Compositions and methods for in VIVO detection, diagnosis and treatment of a disease or a disorder

Abstract

The present disclosure relates to synthetic probes and methods of using the synthetic probes. The synthetic probes include a binding domain that specifically binds a target, a cleavable linker, and a reporter. Cleaving the linker releases the reporter after a sufficient time following administration of a cleaving agent allowing for collection of the released reporter in a sample from the subject. The synthetic probes and methods can be used to diagnose a disease or disorder in a subject having or suspected of having a disease or disorder and for treating a disease or disorder.

Description

COMPOSITIONS AND METHODS FOR IN VIVO DETECTION, DIAGNOSISAND TREATMENT OF A DISEASE OR A DISORDERCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of United States Provisional Patent Application No. 63 / 703,676, filed on October 4, 2024, United States Provisional Patent Application No. 63 / 704,318, filed on October 7, 2024, United States Provisional Patent Application No. 63 / 709,298, filed on October 18, 2024, United States Provisional Patent Application No. 63 / 756,630, filed on February 10, 2025, and United States Provisional Patent Application No. 63 / 818,970, filed on June 6, 2025. the disclosures of which are incorporated by reference herein in their entireties.BACKGROUND OF THE DISCLOSURE

[0002] The present disclosure relates generally to research and medicine. More particularly, the present disclosure relates to synthetic probes, methods for detecting target molecules using the synthetic probes, and methods for diagnosing diseases and disorders using the synthetic probes. Synthetic probes of the present disclosure include a binding domain, a cleavable linker, and a reporter. The methods of the present disclosure use the synthetic probes that specifically bind to a target molecule when present in a subject and releases a reporter upon cleavage of a cleavable linker following administration of a cleaving agent to the subject allowing for collection of the released reporter in a sample from the subject.

[0003] Detection of diseases and disorders allows for accurate treatment.Early detection makes curative treatment more likely. Typically, diagnosis requires obtaining a sample from a subject suspected of having a disease or a disorder, processing that sample, and determining whether the subject has the disease or disorder by detecting a biomarker that is associated with a specific disease or disorder. This can be timely and not allow for probing the sample for several biomarkers at the same time. These tests often occur in a laboratory rather than at home, which adds to the cost and delay to treatment.

[0004] Targeted therapies have been developed to provide more effective and specific treatment. Monoclonal antibodies (mAbs) have emerged as a promising therapeutic approach due to their ability to specifically target cancer cells by binding to antigens expressed on the surface of these cells. To enhance the potency of mAbs, researchers have developed antibody-drug conjugates (ADCs). ADCs consist of a monoclonal antibody specific to a tumor-associated antigen, a cytotoxic drug, and a linker that connects the drug to the antibody. The antibody component of the ADC selectively binds to the target antigen on the cancer cell surface, facilitating the internalization of the ADC into the cell. Once inside the cell, the linker is cleaved, releasing the cytotoxic drug to exert its lethal effect on the cancer cell. Therefore, targeted anti-cancer agents may highly reduce the risk of side-effects, compared to traditional anti-cancer agents. Alternative forms of ADCs include a linker that can be cleaved outside the cell to release the cytotoxic drug to exert its lethal effect on the cancer cell.

[0005] While biopsy is considered the gold standard for cancer diagnosis, it is an invasive procedure that involves the removal of tissue samples for histopathological examination. This method can be painful, carries risks of infection and complications, and may not always be feasible for tumors located in hard-to-reach areas. Imaging modalities like MRI. CT scans, and X-rays are non-invasive but often lack the sensitivity required to detect small or early-stage tumors. Additionally, these techniques can sometimes produce false positives or negatives, leading to unnecessary anxiety or missed diagnoses. Blood-based biomarkers for cancer detection, such as PSA for prostate cancer or CA-125 for ovarian cancer, offer a less invasive alternative. However, these markers are not always specific to cancer and can be elevated in benign conditions, reducing their diagnostic accuracy. Techniques such as liquid biopsy, which analyzes circulating tumor DNA (ctDNA) or circulating tumor cells (CTCs) in the blood, show promise for non-invasive cancer detection. However, these methods are still under development and face challenges related to sensitivity, specificity, and standardization. For signals to reach detectable levels, cancer progression may need to reach certain thresholds for detection. Being able to detect circulating biomarkers for some early-stage cancers is often a challenge, if at all possible.

[0006] Significant advances have been made in the field of bioorthogonal prodrug activation, particularly using 'click-to-release' chemistries. These systems, however, have been developed for therapeutic applications, focusing on the site- specific release of a cytotoxic or otherwise bioactive drug. Consequently, the design of these platforms is governed by principles fundamentally at odds with the requirements for a non-invasive diagnostic. The therapeutic payloads in such systems are typically engineered for local retention, cellular uptake, and potent biological activity, often being hydrophobic and inherently non-inert. These objectives can be at odds with that of a diagnostic reporter, which is often engineered to be biologically inert, highly hydrophilic, and optimized for rapid systemic clearance into a remote biofluid like urine for detection. Therefore, the art of therapeutic click-to-release has not addressed the often distinct technical challenges of designing a system for the efficient generation, release, and remote measurement of a diagnostic molecular signal.

[0007] Achieving a high signal-to-background ratio sufficient for early disease detection is challenging. Existing one-step systems are often hampered by high background signals from unbound probes that are still circulating at the time of activation, thereby obscuring the true target-derived signal. On the other hand, systems that rely on endogenous triggers lack precise temporal control, resulting in a stochastic and temporally diffuse release of the reporter that is difficult to distinguish from noise.

[0008] There is a critical need for improved in vivo cancer detection methods that are non-invasive. highly sensitive, and specific. Such methods should be capable of detecting cancer at its earliest stages, providing real-time results, and being applicable to a wide range of cancer types. The development of such technologies would revolutionize cancer diagnostics, enabling more effective screening, monitoring, and treatment. Detection and diagnosis of other diseases and disorders can also benefit from non-invasive methods. Previous attempts have been made to introduce "synthetic biomarkers’’ to patients. One such attempt taught introducing synthetic biomarkers that release reporters spontaneously when encountering certain conditions, such as when encountering certain proteases in a physiological condition that elicit self-immolating molecular cleavage of the synthetic biomarkers to release reporters that are subsequently detectable in urine, breath and otherwise. While this has provedpromising, the cleavage of synthetic biomarkers to release reporters is stochastic and less predictable thereby resulting in a temporally diffuse signal. Factors that may influence temporal vanability include perfusion rates to certain tumor types. One strategy to counter this temporal dispersion of signal is to introduce larger quantities of synthetic biomarkers. This not only increases costs of the assay, but also may result in higher toxicity to the patient and immunogenic responses.

[0009] Accordingly, a need exists for new detection methods and synthetic probes that can be used to diagnose diseases and disorders. The present disclosure addresses the aforementioned limitations by providing a novel in vivo detection system. This system leverages advanced imaging techniques, molecular probes, and / or biosensors to identify target molecules such as cancerous cells and a range of other disease states that may relate to pathogens or other disease states that distinguish themselves from healthy tissue, that may include having binding epitopes associated with a pathogen or disease state. An example of an application of the present disclosure includes determining the origins of a pneumonia and the presence of a virus, bacteria, fungi or some other disease state present in the patient with high precision and accuracy. The present disclosure offers anon-invasive, rapid, and reliable diagnostic tool that can be used in clinical settings to improve patient outcomes through early detection and timely intervention.

[0010] In addition to cancer applications, the synthetic probes and methods described in the present disclosure can be applied to other diseases and disorders. The synthetic probe described herein includes a binding domain that specifically binds to a target molecule and releases a reporter following administration of a cleaving agent that cleaves a cleavable linker connecting the reporter to the binding domain. In addition to a detection purpose, once the reporter is released from the target what remains behind of the synthetic probe can further include a "flag or stub' domain that can function as a binding domain for the binding of another molecule (referred to herein as a ‘'finishing agent”). This ‘pre-targeting’ of a target molecule can be beneficial in either a second stage of diagnosis, such as binding an imaging agent to the target molecule to allow for in vivo imaging, for example, and / or specific binding a therapeutic by enabling the release of a therapeutic molecule bespoke for the target molecule.

[0011] The present probes and methods can also, in various embodiments, overcome the limitations described above through a novel two-step pretargeting regimen. By introducing a predetermined delay (e.g., hours to days) between the administration of the probe and the cleaving agent (also referred to herein as a “trigger”), the system can allow for substantial clearance of unbound probe from circulation, dramatically reducing background. Subsequently, the administration of a bioorthogonal exogenous cleaving agent initiates a rapid, controlled bolus release of the reporter from the now target-localized probe, creating a sharp and predictable signal peak within a narrow detection window. This unique combination of a clearance phase and on-demand activation provides a robust solution to the signal-to-noise challenge that is not described in known one-step or endogenously -triggered systems.BRIEF DESCRIPTION OF THE DISCLOSURE

[0012] The present disclosure is generally related to synthetic probes and methods for detecting a target molecule when present in a subject. The methods of detecting a target molecule are particularly suitable for diagnosing and treating diseases and disorders using the synthetic probes.

[0013] In one aspect, the present disclosure is directed to a synthetic probe comprising: a binding domain that specifically binds a target molecule; a cleavable linker; and a reporter, wherein the cleavable linker is cleavable by a bioorthogonal reaction to release the reporter.

[0014] In one aspect, the present disclosure is directed to a method for detecting a target molecule when present in a subject, the method comprising: administering a synthetic probe to a subject, wherein the synthetic probe comprises: a binding domain that specifically binds the target molecule; a cleavable linker; and a reporter; allowing time for the synthetic probe to bind to the target molecule if present in the subject and to allow unbound synthetic probe to clear the subject; administering a cleaving agent to the subject, wherein the cleaving agent interacts with the cleavable linker to initiate cleavage of the cleavable linker and release the reporter; allowing time for the reporter to accumulate in a body fluid of the subject; collecting the body fluid;analyzing the body fluid for the reporter; and determining the presence of the target molecule based on the reporter detected in the body fluid.

[0015] In one aspect, the present disclosure is directed to a kit comprising: a synthetic probe, wherein the synthetic probe comprises: a binding domain that specifically binds a target molecule, a cleavable linker, and a reporter; and a cleaving agent.

[0016] In one aspect, the present disclosure is directed to a method of diagnosing and treating a subj ect having or suspected of having a disease, the method comprising: administering a synthetic probe to a subject, wherein the synthetic probe comprises: a binding domain that specifically binds the target molecule; a flag associated with the binding domain; a cleavable linker; and a reporter; allowing time for the synthetic probe to bind to the target molecule if present in the subject and to allow unbound synthetic probe to clear the subject; administering a cleaving agent to the subject, wherein the cleaving agent interacts with the cleavable linker to initiate cleavage of the cleavable linker and release the reporter; allowing time for the reporter to accumulate in a body fluid of the subject; collecting the body fluid; analyzing the body fluid for the reporter; determining the presence of the target molecule based on the reporter detected in the body fluid; diagnosing the subject as having a disease based on the target molecule if detected in the subject; and administering a finishing agent comprising: a reactive group that specifically binds the flag and a therapeutic, wherein the finishing agent specifically binds the binding domain-flag conjugate that remains bound to the target molecule to deliver the therapeutic.

[0017] In one aspect, the present disclosure is directed to use of the synthetic probe for detecting a target molecule in a subj ect having or suspected of having the target molecule, the use comprising: administering the synthetic probe to the subject, wherein the synthetic probe comprises: a binding domain that specifically binds a target molecule; a flag associated with the binding domain; a cleavable linker; and a reporter; allowing time for the synthetic probe to bind to the target molecule if present in the subject and to allow unbound synthetic probe to clear the subject: administering a cleaving agent to the subject, wherein the cleaving agent interacts with the cleavablelinker to initiate cleavage of the cleavable linker and release the reporter and wherein the binding domain and flag form a binding domain-flag conjugate that remains bound to the target molecule, allowing time for the reporter to accumulate in a body fluid of the subject; collecting the body fluid; analyzing the body fluid for the reporter; and determining the presence of the target molecule based on the reporter detected in the body fluid.

[0018] In one aspect, the present disclosure is directed to use of the synthetic probe for diagnosing and treating a disease in a subject having or suspected of having the disease, the use comprising; administering the synthetic probe to the subject, wherein the synthetic probe comprises: a binding domain that specifically binds a target molecule; a flag associated with the binding domain; a cleavable linker; and a reporter; allowing time for the synthetic probe to bind to the target molecule if present in the subject and to allow unbound synthetic probe to clear the subject: administering a cleaving agent to the subject, wherein the cleaving agent interacts with the cleavable linker to initiate cleavage of the cleavable linker and release the reporter and wherein the binding domain and flag form a binding domain-flag conjugate that remains bound to the target molecule; allowing time for the reporter to accumulate in a body fluid of the subject; collecting the body fluid: analyzing the body fluid for the reporter; determining the presence of the target molecule based on the reporter detected in the body fluid; diagnosing the subject as having a disease based on the target molecule if detected in the subject; and administering a finishing agent comprising: a reactive group that specifically binds the flag and a therapeutic, wherein the finishing agent specifically binds the binding domain-flag conjugate that remains bound to the target molecule to deliver the therapeutic.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The disclosure will be better understood, and features, aspects and advantages other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such detailed description makes reference to the following drawings, wherein:

[0020] FIG. 1 is an illustration depicting functional components of an exemplary synthetic probe of the present disclosure.

[0021] FIG. 2 is a flowchart depicting a 4-step assay for administering a synthetic probe, administering a “trigger” that initiates cleavage of the linker of the synthetic probe to release a reporter of the synthetic probe that is collected in a sample and then detection of the reporter.

[0022] FIG. 3 is an illustration depicting and describing temporal considerations of an exemplary embodiment of a synthetic probe diagnostic method of the present disclosure.

[0023] FIG. 4 is an illustration depicting and describing an exemplary molecular design of in vivo detection using a synthetic probe in a multiplexed cancer assay.

[0024] FIG. 5 is an illustration depicting and describing an exemplary molecular design of in vivo detection using a synthetic probe in a multiplexed cancer assay after linker cleavage.

[0025] FIG. 6 is an illustration depicting and describing an exemplary molecular design of in vivo detection using a synthetic probe in a multiplexed cancer assay after linker cleavage where reporter includes antibody for detection of reporter by lateral flow assay and binding domains including universal linker remain bound to target as potential flag site.

[0026] FIG. 7 illustrates an exemplary embodiment of the molecular design of two synthetic probes for two tumor biomarkers for detection by lateral flow each having a universal Lateral Flow Assay strip paired antibody and an additional component (e.g., spacer).

[0027] FIG. 8 illustrates an exemplary embodiment of the molecular design of two synthetic probes for two tumor biomarkers for detection by lateral flow each having a universal Lateral Flow Assay strip paired antibody and an additional component (e.g., spacer).

[0028] FIG. 9 is a graph depicting perfusion-dependent kinetics for tumor uptake versus clearance.

[0029] FIG. 10 is a table summarizing a comparison of various cancer diagnostic modalities.

[0030] FIGS. 11A and 11B are graphs depicting tumor signal -to- background control ratio versus time (FIG. 11 A) and clearance of an inert control probe (lacking a binding domain) (FIG. 1 IB).

[0031] FIG. 12 is table summarizing a pneumonia diagnostic modalities comparison expressing the value-add of the synthetic probe (SynBio) system of the present disclosure.

[0032] FIG. 13 is table summarizing a stage I multiplexed assay for solid tumor detection of the present disclosure.DETAILED DESCRIPTION

[0033] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described below.

[0034] The present disclosure provides synthetic probes and methods for detecting a target molecule when present in a subject. The synthetic probes and methods are particularly useful for detecting target molecules that can further be used for diagnosing diseases and disorders using the synthetic probes and further allow for treating diseases and disorders in subjects having or suspected of having a disease or disorder.

[0035] The present disclosure provides a two-step bioorthogonal synthetic probe system for in vivo diagnostics and therapy that uniquely combines targeted binding with a chemically triggered reporter release. In the detection and diagnosticmode, a synthetic probe of the present disclosure is administered, which binds, for example, to a disease-specific marker via a high-affinity binding domain and carries a cleavable linker attached to a reporter. After allowing time for the synthetic probe to localize and unbound synthetic probe to clear, a biocompatible cleaving agent (also referred to herein as an “activator” and an “activator agent”) (bearing a complementary bioorthogonal reactive group) is administered to the subject. This cleaving agent “clicks” with the synthetic probe’s cleavable linker, inducing cleavage and releasing the reporter in vivo. The released reporter (now a small, freely diffusible molecule) is quickly filtered into a body fluid (e.g., urine, blood, spinal fluid, etc.), which can be non-invasively collected and analyzed ex vivo to detect the reporter. Detecting the presence or identity of the reporter reveals whether the target molecule (and subsequently, the disease or condition) is present in the subject.

[0036] The two-step “detectable release” mechanism represents a diagnostic paradigm shift. Unlike prior one-step enzyme-activated probes or imaging agents, which either rely on endogenous biology or retain signals at the target site, the present disclosure provides an exogenous bioorthogonal trigger to actively amplify and externalize the signal. The approach of the present disclosure is highly specific - the reporter is only liberated if the synthetic probe has bound the target molecule and only upon introducing the matching cleaving agent, minimizing background noise. It is also highly sensitive - each bound synthetic probe can release multiple reporter molecules, resulting in amplified signal in bodily fluids. A synthetic probe can also have more than one target to affect affinity, avidity, and / or specificity through bi-specific or even tri- specific binding capability. This enables detection of microscopic disease (e.g. tiny tumor residuals or low bacterial burden) that would evade conventional imaging (which requires bulk accumulation). Furthermore, the use of noninvasive liquid biopsy readouts (urine, blood, even breath for volatile reporters) makes the diagnostic test simple and repeatable, potentially as easy as a home urine strip or lab kit, rather than requiring expensive scans or biopsies.

[0037] In an exemplary clinical workflow, for example, a subject receives a cocktail including a plurality of synthetic probes (each synthetic probe including a binding domain that specifically binds different target molecules, plus maybe a controlprobe that includes a linker and a reporter). After sufficient time (e.g., about 2 hours to about 7 days) to allow binding and renal clearance of unbound synthetic probes, the subject is administered a cleaving agent (e.g., a TCO solution, in the case of tetrazine- equipped probes). Within minutes, the cleaving agent reacts wherever a synthetic probe is bound, severing the cleavable linker and releasing the reporter. The subject’s urine (or other sample) is collected and sent to a lab (or applied to a point-of-care device or analyzed using an at-home device) where the collected sample is analyzed to detect and identify the reporter if present in the sample. In an exemplary analysis where the reporter is a nucleic acid barcode, a CRISPR-Cas12a detection assay is performed (alternatively, PCR amplification is performed). Cas12a complexes programmed for each barcode reporter sequence produces a fluorescent signal if that barcode is present in the collected sample. The readout reveals which target molecule(s) were present, indicating the presence of tumor and even providing a molecular “fingerprint” of the disease. This two-step approach essentially creates an in vivo signal amplification loop: instead of passively measuring an existing biomarker, it actively generates a synthetic probe in situ and then detects it ex vivo. The result is a highly sensitive “synthetic biopsy” that can detect target molecules and subsequently diseases at stages far earlier than traditional blood biomarkers or imaging, with potential at-home and point-of-care testing applications.

[0038] It should be understood that the subject’s sample(s) can be processed using methods known in the art. For example, the sample can be treated with preservatives, enzymes, inhibitors, chelators, frozen, refrigerated, transported, divided (split) into parts, diluted, concentrated, purified, etc. In some embodiments is may be particularly desirable to concentrate the sample. When using Al exaFluor reporters to be detected in a urine sample (e.g., 200 mL sample collected), methods such as solid phase extraction (SPE), mixed-mode anion exchange, graphitized carbon black (GCB) SPE, large volume disk SPE, SPE plates, adsorptive microextraction, affinity capture (e.g., click chemistry), evaporative concentration, protein precipitation and concentration, liquid-liquid extraction (LLE) / ion pair extraction, membrane-based concentration, and combinations thereof can be used to concentrate the sample.

[0039] Beyond diagnostics, the same “flag-and-finish” concept extends to therapeutics. As described herein, a synthetic probe is designed to leave behind a chemical “flag” (stub) on the target molecule after releasing its reporter. For instance, if the cleavable linker fragment remaining on the bound synthetic probe contains a reactive handle (e.g. a TCO or azide) covalently attached to the target molecule, that handle now acts as an anchoring stub. A subsequent finishing agent - carrying a complementary reactive group and a therapeutic payload - is administered to the subject and will selectively “click” onto the flagged target molecule(s) or otherwise bind (in designs using an alternative to a click chemistry design). The finishing agent can also initiate a response to cause internalization for a drug to affect cancer or carry an antibiotic to affect a pathogen. The synthetic probe can also include different flags and / or multiple flags. Different or multiple flags can allow one flag to function as a binding site for a next-step finishing agent including a second reporter (e.g., an imaging agent such as a radiodense particle for in vivo imaging by MRI and / or X-ray) and a different flag to function as a binding site for a next-step finishing agent including a therapeutic. The finishing agent can be an array of therapies: a cytotoxic drug (chemotherapy) conjugated via a linker, a radioactive isotope for targeted radiotherapy, an immune effector (e.g. cytokine or immunotoxin) that is only delivered to the flagged site, an enzyme that locally activates a prodrug, an imaging agent, a fluorescent reporter, etc. Because the stub does not exist on normal cells or on non-target molecules, the therapy is highly specific, sparing healthy tissues, and next-step imaging provides specific localization. This represents an advanced pretargeted approach, with the difference that the synthetic probe of the present disclosure initially enables a diagnostic readout followed by a targeted therapy and / or a next-step in vivo target localization. In practice, a clinician can first use the diagnostic mode to “flag” and confirm the presence / location of disease (via reporter in urine or via an imaging agent as a finishing step), and then (perhaps immediately or the next day) administer a therapeutic finishing agent to treat the disease. This dual-phase approach (diagnose, then treat) offers an unprecedented level of control and safety: one can ensure the “flag” has properly localized before delivering the potent therapy, reducing off-target effects.

[0040] As used herein, a “subject ", a “patient" , “a subject in need thereof" (also used interchangeably herein with “a patient in need thereof") refers to a subject susceptible to or at risk of a specified disease, disorder, or condition. The methods disclosed herein can be used with a subset of subjects who have, are suspected of having, are susceptible to, and / or at elevated risk for a disease, a disorder, an infection, and / or as a screen. Because some of the method embodiments of the present disclosure are directed to specific subsets or subclasses of identified subjects (that is. the subset or subclass of subjects “in need” of assistance in addressing one or more specific conditions noted herein), not all subjects will fall within the subset or subclass of subjects as described herein for certain diseases, disorders or conditions. Suitable subjects include non-human animals, such as. for example, mammals, non-human primates, rodents (e.g., mice, rats, and hamsters), stock and domesticated animals (e.g., pigs, cows, sheep, horses, cats, and dogs), and birds. Suitable subjects also include humans.

[0041] The term “target molecule” is used according to its ordinary meaning to refer to a specific biological entity that is identified, detected, or measured in a biological sample for the purpose of diagnosing, monitoring, or correlating with the presence, progression, or risk of a disease, disorder, or infection. Target molecules serve as indicators or biomarkers that reflect underlying physiological or pathological processes and are central to clinical diagnostics, therapeutic monitoring, and biomedical research. A non-exhaustive list of examples of target molecules includes cells, viral particles, bacteria, fungi, proteins, nucleic acids, carbohydrates, lipids, small molecules, metabolites.

[0042] The term “synthetic probe (also referred to herein as synthetic biomarker probe and SynBio) refers to herein as a molecule including at least a binding domain, a cleavable linker, and a reporter. A “binding domain” refers to a moiety that specifically binds a target molecule. A “cleavable linker” refers to a linkage that can be cleaved (via a cleaving agent) to release the reporter. A “cleaving agent” refers to an exogenous trigger compound administered to cleave the cleavable linker. A “reporter” refers to a detectable moiety released upon linker cleavage and subsequently measured to infer presence of the target. A “flag” or “stub” refers to a residual handle left on thebinding domain after the linker is cleaved and the reporter is released and serves as an anchor for a finishing agent. A “finishing agent” refers to a secondary reagent that binds to the flag and delivers a therapy (or a reporter).

[0043] In one aspect, the present disclosure is directed to a synthetic probe comprising: a binding domain that specifically binds a target molecule; a cleavable linker; and a reporter.

[0044] The synthetic probe of the present disclosure makes use of an abiotic, bio-orthogonal chemical reaction to provoke release of the reporter. In particular, the present disclosure uses a “click-to-release” ligation strategy between a binding domain, a cleavable linker, and a reporter. The cleavable linker is initially conjugated to the binding domain and the reporter. This conjugation is stable under physiological conditions, ensuring that the reporter remains attached until the desired release event. A click chemistry reaction is used to trigger the reporter release. Upon the activation event, the cleavable linker undergoes a structural change that results in the cleavage of the bond holding the reporter, cleavage of the bond holding the binding domain, or both. This cleavage can occur through various mechanisms, such as hydrolysis, elimination, and intramolecular rearrangement. Suitable click reactions include, for example, azide-alkyne cycloaddition, tetrazine-cyclopropene ligation, and tetrazine-trans-cyclooctene (TCO) ligation (a reaction between a tetrazine and a trans- cyclooctene group). The activation event can, in various embodiments, be triggered by the deployment of a cleaving agent that reacts with the cleavable linker.

[0045] In various alternative embodiments, the synthetic probe of the present disclosure comprises a binding domain and a cleavable linker, but not a reporter. In such embodiments, the cleaving agent can comprise the reporter, which is quenched and thus undetectable. In such embodiments, deployment of the cleaving agent results in a reaction between the cleaving agent and the cleavable linker, resulting in the release and un-quenching of the reporter, rendering it detectable. In such embodiments, the cleavable linker can be masked such that it can only react with the cleaving agent after being unmasked, such as by contact with a disease-related environment or as a direct consequence of interaction between the binding domain and a disease-related epitope.

[0046] It is desirable that the synthetic probe have a total molecular weight less than 100 kDa. Suitably, the molecular weight ranges from about 20 kDa to about 60 kDa, including from about 20 kDa to about 50 kDa, including from about 20 kDa to about 40 kDa, and including from about 20 kDa to about 35 kDa. Decreasing the size of the synthetic probe facilitates clearance by the subj ect’ s body . By contrast, increasing the size of the synthetic probe prolongs its circulation in vivo and promotes hepatic uptake of the synthetic probe. Depending on the desired modality of clearance (renal or by avidin chase), the synthetic probe can vary in size. This is in stark contrast to traditional pretargeting approaches that utilize full-length monoclonal antibodies (mAbs) of approximately 150 kDa, which exhibit long circulation half-lives often measured in days. The desirable size profile described herein facilitates both rapid extravasation into target tissues, such as tumors, and, critically, efficient renal clearance of any unbound conjugate from systemic circulation. For renal clearance, it is also desirable for the overall charge of the synthetic probe to have a neutral or slightly positive charge.This results in the substantial clearance of unbound synthetic probe from the blood within approximately 24 hours post-administration, thereby achieving a high target-to-background signal ratio that enables the subsequent reporter release and detection steps of the methods described herein.

[0047] In one exemplary embodiment, cleavage of the cleavable linker of the synthetic probe is activated by controlled administration of a cleaving agent. As used herein, “a cleaving agent” (also referred to herein as an “activating agent”, a “trigger”, and a “triggering agent”) is a species that reacts with the cleavable linker moiety in the synthetic probe to induce release of a reporter (or another cargo) from the synthetic probe. In some embodiments, the cleavable linker is a caged or masked linker as described below herein but also a pH or hypoxic released tether of the reporter to a dPEG or other. In this exemplary embodiment, the cleavable linker comprises a cage or mask that hinders or blocks cleavage of the cleavable linker until the cage or mask is removed. In this exemplary embodiment, if off-target binding occurs then the reporter remains tethered to the synthetic probe even after the cleaving agent is introduced. The reporter can also be “bi-bound” to the synthetic probe such that it is connected to two separate cleavable linkers. In such an exemplary embodiment, when exposed to a tumormicroenvironment for example, the cleavable linkers connected to the reporter is cleaved, making the other cleavable linker susceptible to the cleaving agent. This can be accomplished by protease cleavage of one of the cleavable linkers to the reporter, for example. Another way is to quench the reporter, rendering it undetectable, unless the reporter is exposed to an environment such as proteases, an hypoxic environment, or pH altered environment. Thus, bi-functional action enables detection of the reporter. In an exemplary embodiment, one can double-bind the reporter with a bi-functional cleavable linker having a first chemical linkage reactive to a trigger release mechanism and a second chemical linkage reactive to a microenvironmental-conditional release. The mechanisms of the microenvironmental-conditional release could be pH, proteases, hypoxic conditions, etc. General mechanisms would thus include a reporter tethered by two cleavable linkers, a masked cleavable linker, the bifunctional linker and the quenched reporter.

[0048] As partly discussed above, in some embodiments, certain domains of the synthetic probe are masked (also referred to herein as “caged”), wherein the mask (or “cage”) is a chemical or structural element that reversibly inhibits, blocks, or reduces the reactivity, recognition, or function of the domain. The domains of the synthetic probe include the binding domain, the cleavable linker, and the reporter. While masked, the domain is essentially inert, and thus the domain only functions in response to a chemical and / or a physiological stimuli that unmasks the domain. When unmasked (or “uncaged”), the domain then performs its function or activity. Suitably, any part of the synthetic probe can be masked such as, for example, a binding domain, a cleavable linker, a spacer, a reporter, a cleaving agent, and combinations thereof.

[0049] In one exemplary embodiment, cleavage of the cleavable linker of the synthetic probe is activated endogenously. Suitable endogenous activation mechanisms include an enzyme, a specific pH, hypoxic conditions, and combinations thereof.

[0050] Suitable cleaving agent groups include azides, alkynes, tetrazines, cyclopropenes, and trans-cyclooctenes, which participate in click reactions such as azide-alkyne cycloaddition and tetrazine-trans-cyclooctene ligation.BINDING DOMAIN

[0051] The binding domain of the synthetic probe specifically interacts with one or more specific molecule(s) of the target (referred to herein as a “target molecule”). Any of the binding domains are engineered so as to not perturb the immune system.

[0052] Suitable binding domains include a wide range of molecular entities. These encompass proteins and peptides, such as general proteins, specific peptides (e.g., cell penetrating peptides, octreotide and its derivatives, VIP, MSH, LHRH, chemotactic peptides, bombesin, elastin), and peptide mimetics. Another category of binding domains includes antibodies, their derivatives, and fragments, such as polyclonal antibodies, monoclonal antibodies, multispecific antibodies (e g., bispecific and tri-specific antibodies or their fragments / derivatives), chimeric antibodies, murine antibodies, camelid antibodies, humanized antibodies, and fully human antibodies. Antibody fragments include heavy chain antibody fragments (e.g., variable domain of a heavy chain, VHH), light chain antibody fragments, Fab, F(ab’)2, Fab’, Fv, rlgG, single-chain variable fragment (scFv), nanobodies (single domain antibodies; “sdAb”), diabodies, linear antibodies, fragments produced by a Fab expression library, anti- idiotypic (anti-Id) antibodies, CDRs (Complementarity Determining Regions), and ECDs (extracellular domains), as well as antibody (fragment) fusions. Nucleic acid- based domains include oligonucleotides and aptamers (e.g., DNA aptamers, RNA aptamers). Small molecules are also suitable as binding domains, along with drugs, chemotherapeutic agents, receptor agonists and antagonists, cytokines, hormones, steroids, and toxins. Lipids and lipid derivatives form another class suitable for use as binding domains, including phospholipids, fatty acids, triglycerides, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides, cholesterol, sterol moieties, and cationic lipids, as well as structures like liposomes. Carbohydrates, such as monosaccharides and polysaccharides, can also serve as binding domains. Various polymers are also included as suitable binding domains, for instance, poly(ethylene) glycol, spermine and its derivatives or analogues, poly-lysine and its derivatives or analogues, polyethyleneimine, diethylaminoethyl (DEAE)-dextran, and general cationic polymers. Larger biological entities like viruses, whole cells, and phage can also suitably function as binding domains. Furthermore, molecules characterized bycertain physicochemical properties, such as cationic molecules, hydrophobic molecules, and amphiphilic molecules, are suitable as binding domains. Binding domains can be created by any methods known to in art to specifically bind any known epitope(s) of an antigen. For example, peptide and protein binding domains (e.g., antibodies and antibody domains) can be produced using recombinant protein expression methods. Peptides can also be chemically synthesized. Nucleic acid binding domains can be synthesized and produced by amplification methods. Full-length antibodies can be chemically treated using known methods such as protease treatments to prepare antibody molecules such as Fab molecules.

[0053] F(ab')2, antibody fragments contain two antigen-binding regions joined at the hinge through disulfides and is void of most, but not all, of the Fc region. Fab' (55,000 daltons) fragments can be formed by the reduction of F(ab')2 fragments and thus may contain a small portion of Fc, contain a free sulfhydryl group that may be alkylated or utilized in conjugation with an enzyme, toxin and other proteins of interest. Fab is a monovalent fragment that is produced from IgG and IgM, and includes the VH, CHI and VL, CL regions, linked by an intramolecular disulfide bond. Fv is produced from IgG and IgM and contains a complete antigen-binding site. Fv VH and VL chains are held together by non-covalent interactions. "rlgG" refers to reduced IgG or half-IgG that is produced by selectively reducing just the hinge-region disulfide bonds. Half-IgG having exposed hinge-region sulfhydryl groups can be targeted for conjugation such as for antibody immobilization and enzyme labeling. Antibodies and antibody fragments can also be produced by recombinant protein expression technologies.

[0054] Suitable binding domains also include peptides. Suitable peptide binding domains include LHRH receptor targeting peptides, EC-1 peptide, RGD peptides, HER2-targeting peptides, PSMA targeting peptides, somatostatin-targeting peptides, bombesin. Suitable binding domains include the ligands of receptors or a part thereof that still binds to the receptor, e.g. a receptor binding peptide in the case of receptor binding protein ligands. Other examples of binding domains of protein nature include insulin, transferrin, fibrinogen-gamma fragment, thrombospondin, claudin, apolipoprotein E, affibody molecules such as for example ABY-025, Ankyrin repeat proteins, ankyrm-like repeat proteins, interferons, e.g. alpha, beta, and gammainterferon, interleukins, lymphokines, colony stimulating factors and protein growth factor, such as tumor growth factor, e.g. alpha, beta tumor growth factor, platelet- derived growth factor (PDGF), uPAR targeting protein, apolipoprotein, LDL, annexin V, endostatin, and angiostatin.

[0055] Particularly suitable antibodies for use as binding domains and / or to obtain their fragments include 3F8, 8H9. Abagovomab. Abciximab, Abituzumab. Abrezekimab, Abrilumab, Actoxumab, Adalimumab, Adecatumumab (MT-201, HD69), Atidortoxumab, Aducanumab, Afasevikumab, Afelimomab, Alacizumab pegol, Alefacept, Alemtuzumab, Alirocumab, Altumomab pentetate, Amatuximab, AMG-531, Anatumomab mafenatox, Andecaliximab. Anetumab ravtansine. Anifrolumab, Anrukinzumab (IMA- 638), Apolizumab, Aprutumab ixadotin, Arcitumomab, Ascrinvacumab, Aselizumab, Atacicept (TACI-Ig), Atezolizumab, Atinumab, Atorolimumab, Aurograb. Avelumab, Azintuxizumab vedotin, Babineuzumab (AAB-001). Bapineuzumab, Basiliximab, Bavituximab. bCD-100, Bectumomab, Begelomab, Belantamab mafodotin, Belatacept (LEA-29Y, BMS 224818), Belimumab, Bemarituzumab, Benralizumab, Berlimatoxumab, Bermekimab, Bersanlimab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bimekizumab, Birtamimab, Bivatuzumab mertansine, Bleselumab, Blinatumomab, Blontuvetmab. Blosozumab, Bococizumab. Brazikumab. Brentuximab vedotin, Briakinumab (ABT-874), Brodalumab, Brolucizumab, Brontictuzumab, Burosumab, Cabiralizumab, Camidanlumab tesirine, Camrelizumab, Canakinumab (ACZ-885), Cantuzumab mertansine, Cantuzumab ravtansine, Caplacizumab, Capromab pendetide, Carlumab, Carotuximab, Catumaxomab, CBR96-doxorubicin immunoconjugate, Cedelizumab, Cemiplimab, Cergutuzumab amunaleukin, Certolizumab pegol, Cetrelimab, Cetuximab, Cibisatamab, Cirmtuzumab, Citatuzumab bogatox, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumab tetraxetan, Codrituzumab, Cofetuzumab pelidotin, Coltuximab ravtansine, Conatumumab, Concizumab, Cosfroviximab, Crenezumab, Crizanlizumab, Crotedumab, CR6261 , Cusatuzumab, Dacetuzumab, Daclizumab, Dalotuzumab, Dapirolizumab pegol, Daratumumab, Dectrekumab, Demcizumab, Denintuzumab mafodotin, Denosumab, Depatuxizumab mafodotin, Derlotuximab biotin. Detumomab, Dezamizumab,Dinutuximab (CH- 14. 18), Diridavumab, Domagrozumab, Dorlimomab aritox, Dostarlimab, Drozitumab, DS-8201, Duligotuzumab, Dupilumab. Durvalumab, Dusigitumab, Duvortuxizumab, Ecromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Eldelumab, Elezanumab, Elgemtumab, Elotuzumab, Elsilimomab, Emactuzumab, Emapalumab, Emibetuzumab, Emicizumab, Enapotamab vedotin, Enavatuzumab, Enfortumab vedotin, Enlimomab pegol, Enoblituzumab, Enokizumab, Enoticumab, Ensituximab, Epitumomab cituxetan. Epratuzumab, Eptinezumab, Erenumab, Erlizumab, Ertumaxomab, Etanercept, Etaracizumab, Etigilimab, Etrolizumab, Evinacumab, Evolocumab, Exbivirumab, Fanolesomab, Faralimomab, Faricimab, Farletuzumab, Fasinumab, FBTA05, Felvizumab, Fezakinumab, Fibatuzumab, Ficlatuzumab, Figitumumab, Firivumab, Flanvotumab, Fletikumab, Flotetuzumab, Fontolizumab, Foralumab, Foravirumab, Fremanezumab, Fresolimumab, Frovocimab, Frunevetmab, Fulranumab, Futuximab, Galcanezumab. Galiximab, Gancotamab, Ganitumab, Gantenerumab, Gatipotuzumab, Gavilimomab, Gedivumab, Gemtuzumab ozogamicin, Gevokizumab, Gilvetmab, Gimsilumab. Girentuximab, Glembatumumab vedotin. Golimumab (CNTO-148). Gomiliximab, Gosuranemab, Guselkumab, lanalumab, Ibalizumab, IBI308, Ibritumomab tiuxetan, Icrucumab, Idarucizumab, IDEC-114 (PRIMATIZED® anti- CD80) Ifabotuzumab, IGN101 (anti-Epithelial cell adhesion molecule (EpCAM)), Igovomab, lladatuzumab vedotin, IMAB362, Imalumab, Imaprelimab, Imciromab. Imgatuzumab, Inclacumab, Indatuximab ravtansine, Indusatumab vedotin, Inebilizumab, Infliximab, Intetumumab, Inolimomab, Inotuzumab ozogamicin, Ipilimumab (MDX-010), lomab-B, Iratumumab, Isatuximab, Iscalimab. Istiratumab, Itolizumab. Ixekizumab, Keliximab, Labetuzumab, Lacnotuzumab, Ladiratuzumab vedotin, Lampalizumab, Lanadelumab, Landogrozumab, Laprituximab emtansinee, Llarcaviximab, Lebrikizumab, Lemalesomab, Lendalizumab, Lenvervimab, Lenzilumab, Lerdelimumab, Leronlimab, Lesofavumab, Letolizumab, Lexatumumab, Libivirumab, Lifastuzumab vedotin, Ligelizumab, Loncastuximab tesirine, Losatuxizumab vedotin. Lilotomab satetraxetan, Lintuzumab, Lirilumab. Lodelcizumab, Lokivetmab, Lorvotuzumab mertansine, Lucatumumab, Lulizumab pegol, Lumiliximab, Lumretuzumab, Lupartumab amadotin, Lutikizumab, Mapatumumab, Margetuximab, Marstacimab, Maslimomab, Mavrilimumab,Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mirikizumab, Mirvetuximab soravtansine, Mitumomab. Modotuximab, Mogamulizumab, Monalizumab, Morolimumab, Mosunetuzumab, Motavizumab (MEDI-524), Moxetumomab pasudotox, Muromonab-CD3, Nacolomab tafenatox, Namilumab, Naptumomab estafenatox, Naratuximab emtansinee, Namatumab, Natalizumab. Navicixizumab, Navivumab, Naxitamab, Nebacumab, Necitumumab, Nemolizumab, NEOD001 , Nerelimomab, Nesvacumab. Netakimab, Nimotuzumab. Nirsevimab, Nivolumab, Nofetumomab merpentan, Obiltoxaximab, Obinutuzumab, Ocaratuzumab (LY2469298), Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Oleclumab. Olendalizumab. Olokizumab, Omalizumab, Omburtamab, OMS721 , Onartuzumab, Ontuxizumab, Onvatilimab, Opicinumab. Oportuzumab monatox, Oregovomab, Orticumab, Otelixizumab (ChAglyCD3, TRX4), Otilimab, Otlertuzumab, Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Pamrevlumab, Panitumumab, Pankomab, Panobacumab, Parsatuzumab, Pascolizumab, Pasotuxizumab, Pateclizumab. Patritumab, PDR001 . Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab. Pexelizumab, Pidilizumab, Pinatuzumab vedotin. Pintumomab, Placulumab, Plozalizumab, Pogalizumab, Polatuzumab vedotin, Ponezumab, Porgaviximab, Prasinezumab, Pregovomab, Prezalizumab, Priliximab, Pritoxaximab, Pritumumab, PRO 140, Quilizumab, Racotumomab, Radretumab, Rafivirumab. Ralpancizumab, Ramucirumab. Ranevetmab, Ranibizumab. Raxibacumab, Ravagalimab, Ravulizumab, Refanezumab, Regavirumab, Relatlimab, Remtolumab, Reslizumab, Rilotumumab, Rinucumab, Risankizumab, Rituximab, Rivabazumab pegol, Robatumumab, Rmab, Roledumab, Romilkimab, Romiplostim, Romosozumab, Rontalizumab, Rosmantuzumab, Rovalpituzumab tesirine, Rovelizumab, Rozanolixizumab, Ruplizumab, SA237, Sacituzumab govitecan, Samalizumab, Samrotamab vedotin, Sarilumab, Satralizumab, Satumomab pendetide, Secukinumab, Selicrelumab, Seribantumab, Setoxaximab, Setrusumab, Sevirumab, Sibrotuzumab, SGN-CD19A, SHP647, Sifalimumab, Siltuximab, Simtuzumab, Siplizumab, Sirtratumab vedotin. Sirukumab, Sofituzumab vedotin. Solanezumab. Solitomab, Sonepcizumab, Sontuzumab, Spartalizumab, Stamulumab, Sulesomab, Suptavumab, Sutimlimab, Suvizumab, Suvratoxumab, Tabalumab, Tacatuzumab tetraxetan, Tadocizumab, Talacotuzumab, Talizumab, Tamtuvetmab, Tanezumab,Taplitumomab paptox, Tarextumab, Tavolimab, Tefibazumab, Telimomab aritox, Telisotuzumab vedotin, Tenatumomab, Teneliximab, Teplizumab (Ala-Ala), Tepoditamab, Teprotumumab, Tesidolumab, Tetulomab, Tezepelumab, TGN1412, Tibulizumab, Ticilimumab, Tildrakizumab, Tigatuzumab, Timigutuzumab, Timolumab, Tiragotumab, Tislelizumab, Tisotumab vedotin, TNX-650, Tocilizumab, Tomuzotuximab, Toralizumab, Tosatoxumab, Tositumomab, Tositumomob-1131, Tovetumab, Tralokinumab, Trastuzumab, Trastuzumab emtansine (herceptin). TRBS07, Tregalizumab, Tremelimumab (CP-675 206), Trevogrumab, Tucotuzumab celmoleukin, Tuvirumab, Ublituximab, Ulocuplumab, Urelumab, Urtoxazumab, Ustekinumab (CNTO-1275), Utomilumab, Vadastuximab talirine, Vanalimab, Vandortuzumab vedotin, Vantictumab. Vanucizumab, Vapaliximab. Varisacumab, Varlilumab, Vatelizumab, Vedolizumab, Veltuzumab, Vepalimomab, Vesencumab, Visilizumab, Vobarilizumab, Volociximab, Vonlerolizumab, Vopratelimab, Vorsetuzumab mafodotin, Votumumab, Vunakizumab, WX-G250 (anti- carboxy anhydrase IX), Xentuzumab, XMAB-5574, Zalutumumab (HuMax-EGFR), Zanolimumab, Zatuximab, Zenocutuzumab, Ziralimumab Zolbetuximab (1MAB362. Claudiximab), Zolimomab aritox, and combinations thereof. It is within the knowledge and capability of one of ordinary skill in the art to select an appropriate antibody for targeting a target molecule and prepare a binding domain to be used in the manufacture of the synthetic probe of the present disclosure.

[0056] Particularly suitable binding domains of the synthetic probe is a VHH, a Fab, and an scFv of any one of the antibodies.

[0057] In some embodiments, the binding domain (or binding component) can release from the target and be excreted or removed by the subject’s body. In other embodiments, the binding domain (or binding component) can remain attached to the target. In embodiments where the binding domain remains attached to the target, it may be used as a binding site for a therapeutic or an additional diagnostic molecule as described herein.

[0058] In an exemplary embodiment wherein the synthetic probe is designed to detect target molecules known to be associated with cancers, suitablebinding domains include commercially available cancer-specific antibodies (and fragments thereof as described in the present disclosure). Combinations of cancer- specific antibodies that bind the same cancer, as well as combinations of cancer-specific antibodies that bind different cancer types are also specifically suitable.

[0059] In exemplary embodiments wherein the synthetic probe is designed to detect target molecules known to be associated with viruses, bacteria, fungi, and classes of viruses, bacteria and fungi, suitable binding domains include commercially available antibodies (and fragments thereof as described in the present disclosure) that specifically bind the viruses, bacteria (e.g., gram positive and gram negative), and fungi, or classes thereof. Combinations of specific antibodies that bind the same viruses, bacteria, fungi, as well as combinations of specific antibodies that bind different viruses, bacteria, and fungi types are also suitable for use as the binding domain of the synthetic probes.CLEAVABLE LINKER

[0060] The cleavable linker (linkage component, also referred to herein as “degradable linker(s)”) links (connects) the binding domain to the reporter via a covalent linkage or by a non-covalent linkage. Cleavable linkers are designed to fragment in response to specific chemical molecules (including direct and indirect, and exogenously administered or endogenously related molecules) and / or physiological stimuli (pH, redox conditions, or enzymes) once the synthetic probe construct reaches its target site and any unbound synthetic probe construct has cleared from the subject. As used herein, a “cleavable linker” is a chemical moiety designed to connect one or more components of a synthetic biological construct and selectively fragment in response to one or more specific physiological or externally applied stimuli, thereby releasing, activating, or altering the function of at least one connected component. Such linkers may react with particular molecules, proteins, or enzymes or classes of molecules, proteins or enzymes and may respond to physiological conditions including changes in pH, redox potential, enzy matic activity, or externally applied stimuli such as photochemical activation, and may function through direct bond cleavage or through triggered fragmentation cascades.

[0061] In various forms, the cleavable linker includes a first reagent that, when triggered, induces fragmentation of the cleavable linker. The first reagent can, as described herein, be triggered by physiological conditions including changes in pH, redox potential, enzymatic activity, or externally applied stimuli such as photochemical activation reaction. The first reagent can also be triggered by reacting with a separate structure that is or comprises a second reagent. The separate structure includes particular molecules, proteins, or enzymes or classes of molecules, proteins or enzymes that are or that comprise the second reagent. The first reagent and the second reagent are structured such that they correspond to and can react with one another. For example, a cleavable linker can comprise tetrazine, wherein administration of a quantity of trans cyclooctene (TCO) cleaving agent results in ("triggers") fragmentation of the cleavable linker via a reaction between the tetrazine and the TCO. In this example, the tetrazine is the first reagent, and the TCO is both the separate structure and the second reagent (and serves as the “cleaving agent'’). In various alternative forms of this example, including some embodiments discussed above, the separate structure can comprise a reporter that is spontaneously eliminated after the reaction of the first reagent and the second reagent. In such an alternative embodiment, the separate structure is not the second reagent per se, but the separate structure comprises the second reagent and the reporter. A particularly suitable cleavable linker / cleaving agent pair utilizes iTCO, a click-cleavable linker that unlocks remarkably efficient release across all tetrazine types.

[0062] The cleavable linker may be masked (also referred to herein as “caged”), wherein the mask (or “cage”) is a chemical or structural element that reversibly inhibits the reactivity, recognition, or function of the cleavable linker. While masked, the cleavable linker is essentially inert, and thus the cleavable linker will only fragment in response to chemical and / or physiological stimuli when unmasked (or “uncaged”).

[0063] Many tumors overexpress unique enzymes that can serve as biocatalytic triggers to unmask a caged tetrazine. In various embodiments, proteases such as matrix metalloproteinases (MMPs) and cathepsins, glycosidases, and other tumor-enriched enzymes can cleave linkers attached to the tetrazine. This approachleverages tumor biomarkers such as 0-galactosidase and 0-glucuronidase that are often elevated in tumor or necrotic tissue.

[0064] A suitable cleavable linker includes a galactose-caged tetrazine that remains inert until encountering senescence-associated P-galactosidase. The P-gal enzyme cleaves the galactoside, releasing a free tetrazine, which then can rapidly react with a trans-cyclooctene (TCO)-conjugate to click-release a cargo. Similarly, enzyme- initiated tetrazine unmasking is possible with other linkers such as self-immolative linkers bearing enzyme-cleavable groups (peptides, phosphates, esters) connected to stable dihydrotetrazines. Upon enzymatic cleavage (by, for instance, cathepsin B or alkaline phosphatase), the linker is unmasked, exposing a reactive tetrazine. These enzyme-triggered approaches exploit a gated activation model, wherein the cleavable linker is only unmasked in an appropriate physiological environment that comprises an unmasking agent. The unmasking agent is a first trigger of two triggers in the gated activation model. Thus, in the present example, the tetrazine will be unmasked in the presence of the first trigger, e.g., a specific tumor enzyme, allowing for the subsequent activation by a second trigger (e.g., exogenously administered TCO). Enzymatic triggers are highly modular. For example, peptide linkers cleavable by MMP-2 / 9 (common in tumor stroma) or a 0-glucuronide linker cleaved by extracellular 0- glucuronidase can be incorporated to achieve tumor-specific tetrazine release. Additionally, a self-immolating release can be further included with this design, wherein the reporter first is uncaged but remains connected to the probe for a period of time to allow for various perfusions followed by spontaneous release later through endogenous circumstances.

[0065] Specific cleavable linker constructions can, when fragmented, release particular chemical functional groups. In various synthetic probes of the present description, the particular chemical functional groups released by cleavable linker constructions can be conjugated to the cargo, such that release of the functional group concomitantly releases the cargo. In other various forms, the particular chemical functional groups released by the cleavable linker constructions can be conjugated to the binding domain; in such embodiments, the cargo can remain bound to the cleavable linker, but not to the binding domain. In various forms, the cargo is the reporter. Inother various forms, the cargo is a drug. Suitable examples include beta-keto ester linkers that are used to release isotopically labeled ketones like D6-Acetone; glucuronide conjugates stabilize isotopically labeled alcohols until triggered; percarbonate precursors release ethers like methyl tert-butyl ether (MTBE); and dimethyl sulfone precursors release dimethyl sulfide under reducing conditions.

[0066] Suitable cleavable linkers include click to release linkers, self-immolative linkers, disulfide-based linkers, hydrazone linkers, enzyme-cleavable peptide linkers, hypoxia-activated linkers, pH-responsive linkers, and bi-functional linkers. Val-Cit linker that can be cleaved in the body by means of Cathepsin B enzyme. Click-to-release linkers rely on click chemistry reactions between the first reagent and the second reagent to induce fragmentation of the click-to-release linker. Suitable click- to-release linkers are named and referred to herein by the first and second reagents pertinent to each system, and include, without limitation, alkene-tetrazine linkers, isonitrile tetrazine linkers, azide alkyne linkers, azide phosphine linkers (or Staudinger linkers), thiol-maleimide linkers. The click-to-release linker mechanism enables prodrug designs that activate specifically when the click reaction occurs.

[0067] The alkene-tetrazine linker is a system in which the first reagent is a tetrazine and the second reagent is an alkene, or vice versa. The reaction between the first reagent and the second reagent in the alkene-tetrazine linker is a bioorthogonal ligation in which an electron-rich or strained alkene (e.g., TCO) undergoes an inverse- electron-demand Diels-Alder (IEDDA) cycloaddition with a tetrazine. This cycloaddition triggers or enables an elimination cascade resulting in formation of a dihydropyridazine adduct and release of the conjugated cargo. A variety of suitable electron rich and / or strained alkenes are known to one of ordinary skill and are within the scope of the present description including TCO with or without allylic substitution (such as TCO-carbamate and TCO-carbonate) and its isomers, cyclopropene, vinyl ethers, silyl-substituted alkenes, norborenes, and bicyclo[6.1.0]non-4-ene. In various forms, the alkene is substituted at an allylic position to control elimination of the cargo, such as in the example of TCO-carbamate. In various forms, the alkene is substituted at a vinylic position to control elimination of the cargo, such as in the example of vinyl ethers.

[0068] The isonitrile tetrazine linker is a system in which the first reagent is an isonitrile and the second reagent is a tetrazine, or vice versa. The reaction between the first reagent and the second reagent in the isonitrile-tetrazine linker is a bioorthogonal ligation in which an isonitrile undergoes an inverse-electron-demand Diels-Alder (lEDDA) cycloaddition with a tetrazine. This cycloaddition triggers or enables an elimination cascade resulting in formation of a pyrazole or dihydropyridazine-derived adduct and release of the conjugated cargo. A variety of suitable isonitrile-containing reagents are known to one of ordinary skill and are within the scope of the present description including primary, secondary , or substituted alkyl isonitriles, as well as sterically encumbered or electronically tuned variants that modulate reactivity or release kinetics. In various forms, the isonitrile is substituted at a position adjacent to the isonitrile group to promote elimination of the cargo, such as in the example of an isonitrile-carbamate construct.

[0069] The azide-alkyne linker is a system in which the first reagent is an azide and the second reagent is an alkyne, or vice versa. The reaction between the first reagent and the second reagent in the azide-alky ne linker is a bioorthogonal ligation in which the azide and the alkyne undergo a cycloaddition to form a stable triazole adduct. This reaction may proceed by any means known to one of ordinary skill in the art including via copper(I) catalysis, as in the Cu-catalyzed azide-alkyne cycloaddition (CuAAC), or under strain-promoted conditions using a strained alky ne, as in the strain- promoted azide-alkyne cycloaddition (SPAAC); or through catalyst-free, proximity - induced cycloaddition where specific functionalities on the azide and alkyne partners (such as an o-azidoarylboronic acid and an alkyne bearing a diol) reversibly form a complex (e.g., a boronate ester) that brings the azide and alkyne moieties into close proximity, thereby triggering the intramolecular cycloaddition (termed BAAC in some literature). In various forms, certain engineered linker designs can incorporate one or more cleavable or degradable handles adjacent to the triazole or embedded within the conjugated moiety to enable conditional release of the cargo under specific chemical or physiological conditions. A variety of suitable azides and alkynes are known to one of ordinary skill and are within the scope of the present description including terminal alkynes, strained cyclic alkynes such as bicyclononynes and dibenzocyclooctynes,azides functionalized with boronic acids (e.g., o-azidoarylboronic acids), alkynes functionalized with diols (e.g., N-propargyldiethanolamine derivatives), and electron- deficient or electron-nch azides.

[0070] The azi de-phosphine linker is a system in which the first reagent is an azide and the second reagent is a phosphine, or vice versa. The reaction between the first reagent and the second reagent in the azide-phosphine linker is a bioorthogonal ligation in which a phosphine reacts with an azide to form an iminophosphorane intermediate that subsequently rearranges to form an amide bond. In various forms, this ligation can be engineered such that the ligation event initiates an elimination cascade that results in release of the cargo. In various forms, resulting byproducts can include a phosphine oxide and a stable amide. A variety of suitable azide- and phosphine- containing constructs are known to one of ordinary skill and are within the scope of the present description including substituted aryl and alkyl phosphines, phosphinoesters, and azide-containing constructs bearing carbamate, ester, or carbonate moieties.

[0071] The thiol-maleimide linker is a system in which the first reagent is a thiol and the second reagent is a maleimide, or vice versa. The reaction between the first reagent and the second reagent in the thiol-maleimide linker is a Michael-type addition in which the nucleophilic thiol undergoes conjugate addition to the electron- deficient double bond of the maleimide to form a thioether adduct. In various forms, the resulting thioether linkage can be engineered to undergo retro-Michael elimination or other reactions, thereby enabling release of the conjugated cargo. A variety of thiol- and maleimide-containing constructs are known to one of ordinary skill and are within the scope of the present description including cysteine-terminated peptides, thiolated polymers, N-substituted maleimides, and maleimide derivatives bearing cleavable linkers.

[0072] In various forms, the linker is a self-immolative linker. In the self- immolative linker system, the linker undergoes a fragmentation cascade following a trigger event. This triggering event can be a specific chemical reaction (such as enzymatic cleavage, reduction / oxidation. or a pH shift) or a biomolecular and / or chemical interaction such as the reaction between the first reagent and the secondreagent. Once triggered, the linker conformationally or electronically rearranges in such a way that it becomes unstable and spontaneously fragments to release the cargo, such as through an elimination reaction (e.g., 1,4-, 1,6-, or 1,8-elimination) or cyclization- elimination. For example, 3-isocyanopropyl carbamate conjugates can undergo [4+2] inverse-electron Diels-Alder (IEDDA) reactions that destabilize the carbamate bond, spontaneously leading to elimination-induced cargo release. The self-immolative linker is thus self-immolative in the sense that once appropriately triggered, the self- immolative linker undergoes a reaction to release the conjugated cargo.

[0073] Self-immolative linkers are triggered by, in various non-limiting exemplary forms, cathepsin B, plasmin, [3-glucuronidase, changes in pH, changes in glutathione concentration, changes in peroxide levels, and changes in the concentration of other chemical and biological moieties known to one of ordinary skill in the art.

[0074] In some systems, a click-to-release linker incorporates a self- immolative linker mechanism. For example, if the click reaction between the first and second reagent directly leads to a species that is unstable and spontaneously fragments (such as certain IEDDA reactions destabilizing a carbamate bond), or if it unmasks a group that initiates a subsequent spontaneous elimination cascade, then it functions as a self-immolative system. However, if the click reaction merely forms a new stable linkage that requires a separate, non-spontaneous stimulus or reaction for cargo release, it would not typically be classified as self-immolative based on the click event alone.

[0075] In some systems, a multiplexed assay includes a plurality of linkers, including click-to-release linkers, self-immolative linkers, and externally triggered linkers. Externally triggered linkers do not spontaneously undergo a fragmentation cascade in response to ordinary in vivo microenvironments or tumor microenvironments, but instead are triggered to undergo a fragmentation cascade in response to a chemical or biological trigger that is deployed into the body of a subject. Although it is possible for a click-to-release linker to be designed and constructed to encounter a corresponding trigger in vivo, the phrase “click-to-release’' is broadly understood to typically refer to linkers that are triggered via administration of a triggering agent.

[0076] The p-aminobenzyl alcohol (PABA) based linker is an exemplary self-immolative system. This system typically features a p-substituted benzyl scaffold with two components: a latent electron-donating group (commonly an amine masked as a nitro group, carbamate, or other enzymatically cleavable moiety) positioned para to a benzylic carbon, and the cargo, which is covalently attached to the benzylic carbon, via linkages such as carbonates (for alcohol / phenol release) or carbamates (for amine release), although direct linkages are also possible. The triggering reaction involves the specific unmasking of the electron-donating group (e.g., reduction of a nitro group to an amine, often under hypoxic conditions, or enzy matic cleavage of a masking group). This unmasking initiates a spontaneous 1,6-elimination reaction, driven by electron donation through the conjugated π-system of the aromatic ring. This cascade leads to the formation of a quinone methide or related species and cleavage of the bond at the benzy lic position, hence the spontaneous release of the cargo. PABA-based constructs include the p-aminobenzyloxycarbonyl (PABC) variant, constructs with different trigger mechanisms, all of whhich are considered within the scope of self-immolative linker technologies. As a non-limiting example of the PABA system unmasking and spontaneously immolating, the PABA system can be masked by a beta-glucoronide until extracellular β-glucuronidase enzymes present in tumor environs cleave the sugar and generate a phenolic benzyl carbamate which spontaneously self-immolates. A similar example is evofosfamide.

[0077] Another self-immolative linker example is the nitropyrrole-based linker, exemplified by derivatives of (5-nitro-2-pyrrolyl)methanol (NPYM). which works by taking advantage of the aza-quinone methide reactivity. NPYM uses a 5- nitropyrrole scaffold comprising a 5-nitro group and the cargo, which is typically directly covalently attached via a heteroatom (S, O, or N), to a methylene carbon in the pyrrole ring. This system is triggered by reduction of the 5-nitro group, such as by enzymatic activity. This results in unmasking of the electron-donating amino group and initiates a spontaneous 1,6-elimination cascade, leading to the formation of an aza- quinone methide species and the cleavage of the bond to the cargo, thereby spontaneously releasing the cargo. This system can directly release cargo with higher pKa values, like thiols, amines, sulfonamides, and amides Variations of the NPYMsystem, including modifications at the pyrrole N1 position, are considered within the scope of the present description.

[0078] In the photochemical click-to-release system, light sensitivity is combined with click chemistry wherein linkers maintain integrity until exposure to photons of a specific frequency triggers a reaction that compromises the linker structure, allowing for spatiotemporal control of cargo release.

[0079] In the redox-triggered cleavage system, oxidation / reduction potential differences between biological compartments are exploited to control the release of cargo. The bloodstream is relatively oxidizing, whereas the inside of cells (especially the cytosol of tumor cells with high glutathione levels) is reducing. Thus, a linker comprising a redox-sensitive construct can be stable in blood but cleaved once the synthetic probe construct (or a portion of the synthetic probe construct) is internalized by the target cell.

[0080] An exemplary redox-responsive self-immolative linker utilizes a disulfide bond, which works by taking advantage of the differential redox potential between the oxidizing extracellular environment and the highly reducing intracellular environment rich in glutathione (GSH). This system typically comprises a disulfide linkage (e.g., within structures like SPDP or SPDB linkers), often connected via a self- immolative spacer (such as a thiobenzyl carbamate) to the cargo or reporter. This system is triggered by the reduction of the disulfide bond by intracellular reducing agents like GSH, primarily occurring after targeted delivery and internalization of the construct into the tumor cell cytosol. This results in the generation of a free thiol or thiolate group, which subsequently initiates a spontaneous self-immolative fragmentation cascade (e.g., 1,6-elimination triggered by the thiolate), leading to the cleavage of the bond to the cargo and thereby spontaneously releasing the free cargo within the cell. The specificity of this system relies on efficient tumor-targeted delivery to ensure internalization into the correct cells, as the reducing environment is also present in normal cells. Variations of disulfide-based linkers, including different disulfide stabilities (e.g.. hindered disulfides), various thiol-triggered self-immolativespacers, and conjugation to diverse targeting moieties, are considered within the scope of the present disclosure.

[0081] In the enzymatic activation system, enzyme recognition moieties are incorporated in the linker that, when cleaved, enable subsequent click reactions or cargo release — (for example, a linker that includes a peptide can be cleaved by cathepsin B or other proteases overexpressed in tumors - this exemplifies that the enzyme that cleaves the linker can advantageously be a disease-related enzyme). Protease-cleavable linkers are peptide-based linkers that are designed to be recognized and cleaved by specific proteases such as matrix metalloproteinases enabling the controlled timing of clearance by exposing hepatic targeting ligands only after the construct reaches its intended target site. Trifluoroethylamine is a linker that remains stable w hen bound as an amide until enzymatic cleavage.

[0082] In the pH-sensitive click linker system, pH differences between blood (neutral pH ~7.4) and certain compartments like endosomes (acid pH —5.6) and tumor microenvironments (acid pH ~6.5 in interstitium) are exploited. These linkers (e.g., hydrazone, cis-aconityl, or acetal bonds) remain stable at physiological pH but hydrolyze in mild acidity, releasing the cargo in acidified environments. Acid-labile functional groups like acetals / ketals, hydrazones, and imines remain intact at physiological pH but hydrolyze in acidic microenvironments. For example, a tetrazine can be masked by linking it via a benzy lidene acetal or hydrazone to a carrier. In the acidic tumor interstitium or upon endocytic uptake, the bond cleaves to free the tetrazine. These linkers have been widely used in drug delivery (e.g. hydrazone-linked doxorubicin in acidic endosomes) and are expected to remain intact in circulation (pH 7.4) but rapidly hydrolyze at pH 5-6.9. Tumor acidosis thus acts as the first gate: only in the tumor (or intracellular vesicles) will the tetrazine be liberated, while in healthy tissue (neutral pH) it stays masked. One advantage is that pH-triggered unmasking does not require a specific enzyme - it exploits a universal tumor trait, though care must be taken that the pH difference is sufficient for selective cleavage. Designing the acid- labile group’s pH threshold to ~6-6.5 ensures it responds to tumor acidity but not to blood pH.

[0083] The hypoxia-activated linker system uses regions of hypoxia (low oxygen tension) in solid tumors that exist due to poor perfusion. Hypoxia is harnessed via chemical groups that undergo bioreduction in low oxygen to activate the tetrazine. One such chemical group is 2-nitroimidazole moiety that is stable under normoxia but is enzymatically reduced in hypoxia to a radical anion and fragments to release a cargo. A tetrazine can be masked with a nitroaromatic or azo linkage that is cleaved only under hypoxic conditions. For instance, a 4-nitrobenzyl-carbamate linker on the tetrazine would be stable in normal tissues (the nitro group prevents release), but in hypoxic tumor regions nitroreductase enzymes convert the nitro to an aniline, causing spontaneous self-immolation of the linker and freeing the tetrazine. This two-step trigger (enzymatic reduction and spontaneous cleavage) acts as an oxygen-sensitive gate. The key benefit is extreme specificity - normal oxygenated tissues quench the activation (the radical intermediate is reoxidized by oxygen, preventing release), whereas in oxygen-poor tumor environments, the reaction proceeds to unmask the tetrazine. Hypoxia-activated strategies thus confine tetrazine activation to necrotic or poorly perfused tumor cores. So the approach may need to be paired with another trigger or tumor-targeting method to cover all cancer cells.

[0084] The reactive oxygen species (ROS)-responsive linker system leverages elevated levels of ROS, such as hydrogen peroxide (H2O2). frequently found within the tumor microenvironment (TME) due to metabolic activity of cancer cells and associated immune cells. Higher ROS concentrations, such as about 50 μM - about 100 pM H2O2compared to low micromolar levels in normal tissue, can be harnessed in linkers by incorporating chemical moieties designed to react specifically with reactive oxygen species. As a non-limiting example, a boronic ester or boronate group can undergo oxidation by H2O2to generate a phenolic intermediate. This transformation then triggers a subsequent spontaneous self-immolative fragmentation cascade, leading to cargo release. Another non-limiting example is the use of thioketal-based linkers, that are cleaved under elevated ROS conditions. The ROS-induced chemical reaction serves as the activation trigger, destabilizing the linker structure for spontaneous release. Inflammatory conditions also involve ROS production, so linker sensitivity isdesirably calibrated so that the linkers respond to the sustained, moderate ROS levels typical of the tumor microenvironment.

[0085] An exemplary ROS-responsive self-immolative linker is the boronic ester-based linker, as noted above. This system includes an aryl boronic ester moiety, often linked via a self-immolative spacer (such as a p-aminobenzyloxycarbonyl (PABC) or related benzyl ether structure) to the cargo or reporter. This system is triggered by the oxidation of the boronic ester group by H2O2present in the tumor microenvironment. This oxidation results in the in situ conversion of the boronic ester to a phenolic intermediate. The generated phenol group then initiates a spontaneous self-immolative fragmentation cascade (e.g., 1,6-elimination), leading to the cleavage of the bond to the cargo and thereby spontaneously releasing the cargo. This system leverages the chemical reactivity of boronates towards oxidation by peroxide. Variations of boronic ester-based linkers include different aryl substitutions, pinacol esters, specific self-immolative spacers, and conjugation to tumor-targeting moieties, are within the scope of the present description.

[0086] The tumor-associated protease-responsive linker system exploits known expression or activity of specific protease enzymes within the tumor microenvironment (TME). In such linkers, the linker directly incorporates one or more specific peptide sequences recognized as substrates by corresponding target tumor- associated proteases (such as MMPs like MMP-2 / 9, cysteine cathepsins B / L / S, Fibroblast Activation Protein (FAP), or legumain). The triggering event occurs when the target protease or proteases, present and active within the TME. enzymatically cleave the linker-incorporated peptide substrate. This cleavage event is designed to initiate linker destabilization, leading to a subsequent spontaneous self-immolative fragmentation or conformational change that results in the release of the conjugated cargo. The specificity of this approach depends on the chosen protease's restricted expression or activity profile; enzymes like FAP, largely absent from normal adult tissues but highly expressed on cancer-associated fibroblasts, provide for tumor- selective activation. While other proteases like certain MMPs or cathepsins may be present in normal physiological processes (e.g.. wound healing, inflammation), differences in their sustained levels, extracellular location, or specific isoforms withinthe TME can still provide a basis for selective activation. Specificity can be further enhanced by designing linkers that require additional TME conditions, such as acidic pH, for optimal cleavage or release following protease action.

[0087] An exemplary protease-responsive self-immolative linker is the Valine-Citrulline (Val-Cit) dipeptide linker, often coupled with a p- aminobenzyloxycarbonyl (PABC) self-immolative spacer, which works by taking advantage of specific enzymatic cleavage followed by spontaneous fragmentation. This system includes the Val-Cit dipeptide sequence, recognized as a substrate by proteases like Cathepsin B (often overexpressed in lysosomes of tumor cells or secreted in the TME), linked to the PABC spacer, which connects to the cargo. This system is triggered by the enzymatic cleavage of the Val-Cit amide bond by Cathepsin B, occurring after the linker and construct have been internalized into tumor cell lysosomes. This results in the unmasking of the electron-donating aniline group on the PABC moiety and initiates a spontaneous 1,6-elimination cascade. This leads to the fragmentation of the PABC spacer and the cleavage of the bond to the cargo, thereby spontaneously releasing the cargo. Variations of protease-responsive linkers including alternative protease substrates (such as Phenylalanine-Lysine) or different spacer combinations are within the scope of the present description.

[0088] Another exemplary protease-responsive self-immolative linker system utilizes Matrix Metalloproteinase (MMP)-cleavable peptide sequences, which work by taking advantage of the elevated MMP activity found in the tumor extracellular matrix (ECM). This system includes a specific peptide sequence (e.g., variants ofor PVGLIG), recognized as a substrate by tumor-associated MMPs like MMP -2 or MMP-9, incorporated into the linker structure. This system is triggered by the enzymatic cleavage of the peptide sequence by active MMPs within the tumor microenvironment. This cleavage event is designed to initiate a subsequent spontaneous fragmentation or conformational change leading to the cleavage of the bond to the cargo. Variations of MMP-responsive linkers including different peptide substrates, incorporation onto platforms, or conjugation with tumor-localizing moieties are within the scope of the present description.

[0089] Another exemplary protease-responsive self-immolative linker system is the Fibroblast Activation Protein (FAP)-cleavable peptide linker, which works by taking advantage of the highly specific FAP enzymatic activity present almost exclusively in the stroma of many tumors. This system includes a specific peptide sequence (e.g., ERGETGP or others containing a Gly-Pro motif recognized by FAP) incorporated into the linker. This system is triggered by the enzymatic cleavage of the peptide substrate by FAP. This results in the generation of a new reactive group which initiates a spontaneous self-immolative fragmentation cascade, leading to the cleavage of the bond to the cargo. The high specificity of this system stems from the restricted expression of FAP primarily on cancer-associated fibroblasts (CAFs) in tumors, with minimal presence in normal adult tissues. Variations of FAP-responsive linkers including different FAP substrate peptides, the type of self-immolative linker used, or conjugation to various scaffolds or targeting moi eties are within the scope of the present description.

[0090] Another exemplary protease-responsive self-immolative linker system is the Legumain-cleavable peptide linker, which works by taking advantage of the specific enzymatic activity of Legumain, an asparagine endopeptidase overexpressed in the tumor microenvironment, particularly by tumor-associated macrophages (TAMs). This system includes a specific peptide sequence containing an asparagine residue (e g., Ala-Ala-Asn), recognized as a substrate by Legumain, incorporated into the linker and connected to the cargo. This system is triggered by the enzymatic cleavage of the peptide substrate by Legumain. This results in the generation of a new N-terminal amine (or other reactive group depending on the subsequent residue) which initiates a spontaneous self-immolative fragmentation cascade, leading to the cleavage of the bond to the cargo and thereby releasing the cargo. The specificity of this system derives from the elevated expression and often extracellular activity of Legumain in the TME, combined with its requirement for an acidic pH (around 5.5) for activity - conditions often met in lysosomes or specific tumor microenvironmental niches but less common in normal tissues. Variations of Legumain-responsive linkers including different Legumain substrate peptides, specific self-immolative spacers, orconjugation to targeting moieties or nanoparticle platforms are within the scope of the present description.

[0091] In various forms, a panel of multiple constructs comprising different linkers according to the above description can be employed as a multiplex assay for detection and / or quantification. A multiplex assay including multiple constructs using different linker types, in various exemplary forms, provide a buffer against false positives by increasing the number of false triggers required to ‘spoof each construct effectively, and would also thereby provide a rough estimate of the specificity of a given multiplex assay for a particular disease.

[0092] For example, a benign ulcer can result in the activation of a linker triggered by FAP, but only weak activation, if any, of a linker specifically triggered by tumor microenvironments. Thus, an exemplary multiplexed assay including at least one protease-response linker and at least one TME-responsive linker that responds to the tumor microenvironment (including pH, hypoxia, and ROS-responsive linkers described above) can, in various forms, provide diagnostic and discriminatory power sufficient to distinguish benign ulcers, inflammations, and other conditions from tumors. If such a multiplexed assay returned reporters corresponding to the protease- responsive linker and the TME-responsive linker with high signal strength, then the presence of a tumor is indicated. If, however, such a multiplexed assays returned reporters corresponding to the protease-responsive linker but not the TME-responsive linker, or there was only a weak reporter response from the TME-responsive linker, then the presence of a tumor is potentially contraindicated. Additionally, the presence of TME-responsive linkers in an exemplary multiplex assay, in addition to increasing the discriminatory power of the assay, increases the scope and / or sensitivity of the assay by detecting tumors that are, for example, antigen-silent, meaning that such tumors may not activate protease-triggered linkers.

[0093] As discussed above, many of the example provided herein discuss activating the cleavable linker to result in release of the cargo. However, in alternative embodiments, the cleavable linker can be activated to result in release of the binding domain.REPORTER

[0094] The synthetic probe of the present disclosure includes a reporter. Suitable types of reporters include reporters that are detectable. The detectable reporter of the synthetic probe is a signaling moiety that generates a detectable output upon release. Suitable reporters include any of those known in the art such as nucleic acids, protein labels, affinity ligands, enzymes, fluorescent reporters, carbon dots, radiolables. and combinations thereof. Suitable reporters include Organic Fluorophores (such as Alexa Fluor dyes e.g., Alexa Fluor 650, Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 647, ATTO dyes and cyanine dyes Cy3 / Cy5), NIR dyes (IRDye 680 / 800, Cy7), quantum dots (e.g., semiconductor nanocrystals), carbon dots (fluorescent carbon nanoparticles); fluorescent peptides, nanotubes (e g., single-walled carbon nanotubes with fluorescent properties), nucleic acid stains (SYBR Green, EvaGreen, PicoGreen, RiboGreen. DAPI, Hoechst). Radiolables such as 3H. 14C, 32P, 33P, 35S, 1251. 1311 (autoradiography / scintillation); 18F, 11C, 64Cu (PET); 99mTc. 1231. Raman / SERS reporters (R6G, cyanines, bipyridyls). Gold nanoparticle reporters. Peptide reporters can include a multi-epitope peptide such as tandem repeats of short unique sequences attached to a backbone. Different proteases can cleave the peptide at different positions releasing peptides of distinct masses that are detectable by mass spectrometry (including tandem MS-MS) to identify the peptide fragments, yielding a " spectral barcode”. Peptide reporters also include those containing unnatural amino acids (e.g., stable isotopes or mass tags) that are separated by cleavage. Particularly suitable nucleic acid reporters are barcode DNA detected using nucleic acid amplification techniques (e.g., polymerase chain reaction, isothermal amplification, qPCR), next-generation sequencing (NGS), and CRISPR / Cas-based detection assays. Each DNA barcode is a single-stranded DNA with modifications such as phosphorothioate linkages and 2’ sugar modifications to resist exonucleases. The DNA barcode can also be a circular mini DNA barcode. Suitably, the DNA barcode is a synthetic DNA ranging from about 10 nucleotides (nt) to about 500 nt. Suitably, the DNA barcode ranges in length of about 15 nt to about 300 nt; from about 20 nt to about 200 nt. Also included are DNA-peptide hybrid reporters that are conjugates of nucleic acids and peptides that allows for protease cleavage to cut the DNA-peptide bond or remove a DNA segment. Forexample, a DNA oligonucleotide with a 5 '-amine linked via a peptide to another DNA wherein cleavage yields two DNA fragments, each of which carries a unique primer- binding region. Here, an intact reporter would be amplification inactive until cleavage generates the amplifiable DNA fragments.

[0095] The detectable reporter (also referred to herein as “cargo" and “cargo component" ) is associated with the binding domain of the synthetic probe until, in various embodiments, the reporter is released upon activation of the cleavable linker. The reporter component released from the synthetic probe complex can be detected by any methods known for in vivo detection or when the reporter is excreted and collected in a subject sample.

[0096] In an exemplary embodiment, a single reporter can be used for a multitude of diseases (e.g., different cancers) to generate an inexpensive solid tumor screening tool.

[0097] Additional techniques can be used to enhance carbon dot fluorescence intensity and sensitivity in bioimaging or biosensing applications. Signal amplification can improve detection limits, which is particularly useful in medical diagnostics. Methods for signal amplification of carbon dots are known in the art and include, for example, surface functionalization involving modifying the surface of carbon dots with functional groups or other materials can enhance their luminescence and make them more sensitive for detection. Doping carbon dotes with elements such as nitrogen, sulfur, and phosphorus can alter their electronic structure, increasing their quantum yield and brightness. Conjugating carbon dots with metals such as gold nanoparticles and silver nanoparticles can improve fluorescence. Coating carbon dots with silica shells enhances their stability, brightness, and compatibility in biological environments, improving their use in diagnostic applications. Forster Resonance Energy Transfer (FRET) can enhance signal strength by transferring energy between a donor (such as a carbon dot) and an acceptor molecule. When FRET occurs between carbon dots and a nearby fluorophore or chromophore, the amplified signal can be utilized for highly sensitive bioimaging. Carbon dots can be used with enzymes (like horseradish peroxidase (HRP) or alkaline phosphatase) that catalyze reactions toamplify the luminescent signal. Conjugating carbon dots with signal amplification polymers such as dendrimers or hyperbranched polymers can amplify fluorescence signals. Aggregation-induced emission enhancement (AIEE) can increase emission intensity when carbon dots are aggregated into larger structures. Multi-photon excitation or dual-excitation techniques allows the carbon dots to emit brighter signals and penetrate deeper into biological tissues. Adjusting the wavelength and intensity of the excitation light, can significantly amplify the emission signal, and thereby improve detection capabilities. Carbon dots can be coupled with luciferase enzymes that generate bioluminescence upon substrate binding. Chemical enhancers such as peroxides or other small molecules can improve the chemiluminescent reaction involving carbon dots. These enhancers are often used in conjunction with carbon dot labels to increase the intensity of the signal. Carbon dots can be engineered to emit light at different wavelengths based on their size or surface chemistry, making it possible to develop multiplexed LFAs that can detect multiple targets in the same test. Each set of carbon dots can be designed to correspond to, and thus, allow for identifying different target molecules by emitting light at distinct wavelengths, allowing for simultaneous detection of multiple markers.

[0098] Volatile organic compounds ( “ VOC”) are as included as reporters for the synthetic probe molecule. VOC offer a noninvasive diagnostic readout in breath of a subject. Suitable VOC include 2,2,2-trifluoroethylamine, pentafluoropropylamine, heptafluorobutylamine, D5-ethanol. D6-acetone, De-isopropanol, pentafluorobenzyl alcohol, nonafluorobutane, methyl tert-butyl ether, dimethyl sulfide, hexafluoropropylene oxide, perfluorobutyl ethanol. VOC detection can include pre- concentration methods such as breath sampling into sorbent tubes, cold traps, microfluidic pre-concentrators. VOC can be detected by gas chromatography -mass spectrometry (GC-MS), photon transfer reaction-MS, Selected Ion Flow Tube-MS, electrochemical sensors, E-Nose (sensor array), and Infrared spectroscopy.MASKED CLEARANCE TAGS

[0099] In particularly suitable embodiments, the cleavable linker of the synthetic probe includes a mask (or cage) that is removed or otherwise degraded(unmasked) endogenously (in the subject). Suitable masks include biotin (which is masked by a protecting group) and glycans (e.g., galactose, mannose) that bind to hepatic receptors.

[0100] As used herein, a mask is a chemical or structural element that reversibly inhibits the reactivity, recognition, or function of a chemically and / or biologically active moiety within a synthetic biological construct until specific conditions trigger a removal or transformation of the mask. Masking is achieved through direct chemical modification of the active moiety, through steric hindrance that physically blocks or hinders access to the active moiety, or through conformational constraints that temporarily alter the spatial arrangement of functional groups on the moiety. The removal or transformation of the mask can be triggered by physiological conditions including changes in pH, redox potential, enzymatic activity, or externally applied stimuli such as the presence and / or deployment of specific chemical reagents or photochemical activation.

[0101] In the enzyme-driven masking system, enzymatic activity is used to unveil reactive functionality. Examples include ester-protected cyclooctynes that require esterase activity to generate the active cycloalkyne for subsequent SPAAC reactions. A non-limiting example of the enzyme-driven masking system is a linker including the first reagent, such as an antigen-binding site (paratope), and a protease- cleavable mask that deactivates, blocks or hinders access to the first reagent. When this exemplary linker enters a TME, tumor-associated proteases cleave the mask, exposing the first reagent. Exemplary protease-activatable masks such as the one described above include peptides, antibody domains, or coiled-coil peptides.

[0102] In the pH-dependent activation masking system, the mask is structurally constrained at neutral pH and undergoes conformational or electronic changes in acidic environments, exposing reactive functionality. A non-limiting example of a pH-dependent masking system includes a linker including the first reagent and a pH-sensitive mask that deactivates, blocks or hinders access to the first reagent at physiologic pH (e.g., —7.4). When this exemplary linker enters an acidic TME, the lower pH environment triggers the deactivation or removal of the mask, exposing thefirst reagent. Exemplary pH-activatable masking strategies such as the one described above include employing chemical caging (like maleic anhydride derivatives on lysines) that hydrolyzes in acid, or using acid-labile linkers (such as hydrazones, cis- aconityl, or acetal bonds) designed to cleave at mildly acidic pH.

[0103] In various forms, an exemplary acid-labile mask includes one or more hydrazone functional groups. In an exemplary mask including a hydrazone group, the mask is attached via a hydrazone bond designed to block or hinder access to the first reagent. This hydrazone linkage is engineered to remain relatively stable and intact at physiological pH (~7.4) found in circulation but is susceptible to hydrolysis upon exposure to the mildly acidic conditions characteristic of certain microenvironments like that of TMEs. To achieve this specific pH-triggered release profile, electronically tuned hydrazones such as anilino hydrazones or those derived from benzoic acid with electron-withdrawing substituents, can be employed, as their properties confer stability at neutral pH while still allowing cleavage at moderately acidic pH, thereby enabling the removal of the mask and exposure of the first reagent predominantly within the target acidic milieu.

[0104] In various forms, another exemplary acid-labile mask includes one or more imine functional groups (Schiff Bases). In an exemplary mask including an imine group, the mask is attached via an imine bond (C=N-) designed to block or hinder access to the first reagent. An imine linkage is engineered to enhance its stability at physiological pH (~7.4) while still enabling rapid hydrolysis upon exposure to the mildly acidic conditions characteristic of certain microenvironments like that of TMEs. often by incorporating aromatic aldehydes (forming aryl imines or benzoic imines). To achieve this specific pH-triggered release profile, stabilized imines, such as benzoic imines or other aryl imines, are employed, as their structure confers sufficient stability at neutral pH while still allowing relatively fast cleavage (potentially within minutes to hours) at moderately acidic pHs. This enables the removal of the mask and exposure of the first reagent, such as an unmasked reactive group like a tetrazine or TCO, predominantly within the target acidic milieu.

[0105] In various forms, another exemplary acid-labile mask includes one or more acetal or ketal functional groups. In an exemplary mask including such a group, the mask is attached via an acetal or ketal bond designed to block or hinder access to the first reagent. While stable at neutral-to-basic pH, this linkage is engineered to undergo hydrolysis upon exposure to acidic conditions. Although many simple acetal s / ketals require stronger acidity (e.g., pH < 6) for rapid cleavage, design tunes the hydrolysis profile to enable cleavage within mildly acidic environments like that of TMEs. To achieve this specific pH-triggered release, tunable structures such as certain cyclic acetals / ketals (e.g., derived from 1,3-dioxolanes), those incorporating electron- withdrawing substituents, or acetals derived from diols with specific substitution patterns (e.g., less substituted for faster hydrolysis near pH 6.5), are employed. These engineered structures confer stability at neutral pH while allowing hydrolysis at moderately acidic pHs, thereby enabling the removal of the mask (liberating a carbonyl compound) and the exposure of the first reagent, such as an unmasked reactive group like a tetrazine, predominantly within the target acidic milieu.

[0106] In various forms, a further exemplary acid-labile mask includes one or more cis-aconityl amide or related β-carboxylate amide (e.g., maleic amide) functional groups. In an exemplary mask including such a group, the mask is attached via an amide bond formed, for example, by conjugating an amine using cis-aconitic anhydride or a similar precursor, designed to block or hinder access to the first reagent. This specialized amide linkage is engineered to remain stable at physiological pH (~7.4) but undergoes acid-catalyzed hydrolysis, significantly accelerated by intramolecular anchimeric assistance from a neighboring carboxylate group, upon exposure to the mildly acidic conditions characteristic of TMEs. To achieve this specific pH-triggered release profile, linkers incorporating cis-aconity l amides or structurally similar maleic amides are employed. Their structure facilitates intramolecular catalysis, conferring sufficient stability at neutral pH (e.g.. having a half-life greater than 12 hours) while allowing hydrolysis (potentially within hours or less) at moderately acidic pHs. This enables the removal of the mask and the exposure of the first reagent predominantly within the target acidic milieu.

[0107] In various forms, another exemplary acid-labile mask includes one or more acid-cleavable aryl sulfonate esters. In an exemplary mask including such a group, the mask is attached via an aryl sulfonate ester linkage, which masks a functional group like a phenolic hydroxyl. This sulfonate linkage is engineered to remain stable at physiological pH (~7.4) but undergoes acid-catalyzed fragmentation, through self- immolative elimination mechanisms, upon exposure to the mildly acidic conditions characteristic of TMEs. To achieve a specific pH-triggered release profile, tailored sulfonate structures, such as ortho-hydroxybenzyl sulfonate (OHPAS) motifs, benzyl sulfonate variants, or other aryl sulfonates strategically placed (e.g., masking a phenol on a tetrazine component), are employed, with their lability typically being tunable via aryl ring substituents. Their structure confers stability at neutral pH while allowing cleavage (e.g., via intramolecular attack or elimination pathways ejecting the sulfonyl group) at moderately acidic pHs. This enables the removal of the mask leading to the exposure of the first reagent.

[0108] In various forms, another exemplary acid-labile mask includes one or more silyl ether functional groups. In an exemplary mask including such a group, the mask is attached by forming a silyl ether bond (R-Si-O-R') with a hydroxyl group, designed to block or hinder access to the first reagent, for instance, via steric or electronic effects. This silyl ether linkage is engineered to remain stable at physiological pH (~7.4) but undergoes acid-catalyzed hydrolysis upon exposure to mildly acidic conditions characteristic of TMEs. To achieve this specific pH-triggered release profile, the stability and cleavage rate is tuned by selecting appropriate silyl group substituents; for example, silyl ethers with less bulky or electron-withdrawing substituents (such as trimethylsilyl or diphenyl-tert-butylsilyl derivatives) can be employed for enhanced lability near pH 6.5, while bulkier groups (like TBDMS) offer more stability. Structures like trialkylsilyl ethers masking a hydroxyl group can be used. This enables the removal of the mask (releasing non-toxic silanols) and the regeneration of the hydroxyl group, which can subsequently trigger further reactions like self-immolation, leading to the exposure of the first reagent.

[0109] The various masking strategies described above and others known to one of ordinary skill in the art can, in various forms, require modification of theconstruct to incorporate one or more bonding sites for the mask. In various forms, the mask is attached to the construct by one bonding site, by two bonding sites, by three bonding sites, or by more than three bonding sites. For example, a VHH linker can first be functionalized with an aldehyde group (e.g., by periodate oxidation of a carbohydrate or via an engineered C-terminal formylglycine tag). This VHH-aldehyde is then reacted with a tetrazine-hydrazide to form a VHH-hydrazone-tetrazine conjugate. In this conjugate, the tetrazine component is rendered unreactive because it is part of the hydrazone bond, which remains stable at healthy physiological pHs. However, upon exposure to the mildly acidic conditions of a TME, e.g., pH 6.5, the hydrazone bond undergoes slow hydrolysis. This cleavage regenerates the aldehyde on the VHH to unmask the tetrazine moiety of the originally conjugated tetrazine-hydrazide. restoring its reactivity. Although the hydrazone bond is cleaved, the tetrazine-containing molecule (e.g., the original tetrazine-hydrazide, such as a 6-methyl-tetrazine- phenylacetic acid hydrazide) remains tethered to the VHH through its initial point of attachment (e.g., via its phenylacetic acid linker to an oxidized VHH glycan or other bifunctional scaffold). This now-active. VHH-tethered tetrazine is then available for bioorthogonal click reaction with an administered TCO, minimizing off-target reactions in circulation while the tetrazine was masked.

[0110] In an alternative strategy, a tetrazine's reactivity can be controlled through dual acid-labile attachment to a linker, creating a cyclic tether that sterically 'locks' or 'cages' the first reagent. As a non-limiting example, a VHH linker can be functionalized to present distinct reaction sites, such as an aldehyde group and a surface carboxyl group. A bifunctional tetrazine derivative, possessing for instance both a hydrazide functionality and an amine group, can then react with the VHH to form two distinct acid-labile linkages: a hydrazone bond with the VHH's aldehyde, and a cis- aconityl amide bond with the VHH's carboxyl group. In this doubly -tethered conjugate, the tetrazine component is rendered unreactive due to the conformational constraint imposed by these dual linkages, which remain stable at healthy physiological pHs. However, upon exposure to the mildly acidic conditions of a TME, e.g., pH 6.5, both the hydrazone bond and the cis-aconityl amide bond are susceptible to hydrolysis. The cleavage of these linkages can occur at different rates, making them potentiallysequentially cleaved, due to the different reactivity of each linkage. This effectively alters the tetrazine's constraint; for example, cleavage of one bond while the other temporarily remains can transition the tetrazine to a less constrained single-point attachment, or cleavage of both can more fully release it from its initial caged conformation. This unmasks the tetrazine moiety, freeing it from its sterically hindered state and restoring its reactivity. Although the original bifunctional tetrazine derivative may still be considered tethered to the VHH (e.g., if one acid-labile bond cleaves significantly slower than the other, or if the entire assembly is part of a larger scaffold with its own VHH attachment), its newly acquired conformational freedom renders it available for bioorthogonal click reaction with an administered TCO. This dual-lock strategy can ensure the tetrazine is particularly inert during circulation, with unmasking dependent on the acidic environment triggering the release of these multiple constraints.

[0111] In a further exemplary non-limiting example, a tetrazine's reactivity can be controlled through attachment via multiple, concurrently acting acid-labile linkages to a VHH linker, using a multivalent scaffold approach. A VHH linker can be functionalized to present multiple aldehyde-bearing residues. A central tetrazine unit, synthetically modified to feature multiple hydrazide arms (forming a 'hub'), can then react with these aldehyde sites on the VHH to form multiple hydrazone bonds. In this multiply -tethered conjugate, the tetrazine component is rendered unreactive. effectively 'tied down' and sterically hindered by these collective bonds, which remain stable at healthy physiological pHs. However, upon exposure to the mildly acidic conditions of a TME. e.g., pH 6.5, these multiple hydrazone bonds dissociate or hydrolyze, ty pically simultaneously or near-simultaneously. The cleavage of a sufficient number of these bonds effectively reduces the multiple points of constraint on the tetrazine hub. Even if the tetrazine hub remains partially tethered to the VHH (e.g., by one of its arms still forming an intact hydrazone bond), its significantly increased conformational freedom unmasks the tetrazine moiety, restoring its reactivity. This now-active, less constrained tetrazine is then available for bioorthogonal click reaction with an administered TCO. This design, functioning somewhat like what is referred to in logic systems as an 'AND gate' where sufficient bond breakages are required for activation, can provide ultra-high specificity for unmasking within the target microenvironment.

[0112] In various forms, exemplary acid-labile masking strategies can utilize acetal or ketal functional groups, which are strategically incorporated to control the reactivity or release of components within a system, such as one requiring a bioorthogonal click reaction for ultimate reporter activation. Such acetal / ketal linkages are engineered to remain relatively stable at physiological pH but undergo hydrolysis upon exposure to mildly acidic conditions (e.g., at or around pH 6.5), thereby priming the system by unmasking for a subsequent reaction.

[0113] In one exemplary configuration, a reactive bioorthogonal linker comprising a trans-cyclooctene can be directly 'caged' by the mask. This can be achieved by converting the TCO's reactive double bond into anon-reactive cyclic acetal or ketal structure (e g., by forming a cyclic adduct from a TCO functionalized with a diol, or a TCO-aldehyde reacted with a glycol). The cyclic adduct renders the TCO inert at neutral pH. Upon acid hydrolysis in a target acidic microenvironment, the acetal / ketal linkage opens, regenerating the reactive TCO on the linker, thereby making the linker available for ligation with an administered tetrazine probe to trigger release of the reporter.

[0114] In alternative forms, an acetal can function as an acid-labile junction that controls a degree of conformational freedom or an attachment point of the linker rather than directly masking the first reagent. For example, a tetrazine (e.g., comprising a benzyl alcohol) can be linked to an aldehyde on a VHH via formation of a benzy lidene acetal. While the tetrazine typically remains attached to the VHH through a secondary, stable linkage, the acetal junction, when intact at neutral pH. may sterically constrain the tetrazine or hold it in an inactive state. Ascid hydrolysis of this acetal junction at mildly acidic pH alters the tetrazine's attachment or relieves the constraint, rendering the now un-constrained or reconfigured tetrazine active for reaction with an administered TCO.

[0115] In further variant forms, an acid-cleavable acetal or ketal can be incorporated within a self-immolative variant of the linker that is part of a construct, for example, connecting an already active bioorthogonal handle (e.g., a tetrazine) to a reporter (e.g., VHH-Tz-Spacer(Ketal)-Reporter). In such a design, the ketal effectively'locks' the linker at neutral pH, preventing its premature collapse and reporter release. On exposure to mildly acidic conditions, hydrolysis of the ketal exposed the first reagent. The subsequent bioorthogonal click reaction of the tetrazine with an administered TCO is required to trigger the now-primed linker to undergo to the fragmentation cascade and release the reporter.

[0116] In another exemplary configuration, a linker comprising a bioorthogonal first reagent, such as a tetrazine or a trans-cyclooctene (TCO) system, can be directly 'capped' or functionally altered by an acid-labile mask involving specialized amide bonds. As a non-limiting example, in various forms, a tetrazine bearing an amine group can be conjugated to a linker via cis-aconitic anhydride, forming an acid-cleavable cis-aconityl amide. The amide caps the tetrazine's amine, rendering the tetrazine system unreactive or sterically hindered at neutral pH ranges. Similarly, in various forms, an amine associated with a TCO component can be reacted with maleic anhydride to form an acid-sensitive maleamic acid (maleic amide derivative). In both instances, the resulting amide linkage is stable at neutral pH. Upon acid hydrolysis in a target acidic microenvironment, the amide bond cleaves, regenerating the free amine on the tetrazine or TCO system.. To prevent diffusion after cleavage, the bioorthogonal first reagent might also be attached to the primary construct via a separate, stable linkage.

[0117] In alternative forms, an acid-labile linkage can function as a plug- and-socket junction controlling the association or conformational freedom of the linker and / or the first reagent, rather than directly modifying the first reagent’s reactivity. For example the linker features a stably attached phenol as the first reagent, forming a linker-phenol moiety. The phenol can be 'plugged' by an acid-cleavable aryl sulfonate which itself is part of, or carries, a tetrazine moiety. At neutral pH, the aryl sulfonate ester linkage (e.g., forming a phenyl sulfate) is intact, tethering the tetrazine-bearing component to the linker-phenol moiety. Upon acid hydrolysis of the aryl sulfonate junction at mildly acidic pH, the tetrazine-bearing fragment is released. While this fragment might be designed to remain proximal to the linker (e. g. , through non-covalent interactions or attachment to a larger linker-tethered scaffold), the above-described cleavage alters the attachment or relieves a conformational constraint, rendering thenow more freely accessible or reconfigured tetrazine active for reaction with an administered TCO.

[0118] In further variant forms, the acid-cleavable characteristic can be intrinsically designed into the linker structure through moieties that undergo intramolecularly assisted cleavage, effectively unmasking upon exposure to acid. For example, the cis-aconityl amides and maleic amides described above comprise some acid sensitivity due to intramolecular mechanisms. In various linkers masked using one or more amide bonds, a neighboring carboxylate group, once protonated in a mildly acidic environment, can facilitate the hydrolysis of the amide bond through anchimeric assistance (e.g., by intramolecular nucleophilic attack forming a cyclic anhydride intermediate). This feature ensures that the linker remains stable at neutral pH but efficiently cleaves when the pH drops.

[0119] In yet another non-limiting exemplary configuration, a linker can be directly 'caged' or have its functionality suppressed by a specifically designed acid- labile chemical group such as an ortho-nitrobenzyl sulfonate, which can be employed to mask a hydroxyl group, such as one on a tetrazine or a phenolic moiety within a self- immolative linker. The sulfonate ester linkage effectively blocks the hydroxyl at neutral pH. Upon exposure to an acidic microenvironment, the ortho-nitrobenzyl sulfonate undergoes acid-catalyzed cleavage (e.g., via Meisenheimer rearrangement or hydrolysis), regenerating the free hydroxyl. In various forms, the hydroxyl is or is a component of the first reagent. The above-described masking scheme ensures that the reporter release mechanism is only activated when both the acidic condition (for sulfonate removal) and the click reaction occur, fulfilling a strict ‘AND’ logic.

[0120] In various alternative forms, a silyl ether group can be strategically placed near a thioether linkage that tethers a TCO linker to a construct, or within a hemithioketal-silyl ether structure associated with the TCO. At neutral pH, the bulky silyl group or the intact hemithioketal-silyl ether structure can sterically hinder the TCO's ability to react or maintain it in a less reactive conformation. Upon exposure to mildly acidic pH, the acid-labile silyl ether is cleaved. This cleavage can directly remove a steric impediment or initiate a rearrangement (e.g., elimination from theresulting hemithioacetal), which in turn frees the TCO from its constraint, restoring its reactivity and making it available for cycloaddition with an administered tetrazine.

[0121] In further variant forms, the concept of pH-selective unmasking can extend beyond covalent chemical modifications to include reversible non-covalent 'molecular cage' or host-guest interactions. For instance, the first reagent can be temporarily sequestered or 'hidden' by complexation within the cavity of a macrocyclic host molecule, such as, without limitation, an appropriately engineered cucurbituril or cyclodextrin. The above-described host-guest complex is designed such that the macrocycle effectively shields the tetrazine from external reactants at neutral physiological pH ranges. On exposure to the mildly acidic conditions of a target microenvironment, protonation can alter the change or conformation of the host or guest, weakening the host-guest binding affinity. This can lead to the dissociation of the complex and the release or expulsion of the first reagent from the macrocyclic host.

[0122] In various forms, the mask, if designed to be acid-labile, can have a half life greater or equal to 12 hours at or around pH 7.4, and have a half-life of less than 2 hours at or about pH 6.5.

[0123] In the redox-dependent activation masking system, the mask is structurally constrained and only undergoes conformational or electronic changes in reducing environments, exposing reactive functionality. A non-limiting example of a redox-dependent masking system is a linker comprising the first reagent, and a redox- responsive mask, attached via disulfide bonds or other thiol-cleavable linkages, that deactivates, blocks or hinders access to the first reagent within oxidizing environments like the bloodstream. When this exemplary linker enters a reducing environment (for example the intracellular space or specific TME locations with high glutathione levels) an elevated concentration of reductants cleaves the redox-sensitive linkage (e.g.. the disulfide bond), releasing the mask and exposing the first reagent. Exemplary redox- activatable masking strategies such as the one described above can utilize, without limitation, disulfide bonds or similar thiol-cleavable moieties to tether blocking groups, for example disulfide-tethered peptide masks, so long as they remain stable and blockthe first reagent under oxidizing conditions but are efficiently cleaved upon exposure to the reducing conditions characteristic of the target microenvironment.

[0124] The dihydrotetrazine activation masking system employs a redox- sensitive functionality where dihydrotetrazines serve as "latent" click reagents, requiring oxidation (such as in ROS-rich environments) to generate reactive tetrazines.

[0125] The photocaged reactants masking system incorporates photolabile protecting groups that sequester reactivity until specific light exposure generates the active click component, allowing for spatiotemporal control.

[0126] Polymeric masks use various polymers (e.g. PEG, dextran, zwitterionic polymers, etc.) function as "masks" that shield the conjugate from immune recognition. In particular, zwitterionic polymers attract a hydrated ‘shell’ to mask the construct from the immune system.

[0127] Enzyme demasking uses peptide segments to block antigen-binding sites until cleaved by disease-associated proteases. Sialic acid "capping" of galactose tags requires neuraminidase enzyme for unmasking by removing the sialic acid and exposing the underlying targeting moiety.

[0128] Unmasking desirably occurs endogenously at a time ranging from several minutes (about 5 minutes) to and about 48 hours, including several minutes (about 5 minutes) to about 24 hours.

[0129] Unmasking occurs when a clearing agent binds an exposed clearance tag and facilitates rapid clearance of unbound synthetic probe. Suitable clearing agents for biotin masked clearance tags include NeutrAvidin and engineered streptavidin derivatives. Suitable clearing agents for glycan masked clearance tags include lectins. The clearing agent is administered to the subject and upon exposure to the masked clearance tag, the clearance tag is unmasked to promote hepatic clearance of all remaining circulating (unbound) synthetic probes. Following sufficient target localization with a synthetic probe having a biotinylated binding domain, avidin (or streptavidin) is administered to the subject. Any unbound, circulating synthetic probehaving the biotinylated binding domain will bind the avidin in blood, forming large avidin-synthetic probe complexes. The avidin-synthetic probe complexes are rapidly sequestered by the reticuloendothelial system (pnmarily liver and spleen), accelerating clearance from blood and normal organs. Alternatively, the synthetic probe can be labeled with avidin or streptavidin followed by administration of biotin to form biotin- synthetic probe complexes with any unbound synthetic probes for clearance. Any bound synthetic probe remains in place at the target.

[0130] In various forms, masks can incorporate self-immolative components, thereby ensuring that once triggered, a spontaneous fragmentation cascade will completely unmask the construct.

[0131] In various forms, implementing a mask can require attachment of the mask to the construct in a way that blocks the first reagent without permanently impairing the construct, which can occur by any means known to one of ordinary skill in the art, including chemical bonding, chemical conjugation, enzymatic or chromoenzymatic ligation, and genetic fusions.

[0132] In various forms, the mask can be applied to the construct via chemical conjugation at specific sites on the construct. For example, a VHH (Variable Heavy domain of a Heavy chain) can be engineered for precise mask attachment adjacent to its antigen-binding site. A site-specific cysteine residue can be introduced onto the VHH framework at a non-critical residue site, allowing a mask (such as a peptide or polymer designed to sterically block the paratope) to be conjugated via, for example, thiol-maleimide chemistry. This conjugation incorporates the desired cleavable linker (e.g., protease-sensitive, pH-labile, or redox-sensitive), which in turn enables the removal of the mask removal under specific conditions. pH-labile making pairs include, for example. Hydrazone / acylhydrazone (C=N-NH-), Imine / Schiff base (C=N-), Oxime (C=N-O-), Acetal / ketal, Orthoester, vinyl ether, cis-aconityl and maleic-acid-based amides, boronate ester (phenylboronic esters / boronate-diol), Carbonate / carbamate with self-immolative triggers, Silyl ether (e.g., TMS, TBDMS), and Acylals / oxazolidines and related hemiaminal acetals. Alternative site-specific strategies include incorporating non-canonical amino acids (like p-azidophenylalanine)for attachment via bioorthogonal 'click' chemistry (e.g., SPAAC), or using enzymatic methods such as Sortase A-mediated ligation (often at the C-terminus) or Microbial Transglutaminase (MTG) conjugation to ensure precise placement and consistent masking.

[0133] In various alternative forms, the mask can be applied to the construct via genetic fusion, where the binding domain and a masking peptide or domain are expressed as a single, continuous polypeptide chain. In this approach, the mask is typically fused to the N-terminus of the binding domain adjacent to its antigen-binding site (paratope), via a genetically encoded linker sequence. This linker sequence contains the desired cleavable motif, most commonly a protease recognition site (e.g., for MMPs, urokinase, or FAP), which in turn enables the removal of the mask upon encountering specific proteases, for instance, within the tumor microenvironment. The mask itself, designed to sterically block the paratope until cleavage includes a peptide derived from the VHH's target, an anti-idiotypic binder (such as an engineered affibody or antibody fragment evolved to bind the VHH paratope), or even a natural masking domain repurposed for this function, ensuring a defined 1 : 1 stoichiometry and precise positioning.SPACERS

[0134] The synthetic probe can further include spacers. As used herein, a spacer is a chemical, biological, or synthetic component incorporated into a synthetic biological construct to establish or modify spatial relationships between functional elements, alter physical properties of the construct, and / or influence the biological interactions of the construct with its environment. Spacers may function to control steric accessibility, modify pharmacokinetic properties, reduce immunogenicity, present multiple ligands in defined spatial arrangements, or otherwise modulate the physical, chemical, or biological characteristics of the construct. Spacers include polymeric structures, peptide segments, small molecule connectors, dendrimeric frameworks, or other moieties that provide defined separation or arrangement of functional elements.

[0135] Any spacer may be used. Suitable spacers include, for example, ethylene glycol and poly(ethylene) glycol, such as hexa-ethylene glycol or penta- ethylene glycol. PEG spacers suitably range in length from 2 ethylene glycol units to about 2500 ethylene glycol units. The spacer may be comprised of one or more ethyl groups, such as a C3 (three-carbon) spacer, a C6 spacer, a C 12 spacer, and a C 18 spacer.

[0136] Spacers (or spacer arms) can link the binding domain to the cleavable linker and link the cleavable linker to the reporter. Spacers can vary in length, which can affect the availability of the binding domain for binding to a target and affect the availability of the reporter to be detected or captured.OTHER SYNTHETIC PROBE COMPONENTS

[0137] The synthetic probe can further include a universal connector that connects the binding component domain to the attenuated conjugate component (cleavable linker). The universal connector can remain bound (connected) to the binding component. The universal connector can act as potential therapeutic flag" site or target site for a therapeutic. For example, the universal connector can be a nanotube to deliver a future therapeutic.

[0138] The synthetic probe can further include an antibody or antibody fragment for use in a lateral flow assay strip, for example. In a multiplexed assay example, both reporter components can bind to a single test stripe on a lateral flow assay membrane by means of the '‘universal lateral flow assay strip paired antibody” binding to a paired antibody striped on a traditional lateral flow assay test line. This will focus the location of detection on the strip for all cancers being tested for excitation fluorescence, thereby increasing assay sensitivity. This technique also allows for relatively large amounts of sample buffer to be tested on a membrane. Alternatively, a drop of sample may be tested by fluorescence.

[0139] An exemplary embodiment particularly useful for renal clearance includes an antibody fragment (e.g.. Fab. Fab’. F(ab’)2, svFc, and a nanobody, and a PEGylated antibody with renal-clearable size) conjugated with a bio-orthogonal group. In general, intact antibodies like IgG are too large (-150 kDa) to be filtered by thekidneys. However, researchers have been exploring smaller antibody fragments and engineered constructs that can fit within the molecular weight limits for renal clearance (typically below 40-60 kDa). Thus, as used herein, “renal-clearable” refers to a molecular weight limit below about 60 kDa. An exemplary synthetic probe complex includes a tumor binding antibody coupled with a spacer coupled with TCO coupled with a scFv coupled with a carbon dot reporter (e g., Ab-spacer-TCO-scFv-Carbon dot). Tetrazine can be used to cleave the synthetic probe complex at the TCO and the scFv can be paired to a LFA for detecting the carbon dot reporter.

[0140] In another aspect, the present disclosure is directed to method for diagnosing a disease or a disorder in a subject having or suspected of having the disease or the disorder by detecting a target molecule of interest in the subject with a synthetic probe, the method comprising: administering to the subject a panel of synthetic probes, each of the synthetic probes comprising: the binding domain that specifically binds a target molecule; the cleavable linker; and the cargo, wherein the binding domain, the cleavable linker, and the cargo are in a complex, and wherein each of the synthetic probes of the panel of synthetic probes comprises a different binding domain and comprises a different cargo; allowing the panel of the synthetic probes to circulate in the subject for a time sufficient to allow the synthetic probe to specifically bind the target molecule if present in the subject via the binding domain and for any synthetic probes that remain unbound to be excreted by the subject; collecting a sample from the subject after a time sufficient for the linker to cleave and thereby release the cargo; and detecting the cargo, if it is or comprises a reporter, in the sample, wherein the reporter identity allows for diagnosing a disease or a disorder. FIG. 2 provides an exemplary flow chart illustrating the flow of the method.

[0141] The method can further include administering to the subject a composition that degrades the linker and causes the reporter to be released from the binding domain.

[0142] The reporter can be detected in any sample and / or combinations of samples. Any method of sampling now known or developed is suitable for the method.

[0143] The unbound synthetic probe can be detected if desired. The unbound synthetic probe is excreted in and detected in a sample obtained from the subject.

[0144] The cleavable linker allows for the release of the reporter after a sufficient time such that the reporter allows for the diagnosis of the disease or disorder. In a multiplexing application, a panel of synthetic probes is administered to the subject. If a specific disease or disorder is present in the subject, a particular synthetic probe will bind to the target whereas other synthetic probes will remain unbound and circulate through the subject’s body until the unbound synthetic probes are cleared (or excreted). As illustrated and described in the figures, the synthetic probe that is bound to the target remains bound for a time sufficient to allow the unbound synthetic probes are cleared by the subject's body. After this time, the cleavable linker degrades to release the reporter, which can then be detected. The identity of the reporter allows for determining the target that was present in the subject.

[0145] In an exemplary prophetic example, a panel of synthetic probes is administered to a patient suspected of having disease 1. The panel of synthetic probes have binding domains that specifically bind to disease 1, disease 2, and disease 3. Each synthetic probe includes a unique reporter of a different color. For example, the disease 1 synthetic probe has the structure: disease 1 binding domain-cleavable linker-yellow reporter. The disease 2 synthetic probe has the structure: disease 2 binding domain- cleavable linker-red reporter. The disease 3 synthetic probe has the structure: disease 3 binding domain-cleavable linker-green reporter. All three synthetic probes are administered to the subject and circulate within the subject’s body. Because the subject has disease 1, the disease 1 synthetic probe having the structure: disease 1 binding domain-cleavable linker-yellow reporter will bind to the disease 1 target, whereas the disease 2 synthetic probe and the disease 3 synthetic probe will remain unbound. The unbound disease 2 synthetic probe and the unbound disease 3 synthetic probe will eventually be cleared by the subject after a period of hours or days. After the time sufficient to clear the unbound disease 2 synthetic probe and the unbound disease 3 synthetic probe, the cleavable linker of the disease 1 synthetic probe will degrade and release the yellow reporter. The yellow reporter can then be detected in a sampleobtained from the subject. Because the yellow reported is detected, the medical professional will be able to diagnose the subject as having disease 1.

[0146] In another exemplary prophetic example, a panel of synthetic probes is administered to a patient suspected of having infection of bacteria 1. The panel of synthetic probes have binding domains that specifically bind to bacteria 1. bacteria 2, and bacteria 3. Each synthetic probe includes a unique reporter of a different nucleic acid (DNA) barcode. For example, the bacteria 1 synthetic probe has the structure: bacteria 1 binding domain-cleavable linker- AA reporter. The bacteria 2 synthetic probe has the structure: bacteria 2 binding domain-cleavable linker-GG reporter. The bacteria 3 synthetic probe has the structure: bacteria 3 binding domain-cleavable linker-CC reporter. All three synthetic probes are administered to the subject and circulate within the subject’s body. Because the subject has bacteria 1, the bacteria 1 synthetic probe having the structure: bacteria 1 binding domain-cleavable linker-AA reporter will bind to the bacteria 1 target, whereas the bacteria 2 synthetic probe and the bacteria 3 synthetic probe will remain unbound. The unbound bacteria 2 synthetic probe and the unbound bacteria 3 synthetic probe will eventually be cleared by the subject after a period of hours or days. After the time sufficient to clear the unbound bacteria 2 synthetic probe and the unbound bacteria 3 synthetic probe, the cleavable linker of the bacteria 1 synthetic probe will degrade and release the AA reporter. The AA reporter can then be detected in a sample obtained from the subject by nucleic acid amplification methods. Because the AA reported is detected, the medical professional will be able to diagnose the subject as having a bacterial 1 infection.

[0147] In another aspect, the present disclosure is directed to a sample processing assembly comprising: a sample collection compartment, an target molecule capture region, and a target molecule detection area.

[0148] The target molecule capture region provides a substrate that captures the target molecule of interest if contained in the sample and allows for the removal of the sample whereby the target molecule of interest is left behind in a concentrated amount. The target molecule capture region can include beads such as paramagnetic beads that can be mixed with the collected sample for a time sufficient for the targetmolecule of interest to bind to the paramagnetic beads. A magnetic force can then be applied to capture and hold the paramagnetic beads while the sample is being removed from the collection compartment. Beads having target molecule bound can be washed using routine methods and solutions. The magnetic force can be removed to allow the beads having bound target molecule to become resuspended in the wash solution. The magnetic force can be reapplied to immobilize the beads for the next step. The beads can be contacted with another solution to remove the target molecule from the beads for detection or other steps. Following removal of the original sample solution, the captured target molecule can be resuspended in smaller volumes to result in concentration of the target molecule as compared to the concentration of the target molecule as in the original sample volume.

[0149] In another aspect, the present disclosure is directed to a method for detecting an extracellular target molecule.

[0150] In this aspect, the assay targets extracellular target molecule (proteins, glycans, toxins) on pathogens or diseased tissues and releases a reporter into a sample. In this application, the target molecule is extracellular and accessible (non- internalizing), stable in vivo > 24 h, minimally cross-reactive, and allow a small reporter to be cleared renally.

[0151] The synthetic probe of the “SynBio” platform can bind an extracellular or secreted target molecule on the pathogen or disease site. The target molecule should not be rapidly and / or be minimally endocytosed (i.e. "non- internalizing”), should persist in circulation or bodily fluids for about 24 hours to about 48 hours, and should be sufficiently unique to the target (minimal host expression or related species to avoid off-target binding). The detected reporter (e.g., fluorescent reporter; quantum dot; carbon dot; small nucleic acid barcode; mini-circle nucleic acid; nucleic acid barcode, etc. as described herein) is cleared via the kidneys into urine, so the assay effectively converts a target molecule binding event into detectable signal by accumulation of a reporter in a urine sample. Thus, target moleculess are accessible by their present in blood or interstitial space (not confined within cells) and not immediately internalized by host cells. Desirably, renal -cl earable reporters allow thetarget molecule-synthetic probe complexes to remain extracellular and not be trapped in tissue or internalized by cells.

[0152] The SynBio platform of the present disclosure using the synthetic probes can be adapted to detect any target of interest. It should be readily understood that the SynBio platform using the synthetic probes and methods of the present disclosure can be adapted to detect, diagnose, and / or treat any target of interest. Where the target of interest is associated with a disease, disorder, infection, pathogen, condition, etc., the SynBio platform using the synthetic probes and methods of the present disclosure can be adapted to detect a target molecule of interest, diagnose a disease, disorder, infection, pathogen, condition, etc., and / or treat the disease, disorder, infection, pathogen, condition, etc. Once the target of interest is identified, one only needs to prepare a binding domain using methods described herein and known to one of ordinary skill. It is well within the abilities of one having ordinary skill in the art to identify a target, identify an epitope of the target, and prepare a binding domain that specifically binds to the target. The binding domain is then incorporated into a synthetic probe having any of the features described herein. For example, the SynBio platform is particularly suitable for assaying for cancer and other diseases, bacterial pathogens, viral pathogens, fungal pathogens, protozoan parasites, helminths and other parasites. The SynBio platform is also particularly suitable for detecting infections and conditions including tuberculosis (detecting lipoarabinomannan), HIV / AIDS (detecting p24 antigen and gp120 glycoprotein), malaria (detecting Plasmodium falciparum erythrocyte membrane protein), viral hepatitis, diarrheal diseases (e.g. cholera, Salmonella), Dengue Fever (detecting non-structural protein 1), Schistosomiasis (detecting circulating cathodic antigen), Hepatitis B (detecting small surface antigen), Hepatitis C (detecting envelope glycoprotein and HCV core antigen), Cryptococcosis (detecting Cryptococcal capsular polysaccharide, glucuronoxylomannan, GXM), Legionnaires’ disease (detecting Legionella pneumophila lipopolysaccharide antigen), Lymphatic filariasis (Wuchereria bancrofti circulating filarial antigen). Chigas disease (detecting secreted T. cruzi antigen) Chlamydia trachomatis (detecting major outer membrane protein and PgP3 protein), Gonorrhea (detecting porin protein PorB), Typhoid (detecting Vi capsular polysaccharide). Syphilis (detecting Treponemalipoproteins Tp47, Tpl5, Tpl7), Pneumococcal pneumonia (C-poly saccharide), Influenza A / B (detecting viral nucleoprotein and hemagglutinin glycoprotein). C. difficile (detecting Toxin B TcdB), Cholera (detecting Cholera toxin B subunit). Respiratory Syncytial virus (RSV; detecting F protein), Strep pneumoniae (detecting Pneumococcal surface protein A), Staph aureus (detecting protein A), Klebsiella pneumoniae (detecting outer membrane protein A), E. coli (uropathogenic; detecting type-1 fimbrial adhesin FimH). Bordetella pertussis (detecting filamentous hemagglutinin), Borrelia burgdorferi (detecting outer surface protein A or outer surface protein C), Helicobacter pylori (detecting BabA adhesin), and neglected tropical diseases. In the US / EU, major burdens include lower respiratory infections (influenza / pneumonia), diarrheal and waterborne diseases, and emerging infections (Lyme disease detecting outer surface protein C; SARS-CoV-2 detecting spike glycoprotein), and thus, the SynBio platform is particularly suitable for assaying for these infections and diseases.

[0153] The method of the present disclosure is particularly suitable for any target molecule of interest, even if circulating in blood, which then permits a synthetic probe to bind to the target molecule. For target molecules below 20 kDa, a dendritic dPEG (for example) with > 1 nanobody can bind multiple target molecules to make a large molecule that would be stable for > 24 hours in circulation to slow or prevent renal clearance and allow for detection. The dedritic dPEG can have a click chemistry stub so that a therapeutic can be applied very specifically. This allows an antibiotic (or anti-virals, etc.) to be applied systemically but only activated very specifically (at the target site). Dosages can be high very locally thereby preserving the patient's microbiome and being much more effective than standard antibiotic use. Additionally, small targets (< 20 kDa) can be detected using other methods such as using two or more nanobodies or other spacer molecules that increase the molecular weight size of the complex to slow or prevent renal clearance and allow for detection.

[0154] Application of the SynBio platform targeting pathogens using bioorthogonal synthetic (SynBio) probes represents a novel extension of pretargeting and click-chemistry technologies. In this system, a first “flagging” agent is a synthetic probe that binds a pathogen-specific extracellular epitope (> 20 kDa protein or anchoredglycan) and installs a stable bioorthogonal stub (e.g. tetrazine, azide, cyclopropene) on the pathogen surface. A second "finishing" agent carries a therapeutic or detectable cargo linked to a complementary bioorthogonal group (e.g. trans-cyclooctene, DBCO, tetrazine) that selectively reacts with the stub (such as via strain-promoted cycloaddition). This enables precise in vivo activation of cargo (drug release or tracer binding) only on flagged pathogens, in principle sparing non-target microbes and tissues. Application of the SynBio platform in this manner combines synthetic probes that install a bioorthogonal “beacon” on diverse pathogens (bacteria, viruses, fungi, protozoa) with systemic delivery of reactive cargo for both therapy and detection. The SynBio platform covers any pathogen class and payload class in a two-step regimen.

[0155] Applications of the SynBio platform include (1) general flagging probes that deposit a covalent bioorthogonal handle on pathogen surfaces; (2) complementary payload conjugates that react in vivo with only the flagged pathogens (therapeutic or diagnostic cargo); and (3) microbiome- / tissue-sparing aspects (since native flora lack the unique epitope-stub combination). For example, the method includes administering a synthetic probe having a binder-stub conjugate, administering a second agent (therapeutic or diagnostic) that “clicks” to the stub on only the pathogen surface.

[0156] In particularly suitable embodiments, the stub is permanently installed on a pathogen surface. Permanent installment includes covalently attaching the stub on a pathogen surface (e.g. via covalent attachment of tetrazine or azide). A later dose of a cargo conjugate (drug or diagnostic reporter linked to the matching reactive handle) then “clicks” only on those pathogens flagged with the stub. This modularity' (exchange of stub chemistries and cargo) offers a unique method of flagging (or “tagging”) pathogens in vivo by depositing a chemical “stub” on their surfaces, and a second agent delivers cargo specifically to those flagged cells.

[0157] Additionally, the synthetic probes of the present disclosure can substantially help ADCs because the stub can have an exceedingly high affinity or avidity to the ADCs and subsequently change the nature of the Drug Conjugate efficacy. After the drug conjugate binds to the flag that remains after cleavage of thesynthetic probe, a second trigger can be administered to result in the release the drug onto the tumor or bacteria (or other pathogen).

[0158] The synthetic flagging probe includes a high-affinity binding ligand (antibody, peptide, aptamer, or engineered cell / phage) and a covalently attached stub chemistry. The stub is stable in vivo until it reacts with the cargo (e.g., therapeutic or diagnostic). Suitable stub chemistries include: tetrazines (which can click with trans- cyclooctenes); azides (which react with cyclooctynes / DBCO); and strained alkenes like cyclopropenes (which react with tetrazines) or bicyclononyne. Each offers fast bioorthogonal ligation without catalysts. For example, TCO-tetrazine IEDDA is among the fastest known reactions, often completing in minutes in vivo. Azide-DBCO SPAAC is slightly slower but very biocompatible. Cyclopropene-tetrazine ligations can also be used to yield a stable conjugate. The synthetic probes may bear one or multiple stubs for signal amplification.

[0159] The finishing agent is a cargo (therapeutic or diagnostic) conjugated to a complementary reactive group: e.g., a tetrazine payload to click with a TCO-stub, or a DBCO-linked drug to click with an azide-stub. Examples of therapeutic cargo include antibiotics (small-molecule drugs), antifungals, antimicrobial peptides, or engineered proteins (antibodies, enzymes, phage-delivered CRISPR). For example, antivirals, quorum -sensing inhibitors, or CRISPR-Cas9 antimicrobials (delivered by phage) are envisioned. Diagnostic cargo are described herein including nucleic acids (e.g., DNA barcodes), peptides, DNA-peptide hybrids, radionuclides (PET / SPECT tracers like18F or111In attached to DBCO or TCO), and fluorescent / optical reporters. The cargo may itself be a prodrug, activated upon click or could simply localize by attaching to the stub.

[0160] Stub-cargo pairs are chosen for bioorthogonal kinetics: e.g. Azide (stub) + DBCO-TLR (cargo) or TCO (cargo) is robust for in vivo use; Tetrazine (stub) + TCO- cargo is ultrafast; cyclopropene (stub) + tetrazine- cargo is also feasible. Dual- modality or cleavable linkers (for click-to-release) can be incorporated. The SynBio platform can encode these chemistries by incorporating noncanonical amino acids orenzy matic tags into the probe. The chemistry building blocks are well-established in literature.

[0161] In another aspect, the present disclosure is directed to a method for collecting and concentrating a target molecule in a sample and diagnosing a disease or a disorder in a subject having or suspected of having the disease or the disorder by detecting a target molecule of interest in the subject with a synthetic probe, the method comprising: administering to the subject a panel of synthetic probes, each of the synthetic probes comprising: a binding domain that specifically binds a target molecule; a cleavable linker; and a reporter, wherein the binding domain, the cleavable linker, and the reporter are in a complex, and wherein each of the synthetic probes of the panel of synthetic probes comprises a different binding domain and comprises a different reporter; allowing the panel of the synthetic probes to circulate in the subject for a time sufficient to allow the synthetic probe to specifically bind the target molecule if present in the subject via the binding domain and for any synthetic probes that remain unbound to be excreted by the subject; collecting a sample from the subject after a time sufficient for the linker to degrade and thereby release the reporter, wherein the sample is collected using a sample collection assembly, the sample collection assembly comprising a sample collection compartment, a target molecule capture region, and a target molecule detection area; and detecting the reporter in the sample, wherein the reporter identity allows for diagnosing a disease or a disorder.Exemplary Embodiment - Design of Synthetic probe Complex

[0162] First Compound (Targeting-Cleavable Linker-Reporter Conjugate)

[0163] The first compound will bind to a specific biomarker or site in the body, such as a tumor cell or other diseased tissue. It will have a bio-orthogonal cleavable linker (i.e., cleavable linker) attached to a reporter molecule (e.g., carbon dots, fluorescent nanoparticles, DNA barcode). This compound will remain stable until the second compound is administered, ensuring that the reporter is only released when the second compound interacts with the cleavable linker.

[0164] Second Compound (Cleaving Agent)

[0165] The second compound is a non-toxic agent that specifically interacts with the first compound through a bio-orthogonal reaction to cleave the linker, releasing the reporter. This compound should be highly specific to the linker, with minimal or no off-target effects.

[0166] Reporter Molecule

[0167] A non-toxic reporter such as a carbon dot, other fluorescent nanoparticle, and / or DNA barcode is linked to the first compound via the cleavable linker. When the second compound is administered, the reporter is cleaved and released into circulation, where it can be excreted (e.g.. via urine) and detected ex vivo using standard analytical techniques such as fluorescence spectroscopy or nucleic acid amplification. Once the cleavable linker is cleaved, reporter molecules (e.g., carbon dots or DNA barcodes) are excreted where they can be detected in a sample.Exemplary Embodiment - Synthetic probe Complex for Renal Clearance

[0168] Use a camelid VHH or nanobody as a binding domain that are 12- 25 kDa and can clear the renal system. The VHlT / nanobody binds to a target in vivo (such as a specific biomarker or antigen in the body). Use a bio-orthogonal linker that allows for precise control over when and where the linker is cleaved. Cleavage of the bio-orthogonal linker releases a detectable reporter (such as a peptide or small molecule) upon cleave that can then be detected ex vivo. Detection can be by lateral flow assay (LFA), for example.

[0169] An VHH is engineered with a bio-orthogonal cleavable linker. This linker should be cleavable by a non-toxic, bio-orthogonal agent administered in vivo, such as tetrazine (Tz) reacting with trans-cyclooctene (TCO). This highly specific reaction ensures that the scFv is only cleaved after it has bound to its target in the body. For example, the VHH can be designed to target a tumor antigen or infectious disease marker. After the VHH binds its target, a bio-orthogonal trigger (such as tetrazine) is administered, which reacts with a TCO group on the VHH. This reaction cleaves the linker, releasing a detectable fragment or reporter molecule.Detection via LFA

[0170] The cleaved fragment from the synthetic probe can be a small peptide or a small molecule that can be detected ex vivo using an LFA. After cleavage in vivo, a biotinylated peptide could be released. This biotin-tagged peptide can be captured on the LFA using streptavidin-coated particles, resulting in a visible signal. Alternatively, fluorescent or gold nanoparticle-conjugated reporter can be used. The cleaved fragment could be a fluorescent molecule or a molecule that can bind to nanoparticles for detection via LFA.Workflow

[0171] Administer the VHH linked to a bio-orthogonal cleavable linker, such as TCO, which binds to a specific target (e.g., a cancer cell surface marker). Administer a tetrazine-containing molecule, which reacts with TCO and cleaves the linker in the VHH. The cleaved fragment (e g., a biotinylated peptide) is excreted into bodily fluids like urine or blood. Collect the sample and use an LFA to detect the cleaved fragment using specific capture agents (e g., streptavidin for biotin-labeled peptides).Exemplary Embodiment - Method of Binding Cancer

[0172] As summarized in FIG. 1, one exemplary embodiment is a method of binding to specific cells using a cancer example. Currently in use nanoparticle tumor therapeutic delivery molecules that bind to specific cancer tumors can be repurposed for use in the method of the present disclosure. A binding molecule such as an antibody can attach to the surface of the tumor. An enhanced permeability and retention effect can be used to passively or actively bind the synthetic probe complex to the tumor. Alternatively, a large linkage cell can be attached to the binding molecule to reduce or prevent penetration of the synthetic probe complex into the tumor cells and maintain the reporter domain of the synthetic probe complex outside of the tumor while binding to the tumor.

[0173] The present disclosure provides a novel in vivo detection system. This system leverages advanced imaging techniques, molecular probes, and biosensors to identify disease with high precision and accuracy. The present disclosure offers a non-invasive, rapid, and reliable diagnostic tool that can be used in clinical settings to improve patient outcomes through early detection and timely intervention.

[0174] The synthetic probes and methods described in the present disclosure can be applied to other diseases and disorders. All that is required to adapt the synthetic probes and methods of the present disclosure is to provide a synthetic probe having a binding domain that specifically binds the a target molecule sought to be detected. For example, the present disclosure can be adapted for viral detection by exchanging a binding domain that is specific to a cancer antigen for a binding domain that is specific to a viral antigen. Similarly, the present disclosure can be adapted for other a target molecules by using a binding domain that is specific to that target molecule.Exemplary Embodiment

[0175] A broad list of over a thousand potential tumor-associated targets for recurrence detection, spanning tumor cell markers, stromal / angiogenic markers, and soluble factors was compiled. Considerations included prevalence across tumor types, specificity to malignant tissue (minimal normal tissue expression), and availability of binders. Early candidates included oncofetal extracellular matrix proteins (oncofetal ED-B fibronectin, oncofetal ED-A fibronectin, as distinguished from other ED-B proteins), angiogenesis markers (CD 105, VEGFR2, a_vβ_3 integrin), immune checkpoints (B7-H3, PD-L1), and known tumor antigens (EpCAM, HER2, etc.). The list was narrowed using a quantitative scoring of each marker's attributes: (a) Tumor specificity - whether the target is largely absent in healthy adult tissues; (b) Expression prevalence - fraction of diverse tumor types and recurrences that express the marker; (c) Association with recurrence / aggressiveness - evidence that marker upregulation correlates with relapse or metastasis; (d) Accessibility - extracellular or vascular location accessible to probes; (e) Availability of high-affinity ligands or binders; and (1) Non-redundancy - covering complementary biological facets (tumor cells,neovasculature, tumor ECM), and (g) binding site stability to maximize chance of detection with no endocytosis. Through this process, ED-B fibronectin, Endoglin (CD105), and B7-H3 were selected as three primary targets covering tumor microenvironment compartments and added an optional pH-activatable ED-B probe to enhance specificity if clinically necessary. Other candidates were dropped: for example, ED- A fibronectin was considered but rejected due to its broader expression in inflammation; integrin targets were deemed less specific (often widely expressed on normal endothelium); PD-L1 was not chosen because its expression is heterogeneous and it can be inducible in inflammatory conditions as well as being shed in the bloodstream and hence may quench the assay signal; moreover circulating markers (like VEGF or IL-6) were not suitable for the probe strategy since the platform relies on localized binding and reporter release rather than measuring an endogenous target molecule. Bi-specific nanobodies may be used to further refine specificity, if demonstrated to be clinically necessary.

[0176] DNA barcodes were selected as they can be uniquely coded for many targets and detected with ultra-sensitivity via PCR or CRISPR. A concern with DNA was susceptibility to nucleases in vivo, but chemically stabilized oligonucleotides (phosphorothioate backbones and 2’ modifications) were adopted. The barcodes were also designed to avoid immunostimulatory motifs (e.g. CpG dinucleotides) to reduce innate immune activation or rapid clearance due to anti-DNA antibodies.

[0177] Masking Efficacy: The pH-masked probe’s performance will be tested in vitro by exposing it to buffers of different pH and then adding TCO to determine if the DNA is released. A negligible release is expected at pH 7.4 (mask intact) and substantial release is expected at pH 6.5 (mask cleaved, tetrazine reacts with TCO to release DNA). Additionally, acidic vs. neutral environments can be simulated in cultured cells and tissue models. If needed, the linker chemistry will be adjusted (e.g. use a different hydrazone with a faster or slower hydrolysis rate) to fine-tune this trigger.

[0178] For the masked probe’s linker, the hydrazone is the only part designed to break, and only at low pH. In circulation (pH 7.4), its half-life is longenough that essentially no DNA should release in the 24h window. This will be confirmed by incubating the probe in human serum at 37°C for 24h and checking if any free barcode appears.

[0179] Negative Control Design: The negative control probe uses a scaffold protein that does not bind to anything in humans. A non-targeting antibody (like an IgG against a plant protein) or a mutated version of one of our scFvs that abolishes binding was considered. But, an irrelevant VHH was instead chosen as the scaffold. Alternatively, an anti-dye or anti-hapten nanobody could be used (with no target present in humans). This probe is conjugated with a DNA barcode that is designated as the reference. In analysis, the ratio: (target DNA Code / circulating control DNA Code) may be used as the key metric. A ratio »1 means target accumulation; ~1 means no specific accumulation. The higher the ratio, the clearer the signal detected.

[0180] In summary, unique synthetic DNA barcodes (~20 nt single-stranded DNA) were chosen as the primary reporters on each probe. As a secondary measure, distinct fluorophores can be added to each probe (e.g. Alexa 488, 568, 647, etc.) for use in preclinical imaging or as an alternative detection mode. The fluorescent readout also provides a useful development tool (for tracking probe distribution in animal models or rapid prototype testing). DNA barcodes are selected as the main analytical output, while fluorescence may be built-in as an alternative.

[0181] Activation Mechanism: A fundamental design decision was how the probes would be triggered to release their reporters at the tumor site. Three mechanisms were considered:1. Passive physical targeting only, where binding to the target is the sole step and reporters are constitutively active (released either continuously or by a simple change in context);2. Protease-mediated cleavage, as used in earlier synthetic probe systems where tumor-associated enzymes cut a linker to release reporters;3. An exogenous click-chemistry trigger, where an administered bioorthogonal reagent prompts release of reporters on-demand.

[0182] Many prototypes by others relied on protease-triggered release of reporters (for example, nanoparticles coated in peptides that are cleaved by matrix metalloproteinases or other tumor proteases, liberating a reporter that is then detected in urine). While elegant in leveraging tumor enzymatic activity, several drawbacks to a protease-dependent strategy were identified:1. Protease expression can vary widely between tumor types and patients, risking inconsistent sensitivity (a patient with low levels of the protease or low- levels in low-perfusion tumors might produce little signal).2. Many proteases (MMPs. cathepsins, etc.) are also present in benign conditions like inflammation and wound healing, which could cause false positives if, for example, a patient has an injury or infection.3. Achieving multiplexing is challenging, as proteases often have overlapping substrate preferences; designing multiple orthogonal peptide substrates that do not cross-react is complex.4. Timing control is poor in enzyme-driven systems - reporters are released continuously at a rate dependent on local enzyme kinetics and degree of perfusion, making it harder to synchronize signal capture. This critical piece would require increasing dosage of reporters and synthetic probes, which may increase potential immunogenicity and costs.5. Designing to minimize administered dosage would reduce any unwanted biological effects in the patient. Targeting bioorthogonal elements for the probe that would target 10 micrograms or less would provide a substantial safetymargin for patient tolerance and regulatory- approval. Control signal release temporally would help with this design constraint.

[0183] Given these concerns, a two-step bioorthogonal chemistry approach C click-to-release") was chosen to exert external control over when and where reporters are liberated.

[0184] In the mechanism of this Example, the probes are administered and allowed to accumulate at any tumor sites via their targeting ligands (passive targeting phase) over the course of hours to compensate for perfusion differences and allow robust binding. A small-molecule trigger is then administered that performs a rapid bioorthogonal click reaction with the probes, cleaving the linkers and releasing the reporters in a relative temporal spike. Specifically, a trans-cyclooctene (TCO) functional group is attached to each probe's linker and tetrazine is administered as the trigger. Tetrazine-TCO cycloaddition is an extremely fast and selective reaction that proceeds readily in vivo without interacting with native biochemistry. The perfusion of the small tetrazine molecule throughout the subject’s body will be fast and specifically reactive with the TCO to release the reporter. By designing the linker such that the tetrazine-TCO ligation causes a bond cleavage (through a retro-Diels-Alder elimination), the DNA barcode payload is released upon click reaction. This click-to- release strategy' is validated by recent advances in pre-targeted imaging and drug delivery, where TCO-tetrazine reactions have been used to release drugs or radionuclides at target sites. This approach provides tight temporal control: the release is initiated at a chosen time point (after probes have had time to localize to tumors and unbound probes have cleared from circulation, perhaps after 24 hours), and the reaction occurs quickly and then stops once the trigger is cleared. Moreover, it amplifies specificity - only probes that bound to a target of interest (and thus remain in the tumor) will end up releasing reporters locally.

[0185] A biotin can be added to the probe, if necessary, to enable an Avidin Chase to be employed, where any remaining circulating control probes that have not bound to a target will be eliminated by hepatic uptake. This would substantially attenuate any background signal of unbound probes. For the control probes, however, there may be an advantage to having some baseline signal as a positive control for the assay itself.

[0186] Additionally, making tetrazine the bifunctional linker and TCO the trigger, as well as the reverse, were considered as this affects reaction kinetics and stability concerns. It was decided to put tetrazine on the probes and use a small TCO molecule as the injected trigger (rather than TCO on the probe and a tetrazine trigger). This decision was based on both chemical and pharmacological rationale: tetrazine- TCO reactions have some of the highest second-order rate constants known in bioorthogonal chemistry (~ 106M1s1). A small TCO can more easily diffuse and reach the tetrazine on a large probe, whereas if the bulky probe earned the TCO, an injected tetrazine might face steric hindrance, slowing the reaction. Additionally, certain optimized TCO variants (e.g. sTCO) are highly water-soluble and have favorable in vivo stability, making them suitable as an injected trigger. The TCO trigger can be given in excess to drive the reaction to completion quickly, and any unreacted TCO will naturally clear from the body within a short time (on the order of an hour or two). This way, the click reaction is essentially self-limiting: it fires off the reporters and then ceases as TCO is eliminated, providing a defined snapshot of reporter release.

[0187] Linker Stability: Before trigger, the linker holding the DNA must be stable (especially for the unmasked probes). Linkers were chosen that are entirely stable in plasma (no enzymatic cleavage sites, all synthetic components). To mitigate susceptibility of tetrazine to reduction by some endogenous factors using a di-fluoro substituted tetrazine that has been shown to be more stable in vivo (published variants with half-lives of several hours in mice). The tetrazine is also somewhat buried in the PEG linker to reduce exposure.

[0188] Reporter selection was made between optical reporters (fluorescent dyes), carbon dots or DNA-based reporters for the probe signals. DNA barcodes were selected as the primary reporters due to their inherent multiplexing capacity and compatibility with ultra-sensitive nucleic acid detection techniques. A DNA barcode (e.g. a unique 20-base oligonucleotide) allows each probe to carry a sequence that can be independently quantified in a single sample, enabling dozens of multiplexed readouts in principle In contrast, using distinct fluorescent dyes may limit multiplexing and each additional dye complicates instrumentation. Moreover, DNA reporters can be massively amplified (via PCR or isothermal amplification) to detect minute quantities.reaching femtomolar or attomolar sensitivity, whereas fluorescent detection may require nanomolar concentrations. An advantage of the Fluorophore is current use and familiarity from a regulatory perspective. The design of the assay requires a trace number of reporters so that this may ease regulatory use. If Human Bar-Coding DNA proves challenging for obtaining regulator)' approval (e.g., FDA & EMA), fluorophores are viable alternatives.

[0189] Stability of DNA in vivo was a concern as unmodified oligonucleotides can be rapidly degraded by nucleases. This was addressed by using chemically stabilized DNA: each barcode is a single-stranded DNA with modifications such as phosphorothioate linkages and 2’ sugar modifications to resist exonucleases. Indeed, prior work has shown that 20-mer phosphorothioate DNA barcodes remain intact in circulation long enough to be collected in urine. Confidence in using a DNA barcode reporter approach also arises from the safety of diagnostic oligonucleotides in humans: FDA-approved antisense drugs and aptamers (typically 15-25 bases, chemically modified) have been administered safely. The DNA barcode reporters here are designed to mimic human genomic sequences lacking immunostimulatory motifs (e.g. avoiding CpG dinucleotides) to minimize any innate immune activation or rapid clearance due to anti-DNA antibodies. Somamers and aptamers provide other options for bioorthogonal reporters that can be detected by CRISPR and PCR.

[0190] Each probe will have a distinct sequence that serves as its “ID” in the readout. As a contingency, probes can be formulated with a fluorescent label (Alexa Fluor or similar) as an alternative or even secondary reporter (“fallback”). In early development (animal studies, benchtop tests), the fluorescence can be useful to trace probe distribution or quickly validate the presence of a signal. Clinically, however, the fluorescence mode is not intended for routine use due to its lower sensitivity and limited multiplexing. Spectrally distinct dyes are assigned to each probe (e.g. Alexa 488, 568. 647, etc., up to 4-5 probes). This optical readout could potentially be used in a simplified point-of-care device if needed or desired. Maintaining the Alexa labels adds minimal cost or complexity and provides an additional or alternative detection channel and a way to visualize probe behavior in preclinical models.

[0191] With DNA barcodes as reporters, how best to detect and quantify them from patient samples was evaluated. The options considered included conventional qPCR, next-generation sequencing (NGS), and novel CRISPR / Cas-based detection assays: CRISPR / Cas12a-based detection. A CRISPR / Cas system (specifically a Cas12a collateral cleavage assay) was chosen as the primary detection modality. CRISPR-based diagnostics (e.g. SHERLOCK, DETECTR) can achieve extremely high sensitivity (attomolar range) with sequence-specific detection.

[0192] In these assays, a Cas enzyme is programmed with a CRISPR RNA (crRNA) to recognize a specific DNA sequence (the DNA barcode Reporter). If the target DNA is present, the Cas enzyme becomes activated and nonspecifically cleaves a reporter probe, producing a detectable signal (fluorescent or colorimetric). These assays often operate isothermally (using techniques like RPA or simple amplification) and can be implemented in paper-strip or microfluidic formats.

[0193] A multiplexed CRISPR assay can be set up with parallel reactions, each one detecting one barcode. A cartridge is envisioned where sample (urine) is introduced, divided into wells or channels, each containing a specific crRNA and Cas 12a targeting one of the DNA barcodes. If that barcode is present above a threshold, the corresponding chamber will light up (fluorescence or lateral flow line).

[0194] CRISPR detection offers several advantages: it does not require thermal cycling (unlike PCR), so devices can be simpler; it has single-base specificity, preventing one barcode’s signal from being misread as another’s; and it can be very fast (results in under an hour). Sensitivity is on par with or even better than PCR - Cas 12 / 13- based methods have reached limits of detection in the low femtomolar to attomolar range with a brief amplification step. For our context, since the DNA barcodes are designed and known, each crRNA is designed to ensure no cross-reactivity.

[0195] Additionally or alternatively, quantitative PCR is used an altemative / backup method. qPCR is widely available in clinical labs and familiar to regulators. TaqMan-style assays can be designed for each DNA barcode (with a unique fluorescent probe per target). In a multiplex qPCR, typically up to 4-5 targets can bemeasured simultaneously with different fluorophores - which aligns with the probe count. qPCR has excellent sensitivity (down to ~ 101-102copies, though practically ~103in a clinical setting), but it requires careful calibration to quantify multiple targets and is slower (~1— 2 hours turnaround including DNA extraction). It also needs more manual sample handling (DNA extraction, etc.).

[0196] Additionally or alternatively, a NGS-based readout (basically sequencing the mixture of barcodes in a sample) may be used. This approach, used by some ctDNA MRD tests, is very sensitive and can distinguish many sequences. But for routine clinical testing of a fixed panel, NGS may introduce higher cost, longer turnaround (days), and complex data handling.

[0197] Additionally or alternatively, mass spectrometry (MS) could be used, but MS is not ideal for multiplex nucleic acids and is not commonly available in clinical labs for this purpose. Similarly, electrochemical DNA sensors, while promising, are not yet at a mature stage for multi-target molecule clinical use.

[0198] In view of these considerations, a Cas12a-based multiplexed assay was chosen as the primary readout, with qPCR as a secondary option. Cas12a was chosen because it targets DNA directly (reporters are DNA) and its collateral cleavage yields a strong fluorescence signal easy to read. crRNAs are designed for each of the 5 DNA barcodes (4 targets, 3 core and one masked, if necessary, + 1 circulating control). Each crRNA will be validated for specificity (ensuring it does not trigger any other barcode even at high concentration). The detection instrument is a relatively small fluorescence reader or even a modified plate reader or PCR machine for initial trials. In parallel, a qPCR protocol will be validated that can serve as a fallback or additional detection method. A Cas12a assay can also be made into a near point-of-care format for use in clinics without specialized labs.

[0199] For probe dosages, microdoses well below therapeutically acceptable levels are initially intended. By design, a diagnostic is being delivered (not attempting to treat or accumulate massively in tumor), so doses can be exceptionally low. Based on modeling of the synthetic probes of the present disclosure, clear signalsmay be obtained. Initial calculations indicate a Rough Order of Magnitude dose in the 10-microgram range per probe may be more than sufficient for detection. For example, even if only a tiny fraction of probes reach a small tumor, the amplification through reporter release and CRISPR detection yields a detectable signal (see Section 3 for detailed PK / sensitivity modeling). This is also low enough to minimize any potential unknown toxicity. For comparison, approved diagnostic antibodies for imaging (e.g. radio-immunotracers) have been given at ~0.1-0.2 mg / kg with excellent safety. The chosen dose of the synthetic probes of the present disclosure is orders of magnitude lower. Additionally, the DNA payload per dose is negligible (perhaps less than a microgram), much less than doses of antisense oligonucleotides that have been safely used (which are on the order of tens of milligrams).

[0200] A pharmacokinetic (PK) model of the probes and released reporters, including assumptions on absorption, distribution, metabolism, and excretion (ADME) of each component was performed. The model was anchored on the latest detection limits of CRISPR / Cas readouts (attomolar sensitivity) and quantified how much reporter can be expected in urine given a ~1 cm tumor across different perfusion scenarios. These analyses guided the synthetic probe dose selection (ensuring even low- perfusion lesions yield signal above the limit of detection) and sampling strategy. Regulatory considerations were also integrated (microdosing and combination-product guidance) into the dosing strategy. Lower sensitivity CRISPR / Cas readouts in the femtomolar range were also considered. FIG. 3 illustrates a timeline from initial synthetic probe administration, to distribution, to tumor binding, to triggering agent injection, to reporter release, and to excretion.Pharmacokinetic Simulation of a 35 kDa ED-B-Targeted SynBio Probe - Preclinical Mouse Model

[0201] Assumptions and physiological characterization were based on NJB2 Nanobody (15 kDa) study results. The first SynBio probe uses an ED B VHH as the binding element for the SynBio probe. The NJB2 nanobody (VHH) is a ~15 kDa single-domain antibody selected for high affinity to the EIIIB extra domain of fibronectin (ED-B). In vitro biolayer interferometry measured a binding affinity KD- 1.9-2.3 nM, with rapid association (ka« 105-106M- 1s-1) and very slow dissociation (kd~ 10-5-10-3s-1). This strong affinity suggests that once NJB2 binds ED-B in vivo, it will remain bound for hours unless the target or nanobody is degraded.

[0202] Immuno-PET imaging in mice showed specific accumulation of radiolabeled NJB2 at tumor sites within 2 h and persistent tumor binding at 24 h post-injection. At ~2.5 h after injection, ex vivo biodistribution analysis found significantly higher uptake of NJB2 in ED-B-positive tumors (~16.9-fold tumorblood ratio). This corresponds to an estimated tumor uptake on the order of several % of the injected dose per gram (%ID / g), versus blood levels below 1 %ID / g at that time. By 24 h, the tumor-to-background contrast remained high (tumor TMR > 4) despite some signal decrease, indicating NJB2 remained bound in the tumor ECM for at least one day. Unbound nanobody cleared rapidly via the kidneys.

[0203] Key kinetic parameters extracted from the study are summarized below (mean values in mice):* KD ≈ 2 nM (high affinity)* Blood half-life (15 kDa VHH) -0.5-2 h* Tumor uptake at ~ 2 h: 5-10 %ID / g* Turnonblood ratio @2.5 h: 16.9* Sustained tumor binding at 24 h

[0204] These data confirm that NJB2's small size yields very fast bloodstream clearance (on the order of an hour) and excellent early tumor contrast. For SynBio probe applications requiring prolonged tumor exposure, a modified probe with delayed clearance is desirable. A 35 kDa SynBio probe was therefore considered using the NJB2 VHH coupled to a PEG extension and DNA barcode (total -35 kDa).

[0205] A two-compartment pharmacokinetic model was used to simulate distribution of the 35 kDa ED-B-targeted probe versus a non-binding control of equal size. The governing differential equations (expressed in probe * amounts*, A_p and A_t) are: dA_p / dt = -k_e • A_p - k_pt • A_p + k_tp • A_t dA_t / dt = k_pt • A_p - k_tp • A_t where k_e is the first-order elimination constant from plasma (dominated by renal filtration), k_pt the rate of distribution from plasma to tumor, and k_tp the reverse rate (effectively the unbinding rate when the probe is targeted).Parameters* Dose: 10 pg IV bolus* Plasma volume Vp: ~2 mL (mouse)* k_e (35 kDa): ln2 / 1.5 h ~ 0.46 h-1(plasma t½ ~ 1.5 h)* k_pt: 0.05 h-1(calibrated to match observed tumor uptake)* k_tp (binder): 0.05 h-1(slow dissociation; unbinding t½ ~ 14 h)* k_tp (control): 0.20 h- 1(rapid interchange; no specific binding)Simulation Results

[0206] The targeted 35 kDa SynBio probe and the non-targeted control exhibit rapid initial distribution and clearance from blood. Plasma %ID falls below 10 % within 5 h for both agents (FIG. 11 A). The SynBio probe shows a slightly faster early decline because a fraction partitions at / into the tumor. A ±20 % variation in k_e (shaded band) illustrates the impact of renal -function variability. As depicted in FIG. 11B, the non-tumor binding control probe approaches complete systemic elimination by 24 h (<0.15 %ID in blood, <0.3 %ID anywhere). In contrast, the SynBio probe accumulates in / at the tumor, peaking at ≈ 7-8 %ID around 4-6 h and still retaining~ 3.6 %ID at 24 h. Thus, by 24 h the SynBio probe localizes roughly an order of magnitude more SynBio probe at the tumor site than the control probe is retained anywhere in the body.

[0207] The hourly %ID values are tabulated in Table 1 below:Table 1. Simulated %ID Distribution (Hourly) Mouse Model.

[0208] Circulating inert control (%ID vs. time). The 35 kDa control is filtered rapidly by the kidneys. Less than -0.40% of the dose remains in circulation at 24 (essentially negligible). Most clearance occurs in the first 8-12 h. This rapid elimination of unbound probe is cntical for reducing background. (For comparison, a larger 50 kDa Fab would clear more slowly, leaving -25% at 24 hours - underscoring why smaller probes give faster background clearance.)

[0209] Increasing the molecular weight from 15 kDa to 35 kDa approximately triples the plasma half-life, allowing a larger cumulative fraction of the dose to find and bind tumor targets. Combined with nanomolar affinity, the 35 kDa ED-B SynBio probe is predicted to maintain -3-4 %ID in tumor 24 h post-dose while systemic levels fall below 1 %ID after -8-12 h. This kinetic profile — rapid targeting, delayed clearance — supports downstream triggered-release detection strategies.

[0210] The inert control probe (35 kDa, no binding) clears mono- exponentially. Baseline signal decays very rapidly after injection - a design goal for clear imaging. By 24 h, the control is >99.7% eliminated, providing a low baseline for contrast. Notably, clearance can vary and may affect absolute and relative results of the control and signal.Pharmacokinetics, Distribution, and Signal Amplification - Adult Human Model

[0211] To project the above probe system into an adult human setting, model parameters were adjusted to reflect typical human physiology and size. Table 2 (Human) summarizes the key pharmacokinetic assumptions with referenced values for a -70 kg adult. Importantly, the probes remain -35-37 kDa VHH-based agents (the ED-B-targeted probe modeled on NJB2 with PEG / DNA extensions, plus analogousCD105 and B7-H3 targeted probes of similar format), and each is administered at a microdose (10 pg per probe). This dose is in line with Phase 0 '‘microdosing'’ regulatory limits ( 1 / 100th of atherapeutic dose) and is orders of magnitude below typical antibody imaging doses, ensuring minimal risk or pharmacologic effect. Thus linear, dose- proportional kinetics and no immune reactions were assumed (the humanized VHH fragments are not expected to be immunogenic in this single-dose context).Table 2. Human Pharmacokinetic Model Parameters (Adult 70 kg)

[0212] Using these parameters, the two-compartment model was adjusted for human dimensions. Plasma volume is expanded (~4 L), so an initial IV dose distributes into a larger space, yielding a lower initial concentration than in a mouse. Glomerular filtration (absolute -7.5 L / h in a 70 kg adult) is much higher than in a -25 g mouse (~0.2 L / h), but humans clear substances more slowly relative to body weight - thus the 35 kDa probe’s half-life is expected to be a few hours (-2.3 h base was used, doubling to -4.6 h if GFR is halved). Negligible non-renal clearance and no FcRn recycling (consistent with a VHH fragment lacking an Fc domain) was assumed. The three active SynBio probes (ED-B, CD105, B7-H3 targeted) are modeled identically in pharmacokinetic terms; any differences in their tissue distribution are assumed to stem from target localization rather than fundamental PK. given their similar size and structure. Each probe is given as a simultaneous IV microdose (with an inert control probe, also -35 kDa).

[0213] Rapid renal elimination of unbound probes is desirable for achieving high contrast. In the human model, the inert control probe (non-binding, 35 kDa) is predicted to behave like a one-compartment system with elimination k_e = 0.30 hA-1 (t_1 / 2~ 2.3 h) under normal renal function. In a typical adult this means the control’s blood concentration falls to -50% within -2.3 h and to <1% by -12-15 h. With impaired renal function (50% GFR), clearance is significantly slower (t_l / 2~ 4.6 h). causing the unbound background probe to linger longer. FIG. 1 1 A illustrates that thenormal-clearing probe is >99% eliminated by 24 h, whereas impaired clearance leaves a small residual (~5— 10% ID at 12 h, -1% at 24 h) in circulation. In practice, patients with moderate renal impairment (e.g. eGFR ~60 mL / min) would likely still clear a microdose probe relatively quickly. For example, clinical data on a 55 kDa BiTE antibody indicated no dose adjustment was needed for mild / moderate impairment. Nonetheless, the SynBio model conservatively accounts for such variability, and in all cases the inert control probe should not accumulate in blood or tissues because it lacks any specific binding mechanism. Ensuring the control’s near-complete clearance also provides a low background signal for the SynBio probe readout.

[0214] For the active, tumor-targeted SynBio probes, a plasma-tumor two compartment model analogous to the mouse simulation was uased, scaling perfusion- related parameters to human values (Table 3). Tumor perfusion can vary widely across cancer types and patients, therefore three perfusion scenarios were simulated: low (e.g. pancreatic ductal adenocarcinoma. PDAC), moderate (e.g. breast carcinoma), and high (e.g. renal cell carcinoma, RCC). The plasma-to-tumor transfer rate k_pt was set for each scenario based on the notional blood flow: in the model, k_pt represents the fraction of plasma content moving into tumor per hour. Using the mouse calibration as a starting point (where k_pt -0.05 hA-l produced a few %ID uptake in a moderately perfused tumor), we assigned approximately: k_pt. low -0.01 hA-l, k_pt. mod -0.03 hA-l, and k_pt ,high -0. 10 hA-l . These values qualitatively reflect that a hypervascular tumor might receive 3 * or more blood flow than a normovascular one. The tumor efflux rate for the binder probes (k_tp was set to a slow value (0.03 hA— 1) for all scenarios, corresponding to an -23 h half-life for dissociation - consistent with the high affinity of the VHH (in vivo, most bound SynBio probe is expected to remain at the tumor target for many hours). In contrast, for the inert control probe (which does not bind the tumor), a perfusion-limited reversible distribution was assumed: effectively no net retention. This was implemented by setting the control’s k_pt equal to its k*_tp (using the moderate perfusion rate), so that the control freely equilibrates between plasma and any tissue but does not build up in tumor. Each probe was administered as a 10 μg IV bolus, and simulations were run from 0 to 24 hours in 1-hour increments.

[0215] Table 3 shows the Human model output for the ED-B targeted SynBio probe's tumor uptake (% of injected dose in tumor) under low, moderate, and high tumor perfusion conditions, alongside the circulating control probe’s blood %ID. The qualitative trends mirror the mouse model, but with notable quantitative shifts:Table 3. Human Simulated %ID Distribution (Hourly) by Perfusion Level

[0216] By extending the SynBio probe half-life to ~2-3 h in humans, more of the SynBio probe dose is available to perfuse tumors before being cleared, leading to higher absolute tumor binding in all perfusion scenarios compared to the mouse model. High-perfusion tumors (magenta) uptake -11-12% ID by -5-8 h and retain -15% ID at 24 h, whereas low-perfusion tumors (orange) slowly accumulate only -2.5% ID by -8 h, plateauing at -1.8% ID by 24 h. The control probe (blue) is almostcompletely cleared from blood by 24 h (0.5% ID remaining with normal renal function). Annotations on the curves indicate the 24 h tumor-to-blood ratio (T:B) for each perfusion class, highlighting that higher perfusion drives dramatically improved contrast (T:B ~3.3x low, 9.5x moderate, and ~28x high in this simulation).

[0217] Several key differences emerge in the human model relative to the mouse baseline. First, clearance is somewhat slower (t_l / 2 ~2.3 h vs. ~1.5 h in mice), so the unbound probe persists longer in circulation. This is evident from the control probe’s curve and contributes to higher total tumor delivery: even low-perfused tumors eventually accumulate -1.8% ID by 24 h in the human, roughly double the -0.96% ID seen in the mouse low-perfusion case. Second, because of the larger blood volume and slower clearance, the peak tumor uptake occurs later in humans. In high-perfusion tumors, the binder reaches -11-12% ID at -6 h (vs. -4-6 h in mouse), and tumor uptake remains elevated for longer. Third, the tumor-to-blood ratios tend to become more extreme by 24 h in the human model: not only is the tumor uptake higher, but the blood background is lower (since even with slower half-life, 99+% of free probe is gone by 24 h). For example, in a well-perfused tumor, -15.3% ID is in tumor vs. -0.54% ID in blood at 24 h. The tumor-to-blood (T:B) ratio rises steadily over 24 h in all perfusion scenarios as the blood pool clears. High perfusion (magenta) yields T:B > 10 after -12 h. reaching > 20 by 24 h. whereas low perfusion (orange) only reaches -3 by 24 h. These results reinforce that perfusion is a determinant of how much probe can accumulate at / in a tumor before clearance - a factor that will vary between tumor ty pes and patients.

[0218] In this human scenario, three active probes were envisioned (ED-B, CD105, B7-H3) plus one inert control probe administered together. The above PK analysis was based on the ED-B probe data, but the other targeted probes are similar in size and administered at the same microdose, so their pharmacokinetics should be very similar. All three would clear from the subject’s body rapidly unless bound to their targets. Minor differences could arise: for instance, CD105 (Endoglin) is expressed on tumor neovasculature, so a CD105targeted probe might effectively experience a higher apparent perfusion (since targets are directly in the blood flow path). In a highly vascular tumor, a CD 105 VHH might bind nearly as soon as it enters the lesion, possiblyenhancing initial uptake rates. B7-H3, in contrast, is an epithelial tumor cell-surface marker; a B7H3 probe relies on extravasation into tumor tissue, akin to the ED-B case, so its kinetics would align with the modeled curves (perfusional delivery, then binding to cell surfaces). We assume none of these binds significantly in normal tissues at the low dose (their targets are tumor-associated antigens). If one probe were to clear slightly faster or slower (due to target-mediated disposition differences), the control probe provides an internal reference: it should clear identically in all patients (assuming normal renal function), serving as a real-time indicator of each patient’s clearance rate. In an impaired-renal-function patient, all probes (including control) would retain longer in blood - possibly necessitating a later urine collection or a correction factor.

[0219] The PK results confirm a crucial design feature: by ~24 h postinjection, the signal -to-noise ratio is strongly in favor of tumor-retained probes versus any circulating background. This timing supports administering the bioorthogonal trigger (the small-molecule TCO) at 24 h to selectively cleave and release DNA reporters from any probes bound in the tumor. Because unbound probes have mostly been cleared, the released reporters overwhelmingly originate from tumor- bound probes. These DNA reporters are ~5 kDa fragments that are rapidly filtered into urine. The inert control probe, having no accumulation, should yield minimal background DNA release upon trigger (<0.5% ID worth of reporter in blood). Any residual circulating probe (e.g. in a renally impaired patient) would release a proportional amount of reporter, which is why the trigger timing may be adjusted for slower clearance individuals or include a second ‘‘background’' DNA barcode to quantify any off-tumor activation. However, given the microdose and short t 1 / 2 it is expected that in most patients a single 24 h time-point trigger will cleanly differentiate tumors. The control probe’s reporter serves as a negative reference - if significant control signal is detected, it indicates either early trigger timing or abnormal retention, prompting data interpretation with caution.

[0220] In parallel to the synthetic probe’s reporter readout from urine, imaging the probes (if radiolabeled) during the distribution phase is contemplated. For instance, an89Zr-labeled ED-B nanobody (with -3.3 d isotope half-life) could be used to image tumor uptake at 24-48 h. Likewise,64Cu (t_l / 2 -12.7 h) is well-matched tonanobody kinetics and has been used with NJB2 for PET imaging of tumors. Radiotracer imaging of the SynBio probes in a Phase 0 clinical study would allow direct confirmation of tumor targeting and clearance in humans. Regardless of imaging modality, the PK simulations indicate that sub-10 pg doses would still achieve detectable uptake (a few percent ID) in tumors by 24 h, even in low perfusion cases - highlighting the signal amplification advantage of the approach of the present disclosure. Each bound SynBio probe can release many reporter molecules (DNA barcodes) upon triggering, and those reporters are detected via ultrasensitive CRISPR / Cas12a assays ex vivo. Thus, even 1% ID retained in a tumor could translate to a robust urine DNA signal well above background. Detection is performed ex vivo using a CRISPR / Cas12a assay, which can sensitively detect specific DNA barcodes in unprocessed urine (attomolar sensitivity in optimized systems). The CRISPR readout has an assumed limit of detection (LoD) of 5><10A5 copies per mL.

[0221] Current clinical practice for post-treatment cancer surveillance relies primarily on imaging and, in select cases, blood biomarkers. For example, a colorectal cancer survivor may undergo periodic CT scans and have CEA (carcinoembryonic antigen) blood tests; a lung cancer patient might get scheduled chest CTs; a breast cancer survivor could receive annual mammograms or MRIs. While imaging can directly visualize tumor regrowth, its sensitivity is constrained by lesion size and contrast - typically on the order of 5-10 mm detectable on CT or MRI under optimal conditions. Very small clusters of cancer cells or diffuse microscopic disease remain invisible on scans. Furthermore, frequent serial imaging is impractical due to cost and, for CT. cumulative radiation exposure. Blood tumor markers, where available, offer a simpler repeated test but suffer from modest performance. Many common solid tumors (lung, colorectal, etc.) lack a highly specific circulating marker; even where one exists (e.g. CEA, CA-125, PSA), background variability and non-malignant elevations undermine reliability. The net result is that a significant fraction of recurrences are not detected until they become symptomatic or sizable enough to image, at which point the window for curative intervention may have closed.

[0222] In the last decade, liquid biopsies have introduced new ways to monitor cancer. The leading approach is detection of circulating tumor DNA (ctDNA)fragments in plasma as a surrogate for minimal residual disease. Personalized, tumor- informed ctDNA assays (where a patient’s tumor mutations are known and tracked) can achieve impressive specificity. In research settings, such assays have identified recurrence an average of 3-6 months before radiographic detection in some cancers3. However, sensitivity remains a limiting factor - if the residual tumor sheds DNA below the detection threshold, the test yields a false-negative. Sensitivity of ctDNA for detecting relapse varies by cancer type and stage, but is often on the order of 50-70% for high-risk Stage II— III disease and much lower for truly tiny lesions. For instance, a large validation study of a multi-cancer early detection (MCED) blood test (Galleri) reported an overall sensitivity of -51% for known cancers, with only -17% detection in Stage I and -40% in Stage II cancers7. This underscores that small tumors frequently evade ctDNA detection - likely because they release minuscule amounts of DNA into circulation. Another drawback of generic MCED tests is that a positive result does not immediately localize the tumor; extensive diagnostic workup (imaging, biopsies) is needed to find the cancer, which can delay intervention. Other liquid biopsy target molecules (e.g., circulating tumor cells, exosomes, microRNAs, etc.) are under investigation but none have yet achieved the sensitivity / specificity balance required for routine surveillance of asymptomatic patients.

[0223] Beyond blood-based tests, some creative strategies are being explored. For example, genetically engineered bacteria or probiotics have been used in experimental settings to detect tumors - by selectively colonizing tumor environments and releasing a reporter (such as an enzyme or chromogen) that can be measured in urinel. These "bacterial diagnostics” and other synthetic biology devicesl highlight the growing trend of actively query ing the body for cancer presence, rather than passively waiting for biomarkers to appear. Nevertheless, such approaches face their own safety and regulatory hurdles (e.g. introduction of live microbes). Similarly, activatable imaging probes (such as pro-drugs or fluorescent molecules that turn “on” in the presence of tumor enzymes) are being studied to enhance imaging sensitivity. While promising, most are in early development and not yet applicable for routine systemic surveillance.

[0224] The DNA-barcoded synthetic probe platform of the present disclosure addresses several unmet needs. It is systemic and proactive - probing the entire body for tumor-related biochemical signals, which means it could detect recurrences regardless of location (unlike imaging which may miss lesions outside the scanned field). It is also highly sensitive by design: even a microscopic tumor can trigger a large, amplifiable signal (via release of many DNA barcodes), overcoming the dilution issue of shed biomarkers. Importantly, the platform is multiplexed; by incorporating multiple distinct probes, it can capture different aspects of tumor biology (angiogenesis, immunosuppressive markers, etc.), which improves the chance of detecting heterogeneous tumors and reduces reliance on any single marker. The use of DNA barcodes enables ultrasensitive detection techniques (CRISPR amplification or PCR) that far exceed the sensitivity of direct imaging or protein assays. Moreover, because the readout is ultimately a simple liquid assay (urine or blood test), it could be done frequently without harm - offering a noninvasive surveillance tool that patients could undergo monthly or quarterly, for example, with minimal inconvenience. This contrasts with CT scans which cannot be safely done at high frequency.Cancer-exclusive Circulating Biomarkers for Synthetic Probe Detection

[0225] The synthetic probes is used for early detection of solid tumors via injected synthetic probes specific to blood-borne targets that are uniquely tumor- associated and plentiful enough to bind multivalent synthetic probes (>60 kDa complexes). Proteins, nucleic acids, and extracellular vesicles (EVs) were surveyed that meet these criteria for the top solid cancers (breast, lung, colorectal, prostate, pancreas, liver, stomach, ovarian, cervical, esophageal, bladder, kidney, thyroid, skin / melanoma, head / neck, etc.). Key candidates include oncofetal glycoforms and viral oncoproteins that do not appear in benign inflammation, wound repair, or pregnancy. Each marker was scored for cancer-specificity, blood prevalence, half-life, and probe accessibility (Table 4). Candidate binding domains of the synthetic probes range from -15-50 kDa (VHH, Fab, scFv, aptamer) to full IgG / Fc fusion (>60 kDa). To exceed the renal filtration cutoff (-60 kDa), multimerization strategies, PEGylation, and other strategies are proposed (e.g. bi / tri-valent constructs, Fc / albumin fusions, PEGylation, or avidin-biotin clustering) so that probe-biomarker complexes are retained in circulation long enough to allow a urine readout.Table 4. Cancer-specific circulating biomarkers as potential target molecules. “Type” indicates Protein (P), Nucleic acid (NA), or Exosome / EV (EV). Specificity “Score” is High if essentially tumor-only. Medium if limited benign expression, Low if common. Half-life is approximate blood residence (short = <6h, med = 6-24h, long = >ld). Accessibility refers to extracellular presence (soluble or EV surface). Binder class examples and multimerization notes included.Scores and accessibility are qualitative estimates. For example, Tn and glypican markers score 'High" for specificity (cancer-only) and have detectable levels in patients: PSA and CA125 are “Low” specificity due to benign causes. Half-lives of small binders (~35 kDa) are typically hours, necessitating same-day (=4-24 h) readout.

[0226] An exemplary SynBio synthetic probe platform uses barcoded probes that bind tumor epitopes and release DNA reporters on chemical trigger; reporters are quantified in urine with attomolar sensitivity (such as the CRISPR / Cas12a strategy provided in the present disclosure). In comparison, standard liquid biopsies detect circulating tumor DNA (ctDNA) or proteins by PCR / sequencing or ELISA (pico- to nanomolar sensitivity). The SynBio approach can incorporate 10-20 barcoded probes in one assay, whereas typical liquid biopsies test a limited panel. Table 5 summarizes the quantitative trade-offs (sensitivity, specificity, clearance, throughput). In short, SynBio synthetic probe assays offer signal amplification and high specificity, at the cost of requiring probe injection and timing, whereas conventional 10 mL blood assays yield immediate analytes but suffer from biological noise (clonal hematopoiesis, benign expression) and lower analytic sensitivity.

[0227] The SynBio platform using synthetic probes of the present disclosure is particularly suitable for detecting cancer-exclusive biomarker categories including:Oncofetal Glycoforms: Tumors often expose cryptic glycan antigens (e.g. Tn, sialyl-Tn) on mucins or ECM proteins that are hidden in healthy tissues. The Tn antigen (GalNAca-O-Ser / Thr on mucins) is a “pan-carcinoma” marker: detected in >90% of breast, pancreas, and lung cancers, and >60% of colorectal, gastric, bladder cancers. In normal adults Tn is virtually absent (masked by further glycosylation), making it highly cancer-specific. Synthetic probes (e.g. anti-Tn antibodies, lectin-like VHH, or glycan-binding aptamers) could bind circulating Tn-bearing glycoproteins or exosomes (especially from adenocarcinomas). Similarly, the Thomsen-Friedenreich (TF) antigen (Gaipi- 3GalNAc) is exposed on many carcinomas and is not found in normal blood, offering another pancarcinoma target.Tumor-Shed Proteoglycans: Certain membrane proteoglycans are overexpressed in cancer and shed on exosomes. Glypican-1 (GPC1) is one such marker: it is highly enriched on exosomes from pancreatic adenocarcinoma (100% of PC patients vs. 0% in benign pancreas). Plasma GPC1 likewise distinguishes prostate cancer from benign prostatic hyperplasia. GPC1 is low / absent in healthy adult tissues, so anti-GPCl probes yield near-zero background. A VHH or scFv against GPC1 could bind these large (>50 nm) exosomes, easily producing >60 kDa complexes. Glypican-3 (GPC3) is another oncofetal proteoglycan secreted by hepatocellular carcinoma (HCC) - it is essentially absent in normal liver and only seen in HCC patients, making soluble GPC3 a promising HCC biomarker.Viral Oncoproteins / DNA: Virus-driven cancers offer truly tumor-specific targets. For example, nasopharyngeal carcinoma and some lymphomas harbor EBV; the EBV latent membrane protein LMP1 is packed into tumor-derived exosomes and released into blood. Clinical NPC patients show elevated exosomal LMP1, whereas uninfected individuals do not. Binders (VHH orantibodies) against LMP1 on exosomes would form huge complexes. Likewise, cervical and oropharyngeal cancers driven by high-risk HPV could be detected via circulating HPV DNA (E6 / E7 oncogenes) or viral capsid proteins on vesicles.Circulating Nucleic Acids (ctDNA / miRNA): Tumor-specific mutations (e.g. KRAS. BRAF. EGFR. TP53, IDH1) in cfDNA are exclusive to cancer cells. Unlike other bioorthogonal sensors, synthetic probes could target mutant DNA or RNA by incorporating DNA-binding domains or aptamers. For instance, an aptamer fused to a protein scaffold might bind a tumor-specific point mutation sequence or fusion transcript in blood. miRNAs enriched in tumors (e.g. miR- 21, miR-203) could also be targets, but most oncomiRs are not entirely cancer- exclusive. Because nucleic-acid probes clear extremely fast (e.g., unmodified aptamers (<15 kDa) are renally filtered within minutes), multimerization and / or chemical stabilization is desirable.Extracellular Vesicles (EVs): Tumor-derived EVs (exosomes / microvesicles) carry tumor antigens (proteins, RNAs) on their surface. Markers like tumor- specific mutant EGFR, PD-L1, or novel epitopes on EVs can be targeted. Unlike normal exosomes, tumor EVs often carry distinctive cargo (e.g. mutant KRAS RNA, oncogenic proteins). A SynBio synthetic probe can bind an EV surface protein unique to cancer (e.g. mutant EGFRvIII on GBM EVs) and thereby tag the entire vesicle (mass >1000 kDa). GPC1 on pancreatic cancer EVs (see above) and LMP1 on NPC EVs are concrete examples of EV targets.

[0228] Synthetic probes use any high-affinity binding domain (e.g., antibodies, fragments, alternative scaffolds, aptamers as described herein) tailored to the chosen target molecule. Particularly suitable examples of binding domains useful in the synthetic probe of the present disclosure include single-domain camelid VHH (~15 kDa), Fab (50 kDa), scFv (25 kDa), diabody (50-60 kDa), minibody (~80 kDa), and non-immunoglobulin scaffolds (DARPins, affibodies).

[0229] Because the synthetic probes are designed to ultimately form >60 kDa complexes, the synthetic probe design multimerizes or enlarges the binder. Strategies include:Fc / Albumin fusion: Fusing a small binder to an Fc or albumin-binding domain yields -100+ kDa constructs with ½ -lives of days.Chemical crosslinking: Binders (e.g. biotinylated VHH) plus an injected multimerizer (streptavidin) to form tetramers.Polymer scaffolds: Linking multiple binders onto a PEG or protein scaffold (like an Fc or dextran).Avidin chase: Injecting avidin to bind excess biotinylated probes, clearing them and effectively multimerizing tumor-bound ones.Self-aggregation at target: Designing bivalent or tetravalent constructs (e.g. IgG or anti-idiotype) so that multiple probes bind adjacent epitopes on the target, forming >60 kDa clusters.

[0230] Each strategy trades off speed and size. For instance, PEGylation of a Fab (50 kDa + 40 kDa PEG) extends half-life to -2 weeks. Albumin-binding peptides can prolong VHH circulation from hours to -1-2 days. Thus, a user of the SynBio platform will consider such tags to match desired detection timing windows.

[0231] Synthetic probe assays according to the SynBio platform dramatically amplify signal in vivo. Each tumor-bound synthetic probe can release many reporter molecules, and Cast 2a readouts reach attomolar sensitivity. In contrast, typical ctDNA NGS detects mutant alleles down to -0.1% variant allele fraction (~pM range) and requires large sequencing depth. For example, early-stage lung cancer yields -30-70% detection by ultra-deep sequencing (many cases false-negative), whereas SynBio can detect a single binding event if amplification and trigger timing are optimal.

[0232] Liquid biopsies suffer “biological noise”: -10-100 ng / mL of normal cfDNA (mostly wild-type), and clonal hematopoiesis introduces false-positivemutations. Similarly, common tumor markers (CEA, CA125, PSA) have high background in benign conditions. In contrast, synthetic probes only release reporter upon binding the specific tumor epitope. A circulating control probe calibrates baseline. Thus, S / B ratio is expected to be higher: e.g. GPC1+ exosomes were undetectable in healthy / bureaucratic controls.

[0233] Synthetic probes of the present disclosure are cleared on the order of hours to days, so cleaving agent (trigger) administration and urine collection can occur about 4 h to about 24 h post-injection. This fits a same-day workflow. In contrast, liquid biopsy analytes such as cfDNA and exosomes have short half-lives (~2-3 h) and must be measured immediately after draw. Protein markers (e.g. AFP. PSA) persist longer, but achieving a same-day lab PCR / NGS readout is often not feasible. The SynBio platform thus exchanges a delayed sampling (hours) time for ultra-sensitivity and multiplexing.

[0234] Clonal hematopoiesis causes up to -20% of healthy individuals to carry somatic mutations (DNMT3A, TET2, etc.) that appear as “tumor” mutations in cfDNA. This yields false positives in cancer screens. Exosome assays can misclassify inflammatory vesicles as tumor-derived. By contrast, synthetic probes target bioorthogonal markers; they do not rely on sequence variants that occur in normal clones. If chosen markers (Tn glycans, viral proteins, GPC1) are truly cancer-exclusive, the false-positive rate of the SynBio platform is expected to be very low.

[0235] A 10 mL liquid biopsy is typically analyzed by NGS or ddPCR panels (throughput -50-500 genes / cancers). The SynBio platform supports dozens of synthetic probes via DNA barcoding (and other reporter ty pes). All SynBio reporters are read out together in a single urine sample by parallel CRISPR detection. This means one injection can “query” many tumor antigens at once (and add more if needed) without additional lab runs. However, SynBio requires probe manufacture and injection logistics; liquid biopsy is non-invasive (just draw blood). The two approaches can be complementary.

[0236] In modeling, simulated human PK for a 35 kDa SynBio probe only a few percent of dose reaches a small tumor by 24 h (-3-5% ID) while >99% of unbound probe is renally cleared. Thus, signal amplification (reporter release) is critical. The modeling shows that with attomolar readout, even minimal tumor targeting can yield detectable urine signal, whereas direct detection (e.g. imaging) would likely fail.

[0237] Synthetic probe-biomarker binding provides ultrasensitive, specific early detection if true cancer-only markers are used. As Table 5 outlines, it can outperform standard liquid biopsy in sensitivity and specificity, with a different profile of practical considerations (dose, timing, multi-analyte design).Table 5. Qualitative comparison of bind-and-bulk synthetic probe assay vs. standard liquid biopsy (ctDNA / exosome / protein panel).

[0238] The SynBio platform of the present disclosure provides a precision surveillance tool that actively scans for tumor resurgence with heightened sensitivity and specificity. It fills the gap left by current methods by providing an early warning system for recurrence that is both comprehensive (whole-body, multi-marker) and feasible for serial use. This approach could fundamentally shift recurrence monitoring from today s reactive paradigm to a proactive, “seek-and-signal” diagnostic paradigm, catching relapses at their inception and enabling earlier therapeutic intervention.

[0239] The SynBio platform described herein is particularly suited for binding to the following biomarkers: 1 -phosphatidylinositol 4,5-bisphosphate phosphodiesterase beta-1 (PLCB1), 1 -phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-1 (PLCG1), 1 -phosphatidylinositol 4.5-bisphosphate phosphodiesterase gamma-2 (PLCG2), 14-3-3 protein sigma (STRATIFIN), 14-3-3 protein zeta / delta (14-3-3 protein zeta / delta), 15-hydroxyprostaglandin dehydrogenase [NAD(+)] (HPGD), 3-dioxygenase 1 (IDO1), 3-ketodihydrosphingosine reductase (KDSR), 3-N-acetylglucosaminyltransferase radical fringe (RFNG), 3- phosphoinositide-dependent protein kinase 1 (PDPK1), 4-1BB (Tumor necrosis factor receptor superfamily member 9), 4EBP1 (Eukaryotic translation initiation factor 4E- binding protein 1), 5'-AMP-activated protein kinase catalytic subunit alpha-1 (AAPK1), 5'-cyclic phosphodiesterase (PDE2A), 5'-Nucleotidase (5 ’-Nucleotidase), 5NT3 (Cytosolic 5'-nucleotidase 3A), 5-bisphosphate 3-kinase catalytic subunit delta isoform (PIK3CD). 5-bisphosphate 3-kinase catalytic subunit gamma isoform (PIK3CG), 5-bisphosphate phosphodiesterase beta-1 (PLCB1), 5-bisphosphate phosphodiesterase gamma-1 (PLCG1), 5-bisphosphate phosphodiesterase gamma-2 (PLCG2), 5-Lipoxygenase (5-Lipoxygenase), 5-trisphosphate 3-phosphatase and dual- specificity protein phosphatase PTEN (PTEN). 6-bisphosphatase 1 (FBP1), 60 kDaheat shock protein (HSP 60), 60S acidic ribosomal protein P2 (RPLP2), 72 kDa type IV collagenase (MMP2), 78 kDa glucose-regulated protein (BiP), A disintegrin and metalloproteinase with thrombospondin motifs 1 (ATS1), A disintegrin and metalloproteinase with thrombospondin motifs 15 (ATS 15), Acetylcholinesterase (ACES), Actin, aortic smooth muscle (actin, alpha 2), Actin, alpha skeletal muscle(Actin, alpha skeletal muscle), Activin A (Activin A), Activin RIA (Activin RIA), Acyl-CoA-binding protein (ACBP). ADAM 28 (ADAM 28), ADAM 8 (ADAM-8), Adapter molecule crk (CRK), Adenine DNA glycosylase (MutY homolog). Adenine phosphoribosyltransferase (APT), Adenylate cyclase type 1 (ADCY1), Adenylate cyclase type 2 (ADCY2), Adenylate cyclase type 7 (ADCY7), Adenylate cyclase type 9 (ADCY9), Adiponectin (Adiponectin), ADP-dependent glucokinase (ADPGK), ADP-ribosylation factor 1 (ARF1). ADP-ribosylation factor-binding protein GGA1 (GGA1), Agouti-signaling protein (ASIP), AH receptor-interacting protein (AIP), Alcohol dehydrogenase IB (ADH1B), Alcohol dehydrogenase class 4 mu / sigma chain (ADH7), Aldehyde dehydrogenase, mitochondrial (ALDH-E2), Aldo-keto reductase family 1 member BIO (Aldose reductase-like), Aldo-keto reductase family 1 member Cl (Aldo-keto reductase 1C1), Aldo-keto reductase family 1 member C2 (AK1C2), Aldo-keto reductase family 1 member C3 (Aldo-keto reductase 1C3), Aldose reductase (Aldose reductase), ALK tyrosine kinase receptor (ALK), Allograft inflammatory factor 1 (AIF1), alpha chain E (HLAE), alpha skeletal muscle (Actin, alpha skeletal muscle), Alpha- 1 -anti chymotrypsin complex (Alpha- 1 -anti chymotrypsin complex). Alpha- 1 -anti trypsin (al -Antitrypsin), alpha-2 subunit (GNAI2), Alpha-2 - macroglobulin (a2-Macroglobulin), Alpha-actinin-4 (ACTN4), Alpha-amylase 2B (Alpha-amylase 2B), Alpha-enolase (Alpha enolase), alpha-Fetoprotein (AFP), Aminoacylase-1 (Aminoacylase-1), AMP Kinase (alphalbetalgammal) (AMPK alblgl), Amphiregulin (AREG), Angiomotin (AMOT), Angiopoietin-1 receptor (Angiopoietin-1 receptor, soluble), Angiopoietin-related protein 4 (ANGL4), Angiotensin-converting enzyme (ACE), Angiotensinogen (Angiotensinogen). Annexin Al (annexin I), Annexin A2 (annexin II), Annexin A4 (annexin IV), Annexin A5 (Annexin V), Anoctamin-1 (ANO1), Anterior gradient protein 2 homolog (AGR2), Anthrax toxin receptor 2 (ANTR2), aortic smooth muscle (Actin, aortic smooth muscle), Apolipoprotein A-I (Apo A-I), Apolipoprotein C-II (Apo C-II), Apolipoprotein E (Apo E). Apolipoprotein E (isoform E2) (Apo E2), Apoptosis regulator Bcl-2 (Bcl-2), Apoptosis-associated speck-like protein containing a CARD (ASC), Apoptotic protease-activating factor 1 (Apaf-1), Appetite-regulating hormone (ghrelin), Arrestin domain-containing protein 3 (ARRD3), Arsenite methyltransferase (AS3MT), Artemin (Artemin), Aryl hydrocarbon receptor nuclear translocator(ARNT), Aryl hydrocarbon receptor nuclear translocator 2 (ARNT2), Arylamine N- acetyltransferase 1 (ARY1), AT-rich interactive domain-containing protein 1A (ARI1A), ATP-dependent DNA helicase QI (RECQ1), Atrial natriuretic factor (ANP), Aurora kinase A (Aurora kinase A), Autophagy protein 5 (Autophagy protein 5), Axin-1 (AXN1), Axin-2 (AXIN2), AXL tyrosine-protein kinase receptor UFO (AXL), AZGP1 (Zinc-alpha-2-gly coprotein), B-cell lymphoma 6 protein (BCL6), B-cell lymphoma / leukemia 10 (Bcl-10), B-cell lymphoma / leukemia 11A (B-cell CLL HA). B7-H3 (CD276), Baculoviral IAP repeat-containing protein 2 (cIAP-1), Baculoviral IAP repeat-containing protein 3 (cIAP-2), Baculoviral IAP repeat-containing protein 5 (Survivin), Baculoviral IAP repeat-containing protein 7 (Livin B), BAG family molecular chaperone regulator 1 (BAG-1), BAX protein, cytoplasmic isoform delta (BAXD), Bcl-2-binding component 3 (BBC3), Bcl-2-like protein 1 (BCL2-like 1 protein), Bcl-2-like protein 11 (BIM), Bcl-2-related protein Al (BFL1), Bcl2- associated agonist of cell death (BAD), BCL2 / adeno virus E1B 19 kDa protein- interacting protein 3 (BNIP3). BDNF / NT-3 growth factors receptor (TrkB). Beclin-1 (BECN 1), Benign Prostate specific Antigen (BPSA). Beta-1. Beta-2-microglobulin (b2-Microglobulin), Beta-casein (Beta-casein), Beta-hexosaminidase alpha chain (Hexosaminidase A), Betacellulin (BTC), BH3 -interacting domain death agonist (BID), Bifunctional arginine demethylase and lysyl-hydroxylase JMJD6 (JMJD6), Bifunctional methylenetetrahydrofolate dehydrogenase / cyclohydrolase, Bile acid receptor (NR1H4), Bis(5'-adenosyl)-triphosphatase (Fragile histidine triad protein), Bone morphogenetic protein 2 (BMP-2), Bone morphogenetic protein 4 (BMP-4), Bone morphogenetic protein 7 (BMP-7). Bone morphogenetic protein receptor type-lA (BMPR1 A), Bone morphogenetic protein receptor type-2 (BMP RII), Bone sialoprotein2 (BSP), Brain acid soluble protein 1 (BASP), BRCA1 -associated RING domain protein 1 (BARD1), Breast cancer anti-estrogen resistance protein 3: Guanine Nucleotide Exchange Factor Domain (BCAR3:Ras-GEF), Breast cancer anti-estrogen resistance protein 3:Src Homology domain (BCAR3:SH2), Breast cancer metastasis- suppressor 1-like protein (BRM1L). Breast cancer resistance protein (BCRP), Breast cancer type 1 susceptibility protein (BRCA1), Bromodomain-containing protein 2 (BRD2), Bromodomain-containing protein 4 (BRD4), Brother of CDO (BOC), Butyrophilin subfamily 3 member A2 (BT3A2), C-C motif chemokine 2 (MCP-1), C-C motif chemokine 20 (MIP-3a), C-ets-1 protein (ETS-1), C-terminal -binding protein 1 (CTBP1), C-terminal -binding protein 2 (CTBP2), C-type mannose receptor 2 (MRC2), C-X-C motif chemokine 10 (IP-10), C-X-C motif chemokine 13 (BLC), C- X-C motif chemokine 14 (BRAK), C-X-C motif chemokine 9 (MIG), Cadherin-1 (Cadherin E), Cadherin-13 (CAD13), Cadherin-2 (Cadherin-2), Cadherin-5 (Cadherin- 5), Calcium / calmodulin-dependent protein kinase type II subunit alpha (CAMK2A), Calcium / calmodulin-dependent protein kinase type II subunit beta (CAMK2B). Calgranulin A (Calgranulin A), Calmodulin-like protein 3 (Calmodulin-related protein NB-1), Calmodulin-like protein 4 (CALL4), Calnexin (Calnexin), Calpain I (Calpain1), Calponin-2 (CNN2), Calreticulin (calreticulin), cAMP-dependent protein kinase catalytic subunit alpha (PRKACA), cAMP-dependent protein kinase catalytic subunit beta (KAPCB), cAMP-dependent protein kinase type I-alpha regulatory subunit (KAPO), Carbonic anhydrase 1 (Carbonic anhydrase I), Carbonic anhydrase 2 (carbonic anhydrase II), Carbonic anhydrase 9 (Carbonic anhydrase 9), Carbonyl reductase [NADPH] 1 (CBR1). Carboxypeptidase Al (CBPA1), Carboxypeptidase A2 (CBPA2), Carboxypeptidase B (Carboxypeptidase Bl), Carboxypeptidase E (CBPE). Carboxypeptidase Q (Aminopeptidase), Carcinoembryonic antigen-related cell adhesion molecule 5 (CEA), CASP8 and FADD-like apoptosis regulator (FLIP), Caspase-10:region 1 (Caspase-10:region 1), Caspase-10:region 2 (Caspase-10:region2), Caspase-2 (Caspase-2). Caspase-3 (Caspase-3), Caspase-7 (Caspase-7), Caspase-8 (Caspase-8), Catalase (Catalase), Catechol O-methyltransferase (Catechol O- methyltransferase), Catenin alpha-1 (CTNA1), Catenin alpha-2 (CTNA2), Catenin alpha-3 (CTNA3), Catenin beta-1 (b-Catenin), Cathepsin B (Cathepsin B), Cathepsin D (Cathepsin D), CCAAT / enhancer-binding protein alpha (CEBPA), CD 109 antigen (CD 109), CD 133 antigen (CD 133), CD 166 antigen (ALCAM), CD27 antigen (CD27), CD28 (CD28), CD29 (CD29), CD44 antigen (CD44), CD48 antigen (CD48), CD59 glycoprotein (CD59), CD63 antigen (CD63), CD70 antigen (CD70), CD9 antigen (CD9), CDK2 (CDK2), CDK4 (CDK4), Cell adhesion molecule 1 (Nectin-like protein 2), Cell division control protein 42 homolog (Cdc42Hs), Cellular retinoic acid-binding protein 1 (CRBP), Cellular tumor antigen p53 (p53), Cellular tumor antigen p53 R175H mutant (p53 R175H), Cerebral cavernous malformations 2 protein (CCM2), Ceruloplasmin (Ceruloplasmin), cGMP-dependent 3',5'-cyclic phosphodiesterase(cGMP -stimulated PDE), Chitinase-3-like protein 1 (YKL-40), Chloride intracellular channel protein 1 (NCC27). Cholinesterase (Pseudocholinesterase), Chymotrypsin-C (CTRC), Claudin-1 (CLD1), Clustenn-hke protein 1 (CLUL1), CMRF35-like molecule 2 (CLM2), Coactosin-like protein (Coactosin-like protein). Coagulation Factor V (Coagulation Factor V), Cofilin-1 (Cofilin-1), Coiled-coil domain-containing protein 6 (CCDC6), Collagen alpha-l(IV) chain (C04A1), Collagen alpha-2(IV) chain precursor (CO4A2), Collagen alpha-5(IV) chain (CO4A5), Complement Clq subcomponent (Clq), Complement component 1 Q subcomponent-binding protein, mitochondrial (C1QBP), Connective tissue growth factor (CTGF), Core-binding factor subunit beta (PEBB), Comulin (CRNN), Corticotropin (ACTH), Creatine kinase B-type (CK-BB), Creatine kinase M-type: Creatine kinase B-type heterodimer (CK-MB), CREB-binding protein (CREB-binding protein), Crk-like protein (CRKL), Cullin-1 (CUL1), Cullin-2 (CUL2), Cyclic AMP-dependent transcription factor ATF-3 (ATF3), Cyclin DI (Cyclin DI), Cyclin D2 (Cyclin D2), Cyclin-Al (CCNA1), Cyclin-A2 (Cyclin A), Cyclin-dependent kinase 1 (p34 protein kinase), Cyclin-dependent kinase LG2 / mitotic- specific cyclin-Bl complex (CDKl / cyclin B), Cyclin-dependent kinase 2:Cyclin-A2 complex (CDK2 / cyclin A), Cyclin-dependent kinase 4 inhibitor B (pl5-INK4b), Cyclin-dependent kinase 4 inhibitor C (CDN2C), Cyclin-dependent kinase inhibitor 1 (p21), Cyclin-dependent kinase inhibitor IB (p27Kipl), Cyclin-dependent kinases regulatory subunit 1 (CKS-1), Cyclin-H (Cyclin H), Cystathionine beta-synthase (CBS), Cystatin B (Cystatin B), Cystatin-C (Cystatin C), Cystatin-M (Cystatin M), Cystatin-SN (CYTN), Cystic fibrosis transmembrane conductance regulator (CFTR), Cytidine deaminase (CD A), Cytochrome c (Cytochrome c). Cytochrome P450 3A4 (Cytochrome P4503A4). Cytokine receptor common subunit beta: Cytoplasmic domain (IL3RB:CD), Cytokine receptor common subunit beta:Extracellular domain (IL3RB:ECD), Cytokine receptor common subunit gamma (IL-2 sRg), Cytokine receptor-like factor 2 (TSLP R), cytoplasmic (HARS1), cytoplasmic 2 (ACTG1), cytoplasmic isoform delta (BAXD), Cytosol aminopeptidase (LAP), cytosolic (SHMT1), Cytosolic 5 '-nucleotidase 3A (5NT3). Cytosolic phospholipase A2 alpha (cPLA2-alpha), Cytotoxic T-lymphocyte protein 4 (CTLA-4), D-3-phosphoglycerate dehydrogenase (SERA), DAN domain family member 5 (DAND5), DAXX (DAXX), DCC (DCC), DCC-interacting protein 13-alpha (DP13A), Death domain-containingprotein CRADD (CRADD), Death-associated protein kinase 1 (DAPK1), Death- associated protein kinase 2 (DAPK2), Death-associated protein kinase 3 (DAPK3), Delta-aminolevulinic acid dehydratase (HEM2), Delta-like protein 1 (DLL1), Delta- like protein 3 (DLL3), Delta-like protein 4 (DLL4), Desmin (Desmin), Desmocollin-3 (DSC3), Dickkopf-related protein 1 (DKK1), Differentially expressed in FDCP 8 homolog (DEFI8), Dihydrofolate reductase (DYR), Disintegrin and metalloproteinase domain-containing protein 10:Cytoplasmic domain (ADAM 10: CD), Disintegrin and metalloproteinase domain-containing protein 10: Extracellular domain (ADAM 10:ECD), DNA damage-binding protein 2 (DDB2), DNA damage-inducible transcript 3 protein (DDIT3), DNA excision repair protein ERCC-1 (ERCC1), DNA mismatch repair protein Mlhl (MLH1). DNA mismatch repair protein Msh2 (MSH2), DNA mismatch repair protein MSH6 (MSH6), DNA polymerase beta (DPOLB), DNA polymerase kappa (POLK), DNA polymerase subunit delta 3 (POLD3), DNA repair endonuclease XPF (XPF), DNA repair protein RAD50 (RAD50), DNA repair protein RAD51 homolog 1 (RAD51), DNA repair protein RAD51 homolog 3 (RA51C), DNA repair protein XRCC1 (XRCC1), DNA repair protein XRCC2 (XRCC2), DNA repair protein XRCC4 (XRCC4), DNA topoisomerase 1 (Topoisomerase I), DNA topoisomerase 2-alpha (TOP2A), DNA-(apurinic or apyrimidinic site) lyase (APEX1), DNA-repair protein XRCC3 (XRCC3), DnaJ homolog subfamily C member 10 (DJC10). Double-strand break repair protein MRE11 (MRE11), Dual specificity mitogen-activated protein kinase kinase 1 (MEK1), Dual specificity mitogen-activated protein kinase kinase 2 (MP2K2), Dual specificity mitogen-activated protein kinase kinase 4 (MP2K4), Dual specificity protein phosphatase 6 (DUS6), DVL1 (DVL1), E3 ubiquitin-protein ligase CBL (CBL), E3 ubiquitin-protein ligase CHFR (CHFR). E3 ubiquitin-protein ligase DTX1 (DTX1), E3 ubiquitin-protein ligase Mdm2 (MDM2), E3 ubiquitin-protein ligase parkin (PRKN2), E3 ubiquitin-protein ligase RNF43 (RNF43), E3 ubiquitin-protein ligase XIAP (XIAP), Echinoderm microtubule- associated protein-like 4 (EMAL4). EGF-containing fibulin-like extracellular matrix protein 1 (FBLN3), Egl nine homolog 1 (EGLN1), Egl nine homolog 2 (EGLN2), Egl nine homolog 3 (EGLN3), Elongation factor 1-alpha 1 (EF-1 -alpha- 1), Elongation factor 1-beta (EF-l-beta), Elongator complex protein 1 (ELP1), Ena / V ASP-like protein (EVL), Endoglin (CD248), Endoplasmic reticulum resident protein 29 (ERP29),Endoplasmic reticulum resident protein 44 (TXNDC4), Endoplasmin (Endoplasmin), Endosialin (CD248), Endothelial cell-specific molecule 1 (Endocan), Endothelial monocyte-activating polypeptide 2 (EMAP-2), Endothelin B receptor (EDNRB), Endothelin-1 (Endothelin 1), Enhancer of zeste homolog 2 (EZH2), Ephrin type- A receptor 2 (Epithelial cell kinase), Ephrin type-A receptor 3 (EPHA3), Ephrin t pe-B receptor 2 (EPHB2), Ephrin type-B receptor 4 (EphB4), Ephrin-Al (Ephrin-Al), Ephrin-B2: Cytoplasmic domain (EFNB2:CD). Ephrin-B2:Extracellular domain (EFNB2:ECD), Epidermal growth factor receptor (ERBB1), Epidermal growth factor receptor variant III (EGFRvIII), Epidermal growth factor: Cytoplasmic domain (EGF:CD), Epidermal growth factor: Extracellular domain (EGF:ECD), Epithelial discoidin domain-containing receptor 1 (discoidin domain receptor 1), ER-b (ER-b), Erythropoietin (Epo), Erythropoietin receptor (EPO-R), ETS domain-containing protein Elk-1 (ELK1), ETS domain-containing protein Elk-3 (ELK3), ETS translocation variant 4 (ETV4), Eukaryotic translation initiation factor 2 subunit 1 (IF2A). Eukaryotic translation initiation factor 2 subunit 2 (eIF-2-beta), Eukaryotic translation initiation factor 2C 2 (12C2), Eukaryotic translation initiation factor 4E- binding protein 1 (4EBP1), Eukaryotic translation initiation factor 5A-1 (eIF-5A-1), Extra Domain B Fibronectin (EDB-FN), Ezrin (Ezrin), F-actin-capping protein subunit beta (CAPZB), FAD-linked sulfhydryl oxidase ALR (HERV1), Fanconi anemia group C protein (FANCC), Fanconi anemia group D2 protein (FACD2), FAS-associated death domain protein (FADD), Fat mass and obesity-associated protein (FTO), Fatty acid synthase (Fatty acid synthase), Fatty acid-binding protein, Fatty-acid amide hydrolase 2 (FAAH2), Ferritin (Ferritin), Ferritin heavy chain (FRIH), Fibrinogen (Fibrinogen). Fibroblast Activation Protein (FAP), Fibroblast growth factor 1 (b- ECGF), Fibroblast growth factor 10 (FGF-10), Fibroblast growth factor 16 (FGF-16), Fibroblast growth factor 17 (FGF-17), Fibroblast growth factor 18 (FGF-18), Fibroblast growth factor 19 (FGF-19), Fibroblast growth factor 2 (bFGF), Fibroblast growth factor 20 (FGF-20), Fibroblast growth factor 21 (FGF21), Fibroblast growth factor 22 (FGF22). Fibroblast growth factor 23 (FGF23), Fibroblast growth factor 3 (FGF-3). Fibroblast growth factor 4 (FGF-4), Fibroblast growth factor 5 (FGF-5), Fibroblast growth factor 6 (FGF-6), Fibroblast growth factor 7 (FGF7), Fibroblast growth factor 8 (FGF-8), Fibroblast growth factor 8 isoform A (FGF-8A), Fibroblast growth factor 8isoform B (FGF-8B), Fibroblast grow th factor 8 isoform F (FGF-8F), Fibroblast growth factor 9 (FGF9), Fibroblast growth factor receptor 1 (bFGF-R), Fibroblast growth factor receptor 2 (FGFR-2). Fibroblast growth factor receptor 3: Cytoplasmic domain (FGFR- 3:CD), Fibroblast growth factor receptor 3: Extracellular domain (FGFR-3:ECD), Fibroblast growth factor receptor 4 (FGFR4), Fibronectin (Fibronectin), Fibronectin Fragment 3 (FNE3), Fibronectin Fragment 4 (FN1.4), Fibronectin-1 Fragment 2 (FN1.2), Filamin-A:Calponin Homology 1 (filamin A:CH1), Filamin-A:Calponin Homology 2 (filamin A:CH2), Fms-related tyrosine kinase 3 ligand (Flt3 ligand), Focal adhesion kinase 1 (FAK1), Folate receptor alpha (FOLR1), Follistatin (FST), Forkhead box protein L2 (FOXL2), Forkhead box protein Ml (F0XM1), Forkhead box protein O1A (F0X01A), Forkhead box protein 03 (F0X03A), Forkhead box protein Pl (F0XP1), Forkhead box protein P3 (F0XP3), Fos-related antigen 2 (F0SL2), Four- jointed box protein 1 (FJX1), Friend leukemia integration 1 transcription factor (FLI1), Frizzled-1 (FZD1), Frizzled-10: Cytoplasmic domain (Frizzled-10:CD), Frizzled- 10:Frizzled domain (Frizzled-10:FZ), Frizzled-2 (FZD2), Frizzled-4 (FZD4), Frizzled- 5 (Frizzled-5). Frizzled-7 (FZD7), Frizzled-8 (FZD8). Frizzled-9 (FZD9), Fructose- 1,6-bisphosphatase 1 (F16P1), Fructose-bisphosphate aldolase A (aldolase A), Fumarate hydratase, mitochondrial (FUMH), G protein beta 2 subunit (G protein beta 2 subunit), G0 / G1 switch protein 2 (G0S2). Gl / S-specific cyclin-D3 (Cyclin D3), Gl / S-specific cyclin-El (Cyclin E), G2 / mitotic-specific cyclin-Bl (Cyclin Bl). Galectin-1 (Gal ectin-1 ), Galectin-3 (Galectin-3), Galectin-7 (Galectin-7), Gamma- enolase (NSE), Gamma-glutamyl hydrolase (GGH), Gamma-synuclein (g-Synuclein), Gap junction alpha-1 protein (CXA1), Gastrin-releasing peptide (Gastrin-releasing peptide), Gastrokine-1 (CA11 protein), GDH / 6PGL endoplasmic bifunctional protein (G6PE), Gelsolin (Gelsolin), General vesicular transport factor pl 15 (US01), Glial cell line-derived neurotrophic factor (GDNF), Glucagon (Glucagon), Glucose-6-phosphate isomerase (PHI), Glutamine synthetase (GLNA), Glutathione peroxidase 1 (Glutathione peroxidase), Glutathione peroxidase 2 (GPX2), Glutathione peroxidase 3 (Glutathione peroxidase 3), Glutathione S-transferase Al (GST Al-1). Glutathione S- transferase A2 (GSTA2), Glutathione S-transferase A3 (GSTA3), Glutathione S- transferase A4 (GSTA4), Glutathione S-transferase A5 (GSTA5), Glutathione S- transferase Mu 1 (GSTM1-1), Glutathione S-transferase Mu 3 (GSTM3-3), GlutathioneS-transferase Mu 4 (GSTM4), Glutathione S-transferase Mu 5 (GSTM5-5), Glutathione S-transferase omega-1 (GST omega-1). Glutathione S-transferase P (Glutathione S- transferase Pi), Glutathione S-transferase theta-1 (GSTT1), Glutathione S-transferase theta-2B (GSTT2), Glutathione synthetase (GSHB), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, liver), Glycine amidinotransferase, mitochondrial (GATM), Glycine N-methyltransferase (GNMT), Glycodelin (Glycodelin), Glycogen synthase kinase-3 beta (GSK-3 beta), Glypican-1 (Glypican 1). GMP reductase 2 (GMPR2). gp75 (gp75), Granulocyte colony-stimulating factor (G-CSF), Granulocyte colony- stimulating factor receptor (G-CSF-R), Granulocyte-macrophage colony-stimulating factor (GM-CSF), Granzyme B (Granzyme B), Gremlin-1 (GREM1), Gro-beta (Gro- b), Gro-gamma (Gro-g), Growth factor receptor-bound protein 2 (GRB2 adapter protein), Growth factor receptor-bound protein 7 (GRB7), Growth / differentiation factor 10 (BMP-3b), Growth / differentiation factor 15 (MIC-1), Growth / differentiation factor 3 (GDF-3), GST mu2 (GSTM2), GTP-binding nuclear protein Ran (RAN), GTP- binding protein RAD (RAD). GTPase HRas (H-ras (WT)), GTPase KRas (K-ras), GTPase NRas (RASN). Guanine deaminase (GUAD), Guanine nucleotide-binding protein alpha- 13 subunit (GNA13), Guanine nucleotide-binding protein G(i) subunit alpha-1 (GNAI1), Guanine nucleotide-binding protein subunit alpha-11 (GNA11), Hairy / enhancer-of-split related with YRPW motif protein 1 (HEY1), Hamartin (TSC1), Haptoglobin (HPT), Haptoglobin isoform 2 (HPT), Heat shock 70 kDa protein IB (HS71B), Heat shock cognate 71 kDa protein (HSP70 protein 8), Heat shock protein beta-1 (HSP 27), Heat shock protein beta-6 (HSPB6), Heat shock protein HSP 90-beta (HSP 90b), Hedgehog-interacting protein (HHIP), Hematopoietically-expressed homeobox protein HHEX (HHEX), Heme oxygenase 1 (HO-1), Hemoglobin (Hemoglobin), Heparanase (HPSE), Hepatic leukemia factor (HLF), Hepatocyte growth factor (HGF), Hepatocyte growth factor receptor (Met), Hepatocyte growth factor-like protein (MSP), Hepatocyte nuclear factor 1 -alpha (HNF1A), Hepatocyte nuclear factor 3-alpha (FOXA1), Heterogeneous nuclear ribonucleoprotein Al (ROA1), Heterogeneous nuclear ribonucleoprotein K. (hnRNP K), Heterogeneous nuclear ribonucleoprotein R (HNRPR), High affinity nerve growth factor receptor (TrkA), High mobility group protein HMG-I / HMG-Y (HMGA1), High mobility group protein HMGI-C (HMGA2), Histidine— tRNA ligase, cytoplasmic (Histidyl-tRNAsynthetase), Histidine-rich glycoprotein (HRG), Histo-blood group ABO system transferase (BGAT), Histone acetyltransferase p300 (EP300), Histone deacetylase 1 (HDAC1), Histone deacetylase 2 (HDAC2), Histone deacetylase 6 (HDAC6), Histone Hl.2 (Histone Hl.2), Histone H2B type 1-K (H2B1K), Histone H3.1 (H31), Histone- lysine N-methyltransferase 2C (KMT2C), Histone-lysine N-methyltransferase 2D (MLL2), Histone-lysine N-methyltransferase EHMT2 (NG36), Histone-lysine N- methyltransferase SETD2 (SETD2), HLA class I histocompatibility antigen, alpha chain E (HLAE), HLA class II histocompatibility antigen gamma chain (HG2A), HLA- C (HLA-C), HMW (Kininogen, HMW), Holo-Transcobalamin-2 (Holo-TC II), Homeobox protein NANOG (NANOG), Homeobox protein 0TX2 (0TX2), Hsp90alpha (HSP 90a). Human Chorionic Gonadotropin (HCG), Hyaluronan and proteoglycan link protein 4 (HPLN4), Hydroxymethylglutaryl-CoA synthase, mitochondrial (HMCS2), Hypoxia-inducible factor 1-alpha (HIF-la), I-kappa-B kinase gamma (IKK-gamma), ICOS ligand (B7-H2), Immunoglobulin-binding protein 1 (IGBP1), Importin subunit alpha-3 (IMA4), Inactive tyrosine-protein kinase transmembrane receptor ROR1 (ROR1). Indoleamine 2.3-dioxygenase 1 (INDO). Induced myeloid leukemia cell differentiation protein Mcl-1 (Mcl-1), Inhibitor of growth protein 1 (ING1), Inhibitor of nuclear factor kappa B kinase beta subunit (IKK- beta). iNOS (iNOS). Insulin (Insulin), Insulin receptor (IR), Insulin-induced gene 1 protein (INSI1), Insulin-induced gene 2 protein (INSI2). Insulin-like growth factor 1 receptor (IGF-I sR), Insulin-like growth factor 2 mRNA-binding protein 1 (IF2B1), Insulin-like growth factor 2 mRNA-binding protein 3 (IF2B3), Insulin-like growth factor I (IGF-I), Insulin-like growth factor ILIsoform 3, Pro-form (IGF-II:Pro-form), Insulin-like growth factor ILMature (IGF-II:Mature), Insulin-like growth factor- binding protein 1 (IGFBP-1), Insulin-like growth factor-binding protein 2 (IGFBP-2), Insulin-like growth factor-binding protein 3 (IGFBP-3), Insulin-like growth factor- binding protein 5 (IGFBP-5), Insulin-like growth factor-binding protein 7 (IGFBP-7), Insulinoma-associated protein 1 (INSMI), Integrin al ibi (Integrin alibi), Integrin a5bl (Integrin a5bl), Integrin alpha L beta 2 (Integrin aLb2), Integrin alpha V beta 3 (Integrin aVb3), Integrin alpha V beta 6 (Integrin aVb6), Integrin alpha V beta 8 (Integrin aVb8), Integrin alpha-10 beta-1 (Integrin alObl), Integrin alpha-2 (Integrin alpha-2), Integrin alpha-5 (ITA5), Integrin alpha-6 (Integrin a6), Integrin alpha-I: beta-1 complex (Integrin albl), Integrin alpha-IIb (ITA2B), Integrin alpha-IIb: beta-3 complex (gpllbllla). Integrin alpha-M (Integrin alpha-M), Integrin alpha-V: beta-5 complex (Integrin aVb5), Integrin beta-2 (LFA-1 beta-2), Integrin beta-5 (ITB5), Inter- alpha-trypsin inhibitor heavy chain H4 (ITI heavy chain H4), Intercellular adhesion molecule 1 (sICAM-1), Intercellular adhesion molecule 5 (sICAM-5), Interferon alpha- 1 / 13 (IFNA1), Interferon alpha-10 (IFN10), Interferon alpha-14 (IFN14), Interferon alpha-16 (IFN16), Interferon alpha-17 (IFN17), Interferon alpha-2 (IFN-aA), Interferon alpha-21 (IFN21), Interferon alpha-4 (IFNA4), Interferon alpha-5 (IFNA5), Interferon alpha-6 (IFNA6), Interferon alpha-7 (IFNA7), Interferon alpha-8 (IFNA8), Interferon alpha / beta receptor 1 (IFN-a / b Rl), Interferon beta (IFN-b), Interferon gamma (IFN-g), Interferon gamma receptor 1 (IFN-g Rl), Interferon regulatory factor 1 (IRF1), Interferon regulatory factor 4 (IRF4), Interferon-induced GTP-binding protein Mxl (Mxl), Interleukin 39 (IL-39), Interleukin-1 alpha (IL-la), Interleukin-1 beta (IL-lb), Interleukin- 1 receptor antagonist protein (IL-IRa), Interleukin- 10 (IL- 10), Interleukin- 12 (IL-12), Interleukin- 12 receptor subunit beta-1 (IL-12 Rbl), Interleukin- 12 receptor subunit beta-2 (IL-12 RB2). Interleukin- 12 subunit beta (IL-12 p40). Interleukin- 13 (IL-13), Interleukin- 13 receptor subunit alpha-1 (IL-13 Rai), Interleukin- 15 (IL-15), Interleukin-17F (IL-17F), Interleukin- 18 (IL-18), Interleukin-2 (IL-2), Interleukin-2 receptor subunit alpha (IL-2 sRa), Interleukin-2 receptor subunit beta (IL-2 sRb), Interleukin-23 (IL-23), Interleukin-23 receptor (IL-23 R), Interleukin-24 (IL24). Interleukin-27 (IL-27), Interleukin-3 (IL-3), Interleukin-3 receptor subunit alpha (IL-3 Ra), Interleukin-32 (IL32), Interleukin-35 (IL-35), Interleukin-4 (IL-4), Interleukin-4 receptor subunit alpha (IL-4 sR), Interleukin-5 (IL-5), Interleukin-5 receptor subunit alpha (IL-5 Ra), Interleukin-6 (IL-6), Interleukin-6 receptor subunit alpha (IL-6 sRa), Interleukin-6 receptor subunit beta (gpl30, soluble), Interleukin-7 (IL-7), Interleukin- 7 receptor subunit alpha (IL-7 Ra), Interleukin-8 (IL-8), Internal Use Only: Heat shock 70 kDa protein 1A (HSP 70), Interstitial collagenase (MMP-1), Isocitrate dehydrogenase [NADP] cytoplasmic (IDH). isoform 121 (VEGF121), isoform B (LYNB). Isoform L-VEGFI65 (L-VEGF165), Junction plakoglobin (PLAK), kainate2 (GRIK2), Kallikrein- 10 (kallikrein 10), Kallikrein-4 (Kallikrein 4), Kallikrein-8 (kallikrein 8), Kelch-like ECH-associated protein 1 (KEAP1), Keratin, Kininogen, Kininogen-1 (Kininogen, HMW), Kininostatin (Kininostatin). Kit ligand (SCF),Kunitz-type protease inhibitor 2 (SPINT2), Kynurenine— oxoglutarate transaminase 1 (KAT1), L-lactate dehydrogenase A chain (LDHA), L-lactate dehydrogenase B chain (LDH-H 1), Lactadherin (MFGM), Lactotransferrin (Lactoferrin), Lamin A / C (Lamin A / C), Lamin-Bl (Lamin-Bl), Laminin beta-2 chain (S-laminin), Laminin subunit alpha-3 (LAMA3), Laminin subunit alpha-4 (LAMA4), Laminin subunit beta-4 (LAMB4), Laminin subunit gamma-2 (Laminin gamma-2), Laminin subunit gamma-3 (LAMC3), Laminin-2 (Laminin-2), Leptin (Leptin), Leptin receptor. Lethal(3)malignant brain tumor-like protein 2 (LMBL2), Leucine-rich repeat serine / threonine-protein kinase 2 (LRRK2), Leucine-rich repeat-containing protein 37A2 (L37A2), Leucine-rich repeat-containing protein 3B (LRC3B), Leucine-rich repeat-containing protein 59 (LRC59), Leukemia inhibitory factor receptor (LIF sR), Leukocyte cell-derived chemotaxin-2 (LECT2), LIM domain-containing protein 2 (LIMD2), Lipoprotein lipase (LPL), Low-density lipoprotein receptor-related protein IB (LRP1B), Low-density lipoprotein receptor-related protein 5 (LRP5), Low-density lipoprotein receptor-related protein 6 (LRP6). LRR and PYD domains-containing protein 3 (NALP3), Lymphocyte antigen 6D (LY6D), Lymphocyte-specific protein 1 (LSP1), Lymphoid enhancer-binding factor 1 (LEF1), Lysine-specific demethylase 4C (KDM4C), Lysine-specific histone demethylase 1A (KDM1A), Lysosome-associated membrane glycoprotein 1 (LAMP1), Lysosome-associated membrane glycoprotein 3 (LAMP3), Lysyl oxidase homolog 2 (Lysyl oxidase-like protein 2), Macrophage colony-stimulating factor 1 (CSF-1), Macrophage colony-stimulating factor 1 receptor (M-CSF R), Macrophage migration inhibitory factor (MIF), Macrophage scavenger receptor types I and II: Cytoplasmic domain (Macrophage scavenger receptorCD), Macrophage scavenger receptor types I and ILExtracellular domain (Macrophage scavenger receptor: ECD), Macrophage-capping protein (CAPG), Macrosialin (CD68), Major prion protein (PRIO), Malate dehydrogenase, mitochondrial (Malate dehydrogenase 2), Mast / stem cell growth factor receptor Kit (SCF sR), Matrilysin (MMP-7), Matrix Gia protein (MGP), Matrix metalloproteinase-14 (MMP-14). Matrix metalloproteinase-9 (MMP-9), MAX gene-associated protein (MGAP). Mediator of RNA polymerase II transcription subunit 28 (MED28), Melanoma-derived growth regulatory^ protein (MIA), Menin (MEN1), Merlin (MERL), Mesothelin (Mesothelin), Metastasis-suppressor KiSS-1 (KISSI), Methylated-DNA-protein-cysteinemethyltransferase (MGMT), MHC class I polypeptide-related sequence A (MICA), Microsomal glutathione S-transferase 2 (MGST2), Microtubule-associated proteins 1 A / 1B light chain 3A (MLP3A), Microtubule-associated proteins 1 A / 1B light chain 3B (MLP3B), Midkine (Midkine), Mismatch repair endonuclease PMS2 (PMS2), MITF (MITF), mitochondrial (HSPD1), Mitogen-activated protein kinase 1 (MK01), Mitogen-activated protein kinase 14 (MAPK14), Mitogen-activated protein kinase 3 (ERK-1). Mitogen-activated protein kinase 8 (MK08), Mitogen-activated protein kinase 9 (JNK2), Mitotic spindle assembly checkpoint protein MAD1 (MD1L1), Monocarboxylate transporter 4 (M0T4), Mothers against decapentaplegic homolog 2 (SMAD2), Mothers against decapentaplegic homolog 3 (SMAD3), Mothers against decapentaplegic homolog 4 (SMAD4), Mucin-16 (CA125), Mucin- 1: region 1 (MUCLregion 1), Mucin- 1: region 2 (MUCLregion 2), Mucin- 1: region 3 (MLJC I :region 3), Myc proto-oncogene protein (c-Myc), Myelin protein zero-like protein 1 (MPZL1), Myeloid leukemia factor 1 (MLF1), Myeloperoxidase (Myeloperoxidase), N-glycosylase / DNA lyase (N-glycosylase / DNA lyase), Na(+) / H(+) exchange regulatory cofactor NHE-RF3 (NHRF3), NACHT. NAD(P)H dehydrogenase [quinone] 1 (NAD(P)H dehydrogenase), NAD-dependent protein deacetylase sirtuin-1 (SIR1), NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 2 (NDUA2), Nanos homolog 2 (NAN02), Nectin-2 (Nectin-2), Neprilysin (Neprilysin), Nesprin-2 (Nesprin-2), Netrin receptor UNC5C (UNC5FI3). Netrin-1 (NET1), Neural cell adhesion molecule LI (NCAM-L1), Neuregulin-1 (NEUREGULIN-1), Neuroendocrine protein 7B2 (7B2), Neuroendocrine secretory protein 55 (GNAS3), Neurogenic locus notch homolog protein 1 (Notch 1), Neurogenic locus notch homolog protein 2 (NOTC2). Neurogenic locus notch homolog protein 3 (Notch-3), Neurogenic locus notch homolog protein 4 precursor (NOTC4), Neuronal cell adhesion molecule (Nr-CAM), Neuropeptide Y (NPY), NF-kappa-B inhibitor alpha (IKB-alpha), NF-kB p65 (NF-kB p65), Nibrin (NBN), Nicotinamide phosphoribosyltransferase (PBEF), Nischarin (NISCH), NOS (NOS), NT-3 growth factor receptor (TrkC), Nuclear factor erythroid 2-related factor 2 (NF2L2), Nuclear factor NF-kappa-B plOO subunit (NF-kappa-B plOO), Nuclear factor NF-kappa-B pl05 subunit (NF-kappa-B pl 05), Nuclear pore complex protein Nup98-Nup96 (NUP98), Nuclear receptor coactivator 1 (NCOA1), Nuclear receptor coactivator 2 (NCOA2),Nuclear receptor coactivator 3 (NC0A3), Nuclear receptor coactivator 4 (NC0A4), Nuclease-sensitive element-binding protein 1 (NSEP1), Nucleophosmin (NPM1), Nucleoside diphosphate kinase A (Nucleoside diphosphate kinase A), Nucleoside diphosphate kinase B (NDP kinase B), Occludin (OCLN), Ornithine decarboxylase (Ornithine decarboxylase), Osteopontin (Osteopontin), Oxytocin-neurophysin 1 (NEU1), P-selectin (SELP), pl6-INK4 (CDK4I). PAI-2 (PAI-2), Paired box protein Pax-3 (PAX3). Paired box protein Pax-8 (PAX-8). Paired mesoderm homeobox protein 1 (PRRX1), Parathyroid hormone-related protein (PTHrP), Partner and localizer of BRCA2 (PALB2), Peptidyl-prolyl cis-trans isomerase A (Cyclophilin A), Peptidyl- prolyl cis-trans isomerase NIMA-interacting 1 (PIN1), Peripheral plasma membrane protein CASK (CSKP), Peroxiredoxin- 1 (Peroxiredoxin- 1), Peroxisome proliferator activated receptor gamma (PPAR gamma), Peroxisome proliferator-activated receptor alpha (PPARa), Phorbol-12-myristate-13-acetate-induced protein 1 (APR), Phosphatidylethanolamine-binding protein 1 (prostatic binding protein), Phosphatidylinositol 3-kinase regulatory subunit alpha (P85A), Phosphatidylinositol 4.5-bisphosphate 3-kinase catalytic subunit delta isoform (PK3CD). Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (PK3CG), Phosphoglycerate kinase 1 (phosphoglycerate kinase 1), Phosphoglycerate mutase 1 (Phosphoglycerate mutase 1), Phosphoribosyltransferase domain-containing protein 1 (PRDC1). Pigment epithelium-derived factor (PEDF). PIK3CA / PIK3R1 (PIK3CA / PIK3R1), Placenta growth factor (P1GF), Plasma kallikrein (Prekallikrein), Plasminogen activator inhibitor 1 (PAI-1), Platelet endothelial cell adhesion molecule (PECAM-1), Platelet-derived growth factor receptor alpha (PDGFRA), Platelet- derived growth factor receptor beta (PDGF Rb). Platelet-derived growth factor subunit A (PDGF-AA), Platelet-derived growth factor subunit B (PDGF-BB), Pleiotrophin (PIN), Poliovirus receptor (Poliovirus receptor), Poly [ADP-ribose] polymerase EBRCAl C-terminal:BRCAl C-terminus domian (PARP:BRCT domain), Poly [ADP- ribose] polymerase Eregion 1 (PARP:region 1), Poly comb complex protein BMI-1 (BMI-1). Polycomb protein EED (EED), Polypeptide N- acetylgalactosaminyltransferase 16 (GLTL1), Porphobilinogen deaminase (HEM3), POU domain, class 5, transcription factor 1 (PO5F1), PPAR delta (PPAR delta), PRA1 family protein 3 (PRAF3), Preferentially expressed antigen of melanoma (PRAME),PRKC apoptosis WT1 regulator protein (PAWR), Pro-form (IGF-II:Pro-form). Pro- opiomelanocortin (Corticotropin-lipotropin), Probable ATP-dependent RNA helicase DDX17 (DDX17), Probable transcription factor PML (PML), Progesterone receptor (PgR), Programmed cell death 1 ligand 1 (B7-H1), Programmed cell death protein 4 (PDCD4), Prolactin (PRL), Prolactin receptor (Prolactin Receptor), Prolactin-inducible protein (PIP), Proliferating cell nuclear antigen (PCNA), Proliferation-associated protein 2G4 (PA2G4), Promotilin (MOTI), Prostaglandin E2 receptor EP2 subtype (PE2R2), Prostaglandin G / H synthase 2 (COX-2), Prostate-specific antigen (PSA), Proteasome activator complex subunit 1 (PSME1), Proteasome subunit alpha type-4 (PSA4), Protein atonal homolog 1 (ATOH1). Protein C-ets-2 (ETS2), Protein CBFA2T1 (MTG8), Protein CYR61 (CYR61), Protein DEPP (DEPP), Protein disulfi de-isomerase A3 (Protein disulfide isomerase A3), Protein FAM19A4 (F19A4), Protein jagged-1: Cytoplasmic domain (JAGECD), Protein jagged-1: Extracellular domain (JAGEECD), Protein jagged-2 (JAG2), Protein kinase B gamma (PKB gamma), Protein kinase C alpha type (PKC-A), Protein kinase C beta type (splice variant beta-11) (PKC-B-I1), Protein kinase C gamma type (PK.C-G). Protein kinase C zeta type (PKC-Z), Protein kinase C-binding protein NELLI (NELLI), Protein lin-28 homolog B (LN28B), Protein lin-7 homolog A (LIN7A), Protein max (MAX), Protein Mdm4 (MDM4), Protein NDRG1 (NDRG1), Protein patched homolog 2 (PTC2), Protein phosphatase ID (PPM1D), Protein polybromo-1 (PB1). Protein regulator of cytokinesis 1 (PRC1), Protein S 100-A10 (Calpactin I light chain), Protein S100-A1 1 (S100A11), Protein S100-A4 (S100A4), Protein S100-A5 (S100A5), Protein S100-A6 (S100A6), Protein S100-A8 / A9 heterodimer (S100A8 / S100A9), Protein TFG (TFG), Protein tyrosine phosphatase type IVA 1 (TP4A1), Protein Wnt-lOa (WN10A), Protein Wnt-lOb (WN10B), Protein Wnt-1 1 (WNT11), Protein Wnt-16 (WNT16), Protein Wnt-2 (WNT2), Protein Wnt-2b (WNT2B), Protein Wnt-3a (WNT3A), Protein Wnt- 5a (WNT5A), Protein Wnt-5b (WNT5B), Protein Wnt-7a (WNT7A), Protein XRP2 (XRP2), Protein-arginine deiminase type-4 (PADI4), Protein-glutamine gamma- glutamyltransferase 2 (Tissue transglutaminase), Protein-methionine sulfoxide oxidase MICAL1 (MICA1), Proteinase activated receptor 1 (PAR-1), Prothrombin (Prothrombin), Proto-oncogene protein c-fos (FOS), Proto-oncogene tyrosine-protein kinase MER (MER), Proto-oncogene tyrosine-protein kinase receptor Ret (RET),Proto-oncogene ty rosine-protein kinase ROS (ROS1), Proto-oncogene ty rosine-protein kinase Src (p60-Src), Proto-oncogene vav (VAV), Protogenin (PRTG), Putative methyltransferase NSUN6 (NSUN6), Pyruvate dehydrogenase El component beta subunit (PDHB), Pyruvate kinase PKM (M2-PK), Quinone oxidoreductase PIG3 (QORX), R-spondin-3 (RSPO3), RAC-alpha serine / threonine-protein kinase (PKB), RAC-beta serine / threonine-protein kinase (PKB beta), RAF proto-oncogene serine / threonine-protein kinase (c-Raf), RalBPl -associated Eps domain-containing protein 2 (REPS2), Rapl GTPase-activating protein 1 (RPGP1), Ras association domain-containing protein 5 (RASF5), Ras GTPase-activating protein 1 (RASA1), RAS guanyl-releasing protein 1 (GRP-1), Ras-related C3 botulinum toxin substrate 1 (RAC 1), Ras-related C3 botulinum toxin substrate 2 (RAC2), Ras-related C3 botulinum toxin substrate 3 (RAC3), Ras-related GTP-binding protein C (RRAGC), Ras-related protein R-Ras2 (RRAS2), Ras-related protein Rab-l lA (RB11A), Ras-related protein Rab-3A (Rab3A), Ras-related protein Ral-A (RALA), Ras-related protein Ral-B (RALB), Ras-related protein Rap- 1 A (RAP 1 A), Receptor tyrosine-protein kinase erbB-2 (ERBB2), Receptor tyrosine-protein kinase erbB-3 (ERBB3), Receptor tyrosine- protein kinase erbB-4 (ERBB4), Receptor-type tyrosine-protein kinase FLT3 (Flt-3). Receptor-type tyrosine-protein phosphatase delta (PTPRD), Receptor-ty pe ty rosine- protein phosphatase eta (PTPRJ), Reticulocalbin-1 (RCN1), Retinal dehydrogenase 1 (Retinal dehydrogenase 1), Retinoblastoma-associated protein (Rb). Retinoic acid receptor beta (RARB), Retinoic acid receptor RXR-alpha (Retinoic acid receptor RXR- alpha), Retinoic acid receptor RXR-gamma (RXRG), Retinol -binding protein 1 (CRBP), Retinol-binding protein 4 (RBP), Rho GDP-dissociation inhibitor 1 (Rho- GDIa), Rho guanine nucleotide exchange factor 1 (ARHG1). Rho guanine nucleotide exchange factor 11 (ARHGB), Rho guanine nucleotide exchange factor 12 (ARHGC), Rho-associated protein kinase 2 (R0CK2), Ribonucleoside-diphosphate reductase subunit M2 (RIR2), Ribosomal protein S6 kinase alpha-3 (RPS6KA3), Ribosomal protein S6 kinase alpha-5 (RSK-like protein kinase). Ribosomal protein S6 kinase beta- 1 (KS6B1). Ribosomal protein S6 kinase beta-2 (KS6B2). Ribosyldihydronicotinamide dehydrogenase [quinone] (QORX), RING-box protein 1 (RBX1), RNA-binding protein3 (RBM3), RNA-binding protein EWS (EWS), RNA-binding Raly-like protein (RALYL), Roundabout homolog 1 (R0B01), Roundabout homolog 2 (R0B02),rRNA 2'-0-methyltransferase fibrillarin (FBRL). Runt-related transcription factor 3 (RUNX3), S-methyl-5'-thioadenosine phosphorylase (MTAP), S-phase kinase- associated protein 1 (SKP1), S-phase kinase-associated protein 2 (SKP2), SAFB-like transcription modulator (SLTM), Sarcoplasmic / endoplasmic reticulum calcium ATPase 3 (AT2A3), SARP-3 (SARP-3), Scavenger receptor class B member 1 (SCRB1), Sciellin (SCEL), Secreted frizzled-related protein 1 (SARP-2), Secreted frizzled-related protein 2 (SARP-1), Secretin (Secretin), Segment polarity protein dishevelled homolog DVL-2 (DVL2), Seizure 6-like protein (SEZ6L), Selenium- binding protein 1 (Selenium-binding protein 1), Semaphorin-4A (SEM4A), sensory and motor neuron-derived factor isoform (SMDF), Sequestosome-1 (SQSTM), Serine hydroxymethyltransferase, cytosolic (cSHMT), Serine / threonine-protein kinase 4 (STK4), Serine / threonine-protein kinase A-Raf (ARAF), Serine / threonine-protein kinase B-raf (BRAF1), Serine / threonine-protein kinase Chkl (CHK1), Serine / threonine-protein kinase Chk2 (Chk2), Serine / threonine-protein kinase DCLK3 (DCLK3), Serine / threonine-protein kinase mTOR (FRAP), Serine / threonine-protein kinase pim-1 (P1M1), Serine / threonine-protein kinase Pim-2 (P1M2), Serine / threonine- protein kinase PLK1 (PLK-1), Serine / threonine-protein kinase Sgkl (SGK1), Serine / threonine-protein kinase / endoribonuclease IRE1 (ERN1), Serotransferrin (Transferrin), Serpin B5 (Maspin). Serum paraoxonase / arylesterase 1 (paraoxonase 1), SET-binding protein (SETBP), SH2B adapter protein 3 (SH2B3). SH3 and multiple ankyrin repeat domains protein 1 (SHAN1), Sialidase-1 (NEUR1), Signal transducer and activator of transcription 1-alpha / beta (STAT1), Signal transducer and activator of transcription 2 (STAT2), Signal transducer and activator of transcription 3 (STAT3), Signal transducer and activator of transcription 5A (STA5A), Signal transducer and activator of transcription 5B (STA5B), Signal transducer and activator of transcription 6 (STAT6), SMAD5 (SMAD5), Small nuclear ribonucleoprotein Sm D3 (SMD3), Sodium / iodide cotransporter (SC5A5), Sodium / potassium-transporting ATPase subunit alpha-1 (AT1A1), Sodium / potassium-transporting ATPase subunit beta-1 (AT1B1), soluble (TEK), soluble form (FASLG). Somatostatin-28 (Somatostatin-28). Somatotropin (HGH), Son of sevenless homolog 1 (SOS1), Sonic hedgehog protein (Sonic Hedgehog), SOX-9 (SOX-9), SPARC (ON SPARC), Speckle-type POZ protein (SPOP), Squamous cell carcinoma antigen 1 (SCCA1). Stanniocalcin-2 (STC2),Stathmin (Stathmin), Steroid hormone receptor ERR1 (ERRalpha), Stress-70 protein, mitochondrial (HSPA9B), Stromal cell-derived factor 1 (SDF-1), Stromal cell-derived factor Ibeta (SDF-lb), Stromelysin-1 (MMP-3), Stromelysin-2 (MMP-10), Succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial (SDHB), Succinate dehydrogenase assembly factor 2, mitochondrial (SDHF2), Sulfotransferase family cytosolic 2B member 1 (ST2B1), SULT 1A12 (SULT 1A12). SUMO-conjugating enzyme UBC9 (UBC9), Superoxide dismutase [Cu-Zn] (SOD). Superoxide dismutase [Mn], Suppressor of fused homolog (SUFU), SWI / SNF complex subunit SMARCC1 (SMRC1), SWI / SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1 (SNF5), Synembryn-A (RIC8A), Syntaxin-binding protein 4 (STXB4), T-box transcription factor TBX3 (Tbx3), T-cell leukemia / lymphoma protein 1A (TCL1A), T-cell leukemia / lymphoma protein IB (TCL1B), T-cell surface glycoprotein CD3 delta chain (CD3D), T-cell surface glycoprotein CD3 epsilon chain (CD3E), T-cell surface glycoprotein CD4 (sCD4), T-cell surface glycoprotein CD8 alpha chain (CD8A), T-complex protein 1 subunit epsilon (TCP- 1 -epsilon), TATA- binding protein-associated factor 2N (RBP56), Tenascin-C (TNC). TGF-beta receptor type-1 (TGFR-1), TGF-beta receptor type-2 (TGF-b R II), Thiopurine S- methyltransferase (TPMT), Thioredoxin (Thioredoxin), Thioredoxin domain- containing protein 15 (TXD15). Thioredoxin reductase 1 (Thioredoxin reductase 1), Thrombin (Thrombin), Thrombopoietin Receptor (Thrombopoietin Receptor). Thrombospondin- 1 (Thrombospondin-1), Thymidine kinase (Thymidine kinase), Thymidine phosphorylase (TP), Thymidylate synthase (TS), Thyrotropin receptor (TSHR), Tight junction protein ZO-1 (ZO1), Tissue Factor (TF), Tissue factor pathway inhibitor 2 (TFPI-2). Titin (TITIN), TNF receptor-associated factor 1 (TRAF1). TNF receptor-associated factor 4 (TRAF4), TNF receptor-associated factor 5 (TRAF5), TNFAIP 3 -interacting protein 1 (TNIP1), TNFR-Cys domains 1-3 (TRAIL R2:ECD), Toll-like receptor 3 (TLR3), Toll-like receptor 4 (TLR4), Toll-like receptor 4:Lymphocyte antigen 96 complex (TLR4:MD-2 complex), TOX high mobility group box family member 3 (T0X3). TRAF family member-associated NF -kappa-B activator (TANK), transcription factor 1 (POU5F1), Transcription factor AP-1 (c-Jun), Transcription factor E2F3 (Transcription factor E2F3), Transcription factor HES-1 (HES1), Transcription factor HES-5 (HES5), Transcription factor PU. 1 (SPI1),Transcription factor SOX-2 (S0X2), Transcriptional activator Myb (MYB), Transcriptional coactivator YAP1 (YAP1), Transcriptional repressor CTCF (CTCF), Transcriptional repressor protein YY1 (TYY1), Transferrin receptor protein I : Cy toplasmic domain (TR:CD), Transferrin receptor protein 1 : Extracellular domain (TR:ECD), Transforming acidic coiled-coil-containing protein 3 (TACC3), Transforming growth factor alpha (TGF-a), Transforming grow th factor beta-1 (TGF- bl), Transforming growth factor beta-3 (TGF-b3), Transforming protein RhoA (Rho A), Transgelin-2 (Transgelin-2), Transketolase (Transketolase), Translationally- controlled tumor protein (TCTP), Transmembrane glycoprotein NMB: Cytoplasmic domain (GPNMB:CD), Transmembrane glycoprotein NMB: Extracellular domain (GPNMB:ECD), Transmembrane protein 132D (T132D), Transmembrane protein 25 (TMM25), Triosephosphate isomerase (Triosephosphate isomerase). Triple functional domain protein (TRIO), Tropomyosin alpha-3 chain (Tropomyosin alpha-3 chain), Tropomy osin alpha-4 chain (Tropomy osin 4), Tuberin (TSC2), Tubulin alpha (Tubulin alpha), Tubulin alpha-lA chain (TBA1A). Tumor necrosis factor (TNF-a), Tumor necrosis factor alpha-induced protein 3 (TNFAIP3), Tumor necrosis factor alpha- induced protein 8 (TFIP8), Tumor necrosis factor ligand superfamily member 10 (TRAIL), Tumor necrosis factor ligand superfamily member 6, soluble form (Fas ligand, soluble), Tumor necrosis factor receptor superfamily member 10A (TRAIL Rl), Tumor necrosis factor receptor superfamily member 10B:Death domain (TRAIL R2:Death), Tumor necrosis factor receptor superfamily member 10B:Extracellular domain (TRAIL R2:ECD), Tumor necrosis factor receptor superfamily member 19 (TAJ), Tumor necrosis factor receptor superfamily member 4 (TNR4), Tumor necrosis factor receptor superfamily member 6 (Fas, soluble), Tumor necrosis factor receptor superfamily member 8 (CD30), Tumor necrosis factor receptor superfamily member 9 (4-1BB), Tumor protein 63 (P73L), Tumor-associated calcium signal transducer 1 (EpCam), Tumor- Associated Glycoprotein 72 (TAG-72), Twist related protein 1 (Twist related protein 1), Two Chain (Kininogen, HMW, Two Chain), type I cytoskeletal 14 (K1C 14). type I cytoskeletal 17 (Keratin 17), type I cytoskeletal 18 (Keratin 18), type I cytoskeletal 19 (Keratin 19), type I cytoskeletal 20 (Keratin 20), type II cytoskeletal 5 (K2C5), type II cytoskeletal 7 (Keratin 7), type II cytoskeletal 71 (K2C71), Type II inositol-3, Tyrosine-protein kinase ABL1 (ABL1), Tyrosine-protein kinase BTK(BTK), Tyrosine-protein kinase Fyn (FYN), Tyrosine-protein kinase JAK1 (JAK1), Tyrosine-protein kinase JAK2 (JAK2). Tyrosine-protein kinase JAK3 (JAK3), Tyrosine-protein kinase Lyn, isoform B (LYNB), Tyrosine-protein kinase receptor UFO (AXL), Tyrosine-protein phosphatase non-receptor type 1 (PTP-1B), Tyrosine- protein phosphatase non-receptor type 11 (SHP-2), Ubiquitin carboxyl-terminal hydrolase BAP1 (BAP1), Ubiquitin carboxyl-terminal hydrolase CYLD (CYLD), Ubiquitin carboxyl-terminal hydrolase isozyme LI (PGP9.5), Ubiquitin D (UBD). Ubiquitin-conjugating enzyme E2 C (UBE2C), Ubiquitin-conjugating enzyme E2 T (UBE2T), Ubiquitin-like domain-containing CTD phosphatase 1 (UBCP1), UDP- glucuronosyltransferase 1-1 (UGT 1A1), Uncharacterized protein C10orf25 (CJ025), Urokinase plasminogen activator surface receptor (suPAR), Urokinase-type plasminogen activator (uPA), Uroporphyrinogen decarboxylase (DCUP), Vacuolar protein sorting-associated protein 53 homolog (VPS53), Vascular endothelial growth factor A (VEGF), Vascular endothelial growth factor B (VEGF-B), Vascular endothelial growth factor C (VEGF-C), Vascular endothelial growth factor D (VEGF- D). Vascular endothelial growth factor receptor 1 (VEGF sRl), Vascular endothelial growth factor receptor 2 (VEGF sR2), Vascular endothelial growth factor receptor 3 (VEGF sR3), Very Late Antigen-4 (VLA-4), Vesicle transport through interaction with t-SNAREs homolog 1A (VTI1A), Vesicle-associated membrane protein 8 (VAMP8), Vimentin (Vimentin), Vinculin (Vinculin), VIP36-like protein (LMA2L), Vitamin D3 receptor (VDR), Von Hippel-Lindau disease tumor suppressor (VHL), WAP four- disulfide core domain protein 2 (HE4), Wnt inhibitory factor 1 (WIF-1), WNT1- inducible-signaling pathway protein 1 (WISP-1), WW domain-containing oxidoreductase (WWOX), X isoform (RS4X), Xanthine dehydrogenase / oxidase (XDH), Zinc finger and BTB domain-containing protein 16 (ZBT16), Zinc finger and BTB domain-containing protein 7 A (ZBT7A), Zinc finger E-box -binding homeobox 1 (ZEB1), Zinc finger protein GLI2 (GLI2), Zinc finger protein SNAI1 (SNAI), Zinc finger protein SNAI2 (SLUG). Zinc-alpha-2-gly coprotein (AZGP1), and combinations thereof.

[0240] EXAMPLE

[0241] This Example describes a Boolean interpretation framework for an 8-target VHH panel in cancer surveillance.

[0242] Each of the 8 VHH-based probes targets a distinct biological compartment of tumor biology , chosen to maximize cancer coverage while minimizing false positives in healthy or benign conditions.

[0243] Tumor Lineage Markers (’‘Tier 1” Markers): CLDN18.2, GPC3, TAG-72. These are largely absent in normal adult tissues, appearing mainly in embryonic / fetal or placental contexts, but re-expressed in specific cancers. For example. CLDN18.2 (a tight-j unction protein) is normally confined to gastric mucosa and is nearly undetectable elsewhere in adults, yet it is frequently overexpressed in gastric and pancreatobiliary cancers. GPC3 (glypican-3) is an oncofetal proteoglycan not expressed in healthy adult liver or benign liver disease, but present in -70-80% of hepatocellular carcinomas and in some germ cell tumors. TAG-72 is a tumor-associated glyco-glycoprotein rarely seen in normal adult tissues (absent except transiently in secretory endometrium), yet found in >80% of colorectal adenocarcinomas and a majority of other adenocarcinomas (gastric, pancreatic, ovarian, lung). When one of these tumor lineage markers is detected in blood, it strongly suggests the presence of tumor cells of a particular lineage, since healthy tissue has virtually no such antigen expression.

[0244] Broad Tumor Cell Marker (“Tier 2” Markers): B7-H3 (CD276). B7- H3 is an immune checkpoint protein with very low basal expression in normal adult tissues (mostly restricted to placenta and some immune cells). In contrast, it is overexpressed in a wide range of solid tumors, including lung, breast, prostate, kidney, brain, and others. For instance, >80% of breast tumors expressed B7-H3 vs <15% of normal breast samples, and 95% of clear-cell renal carcinomas were B7-H3-positive. B7-H3 can also appear on tumor endothelium and activated immune cells in the tumor microenvironment. As a single marker, B7-H3 is high-value but not absolutely specific (e.g. placenta causes circulating B7-H3 in pregnancy). Its presence indicates a broad solid tumor signal, so is best used in combination with more specific markers or interpreted with caution if isolated.

[0245] Tumor Microenvironment / Stromal Markers (“Tier 3” Markers): ED- 13 Fibronectin, FAP. These mark the desmoplastic stroma and neovasculature associated with invasive tumors. ED-B fibronectin is an oncofetal splice vanant of fibronectin; it is almost undetectable in normal adult tissues or mature vessels, but becomes abundant in tissues undergoing repair, fibrosis, or angiogenesis. It is highly expressed around new blood vessels in tumors and in fetal / placental tissue, hence its appearance in cancers (and in healing wounds). FAP (Fibroblast Activation Protein) is a protease on activated fibroblasts; it is not expressed by resting fibroblasts but is induced during wound healing and fibrosis. Crucially, FAP is present on cancer- associated fibroblasts in >90% of epithelial cancers, making it a near-universal marker of tumor stroma. However. FAP also appears in benign conditions of tissue remodeling (e.g. post-injury, inflammatory fibroses). Tumor microenvironment / stromal markers are thus highly sensitive for the presence of an invasive tissue remodeling process, but by themselves are not cancer-specific - context (e.g. recent surgery) is needed to distinguish tumor vs benign causes.

[0246] Cell Death / Intrinsic Markers (“Tier 4” Markers): “Tumor Necrosis” antigen, ORFlp. These capture signals of cell turnover and necrosis. The “tumor necrosis target” is an intracellular antigen unmasked by cell lysis (e.g. a nucleoprotein or cytosolic protein common to dying cells). Such necrosis markers are non-specific - any' significant cell death (tumor or non-tumor) can release them (e.g. LDH is a classic necrosis marker released from dying cells of any type). ORFlp, by contrast, is an oncogenic driver protein (LINE-1 retrotransposon) normally silenced in healthy adult tissues, but aberrantly expressed in many cancers. ORFlp is essentially a “tumor- intrinsic” marker - its presence in the circulation strongly indicates malignant cell activity (e.g. detected via exosomes or necrotic debris). However, ORFlp’s accessibility to the bloodstream often requires tumor cell death or secretion, meaning it often co-occurs with necrosis signals. The cell death / intrinsic markers indicate cell destruction; ORFlp in particular points to malignant origin of that debris, whereas a generic necrosis marker alone could come from non-cancer injury (infarct, trauma, etc).

[0247] The Boolean interpretation rules (below) require combinations of these markers to confirm a cancer signal, ensuring that a malignancy is only called whenthere is evidence from multiple angles (tumor cells + stroma, or tumor cells + necrosis, etc.), whereas benign processes rarely activate more than one compartment at a time.

[0248] After internal normalization to the non-binding control (NBCP) and assay QC, each probe is called Positive (1) or Negative (0). The set of positive markers (pattern) is interpreted via logical rules into one of five result categories: Strong Negative (SN). Conditioned Negative (CN), Indeterminate (IND). Conditioned Positive (CP), or Strong Positive (SP). Tables 6 and 7 below enumerates representative marker patterns (especially all 2-marker and 3-marker combos, plus key singles and 4-marker combos) with their default category, rationale, and recommended action. (‘‘Tier’" notation: A = tumor lineage, B = broad tumor. C = stroma, D = cell death.)Table 6. Two-Marker Positive PatternsPositiveMarkers Category Rationale (Logic) Recommended Action{ED-B, IND Stroma-only: both Tier- Indeterminate. Check forFAP} C markers with no recent surgery' or tumor-cell marker. Could inflammation; if yes, be benign fibrotic / wound categorize as CN and retest response after -8-12 weeks. If no patient h context, retest in 2-4 weeks. healing fibrosis, (benign c{B7-H3. CP Tumor + Stroma: Tier- Conditioned Positive.ED-B} B (broad tumor cell) plus Consider confirmation (e.g.Tier-C. Likely imaging of likely tumor site), malignancy (e.g. solid If patient recently had tumor with desmoplastic surgery (placental or wound stroma), but B7-H3 is context), exercise caution or broad and can rarely arise defer, in reactive endothelium 9],{B7-H3, CP Tumor + Stroma: broad Conditioned Positive.FAP} tumor marker plus CAF Confirm with targeted test marker. Similar to above: (imaging or repeat panel), suspicious for solid Absent a benign explanation, tumor with fibroblast plan to escalate on activation, but each could confirmation, rarely be context-related (e.g. B7-H3 inPositiveMarkers Category Rationale (Logic) Recommended Action inflammation, FAP in fibrosis).{B7-H3, CP Tumor + Cell Death: Conditioned Positive.Necrosis} Tier-B plus necrosis Short-term repeat or do signal. Implies tumor cell imaging to confirm presence with tissue persistence. If confirmed breakdown. However, (and no alternate cause), necrosis could be from a upgrade to SP and begin non-tumor injury (and work-up. B7-H3 alone isn’t organ- specific).{ED-B, CP Stroma + Cell Death: Conditioned Positive.Necrosis} cross-compartment (Tier- Investigate confounders: C + D) pattern but no e.g. recent infarct, abscess. If direct tumor-cell marker. a know n benign cause is Could reflect an injured present, treat as CN (no healing site with necrosis cancer) and retest later. If no (e.g. post-infarction context, retest in ~4 weeks; granulation tissue). consider imaging if pattern persists.{FAP, CP Stroma + Cell Death: Conditioned Positive.Necrosis} similar to {ED-B, Evaluate for benign causes Necrosis}. FAP indicates (e.g. healing myocardial activated fibroblasts; infarction). If found, treat as necrosis could be from a CN and follow up after benign process. No healing. If not, retest or tumor-specific marker image to confirm a hidden present. malignancy.{B7-H3, SP Tumor-cell (broad + Strong Positive. High-CLDN18.2} lineage): B7-H3 (pan- confidence malignancy tumor) and CLDN18.2 (likely upper GI origin). (highly specific Escalate to imaging and gastric / pancreatic oncology consult lineage[2J). Independent immediately. tumor signals - extremely unlikely to both be false.{B7-H3, SP Tumor-cell (broad + Strong Positive. ImmediateGPC3} lineage): B7-H3 plus work-up (e.g. liver MRI and GPC3 (oncofetal liver AFP test) for HCC marker[3j). Strongly recurrence. indicative of HCC or related tumor; benignPositiveMarkers Category Rationale (Logic) Recommended Action causes for this combo are essentially none.{B7-H3, SP Tumor-cell (broad + Strong Positive. Immediate TAG-72} lineage): B7-H3 plus imaging (abdominal / pelvic) TAG-72 to locate recurrence. (adenocarcinoma marker] 51). Suggests an epithelial carcinoma (likely GI or ovarian).{B7-H3, SP Tumor + Tumor- Strong Positive. EscalateORFlp} Intrinsic: B7-H3 promptly - broad imaging indicates tumor presence; (if original site uncertain) or ORFlp is cancer-intrinsic directed work-up to find (LINE-l)i i -H and almost active disease. never released except by malignancy. Co- positivity virtually confirms circulating tumor debris.{CLDN18.2, SP Tumor-cell + Stroma: Strong Positive. Immediate ED-B} CLDN18.2 (specific diagnostic gastric lineage) with ED- endoscopy / imaging for B fibronectin (angiogenic upper GI tumor recurrence. stroma)! ]). Orthogonal markers confirm an invasive tumor (e.g. gastric carcinoma with neovasculature).{ED-B, SP Tumor-cell + Stroma: Strong Positive. Image liver GPC3} GPC3 (specific to liver (MRI) and check tumor tumor) plus ED-B (tumor markers (AFP) at once. ECM). Confirms presence of HCC cells and tumor stroma.{ED-B, SP Stroma + Tumor- Strong Positive. Whole- ORFlp} Intrinsic: ORFlp body or site-directed indicates malignant imaging (if primary cells] 14j; ED-B indicates unknown) to locate invasive context. malignancy (ORFlp has no Combination means organ specificity). tumor cells are present with angiogenic stroma.PositiveMarkers Category Rationale (Logic) Recommended Action{ED-B. SP Tumor-cell + Stroma: Strong Positive. Imaging ofTAG-72} TAG-72 likely sites (e.g. CT of (adenocarcinoma marker) chest / abdomen / pelvis for GI plus ED-B. Points to an malignancy) immediately. adenocarcinoma with desmoplastic reaction (common in colon, pancreatic, etc.).{CLDN18.2, SP Tumor-cell + Stroma: Strong Positive. Immediate FAP} CLDN18.2 plus FAP- work-up (e g. PET / CT or positive CAFs. Highly endoscopic evaluation) for specific for an upper GI gastric or pancreatobiliary tumor with invasive recurrence. stroma (no benign scenario gives this).{GPC3, SP Tumor-cell + Cell Strong Positive. Image the Necrosis} Death: GPC3 (HCC liver urgently for recurrent marker) plus necrosis. tumor. Likely an HCC lesion undergoing necrosis. Benign liver necrosis (e.g. infarct) would not express GPC3.{Necrosis, SP Cell Death + Tumor- Strong Positive. Whole- ORFlp} Intrinsic: ORF Ip’s body search for tumor (if no presence confirms known site). Escalate necrotic debris is from immediately. cancer cells H 41. (E.g. a tumor undergoing necrosis.){CLDN18.2, SP Two lineage markers: Strong Positive. Thorough GPC3} Unlikely combination work-up (could indicate (e.g. concurrent gastric multiple tumors or and liver lineage heterogeneous expression). markers) except in Consider if patient had multi- metastatic setting or cancer hi story. multi-primary scenario. Any dual Tier-A positivity is treated as malignancy by definition.{CLDN18.2, SP Tumor-cell + Tumor- Strong Positive. Direct ORFlp} Intrinsic: CLDN18.2 imaging of stomach / GE confirms tumor of gastric junction (and related areas) - lineage; ORFlp confirmsPositiveMarkers Category Rationale (Logic) Recommended Action actual malignant high likelihood of turnover. recurrence.{GPC3, SP Tumor-cell + Tumor- Strong Positive. Work upORF Ip} Intrinsic: GPC3 liver recurrence indicates liver tumor, immediately; consider biopsyORF Ip confirms confirmation while malignancy. (Orthogonal proceeding to treatment, confirmation within Tier- D and Tier-A.){FAP, SP Stroma + Tumor- Strong Positive. Imaging -ORF Ip} Intrinsic: FAP -positive could be any carcinoma (no stroma plus ORF 1 p lineage marker to pin organ,(malignant cells). Implies so broad survey ), a cancer with active stroma and cell turnover (any solid tumor can fit).{TAG-72, SP Tumor-cell + Tumor- Strong Positive. ImmediateORF Ip} Intrinsic: TAG-72 diagnostic work-up (likely(adenocarcinoma) plus GI or ovarian; use patient ORFlp. Strong evidence history to target imaging), of an adenocarcinoma shedding tumor DNA / protein.Table 7. Three-Marker Positive PatternsPositiveMarkers Category Rationale (Logic) Recommended Action{ED-B, FAP, CP Stroma + Necrosis, multi-hit: Conditioned Positive.Necrosis} Two Tier-C + Tier-D. Strong Search for confounders combined signal of tissue (recent major surgery, remodeling and cell death, but infarction). If found, no tumor-cell marker likely benign (would present. This could still occur be CN); if not, this in a large benign injury (e.g. pattern raises suspicion major surgery with necrotic - retest soon or tissue) - hence not definitive perform imaging to be without a tumor marker. safe.{B7-H3, ED- SP Tumor + rich Stroma: Tier-B Strong Positive. B, FAP) plus both stromal markers. Escalate immediately Indicates a solid tumor with - likely a carcinoma vigorous desmoplastic stroma (organ per patient’s (e.g. an invasive carcinoma). cancer history). With multiple compartments Perform whole-bodyPositiveMarkers Category Rationale (Logic) Recommended Action involved, a benign explanation PET / CT if needed to is highly unlikely (e.g. it’s locate recurrence. hard to get simultaneous B7- H3, ED-B, and FAP elevation without a tumor).{B7-H3, ED- SP Tumor + Stroma + Necrosis: Strong Positive.B, Necrosis} Broad tumor marker, stromal Immediate work-up. marker, and necrosis. Points to For example, in a lung an aggressive tumor (necrotic) cancer survivor, this with stromal response. No pattern would strongly single benign process would suggest recurrent cause all three simultaneously. tumor - proceed to imaging of chest and metastatic sites.{B7-H3, SP Tumor + Stroma + Necrosis: Strong Positive.FAP, Similar to above: B7-H3 plus Immediate imagingNecrosis} CAF activation and necrosis. and intervention -Orthogonal evidence of likely tumor recurrence malignancy. with necrosis (common in larger metastases).{ED-B, SP Stroma + Necrosis + Tumor- Strong Positive.Necrosis, Intrinsic: Even without a Tier- Escalate to full work-ORF Ip} A or B marker, the presence of up. Consider whole-ORFlp along with ED-B and body scan if no prior Necrosis seals specificity (the cancer type predicts ORFlp implies the necrosis is the site. from cancer cells p 41). Tumor vasculature and death are evident.{FAP, SP Stroma + Necrosis + Tumor- Strong Positive.Necrosis, Intrinsic: CAF activation, cell Immediate work-upORF Ip} death, and a cancer-intrinsic (broad search if marker together. Ensures the necessary). necrosis is malignant.{B7-H3, ED- SP Tumor + Stroma + Tumor- Strong Positive.B, ORFlp} Intrinsic: A broad tumor Urgent investigation. marker with ORFlp (cancer- Likely recurrence in specific) plus ED-B. Clear prior tumor site, but indicator of a solid tumor consider second shedding ORFlp. primary if unexpected for that marker combo.PositiveMarkers Category Rationale (Logic) Recommended Action{B7-H3, SP Tumor + Necrosis + Tumor- Strong Positive.Necrosis, Intrinsic: B7-H3 and ORF Ip Immediate escalation.ORF Ip} confirm malignant cells; E.g., in a patient with necrosis suggests tumor prior cancer, this damage. Orthogonal would prompt urgent confirmation of malignancy. imaging of known and common metastatic sites.{B7-H3, SP Tumor + Stroma + Tumor- Strong Positive.FAP, Intrinsic: B7-H3 and ORF Ip Immediate work-upORF Ip} ensure a malignant origin, FAP (target imaging to adds invasive stroma context. likely organ based onHighly specific. history).{TAG-72, SP Tumor (lineage) + Stroma: Strong Positive.ED-B, FAP} TAG-72 (mucinous carcinoma Imaging ASAP - marker) plus both stromal likely sites include markers. Indicates an colon, pancreas, etc., adenocarcinoma with depending on patient’s extensive stromal reaction history,(common in GI cancers). No benign process would have TAG-72 with such stromal activity.{TAG-72, SP Tumor + Stroma + Necrosis: Strong Positive.ED-B. TAG-72 plus ED-B plus Urgent imaging (e.g.Necrosis} necrosis, indicating a tumor if prior colon cancer,(likely adenocarcinoma) that is check liver and lungs necrotic and inducing stroma. for metastatic lesions that may be necrotic).{CLDN18.2, SP Tumor + Stroma + Necrosis: Strong Positive.ED-B, CLDN18.2 (gastric lineage) ImmediateNecrosis} plus ED-B and necrosis endoscopic evaluation suggests an ulcerating gastric and imaging of tumor. All three together are stomach / upper GI. definitive for malignancy.{GPC3, SP Tumor + Stroma + Necrosis: Strong Positive.FAP, GPC3 (HCC) with FAP and Urgent liver imaging;Necrosis} necrosis - likely a necrotic treat as recurrence until hepatocellular carcinoma proven otherwise, nodule with stromal interface.{CLDN18.2. SP Tumor + Stroma + Tumor- Strong Positive.FAP, Intrinsic: CLDN18.2 and Immediate work-upORF Ip} ORF Ip confirm malignant (upper GI imaging).PositiveMarkers Category Rationale (Logic) Recommended Action gastric / pancreatic cells; FAP indicates invasion.{GPC3, ED- SP Tumor + Stroma + Tumor- Strong Positive.B, ORF Ip} Intrinsic: GPC3 and ORF Ip Immediate work-up confirm malignant liver cells; (liver-focused). ED-B indicates angiogenic stroma.

[0249] Virtually any 3-marker pattern that includes ORF Ip or a Tier- A marker is Strong Positive by default, due to the high cancer-specificity. The only 3- marker patterns that were not SP by rules are those lacking any definitive tumor-cell marker - e.g. {ED-B, FAP, Necrosis} as shown above, which is CP pending context / confirmation.

[0250] Selected Four-marker Patterns

[0251] All 4-marker (and higher) positive combinations generally meet Strong Positive criteria, since they represent multi-compartment activation that benign processes rarely replicate. A few illustrative examples:

[0252] {B7-H3, ED-B, FAP, Necrosis} - SP: Broad tumor marker + both stromal markers + necrosis. This comprehensive pattern indicates an advanced malignancy with necrosis and desmoplastic response. No single benign condition could cause all four markers at once. Action: immediate full diagnostic work-up (likely a large invasive tumor).

[0253] {CLDN18.2, B7-H3, ED-B, FAP} - SP: Gastric lineage marker + broad tumor + stroma x2. Strong evidence of an upper GI carcinoma with rich stroma. Action: urgent gastroenterology referral and imaging.

[0254] {ED-B, FAP, Necrosis, ORFlp} - SP: Stroma x2 + necrosis + tumor-intrinsic marker. Even though no Tier-A or B here, the presence of ORFlp alongside ED-B / FAP ensures the necrotic process is cancer (e.g. an occult tumor withlarge infarcted area and stromal reaction). Action: immediate whole-body screening to find the tumor.

[0255] Any pattern including ORF Ip with any other marker is Strong Positive by definition, due to ORFlp’s exclusivity to malignant tissue combined with another signal

[0014] . Similarly, any pattern with two or more Tier-A markers or Tier-A plus any other is SP, as those provide organ-specific malignant evidence. Patterns listed as CP become SP if they persist on repeat or if an orthogonal test (imaging / biomarker) comes back positive corroborating malignancy.

[0256] PatternLikely Organ Mapping

[0257] Certain marker patterns can indicte the likely site or organ system of the recurrence, which can guide confirmatory diagnostics. Table 8 below is a mapping of some positive patterns to the most commonly associated tumor origins, based on known marker expression profiles (incidence-weighted for common cancers):Table 8. Map of positive patterns for commonly associated tumor origins.Positive Pattern (markers) Most Likely Origin(s) & Organ System (with rationale){CLDN18.2 ± X} (any Upper GI tract - Specifically gastric or pattern containing gastroesophageal junction cancer is most likely, as CLDN18.2) CLDN18.2 is expressed in -40-50% of gastric adenocarcinomas] 15] and originates in gastric mucosa. A subset of pancreatic adenocarcinomas (-50%) also express CLDN18.2]2], so pancreatic cancer is a secondary consideration. Next steps: Focus imaging on the stomach (endoscopy) and pancreas (if imaging suggests).{GPC3 ± X} (any Liver (Hepatocellular Carcinoma) - Glypican-3 is pattern with GPC3) highly specific to HCC (positive in -72% of HCC cases, and not in normal liver)] 16] . If the patient has a history of HCC, recurrence in the liver or a metastasis is likely. In the absence of known liver cancer, consider germ cell tumors (yolk sac components) as GPC3 is also expressed in certain testicular / ovarian germ cell tumors] 17], albeit those are far less common. Next steps: Liver imaging (e.g. MRI) and liver function / AFP labs.{TAG-72 ± X} (any Adenocarcinomas of Mucinous lineage: Highest pattern with TAG-72) likelihood is colorectal carcinoma (TAG-72 in -80% of cases)] 18] given its high incidence, followed by gastricPositive Pattern (markers) Most Likely Origin(s) & Organ System (with rationale) and pancreatic adenocarcinomas, and ovarian mucinous carcinoma which frequently express TAG- 72[5j. Many lung adenocarcinomas (NSCLC) also express TAG-72119], Use patient’s prior cancer type if any (e.g. a colon cancer survivor with TAG-72 pattern — > colon recurrence until proven otherwise). Next steps: Imaging of abdomen / pelvis (or chest for lung) based on likely organ; consider serum markers like CEA, CAI 9-9 corresponding to GI malignancies.{B7-H3 + (ED-B Broad solid tumor signal - site indeterminate: B7-H3 is and / or FAP) ± overexpressed across many cancers (lung, breast, prostate, Necrosis} (Tier- kidney, etc.)[ 7 }, so a pattern of B7-H3 with stromal B with Tier-C / D, no and / or necrosis markers doesn’t pinpoint an organ but Tier-A) confirms a malignancy. The likely origin will often be the patient’s previous cancer site (e.g. a breast cancer survivor with {B7-H3, FAP} is likely breast tumor recurrence). If the patient’s prior cancer was B7-H3- negative or a long time ago, consider common adult cancers: lung and colon are high on the list (very prevalent and often B7-H3-positive[7{), followed byprostate, renal, breast depending on demographics. Next steps: Context-driven imaging. If prior cancer exists, scan that organ’s region and typical metastasis locations. If no history-, perform whole-body PET / CT or broad MRI to locate the tumor.{ED-B, FAP + any Invasive carcinoma at the organ indicated by the Tier-A / B marker} tumor-cell marker. The presence of both ED-B and FAP(Stroma markers means a robust desmoplastic response, which solidifies plus a tumor-cell that an invasive tumor is present in the organ signaled by marker) the Tier-A or B marker. Examples: {TAG-72, ED-B, FAP} points to a desmoplastic colorectal or pancreatic carcinoma; {B7-H3, ED-B, FAP} suggests a fibrotic carcinoma of common type (could be lung, breast, etc. - use history-). Next steps: pursue imaging of the organ system flagged by the tumor marker (colonoscopy / CT colon for TAG-72 if colon cancer was original, etc.).{ORFlp + any (no Malignancy of unknown primary - ORFlp is a pan- Tier-A)} cancer marker} 1 A j, so if ORFlp is positive alongside(ORFlp present, only broad or microenvironment markers (no organ- lineage unclear) specific marker), the organ could be anywhere. Statistically, consider high-incidence cancers that commonly have necrosis: lung, colorectal, breast, pancreatic, etc. Next steps: Use all available clinical data - e.g. prior cancer history or symptoms - to guide aPositive Pattern(markers) Most Likely Origin(s) & Organ System (with rationale) whole-body search. A PET-CT is often warranted to localize the ORFlp-shedding tumor. Once any lesion is found, targeted diagnostic biopsy may confirm the tissue of origin.

[0258] These organ likelihoods provide a guide. Patient-specific factors (previous cancer type, genetic risks, clinical symptoms) heavily inform which organ is actually involved. E.g., a {CLDN18.2, ED-B} pattern in a gastric cancer survivor is almost certainly gastric recurrence, whereas the same pattern in someone with no gastric history but a pancreatic cancer history could mean a pancreatic relapse with aberrant CLDN18.2 expression. Always integrate the pattern with the patient's oncologic history and epidemiology. The panel’s role is to narrow the search and prioritize investigations (for instance, an ORFlp-positive result tells one to look for cancer somewhere, even if a wide net must be cast, whereas a TAG-72-positive result directs attention to adenocarcinoma-prone organs).

[0259] Clinician interpretation summary (5-Tier System)

[0260] Every result falls into one of five interpretation categories, which dictate clinical action. It is important to note that these results are an adjunct to clinical judgment - patient context (e.g. recent procedures, known benign conditions) must be considered in interpretation.

[0261] Strong Negative (SN): No evidence of recurrence. Definition: All 8 tumor markers are negative (within normal background), OR only an isolated Tier-C marker is positive with a clear benign reason. Action: Continue routine surveillance as per standard follow-up schedule. No change in management: reassure patient. (Example: all markers 0; or FAP+ only but patient is 6 weeks post-surgery - benign healing.)

[0262] Conditioned Negative (CN): Benign-pattern positive (false alarm due to context. Definition: One or more markers are positive, but the pattern matches aknown benign process in this patient (“explained positive”). E.g. isolated ED-B and / or FAP in a patient vxi th a large healing wound or fibrotic disorder; isolated Necrosis marker after a recent myocardial infarction; transient TAG-72 in a lactating patient, etc. Action: Treat as a negative for cancer - do not initiate cancer work-up. Address the underlying benign cause if needed. Repeat the panel once the confounding condition has resolved (e g. 2-3 months later) to ensure markers return to negative.

[0263] Indeterminate (IND): Uncorroborated positives (ambiguous signal). Definition: A pattern of one or more positives that lacks cancer-specificity and has no clear benign explanation. Typically: Isolated single markers from Tier-B, -C, or -D (e.g. B7-H3 alone: FAP alone; Necrosis alone) without context. Two markers from the same compartment (e.g. ED-B + FAP only - both stroma). A Tier-A lineage marker alone with a plausible benign mimic (e.g. CLDN18.2 alone in a patient with recent gastric ulcer - possible false positive). Action: Do not immediately label as recurrence. Instead, retest in ~2-4 weeks to see if the pattern persists or evolves. Concurrently, investigate potential confounders that might have been missed (e.g. mild fibrosis, subclinical inflammation). In some cases, perform a targeted adjunct test: for example, if CLDN18.2 is isolated IND, one might do an upper endoscopy to rule out a benign ulcer before assuming cancer. IND essentially means “unclear result - watch closely, gather more data.”

[0264] Conditioned Positive (CP): Likely cancer signal, needs confirmation. Definition: A pattern strongly suggestive of malignancy, but warranting a one-step confirmation before treating as definite recurrence. Examples:

[0265] A single high-specificity marker in a fitting clinical context, but by itself (e.g. isolated TAG-72 in a patient with prior colon cancer - likely recurrence, but best to confirm with a scan).

[0266] A broad tumor marker combined with one weaker marker (e.g. B7- H3 + FAP; B7-H3 + Necrosis) - probably cancer, yet confirmation will boost certainty.

[0267] Two cross-compartment moderate markers (e.g. ED-B + Necrosis) - suspicious for tumor, but could be rare benign overlap, so confirm.

[0268] Action: Obtain confirmatory test promptly. Options: imaging of the likely organ (e.g. MRI, CT or organ-specific scan), or repeat the panel in a short interval, or a tumor-specific blood test (if available for that cancer type). Choose based on the pattern and patient’s background. If the confirmatory test is positive (or the pattern persists / worsens on repeat), upgrade the result to Strong Positive and proceed with full diagnostic and treatment work-up. If confirmation is negative and no cancer is found, continue to observe closely (it may revert to IND or even SN on repeat once confounders clear).

[0269] Strong Positive (SP): Definite cancer recurrence signal. Definition: A pattern with high specificity for malignancy - typically multiple markers from different compartments all positive, or one extremely specific marker with at least one corroborator. By rule, any of the following is SP: Two or more markers covering orthogonal compartments (tumor cells + stroma, or tumor + cell death, etc.) - e.g. CLDN18.2 + ED-B, or B7-H3 + FAP + Necrosis, etc. ORFlp positive with any other marker - a near-certain sign of active cancer cells present. Two different lineage (Tier- A) markers simultaneously - indicates possibly multifocal disease (e.g. TAG-72 + CLDN18.2 might mean a gastrointestinal tumor expressing both).

[0270] Action: Act immediately. This result should trigger a full diagnostic work-up without delay: imaging to locate the recurrence, referral to oncology, and staging work-up as appropriate. Inform the patient’s oncology team immediately. Use the pattern to guide where to look first (see Pattem^Organ guide above), but ultimately a comprehensive search may be needed if the site is not obvious. Strong Positive means the probability of true recurrence is extremely high (target >99% specificity), so the priority is to find it and treat it as one would for a confirmed recurrence.

[0271] (Invalid / No-Call: If the NBCP control probe or overall assay quality fails - e.g. abnormally high background, or all probes including control are positive (nonsensical) - then no result category is assigned. The test should be repeated after resolving assay issues. Such QC failure criteria are built into the testing protocol.

[0272] Turnaround and Retesting: An IND or CP typically warrants a short- interval follow-up test (on the order of 2-4 weeks) to catch evolving signals or to ensure a confounder has passed. CN cases should be retested once the benign condition has resolved (e.g. 2-3 months) to confirm a return to SN. After an SP or confirmed recurrence, further surveillance testing is usually suspended while therapeutic intervention is pursued (the panel can be repurposed later to monitor treatment response if applicable).

[0273] This 8-target panel was designed to maximize sensitivity for early- stage recurrence (target >95% detection of Stage I-II tumors) while maintaining extremely high specificity (»98-99%). Achieving both goals required an integrated Boolean approach: no single marker can reach those specificity levels alone without sacrificing sensitivity. By combining markers across different biological “compartments” of tumor biology, the panel exploits the fact that a true cancer will often trigger multiple independent signals, whereas benign processes rarely trigger more than one at a time.

[0274] Marker Selection & Coverage: The included markers cover: tumor cell surface antigens (lineage-specific and broad immune checkpoint), the tumor microenvironment (oncofetal ECM and activated fibroblasts), and cell turnover (necrosis and a tumor-intrinsic protein). This breadth ensures that almost all solid tumors will produce a signal. For example, an early invasive carcinoma might not shed enough cells to release a tumor antigen, but its stromal reaction (ED-B, FAP) could still be picked up - giving a “heads up” Indeterminate result rather than a false negative. Conversely, a small indolent tumor might not incite much stromayet could shed ORFlp via exosomes, or express B7-H3 on a few cells - again flagging the panel. The redundancy is intentional for sensitivity. Notably, each Tier-A marker is tied to certain cancers - by having three (CLDN18.2. GPC3. TAG-72) we cover a wide swath of organs (upper GI, liver, mucinous tumors, etc.), while B7-H3 covers many others as a pan-tumor antigen. ORFlp acts as a “universal” cancer presence marker since LINE-1 activation is a hallmark across epithelial cancers.

[0275] Boolean Logic - Why and / or rules: The decision to use Boolean (binary) interpretation was driven by clinical practicality and consistency. A binary call per marker (after normalization to the control) avoids issues of cross-center calibration - each patient serves as their own control via the NBCP. The interpretation then looks at patterns as sets. By using logical and / or gating, we can set rules like “Marker X AND Marker Y must both be positive to call a Strong Positive.” This drastically reduces false positives: for instance, fibronectin ED-B by itself might elevate from a recent injury, but the chance of both ED-B and a tumor lineage marker (like TAG-72) elevating from coincidental benign causes is extremely low. Thus, requiring an AND (co-positivity) increases specificity non-linearly. The OR gates are used in ensuring sensitivity - e.g., ORF Ip OR a necrosis marker can indicate cell death; we don't insist on both, because a small tumor might not show ORFlp if it’s not necrotic, but could still show the necrosis marker if large enough - we capture either, then require a second corroborating signal somewhere else for specificity.

[0276] Thresholds and Normalization: Each marker is normalized internally to the non-binding control to yield a Boolean positive / negative. This within-sample normalization means that systemic factors (like overall protein levels, or patient- specific matrix effects) are controlled. Only biologically relevant elevations trigger a “Positive.” The fixed cutoff per marker was tuned in development to catch even mild elevations in early-stage recurrence. By avoiding inter-patient quantitative thresholding, we eliminate the need for population-based cutoffs that could miss early recurrence signals - instead, we flag any significant rise above that patient’s normalized background as positive. The trade-off is more false positives, which the interpretation algorithm then triages (IND or CN rather than SP). This approach front-loads sensitivity at the single-marker level, but recoups specificity at the multi-marker interpretation level.

[0277] Handling of Confounders (CN vs IND): The Conditioned Negative category was built in to address scenarios where benign processes mimic cancer signals. Literature and clinical experience tell us these confounders are time-limited or context- specific:

[0278] Surgery / wound healing: induces ED-B and FAP in regenerating tissue. The panel might go positive in Tier-C shortly after major surgery - rather than a false “positive,’' that pattern was classified as CN if the timing fits (<2- 3 months post- op, for example). The recommendation is simply to wait and retest, because as healing completes, those markers should subside.

[0279] Inflammation / fibrosis: chronic inflammatory diseases (active rheumatoid arthritis, pulmonary fibrosis, etc.) can elevate FAP (and sometimes ED-B). If known, those isolated signals are not treated as cancer. Retesting is suggested when the disease is quiescent.

[0280] Placental factors: Pregnancy uniquely can cause positive B7-H3 (placental expression) and oncofetal fibronectin ED-B in maternal blood. The protocol excludes pregnant patients from testing because of this. If inadvertently a pregnant patient was tested and showed those markers, it would be a CN (non-interpretable) - effectively a no-call requiring deferral until postpartum.

[0281] Tissue necrosis: for example, a large myocardial infarction or stroke can release DNA / proteins that trip the “Tumor necrosis” marker. Without any tumor marker present, that pattern is CN if the clinical context (recent MI) is clear. .

[0282] By segregating CN, we ensure the specificity target (98-99%) is maintained not by making the test blandly “negative” in everyone with confounders (which would hurt sensitivity), but by allowing positives to occur and then clinically filtering them out with context. This is safer - it’s easier to explain away a positive that we expected (e.g. “y es, your surgery is causing this marker to rise, it’s not cancer”) than to raise the thresholds so high that those situations always read negative (risking that a true early cancer could then hide). Validation with mixed cohorts confirmed that after applying CN rules, the false positive rate in healthy or benign conditions was extremely low (roughly 1-2%, consistent with specificity -98-99%).

[0283] Indeterminate as a Holding Zone: IND is an important category in our framework to avoid both over-calling and under-calling borderline results. During development, we observed patterns like isolated B7-H3 or isolated FAP that wereneither clearly benign nor confidently malignant. Historically, such cases might lead to unnecessary scans (if overcalled) or be ignored (if undercalled). By labeling them Indeterminate, we formally acknowledge the uncertainty and set a protocol: short- interval follow-up. This was informed by studies in screening where repeating a test after a brief interval can clarify if an initial borderline result w as transient. For instance, a mild B7-H3 elevation alone (IND) - if it’s a nascent tumor, one expects that on a re- draw in a few weeks, perhaps additional markers will turn positive or the B7-H3 will rise further (then it migrates to CP / SP), whereas if it was noise or a benign immune blip, it may disappear. This dynamic assessment improves sensitivity (by not ignoring the signal) and specificity (by not jumping straight to full w ork-up on a single-marker positivity). Our simulated patient pathways showed that IND^CP / SP conversions happened promptly for true tumors, whereas IND-SN (noise resolving) happened in many false positive cases, sparing those patients invasive investigations.

[0284] Escalation Pathways (CP^SP): The Conditioned Positive category ensures a high positive predictive value for SP. By requiring a confirmatory step for patterns that are li...

Claims

1. CLAIMSWhat is claimed is:

1. A synthetic probe comprising: a binding domain that specifically binds a target molecule; a cleavable linker; and a reporter, wherein the cleavable linker is cleavable by a bioorthogonal reaction to release the reporter.

2. The synthetic probe of claim 1. wherein the binding domain is selected from an antibody, an aptamer, a protein; an oligopeptide; an oligonucleotide; an oligosaccharide; an organic drug compound; and combinations thereof.

3. The synthetic probe of claim 2. wherein the antibody is selected from a monoclonal antibody; a polyclonal antibody; a Fab; a F(ab’)2; a single-chain variable fragment (scFv); a single domain antibody / nanobody (VHH); a bispecific antibody; a multispecific antibody; a diabody; and combinations thereof.

4. The synthetic probe of claim 1, wherein the protein is selected from a designed ankyrin repeat protein (DARPin); an affibody; a monobody; a cystine-knot miniprotein (knottin); an avimer, an affimer, a Kunitz-domain binder; a lectin; a receptor ligand; and combinations thereof.

5. The synthetic probe of claim 1, wherein the binding domain is selected from a molecularly imprinted polymer; a small molecule ligand; and combinations thereof.

6. The synthetic probe of claim 1, wherein the target molecule is an antigen, an analyte, a cell, a pathogen, a virus, a bacterium, a fungus, a parasite, and combinations thereof.

7. The synthetic probe of claim 6, wherein the target molecule is a cell surface molecule; a structural protein; and combinations thereof.

8. The synthetic probe of claim 1. wherein the cleavable linker is selected from a cleavable linker responsive to a cleaving agent; a click to release linker; a self-immolative linker; a disulfide-based linker; a hydrazone linker; anenzyme-cleavable peptide linker; an hypoxia-activated linker; a pH-responsive linker; a bi-functional linker; and combinations thereof.

9. The synthetic probe of claim 8, wherein the cleavable linker comprises a first reactive group selected from a tetrazine, trans-cyclooctene, cyclopropene, azide, cyclooctyne, and isonitrile, and the cleaving agent compound comprises a second reactive group that is complementary thereto, selected from trans-cyclooctene, tetrazine, tetrazine, dibenzocyclooctyne (DBCO) or bicyclononyne, phosphine, and tetrazine.

10. The synthetic probe of claim 8. wherein the cleavable linker comprises the first reactive group selected from a tetrazine and the cleaving agent compound comprises a second reactive group that is complementary thereto, selected from trans- cyclooctene.

11. The synthetic probe of claim 8, wherein the cleavable linker comprises the first reactive group selected from a trans-cyclooctene and the cleaving agent compound comprises a second reactive group that is complementary thereto, selected from tetrazine.

12. The synthetic probe of claim 8, wherein the cleavable linker comprises the first reactive group selected from a cyclopropene and the cleaving agent compound comprises a second reactive group that is complementary thereto, selected from tetrazine.

13. The synthetic probe of claim 8, wherein the cleavable linker comprises the first reactive group selected from an azide and the cleaving agent compound comprises a second reactive group that is complementary thereto, selected from dibenzocyclooctyne (DBCO) or bicyclononyne.

14. The synthetic probe of claim 8. wherein the cleavable linker comprises the first reactive group selected from a cyclooctyne and the cleaving agent compound comprises a second reactive group that is complementary thereto, selected from phosphine.

15. The synthetic probe of claim 8, wherein the cleavable linker comprises the first reactive group selected from an isonitrile and the cleaving agent compound comprises a second reactive group that is complementary thereto, selected from tetrazine.

16. The synthetic probe of claim 8. wherein the cleavable linker and the cleaving agent is selected from an azide-alkyne cycloaddition pair; a tetrazine- cyclopropene ligation pair; a tetrazine-trans-cyclooctene (TCO) ligation pair; an alkene-tetrazine pair; and an isonitrile-tetrazine pair.

17. The synthetic probe of claim 1. wherein the cleavable linker comprises one of a chemical mask or a structural mask that reversibly inhibits reactivity of the cleavable linker.

18. The synthetic probe of claim 1. wherein the chemical mask or the structural mask is selected from an enzyme-initiated tetrazine mask; a pH-triggered mask; a biotin-streptavidin mask; and combinations thereof.

19. The synthetic probe of claim 1. wherein the reporter is selected from an organic fluorophore; a nucleotide barcode; a quantum dot; a peptide; a small molecule metabolite; a volatile organic compound (VOC); and combinations thereof.

20. The synthetic probe of claim 1. further comprising a spacer located between the binding domain and the cleavable linker; between the cleavable linker and the reporter; between the binding domain and the flag; between the flag and the cleavable linker; between the cleavable linker and the flag; between the cleavable linker and the reporter; and combinations thereof.

21. The synthetic probe of claim 20, wherein the spacer is a polyethylene glycol (PEG) spacer comprising 2 ethylene glycol units to about 2500 ethylene glycol units.

22. The synthetic probe of claim 1, further comprising a flag that remains coupled with the binding domain after cleavage of the cleavable linker.

23. The synthetic probe of claim 1, wherein the cleavable linker is a masked cleavable linker that is removable by an endogenous condition or an enzy me in a target environment, such that a bioorthogonal reactive group is exposed only at a target site prior to administration of a cleaving agent.

24. The synthetic probe of claim 23, wherein the masked cleavable linker is a galactose-caged tetrazine that is unmasked by P-galactosidase.

25. The synthetic probe of claim 23, wherein the bioorthogonal reactive group is linked via a pH-labile bond that cleaves in an acidic microenvironment (pH < 7) to unmask the cleavable linker.

26. A method for detecting a target molecule when present in a subject, the method comprising: administering a synthetic probe to a subject, wherein the synthetic probe comprises: a binding domain that specifically binds the target molecule; a cleavable linker; and a reporter; allowing time for the synthetic probe to bind to the target molecule if present in the subject and to allow unbound synthetic probe to clear the subject; administering a cleaving agent to the subject, wherein the cleaving agent interacts with the cleavable linker to initiate cleavage of the cleavable linker and release the reporter; allowing time for the reporter to accumulate in a body fluid of the subject; collecting the body fluid; analyzing the body fluid for the reporter; and determining the presence of the target molecule based on the reporter detected in the body fluid.

27. The method of claim 26, further comprising diagnosing the subject as having a disease based on the target molecule if detected in the subject.

28. The method of claim 26, wherein the binding domain is selected from an antibody, an aptamer, a protein; an oligopeptide; an oligonucleotide; an oligosaccharide; an organic drug compound; and combinations thereof.

29. The method of claim 26, wherein the cleavable linker is selected from a cleavable linker responsive to a cleaving agent; a click to release linker; a self-immolative linker; a disulfide-based linker; a hydrazone linker; an enzyme-cleavable peptide linker; an hypoxia-activated linker; a pH-responsive linker; a bi-functional linker; and combinations thereof30. The method of claim 26, wherein the cleavable linker comprises a first reactive group selected from a tetrazine, trans-cyclooctene, cyclopropene, azide, cyclooctyne, or isonitrile, and the cleaving agent comprises a second reactive group that is complementary thereto, selected respectively from trans-cy clooctene, tetrazine, tetrazine, dibenzocyclooctyne (DBCO) or bicyclononyne, phosphine, or tetrazine.

31. The method of claim 26, further comprising administering a control synthetic probe, wherein the control synthetic probe comprises a cleavable linker and a reporter.

32. The method of claim 31, wherein the control synthetic probe comprises a control domain selected from a protein; a peptide; an oligonucleotide; and combinations thereof.

33. The method of claim 31, wherein the reporter of the control synthetic probe is the same reporter as the synthetic probe.

34. The method of claim 31 , wherein the cleavable linker of the control synthetic probe is the same cleavable linker as the synthetic probe.

35. The method of claim 26, wherein the body fluid is analyzed by amplification; clustered regularly interspaced short palindromic repeats (CRISPR); Western blot analysis; enzyme-linked immunosorbent assay (ELISA); lateral flow assay; electrochemical / optical biosensor; immunohistochemistry; flow cytometry; molecular imaging; fluorescence microplate reader; spectrofluorometer; photon detector; mass spectrometry; and combinations thereof.

36. The method of claim 26, wherein the synthetic probe further comprises a flag that remains coupled with the bound binding domain to leave a binding domain- flag conjugate bound to the target molecule after cleavage of the cleavable linker.

37. The method of claim 36, further comprising administering to the subject a finishing agent comprising a reactive group that specifically binds the flag and a therapeutic.

38. The method of claim 37, wherein the therapeutic is selected from a cytotoxic drug; a radiopharmaceutical: an immune effector; an enzyme that locally activates a prodrug; and combinations thereof.

39. The method of claim 26, wherein the body fluid is selected from blood; plasma; urine; saliva; sputum; cerebrospinal fluid (CSF); breath; and combinations thereof.

40. The method of claim 26, wherein the target molecule is selected from a viral target molecule; a bacterial target molecule; a fungal target molecule; an allergen target molecule; a parasite target molecule; a tumor cell target molecule; and combinations thereof.

41. The method of claim 26, wherein administering a synthetic probe to a subject comprises administering a plurality of synthetic probes.

42. The method of claim 41, wherein each synthetic probe of the plurality of synthetic probes comprises at least one of a different binding domain and a different reporter.

43. The method of claim 26, wherein the cleavable linker comprises one of a chemical mask or a structural mask that reversibly inhibits reactivity of the cleavable linker.

44. The method of claim 26, wherein the chemical mask or the structural mask is selected from an enzyme-initiated tetrazine mask: a pH-triggered mask; a biotin-streptavidin mask; and combinations thereof.

45. The method of claim 26, wherein the reporter is selected from an organic fluorophore; a nucleotide barcode; a quantum dot; a peptide; a small molecule metabolite; a volatile organic compound (VOC); and combinations thereof.

46. The method of claim 26, further comprising a spacer located between the binding domain and the cleavable linker; between the cleavable linker and the reporter; between the binding domain and the flag; between the flag and the cleavable linker; between the cleavable linker and the flag; between the cleavable linker and the reporter; and combinations thereof.

47. The method of claim 26, wherein the spacer is a polyethylene glycol (PEG) spacer comprising 2 ethylene glycol units to about 2500 ethylene glycol units.

48. A kit comprising: a synthetic probe, wherein the synthetic probe comprises: a binding domain that specifically binds a target molecule, a cleavable linker, and a reporter; and a cleaving agent.

49. The kit of claim 48, wherein the binding domain is selected from an antibody, an aptamer, a protein; an oligopeptide; an oligonucleotide; an oligosaccharide; an organic drug compound; and combinations thereof.

50. The kit of claim 49, wherein the antibody is selected from a monoclonal antibody; a polyclonal antibody; a Fab; a F(ab’)2; a single-chain variable fragment (scFv); a single domain antibody / nanobody (VHH); a bispecific antibody; a multispecific antibody; a diabody; and combinations thereof.

51. The kit of claim 48, wherein the protein is selected from a designed ankyrin repeat protein (DARPin); an affibody; a monobody; a cystine-knot miniprotein (knottin); an avimer, an affimer, a Kunitz-domain binder; a lectin; a receptor ligand; and combinations thereof.

52. The kit of claim 48, wherein the binding domain is selected from a molecularly imprinted polymer; a small molecule ligand; and combinations thereof.

53. The kit of claim 48, wherein the target molecule is an antigen, an analyte, a cell, a pathogen, a virus, a bacteria, a fungus, a parasite, and combinations thereof.

54. The kit of claim 53, wherein the target molecule is a cell surface molecule; a structural protein; and combinations thereof.

55. The kit of claim 48, wherein the cleavable linker is selected from a cleavable linker responsive to a cleaving agent; a click to release linker; a self-immolative linker; a disulfide-based linker; a hydrazone linker; an enzyme-cleavable peptide linker; an hypoxia-activated linker; a pH-responsive linker; a bi-functional linker; and combinations thereof.

56. The kit of claim 55, wherein the cleavable linker comprises a first reactive group selected from a tetrazine, trans-cyclooctene, cyclopropene, azide, cyclooctyne, and isonitrile, and the cleaving agent compound comprises a second reactive group that is complementary thereto, selected from trans-cyclooctene, tetrazine, tetrazine, dibenzocyclooctyne (DBCO) or bicyclononyne, phosphine, and tetrazine.

57. The kit of claim 55, wherein the cleavable linker comprises the first reactive group selected from a tetrazine and the cleaving agent compound comprises a second reactive group that is complementary thereto, selected from trans-cyclooctene.

58. The kit of claim 55, wherein the cleavable linker comprises the first reactive group selected from a trans-cyclooctene and the cleaving agent compound comprises a second reactive group that is complementary thereto, selected from tetrazine.

59. The kit of claim 55. wherein the cleavable linker comprises the first reactive group selected from a cyclopropene and the cleaving agent compound comprises a second reactive group that is complementary thereto, selected from tetrazine.

60. The kit of claim 55, wherein the cleavable linker comprises the first reactive group selected from an azide and the cleaving agent compound comprises a second reactive group that is complementary thereto, selected from dibenzocyclooctyne (DBCO) or bicyclononyne.

61. The kit of claim 55, wherein the cleavable linker comprises the first reactive group selected from a cyclooctyne and the cleaving agent compound comprises a second reactive group that is complementary thereto, selected from phosphine.

62. The kit of claim 55. wherein the cleavable linker comprises the first reactive group selected from an isonitrile and the cleaving agent compound comprises a second reactive group that is complementary thereto, selected from tetrazine.

63. The kit of claim 55. wherein the cleavable linker and the cleaving agent is selected from an azide-alkyne cycloaddition pair; a tetrazine-cyclopropene ligation pair; a tetrazine-trans-cyclooctene (TCO) ligation pair; an alkene-tetrazine pair; and an isonitrile-tetrazine pair.

64. The kit of claim 48, wherein the cleavable linker comprises one of a chemical mask or a structural mask that reversibly inhibits reactivity of the cleavable linker.

65. The kit of claim 48, wherein the chemical mask or the structural mask is selected from an enzyme-initiated tetrazine mask; a pH-triggered mask; a biotin- streptavidin mask; and combinations thereof.

66. The kit of claim 48, wherein the reporter is selected from an organic fluorophore; a nucleotide barcode; a quantum dot; a peptide; a small molecule metabolite; a volatile organic compound (VOC); and combinations thereof.

67. The kit of claim 48, further comprising a spacer located between the binding domain and the cleavable linker; between the cleavable linker and the reporter; between the binding domain and the flag; between the flag and the cleavablelinker; between the cleavable linker and the flag; between the cleavable linker and the reporter; and combinations thereof.

68. The kit of claim 67, wherein the spacer is a polyethylene glycol (PEG) spacer comprising 2 ethylene glycol units to about 2500 ethylene glycol units.

69. The kit of claim 48, further comprising a flag that remains coupled with the binding domain after cleavage of the cleavable linker.

70. The kit of claim 48, wherein the cleavable linker is a masked cleavable linker that is removable by an endogenous condition or an enzyme in a target environment, such that a bioorthogonal reactive group is exposed only at a target site prior to administration of a cleaving agent.

71. The kit of claim 70, wherein the masked cleavable linker is a galactose-caged tetrazine that is unmasked by P-galactosidase.

72. The kit of claim 70, wherein the masked cleavable linker comprises a bioorthogonal reactive group linked via a pH-labile bond that cleaves in an acidic microenvironment (pH < 7) to unmask the cleavable linker.

73. The kit of claim 48, further comprising a finishing agent comprising a reactive group that specifically binds the flag and a therapeutic.

74. A method of diagnosing and treating a subject having or suspected of having a disease, the method comprising: administering a synthetic probe to a subject, wherein the synthetic probe comprises: a binding domain that specifically binds the target molecule; a flag associated with the binding domain; a cleavable linker; and a reporter; allowing time for the synthetic probe to bind to the target molecule if present in the subject and to allow unbound synthetic probe to clear the subject; administering a cleaving agent to the subject, wherein the cleaving agent interacts with the cleavable linker to initiate cleavage of the cleavable linker and release the reporter; allowing time for the reporter to accumulate in a body fluid of the subject;collecting the body fluid; analyzing the body fluid for the reporter; determining the presence of the target molecule based on the reporter detected in the body fluid; diagnosing the subject as having a disease based on the target molecule if detected in the subject; and administering a finishing agent comprising: a reactive group that specifically binds the flag and a therapeutic, wherein the finishing agent specifically binds the binding domain-flag conjugate that remains bound to the target molecule to deliver the therapeutic.

75. The method of claim 74, wherein the therapeutic is selected from a cytotoxic drug; a radiopharmaceutical; an immune effector; an enzyme that locally activates a prodrug; and combinations thereof.

76. Use of the synthetic probe of any one of claims 1-25 for detecting a target molecule in a subject having or suspected of having the target molecule, the use comprising: administering the synthetic probe to the subject, wherein the synthetic probe comprises: a binding domain that specifically binds a target molecule; a flag associated with the binding domain; a cleavable linker; and a reporter; allowing time for the synthetic probe to bind to the target molecule if present in the subject and to allow unbound synthetic probe to clear the subject; administering a cleaving agent to the subject, wherein the cleaving agent interacts with the cleavable linker to initiate cleavage of the cleavable linker and release the reporter and wherein the binding domain and flag form a binding domain- flag conjugate that remains bound to the target molecule. allowing time for the reporter to accumulate in a body fluid of the subject; collecting the body fluid; analyzing the body fluid for the reporter; and determining the presence of the target molecule based on the reporter detected in the body fluid.

77. The use according to claim 76 further comprising diagnosing the subject as having a disease based on the target molecule if detected in the subject.

78. Use of the synthetic probe of any one of claims 1-25 for diagnosing and treating a disease in a subject having or suspected of having the disease, the use comprising: administering the synthetic probe to the subject, wherein the synthetic probe comprises: a binding domain that specifically binds a target molecule; a flag associated with the binding domain; a cleavable linker; and a reporter; allowing time for the synthetic probe to bind to the target molecule if present in the subject and to allow unbound synthetic probe to clear the subject; administering a cleaving agent to the subject, wherein the cleaving agent interacts with the cleavable linker to initiate cleavage of the cleavable linker and release the reporter and wherein the binding domain and flag form a binding domain- flag conjugate that remains bound to the target molecule; allowing time for the reporter to accumulate in a body fluid of the subject; collecting the body fluid; analyzing the body fluid for the reporter; determining the presence of the target molecule based on the reporter detected in the body fluid; diagnosing the subject as having a disease based on the target molecule if detected in the subject; and administering a finishing agent comprising: a reactive group that specifically binds the flag and a therapeutic, wherein the finishing agent specifically binds the binding domain-flag conjugate that remains bound to the target molecule to deliver the therapeutic.

79. The use according to claim 78, wherein the therapeutic is selected from a cytotoxic drug; a radiopharmaceutical; an immune effector; an enz me that locally activates a prodrug; and combinations thereof.

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