polymers (AFPs) can be utilized in the present invention. An AFP is a polymer containing several chromophores that are linked together. As opposed to isolated chromophores that require 1 : 1 interaction with an analyte in conventional fluorescence detection, the fluorescence of many chromophores in an AFP can be influenced by a single molecule. For example, a single binding event to an AFP can quench the fluorescence ofmany polymer repeat units, resulting in an amplification of the quenching. Quenching is a process which decreases the intensity of the fluorescence emission.

[0169] Molecular beacons and AFPs, including their methods for preparation, can be used in the present invention.

[0170] Aptamers such as those described herein can be used to image living tissue of humans or mammals in vivo or ex vivo. One of ordinary skill in the art would know to link a molecule that can be visualized to an aptamer described herein. The aptamer can then be contacted to cells suspected to contain a complex of interest. The presence of the aptamer linked to a molecule to be detected and bound to a complex of interest thus shows where the complex of interest is located.

[0171] In certain embodiments, the present invention provides a composition comprising an aptamer operably linked to a tag.

[0172] In certain embodiments, the tag is selected from a molecule that can be detected using optical sensors, molecular size sensors, or isotopic sensors.

[0173] Spatial analysis of PD1 and PDL1

[0174] In certain embodiments, the present invention provides a method of quantifying a fraction of PD1 occupied by PDL1 or a fraction of PDL1 occupied by PD1 or PDL1 by PD1 in a region of a sample to assess the heterogeneity of PD1 FO by PDL1 and PDL1 FO by PD1 with the goal of using such information to guide prognosis and therapy.

[0175] General Terminology

[0176] “Synthetic” aptamers are those prepared by chemical synthesis. The aptamers may also be produced by recombinant nucleic acid methods. “Recombinant nucleic acid molecule” is a combination of nucleic acid sequences that are joined together using recombinant nucleic acid technology and procedures used to join together nucleic acid sequences known in the art.

[0177] As used herein, the terms “nucleic acid” and “polynucleotide” refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or doublestranded form, composed of monomers (nucleotides) containing a sugar, phosphate and a base that is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides such those that containing the modifications described herein.

[0178] The term “nucleotide sequence” refers to a polymer of DNA or RNA which can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotidebases capable of incorporation into DNA or RNA polymers. A “nucleic acid fragment” is a portion of a given nucleic acid molecule.

[0179] The terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid fragment,” “nucleic acid sequence or segment,” or “polynucleotide” may also be used interchangeably with gene, cDNA, DNA and RNA encoded by a gene, e.g., genomic DNA, and even synthetic DNA sequences. The term also includes sequences that include any of the known base analogs of DNA and RNA.

[0180] The terms “fragment” or “portion” mean a full length or less than full length of the nucleotide sequence.

[0181] As used herein, the terms “sequence identity” or “identity” in the context of two nucleic acid sequences make reference to a specified percentage of residues in the two sequences that are the same when aligned by sequence comparison algorithms or by visual inspection.

[0182] As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences, wherein the portion of the polynucleotide sequence may comprise additions or deletions ( / .< ., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

[0183] The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%; at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%; at least 90%, 91%, 92%, 93%, or 94%; or even at least 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters.

[0184] “Treating,” as used herein, refers to ameliorating at least one symptom of, curing and / or preventing the development of a disease or a condition.

[0185] The terms “protein,” “peptide” and “polypeptide” are used interchangeably herein. A “vector” is defined to include, inter alia, any viral vector, as well as any plasmid, cosmid, phage or binary vector in double or single stranded linear or circular form that may or may not be self-transmissible or mobilizable, and that can transform a prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g., autonomous replicating plasmid with an origin of replication).The term “immunotherapy” relates to a treatment involving activation of a specific immune reaction. In the context of the present invention, terms such as “protect,” “prevent,” “prophylactic,” “preventive,” or “protective” relate to the prevention or treatment or both of the occurrence and / or the propagation of a disease in a subject and, in particular, to minimizing the chance that a subject will develop a disease or to delaying the development of a disease. For example, a person at risk for a tumor, as described above, would be a candidate for therapy to prevent a tumor.

[0186] The term “molecule” refers to an atom or atoms held together by chemical bonds, including chemical compounds. Chemical bonds include covalent bonds, ionic bonds, metallic bonds, and coordinate covalent bonds. Examples of a molecule include but are not limited to a protein, DNA, RNA, nucleic acids, and certain fluorescent dyes.

[0187] The term “pool” refers to a collection of any number of things of a type or types that is separated from other things of the same or different type or types. For example, a collection of polynucelotides can be split into two or more pools of polynucleotides that are separate from each other, in which the sequence or sequences of polynucelotides in a pool may be identical to or different from another pool or other pools.

[0188] The terms “bind” or “bound” refer to two or more molecules that are in contact through various types of non-covalent interactions that do not involve the sharing of electrons, but rather involve more dispersed variations of electromagnetic interactions between molecules or within a molecule. Examples of such interactions are electrostatic interactions (e.g. ionic bonds, hydrogen bonds and halogen binding), Van der Waals forces (e.g. dipole-dipole, dipole-induced dipole and London dispersion forces), 7t-effects (e.g. TI- TI interactions, cation- TI and anion- TI interactions, and polar- TI interactions), and the hydrophobic effect.

[0189] The term “complex” refers to two or more molecules that are bound to each other, e.g. protein-protein, protein-DNA, and protein-RNA complexes.

[0190] The term “endogenous” refers to a molecule or molecules that are normally produced or synthesized within a cells, system, or organism. An endogenous molecule is native to the cell, system, or organism and not heterologous. For example, an endogenous gene is one that naturally occurs within the genomic or mitochondrial-genomic context of the cell, system, or organism.

[0191] The term “selective” refers to something that has some degree of specificity for one or more things over one or more other things. For example, an aptamer that is selective can bind preferentially, more stably, more strongly, more quickly, with higher affinity, or with higheravidity or with slower reversal of binding to one or more molecules relative to one or more other molecules.

[0192] The term “linked” refers to two or more molecules that associated with each other in such a way so that they stay associated with each other or disassociate with each other as desired to at least a certain extent. Such associations can be covalent or non-covalent binding.

[0193] The term “mixture” refers to a collection of things, e.g. molecules, of one or more types. The term “body fluid” refers to liquids originating from the body of a human or other animal. Body fluids include, but are not limited to, those that are retained in, excreted from, and secreted from the body. Body fluids include, but are not limited to intracellular fluid, extracellular fluid, intravascular fluid, interstitial fluid, lymphatic fluid, transcellular fluid, blood, cerebrospinal fluid, lymph, pericardial fluid, peritoneal fluid, pleural fluid, and urine.

[0194] The term “fractional occupancy” abbreviated “FO” refers to the fraction of a receptor (e.g. PD1) occupied by a ligand (e.g. PDL1) or the fraction of a ligand occupied by a receptor.

[0195] The term “two aptamer” refers to an assay in which the fraction of a receptor (e.g. PD1) occupied by ligand (e.g. PDL1) is determined through use of two aptamers.

[0196] The term “three aptamer” refers to an assay in which the fraction of a receptor (e.g., PD1) occupied by ligand (e.g., PDL1), and of a ligand occupied by receptor is determined through use of three aptamers.

[0197] EXAMPLES

[0198] The following examples are intended to illustrate various embodiments of the invention. As such, the specific embodiments discussed are not to be construed as limitations on the scope of the invention. It will be apparent to one ordinarily skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of invention, and it is understood that such equivalent embodiments are to be included herein. For example, the examples described here focus on selection of aptamers that bind PD1, PDL1 and the PD1-PDL1 complex, and application of these aptamers to quantify PD1 FO by PDL1, and PDL1 FO of PD1. One ordinarily skilled in the art would understand the novel approach outlined here could be applied to other receptor-ligand pairs, other molecular complexes or drug-target complexes where quantifying FO of one by the other would be of use. Non-limiting examples of aptamers and other molecules that may be used are described above. Further, all references cited in the disclosure are hereby incorporated by reference in their entirety, as if fully set forth herein.EXAMPLE 1

[0199] LIRECAP Assay to measure FO of PD1 by PDL1 or PDL1 by PD1 including a two- aptamer LIRECAP assay and three-aptamer LIRECAP assay As discussed in the Detailed Description above, therapy directed at interfering with the interaction of PD1 and PDL1 has generated great clinical and commercial excitement in the field of cancer therapeutics. Nevertheless, only a minority of patients respond to anti-PDl therapy. Measurements of clinical biomarkers, including T cell infiltration or PDL1 expression in the tumor, are currently used as predictive biomarkers for response to anti-PDl therapy, but are far from ideal. Better biomarker assays capable of directly analyzing PD1-PDL1 complexes in tissue could be superior in predicting clinical response to PD1 checkpoint blockade.

[0200] The PD1-PDL1 LIRECAP assay was developed to fill this need. This assay uses two or three RNA aptamers that were selected using a unique approach. The two-aptamer LIRECAP allows for determination of receptor (PD1) FO by ligand (PDL1). The three-aptamer LIRECAP allows for determination of both PD1 FO by PDL1 and PDL1 FO by PD1.

[0201] A novel aspect of this invention is that the PD1-PDL1 LIRECAP assay allows for the quantification of PD1 FO by PDL1 or PDL1 FO by PD1 as a potential biomarker that can be used to predict response to therapy or monitor response to therapy. Addition novel aspects include use of formalin fixed targets for selection of, the use of RNase to enhance specificity during the selection process, the use of three aptamers for a LIRECAP assay to measure FO of receptor by ligand and of ligand by receptor and the specific aptamer and probe sequences for use in PD1-PDL1 LIRECAP assays.

[0202] The scheme of LIRECAP assay is given in Figures 1A-1C. The two-aptamer LIRECAP assay (Figure 1A) uses a pair of receptor-specific RNA aptamers that show differential affinity towards unoccupied receptor vs ligand-occupied receptor complex. In other words, one aptamer in the pair preferentially binds unoccupied receptor (unoccupied receptor-preferring aptamer), while the other aptamer in the pair preferentially binds to ligand-receptor complex (complexpreferring aptamer). The relative binding of two aptamers in a given biospecimen reflects the FO of receptor by ligand in that specimen. The concentration of both bound aptamers is assessed simultaneously in a single assay using a single set of primers. This provides robust internal control, as assessment focuses on the ratio of the two aptamers, as opposed to measurement of each aptamer separately. Thus, the LIRECAP assay can quantify FO even though relative binding of the two aptamers to unoccupied receptor versus receptor-ligand complex is relative. Such paired quantification is not practical with mAb-based assays.

[0203] LIRECAP technology is high-throughput, uses standard molecular biology techniques and iswell-suited for clinical diagnostics. LIRECAP technology can also be expanded to other ligandreceptor complexes that are soluble or tissue based that are clinically significant.

[0204] A three-aptamer LIRECAP assay (Figure IB) uses three RNA aptamers that show preferentially affinity towards unoccupied receptor, unoccupied ligand and the ligand-occupied receptor complex. In other words, one aptamer preferentially binds unoccupied receptor (unoccupied receptor-preferring aptamer), one aptamer preferentially binds to ligand-receptor complex (complex-preferring aptamer) and the third aptamer binds preferentially to the ligand (unoccupied ligand-preferring aptamer). The relative binding of these three aptamers to the given sample reflects the FO of receptors by ligands and FO of ligands by receptors. As with the two-aptamer LIRECAP, preferential binding of aptamers to unoccupied receptor, unoccupied ligand or receptor-ligand complex with the three-aptamer LIRECAP assay can be relative instead of absolute since aptamer binding to a biospecimen and PCR expansion is done simultaneously using the same PCR primers. The three-aptamer LIRECAP assay measures FO of ligand by receptor or receptor by ligand.

[0205] An illustration of how the two-aptamer or three-aptamer PD1-PDL1 LIRECAP assay could reflect various clinical scenarios based on infiltration of tumors by T cells that express PD1 and intratumoral expression of PDL1 is provided in Figure 1C.

[0206] Novel approach to selecting aptamers for the PD1-PDL1 LIRECAP assay.

[0207] Systematic Evolution of ligands by Exponential Enrichment (SELEX) is the accepted method for generating RNA aptamers. SELEX enables one to generate aptamers against targets ranging from simple proteins to complex whole cells. It is believed that the current invention is the first to utilize SELEX to generate aptamers with differential binding property for ligands and their receptors compared to the ligand-receptor complex. For rapid identification of RNA aptamers to be used in LIRECAP technology, significant modifications were needed in SELEX.

[0208] Unique aspects of the present approach towards adapting SELEX to generate aptamers with differential binding affinities to receptor, ligand and the receptor-ligand complex presented here includes:

[0209] • Formalin fixation of targets to assure the ligand-receptor complex (PD1-PDL1) is stabilized and not impacted by the biologic on / off rate of the ligand receptor interaction. An added benefit is that the resulting aptamers can also bind to their target in formalin fixed tissue. This allows for eventual use of the LIRECAP assay on FFPE tissue.

[0210] • Inclusion of Counter-SELEX to reduce the cross-reactivity of aptamers to cross targets and to enhance enrichment of those aptamers with differential binding.• Inclusion of dilute RNase cocktail during washing to enrich high affinity LIRECAP aptamers with fewer rounds of SELEX.

[0211] Identification of aptamers for the LIRECAP assay

[0212] Starting with the same library, parallel and separate repeated selections of aptamers specific for immobilized unoccupied receptor (PD1), unoccupied ligand (PDL1) and ligandreceptor complex (PD1-PDL1) were conducted. A customized bioinformatic workflow to favor selection of aptamers with preferential binding properties was developed. The two-aptamer LIRECAP assay is based on aptamers against PD1 and the PD1-PLD1 complex. The three-aptamer LIRECAP assay is based on aptamers against PD1, PDL1 and the PD1-PDL1 complex.

[0213] Scheme of LIRECAP SELEX

[0214] The scheme for LIRECAP SELEX is illustrated in Figure 2A. Starting with a SEL2-N20 RNA aptamer library, eight rounds of SELEX using un-complexed PD1, un-complexed PDL1 or the PD1-PDL1 complex as the targets were conducted in parallel. Cross-target preclearing (Counter SELEX) was used to remove cross-reacting aptamers. Briefly, the method included the following steps:

[0215] • Creation of unoccupied and complexed targets'. Recombinant human PD1 with His tag or PDL1 with Fc tag were immobilized onto magnetic beads (Dynabeads - ThermoFisher). For creation of ligand-receptor complexes, soluble PDLl-Fc was added to beads coated with His-PDl. The uncomplexed PDL1 was washed away.

[0216] • Creation of starting RNA library. The starting RNA library (SEL2-N20) was created using in vitro transcription with 2'flouro-NTPs. This library was initially pre-cleared to remove non-specifically binding aptamers using magnetic beads coated with His-human serum albumin and Fc-containing human IgGl.

[0217] • Repetitive Selection of aptamers to respective targets'. The pre-cleared library was divided into three equal parts, and each was added to respective target coated beads (PD1, PDL1 or PD1-PDL1). After binding occurred, the aptamer-bound beads were washed to remove non-specifically bound aptamers. Resulting target-bound aptamers were extracted using standard phenol-chloroform methods, followed by RT-PCR amplification and in vitro transcription. The SELEX enrichment was repeated for eight cycles. In parallel, an aliquot of aptamer-bound beads from the first round of SELEX were incubated with a dilute preparation of RNase cocktail (RNase Tl+micrococcal nuclease mix, Epicentre) to speed-up the enrichment of high affinity aptamers. Only one round of such SELEX was done.• Clearing libraries to enhance enrichment of aptamers with preferential binding'. Counter SELEX was used to clear pools of aptamers that cross-bound to other targets as outlined in Table 2:

[0218] Table 2: Proteins used to post-clear cross-reactive aptamers

[0219]

[0220] This step was to ensure that highly cross-reactive aptamers are eliminated and aptamers with preferential binding to the target were enriched in respective libraries.

[0221] LIRECAP SELEX resulted in enrichment of aptamer pools

[0222] Aptamer pools enriched for binding to PD1, PDL1 or the PD1-PDL1 complex were selected using SELEX and the enriched pools were sequenced using Illumina-based high-throughput sequencing. Enrichment efficiency following each round for each pool was calculated using unique sequences relative to total sequences (Figure 2B). A progressive enrichment of unique sequences was seen in all the three SELEX library pools (selected with PD1, PDL1 or PD1-PDL1 complex) with most of the enrichment reached after 4-5 rounds of SELEX. In certain cases, selection was performed using target-coated beads that were fixed with formaldehyde before performing SELEX to enrich for aptamers that recognize their target after formalin fixation.

[0223] • Enhanced specificity. Along with progressive enrichment, the specificity of the binding of aptamers in each pool for PD1, PDL1 and the PD1-PDL1 complex increased with sequential rounds of selection (Figure 2C)

[0224] • RNase digestion to enhance enrichment'. In parallel, an aliquot of aptamer-bound beads were incubated with diluted RNase cocktail (RiboShredder® RNase blend, containing RNase T1 and micrococcal nuclease, Epicentre) to enzymatically digest, and therefore remove, unbound aptamers. RNase cocktail digests 2'fluoro-modified aptamers that are weakly bound or unprotected by target and only aptamers tightly bound to and protected by target are spared. This step is to ensure that only tightly-binding, high affinity aptamers are enriched thus improving the specificity of the aptamers remaining in the pool. RNase method was done for only one SELEX round and found to be very efficient in enriching aptamers, when compared to multiple selection rounds without RNase digestion step.Selection of candidate aptamers from PD1, PDL1 and PD1-PDL1 complex pools Lead candidate aptamer sequences were identified using bioinformatic tools based on their increased prevalence with sequential rounds of SELEX enrichment and preferred binding to their target antigen (Figure 3A). Aptamer sequences that were increasingly prominent in subsequent rounds in the PD1 SELEX pool but minimal or non-existent in the PDL1 or PD1-PDL1 complex aptamer pools were identified and designated “P” aptamers. Aptamer sequences that were increasingly prominent in subsequent rounds in the PDL1 SELEX pool but minimal or absent in the PD1 or PD1-PDL1 complex aptamer pools were identified and designated “L” aptamers. Aptamer sequences that were prominent in subsequent rounds in the PD1-PDL1 complex aptamer pool but minimal or absent in the PD1 or PDL1 aptamer pools were identified and designated “C” aptamer. Sequences selected for further analysis bound to both native targets and formalin-fixed targets and had various predicted secondary structures as illustrated for select lead candidate aptamers with preferred binding to PD1 (Figure 3B), PDL1 (Figure 3C) and the PD1-PDL1 complex (Figure 3D).

[0225] Lead P aptamer, L aptamer and C aptamer sequences were synthesized and evaluated for binding to PD1, PDL1 and PD1-PDL1 complex proteins. These included aptamers P10, P17, Pl 50 and Pl 57, that preferentially bound to unoccupied PD1 (Figure 4A), aptamers L20, L42, L45 and L50, that preferentially bound to unoccupied PDL1 (Figure 4B) and aptamers C9, C27, C52, C88 and Cl 13, that preferentially to the PD1-PDL1 complex (Figure 4C).

[0226] EXAMPLE 2

[0227] Evaluation of aptamers for the LIRECAP assay

[0228] PD1 -Binding Aptamers

[0229] Candidate P aptamers were evaluated for binding to PD1 and the PD1-PDL1 complex protein. Dynabeads were coated with 25 nM PDl-His. Aliquots of PD1 -coated beads were incubated with various concentrations of PDLl-Fc. Non-bound PDLl-Fc was washed off to produce samples with various fractional occupancies (FOs) of PD1 by PDL1 (scheme in Figure 5A). Beads were then fixed in formalin.

[0230] Synthesized P aptamer candidates were added to the beads, non-bound aptamer washed off, and bound aptamer quantified by RT-qPCR.to assess relative binding of candidate P aptamers to samples with varying levels of PD1 FO by PDL1. As illustrated in Figures 5B-5D, aptamers designated as P10, P17 and Pl 50 all preferentially bound to unoccupied PD1, with decreasing binding as PD1 FO by PDL1 increased. Furthermore, P candidate aptamers had only minimal binding to beads coated with PDL1 alone.Aptamer binding to cells was then assessed using cells known to express PD1. HEK cells with inducible PD1 were cultured in plain media resulting in cells with minimal expression of PD1 (identified as “HEK”) or with Doxycycline to induce expression of PD1 (identified as “HEK-PD1”). Various concentrations of Recombinant PDLl-Fc were added to HEK-PD1 cells to form cell surface complexes with various PD1 FO by PDL1. Cells were washed and formalin-fixed. Candidate P aptamers were added, cells were washed, and bound aptamer evaluated by RT-qPCR. As illustrated in Figures 6A-6B, candidate P aptamers preferentially bound to unoccupied PD1 expressed on the surface of HEK-PD1 cells. Decreasing binding of P aptamers was seen with increasing FO of PD1 by PDL1.

[0231] Complex-binding aptamers

[0232] Similar studies were done to assess candidate C aptamers. In contrast to the P aptamers, Cl 8, C52 and Cl 13 bound preferentially to beads coated with the PD1-PDL1 complex (Figures 7A-7C) and C18, C52 and Cl 13 all showed increasing binding to PD1 as PD1 FO by PDL1 increased (Figures 8A-8D). Candidate C aptamers showed preferential binding to beads coated with complex when compared to beads coated with PD1 or PDL1 alone (data not shown).

[0233] Candidate C aptamers also showed preferential binding to PD1-PDL1 complex expressed by HEK-PD1 cells (Figures 9A-9B).

[0234] PDLl-Binding Aptamers

[0235] While the two-aptamer LIRECAP assay is based on P and C aptamers, the three-aptamer LIRECAP assay also requires aptamers that preferentially bind to PDL1 (L aptamers).

[0236] Therefore, similar studies were done to characterize the L aptamers. For initial evaluation of synthesized candidate L aptamers, Dynabeads were coated with 25 nM PDLl-Fc.

[0237] These PDL1 -coated beads were incubated with various concentrations of PDl-His to result in beads with various levels of PDL1 FO by PD1. Target-coated beads were formalin fixed, candidate L aptamers were added, beads were washed and binding was assayed by RT-qPCR. Results with the candidate L aptamers demonstrated the expected specificity. L42, L45 and L50 all bound preferentially to beads expressing unoccupied PDL1 (Figures 10A-10C) and showed decreasing binding to beads as PDL1 FO by PD1 increased (Figures 11A-11D). For the cell based binding assay, A549 cells were cultured in plain media (A549) or with IFNg to induce expression of PDL1 (A549-PDL1). Various concentrations of recombinant PDl-Fc were added to A549-PDL1 cells to form cell surface complexes with various PDL1 FO by PD1. Cells were formalin-fixed and candidate L aptamers added. As illustrated in Figures 12A-12B, candidate L aptamers bound preferentially to unoccupied PDL1, with decreasing binding as that PDL1 was occupied by PD1.EXAMPLE 3

[0238] Conditions to stabilize ligand-receptor complexes and improve selection of high affinity aptamers for LIRECAP assay

[0239] The traditional method of SELEX involves immobilization of native targets onto solid surfaces such as agarose beads, against which aptamers are enriched. The issue with this approach, when developing aptamers against ligand-receptors, is the rapid dissociation kinetics of any ligand-receptor complexes back into individual components. To solve these problems, two approaches were utilized in the LIRECAP SELEX.

[0240] First, cold temperature incubation was used. Briefly, each step involving binding of aptamer pools to targets was performed at 4°C in order to reduce the dissociation of the complex. The cold temperature incubation was included both in the multiple-round conventional SELEX using native targets, as well as in single-round RNase-based SELEX (discussed below).

[0241] Second, formalin fixing was used. The ligand-receptor complex was stabilized by fixing the target-bound beads with 2% formaldehyde before applying the aptamer pool to the beadbound targets. This approach has the added advantage of allowing for selection of aptamers that bind to their target after formalin fixation, which allows for the assay to be done using FFPE tissue.

[0242] Experiments were done to assess whether a more efficient approach could be used to select RNA aptamers that preferentially bound to the unoccupied receptor, the unoccupied ligand or the ligand-receptor complex. This was done using an RNase cocktail to remove unbound aptamers. Briefly, the aptamer library was added to the formalin-fixed beads and incubated at 4°C for 90 minutes and unbound aptamer RNA was washed off with LIRECAP wash buffer twice at 4°C as described above.

[0243] The beads were then incubated with an RNase cocktail (Epicentre) (1 pL RNase cocktail per 100 pL LIREACP binding buffer) and incubated at 4°C for 15 minutes.

[0244] The bound enriched aptamer pools were reverse transcribed to cDNA, amplified and in vitro transcribed to RNA. Counter-SELEX to enhance cross-target post-clearing with PD1 or PDL1 was then performed on these enriched pools once to improve target specificity. This approach allowed for selection of aptamers more efficiently due to more effective removal of non-bound aptamers.EXAMPLE 4

[0245] Aptamer sequences, probes and primers for the PD1-PDL1 LIRECAP assay As described above, synthesized candidate P, C and L aptamers were screened to determine the best candidates for developing a two-aptamer or three-aptamer LIRECAP assay. The sequences for the lead candidate aptamers are as follows.

[0246] P aptamers:

[0247]

[0248] L aptamers:

[0249]

[0250] C aptamers:

[0251]

[0252] Additional sequences that were promising based on sequencing of the PD1, PDL1 and PD1-PDL1 aptamer pools were identified and are available for synthesis.

[0253] Once the candidate aptamers had been identified, probes specific for each candidate aptamer were synthesized based on the complementary nucleic acid sequences of the aptamer variable region sequences. The probes and primers for candidate aptamers for both the 2-color and 3-color LIRECAP assay are provided in Figure 13.EXAMPLE 5

[0254] Two-aptamer PD1 LIRECAP Assay

[0255] The two-aptamer LIRECAP assay was developed using the most promising P and C aptamers. The initial two-aptamer LIRECAP assay was done using beads coated with PD1 and various levels of PDL1 FO as illustrated in Figure 5. This included a variety of combinations of candidate P aptamers and candidate L aptamers. Aptamers P17 and C18 were selected for further development. In Figures 14A-14B, equimolar concentrations of Pl 7 and Cl 8 were added to beads with various levels of PD1 FO by PDL1 and the two-aptamer LIRECAP assay performed. The ratio of bound C to P aptamer, as determined by RT-qPCR, increased as FO of PD1 by increased as expected.

[0256] The Jurkat-Raji PD-1 / PD-L1 assay model from InvivoGen was used to assess the two-aptamer LIRECAP assay using whole cells. This model provides Raji cells that do and do not express PDL1. Furthermore, the Jurkat cells expressing PD1 in this model, known as Jurkat-Lucia TCR-hPD-1 cells, were engineered so they are luminescent when PD1 is not engaged by PDL1. This luminescence is turned off when PD1 is engaged by PDL1 expressed by the Raji cells. Therefore, this model allowed for comparison of PD1 FO by PDL1 using the two-aptamer PD1 LIRECAP assay and evaluation of the functional impact of signaling of PD1 by PDL1 in the same samples. To prepare the cell mixture, equal numbers of Jurkat and Raji cells were mixed. However, the ratio of PDL1(+) and PDLl(-) Raji cells was varied to adjust PD1 FO by PDL1 (Figure 15A). There was a strong correlation between expected PD1 FO by PDL1 and luminescence from the Jurkat-Lucia TCR-hPD-1 cells (Figure 15B). There was also a strong correlation between the expected PD1 FO by PDL1 and the LIRECAP results (Figure 15C). These data demonstrate a clear link between the results of the two-aptamer LIRECAP assay and functional signaling via PD1.

[0257] EXAMPLE 6

[0258] Comparison of the LIRECAP assay to a proximity ligation assay (PLA) using FFPE samples

[0259] Additional studies were done to assess whether the LIRECAP assay could be done on formalin fixed, paraffin embedded (FFPE) tissue similar to that available clinically, and whether LIRECAP results with such samples are consistent with results obtained on the same samples using currently acceptable techniques such as Proximity Ligation Assays (PLAs). PLAs are valuable research tools, although they are not used for routine clinical analysis because they are technically challenging and only semiquantitative. Nevertheless, PLA assays are available todetermine the proximity of receptors to ligands, including PD1 to PDL1. Cell pellets of Jurkat-Raji cells, produced as outlined above, were formalin fixed and paraffin embedded. Scrolls were obtained from these FFPE samples for PLA and LIRECAP (see scheme for sample preparation in Figure 16A). To mimic clinical specimens, FFPE scrolls (10-pm thick) were made from Jurkat-Raji cell pellets using a HistoGel®-based approach. Samples were produced from cell pellets made using PDLl(-) Raji (low PD1 FO by PDL1), PDL1(+) Raji (high PD1 FO by PDLI and various mixtures of PDLl(-) and PDL1(+) Raji. Scrolls were deparaffinized and rehydrated using standard protocols. The PLA indicated increasing evidence for PD1 and PDLI proximity as the number of PDL1(+) Raji cells increased (Figure 16B) demonstrating the cell co-culture approach brings PD1 and PDLI in proximity. Results of the two-aptamer LIRECAP assay on FFPE scrolls were consistent with those of the PD1 / PDL1 PLA assay (Figures 16C-16D) These results demonstrate the LIRECAP assay results are consistent with semiquantitative data obtained from PLA which is a gold standard and that the LIRECAP assay can be done on tissue scrolls obtained from FFPE biospecimens.

[0260] Technical, intratumoral and interpatient variability of two-aptamer LIRECAP assay in clinical samples.

[0261] Human sarcoma biospecimens with known high levels of T cell infiltration were assessed using the two-aptamer LIRECAP assay and PLA. Human FFPE biospecimens were cut into 5-um thick or 10-pm thick sections. Immunohistochemistry for PDLI identified three samples (designated A, B and C) with high PDLI expression, and three samples (designated D, E and F) with low PDLI expression. PLA was done on these biospecimens and compared to immunohistochemistry. The PLA results were consistent with results of immunohistochemistry, with a high proximity signal in those samples with high expression of PDLI and low PLA signal in those samples with low expression of PDLI (examples in Figure 17A). The same clinical samples were evaluated using the two-aptamer LIRECAP assay.

[0262] Multiple scrolls were obtained from each biospecimen. Each scroll was macerated into homogeneous pieces and split into three aliquots per sample. This allowed for evaluation of technical variability (evaluation of multiple aliquots of a single biospecimen at different times), intratumoral variability (evaluation of different scrolls representing different sections from the same biospecimen) and intersubject variability (evaluation of samples from different patients). These results demonstrate that the LIRECAP assay is technically highly reproducible (Figure 17B) There was intratumoral variability among the multiple tissue sections obtained from individual tumor biospecimens, particularly those with high PDLI expression suggesting FO of PD1 by PDLI can be variable within different regions of a given tumor (Figure 17C). Overall,evaluation of interpatient variability suggested higher PD1 FO by PDL1 in those samples with higher PDL1 expression (Figure 17 D).

[0263] Three-aptamer LIRE CAP assay for Pl) I. Pl) LI and the Pl) 1 -Pl) LI complex.

[0264] The three-aptamer LIRECAP assay was performed using samples of Jurkat and Raji cells (Figure 18) that had been co-cultured, pelleted, fixed in formalin and embedded in paraffin, and processed as described above. Jurkat cells were included that did or did not express PD1, and Raji cells were included that did or did not express PDL1 to provide biospecimens with a range of PD1 and PDL1. More specifically, combinations were assessed as shown in Table 3.

[0265] Table 3

[0266]

[0267] The three-aptamer LIRECAP assay using Pl 7, L42 and Cl 8 was performed on five separate occasions using one scroll from each combination per run. The relative proportion of each aptamer was calculated as a percentage of respective total bound aptamer (e.g., amount P, L or C) relative to the sum of all aptamers (Total amounts of P+L+C). Mean and standard deviations across the five independent runs were determined. As illustrated in Figure 19, the most prevalent aptamer was the P aptamer in the sample with PD1(+) Jurkat and PDLl(-) Raji, the L aptamer in the sample with PDl(-) Jurkat and PDL1(+) Raji, and the C aptamer in the sample with PD1(+) Jurkat and PDL1(+) Raji demonstrating the three-color LIRECAP assay can be performed on FFPE biospecimens.

[0268] Although the foregoing specification and examples fully disclose and enable the present invention, they are not intended to limit the scope of the invention, which is defined by the claims appended hereto.

[0269] All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification this invention has been described in relation to certain embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein may be varied considerably without departing from the basic principles of the invention.The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0270] Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein.

[0271] Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

What is claimed is:

1. A PDl-binding aptamer comprising a polynucleotide 45-55 nucleotides in length comprising SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3.

2. A PDLl-binding aptamer comprising a polynucleotide 45-55 nucleotides in length comprising SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

3. APD1-PDL1 complex-binding aptamer comprising a polynucleotide 45-55 nucleotides in length comprising SEQ ID NO:7, SEQ ID NO: 8, or SEQ ID NO:9.

4. The aptamer of any one of claims 1-3, wherein the polynucleotide is 51 or 52 nucleotides in length.

5. A composition comprising the aptamer of any one of claims 1-4 operably linked to a tag.

6. The composition according to claim 5, wherein the tag is selected from a molecule that can be detected using optical sensors, molecular size sensors, or isotopic sensors.

7. The composition according to claim 5, wherein the aptamer is bound to a substrate.

8. The composition according to claim 5, wherein the aptamer is operably linked to a therapeutic moiety.

9. The composition according to claim 8, wherein the therapeutic moiety is selected from nucleic acid-based therapeutics, chemotherapeutics, immunotherapeutics, and small molecule therapeutics.

10. A composition comprising an aptamer bound to a complex, wherein the complex comprises at least two polypeptides, wherein the aptamer is the PD1 / PDL1 complexbinding aptamer of claim 3.

11. The composition according to claim 10, wherein the at least two peptides are selected from the group consisting of PD1 and PDL1.

12. The composition according to claim 10, wherein the aptamer specifically binds to the complex.

13. The composition according to claim 1, wherein the aptamer bound to the complex additionally comprises a tag.

14. A composition comprising an aptamer bound to a PD1, wherein the aptamer is the PD1 aptamer of claim 1.

15. The composition according to claim 14, wherein the aptamer bound to the complex additionally comprises a tag.

16. A composition comprising an aptamer bound to a PDL1, wherein the aptamer is the PDL1 aptamer of claim 2.

17. The composition according to claim 16, wherein the aptamer bound to the complex additionally comprises a tag.

18. A labelled probe comprising(a) a polynucleotide selected from the group consisting of a probe for P17 aptamer (SEQ ID NO: 10), a probe for P150 aptamer (SEQ ID NO: 11), a probe for Cl 13 aptamer (SEQ ID NO: 12), a probe for C18 aptamer (SEQ ID NO: 13), a probe for L42 aptamer (SEQ ID NO: 14) or a probe for L50 aptamer (SEQ ID NO: 15), and (b) a label.

19. A method of quantifying a fractional occupancy (FO) of PD1 by PDL1 or of PDL1 by PD1 in a biospecimen, the method comprising:contacting the sample with a first aptamer that specifically binds to PD1, with a second aptamer that specifically binds to PDL1, and with a third aptamer that specifically binds to PD1 / PDL1 complex, andconcurrently determining the concentration of bound first aptamer, second aptamer and third aptamer.

20. The method of claim 19, wherein the biospecimen is a fluid, a cell sample, or tissue.

21. The method of claim 20, wherein the biospecimen is a fluid, and the fluid is blood, saliva, cerebrospinal fluid, or ascites.

22. The method of claim 20, wherein the biospecimen is a cell sample, and the cell sample is a fine needle aspirate.

23. The method of any one of claims 19-22, wherein the biospecimen is fresh, frozen or fixed in preservative.

24. The method of any one of claims 19-22, where the biospecimen is evaluated without additional manipulation, placed on a slide, minced, macerated, sonicated or enzymatically digested prior to analysis.

25. The method of any one of claims 19-24, wherein the quantification of FO of PD1 by PDL1 or PDL1 by PD1 is performed using a two-aptamer assay.

26. The method according to claim 25, wherein the tag on the first aptamer is distinct from the tag on the second aptamer.

27. The method of any one of claims 19-24, wherein the quantification of FO of PD1 by PDL1 or PDL1 by PD1 is performed using a three-aptamer assay.

28. The method according to claim 27, wherein the tag on the first aptamer is distinct from the tag on the second and third aptamers, the tag on the second aptamer is distinct from the tag on the first aptamer and third aptamer, and the tag on the third aptamer is distinct from the tag on the first aptamer and the second aptamer.

29. The method of any one of claims 19-28, wherein the concentration of bound aptamers is determined by PCR or using a single set of primers.

30. The method of any one of claims 19-29, wherein each aptamer is individually linked to a tag, and the concentration of bound aptamers is determined by detecting the tags.

31. The method of claim 30, wherein the detection is by using optical, molecular or isotopic sensors.

32. The method of any one of claims 19-31, wherein assessment of FO of PD1 by PDL1 or PDL1 by PD1 is done in the biospecimen as a whole.

33. The method of any one of claims 19-32, wherein assessment of FO of PD1 by PDL1 or PDL1 or PD1 is done using spatial techniques to assess FO in multiple areas of a single or multiple biospecimens.

34. A method of quantifying in a biospecimen fractional occupancy (FO) of one member of a molecular complex by another member of the molecular complex, the method comprising:contacting the biospecimen with a first aptamer that specifically binds to the first member of the molecular complex, with a second aptamer that specifically binds to the second member of the molecular complex, and with a third aptamer that specifically binds to the complex of the first member and the second member, andconcurrently determining the concentration of bound first aptamer, second aptamer and third aptamer in the biospecimen.

35. The method of claim 34, wherein the first member is a ligand, the second member is a receptor, and the complex comprises a ligand / receptor complex.

36. The method of claim 34 or 35, wherein the biospecimen is a fluid, a cell sample, or tissue.

37. The method of claim 36, wherein the biospecimen is a fluid, and the fluid is blood, saliva, cerebrospinal fluid, or ascites.

38. The method of claim 36, wherein the biospecimen is a cell sample, and the cell sample is a fine needle aspirate.

39. The method of any one of claims 34-38, wherein the biospecimen is fresh, frozen or fixed in preservative.

40. The method of any one of claims 34-39, where the biospecimen is evaluated without additional manipulation, placed on a slide, minced, macerated, sonicated or enzymatically digested prior to analysis.

41. The method of any one of claims 34-40, wherein the quantification of FO of the receptor by PDL1 or the ligand by the receptor is performed using a two-aptamer assay.

42. The method of any one of claims 34-40, wherein the quantification of FO of the receptor by PDL1 or the ligand by the receptor is performed using a three-aptamer assay.

43. The method of any one of claims 34-43, wherein the concentration of bound aptamers is determined by PCR or using a single set of primers.

44. The method of any one of claims 34-43, wherein each aptamer is individually linked to a tag, and the concentration of bound aptamers is determined by detecting the tags.

45. The method of claim 44, wherein the detection is by using optical, molecular or isotopic sensors.

46. The method of any one of claims 34-45, wherein assessment of FO of the receptor by the ligand or the ligand by the receptor is done in the biospecimen as a whole.

47. The method of any one of claims 34-46, wherein assessment of FO of the receptor by the ligand or the ligand by the receptor is done using spatial techniques to assess FO in multiple areas of a single or multiple biospecimens.

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