Compositions and methods for detecting lung cancer

Abstract

In one aspect, the present disclosure provides a technique for detecting lung cancer, particularly for early detection of lung cancer.In another aspect, the technique provided herein is useful for selecting and / or monitoring and / or evaluating the effectiveness of treatment administered to a subject determined to have or be susceptible to lung cancer.In some embodiments, the technique provided herein is useful for developing companion diagnostics, for example, by measuring tumor burden and changes in tumor burden in conjunction with therapeutic agents.In some embodiments, the technique provided herein is useful for developing companion diagnostics, for example, by identifying biomarkers in a subject's body fluid sample (e.g., blood-derived sample) that are associated with treatment response.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 297684, filed January 7, 2022, and U.S. Provisional Application No. 63 / 417299, filed October 18, 2022, the contents of which are incorporated herein by reference in their entireties. [Background technology]

[0002] background Early detection of cancer greatly increases the chance of successful treatment. However, many cancers, including lung cancer, still lack effective screening recommendations. Typical challenges for cancer screening tests include limited sensitivity and specificity. High false positive results can be particularly concerning, as they can create difficult management decisions for clinicians and patients who do not want to unnecessarily administer (or receive) anti-cancer therapy that potentially has undesirable side effects. Conversely, high false negative results will miss patients who need therapy, resulting in delayed treatment and consequently reduced chances of success, thus failing to meet the purpose of screening tests. Summary of the Invention [Means for solving the problem]

[0003] Abstract The present disclosure provides, among other things, insights and techniques for achieving effective lung cancer screening from biological samples. In some embodiments, such biological samples are or include bodily fluid-derived samples, for example, in some embodiments, blood-derived samples. In some embodiments, the present disclosure provides, among other things, insights and techniques that are particularly useful for non-small cell lung cancer screening. In some embodiments, the provided techniques are effective for detecting early-stage lung cancer (e.g., in some embodiments, non-small cell lung cancer). In some embodiments, the provided techniques are effective even when applied to populations that include or consist of asymptomatic individuals (e.g., due to sufficiently high sensitivity and / or low false positive and / or false negative rates). In some embodiments, the provided techniques are effective when applied to populations that include or consist of individuals (e.g., asymptomatic individuals) without a genetic risk of developing lung cancer (e.g., in some embodiments, non-small cell lung cancer). In some embodiments, the provided techniques are effective when applied to populations that include or consist of symptomatic individuals (e.g., individuals suffering from one or more symptoms of lung cancer). In some embodiments, the provided technology is effective when applied to populations that include or consist of individuals at risk for lung cancer (e.g., individuals with genetic and / or lifestyle-related risk factors for lung cancer). In some embodiments, the provided technology can be or can include one or more compositions (e.g., molecular entities or complexes, systems, cells, collections, combinations, kits, etc.) and / or methods (e.g., methods of making, using, assessing, etc.), as would be apparent to one of skill in the art upon reading the disclosure provided herein.

[0004] In some embodiments, the present disclosure identifies the origin of certain prior technology challenges, including, for example, certain conventional approaches to lung cancer detection and diagnosis. For example, the present disclosure recognizes that many conventional diagnostic assays, such as X-ray imaging, sputum testing, low-dose CT scanning, and / or molecular tests based on cell-free nucleic acids, serum biomarkers (e.g., carcinoembryonic antigen (CEA), cytokeratin 19 fragment (CYFRA 21-1), neuron-specific enolase (NSE), progastrin-releasing peptide (ProGRP), and / or squamous cell carcinoma antigen (SCCA)), and / or bulk analysis of extracellular vesicles, can be time-consuming, expensive, and / or lack sufficient sensitivity and / or specificity to provide a reliable, comprehensive diagnostic assessment. In some embodiments, the present disclosure provides technologies (including systems, compositions, and methods) that solve such problems by detecting the co-localization of a target biomarker signature for lung cancer in individual nanoparticles having a desired size range, including extracellular vesicles, including at least one extracellular vesicle-associated surface biomarker (e.g., including a membrane-bound and / or membrane-associated polypeptide) and at least one target biomarker selected from the group consisting of a surface biomarker, an internal biomarker, and an RNA biomarker. In some embodiments, the present disclosure provides technologies (including systems, compositions, and methods) that solve such problems by detecting such target biomarker signatures for lung cancer using the target entity detection approach developed by the applicant and described in US2020 / 0299780 and WO2020180741, which is based on the interaction and / or co-localization of at least two or more target entities (e.g., target biomarker signatures) in individual nanoparticles, e.g., extracellular vesicles.

[0005] In some embodiments, extracellular vesicles for detection as described herein can be isolated from a subject's bodily fluids by size-exclusion-based methods. As will be understood by those skilled in the art, in some embodiments, size-exclusion-based methods can provide samples containing nanoparticles of a desired size range, including nanoparticles. Thus, in some embodiments, the techniques provided herein involve the detection of colocalization of at least two or more surface biomarkers (e.g., as described herein) forming a target biomarker signature for lung cancer in individual nanoparticles of a desired size range, including nanoparticles (e.g., in some embodiments, about 30 nm to about 1000 nm). Those skilled in the art who read this disclosure will understand that various embodiments described herein in the context of "nanoparticles" may also be applicable in the context of "nanoparticles" as described herein.

[0006] In some embodiments, the present disclosure provides, among other things, insight that screening asymptomatic individuals, e.g., periodic screening before the onset of symptoms or otherwise in their absence, may be beneficial and even important for the effective management (e.g., successful treatment) of lung cancer (e.g., in some embodiments, non-small cell lung cancer). In some embodiments, the present disclosure provides lung cancer screening systems that can be implemented to detect lung cancer (e.g., in some embodiments, non-small cell lung cancer), including early stage cancer, in some embodiments, early stage cancer in asymptomatic individuals. In some embodiments, the provided techniques are implemented to achieve periodic screening of asymptomatic individuals. The present disclosure provides compositions (e.g., reagents, kits, components, etc.), including, for example, strategies involving periodic testing of one or more individuals (e.g., symptomatic or asymptomatic individuals), as well as methods of providing and / or using the same. The present disclosure defines the utility of such systems and provides compositions and methods for implementing them.

[0007] In some embodiments, the provided techniques achieve detection (e.g., early detection, e.g., in asymptomatic individuals and / or populations) of one or more characteristics of lung cancer (e.g., incidence, progression, response to therapy, recurrence, etc.) with sensitivity and / or specificity (e.g., resulting false positive and / or false negative rates) appropriate to enable useful application of the provided techniques to single and / or regular (e.g., periodic) assessments. In some embodiments, the provided techniques are useful in conjunction with regular screening tests, including, but not limited to, physical exams, general practitioner visits, cholesterol / lipid blood tests, diabetes (type 2) screening, colonoscopies, blood pressure screening, thyroid function tests, prostate cancer screening, mammograms, HPV / Pap smears, and / or vaccinations. In some embodiments, the provided techniques are useful in conjunction with treatment regimens, and in some embodiments, the provided techniques may improve one or more characteristics (e.g., success rate by accepted parameters) of such treatment regimens.

[0008] In some aspects, techniques are provided for use in classifying a subject (e.g., an asymptomatic subject) as having or susceptible to lung cancer (e.g., in some embodiments, non-small cell lung cancer). In some embodiments, the present disclosure provides methods or assays for classifying a subject (e.g., an asymptomatic subject) as having or susceptible to lung cancer (e.g., in some embodiments, non-small cell lung cancer). In some embodiments, the provided method or assay includes (a) detecting nanoparticles (having a size range of interest, including extracellular vesicles) that express a target biomarker signature for lung cancer in a biological sample (e.g., in some embodiments, a bodily fluid-derived sample, such as, but not limited to, a blood-derived sample) from a subject in need thereof, wherein the target biomarker signature includes at least one extracellular vesicle-associated surface biomarker (e.g., comprising a membrane-bound and / or membrane-associated polypeptide), and at least one target biomarker selected from the group consisting of a surface biomarker (as described herein), an intravesicular biomarker (as described herein), and an intravesicular RNA biomarker (as described herein), wherein the surface biomarker is: [ka] and combinations thereof; wherein the intravesicular biomarker is selected from: [ka] [ka] and combinations thereof; and the intravesicular RNA (e.g., but not limited to, mRNA, and non-coding RNA such as, for example, orphan non-coding RNA, long non-coding RNA, piwi-interacting RNA, microRNA, circular RNA, etc.) biomarker is selected from: [ka] and combinations thereof; (b) comparing the sample information indicating the level of the target biomarker signature-expressing nanoparticles in the biological sample with reference information comprising a reference threshold level; and (c) classifying the subject as having or susceptible to lung cancer if the biological sample exhibits an elevated level of the target biomarker signature-expressing nanoparticles compared to a classification cutoff that references the reference threshold level.

[0009] In some aspects, techniques are provided for use in classifying a subject (e.g., an asymptomatic subject) as having or susceptible to lung cancer (e.g., in some embodiments, non-small cell lung cancer). In some embodiments, the present disclosure provides methods or assays for classifying a subject (e.g., an asymptomatic subject) as having or susceptible to lung cancer (e.g., in some embodiments, non-small cell lung cancer). In some embodiments, the provided method or assay comprises: (a) detecting nanoparticles expressing a target biomarker signature of lung cancer (e.g., in some embodiments, non-small cell lung cancer) in a biological sample from a subject in need thereof, wherein the target biomarker signature comprises at least one nanoparticle-associated surface biomarker and at least one target biomarker selected from the group consisting of a surface biomarker (as described herein), an intravesicular biomarker (as described herein), and an intravesicular RNA biomarker (as described herein); (b) comparing sample information indicating the level of the target biomarker signature-expressing nanoparticles in the biological sample with reference information comprising a reference threshold level; and (c) classifying the subject as having or susceptible to lung cancer if the biological sample exhibits an elevated level of the target biomarker signature-expressing nanoparticles compared to a classification cutoff referenced to the reference threshold level. In some embodiments, the at least one target biomarker is selected from the group consisting of sialyl Tn (sTn) antigen, sialyl-6T antigen (6-sialyl core 1), T antigen, [ka] and combinations thereof. In some embodiments, at least one such target biomarker is or comprises a surface biomarker selected from: [ka] [ka] In some embodiments, at least one such target biomarker is or comprises a surface biomarker selected from sialyl-6T antigen (6-sialylcore 1), sialyl-T antigen, sialyl Lewis A antigen (CA19-9), Globo H, Gb5 (SSEA-3), lactotriaosylceramide (Lc3), Forssman antigen, and combinations thereof. [ka] and combinations thereof. In some embodiments, at least one such target biomarker is or comprises an intravesicular biomarker selected from: [ka] [ka] [ka] and combinations thereof (e.g., but not limited to, mRNA, and non-coding RNA such as orphan non-coding RNA, long non-coding RNA, piwi-interacting RNA, microRNA, circular RNA, etc.).

[0010] In some aspects, techniques are provided for use in classifying a subject (e.g., an asymptomatic subject) as having or susceptible to lung cancer (e.g., in some embodiments, non-small cell lung cancer such as, for example, lung adenocarcinoma (LUAD) and / or lung squamous cell carcinoma (LUSC)). In some embodiments, the present disclosure provides methods or assays for classifying a subject (e.g., an asymptomatic subject) as having or susceptible to lung cancer (e.g., in some embodiments, non-small cell lung cancer such as, for example, LUAD and / or LUSC). In some embodiments, the provided methods or assays include: (a) detecting nanoparticles expressing a target biomarker signature of lung cancer (e.g., in some embodiments, non-small cell lung cancer, such as LUAD and / or LUSC) in a biological sample from a subject in need thereof, wherein the target biomarker signature comprises at least one nanoparticle-associated surface biomarker and at least one target biomarker selected from the group consisting of a surface biomarker (as described herein), an intravesicular biomarker (as described herein), and an intravesicular RNA biomarker (as described herein); (b) comparing sample information indicative of the level of the target biomarker signature-expressing nanoparticles in the biological sample with reference information comprising a reference threshold level; and (c) classifying the subject as having or susceptible to lung cancer (e.g., in some embodiments, non-small cell lung cancer, such as LUAD and / or LUSC) if the biological sample exhibits an elevated level of the target biomarker signature-expressing nanoparticles compared to a classification cutoff that references the reference threshold level. In some embodiments, at least one such target biomarker is [ka] [ka] and combinations thereof. In some embodiments, at least one such target biomarker is or comprises a surface biomarker selected from: [ka] In some embodiments, at least one such target biomarker is or comprises a surface biomarker selected from sialyl-6T antigen (6-sialylcore 1), sialyl-T antigen, sialyl Lewis A antigen (CA19-9), Globo H, Gb5 (SSEA-3), lactotriaosylceramide (Lc3), Forssman antigen, and combinations thereof. [ka] [ka] and combinations thereof. In some embodiments, at least one such target biomarker is or comprises an intravesicular biomarker selected from: [ka] and combinations thereof (e.g., but not limited to, mRNA, and non-coding RNA such as orphan non-coding RNA, long non-coding RNA, piwi-interacting RNA, microRNA, circular RNA, etc.).

[0011] In some aspects, techniques are provided for use in classifying a subject (e.g., an asymptomatic subject) as having or susceptible to a particular type of lung cancer (e.g., in certain embodiments, lung adenocarcinoma (LUAD)). In some embodiments, the present disclosure provides methods or assays for classifying a subject (e.g., an asymptomatic subject) as having or susceptible to a particular type of lung cancer (e.g., in certain embodiments, LUAD). In some embodiments, the provided methods or assays include: (a) detecting nanoparticles expressing a target biomarker signature of a particular type of lung cancer (e.g., in certain embodiments, LUAD) in a biological sample from a subject in need thereof, wherein the target biomarker signature comprises at least one nanoparticle-associated surface biomarker and at least one target biomarker selected from the group consisting of a surface biomarker (as described herein), an intravesicular biomarker (as described herein), and an intravesicular RNA biomarker (as described herein); (b) comparing sample information indicative of the level of the target biomarker signature-expressing nanoparticles in the biological sample with reference information comprising a reference threshold level; and (c) classifying the subject as having or susceptible to a particular type of lung cancer (e.g., in certain embodiments, LUAD) if the biological sample exhibits an elevated level of the target biomarker signature-expressing nanoparticles compared to a classification cutoff that references the reference threshold level. In some embodiments, at least one such target biomarker that is particularly useful for classifying a subject as having or susceptible to LUAD is: [ka] and combinations thereof. In some embodiments, at least one such target biomarker that is particularly useful for classifying a subject as having or susceptible to LUAD is or comprises a surface biomarker selected from: [ka] and combinations thereof. In some embodiments, at least one such target biomarker that is particularly useful for classifying a subject as having or susceptible to LUAD is or comprises an intravesicular biomarker selected from: [ka] [ka] and combinations thereof (e.g., but not limited to, mRNA, and non-coding RNA such as orphan non-coding RNA, long non-coding RNA, piwi-interacting RNA, microRNA, circular RNA, etc.). In some embodiments, certain of the aforementioned target biomarkers are particularly useful for distinguishing LUAD from LUSC.

[0012] In some aspects, techniques are provided for use in classifying a subject (e.g., an asymptomatic subject) as having or susceptible to a particular type of lung cancer (e.g., in certain embodiments, lung squamous cell carcinoma (LUSC)). In some embodiments, the present disclosure provides methods or assays for classifying a subject (e.g., an asymptomatic subject) as having or susceptible to a particular type of lung cancer (e.g., in certain embodiments, LUSC). In some embodiments, the provided methods or assays include: (a) detecting nanoparticles expressing a target biomarker signature of a particular type of lung cancer (e.g., in certain embodiments, LUSC) in a biological sample from a subject in need thereof, wherein the target biomarker signature comprises at least one nanoparticle-associated surface biomarker and at least one target biomarker selected from the group consisting of a surface biomarker (as described herein), an intravesicular biomarker (as described herein), and an intravesicular RNA biomarker (as described herein); (b) comparing sample information indicative of the level of the target biomarker signature-expressing nanoparticles in the biological sample with reference information comprising a reference threshold level; and (c) classifying the subject as having or susceptible to a particular type of lung cancer (e.g., in certain embodiments, LUSC) if the biological sample exhibits an elevated level of the target biomarker signature-expressing nanoparticles compared to a classification cutoff that references the reference threshold level. In some embodiments, at least one such target biomarker that is particularly useful for classifying a subject as having or susceptible to LUSC is: [ka] and combinations thereof. In some embodiments, at least one such target biomarker that is particularly useful for classifying a subject as having or susceptible to LUSC is or comprises a surface biomarker selected from: [ka] [ka] and combinations thereof. In some embodiments, at least one such target biomarker that is particularly useful for classifying a subject as having or susceptible to LUSC is or comprises an intravesicular biomarker selected from: [ka] and combinations thereof (e.g., but not limited to, mRNA, and non-coding RNA such as orphan non-coding RNA, long non-coding RNA, piwi-interacting RNA, microRNA, circular RNA, etc.). In some embodiments, certain of the aforementioned target biomarkers are particularly useful for distinguishing LUSC from LUAD.

[0013] In some embodiments, the methods or assays described herein may be performed for at least one more additional target biomarker signature (e.g., including at least one, at least two, at least three, or more additional target biomarker signatures). In some such embodiments, the classification cutoff may refer to additional reference threshold levels corresponding to each additional target biomarker signature.

[0014] In some embodiments, nanoparticle-associated surface biomarkers for use in the lung cancer target biomarker signatures used and / or described herein may be or include tumor-specific and / or tissue-specific biomarkers (e.g., lung tissue-specific biomarkers). In some embodiments, such nanoparticle-associated surface biomarkers may be or include non-specific markers, e.g., which are present in one or more non-target tumors and / or one or more non-target tissues. In some embodiments, such nanoparticle-associated surface biomarkers include: [ka] and combinations thereof. In some embodiments, such nanoparticle-associated surface biomarkers may be or include one or more of biomarkers selected from SLC34A2, CEACAM5, CEACAM6, and / or EPCAM. In some embodiments, such nanoparticle-associated surface biomarkers may be or include ALCAM, CD55, CDH1, CDH3, CD274 (PD-L1), CEACAM5, CEACAM6, DSG2, EGFR, EPCAM, FOLR1, IG1FR, MET, MSLN, MUC1, SLC34A2, sTn antigen, Tn antigen, T antigen, TACSTD2, TNFRSF10B, or combinations thereof. In some embodiments, such nanoparticle-associated surface biomarkers may be or include SLC34A2 polypeptide. In some embodiments, such nanoparticle-associated surface biomarkers may be or include CEACAM5 polypeptide. In some embodiments, such nanoparticle-associated surface biomarkers may be or comprise CEACAM6 polypeptides. In some embodiments, such nanoparticle-associated surface biomarkers may be or comprise EPCAM polypeptides. In some embodiments, such nanoparticle-associated surface biomarkers may be or comprise sTn antigen and / or MUC1 polypeptides.

[0015] In some embodiments, the target biomarker signature for lung cancer comprises a nanoparticle-associated surface biomarker (e.g., those described herein) and at least one (e.g., including 1, 2, 3, or more) additional target surface biomarker, which in some embodiments includes: [ka] Or it may be or include a combination thereof.

[0016] In some embodiments, nanoparticle-associated surface biomarkers for use in the lung cancer target biomarker signatures used and / or described herein may be or include tumor-specific and / or tissue-specific biomarkers (e.g., lung tissue-specific biomarkers). In some embodiments, such nanoparticle-associated surface biomarkers may be or include non-specific markers, e.g., present in one or more non-target tumors and / or one or more non-target tissues. In some embodiments, such nanoparticle-associated surface biomarkers may be or include one or more of the surface biomarkers described herein. In some embodiments, such nanoparticle-associated surface biomarkers include sialyl Tn (sTn) antigen, sialyl-6T antigen (6-sialyl core 1), T antigen, [ka] and combinations thereof.

[0017] In some embodiments, such nanoparticle-associated surface biomarkers include: [ka] The surface biomarker may be or may include a surface biomarker selected from sialyl-6T antigen (6-sialylcore 1), sialyl-T antigen, sialyl Lewis A antigen (CA19-9), Globo H, Gb5 (SSEA-3), lactotriaosylceramide (Lc3), Forssman antigen, and combinations thereof.

[0018] In some embodiments, the target biomarker signature for lung cancer comprises a nanoparticle-associated surface biomarker (e.g., those described herein) and at least one (e.g., including 1, 2, 3, or more) additional target surface biomarkers, which in some embodiments include sialyl Tn (sTn) antigen, sialyl-6T antigen (6-sialyl core 1), T antigen, [ka] [ka] and combinations thereof. In some embodiments, at least one (e.g., including 1, 2, 3, or more) additional target surface biomarker is: [ka] The surface biomarker may be or may include a surface biomarker selected from sialyl-6T antigen (6-sialylcore 1), sialyl-T antigen, sialyl Lewis A antigen (CA19-9), Globo H, Gb5 (SSEA-3), lactotriaosylceramide (Lc3), Forssman antigen, and combinations thereof.

[0019] In some embodiments, the target biomarker signature for lung cancer comprises nanoparticle-associated surface biomarkers (e.g., those described herein) and at least one target intravesicular RNA (e.g., but not limited to, mRNA, and non-coding RNA such as, for example, orphan non-coding RNA, long non-coding RNA, piwi-interacting RNA, microRNA, circular RNA, etc.) biomarker, which in some embodiments is a biomarker for one of the following human genes: [ka] [ka] or may comprise at least one RNA transcript (eg, an mRNA transcript) encoded by a sequence encoding the nucleotide sequence of the present invention, or a combination thereof.

[0020] In some embodiments, the target biomarker signature for lung cancer comprises nanoparticle-associated surface biomarkers (e.g., those described herein) and at least one target intravesicular RNA (e.g., but not limited to, mRNA, and non-coding RNA such as, for example, orphan non-coding RNA, long non-coding RNA, piwi-interacting RNA, microRNA, circular RNA, etc.) biomarker, which in some embodiments is a biomarker for one of the following human genes: [ka] [ka] or may comprise at least one RNA transcript (eg, an mRNA transcript) encoded by a sequence encoding the nucleotide sequence of the present invention, or a combination thereof.

[0021] In some embodiments, the target biomarker signature for lung cancer comprises nanoparticle-associated surface biomarkers (e.g., those described herein) and at least one additional target intravesicular biomarker, which in some embodiments may be or may include at least one polypeptide encoded by the following human genes: AOC1, C12orf45, CRABP2, CST1, ETV4, FAM83A, FOXA2, HMGB3, LGALS3BP, MIF, NAPSA, PPP1R14D, S100A14, SBK1, SCGB3A2, SFTA2, SFTPA1, SFTPA2, SFTPB, SPINK1, TGFA, ZC3H11A, or a combination thereof. In some embodiments, the target biomarker signature includes at least one nanoparticle-associated surface biomarker, which is or includes an SCL34A2 polypeptide and / or a CEACAM5 polypeptide; and at least one surface biomarker SLC34A2, CEACAM5, CEACAM6, and / or EPCAM.

[0022] In some embodiments, the target biomarker signature for lung cancer comprises nanoparticle-associated surface biomarkers (e.g., those described herein) and at least one additional target intravesicular biomarker, which in some embodiments is a gene encoding one of the following human genes: [ka] [ka] or may comprise at least one polypeptide encoded by a combination thereof.

[0023] In some embodiments, the reference threshold level for use in the provided methods or assays described herein is determined by the level of target biomarker signature-expressing nanoparticles observed in comparable samples from a population of non-lung cancer subjects.

[0024] In some embodiments, nanoparticle-associated surface biomarkers included in the target biomarker signature may be detected using an affinity agent (for example, but not limited to, an antibody-based agent). In some embodiments, nanoparticle-associated surface biomarkers may be detected using a capture assay comprising an antibody-based agent. For example, in some embodiments, a capture assay for detecting the presence of nanoparticle-associated surface biomarkers on nanoparticles may include contacting a biological sample containing nanoparticles with a capture agent directed to such nanoparticle-associated surface biomarkers. In some embodiments, such a capture agent may include a binding moiety (e.g., as described herein) directed to the nanoparticle-associated surface biomarker, which may optionally be conjugated to a solid substrate. Without limitation, an exemplary capture agent for nanoparticle-associated surface biomarkers may be or include a solid substrate (e.g., magnetic beads) and a binding moiety (e.g., antibody agent) directed to the nanoparticle-associated surface biomarker.

[0025] In some embodiments, target biomarkers included in a target biomarker signature may be detected using suitable methods known in the art, which may vary depending on the type of analyte being detected (e.g., surface analytes vs. intravesicular analytes; and / or polypeptides and / or glycoforms vs. carbohydrates vs. RNA). For example, those skilled in the art reading this disclosure will recognize that in some embodiments, surface biomarkers and / or intravesicular biomarkers may be detected using affinity agents (e.g., antibody-based agents), while in some embodiments, intravesicular RNA (e.g., but not limited to, mRNA, and non-coding RNA such as, for example, orphan non-coding RNA, long non-coding RNA, piwi-interacting RNA, microRNA, circular RNA, etc.) may be detected using nucleic acid-based agents, e.g., using quantitative reverse transcription PCR.

[0026] For example, in some embodiments in which the target biomarker is or includes a surface biomarker and / or an intravesicular marker, such target biomarkers may be detected using a proximity ligation assay to capture nanoparticles exhibiting nanoparticle-associated surface biomarkers (e.g., those used and / or described herein), including the following capture assay (e.g., those described herein). In some embodiments, such a proximity ligation assay may include contacting a biological sample containing nanoparticles with a set of detection probes, each targeting a target biomarker, such that a combination comprising the set of nanoparticles and the detection probe is created, the set including at least two distinct detection probes, each of which includes: (i) a binding moiety targeting the surface biomarker and / or the intravesicular biomarker; and (ii) an oligonucleotide domain coupled to the binding moiety, the oligonucleotide domain including a double-stranded portion and a single-stranded overhang portion extending from one end of the oligonucleotide domain. Such single-stranded overhang portions of the detection probes are characterized in that they can hybridize with each other when the detection probes are bound to the same nanoparticle. This combination, which comprises nanoparticles and a set of detection probes, is then maintained under conditions that allow the set of detection probes to bind to their respective targets on nanoparticles, so that their oligonucleotide domains are close enough to anneal and form double-stranded complexes.This double-stranded complex can be detected by contacting the double-stranded complex with nucleic acid ligase to form ligated template; and detecting the ligated template.In some embodiments, the ligated template can be detected by quantitative PCR.The existence of this ligated template indicates the existence of nanoparticles that are positive for the target biomarker signature of lung cancer.Such proximity ligation assays may perform better, e.g., with greater specificity and / or sensitivity, than other existing proximity ligation assays, although those of skill in the art reading this disclosure will recognize that other forms of proximity ligation assays known in the art may alternatively be used.

[0027] In some embodiments where the target biomarker is or includes an intravesicular RNA (e.g., but not limited to, mRNA, and non-coding RNA such as, for example, orphan non-coding RNA, long non-coding RNA, piwi-interacting RNA, microRNA, circular RNA, etc.) marker, such target biomarker may be detected including a nucleic acid detection assay. In some embodiments, an exemplary nucleic acid detection assay may be or include reverse transcription PCR.

[0028] In some embodiments where the target biomarker is or comprises an intravesicular biomarker (e.g., an intravesicular biomarker and / or an intravesicular RNA (e.g., but not limited to, mRNA and non-coding RNA such as, for example, orphan non-coding RNA, long non-coding RNA, piwi-interacting RNA, microRNA, orphan non-coding RNA, long non-coding RNA, piwi-interacting RNA, etc.) biomarker), such target biomarker may be detected prior to a detection assay (e.g., a proximity ligation assay described herein) including treatment of the sample (e.g., fixation and / or permeabilization) to expose such biomarker within the nanoparticles for subsequent detection.

[0029] The present disclosure recognizes, among other things, that detection of a single lung cancer-associated serum protein or multiple lung cancer-associated biomarkers based on a bulk sample (e.g., a bulk sample of extracellular vesicles), rather than at single extracellular vesicle resolution, typically does not provide sufficient specificity and / or sensitivity in determining whether the subject from whom the sample was obtained is likely to suffer from or be susceptible to lung cancer. The present disclosure provides technologies, including systems, compositions, and / or methods, that solve such problems, including, among other things, by specifically requiring that individual nanoparticles having a size range of interest, including extracellular vesicles for detection, be characterized by the presence of a target biomarker signature that includes a combination of at least one or more extracellular vesicle-associated surface biomarkers and at least one or more target biomarkers. In certain embodiments, the present disclosure teaches techniques that require such individual nanoparticles characterized by the presence (e.g., by expression) of such target biomarker signatures for lung cancer, while nanoparticles that do not include the target biomarker signature do not produce a detectable signal (e.g., a level above a reference level, e.g., at least 10% or more above, where in some embodiments the reference level may be the level observed in a negative control sample, such as a sample lacking the individual nanoparticles comprising such target biomarker signature).

[0030] As will be understood by those skilled in the art, in some embodiments, a sample containing extracellular vesicles may also contain nanoparticles having a size range of interest, including extracellular vesicles. Thus, in some embodiments, the techniques provided herein are applicable to the detection of nanoparticles having a size range of interest, including extracellular vesicles, in the context of extracellular vesicles. Thus, in some embodiments, the present disclosure provides techniques for, among other things, the detection of colocalization of at least two or more surface biomarkers (e.g., as described herein) forming a target biomarker signature for lung cancer in individual nanoparticles having a size range of interest, including extracellular vesicles (e.g., in some embodiments, about 30 nm to about 1000 nm).

[0031] In some embodiments, the disclosure provides a method for detecting co-localization of at least two surface biomarkers, the combined expression levels of which have been determined to be associated with lung cancer, on the surface of the nanoparticles, wherein the surface biomarkers are selected from the group consisting of: (i) a human gene encoding at least one of the following: [ka] [ka] and combinations thereof; and / or (ii) a carbohydrate-dependent marker selected from the following: sialyl Tn (sTn) antigen, sialyl-6T antigen (6-sialylcore 1), T antigen, phosphatidylserine, Tn antigen, sialyl Lewis A antigen (CA19-9), Globo H, Gb5 (SSEA-3), lactotriaosylceramide, (Lc3), Forssman antigen, Lewis X, Lewis Y / B antigen, LY6E, Lewis Y / CD174, sialyl Lewis X (sLex) (also known as sialyl SSEA-1 (SLX)), NeuGcGM3, and combinations thereof; (c) comparing the detected co-localization level with a determined level; and (d) classifying the subject as having or susceptible to lung cancer if the detected co-localization level is at or above the determined level.

[0032] In some embodiments, the first surface biomarker and the second surface biomarker are selected from (i) the following human genes: [ka] [ka] and polypeptides encoded by combinations thereof; and / or (ii) carbohydrate-dependent markers independently selected from the following: sialyl Tn (sTn) antigen, sialyl-6T antigen (6-sialyl core 1), T antigen, phosphatidylserine, Tn antigen, sialyl Lewis A antigen (CA19-9), Globo H, Gb5 (SSEA-3), lactotriaosylceramide, (Lc3), Forssman antigen, Lewis X, Lewis Y / B antigen, LY6E, Lewis Y / CD174, sialyl Lewis X (sLex) (also known as sialyl SSEA-1 (SLX)), NeuGcGM3, and combinations thereof.

[0033] In some embodiments, the first surface biomarker and the second surface biomarker are: [ka] Each antigen is independently selected from sialyl-6T antigen (6-sialyl core 1), sialyl-T antigen, sialyl Lewis A antigen (CA19-9), Globo H, Gb5 (SSEA-3), lactotriaosylceramide (Lc3), Forssman antigen, and combinations thereof.

[0034] Thus, in some embodiments, the techniques provided herein may be useful for detecting the occurrence or recurrence of lung cancer in subjects and / or entire populations of subjects. In some embodiments, target biomarker signatures may be selected for the detection of lung cancer. In some embodiments, target biomarker signatures may be selected for the detection of specific categories of lung cancer, including, but not limited to, lung adenocarcinoma, small cell lung cancer, squamous cell and transitional cell lung cancer, large cell lung cancer, non-small cell carcinoma, other defined carcinoma types, sarcoma, and other defined types of lung cancer known in the art (see, e.g., SEER Cancer Statistics Review 1975-2017). In some embodiments, the techniques provided herein can be used periodically (e.g., annually) to screen human subjects or entire populations of human subjects for early stage lung cancer or recurrence of lung cancer.

[0035] In some embodiments, subjects suitable for the techniques provided herein for detecting the occurrence or recurrence of lung cancer may be asymptomatic human subjects and / or the entire asymptomatic population. Such asymptomatic subjects may include subjects with a family history of lung cancer, subjects previously treated for lung cancer, subjects at risk of lung cancer recurrence after cancer treatment, subjects in remission after lung cancer treatment, and / or subjects previously or periodically screened for the presence of lung cancer by chest X-ray, sputum analysis, low-dose CT, and / or the presence of at least one lung cancer serum biomarker, such as, but not limited to, CEA, CYFRA 21-1, NSE, ProGRP, and / or SCCA serum protein. In some embodiments, such asymptomatic subjects may be subjects whose medical diagnosis has been determined to be normal, for example, by chest X-ray, sputum analysis, low-dose CT, or serum CEA, CYFRA 21-1, NSE, ProGRP, and / or SCCA levels. In some embodiments, such asymptomatic subjects may be subjects who have been determined to have an abnormal medical diagnosis, for example, from a chest x-ray, sputum analysis, low-dose CT analysis, and / or serum levels of CEA, CYFRA 21-1, NSE, ProGRP, and / or SCCA levels, when compared to results typically observed in non-lung cancer subjects and / or normal healthy subjects. Alternatively, in some embodiments, asymptomatic subjects may be subjects who have not previously been screened for lung cancer, have not been diagnosed with lung cancer, and / or have not previously received lung cancer therapy.

[0036] In some embodiments, the subject or population of subjects may be selected based on one or more characteristics such as age, race, geographic location, genetic history, medical history, personal history and / or medical history (e.g., smoking, alcohol, drugs, carcinogens, diet, obesity, diabetes, physical activity, sun exposure, radiation exposure, exposure to infectious agents such as viruses, and / or occupational hazards).

[0037] In some embodiments, the techniques provided herein may be useful for selecting a surgical procedure or therapy for a subject suffering from or susceptible to lung cancer. In some embodiments, lung cancer therapy and / or adjuvant therapy can be selected in light of findings based on the techniques provided herein.

[0038] In some embodiments, the techniques provided herein may be useful for monitoring and / or evaluating the effectiveness of a therapy administered to a subject (e.g., a lung cancer subject).

[0039] In some embodiments, the present disclosure provides a technology for managing patient care, for example, for one or more individual subjects and / or a whole group of subjects.To give some examples, in some embodiments, the present disclosure provides a technology that can be used in screening (for example, time-based or contingency-driven screening and / or non-time-based or contingency-driven screening, for example, periodic screening, once a year, twice a year, once every two years, or some other frequency).For example, in some embodiments, the provided technology for use in time-based driving screening can be useful for screening one or more individual subjects or a whole group of subjects (for example, asymptomatic subjects) that are older than a certain age (for example, 30, 35, 40, 45, 50, 55, 60, 65, 70 years old or older). For example, in some embodiments, provided technology for use in temporally motivated screening may be useful for screening one or more individual subjects or entire populations of subjects (e.g., asymptomatic subjects) with a cigarette pack-year history above a certain number (e.g., 5 pack-years, 10 pack-years, 15 pack-years, 20 pack-years, 25 pack-years, 30 pack-years, and / or more than 35 pack-years; 1 pack-year is equivalent to smoking 1 pack per day for 1 year, while smoking 2 packs per day for 1 year is equivalent to 2 pack-years, or smoking ½ pack per day for 2 years is equivalent to 1 pack-year, etc.). In some embodiments, provided technology for use in incidentally motivated screening may be useful for screening individual subjects who may have experienced an event or occurrence that motivated screening for lung cancer as described herein.For example, in some embodiments, the incidental motivation for determining one or more indicators of cancer or susceptibility to cancer may be or may include, for example, a family history event (e.g., a close relative, such as a blood relative, previously diagnosed with lung cancer), the identification of one or more risk factors associated with lung cancer (e.g., lifestyle history risk factors, including, but not limited to, smoking, alcohol, diet, obesity, occupational hazards, etc.), and / or a prior incidental finding from genetic testing (e.g., genomic sequencing), and / or an imaging diagnostic test (e.g., X-ray, ultrasound, computed tomography (CT), low-dose CT, and / or magnetic resonance imaging (MRI) scan), the occurrence of one or more signs or symptoms characteristic of lung cancer (e.g., abnormal imaging results, and / or symptoms potentially indicative of lung cancer, etc.).

[0040] In some embodiments, the provided technology for managing patient care can inform treatment and / or payment (e.g., reimbursement for treatment) decisions and / or actions. For example, in some embodiments, the provided technology can provide a determination of whether an individual subject has one or more indicators of the development or recurrence of lung cancer, thereby informing a physician and / or patient on when to initiate therapy, taking such findings into account. Additionally or alternatively, in some embodiments, the provided technology can inform a physician and / or patient on treatment selection, for example, based on findings of specific response biomarkers (e.g., lung cancer response biomarkers). In some embodiments, the provided technology can provide a determination of whether an individual subject is responsive to a current treatment, for example, based on findings of changes in the levels of one or more molecular targets associated with lung cancer, thereby informing a physician and / or patient on the effectiveness of such therapy and / or decisions to maintain or modify therapy, taking such findings into account.

[0041] In some embodiments, the provided technology can inform decisions by health insurance providers regarding, for example, (1) whether to reimburse (or not) for the screening itself (e.g., reimbursement available only for periodic / routine screening, or only for time- and / or incidentally-motivated screening); and / or (2) whether to initiate, maintain, and / or modify therapy in light of findings from the provided technology. For example, in some embodiments, the present disclosure provides methods for (a) receiving results of the screenings described herein and also receiving claims for reimbursement for the screening and / or for a particular treatment regimen; (b) approving reimbursement for the screening if the screening was performed on the subject according to the appropriate schedule or response to the relevant event, and / or approving reimbursement for the treatment regimen if the received screening results represent appropriate treatment; and, as appropriate, (c) issuing the reimbursement or providing notification that the reimbursement was denied. In some embodiments, a treatment regimen is applied in consideration of the received screening result if the received screening result detects a biomarker that represents an approved biomarker for the associated treatment regimen (e.g., as may be stated on the prescribing information label and / or via an approved companion diagnostic). Alternatively, or in addition, the present disclosure contemplates a reporting system (e.g., implemented via a suitable electronic device and / or communication system) that permits or facilitates reporting and / or processing of the screening results and / or reimbursement determinations described herein.

[0042] Some aspects provided herein relate to systems and kits for use in the provided technology. In some embodiments, the system or kit may include a detection agent (e.g., as described herein) for a tumor biomarker signature of lung cancer. In some embodiments, such a system or kit may include: (a) a capture agent (e.g., as described herein) for an extracellular vesicle-associated surface biomarker present on nanoparticles associated with lung cancer; and (b) at least one or more detection agents targeting one or more target biomarkers of a target biomarker signature of lung cancer, which may be or include additional surface biomarkers (e.g., as described herein), intravesicular biomarkers (e.g., as described herein), and / or intravesicular RNA (e.g., including but not limited to, mRNA, and non-coding RNA such as orphan non-coding RNA, long non-coding RNA, piwi-interacting RNA, microRNA, circular RNA, etc.) biomarkers (e.g., as described herein).

[0043] In some embodiments, the capture agent included in the system and / or kit may comprise a binding moiety (e.g., as described herein) directed to an extracellular vesicle-associated surface biomarker. In some embodiments, such a binding moiety may be conjugated to a solid substrate, which in some embodiments may be or comprise a solid substrate. In some embodiments, such a solid substrate may be or comprise a magnetic bead. In some embodiments, an exemplary capture agent included in the provided system and / or kit may be or comprise a solid substrate (e.g., magnetic bead) and an affinity reagent (e.g., but not limited to, an antibody agent) directed to an extracellular vesicle-associated surface biomarker conjugated thereto.

[0044] In some embodiments, in which the target biomarkers include surface biomarkers and / or intravesicular biomarkers, the system and / or kit may include a detection agent (e.g., as described herein) for performing a proximity ligation assay. In some embodiments, such a detection agent for performing a proximity ligation assay may include a set of detection probes, each directed to a target biomarker of the target biomarker signature, the set including at least two detection probes, each including: (i) a polypeptide binding moiety directed to the target biomarker; and (ii) an oligonucleotide domain coupled to the binding moiety, the oligonucleotide domain comprising a double-stranded portion and a single-stranded overhanging portion extending from one end of the oligonucleotide domain, the single-stranded overhanging portions of the detection probes being characterized in that they can hybridize to each other when the detection probes are bound to the same nanoparticle.

[0045] In some embodiments, the systems and / or kits provided may include multiple (e.g., 2, 3, 4, 5, or more) sets of detection probes, each set including two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) detection probes. In some embodiments, at least one set of detection probes may be directed to the detection of lung cancer. For example, in some embodiments, the systems and kits provided may include at least one set of detection probes for the detection of lung cancer and at least one set of detection probes for the detection of a different cancer (e.g., pancreatic cancer). In some embodiments, two or more detection probes may be directed to different categories of lung cancer, including, for example, but not limited to, lung adenocarcinoma, small cell lung cancer, squamous cell and transitional cell lung cancer, large cell lung cancer, non-small cell lung cancer, other defined lung cancer types, lung carcinosarcoma, and other defined types of lung cancer known in the art (see, e.g., SEER Cancer Statistics Review 1975-2017). In some embodiments, two or more sets may be directed to the detection of different categories of lung cancer. In some embodiments, two or more sets may be directed to the detection of the same category of lung cancer. In some embodiments, two or more sets may be directed to the detection of different stages of lung cancer. In some embodiments, two or more sets may be directed to the detection of the same stage of lung cancer.

[0046] In some embodiments, the detection probes in the provided kits may be provided as a single mixture in a container. In some embodiments, multiple sets of detection probes may be provided as individual mixtures in separate containers. In some embodiments, each detection probe is provided individually in a separate container.

[0047] In some embodiments, where the target biomarkers include intravesicular RNA (e.g., but not limited to, mRNA, and non-coding RNA such as, for example, orphan non-coding RNA, long non-coding RNA, piwi-interacting RNA, microRNA, circular RNA, etc.) biomarkers, such systems and / or kits may include detection agents for performing nucleic acid detection assays. In some embodiments, such systems and / or kits may include detection agents for performing quantitative reverse transcription PCR, which may include, for example, primers directed to intravesicular RNA (e.g., but not limited to, mRNA, and non-coding RNA such as, for example, orphan non-coding RNA, long non-coding RNA, piwi-interacting RNA, microRNA, circular RNA, etc.) targets.

[0048] Those skilled in the art will understand that systems or kits for detecting extracellular vesicles can also be used to detect nanoparticles having a desired size range, including extracellular vesicles. Thus, in some embodiments, the systems or kits may include (i) a capture agent (e.g., as described herein) for a first surface biomarker of a lung cancer-associated biomarker signature present on the surface of nanoparticles having a desired size range, including extracellular vesicles; and (ii) at least one or more detection agents targeting a second surface biomarker of a lung cancer-specific biomarker signature. In some embodiments, such nanoparticles have a size within the range of about 30 nm to about 1000 nm.

[0049] In some embodiments, the disclosure provides a kit for detecting lung cancer, comprising: (a) a capture agent comprising a target capture moiety directed to a first surface biomarker; and (b) at least one set of detection probes, the set comprising at least two detection probes each directed to a second surface biomarker, the detection probes each comprising: (i) a target binding moiety directed to the second surface biomarker; and (ii) an oligonucleotide domain coupled to the target binding moiety, the oligonucleotide domain comprising a double-stranded portion and a single-stranded overhang portion extending from one end of the oligonucleotide domain, wherein the single-stranded overhang portions of the at least two detection probes are characterized in that they can hybridize to each other when the at least two detection probes are bound to the same nanoparticle having a size in the range of about 30 nm to about 1000 nm; wherein the at least first surface biomarker and the second surface biomarker form a target biomarker signature determined to be associated with lung cancer, the first and second surface biomarkers being selected from the group consisting of: (i) a target binding moiety directed to the second surface biomarker; [ka] [ka] and combinations thereof; and / or (ii) a polypeptide encoded by the following carbohydrate-dependent markers, each independently selected from the following: sialyl Tn (sTn) antigen, sialyl-6T antigen (6-sialylcore 1), T antigen, phosphatidylserine, Tn antigen, sialyl Lewis A antigen (CA19-9), Globo H, Gb5 (SSEA-3), lactotriaosylceramide, (Lc3), Forssman antigen, Lewis X, Lewis Y / B antigen, LY6E, Lewis Y / CD174, sialyl Lewis X (sLex) (also known as sialyl SSEA-1 (SLX)), NeuGcGM3, and combinations thereof.

[0050] In some embodiments, the first surface biomarker and the second surface biomarker are selected from (i) the following human genes: [ka] [ka] and polypeptides encoded by combinations thereof; and / or (ii) carbohydrate-dependent markers independently selected from the following: sialyl Tn (sTn) antigen, sialyl-6T antigen (6-sialyl core 1), T antigen, phosphatidylserine, Tn antigen, sialyl Lewis A antigen (CA19-9), Globo H, Gb5 (SSEA-3), lactotriaosylceramide, (Lc3), Forssman antigen, Lewis X, Lewis Y / B antigen, LY6E, Lewis Y / CD174, sialyl Lewis X (sLex) (also known as sialyl SSEA-1 (SLX)), NeuGcGM3, and combinations thereof.

[0051] In some embodiments, the first surface biomarker and the second surface biomarker are: [ka] Each antigen is independently selected from sialyl-6T antigen (6-sialyl core 1), sialyl-T antigen, sialyl Lewis A antigen (CA19-9), Globo H, Gb5 (SSEA-3), lactotriaosylceramide (Lc3), Forssman antigen, and combinations thereof.

[0052] In some embodiments, the provided systems and / or kits may include at least one chemical reagent, e.g., a chemical reagent for processing a sample and / or nanoparticles therein (e.g., including, in some embodiments, extracellular vesicles). In some embodiments, the provided systems and / or kits may include at least one chemical reagent for processing nanoparticles (e.g., including, in some embodiments, extracellular vesicles) in a sample, including, for example, but not limited to, a fixative, a permeabilizing agent, and / or a blocking agent. In some embodiments, the provided systems and / or kits may include a nucleic acid ligase and / or a nucleic acid polymerase. In some embodiments, the provided systems and / or kits may include one or more primers and / or probes. In some embodiments, the provided systems and / or kits may include, for example, one or more primer pairs for PCR, e.g., quantitative PCR (qPCR), reactions. In some embodiments, the provided systems and / or kits may include one or more probes, such as hydrolysis probes (e.g., TaqMan probes), which, in some embodiments, may be designed to increase the specificity of qPCR. In some embodiments, the provided systems and / or kits may include one or more multiplexing probes, as may be useful, for example, when simultaneous or parallel qPCR reactions are used (e.g., to facilitate or improve readout).

[0053] In some embodiments, the provided systems and / or kits can be used for screening (e.g., periodic screening) and / or other assessment of individuals (e.g., asymptomatic or symptomatic subjects) for detection (e.g., early detection) of lung cancer. In some embodiments, the provided systems and / or kits can be used for screening and / or other assessment of individuals predisposed to lung cancer (e.g., individuals with known genetic, environmental, or experiential risk, etc.). In some embodiments, the provided systems and / or kits can be used for monitoring the recurrence of lung cancer in previously treated subjects. In some embodiments, the provided systems and / or kits can be used as a companion diagnostic in combination with a therapy for a subject suffering from lung cancer. In some embodiments, the provided systems and / or kits can be used to monitor or evaluate the effectiveness of a therapy administered to a subject suffering from lung cancer. In some embodiments, the provided systems and / or kits can be used to select a therapy for a subject suffering from lung cancer. In some embodiments, the provided systems and / or kits can be used to make therapy decisions and / or select a therapy for a subject with one or more symptoms (e.g., non-specific symptoms) associated with lung cancer.

[0054] Complexes formed by performing the methods described herein and / or using the systems and / or kits described herein are also within the scope of the present disclosure. For example, in some embodiments, the complexes include (a) nanoparticles expressing target biomarker signatures, at least two of which include at least one nanoparticle-associated surface biomarker and at least one target biomarker selected from the group consisting of a surface biomarker, an intravesicular biomarker, and an intravesicular RNA biomarker, wherein the surface biomarkers are: [ka] and combinations thereof; wherein the intravesicular biomarker is selected from: [ka] and combinations thereof; and the intravesicular RNA (e.g., but not limited to, mRNA and non-coding RNA such as, for example, orphan non-coding RNA, long non-coding RNA, piwi-interacting RNA, microRNA, circular RNA, etc.) biomarkers are selected from: [ka] and combinations thereof, wherein the nanoparticles are immobilized on a solid substrate comprising binding moieties directed to such nanoparticle-associated surface biomarkers. In some embodiments, such complexes further comprise at least two detection probes directed to at least one target biomarker of the target biomarker signature present on the nanoparticles, each detection probe binding to a respective target biomarker and comprising: (i) a binding moiety directed to the target biomarker; and (ii) an oligonucleotide domain coupled to the binding moiety, the oligonucleotide domain comprising a double-stranded portion and a single-stranded overhang portion extending from one end of the oligonucleotide domain, and the single-stranded overhang portions of the detection probes hybridize to each other.

[0055] In some embodiments, the nanoparticle-associated surface biomarkers present on the complexed nanoparticles comprise one or more of the surface biomarkers described herein. In some embodiments, such nanoparticle-associated surface biomarkers include: [ka] and combinations thereof. In some embodiments, such nanoparticle-associated surface biomarkers and / or surface biomarkers included in a target biomarker signature may be or include SLC34A2 polypeptides. In some embodiments, such nanoparticle-associated surface biomarkers and / or surface biomarkers included in a target biomarker signature may be or include CEACAM5 polypeptides. In some embodiments, such nanoparticle-associated surface biomarkers and / or surface biomarkers included in a target biomarker signature may be or include CEACAM6 polypeptides. In some embodiments, such nanoparticle-associated surface biomarkers and / or surface biomarkers included in a target biomarker signature may be or include EPCAM polypeptides. In some embodiments, such nanoparticle-associated surface biomarkers and / or surface biomarkers included in a target biomarker signature may be or include ALCAM polypeptides. In some embodiments, such nanoparticle-associated surface biomarkers and / or surface biomarkers included in a target biomarker signature may be or include CD55 polypeptides. In some embodiments, such nanoparticle-associated surface biomarkers and / or surface biomarkers included in a target biomarker signature may be or comprise a CDH1 polypeptide. In some embodiments, such nanoparticle-associated surface biomarkers and / or surface biomarkers included in a target biomarker signature may be or comprise a CDH3 polypeptide. In some embodiments, such nanoparticle-associated surface biomarkers and / or surface biomarkers included in a target biomarker signature may be or comprise a CD274 (PD-L1) polypeptide.In some embodiments, such nanoparticle-associated surface biomarkers and / or surface biomarkers included in a target biomarker signature may be or may include a DSG2 polypeptide. In some embodiments, such nanoparticle-associated surface biomarkers and / or surface biomarkers included in a target biomarker signature may be or may include an EGFR polypeptide. In some embodiments, such nanoparticle-associated surface biomarkers and / or surface biomarkers included in a target biomarker signature may be or may include a FOLR1 polypeptide. In some embodiments, such nanoparticle-associated surface biomarkers and / or surface biomarkers included in a target biomarker signature may be or may include an IG1FR polypeptide. In some embodiments, such nanoparticle-associated surface biomarkers and / or surface biomarkers included in a target biomarker signature may be or may include a MET polypeptide. In some embodiments, such nanoparticle-associated surface biomarkers and / or surface biomarkers included in a target biomarker signature may be or may include an MSLN polypeptide. In some embodiments, such nanoparticle-associated surface biomarkers and / or surface biomarkers included in a target biomarker signature may be or may comprise a MUC1 polypeptide. In some embodiments, such nanoparticle-associated surface biomarkers and / or surface biomarkers included in a target biomarker signature may be or may comprise sTn antigen polypeptide glycosylation. In some embodiments, such nanoparticle-associated surface biomarkers and / or surface biomarkers included in a target biomarker signature may be or may comprise Tn antigen polypeptide glycosylation.In some embodiments, such nanoparticle-associated surface biomarkers and / or surface biomarkers included in the target biomarker signature may be or comprise T antigen polypeptide glycosylation. In some embodiments, such nanoparticle-associated surface biomarkers and / or surface biomarkers included in the target biomarker signature may be or comprise TACSTD2 polypeptide. In some embodiments, such nanoparticle-associated surface biomarkers and / or surface biomarkers included in the target biomarker signature may be or comprise TNFRSF10B polypeptide.

[0056] In some embodiments, the nanoparticle-associated surface biomarkers and / or surface biomarkers included in the target biomarker signature may be or may include a MUC1 polypeptide. In some embodiments, the nanoparticle-associated surface biomarkers and / or surface biomarkers included in the target biomarker signature may be or may include a Lewis Y antigen. In some embodiments, the nanoparticle-associated surface biomarkers and / or surface biomarkers included in the target biomarker signature may be or may include an sLex antigen. In some embodiments, the nanoparticle-associated surface biomarkers and / or surface biomarkers included in the target biomarker signature may be or may include a T antigen. In some embodiments, the nanoparticle-associated surface biomarkers and / or surface biomarkers included in the target biomarker signature may be or may include a Tn antigen.

[0057] Also within the scope of the present disclosure are complexes comprising nanoparticles of a desired size range that comprise a lung cancer-specific biomarker signature, the complexes comprising at least two surface biomarkers described herein, the nanoparticles being immobilized on a solid substrate comprising a binding moiety targeting a first surface biomarker of the lung cancer-specific biomarker signature. In some embodiments, such complexes are also bound to at least two detection probes, each targeting a surface biomarker of the lung cancer-specific biomarker signature (which may be the same or different surface biomarkers), each of which binds to a respective surface biomarker and comprises: (i) a binding moiety targeting the surface biomarker; and (ii) an oligonucleotide domain coupled to the binding moiety, the oligonucleotide domain comprising a double-stranded portion and a single-stranded overhang portion extending from one end of the oligonucleotide domain, the single-stranded overhang portions of the detection probes hybridizing to each other.

[0058] In some embodiments, the present disclosure provides (a) nanoparticles having a size in the range of about 30 nm to about 1000 nm and comprising at least a first surface biomarker and a second surface biomarker on their surface, the combination of which has been determined to be a target biomarker signature for lung cancer, wherein the first surface biomarker and the second surface biomarker are selected from the group consisting of (i) a target gene of one of the following human genes: [ka] [ka] and polypeptides encoded by combinations thereof; and / or (ii) the following carbohydrate-dependent markers: sialyl Tn (sTn) antigen, sialyl-6T antigen (6-sialyl core 1), T antigen, phosphatidylserine, Tn antigen, sialyl Lewis A antigen (CA19-9), Globo H, Gb5 (SSEA-3), lactotriaosylceramide, (Lc3), Forssman antigen, Lewis X, Lewis Y / B antigen, LY6E, Lewis Y / CD174, sialyl Lewis (b) a solid substrate comprising a target capture moiety directed to a first surface biomarker, wherein the target capture moiety binds to the first surface biomarker on the nanoparticle such that the nanoparticle is immobilized on the solid substrate; and (c) at least a first detection probe and a second detection probe each coupled to the nanoparticle, wherein each detection probe comprises: (i) a target binding moiety directed to a second surface biomarker; and (ii) an oligonucleotide domain coupled to the target binding moiety, wherein the oligonucleotide domain comprises a double-stranded portion and a single-stranded overhang portion extending from one end of the oligonucleotide domain, and wherein the single-stranded overhang portions of the first and second detection probes hybridize to each other.

[0059] In some embodiments, the first surface biomarker and the second surface biomarker are selected from (i) the following human genes: [ka] [ka] and polypeptides encoded by combinations thereof; and / or (ii) carbohydrate-dependent markers independently selected from the following: sialyl Tn (sTn) antigen, sialyl-6T antigen (6-sialyl core 1), T antigen, phosphatidylserine, Tn antigen, sialyl Lewis A antigen (CA19-9), Globo H, Gb5 (SSEA-3), lactotriaosylceramide, (Lc3), Forssman antigen, Lewis X, Lewis Y / B antigen, LY6E, Lewis Y / CD174, sialyl Lewis X (sLex) (also known as sialyl SSEA-1 (SLX)), NeuGcGM3, and combinations thereof.

[0060] In some embodiments, the first surface biomarker and the second surface biomarker are: [ka] [ka] Each antigen is independently selected from sialyl-6T antigen (6-sialyl core 1), sialyl-T antigen, sialyl Lewis A antigen (CA19-9), Globo H, Gb5 (SSEA-3), lactotriaosylceramide (Lc3), Forssman antigen, and combinations thereof.

[0061] These and other aspects encompassed by the present disclosure are described in more detail below and in the claims. [Brief explanation of the drawings]

[0062] [Figure 1]Figure 1 is a schematic diagram illustrating an exemplary workflow for profiling individual nanoparticles, e.g., extracellular vesicles. The diagram shows the purification of EVs from plasma using size exclusion chromatography (SEC) and immunoaffinity capture of EVs displaying specific EV-associated surface markers (Panel A); detection of co-localized target markers (e.g., intravesicular or surface biomarkers) on the captured EVs using a target entity detection assay according to some embodiments described herein (Panel B).

[0063] [Figure 2]2 is a schematic diagram illustrating a target entity detection assay according to some embodiments described herein. In some embodiments, the target entity detection assay uses a combination of detection probes, which are specific for the detection of cancer. In some embodiments, a duplex system includes a first detection probe for target biomarker 1 and a second detection probe for target biomarker 2, added to a sample containing a biological entity (e.g., extracellular vesicles). In some embodiments, the detection probes each include a target binding moiety (e.g., an affinity agent, such as an antibody agent for the target biomarker) coupled to an oligonucleotide domain, which includes a double-stranded portion and a single-stranded overhang extending from one end of the oligonucleotide domain. When the separate target binding moieties (e.g., affinity agents, such as an antibody agent for target biomarker 1 and target biomarker 2, respectively) of the first and second detection probes are localized in close proximity to the same biological entity (e.g., extracellular vesicles) such that the corresponding single-stranded overhangs hybridize with each other, thus resulting in ligation of the oligonucleotide domains. A detection signal is generated when the separate target binding moieties (e.g., affinity agents, such as an antibody agent for target biomarker 1 and target biomarker 2, respectively) of the first and second detection probes are localized in the same biological entity (e.g., extracellular vesicles) such that the corresponding single-stranded overhangs hybridize with each other, thus resulting in ligation of the oligonucleotide domains. For example, a control entity (e.g., a biological entity derived from a healthy subject sample) does not express one or both of target biomarker 1 and target biomarker 2, and as a result, no detection signal is generated. However, if a biological entity derived from a cancer sample (e.g., a lung cancer sample) expresses target biomarker 1 and target biomarker 2, and the target biomarkers are present within a sufficiently short distance from each other in the same biological entity (e.g., extracellular vesicles), a detection signal is generated.

[0064] [Figure 3]Figure 3 shows a graphical representation of the prevalence of different lung and bronchial cancer histologic subtypes. Data are from the National Cancer Institute's Surveillance, Epidemiology, and End Results (SEER) Program Cancer Statistics Review 1975-2017, the entire contents of which are incorporated herein by reference. The category "Other" includes large cell carcinoma, non-small cell carcinoma, other specified carcinoma types, carcinoma NOS, sarcoma, and other specified types. The determination and grouping of histologic classifications are described within the review.

[0065] [Figure 4] Figure 4 is a graphical representation of the population demographics of the pilot patient cohort.A total of 39 patient plasma samples from this cohort were analyzed, showing the profile of age, gender and cohort size relative to the stage of lung cancer diagnosis of patients.The cohort samples were then evaluated by the exemplary assays described herein.

[0066] [Figure 5] Figure 5 is a graphical representation of an exemplary lung adenocarcinoma (LUAD) diagnostic assay described herein. Normalized signals for healthy control and LUAD patient cohorts using SLC34A2 antibody-based EV capture and CEACAM6+CEACAM6 detection probes. The horizontal cutoff line represents the 100% specificity threshold. Sensitivity of 16.7% for stage I LUAD, 60% for stage II LUAD, 50% for stage III LUAD, and 100% for stage IV LUAD was achieved.

[0067] [Figure 6]Figure 6 is a graphical representation of an exemplary lung adenocarcinoma diagnostic assay described herein. Normalized signals for healthy control and LUAD patient cohorts using SLC34A2 antibody-based EV capture and CEACAM6+EPCAM detection probes. The horizontal cutoff line represents the threshold for 100% specificity. Sensitivity of 20% for stage II LUAD, 50% for stage III LUAD, and 75% for stage IV LUAD was achieved.

[0068] [Figure 7] Figure 7 is a graphical representation of an exemplary lung adenocarcinoma diagnostic assay described herein. Normalized signals for healthy control and LUAD patient cohorts using CEACAM5 antibody-based EV capture and CEACAM6+SLC34A2 detection probes. The horizontal cutoff line represents the 100% specificity threshold. Sensitivity of 16.7% for stage I LUAD, 20% for stage II LUAD, 50% for stage III LUAD, and 75% for stage IV LUAD was achieved.

[0069] [Figure 8] 8 is a graphical representation of the correlation between exemplary lung adenocarcinoma diagnostic assays described herein. The signals from the SLC34A2 antibody-based capture and CEACAM6+CEACAM6 detection probe are represented along the x-axis, while the signals from the SLC34A2 antibody-based capture and CEACAM6+EPCAM detection probe are represented along the y-axis. Correlation was determined using Pearson's product-moment correlation coefficient. Strong correlations were observed, particularly for stage III and stage IV LUAD samples.

[0070] [Figure 9]9 is a graph showing the correlation between exemplary lung adenocarcinoma diagnostic assays described herein. The signals from SLC34A2 antibody-based capture and CEACAM6+CEACAM6 detection probe are shown along the x-axis, while the signals from CEACAM5 antibody-based capture and CEACAM6+SLC34A2 detection probe are shown along the y-axis. Correlation was determined using Pearson's product-moment correlation coefficient. Strong correlation was observed, especially for stage III and stage IV LUAD samples.

[0071] [Figure 10] Figure 10 is a graphical representation of the correlation between exemplary lung adenocarcinoma diagnostic assays described herein. The signals from the SLC34A2 antibody-based capture and CEACAM6+EPCAM detection probe are represented along the x-axis, while the signals from the CAECAM5 antibody-based capture and CEACAM6+SLC34A2 detection probe are represented along the y-axis. Correlation was determined using Pearson's product-moment correlation coefficient. Strong correlation was observed, particularly for stage III and stage IV LUAD samples.

[0072] [Figure 11] Figure 11 is a graphical representation of the population demographics of the expanded patient cohort.A total of 138 patient plasma samples from this cohort were analyzed, showing the profile of age, gender, and cohort size relative to the stage of lung cancer diagnosis of patients.The cohort samples were then evaluated by the exemplary assays described herein.

[0073] [Figure 12] Figure 12 is a graphical representation of an exemplary lung adenocarcinoma diagnostic assay described herein. Normalized signals for healthy control and LUAD patient cohorts using SLC34A2 antibody-based EV capture and CEACAM6+CEACAM6 detection probes. The horizontal cutoff line represents a 99.9% specificity threshold. Sensitivity of 50% for stage II LUAD, 55.5% for stage III LUAD, and 71.4% for stage IV LUAD was achieved.

[0074] [Figure 13] 13 is a schematic diagram illustrating a target entity detection assay according to some embodiments described herein. The diagram shows an exemplary triple target entity detection system, in which, in some embodiments, three or more detection probes for each target biomarker can be added to a sample containing a biological entity (e.g., extracellular vesicles). In some embodiments, the detection probes each include a target binding moiety (e.g., an affinity agent, such as an antibody agent for the target biomarker) coupled to an oligonucleotide domain, which includes a double-stranded portion and a single-stranded overhang extending from one end of the oligonucleotide domain. A detection signal is generated when all corresponding single-stranded overhangs of the three or more detection probes hybridize to each other to form a linear double-stranded complex, allowing ligation of at least one strand of the double-stranded complex to occur, thus allowing the resulting ligated product to be detected.

[0075] [Figure 14] FIG. 14 is a non-limiting example of a double-stranded complex containing four detection probes connected to each other in a linear arrangement by hybridization of their respective single-stranded overhangs.

[0076] [Figure 15]15 is a schematic diagram illustrating a target entity detection assay according to an exemplary embodiment described herein. In some embodiments, multiple detection probes, each for a distinct target, are added to a sample containing a biological entity (e.g., extracellular vesicles). In some embodiments, the detection probes each contain a target-binding moiety (e.g., an antibody agent) coupled to an oligonucleotide domain, which contains a double-stranded portion and a single-stranded overhang extending from one end of the oligonucleotide domain. When all the detection probes are localized in close proximity to the same biological entity (e.g., extracellular vesicles or analytes), the corresponding single-stranded overhangs hybridize to form a linear double-stranded complex, ligating at least one strand of the resulting linear double-stranded complex and allowing the ligated product to be detected, a detection signal is generated.

[0077] [Figure 16A] Figure 16A is a graphical representation of the discriminatory power of biomarker combinations for detecting LUAD by simulating "healthy patients" and "cancer patients." Using bioinformatics analysis, plasma samples from 5,000 simulated "healthy patients" and 5,000 simulated "cancer patients" were randomly selected from normal and cancer tissue databases, respectively. The simulated "cancer patients" were modeled to have tumors of various sizes, ranging from 1 g to 1,000 g. Based on the two pools of "healthy patients" and "cancer patients," the sensitivity of the biomarker combinations for detecting LUAD with 99% specificity was calculated.

[0078] [Figure 16B]16B is an exemplary heatmap showing the ability of each potential biomarker combination from the list in Table 3 to detect LUAD based on simulated sensitivity for 100 g of tumor, as described herein. Each row represents one biomarker combination, and each column represents one LUAD cancer patient. Light gray indicates that no cancer in the LUAD patient was detected using the given biomarker combination, and dark gray indicates that cancer in the LUAD patient was detected using the given biomarker combination. The heatmap shows the sensitivity threshold based on 100 g of tumor.

[0079] [Figure 16C] Figure 16C is a histogram showing the AUC values ​​for each possible LUAD biomarker combination based on the list in Table 3 for other cancer types. To compare with other cancers, the EV score for the biomarker combination (where the EV score is the multiplication of all TPM expression values ​​in a given biomarker combination) was calculated for lung adenocarcinoma and all other tumor types in a cancer molecular database (e.g., Cancer Genome Atlas), excluding lung squamous cell carcinoma, and then the AUC was calculated. The histogram shows the distribution of AUC values ​​for all other cancers. In general, the biomarker combinations described herein show more specificity for LUAD than for other cancer types.

[0080] [Figure 17A] Figure 17A is a graphical representation of a particular biomarker combination compared to a healthy smoker sample pool for the detection of early stage lung adenocarcinoma (LUAD). Biomarker combinations were ranked by their ability to distinguish early stage LUAD sample pools from healthy smoker sample pools (highest rank at the top of the chart). The x-axis represents the difference in Ct values ​​obtained from the healthy smoker pooled samples and the early stage LUAD pooled samples. The y-axis represents a particular biomarker combination (target of capture probe, target of detection probe).

[0081] [Figure 17B] Figure 17B is a graphical representation of a particular biomarker combination compared to a healthy smoker sample pool for the detection of end-stage LUAD. Biomarker combinations were ranked by their ability to distinguish end-stage LUAD sample pools from healthy smoker sample pools (highest rank at the top of the chart). The x-axis represents the difference in Ct values ​​obtained from the healthy smoker pooled samples and the end-stage LUAD pooled samples. The y-axis represents a particular biomarker combination (target of capture probe, target of detection probe).

[0082] [Figure 18A] Figure 18A is a graphical representation of a particular biomarker combination compared to a healthy smoker sample pool for the detection of early stage lung squamous cell carcinoma (LUSC). Biomarker combinations were ranked by their ability to distinguish early stage LUSC sample pools from healthy smoker sample pools (highest rank at the top of the chart). The x-axis represents the difference in Ct values ​​obtained from the healthy smoker pooled samples and the early stage LUSC pooled samples. The y-axis represents a particular biomarker combination (target of capture probe, target of detection probe).

[0083] [Figure 18B] Figure 18B is a graphical representation of a particular biomarker combination compared to a pool of healthy smoker samples for the detection of end-stage LUSC. Biomarker combinations were ranked by their ability to distinguish end-stage LUSC sample pools from healthy smoker sample pools (highest rank at the top of the chart). The x-axis represents the difference in Ct values ​​obtained from pooled healthy smoker samples and pooled end-stage LUSC samples. The y-axis represents a particular biomarker combination (target of capture probe, target of detection probe).

[0084] [Figure 19A]Figure 19A is a graphical representation of a particular biomarker combination compared to a healthy non-smoker sample pool for the detection of early-stage LUAD. Biomarker combinations were ranked by their ability to distinguish early-stage LUAD sample pools from healthy non-smoker sample pools (highest rank at the top of the chart). The x-axis represents the difference in Ct values ​​obtained from the healthy non-smoker pooled samples and the early-stage LUAD pooled samples. The y-axis represents a particular biomarker combination (target of capture probe, target of detection probe).

[0085] [Figure 19B] Figure 19B is a graphical representation of a particular biomarker combination compared to a healthy non-smoker sample pool for the detection of end-stage LUAD. Biomarker combinations were ranked by their ability to distinguish end-stage LUAD sample pools from healthy non-smoker sample pools (highest rank at the top of the chart). The x-axis represents the difference in Ct values ​​obtained from the healthy non-smoker pooled samples and the end-stage LUAD pooled samples. The y-axis represents a particular biomarker combination (target of capture probe, target of detection probe).

[0086] [Figure 20A] Figure 20A is a graphical representation of a particular biomarker combination compared to a healthy non-smoker sample pool for the detection of early LUSC. Biomarker combinations were ranked by their ability to distinguish the early LUSC sample pool from the healthy non-smoker sample pool (highest rank at the top of the chart). The x-axis represents the difference in Ct values ​​obtained from the healthy non-smoker pooled samples and the early LUSC pooled samples. The y-axis represents a particular biomarker combination (target of capture probe, target of detection probe).

[0087] [Figure 20B]Figure 20B is a graphical representation of a particular biomarker combination compared to a pool of healthy non-smoker samples for the detection of end-stage LUSC. Biomarker combinations were ranked by their ability to distinguish end-stage LUSC samples from the pool of healthy non-smoker samples (highest rank at the top of the chart). The x-axis represents the difference in Ct values ​​obtained from the pooled healthy non-smoker samples and the pooled end-stage LUSC samples. The y-axis represents a particular biomarker combination (target of capture probe, target of detection probe).

[0088] [Figure 21A] Figure 21A is a graphical representation of a particular biomarker combination compared to a healthy smoker sample pool for the detection of early-stage LUAD. Biomarker combinations were ranked by their ability to distinguish the early-stage LUAD sample pool from the healthy smoker sample pool (highest rank at the top of the chart). The x-axis represents the difference in Ct values ​​obtained from the healthy smoker pooled samples and the early-stage LUAD pooled samples. The y-axis represents a particular biomarker combination (target of capture probe, target of detection probe).

[0089] [Figure 21B] Figure 21B is a graphical representation of a particular biomarker combination compared to a healthy smoker sample pool for the detection of end-stage LUAD. Biomarker combinations were ranked by their ability to distinguish end-stage LUAD sample pools from healthy smoker sample pools (highest rank at the top of the chart). The x-axis represents the difference in Ct values ​​obtained from the healthy smoker pooled samples and the end-stage LUAD pooled samples. The y-axis represents a particular biomarker combination (target of capture probe, target of detection probe).

[0090] [Figure 22A]Figure 22A is a graphical representation of a particular biomarker combination compared to a pool of healthy smoker samples for the detection of early LUSC. Biomarker combinations were ranked by their ability to distinguish the early LUSC sample pool from the healthy smoker sample pool (highest rank at the top of the chart). The x-axis represents the difference in Ct values ​​obtained from the pooled healthy smoker samples and the pooled early LUSC samples. The y-axis represents a particular biomarker combination (target of capture probe, target of detection probe).

[0091] [Figure 22B] Figure 22B is a graphical representation of a particular biomarker combination compared to a healthy smoker sample pool for the detection of end-stage LUSC. Biomarker combinations were ranked by their ability to distinguish end-stage LUSC sample pools from healthy smoker sample pools (highest rank at the top of the chart). The x-axis represents the difference in Ct values ​​obtained from the healthy smoker pooled samples and the end-stage LUSC pooled samples. The y-axis represents a particular biomarker combination (target of capture probe, target of detection probe).

[0092] [Figure 23A] Figure 23A is a graphical representation of a particular biomarker combination compared to a healthy non-smoker sample pool for the detection of early-stage LUAD. Biomarker combinations were ranked by their ability to distinguish early-stage LUAD sample pools from healthy non-smoker sample pools (highest rank at the top of the chart). The x-axis represents the difference in Ct values ​​obtained from the healthy non-smoker pooled samples and the early-stage LUAD pooled samples. The y-axis represents a particular biomarker combination (target of capture probe, target of detection probe).

[0093] [Figure 23B]Figure 23B is a graphical representation of a particular biomarker combination compared to a healthy non-smoker sample pool for the detection of end-stage LUAD. Biomarker combinations were ranked by their ability to distinguish end-stage LUAD sample pools from healthy non-smoker sample pools (highest rank at the top of the chart). The x-axis represents the difference in Ct values ​​obtained from the healthy non-smoker pooled samples and the end-stage LUAD pooled samples. The y-axis represents a particular biomarker combination (target of capture probe, target of detection probe).

[0094] [Figure 24A] Figure 24A is a graphical representation of a particular biomarker combination compared to a pool of healthy non-smoker samples for the detection of early LUSC. Biomarker combinations were ranked by their ability to distinguish the early LUSC sample pool from the healthy non-smoker sample pool (highest rank at the top of the chart). The x-axis represents the difference in Ct values ​​obtained from the pooled healthy non-smoker samples and the pooled early LUSC samples. The y-axis represents a particular biomarker combination (target of capture probe, target of detection probe).

[0095] [Figure 24B] Figure 24B is a graphical representation of a particular biomarker combination compared to a healthy non-smoker sample pool for the detection of end-stage LUSC. Biomarker combinations were ranked by their ability to distinguish end-stage LUSC sample pools from healthy non-smoker sample pools (highest rank at the top of the chart). The x-axis represents the difference in Ct values ​​obtained from the healthy non-smoker pooled samples and the end-stage LUSC pooled samples. The y-axis represents a particular biomarker combination (target of capture probe, target of detection probe).

[0096] [Figure 25]Figure 25 shows that the lung cancer (LC) training and validation cohorts included individuals with lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSqC), healthy never-smokers, and healthy ever-smokers. All samples were K2EDTA plasma except for those provided by Reprocell (ACD plasma). Cancer samples were collected at the time of diagnosis and before treatment, and disease staging was determined pathologically. Healthy subjects had no history of cancer at the time of blood draw, and smoking status was self-reported. Ever-smokers with COPD were clinically diagnosed with chronic obstructive pulmonary disease. A 1 mL aliquot of each sample was purified using size-exclusion chromatography and fractionated across the various biomarker combinations being evaluated. All samples were tested in duplicate. Biomaterials were provided by a tumor bank.

[0097] [Figure 26] Figure 26 shows that an LC panel comprising a combination of eight biomarkers resulted in a locked classifier that was trained on a cohort of 118 individual patient samples and validated in an independent blinded cohort of 456 samples.

[0098] [Figure 27] (A) Boxplots summarizing the probability of lung cancer based on an exemplary LC panel (e.g., as described herein) and locked classifiers. Each data point represents the median of assay replicates for a unique sample, and the red dotted line represents 80% specificity compared to healthy never-smokers, healthy former smokers, and COPD cohorts. The exemplary LC panel shows similar signal distribution across healthy never-smokers, healthy former smokers, and individuals with COPD, indicating no evidence of smoking was detected. (B) ROC curves for an exemplary LC panel (e.g., as described herein) comparing stage I / II lung cancer to healthy never-smokers, healthy former smokers, and COPD cohorts. The disclosed locked model demonstrates promising discrimination of LUAD and LUSqC from healthy never-smokers, healthy former smokers, and individuals with COPD.

[0099] [Figure 28] FIG. 28 shows the number of lung cancer samples sorted by stage and assay sensitivity.

[0100] [Figure 29] FIG. 29 shows that the signal from an exemplary LC assay (such as that described herein) correlates with tumor size, but not with smoking history.

[0101] [Figure 30] Figure 30 shows a subset analysis of screen-detected stage I, II, and III / IV lung cancer cases compared with healthy never-smokers and healthy former smokers.Each data point represents the median value of assay repeats for a unique sample, and the red dotted line represents 80% specificity compared with healthy never-smokers, healthy former smokers, and COPD cohorts.The performance of exemplary LC assays (such as those described herein) is similar in screen-detected lung cancer cases.

[0102] [Figure 31] FIG. 31 shows the performance of an exemplary LC assay (eg, as described herein) in lung cancer cases detected by routine screening. DETAILED DESCRIPTION OF THE INVENTION

[0103] A specific definition Administering: As used herein, the term "administering" or "administration" typically refers to the administration of a composition to a subject to achieve delivery of the agent itself or the agent contained in the composition to a target site or site to be treated. Those of skill in the art will be aware of various routes that may be utilized for administration to a subject, e.g., a human, in appropriate circumstances. For example, in some embodiments, administration may be parenteral. In some embodiments, administration may be oral. In some embodiments, administration may comprise only a single dose. In some embodiments, administration may comprise the application of a fixed number of doses. In some embodiments, administration may comprise dosing that is intermittent (e.g., multiple doses spaced apart in time) and / or periodic (e.g., individual doses spaced apart in time) dosing. In some embodiments, administration may comprise continuous dosing (e.g., perfusion) for at least a selected period of time.

[0104] Affinity agent: As used herein, the term "affinity agent" refers to an entity that is or includes a target-binding moiety described herein and thus binds to a target of interest (e.g., a molecular target of interest, such as a biomarker or epitope). In many embodiments, an affinity agent according to the present disclosure specifically binds to a biomarker described herein. In many embodiments, an affinity agent according to the present disclosure specifically binds to a surface biomarker described herein. In some embodiments, an affinity agent according to the present disclosure specifically binds to a carbohydrate-dependent marker described herein. In some embodiments, an affinity agent may be or include an antibody agent (e.g., an antibody or other entity that is or includes an antigen-binding portion thereof). Alternatively, or in addition, in some embodiments, an affinity agent may be selected from the group consisting of an affimer, an aptamer, a lectin, a sialic acid-binding immunoglobulin-type lectin (siglec), and combinations thereof, and / or another binding agent that may be considered a ligand. In some embodiments, the target of the affinity agent (e.g., a biomarker target) is or includes one or more polypeptide, nucleic acid, carbohydrate, and / or lipid moieties and / or entities.

[0105] Agent: Generally, the term "agent," as used herein, is used to refer to an entity (e.g., a lipid, metal, nucleic acid, polypeptide, polysaccharide, small molecule, etc., or a complex, combination, mixture, or system thereof [e.g., a cell, tissue, organ]), or a phenomenon (e.g., heat, an electric current or field, a magnetic force or field, etc.). In appropriate circumstances, as will be clear from the context to one of skill in the art, the term may be utilized to refer to an entity that is or includes a cell or organ, or a fraction, extract, or component thereof. Alternatively, or in addition, as the context will make clear, the term may be used to refer to a natural product found in nature and / or obtained from nature. In some instances, again as will be clear from the context, the term may be used to refer to one or more entities that are artificially created, in that they have been designed, engineered, and / or produced by the human hand and / or are not found in nature. In some embodiments, an agent may be utilized in isolated or purified form, and in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents may be provided as a collection or library that can be screened, for example, to identify or characterize active agents therein. In some instances, the term "agent" may refer to a polymer or In some cases, the term "agent" may refer to a compound or entity that is or comprises one or more polymer moieties, and in some cases, the term may refer to a compound or entity that comprises one or more polymer moieties. In some embodiments, the term "agent" may refer to a compound or entity that is not a polymer and / or is substantially free of any polymer and / or one or more specific polymer moieties. In some embodiments, the term may refer to a compound or entity that lacks or is substantially free of any polymer moieties.

[0106] Amplification: The terms "amplification" and "amplifying" refer to a temperature-dependent process that results in an increase in the amount and / or level of a nucleic acid molecule compared to its initial amount and / or level.The temperature-dependent process generally involves the temperature-dependent extension of a primer molecule, and the sequence of the newly synthesized strand of nucleic acid is dictated by the well-known rules of complementary base pairing (see, for example, Watson, JD et al., In: Molecular Biology of the Gene, 4th Ed., WA Benjamin, Inc., Menlo Park, Calif. (1987); for the purposes described herein, it is incorporated herein by reference).

[0107] Antibody agent: As used herein, the term "antibody agent" refers to an agent that specifically binds to a particular antigen. In some embodiments, an antibody agent refers to a polypeptide containing sufficient standard immunoglobulin sequence elements to confer specific binding to a particular target antigen. As is known in the art, naturally occurring intact antibodies are approximately 150 kD tetrameric agents composed of two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about 25 kD each) that associate with each other in a commonly referred to "Y-shaped" structure. Each heavy chain is composed of at least four domains (each about 110 amino acids long): an amino-terminal variable (VH) domain (located at the tip of the Y structure) followed by three constant domains: CH1, CH2, and a carboxy-terminal CH3 (located at the base of the stem of the Y). A short region known as the "switch" connects the heavy chain variable and constant regions. A "hinge" connects the CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to each other in intact antibodies. Each light chain is composed of two domains: an amino-terminal variable (VL) domain followed by a carboxy-terminal constant (CL) domain, separated from each other by another "switch." An intact antibody tetramer is composed of two heavy-light chain dimers in which the heavy and light chains are linked to each other by a single disulfide bond, and two other disulfide bonds connect the heavy chain hinge regions to each other so that the dimers are connected to each other and form a tetramer. Naturally produced antibodies are also typically glycosylated on the CH2 domain. Each domain in a natural antibody has a structure characterized by an "immunoglobulin fold" formed from two beta sheets (e.g., a three-, four-, or five-stranded sheet) packed against each other in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops known as "complementarity determining regions" (CDR1, CDR2, and CDR3) and four somewhat invariant "framework" regions (FR1, FR2, FR3, and FR4).When a natural antibody folds, the FR regions form beta sheets that provide a structural framework for the domains, and the CDR loop regions from both the heavy and light chains come together in three-dimensional space to create a single hypervariable antigen-binding site located at the tip of a Y-structure. The Fc region of a naturally occurring antibody binds to elements of the complement system and also to receptors on effector cells, including, for example, effector cells that mediate cytotoxicity. As is known in the art, the affinity and / or other binding characteristics of the Fc region for the Fc receptor can be modulated by glycosylation or other modifications. In some embodiments, antibodies produced and / or utilized according to the present invention comprise a glycosylated Fc domain, including Fc domains that have been modified or engineered, such as glycosylated. For purposes of the present invention, in certain embodiments, any polypeptide or complex of polypeptides that includes a sufficient immunoglobulin domain sequence found in a natural antibody can be referred to and / or used as an "antibody," whether such polypeptide is naturally produced (e.g., made by an organism in response to an antigen) or produced by recombinant engineering, chemical synthesis, or other artificial systems or methodologies. In some embodiments, an antibody is polyclonal, and in some embodiments, an antibody is monoclonal. In some embodiments, an antibody has constant region sequences characteristic of rabbit, rodent (e.g., mouse, rat, hamster, etc.), camelid (e.g., llama, alpaca), ovine, caprine, bovine, equine, chicken, donkey, shark, primate, human, or in vitro-derived (e.g., yeast, phage) antibodies. In some embodiments, the antibody sequence elements are humanized, primatized, chimeric, etc., as known in the art. Additionally, the term "antibody," as used herein, can, in appropriate embodiments (unless otherwise stated or clear from the context), refer to any of the constructs or formats known or developed in the art for utilizing the structural and functional features of antibodies in alternative presentations.For example, in some embodiments, the antibody utilized in accordance with the present invention is in a format selected from, but not limited to, an IgA, IgG, IgE, or IgM antibody; a bi- or multispecific antibody (e.g., Zybodies®, etc.); an antibody fragment, such as a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fd' fragment, a Fd fragment, and an isolated CDR or set thereof; a single-chain Fv; a polypeptide-Fc fusion; a single domain antibody, an alternative scaffold, or an antibody mimetic (e.g., anticalins, FN3 monobodies, Affibodies, Affilins, Affimers, Affitins, Alphabodies, Avimers, Fynomers, Im7, VLR, VNAR, Trimab, CrossMab, Trident); a nanobody, a binanobody, F(ab')2, Fab', di-sdFv, a single domain antibody, a trifunctional antibody, a diabody, and a minibody. In some embodiments, the relevant format may be or include Adnectins®; Affibodies®; Affilins®; Anticalins®; Avimers®; BiTEs®; camelized antibodies; Centyrins®; ankyrin repeat proteins or DARPINs®; dual affinity retargeting (DART) agents; Fynomers®; shark single domain antibodies, e.g., IgNAR; immune mobilizing monoclonal T cell receptors against cancer (ImmTACs); KALBITOR®; MicroProteins; Nanobodies® minibodies; masked antibodies (e.g., Probodies®); small modular immunopharmaceuticals ("SMIPs™"); single chain or tandem diabodies (TandAb®); TCR-like antibodies; Trans-bodies®; TrimerX®; VHHs. In some embodiments, the antibody may lack covalent modifications (eg, glycan attachments) that it would have if produced in nature.In some embodiments, the antibody may contain a covalent modification (e.g., attachment of a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.) or other pendant group (e.g., polyethylene glycol, etc.).

[0108] Antigen: As used herein, the term "antigen" refers to an entity (e.g., a molecule or molecular structure, such as a peptide or protein, carbohydrate, lipid particle, oligonucleotide, chemical molecule, or combination thereof) that contains one or more epitopes and thus recognizes and binds to an affinity agent (e.g., an antibody, affimer, or aptamer).

[0109] Approximately or about: As used herein, the term "approximately" or "about," when applied to one or more values ​​of interest, refers to a value similar to the stated reference value. Generally, a person skilled in the art familiar with the context will recognize the relevant degree of variation encompassed by "about" or "approximately" in that context. For example, in some embodiments, the term "approximately" or "about" can encompass a range of values ​​that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the reference value.

[0110] Aptamer: As used herein, the term "aptamer" typically refers to a nucleic acid or peptide molecule that binds to a specific target molecule (e.g., an epitope). In some embodiments, nucleic acid aptamers may be described by their nucleotide sequence and are typically about 15-60 nucleotides in length. Nucleic acid aptamers may be or include single-stranded and / or double-stranded structures. In some embodiments, nucleic acid aptamers may be or include DNA. In some embodiments, nucleic acid aptamers may be or include RNA. While not wishing to be bound by any theory, it is contemplated that the strands of nucleotides in an aptamer form intramolecular interactions that fold the molecule into a complex three-dimensional shape, which allows the aptamer to bind tightly to the surface of its target molecule. In some embodiments, peptide aptamers may be described as having one or more peptide loops of variable sequence displayed by a protein scaffold. Peptide aptamers can be isolated from combinatorial libraries and often subsequently improved by rounds of directed mutation or variable region mutagenesis and selection. Given the extraordinary diversity of molecular shapes present within the universe of all possible nucleotide and / or peptide sequences, aptamers can be obtained for a wide range of molecular targets, including proteins and small molecules. In addition to high specificity, aptamers typically have very high affinity for their targets (e.g., picomolar to low nanomolar range for proteins or polypeptides). Because aptamers are typically synthetic molecules, they are amenable to various modifications, which can optimize their function for specific applications.

[0111] Associated with: As used herein, two events or entities are "associated" with each other when the presence, level, and / or form of one correlates with that of the other. For example, a particular biological phenomenon (e.g., expression of a specific biomarker) is considered to be associated with lung cancer (e.g., a specific type of lung cancer and / or stage of lung cancer) if its presence correlates with the occurrence and / or susceptibility of lung cancer (e.g., an entire relevant population).

[0112] Biological entity: Where appropriate, and as will be clear from context to one of skill in the art, the term "biological entity" may, in some embodiments, be or include a cell or organism, e.g., an animal or human, or, in some embodiments, a biological tissue or biological fluid. For example, in some embodiments, the term may be utilized to refer to an entity or component present in a biological sample derived from or obtained from a subject. In some embodiments, the biological entity is or includes a cell or microorganism, or a fraction, extract, or component thereof (e.g., including intracellular components and / or molecules secreted by the cell or microorganism). In some embodiments, the biological entity is or includes a nanoparticle having a size in the range of about 30 nm to about 1000 nm, which, in some embodiments, is obtained from a subject's bodily fluid sample (e.g., but not limited to, a blood-derived sample). In some embodiments, such nanoparticles may be or include protein aggregates, e.g., in some embodiments, including glycans and / or extracellular vesicles. In some embodiments, such nanoparticles may have a size within a range of about 30 nm to about 1000 nm, about 50 nm to about 500 nm, or about 75 nm to about 500 nm. For example, in some embodiments, the biological entity is or comprises a cell. In some embodiments, the biological entity is or comprises an extracellular vesicle. In some embodiments, the biological entity is or comprises a biological analyte (e.g., a metabolite, carbohydrate, protein or polypeptide, enzyme, lipid, organelle, cytokine, receptor, ligand, and any combination thereof). In some embodiments, the biological entity present in the sample is in its native state (e.g., a protein or polypeptide is retained in a naturally occurring conformational structure). In some embodiments, the biological entity is processed, for example, by isolating it from the sample or by derivatizing it from a naturally occurring biological entity.For example, the biological entity can be treated with one or more chemical agents to make it more desirable for detection using the techniques provided herein. By way of example only, the biological entity may be a cell or extracellular vesicle that has been contacted with a fixative (e.g., but not limited to, methanol and / or formaldehyde) to cause crosslinking of proteins and / or peptides present in the cell or extracellular vesicle. In some embodiments, the biological entity is in an isolated or pure form (e.g., isolated from a body fluid sample, such as a blood, serum, or plasma sample). In some embodiments, the biological entity may be present in a complex matrix (e.g., a body fluid sample, such as a blood, serum, or plasma sample).

[0113] Biomarker: The term "biomarker" typically refers to an entity, event, or characteristic whose presence, level, degree, type, and / or form correlates with a particular biological event or state of interest and is therefore considered to be a "marker" for that event or state. To give a few examples, in some embodiments, a biomarker may be or include a marker for a particular disease state or the likelihood that a particular disease, disorder, or condition will occur, occur, or recur. In some embodiments, a biomarker may be or include a marker for a particular disease or treatment outcome or likelihood thereof. In some embodiments, a biomarker may be or include a marker for a particular tissue (e.g., but not limited to, brain, breast, colon, ovaries and / or other tissues associated with the female reproductive system, pancreas, prostate and / or other tissues associated with the male reproductive system, liver, lung, and skin). Such markers for a particular tissue may, in some embodiments, be specific to healthy tissue, specific to diseased tissue, or in some embodiments, be present in normal healthy tissue and diseased tissue (e.g., tumors); those skilled in the art reading this disclosure will recognize the appropriate context for each such type of biomarker. In some embodiments, a biomarker may be or include a cancer-specific marker (e.g., a marker that is specific to a particular cancer). In some embodiments, a biomarker may be or include a non-specific cancer marker (e.g., a marker that is present in at least two or more cancers). In some embodiments, a non-specific cancer marker may be or include a general marker for cancer (e.g., a marker that is typically present in cancer regardless of tissue type), or in some embodiments, a marker for cancer of a specific tissue (e.g., but not limited to, the brain, breast, colon, ovary and / or other tissues associated with the female reproductive system, pancreas, prostate and / or other tissues associated with the male reproductive system, liver, lung, and skin).Thus, in some embodiments, biomarkers are predictive, in some embodiments, biomarkers are prognostic, and in some embodiments, biomarkers are diagnostic for relevant biological events or conditions of interest. Biomarkers may be or include any chemical class of entity, or may be or include a combination of entities. For example, in some embodiments, biomarkers may be or include nucleic acids, polypeptides, lipids, carbohydrates, small molecules, inorganic agents (e.g., metals or ions), or combinations thereof. In some embodiments, biomarkers are or include portions of specific molecules, complexes, or structures; for example, in some embodiments, biomarkers may be or include epitopes. In some embodiments, biomarkers are surface markers (e.g., surface markers) of extracellular vesicles associated with lung cancer. In some embodiments, biomarkers are intravesicular (e.g., protein or RNA markers present in extracellular vesicles). In some embodiments, biomarkers may be or include genetic or epigenetic signatures. In some embodiments, a biomarker may be or include a gene expression signature. In some embodiments, a "biomarker" suitable for use in accordance with the present disclosure may refer to the presence, level, and / or form of a molecular entity (e.g., an epitope) present on a target marker. For example, in some embodiments, two or more "biomarkers" (e.g., epitopes) as molecular entities may be present on the same target marker (e.g., a biomarker, e.g., a surface biomarker present on extracellular vesicles).

[0114] Blood-derived sample: As used herein, the term "blood-derived sample" refers to a sample derived from a blood sample (i.e., a whole blood sample) of a subject in need thereof. Examples of blood-derived samples include, but are not limited to, plasma (including, e.g., fresh frozen plasma), serum, blood fractions, plasma fractions, serum fractions, blood fractions including red blood cells (RBCs), platelets, white blood cells, etc., and cell lysates comprising the fractions (e.g., cells, e.g., red blood cells, white blood cells, etc., can be collected and lysed to obtain cell lysates). In some embodiments, the blood-derived sample used in the methods, systems, and / or kits described herein is a plasma sample.

[0115] Cancer: The term "cancer" is used herein generally to refer to a disease or condition in which cells of a tissue of interest exhibit relatively abnormal, uncontrolled, and / or autonomous growth, such that they exhibit an abnormal growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, cancer can include cells that are pre-cancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. The present disclosure provides techniques for the detection of lung cancer.

[0116] Capture assay: As used herein, the term "capture assay" refers to a process for isolating or separating a biological entity of interest from a sample (e.g., in some embodiments, a bodily fluid-derived sample). In some embodiments, the biological entity of interest is isolated or separated from a sample (e.g., in some embodiments, a bodily fluid-derived sample) using a capture probe described herein. In some embodiments, the biological entity of interest that binds to a capture probe described herein is subjected to a detection assay described herein. In some embodiments, the biological entity of interest suitable for the capture assay described herein is or comprises a nanoparticle having a size range of interest, including extracellular vesicles. In some embodiments, such nanoparticles may have a size within a range of about 30 nm to about 1000 nm, about 50 nm to about 500 nm, or about 75 nm to about 500 nm. In some embodiments, the biological entity of interest suitable for the capture assay described herein is or comprises an extracellular vesicle of interest (e.g., in some embodiments, an exosome).

[0117] Capture probe: As used herein, the term "capture probe" refers to a capture agent for capturing a biological entity of interest from a sample (e.g., in some embodiments, a bodily fluid-derived sample, such as, but not limited to, a blood-derived sample). In many embodiments described herein, the capture agent comprises at least one target capture moiety that binds to a surface polypeptide of the biological entity of interest. In some embodiments, such a biological entity of interest is or comprises a nanoparticle having a size range of interest, including extracellular vesicles. In some embodiments, such nanoparticles may have a size within a range of about 30 nm to about 1000 nm, about 50 nm to about 500 nm, or about 75 nm to about 500 nm. In some embodiments, such a biological entity of interest comprises an extracellular vesicle (e.g., in some embodiments, an exosome). In some embodiments, the capture agent comprises at least one target moiety that binds to a surface biomarker (e.g., those described herein) of a nanoparticle having a size within a range of about 30 nm to about 1000 nm, including, for example, an extracellular vesicle (e.g., in some embodiments, an exosome). In some embodiments, the target capture moiety of the capture agent is or comprises an affinity agent described herein. In some embodiments, the target capture moiety of the capture agent is or comprises an antibody agent. In some embodiments, the target capture moiety of the capture agent is or comprises a lectin or a sialic acid-binding immunoglobulin-type lectin. In some embodiments, the capture agent may comprise a solid substrate to which the target capture moiety is immobilized. In some embodiments, an exemplary solid substrate is a bead (e.g., a magnetic bead). In some embodiments, the capture probe is or comprises a population of magnetic beads comprising a target capture moiety that specifically binds to a surface biomarker described herein.

[0118] Classification cutoff: As used herein, the term "classification cutoff" refers to a level, value, or score, or set of values, or index used to predict a subject's risk for a disease or condition (e.g., lung cancer), for example, by defining one or more boundary lines among two or more subsets of a population (e.g., normal healthy subjects and subjects with an inflammatory condition versus lung cancer subjects). In some embodiments, the classification cutoff may be determined with reference to at least one reference threshold level (e.g., reference cutoff) for a target biomarker signature described herein, optionally in combination with other appropriate variables, such as the subject's age, life history-related risk factors, genetic factors, physical and / or medical condition. In some embodiments where classification is based on a single target biomarker signature (e.g., as described herein), the classification cutoff may be the same as a predetermined reference threshold (e.g., cutoff) for the single target biomarker signature. In some embodiments where the classification is based on two or more (e.g., 2, 3, 4, or more) target biomarker signatures, the classification cutoff may refer to two or more reference thresholds (e.g., cutoffs) each individually predetermined for the corresponding target biomarker signatures, and may optionally incorporate one or more appropriate variables, e.g., the subject's age, life history-related risk factors, genetic factors, physical and / or medical condition. In some embodiments, the classification cutoff may be determined via computer algorithm-mediated analysis that references at least one reference threshold level (e.g., reference cutoff) for the target biomarker signatures described herein, in combination with other appropriate variables, e.g., the subject's age, life history-related risk factors, genetic factors, physical and / or medical condition.

[0119] Proximity: As used herein, the term "proximity" refers to a distance between two detection probes (e.g., two detection probes in a pair) that is sufficiently close that interaction between the detection probes (e.g., via their individual oligonucleotide domains) is likely to occur. For example, in some embodiments, the probability that two detection probes will interact with each other (e.g., via their individual oligonucleotide domains) over a period of time is at least 50% or higher, including, for example, at least 60%, at least 70%, at least 80%, at least 90%, or higher, when they are sufficiently close to each other under defined conditions (e.g., when the detection probes bind to their respective targets in extracellular vesicles). In some embodiments, the distance between two detection probes may range from approximately 0.1 to 1000 nm, or from 0.5 to 500 nm, or from 1 to 250 nm, when they are sufficiently close to each other. In some embodiments, the distance between two detection probes may range from approximately 0.1 to 10 nm, or from approximately 0.5 to 5 nm, when they are sufficiently close to each other. In some embodiments, the distance between two detection probes, when they are sufficiently close to each other, may be less than 100 nm or less, including, for example, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 40 nm, less than 30 nm, less than 20 nm, less than 10 nm, less than 5 nm, less than 1 nm, or less than 100 nm. In some embodiments, the distance between two detection probes, when they are sufficiently close to each other, may range from approximately 40 to 1000 nm, or between 40 nm and 500 nm.

[0120] Comparable: As used herein, the term "comparable" refers to two or more agents, entities, circumstances, sets of conditions, etc. that may not be identical to one another, but are sufficiently similar to permit a comparison between them, such that a person of ordinary skill in the art would recognize that they could reasonably draw conclusions based on the observed differences or similarities. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by one or a fewer number of a number of substantially identical traits and a variety of traits. A person of ordinary skill in the art will understand, in context, the degree of identity required for two or more such agents, entities, circumstances, sets of conditions, etc. to be considered comparable in any given situation. For example, a person of ordinary skill in the art will recognize that sets of circumstances, individuals, or populations are comparable to one another when they are characterized by a sufficient number and variety of substantially identical traits to warrant a reasonable conclusion that differences in results obtained under, or phenomena observed using, different sets of circumstances, individuals, or populations are caused by or indicate variations in the altered traits.

[0121] Complementary: As used herein, the term "complementary" in the context of nucleic acid base pairing refers to oligonucleotide hybridization related by the base-pairing rules. For example, the sequence "CAGT" is complementary to the sequence "GTCA." Complementarity can be partial or total. Thus, any degree of partial complementarity is intended to be included within the scope of the term "complementary," provided that the partial complementarity allows for oligonucleotide hybridization. Partial complementarity is when one or more nucleic acid bases do not match according to the base-pairing rules. Total or complete complementarity between nucleic acids is when each and every nucleic acid base matches another base under the base-pairing rules. In the context of identifying a combination of biomarkers for the detection of a particular cancer, the term "complementary" is used herein in reference to sets of biomarkers that have different information content (e.g., the ability to detect cancer in distinct, substantially non-overlapping subgroups of subjects). For example, two sets of biomarkers, Set 1 and Set 2, are said to be "complementary" to one another if, for example, Set 1 detects cancer in a group of subjects in a population (e.g., Group A) and Set 2 detects cancer in a substantially separate and non-overlapping group of subjects in the same population (e.g., Group B), but not in Group A. Similarly, Set 1 does not detect cancer in a significant number of subjects in Group B.

[0122] Detecting: The term "detecting" is used broadly herein to include any suitable means for determining the presence or absence of extracellular vesicles expressing a target biomarker signature for lung cancer or any form of measurement indicative of such extracellular vesicles. Thus, "detecting" can include determining, measuring, assessing, or assaying the presence or absence, level, amount, and / or location of an entity of interest (e.g., a surface biomarker, an intravesicular biomarker, or an intravesicular RNA biomarker) corresponding to a portion of the target biomarker signature in any manner. In some embodiments, "detecting" can include determining, measuring, assessing, or quantifying a form of measurement indicative of an entity of interest (e.g., a ligated template indicative of a protein biomarker and / or an intravesicular biomarker, or a PCR amplification product indicative of intravesicular mRNA). Quantitative and qualitative determinations, measurements, or assessments are included, including semi-quantitative determinations, measurements, or assessments. Such determination, measurement, or assessment can be relative, for example, when the entity of interest (e.g., a surface biomarker, an intravesicular biomarker, or an intravesicular RNA biomarker) or a form of measurement indicative thereof is detected relative to a control reference, or absolute. Thus, the term "quantifying," when used in the context of quantifying an entity of interest (e.g., a surface biomarker, an intravesicular biomarker, or an intravesicular RNA biomarker) or a form of measurement indicative thereof, can refer to absolute or relative quantification. Absolute quantification can be achieved by correlating the detected level of, or a form of measurement indicative of, the entity of interest (e.g., a surface biomarker, an intravesicular biomarker, or an intravesicular RNA biomarker) to a known control standard (e.g., by generating a standard curve). Alternatively, relative quantification can be achieved by comparing the levels or amounts detected between two or more different entities of interest (e.g., different surface biomarkers, intravesicular biomarkers, or intravesicular RNA biomarkers) to provide a relative quantification of each of the two or more different entities of interest, i.e., relative to each other.

[0123] Detection Label: The term "detection label," as used herein, refers to any element, molecule, functional group, compound, fragment, or moiety that is detectable. In some embodiments, a detection label is provided or utilized alone. In some embodiments, a detection label is provided and / or utilized in association with (e.g., conjugated to) another agent. Examples of detection labels include, but are not limited to, various ligands, radionuclides (e.g., 3 H, 14 C. 18 F, 19 F, 32 P, 35 S, 135 I, 125 I, 123 I, 64 Cu, 187 Re, 111 In, 90 Y, 99m Tc, 177 Lu, 89 Zr, etc.), fluorescent dyes, chemiluminescent agents (such as acridinium esters, stabilized dioxetanes, etc.), bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots), metal nanoparticles (such as gold, silver, copper, platinum, etc.) nanoclusters, paramagnetic metal ions, enzymes, colorimetric labels (such as dyes, colloidal gold, etc.), biotin, digoxigenin, haptens, and proteins for which antisera or monoclonal antibodies are available.

[0124] Detection probe: The term "detection probe" typically refers to a probe intended for the detection and / or quantification of a specific target. In some embodiments, a detection probe is a quantification probe that provides an indication of the level of a specific target. According to the present disclosure, a detection probe refers to a composition comprising a target-binding entity coupled directly or indirectly to an oligonucleotide domain, where the target-binding entity specifically binds to an individual target (e.g., a molecular target), and at least a portion of the oligonucleotide domain is designed to allow hybridization with a portion of the oligonucleotide domain of another detection probe for a distinct target. In many embodiments, oligonucleotide domains suitable for use according to the present disclosure comprise a double-stranded portion and at least one single-stranded overhang. In some embodiments, the oligonucleotide domain may comprise a double-stranded portion and a single-stranded overhang at each end of the double-stranded portion. In some embodiments, the target-binding entity of the detection probe is or comprises an affinity agent described herein. In some embodiments, the target-binding entity of the detection probe is or comprises an antibody agent. In some embodiments, the target binding entity of the detection probe is or comprises a lectin or a sialic acid-binding immunoglobulin-type lectin (siglec).

[0125] Double-stranded: As used herein, the term "double-stranded" in the context of an oligonucleotide domain is understood by those skilled in the art to mean that a pair of oligonucleotides exists in a hydrogen-bonded helical configuration, typically in association with a nucleic acid such as, for example, DNA. In addition to 100% complementary forms of double-stranded oligonucleotides, the term "double-stranded," as used herein, is also meant to refer to those forms that contain mismatches (e.g., partial complementarity) and / or structural features such as bulges, loops, or hairpins.

[0126] Double-stranded complex: As used herein, the term "double-stranded complex" typically refers to a complex comprising at least two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) detection probes (e.g., as provided and / or utilized herein) each directed to a target (which may be the same target or distinct targets) connected or coupled to each other in a linear arrangement by hybridization of complementary single-stranded overhangs of the detection probes. In some embodiments, such double-stranded complexes may comprise extracellular vesicles, and the individual target-binding portions of the detection probes are simultaneously bound to the extracellular vesicles.

[0127] Epitope: As used herein, the term "epitope" includes any moiety that is specifically recognized by an affinity agent (for example, but not limited to, an antibody, affimer, and / or aptamer). In some embodiments, an epitope is composed of multiple chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface-exposed when the antigen adopts the relevant three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically close to each other in space when the antigen adopts such a conformation. In some embodiments, at least some of such chemical atoms that are groups are physically separated from each other when the antigen adopts an alternative conformation (e.g., linearized).

[0128] Extracellular vesicles: As used herein, the term "extracellular vesicles" typically refers to vesicles outside cells, e.g., secreted by cells. Examples of secreted vesicles include, but are not limited to, exosomes, microvesicles, microparticles, ectosomes, oncosomes, and apoptotic bodies. Without wishing to be bound by theory, exosomes are nanometer-sized vesicles (e.g., 40 nm to 120 nm) of intracellular origin that can be formed by budding of multivesicular endosomes (MVEs) into the inner side of the limiting membrane, while microvesicles typically bud from the cell surface and their size can vary from 50 nm to 1000 nm. In some embodiments, extracellular vesicles are or include exosomes and / or microvesicles. In some embodiments, a sample containing extracellular vesicles is substantially free of apoptotic bodies. In some embodiments, the sample containing nanoparticles may include extracellular nanoparticles shed from or derived from one or more tissues (e.g., cancerous tissue and / or non-cancerous or healthy tissue). In some embodiments, the extracellular vesicles in the sample may be shed from or derived from lung cancer tumors, and in some embodiments, the extracellular vesicles are shed from or derived from non-lung cancer tumors. In some embodiments, the extracellular vesicles are shed from or derived from healthy tissue. In some embodiments, the extracellular vesicles are shed from or derived from benign lung tumors. In some embodiments, the extracellular vesicles are shed from or derived from tissue of a subject with symptoms associated with lung cancer (e.g., non-specific symptoms).

[0129] Extracellular vesicle-associated membrane-bound polypeptide: As used herein, this term refers to a polypeptide present in the membrane of an extracellular vesicle. In some embodiments, such a biomarker may be associated with the extracellular side of the membrane. In some embodiments, such a polypeptide may be tumor-specific. In some embodiments, such a polypeptide may be tissue-specific (e.g., lung tissue-specific). In some embodiments, such a polypeptide may be non-specific, e.g., it is present in one or more non-target tumors and / or one or more non-target tissues.

[0130] Hybridization: As used herein, the terms "hybridizing," "hybridizing," "hybridization," "annealing," or "annealing" are used interchangeably to refer to the pairing of complementary nucleic acids using any process in which one strand of nucleic acid is joined to a complementary strand by base pairing to form a hybridized complex. Hybridization and the strength of hybridization (e.g., the strength of the association between nucleic acids) are affected by various factors, including, for example, the degree of complementarity between the nucleic acids, the stringency of the conditions involved, the melting temperature (T) of the hybridization complex formed, and the G:C ratio within the nucleic acids.

[0131] Intravesicular protein biomarker: As used herein, the term "intravesicular protein biomarker" refers to a marker that indicates the status (e.g., presence, level, and / or activity) of a polypeptide present within a biological entity (e.g., a cell or extracellular vesicle). In many embodiments, the intravesicular biomarker is associated with or present within an extracellular vesicle. In some embodiments, the intravesicular protein biomarker may be or comprise a phosphorylated polypeptide. In some embodiments, the intravesicular protein biomarker may be or comprise a mutant polypeptide.

[0132] Intravesicular RNA biomarker: As used herein, the term "intravesicular RNA biomarker" refers to a marker that indicates the status (e.g., presence and / or level) of RNA (e.g., but not limited to, mRNA, and non-coding RNA such as, for example, orphan non-coding RNA, long non-coding RNA, piwi-interacting RNA, microRNA, circular RNA, etc.) present within a biological entity (e.g., a cell or extracellular vesicle). In many embodiments, the intravesicular RNA biomarker is associated with or present within an extracellular vesicle. In some embodiments, the intravesicular RNA biomarker is associated with or specific to cancer. In some embodiments, the intravesicular RNA biomarker is or comprises an mRNA transcript. In some embodiments, the intravesicular RNA biomarker is or comprises a non-coding RNA. Exemplary non-coding RNAs may include, but are not limited to, small nuclear RNAs, microRNAs (miRNAs), small nucleolar RNAs (snoRNAs), circular RNAs (circRNAs), long non-coding RNAs (lncRNAs), small non-coding RNAs, piwi-interacting RNAs, etc. Certain RNA biomarkers for cancer are described in the prior art, for example, Xi et al. "RNA Biomarkers: Frontier of Precision Medicine for Cancer" Noncoding RNA (2017) 3:9, the contents of which are incorporated herein by reference for purposes described herein. In some embodiments, the intravesicular RNA biomarker is or includes an orphan noncoding RNA (oncRNA).Certain cancer-specific oncRNAs have been identified and described in references, for example, Teng et al. "Orphan noncoding RNAs: novel regulators and cancer biomarkers" Ann Transl Med (2019) 7:S21; Fish et al. "Cancer cells exploit an orphan RNA to drive metastatic progression" Nature Medicine (2018) 24: 1743-1751; and International Patent Publication No. WO2019 / 094780, each of which is incorporated herein by reference for purposes described herein. In some embodiments, the intravesicular RNA biomarker is or includes a long noncoding RNA. Certain non-coding RNA biomarkers for cancer have been described in the art, for example, as described in Qian et al. "Long Non-coding RNAs in Cancer: Implications for Diagnosis, Prognosis, and Therapy" Front. Med. (2020) Volume 7, Article 612393, the contents of which are incorporated herein by reference for purposes described herein. In some embodiments, the intravesicular RNA biomarker is or comprises a piwiRNA. In some embodiments, the intravesicular RNA biomarker is or comprises a miRNA. In some embodiments, the intravesicular RNA biomarker is or comprises a snoRNA. In some embodiments, the intravesicular RNA biomarker is or comprises a circRNA.

[0133] Ligase: As used herein, the term "ligase" or "nucleic acid ligase" refers to an enzyme for use in ligating nucleic acids. In some embodiments, the ligase is an enzyme for use in ligating the 3' end of a polynucleotide to the 5' end of a polynucleotide. In some embodiments, the ligase is an enzyme for use in performing sticky end ligation. In some embodiments, the ligase is an enzyme for use in performing blunt end ligation. In some embodiments, the ligase is or comprises a DNA ligase.

[0134] Life history-related risk factors: As used herein, the term "life history risk factors" refers to the activities, experiences, medical history, and / or exposures of an individual in their life that can directly or indirectly increase the individual's risk of a condition, such as lung cancer, compared to individuals who do not have such activities, experiences, medical history, and / or exposures in their lives. In some embodiments, non-limiting examples of life history-related risk factors include smoking, alcohol, drugs, carcinogens, diet, obesity, diabetes, chronic obstructive pulmonary disease (COPD), physical activity, sun exposure, radiation exposure, bitumen smoke exposure, exposure to infectious agents such as viruses and bacteria, and / or occupational hazards (see, for example, Jyoti Malhotra et al., "Risk Factors for Lung Cancer Worldwide" European Respiratory Journal (2016) 48: 889-902; which is incorporated herein by reference for purposes described herein). Those skilled in the art will recognize that the above list of lifestyle-related risk factors that contribute to cancer (eg, lung cancer) susceptibility is not exhaustive and is constantly evolving.

[0135] Ligation: As used herein, the terms "ligate," "ligating," or "ligation" refer to methods or compositions known in the art for joining two oligonucleotides or polynucleotides. Ligation may be or include sticky end ligation or blunt end ligation. In some embodiments, the ligation involved in the provided techniques is or includes sticky end ligation. In some embodiments, ligation refers to joining the 3' end of a polynucleotide to the 5' end of a polynucleotide. In some embodiments, ligation is facilitated by the use of a nucleic acid ligase.

[0136] Nanoparticles: The term "nanoparticles," when used in the context of a sample for a detection assay (e.g., as described herein), refers to particles having a size ranging from about 30 nm to about 1000 nm. In some embodiments, nanoparticles have a size ranging from about 30 nm to about 750 nm. In some embodiments, nanoparticles have a size ranging from about 50 nm to about 750 nm. In some embodiments, nanoparticles have a size ranging from about 30 nm to about 500 nm. In some embodiments, nanoparticles have a size ranging from about 50 nm to about 500 nm. In some embodiments, nanoparticles are obtained from a subject's bodily fluid sample, for example, in some embodiments, by a size-exclusion-based method (e.g., in some embodiments, size-exclusion chromatography). In some embodiments, nanoparticles are or comprise analyte aggregates, which in some embodiments may be or comprise protein or mucin aggregates. In some embodiments, nanoparticles are or comprise protein multimers. In some embodiments, nanoparticles are or comprise extracellular vesicles.

[0137] Non-cancer subject: As used herein, the term "non-cancer subject" generally refers to a subject that does not have a non-benign lung cancer. For example, in some embodiments, the non-cancer subject is a healthy subject. In some embodiments, the non-cancer subject is a healthy subject under the age of 55. In some embodiments, the non-cancer subject is a healthy subject aged 55 or older. In some embodiments, the non-cancer subject is a subject with a non-lung-related health disease, disorder, or condition. In some embodiments, the non-cancer subject is a subject with a benign lung tumor (e.g., a benign mass observed in the chest or lung cavity).

[0138] Nucleic Acid / Oligonucleotide: As used herein, the term "nucleic acid" refers to a polymer of at least 10 or more nucleotides. In some embodiments, a nucleic acid is or comprises DNA. In some embodiments, a nucleic acid is or comprises RNA. In some embodiments, a nucleic acid is or comprises peptide nucleic acid (PNA). In some embodiments, a nucleic acid is or comprises a single-stranded nucleic acid. In some embodiments, a nucleic acid is or comprises a double-stranded nucleic acid. In some embodiments, a nucleic acid comprises both single-stranded and double-stranded portions. In some embodiments, a nucleic acid comprises a backbone comprising one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone comprising both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone comprising one or more phosphorothioate or 5'-N-phosphoramidite linkages and / or one or more peptide bonds, e.g., as in "peptide nucleic acids." In some embodiments, a nucleic acid comprises one or more, or all, naturally occurring residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises one or more, or all, non-naturally occurring residues. In some embodiments, the non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalating bases, and combinations thereof).In some embodiments, the non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those in the natural residue. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product, such as an RNA or a polypeptide. In some embodiments, the nucleic acid has a nucleotide sequence that includes one or more introns. In some embodiments, the nucleic acid is isolated from a natural source, enzymatically synthesized (e.g., in vivo or in vivo). It may be prepared in vitro, for example, by polymerization based on a complementary template, reproduction in a recombinant cell or system, or chemical synthesis. In some embodiments, the nucleic acid is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 20 In some embodiments, the amino acid sequence may be 00, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides in length.

[0139] Nucleotide: As used herein, the term "nucleotide" refers to its art-recognized meaning. When the number of nucleotides is used, for example, as an indicator of the size of an oligonucleotide, a particular number of nucleotides refers, for example, to the number of nucleotides on a single strand of the oligonucleotide.

[0140] Patient: As used herein, the term "patient" refers to any organism suffering from or at risk of a disease or disorder or condition. Typical patients include animals (e.g., mammals, such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is human. In some embodiments, the patient is suffering from or susceptible to one or more diseases or disorders or conditions. In some embodiments, the patient exhibits one or more symptoms of a disease or disorder or condition. In some embodiments, the patient has been diagnosed with one or more diseases or disorders or conditions. In some embodiments, the disease or disorder or condition amenable to the provided techniques is or includes cancer, or the presence of one or more tumors. In some embodiments, the patient is undergoing or has undergone a particular therapy for diagnosing and / or treating the disease, disorder, or condition.

[0141] Polypeptide: As used herein, the term "polypeptide" typically has its art-recognized meaning of a polymer of at least three or more amino acids. Those skilled in the art will recognize that the term "polypeptide" is intended to be sufficiently general to encompass not only polypeptides having the complete sequences recited herein, but also polypeptides that represent functional, biologically active, or characteristic fragments, portions, or domains of such complete polypeptides (e.g., fragments, portions, or domains that retain at least one activity). In some embodiments, polypeptides may comprise L-amino acids, D-amino acids, or both, and / or may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, glycosylation, lipidation, methylation, and the like. In some embodiments, polypeptides may comprise (e.g., be or include) natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof.

[0142] Prevent or Prevention: As used herein, "prevent" or "prevention," when used in reference to the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of the disease, disorder, and / or condition occurring and / or delaying the onset of one or more characteristics or symptoms of the disease, disorder, or condition. Prevention may be considered complete if the onset of the disease, disorder, or condition has been delayed for a pre-specified period of time.

[0143] Primer: As used herein, the term "primer" refers to an oligonucleotide that can act as a point of initiation of synthesis when placed under conditions that induce the synthesis of a primer extension product that is complementary to a nucleic acid strand (e.g., in the presence of nucleotides and an inducing agent, such as DNA polymerase, and at an appropriate temperature and pH). The primer is preferably single-stranded for maximum efficiency in amplification. The primer must be sufficiently long to prime the synthesis of an extension product in the presence of an inducing agent. The exact length of the primer can depend on many factors, such as the desired annealing temperature.

[0144] Reference: As used herein, "reference" describes a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and / or determined substantially simultaneously with the test or determination of interest. In some embodiments, the reference or control is, optionally, a historical reference or control embodied in a tangible medium. In some embodiments, a reference or control in the context of a target reference level refers to the level of the target in a normal, healthy subject or a population of normal, healthy subjects. In some embodiments, a reference or control in the context of a target reference level refers to the level of the target in a subject before treatment. Typically, as will be understood by one of skill in the art, a reference or control is determined or characterized under conditions or circumstances comparable to those being assessed. In some embodiments, cell line-derived extracellular vesicles are used as a reference or control. One of skill in the art will recognize when there is sufficient similarity to justify the reliability of and / or comparison to a particular possible reference or control.

[0145] Risk: As will be understood from the context, "risk" of a disease, disorder, and / or condition refers to the likelihood that a particular individual will develop the disease, disorder, and / or condition. In some embodiments, risk is expressed as a percentage. In some embodiments, risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, up to 100%. In some embodiments, risk is expressed as a risk relative to the risk associated with a reference sample or group of reference samples. In some embodiments, the reference sample or group of reference samples has a known risk of the disease, disorder, condition, and / or event. In some embodiments, the reference sample or group of reference samples is from an individual comparable to the particular individual. In some embodiments, the relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or higher.

[0146] Sample: As used herein, the term "sample" typically refers to an aliquot of material obtained or derived from a source of interest. In some embodiments, a sample is obtained from or derived from a biological source of interest (e.g., a tissue or organism or cell culture). In some embodiments, the source of interest may be or include a cell or organism, e.g., an animal or human. In some embodiments, the source of interest is or includes a biological tissue or biological fluid. In some embodiments, the biological tissue or fluid may be or include amniotic fluid, aqueous humor, peritoneal fluid, bile, bone marrow, blood, breast milk, cerebrospinal fluid, earwax, chyle, chime, ejaculate, endolymph, exudate, stool, gastric acid, gastric juice, lymph, mucus, pericardial fluid, perilymph, peritoneal fluid, pleural effusion, pus, mucosal secretions, saliva, sebum, semen, serum, smegma, sputum, synovial fluid, sweat, tears, urine, vaginal secretions, vitreous humor, vomit, and / or combinations or components thereof. In some embodiments, the biological fluid may be or include intracellular fluid, extracellular fluid, intravesicular fluid (plasma), interstitial fluid, lymph, and / or transcellular fluid. In some embodiments, the biological tissue or biological sample may be obtained by, for example, an aspirate, biopsy (e.g., fine needle or tissue biopsy), swab (e.g., oral, nasal, skin, or vaginal swab), scraping, surgery, lavage, or irrigation (e.g., bronchoalveolar, ductal, nasal, ocular, oral, uterine, vaginal, or other lavage or irrigation). In some embodiments, the biological sample is or comprises a bodily fluid sample or a bodily fluid-derived sample. Examples of bodily fluid samples or bodily fluid-derived samples include, but are not limited to, amniotic fluid, bile, blood, breast milk, bronchoalveolar lavage fluid (BAL), cerebrospinal fluid, dialysate, stool, saliva, semen, synovial fluid, tears, urine, and the like. In some embodiments, a bodily fluid sample or a bodily fluid-derived sample that may be useful in accordance with the present disclosure is or comprises a blood-derived sample, a saliva-derived sample, a sputum-derived sample, or a pleural effusion-derived sample. In some embodiments, the biological sample is or comprises a liquid biopsy. In some embodiments, the biological sample is or comprises cells obtained from an individual.In some embodiments, a sample is a "primary sample" obtained directly from a source of interest by any suitable means. In some embodiments, as expected from the context, the term "sample" refers to a preparation obtained by processing a primary sample (e.g., by removing one or more components and / or adding one or more agents). For example, a sample is a preparation that has been processed by using a semipermeable membrane or an affinity-based method, such as an antibody-based method, to separate a biological entity of interest from other non-target entities. Such a "processed sample" may, for example, in some embodiments contain nanoparticles (e.g., nanoparticles having a size range of interest, including extracellular vesicles), but may also contain nucleic acids and / or proteins extracted from the sample. In some embodiments, a processed sample may be obtained by subjecting a primary sample to one or more techniques, such as nucleic acid amplification or reverse transcription, isolation and / or purification of certain components, etc.

[0147] Selective or specific: The terms "selective" or "specific," as used herein with respect to an active agent, are understood by those skilled in the art to mean that the agent discriminates between potential target entities, conditions, or cells. For example, in some embodiments, an agent is said to "specifically" bind to a target if it preferentially binds to that target in the presence of one or more competing surrogate targets. In many embodiments, the specific interaction depends on the presence of a particular structural feature of the target entity (e.g., an epitope, cleft, binding site). It should be understood that specificity need not be absolute. In some embodiments, specificity may be assessed relative to the specificity of the target-binding moiety for one or more other potential target entities (e.g., competitors). In some embodiments, specificity is assessed relative to the specificity of a reference specific binding moiety. In some embodiments, specificity is assessed relative to the specificity of a reference nonspecific binding moiety. In some embodiments, the target-binding moiety does not detectably bind to a competing surrogate target under conditions that bind to the target entity. In some embodiments, the target binding moiety binds to its target entity with a higher on-rate, a lower off-rate, increased affinity, decreased dissociation, and / or increased stability compared to a competing surrogate target.

[0148] Small molecule: As used herein, the term "small molecule" refers to a low molecular weight organic and / or inorganic compound. Generally, a "small molecule" is a molecule that is less than about 5 kilodaltons (kD) in size. In some embodiments, a small molecule is less than about 4 kD, 3 kD, 2 kD, or 1 kD. In some embodiments, a small molecule is less than about 800 daltons (D), 600 D, 500 D, 400 D, 300 D, 200 D, or 100 D. In some embodiments, a small molecule is less than about 2000 g / mol, less than about 1500 g / mol, less than about 1000 g / mol, less than about 800 g / mol, or less than about 500 g / mol. In some embodiments, a small molecule is not a polymer. In some embodiments, a small molecule does not comprise a polymer moiety. In some embodiments, a small molecule is not a protein or polypeptide (e.g., not an oligopeptide or peptide). In some embodiments, a small molecule is not a polynucleotide (e.g., not an oligonucleotide). In some embodiments, a small molecule is not a polysaccharide. In some embodiments, a small molecule does not comprise a polysaccharide (e.g., not a glycoprotein, proteoglycan, glycolipid, etc.). In some embodiments, a small molecule is not a lipid. In some embodiments, a small molecule is biologically active. In some embodiments, suitable small molecules are selected from a wide variety of methods, including screening of large libraries of compounds (Beck-Sickinger & Weber (2001) Combinational Strategies in Biology and Chemistry (John Wiley & Sons, Chichester, Sussex); structure-activity relationships by nuclear magnetic resonance (Shuker et al. (1996) "Discovering high-affinity ligands for proteins: SAR by NMR." Science 274: 1531-1534); encoded self-assembling chemical libraries (Melkko et al. (2004) "Encoded self-assembling chemical libraries." Nature Biotechnol. 22: 568-574); DNA-templated chemistry (Gartner et al.(2004) "DNA-templated organic synthesis and selection of a library of macrocycles." Science 305: 1601-1605); dynamic combinatorial chemistry (Ramstrom & Lehn (2002) "Drug discovery by dynamic combinatorial libraries." Nature Rev. Drug Discov. 1: 26-36); tethering (Arkin & Wells (2004) "Small-molecule inhibitors of protein-protein interactions: progressing towards the dream." Nature Rev. Drug Discov. 3: 301-317); and speed screening (Muckenschnabel et al. (2004) "SpeedScreen: label-free liquid chromatography-mass spectrometry-based high-throughput screening for the discovery of orphan protein ligands." Anal. Biochem. 324: 241-249). In some embodiments, the small molecule may have a dissociation constant for the target in the nanomolar range. .

[0149] Specific binding: As used herein, the term "specific binding" refers to the ability to distinguish between possible binding partners in an environment in which binding occurs. A target-binding moiety that interacts with one specific target in the presence of other potential targets is said to "specifically bind" to the target with which it interacts. In some embodiments, specific binding is assessed by detecting or determining the extent of association between the target-binding moiety and its partner; in some embodiments, specific binding is assessed by detecting or determining the extent of dissociation of the target-binding moiety-partner complex; and in some embodiments, specific binding is assessed by detecting or determining the ability of the target-binding moiety to compete with an alternative interaction between its partner and another entity. In some embodiments, specific binding is assessed by performing such detection or determination over a range of concentrations.

[0150] Cancer stage: As used herein, the term "cancer stage" refers to a qualitative or quantitative assessment of the level of progression of a cancer (e.g., lung cancer). In some embodiments, the criteria used to determine the stage of a cancer may include, but are not limited to, one or more of the following: where the cancer is located in the body; tumor size; whether the cancer has spread to lymph nodes; whether the cancer has spread to one or more different body parts; and the like. In some embodiments, cancer may be staged using the AJCC staging system. The AJCC staging system is a classification system developed by the American Joint Committee on Cancer to describe the degree of disease progression in cancer patients, which utilizes, in part, the TNM scoring system: tumor size, affected lymph nodes, and metastases. In some embodiments, cancer may be staged using a classification system that includes, in part, the TNM scoring system, where T refers to the size and extent of the main tumor, usually called the primary tumor; N refers to the number of nearby lymph nodes that have cancer; and M refers to whether the cancer has metastasized. In some embodiments, cancer may be referred to as Stage 0 (abnormal cells are present but have not spread to nearby tissues, also called carcinoma in situ or CIS; CIS is not cancer but may become cancer), Stage I-III (cancer is present; the higher the number, the larger the tumor and the more it has spread to nearby tissues), or Stage IV (cancer has spread to distant parts of the body). In some embodiments, cancer may be assigned a stage selected from the group consisting of in situ (abnormal cells are present but have not spread to nearby tissues); localized (cancer is limited to where it started and there are no signs of spread); regional (cancer has spread to nearby lymph nodes, tissues, or organs); distant metastasis (cancer has spread to distant parts of the body); and unknown (there is not enough information to determine the stage).

[0151] Subject: As used herein, the term "subject" refers to an organism from which a sample is obtained, e.g., for experimental, diagnostic, preventive, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals, e.g., mice, rats, rabbits, non-human primates, domestic pets, etc.) and humans. In some embodiments, the subject is a human subject, e.g., a male or female human subject. In some embodiments, the subject is afflicted with lung cancer. In some embodiments, the subject is predisposed to lung cancer. In some embodiments, the subject exhibits one or more symptoms or characteristics of lung cancer. In some embodiments, the subject exhibits one or more non-specific symptoms of lung cancer. In some embodiments, the subject does not exhibit any symptoms or characteristics of lung cancer. In some embodiments, the subject is a subject with one or more features characteristic of susceptibility to or risk of lung cancer. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom and / or to whom a diagnostic and / or therapy will be administered. In some embodiments, the subject is a subject (e.g., a male or female subject) determined to have a chest or lung mass. In some embodiments, the subject is an asymptomatic subject. Such a symptomatic subject may be a subject at average population risk (e.g., a male or female subject) with a life history-related or genetic risk. For example, such an asymptomatic subject may be a subject with a family history of cancer, a subject previously treated for cancer, a subject at risk of cancer recurrence after cancer treatment, a subject in remission after cancer treatment, and / or a subject previously or periodically screened for the presence of at least one cancer biomarker. Alternatively, in some embodiments, an asymptomatic subject may be a subject who has not previously been screened for cancer, a subject who has not been diagnosed with cancer, and / or a subject who has not previously undergone cancer therapy. In some embodiments, subjects suitable for the provided technology are individuals selected based on one or more characteristics, such as age, race, geographic location, genetic history, medical history, personal history (e.g., smoking, alcohol, drugs, carcinogens, diet, obesity, physical activity, sun exposure, radiation exposure, exposure to infectious agents such as viruses, and / or occupational hazards).

[0152] Suffering from: An individual "suffering from" a disease, disorder, and / or condition has been diagnosed with and / or exhibits one or more symptoms of the disease, disorder, and / or condition.

[0153] Surface analyte: As used herein, "surface analyte" refers to an analyte present on the surface of a biological entity (e.g., a cell or nanoparticle from a biological sample). In some embodiments, the surface analyte is or comprises a surface polypeptide or surface protein. In some embodiments, the surface analyte is or comprises a glycan.

[0154] Surface biomarker: As used herein, "surface biomarker" refers to a marker that indicates the status (e.g., presence, level, and / or activity) of a surface analyte (e.g., as described herein) of a biological entity (e.g., a cell or nanoparticle, which in some embodiments includes analyte aggregates (e.g., protein or mucin aggregates) and / or extracellular vesicles). In some embodiments, the surface biomarker is or comprises a surface protein biomarker. In some embodiments, the surface biomarker is or comprises a carbohydrate-dependent marker.

[0155] Surface polypeptide or surface protein: As used interchangeably herein, the terms "surface polypeptide" and "surface protein" refer to a polypeptide or protein that is present in and / or on the surface of a biological entity (e.g., a cell or nanoparticle, including, in some embodiments, analyte aggregates (e.g., protein or mucin aggregates) and / or extracellular vesicles, etc.) through direct or indirect interactions. As will be understood by one of skill in the art, a surface protein may, in some embodiments, include post-translational modifications, including, for example, but not limited to, glycosylation. In some embodiments, a surface polypeptide or protein may be or include a membrane-bound polypeptide. In some embodiments, a membrane-bound polypeptide refers to a polypeptide or protein having one or more domains or regions that are present in or on the surface of the membrane of a biological entity (e.g., a cell, an extracellular vesicle, etc.). In some embodiments, a membrane-bound polypeptide may include one or more domains or regions that span and / or are associated with the cellular membrane of a biological entity (e.g., a cell, an extracellular vesicle, etc.). In some embodiments, a binding polypeptide may comprise one or more domains or regions that span and / or are associated with the cellular membrane of a biological entity (e.g., a cell, an extracellular vesicle, etc.) and project into the intracellular and / or intravesicular space. In some embodiments, a membrane-associated polypeptide may comprise one or more domains or regions associated with the cellular membrane of a biological entity (e.g., a cell, an extracellular vesicle, etc.) via, for example, one or more non-peptide linkages (e.g., by a glycosylphosphatidylinositol (GPI) anchor or lipidation, or by non-covalent interactions). In some embodiments, a membrane-associated polypeptide may comprise one or more domains or regions anchored on either side of the cellular membrane of a biological entity (e.g., a cell, an extracellular vesicle, etc.). In some embodiments, the surface protein is associated with or present on the surface of a nanoparticle (e.g., as described herein).In some embodiments, the surface protein is associated with or present within an extracellular vesicle. In some embodiments, the surface protein may be associated with or present within a lung cancer-associated extracellular vesicle (e.g., an extracellular vesicle obtained from or derived from a body fluid-derived sample (e.g., a blood-derived sample) of a subject suffering from or susceptible to lung cancer). As will be understood by one skilled in the art, detecting the presence of at least a portion of a surface polypeptide or surface protein on / in an extracellular vesicle can facilitate separation and / or isolation of lung cancer-associated extracellular vesicles from a biological sample (e.g., in some embodiments, a body fluid-derived sample, such as, but not limited to, a blood-derived sample) from a subject (e.g., blood or a blood-derived sample). In some embodiments, detecting the presence of a surface polypeptide or surface protein may be or may include detection of an intravesicular portion (e.g., an intravesicular epitope) of such a surface polypeptide or surface protein. In some embodiments, detecting the presence of a surface polypeptide or surface protein may be or may include detection of a membrane-spanning portion of such a surface polypeptide or surface protein. In some embodiments, detecting the presence of a surface polypeptide or surface protein may be or may include detecting the extravesicular portion of such a surface polypeptide or surface protein.

[0156] Surface protein biomarker: As used herein, the term "surface protein biomarker" refers to a marker that indicates the status (e.g., presence, level, and / or activity) of a surface protein (e.g., as described herein) of a biological entity (e.g., a cell or nanoparticle, which in some embodiments comprises an analyte aggregate (e.g., a protein or mucin aggregate) and / or an extracellular vesicle). In some embodiments, a surface protein refers to a polypeptide or protein having one or more domains or regions located in or on the surface of the membrane of a biological entity (e.g., a cell or extracellular vesicle). In some embodiments, a surface protein biomarker may be or include an epitope present on the inside (intravesicular) or outside (extravesicular) of a membrane. In some embodiments, a surface protein biomarker is associated with or present within an extracellular vesicle. In some embodiments, a surface protein biomarker may be or include a mutated polypeptide. In some embodiments, a surface protein biomarker may be post-translationally modified (e.g., without limitation, glycosylation, phosphorylation, etc.). In some embodiments, the surface protein biomarker may be post-translationally processed and may exist in the form of a truncated polypeptide, for example, as a result of proteolytic cleavage. In some embodiments, the surface protein biomarker may be or include an epitope present on the exterior surface of the nanoparticle.

[0157] Susceptible to: An individual who is "susceptible to" a disease, disorder, and / or condition is an individual who has a higher risk of developing the disease, disorder, and / or condition than a member of the general population. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not have been diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition in the future. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition in the future.

[0158] Target-binding moiety: In general, the terms "target-binding moiety" and "binding moiety" are used interchangeably herein to refer to any entity or moiety that binds to a target of interest (e.g., a molecular target of interest, e.g., a biomarker or epitope). In many embodiments, a target-binding moiety of interest is one that specifically binds to its target (e.g., a target biomarker) in the context of a particular interaction, such that it distinguishes that target from other potential binding partners. In general, a target-binding moiety may be or include an entity or moiety of any chemical class (e.g., polymeric, non-polymeric, small molecule, polypeptide, carbohydrate, lipid, nucleic acid, etc.). In some embodiments, a target-binding moiety is a single chemical entity. In some embodiments, a target-binding moiety is a complex of two or more distinct chemical entities associated with each other under relevant conditions by non-covalent interactions. For example, one skilled in the art will recognize that in some embodiments, a target-binding moiety can include a "general" binding moiety (e.g., one of biotin / avidin / streptavidin and / or a class-specific antibody) and a "specific" binding moiety (e.g., an antibody or aptamer to a particular molecular target) linked to a generic binding moiety partner. In some embodiments, such an approach can allow for modular assembly of multiple target-binding moieties by linking different specific binding moieties to the generic binding moiety partner.

[0159] Target biomarker signature: The term "target biomarker signature," as used herein, refers to a combination of biomarkers (e.g., at least two or more, including at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, or more) that correlates with a particular biological event or condition of interest, such that one of skill in the art will recognize that this may appropriately be considered a "signature" of that event or condition. To give a few examples, in some embodiments, a target biomarker signature may correlate with a particular disease or disease state and / or the likelihood that a particular disease, disorder, or condition will develop, occur, or recur. In some embodiments, a target biomarker signature may correlate with a particular disease or treatment outcome, or likelihood thereof. In some embodiments, the target biomarker signature may be correlated with a specific cancer and / or its stage. In some embodiments, the target biomarker signature may be correlated with lung cancer and / or its stage and / or subtype. In some embodiments, the target biomarker signature includes a combination of biomarkers (e.g., at least two or more, including at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, or more) that are together specific for lung cancer, or a subtype and / or disease state thereof, although one or more biomarkers in such a combination may be directed to a target (e.g., a surface biomarker, an intravesicular biomarker, and / or an intravesicular RNA) that is not specific to lung cancer.For example, in some embodiments, a target biomarker signature may include at least one biomarker specific to lung cancer, or a stage and / or subtype thereof (i.e., a lung cancer-specific target), and may further include biomarkers that are not necessarily or completely specific to lung cancer (e.g., that may also be found in some or all biological entities, such as cells, nanoparticles, etc., that are not cancerous, are not of related cancers, and / or are not of the particular stage and / or subtype of interest). That is, as will be recognized by those of skill in the art upon reading this specification, so long as the combination of biomarkers utilized in a target biomarker signature is or includes a number of biomarkers that together are specific for (i.e., sufficiently distinguish) the relevant target biological entity for detection (e.g., lung cancer cells of interest, or nanoparticles secreted by lung cancer cells, e.g., lung cancer-specific extracellular vesicles) of interest, from other biological entities that are not of interest for detection, such a combination of biomarkers will be a useful target biomarker signature in accordance with certain embodiments of the present disclosure.

[0160] Therapeutic Agent: As used interchangeably herein, the phrases "therapeutic agent" or "therapy" refer to an agent or intervention that, when administered to a subject or patient, has a therapeutic effect and / or induces a desired biological and / or pharmacological effect. In some embodiments, a therapeutic agent or therapy is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or features of a disease, disorder, and / or condition. In some embodiments, a therapeutic agent or therapy is a medical intervention (e.g., surgery, radiation, phototherapy) that can be performed to alleviate, alleviate, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or features of a disease, disorder, and / or condition.

[0161] Threshold level (e.g., cutoff): As used herein, the term "threshold level" refers to a level used as a reference for obtaining information about and / or classifying the results of a measurement, e.g., a measurement obtained in an assay. For example, in some embodiments, the threshold level (e.g., cutoff) refers to a value measured in an assay that defines the boundary between two subsets of a population (e.g., normal and / or non-lung cancer vs. lung cancer). Thus, a value equal to or higher than the threshold level defines one subset of the population, and a value lower than the threshold level defines the other subset of the population. The threshold level can be determined based on one or more control samples, or the entire population of control samples. The threshold level can be determined before, simultaneously with, or after obtaining the measurement of interest. In some embodiments, the threshold level can be a range of values.

[0162] Treat: As used herein, the terms "treat," "treatment," or "treating" refer to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or features of a disease, disorder, and / or condition. Treatment may be administered to a subject who does not show signs of a disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who shows only early signs of a disease, disorder, and / or condition, for example, to reduce the risk of developing pathologies associated with the disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject at a later stage of a disease, disorder, and / or condition.

[0163] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art, or as described herein. The techniques and procedures described above may generally be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), which is incorporated herein by reference for purposes described herein.

[0164] Detailed Description of Certain Embodiments Lung cancer caused an estimated 148,869 deaths in the United States in 2016 (US Cancer Statistics Working Group, 2019; which is incorporated herein by reference for purposes described herein). The majority of these deaths are due to delayed diagnosis; from 2001 to 2007, the 5-year overall survival rate for lung cancer in the United States was 15.6%. Patients with localized disease at the time of diagnosis had a 52% 5-year survival rate; however, the majority of patients were initially diagnosed when distant metastases had already formed, and these patients had a dismal 5-year survival rate of approximately 3.6% (Cruz et al., 2011; which is incorporated herein by reference for purposes described herein).

[0165] Unfortunately, there are no inexpensive, widely available, recommended lung cancer screening tests for average-risk individuals. Many individuals with genetic or lifestyle-related risk are currently screened with low-dose CT scanning, but these tests are suboptimal for screening because they are relatively expensive and have limited access. For example, the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Randomized Trial found that relatively cost-effective, widely available chest x-ray imaging and sputum testing were ineffective in altering lung cancer mortality, whereas low-dose CT scanning successfully reduced mortality, which in many cases may be considered costly and of limited availability, increasing the number of unnecessary surgeries.

[0166] The present disclosure identifies the origin of certain prior technology challenges, including, among other things, certain conventional approaches to lung cancer detection and diagnosis. For example, the present disclosure recognizes that many conventional diagnostic assays, such as X-ray imaging, sputum testing, low-dose CT scanning, and / or molecular tests based on cell-free nucleic acids, serum proteins (e.g., CEA, CYFRA 21-1, NSE, ProGRP, and / or SCCA), and / or bulk analysis of extracellular vesicles, can be time-consuming, expensive, and / or lack sufficient sensitivity and / or specificity to provide a reliable, comprehensive diagnostic assessment. In some embodiments, the present disclosure provides technologies (including systems, compositions, and methods) that address such challenges by, among other things, identifying combinations of biomarkers that are predicted to exhibit high sensitivity and specificity for lung cancer based on bioinformatics analysis. In some embodiments, the present disclosure provides technologies (including systems, compositions, and methods) that solve this problem by detecting the co-localization (e.g., identified by bioinformatics analysis) of a target biomarker signature for lung cancer in individual nanoparticles having a desired size range, including extracellular vesicles, including at least one extracellular vesicle-associated surface biomarker and at least one target biomarker selected from the group consisting of a surface biomarker, an internal biomarker, and an RNA biomarker present on the nanoparticle associated with lung cancer. In some embodiments, the present disclosure provides technologies (including systems, compositions, and methods) that solve this problem by detecting such a target biomarker signature for lung cancer using, among other things, a target entity detection approach developed by the applicant and described in U.S. Patent Application No. US2020 / 0299780 and International Application No. WO2020180741, which is based on the interaction and / or co-localization of the target biomarker signature in individual nanoparticles. The contents of each of the foregoing disclosures are incorporated herein by reference in their entirety.

[0167] In some embodiments, extracellular vesicles for detection as described herein can be isolated from a subject's bodily fluids by size-exclusion-based methods. As will be understood by those skilled in the art, in some embodiments, size-exclusion-based methods can provide samples containing nanoparticles of a desired size range, including extracellular vesicles. Thus, in some embodiments, the provided techniques of the present disclosure encompass the detection of colocalization of at least two or more surface biomarkers (e.g., as described herein) forming a target biomarker signature for lung cancer in individual nanoparticles of a desired size range (e.g., in some embodiments, about 30 nm to about 1000 nm), including extracellular vesicles. Those skilled in the art who read this disclosure will understand that various embodiments described herein in the context of "extracellular vesicles" (e.g., assays for detecting individual extracellular vesicles and / or the provided "extracellular vesicle-associated surface biomarkers") may also be applicable in the context of "nanoparticles" as described herein.

[0168] The present disclosure provides, among other things, insights and techniques for achieving effective lung cancer screening, for example, for the early detection of lung cancer. In some embodiments, the present disclosure provides techniques for the early detection of lung cancer in subjects who may be experiencing another symptom associated with lung cancer. In some embodiments, the present disclosure provides techniques for the early detection of lung cancer in subjects who are at genetic risk for lung cancer. In some embodiments, the present disclosure provides techniques for the early detection of lung cancer in subjects who may be at genetic risk for lung cancer and / or who may be experiencing one or more symptoms associated therewith. In some embodiments, the present disclosure provides techniques for the early detection of lung cancer in subjects who may have lifestyle history risk factors (e.g., but not limited to, smoking). In some embodiments, the present disclosure provides techniques for screening individuals, for example, individuals with a certain risk (e.g., genetic risk, lifestyle history-related risk, or average risk) for early-stage non-small cell lung cancer, such as lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). Non-small cell lung cancer is the most common subtype of lung cancer, with 54% of cases detected at an advanced stage (SEER Cancer Statistics Review 1975-2017). In some embodiments, the provided techniques are effective for detecting early-stage lung cancer. In some embodiments, the provided techniques are effective when applied to populations that include or consist of individuals with one or more symptoms that may be associated with lung cancer. In some embodiments, the provided techniques are effective even when applied to populations that include or consist of asymptomatic or symptomatic individuals (e.g., due to results of sufficiently high sensitivity and / or low false positive and / or false negative rates). In some embodiments, the provided techniques are effective when applied to populations that include or consist of individuals without a genetic and / or lifestyle-related risk of developing lung cancer (e.g., asymptomatic or symptomatic individuals).In some embodiments, the provided technology is effective when applied to populations that include or consist of individuals with a genetic and / or lifestyle-related risk of developing lung cancer (e.g., asymptomatic or symptomatic individuals). In some embodiments, the provided technology is effective when applied to populations that include or consist of individuals who are predisposed to lung cancer (e.g., individuals with a known genetic, environmental, or experiential risk, etc.). In some embodiments, the provided technology may be or include one or more compositions (e.g., molecular complexes, systems, collections, combinations, kits, etc.) and / or methods (e.g., methods of making, using, assessing, etc.), as will be apparent to one of skill in the art upon reading the disclosure provided herein.

[0169] In some embodiments, the provided techniques achieve detection (e.g., early detection, e.g., in asymptomatic individuals and / or populations) of one or more characteristics of lung cancer (e.g., incidence, progression, response to therapy, recurrence, etc.) with sensitivity and / or specificity (e.g., resulting false positive and / or false negative rates) appropriate to enable useful application of the provided techniques to single and / or regular (e.g., periodic) assessments. In some embodiments, the provided techniques are useful in conjunction with an individual's regular screening, including, but not limited to, physical exams, general practitioner visits, cholesterol / lipid blood tests, diabetes (type 2) screening, colonoscopies, blood pressure screening, thyroid function tests, prostate cancer screening, mammograms, HPV / Pap smears, and / or vaccinations. In some embodiments, the provided techniques are useful in conjunction with treatment regimens, and in some embodiments, the provided techniques may improve one or more characteristics (e.g., success rate by accepted parameters) of such treatment regimens.

[0170] In some embodiments, the present disclosure provides, among other things, insight that screening asymptomatic individuals, e.g., periodic screening before the onset of symptoms or otherwise in their absence, may be beneficial and even important for the effective management (e.g., successful treatment) of lung cancer. In some embodiments, the present disclosure provides lung cancer screening systems that can be implemented to detect lung cancer, including early stage cancers, in some embodiments, in asymptomatic individuals (e.g., without genetic and / or lifestyle-related risk for lung cancer). In some embodiments, the provided techniques are implemented to achieve periodic screening of asymptomatic individuals (e.g., with or without genetic and / or lifestyle-related risk for lung cancer). In some embodiments, the provided techniques are implemented to achieve periodic screening of symptomatic individuals (e.g., with or without genetic and / or lifestyle-related risk for lung cancer). The present disclosure provides compositions (e.g., reagents, kits, components, etc.), including, for example, strategies involving periodic testing of one or more individuals (e.g., asymptomatic individuals), as well as methods of providing and / or using them. The present disclosure defines the utility of such systems and provides compositions and methods for implementing them.

[0171] I. Lung cancer detection Although there is currently no CDC-recommended lung cancer screening test of any kind for screening average-risk asymptomatic individuals, the age-adjusted incidence rate of lung cancer in the United States is 62 per 100,000 men and women per year. The number of Americans who die from lung cancer each year is roughly equal to the combined deaths from prostate, breast, and colon cancers. In 2010 alone, there were an estimated 240,000 new cases of lung cancer and 161,000 deaths from lung cancer in the United States (Cruz et al., 2011; incorporated herein by reference for purposes described herein). While the total number of lung cancer deaths in the United States has been declining since approximately 1985, the global rate of lung cancer has been steadily increasing, increasing by approximately 51% from 1985 to 2010 (Cruz et al., 2011; incorporated herein by reference for purposes described herein). Globally, lung cancer is the leading cause of new cancer cases, with approximately 1,350,000 new cases of lung cancer (about 12.4% of all new cancer cases) and approximately 1,180,000 deaths (about 17.6% of total cancer-related deaths) worldwide in 2010. Approximately half of these new lung cancer cases occur in developing countries. The 5-year lung cancer survival rate in Europe, China, and developing countries is estimated to be only 8.9% (Cruz et al., 2011; which is incorporated herein by reference for purposes described herein).

[0172] The 2013–2017 Surveillance, Epidemiology, and End Results (SEER) data provide extensive information on the prevalence and epidemiology of lung cancer in the United States over the past 45 years. SEER reported a median age of approximately 71 years at diagnosis for lung and bronchial cancers. Lung cancer arises from cells of the respiratory epithelium and can be divided into two broad categories. Small cell lung cancer (SCLC) is highly malignant and originates from cells that exhibit neuroendocrine features. SCLC comprises approximately 12% of all lung cancer cases (Figure 3). Non-small cell lung cancer (NSCLC) accounts for the remaining 85% of cases and is divided into three pathological subtypes: adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. Adenocarcinoma itself accounts for approximately 50% of all new lung cancer cases in the United States (Figure 3), squamous cell carcinoma accounts for approximately 23%, and large cell carcinoma and other lung and bronchial cancers account for the remaining approximately 15% (Figure 3).

[0173] The 5-year survival rate for all patients with invasive non-small cell carcinoma of the lung and bronchus is approximately 25%. Patients with localized lung cancer at the time of initial diagnosis have a 5-year survival rate of approximately 63%, patients with regional lung cancer metastases at the time of initial diagnosis have a 5-year survival rate of approximately 35%, while patients with distant lung cancer metastases have a poor 5-year survival rate of approximately 7% (SEER 1975-2017 review, Table 15.12). These data indicate that early lung cancer diagnosis is important because it can increase the survival rate of lung cancer patients.

[0174] Certain risk factors for lung cancer include age and smoking history. The seminal 1964 report by the U.S. Surgeon General stated: (1) smoking was associated with a 70% increase in age-specific mortality in men and a smaller increase in mortality in women; (2) smoking was causally related to lung cancer in men, the magnitude of the effect far exceeding all other factors that contribute to lung cancer, and risk increased directly with smoking duration and number of cigarettes smoked per day; (3) smoking was considered more important than occupational exposure in causing lung cancer in the general population; (4) smoking was reported to be an important cause of chronic bronchitis in the United States; and (5) male smokers had a higher mortality rate from coronary artery disease than male nonsmokers.

[0175] The International Agency for Research on Cancer (IARC) has identified at least 50 known carcinogens in tobacco smoke. Examples of such carcinogens include, but are not limited to, tobacco-specific N-nitrosamines (TSNAs), which are formed by the nitrosation of nicotine during tobacco processing and smoking. The chemical 4-(methylnitrosamino)-1(3-pyridyl)-1-butanone (NNK) is known to induce lung adenocarcinoma in experimental animals. NNK is known to bind to DNA, create DNA adducts, and cause DNA damage. Failure to repair this damage can lead to permanent mutations. NNK is associated with DNA mutations that result in activation of the K-ras oncogene, which is detected in 24% of human lung adenocarcinomas.

[0176] It is estimated that one in nine smokers will eventually develop lung cancer. The relative risk of lung cancer in long-term smokers is estimated to be 10 to 30 times higher than in lifelong non-smokers. In the USA, over 80% of lung cancers occur in people with tobacco exposure, but globally, 15% of lung cancers in men and up to 53% of lung cancers in women are not attributable to smoking, and never-smokers account for approximately 25% of all lung cancer cases worldwide.

[0177] High-risk individuals and / or populations, as defined by the CDC, are 55-77 years old, have a cigarette history of >30 pack years, are current smokers, or have quit smoking within the past 15 years. For these individuals, low-dose CT scanning is currently the recommended lung cancer screening tool. However, low-dose CT for high-risk populations (e.g., patients defined by CDC guidelines) can be relatively expensive, have limited access, and be considered to have unreasonably high levels of false positives (e.g., the proportion of all positive tests that are false positives can be as high as 97.5%; Raghu et al., 2020 and Kinsinger et al., 2017, both of which are incorporated by reference for purposes described herein). The present disclosure provides, among other things, cost-effective screening assays with sufficiently high specificity and / or sensitivity.

[0178] Among other things, in certain embodiments, the present disclosure provides the insight that there is a need for the development of a lung cancer liquid biopsy assay for screening subjects who have a genetic and / or lifestyle-related risk for lung cancer and / or who may be experiencing one or more symptoms associated with lung cancer. In certain embodiments, the present disclosure provides the insight that there is a need for the development of a lung cancer liquid biopsy assay for screening symptomatic or asymptomatic subjects, for example, before other screening methods, for example, imaging methods for detecting lung cancer, such as MRI, CT scan, etc.

[0179] In some embodiments, the present disclosure provides techniques for the effective screening of lung cancer in individuals with genetic risk or lifestyle-related risk. In some embodiments, the present disclosure provides techniques for the effective screening of lung cancer in average-risk individuals. In some embodiments, the present disclosure provides techniques for the effective screening of lung cancer in individuals with one or more symptoms associated with lung cancer. In some embodiments, the present disclosure provides techniques for the effective screening of lung cancer in asymptomatic individuals. Despite being the biggest killer of all cancers in men and women, there are currently no recommended lung cancer screening tools that are non-invasive, use only bodily fluids (e.g., including but not limited to, blood), and are suitable for asymptomatic and / or average-risk individuals (e.g., individuals under 55 years of age, or individuals over 55 years of age who have never smoked or have quit smoking for more than 15 years). This is due, in part, to cost, limited availability, potential side effects, and / or the poor performance (e.g., high false positive rates, or ineffectiveness) of existing lung cancer screening technologies. Considering the incidence of lung cancer in average-risk individuals, inadequate test specificity (<99.5%) can result in false-positive results exceeding the number of true-positives by more than an order of magnitude. This places a significant burden on the healthcare system and on the individuals screened for false-positive results, resulting in additional testing, unnecessary surgery, and emotional / physical distress (Wu et al., 2016).

[0180] In some embodiments, the present disclosure provides the insight that a particularly useful lung cancer screening test would be characterized by (1) ultra-high specificity (>99.5%) that minimizes the number of false positives, and (2) high sensitivity (>40%) for stage I and II lung cancer (i.e., when the prognosis is most favorable).

[0181] For example, in some embodiments, a particularly useful lung cancer screening test may be characterized by a specificity of >98% and a sensitivity of >50%, e.g., for stage I and II lung cancer. In some embodiments, a particularly useful lung cancer screening test may be characterized by a specificity of >98% and a sensitivity of >60%, e.g., for stage I and II lung cancer. In some embodiments, a particularly useful lung cancer screening test may be characterized by a specificity of >98% and a sensitivity of >70%, e.g., for stage I and II lung cancer. In some embodiments, a particularly useful lung cancer screening test may be characterized by a specificity of >99.5% and a sensitivity of >65%, e.g., for stage I and II lung cancer. In some embodiments, a particularly useful lung cancer screening test may be characterized by a specificity of >99.5% and a sensitivity of >60%, e.g., for stage I and II lung cancer. In some embodiments, particularly useful lung cancer screening tests may be characterized by a specificity of 99% or higher and a sensitivity of >10% or higher (including, for example, >15%, >20%, >25%). In some embodiments, particularly useful lung cancer screening tests may be characterized by a specificity of 99% or higher and a sensitivity of 50% or higher.

[0182] In some embodiments, the present disclosure provides insight that lung cancer screening tests comprising two or more sets of biomarker combinations (e.g., a combination of at least two orthogonal biomarkers described herein) can increase the sensitivity of such assays compared to that achieved by a single set of biomarker combinations. For example, in some embodiments, lung cancer screening tests comprising a combination of at least two orthogonal biomarkers can achieve a specificity of at least 98% and a sensitivity of at least 50%. In some embodiments, lung cancer screening tests comprising a combination of at least two orthogonal biomarkers can achieve a specificity of at least 98% and a sensitivity of at least 60%. In some embodiments, lung cancer screening tests comprising a combination of at least two orthogonal biomarkers can achieve a specificity of 99% and a sensitivity of 50% or higher.

[0183] In some embodiments, the present disclosure provides insight that a particularly useful lung cancer screening test can be characterized by an acceptable positive predictive value (PPV) at an economically justifiable cost. PPV is the probability that a patient has the disease after a positive test and is affected by sensitivity, specificity, and / or disease prevalence. One clinical consensus on the minimum PPV required to screen for lung cancer is 10%. At a PPV of 10%, there will be 9 false positives for every 1 true positive (Lung Cancer Screening: Recommendation Statement., Am Fam Physician. 2005 Mar 15;71(6):1165-1168). These false positives place a significant burden on both the healthcare system and the screened subjects, as they result in additional testing, unnecessary surgery, and emotional and physical distress. In some embodiments, the assays described herein are particularly useful for early lung cancer detection, achieving a PPV of greater than 10% or higher, including, for example, greater than 15%, greater than 20%, or greater than 25%, or higher, at a specificity cutoff of at least 85%, including, for example, at least 90%, at least 95%, or higher (e.g., a specificity cutoff of at least 98% for subjects at genetic risk for lung cancer, or a specificity cutoff of at least 99.5% for subjects experiencing one or more symptoms associated with lung cancer).

[0184] In some embodiments, the assays described herein are particularly useful as primary screening tests for early lung cancer detection. In some embodiments, subjects who receive a positive test result from the assays described herein are recommended to undergo follow-up testing. In some such embodiments, the assays described herein can be useful for early lung cancer detection, achieving a PPV of more than 2% or higher, including, for example, more than 3%, more than 4%, more than 5%, more than 6%, more than 7%, more than 8%, more than 9%, more than 10%, more than 15%, more than 20%, or more than 25%, or higher. In some such embodiments, the assays described herein can achieve a specificity cutoff of at least 85% or higher, including, for example, at least 90%, at least 95%, or higher (e.g., a specificity cutoff of at least 98% for subjects with a genetic risk for lung cancer, or a specificity cutoff of at least 99.5% for subjects experiencing one or more symptoms associated with lung cancer).

[0185] Several different biomarker classes, including circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), bulk proteins, and extracellular vesicles (EVs), are being investigated for lung cancer liquid biopsy assays. EVs are particularly promising due to their abundance and stability in the bloodstream compared to ctDNA and CTCs, suggesting improved sensitivity for early-stage cancer. EVs also contain cargo (i.e., proteins, RNA, and metabolites) of the same cellular origin, providing superior specificity over bulk protein measurements. While the diagnostic utility of EVs has been investigated, much of this work has focused on bulk EV measurements or low-throughput single-EV analysis.

[0186] II. PROVIDED BIOMARKER AND / OR TARGET BIOMARKER SIGNATURES FOR THE DETECTION OF LUNG CANCER The present disclosure provides, among other things, various target biomarkers for lung cancer or combinations thereof (for example, target biomarker signatures). Such target biomarker signatures that are predicted to show high sensitivity and specificity for lung cancer are discovered through a wide range of bioinformatics analysis and biological approaches, for example, in some embodiments, machine learning and / or computer computational modeling, for example, in some embodiments, computer analysis of a diverse set of data, including one or more of sequencing data, expression data, mass spectrometry, histology, post-translational modification data, and / or in vitro and / or in vivo experimental data.

[0187] In some embodiments, a target biomarker signature for lung cancer comprises at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) surface biomarkers (e.g., surface polypeptides present in extracellular vesicles associated with lung cancer; "extracellular vesicle-associated surface biomarkers") and at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) selected from the group consisting of surface biomarkers, intravesicular biomarkers, and intravesicular RNA biomarkers. and (7, 8, or more) target biomarkers, such that the combination of such surface biomarkers and such target biomarkers presents a targeted biomarker signature for lung cancer that provides (a) high specificity (e.g., greater than 98% or higher, e.g., greater than 99%, or greater than 99.5%) that minimizes the number of false positives, and (b) high sensitivity (e.g., greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%) for stage I and II lung cancer, which have the most favorable prognosis.

[0188] In some embodiments, the present disclosure recognizes that, in certain embodiments, the sensitivity rate and specificity rate for subjects with different lung cancer risk levels may vary depending on the risk tolerance guidelines set forth by the attending physician and / or the medical association of interest. In certain embodiments, subjects at risk of lung cancer may be administered with a specificity rate of 85% or higher (e.g., including at least 90%, at least 95%, or higher), and a sensitivity of 50% or higher (e.g., including at least 60%, at least 70%, at least 80%, or higher). In certain embodiments, subjects with lifestyle-related risk factors may be administered with a specificity rate of 85% or higher (e.g., including at least 90%, at least 95%, or higher), and a sensitivity of 50% or higher (e.g., including at least 60%, at least 70%, at least 80%, or higher). In certain embodiments, symptomatic subjects may be tested at a specificity rate of 85% or higher (including, for example, a specificity rate of at least 90%, at least 95%, or higher) and a sensitivity of 50% or higher (including, for example, a sensitivity of at least 60%, at least 70%, at least 80%, or higher). In certain embodiments, asymptomatic subjects may be tested at a specificity rate of 85% or higher (including, for example, a specificity rate of at least 90%, at least 95%, or higher) and a sensitivity of 50% or higher (including, for example, a sensitivity of at least 60%, at least 70%, at least 80%, or higher). In certain embodiments, subjects at risk for lung cancer may be tested at a specificity rate of 99.5% and a sensitivity of 70%, or a specificity rate of 98% and a sensitivity of 80%. In certain embodiments, subjects with lifestyle-related risk factors may be tested with a specificity rate of 99.5% and a sensitivity of 70%, or a specificity rate of 98% and a sensitivity of 80%.In some embodiments, assays described herein for detecting lung cancer in at-risk subjects (e.g., with lifestyle-related risk factors) may have a set sensitivity ratio that is less than 80% sensitivity, including, for example, a sensitivity ratio of less than 70%, less than 60%, less than 50%, or even lower. In certain embodiments, asymptomatic subjects may be subjected to a specificity ratio of 99.5% and a sensitivity of 70%, or a specificity ratio of 98% and a sensitivity of 80%. In some embodiments, assays described herein for detecting lung cancer in asymptomatic subjects may have a set sensitivity ratio that is less than 80% sensitivity, including, for example, a sensitivity ratio of less than 70%, less than 60%, less than 50%, or even lower. In some embodiments, techniques and / or assays described herein for detecting lung cancer in symptomatic subjects may have lower sensitivity and / or specificity requirements than those for detecting lung cancer in asymptomatic subjects. In some embodiments, the assays described herein for detecting lung cancer in symptomatic subjects may have a set specificity ratio that is less than 99.5% specificity, including, for example, a sensitivity ratio of less than 99%, a specificity ratio of less than 95%, less than 90%, or less than 85%. In some embodiments, the assays described herein for detecting lung cancer in symptomatic subjects may have a set sensitivity ratio that is less than 80% sensitivity, including, for example, a sensitivity ratio of less than 70%, or less than 60%.

[0189] The present disclosure observes, among other things, that the gold standard for screening high-risk smokers is chest CT, which had a reported positive predictive value of 3.8% in such high-risk populations in the National Lung Screening Trial study (National Lung Screening Trial Research Team (2013) "Results of initial low-dose computed tomographic screening for lung cancer. New England Journal of Medicine," 368(21): 1980-1991). In some embodiments, the present disclosure recognizes that lung cancer biomarker signatures that provide, among other things, a positive predictive value (PPV) of 3.8% or higher are particularly useful for screening at-risk individuals for lung cancer. In some embodiments, a target biomarker signature for lung cancer comprises at least one surface biomarker (e.g., a surface biomarker present on the surface of extracellular vesicles associated with lung cancer) and at least one target biomarker selected from the group consisting of a surface biomarker, an intravesicular biomarker, and an intravesicular RNA biomarker, such that the combination of such surface biomarkers and such target biomarkers represents a target biomarker signature for lung cancer that provides a positive predictive value (PPV) of at least 3.8% or higher in a high-risk population, including, for example, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10% or higher, at least 15% or higher, at least 20% or higher, at least 25% or higher, and / or at least 30% or higher.

[0190] Generally, gene identifiers as used herein refer to Gene Identifications as classified by the UniProt Consortium (UniProt.org), and one of skill in the art will understand that a particular gene may be known by multiple names and will readily recognize such multiple names.

[0191] Generally, the carbohydrate identifiers used herein refer to the Kegg Cancer-associated Carbohydrates database (genome.jp / kegg / disease / br08441.html), and one of skill in the art will understand that a particular carbohydrate may be known by multiple names and will readily recognize such multiple names.

[0192] In some embodiments, the target biomarker signature for lung cancer includes at least one extracellular vesicle-associated surface biomarker (e.g., a surface polypeptide and / or carbohydrate-dependent marker present on nanoparticles associated with lung cancer), and at least one target biomarker selected from the group consisting of a surface biomarker, an intravesicular biomarker, and an intravesicular RNA biomarker, such that such extracellular vesicle-associated surface biomarkers and such target biomarker combinations are specific for lung cancer. In some embodiments, the target biomarker signature for lung cancer includes CD166 antigen (ALCAM) polypeptide, N-acetyllactosaminide beta-1, encoded by the UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) gene,3-N-acetylglucosaminyltransferase 3 polypeptide, CUB domain-containing protein 1 polypeptide encoded by the CUB domain-containing protein 1 (CDCP1) gene, cadherin-1 (CDH1) polypeptide, cadherin 3 polypeptide encoded by the cadherin 3 (CDH3) gene, complement decay-accelerating factor (CD55) polypeptide, programmed cell death 1 ligand 1 (CD274; also known as PD-L1) polypeptide, carcinoembryonic antigen cell adhesion molecule 5 polypeptide encoded by the carcinoembryonic antigen cell adhesion molecule 5 (CEACAM5) gene, carcinoembryonic antigen cell adhesion molecule 6 polypeptide encoded by the carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) gene, claudin 3 polypeptide encoded by the claudin 3 (CLDN3) gene, claudin 4 polypeptide encoded by the (CLDN4) gene, desmoglein 2 polypeptide encoded by the desmoglein 2 (DSG2) gene, epidermal growth factor receptor (EGFR) polypeptide, epithelial cell adhesion molecule (EPCAM) ) gene, epithelial cell adhesion molecule polypeptide encoded by the folate receptor alpha (FOLR1) gene, folate receptor alpha polypeptide encoded by the folate receptor alpha (FOLR1) gene, gap junction beta-1 protein polypeptide encoded by the gap junction protein beta 1 (GJB1) gene, gap junction beta-2 protein polypeptide encoded by the gap junction protein beta 2 (GJB2) gene, hepatocyte growth factor receptor (MET) polypeptide, insulin-like growth factor 1 receptor (IG1FR) polypeptide, laminin subunit beta-3 polypeptide encoded by the laminin subunit beta 3 (LAMB3) gene, mesothelin (MSLN) polypeptide, mucin 1 (MUC1) polypeptide, GPI transamidase component PIG-T polypeptide encoded by the phosphatidylinositol glycan anchor biosynthesis class T (PIGT) gene, podocalyxin-like protein 2 polypeptide encoded by the podocalyxin-like 2 (PODXL2) gene, and ROS proto-oncogene 1.The proto-oncogene tyrosine-protein kinase ROS polypeptide encoded by the receptor tyrosine kinase (ROS1) gene, the syndecan 1 polypeptide encoded by the syndecan 1 (SDC1) gene, the sodium-dependent phosphate transport protein 2B polypeptide encoded by the solute carrier family 34 (sodium phosphate) member 2 (SLC34A2) gene, the acidic sphingomyelin-like phosphodiesterase 3b polypeptide encoded by the sphingomyelin phosphodiesterase acid-like 3B (SMPDL3B) gene, and the ST14 transmembrane serine protease matriptase (ST14) gene. The polypeptide may be or comprise a suppressor of tumorigenicity 14 protein polypeptide encoded by the gene, a tumor-associated calcium signal transducer 2 (TACSTD2) polypeptide, a transmembrane protease serine 2 polypeptide encoded by the transmembrane serine protease 2 (TMPRSS2) gene, a tumor necrosis factor receptor superfamily member 10B (TNFRSF10B) polypeptide, a tetraspanin-8 polypeptide encoded by the tetraspanin 8 (TSPAN8) gene, sTn polypeptide glycosylation, Tn polypeptide glycosylation, T polypeptide glycosylation, or a combination thereof.

[0193] In some embodiments, the target biomarker signature for lung cancer is protein tyrosine kinase 7 (PTK7), tetraspanin 8 (TSPAN8), cadherin EGF LAG seven transmembrane G-type receptor 2 (CELSR2), glypican 1 (GPC1), suppressor of tumorigenicity 14 protein (ST14), protein tyrosine phosphatase receptor type Z1 (PTPRZ1), G protein-coupled receptor 87 (GPR87), gap junction beta 5 (GJB5), gap junction beta 2 (GJB2), Ras homolog family member V (RHOV), Ly6 / PLAUR domain-containing protein 3 (LYPD3), claudin 7 (CLDN7), desmoplakin (DSP), serine integration factor 2 (SERINC). 2), Abhydrolase domain-containing 17C (ABHD17C), p53 apoptotic effector (PERP), myelin protein zero-like 2 (MPZL2), integrin beta 4 subunit (ITGB4), mesoderm-specific transcript (MEST), glycoprotein Nmb (GPNMB), solute carrier family 35 member A2 (SLC35A2), alpha 1,4-galactosyltransferase (Gb3; CD77), integrin subunit alpha 6 (ITGA6), multidrug resistance-associated protein 5 (ABCC5), ATPase Na+ / K+ transport subunit beta 3 (ATP1B3), Jagged 1 (JAG1), transmembrane serine protease 11D (TMPRSS11D), adhesion G protein-coupled receptor F1 (ADGRF1) polypeptide, phospholipid-transporting ATPase ABCA3 (ABCA3) polypeptide, multidrug resistance-associated protein 1 (ABCC1) polypeptide, ATP-binding cassette subfamily C member 3 (ABCC3) polypeptide, Golgi-resident protein GCP60 (ACBD3) polypeptide, long-chain fatty acid-CoA ligase 5 (ACSL5) polypeptide, advanced glycation end products-specific receptor (AGER) polypeptide, CD166 antigen (ALCAM) polypeptide, AP-1 complex subunit mu 2 (AP1M2) polypeptide, gamma-secretase subunit APH-1A (APH1A) polypeptide, MICOS complex subunit MIC26 (APOO) polypeptide, phospholipid-transporting ATPase IH (ATP11A) polypeptide,Phospholipid-transporting ATPase IF (ATP11B) polypeptide, sodium / potassium-transporting ATPase subunit beta-1 (ATP1B1) polypeptide, renin receptor (ATP6AP2) polypeptide, lactosylceramide 1,3-N-acetyl-beta-D-glucosaminyltransferase (also known as beta-1,3-N-acetylglucosaminyltransferase 5) (B3GNT5) polypeptide, beta-1,4-galactosyltransferase 4 (B4GALT4) polypeptide, B-cell receptor-associated protein 31 (BCAP31) polypeptide, containing a B box and a SPRY domain BSPRY polypeptide, CD109 antigen (CD109) polypeptide, complement decay-accelerating factor (CD55) polypeptide, CD9 antigen (CD9) polypeptide, cell division control protein 42 homolog (CDC42) polypeptide, cadherin-1 (CDH1) polypeptide, cadherin-3 (CDH3) polypeptide, threonylcarbamoyladenosine tRNA methylthiotransferase (CDKAL1) polypeptide, carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) polypeptide, carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) polypeptide, cadherin EGF LAG seven-transmembrane G-type receptor 1 (CELSR1) polypeptide, protein CIP2A (CIP2A) polypeptide, CDGSH iron-sulfur domain-containing protein 2 (CISD2) polypeptide, cytoskeleton-associated protein 4 (CKAP4) polypeptide, calcium-activated chloride channel regulator 2 (CLCA2) polypeptide, claudin 1 (CLDN1) polypeptide, chloride intracellular channel protein 6 (CLIC6) polypeptide, cleft lip and palate transmembrane protein 1-like protein (CLPTM1L) polypeptide, calsyntenin 1 (CLSTN1) polypeptide, contactin 1 (CNTN1) polypeptide, carboxypeptidase D (CPD) polypeptide, cytochrome P450 2S1 (CYP2S1) polypeptide, cytochrome P450 4F11 (CYP4F11) polypeptide, cytochrome P450 4F3 (CYP4F3) polypeptide, putative C-mannosyltransferase DPY19L1 (DPY19L1) polypeptide,Desmocollin 2 (DSC2) polypeptide, Desmocollin 3 (DSC3) polypeptide, Desmoglein 2 (DSG2) polypeptide, Desmoglein 3 (DSG3) polypeptide, Epidermal growth factor receptor (EGFR) polypeptide, Epithelial cell adhesion molecule (EPCAM) polypeptide, Ephrin type B receptor 3 (EPHB3) polypeptide, FAM241B polypeptide, Protocadherin Fat 2 (FAT2) polypeptide, F-box / SPRY domain-containing protein 1 (FBXO45) polypeptide, Fermitin family homolog 1 (FERMT1) polypeptide, Folate receptor alpha (FOLR1) polypeptide, FXYD domain-containing ion transport regulator 3 (FXYD3) polypeptide, Frizzled 6 (FZD6) polypeptide, polypeptide N-acetylgalactosaminyltransferase 1 (GALNT1) polypeptide, polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) polypeptide, polypeptide N-acetylgalactosaminyltransferase 5 (GALNT5) polypeptide, polypeptide N-acetylgalactosaminyltransferase 6 (GALNT6) polypeptide, polypeptide N-acetylgalactosaminyltransferase 14 (GALNT14) polypeptide, vitamin K-dependent gamma-calcium phosphate dehydrogenase (GDC) boxylase (GGCX) polypeptide, Golgi membrane protein 1 (GOLM1) polypeptide, Golgi phosphoprotein 3-like (GOLPH3L) polypeptide, grainy head-like protein 2 homolog (GRHL2) polypeptide, very long-chain (3R)-3-hydroxyacyl-CoA dehydratase 3 (HACD3) polypeptide, hyaluronan synthase 3 (HAS3) polypeptide, immediate early response 3-interacting protein 1 (IER3IP1) polypeptide, immunoglobulin superfamily member 3 (IGSF3) polypeptide, interleukin-1 receptor accessory protein (IL1RAP) polypeptide, integrin alpha 2 (ITGA2) polypeptide, integrin beta 6 (ITGB6) polypeptide, killer cell lectin-like receptor subfamily G member 2 (KLRG2) polypeptide, importin subunit alpha 1 (KPNA2) polypeptide,Keratinocyte-associated protein 3 (KRTCAP3) polypeptide, rhaginin 1 (LAD1) polypeptide, laminin subunit beta 3 (LAMB3) polypeptide, laminin subunit gamma 2 (LAMC2) polypeptide, lysosome-associated membrane glycoprotein 3 (LAMP3) polypeptide, regulatory factor complex protein LAMTOR2 (LAMTOR2) polypeptide, lysocardiolipin acyltransferase 1 (LCLAT1) polypeptide, lysosome-associated transmembrane protein 4B (LAPTM4B) polypeptide, LARGE xylosyl- and glucuronyltransferase 2 (LARGE2) polypeptide, lysophosphatidylcholine acyltransferase 1 (LPCA) polypeptide T1) polypeptide, lipolysis-stimulated lipoprotein receptor (LSR) polypeptide, MAL2 polypeptide, magnesium transporter protein 1 (MAGT1) polypeptide, MARCKS-related protein (MARCKSL1) polypeptide, hepatocyte growth factor receptor (MET) polypeptide, alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (MGAT1) polypeptide, mesothelin (MSLN) polypeptide, mucin 1 (MUC1) polypeptide, mucin 4 (MUC4) polypeptide, nicastrin (NCSTN) polypeptide, nectin 1 (NECTIN1) polypeptide, nectin 4 (NECTIN4) polypeptide, GTPase NRas (NRAS) polypeptide, neural cell adhesion molecule (Nr-CAM) polypeptide, 5'-nucleotidase (NT5E) polypeptide, nuclear pore membrane glycoprotein 210 (NUP210) polypeptide, presenilin-related rhomboid-like protein, mitochondrial (PARL) polypeptide, peroxisomal membrane protein PEX13 (PEX13) polypeptide, GPI ethanolamine phosphotransferase 1 (PIGN) polypeptide, GPI transamidase component PIG-T (PIGT) polypeptide, cytosolic phospholipase A2 (PLA2G4A) polypeptide, l-phosphatidylinositol 4,5-bisphosphate phosphodiesterase eta-1 (PLCH1) polypeptide, plectin (PLEC) polypeptide,26S proteasome non-ATPase regulatory subunit 2 (PSMD2) polypeptide, phosphatidylserine synthase 1 (PTDSS1) polypeptide, prostaglandin F2 receptor negative regulator (PTGFRN) polypeptide, receptor tyrosine-protein phosphatase F (PTPRF) polypeptide, sulfhydryl oxidase 1 (QSOX1) polypeptide, Ras-related protein Rab-25 (RAB25) polypeptide, Ras-related protein Rab-38 (RAB38) polypeptide, Ras-related protein Rab-6B (RAB6B) polypeptide, Ras-related protein Rap-2b (RAP2B) polypeptide, protein RCC2 (RCC2) polypeptide, GTP-binding protein Rit1 (RIT1) polypeptide, secretory carrier-associated membrane protein 3 (SCAMP3) polypeptide, syndecan 1 (SDC1) polypeptide, protein sel-1 homolog 3 (SEL1L3) polypeptide, protein Shroom2 (SHROOM2) polypeptide, solute carrier family 2, facilitative glucose transporter member 1 (SLC2A1) polypeptide, cysteine Inosinate / glutamate transporter (also known as solute carrier family 7 member 11) (SLC7A11) polypeptide, sodium-dependent phosphate transport protein 2B (SLC34A2) polypeptide, adenosine 3'-phospho-5'-phosphosulfate transporter 1 (SLC35B2) polypeptide, zinc transporter ZIP11 (SLC39A11) polypeptide, small integral membrane protein 22 (SMIM22) polypeptide, testisin (also known as eosinophil serine protease 1 (ESP-1) or serine protease 21) polypeptide, acid sphingomyelinase phosphodiesterase-like phosphodiesterase 3b (SMPDL3B) polypeptide, sterol O-acyltransferase 1 (SOAT1) polypeptide, spastin (SPAST) polypeptide, translocon-associated protein subunit alpha (SSR1) polypeptide, translocon-associated protein subunit delta (SSR4) polypeptide, supernumerary locus protein 4 (SURF4) polypeptide, synaptogyrin 2 (SYNGR2) polypeptide, tumor-associated calcium signal transducer 2 (TACSTD2) polypeptide,calcineurin B homologous protein 3 (TESC) polypeptide, transferrin receptor protein 1 (TFRC) polypeptide, transmembrane channel-like protein 5 (TMC5) polypeptide, calcium load-activated calcium channel (TMCO1) polypeptide, transmembrane emp24 domain-containing protein 2 (TMED2) polypeptide, transmembrane emp24 domain-containing protein 3 (TMED3) polypeptide, transmembrane protein 132A (TMEM132A) polypeptide, , transmembrane protein 33 (TMEM33) polypeptide, transmembrane protease serine 4 (TMPRSS4) polypeptide, protein O-mannosyltransferase TMTC3 (TMTC3) polypeptide, mitochondrial import receptor subunit TOM22 homolog (TOMM22) polypeptide, tosin1A-interacting protein 2, isoform IFRG15 (TOR1AIP2) polypeptide, translocation chain-associated membrane protein 1 (TRAM1) polypeptide, transient receptor potential cation channel subfamily V member 4 (TRPV4) polypeptide peptide, tetratricopeptide repeat protein 33 (TTC33) polypeptide, UDP-glucuronosyltransferase 1-6 (UGT1A6) polypeptide, uroplakin 1b (UPK1B) polypeptide, vesicle-associated membrane protein 8 (VAMP8) polypeptide, vacuolar-type ATPase assembly integral membrane protein VMA21 (VMA21) polypeptide, serine / threonine-protein kinase VRK2 (VRK2) polypeptide, von Willebrand factor A domain-containing protein 1 (VWA1) polypeptide, xenotropic and multitopic Torovirus receptor 1 (XPR1) polypeptide, xyloside xylosyltransferase 1 (XXYLT1) polypeptide, a disintegrin and metalloprotease domain-containing protein 28 (ADAM28) polypeptide, tyrosine-protein kinase receptor UFO (AXL) polypeptide, basigin (BSG) polypeptide, programmed cell death 1 ligand 1 (CD274; also known as PD-L1) polypeptide, leukocyte surface antigen CD47 (CD47) polypeptide, clusterin (CLU) polypeptide, Dickkopf-related protein Protein 1 (DKK1) polypeptide, receptor tyrosine-protein kinase erbB-3 (ERBB3) polypeptide, vascular endothelial growth factor receptor 3 (FLT4) polypeptide, (N-glycolylneuraminic acid (NeuGc, NGNA)-ganglioside GM3) (GM3) polypeptide, hepatocyte growth factor (HGF) polypeptide, insulin-like growth factor 1 receptor (IGF1R) polypeptide, interleukin-6 (IL6) polypeptide, vascular endothelial growth factor receptor 2 (KDR) polypeptide, lymphocyte activation gene 3 protein (LAG3) polypeptide,Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1) polypeptide, Lewis X antigen, Lewis Y / B antigen, lymphocyte antigen 6E (LY6E) polypeptide, neurogenic locus notch homolog protein 2 (NOTCH2) polypeptide, neurogenic locus notch homolog protein 3 (NOTCH3) polypeptide, phosphatidylserine-presenting polypeptide, T cell immunoreceptor with Ig and ITIM domains (TIGIT) polypeptide, tumor necrosis factor receptor superfamily member 10A (TNFRSF10A) polypeptide, tumor necrosis factor receptor superfamily member 10B (TNFRSF10B) polypeptide, tumor necrosis factor ligand superfamily member 18 (TNFSF18) polypeptide, trophoblast glycoprotein (TPBG) polypeptide, vascular endothelial growth factor A (VEGFA) polypeptide, sialyl Tn (sTn) antigen, sialyl-6T antigen (6-sialyl core 1), T antigen, phosphatidylserine, αGalNAc-Ser / Thr(Tn) antigen, sialyl Lewis A antigen (CA19-9), Globo The surface biomarker is or comprises a surface biomarker selected from the group consisting of H, Gb5 (SSEA-3), lactotriaosylceramide, (Lc3), Forssman antigen, Lewis X, Lewis Y / B antigen, LY6E, Lewis Y / CD174 antigen, sialyl Lewis X (sLex) (also known as sialyl SSEA-1 (SLX)) antigen, N-glycosyl GM3 ganglioside (NeuGcGM3), and combinations thereof.

[0194] In some embodiments, the extracellular vesicle-associated surface biomarkers included in the target biomarker signature for lung cancer are or include SLC34A2 polypeptide, CEACAM5 polypeptide, CEACAM6 polypeptide, EpCAM polypeptide, and / or a combination thereof. SLC34A2 polypeptide is a multi-transmembrane transporter that is being investigated as a therapeutic target for non-small cell lung cancer (Lin et al., 2015; incorporated herein by reference for purposes described herein). CEACAM5 polypeptide, a member of the carcinoembryonic antigen (CEA) family of cell adhesion molecules (CAMs), is a cell surface glycoprotein that is associated with gastrointestinal cancer and appears to be involved in cell differentiation, apoptosis, and polarity. CEACAM6 polypeptide is a member of the same protein family as CEACAM5, is associated with Crohn's disease and pancreatic adenocarcinoma, and appears to be involved in the innate immune system and cell-surface interactions. EpCAM polypeptide is associated with gastrointestinal cancer and appears to function as a homotypic calcium-independent cell adhesion molecule. In some embodiments, the SLC34A2 polypeptide, the CEACAM5 polypeptide, the CEACAM6 polypeptide, and / or the EpCAM polypeptide are detected as intact EV-associated transmembrane proteins. In some embodiments of the present disclosure, the SLC34A2 polypeptide, the CEACAM5 polypeptide, the CEACAM6 polypeptide, and / or the EPCAM polypeptide are detected as EV-associated transmembrane polypeptides.

[0195] In some embodiments, the surface biomarkers included in the target biomarker signature for lung cancer are an adhesion G protein-coupled receptor F1 polypeptide encoded by the ADGRF1 gene, a CD166 antigen (ALCAM) polypeptide, a bile canalicular multispecific organic anion transporter 2 polypeptide encoded by the ATP-binding cassette subfamily C member 3 (ABCC3) gene, an arylsulfatase L polypeptide encoded by the arylsulfatase L (ARSL) gene, an N-acetyllactosaminide beta-1,3-N-acetylglucosaminyltransferase 3 polypeptide encoded by the UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) gene, and a UDP-GlcNAc:beta-Gal beta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) gene. lactosylceramide 1,3-N-acetyl-beta-D-glucosaminyltransferase polypeptide encoded by the B3GNT5 gene, CUB domain-containing protein 1 polypeptide encoded by the CUB domain-containing protein 1 (CDCP1) gene, cadherin 1 polypeptide encoded by the cadherin 1 (CDH1) gene, cadherin 3 polypeptide encoded by the cadherin 3 (CDH3) gene, complement decay-accelerating factor (CD55) polypeptide, programmed cell death 1 ligand 1 (CD274; also known as PD-L1) polypeptide, carcinoembryonic antigen cell adhesion molecule 5 polypeptide encoded by the carcinoembryonic antigen cell adhesion molecule 5 (CEACAM5) gene, carcinoembryonic antigen cell adhesion molecule 6 polypeptide encoded by the carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) gene, and cadherin EGF Cadherin EGF LAG seven-transmembrane G-type receptor 1 polypeptide encoded by the LAG seven-transmembrane G-type receptor 1 (CELSR1) gene, claudin 18 polypeptide encoded by the claudin 18 (CLDN18) gene, claudin 3 polypeptide encoded by the claudin 3 (CLDN3) gene, claudin 4 polypeptide encoded by the (CLDN4) gene, claudin 7 polypeptide encoded by the claudin 7 (CLDN7) gene,Chloride intracellular channel protein 6 polypeptide encoded by the chloride intracellular channel 6 (CLIC6) gene, deletion-type malignant brain tumor 1 protein polypeptide encoded by the deletion-type malignant brain tumor 1 (DMBT1) gene, desmoglein 2 polypeptide encoded by the desmoglein 2 (DSG2) gene, epidermal growth factor receptor (EGFR) polypeptide, epithelial cell adhesion molecule polypeptide encoded by the epithelial cell adhesion molecule (EPCAM) gene, epoxide hydrolase 3 polypeptide encoded by the epoxide hydrolase 3 (EPHX3) gene, eva-1 homolog A polypeptide encoded by the eva-1 homolog A gene, regulator of programmed cell death (EVA1A) gene, FAM241B protein encoded by the FAM241B gene, folate receptor alpha polypeptide encoded by the folate receptor alpha (FOLR1) gene, FXYD domain-containing ion transport regulator 3 polypeptide encoded by the FXYD3 gene, and N-acetylgalactosaminyltransferase polypeptide encoded by the GALNT14 gene. Gap junction beta 1 protein polypeptide encoded by the gap junction protein beta 1 (GJB1) gene, gap junction beta 2 protein polypeptide encoded by the gap junction protein beta 2 (GJB2) gene, hepatocyte growth factor receptor (MET) polypeptide, insulin-like growth factor 1 receptor (IG1FR) polypeptide, glypican 4 polypeptide encoded by the glypican 4 (GPC4) gene, hyaluronan synthase 3 polypeptide encoded by the HAS3 gene, heparan sulfate 6-O-sulfotransferase 2 polypeptide encoded by the heparin sulfate 6-O-sulfotransferase 2 (HS6ST2) gene, ER luminal protein retention receptor 3 polypeptide encoded by the ER luminal protein retention receptor 3 (KDELR3) gene, keratinocyte-associated protein 3 polypeptide encoded by the keratinocyte-associated protein 3 (KRTCAP3) gene, laminin subunit beta 3 polypeptide encoded by the laminin subunit beta 3 (LAMB3) gene,Lysosome-associated transmembrane protein 4B polypeptide encoded by the LAPTM4B gene, LARGE xylosyl- and glucuronyltransferase 2 polypeptide encoded by the LARGE2 gene, LFNG beta-1,3-N-acetylglucosaminyltransferase lunatic fringe polypeptide encoded by the O-fucosylpeptide 3-beta-N-acetylglucosaminyltransferase (LFNG) gene, lipolysis-stimulated lipoprotein receptor polypeptide encoded by the lipolysis-stimulated lipoprotein receptor (LSR) gene, protein MAL2 encoded by the MAL2 gene, glycoprotein endo-alpha-1,2-mannosidase-like protein polypeptide encoded by the mannosidase endo alpha-like (MANEAL) gene, mesothelin polypeptide encoded by the mesothelin (MSLN) gene, mucin 1 polypeptide encoded by the mucin 1 cell surface-associated (MUC1) gene, mucin 21 polypeptide encoded by the mucin 21 cell surface-associated (MUC21) gene, neural cell adhesion molecule (Nr-CAM) polypeptide encoded by the NRCAM gene, phosphatidylinositol phosphate phosphate phosphate phosphate GPI transamidase component PIG-T polypeptide encoded by the citolglycan anchor biosynthesis class T (PIGT) gene, podocalyxin-like protein 2 polypeptide encoded by the podocalyxin-like 2 (PODXL2) gene, testisin polypeptide encoded by the PRSS21 gene, transmembrane gamma-carboxyglutamic acid protein 4 polypeptide encoded by the proline-rich and Gla domain 4 (PRRG4) gene, proto-oncogene tyrosine-protein kinase ROS polypeptide encoded by the ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) gene, syndecan 1 polypeptide encoded by the syndecan 1 (SDC1) gene, serine incorporation factor 2 polypeptide encoded by the serine incorporation factor 2 (SERINC2) gene, and epilepsy seizure 6-like protein 2 polypeptide encoded by the epileptic seizure-related 6 homolog-like 2 (SEZ6L2) gene.Sodium-dependent phosphate transport protein 2B polypeptide encoded by the solute carrier family 34 (sodium phosphate) member 2 (SLC34A2) gene, choline transporter-like protein 4 polypeptide encoded by the solute carrier family 44 member 4 (SLC44A4) gene, sodium- and chloride-dependent neutral and basic amino acid transporter B(0+) polypeptide encoded by the solute carrier family 6 member 14 (SLC6A14) gene, Y+L amino acid transporter 1 polypeptide encoded by the solute carrier family 7 member 7 (SLC7A7) gene, cysteine / glutamate transporter polypeptide encoded by the solute carrier family 7 member 11 (SLC7A11) gene, small integral membrane protein 22 polypeptide encoded by the small integral membrane protein 22 (SMIM22) gene, acid sphingomyelin phosphodiesterase acid-like 3B (SMPDL3B) gene, and ST14 transmembrane serine / sphingomyelinase polypeptide. suppressor of tumorigenicity 14 protein polypeptide encoded by the protease matriptase (ST14) gene, tumor-associated calcium signal transducer 2 (TACSTD2) polypeptide, transmembrane channel-like protein 4 polypeptide encoded by the transmembrane channel-like 4 (TMC4) gene, transmembrane channel-like protein 5 polypeptide encoded by the transmembrane channel-like 5 (TMC5) gene, transmembrane protein 45B polypeptide encoded by the transmembrane protein 45B (TMEM45B) gene, transmembrane protease serine 2 polypeptide encoded by the transmembrane serine protease 2 (TMPRSS2) gene, transmembrane protease serine 4 polypeptide encoded by the transmembrane serine protease 4 (TMPRSS4) gene, tumor necrosis factor receptor superfamily member 10B (TNFRSF10B) polypeptide, tetraspanin 1 polypeptide encoded by the tetraspanin 1 (TSPAN1) gene, and tetraspanin 8 polypeptide encoded by the tetraspanin 8 (TSPAN8) gene;The surface biomarker is or comprises a surface biomarker selected from the group consisting of a ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCH-L1) polypeptide encoded by the UCHL1 gene, a Lewis X antigen, a sialyl Lewis X antigen, an sTn antigen, a Tn antigen, a T antigen, and a combination thereof.

[0196] In some embodiments, the target biomarker included in the target biomarker signature for lung cancer is or comprises a surface biomarker selected from the group consisting of an SLC34A2 polypeptide, a CEACAM5 polypeptide, a CEACAM6 polypeptide, an EpCAM polypeptide, and combinations thereof.

[0197] In some embodiments, the target biomarker included in the target biomarker signature for lung cancer is or comprises a surface biomarker selected from the group consisting of an ALCAM polypeptide, a CD55 polypeptide, a CDH1 polypeptide, a CDH3 polypeptide, a CD274 (PD-L1) polypeptide, a CEACAM5 polypeptide, a CEACAM6 polypeptide, a DSG2 polypeptide, an EGFR polypeptide, an EPCAM polypeptide, a FOLR1 polypeptide, an IG1FR polypeptide, a MET polypeptide, an MSLN polypeptide, a MUC1 polypeptide, an SLC34A2 polypeptide, an sTn antigen, a Tn antigen, a T antigen, a TACSTD2 polypeptide, a TNFRSF10B polypeptide, and a combination thereof.

[0198] In some embodiments, the target biomarker signature is selected from the group consisting of an ADGRF1 polypeptide, an ABCA3 polypeptide, an ABCC1 polypeptide, an ABCC3 polypeptide, an ACBD3 polypeptide, an ACSL5 polypeptide, an AGER polypeptide, an ALCAM polypeptide, an AP1M2 polypeptide, an APH1A polypeptide, an APOO polypeptide, an ATP11A polypeptide, an ATP11B polypeptide, an ATP1B1 polypeptide, an ATP6AP2 polypeptide, a B3GNT5 polypeptide, a B4GALT4 polypeptide, a BCAP31 polypeptide, a BSPRY polypeptide, a CD109 polypeptide, a CD55 polypeptide, a CD9 polypeptide, a CDC42 polypeptide, a CDH1 polypeptide, a CDH3 polypeptide, a CDKAL1 polypeptide, a CEACAM5 polypeptide, a CEACAM6 polypeptide, a CELSR1 polypeptide, a CIP2A polypeptide, a CISD2 polypeptide, a CKAP4 polypeptide, a CLCA2 polypeptide, a CLDN1 polypeptide, a CLIC6 polypeptide, a CLPTM1L polypeptide, a CLSTN1 polypeptide, a CNTN1 polypeptide, a CPD ... a polypeptide, a CYP2S1 polypeptide, a CYP4F11 polypeptide, a CYP4F3 polypeptide, a DPY19L1 polypeptide, a DSC2 polypeptide, a DSC3 polypeptide, a DSG2 polypeptide, a DSG3 polypeptide, an EGFR polypeptide, an EPCAM polypeptide, an EPHB3 polypeptide, a FAM241B polypeptide, a FAT2 polypeptide, an FBXO45 polypeptide, a FERMT1 polypeptide, a FOLR1 polypeptide, an FXYD3 polypeptide, an FZD6 polypeptide, a GALNT1 polypeptide, a GALNT3 polypeptide, a GALNT5 polypeptide, a GALNT6 polypeptide, a GALNT14 polypeptide, a GGCX polypeptide, a GOLM1 polypeptide, a GOLPH3L polypeptide, a GRHL2 polypeptide, a HACD3 polypeptide, a HAS3 polypeptide, an IER3IP1 polypeptide, an IGSF3 polypeptide, an IL1RAP polypeptide, an ITGA2 polypeptide, an ITGB6 polypeptide, a KLRG2 polypeptide, a KPNA2 polypeptide, a KRTCAP3 polypeptide, a LAD1 polypeptide, a LAMB3 polypeptide, a LAMC2 polypeptide,LAMP3 polypeptide, LAPTM4B polypeptide, LAMTOR2 polypeptide, LARGE2 polypeptide, LCLAT1 polypeptide, LPCAT1 polypeptide, LSR polypeptide, MAGT1 polypeptide, MAL2 polypeptide, MARCKSL1 polypeptide, MET polypeptide, MGAT1 polypeptide, MSLN polypeptide, MUC1 polypeptide, MUC4 polypeptide, NCSTN polypeptide, NECTIN1 polypeptide, NECTIN4 polypeptide, NRAS polypeptide, NRCAM polypeptide, NT5E polypeptide, NUP210 polypeptide, PARL polypeptide, PEX13 polypeptide, PIGN polypeptide, PIGT polypeptide, PLA2G4A polypeptide, PLCH1 polypeptide, PLEC polypeptide, PRSS21 polypeptide, PSMD2 polypeptide, PTDSS1 polypeptide, PTGFRN polypeptide, PTPRF polypeptide, QSOX1 polypeptide, RAB25 polypeptide, RAB38 polypeptide, RAB6B polypeptide, RAP2B polypeptide, RCC2 polypeptide, RIT1 polypeptide, SCA MP3 polypeptide, SDC1 polypeptide, SEL1L3 polypeptide, SHROOM2 polypeptide, SLC2A1 polypeptide, SLC34A2 polypeptide, SLC35B2 polypeptide, SLC39A11 polypeptide, SLC7A11 polypeptide, SMIM22 polypeptide, SMPDL3B polypeptide, SOAT1 polypeptide, SPAST polypeptide, SSR1 polypeptide, SSR4 polypeptide, SURF4 polypeptide, SYNGR2 polypeptide, TACSTD2 polypeptide, TESC polypeptide, TFRC polypeptide, TMC5 polypeptide, TMCO1 polypeptide, TMED2 polypeptide, TMED3 polypeptide, TMEM132A polypeptide, TMEM33 polypeptide, TMPRSS4 polypeptide, TMTC3 polypeptide, TOMM22 polypeptide, TOR1AIP2 polypeptide, TRAM1 polypeptide, TRPV4 polypeptide, TTC33 polypeptide, UGT1A6 polypeptide, UPK1B polypeptide, VAMP8 polypeptide, VMA21 polypeptide, VRK2 polypeptide, VWA1 polypeptide, XPR1 polypeptide,The extracellular vesicle-associated surface biomarkers include one or more extracellular vesicle-associated surface biomarkers selected from the list consisting of XXYLT1 polypeptide, ADAM28 polypeptide, AXL polypeptide, BSG polypeptide, CD274 polypeptide, CD47 polypeptide, CLU polypeptide, DKK1 polypeptide, ERBB3 polypeptide, FLT4 polypeptide, GM3 polypeptide, HGF polypeptide, IGF1R polypeptide, IL6 polypeptide, KDR polypeptide, LAG3 polypeptide, UCHL1 polypeptide, Lewis X antigen, Lewis Y / B antigen, LY6E polypeptide, NOTCH2 polypeptide, NOTCH3 polypeptide, phosphatidylserine-presenting polypeptide, TIGIT polypeptide, TNFRSF10A polypeptide, TNFRSF10B polypeptide, TNFSF18 polypeptide, TPBG polypeptide, VEGFA polypeptide, Tn antigen, Lewis Y / CD174 antigen, sialyl Lewis X (sLex) (also known as sialyl SSEA-1 (SLX)) antigen, NeuGcGM3 ganglioside, and combinations thereof.

[0199] In some embodiments, the target biomarker signature comprises one or more extracellular vesicle-associated surface biomarkers selected from the list consisting of an HS6ST2 polypeptide, a CYP2S1 polypeptide, a HAS3 polypeptide, a LAMC2 polypeptide, an ADAM23 polypeptide, an ABCA13 polypeptide, a TMPRSS4 polypeptide, a UGT1A6 polypeptide, an ILDR1 polypeptide, a CYP4F11 polypeptide, a PIGT polypeptide, a LAMB3 polypeptide, a PRSS21 polypeptide, a DSG3 polypeptide, an SDK2 polypeptide, and combinations thereof.

[0200] In some embodiments, the target biomarker signature comprises one or more extracellular vesicle-associated surface biomarkers selected from the list consisting of an HS6ST2 polypeptide, a CYP2S1 polypeptide, a HAS3 polypeptide, a LAMC2 polypeptide, an ADAM23 polypeptide, an ABCA13 polypeptide, a TMPRSS4 polypeptide, a UGT1A6 polypeptide, an ILDR1 polypeptide, a CYP4F11 polypeptide, a PIGT polypeptide, a LAMB3 polypeptide, a PRSS21 polypeptide, a DSG3 polypeptide, an SDK2 polypeptide, a FERMT1 polypeptide, an EPCAM polypeptide, a SDC1 polypeptide, a PANX2 polypeptide, a ULBP2 polypeptide, an ECE2 polypeptide, a KRTCAP3 polypeptide, a CLCA2 polypeptide, a KPNA2 polypeptide, a TMEM132A polypeptide, an ABCC1 polypeptide, a UPK1B polypeptide, a DSG2 polypeptide, a NECTIN1 polypeptide, a SHISA2 polypeptide, and combinations thereof.

[0201] In some embodiments, the target biomarkers included in the target biomarker signature for lung cancer are an amiloride-sensitive amine oxidase [copper-containing] polypeptide encoded by the amine oxidase copper-containing 1 (AOC1) gene, an unidentified protein C12orf45 polypeptide encoded by the chromosome 12 open reading frame 45 (C12orf45) gene, a cellular retinoic acid binding protein 2 polypeptide encoded by the cellular retinoic acid binding protein 2 (CRABP2) gene, a cysteine ​​SN polypeptide encoded by the cysteine ​​SN (CST1) gene, an ETS translocation variant 4 polypeptide encoded by the ETS variant transcription factor 4 (ETV4) gene, a protein FAM83A polypeptide encoded by the family with sequence similarity 83 member A (FAM83A) gene, a hepatocyte nuclear factor 3-beta polypeptide encoded by the forkhead box A2 (FOXA2) gene, a high mobility group protein B3 polypeptide encoded by the high mobility group box 3 (HMGB3) gene, and a galactolipase A polypeptide encoded by the ... a galectin-3-binding protein polypeptide encoded by the galectin-3-binding protein (LGALS3BP) gene, a macrophage migration inhibitory factor polypeptide encoded by the macrophage migration inhibitory factor (MIF) gene, a napsin A polypeptide encoded by the napsin A aspartic peptidase (NAPSA) gene, a protein phosphatase 1 regulatory subunit 14D polypeptide encoded by the protein phosphatase 1 regulatory inhibitor subunit 14D (PPP1R14D) gene, a protein S100-A14 polypeptide encoded by the S100 calcium-binding protein A14 (S100A14) gene, a serine / threonine-protein kinase SBK1 polypeptide encoded by the SH3 domain-binding kinase 1 (SBK1) gene, a secretoglobin family 3A member 2 polypeptide encoded by the secretoglobin family 3A member 2 (SCGB3A2) gene, and a surfactant-associated protein 2 polypeptide encoded by the surfactant-associated 2 (SFTA2) gene.The intravesicular biomarker is or comprises a pulmonary surfactant-associated protein A1 polypeptide encoded by the surfactant protein A1 (SFTPA1) gene, a pulmonary surfactant-associated protein A2 polypeptide encoded by the surfactant protein A2 (SFTPA2) gene, a pulmonary surfactant-associated protein B polypeptide encoded by the surfactant protein B (SFTPB) gene, a serine protease inhibitor Kazal type 1 polypeptide encoded by the serine peptidase inhibitor Kazal type 1 (SPINK1) gene, a protransforming growth factor alpha polypeptide encoded by the transforming growth factor alpha (TGFA) gene, a zinc finger CCCH domain-containing protein 11A polypeptide encoded by the zinc finger CCCH type-containing 11A (ZC3H11A) gene, and combinations thereof.

[0202] In some embodiments, the target biomarkers in the target biomarker signature for lung cancer are an ABRACL polypeptide, an ACP5 polypeptide, an ADH7 polypeptide, an AGR2 polypeptide, an AIF1 polypeptide, an AKR1C1 polypeptide, an AKR1C2 polypeptide, an AKR1C3 polypeptide, an ALDH1A1 polypeptide, an ALDH3AI polypeptide, an ALDH3B2 polypeptide, an ALG1L polypeptide, an AP1M2 polypeptide, an APOBEC3B polypeptide, an APOBEC3C polypeptide, an ARNTL2 polypeptide, an ASF1B polypeptide, an AURKB polypeptide, a BAIAP2L1 polypeptide, a BIRC5 polypeptide, a C15orf48 polypeptide, a C19orf33 polypeptide, a C1S polypeptide, a C8orf4 polypeptide, a CA9 polypeptide, a CALML3 polypeptide, a CAPNS2 polypeptide, a CBLC polypeptide, a CCL19 polypeptide, a CCNB2 polypeptide, a CDC20 polypeptide, a CDC45 polypeptide, a CDCA4 polypeptide, a CDCA5 polypeptide, a CDK1 polypeptide, a CDKN2A polypeptide, a Tide, CDKN2B polypeptide, CENPW polypeptide, CEP55 polypeptide, CES1 polypeptide, CHMP4C polypeptide, CNN2 polypeptide, CPA3 polypeptide, CRABP2 polypeptide, CSTA polypeptide, CTSC polypeptide, CTSE polypeptide, CYP2S1 polypeptide, DPYSL3 polypeptide, EFS polypeptide, EGLN3 polypeptide, EHF polypeptide, ELF3 polypeptide, ELF4 polypeptide, ENAH polypeptide, ESRP1 polypeptide, EVPL polypeptide, FAM129B polypeptide, FAM60A polypeptide, FAM83D polypeptide, FAM83H polypeptide, FBP1 polypeptide, FERMT1 polypeptide, FOXE1 polypeptide, FOXM1 polypeptide, GBP6 polypeptide, GNA15 polypeptide, GPX2 polypeptide, GRHL2 polypeptide, GSTA1 polypeptide, HCK polypeptide, HOXB7 polypeptide, ID1 polypeptide, IGF2BP2 polypeptide, IMPA2 polypeptide, IRF6 polypeptide, IVL polypeptide, JUP polypeptide,KIAA1522 polypeptide, KIF2C polypeptide, KIFC1 polypeptide, KLF4 polypeptide, KLF5 polypeptide, KRT13 polypeptide, KRT14 polypeptide, KRT15 polypeptide, KRT16 polypeptide, KRT17 polypeptide, KRT18 polypeptide, KRT19 polypeptide, KRT5 polypeptide, KRT6A polypeptide, KRT6B polypeptide, KRT6C polypeptide, KRT7 polypeptide, KRT8 polypeptide, LGALS7B polypeptide, LSP1 polypeptide, MAGEA4 polypeptide, MAGEA6 polypeptide, MCM2 polypeptide, MDFI polypeptide, MYBL2 polypeptide, MYH14 polypeptide, MZB1 polypeptide, NCF2 polypeptide, NNMT polypeptide, NRARP polypeptide, NUP210 polypeptide, NUSAP1 polypeptide, OSGIN1 polypeptide, PALLD polypeptide, PITX1 polypeptide, PKP1 polypeptide, PKP3 polypeptide, PLEK polypeptide, PLEK2 polypeptide, POSTN polypeptide, PPP1R14C polypeptide, PRAME polypeptide Tide, PTPN6 polypeptide, RBP1 polypeptide, RIN2 polypeptide, RIPK4 polypeptide, RPS4Y1 polypeptide, RRM2 polypeptide, S100A11 polypeptide, S100A14 polypeptide, S100A16 polypeptide, S100A2 polypeptide, S100P polypeptide, SERPINB13 polypeptide, SERPINB3 polypeptide, SERPINB5 polypeptide, SH3BP4 polypeptide, SNAI2 polypeptide, SOX2 polypeptide, SPI1 polypeptide, SPINT1 polypeptide, SPRR1A polypeptide, SPRR1B polypeptide, SPRR2A polypeptide, SPRR2D polypeptide, SPRR2E polypeptide, SPRR3 polypeptide, SULF1 polypeptide, SYK polypeptide, SYTL1 polypeptide, TBC1D2 polypeptide, TEAD2 polypeptide, TEAD3 polypeptide, TFAP2C polypeptide, THBS2 polypeptide, TK1 polypeptide, TOP2A polypeptide, TP63 polypeptide, TPD52 polypeptide, TPX2 polypeptide, TRIM29 polypeptide, TRIP13 polypeptide,The intravesicular biomarker is or comprises a vesicular biomarker selected from the group consisting of a UBE2C polypeptide, a YAP1 polypeptide, a ZC3H11A polypeptide, a ZNF217 polypeptide, a ZNF750 polypeptide, and combinations thereof.

[0203] In some embodiments, the target biomarkers included in the target biomarker signature for lung cancer include: [ka] and combinations thereof.

[0204] In some embodiments, the target biomarker signature is [ka] [ka] [ka] and combinations thereof.

[0205] In some embodiments, the target biomarker signature for lung cancer comprises at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) extracellular vesicle-associated surface biomarkers (e.g., those described herein) and at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) surface biomarkers (e.g., those described herein). In some embodiments, at least one extracellular vesicle-associated surface biomarker and at least one surface biomarker are the same. In some embodiments, the at least one extracellular vesicle-associated surface biomarker and at least one surface biomarker of the target biomarker signature for lung cancer are distinct. For example, in some embodiments, the target biomarker signature for lung cancer comprises at least one extracellular vesicle-associated surface biomarker that is or comprises an SLC34A2 polypeptide, and at least one surface biomarker that is or comprises a CEACAM6 polypeptide and / or an EpCAM polypeptide. In some embodiments, the target biomarker signature for lung cancer comprises at least one extracellular vesicle-associated surface biomarker that is or comprises a CEACAM5 polypeptide, and at least one surface biomarker that is or comprises a CEACAM6 polypeptide, and / or an SLC34A2 polypeptide.

[0206] In some embodiments, the target biomarker signature for lung cancer comprises at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) extracellular vesicle-associated surface biomarkers (e.g., those described herein) and at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) intravesicular biomarkers (e.g., those described herein). In some such embodiments, the extracellular vesicle-associated surface biomarkers and the intravesicular biomarkers may be encoded by the same gene, but the former are expressed in the membrane of the extracellular vesicles and the latter are expressed within the extracellular vesicles. In some such embodiments, the extracellular vesicle-associated surface biomarkers and the intravesicular biomarkers may be encoded by different genes.

[0207] In some embodiments, the target biomarker signature for lung cancer comprises at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) extracellular vesicle-associated surface biomarkers (e.g., those described herein) and at least one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) intravesicular RNA (e.g., mRNA) biomarkers (e.g., those described herein). In some such embodiments, the extracellular vesicle-associated surface biomarkers and the intravesicular RNA (e.g., mRNA) biomarkers may be encoded by the same gene. In some such embodiments, the extracellular vesicle-associated surface biomarkers and the intravesicular RNA (e.g., mRNA) biomarkers may be encoded by different genes.

[0208] In some embodiments, the target biomarker signature for lung cancer comprises a combination of biomarkers shown in Table 4A and / or Table 4B. In some embodiments, the biomarkers in such combinations are used as capture probe polypeptide targets (as extracellular vesicle-associated surface biomarkers), for example, as shown in Table 4A and / or Table 4B. In some embodiments, the biomarkers in such combinations are used as detection probe polypeptide targets (as target surface biomarkers), for example, as shown in Table 4A and / or Table 4B.

[0209] In some embodiments, the target biomarker signature for lung cancer includes at least an SLC34A2 polypeptide (as an extracellular vesicle-associated surface biomarker) and a CEACAM6 polypeptide (as a target surface biomarker).

[0210] In some embodiments, the target biomarker signature for lung cancer includes at least an SLC34A2 polypeptide (as an extracellular vesicle-associated surface biomarker) and an EpCAM polypeptide (as a target surface biomarker).

[0211] In some embodiments, the target biomarker signature for lung cancer includes at least a CEACAM5 polypeptide (as an extracellular vesicle-associated surface biomarker) and a CEACAM6 polypeptide (as a target surface biomarker).

[0212] In some embodiments, the target biomarker signature for lung cancer includes at least a CEACAM5 polypeptide (as an extracellular vesicle-associated surface biomarker) and an SLC34A2 polypeptide (as a target surface biomarker).

[0213] In some embodiments, the target biomarker signature for lung cancer includes at least two target surface biomarkers, which may be or may include at least SLC34A2 polypeptide (as an extracellular vesicle-associated surface biomarker), and CEACAM6 polypeptide and EpCAM polypeptide.

[0214] In some embodiments, the target biomarker signature for lung cancer includes at least two target surface biomarkers, which may be or may include at least a CEACAM5 polypeptide (as an extracellular vesicle-associated surface biomarker), and a CEACAM6 polypeptide and an SLC34A2 polypeptide.

[0215] In some embodiments, the target biomarker signature for lung cancer comprises at least one of the following: (i) sTn antigen in combination with CEACAM5, CEACAM6, and / or MUC1; and (ii) MUC1 in combination with CEACAM5, sialyl Lewis X, and / or Lewis Y.

[0216] In some embodiments, the target biomarker signature for lung cancer comprises at least one of the following combinations: (i) (sTn antigen, CEACAM5); (ii) (sTn antigen, CEACAM5, MUC1); (iii) (sTn antigen, MUC1); (iv) (sTn antigen, CEACAM6); (v) (sTn antigen, CEACAM5, CEACAM6); (vi) (sTn antigen, MUC1, CEACAM6); (vii) (MUC1, CEACAM5); (viii) (MUC1, CEACAM5, sialyl Lewis X antigen); and (ix) (MUC1, CEACAM5, Lewis Y antigen).

[0217] In some embodiments, any one of the provided biomarkers can be detected and / or measured by protein and / or RNA (eg, mRNA) expression levels of the wild-type form.

[0218] In some embodiments, any one of the provided biomarkers can be detected and / or measured by protein and / or RNA (e.g., mRNA) expression levels of mutant forms. Thus, some embodiments can include mutant-specific detection of a provided biomarker (e.g., protein and / or RNA, such as, e.g., mRNA).

[0219] As described herein, in some embodiments, a biomarker is or comprises a particular form of one or more polypeptides or proteins (e.g., proforms, truncated forms, modified forms, e.g., glycosylated, phosphorylated, phosphatidylated, lipidated forms, etc.). In some embodiments, detection of such forms detects a majority (and in some embodiments, substantially all) of the polypeptides present in that form (e.g., containing a particular modification, e.g., a particular glycosylation, e.g., sialyl-Tn (sTn) glycosylation, e.g., a truncated O-glycan containing sialic acid α-2,6 linked to GalNAc α-O-Ser / Thr, etc.). In some embodiments, the surface biomarkers described herein may include glycosylated forms of the surface biomarkers. For example, in some embodiments, the surface biomarkers ADGRF1, B3GNT3, B3GNT5, HAS3, LARGE2, MAL2, NRCAM, PODXL2, PRSS21, and SCL7A11 may comprise glycosylation. In some embodiments, the surface biomarkers described herein may comprise lipidated forms of the surface biomarkers. For example, in some embodiments, the surface biomarkers PRSS21 and UCHL1 may comprise lipidation.

[0220] Thus, in some embodiments, a surface biomarker may be or include a glycosylated moiety (e.g., an sTn antigen moiety, a Tn antigen moiety, or a T antigen moiety). The Thomsen-Noubel (Tn) antigen is an O-linked glycan that is thought to be associated with a wide range of tumors. Tn is a single alpha-linked GalNAc attached to Ser or Thr as the first step in the major O-linked glycosylation pathway. Those skilled in the art will understand that in certain embodiments, T antigen typically refers to an O-linked glycan having the structure Galβ1-3GalNAc-.

[0221] In some embodiments, the surface biomarker may be or include a tumor-associated post-translational modification, such as a tumor-specific glycosylation pattern, such as mucins with aberrantly truncated glycans at the first GalNAc (e.g., Tn), or a combination thereof.

[0222] In some embodiments, the target biomarker signature includes a combination of targets represented in Table 4A and / or Table 4B, where the targets may be used in the capture probes and / or detection probes. In some embodiments, the target biomarker signature includes a target of a capture probe represented in Table 4A and / or Table 4B and at least one or more (e.g., including at least two or more) targets of a detection probe (e.g., Detection Probe 1 and / or Detection Probe 2).

[0223] In some embodiments, certain biomarker combinations represented in Table 4A and / or Table 4B that may be particularly useful for lung cancer detection (e.g., have higher sensitivity, specificity, and / or PPV) may undergo an initial round of screening using an advanced stage (e.g., late stage, e.g., stage III and / or IV) lung cancer sample pool and a healthy control sample pool as a reference. In some embodiments, the selected combinations may be further tested using an early stage lung cancer sample pool (e.g., stage I and / or II, appropriately differentiated as needed), a benign lung tumor plasma sample pool (e.g., as described herein), a non-lung cancer sample pool (e.g., as described herein), and / or any combination thereof. In some embodiments, the performance of the biomarker combination may be determined by calculating the difference in assay signal (e.g., Ct criterion) between the healthy sample pool and the lung cancer sample pool.

[0224] In some embodiments, certain biomarker combinations for detecting lung cancer can be selected with a delta Ct greater than the inter-assay variation. For example, in some embodiments, biomarker combinations with a delta Ct greater than 2.0 (corresponding to a 4-fold difference) or 1.0 (corresponding to a 2-fold difference) are considered to provide particularly effective diagnostic utility (e.g., provide a signal greater than the inter-assay variation). For example, see Example 10, which provides an exemplary analysis of certain combinations described herein.

[0225] In some embodiments, a target biomarker signature for lung cancer may be or include a target of a combination described in Table 4A and / or Table 4B. In certain embodiments, a target biomarker signature for lung cancer comprises a set of markers that distinguish late-stage lung cancer samples from control samples (e.g., compared to healthy smoker samples and / or compared to healthy non-smoker samples; see, e.g., Table 4A and / or Table 4B). In certain embodiments, a target biomarker signature for lung cancer comprises a set of markers that distinguish early-stage lung cancer samples from control samples (e.g., compared to healthy smoker samples and / or compared to healthy non-smoker samples; see, e.g., Table 4A and / or Table 4B). In some embodiments, an assay directed to detecting a target biomarker signature for lung cancer may comprise a combination of capture and detection probes described in Table 4A and / or Table 4B.

[0226] In some embodiments, a target biomarker signature for lung cancer may be or may include a combination of targets described in Example 10, Table 4A and / or Table 4B that distinguish subjects with early stage lung cancer (e.g., early stage non-small cell lung cancer, e.g., LUAD and / or LUSC) from subjects who do not have lung cancer (e.g., healthy subjects, or subjects who have a condition that is not lung cancer or is not associated with a lung condition). In some embodiments, a target biomarker signature for lung cancer may be or may include a combination of targets described in Example 10, Table 4A and / or Table 4B that distinguish subjects with end-stage lung cancer (e.g., end-stage non-small cell lung cancer, e.g., LUAD and / or LUSC) from subjects who do not have lung cancer (e.g., healthy subjects, or subjects who have a condition that is not lung cancer or is not associated with a lung condition). In some embodiments, a target biomarker signature for lung cancer may be or may include a combination of targets described in Example 10, Table 4A and / or Table 4B that distinguish subjects with early stage lung cancer (e.g., early stage non-small cell lung cancer, e.g., LUAD and / or LUSC) from subjects with late stage lung cancer (e.g., late stage non-small cell lung cancer, e.g., LUAD and / or LUSC).

[0227] In some embodiments, a target biomarker signature for lung cancer may be or may include a combination of targets that distinguish lung cancer from healthy samples in at least eight of the eight conditions tested as described in Example 10, Table 4A and / or Table 4B. In some embodiments, a target biomarker signature for lung cancer may be or may include a combination of targets that distinguish lung cancer from healthy samples in at least seven of the eight conditions tested as described in Example 10, Table 4A and / or Table 4B. In some embodiments, a target biomarker signature for lung cancer may be or may include a combination of targets that distinguish lung cancer from healthy samples in at least six of the eight conditions tested as described in Example 10, Table 4A and / or Table 4B. In some embodiments, a target biomarker signature for lung cancer may be or may include a combination of targets that distinguish lung cancer from healthy samples in at least five of the eight conditions tested as described in Example 10, Table 4A and / or Table 4B. In some embodiments, a target biomarker signature for lung cancer may be or may include a combination of targets that distinguish lung cancer from healthy samples in at least four of the eight conditions tested as described in Example 10, Table 4A and / or Table 4B. In some embodiments, a target biomarker signature for lung cancer may be or may include a combination of targets that distinguish lung cancer from healthy samples in at least three of the eight conditions tested as described in Example 10, Table 4A and / or Table 4B. In some embodiments, a target biomarker signature for lung cancer may be or may include a combination of targets that distinguishes lung cancer from healthy samples in at least two of the eight conditions tested as described in Example 10, Table 4A and / or Table 4B.In some embodiments, a target biomarker signature for lung cancer may be or may include a combination of targets that distinguishes lung cancer from healthy samples in at least one of the eight conditions tested as described in Example 10, Table 4A and / or Table 4B.

[0228] In certain embodiments, the target biomarker signature for detecting lung cancer is a TNFRSFlOB biomarker and a PD-L1 biomarker; or a TNFRSFlOB biomarker and a CEACAM6 biomarker; or a TNFRSFlOB biomarker and an EGFR biomarker; or a TNFRSFlOB biomarker and an IGF1R biomarker; or an ALCAM biomarker and an EPCAM biomarker; or a CEACAM6 biomarker and a MUC1 biomarker; or an EGFR biomarker and a T antigen biomarker; or an EPCAM biomarker and a T biomarker; or a FOLR1 biomarker and a T antigen biomarker; or a Tn antigen biomarker and a TACSTD2 biomarker; or a TNFRSFlOB biomarker and a FOLR1 biomarker; or a Tn antigen biomarker and a sTn antigen biomarker; or an ALCAM biomarker and a PD-L1 biomarker; or an EPCAM biomarker and a MUC1 biomarker; or a TNFRSF10B biomarker and a CD55 biomarker; or a TNFRSF10B biomarker and a MUC1 biomarker; or a FOLR1 biomarker and a TACSTD2 biomarker; or a MET biomarker and a MUC1 biomarker; or a MET biomarker and a sTn antigen biomarker; or a MUC1 biomarker and a TACSTD2 biomarker; or a PD-L1 biomarker and a MUC1 biomarker; or a PD-L1 biomarker and a Tn antigen biomarker; or a SLC34A2 biomarker and a MET biomarker; or a SLC34A2 biomarker and a T antigen biomarker; or a TNFRSF10B biomarker and a CEACAM5 biomarker; or a TNFRSF10B biomarker and an MSLN biomarker; or a TNFRSF10B biomarker and a Tn biomarker; or combinations thereof.

[0229] In certain embodiments, a target biomarker signature for detecting lung cancer comprises the TNFRSF10B biomarker and the PD-L1 biomarker. In certain embodiments, a target biomarker signature for detecting lung cancer comprises the TNFRSF10B biomarker and the CEACAM6 biomarker. In certain embodiments, a target biomarker signature for detecting lung cancer comprises the TNFRSF10B biomarker and the EGFR biomarker. In certain embodiments, a target biomarker signature for detecting lung cancer comprises the TNFRSF10B biomarker and the IGF1R biomarker. In certain embodiments, a target biomarker signature for detecting lung cancer comprises the ALCAM biomarker and the EPCAM biomarker. In certain embodiments, a target biomarker signature for detecting lung cancer comprises the CEACAM6 biomarker and the MUC1 biomarker. In certain embodiments, a target biomarker signature for detecting lung cancer comprises the EGFR biomarker and the T antigen biomarker. In certain embodiments, a target biomarker signature for detecting lung cancer comprises an EPCAM biomarker and a T antigen biomarker. In certain embodiments, a target biomarker signature for detecting lung cancer comprises a FOLR1 biomarker and a T antigen biomarker. In certain embodiments, a target biomarker signature for detecting lung cancer comprises a Tn antigen biomarker and a TACSTD2 biomarker. In certain embodiments, a target biomarker signature for detecting lung cancer comprises a TNFRSF10B biomarker and a FOLR1 biomarker. In certain embodiments, a target biomarker signature for detecting lung cancer comprises a TNFRSF10B biomarker and a sTn antigen biomarker. In certain embodiments, a target biomarker signature for detecting lung cancer comprises an ALCAM biomarker and a PD-L1 biomarker. In certain embodiments, a target biomarker signature for detecting lung cancer comprises an EPCAM biomarker and a MUC1 biomarker.In certain embodiments, a target biomarker signature for detecting lung cancer comprises the TNFRSF10B biomarker and the CD55 biomarker. In certain embodiments, a target biomarker signature for detecting lung cancer comprises the TNFRSF10B biomarker and the MUC1 biomarker. In certain embodiments, a target biomarker signature for detecting lung cancer comprises the FOLR1 biomarker and the TACSTD2 biomarker. In certain embodiments, a target biomarker signature for detecting lung cancer comprises the MET biomarker and the MUC1 biomarker. In certain embodiments, a target biomarker signature for detecting lung cancer comprises the MET biomarker and the sTn antigen biomarker. In certain embodiments, a target biomarker signature for detecting lung cancer comprises the MUC1 biomarker and the TACSTD2 biomarker. In certain embodiments, a target biomarker signature for detecting lung cancer comprises the PD-L1 biomarker and the MUC1 biomarker. In certain embodiments, a target biomarker signature for detecting lung cancer comprises a PD-L1 biomarker and a Tn antigen biomarker. In certain embodiments, a target biomarker signature for detecting lung cancer comprises a SLC34A2 biomarker and a MET biomarker. In certain embodiments, a target biomarker signature for detecting lung cancer comprises a SLC34A2 biomarker and a T antigen biomarker. In certain embodiments, a target biomarker signature for detecting lung cancer comprises a TNFRSF10B biomarker and a CEACAM5 biomarker. In certain embodiments, a target biomarker signature for detecting lung cancer comprises a TNFRSF10B biomarker and an MSLN biomarker. In certain embodiments, a target biomarker signature for detecting lung cancer comprises a TNFRSF10B biomarker and a Tn antigen biomarker.

[0230] In some embodiments, the target biomarker signature comprises a combination of at least two target biomarkers, the combination being: a CYP2S1 polypeptide and an HS6ST2 polypeptide; or an ADAM23 polypeptide and a CYP2S1 polypeptide; or an ADAM23 polypeptide and a CYP4F11 polypeptide; or an ADAM23 polypeptide and a UGT1A6 polypeptide; or an ADAM23 polypeptide and a TMPRSS4 polypeptide; or an ADAM23 polypeptide and an ILDR1 polypeptide; or a DSG3 polypeptide and a UPK1B polypeptide; or an ABCA13 polypeptide and a CYP2S1 polypeptide; or an ABCA13 polypeptide and an ADAM23 polypeptide; or an ADAM23 polypeptide and a LAMC2 polypeptide; or an ADAM23 polypeptide and a HAS3 polypeptide; or an HS6ST2 polypeptide and a LAMC2 polypeptide; or a CYP4F11 polypeptide and an HS6ST2 polypeptide; or an ADAM23 polypeptide and and a ULBP2 polypeptide; or an ABCA13 polypeptide and an HS6ST2 polypeptide; or a CYP4F11 polypeptide and a LAMC2 polypeptide; or an ABCA13 polypeptide and a UPK1B polypeptide; or an ABCA13 polypeptide and a CYP4F11 polypeptide; or a CYP2S1 polypeptide and a LAMC2 polypeptide; or a CYP2S1 polypeptide and a CYP4F11 polypeptide; or an HS6ST2 polypeptide and a PIGT polypeptide; or a CYP4F11 polypeptide and a SHISA2 polypeptide; or an ABCA13 polypeptide and a DSG2 polypeptide; or an ADAM23 polypeptide and a LAMB3 polypeptide; or a CYP2S1 polypeptide and a VTCN1 polypeptide; or a CYP4F11 polypeptide and a HAS3 polypeptide; or a CYP4F11 polypeptide and an SLC7A11 polypeptide; or an ADAM23 polypeptide and a DSG3 polypeptide; or an ADAM23 polypeptide and a FERMT1 polypeptide;or an ADAM23 polypeptide and an HS6ST2 polypeptide; or an HS6ST2 polypeptide and a ULBP2 polypeptide; or a CYP4F11 polypeptide and a RAP2B polypeptide; or a RACGAP1 polypeptide and a TFRC polypeptide; or a CYP2S1 polypeptide and a ULBP2 polypeptide; or a KLRG2 polypeptide and a UPK1B polypeptide; or a CLCA2 polypeptide and a CYP2S1 polypeptide; or an ADAM23 polypeptide and a PANX2 polypeptide; or an ABCA13 polypeptide and a HAS3 polypeptide; or an ADAM23 polypeptide and an SLC7A11 polypeptide; or a CYP4F11 polypeptide and a DSG3 polypeptide; or an ADAM23 polypeptide and a CYP4F3 polypeptide; or a CYP2S1 polypeptide and a KLRG2 polypeptide; or an HS6ST2 polypeptide and a SLC7A11 polypeptide; or an ADAM23 polypeptide and a RAP2B polypeptide; or a CYP4F11 polypeptide tide and a PIGT polypeptide; or an ADAM23 polypeptide and an SDC1 polypeptide; or an ADAM23 polypeptide and an ECE2 polypeptide; or a DSG2 polypeptide and a MARVELD2 polypeptide; or a CYP2S1 polypeptide and a SHISA2 polypeptide; or an ABCA13 polypeptide and a UGT1A6 polypeptide; or an ABCC1 polypeptide and a CYP4F11 polypeptide; or an ADAM23 polypeptide and a PIGT polypeptide; or a CYP2S1 polypeptide and a PIGT polypeptide; or a CYP2S1 polypeptide and an ILDR1 polypeptide; or a CYP2S1 polypeptide and a NECTIN1 polypeptide; or a CYP4F11 polypeptide and a RAB6B polypeptide; or a CYP4F11 polypeptide and a KLRG2 polypeptide; or an ADAM23 polypeptide and an FXYD3 polypeptide; or a CLCA2 polypeptide and a CYP4F11 polypeptide; or an ABCA13 polypeptide and a DSG3 polypeptide;or a CYP4F11 polypeptide and a XXYLT1 polypeptide; or an ADAM23 polypeptide and a PRSS21 polypeptide; or an FGFBP1 polypeptide and a MARVELD3 polypeptide; or a CYP2S1 polypeptide and a HAS3 polypeptide; or an ADAM23 polypeptide and a CLCA2 polypeptide; or a CYP4F11 polypeptide and a NECTIN1 polypeptide; or a CLCA2 polypeptide and an HS6ST2 polypeptide; or an HS6ST2 polypeptide and a PRSS21 polypeptide; or a CYP2S1 polypeptide and a DSG2 polypeptide; or a CD9 polypeptide and a CYP4F11 polypeptide; or a CYP4F11 polypeptide and a FERMT1 polypeptide; or a CYP4F11 polypeptide and a TFRC polypeptide; or an ADAM23 polypeptide and an EPCAM polypeptide; or a DSG2 polypeptide and an FBXO45 polypeptide; or a CYP2S1 polypeptide and a PANX2 polypeptide; or a CYP2S1 polypeptide and a PRSS21 polypeptide or a CYP4F11 polypeptide and an SDK1 polypeptide; or a CYP4F11 polypeptide and a ULBP2 polypeptide; or an ABCA13 polypeptide and a PIGT polypeptide; or an HS6ST2 polypeptide and a RAP2B polypeptide; or a CYP2S1 polypeptide and an ECE2 polypeptide; or an ADAM23 polypeptide and a NECTIN1 polypeptide; or a CYP2S1 polypeptide and an SLC7A11 polypeptide; or an ECE2 polypeptide and an HS6ST2 polypeptide; or a CYP4F11 polypeptide and an SDK2 polypeptide; or a PACC1 polypeptide and a TFRC polypeptide; or a CYP4F11 polypeptide and a KPNA2 polypeptide; or an ADAM23 polypeptide and an SLC12A8 polypeptide; or an ADAM23 polypeptide and an APOO polypeptide; or an APOO polypeptide and a MARVELD2 polypeptide; or a CD9 polypeptide and a PACC1 polypeptide; or an ECE2 polypeptide and a UPK1B polypeptide;Or an ILDR1 polypeptide and a MARVELD2 polypeptide; or an ITGA2 polypeptide and a TMEM158 polypeptide; or an ADAM23 polypeptide and a UCHL1 polypeptide; or a CYP4F11 polypeptide and a PANX2 polypeptide; or a CYP4F11 polypeptide and an ILDR1 polypeptide; or a CYP4F11 polypeptide and an NRCAM polypeptide; or an ADAM23 polypeptide and a CDH1 polypeptide; or a CYP4F11 polypeptide and an ECE2 polypeptide; or combinations thereof. In some embodiments, the target biomarkers in the aforementioned combinations may be used as targets for capture probes and / or detection probes in the assays described herein.

[0231] In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or comprise a CYP2S1 polypeptide and an HS6ST2 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or comprise an ADAM23 polypeptide and a CYP2S1 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or comprise an ADAM23 polypeptide and a TMPRSS4 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or comprise an ADAM23 polypeptide and an ILDR1 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or comprise an ABCA13 polypeptide and a CYP2S1 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or comprise an ABCA13 polypeptide and an ADAM23 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or comprise an HS6ST2 polypeptide and a LAMC2 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or comprise a CYP4F11 polypeptide and a HS6ST2 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or comprise an ABCA13 polypeptide and a HS6ST2 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or comprise an ADAM23 polypeptide and a CYP4F11 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or comprise an ADAM23 polypeptide and a UGT1A6 polypeptide.In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or comprise an ADAM23 polypeptide and a LAMC2 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or comprise an ADAM23 polypeptide and a HAS3 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or comprise an ADAM23 polypeptide and a LAMB3 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or comprise an ADAM23 polypeptide and a ULBP2 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or comprise an ADAM23 polypeptide and a DSG3 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or comprise a CYP4F11 polypeptide and a LAMC2 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include a CYP4F11 polypeptide and an SLC7A11 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include an ADAM23 polypeptide and an FERMT1 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include an ADAM23 polypeptide and a CYP4F3 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include a RACGAP1 polypeptide and a TFRC polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include a CYP4F11 polypeptide and an HAS3 polypeptide.In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or comprise a CD9 polypeptide and a DSG3 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or comprise a DSG3 polypeptide and a UPK1B polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or comprise a DSG3 polypeptide and a RACGAP1 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or comprise an EPCAM polypeptide and a LAMP3 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or comprise a DSG3 polypeptide and an ITGA2 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or comprise a CDH1 polypeptide and a DSG3 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or comprise a CDH3 polypeptide and a DSG3 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include a KPNA2 polypeptide and a ULBP2 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include a TFRC polypeptide and a ULBP2 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include a DSG3 polypeptide and a VTCN1 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include a ULBP2 polypeptide and a UPK1B polypeptide.In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include a DSG3 polypeptide and a NECTIN1 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include a DSG3 polypeptide and a PTPRZ1 polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include a KPNA2 polypeptide and a UPK1B polypeptide. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include a RACGAP1 polypeptide and a ULBP2 polypeptide. In some embodiments, the target biomarkers in the above combinations may be used as targets for capture probes and / or detection probes in the assays described herein.

[0232] In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include TNFRSF10B and PD-L1 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include TNFRSF10B and CEACAM6 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include TNFRSF10B and EGFR biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include TNFRSF10B and IGF1R biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include ALCAM and EPCAM biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include CEACAM6 and MUC1 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include EGFR and T antigen biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include EPCAM and T biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include FOLR1 and T biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include Tn antigen and TACSTD2 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include TNFRSF10B and FOLR1 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include TNFRSF10B and sTn antigen biomarkers.In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include ALCAM and PD-L1 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include EPCAM and MUC1 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include TNFRSF10B and CD55 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include TNFRSF10B and MUC1 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include FOLR1 and TACSTD2 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include MET and MUC1 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include MET and sTn antigen biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include MUC1 and TACSTD2 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include PD-L1 and MUC1 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include PD-L1 and Tn antigen biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include SLC34A2 and MET biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include SLC34A2 and T antigen biomarkers.In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include the TNFRSF10B and CEACAM5 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include the TNFRSF10B and MSLN biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include the TNFRSF10B and Tn antigen biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include the ALCAM and T antigen biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include the ALCAM and TACSTD2 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include the CD55 and PD-L1 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include CDH3 and CDH1 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include CEACAM5 and MUC1 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include CEACAM6 and EGFR biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include CEACAM6 and EPCAM biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include CEACAM6 and FOLR1 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include CEACAM6 and MSLN biomarkers.In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include CEACAM6 and sTn antigen biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include CEACAM6 and TACSTD2 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include DSG2 and CEACAM6 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include EGFR and MUC1 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include FOLR1 and MUC1 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include MUC1 and sTn antigen biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include SLC34A2 and MSLN biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include T and CDH1 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include Tn antigen and IGF1R biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include TNFRSF10B and ALCAM biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include CD55 and EPCAM biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include CEACAM6 and PD-L1 biomarkers.In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include CEACAM6 and SLC34A2 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include DSG2 and CEACAM5 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include DSG2 and EPCAM biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include DSG2 and MET biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include DSG2 and T biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include EGFR and sTn antigen biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include EGFR and TACSTD2 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include EPCAM and TACSTD2 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include IGF1R and T biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include MET and Tn antigen biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include MSLN and T antigen biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include PD-L1 and sTn antigen biomarkers.In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include PD-L1 and T antigen biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include SLC34A2 and MUC1 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include PD-L1 and T antigen biomarkers. The biomarker signature comprises at least two target biomarkers that are or include SLC34A2 and PD-L1 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include SLC34A2 and sTn antigen biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include SLC34A2 and Tn antigen biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include TACSTD2 and sTn antigen biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include TNFRSF10B and CDH3 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include TNFRSF10B and EPCAM biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include TNFRSF10B and MET biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include ALCAM and CDH3 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include ALCAM and MET biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include ALCAM and MUC1 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include ALCAM and SLC34A2 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include ALCAM and sTn antigen biomarkers.In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include CD55 and CDH1 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include CD55 and MET biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include CDH3 and T biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include CEACAM5 and MSLN biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include CEACAM5 and TACSTD2 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include CEACAM6 and Tn antigen biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include DSG2 and CDH1 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include DSG2 and CDH3 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include DSG2 and MUC1 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include DSG2 and SLC34A2 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include DSG2 and sTn antigen biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include EGFR and MSLN biomarkers.In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include FOLR1 and sTn antigen biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include FOLR1 and Tn antigen biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include MET and T antigen biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include MSLN and sTn antigen biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include MUC1 and T antigen biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include MUC1 and Tn antigen biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers that are or include SLC34A2 and EGFR biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers, which are or include TNFRSF10B and CDH1 biomarkers. In some embodiments, the target biomarker signature comprises at least two target biomarkers, which are or include TNFRSF10B and SLC34A2 biomarkers. In some embodiments, the target biomarkers in the above combinations may be used as targets for capture probes and / or detection probes in the assays described herein.

[0233] In some embodiments, the target biomarker signature comprises a combination of at least three target biomarkers, the combination being: an ADAM23 polypeptide, a CYP2S1 polypeptide, and a LAMC2 polypeptide; or a CYP2S1 polypeptide, an HS6ST2 polypeptide, and a LAMC2 polypeptide; or a CYP2S1 polypeptide, an HS6ST2 polypeptide, and a PIGT polypeptide; or an ADAM23 polypeptide, an ILDR1 polypeptide, and a LAMC2 polypeptide; or an ABCA13 polypeptide, a CYP2S1 polypeptide, and a DSG2 polypeptide; or a CYP2S1 polypeptide, an HS6ST2 polypeptide, and a KPNA2 polypeptide; or an ABCA13 polypeptide, an ADAM23 polypeptide, and a UGT1A6 polypeptide; or a CYP2S1 polypeptide, an HS6ST2 polypeptide, and a ULBP2 polypeptide; or an ADAM23 polypeptide, a UGT1A6 polypeptide and a ULBP2 polypeptide; or an ADAM23 polypeptide, a CYP2S1 polypeptide, and a VTCN1 polypeptide; or an ABCA13 polypeptide, an ADAM23 polypeptide, and a CYP2S1 polypeptide; or a CLCA2 polypeptide, a CYP2S1 polypeptide, and an HS6ST2 polypeptide; or an ABCA13 polypeptide, a CYP2S1 polypeptide, and a UGT1A6 polypeptide; or a CYP2S1 polypeptide, an HS6ST2 polypeptide, and a NECTIN1 polypeptide; or an ABCA13 polypeptide, a CYP2S1 polypeptide, and a FERMT1 polypeptide; or an ABCA13 polypeptide, a DSG2 polypeptide, and a HAS3 polypeptide; or an ABCA13 polypeptide, an ADAM23 polypeptide, and a DSG3 polypeptide; or an ADAM23 polypeptide, a RAP2B polypeptide, and a TMPRSS4 polypeptide; or an ADAM23 polypeptide, a PIGT polypeptide, and a TMPRSS4 polypeptide;or an ADAM23 polypeptide, a TMPRSS4 polypeptide, and a ULBP2 polypeptide; or a CYP2S1 polypeptide, a HAS3 polypeptide, and a PIGT polypeptide; or a CYP2S1 polypeptide, an HS6ST2 polypeptide, and a XXYLT1 polypeptide; or an ADAM23 polypeptide, an ILDR1 polypeptide, and a ULBP2 polypeptide; or an ADAM23 polypeptide, a LAMC2 polypeptide, and a UGT1A6 polypeptide; or an ADAM23 polypeptide, a CLCA2 polypeptide, and a CYP2S1 polypeptide; or an ABCA13 polypeptide, an ADAM23 polypeptide, and a FERMT1 polypeptide; or an ADAM23 polypeptide, a CYP2S1 polypeptide, and an SDC1 polypeptide; or an ABCA13 polypeptide, an ADAM23 polypeptide, and a HAS3 polypeptide; or an ABCA13 polypeptide, a CYP2S1 polypeptide, and a HAS3 polypeptide; or an ABCA13 polypeptide, a DSG2 polypeptide, and an HS6ST2 polypeptide; or a CYP2S1 polypeptide, an HS6ST2 polypeptide, and an SDK2 polypeptide; or a CYP2S1 polypeptide, an HS6ST2 polypeptide, and a KLRG2 polypeptide; or an ADAM23 polypeptide, a CYP2S1 polypeptide, and a FERMT1 polypeptide; or an ABCA13 polypeptide, an ADAM23 polypeptide, and a CDH3 polypeptide; or an ABCA13 polypeptide, a CYP2S1 polypeptide, and an ECE2 polypeptide; or an ADAM23 polypeptide, a CDH3 polypeptide, and an EPCAM polypeptide; or a CELSR1 polypeptide, a CYP2S1 polypeptide, and an HS6ST2 polypeptide; or an ADAM23 polypeptide, a CDH3 polypeptide, and an ILDR1 polypeptide; or an ADAM23 polypeptide, a CYP2S1 polypeptide, and an ILDR1 polypeptide; or an ABCA13 polypeptide, a CYP2S1 polypeptide, and a NECTIN1 polypeptide; or an ADAM23 polypeptide, a LAMC2 polypeptide, and a TMPRSS4 polypeptide;or an ABCA13 polypeptide, a CYP2S1 polypeptide, and a DSG3 polypeptide; or an ADAM23 polypeptide, a LAMP3 polypeptide, and a UGT1A6 polypeptide; or a CYP2S1 polypeptide, an HS6ST2 polypeptide, and a RAP2B polypeptide; or a CYP2S1 polypeptide, an ECE2 polypeptide, and an HS6ST2 polypeptide; or an ADAM23 polypeptide, a CYP2S1 polypeptide, and a HAS3 polypeptide; or an ADAM23 polypeptide, an ILDR1 polypeptide, and a LAMB3 polypeptide; or an ADAM23 polypeptide, an ILDR1 polypeptide, and a RAP2B polypeptide; or a CYP2S1 polypeptide, an FBXO45 polypeptide, and an HS6ST2 polypeptide; or an ABCA13 polypeptide, a CYP2S1 polypeptide, and a HS6ST2 polypeptide; or a CYP2S1 polypeptide, a LAMC2 polypeptide, and a PIGT polypeptide; or an ADAM23 polypeptide, an ECE2 polypeptide, and an ILDR1 polypeptide. or an ADAM23 polypeptide, a KLRG2 polypeptide, and a TMPRSS4 polypeptide; or an ABCA13 polypeptide, a FERMT1 polypeptide, and an HS6ST2 polypeptide; or an ABCA13 polypeptide, a DSG2 polypeptide, and a PIGT polypeptide; or a CDH3 polypeptide, a CYP2S1 polypeptide, and an ILDR1 polypeptide; or an ADAM23 polypeptide, an ILDR1 polypeptide, and a PIGT polypeptide; or an ADAM23 polypeptide, a CYP2S1 polypeptide, and an ECE2 polypeptide; or a CYP2S1 polypeptide, an HS6ST2 polypeptide, and a PRSS21 polypeptide; or an HS6ST2 polypeptide, a LAMC2 polypeptide, and an LSR polypeptide; or an ADAM23 polypeptide, a CYP2S1 polypeptide, and HS6ST2 polypeptide; or an ADAM23 polypeptide, an ECE2 polypeptide, and a TMPRSS4 polypeptide; or an HS6ST2 polypeptide, a LAMC2 polypeptide, and a RAP2B polypeptide;or an ADAM23 polypeptide, a HAS3 polypeptide, and an ILDR1 polypeptide; or an ABCA13 polypeptide, an ADAM23 polypeptide, and a NECTIN1 polypeptide; or a ...

Claims

1. A method for detecting lung cancer-associated nanoparticles, comprising: capturing nanoparticles having a size of about 30 nm to about 1000 nm from the biological sample using a capture agent linked to a solid substrate, wherein the capture agent binds to a surface biomarker for lung cancer on the nanoparticles; contacting the captured nanoparticles with at least two detection probes directed to biomarkers for lung cancer, each comprising an oligonucleotide domain with a single-stranded overhang, wherein when the detection probes bind to the same nanoparticle, the overhangs hybridize to form a double-stranded complex; contacting the double-stranded complex with a nucleic acid ligase to produce a ligated template; and detecting the ligated template, wherein the presence of the ligated template indicates co-localization of the biomarker for lung cancer on the nanoparticle. A method comprising:

2. The method of claim 1, wherein the capture agent comprises an antibody agent directed against a membrane-associated polypeptide.

3. The method described in claim 1, wherein the detection probes include antibody agents targeting different surface biomarkers.

4. The method described in claim 1, wherein the first detection probe targets a surface biomarker and the second detection probe targets an intravesicular protein biomarker.

5. The method of claim 1, wherein the first detection probe comprises an antibody agent directed to a surface biomarker, and the second detection probe comprises a nucleic acid-based agent directed to an intravesicular RNA biomarker.

6. The method of claim 1, wherein the solid substrate is a magnetic bead.

7. The method of claim 1, wherein each oligonucleotide domain comprises a double-stranded portion and the single-stranded overhang extending from one end of the double-stranded portion.

8. The method described in claim 1, wherein the single-stranded overhangs of the detection probes are complementary to each other.

9. The method of claim 1, further comprising amplifying the ligated template prior to detection.

10. The method of claim 9, wherein the amplifying comprises quantitative PCR.

11. The method of claim 1, wherein the capture agent targets a membrane-bound polypeptide and the detection probes each target a biomarker selected from a surface protein biomarker, an intravesicular protein biomarker, or an intravesicular RNA biomarker.

12. The method of claim 1, wherein the surface biomarker bound by the capture agent is a membrane-bound polypeptide, a first detection probe is directed to a surface protein biomarker, and a second detection probe is directed to an intravesicular RNA biomarker.

13. The method of claim 1, further comprising a step of washing to remove unbound detection probe prior to the step of contacting with the nucleic acid ligase.

14. The method described in claim 1, wherein the biological sample is subjected to size exclusion chromatography to isolate nanoparticles containing extracellular vesicles.

15. The method described in claim 1, further comprising repeating the method using an orthogonal target biomarker signature for lung cancer.

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